(Toxinology) P. Gopalakrishnakone, Anita Malhotra (Eds.) - Evolution of Venomous Animals and Their Toxins-Springer Netherlands

Evolution of Venomous Animals and Their Toxins
DOI 10.1007/978-94-007-6727-0_1-1
# Springer Science+Business Media Dordrecht 2015
Evolution, Morphology and Development of the Centipede Venom System

Michel M. Dugon*
Discipline of Zoology – School of Natural Sciences, National University of Ireland Galway, Galway, Ireland
Abstract
With approximately 3,500 species distributed across five extant orders, centipedes (class Chilopoda)
make the second most speciose class among the subphylum Myriapoda. The most conspicuous
synapomorphic character of centipedes is certainly the modification of the first pair of legs into powerful
venomous forceps (the forcipules). The venom gland encased in each forcipule produces a potent cocktail
of paralytic toxins delivered into prey and opponents via a cuticular duct which opens on the subterminal
part of the apical claw. It has been hypothesized that this modification, unique in the animal world, results
from the folding of the outer cuticle of the walking legs and the transformation of related subepidermal
gland units into venom-producing cells as an adaptation to a new terrestrial predatory niche over
430 million years ago, thus making centipedes one of the most ancient known clade of terrestrial
venomous organisms. However, despite their global distribution, synanthropic habits, and reputation
for inflicting painful stings, little is known about centipedes and their venom system. This chapter reviews
the current knowledge on the development, the evolutionary trajectory, the anatomy, the physiology, and
the predatory ecology of centipedes, with a strong emphasis on the forcipular apparatus.
Keywords
Centipedes; Forcipules; Maxillipeds; Venom system; Chilopoda
Introduction
For because Minos cohabited with many women, Pasiphae bewitched him, and whenever he took another woman to his
bed, he discharged scorpions, serpents and centipedes at her joints, and so the women perished.
Pseudo-Apollodorus, Bibliotheca 3.15.1
Feared and revered for their venomous sting and feisty nature, centipedes have been the center of several
myths and tales around the world. In ancient Egypt, the Goddess Sepa had the shape of a centipede and
was implored by prayers to cure snake bites and increase fertility. Her cult developed particularly in
Heliopolis and later merged with the cult of Osiris. Several thousand kilometers away, the Chinese
centipede (Wu-Gong) was linked to tales of dragons and thunder as a symbol of strength and power. In
Indonesia, “batu mustika lipan” (allegedly centipede bezoars, sometimes said to be worn as a crown by the
animal) are said to hold powerful mystical attributes. In the state of Seremban in Peninsular Malaysia, a
temple has been built on the site of the Wu-Gong San, for devotees to ask favors of the spirit of a giant
centipede roaming the local hills.
*Email: micheldugon@hotmail.com
*Email: mailto:michel.dugon@nuigalway.ie
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In Western literature, centipedes are probably first presented as venomous creatures by Pseudo-
Apollodorus in the Bibliotheca, a text attributed to the first or second century AD. However, the myth
of Minos’ infidelities to his wife Pasiphae is much older and brings us back deep into the mythical roots of
the Hellenist Era and the Minoan culture.
One and half millennia later, Leeuwenhoek (1719) was the first European scientist to write about the
venom claws of centipedes. In a surprisingly successful attempt, Leeuwenhoek provides a rather precise
account of the external morphology of the claws, taking much care in locating the duct opening on the
subterminal end of the claw from which venom is secreted. Leeuwenhoek’s description appears under the
term “Millepaeda.” The distinction between millipedes and centipedes was to be clarified by Linnaeus
four decades later (1758), with the description (still valid to this day) of a large Mediterranean centipede,
giving it the appropriate name of “biting centipede” (Scolopendra morsitans).
Despite detailed account of the effects of a centipede bite and accurate morphological descriptions (e.g.,
Newport 1844), the existence of a venom apparatus linked to the claws was debated until the ultrastruc-
tural description of the venom gland by MacLeod (1878) and Duboscq (1898). The two latter authors are
recognized as the pioneers in the study of the venom claws, and both their publications are landmarks and
starting points for all those interested in the study of the venom system of centipedes.
This chapter explores some of the current knowledge on centipedes with a strong emphasis on the
evolution, the development, the morphology, and the functionality of their venom system, the forcipular
apparatus.
Phylogeny and Diversity

Along with millipedes (Diplopoda), pauropods (Pauropoda), and symphylans (Symphyla), centipedes
(class Chilopoda) form the subphylum Myriapoda. Myriapods are terrestrial, mainly ground dwelling,
animals present on all continents except Antarctica. Myriapods are easily recognizable by their body
divided in two tagmata (head + abdomen and absence of thorax), and their multisegmented trunk bearing
one or two pairs of legs per segment. The number of trunk segments and related appendages is highly
variable (12–191 trunk segments). The head always bears four pairs of cephalic appendages (antennae,
mandibles, first maxillae, and second maxillae). The gas-exchange system is composed of a tight network
of tracheae and spiracles. When present, the eyes are usually composed of a variable number of simple
ocelli, with at least one order of centipedes possessing compound eyes.
However, while millipedes, pauropods, and symphylans feed almost entirely on plant and decaying
matter, centipedes are fundamentally predators, using the highly modified legs of the post-cephalic trunk
segment to subdue their prey before devouring them. This is arguably the most conspicuous feature of the
clade and the only known example in the animal kingdom of the modification of legs into venom-injecting
appendages.
Morphologically, centipedes are generally characterized by a long and slender, dorsoventrally com-
pressed body protected by rigid cuticular plates (ventral sternites and dorsal tergites) separated by flexible
membranes. Upon maturation, centipedes bear 15–191 pairs of legs, as one pair per trunk segment
(Minelli et al. 2000). Most specimens from temperate areas measure from 1 to 10 cm, with larger tropical
species attaining lengths in excess of 30 cm (Lewis 1981).
With approximately 3,500 described species, centipedes make the second most speciose class among
the subphylum Myriapoda. The class Chilopoda is further divided into two subclasses containing a total of
five extant orders and one extinct order. The subclass Notostigmophora contains the archaic order
Scutigeromorpha, while the subclass Pleurostigmophora contains the orders Lithobiomorpha, Crateros-
tigmomorpha, Scolopendromorpha, Geophilomorpha, and the extinct Devonobiomorpha.
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Fig. 1 Representatives of the four main extant orders of centipedes: (a) Thereuopoda longicornis (Scutigeromorpha), (b)
Lithobius variegatus (Lithobiomorpha), (c) Scolopendra subspinipes (Scolopendromorpha), and (d) Strigamia maritima
(Geophilomorpha)
– The order Scutigeromorpha (house and cave centipedes, Fig. 1a) is a small (c. 200 species) order of
centipedes distributed mainly in the tropical and subtropical belt, with a cosmopolitan synanthropic
species (the house centipede Scutigera coleoptrata). The body is proportionally short and cylindrical,
supported by long and thin appendages. Development is anamorphic: the hatching larva has four pairs
of legs, and new segments are added in subsequent molts to reach 15 pairs of legs upon maturity.
Scutigeromorph centipedes display many features differentiating them from other centipedes, such as
dorsal spiracles (as opposed to lateral spiracles in other orders), compound eyes (as opposed to simple
ocelli or complete absence of eyes), and leg-like, stiletto-shaped venom claws (as opposed to stout
forceps-like claws).
– Members of the order Lithobiomorpha (Fig. 1b, stone centipedes, c. 1,150 species) are small
(8–40 mm), dorsoventrally compressed centipedes distributed worldwide, with some notable
synanthropic species (e.g., Lithobius forficatus). Development is anamorphic with hatchlings bearing
four pairs of legs. Mature specimens have 15 pairs of legs.
– The order Craterostigmomorpha comprises only two small to medium (c. 50 mm) species confined to
Tasmania and New Zealand. Egg clutches are guarded by the mother. Hatchings possess 12 pairs of
legs and add the remaining three pairs in a single molt.
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– Centipedes from the order Scolopendromorpha (“giant” centipedes, c. 700 species, Fig. 1c) are
distributed worldwide and range in size from 10 to 300 mm. Large species (over 150 mm) are mainly
distributed within the tropical belt. Development is epimorphic: the number of legs (normally 21 or
23 pairs, with a single species having 39–43 pairs) is family-specific and remains fixed throughout
lifetime. Simple eyes (ocelli) can be present or absent. This is the only order of centipedes known to
inflict medically significant stings.
– Geophilomorpha (earth centipedes, Fig. 1d) is the most speciose order, comprising small to long
thread-like burrowing species and is distributed worldwide. Development is epimorphic with extended
maternal care. The number of leg-bearing segments (LBS) varies greatly within and between species
(27–191 LBS).
– Devonobiomorpha is an order created to accommodate the Devonian centipede Devonobius delta
originally described by Shear and Bonamo (1988) from the Middle Devonian sediments of Gilboa
(USA).
The Forcipular Apparatus

External Structure
The venom claw (or forcipular) segment is a modification of the first post-cephalic, leg-bearing trunk
segment. Modifications involve the whole internal and external structure of both the segment and the pair
of appendages. It has been hypothesized that this modification, unique in the animal world, results from
the folding of the outer cuticle of the walking legs and the transformation of related subepidermal gland
units into venom-producing cells as an adaptation to a new terrestrial predatory niche over 430 million
years ago (Dugon and Arthur 2012a).
The segment’s outer cuticle is heavily sclerotized. On the dorsal aspect, the head capsule largely
overlaps with the forcipular dorsal plate (tergite), except in the order Scutigeromorpha. On the ventral
aspect, the two sternal plates are fused into a strong sternite (Fig. 2a), except in the order Scutigeromorpha
where they remain separated by a thin membrane providing flexibility to the sternal shield (Fig. 2b). In all
other centipedes, the sternite curves laterally and protects the anchorage points of the forcipules located on
the sides of the segment. The sternal plates usually bear notches (replaced by long bristles in
Scutigeromorpha) projecting anteriorly under the mouth.
Each forcipule is composed of four segments. From proximal to distal, these four segments are (1) the
trochantero-prefemur (resulting from the fusion of the trochanter and the prefemur), (2) the femur, (3) the
tibia, and (4) the tarsungulum. The tarsungulum is composed of two fused segments, the tarsus and the
apical claw (Dugon et al. 2012a). Each forcipules can move independently from the other. A set of strong
Fig. 2 Ventral aspects of the forcipular apparatus of (a) the scutigeromorph Scutigera coleoptrata and (b) the lithobiomorph
Lithobius forficatus. SEM micrographs. Scale = 1 mm
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Fig. 3 Details of the head capsule and forcipules of the scolopendromorph Scolopendra hainanum (a) and the scutigeromorph
Scutigera coleoptrata (b)
condyles and muscles provide mobility (although limited) to the trochantero-prefemur in the three plans;
the femur and the tibia are limited to lateral movements (Pleurostigmophora) or vertical movements
(Notostigmophora) (Fig. 3).
Careful examination of the tarsungulum reveals the presence of a small opening (meatus) from which
venom is secreted on the outer subterminal part of the apical claw. The diameter of the meatus varies
greatly from species to species and seems positively correlated to the overall size of the specimen, ranging
from 2 mm in smaller species of geophilomorphs to over 50 mm in large scolopendromorphs. The meatus
is prolonged by a groove extending distally toward the tip of the claw which may increase the effective-
ness of venom delivery (Dugon et al. 2012a).
The outer cuticle of the forcipules is covered with a tight network of microscopic sensillae involved in
the reception of mechanical and chemical stimuli. The apical claw bears three types of short sensilla
coeloconica emerging from depressions in the cuticle. Sensilla coeloconica bear a small pore on their apex
and their density increase on the most distal part of the apical claw, thus suggesting a chemoreceptive role.
The more proximal articles bear various trichomes embedded in large socket, presumably fulfilling
mechanoreceptive functions (Dugon et al. 2012a; Ernst and Rosenberg 2003). Scutigeromorpha possess
two rows of club-shaped trichomes on the inner curvature of the tarsus which are involved in the preening
of the antennae and legs (Rosenberg et al. 2004).
The Venom Gland
Location and Shape of the Venom Gland

The venom gland encased in each forcipule produces a potent cocktail of paralytic toxins delivered into
the prey via a cuticular duct which opens on the subterminal part of the apical claw (Undheim and King
2011). The venom duct is an invagination of the exoskeleton penetrating the mass of the forcipule (Dugon
and Arthur 2012a).
The size and location of the venom glands within the forcipules are very variable between and within
orders. In the anamorphic species (scutigeromorphs and lithobiomorphs), the venom gland usually
extends distally in the tarsungulum and reaches proximally down to the trochantero-prefemur
(Undheim and King 2011). In scolopendromorphs, the length of the venom glands differs between
families, but it seems always contained within the forcipular segment. Variations are greatest in
geophilomorphs. Among the members of the family Schendylidae, the venom gland is contained in the
tarsungulum. In the Dignathodontidae Henia vesuviana, the venom gland is located between the 12th and
18th trunk segments (Duboscq 1898).
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Fig. 4 Cross section through the venom gland of Scolopendra subspinipes mutilans. The venom secretory units are arranged
around the porous proximal part of the cuticular duct (calyx)
Ultrastructure and Function of the Venom Gland

The venom gland is composed of a glandular epithelium arranged radially around the proximal porous
part of the venom duct (the calyx) (Fig. 4). During injection, the venom stored in the glandular epithelium
is released into the lumen of the duct via small pores on the calyx. Each pore measures 1–2 mm in diameter
and connects a single-venom secretory unit to the lumen of the venom duct. In Geophilomorpha, the short,
bulbous calyx bears pores on its entire circumference. In the other orders, pores are not present on the side
of the calyx closest to the cuticle. The distal part of the duct is smooth, without perforation, and follows the
outer curvature of the forcipule to open into the meatus on the outer subterminal part of the apical claw
(Dugon et al. 2012a).
The venom gland is surrounded by an epithelial basal lamina attached on the outer-lateral side of the
venom duct. In Scolopendromorpha and Scutigeromorpha, peripheral muscles surround the glandular
epithelium. Additionally, centripetal muscles attached to the calyx and the peripheral muscles run
between the secretory units of the venom gland, thus suggesting that venom release is under muscular
control (Antoniazzi et al. 2009; Dugon and Arthur 2012a).
The glandular epithelium is composed of hundreds of secretory units, each comprising four cells: (1) a
proximal canal cell, (2) a distal canal cell, (3) an intermediary cell, and (4) a secretory cell (Rosenberg
and Hilken 2006).
The proximal canal cell forms the clove-shaped valve, occupying the cuticular atrium and the more
cuticular pads forming the cap of the pore. It is thought to control the secretion of venom into the lumen of
the duct. The distal canal cell encloses the cuticular atrium opening on the main lumen of the calyx. The
long and thin intermediary cell encloses a large extracellular space filled with the solubilized venom
produced under the form of granules by the secretory cell. The granules produced by the secretory cell are
thought to be released in the extracellular space via exocytosis.
The venom stored in the extracellular vacuoles is squeezed through the cuticular pads of the proximal
canal cell, enters the atrium of the cuticular pore formed by the proximal canal cell, and is released into the
main lumen of the venom duct. The clove-shaped atrial valve stops the venom from rushing back into the
glandular structure and may also act as barrier against bacterial infections (Dass and Jangi 1978).
The observations conducted on scolopendromorphs by early authors (e.g., Duboscq 1898; Barth 1967)
led them to believe that the venom gland was holocrine in nature. However, more recent studies (Menez
et al. 1990; Antoniazzi et al. 2009) did not find any evidence of cell degeneration. It is likely that the
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extracellular venom vacuoles were misinterpreted as degenerated cells (because of the absence of
organelles). The venom secretion may in fact be merocrine (Undheim and King 2011).
The venom production cycle in centipedes has not been extensively investigated. From various TEM
investigations in scolopendromorphs, it seems that electron-dense granules are formed in the secretory
cell (Dass and Jangi 1978; Menez et al. 1990; Antoniazzi et al. 2009). The occurrence of these granules is
preceded by the multiplication of large strains of rough endoplasmic reticulum. The granules show
sometimes lighter rod-like inclusions, which have been interpreted as a possible sign of solubilization
before exocytosis (Menez et al. 1990).
Venom Regeneration
Venom regeneration has been investigated in the common desert centipede Scolopendra polymorpha
(Cooper et al. 2014) and to a lesser extent in the Chinese red-head centipede Scolopendra subspinipes
mutilans (Dugon and Arthur 2012b).
In captivity, the predatory behavior of Scolopendra subspinipes mutilans is significantly altered,
following venom extraction by electrostimulation. Usual prey (i.e., small crickets and larger migratory
locusts) are refused in the hours following complete depletion of the venom gland. Crickets are accepted
again 24 h after venom extraction and locusts 48 h after venom extraction (Dugon and Arthur 2012b).
Cooper et al. (2014) found that venom regeneration in the common desert centipede Scolopendra
polymorpha occurs most rapidly in the first 48 h following complete depletion (65–86 % regeneration)
and then plateau for several weeks. While the volume of venom is rapidly restored after secretion, protein
mass remains low and protein compounds appear to be produced asynchronously. Near-full regeneration
occurs only several months after extraction, although this might have been due to structural damages to
the venom gland during the extraction procedure. The long regeneration cycle would suggest a period of
latency during which centipedes may be vulnerable to predators; however, it is unlikely that centipedes
use the full content of their venom gland during a predation episode.
Ontogeny of Centipedes with Reference to the Forcipular Apparatus

In general, centipedes are sexually reproducing arthropods, although some geographically restricted
populations of some geophilomorph and lithobiomorph species are suspected to be parthenogenetic
(Enghoff 1975; Bonato et al. 2005). The life cycle of centipedes is relatively slow when compared to
most other arthropods. For many taxa, life expectancy is estimated to be at least 2–6 years (Minelli and
Sombke 2011) but it may actually be longer for some large scolopendromorphs. Sexual maturity is
reached after approximately 1 year for Lithobius erythrocephalus (Voigtländer 2006) and after 2 years for
Strigamia maritima (Lewis 1981). The scolopendromorph Rhysida nuda matures within 2 years (Lewis
1981). The ontogeny of centipedes is strongly influenced by the presence (Craterostigmomorpha,
Scolopendromorpha, and Geophilomorpha) or absence (Scutigeromorpha and Lithobiomorpha) of
maternal care.
Reproduction, Egg Laying, and Maternal Care

Usually, a courting ritual takes place, involving a careful and long (sometimes several hours) approach,
defensive postures, and tapping with the legs and/or antennae on the extremities of the partner (cf. Lewis
1981 for a review). In lithobiomorphs, scolopendromorphs, and geophilomorphs, the male produces a
web on which the sperm is deposited before being collected by the female. Male scutigeromorphs,
lithobiomorphs, and scolopendromorphs produce spermatophores, while some geophilomorphs appear
to deposit an uncased sperm droplet (Lewis 1981). According to Minelli (2011) females are unlikely to
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receive sperm more than once a year and most probably are impregnated only once in their lifetime.
A female captive specimen of Ethmostigmus trigonopodus (Scolopendromorpha) mated only once
produced two clutches 144 days apart (Iorio and Ythier 2007).
Scutigeromorpha, Lithobiomorpha, and Craterostigmomorpha present an anamorphic development. The
hatchlings are fully mobile and leave the egg with an incomplete number of trunk segments. The remaining
segments develop after successive molts. The eggs are laid in a small cluster or individually, covered with
soil, and then abandoned by the mother. Scolopendromorphs and geophilomorphs are incapable of
movements when hatching, but possess already their final number of trunk segments (epimorphic devel-
opment). In these two orders plus Craterostigmomorpha, the mother takes care of the brood for several
weeks or months, until the young are sufficiently developed to move and hunt on their own.
Development of the Venom Apparatus

Little is known about the development of the venom system during embryogenesis. At the early stages of
development, the forcipular segment is morphologically similar to other trunk segments. Enlargement and
repositioning of the forcipules become noticeable only in mid to late pre-hatching developmental stages
(Fig. 5). However, Hox gene expression patterns in the forcipules of the geophilomorph Strigamia
maritima reveal a forcipule-specific Hox expression (Hayden and Arthur 2013) in earlier germ-band
developmental stages.
The forcipular appendages and the venom gland are not functional in newly hatched centipedes. In
geophilomorphs and scolopendromorphs, the forcipules of freshly hatched specimens are somewhat
enlarged compared to the leg buds and held perpendicular to the body. The forcipules are blunt and soft
and lack articulations and sclerotization. The venom gland and the venom duct are absent. The forcipules
of hatching lithobiomorphs and scutigeromorphs appear to be segmented. Although there is no maternal
care, the developmental stage immediately following hatching is believed to be nonfeeding, indicating
that the venom system is still nonfunctional at this stage (Dugon et al. 2012b).
The development of the venom glands of the Chinese centipede Scolopendra subspinipes mutilans has
been reported by Dugon and Arthur (2012b). In the second postembryonic stage, the forcipules are still
held perpendicular to the trunk, and the venom gland is absent. At the third postembryonic stage (about
35 days after hatching), a cuticular fold on the dorsolateral aspect of the apical claw is shaping into the
Fig. 5 (a) Embryo of the scutigeromorph Scutigera coleoptrata (anamorphic development) at mid-development. (b) Embryo
of the scolopendromorph Scolopendra subspinipes mutilans (epimorphic development) at the hatching stage. The arrows point
to the forcipular buds
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venom duct. At the same time, the glandular epithelium emerges from the internal wall of this fold and
grows posteriorly toward the trochantero-prefemur. The full extension of the venom gland is reached
within 4–5 days. Although most of the venom duct is still embedded in the dorsolateral cuticle of the claw,
the forcipules and the internal venom apparatus appear to be fully functional at the adolescence stage,
approximately 7–8 weeks after hatching. In the adult Scolopendra subspinipes mutilans, an indent in the
cuticle is visible on the dorsal aspect of the tarsungulum, where the duct sank into the mass of the forcipule
during development. On the basis of these developmental observations and the evolutionary trajectory of
the venom claw morphology from a Scutigera-like ancestor some 420 million years ago, it has been
suggested that the development of the venom apparatus of Scolopendra recapitulates, at least partially, the
evolution of the apparatus (Dugon et al. 2012b).
Fossil Records of Centipedes with Special Reference to the Forcipular

Apparatus
The fossil record of myriapods is sparse and very incomplete. However, several major discoveries have
been made in the last three decades, providing an insight into the deep phylogenetic nodes of the
subphylum. Most of these discoveries have been compiled and discussed in four major reviews
(Almond 1985; Shear 1997; Shear and Edgecombe 2010; Edgecombe 2011) which, when read in
chronological order, offer an interesting view of the large progress made in reconstructing the evolution-
ary history of the myriapods.
The absence of calcium carbonate in the cuticle of chilopods is the main reason for the low number of
fossilized remains (Shear and Edgecombe 2010). No stem-group myriapod fossils have been definitely
assigned so far. The earliest confirmed myriapod records have been dated to the early and mid-Silurian
and described as primitive diplopods (Almond 1985). However, speculations on a Cambrian-Ordovician
myriapod have not been dismissed and are sometimes debated (Shear 1997; Wilson 2006). Using the
fossil record, combined with morphological features and molecular data, Murienne et al. (2010) were the
first to propose a chronogram for the centipede tree of life.
The oldest confirmed chilopod fossils belong to the genus Crussolum, a scutigeromorph recovered
from the Scottish Rhynie Chert, a hot-spring vent location dated from the Early Devonian (407 MYA)
(Anderson and Trewin 2003) and from the Gilboa site in New York State (Shear et al. 1998). The Scottish
specimen presents perfectly preserved forcipular coxal plates and partially preserved forcipules.
Crussolum has forcipular attributes that are typical of the extant Scutigeromorpha: separated coxal plates,
insertion point of the elongated trochantero-prefemur, long setae on the distal end of the coxal plates, and
absence on the posterior end of the coxae of apodemal insertions into the first trunk segment.
Later Scutigeromorpha fossils were identified in the Mazon Creek deposits of Illinois (Upper Carbon-
iferous), USA (Shear and Edgecombe 2010), and in the Crato Formation of Northeast Brazil
(Fulmenocursor tenax Wilson 2001). Fulmenocursor tenax (Lower Cretaceous) has been assigned to
the family Scutigeridae on the base of the possession of antennomeres that are longer than wide.
Devonobius delta (Chilopoda: Devonobiomorpha: Devonobiidae), a member of an extinct order of
centipede from the Middle Devonian, has been described from the Gilboa deposits (Shear and Bonamo
1988). Upon examination, Devonobius was placed between the Craterostigmomorpha and the Epimorpha
(Scolopendromorpha + Geophilomorpha). This central position is interesting, as some transitional
morphological features may be present in the specimens.
The forcipules of the Devonobius specimen are relatively well conserved. The coxosternite is in one
block (while all the previous centipede fossils presented two separated coxal plates as in
Scutigeromorpha), with a ridge visible where the fusion between the two coxae occurred. The coxal
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tooth plates project forward in a manner similar to some Scolopendromorpha. The forcipules are long and
project far distally. The general shape and organization are very close to that of Craterostigmus
tasmanianus, a parallel noted by Shear and Edgecombe (2010). The forcipules possess a long
trochantero-prefemur and a long tarsungulum, but a much reduced femur and tibia. However, and unlike
the Epimorpha, the trochantero-prefemur and the tarsus do not share an articulation point. Each forcipular
article forms a complete ring around the limb.
Scolopendromorpha representatives have been found in the Crato Formation of Brazil and the Mazon
Creek of Illinois, with three named species: Mazoscolopendra richardsoni (Mundel 1979) Cratoraricus
oberlii (Wilson 2003), and Velocipede betimari (Martill and Barker 1998). The forcipules of the four
specimens present the typical form of scolopendromorph forcipules: enlarged trochantero-prefemur and
reduced and incomplete femur and tibia, with a stout tarsungulum. The tarsungulum and trochantero-
prefemur touch each other on the external lateral part of the forcipule, a synapomorphic character of the
modern epimorphs.
According to Shear and Edgecombe (2010), the fossil records of both Lithobiomorpha and
Geophilomorpha are very limited. Regarding Lithobiomorpha representatives, the authors mention that
the “fossil record is confined to the Cenozoic, with several taxa having been named from Baltic amber,
though none has received modern study.” Considering the rather “young” age of such specimens, it is
unlikely that the forcipular system looked any different from the one existing today in Lithobiomorpha.
As for the Geophilomorpha, the earliest fossil is a single specimen from the Upper Jurassic,
Eogeophilus jurassicus (Schweigert and Dietl 1997). The minute size of the specimen and the rather
bad preservation make the photographic material that is available difficult to interpret in terms of forcipule
shape.
Another single specimen of geophilomorph from French amber, Buziniphilus antiquus (Edgecombe
et al. 2009), was dated from the early Cenomanian (Upper Cretaceous, 93–100 MY). This specimen is
interesting for the very clear view it offers on an undamaged forcipule. It appears that the trochantero-
prefemur and the tarsungulum are linked by a joint as in all known living Geophilomorpha. The pleuritis
seems very developed. Interestingly, the embedment in amber permits distinguishing the venom duct and
the porous proximal extremity, the calyx. The calyx appears very developed and large in comparison to all
of the living species I have examined. However, the calyx is mostly confined to the femur/tarsungulum
part of the claw, a common occurrence in living geophilomorphs. The specimen was placed into the
suborder Adesmata and belongs to either of the families Geophilidae or Schendylidae.
Predatory Behavior and Prey Choice

Although all centipedes are thought to be opportunistic predators rather than specialist feeders, their
predatory behavior is strongly correlated to their morphotype and the specific ecological niche they
occupy (Voigtländer 2011). Three main morphotypes were identified by Manton (1977): (1) running type
(Scutigeromorpha and Lithobiomorpha), (2) burrowing type (Geophilomorpha), and (3) intermediate
type (Scolopendromorpha and Craterostigmomorpha).
In all five orders the anterior legs and forcipules are all involved in holding and subduing prey. In
scutigeromorphs, the stiletto-like forcipules are only capable of stabbing motion and are solely involved
in prey envenomation. In the remaining four orders, the forcipules bear a cutting edge on the inner
curvature of the tarsungulum which allow for the dissection and the mastication of prey items.
Scutigeromorphs are fast-running ambush predators using their long legs to “cage” their prey before
stabbing it with a vertical motion of the forcipules. Prey are located through olfactory and tactile cues and
attacks usually take place on open grounds (Voigtländer 2011). The diet of the common house centipede
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Fig. 6 Scolopendra subspinipes mutilans injecting venom in (a) the thorax and (b) the head of field crickets Gryllus assimilis
Scutigera coleoptrata is composed of a variety of flying and terrestrial arthropods, including spiders and
small centipedes (Lewis 1981). The species demonstrates territorial habits, with spatial segregation
between males and females (Lewis 1981). Cannibalism is frequent, and adult females have a particular
liking for freshly molted males (Lewis 1981).
Lithobiomorphs are opportunistic ambush and foraging predators, locating their prey through direct
contact with their legs or their antennae (Voigtländer 2011). Prey are seized with the forcipules and kept
firmly close to the mouth. Although most lithobiomorphs hunt small prey under the cover of the leaf litter,
stones, and logs, some species are known to climb trees in search of small insects (Voigtländer 2011). In
British woodlands, a large array of insects, arachnids, and worms seem to constitute the diet of Lithobius
forficatus (Lewis 1981).
Scolopendromorphs prey mainly upon arthropods, but large specimens are known to occasionally feed
on small vertebrates (bats, Molinari et al. 2005; toads, Carpenter and Gillingham 1984; rodents, Clark
1979; snakes, Okeden 1903). The Chinese red-head centipede Scolopendra subspinipes mutilans is
capable of choosing prey depending on the amount of venom available in its venom glands and the
sensitivity of the prey to its venom, thus suggesting the presence of a complex prey detection system. The
prey is usually manipulated and oriented before envenomation which usually occurs in the head or thorax
(Fig. 6) (Dugon and Arthur 2012b). Although a few species of scolopendromorphs have shown aggre-
gation behavior in captivity (e.g., Scolopendra subspinipes mutilans, Alipes grandidieri), most scolopen-
dromorphs are solitary and highly territorial. Intraspecific and intergenerational cannibalism is common
(Siriwut et al. 2014) and may be involved in the regulation of population density.
Geophilomorphs are opportunistic burrowing predators of the topsoil layers. These blind centipedes
rely on the mechanoreceptors and chemoreceptors located on their antennae and forcipules to locate their
prey at a distance. Geophilomorphs have been observed feeding on small arthropods, including ants,
woodlice, coleopteran larvae, and dipteran larvae, but small earthworms are likely to make the bulk of
their diet (Lewis 1981). Reports suggest that they occasionally feed on plant material (Voigtländer 2011),
although this intake may be marginal. Strigamia maritima, a North-Atlantic littoral species of the supra-
tidal fringes, forms colonies of hundreds of specimens and has been observed feeding in groups of up to
20 individuals (Lewis 1981). Geophilomorphs use their strong forcipules and cephalic shield to cut
through the cuticle of prey and insert their head and anterior trunk segment to devour the prey from the
inside (Lewis 1960).
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Conclusion and Future Directions

The forcipular apparatus of centipedes is a synapomorphic character which was already possessed by the
genus Crussolum during the Late Silurian (418 MY). It most likely predates the split between
Notostigmophora and Pleurostigmophora which may have occurred 430–450 million years ago
(Murienne et al. 2010). If the venom gland was already active at this point – and it may have
been – centipedes could compete for the title of “oldest terrestrial venomous animal” alongside archaic
arachnids (Undheim et al. 2014).
Evidence tends to demonstrate that the forcipular apparatus is the result of the individualization of the
first trunk segment. Because of its strategic post-cephalic position, this segment may have been under an
important selective pressure. It was first reassigned to perform a multitude of novel tasks, from preening to
prey capture and feeding. Once established in these roles, it probably underwent further specialization
when centipedes diversified and occupied new ecological niches, from open spaces to subterranean
crevices.
The venom glands may have evolved shortly after the reassignment of the forcipules from locomotory
to prey-seizing appendages. The venom glands start developing on the dorsolateral part of the forcipules’
apical claws and then penetrate the forcipules more posteriorly, down to the trochantero-prefemurs.
The broad developmental sequence of the forcipules can be summarized in three major steps: (1) the
forcipular segment develops following the general anteroposterior segmental direction; (2) the forcipules
gain the typical antero-median orientation at a late developmental stage; and (3) the venom gland develops
once the forcipules are already formed into sclerotized prehensile appendages and before the centipede
starts to hunt.
The presence of nerve endings in the core of the venom gland suggests that (1) the animal can possibly
regulate the amount of venom it delivers and (2) the animal may “know” how much venom is available in
the venom glands and adapt its foraging behavior accordingly. Also, venom is preferably injected into the
body parts where it is most efficient (Dugon and Arthur 2012b), thus confirming a parsimonious use of
venom.
The current literature on the venom system of centipedes is largely based on the interpretation of
morphological observations, ecological studies, and subsequent deductions on the possible evolutionary
and developmental trajectories of the apparatus. This approach has its shortcomings, notably the lack of
comparative molecular data which would permit insights into the causality of developmental patterns,
both general ones and ones that are distinct to each species (e.g., due to heterochrony).
For that reason, further comparative studies of the gene cascade involved in the formation and identity
of the forcipular segment are needed. Some of these genes have already been identified for the
lithobiomorph Lithobius atkinsoni (Hughes and Kaufman 2002) and the geophilomorph Strigamia
maritima (Brena et al. 2006; Hayden and Arthur 2013). However, information is fragmentary and no
in-depth comparative analysis has been performed so far. An insight from the developmental genetics of a
scutigeromorph, a craterostigmomorph, and a scolopendromorph would be very valuable.
A second topic of research directly related to the venom apparatus would be an in-depth comparative
study of the venom following two approaches: (1) the creation of cDNA libraries to investigate the venom
gland transcriptomes across the five orders and trace back the evolution of the venom gland on the basis of
molecular evidence and (2) an assessment of the spectrum of venom components, not only between
medically significant species but also at the inter-order, intrageneric, and intraspecific levels. Such
population venomic studies may shed some light on the interactions between occupation of ecological
niches, venom evolution, and speciation events.
While functional venom studies trigger the interest of both the academic world and the pharmaceutical
industry, the evolution of venom systems in invertebrate organisms has attracted relatively little attention
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so far. Here, there is a virtually untapped potential for important discoveries in many animal phyla. Such
work would produce very interesting comparative material to address conceptual questions related to
evolutionary novelties, gene co-option, and the functional shift of preexisting structures. Also, from a
more pragmatic perspective, a better understanding of the origin, evolution, and development of venom
systems would profit applied research by providing a new insight into the evolution of complex proteins
and the way they are produced.
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Parasitoid Wasps and Their Venoms
Mrinalini and John H. Werren
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Evolution of Parasitic Wasps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
The Parasitic Lifestyle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Parasitic Wasp Venom System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
The Venom Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
The Ovipositor or Stinger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Venom Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
How Parasitoid Venoms Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Venom Functions in Insect Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Parasitoid Venoms and Arachnid Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Venom Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Models of Venom Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Questions to Explore in Parasitic Venom Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Recent Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Abstract
Parasitoid wasps are a unique group among venomous organisms. In contrast to
the common venom functions of predation and defense, female parasitoid wasps
use venom to manipulate the metabolism, development, and behavior of other
arthropods for reproductive purposes. This provides a safe environment and
nutrition for the next generation of wasps to feed and develop. Parasitoid wasp
species diversity is estimated to be between 150,000 and 600,000 species, likely
Mrinalini (*)
Department of Biological Sciences, National University of Singapore, Singapore, Singapore
e-mail: dbsmrin@nus.edu.sg; mrinalini.urs@gmail.com
J.H. Werren
Department of Biology, University of Rochester, Rochester, NY, USA
# Springer Science+Business Media Dordrecht 2016 1

P. Gopalakrishnakone, A. Malhotra (eds.), Evolution of Venomous Animals and Their
Toxins, Toxinology, DOI 10.1007/978-94-007-6727-0_2-2
2 Mrinalini and J.H. Werren
making them the largest group of venomous organisms. They parasitize all
orders of Insecta and several taxa from Arachnida. Parasitoids display highly
diverse morphologies and parasitic lifestyles. This diversity likely plays a strong
role in the adaptive evolution of venom apparatus structures, venom genes, and
venom functions. However, parasitoid wasps are underexplored and little
represented in toxinology.
This chapter provides a background into evolution of parasitoid wasps and
their parasitic lifestyle. The evolution of parasitoid venoms and their functions
are discussed, and a comparison of venom functions in two major ecological
categories, ectoparasitoids and endoparasitoids, is provided. Expanding on the
standard gene duplication and recruitment model of toxin gene evolution,
additional mechanisms are proposed. These include co-option, multifunctiona-
lization, alternate splicing, and origins from lateral gene transfers or noncoding
DNA. Novel tools such as RNA interference (RNAi) knockdown of parasitoid
venom genes, combined with RNA sequencing of envenomated hosts, are
proposed for venom function hypothesis testing and hypothesis generation.
This chapter also addresses key questions concerning the future directions of
parasitoid venom research.
Keywords
Hymenoptera • Insect • Endoparasitoid • Ectoparasitoid • Nasonia • Ovipositor
Introduction
Parasitoid wasps (also known as parasitic wasps) (Hymenoptera) are a distinctive,

but lesser-known, group of venomous organisms. They are typically referred to as
parasitoids because although the adult wasps are free-living, their juvenile (larval)
developmental stages are completed within or upon other insects or arachnids,
eventually killing the “host.” In contrast to other venomous animals that use
venom for prey capture and/or defense against predators (e.g., snakes, spiders,
scorpions, bees, and jellyfish), parasitoid wasps use venom for reproduction and
completion of their parasitic life cycle. These wasps parasitize insect or arachnid
hosts by injecting venom into them and then laying eggs on or inside the host tissue.
Typically, the venom does not immediately kill the host. Rather, it manipulates the
host in several ways, such as changing host physiology, metabolism, and behavior
and causing paralysis, developmental arrest, and immune suppression (Rivers and
Denlinger 1994a, b; Weisel-Eichler et al. 1999; Eberhard 2000; Rivers et al. 2002;
Danneels et al. 2013; Martinson et al. 2014; Mrinalini et al. 2014). These changes
render the host incapable of performing normal bodily functions and completing
normal development. Thus, the wasp ensures a nutritional supply and a safe
environment for its larvae, which develop and consume the host.
Despite this evolutionarily innovative use for venom, parasitoid wasps are little
acknowledged in toxinology. The often-used definition of venom as “a secretion
that disrupts normal physiological or biochemical processes to facilitate feeding or
Parasitoid Wasps and Their Venoms 3
Fig. 1 Parasitic wasps are

usually very small in size.
Nasonia vitripennis, a model
parasitoid species, is seen
here on a human fingernail.
The small size of parasitic
wasps has made venom
collection and analysis a
challenge until recently
(Photo courtesy John (Jack)
Werren)
defence by the producing animal” (Fry et al. 2009) does not include the function of
parasitic wasp venom. Parasitoid venoms have evolved to promote the growth,
development, and the survival of the parasitoid offspring, whereas references to the
most common functions of venom are usually foraging and defensive adaptations of
the venom producer (Casewell et al. 2013). The biodiversity of parasitoid wasps is
estimated at several hundreds of thousands of species (Noyes 2000; Heraty and
Gates 2003; Whitfield 2003; Pennacchio and Strand 2006; Heraty 2009; Munro
et al. 2011; Noyes 2014). This outnumbers species diversity in every other group of
venomous organism and might even outnumber all groups put together. Therefore,
the most common function of venom in nature may well be to facilitate the
successful completion of the parasitoid life cycle.
The majority of parasitoid wasps are of no danger to humans, which may be one
reason they are understudied in toxinology. Added to this is their typically very
small size (Fig. 1), which has made venom collection and analysis a challenge until
recently. However, these characteristics also make them easier to work with
compared to, say, venomous snakes or jellyfish. Parasitoid wasps are ubiquitous
around the world, and many species are cosmopolitan in their distribution. More-
over, parasitoid species such as Nasonia vitripennis have been established as
efficient genetic models due to their short generation time, ease of maintenance
in the lab, and interspecies fertility (Werren and Loehlin 2009a, b; Werren
et al. 2010). Genotyping and characterization of gene expression are easier, as
whole wasps can be used for sequence analysis. Parasitoid wasps are easily
genetically manipulated using RNA interference (RNAi) knockdowns, allowing a
subtractive approach to venom functional studies via RNAi-mediated
downregulation of venom gene expression (Lynch and Desplan 2006; Werren
et al. 2009; Colinet et al. 2014b).
Recently, the potential uses of parasitoid venom in pharmacology and agricul-

ture have been recognized (Beckage and Gelman 2004; Zhang et al. 2005; Danneels
et al. 2010; Werren et al. 2010; Zhu et al. 2010; Heavner et al. 2013, 2014; Moreau
2013; Colinet et al. 2014a). Given their incredible species diversity and the wide
range of parasitic lifestyles, parasitoid wasps are a potential gold mine of novel
bioactive peptides. For example, a large proportion of proteins in N. vitripennis
venom are not found in any other organism (Danneels et al. 2010; de Graaf
et al. 2010; Werren et al. 2010). Parasitoid venom functions of targeted manipula-
tions of host immunity, physiology, metabolism, development, and behavior are not
found in other venomous organisms (Rivers and Denlinger 1994b; Korenko and
Pekar 2011; Danneels et al. 2013; Martinson et al. 2014; Mrinalini et al. 2014).
Given this background, a review of parasitoid wasps and their venoms is highly
relevant and essential for complete representation of venomous organisms in
toxinology. This chapter presents to the reader the evolutionary history of parasitoid
wasps and their parasitic lifestyle and insights into the evolution of parasitoid
venoms and their functions and, finally, notes on recent advances and future
directions in parasitoid venom research.
Evolution of Parasitic Wasps
A recent phylogeny using ~1500 protein-coding genes estimates the origin of

Hymenopteran stem lineages, including those of parasitoid wasps, in the Late
Carboniferous period (Misof et al. 2014). The first parasitoids appeared around
160 mya (Whitfield 1998), and the parasitic lifestyle had a single origin in the
common ancestor of sister groups Orussoidea and Apocrita (Whitfield 1992;
Pennacchio and Strand 2006; Heraty et al. 2011). Extensive diversification of
established Hymenopteran lineages occurred during the Early Cretaceous period
(Misof et al. 2014). Present-day parasitoid wasps are highly speciose, representing
10–20 % of known insect species and >75 % of the ~320,000 known species in
Hymenoptera (Whitfield 2003; Pennacchio and Strand 2006). However, social
Hymenopterans (e.g., ants, bees, and wasps), which make up 2 % of all insects
(Holldobler and Wilson 2008), are the more frequent representatives of the ven-
omous Hymenoptera in toxinology.
High levels of species diversity and cryptic morphology have made the resolu-
tion of parasitoid wasp evolution and taxonomy exceptionally difficult but also very
interesting. The extent of biodiversity in parasitoid Hymenoptera is being realized
only in recent years. There are two major Hymenopteran superfamilies consisting
mostly of parasitoid wasps: Chalcidoidea and Ichneumonoidea. Chalcidoidea is
estimated to contain ~500,000 species with only 23,000 currently described (Noyes
2000; Heraty and Gates 2003; Heraty 2009; Munro et al. 2011; Noyes 2014).
Ichneumonoidea is predicted to consist of ~100,000 species (Gauld et al. 2002),
although this could likely be an underestimate since a single family Braconidae
(Ichneumonoidea) was recently estimated to consist of 46,000 species (Rodriguez
et al. 2012).
Given that each parasitoid wasp species synthesizes around 100–150 venom
proteins on average (de Graaf et al. 2010; Goecks et al. 2013), cataloging and
investigating venoms from all parasitoid species is a monumental task, regardless of
venom sharing among lineages. To date, comprehensive knowledge of venom
protein repertoires exists for only seven parasitoid species (de Graaf et al. 2010;
Vincent et al. 2010; Werren et al. 2010; Zhu et al. 2010; Goecks et al. 2013; Burke
and Strand 2014; Colinet et al. 2014a). This barely scratches the surface of the
diversity in parasitoid wasp species.
The Parasitic Lifestyle
Parasitoid wasps are holometabolous or metamorphosing insects, and they com-

plete their development on or inside the hosts that they parasitize. Parasitoid species
are highly diverse in morphology, life strategies, host preferences, venom systems,
and venoms. Parasitoid wasps can be generalists, parasitizing a range of host
species spanning diverse orders of Insecta, or they can be specialists, using a single
host genus or species. They can be gregarious, laying large clutches of eggs per
host, or solitary, laying a single egg per host. Different species specialize on hosts
that are at different life stages and can be categorized as egg, larval, pupal, or adult
parasitoids.
Parasitoids can be further characterized by the stage at which their hosts are
killed. Venom injection by idiobionts immediately arrests host development,
whereas following venom injection by koinobionts, the host continues to develop
until the growing parasitoid larvae kill it at a later stage. For instance, some
parasitoids such as Chelonus inanitus (Braconidae) inject venom and oviposit
inside host eggs; however, the adult wasps emerge when hosts are at late larval
stages (egg-larval parasitoids). In such cases, different parasitic strategies are used
to survive as the host continues to develop (Kaeslin et al. 2005). Koinobionts are
typically endoparasitoids and they lay their eggs within the host body cavities or in
specific host tissues. In contrast, idiobionts are mostly ectoparasitoids that lay their
eggs on the surface of host integument, and their developing young feed by
puncturing the host integument. Combined analysis of genetic and morphological
data indicates that the basal parasitic clades in Apocrita were ectoparasitic, and the
endoparasitic lifestyles were derived later (Dowton and Austin 2001).
Because of the differences in host interactions, venom systems in ectoparasitoids
and endoparasitoids are expected to be under different selective pressures. More-
over, generalist idiobionts can also display host-switching behavior, and this can
cause rapid adaptations that work toward increasing offspring survival in short
durations (Tschopp et al. 2013; Jones et al. 2015). Therefore, venom protein
compositions of ectoparasitoids and endoparasitoids can generally be expected to
be different. In addition, parasitic venom action is often supplemented by various
non-venom secretions and developmental strategies to ensure maximal survival and
fitness of parasitoid young. In the case of endoparasitoids, where development
inside the host necessitates overriding host immunity, venoms contain mutualistic
polydnaviruses (PDVs) and virus-like particles (VLPs) that play a role in host
immune suppression (Strand and Pech 1995; Asgari and Rivers 2011). Host feeding
and salivary secretions of ectoparasitic larvae are known to modulate host immu-
nity and metabolism (Periquet et al. 1997; Richards and Edwards 2002; Nakamatsu
and Tanaka 2004), whereas endoparasitic larvae secrete teratocytes into the host to
manipulate host growth and metabolism (Dahlman et al. 2003; Basio and Kim
2005; Strand 2014). The endoparasitoids use developmental strategies such as
polyembryony (where a single egg clonally divides into multiple genetically iden-
tical embryos), since protection and nutritional supply are readily available inside
the host (Segoli et al. 2010).
These different lifestyles are likely to influence parasitoid venom repertoires and
the modes of parasitoid venom action. The implications of parasitic lifestyles on
venom evolution and the contrasts and similarities in ecto- and endoparasitic venom
systems are discussed in subsequent sections.
Parasitic Wasp Venom System
The Venom Apparatus
Parasitoid wasps display the greatest diversity in venom apparatus structures than in
any other group of venomous organisms. Adaptation to different host species and
parasitic lifestyles likely drives this extreme diversity. However, the venom appa-
ratus shares a set of common features across species. The venom apparatus is found
only in the female wasp. It is located at the posterior, dorsal surface of the wasp, and
is usually well protected under chitinous ovipositor plates that are part of the
exoskeleton. Internally, it is attached to the vagina at the proximal end of the
ovipositor or stinger (de Graaf et al. 2010). This close interaction between the
venom apparatus and the female reproductive system complements their function-
ing, since venom is injected at the time of oviposition (Fig. 2a).
A typical venom apparatus comprises of one or more venom glands (also known
as acid glands), a venom reservoir, and a Dufour gland (also known as alkaline
gland) (Fig. 2b). Venom glands are highly variable in size and can be elongated,
cylindrical, saclike, or branched (Ferrarese et al. 2009). They are lined with
glandular columnar epithelial cells containing secretory granules and vesicular
structures that secrete venom proteins (Ferrarese et al. 2009; Formesyn
et al. 2012). The Dufour gland is also secretory in nature but is thought to perform
lubricating functions in most parasitoids (Formesyn et al. 2012). The venom
reservoir is a muscular saclike structure that collects and stores venom, but it may
also secrete some venom components (Formesyn et al. 2012). Venom synthesis
begins in the venom gland when the adult female wasp emerges from its puparium.
Venom accumulates in the reservoir, reaching maximum capacity within the first
few days after adult female eclosion (Zhang et al. 2013).
Fig. 2 Venom delivery and venom apparatus in Nasonia vitripennis. (a) N. vitripennis
injecting venom into the pupa of its flesh fly host Sarcophaga bullata. (b) A typical venom
apparatus consists of the venom or acid gland that secretes venom, the Dufour or alkaline gland
that produces lubricating fluids, and the sac-like venom reservoir that collects and stores venom
(Photo courtesy (a) Michael Clarke (b) Amanda Dolan)
The Ovipositor or Stinger
Venom is delivered into the host by the ovipositor or stinger (Fig. 2a). On a
mechanical level, the parasitoid ovipositor is a foldable multitool – it is used as a
drill to perforate host structures, a hypodermic needle to inject venom, and a duct to
convey and deposit eggs during egg laying. Additionally, when a space separates
the adult wasp from the host body, the ovipositor is used to build a feeding tube that
allows the wasp to feed on host hemolymph. For example, pupal parasitoids in the
genus Nasonia build a feeding tube that bridges the space between the puparium
and the host integument. When the ovipositor is not in use, it is usually folded back
or reeled in and put away.
However, the ovipositor is not just a stinger. It is a highly complex structure that
has evolved sensory structures and functions that are adaptations to the wide range
of hosts and lifestyles in parasitic Hymenoptera (Quicke et al. 1994; LeRalec
et al. 1996; Quicke 1997). The parasitoid ovipositor is comprised of a complex
system of valves, ridges, grooves, and stops, which enable steering and orientation
during egg laying (Quicke and Fitton 1995; Quicke et al. 1995). Nasonia vitripennis
uses its ovipositor tip to examine the host puparium and tissues to assess host
quality (King and Rafai 1970). It can discriminate between unparasitized and
parasitized hosts and the relative time since prior parasitization (Werren 1984).
Ovipositor stylets contain different types of sensilla that can be gustatory for
discriminating hosts and secretory for lubrication and for thermo-hygroreception
(Shah 2012). Endoparasitic Leptopilina sp. possess a clip on their ovipositor to grip
host larvae and prevent escape until the venom induces paralysis (Lenteren
et al. 1998). In the jewel wasp, Ampulex compressa, the ovipositor performs
mechanosensory functions that help locate and directly inject venom into the
brain of its cockroach host (Gal et al. 2014).
Venom Components
Parasitic wasp venom can be a complex mixture of proteins, polydnaviruses

(PDVs), virus-like particles (VLPs), microRNAs, small molecules, and ovarian
fluids. Given the variety of parasitoid ecologies and host interactions, it is attractive
to compare venom compositions among parasitoids with contrasting lifestyles.
However, with the limited parasitoid venom analyses currently available, such a
synthesis is premature and would likely result in artifacts of incomplete knowledge
rather than realistic representations. Therefore, the following sections provide a
brief account of venom components in the context of two broad parasitoid catego-
ries, ectoparasitoids and endoparasitoids. Specific examples demonstrating host
diversity and modes of venom action in the two parasitoid groups are discussed
later in the chapter.
Venom Proteins
Parasitic wasps secrete anywhere between 0.04 and 180 ug of venom protein,
depending on the species (Parkinson and Weaver 1999; Uçkan et al. 2004). So
far, venom proteins in 17 parasitoid wasp species have been examined at some level
(Poirie et al. 2014), although comprehensive proteomic analysis has been under-
taken in only seven species (de Graaf et al. 2010; Vincent et al. 2010; Werren
et al. 2010; Zhu et al. 2010; Goecks et al. 2013; Burke and Strand 2014; Colinet
et al. 2014a). A large proportion of these proteins are enzymatic in nature and
resemble insect metabolic enzymes (Asgari and Rivers 2011); however, the func-
tions of the vast majority are still unknown (Poirie et al. 2014). Moreover, many
parasitoid venom proteins have no homology to proteins in other organisms
(de Graaf et al. 2010; Werren et al. 2010; Poirie et al. 2014).
Ectoparasitoid venoms have been studied for years (Rivers and Denlinger 1994a,
b, 1995; Coudron and Brandt 1996; Periquet et al. 1997; Rivers et al. 2002, 2006;
Nakamatsu and Tanaka 2003; Tian et al. 2010; Ye et al. 2010); however few
comprehensive analyses of ectoparasitoid venom protein repertoires have been
performed so far. The most complete ectoparasitoid venom data exists for
N. vitripennis, which contains at least 79 venom proteins (de Graaf et al. 2010;
Werren et al. 2010). These venoms have been categorized into eight functional
categories: proteases/peptidases, protease inhibitors, esterases, carbohydrate
metabolism, DNA metabolism, glutathione metabolism, recognition and binding
proteins, and immune-related proteins (de Graaf et al. 2010). Twenty-three venom
proteins of N. vitripennis do not share homology to any known protein and therefore
are of unknown origin and function (de Graaf et al. 2010). The role of specific
ectoparasitoid venom proteins in causing developmental arrest, immune suppres-
sion, and increased lipid content in the host has been investigated in species from
the genus Euplectrus (Coudron and Brandt 1996; Nakamatsu and Tanaka 2003) and
in Eulophus pennicornis (Price et al. 2009).
Among endoparasitoids, however, comprehensive venom protein repertoires of
six species have become available in recent years: Chelonus inanitus (Vincent
et al. 2010), Microplitis demolitor (Burke and Strand 2014), Aphidius ervi (Colinet
et al. 2014a), Leptopilina boulardi and Leptopilina heterotoma (Goecks

et al. 2013), and Pteromalus puparum (Zhu et al. 2010). It is also well established
that venoms of some endoparasitoids contain mutualistic polydnaviruses and virus-
like particles that are involved in host immune suppression (discussed in detail in
the next section). Endoparasitoid venom proteins include neurotoxin-like/paralytic
factors, such as pimplin in Pimpla hypochondriaca, that induce transient paralysis
in contrast to the permanent host paralysis induced by ectoparasitoids (Parkinson
et al. 2002; Asgari and Rivers 2011). However, several ectoparasitic venom cate-
gories and specific venoms are shared with the more derived endoparasitic wasps
(Asgari and Rivers 2011; Poirie et al. 2014). Among the immune-regulating
venoms in the ectoparasitoid N. vitripennis, serine proteases are the largest group
and are also found in endoparasitoids (Asgari et al. 2003; de Graaf et al. 2010; Zhu
et al. 2010; Asgari and Rivers 2011). In the endoparasitoid, Cotesia rubecula, serine
proteases inhibit host defense via downregulation of prophenoloxidase cascades
(Asgari et al. 2003; Zhang et al. 2004). On the other hand, Pimpla hypochondriaca
has evolved an additional three phenol oxidases to mediate host immunity
(Parkinson et al. 2001). Poirie et al. (2014) provide a review of specific venom
proteins in 2 ectoparasitoid species and 15 endoparasitoid species.
Viruses and Virus-like Particles

Endoparasitic wasps, which lay their eggs within host tissues, are at a high risk of
losing their eggs to host immune responses. Insect immune systems attack foreign
bodies by encapsulating them in hemocytes and coating them in a thick melanin
layer, a process called the encapsulation and melanization reaction. To override this
host defense and ensure survival of their offspring, endoparasitic wasps have
evolved novel mutualistic relationship with viruses (Strand and Pech 1995; Asgari
and Rivers 2011). Polydnaviruses (PDVs) and virus-like particles (VLPs) are
injected into the host along with venom proteins to suppress host immunity.
Research into the evolution and function of PDVs and VLPs is relatively recent
but has garnered much interest. PDVs are an endogenous and integral part of
endoparasitoid genome (Belle et al. 2002) and are produced in the ovarian calyx
of the wasp (Wyler and Lanzrein 2003). PDVs can deliver non-viral wasp genes
into hosts to perform immune suppressive functions (Webb 1998; Roossinck 2011;
Drezen et al. 2014). PDVs became incorporated into certain groups of braconid and
ichneumonid genomes in two independent events (Webb 1998), with braconid
PDVs or bracoviruses originating ~74 mya (Whitfield 2002). PDVs function syn-
ergistically with the venom proteins or overlap with them in function, although the
exact mechanisms are still unclear (Asgari and Rivers 2011).
VLPs are produced in actin-lined canals of the venom gland secretory cells
(Ferrarese et al. 2009). They suppress host cellular immune responses by reducing
the spreading and adhesive properties of host hemocytes and inducing apoptosis
(Suzuki and Tanaka 2006; Suzuki et al. 2008; Asgari and Rivers 2011). Mecha-
nisms of host immune suppression can differ between parasitoids adapted to
different ecologies (Schlenke et al. 2007). For example, the generalist drosophilid
parasitoid Leptopilina heterotoma produces highly immune-suppressive venom,
whereas the venom of specialist Leptopilina boulardi interferes further downstream

of the immunity pathways (Schlenke et al. 2007). The role of VLPs and their
evolution with respect to host range warrants further investigation in such cases.
Small Molecules
Venoms of parasitic wasps may also contain small molecules. Scoliid wasps secrete
peptides such as bradykinins to block the synaptic transmission of nicotinic acetyl-
choline receptors and induce paralysis (Konno et al. 2002). Ampulex compressa
produces and injects dopamine (an amine) or a dopamine-like substance directly
into the brain and ganglia of its cockroach host to inhibit locomotion and escape
behavior (Weisel-Eichler et al. 1999). However, more comprehensive analyses of
the small molecules present in parasitoid venoms are yet to be done.
Gene Regulatory Elements

Recent evidence has shown that venom glands produce gene regulatory elements
such as microRNAs (miRNAs) that can control expression of venom genes to alter
venom protein secretion (Rendon-Anaya et al. 2012; Durban et al. 2013; Vonk
et al. 2013). In snakes, venom gland miRNAs are co-opted from regulatory net-
works of other organs (Vonk et al. 2013), to cause shifts in venom profiles through
regulation of gene expression (Durban et al. 2013). miRNA in scorpions is hypoth-
esized to regulate toxin secretion via post-translational control mechanisms and
transcript degradation to reduce RNA abundance (Rendon-Anaya et al. 2012).
Colinet et al. (2010) suggest that intraspecific venom variation in parasitic wasps
may result from differential binding levels of transcription factors to cis-regulatory
sequences. In the genome of the ectoparasitoid wasp N. vitripennis, ~100 miRNAs
have been found, although tissue specificity and function of these are yet to be
established (Werren et al. 2010).
In contrast, little is known about the role of miRNA themselves as venom
components for modulating gene expression in the target envenomated species.
This is clearly an interesting topic for future exploration.
How Parasitoid Venoms Function
Venom Functions in Insect Hosts
The earliest parasitoid wasps in Apocrita parasitized larvae of Coleoptera (beetles)

(Dowton and Austin 2001). The range of insect host species rapidly expanded along
with the evolution and diversification of Insecta. Modern-day parasitoid wasps are
known to attack every order of Insecta, and insect hosts are parasitized in many
diverse ways.
A substantial number of studies have been aimed at understanding differences in
ecto- and endoparasitic wasp interactions with hosts. Insect ecto- and
endoparasitoids employ different strategies of host manipulation. Most
ectoparasitoids are idiobionts, i.e., they arrest host growth and development.
Endoparasitoids are usually koinobionts – they allow hosts to continue to grow and
develop. The following sections provide an overview of mechanisms by which
ecto- and endoparasitoids manipulate their hosts.
Ectoparasitic Host Manipulations

Most ectoparasitoids are idiobionts. It is a general perception that ectoparasitoid
venom has mainly evolved to cause developmental arrest in hosts and that
ectoparasitoids harvest nutrition from hosts in this arrested state. However, ecto-
parasitic manipulations of the host are more complex and include alteration of
metabolism and physiological state of the host, presumably in order to produce a
more nutritious environment for the developing young. Venom functions are best
characterized in the model ectoparasitoid N. vitripennis, which secretes at least
79 venom proteins and parasitizes a range of flesh fly, blowfly, and muscoid fly
pupae (Rivers et al. 2006; Werren et al. 2010). N. vitripennis venom induces a
diapause-like developmental arrest in host pupae (Rivers and Denlinger 1994a).
Eye pigment deposition and bristle formation normally seen in developing hosts fail
to occur in envenomated hosts. However, rather than completely arresting bodily
functions, venom causes targeted changes in physiology and metabolism of the host
(Danneels et al. 2013; Martinson et al. 2014; Mrinalini et al. 2014). The set of
metabolic symptoms produced by N. vitripennis venom emulates some aspects of
natural developmental arrest of insects during overwintering and diapause, but
there also are distinct differences (Mrinalini et al. 2014). Venom suppresses oxi-
dative respiration, by manipulating glycolytic and Krebs cycle pathways, and
amplifies select pathways (Rivers and Denlinger 1994b; Martinson et al. 2014;
Mrinalini et al. 2014). Amino acids and sugar derivatives are enriched (Rivers and
Denlinger 1994b; Mrinalini et al. 2014). The fat bodies of hosts increase in lipid
content (Rivers and Denlinger 1994b, 1995) and could be compensating for evolu-
tionary loss of liposynthetic abilities in parasitoids (Visser and Ellers 2008; Visser
et al. 2010, 2012; Ellers et al. 2012). The extent to which these changes increase
host nutritive value and enhance parasitoid fitness is still not clear, although it is
assumed.
N. vitripennis venom effects developmental arrest by altering developmental
pathway signaling and gene expression (Danneels et al. 2013; Martinson
et al. 2014). The central nervous system undergoes neuronal apoptosis (Ratcliffe
and King 1969), and biosynthesis of neurotransmitters such as L-DOPA and GABA
is dysregulated (Mrinalini et al. 2014). Adult wasp structures never develop as
chitin biosynthesis, essential for insect metamorphosis, is downregulated (Mrinalini
et al. 2014). Some ectoparasitic venoms can also modulate development of a
growing host. Species in Eupelmidae modulate hormonal physiology of ecdysis
in their lepidopteran hosts (Nakamatsu and Tanaka 2003). Host larvae never molt,
but continue to feed and grow, thereby diminishing the risk of parasitoid larval
detachment while providing a food source enriched in proteins and lipids
(Nakamatsu and Tanaka 2003).
Ectoparasitoid venoms also manipulate both cellular and humoral immunity of
the host. Venom suppresses cellular immunity via reduced melanization, phenol
oxidase activity, and programmed cell death/apoptosis of hemocytes (Rivers

et al. 2002; Abt and Rivers 2007; Danneels et al. 2013). Humoral immunity of
the host is modulated to increase antimicrobial peptide synthesis, possibly
preventing bacterial growth and host decay (Martinson et al. 2014). A more
complete understanding of ectoparasitoid venom function will emerge from studies
using RNAi knockdown of venom genes combined with metabolic analyses and
gene expression analyses in the host (Siebert et al. 2015).
Endoparasitic Host Manipulations

Endoparasitoid eggs are laid inside the host. Once the eggs hatch, the larvae may
spend their life partially or completely inside the host. Therefore endoparasitoids
target host immune response, development, physiology, as well as behavior.
Endoparasitoids regulate host immunity using venom as well as parasitoid larval
secretions (Basio and Kim 2005). The foremost function of endoparasitoid venom
is to ablate immune responses for immediate protection of eggs. Venom proteins,
PDVs, and VLPs injected into the host suppress encapsulation and melanization
responses (Asgari and Rivers 2011). Some species lack PDVs and VLPs, and
venom alone is sufficient (Er et al. 2011). Immune suppression by venom can
occur at the initial stages or further downstream of immune pathway cascades
(Schlenke et al. 2007). A comprehensive review of endoparasitoid venom-induced
changes to host immunity has been provided by Er et al. (2011) and Asgari and
Rivers (2011). A comparative study of endoparasitoid (Pteromalus puparum) and
ectoparasitoid (N. vitripennis) venom has shown that toxicity to host hemocytes has
a broader host species range among endoparasitoids (Zhang et al. 2005).
When endoparasitoid eggs hatch and larvae emerge, they continue to feed and
grow inside the hosts. This requires parasitoid larvae to mitigate any remaining host
immunity that can kill them. Larvae of parasitoids produce teratocytes – cells
derived from the embryo that detach and grow into giant cells while inside the
host. Teratocytes may inhibit host phenol oxidase activity in host hemolymph and
thereby suppress host immunity (Basio and Kim 2005).
Parasitoids may also capitalize on defensive aposematic coloration of hosts.
Adult ladybird beetles are toxic, and they advertise their toxicity with brightly
colored dorsal surfaces. Dinocampus coccinellae is an endoparasitoid of the
ladybird beetle. The parasitoid larva emerges from its host, builds a cocoon around
host legs while it is still alive, and therefore decreases the chances of predation by
remaining under the aposematic host (Maure et al. 2011).
Endoparasitoids adopt different host manipulation strategies based on their
clutch size (Harvey 2000). Solitary parasitoids generally suppress development
and the behavior of their host. Hosts of gregarious parasitoids (multiple egg layers)
continue to grow and increase in size to provide nutrition for a larger brood.
Behavioral Manipulations of Endoparasitoid Hosts

An interesting aspect of endoparasitism is manipulation of host behavior. Larval
secretions of endoparasitoids are known to regulate behavior in spider hosts
(Eberhard 2010b); however, adult female parasitoids also secrete venom
components that manipulate host behavior. For example, when Ampulex compressa
injects venom into its cockroach host, the cockroach shows no escape behavior but
grooms itself excessively (Weisel-Eichler et al. 1999; Libersat and Gal 2014).
Some parasitoids can induce guarding behaviors that protect developing parasit-
oids. White cabbage butterfly caterpillars protect Cotesia glomerata pupa
(Braconidae) against predators using their own silk to build cocoons (Harvey
et al. 2008). The caterpillar host (Thyrinteina leucocerae) reduces mortality in
Glyptapanteles sp. (Braconidae) by actively defending them against predators
using violent head swings (Grosman et al. 2008). Aphidius nigripes induces migra-
tion of aphid hosts to concealed and protected locations if parasitoid larvae are in
diapause (Brodeur and McNeil 1989). It remains to be seen whether these behav-
ioral regulations are induced by venom injection or larval secretions.
Parasitoid Venoms and Arachnid Hosts
Parasitic wasps are known to parasitize ticks and spiders, but the knowledge base on
Arachnid host diversity or venom-induced host manipulations is still relatively
limited. Ixodiphagus wasps (Encyrtidae) parasitize tick species from several genera
and have been proposed as a natural means for controlling tick populations and tick-
transmitted diseases (Hu et al. 1998). Spiders are mostly parasitized as eggs and
adults, and recently ichneumonid and eupelmid wasps were found to parasitize
juvenile ant-eating spiders (Zodariidae) (Korenko et al. 2013). Whether venom can
also manipulate development in spiders as in insects is not known.
Spider eggs provide a food source and egg cases provide a protective mantle
until the adult wasps emerge. Parasitization of adult spiders is, however, more
complex. Adult spiders are exploited as a source of nutrition as well as for their
characteristic web-weaving skills (Eberhard 2000). Orb-weaving spiders
(Araneidae) normally weave orb webs, but when parasitized by Polysphinctine
wasps (Ichneumonidae), they weave “cocoon webs” for the wasp larva to pupate
(Eberhard 2000, 2010a; Gonzaga and Sobczak 2011; Korenko et al. 2014).
Neottiura species (Theridiidae) are used by ichneumonid wasps for their
overwintering web-weaving behavior (Korenko and Pekar 2011). Dense webs,
originally meant as a survival mechanism against cold temperatures, are instead
built around wasp larvae (Korenko and Pekar 2011). Spider species that share the
same web type build different types of webs when parasitized by different wasp
species (Korenko et al. 2014). In these cases, it appears that venom from adult
wasps only serves to temporarily paralyze spider hosts (Eberhard 2010b). The wasp
larva, however, controls web-weaving behavior possibly via introduction of
neuromodulatory substances into the spider while ingesting its hemolymph
(Eberhard 2010b).
Perhaps the most charismatic of Arachnid hunters is the tarantula hawk wasp
(Pompilidae). It stings and paralyzes tarantulas (Theraphosidae) and stores them in
burrows for its solitary offspring (Cazier and Mortenson 1964). At 3–5 cm in
length, it is rather large compared to other parasitic wasps, and it is also relatively
long-lived (Schmidt 2004). Its venom performs the dual functions of paralyzing the
tarantula host as well as defense. The tarantula hawk wasp is infamous for its sting.
Scoring 4.0 on Schmidt’s pain index, its sting is considered the most painful yet to
be delivered to humans by an insect (Schmidt 1990). The pain is said to cause such
an uncontrolled physical reaction that it is recommended the victim “lie down and
scream” to avoid risk of injury (Schmidt 2004). The venom, however, lacks other
toxic effects, indicating that these wasps likely evolved solely pain-inducing venom
components for defense against vertebrate predators (Schmidt 2004).
Venom Evolution
Parasitoid-host interaction can be an arms race between successful parasitization

and host resistance to parasitism. This can drive adaptive evolution of both para-
sitoid venom proteins and host immunity (Kraaijeveld and Godfray 1997; Fellowes
et al. 1998; Keebaugh and Schlenke 2012; Goecks et al. 2013). Venom proteins are
known to evolve rapidly in closely related parasitic wasp species with different
lifestyles. For example, generalist and specialist congeners produce venoms
containing unique as well as shared toxins (Schlenke et al. 2007; Goecks
et al. 2013). Moreover, the function of parasitic wasp venom in host manipulation
for the benefit of its offspring (as opposed to defense and feeding) brings into
consideration several distinct aspects of parasitic lifestyle that likely exert evolu-
tionary pressures on venom toxins. Host-switching behavior in generalist
idiobionts, due to relative abundance of hosts, can cause adaptive venom evolution
within short durations to increase offspring survival (Tschopp et al. 2013; Jones
et al. 2015).
In endoparasitic wasps, clutch size, egg mobility inside the host, and time to
larval emergence could drive variations in venom effects on host growth and
immunity (Harvey 2000; Schlenke et al. 2007). In particular, host immune
responses can result in encapsulation of the parasitoid egg through melanization
responses. There is ample evidence for genetic variation in ability to encapsulate
the eggs of different wasp species, and this genotype matching could be a driving
force for antagonistic coevolution of parasitoid venoms and host immune responses
(Kraaijeveld and Godfray 1997; Fellowes et al. 1998). In contrast, because
ectoparasitoid eggs are external to the host, they avoid the need to evade host
immune responses. Nevertheless, ectoparasitoid venom could evolve rapidly fol-
lowing host shifts and changes from specialist to generalist host use.
Models of Venom Evolution
Key questions in venom biology are: what is the origin of venom genes and how do
venoms evolve? The gene birth-death model (Nei and Rooney 2005) is widely
invoked to explain the evolution of venom genes. In their review paper, Casewell
et al. (2013) provide an excellent scheme of genomic mechanisms for toxin gene
Fig. 3 Models of venom evolution. Schematic representations are provided for six different
mechanisms by which venoms can evolve: (a) gene duplication and neo-functionalization
(Adapted from Casewell et al. 2013), (b) multifunctionality, (c) co-option of non-venom genes
for venom function, (d) evolution of alternative splicing of venom transcripts, (e) evolution from
noncoding DNA, and (f) venom gene evolution following lateral gene transfer (LGT) from
microorganisms
evolution via gene duplication and recruitment of one paralog as a venom protein
through expression in the venom gland. They also discuss gene-level processes,
such as toxin domain duplication/loss and alternative splicing (Casewell
et al. 2013). However, additional mechanisms are possible. Building on Casewell
et al. (2013), the authors here propose six general mechanisms of venom gene
evolution (Fig. 3). These are (a) gene duplication and neo-functionalization,
(b) multifunctionality, (c) co-option of non-venom genes for venom function,
(d) evolution of alternate splicing of venom transcripts, (e) evolution from noncod-
ing DNA, and (f) venom gene evolution following lateral gene transfer (LGT) from
microorganisms.
Gene Duplication and Neo-functionalization

The gene birth-death model is a common mechanism for the origin of new genes
and evolution of novel functions via duplication of existing genes and divergence
of the paralogs (Nei and Rooney 2005) (Fig. 3a). Asgari and Rivers (2011)
propose that parasitoid venoms are likely to have evolved by this mechanism,
where normal metabolic insect genes undergo duplication, which is followed by
recruitment of a paralog to venom gland expression and its functional divergence.
However, a systematic analysis of the parasitoid venom proteomes has not yet
been done to determine the origins of venom proteins. Therefore, it remains an
open question whether the classic model is the most common means of venom
evolution.
Multifunctionality
The second method of venom evolution is multifunctionalization, which can
occur via regulation of gene expression (Fig. 3b). In this scenario, a preexisting
non-venom protein (e.g., expressed in the whole body or other tissues) is addi-
tionally recruited for venom function via expression in the venom gland. For
example, enzymatic genes that are normally expressed in the body can acquire
toxin functions when they start becoming expressed in the venom glands. Such
venom proteins that have both “normal” physiological function and venom
function will be constrained in optimization as a venom protein due to the
requirement of also maintaining their standard role. Two mechanisms by which
this constraint could be removed over evolutionary time are (a) co-option and
(b) alternative splicing.
Co-option of Non-venom Genes for Venom Function

Venom evolution via co-option occurs when gene expression becomes
restricted to the venom gland, and there is a loss of expression or decreased
expression in other parts of the body (Fig. 3c). Co-option can result from
disruptive selection induced when the fitness benefit of producing a venom
protein is high, but there is a lesser loss of fitness from eliminating expression
in other tissue(s). Therefore co-option is only likely when the normal physio-
logical function of the gene is non-vital. The authors thereby hypothesize that
co-option may be the most frequent mechanism of venom gene recruitment
because it can occur by rapid evolution of cis regulation of gene expression.
However, demonstrating co-option requires detailed knowledge of expression
patterns of newly evolved venom genes in different tissue types and life stages
from a set of related species.
Evolution of Alternative Splicing of Venom Transcripts

Disruptive selection on a gene serving both “normal” and venom function can lead
to the evolution of alternative splicing in different tissues (Fig. 3d). How quickly
alternative splicing can evolve remains an open question, but once it has occurred,
venom-specific exons are freed to specialize for toxic function. In addition, post-
genomic mechanisms such as post-translational modifications can cause differences
in venom protein compositions among species in spite of shared genotypes
(Casewell et al. 2013). For example, viperid snake species can regulate venom
protein compositions at transcriptional (alternative splicing), translational, and
post-translational levels (Casewell et al. 2014).
Evolution from Noncoding DNA

Sometimes, novel protein-coding genes can evolve from noncoding DNA that is not
a preexisting gene in the organism (Tautz and Domazet-Loso 2011). New func-
tional genes can be derived from noncoding DNA via acquisition of transcription
start sites and gene expression (Fig. 3e). Noncoding DNA could therefore be a
potential source of genetic material for evolution of toxin genes that subsequently
acquire venom function. However, the frequency with which this occurs among
venom genes is unknown.
Venom Gene Evolution Following Lateral Gene Transfer (LGT) from

Microorganisms
LGTs are horizontal transfers of genetic material from one organism to another.
Lateral gene transfers from prokaryotes and single-celled eukaryotes to metazoans
were considered to be rare but are being increasingly discovered in insects
(Dunning Hotopp et al. 2007; Dunning Hotopp 2013), including in the
ectoparasitoid N. vitripennis (Werren et al. 2010). This genetic material can become
incorporated into eukaryotic host genomes and acquire novel functions when there
is a selective advantage. Therefore prokaryotic LGTs can be a source of genetic
material for the evolution and recruitment of genes for venom function (Fig. 3f).
Recently, endochitinase, a protein synthesized by the venom gland of parasitoid
wasps, was found to be horizontally transferred from microsporidia (Martinson
et al. 2016). An RNAi knockdown of the gene suggests that it may play a role in
dysregulating chitin metabolism of hosts and in defence of hosts against opportu-
nistic fungi (Martinson et al. 2016).
Questions to Explore in Parasitic Venom Evolution
As discussed previously, parasitoids are among the richest group in terms of species
diversity, morphology of venom glands, and lifestyles. Adaptations to different
hosts and parasitic ecologies likely have implications for venom protein evolution.
Among the several questions that arise are: how are new venom proteins recruited
and from what sources? What is the origin of parasitoid toxins that have no
homology to proteins in other organisms? How does the venom repertoire change
when parasitoids switch from a generalist to a specialist host range (or vice versa)?
What are the venom compositional differences in ectoparasitoids and
endoparasitoids that share the same host? Is there geographic variation in venom
within the same species and do individual parasitoids alter their venom profiles in
response to host use? How do the repertoires differ between egg, larval, pupal, and
adult parasitoids? What venom proteins are conserved across diverse parasitic
species, and finally, is there evolutionary convergence of venom components
among parasitoids, bees, ants, wasps, arachnids, and other venomous organisms?
The first step toward answering these questions is cataloging the venom reper-
toires in different species of parasitoids. To date, investigation of parasitic venoms
has mostly focussed on functions of individual toxins, usually of high abundance,

whereas relatively complete venom repertoires are known for only seven species
(de Graaf et al. 2010; Vincent et al. 2010; Werren et al. 2010; Zhu et al. 2010;
Goecks et al. 2013; Burke and Strand 2014; Colinet et al. 2014a). Parasitic wasp
venom protein analysis was difficult until recently due to their small size and
smaller venom yield. However, high-throughput mass spectrometry-based proteo-
mic analysis has now made de novo sequencing of parasitoid venom proteomes
possible. When used in tandem with venom transcriptomes, locus-based identifica-
tion of venom proteins can be performed. These discovery-based approaches are
key to cataloging and understanding parasitic venom evolution and are now an area
of active research (de Graaf et al. 2010; Zhu et al. 2010; Colinet et al. 2013; Goecks
et al. 2013).
Recent Advances
Technological advances in recent years have made venom analysis in parasitoid

wasps possible. Liquid chromatography coupled with mass spectrometry is a high-
throughput proteomic method that can obtain protein sequences from a quantity as
small as 5–10 ng (Dr. Sheng Zhang, personal communication). Additionally, two
new methods of venom functional analysis have recently been used very success-
fully: metabolomics and RNAi knockdowns. Metabolomics is a recently developed
toolset in the “omics” field. Mrinalini et al. (2014) used metabolomic analysis to
characterize the changes in 249 biochemicals in the insect host Sarcophaga bullata
after envenomation by N. vitripennis. The data from this study, used in conjunction
with pathway analysis, provided a global understanding of the biochemical changes
brought about by the venom of this ectoparasitoid (Mrinalini et al. 2014). Atypical
regulation of host glucose and sorbitol metabolism was found in this study and
provides a basis for exploring applications of N. vitripennis venom in diabetes
research (Mrinalini et al. 2014).
The second new approach in venom function studies combines RNAi knock-
down of venom genes in the venomous species with RNA sequencing in the
envenomated target species (Siebert et al. 2015). The RNAi method uses double-
stranded RNA to severely reduce (or knock down) the expression of genes of
interest and has been successfully developed in the ectoparasitoid N. vitripennis
(Lynch and Desplan 2006; Werren et al. 2009). After a venom gene of interest is
knocked down, the wasp is allowed to sting the host, and venom function is
assessed by performing RNA-seq analysis of the host to characterize gene expres-
sion changes. When this data is compared to gene expression of hosts injected with
whole venom (Martinson et al. 2014), it is possible to subtractively assess the
function of the knocked-down venom gene. Therefore, RNAi/RNA-seq method
can be used both to test hypotheses concerning functions of particular venom
components and as a potent hypothesis generation tool.
One finding of RNAi/RNA-seq studies is that some venom components have
compensatory effects, i.e., reducing alteration in the host by total venom of certain
target genes and pathways. Therefore the authors propose that compensatory effects
are likely an important function of different components in the venom repertoire.
An analogy to the “drug treadmill” can be made. That is, some venom proteins play
a role in reducing and modulating negative effects of other venom proteins. In the
case of parasitoid venoms, this occurs in order to keep the host alive long enough
for the desired alternations in host metabolism to occur.
Finally, the application of parasitic wasp venom for pharmacological purposes is
being explored using mammalian cell lines. N. vitripennis venom applied to
fibrosarcoma cells has been found to suppress NF-kB gene activity that drives
deleterious inflammatory responses, tissue damage, and tumor growth (Danneels
et al. 2014).
Future Directions
Rapid advancement of parasitic venom research is needed to establish parasitoid

wasps in toxinology. Several studies have already established discovery-based
analysis as a proof of concept for characterization of parasitoid venom repertoires.
While the need of the day is to collect and analyze venom from different lineages of
parasitoids, it is important to recognize that the extent to which venom can be
characterized is dependent on the technology used. Therefore, it may be essential to
draw up guidelines for analysis of parasitic venom. For example, the venom
repertoire of a particular species should not be considered completely known unless
a locus-based characterization using venom gland transcriptome and/or species
genome coupled with venom proteomic analysis has been performed.
Perhaps the greatest challenge in parasitoid wasp toxinology is the sheer number
of species and rapid turnover of venom proteins. However, this also presents great
opportunities for functional studies and exploring pharmacological uses of parasit-
oid venoms. Because these insects are tractable for genetic manipulations (e.g.,
systemic RNAi) and their venoms have evolved specifically to manipulate physi-
ological and metabolic processes in subtle ways, they present an outstanding
resource for toxinological research and drug discovery.
Acknowledgments We thank the National University of Singapore, South East Asian Biodiver-
sity Genomics (R-154-000-648-646), and the National Institutes of Health (RO1GM098667) for
resources and support.
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DOI 10.1007/978-94-007-6727-0_3-1
Venom as a Component of External Immune Defense in Hymenoptera

David Baracchia* and Simon Tragustb
a
Research Centre for Psychology, School of Biological and Chemical Sciences, Queen Mary University of London,
London, UK
b
Animal Population Ecology, Animal Ecology I, Bayreuth Center for Ecology and Environmental Research (BayCEER),
University of Bayreuth, Bayreuth, Germany
Abstract
An intriguing feature of most hymenopteran venoms is that they display broad antimicrobial activity. In
particular, the venoms of social Hymenoptera (ants, wasps, and bees) represent the most conspicuous
source of antimicrobial secretions. In solitary and parasitic species, venom is used to immobilize or kill
prey and to preserve them as stored food for their immature brood. In social species, venom is frequently
also externalized both onto the cuticle and the nest surface. This indicates that venom use in Hymenoptera
is not just restricted to hunting activities or to deter predators, but is also actively used as an externalized
defensive agent, providing a first chemical barrier against microorganisms present in the environment.
This chapter will discuss the importance and biological significance of venom as part of an external
immune defense in Hymenoptera with special emphasis on social species. In addition ecological and
environmental factors constraining the use of venom as external immune defense will be highlighted.
Keywords
Antimicrobial peptides; Social insects; Ecological immunology; Social immunity
Introduction
A variety of venom systems have evolved across the animal kingdom. This taxonomic diversity highlights
the importance of venom as an evolutionary innovation (Casewell et al. 2013). Unsurprisingly, many
studies have been conducted to understand the evolutionary processes that drove the generation of these
venomous systems and of venom complexity. From this wealth of data, the insight emerged that the
complex composition and targeting of venom reflects the multiple functions and biological roles venom
has in different animals. From an evolutionary perspective, venoms are commonly regarded as either
foraging adaptations to subdue prey or as defensive adaptations against predators (Casewell et al. 2013).
Venoms found in the insect order Hymenoptera are certainly not an exception from this point of view
(Piek 1986). As in other venomous animals, the composition and function of venom in Hymenoptera are
well adapted to immobilize or kill prey, and in many other cases, it serves as a defensive adaptation against
enemies such as invertebrate and vertebrate predators. Defense is often also a common secondary function
of venom in many species in which foraging is its primary purpose. This conception has led to neglect the
fundamental role that venoms play in the interactions with pathogenic, parasitic, commensal, or mutual-
istic microorganisms. Yet, these microorganisms certainly also represent a strong selective pressure for
*Email: david.baracchi@gmail.com
*Email: d.baracchi@qmul.ac.uk
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the maintenance of venom for defensive purposes (Moreau 2013). Indeed, a characteristic of venomous
secretions in Hymenoptera is the strong antimicrobial activity that they exert (Kuhn-Nentwig 2003;
Moreau 2013). Although this characteristic of venom is broadly distributed among distant hymenopteran
species, it has so far been considered to be only of secondary importance. Only recently it became clear
that many hymenopteran species, whatever their life styles, have evolved venom features that actively
participate in the regulation of microbial infections. This view has come from the recognition that many
insects deploy antimicrobials to their immediate environment in order to manipulate the composition of
the microbial community surrounding them. These antimicrobials often originate from exocrine glands,
especially from venom glands (Otti et al. 2014).
In this chapter the importance and biological significance of venom as part of an external immune
defense in Hymenoptera will be highlighted with special emphasis on those species characterized by
social habits. Venom of vertebrates and invertebrates is thought to be metabolically costly and the
energetic cost of venom might constrain both its synthesis and use (Casewell et al. 2013; Nisani
et al. 2012; but see Smith et al. 2014). Despite that, most social hymenopterans use considerable quantities
of venom to sanitize themselves, related group members, and the nest surface, implying that the
advantages overcome the metabolic cost.
Immune Defenses in Solitary and Social Hymenoptera

Like all animals, Hymenoptera enlist a variety of immune defenses against disease agents (Schmid-
Hempel 2011). From a molecular perspective, the insect immune system involves three core signal
transduction pathways, two of which are regulated by pattern recognition receptors (Toll and Imd) and
the third one by stress signals from tissues (JAK/STAT). These pathways orchestrate a huge number of
molecular effectors, including antimicrobial peptides, reactive oxygen species, and lectins. The system,
however, also involves physical barriers to infection such as the integument and the gut. Furthermore
coordinated responses of several subpopulations of hemocytes are activated in the hemolymph when
these barriers are breached by a putative pathogen.
Apart from these internally expressed immune defenses, there are several other defense mechanisms
existing outside of what is traditionally considered to be part of the immune system. Those mechanisms
involve, for example, changes in life-history traits (Michalakis 2009) or behavioral avoidance and self-
medication (de Roode and Lefèvre 2012; Moore 2002) and clearly contribute to an organism’s defense
against parasites and pathogens. Social insects also benefit from the fact that they show cooperative
defenses that complement the defense of the individual. Thus, insects living in a society can rely on both
individual and collective defenses, with selection for immunity acting simultaneously on both these
levels, encompassing complex interactions and different selective constraints. One of the most illustrative
examples of cooperative defense is the social fever exhibited by honeybees, where an increase of comb
temperature is induced by adults in response to infestation by the fungal pathogen Ascosphaera apis,
preventing disease development (Starks et al. 2000). Other defenses in insect societies include organiza-
tional properties of the colony that are critical in shielding infectious diseases (Schmid-Hempel 1998;
Stroeymeyt et al. 2014). For example, in the colonies of ants and bees, the inner region of the nest
containing immature brood, young workers, and the queen is spatially and behaviorally segregated from
older workers, which are mainly active outside the nest foraging or in the nest periphery disposing of dead
bodies and garbage (Baracchi and Cini 2014; Mersch et al. 2013). The spatial segregation emerging from
division of labor and preferential age and task-based interaction leads to a form of organizational
immunity protecting the more important and delicate region of the nest from possible infections.
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Besides indirect effects of behaviors through organizational immunity, behaviors can have a more
direct effect on immune defense. Behaviors targeted at decreasing disease transmission and increasing
resistance to parasites and pathogens within a social insect colony have been referred to as antiseptic
behaviors (Wilson-Rich et al. 2009). Antiseptic behaviors include a large repertoire ranging from the
hygienic removal and undertaking of diseased brood and young adults in ants and bees (Baracchi
et al. 2012a; Sun and Zhou 2013; Tragust et al. 2013a, b) to mutual grooming behavior (Evans and
Spivak 2010; Tragust et al. 2013a).
The use of antimicrobials against parasites and diseases in insect societies is intimately linked to
behavioral adaptations as they are required to apply and distribute antimicrobial compounds as a first line
of defense. Antimicrobials acting in the environment of a social insect colony might be environment
derived, derived from symbiotic relations, or self-produced.
Ants and bees often disinfect their nest material with resins, i.e., complex plant secretions with diverse
antimicrobial properties, derived from the environment. In the wood ant, Formica paralugubris, resins
have been shown to inhibit the growth of microbes, and nests rich in resins have fewer bacteria and fungi
than ant nests containing only very little resin (Christe et al. 2003). Even if resin collection might be costly
in term of time and effort, there are indications that wood ants benefit directly from the antimicrobial
property of resin as they survive longer if infected by bacteria or fungi (Chapuisat et al. 2007). Similar
behaviors are also common in the honeybee, Apis mellifera, and other honeybee species where resins are
actively included into the wax of the nest to form what has been called propolis. This behavior is clearly an
adaption to fight pathogens, as colonies of Apis mellifera increase resin foraging rate after a challenge with
the fungal pathogen Ascophera apis. Additionally, colonies experimentally enriched with resin had
decreased infection intensities of this fungal pathogen (reviewed in Simone et al. 2009).
In addition to antimicrobial active plant resins, the antimicrobial immune defense of social insects also
relies on antimicrobials gained through symbiotic relationships. It has recently been shown that members
of all nine recognized honeybee species, plus stingless bee species, harbor diverse symbiotic lactic acid
bacteria that are involved in food preservation. In addition those symbiotic bacteria likely also contribute
to host defense against pathogens and parasites intercepted during foraging (Vásquez et al. 2012).
Besides antimicrobial compounds derived from the environment and from symbionts, social insects
produce a variety of antimicrobial secretions in their exocrine glands, especially ants, and use them to
sanitize their own body and their nest. Until recently, the role of venom as a major source of self-produced
antimicrobial compounds has often been neglected, despite the fact that most venoms show a strong
antimicrobial activity (Kuhn-Nentwig 2003).
Altogether, organizational, behavioral, and physiological adaptations of social insects to prevent the
establishment and spread of parasites and pathogens have been referred to as social immunity (Cremer
et al. 2007). The key idea is that by acting collectively, individuals are better able to mount a defense than
is possible acting independently. The idea of a social immune system has been later expanded to include
immune services targeting one or more recipients not only in social insects but also in other animal family
structures, in social microbes, or in the context of herd immunity, i.e., the reduction of the risk of infection
among susceptible individuals by the presence and proximity of immune individuals (Cotter and Kilner
2010). With the focus on immune defense of organisms in general, it was recently proposed to view any
heritable trait acting outside an organism and improving the protection from pathogens or manipulating
the composition of the microbial community in favor of an organism, as external immune defense (Otti
et al. 2014). This broad definition of immune defense integrates ideas on social immunity and proposes
that the expression of internal or external immune defenses will depend on the ecological niche or life
history of an organism. Furthermore it provides a framework in which costs and benefits of immune
defense traits can be evaluated from an evolutionary and ecological perspective. In particular the
framework proposes that variation in the level of microbe pressure present in a given environment and
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Fig. 1 Selection for external immune defense. Three gradients of important ecological factors, in combination with microbe
pressure and spatial or temporal variation in the environment, favor the evolution of external immune defenses. Selection
pressure will increase (i) from small to large group size, (ii) from temporary/open to permanent/confined nests, and (iii) from no
food storage/slow decay to permanent food storage/fast decay (Reprinted from Otti et al. (2014) with permission of Cell Press)
the temporal or spatial variation of the environment itself represent the two most important factors in the
evolution of external immune defense and its effectiveness (Otti et al. 2014), (Fig. 1).
Focusing on antimicrobially active venoms, the following sections of this chapter will explore whether
the evolution of external immune defense has indeed been favored due to life-history traits found in
solitary and social Hymenoptera, i.e., the storage of food, the use of a stable and confined nest, and group
living. However, first, the antimicrobially active venom of Hymenoptera and its biological role and
function as external immune defense will be described.
Hymenoptera Venoms: A Complex Multifunctional Secretion

The majority of Hymenoptera have a venom gland associated with the ovipositor or the sting (Piek 1986),
(Fig. 2). Details on the function and composition of the secretions of these glands are known for only a
part of the over 150,000 hymenopteran species, and for the sawflies (Symphyta) such knowledge is almost
completely lacking. Hymenoptera venom glands produce extremely complex cocktails of diverse bioac-
tive compounds. It is possible to distinguish at least three different groups of chemical substances
according to their molecular weight (Kuhn-Nentwig 2003; Piek 1986). The first group of heavy com-
pounds (higher than 10 kDa) consists of proteins, including several enzymes such as phospholipases
(responsible for cleaving the membrane phospholipids), hyaluronidases (which degrade the matrix
component hyaluronic acid), acid phosphatases (acting on organic phosphates), and sphingomyelinases
(involved in sphingolipid metabolism reactions). The second group of intermediate molecular weight
(around and lower than 10 kDa) is represented by a peptide fraction, including several cytolytic and
neurotoxic compounds. A third group is composed of low-molecular-mass substances such as ions, free
amino acids, biogenic amines (commonly histamine, serotonin, dopamine, and noradrenaline),
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1 Symphyta 11
8
7
10
Sphecoidea
12 13 14
2 Ichneumonoidea Vespoidea
17
4 5
15 16
Scolioidea
Pompiloidea Formicoidea
6 21 18 19 20
Apoidea
Fig. 2 A selection of types of glandular venom apparatus in Hymenoptera. All representatives show a venom gland, mostly
paired and highly branched, and a venom reservoir. The venom reservoir is part of the ductus venatus, except in Braconidae (3).
Nearly all show a second gland, the Dufour’s gland, which is smaller, unpaired, and not branched, except in some Apoidea
(15, 16). In the Sphecidae, a third gland is frequently present (7–10). In some groups the venom bladder is muscular 2, 3, 4, 12,
13, 14 (Reprinted from Piek (1986) with permission of Academic Press)
neurotransmitters, polyamines, heterocyclic compounds, and alkaloids. Understanding why venoms are
such complex mixtures of compounds requires a clear understanding of what is the evolutionary history of
venom and what functions it holds in living species.
The Evolutionary History of Venom in Hymenoptera

Traditionally, the order of Hymenoptera has been taxonomically partitioned into two major groups: the
Symphyta or sawflies, most of which are phytophagous, and the Apocrita, most of which are entomoph-
agous. The Apocrita can be further divided into the Terebrantia and Aculeata that share common parasitic
ancestral origins. Terebrantia have an ancestral ovipositor (terebra or drill) that is also used as venom duct,
while Aculeata have an ovipositor (aculeus or sting) that is fully modified for injecting venom into a host
and has lost its association with the reproductive system. Terebrantia use their stinging organ to transiently
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or permanently immobilize prey for their developing offspring and to deposit their eggs inside
(endoparasitoids) or outside (ectoparasitoids) the prey’s body. In many solitary aculeate species, venom
compounds retained their nonlethal paralytic function for the storage and capture of prey while acquiring a
new one for use in self-defense (Hermann and Blum 1981). In the social Hymenoptera Aculeata, the
venom, originally used as a tool for capturing and storing prey in solitary species, essentially became a
weapon for defending the colony from predators and competitors. In addition to serve as injectable or
topically applied defensive agent, ant venoms are used also as trail, alarm, sex, queen-recognition,
aggregation, attractant-recruitment, and recognition pheromones, as repellents, and even as toxic agents
for prey capture (Piek 1986).
Venom Use in Solitary and Parasitic Hymenoptera

Besides the well-studied venomous functions of prey capture and defense, the antimicrobial properties of
hymenopteran venoms have often been considered of secondary importance although they constitute a
function broadly distributed among distant hymenopteran species (Moreau 2013). A hypothesis that could
explain the antimicrobial activity in hymenopteran venom is that it serves to prevent the contamination of
the venom apparatus by opportunistic pathogens, contracted at the occasion of stinging events. Data in
support of this hypothesis are however completely lacking except for a recent survey of bacteria, fungi,
and viruses associated with the venom apparatus of Hymenoptera. This survey revealed that the venom
apparatus of Hymenoptera is a suitable organ for the development of viruses only and not for other
microbes (Moreau 2013). An alternative hypothesis to explain the adaptive significance of antimicrobial
venom in solitary and parasitic Hymenoptera is its use to control infection by opportunistic pathogens in
stung prey. This makes intuitive sense, especially for parasitoid and solitary species, which need to keep
the paralyzed prey alive or from decomposing during the development of their offspring. Furthermore,
protection of stored food has been outlined as a likely selective pressure for the evolution of external
immune defense traits such as antimicrobially active venom (Otti et al. 2014). Indeed, evidence points to
the fact that Hymenoptera, especially parasitoids, appear to have evolved venom-based strategies that
limit the opportunity for microorganisms to establish a secondary infection in their host (reviewed in
Asgari and Rivers 2011; Moreau 2013). These include the injection of venom antimicrobial proteins and
peptides, but also the selective manipulation of the host’s immune reactions to the benefit of the
parasitoid’s offspring. For example, the venom components of the endoparasitic hymenopteran
Leptopilina boulardi specifically target their dipteran host’s encapsulation and melanization responses,
but parasitized hosts keep their ability to produce antibacterial and antifungal peptides (Moreau 2013).
Another example is the venom of the jewel wasp Ampulex compressa, which induces excessive grooming
behavior in the stung prey (Libersat and Gal 2014). Both venom-based strategies presumably function to
counteract the increased risk of infection, resulting from a complete suppression of the host’s immune
responses in the case of Leptopilina boulardi or from pathogens on the host’s cuticle in the case of
Ampulex compressa, which may be harmful for the wasp’s egg or developing larva. Similar to parasitic
Hymenoptera, several antimicrobial peptides in the venoms of solitary predatory Hymenoptera are known
(Moreau 2013). Although the potential to regulate infections in animals they sting can be envisaged, the
exact biological roles are still unclear.
Taken together, the venom in many solitary and parasitoid hymenopteran species holds functions as
external immune defense in addition to that of paralyzing hosts. The following sections will show that the
antimicrobial activity of venom has been retained in social Hymenoptera and that venom has a biological
function as external immune defense also in social species.
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Rise of Sociality and the Threat of Predators and Pathogens

In the escalation of parental care, species in which the females of parasitoid Hymenoptera lay their eggs on
paralyzed prey, to species in which a solitary female builds a shelter before capturing a prey on which to
lay an egg, and then to species in which the growing larvae are kept in a nest and progressively furnished
with prey in social Hymenoptera can be found. The nest provides social insects with an element of control
over the environment, improving colony capacities for rearing the immature brood through storage of
food reserves. Apart from cooperative brood care, living in a society has many other benefits. The fitness
of each individual in a group is thought to increase by decreasing the costs associated with important life-
history activities such as foraging efficiency, colonizing and competitive abilities, and the ability to
adaptively modify the environment. In turn, the social lifestyle requires highly developed defense
abilities. The amount of resources offered by insect colonies is likely not only to attract a wide array of
potential predators, notably mammals, birds, and various other arthropods, but also several microorgan-
isms to take advantage of it. The high number of, often closely related, individuals living in high densities
with frequent physical contact and the shared use of space is predicted to significantly increase the
vulnerability of societies to the establishment and spread of infectious diseases. This hypothesis is
generally supported by the observation across many different species that the prevalence of pathogens
and parasites increases with the size of host social groups (Côté and Poulin 1995; Rifkin et al. 2012) and
that numerous parasites and pathogens exist in social insect societies (Schmid-Hempel 1998).
Venom as Externalized Immune Defense in Social Hymenoptera

Several antimicrobial compounds acting against a wide range of bacteria and fungi have been described in
the venom of eusocial bees, bumblebees, social wasps, hornets, and ants. The presence of a range of
antimicrobial peptides which are used also for internal immune defense is notable. For example, the
venom of the honeybee Apis mellifera contains melittin, a basic 26-amino acid peptide that accounts for
45–50 % of the venom dry weight and exhibits strong antimicrobial activity. Similarly, several antimi-
crobial peptides named mastoparans have been described in social wasp genera such as Agelaia, Vespula,
Protonectarina, Protopolybia, Parapolybia, Polybia, and Polistes (Kuhn-Nentwig 2003; Moreau 2013).
In ants, the metapleural glands have long been considered to be one of the most important sources of
antimicrobial compounds, active against a wide range of bacteria and fungi (Yek et al. 2013). Nonetheless,
several antimicrobial peptides have been described also in the venoms of ants, for example, in the
Australian jumper ant Myrmecia pilosula and in the ponerine ant Pachycondyla goeldii. Furthermore,
other venom compounds with strong antimicrobial activity (e.g. alkaloids or formic acid (Morgan 2008))
are known from ants, such as the fire ant Solenopsis invicta (Storey et al. 1991) or species belonging to the
ant subfamily Formicinae (Tragust et al. 2013a).
Venom on the Cuticle

Interestingly, venom components can be found on the cuticle of social bees, wasps, and ants. The primary
function of the epicuticle, the most external layer of the insect cuticle, and the complex mixtures of lipids
on it, is to protect against dehydration and to provide a mechanical barrier against invasion of foreign
matter. The presence of venom compounds with strong antimicrobial activity on insect surfaces suggests
that the venom acts also as a chemical barrier providing a first line of protection against microorganisms.
Besides Polistes paper wasps (Turillazzi 2006; Turillazzi et al. 2006), the presence of venom components
with strong antimicrobial activity on the epicuticle has been recently documented in Stenogastrinae wasps
(Baracchi et al. 2010, 2012b). Stenogastrinae wasps are a subfamily of tropical facultative eusocial wasps,
closely related to Polistinae and Vespinae, forming simple societies that are very small in size. The
Page 7 of 17
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medium-molecular-weight polar substances found on the wasp epicuticle (roughly from 900 to 4000 Da)
were identical to those found in the venom of all the ten studied species from four different genera,
suggesting the venom reservoir as the primary source of cuticular polar substances. Support for the idea
that the venom reservoir is the source of antimicrobial compounds on the cuticle comes also from the
study of different social bees of the genus Apis (Baracchi et al. 2011; Baracchi and Turillazzi 2010). While
venom peptides are present on the cuticle of females, irrespective of their colony duties, they can be found
only in traces on the cuticle of drones, which lack the sting apparatus (Fig. 3). The fact that newly emerged
bees lack venom antimicrobial peptides, both in the venom reservoir and on the cuticle, further confirms
this hypothesis. The presence of antimicrobial venom components on the cuticle of ants is known only for
the fire ant Solenopsis invicta. In this ant species, small quantities of venom are dispensed on the brood
surface during a behavior called “gaster flagging” (Obin and Vander Meer 1985) (Fig. 4), and venom
components are also deposited on eggs by queens during the egg-laying process (Vander Meer and Morel
1995) (Fig. 5).
The behavioral mechanisms responsible for the presence of venom compounds on the cuticle of bees
and wasps are still not completely clear. The most likely explanation is the use of cleaning movements
during grooming to smear venom on the body. Self-grooming observations in Stenogastrinae wasps
suggest the possibility that little drops of venom released from the sting can be collected with the legs by
the wasps and applied all over the body surface (Baracchi et al. 2012b). The importance of grooming for
the spread of antimicrobial active substances derived from the venom gland has recently also been shown
in the ant Lasius neglectus (Tragust et al. 2013a). In this species, adults continuously apply antimicrobial
venom onto their pupae. While direct spraying of their venom onto the pupae can be occasionally
observed, the predominant mode of application is indirect. Venom is first taken up orally during a
behavior called “acidopore grooming” and subsequently applied to pupae during grooming.
Although it is likely that antimicrobial venom components on the cuticle of adults and brood of social
bees, wasps, and ants serve as a protection against microorganisms, direct evidence for this hypothesis
exists only for ants. Blockage of the venom gland opening in the weaver ants Polyrhachis dives, in the
fungus-growing ant Acromyrmex echinatior, and in the garden ant Lasius neglectus all resulted in a
reduced survival of adults and pupae cared by them when challenged with the entomopathogen
Metarhizium anisopliae (Graystock and Hughes 2011; Tragust et al. 2013a; Tranter et al. 2014) (Fig. 6).
In the ant Lasius neglectus, the authors could show that formic acid from the venom gland is the active
agent inhibiting fungal growth and that venom-depleted ants had a significantly reduced ability to resist
such growth (Fig. 7). These authors could also show that application of venom on pupae is amplified
under pathogen pressure, indicating that it is an adaptive behavior.
Although, so far, brood care in the ant Lasius neglectus is the only example of therapeutic use of the
venom in response to pathogens reported in all Hymenoptera, it is likely that future work will reveal that
other species of social insects are also capable to therapeutically defend themselves and related group
members from a wide array of pathogens using their antimicrobial secretions.
Venom on the Nest Surface

Venom components are found not only on the cuticle of social bees, wasps, and ants but also on the nest
surface, likely also serving as a first-line chemical barrier against microorganisms. For example, the
antimicrobial peptide melittin has been described from the nest surface of several species of the genus
Apis (Baracchi et al. 2011; Baracchi and Turillazzi 2010), and the antimicrobial mastoparan peptides
Dominulin A and Dominulin B have been described from the nest surface of the social paper wasp
Polistes dominula (Turillazzi et al. 2006). In ants, there is only indirect evidence that antimicrobial active
venom compounds are found on the nest surface; for example, greater fungal abundance but lower fungal
species richness and diversity were detected in mounds of the fire ant Solenopsis invicta and in
Page 8 of 17
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a
400 Nurses
Arb. U.
300
200
100
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z
b 400 Guards
Arb. U.
300
200
100
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z
c 400 Foragers
Arb. U.
300
200
100
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z
d 400 Queens
Arb. U.
300
200
100
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z
e
400 Drones
Arb. U.
300
200
100
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
m/z
Fig. 3 Average mass spectrometry spectra of 950–4000 Da fraction of cuticular methanol extracts of individuals belonging to
different sexes and castes of honeybee (Apis mellifera). The highest peaks at ~2000 Da (apamin) and ~2850 Da (melittin) of
each spectrum accounts for ~45–50 % and ~2 % of the venom dry weight, respectively, but only melittin has proven
antimicrobial activity (Baracchi et al. 2013) (Reprinted from Baracchi and Turillazzi (2010) with permission of Elsevier)
Aphaenogaster texana nests (Zettler et al. 2002). An involvement of venom compounds in the sanitation
of nests is likely for the weaver ant Polyrhachis dives. In this species, the blockage of the venom gland
opening resulted in an increased risk of the nest material being overgrown by fungi, compared with nest
material that was tended by workers with a functional gland (Tranter et al. 2014) (Fig. 8).
Page 9 of 17
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Fig. 4 Gas chromatogram demonstrating the presence of worker-derived venom alkaloids on the surface of S. invicta brood.
(a) S. invicta venom alkaloids from dissected worker poison sac (b) S. invicta brood rise. Std internal standard, un. pk.
unidentified peak (Reprinted from Vander Meer and Morel (1995) with permission of Springer)
Fig. 5 Comparison of venom alkaloid gas chromatogram profiles: (a) worker, (b) queen, (c) hexane rinse of eggs. QA queen-
specific piperidine alkaloid, WA worker-specific alkaloids. Chromatograms (a, b) are from worker and queen venom sac
extracts, respectively, and are very concentrated compared to chromatogram (c) (Reprinted from Vander Meer and Morel
(1995) with permission of Springer)
Venom on the Cuticle and the Nest Surface as Externalized Immune Defense
Recently, venom components on the nest surface and on the cuticle of several species belonging to the
genus Apis (A. mellifera, A. dorsata, A. cerana, and A. andreniformis) have been investigated with respect
to their nesting ecology and environmental constraints (Baracchi et al. 2011). According to their nesting
Page 10 of 17
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a 1
0.9
0.8
Proportion surviving 0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time after treatment (days)
b 1
0.9
0.8
Proportion surviving
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time after treatment (days)
Fig. 6 Survival of Acromyrmex echinatior leaf-cutting ants (a) and Polyrhachis dives weaver ants (b) that had either their
venom gland (squares) or metapleural gland (triangles; A. echinatior only as P. dives lacks a metapleural gland) blocked with
nail varnish or had nail varnish applied to the pronotum as a control (circles) and which were then treated with either the
Metarhizium anisopliae fungal parasite (solid lines, filled symbols) or with 0.05 % Triton-X control solution (dashed lines,
open symbols) (Reprinted from Graystock and Hughes (2011) with permission of Springer)
habits, the species can be divided into two groups: cavity-dwelling species (Apis cerana and Apis
mellifera) and open-nesting species (dwarf honey bees Apis andreniformis and giant honey bees Apis
dorsata). Using an analytical survey of medium-weight polar venom compounds, it was found that the
major difference between these Apis species corresponds to nesting habit, i.e., between the cavity-
dwelling and the open-nesting species. While the former have venom compounds on the cuticle,
venom peptides are almost absent on those of A. dorsata and A. andreniformis. Similarly, the antimicro-
bial venom compound melittin is present on the nest surface of both the cavity-dwelling species but not
evident on the nest surface of the open-nesting giant honeybee and dwarf honeybee. This result is exactly
what would be expected for the conditions favoring the evolution of external immune defense such as the
use of externalized antimicrobial active venom suggested by Otti et al. (2014): i.e., a highly stable and
confined environment with constant or high microbe pressure. In this context, it is interesting to note that
extracts from the cuticle of social wasp species with paper nests show a higher antimicrobial activity than
Page 11 of 17
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a 1.0
Proportion germination inhibition

0.8
a a
0.6
0.4
b
0.2
b b
0.0
ka ore
nc r
n t er
ka h
se ke
oc G
ge
oc t
ge
ge
oc p
e
co ork
bl Mou
l
bl MP
bl ido
ab or
ro
ka
W
W
Ac
Worker treatment
b 1.0
Proportion germination inhibition
0.8 a
0.6
0.4
b
0.2
c
0.0
nt er
nc r
pl on
se ke
n
e
co ork
l
de ois
io
ab or
ro
et
W
W
P
Fig. 7 (a) Workers of Lasius neglectus inhibited germination of conidiospores on the surface of pupae, as revealed by
germination checks of conidiospores washed off after 24 h of tending and subsequently plated on agar. MPG-blocked workers
inhibited fungal growth to the same extent as control workers. In contrast, blockage of the acidopore and the mouth prevented
this antifungal effect. (b) Venom-depleted ants also had a significantly reduced ability to inhibit fungal growth in comparison to
control workers, but they still showed some antifungal effect compared to the worker-absence control. Bars in panels (a–c)
show means + SEM. Different letters indicate statistically significant differences at a = 0.05 (Reprinted from Tragust
et al. (2013a) with permission of Cell Press)
those of solitary species which excavate burrows, while extracts of solitary mud-nesting species show no
antimicrobial activity at all (Hoggard et al. 2011) (Table 1). It might be argued that the environmental
conditions found in excavated burrows and mud are much more variable than the conditions found in
paper nests, thus not favoring the evolution of external immune defense. On the other hand, factors such as
the relative contribution of social lifestyle and of phylogenetic relationships to the evolution of external
immune defense clearly need to be considered and disentangled. For example, the primitive social hover
wasps Stenogastrinae lack venom compounds on the nest surface, despite the fact that not a single species
excavates burrows (Baracchi et al. 2012b). The following section of this chapter will explore whether life-
history traits of social insects, namely, the high number of often closely related individuals living in high
Page 12 of 17
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1.0
A. fumigatus
A. tamarii
0.9
A. nomius
A. sclerotiorum
Proportion of Leaf-cutting ant trials 0.8 Fusarium sp.
Trichodermo sp.
0.7
Escovopsis sp.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
No Venom Metapleural Both Worker
block block block block absent
Treatment
Fig. 8 Proportion of trials where foreign fungus overgrew leaf-cutting ant nest material, grouped by treatment. Foreign fungal
species were Aspergillus fumigatus (white), A. tamarii (light gray), A. nomius (dark gray), A. sclerotiorum (black), Fusarium
sp. (leftward diagonals), Trichoderma sp. (cross-hatched), and Escovopsis sp. (rightward diagonals) (Reprinted from Tranter
et al. (2014) with permission of Springer)
Table 1 Antimicrobial activity of cuticular extracts from several solitary, communal, and social wasp species
Species (family) n Sociality Nest type IC50 (95 % CI) nr
Polistes humilis (Vespidae) 1077 (10) Soc. Paper 6.03 (2.26) 28
Ropalidia plebeiana (Vespidae) 49 (2) Soc. Paper 7.58 (5.91) 5
Bembix sp. (Crabronidae) 83 Com Burrow 31.97 (27.62) 6
Austroscolia sp. (Scoliidae) 47 Sol. Burrow 158.27 (152.82) 5 (3)
Cryptocheilus sp. (Pompilidae) 4 Sol. Burrow 14.47 1
Pepsinae Sp1 (Pompilidae) 1 Sol. Burrow 90.26 1
Abispa ephippium (Vespidae) 1 Sol. Mud No inhibition 1
Sceliphron laetum (Sphecidae) 5 Sol. Mud No inhibition 2
Delta sp. (Vespidae) 1 Sol. Mud No inhibition 1
n number of individuals (number of colonies for social species). Sociality: social (Soc.), communal aggregator (Com.), solitary
(Sol.). IC50: mean equivalent surface area (mm2) of wasp cuticle required to kill or inhibit 50 % of S. aureus growth. nr number
of replicates per species (Reprinted from Hoggard et al. (2011) with permission of Plos Library of Science)
densities with frequent physical contacts, have indeed favored the use of antimicrobial active venom as
external immune defense.
Social Lifestyle and the Evolution of Venom as External Immune Defense

Since the discovery of antimicrobial properties of hymenopteran venoms, it has been argued that the
adaptive significance of this trait relies on protection from commensal pathogen infections during stinging
Page 13 of 17
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events. However, experimental data supporting this hypothesis are lacking to date (Moreau 2013).
Instead, researchers have started to shed light on the evolutionary significance of antiseptic venoms in
social insects. Stow and coworkers (Stow et al. 2007) explored whether the evolution of sociality required
the synchronous evolution of increased chemical defenses against pathogens in social bees. They found
that the strength of antimicrobial compounds on the cuticle of bees was positively correlated to group size
and genetic relatedness along a gradient of sociality ranging from solitary (Amegilla bombiformis and
Amegilla asserta) and semi-social (Exoneura robusta and Exoneura nigrescens) to eusocial (Exoneurella
tridentata and Trigona carbonaria). This indicates that the evolution of sociality was accompanied by the
evolution of stronger antimicrobial compounds. The link between the levels of antimicrobial compounds
on the cuticle and the levels of social complexity was also revealed by Hoggard and coworkers (Hoggard
et al. 2011) in wasps. Besides trends of increasing antimicrobial activity along social complexity, within a
single species, correlations between antimicrobial activity on the cuticle and both colony size and the level
of within-colony genetic variation were also found (Hoggard et al. 2013). More precisely, in the paper
wasp Polistes humilis, the effectiveness of antimicrobial activity on the cuticle increases with genetic
diversity and decreases with colony size (i.e., the number of wasps forming the colony). It is most likely
the venom that is responsible for the antimicrobial activity found on the cuticle, as venom components of
bees and wasps are commonly found on the cuticle (see previous sections). Since the increase in
antimicrobial strength on the cuticle found in the study of Stow and coworkers (Stow et al. 2007) was
not linear, with the greatest increment being between smaller group sizes, it was suggested that selection
pressure from microbial pathogens is so intense that even minimal sociality requires substantially stronger
antimicrobials. Support for this hypothesis comes from the fact that even minimal societies such as those
of the hover wasps Metischnogaster drewseni, whose colonies count a maximum of two to three females,
have strong antimicrobial venoms (Baracchi et al. 2012b).
The same link between the strength of antimicrobial compounds and level of sociality has been
established in both wasps (Hoggard et al. 2011) and bees (Stow et al. 2007), but information is lacking
for ants. However, it is known that in fungus-growing ants, there is a positive correlation between the size
of metapleural gland reservoirs, an important source of antimicrobial compounds on the cuticle of ants
(Yek et al. 2013), and social complexity. The relationship between antimicrobials compounds and the
level of sociality might thus hold throughout the social Hymenoptera.

This chapter has summarized the evidence that predatory and social lifestyles found in Hymenoptera have
resulted in the increased use of venoms for defensive and offensive purposes. Intriguingly, a background
antimicrobial function has been conserved or recruited in these venoms, indicating that microbial
pressures have been important in shaping the evolution of the composition and the use of hymenopteran
venoms. However, until recently, this has almost never been taken into consideration. Recent research has
proposed that any heritable trait acting outside an organism and improving protection from pathogens or
manipulating the composition of the external microbial community should be viewed as external immune
defense (Otti et al. 2014). As outlined in this chapter, antimicrobial venom of Hymenoptera is frequently
externalized for the purpose of self-sanitation, sanitation of related group members and the nest, and for
the preservation of stored food. Thus, there is no doubt that antimicrobial venoms represent an important
component of external immunity in Hymenoptera.
However, many facets of the ecological immunology of the venom remain insufficiently understood.
External immune defenses come at a cost and are often tightly linked to the physiology of an organism and
its internal immune system. Elucidating the costs related to the use of venom as external immune defense
Page 14 of 17
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is thus required to clarify potential trade-offs in a more precise way. For example, it is known that the use
of environment-derived antimicrobials as external immune defense in ants and bees reduces the expres-
sion of the internal immune response (Castella et al. 2008; Simone et al. 2009). Pros and cons of relying
more on external rather than internal immunity clearly depend on different ecological and environmental
factors, but this needs to be evaluated in more detail. Potential trade-offs between different external
immune defense traits will also have to be taken into consideration, while recent advances in many
technologies and analytical techniques will undoubtedly help researchers in this endeavor. However,
insights from the fields of ecological immunology, chemical ecology, biochemistry, and molecular
biology clearly need to be combined in order to complete our understanding of hymenopteran venom
compounds and functions.
Cross-References
▶ Venom in Parasitoids
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DOI 10.1007/978-94-007-6727-0_4-1
A Critique of the Toxicoferan Hypothesis

Adam D. Hargreavesa*, Abigail S. Tuckerb and John F. Mulleyc
a
Department of Zoology, University of Oxford, Oxford, UK
b
Department of Craniofacial Development and Stem Cell Biology, King’s College London, Guy’s Hospital, London, UK
c
School of Biological Sciences, Bangor University, Bangor, Wales, UK
Abstract
Historically, venom was believed to have evolved twice independently in squamate reptiles, once in the
advanced snakes and once in venomous lizards. The presence of putative toxin proteins in the saliva of
species usually regarded as non-venomous, and the expression of venom gene homologs in their salivary
glands, led to the hypothesis that venom evolved a single time in reptiles. As the single, early origin of
venom is synonymous with the Toxicofera clade (Serpentes, Anguimorpha and Iguania), it will subse-
quently be referred to as the Toxicofera hypothesis. This hypothesis has proved to be remarkably
pervasive for almost a decade, but has until recently never been tested. Here, evidence used in support
of the Toxicofera hypothesis is reviewed and critically evaluated. Taking into account both new and old
data, it appears that this hypothesis is unsupported, and should be subject to further scrutiny and
discussion. Finally, the implications of the rejection of the Toxicofera hypothesis are discussed, with
respect to the knowledge of venom evolution in the Reptilia and also the practical implications of this
knowledge.
Keywords
Toxicofera; Reptiles; Venom; Oral glands; Venom glands
Introduction
Venomous reptiles have long been the source of fear and fascination in roughly equal measure, not least
because of the extensive annual global mortality and morbidity caused by reptile envenomation, partic-
ularly in the developing world (Kasturiratne et al. 2008; Harrison et al. 2009). Research effort has
traditionally focused on the characterisation of venom toxins and the development of treatments to
counteract their clinical effects, and so species considered to be medically important have received the
most attention (for example, the saw scaled vipers (Wagstaff and Harrison 2006; Wagstaff et al. 2009;
Casewell et al. 2009)). As a consequence, the full evolutionary history of venom in the Reptilia has
remained unknown, and to this day poses unanswered questions, including fundamental topics such as the
origin of venom toxins, what constitutes venom and a venomous animal and even the timing of the
evolution of venom itself.
Hypotheses concerning the evolution of venom within reptiles have undergone dramatic revision
within the last decade, and are currently in a state of flux. Historically, venom within reptiles was believed
to have evolved twice independently: once in the Caenophidia (advanced snakes) and once in the
Helodermatid lizards (Gila monsters and beaded lizards) (Kochva 1978; Pough et al. 2004) (Fig. 1).
*Email: adam.hargreaves@zoo.ox.ac.uk
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Fig. 1 Simplified Reptile cladogram. The phylogenetic position of venomous Helodermatid lizards and the Caenophidia
(advanced snakes) are indicated. The phylogenetic position of the proposed venomous Toxicoferan ancestor also indicated
along with the three proposed punctuated toxin gene recruitment events. Proposed recruited toxin gene families are also shown
This belief was mainly due to the distant phylogenetic relatedness of these animals and clear differences in
the morphology of their respective venom delivery systems (Kochva 1978; Saintgirons 1988). A more
recent, alternative hypothesis (which we refer to as the “Toxicofera hypothesis”) has become widely
accepted within (and seemingly far beyond) the toxinological community. The Toxicofera is a clade of
squamate reptiles comprising Iguania, Anguimorpha and Serpentes, whose name refers to the presence of
venom within at least some members of these groups (Vidal and Hedges 2005). Phylogenetic analysis
utilising nine nuclear genes (a-enolase, amelogenin, c-mos, hoxa13, jun, mafb, rag1, rag2 and r35) found
this clade to be strongly supported (Vidal and Hedges 2005), and this support has been reproduced in
subsequent studies (e.g., Pyron et al. 2013). However, phylogenetic relationships within the Toxicofera
are unresolved based on nuclear data, although the use of SINEs (short interspersed nuclear elements) has
suggested a clustering of snakes with anguimorph lizards (Piskurek et al. 2006) which is also supported by
a more recent analysis (Hsiang et al. 2015).
The majority of the roughly 2,500 species of snake are classified within the Caenophidia, a sub-order
containing four major lineages: Atractaspidinae; Viperidae (vipers, pit vipers); Elapidae (such as cobras
and mambas) and Colubridae (a polyphyletic group which is constantly undergoing taxonomic revision)
(Quijada-Mascarenas and W€ uster 2009). Approximately 600 species, all belonging to the former three
lineages, were traditionally considered to be venomous in that they possessed venom glands surrounded
by compressor muscles, tubular fangs at the front of the mouth and are of medical significance to humans
(although medical significance to humans is obviously a poor criterion on which to base classification of
toxicity). Whilst some members of the Colubridae are opisthoglyphous (rear fanged), they do not
generally pose a threat to humans and have historically not been considered to be venomous.
Evidence for a wider use of venom within advanced snakes was initially based on proteomic analysis of
the saliva of the radiated rat snake (Coelognathus radiatus), a snake reliant on constriction for prey
capture, where a post-synaptic neurotoxin belonging to the three finger toxin (3Ftx) family was discov-
ered (Fry et al. 2003a). This protein was found to possess the typical ten conserved cysteine residues of
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elapid 3Ftxs and when functionally tested led to antagonism of nicotinic acetylcholine receptors. This
protein was therefore considered to be structurally and functionally homologous to the elapid three finger
toxins (Fry et al. 2003a) and phylogenetic analysis showed strong support for the nesting of the rat snake
3Ftx within a clade of previously categorised 3Ftxs (Fry et al. 2003b). On the basis of these results it was
suggested that three finger toxins were recruited into the venom repertoire prior to the divergence of the
Elapidae and Colubridae (Fry et al. 2003a). Indeed, the analysis of other colubrid “venoms” (Mackessy
2002) added further support that the use of venom in the advanced snakes pre-dated their radiation in the
Cenozoic era (Vidal and Hedges 2002). More interestingly, the presence of putative toxin proteins in the
saliva of lizard species usually regarded as non-venomous (such as the lace monitor, Varanus varius), and
the expression of venom gene homologs in their salivary glands, led to the proposed hypothesis that
venom evolved a single time in squamate reptiles approximately 170 Mya (Fry et al. 2006), and not twice
independently as had been previously believed (Pough et al. 2004; Kardong et al. 2009).
The timing of venom gene recruitment events within reptiles has undergone significant modification
over the course of subsequent Toxicofera-related studies, with further sampling leading to the detection of
an increased number of putative venom genes in a diverse collection of species (Fry et al. 2009, 2010,
2012a, 2013). These findings suggest an increasingly complex view of venom gene recruitment through-
out the evolution of the Toxicofera, which has even extended to include the Komodo dragon (Varanus
komodoensis). This species was previously considered to be reliant on oral bacteria (e.g., see Bull
et al. 2010) to induce septicaemia in prey items, but is now considered to be venomous (Fry et al. 2009).
Here, the foundation and expansion of the Toxicofera hypothesis and the proposed single, early
evolution of venom in reptiles are discussed and examined. The assumptions and key shortcomings of
the evidence used in support of this hypothesis are reviewed, taking into account more recent findings and
novel interpretations.
The Toxicofera Hypothesis

The first proposal of the single, early origin of venom in reptiles occurred in 2006 based upon the detection
of genes homologous to those previously identified in the venom glands of venomous snakes expressed in
the mandibular salivary glands of four Varanid lizards (Varanus acanthurus, V. mitchelli, V. panoptes
rubidus and V. varius) and a single Iguanian (Pogona barbata) (Fry et al. 2006). Phylogenetic analysis
demonstrated that nine toxin families were shared between these non-venomous lizards and advanced
snakes: AVIT peptide; B natriuretic peptide; cysteine-rich secretory protein (CRISP); cobra venom factor
(which is in fact complement component C3 (Alper and Balavitch 1976)); crotamine; cystatin; kallikrein;
nerve growth factor and vespryn. Additionally, a type III phospholipase A2 (PLA2) was detected in the
mandibular salivary glands of Varanus varius (Fry et al. 2006).
Subsequent Toxicofera-related studies mainly focused on the inclusion of additional lizard species (Fry
et al. 2009, 2010, 2013). A more recent study sequenced cDNA derived from the oral glands of Iguanian
lizards and Henophidian snakes using 454 pyrosequencing (Fry et al. 2013). The detection of apparent
homologs of several Toxicoferan genes in these species led to a number of proposed gene recruitment
timing events being shifted even earlier in Toxicoferan evolution, in some cases by up to 112 million
years, and the adoption of a punctuated evolutionary history of toxin recruitment. In this scenario, three
rounds of toxin gene recruitment have been proposed to have occurred in the Toxicofera: up to ten at the
base of the Toxicofera (cysteine-rich secretory protein (CRISP), crotamine, cystatin, cobra venom factor,
kunitz, L-amino acid oxidase, lectin, renin aspartic protease, veficolin, vespryn), six in the ancestor of
Serpentes and Anguimorpha (AVIT peptide, epididymal secretory protein, hyaluronidase, kallikrein,
nerve growth factor, ribonuclease) and eight (acetylcholinesterase, lipocalin, C-type natriuretic peptide,
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snake venom metalloproteinase, phosphodiesterase, phospholipase B, vascular endothelial growth factor,

waprin) in the common ancestor of the Caenophidia (Fry et al. 2013) (Fig. 1).
The Toxicofera hypothesis proposes the existence of an early venomous squamate that would have
possessed toxin-secreting glands on both the upper (maxillary) and lower (mandibular) jaw (Fry
et al. 2006). The venom delivery systems in advanced snakes and lizards are therefore homologous but
morphologically distinct derivatives of this primitive system, with snakes retaining the maxillary venom
glands and venomous lizards maintaining the mandibular glands (Fry et al. 2006), with the opposing
glands being secondarily lost by each lineage. It has been proposed that members of the Iguania (such as
the green anole lizard, Anolis carolinensis) diverged whilst this venom system was in an incipient stage,
and so lack any form of specialised toxin secreting glands. Furthermore, snakes which use alternative prey
capture methods such as constriction are proposed to have secondarily lost venomous function (Fry
et al. 2006).
Alongside the conserved shared expression of homologous genes, the conserved structure of homol-
ogous proteins has also been used to support the Toxicofera hypothesis, namely the conserved cysteine
structure and functional residues (Fry et al. 2006).
Several Toxicofera-related studies have also included functional tests on the mandibular oral secretions
of two varanid species, Varanus komodoensis and V. varius (Fry et al. 2006, 2009). Samples of crude oral
secretion and purified natriuretic peptide were injected intravenously into anaesthetised male rats, which
resulted in a drop in mean arterial pressure (MAP). Platelet aggregometry was also carried out using
purified type III PLA2 from V. varius which showed inhibition of platelet aggregation when tested on
human blood samples.
Shortcomings of the Toxicofera Hypothesis

The Toxicofera hypothesis assumes that shared expression of a gene between what were previously
considered non-venomous species and more derived venomous species implies shared toxicity (or at least
a shared venomous ancestry) (Fry et al. 2006). It is of course plausible that homologous tissues (e.g., the
venom gland and other oral glands) within related species will express similar complements of genes, and
therefore presence alone does not provide any evidence of toxicity. Indeed, many of the proposed toxins
which have been used to support the Toxicofera have never been functionally characterised. Moreover,
the products of several of these genes have never been suggested to be toxic (for example cystatin type
E/M (Ritonja et al. 1987)) or have been shown to not be toxic, even up to high doses, through functional
tests (for example, acetylcholinesterase (Cousin et al. 1996)). Therefore these genes have been used to
support shared ancestral toxicity, without actually functioning as toxins. Additionally, it now seems
certain that many of the proposed shared venom toxins within the Toxicofera actually result from the
confusion of orthologs and paralogs, where non-toxic relatives of toxin genes have been identified
(Hargreaves et al. 2014a). For example, genes encoding complement c3 and nerve growth factor have
been shown to have undergone an Elapid-specific gene duplication (Sunagar et al. 2013; Hargreaves
et al. 2014a, b) to give rise to the putatively toxic “cobra venom factor” and nerve growth factor b
(Hargreaves et al. 2014b). This mis-identification of physiological orthologs as toxin-encoding paralogs
has led to the conclusion that all Toxicoferan reptiles produce toxins in their oral secretions, and are
therefore descended from a common venomous ancestor. In addition, many previous studies (e.g.,
Casewell et al. 2012) have been based on a flawed assumption – that phylogenetic trees containing
monophyletic clades of reptile sequences that include a known (or hypothesised) toxin from venomous
snakes constitute venom toxin clades. The true evolutionary history of these genes (which have duplicated
to possibly give rise to toxic versions in some species), and these clades (which contain both genes
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encoding toxic products in some species, along with related genes encoding non-toxic products in other
species), has therefore been obscured by being labelled as toxins by default. This is further confounded by
a lack of data, both for the tissue being studied and also for other tissues and species (the majority of
Toxicofera-related studies (Fry et al. 2006, 2010, 2012a) used only “up to 384” individual venom gland
cDNA library colonies per species, a minimal amount of sequencing considering the frequently cited
complexity of snake venoms (Li et al. 2005b; Kini and Doley 2010; Casewell et al. 2013)). This paucity of
data, whilst understandable given the technology and resources of the time, has seemingly led to errors of
interpretation, and, possibly more seriously, over-interpretation of results. Indeed, few genes were found
expressed in all species surveyed (for example out of nine genes, only Kallikrein was detected expressed
in the mandibular salivary gland of all four species of varanid (Fry et al. 2006)). With increased taxon
sampling, only Kallikrein and CRISP were detected in all 18 species of lizard sampled (Fry et al. 2010)
which included 13 species of varanid. Whilst this may be an artefact of low sequencing depth, the lack of
consistent expression should have precluded these genes being used to support a conserved repertoire of
“venom” genes across the Toxicofera.
Perhaps the most significant issue with the evidence used to support the Toxocifera hypothesis is that all
samples used for sequencing were derived from either salivary or venom glands, and no “body” tissues
were included with which to compare gene expression. Transcriptomic analysis of solely venom gland is
perfectly acceptable for descriptive studies which seek to characterise the transcriptome of this tissue.
However, in order to assign a potential toxic role to a gene (and especially to infer its true evolutionary
history, or the evolution of the venom repertoire in an entire lineage), sequencing the venom gland alone is
insufficient. It has long been known that tissues all express a repertoire of “housekeeping” or maintenance
genes (Butte et al. 2001) and as a result the sequencing of the entire venom or salivary gland will result in
the identification of genes associated with a diverse range of functions (e.g., protein synthesis, cell-cell
signalling and energy metabolism), not to mention that the sample will likely contain traces of other
tissues such as muscle and blood. Consequently, genes cannot be inferred to encode toxins simply because
they happen to be expressed in the venom or salivary gland.
Conservation in the structure of proteins detected in lizard oral secretions has also been used in support
of the Toxicofera hypothesis. However, many secreted proteins, particularly members of the same gene
family, have a conserved cysteine-rich “scaffold” (Anantharaman et al. 2003). It should not be too
surprising that related proteins have similar structures, especially as alterations to this scaffold, or to the
conserved residues, would likely result in a disruption of the protein structure and function. Similarity of
structure should not necessarily always be considered to reflect shared toxicity. When using the Australian
snake venom detection kit, Jelinek et al. (2004) found cross-reactivity between several snake species,
most notably the tiger snake (Notechis scutatus) and the black-headed python (Aspidites
melanocephalus). This has been used as evidence that putative toxin genes are translated into proteins
in the venom or oral glands of these species, and that these proteins represent relics of an ancestral venom
system which has been down-regulated in Henophidians (boas, pythons and several other families of
basal snakes) (Fry et al. 2013). However, such cross-reactivity has been observed many years previously,
with cross-reactivity demonstrated between colubrid oral secretions and antivenoms raised against
African and Australian elapids (Minton and Weinstein 1987). Interestingly, the authors also found
some antigenic cross-reactivity between a Henophidian snake (Epicrates striatus strigilatus) oral secre-
tion when tested using a polyvalent antivenom raised against three Dendroaspis (mamba) species. Some
of the responsible antigens were shown to be present in both venom and plasma, whilst some were present
only in venom. Therefore, it is likely that some of this cross-reactivity between species is due to antigens
present in secretions common to many species, as well as to cross-reaction between related members of
protein families and cannot be taken as representative of any shared toxicity.
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Whilst several Toxicofera-related studies commendably attempted to functionally test the oral secre-
tions of some varanid lizard oral secretions, the results must be interpreted carefully. Purified group III
PLA2 from V. varius appears to have caused inhibition of platelet aggregation, although it is unclear why
this was tested on human blood instead of the blood of native prey items such as birds or rabbits (Weavers
1989). It is also unclear whether physiological concentrations (within a range of concentrations which
occur naturally in oral secretions) of this protein were used in this assay or if an increased dosage was
required to achieve this inhibition of platelet aggregation.
Crude mandibular oral secretion and synthesised natriuretic peptide from V. varius and V. komodoensis
caused a drop in mean arterial pressure when injected intravenously into anaesthetised rats (Fry
et al. 2006, 2009). However, intravenous (I.V.) administration is an unlikely delivery method in the
event of a lizard bite, and the depressor effects of I.V. administration of saliva has been noted in previous
experiments (Gibbs 1935; Levy and Appleton 1942). Therefore, physiological effects noted in a con-
trolled laboratory experiment may not be translated in a real life scenario. For crude V. varius mandibular
secretion, a concentration of 1 mg kg 1 was required to cause a drop in blood pressure in an anaesthetised
rat (Fry et al. 2006) whilst a decrease in blood pressure was seen at doses above 100 mg/kg for synthesised
natriuretic peptide (from V. komodoensis) with 400mg/kg required to induce hypotensive collapse (Fry
et al. 2009). Conversely, in a similar experiment, 10mg/kg of crude Papuan taipan (Oxyuranus scutellatus
canni) venom caused a complete respiratory and cardiovascular collapse (Crachi et al. 1999). It is safe to
say that lizard “venom” is much more inefficient, and coupled with the inefficient delivery method in these
species, is it realistic that they will administer sufficient amounts of toxin in a single bite?
Casting Doubt on the Toxicofera Hypothesis

The Toxicofera hypothesis has been widely accepted for almost a decade, and has proved to be pervasive
and attractive. However, the downside of these qualities is that it has also avoided scrutiny and testing.
There have recently been several studies which have cast doubt on the Toxicofera hypothesis (Hargreaves
et al. 2014a; Reyes-Velasco et al. 2015), although their interpretation has led to alternative conclusions.
Several phylogenetic analyses incorporating non-venom gland transcriptomic data have shown that
non-toxin sequences nest within clades of toxin genes, and it has been acknowledged that such findings
provide “. . .strong evidence for the non-monophyly of Toxicoferan toxins” and that “. . .the results of
[these] phylogenetic analyses would strongly refute the key prediction of the ‘SEO’ (single early origin)
hypothesis. . .” (Casewell et al. 2012). Rather than accepting these conclusions, it has instead been
proposed that venom gene recruitment may not be one-way, and that genes encoding venom toxins
undergo a dynamic to-ing and fro-ing between toxin and physiological protein, whereby a venom toxin
may undergo additional duplication, with subsequent recruitment back into a body tissue to fulfil a
non-toxic physiological role. However, the more parsimonious hypothesis that these sequences actually
represent reptile body sequences (which have never been toxins) forming reptile clades rather than body
sequences nesting within venom clades is not considered. Similarly, Koludarov et al. (2012) investigated
the oral secretions of the lizard Abronia graminea and determined that “the NGF [nerve growth factor]
expressed in venom may be the same gene as is used in the body and therefore may be a rare case of a
venom protein resulting from a non-duplicated gene.” It is possible that the product of a gene may be used
pleiotropically as a toxin (fulfilling a toxic and non-toxic role simultaneously), but unless its expression is
elevated in the salivary gland, there would be little evidence to suggest that it was anything more than a
non-toxic physiological protein encoded by a housekeeping or maintenance gene.
More recent analyses incorporating an increased number of non-venom gland samples has further cast
doubt on the Toxicofera hypothesis. A large scale test of the robustness of this hypothesis found that many
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of the genes used to support the single, early evolution of venom in squamates are in fact expressed in
multiple body tissues including the salivary gland of a non-Toxicoferan lizard, the leopard gecko
(Eublepharis macularius) (Hargreaves et al. 2014a). No evidence has been found of either a venom-
specific splice variant or significantly elevated expression level in the venom or salivary gland. Therefore,
it is likely that these genes are simply encoding maintenance or “housekeeping” proteins, and are
expressed in multiple tissues at low levels. Many of these genes were also found expressed in several
other body tissues in Echis coloratus (Hargreaves et al. 2014b), adding further support that these are
housekeeping genes due to their ubiquitous expression pattern. Several of these genes are also only
present as a single copy in the genome of this species, and so there is no evidence of duplication and
recruitment of a toxic version to the venom gland (Hargreaves et al. 2014a). Indeed, genes homologous to
known toxins have been found expressed in the rictal gland, brain, intestine, kidney, testes, spleen, ovary,
heart, stomach, liver, blood and muscle of the Burmese python (Python molurus bivittatus) and the venom
gland, liver, pancreas, kidney, brain and heart of Bothrops jararaca (Junqueira-de-Azevedo et al. 2014;
Reyes-Velasco et al. 2015). Whilst these results have been interpreted in different ways, they demonstrate
that genes which are homologous to putative venom genes are expressed in many different tissues outside
of the oral glands, and that sequencing solely the venom or salivary gland without other body tissues to
use as a reference for gene expression is not enough. Interestingly, when the genome of the Burmese
python was surveyed for genes orthologous to putative toxin genes, only one or two orthologs were
detected for each toxin gene family. The authors suggest that the Burmese python is representative of the
ancestral state, prior to the expansion of toxin gene families in the Caenophidia (Reyes-Velasco
et al. 2015).
If the proteins encoded by these genes are not being used to fulfil a venomous function, why are they
still being expressed in the oral secretions of these reptiles? Given the metabolic cost of producing venom
(McCue 2006) it would be more logical that natural selection would act to end any unnecessary gene
expression and protein synthesis. Indeed, this process has been shown to occur in the marbled sea snake,
Aipysurus eydouxii, following a switch in diet from fish to sedentary fish eggs (Li et al. 2005a, b), whereby
several toxin genes have become pseudogenized (rendered non-functional via mutation). Why then has
this not occurred in a plethora of reptile species which have no use for venomous function? Since many of
the proposed toxins secreted by these glands are nothing of the sort, these oral secretions and the proteins
they contain must have alternative functions, incorporating aspects of lubrication, pre-digestion and the
stimulation of digestive processes and anti-microbial activity (Weinstein et al. 2012).
Glands and Fangs

Reptiles possess many salivary glands that secrete into the oral cavity, with a key role in the lubrication of
food. Many are mucous in nature, however, some glands also have serous secretions which, in some cases,
have become adapted as venom producing glands, as observed in venomous (Helodermatid) lizards,
front-fanged snakes and some rear-fanged snakes.
In front-fanged snakes (such as elapids and vipers) and rear–fanged snakes, the fang and venom gland
develop from a region at the back of the maxillary dental lamina (Vonk et al. 2008). The final position of
the fangs is therefore attained by movement of the growing fangs, forward or backwards in the mouth,
after initiation. Importantly in the venomous snakes, the venom gland and the fang appear to form from a
united primordium that starts as an epithelial thickening below the eye on the upper jaw. This thickening
has been called the primitive dental ridge (Martin 1899). In Vipera palaestinae, the thickening splits into
an anterior gland and more posterior fang, with the venom gland extending first anteriorly before turning
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posteriorly and branching (Kochva 1963). In contrast to the serous venom gland, the nearby supralabial
glands develop from independent placodes and are generally mucous.
In the rear-fanged snakes (Colubridae) the fang is associated with the Duvernoy’s gland, which appears
not to act as a venom gland and has instead been proposed to have an anti-bacterial role in coating dental
surfaces (Jansen 1983). Secretion from the Duvernoy’s gland in Thamnophis elegans vagrans was found
to have enhanced anti-bacterial properties when compared to supralabial glands (Jansen 1983). In
addition to a similar position of the fang primordium when compared to front-fanged snakes, the fang
and venom gland of rear-fanged snakes also develops from a united primordium, as has been described in
the opisthoglyph Telescopus fallax and aglyph Thamnophis sirtalis (Kochva 1965). Telescopus has a
complete row of maxillary teeth with the fang primordia and gland forming at the posterior end. In
contrast to the viperidae the venom gland does not first grow anteriorly before growing posteriorly. The
fact that in these different snakes the venom gland and fang initiate from a common primordium that forms
at the back of the maxillary dental lamina indicates that these front and rear fangs are homologous
structures (see also Vonk et al. 2008). Importantly, Duvernoy’s glands do not appear to form at all in many
colubrids, for example some species of the genus Elaphe, genera Lampropeltis, Pituophis, Pseuetes,
Rhinocheilus and Spilotes (Taub 1967). A variety of Elaphe species used in this study (although some of
these have since been assigned to different genera) have no Duvernoy’s gland and their supralabial glands
are purely mucous (Taub 1967). In general such snakes without a Duvernoy’s gland are constrictors who
suffocate their prey before digestion. The lack of large serous glands in these species has been suggested
to be due to secondary loss (Underwood and Kochva 1993; Vidal 2002). Although this may well be
correct in some derived forms it is also possible that the Duvernoy’s gland may not have evolved in all
snakes, indicating independent evolution of this gland. Supporting this idea, Boidae and other primitive
snakes have mainly mucous salivary glands, which are found at a range of positions in the oral cavity
(Kochva and Gans 1970) In Boidae, anterior temporal glands composed of serous cells have been
described at the back of the maxilla (Taub 1966). Supralabial glands are generally thought of as mucous
in most snakes but some Colubrids have serous cells included in the supralabial glands (Taub 1967). Thus
whether a gland is mucous or serous is subject to some variation across reptiles and, in keeping with this,
Duvenoy’s glands can be mucous in part in some Colubridae (Taub 1967). Whether a gland is serous or
mucous, therefore, cannot be necessarily used to infer evolutionary relationships.
In both front and rear fanged snakes, the fangs are associated with a gland that forms from the same
dental primordium as the tooth. These are true dental glands. Any homologous structures would
consequently be proposed to share this joint origin. It is therefore important to know whether venom
glands in Toxicoferan lizards also develop from a united dental placode. If not, they are unlikely to be
homologous, but instead would represent independent adaptations to venom formation in other oral
glands. Some oral glands in lizards do indeed appear to develop from a lamina linked to the dental lamina.
For example in chameleons the tooth and dental gland appear to share a similar origin (Tucker 2010).
However in helodermatids, where venom glands are found on the lower jaw, the glands lie adjacent to the
tooth with the duct at a slight distance (Kardong et al. 2009), indicating that the tooth and gland develop
from separate placodes. Supporting this view, the ducts have been proposed to run to an opening between
the lip and the jaw, rather than to the base of the teeth (Shufeldt 1891) and the location of the gland appears
more similar to an infralabial gland. If this is the case, then the venom glands of helodermatids are not
homologous to those of snakes.
The lack of a developmental link between dental glands and teeth in venomous lizards compared to
snakes, and the lack of a large serous gland associated with the maxillary dental lamina in primitive snakes
and some colubrids strongly suggests that the venom delivery system in snakes and lizards evolved
independently. From the presence of Duvernoy’s glands in snakes without venom, it would appear that the
Duvernoy’s gland first evolved as a branch of the forming dental lamina and then was adapted into a
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venom-producing gland in both front and rear-fanged snakes. A clear understanding of the embryonic
development of the venom glands in venomous lizards will be important to clarify such points.
Varanid Venom
Many Toxicofera-related studies suggest that lizards belonging to the genus Varanus are in fact venom-
ous, in particular the Komodo dragon V. komodoensis (Fry et al. 2006, 2009, 2013). A review of the
available evidence found it unlikely that the Komodo dragon utilises venom as a prey capture method,
instead suggesting that if it did use venom it was used as a pre-digestion method (Arbuckle 2009).
Historical field observations have suggested that blood loss due to injury is the main prey capture strategy
utilised by Komodo dragons (Auffenberg 1981). Whilst many Varanus species have been kept in captivity
for many years, there have been almost no reports of any symptoms concurrent with envenomation
following a bite. In the original Toxicofera paper (Fry et al. 2006) there are anecdotal reports of bites from
three species of Varanus which resulted in symptoms such as dizziness and rapid swelling. Most recently,
a bite by a Bengal monitor (Varanus bengalensis) reportedly caused acute kidney injury to a human
patient, which ultimately (and most unfortunately) resulted in death (Vikrant and Verma 2014). However,
no positive identification was made of the offending animal, other than the name given by the patient.
Perhaps more dubious is that the bite symptoms were more in line with envenoming from a Russell’s viper
(Daboia russeli) (White and Weinstein 2015), a member of the so-called “Big four” and a main cause of
mortality due to snakebite in India (Simpson and Norris 2007). Unfortunately no mention is made of the
bite wound itself which may aid in distinguishing between a lizard or snake as the culprit. Additionally, a
recent bite by a Komodo dragon reportedly resulted in no symptoms of envenomation (Borek and
Charlton 2015). Therefore, the status of varanid lizards as venomous is uncertain, particularly when
compared to known venomous lizards such as the Gila monster and beaded lizards.
Conclusions and Future Directions

Venom Evolved Multiple Times in Reptile Evolution
Whilst the Toxicofera hypothesis represents a parsimonious explanation of the evolution of venom in
reptiles (one character evolving a single time), the inclusion of non-venom-gland derived transcriptomic
data in phylogenetic analyses along with the quantification of gene expression would strongly suggest that
the Toxicofera hypothesis is unsupported (Hargreaves et al. 2014a). This would prompt a move back to
the previous hypothesis that venom has evolved multiple times within squamate reptiles, once in the
advanced snakes, once in the helodermatid lizards, and potentially another time in varanid lizards
(although more evidence is needed to confirm this). This is in keeping with the large phylogenetic
distance between venomous snakes and venomous lizards, the differing morphology of venom delivery
systems between these animals (e.g., gland location, teeth/fangs), and the differing uses for their venoms
(i.e., snakes predominantly for prey capture and helodermatid lizards for defence).
Simplified Complexity of Reptile Venom

The rejection of the Toxicofera hypothesis and the ruling out of many of the genes used to support it as
toxins leads to an inescapable conclusion, that snake venom is not as complex as previously suggested
(Li et al. 2005b; Kini and Doley 2010; Casewell et al. 2013). A review of venom proteome data from
several species (Calvete et al. 2007; Wagstaff et al. 2009; Vonk et al. 2013) shows that snake venom is
composed of a relatively small number of gene families encoding a few dozen different proteins, with
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most extensive diversity found in only one or a few of these families (Calvete 2013; Hargreaves
et al. 2014a). Whilst post-translational modifications may prove to play a significant role in generating
more extensive diversity from a limited genetic background (Casewell et al. 2014), the idea that snake
venom is a “complex cocktail” (Casewell et al. 2013) of hundreds of different proteins encoded by many
gene families seems to be unsupported by experimental evidence. The low number of products in snake
venom makes perfect sense as (1) a complex proteinaceous mixture would be metabolically expensive to
produce and (2) natural selection will act to streamline the venom, tailoring it to the snakes’ prey items. In
short, a simple venom is efficient; a complex venom is overkill. The implications of this reduced
complexity are significant, particularly for the development of the next generation of antivenom treat-
ments utilising methods such as “string of beads” (Whitton et al. 1993) and “epitope-string” (Casewell
et al. 2013). A reduction in the number of likely toxins inherently means a reduction in the number of
targets requiring neutralisation by antivenom, and as a consequence the reduced number of components
contained in the antivenom would mean a reduction in antigenicity, meaning a reduced chance of adverse
reactions to treatment such as anaphylaxis and serum sickness (Nuchprayoon and Garner 1999).
From an evolutionary perspective, the reduction in the number of toxins does not detract from the
fascination or specialization of venoms, in fact the opposite is true. The occurrence of lineage-specific
gene duplications (for example complement c3 and nerve growth factor in Elapids (Sunagar et al. 2013;
Hargreaves et al. 2014a; Hargreaves et al. 2014b)) would indicate that these genes may confer some prey-
specific effects (as seen in the Mangrove catsnake, Boiga dendrophilia (Pawlak et al. 2006)), or may have
allowed adaptation to a new ecological niche.
The Changing Definition of Venom

The Oxford English dictionary defines venom as “a poisonous substance secreted by animals such as
snakes, spiders, and scorpions and typically injected into prey or aggressors by biting and stinging.”
A more specific and long-standing definition would be “a complex substance produced in a specialized
gland and delivered by an associated specialized apparatus that is deleterious to other organisms in a given
dosage and is actively used in the subjugation and/or digestion of prey and/or in defence” (Mebs 2002).
More recently, the quest for a catch-all term that encompasses the diverse uses of venom by insects,
molluscs, reptiles and mammals has led to increasingly broad definitions of venom, such as “a secretion,
produced in a specialised tissue (generally encapsulated in a gland) in one animal and delivered to a target
animal through the infliction of a wound (regardless of how tiny it is). A venom must further contain
molecules that disrupt normal physiological or biochemical processes so as to facilitate feeding or defence
by/of the producing animal” (Fry et al. 2012b). It is perhaps time to discard this quest in favour of more
restricted, possibly even lineage-specific, terminology with emphasis on the biological role of the venom
to the survival of the animal. As an example, human saliva contains many of the proteins encoded by the
same gene families which are also found present in the snake venom proteome, including cystatins,
disintegrin-like metalloproteinases, epididymal secretory protein E1, group IIA PLA2s, b-defensins, and
kallikrein (Hu et al. 2005; Guo et al. 2006). Human saliva has also been shown to be toxic (Bonilla
et al. 1971). However, humans are not considered to be venomous, we do not use these secretions to kill or
otherwise incapacitate prey, and so these proteins must fulfil some other biological role, such as
pre-digestion and lubrication. Therefore, the presence of proteins homologous to known (or proposed)
toxin proteins in oral secretions does not automatically mean that the organism is venomous. Moreover,
considering the presence of homologous proteins in the oral secretions of basal snakes as toxins based on
their use as toxins in more derived species, without evidence of these proteins showing any functional
significance, is an erroneous and premature assumption, which has been stated previously by other
authors (Kardong 2012; Weinstein et al. 2012).
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Future Directions
The increased application of second generation DNA sequencing technologies and the integration of
multiple types of ‘omic data (genomic, transcriptomic, proteomic) is revolutionising the study of the
evolution and composition of venom in reptiles, with implications not only for our understanding of this
evolutionary innovation, but also for the treatment of snakebite and development of novel pharmaceu-
ticals. Once the genome to proteome path of toxin expression is completely elucidated, this leaves the
fundamentally important question: what do these proteins actually do? Perhaps more pertinent, is the
functional property of these proteins relevant to the biological role of the venom and to the survival of the
animal? Oral secretions are likely to have several biological roles, such as pre-digestion and lubrication,
and so some proteins are likely to fulfil these rather than act as venom toxins. Only with functional
characterisation (which can be a long and arduous task, particularly compared to the “one-shot” nature of
high throughput sequencing) of these putative toxins can a true role be assigned to them. Moreover,
functional testing of proteins should be performed at physiological concentrations on native prey items.
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Mutation, Duplication, and More in the Evolution of Venomous Animals

and Their Toxins
Anita Malhotra*
School of Biological Sciences, Bangor University, Bangor, Gwynedd, UK
Abstract
Toxins represent one of the fastest evolving types of protein to be found in animal systems, sharing many
of their features with other protein families that respond to extrinsic factors, such as those involved in
immunity, and detecting and responding to the environment in which they live. However, studies on toxin
genes have been lagging behind those on other gene families as until very recently, no fully sequenced
genomes from venomous animals have been available. In this chapter, the molecular forces acting on
toxin gene sequences are compared to those acting on other non-toxin genes, addressing in particular
several features that have been stressed in the toxinological literature, i.e., their hypervariability, accel-
erated evolution, and apparent focal mutagenesis centering on the active site of the toxins. The accepted
paradigm that the birth-and-death model underlies toxin multigene family evolution is challenged by
studies that show both concerted evolution and birth-and-death can give rise to similar patterns following
gene duplication and that both models may operate simultaneously. Much of the dynamics of gene
duplication and the fate of duplicated genes seem to depend on the genomic and biological context in
which they occur. Therefore, there is no reason to expect toxin-encoding genes from diverse animal
groups to show common mechanisms of evolution.
Introduction
A major endeavor of evolutionary biologists is to understand variability in rates of evolution of different
classes of genes (Hirsh and Fraser 2001). Toxin evolution represents a unique example of protein
evolution with applicability to an increasingly broad range of venomous taxa (Casewell et al. 2013;
von Reumont et al. 2014) but also to other classes of rapidly evolving genes in most organisms, such as
those involved in the response to external stimuli (Bowmaker and Hunt 2006; Niimura 2012). Although
Brookfield (2000) cautions that fast-evolving is not necessarily equivalent to evolving under positive
selection, Endo et al. (1996) identified neurotoxins from snakes among only 17 out of almost 4000 gene
groups surveyed, which showed evidence of being under positive selection. To what extent are the
mechanisms operating on proteins expressed in venom glands similar to those acting on proteins
expressed in other body tissues? The purpose of this chapter is to review the features of toxin evolution
in the context of recent findings about protein evolution in general.
Dating from the 1970s (Hirsh and Fraser 2001), the predominant view of protein evolution has been
that the conservation of protein sequence is largely determined by the dispensability of the protein in
biochemical networks. However, this largely applies to single copy genes, which are more easily studied,
and gene duplication can act as a trigger for innovation in protein function. For example, Hahn
et al. (2007) provided evidence for excess positive selection on coding sequence gene families that had
experienced rapid expansion in primates. Other factors affecting the tendency to evolve fast may include
*Email: a.malhotra@bangor.ac.uk
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differences in expression level (Reyes-Velasco et al. 2015), number of duplicate copies, and rates of gene
turnover within duplicated gene families (Katju and Bergthorsson 2013). In addition, particularly
pertinent to tissue-specific expression as seen in toxins that are expressed only in the venom gland, a
major cause of evolutionary innovation is expression shifts caused by mutations in regulatory noncoding
regions (Margres et al. 2015).
The main features of toxin families highlighted in the extensive literature on this topic are their
hypervariability (Conticello et al. 2001; Zhang et al. 2014), signature of strong positive selection (often
referred to as accelerated or Darwinian evolution ), an excess of non-synonymous substitutions compared
to synonymous substitutions, and the nonuniform distribution of mutations over the length of the protein
sequence. These are the features on which the following chapter will focus, followed by a discussion of
evolutionary phenomena seen in other similar gene families and the extent to which they might apply to
toxin gene families. The recent inclusion of venomous animals into the select group of organisms that
have had their genomes sequenced may soon allow some outstanding questions about toxin evolution to
be answered.
Positive Selection/Accelerated Evolution

As toxinologists and molecular evolutionists have described for over two decades, a common feature of
toxin genes is that the exons are considerably more variable than the introns (Ogawa et al. 1996), in direct
contrast to the usual observations for proteins. Moreover, the exons frequently show a signal of positive
selection in that non-synonymous substitutions (KA, sometimes written as dN) occur more often than
expected under the null hypothesis of neutral evolution (where the number of non-synonymous and
synonymous substitutions, KS or dS, would be expected to be equal).
However, excessive reliance on between-species KA/KS tests for detecting selection has been
highlighted by Brookfield (2000). A high KA/KS ratio, indicating positive selection, might be found
where substitution is actually neutral (McAllister and McVean 2000), and a ratio less than one, which
might be taken to indicate purifying selection, may instead be the result of adaptive substitution in some
regions of the protein being masked by purifying selection in others. Indeed, Bazykin and Kondrashov
(2012) have shown that, in the Drosophila genome, the strongest positive selection observed is that which
drives allele replacements at conservative sites, accelerating evolution by a factor of approximately 40, as
opposed to a factor of approximately 5 at rapidly evolving sites.
A less frequently used alternative to the KA/KS tests for detecting selection are those that rely on
examination of within-species polymorphism in comparison to between-species fixed differences, such as
the McDonald-Kreitman test (McDonald and Kreitman 1991). This test has been applied to a wide range
of multigene families in plants and animals and had been extended to account for background selection
and selective sweeps that may affect genome-wide patterns of polymorphism (Messer and Petrov 2013).
Another complicating factor is that selection is likely to be episodic and may even change direction
over time. It has to be borne in mind that the result of positive directional selection is to drive a rare allele
toward increasing in frequency at the expense of the existing most frequent allele, while purifying
selection will favor the most frequent existing allele against newly introduced rare alleles. Thus,
directional selection may only act when changing conditions favor switching or tweaking of toxin targets.
The effectiveness of the process is linked to the effective population size, as in small populations the
chances of fixation or elimination by random fluctuation are higher than in large ones. Luckily, more
biologically realistic methods that are capable of detecting such episodes of selection at individual sites
and/or lineages have been developed and are being constantly refined (e.g., the plethora of methods
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available in the HyPhy package (Kosakovsky Pond et al. 2005; Murrell et al. 2015)) and are now being
applied to toxin evolution (Sunagar et al. 2015).
Evolution of Hypervariability
Ever more sensitive methods of studying the small amounts of venom produced by individual specimens
of some species rather than traditional pooled venom approaches, for example, have revealed that each
individual Conus may be synthesizing up to 1000 bioactive peptides, and there may be virtually no
overlap between species and even rather little overlap between individuals of the same species (Dutertre
et al. 2010). When the diversity of venomous animals is taken into account (over 700 species of Conus
alone), the true number of toxins generated by animals is staggering. This hyperdiversity is not restricted
to toxin genes but applies to any genes whose products interact with other species, whether as predators,
prey, or pathogens. This panel of interacting organisms is likely to be different for each species or even
populations within species, and thus genes of this type are likely to be exposed to different selection
pressures on a regular basis. As a result, they are often hyperdiverse, encoding hundreds if not thousands
of different variants within the same species or even individual. Classic examples are the major
histocompatibility complex (MHC) (Bernatchez and Landry 2003) and defensin genes (Das et al. 2010)
in vertebrates and surface antigens of microbial pathogens (Zilversmit et al. 2013), which are involved in
evading the innate immune response of their hosts. Other examples include plant genes involved in
pathogen resistance (Bergelson et al. 2001) and proteins involved in sensing of the environment, such as
visual (Bowmaker and Hunt 2006) and olfactory genes (Niimura 2012), and in communication (Wilburn
et al. 2012). As well as its practical relevance, this hyperdiversity raises some very interesting theoretical
questions. How is it generated and maintained? How is the expression of particular toxin variants
controlled?
Many toxins belong to multigene families (MGF), in which genes (often coding for compounds
performing a basic physiological function in the organism originally) are duplicated and inserted into
the genome (Wong and Belov 2012). Gene duplication has been recognized as an important source of
evolutionary innovation in eukaryotes for decades, and recent work suggests that it may have been
fundamental to the successful radiation of early eukaryotes (Zhou et al. 2010). The original model
proposed to explain the contribution of gene duplication to evolution was that the presence of a duplicate
copy of a gene allowed the development of new functions as it was free from functional constraint
(neofunctionalization model). However, there are a number of problems with this model, outlined by
Bergthorsson et al. (2007), largely relating to the fate of the nonfunctional copy while free from selection.
While purifying selection may act on a duplicated gene to maintain it in the population, its ability to
acquire new functions would then be limited. However, the acquisition of new functions through random
mutation while remaining free from more common deleterious mutations and avoiding elimination from
the population through drift would seem to require unrealistically large populations. This has been
confirmed by simulation studies using point mutation as the predominant process involved in change
and assuming that the new function requires more than one mutation. However, other, more complex,
processes by which a larger number of changes may occur in a single step are also known to occur,
including recombination and insertion-deletion events (see section “Concerted Evolution by Gene
Conversion” below).
A number of variants of the basic model have been proposed (reviewed by Innan and Kondrashov
2010) which differ, sometimes rather subtly, in aspects such as the type and timing of selection acting on
one or both of the duplicated genes, during the process of spread and fixation in the population. Despite
this, Innan and Kondrashov (2010) pointed out that most of the critical information that would allow one
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model to be favored over others come from the early stages of the process when distinguishing different
types of relationship between duplicated copies, which are essential for proper understanding of the
evolutionary dynamics, can be difficult (Mendivil Ramos and Ferrier 2012). While phylogenetic methods
(e.g., Han et al. 2009) may provide greater power to distinguish recent paralogs, lineage-specific
duplications may still represent relatively old events if the lineages diverged a long time ago and
duplication rates are high. In addition, phylogenetic methods depend quite heavily on the accuracy of
the phylogenetic hypothesis being used. Moreover, such studies often rely on analyzing protein or
complementary deoxyribonucleic acid (cDNA) sequence of toxins, rather than gene sequences, as this
is still more readily available than genomic data. However, if protein products are exposed to strong
positive selection, phylogenetic analysis on coding regions may well give misleading results about the
relationships of the genes themselves (Malhotra et al., 2015). The large size of such datasets also
frequently leads to simplification of phylogeny reconstruction methods. As a consequence, complex
evolutionary phenomena such as recombination and rate variation (both among-site and among-lineage)
may not be adequately controlled (Arenas 2015). The incorporation of biologically reasonable variation in
processes among sites may often account for the apparent derived trends predicted by simpler methods
(Goldstein and Pollock 2006). Further, rate calculations depend on the availability of known divergence
times among taxa. As a result, few studies of toxin families have attempted to quantify duplication rates
and those that have usually employ multiple assumptions to produce a range of rates (Binford et al. 2009).
However, it seems highly probable that toxin genes are evolving at some of the fastest rates yet recorded
(Chang and Duda 2012).
The “birth-and-death” model of gene evolution (Eirín-López et al. 2012) was first proposed in relation
to MHC genes, and this model has been applied frequently to the evolution of specific gene families
thereafter. Birth of new genes is relatively easy to observe, but death (gene loss through deletion or
pseudogenization) is far less so. Gene loss is often ignored as it is assumed that most duplications will in
fact be deleted as they are more likely to be slightly deleterious than beneficial. However, gene loss may
play an important role in reshaping the venom arsenal of similar organisms (Rachamim et al. 2015).
Additionally, when it results from pseudogenization rather than deletion from the genome, it may in fact
still provide fodder for further evolutionary change, as several studies have now indicated that
pseudogenes retain the potential to become new genes (Balakirev and Ayala 2003; Duda and Remigio
2008).
Although the sequencing of more and more genomes will make the job of studying gene gain and loss
much more robust, presently, gene loss can only be predicted by analytical methods such as gene-species
tree reconciliation (Szöllősi et al. 2015), which may suffer from errors and, in large gene families, cannot
distinguish between unsampled genes and genes which have really been lost from the species. There may
also be hidden genes, those that are still functional but are not normally transcribed. Casewell et al. (2014)
suggested that between 44 % and 70 % of toxin genes may not be transcribed, although this is complicated
by possible translational controls on functional genes as well.
Nevertheless, some carefully conducted studies (e.g., Chang and Duda 2012) have supported the
hypothesis of relatively constant and high rates of gene turnover in toxin gene families. This process
would provide a constant supply of new genes, ready to undergo selection for new functions, which is
likely to far outstrip the rate of novel mutation (Katju and Bergthorsson 2013). In fact, the number of gene
copies circulating in a population at any one time is likely to be much higher than estimated, since
phylogenetic analyses cannot separate the rate of duplication of genes and the rate of their subsequent
fixation in the population, and instead represents a combination of both these processes (Innan and
Kondrashov 2010). It is also likely that a greater number of duplicate copies coding for a particularly
important toxin in the venom might in itself be beneficial, since snakes need to rapidly replenish the
venom contained in the lumen of the venom gland once it has been expended, and the rate of transcription
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gene
More of the same toxin increases fitness
n n
Multiple identical copies arise Multiple identical copies arise

as a result of high birth rate as a result of gene conversion
Positive selection maintains copies in population
Mutation probability in gene increased

by presence of multiple copies
If mutation causes favourable change,

n may gain selective advantage
n
Mutation spreads through population Mutation spreads through population driven by
driven by selection or further gene conversion
amplification
a BIRTH AND DEATH b GENE CONVERSION
Fig. 1 The fate of duplicated genes depends on a complex interplay of processes that tends to fix them in, or eliminate them
from, the population (including drift, selection, pseudogenization, and gene conversion). One reason that duplicates might be
maintained in the population by selection is through the dosage effect, if an increased amount of gene product (e.g., a toxin that
is particularly effective at subduing a commonly available prey type) provides a fitness benefit to the organism. The larger
number of copies then provides an increased target for point mutation, which occurs much more slowly. However, a similar
pattern may be produced by both a birth-and-death model with a rapid rate of gene duplication (a) and gene conversion (b)
when the presence of additional gene copies similar to the advantageous one is favored by selection, although the probability
and speed of fixation of a favorable mutation in the population will be faster in the case of concerted evolution when direct
selection on gene duplicates is weak. Gene conversion is also known to maintain multiple gene copies that are already fixed in
the population, with or without selection favoring these duplicates.
will be limited by the number of genes encoding the toxin. In other words, gene dosage effects might be
beneficial rather than detrimental in this case (Kondrashov 2012). However, it is not yet very clear
whether this would provide a temporary advantage only, with the duplicates being lost once the advantage
of that particular isoform is lost (e.g., by a change in diet), or whether these duplicates then provide fodder
for further functional divergence (Katju and Bergthorsson 2013). More recent studies have provided
evidence for the latter in genes that have environmental response functions (Chain et al. 2014).
Some evidence to support the importance of gene dosage in the preservation of duplicated toxin genes
has been found in venomous snakes. Malhotra et al. (2013) found a number of distinct gene copies
encoding the same protein, which in all cases corresponded to the most abundant isoform detected in the
venom by mass spectrometry, in a study of phospholipase A2 (PLA2) evolution in pit vipers. Oguiura
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et al. (2009) found up to 32 copies of genes encoding crotamine in Crotalus durissus per haploid genome
and detected a positive correlation between the number of copies of the crotamine gene and the
concentration of crotamine in the venom. Even a slight advantage provided by overexpression of
particular toxin variants might be enough for selection to be the primary force acting to fix a new duplicate
in the population, rather than neutral drift. Bergthorsson et al. (2007) also pointed out that genes often
possess auxiliary functions when present in high copy number that are not seen when present in low copy
number. Although evidence for this is mostly from prokaryotes, it has been shown that a given snake
venom protease may behave both as an anticoagulant and coagulant, depending on its concentration in the
venom (Matsui et al. 2000).
Snake venom PLA2 evolution was one of the case studies cited in defense of the innovation-
amplification-divergence (IAD) model (Fig. 1) developed by Bergthorsson et al. (2007). The IAD
model additionally predicts that the emergence of a highly functional allele with a novel function
would lead to removal of selection on paralogous copies with increased loss through pseudogenization
or deletion. This model may help to explain the “streamlining” of venom that has recently been reported
from sea snakes (Pahari et al. 2007) and cone snails (Duda and Remigio 2008), where a relationship
between specialization of diet and the number of toxin isoforms expressed in individual venoms was
observed. In addition, it may also be applied to the observed restriction of PLA2 isoform expression in
populations of Taiwanese Viridovipera stejnegeri that are known to feed on more challenging prey than
frogs (Creer et al. 2003). These patterns could result from increased levels of purifying selection on
mutations which take the “adaptive” isoform away from its optimal form, or, conversely, relaxation of
positive selection in the case of moving to a less challenging prey item.
Many studies of toxin families in a diverse range of organisms have also provided evidence that
adaptive molecular evolution is directed toward the active site of toxins, regions that are implicated in
protein-protein interactions, which are usually located on the surface of the protein (Casewell et al. 2013).
Conversely, purifying selection is usually detected in residues important for maintaining the structural
stability of the protein. In large proteins, such as snake venom PLA2s (Kini and Chan 1999), these may
form a conserved core, while in smaller toxins, they are surface-exposed residues which are clustered on
the opposite side to the active surface (Kozminsky-Atias and Zilberberg 2012). It is often not stated
explicitly what mechanism might bring about this pattern. Most authors implicitly refer to a mechanism
whereby random mutations occurring in active sites become fixed more rapidly than random mutations
occurring elsewhere due to the action of positive selection when they confer an advantage, such as the
ability to subdue a novel prey type. However, directed mutagenesis, whereby the mutation rate is
hypothesized to vary according to the conformational position of the residues, is also implicitly or
explicitly invoked in many discussions. This is based on the apparent adaptive mutability observed in
bacterial experimental systems (Bergthorsson et al. 2007), which has been a subject of intense debate and
controversy for decades. It is now thought that the original “adaptive mutability” experiment can be
explained by the presence of multiple copies of plasmids carrying the mutant allele (Sano et al. 2014),
without the need to invoke the action of error-prone polymerases (which moreover, have never been found
in eukaryotes). Thus, explanations of variation in amount of change observed at different sites must be
based on varying rates of fixation in the population or species, rather than varying rates of mutation in
individual genomes.
Genomic Location
One possible explanation for rapid gene turnover rates is the physical location of the genes in question
(i.e., among the chromosomes). It has been observed that many hypervariable gene families in primates,
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which are evolving in a similar birth-and-death manner, are located in subtelomeric regions of chromo-
somes. In primates these regions are known to be subjected to higher rates of recombination, duplication,
translocation, and other diversifying phenomena than other chromosomal regions (Das et al. 2010;
Linardopoulou et al. 2005). At present, we only have a few clues about the role that this might play in
toxin evolution. Recently, Jiang et al. (2011) identified five putative tandem duplicates of three-finger
toxin genes in Bungarus multicinctus and seven in Naja atra, while Ikeda et al. (2010) demonstrated the
presence of five tandemly arranged PLA2 genes (including two pseudogenes) located in a
microchromosome in the Japanese habu Protobothrops flavoviridis. The location could be significant
as reptilian microchromosomes show high rates of recombination leading to tandem duplications arising
frequently (Janes et al. 2010).
Concerted Evolution by Gene Conversion

One major model of evolutionary change that has not yet been mentioned is concerted evolution. In
contrast to the birth-and-death model, in this model members of a gene family tend to be more similar to
each other within a species than they are between species. This is because gene conversion acts as a
homogenizing factor (acting via homologous recombination-based repair of double-strand breaks trans-
ferring genetic information among paralogs or unequal crossing-over events during meiosis between
tandemly arranged copies) within a population or species. Although the evidence to date from toxin gene
families has favored the birth-and-death model, this might be because sampling has been biased toward
interspecific rather than intraspecific comparisons and is usually incomplete to an unknown extent.
Moreover, few studies have examined polymorphisms within species (Oguiura et al. 2009). More recent
evidence suggests that these two models are not mutually exclusive, and mixed-effect evolution of certain
large gene families, such as 5S ribosomal RNA, has been proposed (Eirín-López et al. 2012). Gene
conversion has been shown to be active in large gene families and those where increased gene dosage is
advantageous, meaning that quite sophisticated analyses are required to distinguish the underlying model
in such cases. This suggests that future studies of toxin family evolution should not automatically assume
that birth-and-death evolution is the force in action without explicitly considering concerted evolution
through gene conversion (Arguello and Connallon 2011). Ignoring gene conversion when it has occurred
will lead to misinterpretation of temporal patterns of selection and an overestimation of the rates of
duplication.

Although the evolution of toxins is often discussed as if they were a homogenous group of biomolecules,
they are of course very diverse in their structural features and genomic backgrounds. Casewell
et al. (2014) highlighted that some toxin families, such as cysteine-rich secretory proteins (CRISP), and
L-amino acid oxidase (LAAO) show very different features to PLA2s, snake venom metalloproteinases
(SVMP), three-finger toxins (3FTx), and other toxins that are encoded by multigene families with high
number of paralogs. Instead, they appear to be subject to considerable control at transcriptional and
translational stages, as well as being different in their evolutionary dynamics as revealed in the genome of
the king cobra (Vonk et al. 2013). Posttranslational processes, such as proteolytic cleavage to produce
multiple toxins from a single gene product, are also much more common in some gene families (such as
serine proteases and SVMP) than others (such as PLA2) and are bound to considerably affect their
evolutionary dynamics. Notably, Casewell et al (2014) showed that several highly transcribed genes
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(including SVMP and C-type lectins, which belong to generally highly expressed protein families in the
venom gland of the Echis species under investigation) were present at very low levels in the proteome.
Durban et al. (2013) also showed, in relation to ontogenetic changes in the venom of Crotalus simus, that a
single transcriptome could give rise to distinctly different proteomes with drastically different functional
capabilities and provided evidence that this was mediated by micro-ribonucleic acids (miRNA). They
may also play a similar role in modulating the plastic response of adult snakes to changing environmental
conditions.
Moreover, work on other, non-toxin, gene families shows that genomic architecture and signaling
pathways, which may vary dramatically across the range of venomous taxa, can influence the evolution-
ary dynamics substantially. For example, Nozawa and Nei (2007) showed that olfactory receptor
(OR) genes show very different patterns in Drosophila compared to mammals, which they attributed to
differences in expression patterns of OR genes in the two groups, as well as more flexible signaling
pathways involving ORs in mammals. Transcription factors (proteins which bind to deoxyribonucleic
acid (DNA) thus affecting their expression patterns) have been shown to display lineage-specific
expansion associated with adaptive niche changes in various groups including Archaea and primates
(Iskow et al. 2012). The dynamic evolution of conotoxin expression patterns is also responsible, at least
partly, for the differentiation of venoms of Conus (Duda and Remigio 2008), and Dutertre et al. (2014)
recently demonstrated that the genetic complement of conopeptides can be combined very differently in
different parts of the Conus venom gland to produce two distinct types of venom that are deployed
differentially in defensive and predatory contexts. While the venom gland and the neighboring accessory
gland in snakes appear to make different contributions to the venom (Vonk et al. 2013), the role of the
accessory gland secretion is not yet entirely clear. It may be that in snakes, venom plays a lesser role in
defense as snakes display much more flexible and varied, context-dependent, antipredator responses
(Llewelyn et al. 2010). Thus, it is apparent that there may be sound biological reasons for not over-
generalizing across toxin groups and venomous animals. Toxinologists will, in the future, need to
examine the evolutionary history of toxins in different animal groups in much more detail, making full
use of the novel analytical tools now available, to fully understand the forces which have shaped the
generation of these highly sophisticated bioweapons. Luckily, rapidly advancing techniques and tools,
from next-generation sequencing to large increases in computational power, that make assumption-free
analysis more readily available, will assist in addressing this challenging task in the near future (Wong and
Belov 2012).
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Page 11 of 11
Evolution of Resistance to Toxins in Prey
Thomas M. McCabe and Stephen P. Mackessy
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Coevolution of Predator Venoms and Prey Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Resistance to Snake Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Resistance to Snake α-Neurotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Resistance in Woodrats (Genus Neotoma) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Resistance of Ground Squirrels (Genus, Otospermophilus) to Snake Venom
Metalloproteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Resistance to Snake Venoms in the Opossums (Family Didelphidae) . . . . . . . . . . . . . . . . . . . . . . . 9
Correlational Evidence for Resistance/Toxicity Coevolution in Venomous Snakes . . . . . . . . 9
Natural Resistance in Prey of Other Venomous Taxa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Explanations of a Limited Literature on Natural Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Conclusion and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Abstract
Venoms, as simple to complex mixtures of toxic components, are well understood
to be used as trophic weapons by a range of predator species. Ecological pre-
dictions obviate the response of putative prey species against predator attacks,
such as the development of biochemical defenses that allow prey species to evade
predation, namely, resistance. Current hypothetical predictions indicate that
venom toxicity and resistance form an antagonistic dyad that may be described
as a coevolutionary chemical arms race. The development of resistance in prey
populations is expected to drive the evolution of novel toxicities in predator
populations and vice versa, given that predator-prey pairs are stably associated
through evolutionary time. The utility of a chemical arms race model to describe
T.M. McCabe (*) • S.P. Mackessy

School of Biological Sciences, Ross Hall, Room 2480, Campus Box 92, University of Northern
Colorado, Greeley, CO, USA
E-Mail: thomas.mccabe@unco.edu; stephen.mackessy@unco.edu
# Springer Science+Business Media B.V. 2016 1

2 T.M. McCabe and S.P. Mackessy
toxicity-resistance systems as well as known information about natural resistance

mechanisms derived against venomous predators are discussed across prey spe-
cies of a wide range of venomous predators. The efficacy of resistance, mecha-
nism(s) of resistance, phylogenetic breadth of resistance, and phylogeographic
distribution of resistance are provided where information is available. For many
predator groups, known prey resistance is not well described, and we discuss the
cause(s) of such a gap in understanding, as well as future directions for resistance
research and application of known resistance information for practical and theo-
retical purposes.
Keywords
Predator prey interactions • Resistance • Mechanism • Evolution • Chemical arms
race
Introduction
Venoms are simple to complex mixtures of toxic components that are conveyed
through specialized delivery systems to subdue prey (Mackessy 2002, 2010), and
possibly to aid in predigestion of prey tissues (Pough and Groves 1983; Mackessy
1988). For prey species, on the defensive side of the predator/prey dyad, becoming a
meal greatly decreases lifetime fitness, and predictably many forms of predator
evasion have been documented. This essay discusses the nature of chemical defenses
against predator venoms, often described as venom resistance, that have arisen in
response to the selective pressure imposed by the chemical weapons of predators.
For the purpose of this discussion, venom resistance is defined as the endogenous
chemical/physiological capacity of a prey species to prevent or hinder the pathologic
consequences of envenomation by a predator species. By this definition, in the
absence of resistance mechanisms, venoms are pathological to prey species. This
venom antagonism is in contrast to cases where a predator’s venom has no bioactive
effect on one or a group of potential prey species, but may be lethal to other species
or groups of species (i.e., prey-specific venoms: Heyborne and Mackessy 2013;
Mackessy and Saviola 2016; Pawlak et al. 2006, 2009). Venoms represent complex
molecular weapons to defend against, and venom resistance is assumed to be
conferred by venom-resistant molecules or mechanisms that are able to neutralize
partially or fully the negative effects of a venom and its toxic constituents. Success-
ful resistance should allow prey species to evade capture and digestion. There is
evidence that in some cases, chemical neutralization of venomous components may
not be sufficient to allow prey species to escape predator behaviors that enable prey
capture, regardless of the effectiveness of venoms. However, behavioral responses
that allow prey species to evade predators, or allow predators to successfully capture
prey species, independent of the role of venom, will not be discussed.
This chapter focuses on known cases of prey resistance to predator venoms.
Resistance in some groups, such as prey species of venomous snakes, is well
described, but resistance in other groups, such as prey species of venomous insects,
Evolution of Resistance to Toxins in Prey 3
is not well understood, and little information appears to be available even after
extensive literature searches. Instances of resistance are discussed in relation to the
venoms they are able to neutralize. Each section provides information regarding
efficacy of resistance, mechanism(s) of resistance, phylogenetic breadth of resis-
tance, phylogeographic distribution of resistance, as well as other relevant informa-
tion about the nature of the predator/prey pairs in question. The discussion here
centers on chemical arms races between venomous predators and resistant prey; that
is, the focus remains only on animal/animal interactions, as there are no known cases
of an animal venom used to subdue plant or prokaryote prey, or a plant that uses
venom to dispatch prey species. Following the predator-specific sections is a con-
cluding discussion of our current understanding of prey resistance to natural toxins,
future directions for resistance research, and possible applications of resistance
systems for practical and theoretical purposes.
Coevolution of Predator Venoms and Prey Resistance
When considering prey resistance, the underlying issue is whether a coevolutionary

response to the selective pressure of predator venom exists within the system.
Venoms, as derived trophic adaptations, are expected to experience selection pres-
sure from mechanisms that allow prey species to evade predation. The appearance of
resistance molecules in response to the derivation of new snake venom toxicities is
expected to follow Dawkins and Krebs’ (1979) model for an arms race between two
taxa in an antagonistic coevolutionary relationship. A predator develops a chemical
weapon (venom), which is used to subdue a prey species. As predators capitalize on
susceptible individuals, the diversity of the prey population becomes limited to those
individuals who are able to evade predation. These remaining individuals may
persist because of phenomena like behavioral modifications, changes in microspatial
distribution, or the appearance of a chemical mechanism that inhibits the toxic action
of the predator’s venom, namely, resistance. This resistance phenotype is expected to
increase over time as the snake predator becomes increasingly incapable of incapac-
itating prey with the new resistance phenotype. Variations in predator and prey
phenotypes are expected to follow each other through time in a frequency-dependent
manner that creates new resistances to new toxicities and vice versa.
Several expectations follow from this scenario of the development and mainte-
nance of resistance in prey. First, predator/prey pairs are expected to associate with
each other for stable periods of time. By definition, predators and prey should
respond in sequential and reciprocal manners as the opposing partner develops a
new offensive or defensive strategy to the other. Van Valen (1973) described this
stable reciprocity in his postulation of the Red Queen hypothesis. Much as the Red
Queen in Lewis Carroll’s Through the Looking Glass tells Alice that to stay in one
place she must keep running, Van Valen hypothesized that for either predator or prey
to “stay in one place” (i.e., persist through evolutionary time), they must continue to
evolve. By extension, if one of the predator/prey pair was unable to continue to
respond to a newly derived trait in the other partner, they would soon become
extinct, assuming intense predation pressures on the susceptible prey phenotype.

Extant predator/prey pairs should demonstrate some balance between the relative
abundance of resistant and susceptible individuals, keeping in mind that this balance
may be skewed toward one partner or the other at any given time point.
In addition to stable reciprocity, the timeline of coevolutionary relationships is
expected to develop over longer rather than shorter timescales. When investigating
the frequency and mechanism of resistance, it may be that the newly evolved
resistance or toxicity is at such low abundance that detection of this functionality
is nearly impossible. In the real time of academic research, the turnover of enough
generations of predator or prey species to produce a new functionality may be too
slow for any given researcher to describe in a lifetime. Additionally, whether novel
toxicity or resistance are diversifying or are being selected against may depend on
the historic length of predator/prey associations. Sunagar and Moran (2015) com-
pared the rate of diversification of a variety of toxin groups against the relative age of
a number of venomous species’ lineages. These authors proposed a “two-speed”
mode of venom evolution, where more recent lineages of venomous predators, such
as cone snails and venomous snakes, show increased diversifying selection, and
older lineages appear to be under increased levels of purifying selection. The authors
proposed that diversifying selection for venomous predators would be associated
with prey base or niche expansion; however, it is possible that diversifying selection
may allow for maintenance of a stable relationship with current prey species and
simply throw frequency-dependent selection of a chemical arms race into another
round of novel toxicity and resistance development. In any case, younger or older
lineages are not fixed in a selective regime and may experience a switch from
purifying to diversifying selection and vice versa. Thus, it appears that the age of
the lineage in question may increase the likelihood that resistance is a prominent
feature of prey populations or that the toxicity of the predator may have an advantage
over prey defenses (such as in Holding et al. 2016), again making resistance more
difficult to detect.
It is cogent to note that while a chemical arms race scenario is presently a “best
guess factor” as the driving force for biochemical diversification of venoms over
evolutionary time, numerous cases of prey-specific toxicities and venom resistances
are documented in the literature, which lends support to a coevolutionary relation-
ship between toxicity and resistance. In support of the chemical arms race scenario,
research into the relationships between venomous snakes and their resistant prey will
serve as a test case. Current information about a diversity of resistant prey is prefaced
by a discussion of theoretical and methodological approaches to evaluating the
importance of coevolutionary processes in the development of resistance.
Resistance to Snake Venoms
Natural resistance to predator venoms is best described in prey species of venomous

snakes, particularly mammals. The impetus for this wealth of knowledge comes
from the attempt by snake venom researchers to elucidate the merits of the
hypothesis that diet has served as a major selective pressure shaping snake venom
composition. Over the past several decades, researchers have demonstrated that
venom composition may vary across geographic space and ontogenetically (see
Mackessy (2010)) and has been purported to vary with diet (e.g., Gibbs and
Mackessy 2009; Sanz et al. 2006). The more recent championing of diet as a
major driver for venom compositional change is born out of an institutional debate
over the origin of venom, i.e., whether venom is the product of neutral or selective
processes over evolutionary time.
Near the end of the twentieth century, the issue of the origin of snake venoms as
the product of neutral or selective processes became a major theoretical divide
between venomous snake biologists. Scientists such as Dietrich Mebs (2001) and
Mahmood Sasa (1999) argued that because snakes delivered venom in such large
quantities, many times more than was sufficient to incapacitate prey, venom must not
have arisen from selective processes and was “overkill.” Considering the discrep-
ancy between the minimum amount of venom required for prey capture and the
actual amount delivered, they argued that venom components were too metabolically
costly to be used in such large quantities. Additionally, they noted that the individual
components of venom were so toxic across a variety of possible prey species that
there did not appear to be a selection for specific toxicities. To these authors, venom
arose out of neutral evolutionary processes that allowed for the sequestration and
concentration of modified somatic molecules into what we observe today as the
components of snake venom.
This neutral view was quickly challenged by research showing that the notion of
overkill was unlikely. Hayes et al. (2002) demonstrated that venomous snakes had
control over the amount of venom released in striking a prey item. The amount of
venom delivered was more than absolutely necessary to subdue prey items, but
control over venom delivery indicated that there was a functional role for allowing
large volumes to be expressed in snakebite envenomation. Saviola et al. (2013)
demonstrated that, at least in venomous snakes from the family Viperidae (vipers, pit
vipers, and other solenoglyph venomous snakes), a large bolus of venom was
required in order to deliver a particular molecule in high enough concentration to
allow the snakes to recover their envenomated prey item. Viperid snakes often use a
sit-and-wait ambush strategy and strike prey as they cross the snake’s path; prey that
has fled the ambush site and succumbed to the effects of the venom is then recovered,
often at some distance to the ambush site. The process of prey relocation may be
challenging because prey may escape in any direction in three-dimensional space,
and thus a relocator molecule is needed to track the envenomated prey item effec-
tively. At this point, an arms race hypothesis was explored to explain the evolution of
the complex phenotype of snake venom and associated delivery systems.
A number of prey species groups show resistance to snake venoms, and a wide
variety of evidence helps to corroborate a chemical arms race scenario. Each species
group will be treated separately, and data has been compiled on the prevalence and
mechanism of resistance. Any study attempting to uncover coevolutionary relation-
ships between species pairs faces the challenge of using extant and historical
evidence to infer reciprocity across evolutionary time. A number of approaches are
often used and synthesized to confirm coevolution (Futuyma and Slatkin 1983). In
the case of resistance/toxicity systems, the demonstration of resistance through
standard toxicity assays is required. Anecdotal evidence for prey ability to avoid
predation may not be explained by chemical resistance; resistance must be confirmed
through direct challenges with physiologically and biologically relevant doses of
venom. As novel phenotypes should appear in a single individual or small popula-
tion of individuals and radiate out in the direction of gene flow, locality of both
predator and prey must be taken into account. A record of the geographic distribution
of populations with resistance or susceptibility may further allow for spatial corre-
lation with the range of the venomous partner species. Thus, a biogeographic
account of current resistance may be constructed. Longitudinal documentation of
the biogeography of a particular resistance mechanism may offer some insight into
the rate of change in the dynamics of resistance and toxicity for a given species pair.
To date, it does not appear that this type of long view has been established for any
system involving snakes, and even if one could be constructed, if reciprocal
responses occur over evolutionary time, this may preclude any detection of active
flux in the relationship between toxicity and resistance within the lifetime of a given
researcher.
Following initial screening for resistance, mechanistic descriptions are often
elucidated that demonstrate the direct ability of prey physiologies to negate the
pathologic effects of venoms. As mentioned earlier, prey species are challenged by
the (often) complex phenotype of predator venom, and their responses may range
from a wholesale attempt to neutralize the diversity of toxins in a venom to
mechanisms that attack a limited number of toxins. Finally, some attempt must be
made to connect species pairs in evolutionary time and demonstrate stepwise
evolutionary change. This correlation through time is the most difficult line of
evidence to obtain, as current technologies limit these types of studies to phyloge-
netic comparisons between predator and prey species complexes (Filipiak et al.
2016; Page 2002; Suchan and Alvarez 2015). Correlation between the divergence
of predator and prey clades would seem to indicate reciprocal evolutionary diver-
gence; however, correlational analyses are limited in their ability to confirm causality
between the coevolution of toxicity and resistance and speciation or divergence in
predator and prey taxa. It is also possible that some common biotic or abiotic
pressure, unrelated to potential coevolutionary scenarios, caused cladogenesis in
both predator and prey species, and resistance is secondarily derived.
Resistance to Snake a-Neurotoxins
A resistance mechanism that has been confirmed across a diversity of mammalian

predators and prey is the ability to tolerate snake α-neurotoxins, acetylcholine
receptor (AChR) agonists. Ovadia and Kochva (1977) demonstrated that mongoose
sera challenged with venoms from snakes in the family Elapidae (cobras, kraits,
and other opisthoglyphous snakes) was able to neutralize the effects of the venom.
Later research uncovered that this resistance to elapid venoms is directed against
α-neurotoxins that make up a significant portion of the total venom protein. Barchan
et al. (1992) sequenced the mongoose AChR and detected a number of
non-synonymous mutations in the ligand binding site of the AChR. Hypothesized
structures for these mutations indicate a confirmation change in the ligand binding
site that prevents α-neurotoxins from binding while still allowing acetylcholine
(ACh) to bind its receptor. Later work (Asher et al. 1998) further demonstrated
that the mongoose’s resistant AChR prevented α-neurotoxins from binding while
still allowing ACh to bind with higher affinity than non-resistant type AChR found
in rats. This elevated binding affinity indicated that mongoose AChR was able to
prevent complete binding of α-neurotoxins while allowing ACh to bind with little
steric or concentration-dependent competitive hindrance from α-neurotoxins that
had inundated synaptic junctions. A slight conformational change was sufficient to
create near complete resistance to α-neurotoxins.
In addition to mongooses, similar conformational changes in acetylcholine recep-
tors have been documented in the Chinese cobra (Naja atra), the Javelin sand boa
(Eryx jaculus), the dice snake (Natrix tessellata), and also in the European hedgehog
(Erinaceus europaeus) (Barchan et al. 1992; Neumann et al. 1989). Resistance in
N. atra is most likely protection against auto-envenomation; however, it is possible
that this resistance may allow evasion from cannibalism or predation by other
sympatric elapid snakes. The example of E. europaeus provides an additional
mammalian example of resistance to α-neurotoxins, but perhaps the most intriguing
example of resistance is the case of the three non-venomous snakes. Considering the
ongoing debate among snake venom toxinologists about the ultimate origin of snake
venom proteins and the delivery apparatus (e.g., Fry et al. 2012), the appearance of
α-neurotoxin resistance across more basal snake taxa begs the question of whether
resistance is intrinsic to snake physiology or has appeared independently several
times throughout the radiation of the snakes. In any case, a better understanding of
the molecular origin of snake resistance to snake venoms could indicate a coevolu-
tionary predator-prey situation if the hypothesis that resistant, non-venomous snakes
were once or are currently preyed upon by venomous snakes is supported.
Resistance in Woodrats (Genus Neotoma)
As a follow-up study to anecdotal evidence of resistance in Southern Plains woodrats

(Neotoma micropus), Perez et al. (1978) challenged woodrats with venom from the
western diamondback rattlesnake (Crotalus atrox), showing that these rodents had
greatly elevated tolerance to the venom compared to a laboratory mouse control.
Perez et al. (1979) further showed that this resistance mechanism was able to
significantly decrease the hemorrhagic effects of C. atrox venom for N. micropus.
De Wit (1982) screened a second Neotoma species, the eastern woodrat (Neotoma
floridana), with the venom from Osage copperhead (Agkistrodon contortrix
phaeogaster) and detected a similar resistance to hemorrhagic toxins. It appeared
that venom resistance was shared across the genus. Using electron microscopy,
Huang and Perez (1982) further showed that N. micropus suffered little hemorrhage
or muscle damage following envenomation. Some mitochondrial and myofibril
damage were detected, but it appeared that resistance also prevented myotoxic
pathologies, especially in comparison to laboratory mouse controls. A candidate
antihemorrhagic resistance molecule was purified and partially described by Garcia
and Perez (1984). This single, non-enzymatic resistance molecule was able to bind
and neutralize C. atrox toxins. Binding was shown to be non-polyvalent, and the
authors concluded that this candidate molecule was not an immunoglobulin. Unfor-
tunately, it does not appear that further descriptive work has been completed on this
resistance molecule, and no biogeographic or further phylogenetic information is
available regarding the distribution and prevalence of this resistance mechanism in
Neotoma.
Resistance of Ground Squirrels (Genus, Otospermophilus) to Snake

Venom Metalloproteases
Another well-described example of snake venom resistance are endogenous snake

venom metalloprotease inhibitors (SVMPIs), best documented in a number of
squirrel species in the genus Otospermophilus (formerly Spermophilus). Biardi
and Coss (2011) showed that rock squirrel (Otospermophilus variegatus) serum
was able to neutralize the pathological effects of venom from two species of
rattlesnake, the western diamondback rattlesnake (Crotalus atrox) and prairie rattle-
snake (Crotalus viridis viridis), which were sympatric to assayed squirrel
populations. Challenges with venom from an allopatric species of rattlesnake, the
northern Pacific rattlesnake (Crotalus oreganus oreganus), were not successfully
neutralized. Interestingly, the venom used in these experiments was commercially
purchased; however, even without a confirmation of matching locality between
predator and prey samples tested, there still appeared to be an inhibitory effect
against individuals from a sympatric predator species. In the same year, another
team (Biardi et al. 2011) published a description of an SVMPI isolated from
O. beecheyi serum. This molecule was able to prevent tissue damage and hemor-
rhage normally expected from envenomation by the sympatric C. o. oreganus.
Further, resistance was positively correlated with the proximity of rattlesnake pop-
ulation to resistant O. beecheyi; that is, resistance was ineffective against distant
populations of C. o. oreganus, indicating that resistance is geographically localized
and requires predation (or at least offensive) pressure from the colocalized rattle-
snake population to select for resistance. The authors recognized that while other
mammals do not have similar SVMPIs that serve as resistance molecules, there
appears to be convergence of defenses against hemorrhagic toxins, a hallmark of
many viperid snake venoms. Future work in mammalian resistance to viperid
venoms will confirm or reject convergence to defenses against hemorrhagic toxin
classes of snake predators.
Resistance to Snake Venoms in the Opossums (Family Didelphidae)
A final group of prey items with described resistance to venomous snake predators
are the opossums (Mammalia: Didelphidae). Jansa and Voss (2011) reported an
increased number of non-synonymous changes in gene sequences of a hemostatic
protein, von Willebrand factor (vWF), in opossums known to exploit venomous
snakes as prey items. These researchers found that these non-synonymous changes
are associated with binding sites for C-type lectin-like proteins found in some viperid
snake venoms; changes to these regions were inferred to decrease binding affinity
with these toxins. These data do not indicate that opossums preyed upon by
venomous snakes have similar resistance, but later work (Voss 2013) found that a
number of opossum species could be confirmed as venomous snake prey and that
their relationships to known, resistant species of opossums make it plausible that
they would also likely show changes to vWF. However, beyond these types of
phylogenetic correlations, evidence for resistance against venom challenges is not
available, and physiological data would be required to verify that resistance to
C-type lectin-like proteins is sufficient to allow for evasion from predation by
venomous snakes.
Correlational Evidence for Resistance/Toxicity Coevolution

in Venomous Snakes
The extent of information regarding resistance to snake venoms varies depending on

the species group of interest and may include as little as an initial confirmation of
resistance to a full description of the resistance mechanism. In relatively few cases,
functional information can be paired with evolutionary analyses to test the underly-
ing assumptions of a chemical arms race. Barlow et al. (2009) investigated a
potential coevolutionary relationship between venom specificity toward scorpion
prey in four species groups of the genus Echis (saw-scaled vipers). They used a
Bayesian inference method to plot a phylogeny of these four groups and compared
the relative amounts of scorpion versus rodent prey found in the stomach contents of
museum specimens, as well as toxicity assays (LD50) toward scorpions (Scorpio
maurus), to species relationships. Venoms of species groups with the highest
amounts of scorpions in their diet were the most toxic against scorpion prey, while
the E. coloratus group, rodent specialists, showed the lowest toxicity. Relative
abundance of a particular type of prey scaled with the relative toxicity of the
venom; for example, the E. ocellatus group had an intermediate amount of dietary
scorpions and showed an intermediate toxicity toward live scorpion prey. The
implication of this increased toxicity toward preferred prey group was that Echis
venom has undergone selection favoring increased toxicity toward a preferred prey
type. While Barlow et al. (2009) did not test for scorpion resistance, the demonstra-
tion of prey specificity that follows the best resolution of Echis phylogenetic
relationships indicated a positive selective pressure for enhanced toxicity, perhaps
driven by prior prey resistance mechanisms. For example, a common ancestor to

Echis may have retained toxicity toward scorpions, while sympatric Rodentia
developed resistance, to the point that only Echis phenotypes that could shift to
non-rodent prey were able to persist. Secondary diversification of the venom toxins
may have restored high toxicity toward rodent prey, favoring a shift in those lineages
to specializing on rodents. The availability of non-scorpion taxa, preference toward
these taxa (how often they attempt to predate), and the relative resistance or
susceptibility of these taxa would be needed to corroborate reciprocal selectivity of
venom and resistance.
In the case of opossums, antihemorrhagic toxicity has been correlated with
phylogenetic comparisons of predator and prey species. Voss and Jansa (2012)
compared South American opossums and vipers, revealing that species of opossums
that were too large as adults to be ingested by vipers showed no resistance to venom.
Nonresistance in larger prey taxa was interpreted as the result of non-predation that
venomous snakes had no behavioral inclination to attempt predating these overly
large meals and thus no selective pressure to develop resistance was present.
Verifying the assumption of reciprocity between predator and prey, resistance may
arise or be maintained only in prey lineages that are likely targets of venomous snake
predators.
Natural Resistance in Prey of Other Venomous Taxa
Presently, little information is available regarding the appearance or mechanisms of

resistance in prey species of cone snails, insects, helodermatid lizards, cnidarians,
centipedes, shrews, scorpions, arachnids, and anemones. The sporadic and some-
times tangential evidence that exists for resistance against a number of these
venomous predators will now be discussed. Literature searches for documented
cases of resistance in prey species of insects were unproductive, but protective
immune reactions in non-prey species may indicate a set of mechanisms that pro-
vides resistance for prey. Metz et al. (2006) described the ability of mast cells in
inbred laboratory mice to confer protection against hypothermia and death associ-
ated with envenomation by the European honeybee (Apis mellifera). Palm and
Medzhitov (2013) later demonstrated that whole honeybee venom and the isolated
pore-forming toxin, melittin, was able to induce inflammatory pathways in in vitro
and in vivo experiments. The honeybee does not use its venom for prey capture;
however, it may be that resistance to venoms of bee relatives in the order Hyme-
noptera, such as predatory wasps, rely on the escalation of similar immune and
allergic responses to evade predation.
Immune responses conferring resistance to envenomation have been documented
for some arachnids. Schenone et al. (1970) induced resistance to challenge doses of
venom in laboratory rabbits through repeated sublethal doses of venom from the
Chilean recluse spider (Loxosceles laeta). A ramping of immune response to venom
dosing was detected by observing the increasing presence of antibodies in rabbit
serum across the dosing period. Similarly, Njau et al. (1986) induced resistance to
paralysis in laboratory rabbits through repeated sublethal infestations of red-legged

ticks (Rhipicephalus evertsi evertsi). Later, Reck et al. (2009) used serum from tick-
infested cattle to confer protection again the anti-hemostatic properties of tick saliva
in in vitro and in vivo assays. While defenses to parasitism by tick species do not fit
with a definition of prey resistance to venom, the apparent excitation of the immune
system in cattle speaks to a convergent mechanism by which arachnid venoms may
be neutralized. As arachnid toxins are quite diverse, hypothesizing a general con-
vergent mechanism may be too simplistic, but it stands to reason that in the absence
of other candidate resistance mechanisms to explore, immune responses to arachnid
venoms are plausibly productive.
Other than immune-based resistance to arachnid venoms, research into the
application of arachnid toxins as insecticides has revealed another possibly fruitful
avenue of study regarding prey resistance to arachnid venoms: the prevention of
toxin binding to nervous cell receptors by structural interference. Bende et al. (2014)
identified two residues in a particular region of American cockroach (Periplaneta
americana) voltage-gated sodium channels that conferred resistance against
β-Diguetoxin-DC1a from the desert bush spider (Diguetia canities). These
researchers were attempting to discover novel targets for insecticide development
and in the process uncovered the mechanism whereby some insects may avoid
envenomation by desert bush spiders. Differential toxicity to prey nervous tissue
has been identified for other spider predators. For example, Liu et al. (2016)
documented the ability of Araneus ventricosus venom to block cockroach, but not
mouse, voltage-gated sodium channels, suggesting the binding mechanism causes
lethal effects in insects while inactive toward vertebrates. In both cases, the exper-
iments were motivated by the development of insecticides that are insect-specific;
however, these lines of inquiry reveal possible candidate resistant prey species.
Another group with preliminary evidence for resistance in prey is the sea anem-
ones (phylum, Cnidaria; class, Anthozoa). Some species of this group capitalize on
prey species that are powerful enough to escape the grasp of an anemone, such as
teleost fishes, or have durable defenses to infiltrate, such as mollusks, which
necessitate the use of venom for prey capture (Frazão et al. 2012). While direct
evidence of the development of resistance in putative prey species is not available,
there are a number of studies that indicate two mechanisms that confer resistance to
mutualistic anemone fishes (genera Amphiprion and Premnas) and crustaceans
(representatives from several genera; Mebs 2009). First, mutualistic partners may
develop or acquire a mucus coat that neutralizes defensive compounds on the surface
of the anemone, or else allow the partner to associate closely with the anemone
without eliciting the firing of venom-delivering stinging cells, nematocysts (Frazão
et al. 2012). A second line of defense in mutualistic partners of sea anemones are
internal defenses that allow the partners to neutralize venom toxins, should the
nematocysts fire. Mucus coat defenses appear to be the main defense for mutualistic
crustaceans (Mebs 2009), and mutualistic anemone fish appear to use combinations
of both strategies. Mebs (1994) tested three mutualist Amphiprion anemone fish
species against the venom of four sea anemone species, finding limited endogenous
resistance in cohabitating fish species. In some cases, the mutualist fish was not
resistant to the venom of its own host anemone. Together, these trends indicated that
the development of a protective mucus coat was the main defense against host
venom for anemone fish and that resistance may or may not be necessary for
successful mutualistic relationships. A survey by Nedosyko et al. (2014) of the
number of associations between all 26 species of mutualist anemone fishes and all
ten species of host anemones indicated that anemones with the least and most toxic
venoms were inhabited by the fewest numbers of mutualist species. Intermediate
toxicity was associated with the greatest diversity of mutualist species, and these
authors concluded that there must be a trade-off in the amount of protection versus
the amount of risk for potential mutualist species. For putative prey species of
anemones, differential toxicity across anemones may reflect a variegated landscape
of selective pressures that could lead to the development or refinement of resistance
mechanisms. However, no evidence of resistance in prey species is currently avail-
able. One mechanism of resistance that may be of interest for future investigation is
changes in the architecture of ion channels of sea anemone prey species. Gasparini
et al. (2004) compared the previously documented ability of scorpion and sea
anemone venoms to block voltage-gated potassium channels, indicating conver-
gence on the same toxic mechanism, i.e., binding a specific portion of the pore
complex to prevent the passage of current through these channels. Thus, candidate
resistance mechanisms to sea anemone venoms may arise as the result of
non-synonymous changes to exposed surfaces of ion channels that reduce the ability
of toxins to bind and block physiological currents. This kind of change has given rise
to the tetrodotoxin resistance seen in red-sided garter snakes (Thamnophis sirtalis),
allowing the predator to capitalize on otherwise deadly prey (Feldman et al. 2012;
McGlothlin et al. 2014).
Finally, resistance to scorpion venoms has been documented, but further inves-
tigations of the mechanisms or biogeography of resistance have yet to appear in the
literature. Israeli-Zindel et al. (1973) derived LD50 values for venom of the yellow
scorpion (Leiurus quinquestriatus) toward seven species of beetles and a strain of
laboratory mouse. They found a wide range of susceptibility and resistance and
demonstrated that several beetle species tested had several orders of magnitude
greater tolerance to the venom than the laboratory mouse. When the hemolymph
of the most resistant beetle was analyzed 24 h following envenomation, detectable
venom concentration had dropped to 40% of the original level. A further assay
testing the specificity of resistance revealed that an enzyme-deactivating mechanism
confers resistance to this beetle species. However, beyond this early study, few have
tested the ability of plausible prey species to defend against scorpion envenomation,
and most studies focus on species that are unlikely prey of scorpions, such as rodent
predators of scorpion (Rowe and Rowe 2008). As in other non-snake predators
mentioned above, there is evidence from tests in model organisms that immune
responses may be likely resistance mechanisms for some prey items (see Akahoshi
et al. 2011; Kamon and Shulov 1965), but it remains to be seen whether these are
mechanisms present in scorpion prey species. Collectively, the literature presents a
range of possible resistance mechanisms to venomous predators, and future research
may confirm the presence of resistance in prey species.
Explanations of a Limited Literature on Natural Resistance
In general, it appears that natural resistance to predator toxins should appear, yet
available information is limited. Reaffirming the likelihood that predation pressures,
particularly the trophic adaptation of venom, should drive coevolutionary develop-
ment of resistance, several explanations for a lack of information on resistance
emerge. First, a dearth of reported resistance may result from variable and insuffi-
cient research effort: the simplest explanation would be that little or no effort has
been made to screen candidate resistant prey. Even in the most well-described
resistance systems, resistance to venomous snakes, mammalian resistance dominates
the literature, despite abundant natural history accounts of venomous snakes con-
suming nonmammalian prey (but see Mackessy and Saviola (2016)). Second, while
some effort may have been made to investigate predator/prey interactions, the
documentation of local specificity in some of the prey resistance systems discussed
suggests that analyses may not detect resistance because of mismatches between the
localities of predator and prey that are tested. The maintenance of resistance in a
population of prey species may be dependent on the presence of a particular venom
profile that in turn is delimited by the overlapping ranges of local populations of
predator and prey. Thus, assaying for resistance using a venom from outside of
assayed individuals’ local area may lead to the false conclusion that resistance is not
present in a prey species or population. Third, beyond mismatching of predator/prey
populations, small sample sizes also may allow resistant prey to be overlooked.
Under a Red Queen dynamic, the frequency of resistance is expected to cycle
through periodic minima. Low-frequency resistance phenotypes would be increas-
ingly harder to detect by random sampling. All in all, future investigations in these
least described predator/prey systems and continuing investigations in known resis-
tance systems must consider that limitations in research design and effort may not
capture the evolutionary processes driving reciprocal flux between resistance and
toxicity.
Another explanation for limited information on prey resistance is the possibility
that these predators do not exert enough predation pressure to cause selection for
prey resistance. Simply, prey resistance may not exist, despite the logic of coevolu-
tion under a chemical arms race hypothesis, because venomous species are not
significant predators. If predators move from specialist to more generalist diets
over time, selection of novel toxicities may be favored, and therefore reciprocal
resistance may not appear. Initial development of toxicity against a limited number
of prey species may allow predators to capitalize later on a wider range of related
prey species with similar physiologies. With a wider prey base, predators would be
able to take advantage of other food sources in the event that resistance does appear
in some prey individuals. Therefore, if selective pressure from venomous predators
is negligible, and the appearance of resistance alleles in a population only happens as
a result of random mutation, the fixation of prey resistance in the population is
unlikely, because these rare resistance alleles risk early extinction due to their low
abundance. Finally, over time, overcoming the toxic action of venom by prey may
prove insurmountable, and our present-day analysis would detect venom toxicity to a
variety of locally available prey, but no or extremely small numbers of resistance

mechanisms in prey. The present discussion only considers chemical resistance to
predators’ venoms, but other strategies may evolve in response to the selective
pressure of venom toxicity. Behavioral modifications, and/or reproductive strategies
that allow further generations of prey to persist in an area, may subvert the predation
pressures of venomous animals and bypass chemically based coevolutionary pro-
cesses. For example, in one of the better described toxicity/resistance dyads
(between Pacific rattlesnakes and ground squirrels), several behaviors that prevent
predation are documented. Certain populations of squirrels are known to tail flag to
signal their awareness of a nearby predator, resulting in the retreat of the approaching
rattlesnake (Putman and Clark 2014); others bombard approaching rattlesnakes with
substrate to motivate predator retreat (Goldthwaite et al. 1990), and some rub
themselves against shed skins of local rattlesnakes to mask individual scent and
evade chemosensory detection (Clucas et al. 2008). While these populations may
also have chemical defenses against predator venoms, behavioral modifications that
disrupt predatory episodes exist as well, demonstrating that other prey species may
not require physiological resistance mechanisms if behavioral modifications are
sufficient to elude detection and/or envenomation.
The diversity and efficacy of prey resistance appears to be shaped by the selective
pressure of predator toxicity as predicted by chemical arms race hypotheses. How-
ever, the fact that only a handful of well-described resistance systems exist in the
literature demonstrates the need for further investigations into the diversity and
extent of prey resistance. Future directions in the study of natural resistance to
venoms must include screens for resistant prey species, using in vitro or in vivo
assays to identify the capacity of prey species to avoid the normally pathological
consequences of envenomation. Development of a well-supported alternative to
LD50 determinations is crucial to reduce the number of native prey animals needed
to demonstrate resistance and increase throughput, but at present there is no suffi-
cient model to replace whole animal toxicity tests, particularly for unknown systems.
Special attention should be paid to the interaction of local populations of predators
and prey versus the effects of predator venoms on nonlocal populations of (possible)
prey. Further, the prevalence of resistance mechanisms that appear specific to local
predators indicates that the development and propagation of resistance genotypes
could be modeled to predict or detect the appearance of new resistance mechanisms
or to track the spread of resistance mechanisms through prey populations across
large landscapes that connect multiple populations. The detection of local resistance
also may indicate that current information about the relative abundance of resistance
in a given prey species is underestimated; multiple pairwise comparisons between
local predator and prey populations would be required across a significant portion of
their sympatric range to document resistance or susceptibility unequivocally. Under-
standing that evolutionary processes are adequate but not necessarily ideal,
reciprocal stepwise modifications to either toxicity or resistance mechanisms are

expected to be the norm in coevolutionary systems, rather than wholesale changes to
composition. The recent use of genome/transcriptome/proteome comparisons (i.e.,
Cardoso et al. 2010; Gibbs et al. 2009) could shed light on underlying trends in
molecular evolution: how often do resistance genotypes change, how often do novel
genotypes appear, and what resistance mechanisms are likely to experience the
strongest selection?
Beyond research opportunities focusing on the evolutionary history and devel-
opment of prey resistance, a better understanding of resistance mechanisms may
provide a source for future biomedical innovation. Currently, clinical treatment, both
medical and veterinary, of envenomation by venomous species commonly relies on
the use of antivenom therapeutics and complementary treatment regimens to combat
systemic pathologies such as hypofibrinogenemia, thrombocytopenia, myotoxicity,
neurotoxicity, and many other symptoms (Chippaux and Goyffon 1998; Diaz 2004;
Rhoads 2007). The incidence of envenomation by spiders, scorpions, and snakes are
of particular concern considering their common occurrence, dramatic impacts to
global health, and significant financial impacts to health systems. In an attempt to
improve treatment, the World Health Organization (WHO 2007) deemed envenom-
ation by snakes and scorpions to be a neglected public health issue and has suggested
strategies to develop better antivenom therapeutics. While improvement of existing
antivenom therapeutics promises to increase the efficacy of envenomation treatment,
the addition of venom resistance molecules to treatment protocols may further
improve clinical outcomes. Resistance molecule therapeutics are not intended to
replace antivenom therapies, but instead work synergistically with existing treatment
protocols to combat venom toxicities. As proof of concept, two classes of anti-snake
venom compounds derived from resistant prey species have been cited as promising
candidates for drug discovery. Thwin et al. (2010) provide a summative review of a
number of these molecules, including a group of phospholipase A2 inhibitors (PLIs)
derived from venomous snake blood sera (Viperidae, Elapidae). The biological roles
of these molecules is to prevent complications from auto-envenomation or enven-
omation by other sympatric (intra- and interspecific) venomous snakes. Hypotheti-
cally, clinicians could administer the appropriate antivenom to combat broad
spectrum effects of envenomation and additionally employ a derived PLI in cases
where patients present with envenomations from snakes with PLA2-rich venoms.
Treatment schedules that incorporate such molecules could be better tailored to
individual patient needs to improve the efficacy of medical intervention and patient
health outcomes.
In addition to PLIs, another promising class of resistance molecules for drug
development are snake venom metalloprotease inhibitors (SVMPIs). As mentioned
earlier, SVMPIs have been isolated from a wide range of mammalian prey species of
snake predators. Especially in the Americas, SVMPIs promise an excellent addition
to combat the hematologic pathologies experienced in a large number of snakebite
envenomations (owing to a higher proportion of venomous taxa with snake venom
metalloprotease-rich venoms; Mackessy 2010). Metalloproteases have been
described as “gateway toxins” (Biardi et al. 2011) because they break down
structural elements within tissues, potentially increasing the rate that other toxic
components of the venom may infiltrate and access the bloodstream. Biardi et al.
(2011) postulated that the therapeutic use of an SVMPI would limit access of venom
components by destroying the ability of the venom to spread from the envenomation
site. The biochemical functions of metalloproteases (hemorrhage, tissue destruction)
would be blocked, and spread of venom would be attenuated, and the hope is that
this temporary neutralization of one part of the venom and subsequent sequestration
of other toxins would allow antivenom therapeutics time to propagate to and
neutralize the locally envenomated tissue. In short, resistance molecules such as
PLIs and SVMPIs are expected to shorten treatment regimens by increasing imme-
diate efficacy of antivenom therapeutics.
In conclusion, our understanding of the prevalence and mechanisms of prey resis-
tance to natural toxins remains limited to a small number of predator/prey systems.
However, the prediction that prey species in tightly coupled predator/prey relationships
should develop reciprocal chemical arms against predator toxins motivates a continued
effort to discover and describe resistance. Future studies should focus on assessing not
only the mechanistic nature of resistance but also the demography of resistance in
natural populations of prey. Dedication to interdisciplinary approaches that couple
molecular and ecological information will exponentially increase what we understand
of the interactions between venomous predators and their resistant prey.
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DOI 10.1007/978-94-007-6727-0_7-1
# Her Majesty the Queen in Right of Australia 2015
Toxicity in Cephalopods
Ira R. Cookea,b*, Brooke Whitelawc, Mark Normand, Nikeisha Caruanac and Jan M. Strugnellc
a
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, VIC,
Australia
b
Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Melbourne, VIC, Australia
c
Department of Ecology, Environment and Evolution, La Trobe University, Melbourne, VIC, Australia
d
Sciences, Museum Victoria, Carlton, VIC, Australia
Abstract
Cephalopods are a morphologically diverse molluscan class that includes octopuses, cuttlefishes, squids, and
nautiluses. The behavior, morphology, and sometimes aposematic appearance of coleoid cephalopods
(octopuses, cuttlefishes, and squids) are highly suggestive of the widespread use of toxins for predation
and/or defense. Many cephalopods use a combination of their parrot-like beak and/or toothed radula to inject
venomous saliva, thought to be produced in the posterior salivary gland, into prey through a bite wound or a
hole drilled into the shell. However, relatively few toxins have been studied to date from only a narrow range
of cephalopods. Active components that have been identified from cephalopod posterior salivary gland
extracts (or saliva) include neurotoxins such as tetrodotoxin (also found in body tissues), tachykinins and
cephalotoxins, biogenic amines such as serotonin and octopamine, and a diverse range of enzymes including
serine proteases, phospholipase A2, hyaluronidases, and chitinases. Coleoid cephalopods represent excellent
candidates for biodiscovery, being taxonomically distinct from heavily studied venom-producing organisms,
and because their venoms appear to be complex mixtures of proteins and small molecules. Understanding the
evolutionary history of toxicity in cephalopods remains a challenge, with many major taxa remaining
unstudied and very little specific functional information available on most cephalopod toxins. The application
of “omics” technologies to research on venoms and other toxic secretions (“venomics”) is an important and
powerful way of characterizing entire suites of proteinaceous toxins from pure venom or gland extracts in
cephalopods and is likely to yield future insights into the evolution of toxicity in this class.
Keywords
Cephalopoda; octopus; squid; venom; coleoid; tachykinin; cephalotoxin
Introduction
The molluscan class Cephalopoda contains the octopuses, squids, cuttlefishes, and nautiluses. Although the
greatest diversity of cephalopods is found in tropical shallow oceans, they occur in all marine habitats
including the deep sea and polar regions. Cephalopods do not occur in freshwater environments. There are
about 800 known species encompassing a large size range, from the giant squids (14 m) to the pygmy
squids (2 cm). Most extant cephalopods do not possess an external shell, the exception to this being the
nautiluses (subclass Nautiloidea). The remaining cephalopods (subclass Coleoidea) possess an internal
shell, which has been reduced or lost in many cases throughout the course of evolution and thus they are
*Email: I.Cooke@latrobe.edu.au
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Fig. 1 Cephalopods with (possibly) aposematic coloration and displays. Photograph A is Metasepia pfefferi and B is
Hapalochlaena sp. (Photograph A was taken by Roger Steene and B by Mark Norman)
vulnerable to predation. This lack of an external shell has likely driven the evolution of the remarkable
ability of coleoids to change texture and color to blend in with their surroundings or, in some cases, to
display warning colorations – potentially advertising their toxicity. In addition to the use of toxins in a
defensive role, the behavior and feeding morphology of many coleoids is highly suggestive of the use of
venom in subduing and/or externally digesting prey. The existence and use of toxins are so far unknown for
nautiluses, and this review will therefore focus exclusively on coleoid taxa.
Coleoids are carnivorous, with many species, especially those that are benthic or demersal, specializing
in hard-shelled prey such as bivalves, gastropods, crustaceans, and nautiluses (Pilson and Taylor 1961;
Chichery and Chichery 1988; Saunders et al. 1991). The feeding apparatus of all coleoids is a small parrot-
like beak, which would appear to be unsuitable for dealing with hard-shelled prey by crushing. Although
brute force dismemberment is possible for smaller prey, it seems likely that venoms are often employed to
at least weaken the prey, if not to kill it. This is well documented among many octopods, whereby the hard
shell of the prey is penetrated by drilling a small hole through which a venom is injected (Grisley
et al. 1996; Mather and Nixon 1995; Pilson and Taylor 1961; Runham et al. 1997; Saunders et al. 1991;
Wodinsky 1969). Close observation of prey capture by cuttlefishes (Chichery and Chichery 1988)
suggests that they also inject toxins through a small wound inflicted in a soft joint area of their prey
(crabs). Likewise, the tiny (7–12 mm) Japanese pygmy squid (Idiosepius paradoxus) is capable of
paralyzing shrimp much larger than itself (20–30 mm) within 1 min of capture (Kasugai et al. 2004)
and also uses salivary secretions for external digestion.
The brightly colored, or otherwise striking, appearance of some octopus and squid species suggests that
they may use toxins for defense (based on the aposematic warning colorations of diverse vertebrates and
invertebrates). In some cases, such as in the brightly colored flamboyant cuttlefish (Metasepia pfefferi, see
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Fig. 2 Taxonomic distribution of 6,306 verified proteins in the UniProt toxin database. The database contains just a single
cephalopod protein (SE-cephalotoxin). Note that at least one other cephalopod toxin (eledoisin) was erroneously missing from
the UniProt toxin database at the time of writing
Fig. 1a), this striking coloration is coupled with diurnal activity in environments with high predator
pressure (in contrast, many less colorful cephalopods are primarily nocturnal). The best-studied example
of a cephalopod with a striking appearance is that of the blue-ringed octopuses (genus Hapalochlaena, see
Fig. 1b), which flash iridescent blue markings when agitated (these cephalopods are also capable of
excellent camouflage in order to stalk prey) (Norman 2000). Hapalochlaena species possess tetrodotoxin
(TTX) in both the salivary glands and distributed throughout body tissues (Yotsu-Yamashita et al. 2007).
The dramatic warning colorations and presence of the powerful neurotoxin throughout the body suggest
more than a purely predatory role. Other examples include the striped pyjama squid (Sepioloidea
lineolata) whose striking black-and-white striped color pattern suggests a warning to potential predators
(Norman 2000). In addition, many coleoids that inhabit relatively shallow water environments produce
ink that is released when attempting to evade a predator. Studies on the gastropod slugs known as sea
hares (Aplysiomorpha), which also produce a form of ink, have discovered a complex cocktail of
compounds in the ink that affect the behavior of predators such as lobsters (Derby 2007). Far less is
known about the chemical composition and ecological role of cephalopod inks. Although some form of
chemical defense in inks seems likely, this is yet to be demonstrated (Derby 2014; Wood et al. 2008).
These life history traits (hole drilling, aposematic coloration, lack of a hard protective shell, ink and
slime production) suggest that cephalopods may possess a rich arsenal of toxic compounds in their venom
and tissues. Nevertheless, the number of cephalopods for which any form of toxin research has been
conducted remains low (fewer than 30 species), and the majority of these species appear in just four
studies (Fry et al. 2009; Ruder et al. 2013; Ueda et al. 2008; Undheim et al. 2010). These four recent
studies provide a glimpse into the diversity and potency of proteinaceous cephalopod toxins. Ueda
et al. (2008) observed potent toxicity against crabs for posterior salivary gland (PSG) extracts of all
three species of cuttlefish (Sepia esculenta, Sepia lycidas, Sepia japonica) studied and toxicity against
both crabs and mice for PSG extracts of all three species of loliginid squids (Loliginidae) studied. This
suggests that the use of potent venoms is widespread in cuttlefish and squids and that examination of a
range of cephalopod taxa is likely to reveal different compounds (or variants) to account for varying target
specificity. This is underscored by the studies by Ruder et al. (2013) and Fry et al. (2009) who provide a
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list of nine putative toxin protein families coded for in the transcriptomes of cephalopod posterior salivary
glands.
Despite the high likelihood that cephalopods exhibit a diverse proteinaceous toxin arsenal, the number of
well-characterized cephalopod toxins is extremely low. This is reflected in Fig. 2 which shows the number
of proteins in the UniProt toxin database (verified toxins or components of venom) according to the
taxonomic group from which the proteins were isolated. In this database, cephalopods, a taxonomic
group of some 800 species, are represented by just one protein (SE-cephalotoxin), whereas a single well-
studied genus of gastropods (Conus) accounts for 1,022 entries.
Although recent venomics work provides strong evidence that cephalopod posterior salivary gland
secretions are complex mixtures of many molecules (Cornet et al. 2014; Ruder et al. 2013), the effects of
these compounds on potential prey (or predators) have not been established in most cases. In this review,
we first focus on the most studied classes of molecules, including small molecules such as tetrodotoxin
(TTX) and biogenic amines, as well as proteins such as cephalotoxin, chitinases, and peptidases. Later
sections of the review summarize current knowledge on the evolution of toxicity in cephalopods and
technological developments including the potential insights to be gained from venomics studies.
Since cephalopods produce a range of potentially toxic substances (salivary venom, ink, body tissues),
it is important to clarify our use of the terms venom, venomics, toxin, and toxic. In this review we use the
term venom to refer to substances injected via a bite or drill hole (i.e., salivary secretions) but accept that
the term “venomics” can sometimes be applied to studies of other toxic secretions. We use the term toxin
to refer to specific molecules that confer toxicity and emphasize that toxins may be contained within
various tissues in the cephalopod body or secreted as part of venoms or inks. All substances that contain
toxins are referred to as toxic.
Significant Toxin Classes in Cephalopods

Tetrodotoxin
Tetrodotoxin (TTX) is an extremely potent low molecular weight neurotoxin. In cephalopods, TTX is
known to be present in members of the genus Hapalochlaena (blue-ringed octopuses). Many studies have
focused their attention on the genus Hapalochlaena due to the immediate threat it poses to humans. The
first documented fatal envenomation from a blue-ringed octopus occurred in 1954 in Darwin, Northern
Territory, Australia (Jacups and Currie 2008). Studies into the posterior salivary gland (PSG) of
H. maculosa identified the fatal component as maculotoxin (Crone et al. 1976), which was later revised
to TTX (Sheumack et al. 1978). A similar toxin to TTX was isolated from H. maculosa called
hapalotoxin, distinguishable from TTX due to its higher polarity (Savage and Howden 1977).
Aside from cephalopods, TTX also occurs in a diverse range of other organisms including fishes,
amphibians, arthropods, nemerteans, flatworms, arrow worms, echinoderms, and other molluscs. The
origins of tetrodotoxin are not fully understood, but its sporadic appearance across unrelated taxa suggests
that it is either ingested or produced by bacterial symbionts (Chau et al. 2011). Studies on puffer fish have
shown that captive animals lose toxicity over time if fed a non-TTX containing diet (Noguchi et al. 2006),
which suggests accumulation via ingestion. However, the newt, Taricha granulosa, is able to maintain
and even increase TTX levels on a non-TTX containing diet (Hanifin et al. 2002), which suggests that it is
produced by symbionts or even endogenously by the newts themselves. Studies on puffer fish
(Yu et al. 2004; Wu et al. 2005) have identified TTX-producing bacteria from the tissues of
TTX-bearing host species. TTX in blue-ringed octopuses is thought to be bacterial in origin, but it is
not known whether these bacteria are housed within a particular organ, spread throughout the body, or
ingested. Hwang et al. (1989) cultured 22 strains from the tissues of H. maculosus, [sic] of which 16 were
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shown to produce TTX. TTX-producing strains were found in cultures made from the posterior salivary
gland, tentacle, intestine, and ink sac and were identified as species of Vibrio, Alteromonas, Pseudomonas,
and Bacillus.
Blue-ringed octopus are one group of only a small number of animals believed to use toxins for defense
as well as to subdue prey. Evidence for a predatory role comes from the presence of TTX in the posterior
salivary glands, which are the usual venom-producing organs in octopods, but its efficacy against typical
prey has not been quantified (Williams 2010). Evidence for a defensive role comes from the characteristic
iridescent blue warning rings that the animal displays when distressed, as well as from the relative
distribution of TTX across tissues, eggs, and larvae. This intraorganismal distribution of TTX varies
depending on species and geographic location (Hwang et al. 1989; Williams and Caldwell 2009). For
example, Hwang et al. (1989) examined the relative concentrations of TTX in the posterior salivary glands
versus other soft tissues, for two specimens collected at different locations. In one specimen (Cebu Island,
Philippines), they found that the majority of total TTX was located in the soft tissues, with only around a
quarter of total TTX being located in the PSG. In contrast, a specimen from Izu Peninsula (Japan) had
concentrated almost all TTX in its posterior salivary gland. This suggests that the relative role (defense or
predation) of TTX in blue-ringed octopus may vary between individuals or subpopulations. Further
evidence for a defensive role for TTX comes from Yotsu-Yamashita et al. (2007), who examined six
specimens from South Australia and found much higher levels of TTX in bulk body tissues (arms,
cephalothorax, abdomen) than in the posterior salivary gland. Williams et al. (2011) found that while
H. maculosa larvae contained TTX, it was not at a sufficient concentration to intoxicate or deter predators
in controlled feeding experiments. Interestingly, they found that H. maculosa larvae were indeed
distasteful to predators but speculate that this is due to some compound other than TTX, since
TTX-spiked control items were not distasteful to representative fish or stomatopod predators.
Utilization of a toxin necessarily requires an organism to also contain these highly detrimental proteins
or compounds. As a result, the organism requires a degree of resistance to these toxic elements
(Flachsenberger and Kerr 1985). The tolerance of one Hapalochlaena species (most likely H. maculosa
based on the collection site) was examined by Flachsenberger and Kerr (1985), who found no negative
effects when the specimen was injected with high doses of purified TTX or salivary extract. This suggests
that any predatory or defensive function of Hapalochlaena venom is not directed at others of the same
genus. In contrast, the male platypus utilizes its venom almost exclusively for intraspecies competition
(Wong et al. 2012).
Cephalotoxin
The term cephalotoxin was first used by Ghiretti (1959) to describe a protein purified from the posterior
salivary gland of Sepia officinalis. The defining characteristic of this protein was its ability to induce
complete paralysis in crabs, a phenomenon that had previously been observed for whole octopus saliva as
early as 1897 (Krause 1897). Later work by Ghiretti (1960), McDonald and Cottrell (1972), Songdahl and
Shapiro (1974), and Cariello and Zanetti (1977) isolated protein fractions with similar neurotoxic effects
from the common octopus (Octopus vulgaris), the curled octopus (Eledone cirrhosa), and the giant
Pacific octopus (Octopus dofleini, now Enteroctopus dofleini). Although all of the proteins in these early
works have been referred to as cephalotoxin, no sequencing or other detailed molecular characterization
was performed, and it is not clear that they were homologues. Indeed, Songdahl and Shapiro (1974) noted
that the molecular weight of their protein (23 kDa) was very different from the protein studied by
McDonald and Cottrell (30–70 kDa) and suggested that cephalotoxins may be a diverse group.
Much more recently, Ueda et al. (2008) performed the first direct protein sequencing on a compound
with the neurotoxic effects characteristic of cephalotoxin. This protein, purified from the posterior
salivary glands of the golden cuttlefish (Sepia esculenta), was named SE-cephalotoxin and is one of
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only a small number of cephalopod toxins that have been purified and sequenced. At 1,052 amino acids, it
is exceptionally large for a venom protein. Currently, the Tox-Prot database of over 6,000 proteins
contains just 14 longer sequences and has a median length of 79 amino acids (http://www.uniprot.org/
program/Toxins). Other features of the protein sequence are the presence of a signal peptide, pro-peptide,
and multiple cysteine-rich regions, all of which are commonly observed in other venom proteins (Ueda
et al. 2008). It is likely that SE-cephalotoxin is indeed a homologue of at least some of the proteins
previously called cephalotoxin. It is highly glycosylated, which is consistent with observations made by
Cariello and Zanetti (1977) on cephalotoxins isolated from the O. vulgaris.
The evolutionary origin of cephalotoxin remains unclear as very few homologues have been
sequenced. SE-cephalotoxin homologous sequences have been found in Sepia officinalis (Cornet
et al. 2014), Hawaiian bobtail squid (Euprymna scolopes) (Collins et al. 2012), the coral Acropora
millepora (Ramos-Silva et al. 2013), and most recently the Australian ghost shark (Callorhinchus milii)
(Venkatesh et al. 2014). In addition, Ruder et al. (2013) sequenced cephalotoxin homologues in the
broadclub cuttlefish (Sepia latimanus), the pharaoh cuttlefish (Sepia pharaonis), and the southern reef
squid (Sepioteuthis australis) but did not publish the assembled sequences or present a phylogenetic
analysis for cephalotoxin. The existence of homologues in the coral and ghost shark genomes indicates an
ancient (pre-molluscan) origin for the protein, and functional work (Collins et al. 2012) indicates that
within Euprymna scolopes, the protein plays an important role in the organism’s immune system that is
possibly unrelated to its use as a venom.
Tachykinins
Tachykinins comprise a large family of highly conserved neurotransmitters found in vertebrates and
invertebrates including cephalopods. They participate in both the peripheral and central nervous systems
via afferent and efferent pathways, being involved in smooth muscle contraction, peripheral sensing, and
neurogenic inflammation (Khawaja and Rogers 1996). Tachykinin receptor proteins (TKRP) mediate the
effects of tachykinins via specific binding between each tachykinin and its associated receptor.
The first peptide toxin to be isolated and sequenced from cephalopod venom was the tachykinin
eledoisin (Erspamer and Anastasi 1962), from the musky and curled octopuses (Eledone moschata and
Eledone cirrhosa, respectively). This followed the discovery of substance P, a mammalian tachykinin in
1931 (von Euler and Gaddum 1931), and it was soon realized that these molecules were structurally and
pharmacologically similar, being part of a large family widespread across many taxa (Khawaja and
Rogers 1996). Eledoisin is present in the salivary glands of octopods from the genus Eledone (Anastasi
and Erspamer 1963; Erspamer and Anastasi 1962). It induces hypotension and contraction of gut muscles
in vertebrate (dog and guinea pig) assays (Anastasi and Erspamer 1962) but its effects on invertebrates
have not been assayed.
More recent studies on O. vulgaris have revealed considerable additional detail about the role of
tachykinins in octopods. Kanda et al. (2003) isolated two tachykinins from the posterior salivary glands of
O. vulgaris (Oct-Tk-I, Oct-TK-II), and subsequently, Kanda et al. (2007) detected a total of seven
tachykinin-related peptides in O. vulgaris brain tissue. The first octopod tachykinin receptor
(Oct-TKRPR) was identified by Kanda et al. (2007) from heart tissue of O. vulgaris, and it was found
that this receptor is not responsive to tachykinins expressed in salivary glands (Oct-Tk-I), but that it is
responsive to tachykinin-related peptides expressed in brain tissue (Oct-TKRP’s I–IV). This suggests that
production of tachykinins in saliva is likely to be specifically related to venom production rather than
participating in the octopus’ endogenous neurologic pathways.
Tachykinins and tachykinin-related peptides are usually small (approx 10–150 amino acids) and are
often produced by cleavage of a larger precursor protein. It has been noted that vertebrate tachykinins
often possess a motif of the form FXGLM, whereas in invertebrates it is FXGXR. A study by Ikeda
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et al. (1999) showed that the C-terminal residue within this motif (M for vertebrates, R for invertebrates)
was crucial to the potency of neuromuscular effects for a tachykinin from an echiuroid worm and one of
mammalian origin. In particular, they assayed invertebrate and vertebrate tachykinins against invertebrate
and vertebrate assays and found that substitution of the C-terminal residue (R to M for invert tachykinin
and M to R for vertebrate) could induce a switch in assay specificity from invertebrate to vertebrate and
vice versa. Interestingly, tachykinins from the O. vulgaris posterior salivary gland and Eledoisin (found in
posterior salivary glands of Eledone) all possess the vertebrate motif (FXGLM) despite the fact that their
dominant prey items are invertebrates. Ruder et al. (2013) assayed the relative activity of three octopus
tachykinins (two from O. vulgaris and one from Octopus kaurna), all of which had an FX[SG]LM motif
using vertebrate and invertebrate assays. They found that all peptides elicited a response from both assays
and that their relative potency was the same on invertebrate and vertebrate tissues. These results and others
(e.g., Poels et al 2009) point to a deficiency in our current understanding of the differences between
vertebrate and invertebrate tachykinins and their receptors, and that vertebrate vs invertebrate tachykinin
specificity cannot simply be induced by analysis of the tachykinin motif amino acid sequence alone.
Chitinases, Peptidases, and Other Degradative Enzymes

Many venomous animals include degradative enzymes as components of venom and which may be
classed as toxins due to their ability to cause cell or tissue damage or even neurotoxic effects (Kini 2003).
These often target specific structurally important molecules whose degradation may have anticoagulant
effects, leading to increased tissue permeability, cell lysis, or hemorrhage (Gutierrez and Rucavado 2000;
Kini 2003; Kang et al. 2011; Wong and Belov 2012). This in turn can enhance the spread of other venom
components and/or hasten the immobilization or death of the victim. It is likely that cephalopod venoms
include molecules to perform these functions, but little is known about them. Transcript sequences from
several major venom enzyme classes have been identified in cephalopod posterior salivary gland extracts
(Fry et al. 2009; Ruder et al. 2013) including hyaluronidase (H. maculosa, O. cyanea), serine peptidases
(probably ubiquitous), and phospholipase A2 (Loliolus, Sepia, Sepiotheuthis). In addition, whole PSG
extracts of four Antarctic octopus species (Adelieledone polymorpha, Megaleledone setebos, Pareledone
aequipapillae, and Pareledone turqueti) have been assayed for alkaline phosphatase, acetylcholinester-
ase, phospholipase A2, hemolytic, and proteolytic activities (Undheim et al. 2010) with three species
showing some activity in all assays. That these degradative enzymes are important components of
cephalopod venoms is suggested by the fact that they have undergone diversification (in the case of
serine proteases) within the cephalopod lineage (Fry et al. 2009) and have acquired adaptations to cold
temperatures within Antarctic species (Undheim et al. 2010).
Chitinases and peptidases appear to be a ubiquitous component of cephalopod posterior salivary gland
extracts, being found in all species for which transcriptomic sequencing has been performed (Fry
et al. 2009; Ruder et al. 2013; Cornet et al. 2014), as well as being identified in numerous other species
via bioassays (Grisley and Boyle 1987; Undheim et al. 2010; Grisley 1993) or by purification and direct
sequencing (Ogino et al. 2014). As components of venom, they are likely to cause considerable damage to
prey, breaking down muscle or connective tissue and/or assisting with cephalopod hole boring into the
exoskeletons of crustaceans. A key role for chitinases, peptidases, and other digestive enzymes in
cephalopod venom is likely to be external digestion. The cephalopod body plan includes a very small
mouth and a narrow esophagus that passes through the doughnut-shaped brain, thereby placing strong
constraints on the size of food particles that can be ingested. External digestion is particularly well
documented for octopus, where the injection of salivary secretions greatly increases the ease with which
crustacean prey can be dismembered (Grisley and Boyle 1987; Nixon 1984). By devising a specific
bioassay for detachment of crab muscle from carapace, Grisley and Boyle (1987) were able to attribute
this activity to a proteolytic enzyme contained in milked salivary extracts from Eledone cirrhosa.
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Amines
Amines are low molecular weight organic compounds that are ubiquitously found in biological systems.
Many amines are neurotransmitters, including epinephrine, norepinephrine, dopamine, serotonin, and
histamine. They are frequently encountered as components of invertebrate venoms, and, while they are
often responsible for producing an acute pain response (Welsh 1964), they are not often the cause of more
serious effects such as paralysis or death (Welsh 1964).
Early work on cephalopod venoms identified a substance with the ability to induce smooth muscle
contractions in mammals from posterior salivary gland extracts of O. vulgaris. This substance was then
named enteramine, but in 1952 (Erspamer 1952) it was found to be identical to serotonin, a substance that
had been studied independently by another group of researchers working on mammalian blood
(Whitaker-Azmitia 1999). In addition to identifying enteramine/serotonin, Erspamer (1952) also discov-
ered octopamine from the posterior salivary gland extracts of O. vulgaris. The effects of octopamine in
invertebrates and in vertebrates have been extensively studied (David and Coulon 1985). It acts as a
neurotransmitter, neurohormone, and neuromodulator with effects on both the central and peripheral
nervous systems (David and Coulon 1985). It has been shown that both octopamine and serotonin elicit a
neurophysiological response from crustaceans (Livingstone et al. 1980), but because these molecules are
so ubiquitous in biological systems, it is unclear whether their presence in posterior salivary glands
indicates an active role in octopus venom itself.
Taxonomic Coverage and the Evolution of Toxicity in Cephalopods

The evolutionary history of toxicity in cephalopods remains relatively unknown, and in order to better
understand it, two major challenges need to be overcome. The first is poor taxonomic coverage. Several
taxonomic groups have received very little (e.g., cirrate octopuses [Cirrata], vampire squid
[Vampyroteuthidae]) or no (e.g., argonauts, blanket octopus [Argonautoidea], bottletail squids
[Sepiadariidae]) investigation. Also, relatively few species of some speciose groups have been investi-
gated. For example, around six species from the family Sepiidae (cuttlefishes) have been investigated to
date, yet the family is known to contain around 100 species. Similarly there are over 300 species known
from the super family Octopodoidea, Strugnell et al. (2013), but only 7 species have been investigated at
the present time. Only a single species (Todarodes pacificus) from the order Oegopsida has been studied,
yet at least 250 species are known to belong to the order. Therefore, we cannot be certain that the toxins
identified to be present within those species studied are “representative” of the broader taxonomic group.
The second challenge is that almost no cephalopod-specific functional information exists regarding the
molecular components of venoms. This means that although we may be able to identify protein variants
that are specific to cephalopods, or perhaps to a particular cephalopod clade, we are limited in our ability
to draw links with feeding behaviors or life history traits that would explain these evolutionary events. In
the absence of cephalopod-specific functional information (e.g., studies on the specific effects of pure
venom fractions), the best that can be achieved is to infer function based on sequence similarity with
venom components from well-studied species such as cone snails, snakes, and arachnids. This is
problematic because some families of venom proteins (e.g., PLA2, Kini 2003) include many variants
that have evolved diverse and highly specific functions that cannot readily be determined from the amino
acid sequence alone. Furthermore, since all venomics studies on cephalopods to date have used posterior
salivary gland extracts as a proxy for venom, it is sometimes difficult to rule out the possibility that a
putative venom component is in fact merely part of the salivary gland apparatus and not present in the
actual saliva produced.
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Several recent studies have made progress toward addressing these challenges and have provided the
first significant insights into toxin evolution in cephalopods. A study by Ueda et al. (2008) assayed
posterior salivary gland extracts from three cuttlefish species (Sepia), three loliginid squid species (Loligo,
Sepioteuthis), and one oegopsid squid species (Todarodes pacificus) for lethal activity against mice and
crabs (Potamon dehaani). They found that extracts from all cuttlefish species were most potent against
crabs but had no activity against mice, whereas all the loliginid squids exhibited some potency against
both mice and crabs, and the oegopsid squid had no lethal activity in either assay. This finding suggests
that Sepia and loliginid squids possess specialized toxins to suit their preferred prey, with loliginids
consuming a greater proportion of vertebrates (fish) than Sepia whose main diet is crustaceans (Hanlon
and Messenger 1996). The complete lack of lethal activity from PSG extracts of Todarodes pacificus is
also interesting because this species is from a different taxonomic group (order Oegopsida), contained
within the Decapodiformes, from the six toxic species studied (which belong to the order Myopsida and
family Sepiidae). Another recent study that used assays on whole PSG extracts to demonstrate toxin
evolution was that of Undheim et al. (2010). They provide evidence that some Antarctic octopods have
evolved cold-adapted enzymes as part of their venom. In particular, they showed that alkaline phosphatase
activity was cold adapted (higher activity at 0 C than at 37 C) in all species and proteolytic activity was
also cold adapted in three of four species tested.
Recently, two studies (Fry et al. 2009; Ruder et al. 2013) have specifically attempted to gain insights
into toxin evolution in cephalopods by applying high-throughput transcriptome sequencing to posterior
salivary gland extracts across ten cephalopod species from major octopod and decapod groups. These
offer the first molecular phylogenetic insights into toxin evolution in these taxa, in addition to providing a
broad overview of the protein composition of cephalopod venoms. Of particular interest is the finding by
Fry et al. (2009) (later expanded upon by Ruder et al. (2013)) that a large number of S1 peptidases exist in
the venoms of all ten species studied. At least for H. maculosa, O. kaurna, and S. latimanus, these
molecules are collectively distinct from non-cephalopod taxa while being present throughout the ceph-
alopod clade (Fry et al. 2009). Fry et al. (2009) argue that at least four successive gene duplication events
have occurred prior to the divergence of the decapodiform and octopodiform lineages (revised to six
events by Ruder et al. 2013) and that this suggests an ancient origin of the posterior salivary gland organ in
cephalopods and, by extension, its use in venom production. In addition, Ruder et al. (2013) applied site-
specific selection analyses to serine proteases and pacifastins. They found that although most sites were
under negative selection, 26 serine protease sites and three pacifastin sites were under positive selection,
with the majority of positively selected serine protease sites likely to be at the surface of the folded protein.
Since changes at the protein surface are most likely to affect receptor binding and/or enzymatic function,
positive selection at these sites agrees with a model for toxin evolution in which genes are duplicated and
then one of the copies takes on a role as a toxin (Wong and Belov 2012). Given that venom proteins are
injected into a foreign victim, they are likely be subject to some positive selection in order to optimize
efficiency against novel receptors or substrates.
New Technologies and Shifting Emphasis

Over the past 50 years, technologies for separating, assaying, and characterizing venom extracts and toxin
molecules have become more sensitive and are providing increasing quantities of data. Constrained by
older technologies, early studies tended to focus on a small number of abundant species (available
commercially), used large volumes of starting material, and attempted to isolate a single molecule for
detailed study. For example, Erspamer and Anastasi (1962) used salivary glands from 10,000 Eledone
individuals in order to characterize the tachykinin peptide, eledoisin, and 30,000 O. vulgaris individuals
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were used by Erspamer and Asero (1953) to isolate enteramine. Such large quantities of material are
astonishing by the standards of most modern studies where less than ten individual animals is the norm
(e.g. Ruder et al. 2013; Ueda et al. 2008; Undheim et al. 2010). This trend toward the use of fewer animals
is undoubtedly a positive one as it enables study of less abundant species, is significantly cheaper, and is
more ethically sound.
Despite using reduced sample volumes, modern studies are able to generate vastly more data than ever
due to the adoption of modern “omics” technologies. These allow whole genomes and transcriptomes to
be sequenced via next-generation nucleic acid sequencing technology and for whole proteome surveys to
be conducted using mass spectrometry. Collectively termed “venomics” (Escoubas and King 2009), these
techniques allow data to be obtained on whole suites of molecules (e.g., whole animal or whole tissue), at
low cost, from a small (milligram to gram) quantity of sample. Techniques related to venomics (indicated
by arrows l, m, n, and o in Fig. 3) have so far seen very little application to cephalopod venom research.
Notable exceptions are the studies by Fry et al. (2009) and Ruder et al. (2013), which performed
transcriptome sequencing on posterior salivary gland (PSG) extracts of several species, and the study
of Cornet et al. (2014), which performed both transcriptomic and proteomic analyses on PSG of
S. officinalis. These studies identified numerous transcripts with homology to venom proteins observed
in other taxa (snakes, arachnids, and cone snails) including CAP (CRiSP/Allergen/PR-1) proteins,
carboxypeptidases, chitinases, hyaluronidases, pacifastins, phospholipase A2 proteins,
SE-cephalotoxin, and serine proteases (Table 1).
Notable missing lines of inquiry from Fig. 3 include those corresponding to direct protein analysis, for
example, by mass spectrometry-based proteomics, and particularly analysis of venom or pure toxic
fractions. These are important because venom proteins are often posttranslationally modified (Buczek
et al. 2005) and because transcripts may be expressed in venom gland tissue without necessarily being
Fig. 3 Summary of research on proteinaceous cephalopod toxins to date. Toxin purification is shown in along the center line,
with methods for measuring toxin effects shown on the right, and for molecular characterization on the left. Notable missing
lines of inquiry are shown in thick red (no studies) or as dashed lines (very few studies). Full details describing each labeled
arrow are given in Table 1 with example studies
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Table 1 Methodologies and lines of inquiry pursued in cephalopod toxin research. Labels correspond to arrows in Fig. 3
Label Description Examples
a Behavioral and morphological studies demonstrating venom Fiorito and Gherardi 1999; Kasugai et al. 2004
use by cephalopods
b, c Injection of salivary extract or milked venom into Ghiretti 1960; Songdahl and Shapiro 1974
representative prey species (as an assay)
d, e Model assays on milked venom or posterior salivary gland Erspamer 1948; Grisley and Boyle 1987; Key
extract et al. 2002
f, g Investigation of potential medicinal effects of whole venom or Karthigayan et al. 2007
a pure toxin isolated from venom
h Extraction of pure saliva via milking or dissection Ballering et al. 1972; Ghiretti 1960
j Dissection of posterior salivary glands and homogenization Erspamer 1948; Cariello and Zanetti 1977
and/or extraction with solvent
k, i Isolation of pure active fraction via bioassay-guided Anastasi and Erspamer 1962; Cariello and Zanetti
fractionation 1977; Ghiretti 1959
l, m mRNA sequencing (transcriptomics) or proteomics (via mass No cephalopod studies. See Corrêa-Netto
spectrometry) on pure cephalopod saliva et al. 2011 for an example in other taxa
n Transcriptomic sequencing on posterior salivary gland extracts Fry et al. 2009; Ruder et al. 2013
o Proteomics on posterior salivary gland extracts Cornet et al. 2014
p Direct sequencing of pure toxic peptides or proteins Anastasi and Erspamer 1963; Ueda et al. 2008
q Secondary or tertiary structure determination Grace et al. 2003
translated to protein and/or secreted into the mature venom. Posttranslational modifications can include
modifications to the amino acid sequence (e.g., cleavage of pro-peptides, signal peptides, etc.) and site-
specific modifications (Buczek et al. 2005; Kapono et al. 2013).
An additional issue identified by Fig. 3 is that no venomics studies have been performed on pure
cephalopod saliva (as opposed to posterior salivary gland extracts). Obtaining pure saliva is clearly a
technical challenge, but one that a number of studies in the past have overcome through milking (Grisley
and Boyle 1987) or careful dissection of the venom ducts (Ghiretti 1960). Studies on pure saliva are
important because they allow unambiguous identification of venom products without the presence of
proteins related to other functions of the posterior salivary gland. Nevertheless, future studies to tackle this
issue will need to employ a combined transcriptomic/proteomic strategy since protein production (and
hence related mRNA) occurs in the gland and not in the saliva/venom itself. Thus, proteomics of saliva/
venom combined with a database of transcript sequences obtained from a variety of body tissues including
posterior salivary gland is required.
While venomics studies can identify the protein sequences of many potential toxins simultaneously,
their ability to make inferences about the biological significance of these molecules is heavily reliant on
homology with sequences where detailed functional studies, or bioassays, have previously been
conducted. One of the few recent studies to pursue such detailed work is that of Ueda et al. (2008),
who used between three and 18 specimens per species to study posterior salivary gland toxins across three
cuttlefish and four squid species. This study observed toxic activity in six of seven species and was able to
fully isolate and sequence one toxic protein (SE-cephalotoxin) from Sepia esculenta. Unfortunately, toxic
proteins from squids in this study were not amenable to separation on the basis of salt concentration;
however, it is clear that those proteins were potent against both mice and crabs (whereas cuttlefish venom
was effective only against crabs) and would be interesting targets for future purification and sequencing
efforts.
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Research into the nature of toxicity in cephalopods dates back over 100 years with detailed biochemical
studies dating from the 1950s. Despite this long history, our understanding of cephalopod venoms, and the
toxins they contain, remains extremely rudimentary. In particular, very few examples exist where a
particular molecule has been isolated as a pure (or near pure) fraction, assayed to determine its physio-
logical effects, and then characterized to determine its structure or amino acid sequence. Notable
exceptions are the tachykinin eledoisin, SE-cephalotoxin, and TTX. Toxins of octopuses, squids, and
cuttlefishes show a very high potential for biodiscovery. The application of “venomics” technologies to
toxin research is emerging as an important and powerful way of characterizing entire suites of protein-
aceous toxins from pure venom or gland extracts in cephalopods.
Cross-References
▶ A Critique of the Toxicoferan Hypothesis
▶ Mutation, Duplication, and More in the Evolution of Venomous Animals and Their Toxins
▶ Systematics of Cephalopods
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DOI 10.1007/978-94-007-6727-0_8-1
Systematics of Cephalopods
A. L. Allcock*
School of Natural Sciences, National University of Ireland Galway, Galway, Ireland
Abstract
Cephalopoda is an extremely diverse class of mollusks that has been evolving since the Cambrian. The
extant lineages arose in the late Silurian and diverged into Nautiloidea and Coleoidea in the
mid-Palaeozoic. Nautiloidea is represented by a handful of Recent species only. In contrast, Coleoidea
has diverged into two superorders, Decapodiformes and Octopodiformes, which together comprise
around 800 Recent species. The relationships among orders of Decapodiformes are not well understood,
and molecular systematics has failed to provide much resolution, although there is some evidence for a
sister-taxon relationship between Spirulida, the ram’s horn squid, and Sepiida, the cuttlefishes. A sister-
taxon relationship between Bathyteuthida and Oegopsida is well established. The relationships among
Octopodiformes are better understood. The vampire squid is placed in a separate order, and all other
octopods are placed in Octopoda. Within Octopoda there are well-understood clades: Octopoda is divided
into Cirrata and Incirrata; Incirrata is further divided into Argonautoidea and Octopodoidea. Several
lineages of cephalopods have been evolving independently for a long time: for example, Spirulida,
represented by a single extant species, appears to have diverged from other groups 150 million years
ago, nautiloids appear little changed since 300 million years ago, and Vampyromorphida, also represented
by a single extant species, appears little changed since 200 million years ago. In contrast, several groups,
for example, the sepiids, appear to have undergone recent radiations.
Keywords
Evolution; Paleontology; Molecular phylogenetics; Coleoidea; Nautiloidea; Octopodiformes;
Decapodiformes; Squid; Cuttlefish; Octopus
Introduction
The molluscan class Cephalopoda arose in the Cambrian (Young et al. 1998; Kröger et al. 2011).
Arguably the most diverse of all molluscan lineages, cephalopods have adapted to occupy multiple
niches in the marine environment, from estuarine bays to shallow neritic shelf seas, to the open ocean, and
to benthic abyssal plains.
Cephalopods emerge in the fossil record in Cambrian strata, although there is some dispute over which
fossils represent stem cephalopods, a not uncommon problem in paleontology where fossils are rare and
where preservation of soft parts is usually far from complete. Reviewing available literature, Kröger
et al. (2011) suggested that stem cephalopods were present in the early Cambrian (e.g., Tannuella, a
mollusk with a chambered shell) and that the earliest undisputed cephalopod fossil is Plectronoceras
cambria, whose chambered shell probably facilitated buoyancy control as seen in modern nautiloids.
*Email: louise.allcock@nuigalway.ie
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Kröger et al. (2011) place the origin of the lineage that contains all modern (as well as some extinct)
cephalopods in the late Silurian. They suggest that this lineage arose from the orthocerids, cephalopods
with straight but chambered external shells, and that, in the mid-Palaeozoic, it diverged into the two
recognized subclasses that have Recent representatives, Nautiloidea and Coleoidea. The nautiloids found
in the Indo West Pacific today are markedly similar to their Palaeozoic ancestors and are widely
recognized as living fossils. They retain the external chambered shell and have simple “pinhole camera”
eyes (without a lens). In contrast, the coleoids have diverged substantially. Molecular divergence
estimates (Kröger et al. 2011) place the initial divergence of coleoids into two extant (Decapodiformes
and Octopodiformes) and three extinct (Phragmoteuthida, Belemnitida, and Diplobelida) lineages in the
Permian, although undisputed fossils of the extant coleoid lineages are only found in much more recent
strata. Further divergence of Decapodiformes and Octopodiformes then appears to have occurred
throughout the Jurassic and Cretaceous to provide the variety of forms present in the ocean today (Fig. 1).
Decapodiformes comprises about 500 Recent species in between five and seven orders (see Table 1),
depending on taxonomic opinion, whereas Octopodiformes comprises approximately 300 species in two
orders, with all but one species within the order Octopoda. Within the general bauplan of mantle, head,
and arms, the diversity of forms is remarkable: animals may be streamlined or robust, dorsoventrally
flattened or not, and may be adapted to benthic, demersal, or pelagic lifestyles. All coleoids, in contrast to
nautiloids, have an internalized shell, but this takes many forms. Cuttlefishes (order Sepiida, superorder
Decapodiformes) have a dorsoventrally flattened phragmocone, the “cuttlebone.” Ram’s horn squids
(order Spirulida, superorder Decapodiformes) have a small internal calcareous-chambered open
planispiral shell which is used in buoyancy. Most other Decapodiformes have a pen-like gladius, but
this may be reduced or absent in some groups. Among Octopodiformes, the vampire squid (which is
actually more closely related to octopuses and is the sole representative of the order Vampyromorphida)
has a thin gladius, cirrate octopuses have reasonably robust internalized cartilaginous shells, and incirrate
octopuses tend to have small vestigial internal shell remnants, the “stylets”. There is some relationship
between shell type and habitat/lifestyle: for example, the buoyancy controls provided by the
phragmocones of cuttlefishes and ram’s horn squids allow them to exploit the water column, the
cartilaginous shells of cirrate octopuses support the fins they use for swimming, and the gladius reflects
the streamlined form of squids that facilitates their speed in their pelagic environment. In many cases, the
diversity among groups is so great that establishing which groups are most closely related has been a
difficult task for systematists and evolutionary biologists.
Nautiloidea
Just a handful of extant nautiloid species exist today, although there is some dispute over the actual
number. They are considered to be living fossils, shell morphology having changed little since the late
Carboniferous (Wani and Mapes 2010), and they exhibit many of the cephalopod characteristics thought
to be plesiomorphic, including an external chambered shell and pinhole eyes as detailed above.
Biogeographically, modern nautiloids are restricted to the Indo West Pacific, but nautiloids were more
widespread in their earlier history, being both abundant and distributed worldwide from the Jurassic to the
Miocene (Teichert and Matsumoto 1987).
Today, they are found on reefs at approximately 100–700 m depth, where they are both scavengers and
predators. They are slow growing, and are estimated to reach maturity after about 15 years, and have a life
span in excess of 20 years (Dunstan et al. 2011). In captivity, at least, they are slow to reproduce, laying
very few eggs throughout the year (Arnold et al. 1990), and this k-selected life history strategy makes
them particularly susceptible to overfishing (in support of the ornamental shell trade) to which they are
subjected through much of their range.
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Fig. 1 Representatives of major extant lineages of cephalopods. (a) Nautiloidea, Nautilus macromphalus; (b) Sepiida, Sepia
officinalis; (c) Spirulida, Spirula spirula; (d) Sepiolida, Sepiola affinis; (e) Idiosepiida; (f) Myopsida, Loligo vulgaris;
(g) Oegopsida, Ommastrephes bartramii; (h) Bathyteuthida, Bathyteuthis abyssicola; (i) Vampyromorphida, Vampyromorpha
infernalis; (j) Cirrata, Cirroctopodidae; (k) Argonautoidea, Argonauta hians; (l) Octopodoidea, Octopodidae
(Figures reproduced with kind permission of the FAO, Rome, from Jereb and Roper (2005, 2010), and Jereb et al. (2014))
Modern nautiloids are placed in two genera, Nautilus and Allonautilus, discriminated by differences in
gill structure and the male reproductive system, among other characters (Ward and Saunders 1997), within
a single family, Nautilidae. The currently recognized species are Allonautilus scrobiculatus (Lightfoot,
1786), known from Papua New Guinea and surrounding islands; Nautilus pompilius Linnaeus, 1758,
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Table 1 Major lineages within Decapodiformes. A minimal number of defining characters are provided. For additional
characters, see Young et al. (2012a)
Number
of
Order Common name species Defining characters Nomenclatural notes
Spirulida Ram’s horn squid 1 Shell as coiled phragmocone, Some authors treat at
without cornea family level as Spirulidae
Sepiida Cuttlefishes 120 Shell as flattened Some authors treat at
phragmocone, with cornea family level as Sepiidae
Sepiolida Bob-tailed squids and bottle- 70 Shell as rudimentary gladius,
tailed squids with cornea
Myopsida Squids, often “neritic squids” 50 Shell as gladius, with cornea
because of their habitat
preferences
Idiosepiida Pygmy squids 6 Shell as gladius, with cornea, Some authors treat at
with adhesive organ family level as Idiosepiidae
Oegopsida Squids, often “oceanic squids” 230 Shell as gladius, without
because of their habitat cornea, with carpal locking
preferences apparatus
Bathyteuthida Squids 6 Shell as gladius, without Some authors treat at
cornea, without carpal locking superfamily level as
apparatus Bathyteuthoidea
originally described from Ambon in Indonesia, with an extensive distribution and possibly comprising a
species complex; Nautilus belauensis Saunders, 1981, from Palau; Nautilus macromphalus Sowerby,
1849, from New Caledonia; Nautilus repertus Iredale, 1944, from Western Australia; and Nautilus
stenomphalus Sowerby, 1849, from the Great Barrier Reef. Allonautilus perforatus, which is known
from drift shells in Bali, Indonesia, is probably synonymous with A. scrobiculatus.
Molecular genetic work on N. pompilius across a wide geographic range has shown several populations
to be extremely divergent (Sinclair et al. 2007; Bonacum et al. 2011; Williams et al. 2012). Samples from
Vanuatu, Fiji, and American Samoa were more closely related to N. macromphalus than to other
N. pompilius, and species from Eastern Australia and Papua New Guinea were clearly divergent from
those from the Philippines, Indonesia, Western Australia, and Palau. Bonacum et al. (2011) suggested that
several of these populations actually represented phylogenetic species but did not tackle the nomencla-
ture. Sinclair et al. (2007) found further divergence between N. pompilius samples from the Great Barrier
Reef and the Coral Sea separated by small geographic distances, and Williams et al. (2012) also found this
pattern, as well as separation of these populations from samples from Western Australia, the Philippines,
and Indonesia.
Other species not currently considered valid have been described, and Young (2010) provides a
complete list of nominal species. Many of these names do not, in fact, refer to nautilids and some are
nomen nudum, but a few are nomenclaturally valid and may be applied in the future, particularly to widely
separated populations that are found to merit specific status. Thus, it is important to note that nautilid
systematics is in a state of flux. Given their status as living fossils and the fact that they have been evolving
independently for hundreds of million years, nautilids may prove very interesting in comparative studies
of venom with other cephalopods and indeed other mollusks. Therefore, knowledge of their more recent
radiations and the current diversity within this group may be important for venom studies.
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Coleoidea: Decapodiformes
There are seven main groups of decapodiforms (Table 1). Herein they are all recognized as separate
orders, but differing opinions exist as to their ranks. In two of these groups, Spirulida and Sepiida, the shell
is a phragmocone (i.e., chambered as in nautiloids, although internal as in all coleoids). However, the form
of the phragmocone is highly divergent between Spirulida and Sepiida. Spirulida comprises the single
species Spirula spirula Linnaeus, 1758. It is a midwater species that seems to be most abundant over
bottom depths of 1,000–2,000 m (e.g., continental slopes or slopes associated with volcanic islands).
Spirula has a long fossil record, extending back to the latest Jurassic (Kröger et al. 2011). Often also
referred to as a “living fossil”, its lineage is estimated to have diverged from that of other decapodiforms
150 mya (Warnke et al. 2011). Species of the order Sepiida are characterized by the presence of a
cuttlebone. Sepiida comprises more than 100 species of cuttlefishes that live in continental shelf waters
although they may extend onto the slope to depths of about 600 m. They are present along tropical and
temperate coasts of the world including Australasia, Asia, Africa, and Europe but are totally absent from
North and South America. Sepiids have a benthic or demersal lifestyle, are short lived, and spawn large
eggs for an extended period once they reach maturity. Cuttlefishes are dorsoventrally flattened as is their
internal phragmocone, the cuttlebone, which runs the length (or most of the length) of the body. The
phragmocone of spirulids is superficially very different: the planispiral calcareous phragmocone is
situated rostrally and occupies a much smaller portion of the body.
The temptation to unite these two groups on morphological grounds is resisted not only because of
superficial differences but also because the phragmocone is a plesiomorphic structure. Nonetheless, the
wall structure of their phragmocones is similar and differs from that of extinct coleoids such as
Belemnitida and non-coleoid ectocochleate cephalopods (Doguzhaeva 1996; Young et al. 1998). Fur-
thermore, Spirulida and Sepiida share other morphological characters such as statolith shape and the
structure of the tentacular clubs (Clarke and Maddock 1988; Young et al. 1998).
Young and Vecchione (1996) conducted a cladistics analysis of extant coleoid cephalopods based on
50 morphological characters. Their analysis neither resolved relationships within Decapodiformes nor
found a sister-taxon relationship between Sepiida and Spirulida. In fact, in their analysis, all decapodiform
taxa branched as a polytomy from a single node in a strict consensus tree. Molecular studies have often
also failed to recover deep cephalopod relationships. However, two studies (Strugnell et al. 2005;
Lindgren and Daly 2007) have found support for a sister-taxon relationship between Spirulida and
Sepiida. These studies used different nuclear genes (Pax6, octopine dehydrogenase, rhodopsin versus
18S rRNA), but it should be noted that bootstrap support values for the 18S rRNA tree were relatively low
(66 %). Nonetheless, conflicting topologies have not been found either. The major problem with
molecular studies to date is that they have failed to yield topologies with notable bootstrap or posterior
probability support on deep nodes.
Khromov (1998) discussed the biogeography of Recent sepiids (which are absent from the Americas)
in light of paleoceanography and concluded they had a very recent origin in the Old World, a conclusion
that fits with the available fossil data (Young et al. 1998). Naef (1921–1923) concurred, suggesting
similarly recent origins for idiosepiids and sepiolids. Although a close relationship between these taxa is
far from certain, if correct, these conclusions have implications for the length of time the various branches
have been diverging when considering comparative studies of their venom.
Naef (1921–1923) studied fossil, morphological, and embryological evidence and recognized a close
relationship between Spirulida and Sepiida but also considered these taxa to be closely united with
Idiosepiida and Sepiolida. Idiosepiida is a monogeneric taxon of pygmy squids. These tiny squids are
circa 2 cm long as adults, and the synapomorphic character for the group is an adhesive organ on the dorsal
surface of their mantle, which they use to attach to sea-grass blades or other algae in their inshore habitat.
Sepiolida is a more diverse order, comprising the bob-tailed and bottle-tailed squids. These small round
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cephalopods have broad posteriorly placed fins and may be benthic, demersal, or pelagic. Naef treated
Spirulida, Sepiida, Idiosepiida, and Sepiolida as families and placed them together in the superfamily
Sepioidea. Although sepiolids have a gladius rather than a phragmocone, Naef (1921–1923), who
conducted meticulous embryological studies, concluded that a phragmocone anlage could be deduced
from the form of the embryological shell sac. Naef did not conduct embryological studies on Idiosepius
but concluded that a similar form would be seen. Naef (1921–1923) noted other similarities between
Sepiolida and Idiosepiida, including the similarity of the shell, and the presence of an adductor pallii
medialis and suggested that Sepiolida developed from an “Idiosepius-like predecessor.” Strugnell
et al. (2005) did find some support from nuclear genes for a sister-taxon relationship between Idiosepius
and sepiolid species, particularly when just the third codon positions were used in the analysis. Note that
the nomenclature is somewhat confusing, since more recent authors (e.g., Young et al. 2012a) use the
name Sepioidea to describe a clade containing just Sepiolida and Sepiida, although this two-taxon
grouping is not widely accepted.
The neritic squids, Myopsida, and the oceanic squids, Oegopsida, the latter sometimes assumed to
include the bathyteuthids, have often been combined into the taxon Teuthida. However, support for this
taxon is equivocal. Superficially, the morphology of Myopsida and Oegopsida is similar. The shell
(or gladius) is similar, they share the same long streamlined body, and they have similar tentacular
clubs. However, myopsids share several characters with sepiids and sepiolids that oegopsids do not have,
the most notable of which is the presence of a cornea. Thus, despite their superficial similarity to
oegopsids, some authors consider myopsids to be more closely related to sepiids and sepiolids. Once
again, molecular data have failed to resolve this issue. In many cases, phylogenies have even failed to
recover these orders as monophyletic. This is not believed to reflect confused systematics as these orders
are well defined morphologically. Furthermore, these orders tend to resolve as well-supported clades in
studies based on multiple nuclear genes (e.g., Strugnell et al. 2005; Lindgren and Daly 2007; Strugnell
and Nishiguchi 2007; Lindgren 2010; Lindgren et al. 2012). Molecular studies do recognize the close
relationship between Bathyteuthida (which many authors do not treat at order level) and Oegopsida, but
this has anyway long been recognized. Naef (1921–1923) suggested that Bathyteuthidae possesses
characters primitive for all Oegopsida and Bathyteuthis has historically been placed in Oegopsida.
Multigene phylogenies (Strugnell et al. 2005; Strugnell and Nishiguchi 2007; Lindgren 2010; Lindgren
et al. 2012), combined morphological and molecular analyses (Lindgren et al. 2004), and analyses based
on mitochondrial genome rearrangement (Allcock et al. 2011) have failed to resolve deep relationships
among Decapodiformes lineages (for review see Allcock et al. 2015). It is likely these will only be
resolved by genomic studies (Albertin et al. 2012). Fortunately, relationships within some of these orders
are better understood.
Sepiida
Cuttlefishes are perhaps best known for their remarkable camouflage, signaling patterns, and behavior, all
of which are relatively easy to study, given their presence in shallow coastal waters. There are just three
genera in a single family Sepiidae within the order Sepiida, with species distributed unevenly among the
genera. There are two species within the genus Metasepia, which is characterized by a reduced cuttlebone.
Species of Metasepia are only found around Australia and in the Western Pacific. There are seven species
with the genus Sepiella, which is characterized by a subcutaneous gland opening through a pore between
the fins, the function of which is unknown. Sepiella is also found in the Pacific with species as far north as
Japan and Korea and as far south as northern Australia, but the distribution of this genus also extends
westward with two species known from the Mozambique coast and a third species extending down the
west coast of Africa from Mauritania to Namibia (Barratt and Allcock 2012).
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All other species of sepiid are grouped in the large (with more than 100 species) genus Sepia. Khromov
(1998) attempted to diagnose subgroups within the genus Sepia. He defined and provided keys to six
species complexes within the genus: Hemisepius, Acanthosepion, Sepia sensu stricto, Anomalosepia,
Rhombosepion, and Doratosepion. He further allocated all (at that time) recognized species to one of
these species complexes and provided keys to the species within each complex. There have been no large-
scale molecular studies with extensive taxon sampling of sepiids to date to verify these subdivisions.
Nonetheless, Yoshida et al. (2010), in a relatively small-scale study, did find molecular support for
Doratosepion and Acanthosepion. However, molecular phylogenies have also highlighted an unexpected
relationship between Sepia officinalis and Sepiella (Bonnaud et al. 2006; Yoshida et al. 2010; Lindgren
et al. 2012), suggesting that our current understanding of generic level relationships is not totally correct.
The genus Sepia has the widest distribution of all the sepiid genera, perhaps not surprisingly, since it
contains very many more species than the other genera. It occurs off the coasts of Europe, Asia, Africa,
and Australasia; however, as mentioned above, it is absent from the coasts of North and South America.
Khromov (1998) analyzed biogeographic patterns in sepiids. He found greatest diversity in the Indo West
Pacific, which was home to 91 species, 86 of which were endemic to the region. In contrast, the Northeast
Atlantic, including the Mediterranean Sea, was home to just nine species, five of which were endemic. He
concluded that the northern and southern limits of the family were governed by temperature. Low
diversity in parts of Indonesia was attributed to poor faunal knowledge of that region; however, Khromov
(1998) found clear evidence of a decrease in species numbers from inshore waters of the Pacific and
Indian Oceans to more remote island habitats, with sepiids absent from Hawaii, the Seychelles, and the
Chagos Archipelago. The inability of sepiids to colonize habitats separated by deep water likely reflects
their lack of dispersal phase: adults lay large eggs that give rise to miniature benthic hatchlings which
inhabit the same habitat as the adult phase. Molecular investigations of island endemics from places such
as Guam and the Marshall islands may therefore show evidence of founder populations.
The earliest fossil sepiids are found in US deposits but their identification is disputed. Voltzia from the
Upper Jurassic and Actintosepia from the Cretaceous have been considered not to be sepiids by some
authors (e.g., Waage 1965). However, of the five genera reported from the Eocene (Archaeosepia,
Belosepia, Pseudosepia, Sepia, and Stenosepia), two are known from US deposits (Roeleveld 1972;
Khromov 1998), but only Sepia is known from more recent strata (Roeleveld 1972). Hence, it is likely that
low water temperatures, particularly in the western Atlantic in the Oligocene, led to the extinction of
sepiids from the Americas. Although the genus Sepia later radiated out of Europe, Roeleveld (1972)
suggests that temperatures would have been too low on the only suitable routes for recolonization (i.e., via
the Bering Straits or from Europe to Greenland via the Faroe Islands and Iceland). Khromov (1998)
proposed that Sepia subsequently radiated from the Mediterranean Ocean, spreading to the developing
Indian Ocean in the Oligocene and then radiating throughout Southeast Asia. He suggests that the African
fauna and that of the Japan Sea (i.e., those species on the periphery of the range) are therefore the youngest
in evolutionary terms. There is considerable concordance between the subgeneric classification and
geographic location, presumably reflecting these different radiations.
Sepiolida
There are two families within Sepiolida: the eponymous Sepiolidae and Sepiadariidae. Sepiadariidae is a
small family of bottle-tailed squids in just two genera: Sepioloidea and Sepiadarium. Neither genus has a
shell remnant, and the members of the genera are colorful small squids that live on the seafloor in mostly
shallow tropical seas of the Indo West Pacific. Sepioloidea is known from Australia, Indonesia, and New
Zealand, with the distribution of one species extending along submarine ridges as far as Easter Island
(Reid 2005, 2009). Sepiadarium has a slightly broader distribution with species occurring from South
Australia northward through the Pacific to Japan and westward to east India and Sri Lanka.
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In contrast, the family Sepiolidae is more diverse, comprising 16 genera divided into three subfamilies.
Sepiolids are small-rounded squids, with a rudimentary gladius, which may be absent, and posterior fins,
not dissimilar in overall shape to the sepiadariids. Members of two of the subfamilies, Rossiinae and
Sepiolinae, are benthic, while members of Heteroteuthinae are pelagic or benthopelagic. All
heteroteuthins have a large visceral photophore, and Naef (1921–1923) believed this subfamily to be
the most derived form. Unfortunately, because of the very delicate shell in this group, there is no fossil
evidence with which to consider evolutionary pathways.
The relationships among genera are not completely clear. Young (2007) placed Sepiola, Euprymna,
Inioteuthis, Rondeletiola, and Sepietta in Sepiolinae; Heteroteuthis, Nectoteuthis, Stoloteuthis,
Iridoteuthis, Amphorateuthis, and Sepiolina in Heteroteuthinae; and Rossia, Austrorossia, Neorossia,
and Semirossia in Rossiinae. This reflects Naef’s (1921–1923) placements with the exception that
Sepiolina is placed in Heteroteuthinae rather than Sepiolinae. Young (2007) left Choneteuthis unplaced,
noting its similarity to Sepiolina but also the differences of Sepiolina to other Heteroteuthinae. Young
(2007) also highlighted the presence of an as-yet undescribed subfamily whose affinities are not clear,
specimens of which are currently only known from fish stomachs.
A multigene molecular phylogeny of Decapodiformes (Lindgren et al. 2012) suggests that our
understanding of sepiolid relationships may not yet be complete. The phylogeny supported monophyly
of Sepiadariidae and confirmed Sepiadariidae and Sepiolidae to be sister taxa. This study, which included
representatives of eight genera of Sepiolidae (Rossiinae, Rossia; Heteroteuthinae, Heteroteuthis,
Stoloteuthis, Sepiolina; Sepiolinae, Euprymna, Sepiola, Sepietta, Rondeletiola), found Stoloteuthis as
sister taxon to Rossia and consequently did not support the monophyly of Heteroteuthinae. Only two
species of Rossiinae were included, but they did group as sister taxa. The 13 included species of
Sepiolinae formed a clade, but within this clade, the genera Sepiola and Sepietta were not monophyletic,
although included members of the genus Euprymna did form a clade. Groenenberg et al. (2009) also failed
to recover monophyletic genera within Sepiolinae in a study using the cytochrome oxidase subunit
I (COI) barcode gene. Importantly, Groenenberg et al. (2009) highlighted the presence of several
misidentified sequences on GenBank that could confound future studies: see Groenenberg et al. (2009)
and Lindgren et al. (2012) for details.
Idiosepiida
Represented by the single genus Idiosepius in the family Idiosepiidae, pygmy squids also have an Indo
West Pacific distribution. However, the delimitation of species is not clear, and researchers conducting
comparative studies on venom should be aware of this. The currently known specimens probably
comprise a single species (Idiosepius minimus) off the coasts of Africa, a species endemic to Australia
(Idiosepius notoides), and at least four species in the Indo West Pacific whose precise ranges and overlaps
have not been elucidated (I. picteti, I. thailandicus, I. paradoxus, and I. pygmaeus). Idiosepius biserialis is
probably a junior synonym of I. minimus and confined to African coasts, and species treated under this
name from the Indo West Pacific likely refer to I. thailandicus. DNA barcoding has helped clarify the
distribution of some species (Byern et al. 2012), but much further work is required.
Myopsida
Myopsid squids are found in neritic zones where they inhabit pelagic or demersal waters. They number, in
total, about 50 species and there have been several major rearrangements of myopsid systematics. The
currently accepted classification has all species except one in the family Loliginidae, with a second
monospecific family Australiteuthidae restricted to northern Australia and Papua New Guinea. Eleven
genera, and a number of subgenera, are currently recognized as valid within Loliginidae. Intriguingly,
most of these genera appear to affiliate with particular geographic regions, much as the subgenera of
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sepiids do. Loligo, Afrololigo, and Alloteuthis are associated with Europe and Africa. Doryteuthis,
Lolliguncula and Pickfordiateuthis are associated with the Americas. Heterololigo is associated with
the northern Pacific. Loliolus, Uroteuthis, and Photololigo are associated with the Indo West Pacific. The
exception is Sepioteuthis which is distributed in the western Atlantic, the Mediterranean, the Indo West
Pacific, and around the coasts of Australia as far south as Tasmania. This distribution led Brakoniecki
(1986) to suggest that Sepioteuthis is a Tethyan relic, and molecular data (Anderson 2000; Sales
et al. 2013) support this, indicating Sepioteuthis to be the sister taxon to all other loliginids. Anderson
(2000) further suggested subsequent dispersal from the Indo West Pacific to East Pacific American waters,
possibly along the continental shelf on the northern periphery of the Pacific, and then further radiation in
the Americas, possibly driven by the uplift of the isthmus of Panama. This could explain the sister-taxon
relationship between Heterololigo and Doryteuthis found in molecular studies and the sister-taxon
relationship between Doryteuthis opalescens (Berry, 1911) in American Pacific waters and Doryteuthis
pealeii (Lesueur, 1821) in American Atlantic waters. To date, no molecular studies have included
Australiteuthidae.
There is marked genetic structure among populations of some loliginid species (Dai et al. 2012; Sales
et al. 2013; Zheng et al. 2012), possibly indicative of a number of cryptic species or ongoing speciation in
these taxa.
Oegopsida
Oegopsid squids are found in the open ocean, and this taxon comprises the largest order of cephalopods
with more than 200 species in 24 families and 69 genera. Seven families are monospecific
(Architeuthidae, Architeuthis dux Steenstrup, 1857; Batoteuthida, Batoteuthis skolops Young and
Roper, 1968; Joubiniteuthidae, Joubiniteuthis portieri (Joubin, 1912); Ancistrocheiridae, Ancistrocheirus
lesueurii (d’Orbigny, 1842); Psychroteuthidae, Psychroteuthis glacialis Thiele, 1920; Lepidoteuthidae,
Lepidoteuthis grimaldii Joubin, 1895; Thysanoteuthidae, Thysanoteuthis rhombus Troschel, 1857), and a
further three families are monogeneric (Promachoteuthidae, Magnapinnidae, Pholidoteuthidae). The
families with greatest diversity are Cranchiidae (13 genera, circa 60 species), Ommastrephidae (11 genera,
circa 22 species), Onychoteuthidae (7 genera, circa 25 species), and Gonatidae (4 genera, circa 19 species).
Relationships among families are not well understood, but some families are believed to be closely
associated.
Young and Vecchione (2004) propose four groupings of multiple families. They highlight loss of the
true tentacular club as a feature shared by Batoteuthidae, Chiroteuthidae, Joubiniteuthidae,
Magnapinnidae, Mastigoteuthidae, and Promachoteuthidae. However, they note that monophyly of this
group is far from certain. Lindgren et al. (2012), in a total evidence molecular study, found five of these
families formed a clade that also included Pholidoteuthidae; Promachoteuthidae was not included in the
study. This clade was not strongly supported, but a subclade containing Batoteuthidae and Chiroteuthidae
received bootstrap support >90 %, and a subclade containing Joubiniteuthidae, Mastigoteuthidae, and
Magnapinnidae received bootstrap support >50 %.
Small mesopelagic squids in the families Ancistrocheiridae, Enoploteuthidae, Lycoteuthidae, and
Pyroteuthidae may be related (Young and Vecchione 2004). Lindgren et al. (2012) found that members
of Enoploteuthidae and Pyroteuthidae formed a clade, which had good bootstrap support >90 %.
However, members of Lycoteuthidae fell together, but no support was found for a relationship between
Lycoteuthidae and any other family. Ancistrocheiridae was not included in the study.
Histioteuthidae and Psychroteuthidae share the same tentacular club structure, and their relationship is
supported by 100 % bootstrap support in a total evidence molecular study (Lindgren et al. 2012).
The fourth family-level relationship is among Lepidoteuthidae, Octopoteuthidae, and
Pholidoteuthidae. There is no single uniting character for this grouping (Young and Vecchione 2004),
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and it is not supported as a monophyletic group by molecular work. Nevertheless, there is some bootstrap
support (>50 %) for a relationship between Lepidoteuthidae and Octopoteuthidae (Lindgren et al. 2012).
Relationships within some of the four most diverse families are better understood than others.
Cranchiidae is divided into two subfamilies: Cranchiinae and Taoniinae. While there have been no
dedicated molecular studies on Cranchiidae, Lindgren et al. (2012) included three cranchiins and seven
taoniins and found the family and both subfamilies to be monophyletic.
In contrast, a molecular study of Gonatidae, using three mitochondrial genes and including 14 species
representing all four genera, found all genera, except Eogonatus which is monospecific, to be polyphyletic
(Lindgren et al. 2005).
Similar issues have been found within Onychoteuthidae. Early molecular work (Bonnaud et al. 1998)
suggested that the genera Onychoteuthis and Moroteuthis were in need of taxonomic revision. Subse-
quently Wakabayshi et al. (2007) used DNA barcoding to show that adults identified as belonging to the
genus Moroteuthis fell in a clade with paralarvae known as Onykia (a genus where adults were unknown).
However, to complicate matters further, Onykia does not appear to be monophyletic, since Onykia
carriboea Lesueur, 1821, has fallen outside the main Onykia clade in molecular studies
(Lindgren 2010; Lindgren et al. 2012) and resolved as sister taxon to Ancistroteuthis lichtensteini
(Férussac [in Férussac and d’Orbigny], 1835).
Ommastrephidae has been divided into three subfamilies, Illicinae, Ommastrephinae, and Todarodinae
which can be diagnosed on a few simple characters such as the sucker seriation on the dactylus of the
tentacular club and whether or not photophores are present (Young et al. 2012b). A study using two
mitochondrial genes and including 15 species in ten genera provided support for these divisions,
recovering all the subfamilies as monophyletic in nearly all analyses (Wakabayashi et al. 2012). However,
within Todarodinae, two genera (Todarodes and Nototodarus) were found to be polyphyletic, casting
doubt on the characters used to separate them.
Bathyteuthida
This small group of deep-water squids currently comprises six species: three in the genus Chtenopteryx
(family Chtenopterygidae) and three in the genus Bathyteuthis (family Bathyteuthidae). A molecular
study including two Chtenopteryx species and three Bathyteuthis species recovered both genera as
monophyletic and also as sister taxa (Lindgren et al. 2012).
Coleoidea: Octopodiformes
Octopodiformes comprises approximately 300 species in two orders, with all but one species within
Octopoda. The order Vampyromorphida is represented by the single species Vampyroteuthis infernalis
Chun, 1903 – the enigmatic vampire squid, widely recognized as a living fossil. It inhabits mesopelagic
depths in the temperate to tropical zones of the world’s oceans. Until recently, there was much debate as to
whether the vampire squid was a representative of Decapodiformes or Octopodiformes. However, its
placement in Octopodiformes is confirmed by morphological evidence from hatchlings (Young and
Vecchione 1999) and from embryological evidence of octopods (Boletzky 1978–1979, 2006). Molecular
evidence has given conflicting results, but an increasing body of molecular evidence also supports the
placement of Vampyromorphida in Octopodiformes (see, e.g., Yokobori et al. 2007; Strugnell and
Nishiguchi 2007; Allcock et al. 2011). Kröger et al. (2011) place the earliest vampyromorph fossils in
the late Triassic/early Jurassic, suggesting that this lineage has been evolving independently for about
200 million years.
The order Octopoda comprises two suborders, Cirrata (or Cirrina) and Incirrata (or Incirrina), and
molecular evidence provides strong support for monophyly of these groups and for a sister-taxon
relationship between them (Carlini et al. 2000; Strugnell et al. 2004, 2005, 2014; Lindgren et al. 2012).
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The earliest evidence of these taxa in the fossil record is found from Late Cretaceous deposits (Tanabe
et al. 2008; Fuchs et al. 2009), although divergence time estimates (Kröger et al. 2011) place their
separation in the Late Jurassic.
Cirrata comprises the finned octopods, which tend to have a gelatinous body and a deep web. The name
Cirrata derives from the cirri that extend down the arms alongside the suckers. The fins are supported by
an internal cartilaginous shell and the animals use these to swim, and while some finned octopods are
primarily benthic, they may also be demersal or entirely pelagic.
Cirrata was reviewed by Collins and Villanueva (2006). In their systematic section, they followed the
proposals made by Piertney et al. (2003), based on molecular work involving a single mitochondrial gene,
that Cirrata comprises four families. Revising some of the existing taxonomy and clarifying the status of
some difficult taxa, Collins and Villanuevea (2006) suggested the following families were valid:
Cirroteuthidae for the pelagic genera Stauroteuthis, Cirroteuthis, and Cirrothauma; Grimpoteuthidae
for the genera Grimpoteuthis, Cryptoteuthis, and Luteuthis; Opisthoteuthidae for the genus
Opisthoteuthis; and Cirroctopodidae for the genus Cirroctopus. However, other arrangements are also
followed. For example, Vecchione et al. (2014) place the genera in three families as follows:
Opisthoteuthidae (Cirroctopus, Grimpoteuthis, Luteuthis, Opisthoteuthis, Cryptoteuthis), Cirroteuthidae
(Cirroteuthis, Cirrothauma), and Stauroteuthidae (Stauroteuthis). They also note the morphological
similarity between Cirroteuthidae and Stauroteuthidae (united as Cirroteuthidae by Collins and
Villanueva), which have similar body shape, long cirri, and a secondary web, but which differ markedly
in shell shape. Although these classifications are not widely different, further molecular work with
additional markers would be extremely useful.
Incirrata comprises the benthic octopuses familiar from shallow waters, as well as some more unusual
groups. Young et al. (1998) discussed the unusual “oral-end-down” habit of benthic octopuses, which
mostly crawl on the seafloor using their arms. They noted that the brain of Vampyroteuthis indicates that it
is capable of processing complex chemotactile signals from the arms and wondered whether the arms
played some important role in Vampyroteuthis or its ancestors that might have facilitated oral exploratory
behavior of the seafloor so as to lead to the evolution of benthic octopods. In fact, recent work (Hoving and
Robison 2012) shows that vampire squids are detritivores and use their retractile filaments to accumulate
food in a sticky matrix which is then passed to the mouth, so oral exploration of the benthos in a
hypothetical ancestor is not an unreasonable proposition. However, this mode of feeding also raises
interesting questions as to the role of venom in vampyromorphs, and comparisons between
vampyromorphs and octopods would be interesting from an evolutionary point of view.
Incirrata is divided into two superfamilies: Argonautoidea, comprising the genera Haliphron,
Argonauta, Ocythoe, and Tremoctopus, each placed in their own family, and Octopodoidea, comprising
all other genera of incirrate octopods. The argonautoid families are unusual and highly diverse but are
united by an unusual feature: that males have a detachable hectocotylus. The only known species of
Haliphron inhabits deep waters around the world. It shows exceptional sexual dimorphism, with females
reaching a total length of 2 m versus about 30 cm in males. The four Tremoctopus species, known as
blanket octopuses, have extensive but thin webs. They float in the upper layers of subtropical and tropical
oceans. They are also sexually dimorphic, with males about 5 % the size of females, which can reach more
than a meter in total length. The four Argonauta species are also found in the upper layers of subtropical
and tropical oceans. They are unique in that the female secretes a calcareous shell, in which it lives and
lays its eggs. It is from the delicate nature and shape of the shell that the animal gets its common name
“paper nautilus.” Males are also dwarf and have been reported associated with salps. The single known
species of Ocythoe is also found in upper water layers but of temperate oceans. The males are also dwarf.
Several molecular studies confirm Argonautoidea as sister group to all other incirrate octopuses
(Strugnell et al. 2004, 2014; Lindgren et al. 2012). Naef (1921/1923) suggested that Alloposidae and
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Tremoctopodidae were closely related to one another, as were Argonautidae and Ocythoidae, based on the
structure of the hectocotylized arm. Bizikov (2004) supported this arrangement, but based on the
structure, or absence, of the stylets (shell remnants). Subsequent molecular work (Strugnell and Allcock
2010), based on several mitochondrial genes, supported this arrangement.
Argonautoidea has been hypothesized to have a benthic ancestry, because of the morphological
resemblance of species to benthic octopuses in Octopodoidea (Naef 1921–1923). Relevant characters
include the absence of fins and cirri, and a well-developed frontal lobe system, and evidence of corneas
(Young et al. 1998).
Octopodoidea is easily the most speciose group of Octopodiformes, and relationships within it are still
not well understood, although some advances have been made in recent years. Until recently, all benthic
Octopodoidea were placed in the family Octopodidae, with the four valid pelagic genera distributed in the
families Amphitretidae (Amphitretus), Vitreledonellidae (Vitreledonella), and Bolitaenidae (Bolitaena,
Japatella) and often combined into a suborder Ctenoglossa on the basis of the structure of their radulae.
However, early molecular work (Carlini and Graves 1999; Carlini et al. 2001) had suggested that the
family Octopodidae was not monophyletic, and further evidence for this was provided in a study showing
that the pelagic genera were a derived branch within Octopodidae, leading the authors to suggest these
genera had neotonous origins (Strugnell et al. 2004). This topology has since been recovered in multiple
studies (Strugnell et al. 2008, 2014; Lindgren et al. 2012), which led Strugnell et al. (2014) to propose a
revised taxonomy based on a molecular study utilizing three nuclear and four mitochondrial genes and
including representatives of 25 Octopodoidea genera as well as Vampyroteuthis and representatives of the
argonauts and cirrates. They combined the four pelagic genera into a single family Amphitretidae, with
the genera placed within the subfamilies Amphitretinae, Vitreledonellinae, and Bolitaeninae. They placed
the genus Bathypolypus in a family of its own, Bathypolypodidae. Eledone and Aphrodoctopus, two
genera with uniserial suckers and heteromorphic arm tips in males, were combined in the family
Eledonidae. Southern Ocean and deep-sea octopuses with uniserial suckers were combined in the family
Megaleledonidae. The origins of this clade were explored by Strugnell et al. (2008), who showed that
changing environmental conditions in the mid-Miocene led to strengthening of the thermohaline circu-
lation and the consequent spreading of Antarctic bottom water northward, allowing radiation of a clade of
octopuses out of Antarctica. Muusoctopus, Enteroctopus, Sasakiopus, and Vulcanoctopus were placed in
a new family Enteroctopodidae. All other genera remained in the family Octopodidae.
A summary of octopod higher-level systematics is provided in Table 2. Although our understanding of
octopus systematics has improved substantially in recent years, there are still many species whose generic
affinities remain unclear. The tightening of the diagnosis of the genus Octopus, which for many years had
contained a large number of widely divergent species, left many species without a generic placement. The
work of Strugnell et al. (2014) shows that currently available molecular markers are well suited to solving
such problems.
Table 2 Major lineages within Octopodiformes

Order Suborder Superfamily Included families
Vampyromorphida Vampyroteuthidae
Octopoda Cirrata Cirroctopodidae, Cirroteuthidae, Grimpoteuthidae, Opisthoteuthidae
Incirrata Argonautoidea Alloposidae, Argonautidae, Ocythoidae, Tremoctopodidae
Octopodoidea Amphitretidae, Bathypolypodidae, Eledonidae, Enteroctopodidae,
Megaleledonidae, Octopodidae
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Conclusions
Cephalopoda is a particularly interesting class from an evolutionary point of view and therefore provides
extensive opportunities for comparative studies. It is highly divergent, with widely differing body forms
and species that inhabit widely differing environments. It is not particularly speciose, making comparative
studies across the whole group a real possibility. Furthermore, it contains species on long branches, which
have been evolving independently for hundreds of millions of years such as the nautiluses, Spirula and
Vampyromorpha, as well as groups that have undergone recent radiations such as the sepiids, possibly
Idiosepius, and some myopsid genera. Our understanding of deep phylogenetic relationships is still poor
and may only be solved by genomics, but suitable molecular markers do exist to solve many of the
remaining taxonomic issues at more shallow nodes, and progress in these areas is likely to be rapid in the
coming years, dependent only on sampling opportunities and resources.
Cross-References
▶ Toxicity in Cephalopods
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Evolutionary History of Venom Glands in the Siluriformes

Jeremy J. Wright*
New York State Museum, Albany, NY, USA
Abstract
The order Siluriformes represents a hyperdiverse group of fishes (>3,000 currently recognized species),
which has been known to contain venomous species diversity for over 250 years. In spite of this historical
knowledge, scientific examinations of the basic characteristics and evolutionary history of these species’
venom glands, and their products, have been extremely sparse compared to those of terrestrial venomous
organisms, or even venomous fishes in general. Here, the current state of knowledge regarding the venom
glands of catfishes and their products is examined in a review of morphological, pharmacological, and
chemical studies of these structures. Several hypotheses regarding the evolution of siluriform venom
glands are able to be drawn from the information contained in these studies as well as the limited work that
has attempted to study the evolution of these structures in detail. These include selective scenarios to
explain the secondary losses of venom glands in several catfish species and families, compositional
variation in siluriform venom chemistry, and the derivation of venom glands from secretory cells of the
epidermis. Future work directly addressing multiple issues of venom production and composition in
catfishes is necessary before investigations of the evolution of siluriform venoms and delivery structures
can reach the levels of detail and sophistication seen in other venomous groups. These studies will benefit
greatly from the advent of genomic, transcriptomic, and proteomic methods, which have seen wide use in
examinations of venoms produced by other taxa, but have yet to be widely applied to analyses of piscine
venoms.
Keywords
Catfish; Defense; Proteins; Epidermal; Crinotoxins
Introduction
Species falling under the general classification of “fishes” (a paraphyletic assemblage including the
classes Myxini (hagfishes), Petromyzontida (lampreys), Chondrichthyes (sharks, rays, and chimaeras),
Actinopterygii (ray-finned fishes), Sarcopterygii (coelacanths and lungfishes)) represent more than half of
the world’s known vertebrate species (Nelson 2006). Many species within the Chondrichthyes and
Actinopterygii have long been known to utilize venoms in a natural defensive capacity as well as in
interactions with bathers and fishermen (Halstead 1988). Human envenomations by fishes are a relatively
common occurrence; globally, incidents involving venomous spiny-rayed fish species (superorder
Acanthomorpha) alone number over 50,000 cases annually (Smith and Wheeler 2006), which, due both
to unreported incidents and exclusion of several venomous groups, likely severely underestimates the
actual number of cases. In one study, nearly 70 % of marine fish and 90 % of freshwater fish
envenomations of humans were caused by non-acanthomorph species (Haddad and Martins 2006);
*Email: Jeremy.Wright@nysed.gov
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when extrapolated to global estimates, this would elevate the estimated number of incidents to over
100,000 per year.
As would be expected of a venom whose putative purpose is the rapid deterrence of predators, the most
common result of envenomation by fish species is intense pain that is highly disproportionate to the
magnitude of the injury, suggesting that components of these venoms target nociceptive sensory neurons
(Church and Hodgson 2002; Trim and Trim 2013). In addition to the elicitation of this intense pain
response, fish venoms are known to cause a number of other physiological symptoms, including
cardiovascular, hemolytic, and neuromuscular effects (Halstead 1988; Church and Hodgson 2002;
Sivan 2009). Despite their clear ramifications for human health, fewer than a dozen toxic compounds
have been characterized from this highly diverse assemblage (Halstead 1988; Church and Hodgson 2002;
Smith and Wheeler 2006). In addition to medical interest in their native physiological effects, fish venoms
represent an untapped reservoir of potentially pharmaceutically valuable compounds, particularly as lead
compounds in the development of new analgesics, due to their possible ability to directly interact with
neuronal signaling pathways (Trim and Trim 2013).
Until recently, however, even the most basic information regarding venomous fishes, such as the
number and phylogenetic distribution of venomous taxa, has been unavailable to researchers interested in
the evolutionary history of these compounds and the structures that produce them. In the last decade,
phylogenetic analyses of acanthomorph species have estimated that 585–650 of the species in this group
should be presumed to be venomous, a substantial increase from previous estimates of approximately
200 species (Halstead 1988; Smith and Wheeler 2006). When other types of venomous fishes
(Chondrichthyes, Siluriformes) are included, this estimate potentially increases to over 2,500 species,
or just under 10 % of all known fish species (Wright 2009). Though this level of species diversity is greater
than that of all other venomous vertebrates combined, venomous fishes remain severely understudied
relative to venomous terrestrial organisms, as evidenced by a recent review of venom evolution that
mentions fishes only in passing, and without providing any detailed information regarding the toxic action
of their venoms (Casewell et al. 2012).
The order Siluriformes, commonly known as catfishes, is a globally distributed, highly diverse clade
containing over 3,000 currently recognized species in 36–38 families (Sullivan et al. 2006; Ferraris 2007).
The order has been known to contain venomous representatives for nearly 300 years, beginning with
Johann Richter’s description of Spanish fishermen’s fear of stings from marine catfishes belonging to the
family Ariidae (Halstead 1988). Although the stings of most catfish species are relatively harmless, albeit
very uncomfortable, fatalities have been reported as the result of envenomations by members of the
families Plotosidae (Plotosus lineatus) and Clariidae (Heteropneustes fossilis) (Halstead 1988). These
species undoubtedly possess notably potent venoms, but these fatalities, which occurred in the late
nineteenth and early twentieth centuries, were likely due to poor medical care and/or secondary infection
of the wound (a common complication of siluriform envenomations) (Halstead et al. 1953; Haddad and
Martins 2006). Only one modern fatality involving a catfish sting has been recorded, a freak accident in
which a fisherman’s heart was penetrated by the spine of a large individual (Haddad et al. 2008).
While venomous fishes in general have received little research attention relative to other venomous
groups of organisms, catfishes in particular have suffered from a dearth of focused studies. Until recently,
few families had been confirmed to contain venomous species, although several had been suspected to
harbor venom-producing representatives (Halstead 1988). Wright (2009) performed an extensive histo-
logical survey of nearly 150 catfish species, sampling over 100 genera (25 % of the genus-level diversity
in the order) in 32 families, demonstrating the presence of venomous taxa in 20 siluriform families
(Table 1), and arriving at a total estimate of 1,250–1,625 venomous species, a significant majority of
venomous actinopterygian diversity. The upper end of this estimate would make catfishes the most diverse
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Table 1 Taxonomic distributions and estimates of venomous catfish diversity. Estimates reproduced from Wright (2009)
Taxon # Presumed venomous
Siluriformes – catfishes 1,250–1,625 species
Akysidae – Asian stream catfishes 48
Amblycipitidae – torrent catfishes 26–28
Anchariidae – Madagascan catfishes 4–6
Ariidae – sea catfishes 67–134
Bagridae – bagrid catfishes 176–198
Callichthyidae – armored catfishes 182–194
Chacidae – angler catfishes 3
Clariidae – labyrinth catfishes 79–114
Claroteidae – claroteid catfishes 56–84
Cranoglanididae – armorhead catfishes 3
Doradidae – thorny catfishes 48–81
Heptapteridae – shrimp catfishes 91–160
Ictaluridae – North American catfishes 57–64
Mochokidae – squeakers 166–189
Pangasiidae – shark catfishes 27–30
Pimelodidae – antennae catfishes 41–79
Plotosidae – eel-tailed catfishes 17–37
Pseudopimelodidae – bumblebee catfishes 21–31
Schilbeidae – glass catfishes 48–62
Siluridae – sheat catfishes 74–83
single group of venomous vertebrates known (Wright 2009; Egge and Simons 2011) and continues to
increase each year, due to descriptions of new species in venomous families and genera.
Examinations of the evolutionary history of venoms and venom production in catfishes are currently
hampered by a lack of resolution in higher-level siluriform phylogeny (Sullivan et al. 2006) and basic
knowledge regarding the identity of venom components and the genetic architecture underlying their
production (Wright 2009; Egge and Simons 2011) as well as selective factors driving the compositional
evolution and properties of defensive venoms (Casewell et al. 2012). Nonetheless, sufficient progress has
been made to be able to generate inferences regarding several aspects of catfish venom gland evolution.
This chapter attempts to provide a review of our current knowledge and hypotheses regarding the
evolution of siluriform venom glands, as developed through an examination of relevant literature
concerning the identification and anatomy of siluriform venom glands and delivery systems; the toxicol-
ogy, pharmacology, and basic chemistry of the venoms of species investigated thus far; and the few
studies that have attempted to directly address the ecology and evolution of the venom systems of
catfishes. Such a survey serves as an illustration of not only the surprising amount of evolutionary
information that can be gleaned from the existing literature but how far the study of venomous catfishes,
and venomous fishes in general, must proceed before reaching the levels of detail and sophistication seen
in other groups of venomous organisms.
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Fig. 1 The venom delivery system of catfishes. (a) The venomous species Noturus stigmosus (Northern madtom), with red
arrows indicating the position of the dorsal and pectoral fin spines. (b) The pectoral girdle of N. stigmosus with articulated fin
spines, illustrating the increased levels of spine serration found in this species. (c) Cross section of the pectoral fin spine of
N. stigmosus, showing the association of venom glands with the fin spine. Abbreviations: ps pectoral fin spine, cle cleithrum,
cor coracoid, cor-pp posterior process of coracoid, vgc venom gland cells (Figure reproduced from Wright (2009))
Siluriform Venom Gland and Delivery System Morphology

Gross Morphology
Venoms, by definition, require a method by which their bearer is able to introduce them into the body of a
target organism. In all known venomous fishes (with the exception of Meiacanthus sp. and members of the
deep-sea family Monogathidae), this is accomplished via spiny elements associated with the fins and/or
opercular and cleithral bones (Halstead 1988; Smith and Wheeler 2006). These spiny elements contain
grooves that facilitate the flow of venom along the spin; in most cases, the glandular tissue rests within the
groove itself. The association of these venom glands with spiny elements led Perrière and Goudey-
Perrière (2003) to name their toxic secretions acanthotoxins. In Meiacanthus sp. (saber-toothed blennies),
injection is achieved by the use of enlarged fangs in the bottom jaw rather than spines, with buccal venom
glands surrounding the proximal two thirds of the fang (Halstead 1988; Smith and Wheeler 2006). Venom
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flows toward the site of envenomation through grooves along the anterior fang margins. Monognathids,
which lack upper jaws, apparently inject venom via a single, hollow rostral fang, which has paired glands
at its base (Bertelsen and Nielsen 1987). These species are unique among venomous fishes, in that they
appear to use their venoms to subdue their prey, shrimps that are very large relative to their own size
(Bertelsen and Nielsen 1987), and which would have the potential to cause significant damage to these
relatively fragile fishes.
The venom glands of catfishes are composed of aggregations of glandular cells associated with bony
spines in the dorsal and pectoral fins (Fig. 1a–c), which can be erected and locked into place via frictional
forces and/or muscular action when the fish is threatened, effectively increasing the individual’s cross-
sectional area and leading to increased handling difficulty for potential predators (Bosher et al. 2006;
Fine et al. 2011; Emmett and Cochran 2010; Wright 2012a). The pectoral and, occasionally, dorsal spines
of many species are additionally armed with retrorse serrations along one or both of the spine margins
(Fig. 1b), the presence and orientation of which can vary both between and within different catfish
families (Wright 2009; Egge and Simons 2011). When the spine enters a potential predator, the glands are
torn, releasing the largely proteinaceous venom into the wound.
This passive method of venom delivery appears to represent a rather primitive condition, which is
found across multiple groups of venomous fishes; members of only a few families (e.g., Batrachoididae,
Scorpaenidae) show a more specialized, hypodermic-style apparatus characteristic of most other venom-
ous vertebrates (Birkhead 1972; Halstead 1988; Smith and Wheeler 2006). It also results in potentially
significant damage to the integumentary and glandular tissue surrounding the spine, which can take a
significant amount of time (over a week) to heal (Birkhead 1972). Nonetheless, such compromised spines
still represent a potent antipredatory defense; multiple experiments presenting North American catfish
species (family Ictaluridae) to largemouth bass (Micropterus salmoides), a common piscivorous species,
have demonstrated that the presence of spines increases predator handling time and catfish survivorship
relative to individuals in which the spines had been removed (Bosher et al. 2006; Emmett and Cochran
2010; Wright 2012a). Wright (2012a) further demonstrated, however, that the presence of venom glands
significantly increases the antipredatory capabilities of spines in intact tadpole madtoms (Noturus
gyrinus), relative to individuals in which the venom glands had been surgically removed.
The extent and orientation of the venom glands in relation to spine serrations, as well as grooves within
the spine itself, varies significantly between different catfish species (Halstead 1988; Wright 2009; Egge
and Simons 2011). Serrated spines may increase the amount of mechanical damage produced when the
spine enters a potential predator, increasing the surface area exposed to the concomitantly released venom
(Reed 1907; Birkhead 1972; Egge and Simons 2011). There is little evidence, however, to suggest that the
venoms of species with greater levels of spine ornamentation possess significantly greater toxicity
(Birkhead 1972), nor have experiments been performed to demonstrate increased predator deterrent
ability in those species. In fact, Egge and Simons (2011) found that of five evolutionary changes in
sting morphology in the genus Noturus, four involved decreases in morphological complexity, including
the loss of spine serrations, loss of venom gland tissue associated with serrations, or, in one case, the total
loss of the venom gland.
These results suggest that certain ecological and life history traits may result in the relaxation of
selective pressures related to the maintenance of venom glands, leading to their eventual loss, which
appears to be a relatively widespread phenomenon throughout the order (Wright 2009). Such scenarios
may include ontogenetic loss of venom glands in species obtaining body sizes that effectively protect
them from natural, gape-limited predators (Egge and Simons 2011) or the secondary loss of venom glands
in members of families that have lost ossified fin spines (e.g., Malapteruridae, many amphiliids) and, thus,
an effective delivery system for metabolically expensive venom compounds (Wright 2009). Wright
(2009) also found that members of the families Sisoridae and Erethistidae have secondarily lost venom
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glands, while maintaining their fin spines. Many of the species in these families occupy highly rheophilic
habitats (as does Ameiurus brunneus, another species that has secondarily lost venom glands), where
effective foraging by large-bodied predatory species would be highly difficult, if not impossible, offering
a possible explanation for the lack of venom production in these species.
Cellular Morphology
The cellular morphology of venom glands in fishes is very similar across broad taxonomic categories,
indicating possible widespread convergent evolution of these cells. Venom-producing cells are enclosed
within an integumentary sheath composed of epithelial cells. The venom gland cells are large and
polygonal, with prominent nucleoli and highly granulous cytoplasm, presumably due to high concentra-
tions of venomous peptides (Reed 1907; Halstead et al. 1953; Halstead 1988); in catfishes, the cells of the
venom gland are also binucleate (Reed 1907; Halstead et al. 1953; Halstead 1988). As the cells mature,
organelles and nuclear structures are lost and only the cytoplasmic granules are visible. Venomous
secretions are either held within the cells or the cells undergo holocrine secretion, whereby the secreting
cells are lysed and release the venomous secretions (along with cellular fragments) into the intercellular
space, where they are held until being used.
Cameron and Endean (1973) hypothesized that the venom gland cells of fishes and the acanthotoxins
that they contain are evolutionarily derived from the clavate or club cells of the epidermis, which secrete
proteins known as crinotoxins (Halstead 1988). While crinotoxic secretions are released into the water
when the cells are ruptured, ostensibly to repel predators or fouling organisms (Cameron and Endean
1973), the direct injection of these compounds into other organisms has also been shown to have toxic
effects (Al-Hassan et al. 1987; Shiomi et al. 1987, 1988). A preliminary study of the catfish Plotosus
lineatus offers some support for Cameron and Endean’s hypothesis, as the club cells of this species were
found to produce a substance that is similar, and possibly identical, to one of the toxic fractions found in
the venom gland, based on immunological reactions (Shiomi et al. 1988).
Perrière and Goudey-Perrière (2003), however, point out that common production of a single toxic
component is not sufficient evidence to prove the homology of these cell types. While certain crinotoxins
and acanthotoxins produced by P. lineatus show similar histochemical and pharmacological activities,
Whitear et al. (1991a) found distinct differences in the ultrastructure and histochemistry of the venom
gland cells and club cells in the skin of Heteropneustes fossilis (Indian stinging catfish) that, in their
estimation, precludes the homology of the two cell types. Specifically, club cells were found to contain
helical filaments and a division of the cytoplasm into perinuclear and peripheral zones, both of which were
lacking in the venom cells. Additionally, while a previous study (Zaccone et al. 1990) had shown a
positive immunohistochemical reaction for serotonin in the club cells of this species, Whitear
et al. (1991a) found that this reaction was lacking in the venom cells.
Whitear et al. (1991a) did not address why these differences should mean that the venom gland cells
could not possibly have been derived from epidermal club cells. If venom glands are indeed adaptive
structures, one might expect their cellular morphology and the secretions that they produce to be subject to
selection pressures that differ from those experienced by secretory cells in other locations. The differences
reported by Whitear et al. may simply reflect this history. Additional comparative morphological and
transcriptomic studies of venom glands and secretory epidermal cells from different groups of venomous
catfishes should serve to clarify these issues.
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Fig. 2 Gross morphology of the axillary glands and associated structures in catfishes. (a) Anterior half of Ariopsis felis, with
cleithral region and axillary pore indicated by white box. (b) Close-up of cleithral region from the same specimen, with the
axillary pore indicated by the white arrow. (c) Cleithral region of Bagre marinus with skin removed, showing the position of
the axillary gland relative to the cleithrum. Black arrow indicates glandular tissue, which extends further upward behind the
cleithrum. (d) The axillary gland of the same specimen, removed from behind the cleithrum
Siluriform Axillary Glands

Gross Morphology
In addition to the venom glands lining the spinous elements of the fins, many siluriform species possess
secretory glands situated in the axil of the pectoral fin (Reed 1907; Halstead et al. 1953; Halstead and
Smith 1954; Greven et al. 2006). These structures are known from several families, including the
Akysidae, Ariidae, Callichthyidae, Ictaluridae, Mochokidae, and Plotosidae, but to date, no comprehen-
sive survey has been performed to document the distribution of axillary glands throughout the
Siluriformes. Various authors have considered the axillary glands to be part of the venom apparatus
(Reed 1907; Halstead et al. 1953; Birkhead 1967; Cameron and Endean 1971), and, as such, they are
briefly discussed here.
The axillary glands of catfishes are small pouch-like structures that release their secretions via a pore
located below the postcleithral process, near the base of the pectoral fin spine (Fig. 2a, b). In most species,
the gland itself is roughly triangular in shape, with its upper half covered by, and the long axis oriented at a
perpendicular to, the postcleithrum (Fig. 2c, d). The interior of the gland is divided into several lobes, with
each lobe being separated from the others by a layer of connective tissue (Reed 1907; Halstead
et al. 1953). Recent studies of callichthyid catfishes have revealed a simple, tubular morphology of the
axillary gland in these species (Greven et al. 2006).
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Fig. 3 Cellular morphology of the axillary gland of Bagre marinus. Photomicrographs of (a) a histological section of the
axillary gland pictured in Fig. 2d and (b) a close-up view of the glandular cells
Cellular Morphology
The secretory cells of siluriform axillary glands are located within further subdivisions of the axillary
gland lobes (Fig. 3a). In all species thus far studied, these cells are large and polygonal and contain large
quantities of a granular, secretory product, which has been shown by multiple authors to be proteinaceous
in nature (Cameron and Endean 1971; Al-Hassan et al. 1987; Kiehl et al. 2006). The cellular ultrastructure
resembles that of the venom gland, with the cells originating as binucleate cells with prominent nucleoli
and large amounts of endoplasmic reticulum (Whitear et al. 1991b). The cells become completely filled
with secretory product as they mature, to the point that most subcellular structures are no longer visible
(Halstead et al. 1953; Cameron and Endean 1971; Kiehl et al. 2006) (Fig. 3b). Release of the secretory
product appears to be holocrine in nature, which is indicated by the presence of burst cells in secretions
drawn directly from the axillary pore (Reed 1907; Cameron and Endean 1971; Whitear et al. 1991b) and
lack of evidence for other methods of secretion.
Possible Function
The earliest mention of axillary glands in catfishes was made by G€ unther (1880). He assumed that
secretions issuing from the axillary pore anoint the pectoral fin spine, allowing them to be injected along
with secretions from the pectoral venom glands. Many works that followed (i.e., Reed 1907) accepted this
statement without experimental confirmation. More recently however, several additional, more likely,
hypotheses have been proposed for the function of these structures. While later studies showed that
axillary gland extracts are toxic when injected into other organisms (Cameron and Endean 1971; Birkhead
1967), the water-soluble nature of axillary pore secretions is difficult to reconcile with the venomous
scenario envisioned by earlier authors. Current hypotheses regarding the function of the axillary gland
secretions include antimicrobial (Kiehl et al. 2006), ichthyotoxic (Greven et al. 2006), pheremonal, and
ionoregulatory roles, though only the first two are supported by empirical evidence.
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While it appears that the axillary glands of catfishes do not function as part of the venom delivery
apparatus, their true function and the action of their products remains a potentially fruitful area for future
research. Fairly simple procedures, such as comparative electrophoresis, HPLC, or mass spectrometry of
venom and axillary gland extracts, could be used to more conclusively rule out the presence of axillary
gland secretions on the pectoral spine. Further investigations of the antimicrobial and ichthyotoxic
hypotheses that have thus far received preliminary support are also warranted.
Pharmacology and Toxicology of Siluriform Venoms

Wright (2009, 2011, 2012a, b) has demonstrated that the crude venom extracts of a phylogenetically
diverse group of catfish species produce a wide array of symptoms when injected into a model predatory
species (largemouth bass), most notably the rapid loss of color pattern throughout the body, which has
been observed from the venoms of nearly every catfish species studied thus far. This suggests the presence
of conserved venom function that acts in some way on the nervous system, which controls chromatophore
and melanophore activity. Suites of additional envenomation symptoms observed by Wright (2009, 2011,
2012a, b) were highly species specific and included the expansion of melanophores at the injection site,
rapid loss of color pattern elsewhere on the body, muscle spasms of varying degrees of intensity and
duration, hemorrhage, loss of equilibrium, and, in the case of Plotosus lineatus, rapid mortality. Earlier
work by Birkhead (1967, 1972) examined the venoms of several species from the North American family
Ictaluridae that were also studied by Wright (2012b) and found that they produced some of the same
symptoms, including melanophore expansion and hemorrhage. Several additional symptoms were found,
however, including notable edema, necrosis, and death. Some of these differences may be attributable to
Birkhead’s use of a different assay organism (Gambusia affinis) in his assessments of venom toxicity. It
must be noted, however, that the effects demonstrated by both Birkhead (1967, 1972) and Wright (2009,
2011, 2012a, b) were likely elicited by the injection of much higher doses of venom than would be
encountered in a natural situation. These encounters usually result in violent ejection of the catfish from
the buccal cavity of the bass, accompanied by rapid gaping of the mouth and flaring of the gills (Wright
2011, 2012a).
As naturally occurring substances which are able to elicit potent responses in vertebrate physiological
systems, the venoms of fishes have come under increased scrutiny as possible sources of future biomed-
ical compounds. Studies of the toxic effects elicited by fish venoms in other organisms have revealed a
high degree of similarity in these effects and the mechanism of their production, providing an additional
example of apparent convergent evolution of fish venom glands and the substances they produce. The
most common sites of human envenomation are the hands or feet, and in many cases, the pain has been
known to travel up the entire length of the affected appendage (Halstead et al. 1953; Calton and Burnett
1975; Halstead 1988; Church and Hodgson 2002; Sivan 2009). The pharmacological actions of the
venoms of a select few catfish species have been studied and have been shown to have cardiovascular,
neuromuscular, and general cytolytic effects in various assays (Church and Hodgson 2002; Sivan 2009).
The widespread elicitation of cardiovascular effects by piscine venoms in experimental tissue prepa-
rations indicates convergence in venom target systems, although the nature of these effects and the
mechanisms by which they are produced vary between species and taxonomic groups. The venoms of
Plotosus canius and Heteropneustes fossilis are thought to either contain or cause the release of
prostaglandins, contributing to their production of smooth muscle contractile responses in a number of
tissue preparations (Church and Hodgson 2002; Sivan 2009). In contrast, the smooth muscle contraction
produced by the venom of Arius thallasinus appears to be produced through effects on muscarinic
acetylcholine receptors (Church and Hodgson 2002; Sivan 2009). Effects on cardiac muscle preparations
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are similarly variable, with the venom of H. fossilis producing inotropic increases in guinea pig and toad
hearts, while toxin-PC isolated from P. canius causes cessation of heartbeat in guinea pig preparations
(Auudy and Gomes 1996; Church and Hodgson 2002; Sivan 2009). The combined effects of siluriform
venoms on blood vessel and cardiac function have also produced alternate results in in vivo preparations.
The venom of P. canius has been shown to produce a hypertensive response, while that of H. fossilis
produces a hypotensive effect (Auddy and Gomes 1996; Church and Hodgson 2002; Sivan 2009).
Potent neuromuscular activities have been reported from several catfish venoms, in addition to the
systemic, neurologically mediated color loss and muscle spasms observed in toxicity assays using living
predators. The crude venom of Plotosus canius has been shown to irreversibly inhibit electrically induced
muscle contractions in rat and chick muscle preparations, as has an isolated preparation of toxin-PC, the
lethal component of that species’ venom (Church and Hodgson 2002; Sivan 2009). It is thought that toxin-
PC prevents neurotransmitter release presynaptically, as it produces sustained muscular contraction
without affecting muscular preparations’ responses to acetylcholine or carbachol, although its blockage
of neuromuscular activity apparently does not result from K+-channel or cholinesterase modulating
abilities (Auddy and Gomes 1996; Church and Hodgson 2002; Sivan 2009). The venom of a related
species, P. lineatus, has also been shown to produce neurotoxic symptoms upon intraperitoneal injection
into mice (Fahim et al. 1996). In another case of interspecific divergence in siluriform venom effects,
however the venom of Heteropneustes fossilis has been found to display no appreciable neuromuscular
effect (Church and Hodgson 2002; Sivan 2009).
Nearly all piscine venoms exhibit cytolytic properties, and the venoms of catfishes are no exception. In
fact, local necrosis is one of the most common clinical symptoms of piscine envenomations (Sivan 2009)
and has also been documented in Birkhead’s (1967, 1972) envenomations of Gambusia with ictalurid
species’ venoms. The lack of such symptoms in Wright’s (2009, 2011, 2012a, b) experiments, however,
may indicate that these necroses are largely due to secondary bacterial infections. Nonetheless, the
venoms of several siluriform species have produced hemolysis in rabbit (Plotosus canius), rat
(P. canius, P. lineatus), human (Arius thallasinus), mouse (P. canius), cow (A. thallasinus, P. canius),
and sheep (A. thallasinus) erythrocytes (Church and Hodgson 2002). The venom of P. lineatus has
additionally been shown to be cytotoxic to cultured Ehrlich ascites tumor cells as well as a number of other
cell types (Fahim et al. 1996). The cytolytic action of these venoms is thought to contribute to other
negative effects of envenomation, through forming pores in the plasma membranes of target cells,
allowing the influx of Ca2+ which triggers the release of several biologically active compounds from
the cell (Church and Hodgson 2002). Such an action is also known from bee (Pawlak et al. 1991) and
platypus venoms (Kourie 1999), both of which are primarily pain-producing venoms, like those of
catfishes.
Chemistry of Siluriform Venoms

Proteins
The majority of existing information regarding the toxic proteins found in siluriform and other piscine
venoms concerns the sizes of these compounds in various species. Of the ten fish species’ venoms detailed
by Church and Hodgson (2002), the sizes of the toxic compounds ranged from 15 to 324 kDa. Catfish
venoms generally fall within the lower end of this range (10–15 kDa) (Calton and Burnett 1975; Auddy
and Gomes 1996), although Wright (2009) identified an additional putative toxin of approximately
110 kDa in the venoms of several species. Siluriform venoms appear to display a high degree of
conservatism in at least some of their toxic components, as this putative 110 kDa toxin has been found
in the venom electrophoretic profile of nearly every siluriform species thus far examined (Wright 2009,
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Fig. 4 SDS-PAGE profiles of venom extracts from several catfish species. Left lanes represent venom extracts, right lanes
represent extracts prepared from fin tissue. Arrows indicate positions of unique venom protein bands or proteins found in
greater concentrations in venom extracts than in fin tissue extracts. (?) represents ambiguity between smearing and an
additional, unique venom peptide band. Large quantities of a 110 kDa peptide are found in the venom extracts of nearly all
species shown, with the exception of Pimelodus. The presence and variation of venom peptides in the size range of 10–20 kDa
is also clearly visible. Samples from non-venomous Ameiurus melas are shown for comparison (Figure reproduced from
Wright (2009))
2011, 2012a, b; Fig. 4). Without additional information regarding the actual amino acid sequence and
structure of this protein, however, it is not possible to state conclusively that the identity of this venom
protein is the same between all species in which it has been found. Nonetheless, the widespread presence
and apparent conservation of a toxic peptide of this size in catfish venoms indicates that these proteins are
likely to be involved in the rapid loss of coloration seen when a natural predator is injected with catfish
venom extracts (Wright 2009, 2011, 2012a, b).
Additional putatively toxic peptides, generally falling within the size range of 10–20 kDa (Calton and
Burnett 1975; Church and Hodgson 2002; Auddy and Gomes 1996; Wright 2009), have been identified in
the venoms of many siluriform species, although putative toxins of 40–50 kDa have been indicated in
some ariid species (Junqueira et al. 2007; Wright 2009). The lethal fraction of the venom of Plotosus
canius (toxin-PC) is one such protein, having a molecular weight of approximately 15 kDa (Auddy and
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Gomes 1996; Wright 2009). These smaller venom components show significant variation in number and
size over interspecific, intergeneric, and interfamilial scales, identifying them as likely candidates
underlying the variation observed in the effects elicited by different species’ venoms in toxicological
assays (Wright 2009, 2012b). This high degree of variation, even between relatively closely related
species, would seem to strongly indicate that selective forces associated with different habitats and/or
predatory regimes have contributed to the establishment of differing levels of venom protein identity and
complexity between different siluriform lineages. Our current lack of information regarding the genes
coding for these proteins, as well as their structure and physiological targets, precludes the testing of
further hypotheses regarding the evolution of these compounds. The molecular weight data obtained thus
far for siluriform venoms is nonetheless valuable, as it offers an independent check on the identities of the
potentially novel toxin-related sequences that will undoubtedly be uncovered by future evolutionary
studies utilizing omics-scale technologies and analytical methods to identify catfish venom toxin genes,
transcripts, and proteins.
Chemical Complexity of Siluriform Venoms

In contrast to the venoms of organisms that utilize these secretions in prey capture, which can potentially
contain hundreds of toxic components per species, the venoms of fishes and other organisms that utilize
venom in a strictly defensive capacity appear to contain only one or a few toxic components (Church and
Hodgson 2002; Wright 2009; Casewell et al. 2012). This has been confirmed for several catfish species
using comparative electrophoresis of extracts prepared from fin spines and associated tissues, which
showed only one to three unique peptides being expressed in spine extracts relative to control extracts
prepared from histologically similar fin tissues (Wright 2009, 2011, 2012a, b). The toxic nature of these
peptides was confirmed using toxicity assays performed in largemouth bass (Micropterus salmoides),
which showed marked, species-specific effects associated with injection of fin spine extracts, but no
toxicity associated with the injection of fin tissue extracts. An interesting parallel to this condition of
reduced venom toxin diversity is found in the venoms of sea snakes, which have also been shown to
contain a highly reduced number of toxic components relative to other venomous snakes (Fry et al. 2003).
The similarities become even more striking when one considers that venomous marine snakes represent
two evolutionary radiations that have independently arrived at a state of reduced venom complexity
(Scanlon and Lee 2004), while compositionally simple venoms have been independently derived in
acanthomorph fishes no fewer than 11 times (Smith and Wheeler 2006), and at least twice in catfishes
(Wright 2009).
The white catfish (Ameiurus catus) may represent an exception to the generalization that piscine
venoms exhibit low toxin diversity. The venom of this species was found to contain two to eight fractions
that showed lethal activity in mice (Calton and Burnett 1975). The additional finding that A. catus venom
lost little to no activity following treatment with trypsin and elevated temperature indicates that additional,
non-proteinaceous compounds may be present in the venomous secretions of this species. These results
are questionable however, as different methods of analysis yielded proteinaceous fractions of varying
weights and biological activities. Wright (2012b) also examined the venom of this species, using the
comparative electrophoretic methods discussed above, and found evidence for only two putative toxic
peptides in prepared fin spine extracts; these produced a significant toxic effect in injected largemouth
bass. While this study could not speak to the possible presence of non-proteinaceous toxins in A. catus
venom, it clearly supports the lower value from Calton and Burnett’s (1975) estimate of the number of
lethal fractions in the venom of A. catus and is much more consistent with what is known from other
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species. It is possible, however, that siluriform venoms show intraspecific regional variation and that the
conflicting results of these studies resulted from drawing individuals from geographically distant
populations (Chesapeake Bay tributaries in the case of Calton and Burnett, an inland North Carolina
lake in the case of Wright).
The low number of toxic compounds found in fish venoms would appear to be an asset to studies of
their evolution, as the problems of homology inherent in evolutionary studies of species that produce
many different toxins should be easily addressed. It is tempting to suggest that the parallel streamlining of
these species’ venoms is due to selection associated with a common target: piscine physiological systems.
Little empirical evidence exists to support this hypothesis however, as few studies of the action of sea
snake and piscine venoms on their (presumed) natural targets exist. The few studies of sea snake venoms
performed in this context have indicated that likely prey species possess high levels of resistance to sea
snake venoms (Heatwole and Powell 1998). This would appear to run counter to a selective streamlining
hypothesis, as one might expect these species of sea snakes to possess more complex venoms to overcome
prey resistances to particular toxic compounds. Preliminary results from studies on ictalurid catfishes
(Wright 2012b) indicate that the venoms of bullheads have little effect on potential predators with which
they share a habitat type. These results may indicate that coevolution between predator and prey is
occurring in these systems, leading to these somewhat counterintuitive results. Further studies are clearly
necessary to examine possible correlations between the low number of toxic compounds in catfish
venoms and the selective factors influencing siluriform venom evolution.
Evolutionary Origins of Siluriform Venom Glands

Phylogenetic Distribution
Our lack of knowledge regarding basic characteristics of siluriform venoms and their targets represents a
significant obstacle to the study of their evolution, which is compounded by our incomplete understanding
of siluriform phylogeny. Current classifications divide modern catfishes into two monophyletic suborders,
the Loricarioidei (South American armored, sucker-mouthed, pencil, and parasitic catfishes) and the
Siluroidei (all remaining catfish families). The morphologically primitive family Diplomystidae has been
alternatively recovered as the sister group to all catfishes, or the sister group of the Siluroidei, with the
Loricariodei sister to all other catfishes. Multiple higher-level phylogenetic analyses of catfishes, using
both morphological and molecular data (e.g., Diogo 2004; Hardman 2005; Sullivan et al. 2006), are
available, but these studies nearly universally suffer from poorly supported resolution of the early
evolutionary relationships between catfish families, particularly within the Siluroidei.
Wright (2009) mapped the presence of venom glands (as determined from histological surveys) onto
available siluriform phylogenies (Fig. 5), generating reasonable inferences regarding the number of times
venom glands have been evolved within the order, in spite of these poorly resolved basal relationships.
The presence of venom glands in the Callichthyidae almost certainly represents an independent origin of
venom glands within the Loricarioidei, as none of the other families within the suborder showed any
evidence of venom gland tissue associated with their fin spines. Venom glands are widespread in the
Siluroidei (19 of the 20 known venomous siluriform families), indicating that a single, relatively basal
origin of venom glands within this suborder is the most parsimonious hypothesis, although the exact
evolutionary placement of this event awaits further resolution of relationships within the clade. Sullivan
et al.’s (2006) proposed phylogeny requires a third evolutionary derivation of venom glands in the South
American family Doradidae due to the recovery of this family in a sister relationship with the
nonvenomous Auchenipteridae, within a clade also containing the Aspredinidae, another nonvenomous
family. The venom glands of doradids do vary significantly from those of other siluroid groups in terms of
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Fig. 5 Results of mapping the presence of venom glands onto a siluriform molecular phylogeny. Phylogeny from Sullivan
et al. (2006). Red branches indicate venomous lineages; black branches indicate non-venomous lineages. An independent
origin of venom glands in the Callichthyidae is clearly supported. The possible independent origin of venom glands in the
Doradidae is also depicted. The evolutionary history of venom glands at the base of the Siluroidei is obscured, due to poor
phylogenetic resolution, but a single origin in the early history of the suborder remains the most parsimonious hypothesis
(Figure reproduced from Wright (2009))
their organizational structure, orientation relative to the fin spine, and visibility without magnification,
offering morphological support for a hypothesis of an independent redevelopment of venom glands
within the family following their secondary loss during the origins of this clade.
Evolution from Epidermal Secretory Cells

There is strong evidence that the venom glands of several previously studied catfish species produce
similar compounds to epidermal glandular cells. Immunocytochemical assays of epidermal cells taken
from Plotosus lineatus have indicated that these cells produce a highly similar protein to one of the toxic
fractions identified from the venom gland of that species (Shiomi et al. 1988). Further evidence for this
similarity is provided by the results of SDS-PAGE analyses performed by Wright (2009). This study
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indicated the presence of major toxin bands in the venom of P. lineatus, at 15–16 kDa and 13–14 kDa, in
addition to the conserved 110 kDa putative toxin found in the venoms of most catfishes. The larger peptide
is likely to represent toxin-PC, which showed a similar molecular weight in previous characterizations by
Auddy and Gomes (1996) in the related species P. canius. The smaller peptide, however, is very similar in
molecular weight to the toxic fraction isolated from epidermal secretions of P. lineatus by Shiomi
et al. (1987, 1988). Wright (2009) also identified a 39 kDa putative toxin in the electrophoretic profile
of the venom of Arius jordani. This corresponds closely with the major toxic factor of the skin secretion of
the congeneric A. bilineatus, which has been isolated and shown to have a molecular weight of
approximately 39 kDa (Thomson et al. 1998).
Two scenarios have been proposed to explain the evolutionary origins and derivation of venom glands
and their products in catfishes, both of which theorize that these structures and their products are derived
from epidermal secretory cells. The first of these, developed by Cameron and Endean (1973) and outlined
in the above discussion of siluriform venom gland cellular morphology, hypothesizes that the venom
glands of all fishes, including catfish species, are derived from crinotoxin-producing epidermal cells. In
this scenario, the early stages of venom gland development would consist of a thickening of crinotoxin-
producing epidermal tissue surrounding the spines of early venomous catfish species, offering a selective
advantage to these individuals in deterring potential predators. Subsequent evolutionary changes, includ-
ing further increased concentrations of toxic protein-secreting cells and their segregation from the
epidermal tissue by an integumentary sheath; the suppression of other epidermal cell types from being
produced within this tissue; and the movement of this glandular tissue closer to the fin spines, thereby
achieving efficient delivery of cellular products during envenomations, then established the morphology
of siluriform venom glands as they are seen today. Modifications to fin spines facilitating the delivery of
venom into wounds, such as the grooves found in the spines of akysid, amblycipitid, and some ictalurid
catfishes (Wright 2009, 2012a, b; Egge and Simons 2011), are hypothesized to have occurred secondarily
to the development of venom glands in ancestral species.
It is true that crinotoxins are released when epidermal cells are damaged during predation attempts on
catfishes, which is evocative of the manner in which venom is released when the spines of a catfish enter a
potential predator. The actual function of these toxins appears to be in the deterrence of fouling organisms,
however, as ichthyocrinotoxic species are characteristically sedentary and possess decreased or absent
squammation (Cameron and Endean 1973). Crinotoxins have also never been shown to have any
appreciable predator deterrent effects, and, in fact, predatory species will readily attack and feed on
damaged and distressed catfishes (Bosher et al. 2006; Emmett and Cochran 2010; Wright 2012a) as well
as baits coated with stress-related epidermal secretions (Al-Hassan et al. 1985). Studies of the skin
secretions of several Arius species have indicated that compounds contained therein are able to accelerate
healing of wounds and may also have antimicrobial properties (Al-Hassan et al. 1983, 1985, 1987;
Robinette et al. 1998). Antimicrobial capabilities have also been demonstrated from the axillary gland
secretions of callichthyid catfish species, which are also thought to be derived from epidermal stress-
related secretions (Greven et al. 2006; Kiehl et al. 2006), suggesting that secretory cells producing
antimicrobial products have already been co-opted into other siluriform secretory structures.
This information led Wright (2009) to propose an alternative selective scenario to the one proposed by
Cameron and Endean, centering on the apparent healing and antimicrobial properties of catfish epidermal
secretions (see also ▶ Venom as a Component of External Immune Defense in Hymenoptera, this
volume). The epidermal tissue covering the spines of catfishes is frequently damaged during interactions
with predators and with their physical environment. It is therefore conceivable that higher concentrations
of epidermal secretory cells surrounding the spine could confer a selective advantage, through improved
healing times and decreased opportunities for infection of compromised tissues. This selection would lead
to increased aggregations of these cells around the fin spines, with the toxic, antipredatory effects of their
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secretions being either an epiphenomenon to their primary healing benefits in catfish species or second-
arily developed to augment existing defensive structures. Once venomous secretions had been established
and associated with their delivery devices, lineage-specific selective regimes could then act on venom
toxicity and composition in catfishes to produce the compositional and toxicological variation found in
the venoms of modern siluriform species as well as the conservation of the primary, pain-producing
peptides that form the basis of their predator deterrent abilities.

The evolutionary study of siluriform venom glands and their products represents an important but
understudied area of inquiry, due both to their human impacts and potential untapped benefits as well
as the fact that they represent an important antipredatory trait in a globally ubiquitous group of organisms,
which often represent a significant portion of a given region’s aquatic vertebrate biodiversity. Though
these structures have been shown to provide a formidable defense against predators in several cases, even
influencing other aspects of morphological evolution in some genera, secondary losses of venom glands
are evident in several groups, most likely due to relaxation of predation pressures due to different aspects
of life history and habitat choice. Venomous catfishes comprise a highly diverse group of organisms,
possibly outnumbering all other venomous vertebrates combined, and display a correspondingly high
degree of variation in venom delivery apparatus morphology and venom effects. In natural predators and
laboratory organisms, the venoms of catfishes have been shown to elicit symptoms consistent with
cardiovascular, neurotoxic, hemolytic, and/or lethal effects, with a high degree of taxonomic variation
in the suite of effects induced by different species’ venoms. Despite this, siluriform venoms appear to be
quite simplified, consisting of only a few toxic venom proteins per species. Very few siluriform venoms
have been studied in any detail, however, and future examinations of inter- and intrafamilial variation in
venom toxicity and composition would have great potential to uncover additional venom toxin diversity
within catfishes, as well as to generate insights into ecological differences influencing species-specific
venom characteristics.
Venom glands have arisen independently at least twice within the order Siluriformes, with the potential
for a third origin in the South American family Doradidae. Additional higher-level analyses of siluriform
phylogeny are also required to provide greater resolution of basal siluroid relationships and a well-
supported consensus of internal relationships within this suborder, which will allow stronger conclusions
regarding the developmental history of catfish venom glands to be drawn. Histological, toxicological, and
electrophoretic evidence all suggest that the venom glands of catfishes are evolutionarily derived from
epidermal secretory cells. Whether catfish venoms are derived from crinotoxins or healing and antimi-
crobial substances produced by epidermal cells in the skin is unclear, however. Further studies of both
epidermal secretion types and the venoms of catfishes are required at a proteomic and genetic level in
order to determine the relationships between these different substances. It is entirely possible that these
crinotoxins and antimicrobial agents are one and the same, leaving little hope for possible resolution to the
debate regarding which defensive selective force, increased predator deterrence, or rapid healing and
infection defense initiated the process of venom evolution in catfishes.
Studies making use of next-generation proteomic, transcriptomic, and genomic technologies and
analyses have great potential to generate desperately needed data regarding the identity and structure of
siluriform venom toxins, their physiological targets, their genetic origins, and the selective forces driving
their evolution. Continued studies of catfish venoms have the potential to greatly increase our under-
standing of the general ecology and evolution of this hyperdiverse order of fishes as well as to generate
insights into the evolution of venoms as defensive traits. The chemical complexity of the venoms of
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species utilizing these secretions in prey capture makes it exceedingly difficult to determine whether and
how selection for prey capture or predator defense has influenced any particular venom component, as
these differing selective forces have the potential to be non-complimentary. The relatively simple
composition of catfish venoms, and fishes in general, as well as their use in a strictly defensive capacity,
therefore presents an outstanding opportunity to study the selective factors influencing defensive venom
evolution, examinations of which are largely absent from the literature.
Cross-References
▶ Venom as a Component of External Immune Defense in Hymenoptera
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Evolution of the Snake Venom Delivery System

Harald M. I. Kerkkampa*, Nicholas R. Casewellb and Freek J. Vonka,c,d
a
Institute of Biology Leiden, Sylvius Laboratory, Leiden University, Leiden, The Netherlands
b
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK
c
Naturalis Biodiversity Center, Leiden, The Netherlands
d
Molecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor, UK
Abstract
There are over 3,000 species of snakes known to man. These limbless predators have been divided into
two groups, the basal snakes (Henophidia) and the advanced snakes (Caenophidia). Venom evolved prior
to the advanced snake radiation and, consequently, many use venom to subdue their prey. To do so, venom
is injected via the use of a venom delivery system. The venom delivery system includes a postorbital
venom gland on each side of the upper jaw that is associated with specialized venom-conducting fangs or
teeth. Both the venom gland and fangs are considered to have originated from a common ancestor and are
thought to be developmentally linked to one another. Even though the venom gland has a common
ancestral origin, it can exhibit considerable morphological variation among the main snake families.
Similarly, the fangs can occupy various positions on the upper jaw but are always found on the maxilla.
Caenophidians are often referred to by the position of their fangs as either rear- or front-fanged snakes.
The vast majority of snakes that are medically important to humans are front-fanged, and this character
has evolved independently on at least three occasions. In addition, some front-fanged snakes have evolved
a secondary gland associated with the venom system, known as the accessory gland. The venom glands,
accessory glands, and fangs of different caenophidian snake families exhibit substantial morphological
differences reflecting their evolutionary history. However, further studies are required to fully elucidate
the ecological significance of differences in fang position, the function of the accessory gland, and the
driving forces underpinning the convergent evolution observed in the snake venom delivery system.
Keywords
Venom delivery systems; Evolution; Advanced snakes; Venom glands; Accessory gland; Front-, rear-fang
Introduction
Snakes have been both feared and revered by humans throughout the ages, having been a symbol to the
gods in Mayan culture and a symbol of evil in Christian faith. Indeed, these venomous animals have left,
with their frequently lethal encounters (Kasturiratne et al. 2008), an everlasting impression on humans. It
is even believed that they might have played a prominent part in the evolution of the primate brain and
sensory system (Isbell 2006). Snakes themselves are limbless tetrapods that are represented on earth today
by some 3,150 species (Vidal et al. 2007; Vonk et al. 2011); they are highly specialized predators and they
inhabit most major ecosystems. All snakes share a unique body plan having undergone an elongation of
the body, a loss of most clear morphological boundaries, and a substantial increase of the number of
*Email: h.m.i.kerkkamp@biology.leidenuniv.nl
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Fig. 1 Phylogeny from Vonk et al. 2008. The evolutionary changes leading from an unmodified maxillary dentition to the
different fang types in advanced snakes are indicated at the nodes: (1) continuous maxillary dental lamina, no specialized
subregions – ancestral condition for advanced snakes; (2) evolution of posterior maxillary dental lamina – developmental
uncoupling of posterior from anterior teeth; (3) starting differentiation of the posterior teeth with the venom gland; (4) loss of
anterior dental lamina and development of front fangs. Asterisks indicate the families represented by the traditional
“colubrid” name
vertebrae (up to 500) (Vonk and Richardson 2008). Though their different body plan gives snakes a
distinctive appearance, it is the incongruity between their often fragile appearance and the devastating
damage they can inflict with their venom that is notable. Whereas the Henophidia (basal snakes such as
pythons and boas) rely on constriction for prey capture, the vast majority (ca. 2,700 species) of snakes
utilize venom. This large group, known as the Caenophidia (advanced snakes), represents a single
massive diversification event that occurred after the Cretaceous-Tertiary boundary and the extinction of
the dinosaurs around 64 million years ago (Vidal et al. 2009; Vonk et al. 2011). Most of the advanced
snakes (Caenophidia) have the ability to inject venom into prey using their venom delivery system
(Warrell 2010). The major snake families within the Caenophidia are the Viperidae (e.g., vipers, adders,
pit vipers, and mocassins), the Elapidae (e.g., cobras, mambas, kraits, coral snakes, taipans, and sea
snakes), the Atractaspididae (burrowing asps), and the Colubridae sensu lato (Fig. 1). The term
Colubridae sensu lato represents a traditional name for all snakes with a venom gland whose venom
poses no danger to humans (i.e., any species of advanced snake that is not a member of the Viperidae,
Elapidae, or Atractaspididae). Recently, the Colubridae have been shown to be paraphyletic and have
been redefined with many subfamilies (e.g., Dipsadidae, Pseudoxenodontidae, Colubridae, Natricidae,
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Fig. 2 Schematic ventral views of adult skull and in situ of venom delivery system of (a) “colubrids,”( b) Viperidae, (c)
Elapidae, and (d) Atractaspidinae. Maxilla colored gray, teeth colored green, fangs colored orange, AG accessory gland,
D duct, SD secondary duct, PD primary duct, LU lumen, VG venom gland (Redrawn from Kochva (1987) and Vonk
et al. (2008))
Lamprophiidae, Homalopsidae, Pareatidae, and Xenodermatidae) being elevated to a family level to

reflect their evolutionary distinctiveness (Fig. 1) (Vidal et al. 2007; Lawson et al. 2005). However, for
clarity of description, the traditional “colubrid” name will be resurrected to represent these families here.
While venom delivery systems have evolved independently among vertebrates on multiple occasions
(Casewell et al. 2013), advanced snakes exhibit the most complex, specialized, and variable venom
system. Its main function is the production of a specialized toxic secretion in the venom gland and its
delivery from this gland into prey (and occasionally predators or aggressors, such as humans). The venom
delivery system of advanced snakes primarily consists of a postorbital venom gland associated with
specialized venom-conducting fangs (Fig. 2). Though the fangs of advanced snakes can occupy various
positions on the upper jaw (e.g., at the caudal or rostral end), they are always found on the maxilla (Vonk
et al. 2008). In this chapter, the components that comprise the snake venom delivery system will be
discussed. First, the venom itself will be examined as a secretion, before discussing the venom-producing
glands, the venom ducts, the fangs, and the specialized accessory gland found downstream of the venom
gland in some species (Fig. 2). Second, a comparative analysis of the development of the venom glands
and fangs for each of the advanced snake families will be provided and, finally, an overview of the current
understanding of the evolution of the venom system of advanced snakes.
Venom
As limbless predators, the majority of advanced snakes rely on venom to immobilize or incapacitate their
prey. Snake venom is a complex cocktail of bioactive components, largely consisting of a mixture of
proteins and peptides (referred to as toxins) but also containing salts and organic compounds such as
amino acids and neurotransmitters. Venom toxins are biologically active proteins most of which are
encoded by several multilocus gene families, which function synergistically to facilitate prey incapaci-
tation and capture (Casewell et al. 2013; Vonk et al. 2013). A few venom toxins appear to be modified
salivary gland secretions, whereas many venom genes have originated from housekeeping genes
co-expressed in other organs, which have either been recruited for, or restricted to, expression in the
venom gland (Fry 2005; Vonk et al. 2013; Hargreaves et al. 2014; Junqueira-de-Azevado et al. 2015;
Reyes-Velasco et al. 2015). Following their recruitment to the venom gland, it is apparent that gene
duplication events are critical for the expansion of the toxin repertoire found in the venom of many of the
advanced snakes (e.g., Casewell et al. 2011, 2014; Vonk et al. 2013; Sunagar et al. 2013). Once injected,
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venom toxins permeate from tissue and become systemic via dispersal by the bloodstream and lym-
phatics. Venom toxins are capable of interacting with a wide variety of physiological proteins and
receptors present in their prey, ultimately resulting in disruptions to the central and peripheral nervous
system, the blood coagulation cascade, the cardiovascular and neuromuscular system, and/or the homeo-
stasis in general. Synergism between different venom toxins also appears to be commonplace, with
distinct or related proteins present in venom capable of targeting multiple different steps of the same
physiological pathway, such as the coagulation cascade or neurotransmission.
Venom Glandular Apparatus

The venom glandular apparatus is a compilation of all the components utilized by advanced snakes to
produce, store, and deliver venom to the fangs. Though there is a high degree of variability in the
apparatus components, all advanced snakes possess a pair of postorbital glands in which venom is
secreted and stored. These structures are naturally referred to as venom glands. The glands are enclosed
in a fibrous sheath that, in some species, permits the attachment of muscles. These venom glands are
considered to be specialized postorbital oral glands that produce venom and are developmentally coupled
to fang formation (Kochva and Gans 1965). Through morphological, embryological, and developmental
biological studies, the venom glands found in the different caenophidian snake families are considered to
be homologous and therefore to be the result of a single origin at the base of the colubroid radiation
between 60 and 80 million years ago (Fry 2005).
From this early common origin of the venom gland, the venom glandular apparatus has undergone
severe evolutionary tinkering in each of the Colubridae, Viperidae, Elapidae, and Atractaspidinae snake
families. In “colubrids,” the venom gland is positioned posterior to the eye, usually encased in a thin cover
of connective tissue, and consists mostly of serous cells (i.e., specialized to secrete an enzyme solution).
A single, short duct extends anteromedially from the lumen of the gland to the base of the posterior fang
(Fig. 2a). In contrast, the viperid venom gland is large and generally triangular in shape, with the longest
side of the triangle found along the upper lip and the rounded apex directed dorsally. The main venom
gland has a complex tubular structure and is divided into several lobules by infoldings of the outer sheath.
The lumen is voluminous and can store a large quantity of venom. Anteriorly, the triangle comes to a point
as the lumen forms the primary venom duct. This duct then passes through an accessory gland, leaving it
as the secondary duct, which extends to the sheath of the fangs. Just proximal to the accessory gland, the
primary duct narrows, forming a glandular isthmus, which restricts the passage of venom to the distal
components of the venom gland apparatus (Fig. 2b) (Mackessy and Baxter 2006). The elapid venom
gland is oval in shape. It is made up from a main venom gland and an accessory gland, but, in contrast to
vipers, does not have a primary duct connecting the two. Instead, the accessory gland forms the distal part
of the venom gland. The main venom gland is made up of many simple or branching tubules. The lumen is
narrow and most of the venom is stored within secretory cells rather than the lumen. The accessory gland
surrounds the entire duct, so that the narrow lumen continues through it (Fig. 2c). The atractaspid venom
gland is cylindrical. A central lumen extends along the length of the gland with a characteristic pattern of
unbranched tubules radiating outwards. There is no distinctive accessory gland, but mucous cells line the
lumen along most of the length of the gland, in contrast to the serous cell lining observed in the vipers and
elapids (Fig. 2d) (Jackson 2002).
Venom Gland
Venom glands reside next to the upper jaw behind the eye, although in some taxa they can be highly
elongated, extending posteriorly along the body well beyond the head (Fry et al. 2008). The main venom
gland contains at least four distinct cell types: secretory cells (79 %), mitochondria-rich cells (2 %),
horizontal cells (10 %), and dark cells (9 %) (Mackessy 1991). Venom glands are innervated by the
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maxillary branch of the trigeminal nerve (V2) with contributions from the facial nerve (VII) (Kochva
1965; Taub 1966), and their vascular supply is from branches of the internal carotid artery (Phisalix 1922;
Kochva 1965). Depletion of stored venom stimulates a new cycle of venom synthesis, and the secretory
epithelium undergoes morphological and biochemical changes. In response, the epithelial cells change
their shape from cuboid to columnar; the cistern of the rough endoplasmic reticulum expands and venom
is synthesized (Carneiro et al. 1991; Kochva et al. 1980; Mackessy 1991; Warshawsky et al. 1973). This
happens in response to stimulation by the autonomic nervous system (Kerchove et al. 2004; Yamanouye
et al. 1997). The venom production cycle lasts around 30–50 days and, within 4–8 days, proliferation of
the rough endoplasmic reticulum and messenger ribonucleic acid (mRNA) levels have reached their peak
(Carneiro et al. 1991; Kochva et al. 1980; Mackessy 1991; Rotenberg et al. 1971; Yamanouye et al. 1997),
and subsequent merocrine exocytosis results in the replenishment of venom in the epithelial ductules and
large basal lumen. Completion of the synthesis and secretion stage occurs approximately 16 days after
depletion of the gland, and during this period, cells cycle from cuboidal to columnar and back to cuboidal
morphology (Kochva 1987; Kochva et al. 1975; Mackessy 1991). The venom is then stored in the basal
lumen and ductules of the venom gland for varying periods of time and is therefore available when needed
(Mackessy and Baxter 2006).
Compressor Musculature
As previously described, the venom glands are enclosed in a fibrous sheath that, in some snakes, facilitates
the attachment of muscles. The musculaturization of the venom glands observed in Viperidae, Elapidae,
and Atractaspidinae snakes allows the ejection of venom from the glands and injection into prey in a high-
pressure manner (Vonk et al. 2011). The contracting muscle that compresses the venom gland is the
muscularis compressor glandulae (Rosenberg 1967; Jackson 2007; Warrell 2010). Though all three
families possess this musculature, they are not homologous. The elapid compressor glandulae muscle is
derived from the adductor externus superficialis muscle, the viperid from the adductor externus profundus
muscle, and the atractaspididae from the adductor externus medialis muscle (Jackson 2007).
Accessory Gland
The accessory gland, a glandular structure found in Viperidae and Elapidae snakes, is associated with the
main venom gland and has two distinct regions: the anterior and posterior. The anterior region has a
simple secretory epithelium with at least six types of cells: two types of secretory cells, mitochondria-rich
cells without secretion, horizontal cells, dark cells, and basal cells. The posterior region has a simple
epithelium with two types of cells: seromucous cells and horizontal cells (Sakai et al. 2012). In elapids, the
accessory gland begins immediately anterior to the main gland and completely surrounds the entire duct
of the venom gland. In viperids, the accessory gland is considerably smaller and surrounds the anterior
end of a primary duct (Kochva and Gans 1970). As in the main venom gland, the anterior region of the
accessory gland displays a long secretion production cycle lasting around 15 days. The peak of secretion
occurs 4 days after venom extraction, and cells are replenished with secretory vesicles approximately
15 days after venom extraction (Sakai et al. 2012).
The primary function of the accessory gland remains unknown. It has been postulated that it may
condition or activate venom passing through it during injection (Gans and Elliott 1968). The presence of
serous cells caudally, followed rostrally by mucus-secreting epithelium (Hattingh et al. 1984; Mackessy
1991), implies that lytic venom components passing through the accessory gland are activated by the
caudal portion (Mackessy and Baxter 2006). The accessory gland, especially the rostral part, may actually
contribute substances to the venom during injection. Supporting this hypothesis is evidence that the
accessory gland appears to express most toxins that are expressed in the venom gland, although it does so
at a much lower level (Vonk et al. 2013). However, electrophoresis and reverse-phase high-performance
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liquid chromatography analyses have yet to identify any peptide or protein components that have been
added to the venom bolus exiting the intact apparatus, when compared with samples taken extracted from
the main venom gland (Mackessy and Baxter 2006; Weinstein et al. 2010).
Fangs
Fangs are grooved or hollow teeth used to deliver venom. There is extensive variation in both the
morphology and the position of the fangs throughout the advanced snakes, but the fangs are always
located on the maxilla. In addition, the length of the maxilla is also variable. For example, in viperids and
atractaspids, it is simply a stub serving as a base for the fang, whereas in “colubrids” the maxilla is long
and forms the base for teeth in addition to the fangs. Like all teeth, fangs are continuously replaced
throughout the life of the snake. The functional fang is ankylosed to the maxilla, with a series of
replacement fangs, which are not attached to any dentigerous bone, caudal to it. These replacement
fangs are formed starting with the distal tip and growing by gradual accretion of material to the proximal
end. When the functional fang is shed, the replacement fang will ankylose to the maxilla and take its place
(Jackson 2007; Zahradnicek et al. 2008).
Snakes can be divided into three groups based on their fang position: (i) snakes with no fangs, (ii) rear-
fanged snakes, and (iii) front-fanged snakes. The Henophidia have no fangs and instead only have teeth on
the maxilla, the “colubrids” are rear-fanged snakes, and the Viperidae, Elapidae, and Atractaspidinae are
front-fanged snakes. Unfanged snakes have no specialized teeth on the maxilla. The maxillary teeth
develop from a single continuous maxillary odontogenic band, and this band invaginates to form one
dental laminae, from which all teeth develop.
In rear-fanged snakes, the fangs are positioned at the caudal end of the maxilla, whereas there are
normal teeth present on the rostral end of the maxilla. The fangs can be ungrooved and resemble enlarged
teeth (with some slight morphological differences) or grooved to facilitate venom delivery by possessing
an open channel along the lateral or anterolateral surface (Jackson 2007). The maxillary teeth and fangs of
the rear-fanged snakes develop from two dental laminae which invaginate separately and fuse during
development. The anterior and posterior dentitions have been found to be developmentally independent
from each other: the anterior dental laminae only bear teeth, similar to those found in non-fanged snakes,
whereas the posterior laminae bear both teeth and the common primordium with the postorbital gland that
develops into both the rear fangs and the venom gland (Vonk et al. 2008).
In the front-fanged snake families, the fang is positioned at the rostral end of the maxilla, and there are
no normal teeth present. The front-fangs are more or less closed, hypodermic-like, tubular venom-
injecting fangs that are open through two orifices situated at the top and bottom on the ventral tooth
side. At the fang base, one orifice is connected to the venom duct, and at the apex of the fang, the channel
that runs through it does not extend all the way to the distal tip of the fang but instead ends somewhat more
proximally to the tip, thereby forming a beveled tip like that of a hypodermic needle (Jackson 2007;
Zahradnicek et al. 2008). The maxillary front fangs develop from a single maxillary odontogenic band.
This maxillary odontogenic band is found in the posterior part of the upper jaw, and there is no dental
lamina in the anterior region of the jaw. The odontogenic band invaginates normally and forms one dental
lamina; the fangs develop from the posteriormost part of this lamina, and no other teeth develop on the
anterior part of it. The anterior toothless part of the dental lamina has been termed the dental ridge and has
been described in both the Viperidae and Elapidae. The fangs then displace from their embryonic posterior
position into its adult front position by ontogenetic allometry (Vonk et al. 2008). Front fangs initially
develop in a very similar fashion to the rest of the dentition, going through the bud, cap, and bell stages of
development. However, at the bell stage, an invagination is observed on the ventral side of the developing
tooth. On the ventral side, the epithelial wall of the developing tooth germ invaginates, and epithelial cells
invaginate into the dental mesenchyme, and the sides of the invaginating wall make contact and fuse to
Page 6 of 11
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form an enclosed canal. The epithelial cells proliferate to enlarge the canal and then die by apoptosis,
forming an empty tube through which the venom flows. The orifices at each end of the canal develop by a
similar invagination, but the initial width of the invagination is different from that in the middle of the
tooth, and it is associated with higher proliferation. The two sides of the invaginating epithelium never
come into contact, leaving the orifice open. Once the infolding process has occurred, hard tissue is laid
down; fangs are formed from the distal tip and grow by gradual accretion of material to the proximal. The
fang is thus built up from the tip to the base (Zahradnicek et al. 2008).
Although all three front-fanged families share these tubular fangs, elapid, viperid, and atractaspidid,
snakes are not monophyletic and, consequently, their fangs exhibit some differences. Similar to “colu-
brids” the Elapidae have a fixed maxilla to which the front fangs are attached, whereas the Viperidae have
front fangs that are mounted on moveable maxilla that allows the fang to be erected when biting and
remain parallel to the jaw when relaxed. Similarly, the Atractaspidinae have moveable maxillae with fangs
attached (Jackson 2007; Zahradnicek et al. 2008). In addition, in viperids the surface of the anterior side of
the fang between the entrance and discharge orifices is smooth, whereas elapids and atractaspids have an
anterior suture line connecting the two orifices, although the venom canal is still enclosed (Jackson 2007;
Zahradnicek et al. 2008).
A further adaptation of the fangs is seen within the Elapidae family, where the fangs of some cobras
(genus Naja) have been modified such that they are capable of spitting their venom toward an aggressor,
to a distance of at least 3 m. While most elapid snakes have a fang exit orifice appearing as an elongated/
hypodermic-like opening, the exit orifice of spitting cobras is directed more cranial and is more or less tear
shaped in outline with the lower edge of the opening appearing rounded, while the upper end of the
opening terminates where the walls of the venom canal meet the suture (Young et al. 2004; Westhoff
et al. 2005; Bogert et al. 1943). This fang morphology has been observed in members of both the African
and Asian cobras, and the spitting cobras are therefore not monophyletic (W€ uster et al. 2007). In addition,
the African rinkhals (Hemachatus haemachatus), an elapid near relative of the cobras, has also developed
the ability to spit its venom. Spitting is therefore considered to have arisen several times independently
(on at least three occasions) in the elapid family (Young et al. 2004; Westhoff et al. 2005; W€uster
et al. 2007).
View on Evolution
It is thought that the venom delivery system of all advanced snakes shares a common ancestral origin (Fry
et al. 2008) (Fig. 1). This ancestor likely possessed a single continuous maxillary dental lamina with no
specialized subregions and a mucous secreting post orbital gland from which the ancestral venom delivery
system evolved. It was previously hypothesized that the snake venom gland evolved by evolutionary
modification of the pancreatic system (Kochva 1987). While this hypothesis has not been borne out (e.g.,
the toxins found produced in the snake venom gland do not appear to be predominately from a pancreatic
origin Fry 2005; Hargreaves et al. 2014; Junqueira-de-Azevedo et al. 2015; Reyes-Velasco et al. 2015), a
recent analysis of micro-ribonucleic acid (miRNA) libraries of the venom gland showed molecular
similarities between venom gland miRNAs and those previously identified from the human and mouse
pancreas (Vonk et al. 2013). These results suggest that certain components of a core genetic network
regulating secretion may have been co-opted from an ancestral role in the pancreas during the evolution of
the snake venom gland (Vonk et al. 2013). Regarding fangs, it is believed that at the base of the advanced
snakes, a posterior subregion of tooth-forming epithelium became developmentally uncoupled from the
remaining dentition and has become developmentally linked to the primordium of the venom gland. This
has allowed the posterior teeth to develop in close association with the venom gland and independently
from the rest of the maxillary teeth. Subsequently, the posterior maxillary dental lamina became fang
bearing and the venom gland became protein secreting, resulting in the first venom delivery system.
Page 7 of 11
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Taking into account the structural homologies observed in front-fanged snakes, extant rear-fanged snakes
appear to represent the venom system most closely related to the ancestral state. For example, derived
characters observed in front-fanged snakes include the toothless dental ridge (observed in elapids and
viperids), which is similar to the part of the posterior dental lamina that fuses with the anterior dental
lamina in rear-fanged snakes. In addition, the posterior developmental origin of the front fangs in both
elapids and viperids, along with the invagination mechanisms by which the orifices in the front fang are
formed, are similar to the grooving of the rear fangs (Vonk et al. 2008; Zahradnicek et al. 2008). It
therefore appears that, from the ancestral state, the venom delivery system has evolved tubular anterior
fangs with an associated muscularized venom gland in at least three different lineages (viperids, elapids,
and atractaspids) (Vonk et al. 2008; Chipman 2009). It is notable that these three lineages contain the vast
majority of venomous snakes that are known to be medically important to humans. The muscularized
front-fang system facilitates the rapid injection of voluminous venom absent in most rear-fanged snakes.
In addition, it is apparent that two unrelated lineages (viperids and elapids) have independently evolved an
accessory gland downstream of the venom gland, but it remains unclear what the function of these glands
are in these families.

Snakes are vertebrates that have undergone a number of notable adaptations, from their loss of limbs and
elongation of their body plan, to the physiological extremes associated with digesting large intact prey
items (Di Poi et al. 2010; Castoe et al. 2013). However, it is the venom delivery system which is perhaps
their most renowned adaptation, and it represents one of the most sophisticated weapon systems found in
the animal kingdom. The main components of this system are the venom-secreting gland and the venom-
conducting fangs (Fig. 2). Interestingly, the front-fanged system of viperid, elapid, and atractaspid snakes
appears to have evolved by the process of convergent evolution. Convergent processes also appear to
underlie the fang adaptations observed in the spitting cobras.
The driving forces underlying the convergent adaptations observed in the snake venom system remain
predominately unknown. Therefore, it is apparent that to better understand the evolutionary mechanisms
acting on the venom delivery system, we need additional information. For example, the ecological
significance of front versus rear fangs remains ambiguous. It is highly plausible that selection would
favor tooth morphologies that are more efficient at introducing venom into prey, such as grooved over
ungrooved fangs or fangs with a closed channel over deeply grooved fangs. Furthermore, whereas rear-
fanged snakes utilize a bite-and-hold method of prey capture, front-fanged systems appear to have
enabled certain snakes to feed on larger (and potentially more dangerous) prey through a bite-and-
release method of venom injection – this adaptation may therefore have allowed those snakes to occupy
different niches to many rear-fanged snakes. In summary, it is plausible that the front-fang venom systems
have evolved convergently because of increased efficiencies, or in response to different lineages
exploiting new predator niches in a similar manner, or perhaps simply because the existing fang
developmental pathways allow limited adaptation (Zahradnicek et al. 2008).
Secondly, the venom system of elapids and viperids both contain an accessory gland (Gans and Kochva
1965; Mackessy 1991; Mackessy and Baxter 2006) (Fig. 2). The function of this small enigmatic gland in
these different lineages remains unclear. It has been speculated that accessory gland secretions keep
venom from continuously draining into the mouth of the snake, that they facilitate the venom flow through
the fang (Gans and Kochva 1965), or are a source of toxins, or that they function to activate the toxins of
the venom gland (Gennaro et al. 2007). The conserved morphology of the accessory glands and the many
cell types present do suggest that they possess an important functional role (Mackessy and Baxter 2006).
Page 8 of 11
DOI 10.1007/978-94-007-6727-0_11-1
However, considering that the accessory gland is only found in the Elapidae and the Viperidae, which do
not form a monophyletic group (Fig. 1), it remains unclear whether these accessory glands are homol-
ogous, share a similar function, and have been lost from other advanced snakes over evolutionary time, or
whether they have evolved independently in elapids and vipers and therefore potentially function in a
differential manner.
Through the application of a variety of new molecular, genetic, and genomic methods available to
scientists today, it is possible that these fundamental questions can be answered to shed light on the
evolutionary mechanisms that underlie the fascinating diversity of venom delivery systems observed in
snakes.
Cross-References
▶ Mutation, Duplication and More in the Evolution of Toxins
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Page 11 of 11
DOI 10.1007/978-94-007-6727-0_12-2
Independent Origins of Scorpion Toxins Affecting Potassium and

Sodium Channels
Shangfei Zhang, Bin Gao and Shunyi Zhu*
Group of Peptide Biology and Evolution, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute
of Zoology, Chinese Academy of Sciences, Beijing, China
Abstract
Peptide neurotoxins targeting sodium (Na+) and potassium (K+) channels are two major components of
scorpion venom for capturing prey (e.g., insects) and deterring competitors (e.g., small mammals).
Although a great amount of information in terms of their sequences, structures and pharmacological
functions is available currently, the origin of these toxins remains unsolved. Based on the genomic
organization and three-dimensional structure similarities together with close functional relatedness, it has
been proposed that these two types of molecules could arise from a common ancestor. However, recent
studies have provided convincing experimental evidence in favor of their independent origins, in which
an ancestral K+ channel toxin firstly evolved from an antibacterial insect defensin-like molecule via a
small deletion of the amino-terminal loop (n-loop) to remove steric hindrance between peptide-channel
interaction whereas scorpion Na+ channel toxins originated from an antifungal drosomycin-like ancestor
through the insertion of a small amino-terminal turn and the extension of a carboxyl-terminal tail to reach
a new receptor region on the channels, in line with the discovery that drosomycin can bind to the
Drosophila’s own Na+ channels. These studies highlight the importance of insertion/deletion (indel)
mutations in toxic origin from ancestral scaffolds of physiological functions.
Keywords
Scorpion toxin; Voltage-gated ion channel; Drosomycin; Insect defensin; Evolution
Introduction
Scorpions are one of the most ancient arthropods and have existed on earth for more than 400 million
years without obvious morphological change (Possani et al. 1999). They developed polypeptide-rich
venom as weapon for predation and defense. These toxic peptides impair functions of a variety of ion
channels (Na+, K+, Cl and Ca2+) present in excitable membranes via interacting with their voltage-sensor
domain (VSD) or the channel pore (Zhu et al. 2005; Wang et al. 2011; Zhang et al. 2011; Banerjee
et al. 2013). Among them, toxins affecting Na+ and K+ channels are the two most abundant peptide
components in the venom. These neurotoxins comprise about 28–76 amino acids with two to four
disulfide bridges and the majority of them adopt a cysteine-stabilized a-helical and b-sheet (CSab) fold.
Although a great amount of information in terms of their sequences, structures and pharmacological
functions is available currently, the origin of these toxins remains an unsolved issue. Similarities in the
genomic organization and three-dimensional structure together with close functional relatedness appear to
*Email: zhusy@ioz.ac.cn
Page 1 of 16
DOI 10.1007/978-94-007-6727-0_12-2
a
NHD
CDD
ChTx (α-KTX) MeuTxKβ1 (β-KTX)
d
c
OmTx3 (κ-KTX) λ-MKIa (λ-KTX)
Fig. 1 The fold diversity of scorpion toxins affecting K+ channels. (a). CoTx1 (a-KTx) (pdb entry 1PJV); (b) MeuTXKb1
(b-KTx) (Zhu et al. 2010a). NHD, N-terminal a-helical domain. CDD, C-terminal defensin domain; (c) OmTx3 (k-KTx) (pdb
entry 1WQE); (d) l-MKIa (l-KTx) (Gao et al. 2013). Ribbon models are generated by MolMol (http://www.gunda.hu/
mol2mol/index.html)
support an opinion of common origin for these two types of molecules (Froy et al. 1999). In particular, it
was proposed that the scorpion short-chain Kv channel toxins might be evolved from a long-chain Nav
channel toxin via genetic “CC” deletion (Céard et al. 2001). Unfortunately, this proposal is only based on
their isolated cDNA sequences and thus genetic polymorphism of the Nav channel toxin gene cannot be
ruled out. By using experimental evolution studies, we provide convincing evidence in favor of their
independent origins, in which the ancestor of K+ channel toxins is traced to an antibacterial insect
defensin-like molecule via evolutionary deletion of a loop to remove steric hindrance between the
peptide-channel interaction (Zhu et al. 2014) and an ancestor similar to the antifungal drosomycin is
proposed to have evolved into scorpion toxins affecting Na+ channels via assembly of a functional
subdomain on a conserved scaffold to target new receptor site on the channels (Zhu et al. 2010b). This
work summarizes current progress on the origin and evolution of these two types of neurotoxins.
Classification and Structures of Scorpion Potassium Channel Toxins

K+ channels are the most diverse type of ion channels with extensive distribution in nearly all cells of
prokaryotic and eukaryotic organisms. They form K+-selective pores spanning cellular membranes and
regulate a wide variety of physiological functions of excitable and non-excitable cells, such as regulation
of action potential of neurons and muscles as well as secretion of hormones. Voltage-gated K+ channels
(Kv) are homo-tetramer comprising four a-subunits, each containing six transmembrane helices (S1-S6).
The S1 to S4 helices comprise the VSD and S5 to S6 constitute the pore domain for specific K+ ion
conduction. Apart from a-subunits, K+ channel complex usually contain additional b-subunits for
modulation of the kinetics and voltage dependence of the a-subunits and the T1 linker domain that
restricts Kv channel subunit heteromultimerization (Chen et al. 2010). Given key physiological functions,
Page 2 of 16
DOI 10.1007/978-94-007-6727-0_12-2
K+ channels have been frequently selected as targets of a diversity of venomous animals, in which the pore
domain is a major site for toxin binding to inhibit the passage of K+ ions (Banerjee et al. 2013).
Scorpion venom is a rich source of peptides targeting various types of K+ channels (KTxs). To date,
about 120 KTxs have been characterized via a combination of molecular and biochemical techniques
(Rodriguez de la Vega et al. 2003). On the basis of sequence similarity as well as phylogenetic relationship,
KTxs have been divided into three major groups: a, b, and g, which all adopt a conserved CSab fold
(Tytgat et al. 1999): (1) Alpha-KTxs are the largest group that contains at least 26 different subfamilies.
They are short-chain peptides composed of 28–45 residues with three to four disulfide bridges and block
several types of K+ channels, such as voltage-gated Shaker-related K+ channels (Kv), ether-a-go-go-related
(ERG) K+ channels, Ca2+-activated K+ channels of high (BK), intermediate (IK) and small
(SK) conductance (Rodriguez de la Vega and Possani 2004). Figure 1a presents a representative structure
of a-KTxs (Fig. 1a); (2) Beta-KTxs are a group of long-chain toxins composed of 50–75 amino acids cross-
linked by three disulfide bridges. In comparison with a-KTxs, all the b-KTx members possess an
N-terminal extension that folds into a-helical conformation and thus named N-terminal helix domain
(NHD) (Fig. 1b) (Zhu et al. 2010a). Their carboxyl-terminal domain is similar to a-KTxs and defensins
from multicellular organisms, called C-terminal CSab domain (CCD domain). Functionally, some b-KTxs
exhibit dual activities as Kv channel blockers and microbicidel agents; (3) Gamma-KTxs consist of 36–43
amino acids with three or four disulfide bridges. These peptides specifically affect the ERG family of K+
channels and have an additional helix in their N-terminus (Frenal et al. 2004).
In addition to these major groups, scorpion venom also contains several minor KTx components with
different folds (Gao et al. 2013), including: (1) k-KTxs are short peptides with extremely low sequence
similarity to other families. Members in this family adopt an uncommon cysteine-stabilized a-helix-loop-
helix (CSa/a) scaffold with two disulfide bridges (Fig. 1c). OmTx1 to OmTx3 are three k-KTx
members purified from Opisthacanthus madagascariehsis with inhibitory effect on Kv1.1, Kv1.2,
and Kv1.3 channels (Chagot et al. 2005); (2) l-KTxs. Toxins in the l-KTx family adopt an inhibitor
cysteine knot (ICK) fold composed of a three-strand antiparallel b-sheet and a 310-helix (Fig. 1d).
l-MeuKTx-1 is a representative of this family and the first number isolated from the scorpion venom
of Mesobuthus eupeus. l-MeuKTx-1 specially blocks the Drosophila Shaker K+ channel (Gao
et al. 2013); (3) The Kunitz-type K+ channel toxin family. All members share a common cysteine pattern
and act as specific inhibitors of Kv channels (Chen et al. 2012); (4) k-BUTX-Tt2b and Ts16. These two
peptides are weak inhibitors of Kv channels isolated from the Tityus trivittatus venom and have a similar
cysteine spacing to a-KTxs but unconventional disulfide bridge pattern. They fold into an uncommon
cysteine-stabilized helix-loop-helix (CSaa) structure cross-linked by three disulfide bridges (Saucedo
et al. 2012).
Interactions Between Scorpion Toxins and Potassium Channels

In line with their molecular diversity, scorpion KTxs employ multiple functional regions to bind to
different types of K+ channels, which can be summarized as follows: (1) Alpha-helix. Toxins derived from
the fifth subfamily of a-KTxs, such as BmP05 and P05, block SK channels mainly by a-helical residues,
including a positively charged area (two Arg residues in the RRCQ motif) (Wu et al. 2002). The g-KTx,
BeKm1, also uses its a-helix to block the ERG channels where four amino acid residues (Tyr11, Phe14,
Lys18, Arg20) are located on the interface of the toxin-channel complex (Korolkova et al. 2002). Two
basic residues (CKKX, CKXK, or CXKKX) in the a-helix of several a-KTxs constitute a “hot spot” for
the pore blockade of the ERG channels (Abdel-Mlttaleb et al. 2008); (2) Beta-strands. It is known that a
conserved dyad motif constitutes a minimum functional unit of many toxins to interact with the channel
Page 3 of 16
DOI 10.1007/978-94-007-6727-0_12-2
pore, which is composed of a basic Lys located in a b-strand together with a neighboring aromatic (Phe
and Tyr) or hydrophobic (Leu) residue within about 7 Å of distance (Dauplais et al. 1997; Rodriguez de la
Vega et al. 2003). The dyad has been convergently evolved in many toxins from different venomous
species, such as sea anemones, snakes and cone snails. The same Lys in combination with an Asn located
between two b-strands was also found functionally important, in which the Asn is proposed to bind to an
acidic Asp in the pore helix via H-hydrogen interaction while Lys directly inserts into the channel to
interact with Tyr on the filter region of the channel (Lange et al. 2006; Zhu et al. 2014); (3) C-tail. Hg1, a
Kunitz-type toxin from the Mexican scorpion Hadrurus gertschi, was found to interact with K+ channels
by its C-terminal region (Chen et al. 2012); (4) Multiple domains. Pi1, a scorpion toxin isolated from the
venom of Pandinus imperator, uses four residues from different domains of the toxin to assemble a basic
ring interacting with Kv1.2 (Mouhat et al. 2004). In ErgTx1, a g-KTx purified from the venom of the
scorpion Centruroides noxius, four residues (Tyr14, Tyr17, Met35 and Phe37) derived from different
secondary structural elements constitute a hydrophobic patch to bind to the hERG1 K+ channel (Frenal
et al. 2004; Jimenez-Vargas et al. 2012).
The dyad-mediated toxin-channel interaction is the most common mode between a-KTxs and Kv
channels. Thanks to the work of Banerjee et al., the first experimental complex structure between a KTx
and a channel pore is available currently. This crystal structure includes a chimeric Kv channel and the
scorpion toxin CTX from Leiurus quinquestriatus. In this complex, CTX binds into the extracellular side
of the pore of the channel in a key and lock manner, in which Lys27 in CTX inserts into the pore to make
direct contact with the backbone carbonyl oxygen of Tyr445 in the paddle chimera (Banerjee et al. 2013).
KTX, an a-KTx of 37-residues originally isolated from the venom of the scorpion Androctonus
mauretanicus mauretanicus, uses similar residues CTX to bind a channel pore. However, it is remarkably
different from the CTX’s rigid mode, KTX’s binding induces conformational changes in the selectivity
filter of the pore of a K+ channel chimera (KcsA-Kv1.3), as revealed by a solid-state NMR study (Lange
et al. 2006).
From Defensins to Potassium Channel-Targeted Neurotoxins: Evolutionary

Deletion of n-Loop
The study of the origin of KTxs is a great challenge as their enormous sequence diversity and multiple
action modes. More recently, based on experimental evolution strategy guided by the concept of
evolutionary intermediate, Zhu et al. have provided convincing evidence in favor of the origin of
a-KTxs from ancestral CSab-type antibacterial defensins (Zhu et al. 2014). This kind of defensins
constitute essential innate immunity components of insects and other arthropods in fighting against
microbial infection. In addition to their structural similarity, scorpion a-KTxs and insect defensins are
also functionally related, both involved in either defense against competitors or invasive microbes by
disrupting their cellular membrane functions. To establish evolutionary link between these two types of
molecules, the authors firstly defined “Scorpion Toxin Signature” (STS) comprising the evolutionarily
conserved and structurally/functionally important residues, described as
“C. . .CXXXC. . .KCXN. . .CXC” (C, Cys; X, any non-cysteine amino acid; K, Lys; N, Asn) (Zhu
et al. 2014). By using the STS, they searched for defensins from six insect Orders to find potential
evolutionary intermediates (i.e., defensins containing the STS) for experimental study. As a result, eight
classical insect-type defensins (CITDs) were identified to contain the STS, which all are restricted to two
venomous insect Orders (Hemipteran and Hymenopteran). Of them, navidefensin2-2 was found to recruit
into the venom gland of Nasonia vitripennis, suggesting its evolutionary potential in developing Kv
channel-targeted neurotoxins.
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Structurally, these CITDs have a conformationally flexible amino-terminal loop (n-loop) that is lacking
in a-KTxs. This region exhibits variable sizes among different members, indicating its genetic variability
in tolerance of indel mutations. More importantly, when a CITD was placed on the interface of toxin-
channel interaction according to the mode of a-KTxs, this loop gave rise to severe steric hindrance. Based
on these findings, it was proposed that the deletion of the n-loop of an evolutionary intermediate to remove
the steric hindrance could be a key evolutionary event mediating the emergence of a-KTxs. To validate
this deduction, the authors deleted the n-loop of navidefensin2-2 and named this new peptide navitoxin
(Fig. 2) (Zhu et al. 2014). As expected, navitoxin folds into an a-KTx’s structure and obtains capability in
blocking several subtypes of Kv channels with nanomolar affinity accompanying the loss or reduction of
antibacterial activity due to the deletion of the functional n-loop. Similar to a-KTxs, navitoxin also uses
two key residues in the STS (Lys21 and Asn24) to interact with the channel pore (Fig. 2) (Zhu et al. 2014).
The removal of steric hindrance of a venom CITD for interacting with the Kv channel via small loop
deletion induces a switch from antibacterial function to Kv channel blockade, providing key functional
evidence for their evolutionary relationship (Zhu et al. 2014). This is further strengthened by the action
mode similarity between the CITD-derived peptide and a-KTxs, in which the ancestral form of CITDs
firstly evolved a Lys-Asn motif in venomous animals and then the loop was deleted in the scorpion lineage
to evolve Kv channel-targeted toxins (Fig. 2). Considering extensive distribution of the a-KTxs in many
scorpion species, it is reasonable to infer that they represent the earliest components of KTxs. Subsequent
accelerated substitutions at key sites expand their targets to other types of K+ channels, such as ERG and
SK channels. BmTx3, an a-KTx isolated from the venom of Mesobuthus martensii, represents such an
example (Huys et al. 2004). This toxin has two functional surfaces acting on Kv and ERG channels,
respectively, and is considered as an intermediate between a-KTxs and g-KTxs. In addition to evolution-
ary divergence from a conserved scaffold, the expansion of scorpion KTx arsenal may also occur through
evolutionary convergence to recruit endogenous body proteins with various folds into the venom.
A specific example is that toxins from different families (e.g., l-KTxs and k-KTxs) convergently
developed a functional dyad, initially recognized in a-KTxs (Dauplais et al. 1997; Rodriguez de la
Vega et al. 2003), to target Kv channels.
Classification and Structures of Scorpion Sodium Channel Toxins

Scorpion toxins affecting voltage-gated Na+ (Nav) channels (ScNaTxs) are major toxic components of the
venom with lethal effect on both insects and mammals. They are polypeptides of 61–76 residues typically
with four disulfide bridges (Possani et al. 1999). ScNaTxs are divided into two distinct pharmacological
classes: a-toxins that slow the inactivation process of Na+ currents to prolong the action potential by
binding to the receptor site 3 of Nav channels; and b-toxins that shift the voltage-dependent activation
toward more hyperpolarizing potentials by binding to receptor site 4. According to their preference for
insect and mammalian Nav channels, the a-toxin group can be further divided into three distinct sub-
groups (Bosmans and Tytgat 2007): (1) Classical anti-mammalian toxins (e.g., AaHII) that are highly
active on mammalian Nav channels; (2) Anti-insect toxins (e.g., Lqq3) that strongly affects insect Nav
channels; (3) Alpha-like toxins (e.g., Lqh3) that are highly toxic to both mammalians and insects. Based
on the same criterion, the scorpion b-toxin group is also further divided into these three subgroups:
(1) Classical anti-mammalian b-toxins (e.g., Cn2) that are exclusively found in scorpions of the genus
Centruroides and highly toxic to mammals; 2) Anti-insect b-toxins, including depressant toxins (e.g.,
LqhIT2) that induce flaccid paralysis and excitatory toxins (e.g., Bj-xTRIT) that induce contraction
paralysis in fly larvae; (3) Beta-like toxins highly active on both insect and mammalian Nav channels
(e.g., Ts1) (Possani et al. 1999).
Page 5 of 16
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Antibacterial
n-Loop Defensin
G
Y
Evolution of KN
Antibacterial
n-Loop Evolutionary
Intermediate
N K
Evolutionary
Deletion of n-Loop
K+ Channel Toxin
N K
rKv1.1
Fig. 2 The origin of a scorpion K+ channel toxin from an antibacterial defensin. The antibacterial defensin is sapecin (pdb
entry 1L4V) and its two residues corresponding to the KN motif shown. The evolutionary intermediate used here is
navidefensin2-2 and the K+ channel toxin is navitoxin (Zhu et al. 2014). Dotted circle indicates the removal of steric hindrance
between the n-loop and the turret region of the channel pore. KN, Lys and Asn, two key functional residues belonging to the
STS (Zhu et al. 2014). Amino acids in the sphere model are key channel residues involved in toxin binding
Despite remarkable pharmacological diversification associated with differential phyletic selectivity,

these toxic molecules overall adopt a conserved three-dimensional structure, composed of a cysteine-
stabilized a-helical and b-sheet (CSab) molecular scaffold and a perpendicular NC-domain comprising
an amino-terminal turn and a carboxyl-terminal tail (Fig. 3). The CSab scaffold is shared by other
Page 6 of 16
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a-toxins b-toxins
Anti-mammalian [AaHII, 1AHO] Anti-mammalian [Cn2, 1CN2]
C-tail
N-turn
Anti-insect [Lqq3, 1LQQ] Depressant anti-insect Excitotory anti-insect

[LqhIT2, 2I61] [Bj-xTRIT, 1BCG]
α-like [Lqh3, 1BMR] β-like [Ts1, 1NPI]
Fig. 3 Representative structures of scorpion a-toxins and b-toxins. Toxin names and their pdb entry numbers are provided in
brackets and each pharmacological subgroup is shown in different colors. The core- and NC-domain are boxed in orange and
light blue, respectively
scorpion toxins affecting K+, Cl and Ca2+ channels and is even present in an array of polypeptides with
diverse origins and biological functions, but the NC-domain appears to be unique to scorpion Nav channel
toxins (Zhu et al. 2005).
Functional Domains of Scorpion Sodium Channel Toxins

Previous extensive mutational studies have indicated that ScNaTxs from different pharmacological
subgroups employ a similar strategy to assemble their functional surfaces into two distinct subdomains:
(1) A core domain that is conserved across all specific pharmacological subgroups and contains the
so-called pharmacophore; (2) A variable domain that is most likely determinant of toxin specificity on
different Nav channel subtypes (Gurevitz 2012). For the a-toxins, there exists a common bipartite
bioactive surface where the core domain is composed of short loops connecting the conserved secondary
structure elements of the molecules, and the NC-domain formed by a five-residue N-turn and a C-tail.
Similar active sites in the core domain of Lqh2, Lqh3 and LqhaIT provide structural basis for different
toxins binding to the same site in Nav channels (Gurevitz 2012). By contrast, the difference in the shape of
Page 7 of 16
DOI 10.1007/978-94-007-6727-0_12-2
NC-domain among members has been considered to be a factor relevant to the selectivity of a-toxins. This
appears true in that in mammal-specific a-toxins, the N-turn is more mobile and it moves with the C-tail in
a concerted manner with respect to the core module, but this has not been observed in insect a-toxins
(Chugunov et al. 2013).
For the b-toxins, their functional surfaces are also composed of two parts: one situated in the a-helix
and its vicinity with a common “pharmacophore”; and another differing among subgroups that determines
the selectivity of b-toxins (Gurevitz 2012). The pharmacophore is formed primarily by a negatively
charged residue (E30 in Bj-xtrIT, E24 in LqhIT2, E28 in Css4, and E26 in Lqhb1) flanked by hydrophobic
residues (Gurevitz 2012). While this common feature is related to activity, amino acids in variable
domains seem crucial for the selectivity of toxins. For example, the b2-b3 loop in Css4; the C-terminal
region in Bj-xTRIT; and residues forming a hydrophobic bed and the C-terminal region (especially R58) in
LqhIT2, are associated with preference of the toxins for insect Nav channels (Gurevitz 2012).
Two Distinct Sodium Channel Receptor Sites for Toxin Binding

The Nav channels are transmembrane protein complexes mainly comprising one pore-forming a-subunit
and one or two smaller auxiliary b-subunits (b1-b4) in mammals, or TipE in insects, which modulate the
kinetics and voltage dependence of channel gating (Catterall et al. 2005). The a-subunit constitutes an
essential functional unit of the channel and usually contains more than 2,000 amino acids with four highly
homologous structural domains (DI to DIV), each of which includes six a-helical segments (S1-S6) long
enough to cross the membrane and a reentrant pore loop (P) between S5 and S6 (Fig. 4a). When viewed
from the outside, DI to DIV are arranged in a clockwise pattern (Fig. 4b). The first four-helix (S1-S4)
bundle forms a modular VSD to initiate channel activation, and S5-S6 and the intervening P-loop form the
pore domain allowing Na+ to across the membrane. The conformational change of the VSD of one domain
in response to depolarization can be transmitted to the pore module of the neighboring domain (Zhang
et al. 2011). The S4 segment is a voltage sensor of the channels with four to seven conserved positively
charged residues separated by two hydrophobic residues (Catterall 2000). They move outward upon
depolarization and transport the gating charges from inner membrane to out membrane (Wang et al. 2011;
Zhang et al. 2011).
A variety of peptide toxins exert effect on Nav channels via binding to specific receptor sites (Zlotkin
1999), among which site 3 is targeted by scorpion a-toxins and site 4 by scorpion b-toxins. With the
exception of the Na+ channel blocker Cn11 isolated from the venom of Centruroides noxius (Ramirez-
Dominguez et al. 2002), all ScNaTxs affect gating of the channels (Possani et al. 1999). Site 3 is formed
by residues in the extracellular linker in DI (SS2-S6) and the extracellular linker connecting segments
S1-S2 and S3-S4 in DIV; and site 4 consists of residues in the SS2-S6 loop in DIII and S1-S2 and S3-S4
loops in DII (Zhang et al. 2011) (Fig. 4c). Structural studies indicate that a S5-S6 loop in a domain is in
close proximity to S1-S2 and S3-S4 loops in its adjacent domain (Payandeh et al. 2011).
The division of bioactive surfaces of ScNaTxs into two distinct domains mirrors their receptor site
division, as supported by two recent toxin-channel complex models built based on a series of mutational
data both from toxins and channels (Lqh2-rNav1.2 and Css4-rNav1.2) (Wang et al. 2011; Zhang
et al. 2011; Zhang et al. 2012). In these models, although sites 3 and 4 are located on different domains
of Nav channels, S1-S2 and S3-S4 loops from one domain are required for binding to ScNaTxs via the
core-domain of these toxins; whereas SS2-S6 loop derived from the adjacent domain may contact the
NC-domain of toxins and play a secondary role.
Page 8 of 16
a
Out
+
+
S1 S2 S3 S4 S5 S6
+
DOI 10.1007/978-94-007-6727-0_12-2
+
In
DI DII DIII DIV
NH3+ COO-
b c
DI DII Site 3 Site 4
LDIIIS5-S6
DIII
Site 4
LDIS5-S6 b-Tx
a-Tx S2
LDIIS1-S2
DI
LDIVS3-S4
LDIIS3-S4
Site 3
LDIVS1-S2
S3
S4
S6 S1
S2 S5
S5
S4 S1 S3
DIV DII S6
DIV DIII
Fig. 4 The structure of Nav channel a-subunit. (a) The topology showing four repeating domains (DI-DIV), each consisting of six membrane-spanning segments
(S1-S6). S1-S6 segments and their connecting loops are respectively colored in yellow, cyan, green, and gray, except the loops comprising sites 3 and 4 that are colored
in blue and red, respectively; (b) The model structure of human Nav1.7 (Yang et al. 2012). The color code of four domains is in accordance with that of Fig. 4a; sites 3 and
4 are circled in blue and red, respectively; (c) Sites 3 and 4 shown to bind to a toxin, in which three loops (LDIVS1-S2, LDIVS3-S4 and LDIS5-S6) comprising site 3 are marked
and colored in blue whereas the loops (LDIIS1-S2, LDIIS3-S4 and LDIIIS5-S6) forming site 4 marked and colored in red
Page 9 of 16
DOI 10.1007/978-94-007-6727-0_12-2
a
Size
Drosomycin DCLSGR---YKGPCAVWDNETCRRVCKEEGRSSGHCS-PSLKCWCEGC-------------- 44
LqhIT2 DGYIKRRDGCKVACLIG-NEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG 61
N-turn C-tail
b Drosomycin/
LqhIT2
C-tail
N-turn
Drosomycin
Core domain
NC-domain
Fig. 5 Sequence and structural similarity between drosomycin and LqhIT2. (a) Sequence alignment. Identical residues are
shadowed in yellow and identical disulfide bridges shown in black lines. Amino acid residues identified crucial for activity in
LqhIT2 (Karbat et al. 2007) are shown in green in the core domain and blue in the NC-domain; the structurally equivalent
residues to those of LqhIT2 in drosomycin colored in green and highlighted in gray if they display conservative replacement;
(b) Structural superimposition of drosomycin (pdb entry 1MYN) with LqhIT2 (pdb entry 2161)
Sequence and Structural Similarity between Drosomycin and Scorpion Sodium

Channel Toxins
Drosomycin is the first inducible antifungal peptide identified in insects, which was initially isolated from
Drosophila melanogaster (Fehlbaum et al. 1994) and recently found in some ecdysozoans (Zhu and Gao
2014). This cationic peptide of 44 amino acids adopts a compact CSab structure composed of one a-helix
and an anti-parallel three-stranded b-sheet (Landon et al. 1997). Drosomycin shares significant sequence
similarity (>50%) to the core region of scorpion depressant toxins (Zhu et al. 2005; Zhu et al. 2010b).
When compared with the depressant toxin LqhIT2, a total of 19 identical residues and five conservative
replacements were identified. In particular, six functional residues previously characterized to be impor-
tant for channel binding of LqhIT2 (Karbat et al. 2007) are also conserved in drosomycin, including L3,
K8, P10, A12, V13 and R21 (Fig. 5a). Structural comparison revealed that the core of LqhIT2 accurately
matches drosomycin but its extra NC-domain is lacking in drosomycin (Fig. 5b). Sequence and structural
similarities along with functional relatedness (defense against multicullular organisms) support an
orthologous relationship between depressant b-toxins and drosomycins (Zhu et al. 2005).
From Drosomycin to Toxins: Evolutionary Gain of an NC-Domain

To provide functional evidence to establish a reliable evolutionary link, the NC-domain of a depressant
scorpion toxin BmKITc was grafted onto the scaffold of drosomycin and the engineered chimeric peptide,
termed drosotoxin, changes its target from fungi to rat ion channels (Zhu et al. 2010b). This discovery is
further strengthened by two remarkable observations: (1) The deletion of N- and C-terminal sequences of
a scorpion b-toxin restored its antifungal activity (Cohen et al. 2009); (2) Drosomycin can bind to its own
Nav channel (DmNav1) to induce a conformational change (Cohen et al. 2009).
Page 10 of 16
DOI 10.1007/978-94-007-6727-0_12-2
Drosomycin LqhIT2
C-tail
No
Evolution
NC-domain
N-turn
A13
SS2 SS2
A12 I16
V13
P10 L15
SS1 SS1
S4 S4
S3 S5 S3 S5
S2 S2
S6 S6
S1 S1
DII: S1-S4 DIII: S5-S6 DII: S1-S4 DIII: S5-S6
DmNav1 DmNav1
Fig. 6 Structural models in favor of drosomycin and LqhIT2 interacting with a common site on the voltage-sensor domain
(VSD) from DII of DmNav1. Drosomycin is colored in green and LqhIT2 in blue. The absence and presence of an NC-domain
in drosomycin and LqhIT2 are indicated by dotted and solid boxes, respectively. Three hydrophobic residues conserved
between drosomycin and LqhIT2 and hypothesized to be implicated in channel binding are shown in their Ca atoms. The
structural models were prepared with WebLab ViewerLite 4.0 (Molecular Simulations Inc.)
The Nav channel binding feature and high sequence and structural similarities to the depressant
b-toxins suggest that this Nav channel-targeted molecule might bind to site 4 of DmNav1 in a manner
similar to LqhIT2. The constructed complex models of drosomycin or LqhIT2 with the DmNav1 site 4 in
reference with the structure of Css4-rNav1.2 (Zhang et al. 2011; Zhang et al. 2012) indicate that
drosomycin interacts with the loops connecting S1-S2 and S3-S4 in DII via three hydrophobic residues
(P10, A12 and V13) which are structurally equivalent to A13, L15 and I16 in the interface of LqhIT2 and the
VSD of DmNav1 (Karbat et al. 2007). Different from drosomycin, LqhIT2 also binds to the SS2-S6 loop
in DIII via its NC-domain (Fig. 6).
Taken together, it is becoming clear that evolutionary gain of an NC-domain on a Nav channel-targeted
ancestral scaffold (drosomycin) represents a key event that mediates the emergence of ScNaTxs through
extending its interacting region around the two loops of DII to the SS2-S6 loop in DIII (Fig. 6). In other
words, a drosomycin-like molecule acts as a toxin only when it can simultaneously interact with the two
regions of a Nav channel (the two loops in DII: S1-S4 and the SS2-S6 loop in DIII: S5-S6) after the gain of
the NC-domain (Fig. 6). According to the current viewpoint, most of animal toxins are developed from
related normal body proteins by gene duplication and subsequent mutations to modify their structure and
function (Fry 2005). However, it appears that evolution of the first ScNaTx is a result of genetic
modification of an ancestral drosomycin-like peptide without gene duplication, as evidenced by the absence
of the antifungal peptide in the scorpion genome (Cao et al. 2013b). Froy and Gurevitz hypothesized that an
ancestral long-chain Nav channel toxin first developed into b-like toxins in the New World scorpions and
then evolved into a- and b- toxins, including depressant toxins, in the Old World scorpions (Froy and
Gurevitz 2003). However, the establishment of evolutionary link between drosomycin and depressant
b-toxins suggests that the first ScNaTx evolved should be a depressant b-toxin in the Old World scorpions
and subsequent gene duplication combined with speciation generates multiple pharmacological groups.
Page 11 of 16
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Drosomycin Binding to Fly Sodium Channels as Endogenous Ligands?

The Drosophila fat body, a functional equivalent of the mammalian liver, is a major site of the expression
of a series of antimicrobial genes. When Drosophila is challenged by fungi, drosomycin is rapidly
synthesized in the fat body and secreted into the haemolymph (Fehlbaum et al. 1994). This systemic
response is controlled by the Toll signal pathway (Lemaitre et al. 1996). Apart from its antifungal role, the
discovery that drosomycin binds to its own Nav channel gives a clue to other biological functions of this
immune molecule. It is known that glia are major contributors to central nervous system immunity in both
Drosophila and mammals and that their activation is largely dependent on the activity of Nav channels
(e.g., Nav1.6). Moreover, in a recent study, Cao et al. found that drosomycin is expressed in neurons and
glia of Drosophila brain (Cao et al. 2013a). Co-expression of drosomycin and DmNav1 in brain suggests
this gene could exert specific effects on brain immunity, presumably acting as an endogenous ligand of
DmNav1 to activate glia-dependent innate immunity. In addition, Nav channels have been shown to play a
vital role in development, aging and neurodegeneration of flies and their adaptation to temperatures
(Garber et al. 2012). These discoveries are very attractive as in addition to inducible expression in
response to fungal infection, drosomycin is also constitutively expressed in three developmental stages
of Drosophila (i.e., larva, pupa and adult) (Tian et al. 2008). It has also been shown that diapause can lead
to increased transcription of drosomycin in the absence of infection, and that drosomycin is a target gene of
insulin signaling that controls body size and some life history traits, such as fertility and lifespan (Becker
et al. 2010; Kubrak et al. 2014). All these provide evidence supporting other physiological roles of
drosomycin beyond immunity. Experimental confirmation of Nav channel ligand function of drosomycin
will help elucidate the explicit mechanism behind these biological processes.

In conclusion, there is multidimensional evidence for independent origins of scorpion toxins targeting K+
and Na+ channels. This appears to be obviously different from the sea anemone venom K+ and Na+
channel toxins which are considered to arise from a common ancestor (Jouiaei et al. 2015). Evidences for
a CITD as the ancestor of scorpion KTxs can be found: (1) These toxins share high structural similarity to
CITDs but the lack of an n-loop; (2) Only CITDs contain the STS and they all are restrictedly distributed
in venomous insects; (3) At least one member was found to recruit into the venom; (4) Conformational
flexibility in the n-loop of CITDs is associated with steric hindrance between peptide-channel interaction;
(5) Frequent genetic deletion in the n-loop of CITDs; (6) Experimental deletion of a venom-derived CITD
gives rise to a toxin with structural, functional and mechanical similarities to scorpion toxins.
Also, the hypothesis that ScNaTxs might originate from an ancestral antifungal drosomycin is
supported by the following evidences: (1) At the sequence level, drosomycin and scorpion depressant
toxins share >50 % similarity and three identical disulfide bridges; (2) The structure of drosomycin
exactly corresponds to the core domain of depressant b-toxins; (3) The transfer of the NC-domain of a
depressant b-toxin onto the drosomycin scaffold led to the emergence of Nav channel toxicity whereas a
b-toxin with N- and C-terminal sequences deleted exhibited antifungal effect; (4) They both commonly
target Nav channels, although in Drosophila such binding could induce an “endogenous ligand” function;
(5) Structural studies suggest that drosomycin and depressant b-toxins both use a conserved region to
interact with the VSD region of site 4 from DII, and the toxins’ NC-domain is close to the adjacent region,
SS2-S6 loop in DIII; (6) At the phylogenetic level, the history of drosomycin has been traced to the
common ancestor of the Ecdysozoa (Zhu and Gao 2014). Therefore, the evolution of Nav channel toxins
appears to have occurred after the divergence of scorpions from other arthropods. Further investigation of
Page 12 of 16
DOI 10.1007/978-94-007-6727-0_12-2
the binding mode between drosomycin and Na+ channels may provide new insights into the evolutionary
mechanism responsible for the origin of toxicity in an ancient scaffold carrying physiological functions.
In recent years, the evolutionary origin of toxins from venomous animals is becoming a hot research
topic and the history of many toxins from different species is being uncovered. For example, the origins of
shrew and lizard toxins from ancestral serine proteases was achieved via small insertions in several
regulatory loops and subsequent accelerated sequence evolution to create new chemical environment and
functional changes (Aminetzach et al. 2009). In addition, it was proposed that two arthropod predators
(spider and centipede) convergently employed an ancestral hormone scaffold to develop their venom
(Undheim et al. 2015). With more genomes of venomous arthropods sequenced, it is expected that more
examples of toxin origin from nontoxic physiological peptides will be uncovered. This will enhance our
understanding of toxic peptide evolution and help design novel molecules with specific activity and
selectivity.
Cross-References
▶ Evolutionary Context of Venom in Animals
▶ Mutation, Duplication, and more in the Evolution of Venomous Animals and their Toxins
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DOI 10.1007/978-94-007-6727-0_13-1
The Strategic Use of Venom by Spiders

Allen M. Coopera*, David R. Nelsenb* and William K. Hayesa
a
Department of Earth and Biological Sciences, Loma Linda University, Loma Linda, CA, USA
b
Department of Biology and Allied Health, Southern Adventist University, Collegedale, TN, USA
Abstract
Understanding the behaviors by which animals deploy their venoms has been largely neglected compared
to other aspects of the evolution and biology of venomous organisms and their venoms. Yet, behavior has
long been recognized as a pacemaker for the evolution of morphological, ecological, life history, and other
traits, in large part because behavioral responses can expose organisms to or protect them from novel
selection pressures. The importance of behavior is especially evident in that venom most often functions
through a behavioral act that generates a wound in a target animal through which the toxic secretion must
be introduced. As a limited and costly commodity, venom should be deployed strategically and judi-
ciously by those animals that possess it. The chapter summarizes the major aspects of adaptive venom use
in animals, and highlights the best documented examples of strategic venom deployment among spiders.
These animals, like other venomous taxa, exhibit four major behavioral strategies. First, they are often
highly selective when using their venom, discharging it only under certain conditions. Second, they can
modulate the quantity of venom they expend in both predatory and defensive contexts, delivering multiple
bites or variable quantities within individual doses. Third, at least one study suggests that spiders possess
venom gland heterogeneity and therefore deliver varying venom composition with successive venom
expulsions. Finally, some evidence suggests that spiders can strategically target the delivery of their
weapon at a particularly vulnerable region of their target. Collectively, the evidence suggests a common
theme among spiders and other venomous animals for economization and optimization of venom
deployment.
Keywords
Chemical ecology; Defense; Predation; Venom economization; Venom metering; Venom optimization
Introduction
Much of the research on venomous animals and their venoms has focused on the venom delivery systems
and the biochemistry, pharmacology, and toxicology of the venoms. Implications for human health, quite
naturally, have driven this intense interest and investment. The impetus is evident, for example, among the
most studied groups of snakes, for which venomous genera have received far more attention than
nonvenomous genera, and the overwhelming majority of publications for the venomous taxa have
addressed aspects related to venom rather than other facets of their biology (Beaman and Hayes 2008).
*Email: acooper@llu.edu
*Email: dnelsen@southern.edu
*Email: dnelsen@llu.edu
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Researchers have also investigated the behaviors associated with venom deployment, but this impor-
tant aspect of the evolution and biology of venomous animals remains relatively neglected. The notion
that behavior contributes substantially to the evolutionary process dates to Lamarck (1809) and has been
developed further by subsequent Darwinian expositors (reviewed by Corning 2014). Behavior has been
viewed by many as the “pacemaker of evolution” because behavioral responses can expose organisms to
or protect them from novel selection pressures, thereby influencing the evolution of morphological,
physiological, life history, and other traits (Duckworth 2009; Wcislo 1989; Wolf and Weissing 2012).
Indeed, the individual’s environment is influenced substantially, for example, by its movements, choice of
habitat, feeding behavior, predator avoidance, mating strategy, and social behavior. Moreover, the
behavior of individuals affects population-level properties such as spatial distribution, social structure,
trophic interactions, community structure, and ecosystem function. The importance of behavior to the
evolution of venomous animals is especially significant because venom most often functions through a
behavioral act that generates a wound in a target animal through which the toxic secretion must be
introduced (Nelsen et al. 2014b). Clearly, the study of behavioral phenotypes, mechanisms, and adapta-
tions of venomous animals is critical to understanding the factors that shape the evolution of venom and its
associated non-behavioral traits.
Venoms comprise toxic secretions that organisms deliver directly to the tissues of other organisms via
physical contact and generation of a wound (Nelsen et al. 2014b). Venom can be distinguished from other
toxic secretions that are administered passively by contact or ingestion (poisons) or are transferred by a
delivery mechanism to a target’s surface without creation of a wound (toxungens). These distinctions
underscore the importance of behavior in toxin delivery, but a given secretion may nonetheless function in
two or all three of these modes (Nelsen et al. 2014b). Venoms, which may be comprised of a single toxin
or a mixture of different toxins, and can be synthesized autogenously or acquired exogenously, interact
with the target organism’s internal milieu to bring about rapid pathophysiological changes. Animals
employ venom for a variety of purposes, including predation, defense, competition for space and mates,
and, secondarily for some animal groups, hygiene, communication, and potentially other roles (Mebs
2002; Nelsen et al. 2014b).
Because venom is both a valuable resource and a limited commodity, selection should favor the
behavioral use of venom in ways that maximize effectiveness and minimize waste (Hayes 2008; Hayes
et al. 2002; Hostettler and Nentwig 2006; Morgenstern and King 2013). Cost-benefit analyses are
essential for understanding the adaptive value of behavior because natural selection favors strategies
that have a propitious cost-benefit ratio (Cuthill and Houston 1997). The benefits of venom are obvious,
particularly for predation and defense (e.g., Mebs 2002; Nelsen et al. 2014b). However, the synthesis of
venom, its storage, and its compartmentalization to avoid autotoxicity all entail costs that have only
recently been analyzed (reviewed by Morgenstern and King 2013). Regeneration of spent venom may
also require a few days or weeks, during which time the animal may be disadvantaged (Morgenstern and
King 2013). Beyond the metabolic cost of generating venom, there are ecological costs to venom use as
well. These include unnecessary or excessive discharge that can temporarily impair an organism’s ability
to defend itself or take advantage of future opportunities to procure prey (Hayes 2008; Malli et al. 1998).
Venom use is also associated with serious risk of bodily injury, and possibly death, as envenomation
requires direct physical contact that can lead to retaliation (Schmidt 1990).
For an organism to deploy venom in an optimal or strategic manner, it must accurately assess both its
external environment, including the intended recipient of the venom, and its own internal state (Hostettler
and Nentwig 2006; Wullschleger and Nentwig 2002). External factors assessed for predatory venom
deployment may include type of prey (Hayes 1992; Wigger et al. 2002), prey size (Edmunds and Sibly
2010; McCormick and Polis 1990; Steiner 1986), and prey struggle intensity or duration (Djieto-Lordon
et al. 2001; Malli et al. 1999; Steiner 1986). External factors assessed for defensive venom deployment
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may include type of predator or aggressor (Carlin and David 1989), degree of threat (Haight 2006; Nisani
and Hayes 2011), and persistence of threat (Fink 1984). Internal factors that may influence venom
deployment include the amount of venom remaining in storage (Wullschleger and Nentwig 2002) and
satiety level (Hayes 1993). In many cases, the venomous animal may physically interact with the intended
venom recipient prior to venom deployment, thus enabling assessment (e.g., struggling prey receive more
bites or stings; Malli et al. 1999; Steiner 1986). However, in other cases, such as with snakes that inject
venom during a single brief bite, assessment of the target and decisions regarding deployment may be
made prior to contact with the receiver (Hayes et al. 2002).
The act of venom deployment, whether it occurs via a simple stimulus-response reflex or by more
complex decision making (Jackson and Cross 2011), potentially including cognition, which remains an
ill-defined concept (Menzel 2013; Perry et al. 2013; Shettleworth 2013), is generally triggered by stimuli
that exceed a threshold. In the case of predation, subthreshold stimuli arising from relatively small,
harmless, or unresponsive prey may result in prey capture and consumption without envenomation
(Hayes et al. 2002; Malli et al. 1998; Wigger et al. 2002). For defense, subthreshold stimuli may evoke
alternate presumably less costly behaviors, including fleeing (Gibbons and Dorcas 2002; Nelsen
et al. 2014a), leg autotomy (Nelsen et al. 2014a), threat displays (Gibbons and Dorcas 2002), retaliatory
pinching/biting (Heatwole 1967; Schmidt 1990), sham strikes (Hayes 2008), and dry bites or stings
(Herzig 2010; Morgenstern and King 2013; Nisani and Hayes 2011).
Venomous animals can strategically deploy their venom in four ways. First, presumably all venomous
animals are highly selective when using their venom, discharging it only under certain conditions. In some
cases, behavioral regulation of venom release is sufficient that the full act of venom delivery may ensue
without actual release of venom, resulting in a dry bite or sting. Second, animals can modulate the amount
of venom they expend, delivering more in some circumstances and targets and less in others. This can be
accomplished in either of two ways: by varying the number of bites or stings or by varying the quantity of
venom expended with each bite or sting. The capacity of animals to modulate venom quantity has been
described as the “venom metering” (Hayes 2008; Hayes et al. 2002) or “venom optimization” (Wigger
et al. 2002) hypotheses. Third, at least some venomous animals can alter the composition of venom
depending on context of use, as occurs most notably in cone snails (Dutertre et al. 2014). Scorpions also
possess venom heterogeneity, but venom composition during successive stings covaries with quantity of
venom (Nisani and Hayes 2011). Finally, venomous animals may strategically target their venom by
aiming it in the direction of an intended victim, or even delivering it to a particularly vulnerable body part
(Libersat and Gal 2014; Malli et al. 1998, 1999). This targeting can potentially reduce the quantity of
venom used, particularly for predation.
The purpose of this review is to summarize the major aspects of adaptive venom use in spiders and
highlight the best documented examples of strategic venom deployment among this diverse group.
Spiders (class Arachnida, order Araneae) comprise one of the most studied groups of venomous animals
and serve as excellent models for exploring the behavioral use of venom. Almost all spiders are
venomous, with only a few families having lost their venom glands. Many species, particularly those
among the araneomorphs, have invested heavily in the use of venom. Venom has allowed spiders to
become very successful predators. They are often among the top terrestrial predators of arthropods, but
some species are capable of capturing much larger vertebrate prey. Sufficient evidence has now accumu-
lated to provide compelling evidence that, despite their relative neurological simplicity (compared to
venomous vertebrates), spiders can utilize each of the four aforementioned aspects of strategic venom
delivery, as summarized in Table 1.
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Table 1 Documented examples of strategic venom use by spiders. Parenthetic examples are explained in footnote. See text for
references
Strategic venom deployment Predation Defense
Selective venom use ✔ ✔
Number of doses ✔ ✔
Volume per dose ✔ ✔
Venom composition (✔) (✔)
Delivery location ✔
Spider venom composition, like that of scorpions, appears to be heterogeneous (Morgenstern et al. 2012); thus, their venom
composition presumably covaries with venom quantity and therefore is indirectly modulated
Spider Venom Apparatus and Venom

Spiders possess a pair of fangs attached to the chelicerae that they use to deliver venom via biting. The
venom gland may be exclusively located within the chelicerae (as in the mygalomorphs), but may also
extend well into the cephalothorax, occupying a significant proportion of the total volume (as in the
araneomorphs; Nentwig and Kuhn-Nentwig 2013). Cheliceral fangs act as hypodermic needles, typically
opening at their distal tip and usually moving in one of two planes: either dorsoventrally in the
mygalomorphs (infraorder Mygalomorphae, or Orthognatha) or mediolaterally in the araneomorphs
(infraorder Araneomorphae, Labidognatha). Spider venoms comprise complex, multi-component mix-
tures of biologically active substances, including neurotransmitters and salts, acylpolyamines, peptides,
and proteins (including some of large molecular weights; Casewell et al. 2013; Fry et al. 2009; Kuhn-
Nentwig et al. 2011), that play important roles in both predation (killing or paralyzing prey) and defense
(Foelix 1996; Nentwig and Kuhn-Nentwig 2013; Quintero-Hernandez et al. 2011).
Cost of Venom in Spiders

Despite the value of venom to spiders, no quantitative data exist on the metabolic cost of its regeneration,
although Malli et al. (1999) suggested venom production is energetically expensive. Nevertheless, data on
other aspects of venom regeneration suggest that spiders incur an ecological cost for venom expenditure.
Because venom regeneration may take weeks (Boeve et al. 1995; Perret 1977b) to months (Freyvogel
et al. 1968), and spiders may capture several prey items per day, spiders should modulate venom release to
avoid the metabolic expense of regenerating depleted reserves, which could leave the spider vulnerable to
predators or unable to deal with subsequent prey (Boeve and Meier 1994; Malli et al. 1998). The
secondary loss of venom in uloborid spiders, which kill their prey instead by wrapping them tightly in
hackled silk, further suggests that venom use comes with a considerable biochemical price (Morgenstern
and King 2013).
Selective Venom Use by Spiders

Strategic deployment of venom by spiders includes using this valuable commodity selectively. Spiders
appear to be capable of choosing whether to use any venom at all. Withholding venom can occur in two
contexts: by crushing prey with the chelicerae without employing the fangs, or by using the fangs without
concomitant venom expulsion. The ability to use or withhold venom independent from fang use stems
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DOI 10.1007/978-94-007-6727-0_13-1
from spider anatomy. The venom glands are surrounded by striated muscle under nervous control,
allowing the deployment of venom via muscular contraction of the gland at the volition of the spider
(Boeve et al. 1995; Bucherl 1971; Malli et al. 2000; Schenberg and Pereira-Lima 1978).
In the context of predation, the interplay between prey size, prey defensive capabilities, and capacity of
prey to struggle will influence whether spiders deploy their venom. In a number of species investigated,
prey size represents an important factor influencing venom deployment, with spiders routinely seizing and
chewing small arthropods without applying venom, relying instead on the chelicerae to crush or chew
them, thereby reserving venom for larger prey (Cupiennius salei [Ctenidae]: Malli et al. 1998; Nentwig
and Kuhn-Nentwig 2013; Wigger et al. 2002; Argiope argentata [Araneidae]: Robinson 1969; Phoneutria
nigriventer [Ctenidae]: Schenberg and Pereira-Lima 1978). Malli et al. (1998) quantified the venom dose
injected by C. salei (Ctenidae) into crickets of various size classes ranging from 100 to 660 mg. They
found that the spiders did not inject venom into 22 % (7/32) of the crickets bitten in the smallest size class
(100–110 mg). The authors contended that C. salei does not rely exclusively on its venom when feeding
on small prey, but can accomplish the job through mechanical damage alone inflicted by the chelicerae.
Prey size is also important for P. nigriventer (Ctenidae), which only injects venom into excessively large
prey, relying on mechanical damage caused by the chelicerae to kill small insects (Schenberg and Pereira-
Lima 1978). According to Wigger et al. (2002), the difficulty a spider encounters in overwhelming prey,
which can vary with prey species, may also determine whether spiders use their venom. Wigger
et al. (2002) documented selective venom use by C. salei while using an enzyme-linked immunosorbent
assay (ELISA) to quantify the amount of venom the spider injected into four different prey (blowflies,
crickets, stick insects, and ground beetles). The authors found that no venom could be detected in 32 %
(6/19) of the crickets attacked, whereas all individuals of the other prey types that were attacked had been
envenomated. The authors suggested that sometimes the spider relies on its strong chelicerae to kill soft
prey susceptible to mechanical damage. However, it remains unclear why, if C. salei often withheld
venom from crickets, it did not also occasionally withhold venom from stick insects, which the authors
argued were also a soft, unproblematic prey type. Other investigators have also noted the influence of prey
struggle (B€ucherl 1971) and the prey’s defensive capabilities (Kuhn-Nentwig et al. 2011) on whether
venom is used.
Spiders may selectively deploy venom in defense as well. For example, P. nigriventer employs venom
only when the spider finds no way to escape attack (Schenberg and Pereira-Lima 1978). In female mouse
spiders, Missulena bradleyi (Actinopodidae), aggravation by experimenters led to voluntary expression
of venom from only 15 % of spiders, suggesting subthreshold stimulation for venom expenditure in most
cases (Herzig et al. 2008). Defensive dry bites represent another example of selective use of venom.
Herzig (2010) argued that some dry bites by the mouse spider (Missulena spp.) investigated by Isbister
(2004) could be explained by the voluntary decision of the spider not to deploy venom during a bite in
order to save the metabolic expense of venom synthesis. While analyzing methods of venom extraction
from the African tarantula Scodra griseipes (Theraphosidae), Celerier et al. (1993) observed that the
spiders could bite a lure many times without emitting venom. Freyvogel et al. (1968) similarly noted that
the baboon spider Pterinochilus sp. (Theraphosidae) often actively withheld venom during milking
attempts. Nelsen et al. (2014a) investigated defensive venom use in the western black widow spider
(Latrodectus hesperus) and found that, when pinched, at least 50 % of the bites to three successive
presentations of parafilm-covered tubes appeared to be dry. The proportion of dry bites did not decline
among the three targets in succession, and dry bites often preceded wet bites (Table 2), suggesting that the
spiders deliberately withheld their venom. Taken together, these data indicate that decisions about venom
deployment depend on several factors, including prey size, prey type, and threat level.
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Table 2 Selective venom use. Sequence of venom usage (dry vs. wet bites) by western widow spiders (Latrodectus hesperus)
when defensively biting three targets in succession (N = 80 trials) separated by brief (5 s) or lengthy (5 min) intervals
(Adapted from Nelsen et al. 2014a)
Frequency
Target 1 Target 2 Target 3 Brief intervals Lengthy intervals Plausible interpretation
Dry Dry Dry 15 7
Dry Dry Wet 8 7 Venom metering
Dry Wet Dry 7 6 Venom depletion
Dry Wet Wet 1 9 Venom metering
Wet Dry Dry 5 3 Venom depletion
Wet Dry Wet 3 4 Venom metering
Wet Wet Dry 1 1 Venom depletion
Wet Wet Wet 0 3
Venom metering: dry bites preceded wet bites, indicating available venom and decision making
Venom depletion: dry bites resulted from prior venom use that depleted reserves
Amount of Venom Deployed by Spiders

Spiders have been compared to snakes in their ability to control the amount of venom delivered
(Schenberg et al. 1970). Evidence suggests that the degree of venom gland emptying is at the spider’s
volition (Boeve et al. 1995; Maretic 1987). Using indirect measures of the amount of venom deployed,
investigators have found that prey size and struggle intensity are important factors influencing predatory
venom expenditure. For example, Perret (1977a), comparing volume of venom electrically milked before
and after spiders were fed, found that the tarantulas Aphonopelma chalcodes and Dugesiella hentzi
released more venom in the first bite when feeding on adult (1–2 g) cockroaches (Periplaneta americana)
than when feeding on adult (0.1 g) mealworm beetles (Tenebrio molitor). When attacking cockroaches,
A. chalcodes injected, on average, 1.7 mL of venom (25 % of available venom), but injected no venom into
mealworm beetles. Similarly, D. hentzi injected 1.7 mL of venom (28 % of available venom) into
cockroaches, but only 0.1 mL of venom (2 % of available venom) into mealworm beetles. As the
venom was delivered in a single bite, the mechanism of venom deployment is likely related to the extent
of venom gland compression or the number of compressions. In another study, based on mass gain of
bitten prey, Pollard (1990) found that the New Zealand crab spider Diaea sp. (Thomisidae) injected more
venom into a larger fly, Pegohylemyia sp., than into the much smaller fruit fly Drosophila immigrans
(0.108 vs. 0.067 mg venom, respectively). In only 7 % (3/43) of cases was a prey item bitten more than
once, indicating that number of bites was not the primary mechanism for controlling amount of venom
deployed. Although the author noted that Diaea can regulate the amount of venom injected based on prey
size, he also suggested that the spider may use tactile information from captured, struggling prey to help
assess prey size.
Boeve (1994) investigated the mortality rates of multiple series of crickets (Acheta domesticus) of
different mass classes attacked by C. salei. He found that for prey of small or medium mass (less than
40 mg), the spider injected an amount of venom proportional to the mass of the prey, whereas large prey
items were injected with all its venom. Furthermore, by interrupting bites on different prey size classes at
various intervals and analyzing the state of bitten prey, the author showed that the spiders varied the rate of
venom delivery in a single bite based on prey mass. The author further speculated that each escape attempt
may have stimulated the spider to inject a discrete amount of additional venom. In another study using
C. salei, and based on similar methods, Boeve et al. (1995) demonstrated that larger crickets received
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Fig. 1 Modulation of venom quantity based on prey size and species. (a) Volume of venom injected by the spider Cupiennius
salei (N = 16) into four size classes of crickets, as determined by enzyme-linked immunoassay (ELISA) of whole-cricket
homogenates. Box plots show the first quartile, median, and third quartile. Error bars represent the 10th and 90th percentiles.
Note the correspondence between prey size and venom expended (Adapted from Malli et al. (1998)). (b) Volume of venom
injected by C. salei into four different prey genera, adjusted for prey body mass. Box plots are as above, except that error bars
indicate minimum and maximum values. Asterisks represent median lethal dose (LD50) of venom for each prey type. Spiders
expended venom doses similar to the LD50 value when feeding on larval crickets (Acheta domesticus) and stick insects
(Carausius morosus), but delivered greater doses of venom relative to LD50 values for blowflies (Protophormia sp.) and
ground beetles (Poecilus cupreus), which were more difficult to subdue (Adapted from Wigger et al. (2002))
larger venom doses than smaller crickets. Furthermore, the authors showed that C. salei injected larger
venom doses into “difficult-to-handle” prey (cricket Gryllodes sigillatus) than into “easy” prey (cricket
Gryllus bimaculatus), with the dichotomy essentially reflecting a difference (not quantified) in struggle
intensity after attack. The authors concluded that C. salei could empty its glands partially or completely,
resulting in dosed or metered injections of venom. Thus, several studies using indirect measurements of
venom expenditure suggest that the amount of venom deployed by spiders varies with prey size and
struggle intensity.
Malli et al. (1998) were the first to directly quantify spider venom expenditure into various size classes
of prey. The authors performed ELISA on whole-cricket (A. domesticus) homogenates using monoclonal
antibodies to the main toxin in C. salei venom, CSTX-1. Their results (Fig. 1a) revealed that, when mature
C. salei females attacked crickets (N = 128 attacks by 16 spiders) of four size classes (12, 15, 18, 22 mm),
a significant relationship existed between the size of prey and the quantity of venom expended (r = 0.80),
with mean venom quantities ranging from 0.15 mL for the smallest prey to 1.53 mL for the largest. Multiple
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comparisons indicated that C. salei released significantly more venom with increasing size of cricket
(p < 0.01 for all comparisons). Although a clear relationship between venom dose and prey size was
found, the authors acknowledged it remained unclear whether more venom was injected into larger prey
simply because of size, or as a consequence of greater struggle by larger prey. The authors also suggested
that the pattern of venom deployment could result from some combination of C. salei injecting venom
gradually until prey is motionless and a size-based difference in venom susceptibility. The authors did not
emphasize number of bites delivered to prey, although multiple bites did occur in at least 5 % of attacks.
Thus, a metering mechanism based on a gradually delivered dose of venom from a single bite was more
common than one based on multiple bites. In a follow-up study, Malli et al. (1999) further investigated the
influence of prey size on venom expenditure by mature female C. salei, once again using an ELISA. To
disentangle the effects of prey size and struggle intensity on venom dosage, the authors used anesthetized
crickets (A. domesticus) in four size classes (100–110, 290–320, 420–460, and 600–660 mg) that were
artificially induced to struggle at the same intensity for a set duration (5 min). Quantity of venom released
varied widely within a size class, and prey size and quantity of venom expended were weakly correlated
(r = 0.23, p < 0.05). Multiple comparisons revealed that C. salei injected significantly less venom into
the smallest size class, whereas no significant differences were found among the other size classes. The
authors concluded that prey size alone is not likely to be an important cue for regulating venom injection
in this spider. Further, they argued that the results of Malli et al. (1998), in which larger prey received
larger venom doses, were a consequence of predator-prey interactions during envenomation which,
though increasing with size of prey, did not depend on the size of the prey itself.
In addition to prey size, Malli et al. (1999) investigated the effects of prey struggle intensity and prey
struggle duration on the amount of venom C. salei injected. Using ELISA to quantify the venom injected
into anesthetized crickets of the same mass (290–320 mg) that were experimentally manipulated to
struggle at four different intensities (no movement [control], low, medium, and high), the authors found a
highly significant relationship between intensity of prey movement and quantity of venom expended.
Multiple comparisons indicated that, with the exception of the difference between the control and the
low-intensity prey movement condition, C. salei released significantly more venom as the intensity of
prey movement increased. The authors suggested that injection of larger quantities of venom into
vigorously resisting prey would induce rapid immobilization, thus preventing injuries to the spider
and/or lost prey. Additionally, the authors noted that C. salei saved up to 50 % of its venom by
discriminating between high- and low-intensity prey movements.
Malli et al.’s (1999) study of the influence of prey struggle duration on venom expenditure by C. salei
yielded similar results. In this experiment, the crickets (290–320 mg) were vibrated at the same intensity
(medium) but for different lengths of time (0 [control], 1, 2.5, or 5 min) following the initial bite. Data
indicated that the duration of prey movement and quantity of venom expended were positively correlated
(r = 0.61, p < 0.01). Multiple comparisons showed that, with the exception of the difference between
the control and 1-min treatments, C. salei released significantly more venom with increasing duration of
prey movement. Malli et al. (1999) concluded that C. salei injects venom gradually in response to stimuli
generated during the course of envenomation. The authors speculated that perhaps tactile hairs and slit
sense organs found on the chelicerae and base of the claws serve a vibrosensitive function in controlling
the release of venom during envenomation. For all experiments, the mechanism for varying venom
expenditure was independent of number of bites, because the prey were held in the chelicerae (i.e., single
bite) for the duration of each trial.
Following in the footsteps of Malli et al. (1999), Wigger et al. (2002) demonstrated differential venom
expulsion by C. salei based on prey species. The authors used ELISA to quantify venom injected by adult
female C. salei into four prey species: blowflies (Protophormia sp.), larval crickets (A. domesticus), stick
insects (Carausius morosus), and ground beetles (Poecilus cupreus). All prey were of a uniform (but
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unreported) size class. Their results (Fig. 1b) indicated that ground beetles received significantly more
venom than the other three prey species. The authors argued that the blowflies, crickets, and stick insects
were relatively soft and thus unproblematic prey types, resulting in a relatively low dose of venom. In
contrast, the heavily sclerotized ground beetles represented difficult-to-overwhelm prey because spiders
were forced by the beetles’ mechanical protection to inject their neurotoxic venom into the prey’s
abdomen, an injection site requiring more venom to subdue the prey than a bite to the head or thorax
normally would. In fact, the authors suggested that the lengthy handling time for ground beetles may have
been the stimulus leading to greater venom expenditure. Although the number of bites C. salei delivered
to prey was not stated, spiders appeared to hold prey in their chelicerae (i.e., single bite) for each 5-min
trial, indicating that the mechanism controlling venom expenditure was independent of number of bites.
Risk of prey escape, which may vary with prey species, may also influence venom deployment.
Robinson’s (1969) findings suggest this possibility, and Boeve et al. (1995) interpreted them in this
way, citing the study as evidence that more venom is injected into easily escaping insects. Robinson
(1969), while studying the predatory behavior of A. argentata, noted that lepidopteran prey, which were
bitten prior to silk wrapping, received a statistically longer bite than other prey types, which were first
wrapped in silk and then bitten. It was suggested that the short bite might deliver a smaller dose of venom
(Robinson 1969; Robinson et al. 1969; Robinson and Olazarri 1971) because it would be wasteful to use
biologically expensive secretions unnecessarily on a wrapped prey item (Robinson 1969). However, the
long bite may be long simply because the spider must wait for the venom to take effect before it can safely
release the prey and commence wrapping (Robinson et al. 1969). The adaptive significance of the long
bite lies in its ability to cause the most rapid restraint of prey with high escape potential, such as
lepidopterans (Robinson 1969; Robinson and Olazarri 1971). Although the duration of the long bite
delivered to lepidopterans varied dramatically (e.g., from 1 to 527 s when attacking live moths; Robinson
and Olazarri 1971), there was no systematic relationship between length of bite and weight of prey for
either the long or short bite (Robinson 1969).
Wullschleger and Nentwig (2002) experimentally examined whether adult females of C. salei “know”
how much venom is available in their venom glands and make predatory decisions accordingly (Fig. 2).
They emptied the venom glands of their contents by either electrical milking or by allowing the spider to
bite three crickets and compared these spiders to control animals having replete (full) venom glands.
When presented with two prey items simultaneously, adult female C. salei spiders shifted their attacks
toward the cockroach species that was more easily subdued by venom (Blatta orientalis) and avoided the
more venom-resistant species (Nauphoeta cinerea). Hostettler and Nentwig (2006) subsequently showed
that C. salei uses olfactory information to identify prey type and distinguish venom sensitivity, presum-
ably to conserve venom.
Although much of the work examining venom deployment in spiders has focused on the circumstances
of a single sustained bite, spiders do sometimes bite prey multiple times (Gilbert and Rayor 1985; Malli
et al. 1998; Parks et al. 2006; Schenberg and Pereira-Lima 1978; Willey et al. 1992). Thus, varying the
number of bites represents an additional means by which spiders could control the amount of venom
deployed. In such cases, continued prey struggle may be the stimulus for additional bites (Gilbert and
Rayor 1985).
In the context of defense, venom metering is less well studied. Fink (1984) found that the green lynx
spider Peucetia viridans (Oxyopidae) varies the amount of venom expended based on extent of provo-
cation and may vary the amount of venom in individual spits. Female spiders ejected venom forward up to
20 cm when approached or when their legs were pulled. Although a single spit was most common, spiders
would spit several times in succession if repeatedly provoked. The quantity of venom in a spit was
variable, from trace amounts to more than 5 mL. In tarantulas, Perret (1977a) investigated the amount of
venom released in a single bite. In defensive bites against mice (30 g; n = 2 cases), A. chalcodes injected,
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Fig. 2 Modulation of predatory attack based on venom supply. When presented with two prey items simultaneously, adult
female Cupiennius salei spiders shifted their attacks toward the cockroach species more easily subdued by venom (Blatta
orientalis, white bars) and avoided the more venom-resistant species (Nauphoeta cinerea, black bars), after experimental
depletion of their venom. Venom glands were emptied by either electrical milking or by allowing the spider to bite three
crickets. *p < 0.05, **p < 0.01 (chi-square tests) (Adapted from Wullschleger and Nentwig (2002))
on average, 2.3 mL of venom (36 % of available), about the same amount of venom (1.7 mL, 25 % of
available) that they injected, when feeding, into cockroaches, but more than they injected into mealworm
beetles. The author suggested that since cockroaches and mice were of considerably different sizes, and
spiders displayed typical defensive behavior toward mice, it was possible that the spider calculated venom
injection differently in defensive compared to predatory situations.
More recently, Nelsen et al. (2014a) used ELISA to quantify the venom delivered by L. hesperus during
defensive encounters, investigating how presumed level of threat (body vs. leg pinch) and persistence of
threat (brief vs. lengthy intervals between three successive simulated attacks) affected venom expenditure.
The spiders injected 1.8-fold more venom per bite when pinched on the body compared to a leg (Fig. 3).
Body pinches presumably would be perceived as a higher threat than leg pinches, because the body
contains the vital organs and leg autotomy would be an alternative strategy to venom use. The spiders also
deployed 2.3-fold more venom when the three successive threats were separated by brief (5 s) compared
to lengthy (5 min) intervals. The authors suggested that the spiders treated the brief intervals as a single
predatory encounter and the lengthy intervals as separate events requiring additional venom for each new
attacker. Nelsen et al. (2014a) concluded that their results were consistent with risk assessment and the
capacity to modulate venom expenditure during defensive encounters.
Considering this large body of evidence, there can be little doubt that some spiders have the capacity to
vary the amount of venom expended during predatory contexts. Perhaps unsurprisingly, the responses of
bitten prey comprise an important cue influencing how much venom spiders inject. The evidence for
venom metering within a defensive context is weaker, with fewer studies having examined the possibility.
Nevertheless, spiders appear capable of risk assessment and making decisions about the quantity of
venom to use.
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Fig. 3 Modulation of venom quantity based on presumed threat assessment. Mean (1 S.E.) mass of venom expended during
defensive bites by adult females of the western black widow spider (Latrodectus hesperus) when pinched on different body
parts by investigators. Spiders delivered 1.8-fold more venom when the more vulnerable body was pinched compared to when
an expendable leg was pinched (Data from Nelsen et al. (2014a))
Modulation of Venom Composition in Spiders

Recent research hints that certain (and possibly all) spider species, similar to scorpions (Nisani and Hayes
2011), are capable of expulsing venom of heterogeneous composition. To date, a single study has found
that venom of the funnel web spider (Hadronyche infensa) ejected early in a bite sequence differs
significantly in protein composition from venom delivered later in a bite sequence (Morgenstern
et al. 2012). If venom expulsion heterogeneity is widespread in spiders, then venom composition may
simply covary with quantity of venom expended and therefore be modulated only indirectly, as occurs in
scorpions (Nisani and Hayes 2011). Venom expulsion in this fashion results presumably from a relatively
large, fairly stationary, slowly replenished venom pool that is differentially synthesized and stored
regionally along the length of the venom gland. However, further study may reveal rapid and reversible
venom composition changes associated with venom use, which has been documented in cone snails
(Dutertre et al. 2014). In the snails, differences in stimulation between predatory and defensive contexts
apparently promote rapid venom secretion from different regions of the gland into a relatively small
lumen, which is then flushed with each venom expulsion event. The different regions of the gland
synthesize unique venom components, resulting in substantially different venom composition among
successive venom expulsions. One spider group, the spitting spiders (Scytodes, Scytodidae), also pos-
sesses regional heterogeneity of their venom gland, with the anterior portion producing adhesive, silken
strands apparently devoid of toxicity that are spat to immobilize prey, and the posterior portion producing
a toxic venom that is delivered by injection to further incapacitate or kill their prey (Zobel-Thropp
et al. 2014). Both secretions are extruded through the same duct. Because the glue-like material must pass
through the anterior portion of the gland without picking up toxicity, this suggests a mechanism similar to
that of cone snails, and the widespread possibility among spiders of rapid and reversible venom
composition changes with successive bites.
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Strategic Targeting of Venom by Spiders

Delivery location of venom may be an important part of strategic venom deployment in some spiders.
However, without data on prey morphometrics, it can be difficult to distinguish spider targeting preference
from an unequal, but still random, bite distribution stemming from unequal surface areas of body parts
available for biting (Morse 1999). Furthermore, for spiders, evidence suggests that initially attempted bite
location may often play a smaller role in prey incapacitation than the final location of envenomation (Malli
et al. 1998; Pollard 1990). One might suspect that a strategic site of venom injection used by spiders, given
the potent neurotoxins present in their venoms (King and Hardy 2013), would be near the prey’s central
nervous system, typically the thorax or head. In general, thorax envenomations are common (Foelix
1996), and several investigators have contended that targeting the thorax or head produces the fastest
effects (Malli et al. 1998, 1999; Pollard 1990; Wigger et al. 2002). In fact, as Morse (1999) pointed out,
such a “neckbite” pattern of envenomation is often reported by general spider sources. Even so, data on
prey bite location remain relatively scarce.
Much of the limited data on location of predatory spider bites comes from the spider C. salei. Malli
et al. (1999) reported that when C. salei was offered CO2-anesthetized crickets (A. domesticus), the
majority of the prey were bitten in the thoracic region (87.4 % [420/480] of bites), whereas only 12 % and
0.6 % of bites were delivered to the abdomen and head, respectively. In a separate study, Malli et al. (1998)
reported the frequency of bites to a given body region varied among prey size classes, but most crickets
were bitten either in the thorax (66–85 %) or the pronotum right behind the head (9–28 %). In contrast,
few bites occurred on the abdomen (3–9 %) or the chitinized head capsule (0–3 %). Furthermore, except in
one case, all crickets first bitten in the abdomen were subsequently bitten also in the thorax. Given that a
cricket’s thorax is smaller than its abdomen, the reported distribution of bites suggests a preference for
thorax envenomation. Malli and colleagues (Malli et al. 1998, 1999) argued that bites to the thorax may
decrease the amount of venom needed for paralyzation and reduce time to immobilization, thereby
reducing the spider’s risk of injury. In contrast to the high frequency of thorax bites to crickets, when
C. salei preyed on heavily sclerotized ground beetles (P. cupreus), the spiders, after attempts to deliver a
bite to the thorax or head, most often ended up biting the abdomen (Wigger et al. 2002). This study
demonstrates that strategic deployment of venom by delivery location can be constrained by prey
characteristics.
Other spiders besides C. salei appear capable of targeting their venom. Pollard (1990) noted that more
than half a dozen species of crab spiders are known to envenomate prey principally in the head and thorax,
and several species have been observed to re-envenomate prey in the head or thorax following initial
capture by envenomation of the abdomen. This author also hypothesized that crab spiders re-envenomate
prey in the thorax to achieve faster immobilization. The orb-web spider Argiope aurantia may likewise
strategically envenomate certain prey (Harwood 1974). After wrapping orthopteran prey, this spider
delivers a series of short bites and a single sustained bite. Data for 51 bites indicated the majority (~80 %)
of sustained bites were on the anterior half of the prey. For the orb-web spider A. argentata,
non-lepidopteran prey were bitten after wrapping, and bites were often directed at the head or thorax
(Robinson and Olazarri 1971). However, in some cases initial bite location is more a matter of happen-
stance; when prey were bitten before wrapping, which is common for lepidopteran prey, the bite was often
delivered to the first point of prey contact. Even so, if the initial bite occurred on a wing or other
appendage, the bite was transferred to a more “substantial” part of the prey, possibly due to sensory
information received directly by the chelicerae.
In contrast to the above examples in which spiders targeted their venom toward the prey’s central
nervous system, the bites of other spiders may be directed toward peripheral targets such as legs or
antennae (Parks et al. 2006; Suter and Stratton 2012), or directed seemingly randomly toward prey in the
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same proportion as the surface area of the prey’s body parts (Morse 1999). Bites directed toward the legs
appear to be particularly efficacious for the ant-eating spider Zodarion cyrenaicum (Zodariidae). Both the
young and adults of this species subdued exceptionally large prey, more than 30 times their own mass,
with one or two highly toxic bites that were delivered 75 % of the time to a leg (Pekar et al. 2014). Most of
the bites to legs (60 %) were delivered to a rear leg, which presumably afforded greater safety.
Taken together, sufficient evidence indicates that many spiders demonstrate a preference for
envenomating certain types of prey in a particularly vulnerable region, presumably to effect rapid prey
immobilization, or in a relatively safe region to avoid retaliatory injury. However, targeted venom delivery
in spiders is neither strict nor universal.

The widespread reliance on venom for predation and defense among spiders underscores the important
role of this toxic secretion in the biology of this group. Two common themes emerge from this review of
venom deployment: economization and optimization of venom. Accumulating evidence from spiders
supports the view that venom represents a valuable but limited commodity. Although spiders are ideally
suited to measure the metabolic costs associated with synthesis, storage, and discharge of their venom, no
empirical study has been conducted in this group to date. However, evidence from behavioral and
ecological perspectives supports the importance of venom economization in spiders. Compelling evi-
dence also establishes that spiders typically deploy their venom in an optimal manner, as documented for
four major strategies. Many spiders are highly selective in their use of venom; many appear capable of
metering their venom by number of doses or quantity within a single dose; at least some and perhaps many
may be capable of modulating the composition of their venom gland secretions during successive
episodes of deployment; and many are capable of selectively targeting the delivery of their venom to a
particularly vulnerable region of their target, or to a relatively safe region to avoid retaliatory injury.
Although these four behavioral strategies of spiders are supported by the current body of research, the
behaviors of only a very small proportion of the 45,000-plus species (Natural History Museum Bern
2015) have been studied thus far. By contrast, researchers have generated substantial details on other
aspects of venom (e.g., biochemistry, pharmacology, toxicology) for a much greater diversity of spiders
(Nentwig and Kuhn-Nentwig 2013). Again, researchers have to a large extent neglected to study the
behavioral roles and usage of venom. Investigators need not only to examine venom use for a greater
variety of species but also to identify possible sex differences and ontogenetic changes. Further studies of
economization and optimization in spiders will shed light on the many opportunities and constraints
associated with venom deployment that are afforded by the diverse trophic specializations within this
group.
The possible role of cognition in spider venom deployment will no doubt receive further attention.
Unfortunately, authors continue to differ in how they define cognition, which ranges from more general
concepts, including any use of information from the environment, to the need for higher-order processing
(beyond the normal stimulus-response pathway), such as cognitive maps, planning, and concept learning
(e.g., Menzel 2013; Perry et al. 2013; Shettleworth 2013). In part due to their relative structural and
neurological simplicity, spiders have proven to be useful models for exploring the features of behavioral
decision making and cognition (Jackson and Cross 2011). Spiders exhibit complex, flexible behavior that
sometimes closely parallels that of much bigger animals. Although better documented in larger animals,
spiders are able to decide between different options in reference to the expected outcome of potential
actions. Venom deployment by spiders may well satisfy some or many definitions of cognition, but more
careful assessment is required to rule out simpler explanations.
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The venom delivery system and venom deployment strategies of spiders invite comparisons to other
animal groups. Many venomous taxa possess a single venom delivery system, which is often placed
posteriorly on the body (e.g., the stinger of scorpions and hymenopterans). Spiders share more in common
with snakes, however, in having a paired venom delivery apparatus placed anteriorly, which includes two
chelicerae with fangs. In both snakes and spiders, the right and left venom delivery systems can function
independently, which creates unique opportunities for prey capture and handling (e.g., backward stabbing
by atractaspidid snakes) and fang repositioning during venom deployment (Hayes 2008). At least one
group of spiders delivers venom into prey with a single fang at a time (Rezac et al. 2008), but the
functional aspects of two independent venom delivery systems warrant further consideration. Some
animals, including certain groups of ants, wasps, scorpions, and snakes, are capable of modulating the
mechanism of venom expulsion, delivering toxins via either biting or spraying. One araneomorph group,
the spitting spiders, is similarly capable of both biting and spitting. However, the content of the spider
secretions differs, with spits comprised mostly or entirely of adhesive, glue-like strands produced in the
anterior portion of the venom gland, which immediately immobilize the prey, and subsequent bites
delivering venom produced in the posterior portion of the venom gland, which further incapacitates or
kills the prey (Zobel-Thropp et al. 2014). Stimuli relied on for decisions regarding venom use also
appear to differ among venomous animals. Whereas many snakes bite and then immediately release their
prey, which presumably requires a decision on the snake’s part prior to attack (Hayes et al. 2002), spiders
generally maintain contact with their prey during and/or subsequent to envenomation and evaluate
the optimal venom dose based on the prey’s struggle. However, considering the apparent ability to
perceive venom availability (Wullschleger and Nentwig 2002) and threat level (Nelsen et al. 2014a) in
ways that influence venom use, spiders may also have the ability to decide venom dose prior to attack.
Ant-eating spiders of the genus Zodarion offer an excellent model to study this possibility, as they
typically deliver a single bite or two to their giant, dangerous prey and then wait for envenomation to take
effect (Pekar et al. 2014). Finally, some groups of spiders share with hymenopteran insects the capacity to
deliver venom as a social group. In doing so, these animals are able to procure larger prey items than
otherwise possible and are better able to defend themselves against attack (e.g., Campon 2007; Grinsted
et al. 2013).
Behavioral opportunities and constraints related to venom deployment are associated with morpho-
logical and biochemical traits that act in concert to achieve a desired outcome (Pekar and Toft 2014).
The capacity of spiders to meter their venom, or to target vulnerable body regions of a target animal, can
presumably influence selection on a number of traits, and vice versa. Examples include size and shape of
structures related to venom storage and delivery; neurological complexity to support decision
making; and venom composition, since different peptides and proteins may be more effective when
delivered to or near ganglia in the head or thorax compared to delivery into the abdomen. Spiders within a
single genus (Dysdera) specialized to feed on woodlice (Crustacea, Oniscoidea) illustrate how multiple
solutions can evolve in response to some of these complex relationships (Rezac et al. 2008). Species
having elongate chelicerae insert a single fang into the soft ventral side of their woodlouse prey and
place the other chelicera on the dorsal side of their prey; species with dorsally concave chelicerae
quickly tuck their chelicerae under their prey to bite the ventral surface; and species with flattened
chelicerae insert their chelicerae between the sclerites into the armor of the woodlouse. Future studies
with diverse spider groups will offer further insights on how venom deployment relates to other
non-behavioral traits.
Page 14 of 18
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Cross-References
▶ Slow Loris (Evolution of Venomous Animals and Their Toxins)
▶ Venom as a Component of External Immune Defense in Hymenoptera
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DOI 10.1007/978-94-007-6727-0_14-1
Systematics of Siphonophores
Gillian M. Mapstone*
Department of Life Sciences, The Natural History Museum, London, UK
Abstract
Siphonophores are the most complex of all pelagic medusozoan hydrozoan cnidarians, bearing various
types of zooids on a long stem and often termed “string jellyfish.” They are extremely fragile and live
almost exclusively in the open ocean. They vary in length from 50 m down to 10–20 mm. Most species
bear swimming bells (nectophores) for locomotion, some have a float (pneumatophore), and all have a
long stem of iterative units termed cormidia for feeding, reproduction, and also protection and buoyancy.
Tentacles from the cormidia bear stinging cells (nematocysts) for prey capture, either in simple groups or
lines or in more complex nematocyst batteries on side branches known as tentilla. In life, tentacles and
their side branches extend into a three-dimensional net for fishing, into which prey either blunders by
accident or, in a few species, is attracted by lures. Such great diversity has led to a complex systematics
based on a range of morphological characters, recently enhanced by the first molecular study of the group.
From this a new phylogeny has been proposed, for 17 valid families (one semi-benthic) and 177 valid
species (some unassigned). Characters of these families are reviewed in two tables and 17 summaries,
including diagnostic characters, number and variety of species, and, where appropriate, habitat prefer-
ences and relative success in today’s seas. Figures and images showing different types of siphonophores,
their morphology, stinging organs, and appearance in life accompany the main text.
Keywords
Siphonophores; Cystonects; Physonects; Calycophorans; Hydrozoa; Nematocysts; Nectophores;
Cormidia, pneumatophore; Tentacles; Eudoxid
Introduction
Siphonophores are complex pelagic cnidarians in the medusozoan group Hydrozoa. They are carnivorous
“sit and wait” predators, some of which can lure prey into their tentacles by mimicking shoals of small
copepods, medusae, or fish. They rapidly stun their prey with toxins delivered from batteries of
nematocysts on the tentacles or tentacle side branches. This is essential because, otherwise, the siphono-
phore itself would be damaged by struggling prey attempting to escape.
Individuals possess enormous powers of extension, contracting their stem and tentacles completely for
swimming and then relaxing them to the maximum for feeding. This relaxation allows formation of an
enormous three-dimensional fishing net, or web, often with the aid of swirling swimming movements to
“set the trap.” Once set, many siphonophores simply remain stationary in the water until prey blunders
into their net. Others may lure them in either by drawing copepod-like stinging batteries through the water
or by flicking red lures resembling shoaling fish to attract prey.
*Email: g.mapstone@nhm.ac.uk
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Cnidaria are an ancient lineage, characterized by the presence of cnidae, or stinging cells, most of which
are nematocysts. In siphonophores, nematocysts are grouped into either pads on the tentacles
(e.g., Physalia, the Portuguese man-of-war) or, in most other species, complex nematocyst batteries on
side branches from the tentacles termed tentilla.
Siphonophores are often called string jellyfish, or chain jellyfish, to distinguish them from true jellyfish
and hydromedusae, which are mostly disk shaped. All belong to one of two major clades which comprise
the Cnidaria, namely, the Medusozoa. This clade includes Hydrozoa, Scyphozoa, Cubozoa, and
Staurozoa. Most jellyfish in these groups have a bottom-living stage in their life cycle that restricts
them to the shelves around most continents. Siphonophora, however, are holopelagic (except one family),
meaning that they pass through their entire life cycle in the water column, without having a benthic
stage. This has enabled them to penetrate all oceans, and most exhibit a worldwide, or cosmopolitan,
distribution. Some species are restricted to tropical latitudes, others to temperate latitudes, and a few are
truly cosmopolitan, with records from all around the globe from Arctic to Antarctic waters (Mapstone
2014, Tables 2 and 3).
Siphonophore specimens are difficult to obtain, because they inhabit the deep sea, and are therefore
absent from net catches used to monitor coastal waters. Today most are collected by either blue-water
SCUBA (self-contained underwater breathing apparatus) or remotely operated vehicles (ROVs), but in
the past when nets were used, specimens were often fragmented and damaged and the importance of
siphonophores in pelagic assemblages not fully appreciated. Gelatinous animals have traditionally been
preserved in buffered formaldehyde to preserve their shape, and vast collections are present at a few
locations around the world, including the Natural History Museum, London, but these are of little use for
molecular work, which requires alcohol-preserved material.
Siphonophores are colonial polymorphic hydrozoans with physiological integration of zooids and a
complex morphology. They also exhibit a diversity of body form. An understanding of their morphology
is therefore needed to investigate their systematics, and morphology was very well explained in a seminal
monograph of 1965 by A.K. Totton. Little then changed until the first molecular analyses of the group by
Dunn et al. (2005b), which revealed some new relationships and diagnostic characters within the group.
Characters not previously thought to be important were found to be diagnostic, including the presence or
absence of swimming bells, the sexual state of the family or species, the presence or absence of a muscular
free zone in the wall of the nectosac of the nectophores, and the type of cormidia present on the stem.
These, and other more traditional characters, are reviewed below.
Taxonomic Status
Siphonophora are a small monophyletic group in the phylum Cnidaria and members of the diverse clade
Hydrozoa, comprising c. 3,500 species (Fig. 1a). The Hydrozoa includes the large subclass Hydroidolina
(3,317 species) and the much smaller subclass Trachylina (155 species) (Fig. 1b). The Siphonophora are a
small group of c. 177 species within the Hydroidolina, while the remainder of this subclass comprises the
much larger groups Anthoathecata and Leptothecata (c. 3,140 species). These latter two clades are mostly
meroplanktonic, with a bottom-living polypoid “hydroid” stage in the life cycle. As noted above, this
restricts their geographical distribution and makes them species-rich. Siphonophores, in contrast, are a
depauperate species-poor clade and in this respect resemble the other two holopelagic hydrozoan groups
Trachymedusae and Narcomedusae, which together comprise c. 96 species (Fig. 1b) (World Hydrozoa
Database).
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Fig. 1 Position of Siphonophora within the phylum Cnidaria. (a) the c. 10,000 Cnidaria species (excluding Myxozoa)
subdivided into clades; (b) the c. 3,500 Hydrozoan species, subdivided by ranks (Modified from # Gillian Mapstone (2014),
Fig. 2A, B; see legend for this figure and Mapstone (2015) for origins of other numbers used in a and b)
Typical Body Plans and Cormidia

The basic morphology of siphonophores is best explained with reference to the body plans of three typical
species from the groups Cystonecta, Physonectae, and Calycophorae (Fig. 2). An anterior-posterior axis
related to the direction of swimming is recognized in all siphonophores (reviewed by Mapstone 2009),
which in Fig. 2 is oriented vertically, with the anterior end uppermost.
All three types have a long stem, the siphosome, which bears repeating, or iterative, groups of zooids
known as cormidia. One cormidium always includes a gastrozooid with tentacle for feeding and one or
more sexual zooids for reproduction, often on a tree-like stalk known as a gonodendron. In addition,
the cormidia of physonects and calycophorans also include one or more bracts (Fig. 2b, c). A float, the
pneumatophore, is present in cystonects and physonects (Fig. 2a, b), and swimming bells, the
nectophores, are present in physonects and calycophorans (Fig. 2b, c). An additional length of stem,
the nectosome, carries the nectophores in Fig. 2b (see definition in Mapstone 2009, p. 72).
The pneumatophore is filled with carbon monoxide secreted by a gas gland, and this provides buoyancy
for cystonects and physonects. Additional buoyancy is gained from gelatinous tissues in physonects and
calycophorans, particularly from bracts in the cormidia; these tissues have a lowered specific gravity from
the partial replacement of sulfate ions by chloride ions in the mesoglea (Mackie et al. 1987).
Propulsive zooids, the nectophores, are characteristic of physonects and calycophorans, and each
contains a muscular nectosac which undergoes repeated contractions during swimming to facilitate active
locomotion through the water (water exits via the ostium). Nectophores are absent in cystonects, which
can only drift passively, and, by repeated contraction and expansion of the stem and tentacles, extend their
tentacles to form a very basic fishing net for feeding.
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Fig. 2 Siphonophore morphology. Three typical body plans: (a) long-stemmed cystonect Rhizophysa eysenhardti,
(b) long-stemmed physonect Nanomia bijuga, and (c) typical calycophoran Lensia conoidea; inset Cc shows two tentilla
attached to one tentacle. Labels: b bract, c cormidium, gz gastrozooid, gd gonodendron, h hydroecium, n nectophore
(swimming bell), nb nematocyst battery (a cnidoband), o ostium, p pedicel, pn pneumatophore (float), s stem, sh siphosomal
horn (growth or budding zone), som somatocyst, t tentacle, tf terminal filament (Derived from # Gillian Mapstone (2014),
Fig. 3; refer to this figure legend for details of published figures and references from which drawings derived)
All zooids are formed by budding in budding (or growth) zones present in various parts of the
siphonophore individual or “colony.” Buds mature into particular zooids, for example, those in a
cormidium, as they move down the stem toward the posterior end and the stem simultaneously lengthens.
The budding zone is often contracted and difficult to identify in preserved material, but in Fig. 2b of a
typical physonect, the siphosomal budding zone is clearly visible.
Molecular Phylogeny and Valid Taxa

The new molecular phylogeny of Siphonophora by Dunn et al. (2005b) is based on the nuclear small
subunit ribosomal RNA gene 18S and the mitochondrial large subunit ribosomal RNA gene 16S. It shows
that Cystonecta (as Cystonectae), without nectophores, is sister to all other siphonophores, termed the
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Fig. 3 Molecular phylogeny of Siphonophora from Dunn et al. (2005b, Fig. 6). Consensus tree of all trees for the Bayesian
analysis of the combined data set (from an initial 20 million trees). The left score above the branch is the Bayesian posterior
probability (%), the right score above the branch is the ML bootstrap support value (%), and the score below the branch is the
MP bootstrap support value (%). The bars to the right of the species names indicate clades and grade taxa. Abbreviations: Atl
Atlantic, Med Mediterranean, Pac Pacific. For full details of analyses and consensus tree computations, refer to Dunn
et al. (2005b) (# Mapstone 2014, Fig. 9)
Codonophora, or bell-bearers, with nectophores (Fig. 3). The phylogeny also shows the Physonectae to be
paraphyletic, whereas the Calycophorae are a monophyletic clade within the Codonophora.
Sexual state has been found to be an important character in determining relationships within the
Codonophora. Cystonects are all dioecious (separate sexes), and five discrete physonect families and
one unascribed genus within the Codonophora also display dioecy (Table 1). The remaining four discrete
physonect families, three further unascribed physonect genera, and all calycophoran families are mon-
oecious, with both sexes developing on the same individual, albeit at different times, to prevent self-
fertilization. Monoecy enables cross fertilization between individuals in the deep sea where populations
can be very small and mating opportunities limited.
In codonophorans, cormidia first arise as “pro-buds” on a swelling at the anterior end of the siphosome
known as the “horn” or siphosomal growth zone (Fig. 2b). In cystonects there is no horn, and zooids arise
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Table 1 Siphonophora systematics: higher ranks, valid families, and genera (Extracted from # Mapstone 2014, Table 3, with
Stephanomiidae added and a question mark added to Rudjakovia; see Table 2)
High rank Family and subfamily Genera
I – Cystonecta 01. Physaliidae Physalia
02. Rhizophysidae Bathyphysa, Rhizophysa
II – Codonophora
Physonectae
Dioecious families 03. Apolemiidae Apolemia
04. Erennidae Erenna, Parerenna
05. Pyrostephidae Bargmannia, Pyrostephos
06. Rhodaliidae Angelopsis, Aranciala, Dromalia, Archangelopsis, Steleophysema,
Stephalia, Thermopalia, Tridensa
07. Stephanomiidae Stephanomia
08. Unascribed Marrus
dioecious genus
Monoecious 09. Forskaliidae Forskalia
families
10. Physophoridae Physophora
11. Resomiidae Resomia
12. Agalmatidae Agalma, Athorybia, Melophysa
sensu stricto
13. Unascribed Cordagalma, Frillagalma, Lychnagalma, and maybe Rudjakovia
monoecious genera
Calycophorae
Prayomorphs 14. Prayidae
S-f Amphyicaryoninae Amphicaryon, Maresearsia
S-f Prayinae Craseoa, Desmophyes, Rosacea, Gymnopraia, Lilyopsis, Mistoprayina,
Praya, Prayola, Stephanophyes
S-f Nectopyramidinae Nectadamas, Nectopyramis
15. Hippopodiidae Hippopodius, Vogtia
Diphyomorphs 16. Clausophyidae Chuniphyes, Clausophyes, Crystallophyes, Kephyes, Heteropyramis
17. Sphaeronectidae Sphaeronectes
18. Diphyidae
S-f Sulculeolariinae Sulculeolaria
S-f Diphyinae Chelophyes, Dimophyes, Diphyes, Eudoxoides, Lensia, Muggiaea
S-f Giliinae Gilia
19. Abylidae
S-f Abylinae Abyla, Ceratocymba
S-f Abylopsinae Abylopsis, Bassia, Enneagonum
as independent buds directly on the stem (Dunn and Wagner 2006). This synapomorphy makes the
cormidia into integrated units in Codonophora and may explain the huge radiation and diversity within
this group, in contrast to the Cystonecta.
One hundred seventy-seven species of siphonophore are considered valid at the time of writing
(WoRMS Siphonophora List), and these are shown in relation to families, higher ranks, and sexual
state in Fig. 4.
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Fig. 4 Siphonophora species. The 177 valid Siphonophora species subdivided into ranks based on Table 1 (Derived from #
Gillian Mapstone 2014, Fig. 2C)
Proposed Morphological Phylogeny of the Siphonophora

A tentative phylogeny of the Siphonophora derived from the molecular phylogeny in Fig. 3 is given in
Fig. 5. It was proposed by Pugh (2006a) and is based on sexual state and several other morphological
characters not previously considered important.
This phylogeny shows that particular diagnostic characters, including those discussed above, might
have been important during siphonophore evolution. Other significant characters could have been the
position of origin of the zooids on the siphonophore stem, the type of canals on the proximal surface of the
nectophore, and the amount of musculature in the nectophore nectosac. Studies on budding (growth)
zones and cormidial development in seven siphonophore taxa, together with earlier studies on three other
taxa, led Dunn and Wagner (2006) to suggest several key transitions that could have occurred during
siphonophore evolution. These include the appearance of a siphosome and pneumatophore in the
ancestral siphonophore, the origin of the nectosome in the ancestral codonophoran, and a change from
dioecy to monoecy during the evolution of the four monoecious physonect families (and three unascribed
genera) and all calycophoran families (Fig. 5, and see Dunn and Wagner 2006, Fig. 7).
Dunn and Wagner (2006) also suggest that the nectosome of the ancestral codonophoran might first
have appeared as a tandem duplication of the siphosome. Siphosomal zooids are taken, by convention, to
arise from the ventral side of the stem (Haddock et al. 2005a), and nectosomal nectophores are also
thought to have arisen from this same meridian in the first codonophorans (Fig. 5). Later during evolution,
the nectosome appears to have twisted relative to the siphosome with the result that, in the family
Agalmatidae sensu stricto, nectophores arise from the dorsal side of the stem (Fig. 5; Dunn and Wagner
2006).
Another character of importance, which appears to have been modified during codonophoran evolu-
tion, is the type of canals present on the proximal surface of the nectophore. These canals, known either as
the pallial canals (Mapstone 2014), the mantle canals (Pugh and Baxter 2014), or the ascending and
descending diverticula (Mapstone 2009), are important for shedding, or self-amputation, of nectophores
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Fig. 5 Possible phylogeny of the Siphonophora (Derived from Pugh 2006a, Fig. 21; Siebert et al. 2013; Pugh and Baxter
2014; # Mapstone 2014, Fig. 10); MFZ – muscle-free zone on nectophore; * – dorsal nectosome and some undescribed
species monoecious; ** – one species monoecious
and other zooids during autotomy (Mapstone 2009). Thus, they form what is sometimes termed an
“autotomy joint.” Nectophores senesce (age) as they pass down the nectosome to the posterior end
where they split off from the muscular lamella along the line of this canal (Fig. 6) one by one, as more
nectophores are added in the nectophoral budding zone at the anterior end of the nectosome (Mapstone
2009). Nectophores may also be shed for defense, if startled by a predator, leaving loose zooids in
the water to create confusion and enabling the siphonophore to retreat (Mackie et al. 1987). Only an
ascending pallial canal is present in the dioecious Apolemiidae, Erennidae, Pyrostephidae, Rhodaliidae,
and Stephanomiidae, whereas in monoecious physonects a second descending pallial canal is also
developed (Figs. 5 and 6).
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Fig. 6 Pedicular and radial canal arrangements in nectophores of four physonect species. All diagrams through
nectophore midline. (a, b) Dioecious Apolemia uvaria; (c) dioecious Bargmannia sp.; (d, e) monoecious Agalma elegans;
(f, g) monoecious Nanomia bijuga; apc ascending pallial canal (surface diverticulum), dpc descending pallial canal (surface
diverticulum), loc lower radial canal, lrc lateral radial canal, ns nectosac, pe external pedicular canal, pi internal pedicular
canal, st stem, urc upper radial canal; nectophoral muscular lamella shown in dark gray in (a, d, f); nectosac shown in pale gray
in (b, c, e, g) (Derived from # Gillian Mapstone 2009, Figs. 5 and 6)
Other morphological characters of importance during siphonophore evolution are summarized in

tabular form and discussed under family headings below (see Table 2).
Cystonecta
A monophyletic clade that is sister to all other siphonophores, as noted above. Cystonects are dioecious,
without a nectosome or any bracts in the siphosomal cormidia. The two families referable to this clade are
Physaliidae and Rhizophysidae, and typical cystonect morphology is shown in Fig. 2a (long-stemmed
cystonect) and Fig. 7 (Cystonect morphology).
The well-known and enigmatic Portuguese man-of-war cystonect, Physalia, is large, is pleustonic
(lives at the surface) when mature, and has a much enlarged crested float (pneumatophore) propelled by
the wind, but no stem (Fig. 7a). It is the only siphonophore with toxins sufficiently powerful to harm
humans, although the envenomations reported worldwide in warmer waters rarely lead to death (Fenner
2000). Cormidia arise directly on the underside of the float and the long tentacles stream out to windward
(Fig. 7b). Cormidia bud one from another in a series, each termed a “cormidial complex” (Fig. 7c). Indeed,
Physalia displays the most prolific budding of any siphonophore. A mature specimen has 12 cormidial
complexes arising in two groups (Fig. 7b), with, for example, one simpler complex in the oral region,
giving a total of c. 13 cormidia. Most cormidia are tripartite, with a gastrozooid and gonodendron and a
basigaster separated from the column of the gastrozooid to form a separate ampulla with tentacle (Fig. 7d);
in the gastrozooids of all other siphonophores, the basigaster forms a thickened ring around the proximal
end of this zooid itself (where nematocyst formation occurs). Most tentacles are convoluted (Fig. 7e) and
supported by an extensible membrane, which allows them to contract up near to the float when not
feeding. Nematocysts cover the tentacles and are particularly concentrated in the convoluted tentacles
(Fig. 7f); however, these concentrations do not constitute true nematocyst batteries, which, in siphono-
phores, occur on the tentilla (side branches) of the tentacles of all codonophorans except the Apolemiidae
(Mapstone 2009, p. 74). In Rhizophysa, nematocysts are variably distributed, forming a simple line along
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Table 2 Characters for cystonect and “physonect” families (Derived from # Mapstone 2014, Table 4, with Stephanomiidae
added; see this paper also for additional references omitted below; see Fig. 5 for details of fundamental siphonophore
characters mentioned below)
Family Comments
01. Physaliidae Monotypic for Physalia physalis (P. utriculus considered a junior synonym, Bardi and
Marques 2007)
02. Rhizophysidae Long-stemmed family of four valid species; Bathyphysa japonica a junior synonym of
B. conifera; SEM (scanning electron microscope) studies of budding sequences described
for B. sibogae, Rhizophysa filiformis, and R. eysenhardti (Dunn and Wagner 2006)
03. Apolemiidae Long-stemmed family; monophyletic and sister to all other Codonophora, with unique
nectophoral palpons on the nectosome. Nectophores distinctive and ridgeless, cormidia
dispersed or discrete (pedunculate); gastrozooids with simple tentacles (no tentilla)
resembling palpacles of palpons. Monogeneric for Apolemia. Two new species include
A. lanosa and A. rubriversa (Siebert et al. 2013) and three older species A. contorta,
A. uvaria, and A. vitiazi (Tottonia contorta sensu Mapstone 2003 now referable to
A. lanosa). A number of other species are known to exist (Dunn et al. 2005b; Mapstone
2003, 2009; Siebert et al. 2013) and await full description
04. Erennidae Long-stemmed family erected for four species with large prominent straight tentilla, no
involucrum, and a rigid terminal process lacking nematocysts. Two genera: Erenna (three
species) and Parerenna (one species). E. richardi Bedot, 1904, and a new species
E. laciniata have large flattened nectophores and large tentilla held close to the body and
vibrated to attract prey; two further new species E. cornuta and Parerenna emilyae have
different and also unique tentilla and gastrozooids (Pugh 2001)
05. Pyrostephidae Long-stemmed family of five species in two genera: Bargmannia (four species),
Pyrostephos (one species). Pugh (1999a) reviewed the family, introducing two new species
(B. amoena, B. gigas) and revising two others (B. elongata, B. lata); Mica micula shown to
be putative post-larva of a pyrostephid (Grossmann et al. 2013). Nectophores with unique
lower-lateral wings and much enlarged triangular thrust block; in B. elongata two growth
zones on stem and composition of the cormidia studied using SEM (Dunn 2005);
pyrostephid cormidia either have oleocysts (modified tentacle-less palpons) (in Pyrostephos)
or none (in Bargmannia) (Pugh 1999a)
06. Rhodaliidae Short-stemmed family of eight genera, with four new species including Archangelopsis
jagoa, Arancialia captonia, and two in the genus Tridensa, including T. sulawensis and
T. rotunda. Genus Sagamalia reduced to junior synonym of Steleophysema (WoRMS
Siphonophora List). First in situ feeding observations on four species (Hissmann 2005).
Dromalia alexandri redescribed (Mapstone and Ljubenkov 2013)
07. Stephanomiidae Disbanded family reintroduced for single large long-stemmed dioecious species
Stephanomia amphytridis (Pugh and Baxter 2014); nectosome ventral, nectosac of mature
nectophores with muscle-free zone, and other characters (Fig. 5)
08. Unascribed dioecious Long-stemmed genus Marrus Totton, 1954, with muscle-free zone on nectosac and other
genus characters (Fig. 5); new species M. claudanielis introduced (Dunn et al. 2005a)
09. Forskaliidae Long-stemmed and delicate monotypic family, probably sister to the Physophoridae
(Dunn et al. 2005b). Recently revised (Pugh 2003) with two new species added
(Forskalia asymmetrica, F. saccula) and one reduced to a species inquirenda
(WoRMS Siphonophora List)
10. Physophoridae Family with long nectosome but short corm-like siphosome; previously monotypic for
Physophora hydrostatica with bract present only in larva; second species P. gilmeri added by
Pugh (2005). Smaller, less colorful, and with bracts retained on adult colony. Tentilla of this
family unique
11. Resomiidae Long-stemmed family newly introduced for two species previously referred to the
Agalmatidae (Moseria convoluta, M. similis) and now transferred to a new monotypic genus
Resomia (Pugh 2006a); two tentilla types uniquely present on each tentacle. Three new
species R. dunni, R. ornicephala, and R. persica added by Pugh and Haddock (2010)
(continued)
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Table 2 (continued)
Family Comments
12. Agalmatidae Mostly long-stemmed and recently restricted to genera with dorsal nectosome (Fig. 5) and
sensu stricto involucrate tricornuate or unicornuate tentilla with typically tightly coiled cnidoband which
now includes two short-stemmed genera (Athorybia, Melophysa) (Dunn et al. 2005b), with
two new species of Halistemma, H. transliratum, and H. maculatum introduced and four
other Halistemma species redescribed (see Pugh and Baxter 2014 for details)
13. Unascribed Long-stemmed monotypic genera Cordagalma, Frillagalma, and Lychnagalma now
monoecious genera removed from the Agalmatidae for their ventral nectosomes (Fig. 5); new species C. tottoni
described; Rudjakovia plicata considered a valid species, with a dorsal nectosome but sex
unknown; it may be transferred to Agalmatidae when more characters are elucidated (Pugh
2006a; Mapstone 2015)
each tentacle side branch in R. eysenhardti (Fig. 2a), and concentrated into small button-like clusters on
the trifid tips of each tentacle side branch in R. filiformis (Fig. 7h, i) (Totton 1965).
Apolemiidae
The main characters of this family are summarized in Table 2, and the nectosomal palpons are shown in
Fig. 8f. Apolemiids are unusual in growing to lengths of 30 m or more (Siebert et al. 2013), longer than
any other known siphonophore (Fig. 8a, b). All zooids arise from the ventral meridian of the stem
(Fig. 8c), giving apolemiids a ventral nectosome (Figs. 5 and 8c). The nectosome and cormidia are more
complex than in other codonophoran families, with at least two different patterns of cormidial organiza-
tion apparent in the three different Apolemia taxa so far investigated (Siebert et al. 2013). Dispersed
cormidia occur in A. lanosa (Fig. 8a, b) where zooids spread out along the stem (Fig. 8d, g) as soon as the
pro-bud leaves the siphosomal horn. In A. rubriversa (Fig. 8f) and A. uvaria (Fig. 8e), cormidia are
pedunculate, with all zooids of one cormidium arising from the peduncle (or pedicel) of the first
gastrozooid to be formed, on the siphosomal horn (Siebert et al. 2013). These fundamental features of
zooid budding are concluded by Siebert et al. (2013) to be homoplastic in codonophorans.
In young specimens of Apolemia uvaria, pedunculate cormidia are clearly separated from others by
bare lengths of stem (Fig. 8e). As growth proceeds, the naked stem portions become partially or
completely obscured by prodigious budding of the pedunculate cormidia, as well shown by Siebert
et al. (2013, Fig. 18c). In A. lanosa the siphosome becomes very long and extends a prominent curtain of
“tentacles” for feeding (Fig. 8g). This curtain comprises mainly palpacles from the numerous palpon
clusters on the stem, and also many fewer thin tentacles from the gastrozooids (Siebert et al. 2013). Each
simple palpacle or tentacle bears a narrow band of nematocysts down one side for catching prey
(Mapstone 2003, Fig. 12e, f) but no true tentilla. Stem length is further increased in A. lanosa during
growth by interpolation of secondary gastrozooids and more palpon clusters, as shown in Fig. 8d.
Erennidae
In this, and all remaining codonophorans, taxa are characterized by the presence of tentilla, or complex
stinging batteries, on the side branches of their tentacles. For physonects, these tentilla are diagnostic, but
calycophoran tentilla are rather uniform and of little or no diagnostic value. Each tentillum includes a
pedicel, thickened cnidoband, and (usually) a thin extensible terminal filament. Each cnidoband is packed
with rows of nematocysts, with more on the terminal filament(s) of almost all species. It is the nemato-
cysts, or cnidae, which deliver toxins to the prey, either by penetration or entanglement, and more
nematocyst types are found in siphonophores than in any other hydrozoans. Different types of
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Fig. 7 Cystonect morphology. (a) Atypical Physalia physalis, pleustonic (lives at surface), with much enlarged pneumat-
ophore, no stem, cormidia arising directly from underside of pneumatophore; (b) left-handed drifting specimen viewed from
above – added numbers 1–5 identify oral cormidial groups, while numbers I–VII identify main cormidial groups – note how
Physalia’s surface float drifts to starboard with the wind on a broad reach; (c) oral cormidial complex number 2 viewed from
inside the float – note groups 3–8 are tripartite, with more tripartite groups on oral and aboral side branches – with numbers in
brackets added to identify tripartite groups; (d) a developing tripartite group from main cormidial complex number VI; (e) SEM
of part of contracted Physalia tentacle; (f) detail from (e), with darker holes marking discharged nematocysts; (g) Rhizophysa
filiformis (see Fig. 2 legend for long-stemmed cystonect features); (h, i) R. filiformis trifid nematocyst pads; Labels: a ampulla
(basigaster), fw float wall, gd gonodendron, gz gastrozooid, np nematocyst pad, pn pneumatophore (float), t tentacle, ta tentacle
with ampulla (basigaster), tg tripartite group (a # Casey Dunn Brown; b–d, i # Gillian Mapstone 2014, Fig. 2 insets Aa, Ab,
Fig. 7; for details of published figures and references from which drawings were derived, see relevant figure legends; e, f from
Bardi and Marques 2007, with permission from Iheringia Série Zoologia; g # Larry Madin WHOI; h: Kawamura 1910,
Fig. 5d)
nematocysts, their distribution across siphonophore families, and suitability for different types of prey are
summarized by Mapstone (2014, Table 6).
Erennids have unique and exceptionally large tentilla, with a straight cnidoband of small haploneme
nematocysts, but no larger heteroneme nematocysts (typical of other codonophorans). Also, uniquely, the
thick terminal filament completely lacks nematocysts and instead, in one species at least, bears a pair of
distal red lures to attract prey (Table 2, Fig. 9b). Species are dioecious, with nectophores having only an
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Fig. 8 Apolemiid morphology. (a, b, d, g) Apolemia lanosa from Monterey Bay, (a) whole colony, from 1,150 m (# 2005
MBARI); (b) whole colony, from 670 m (# 2011 MBARI); (c) A. uvaria TS through stem showing ventral zooid meridian
(Korotneff 1883, pl. 14 Fig. 9); (d) A. lanosa detail of cormidium with two large gastrozooids (large red zooids), many longer
palpons, and a secondary gastrozooid (small red zooid) (# Stefan Siebert); (e) A. uvaria young colony, showing pedunculate
cormidia (Gegenbaur 1853, pl. 18 Fig. 1); (f) A. lanosa nectosome with siphosome fragment; note nectophoral palpons
emerging from between the nectophores (# Stefan Siebert); (g) A. lanosa part of siphosome with extended “tentacle” curtain
(no tentilla); note many red gastrozooids (# Stefan Siebert)
ascending pallial canal on the proximal surface (see Fig. 6c) and a muscle-free zone (MFZ) at the proximal
end of the nectosac (Fig. 5). The nectosome is typically long (Fig. 9a) to very long, without any
nectosomal polyps, and the nectophores typically large and flattened, often with black pigment on the
radial canals and other zooids (Pugh 2001). Erennids live at great depths, in the twilight zone where prey
is scarce. In the species with red lures, and probably also the other Erenna species (Pugh 2001), the
siphosome is permanently contracted, and the tentilla always held close to the body. The lures emit red
light (from bioluminescence) and together resemble a shoal of small deep-sea fish in the water. These
attract deep-sea bristlemouth fish, which are of similar size and color. These fish are thought to swim into
the lures, to be immediately stunned by nematocysts on the long straight cnidobands and then ingested by
the gastrozooids, together with some lures (Haddock et al. 2005b).
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Fig. 9 Morphology of other dioecious physonects. (a) Erenna laciniata (# JAMSTEC, https://fbox.jamstec.go.jp/public/
XMH0AAdKtcvAt7cBnXVJOhXGd3gQe6hkGsJUttHV-l Si; the organism in this image was identified by Dr. Dhugal
Lindsay based on examination of the collected specimen); (b) Erenna richardi tentillum; (c) Bargmannia elongata tentillum;
(d) Marrus claudanielis colony, nectophores autotomized (# Marsh Youngbluth); (e) Marrus orthocanna, nectosome, and
first part of siphosome (# Casey Dunn); (f) Marrus orthocanna tentillum; (g) Dromalia alexandri (permissions@wiley); (h)
Tridensa sulawensis tentillum (b, c, f, and h # Gillian Mapstone 2014, from Fig. 12). Labels: cb cnidoband, gv gastrovascular
canal, pe pedicel, rl red lure, st stenotele nematocysts, t tentacle, tf terminal filament
Pyrostephidae
Most family characters are summarized in Fig. 5 and Table 2. Additionally, pyrostephids lack palpacles
(palpon tentacles), and each cormidium contains only either a single unique siphosomal tentacle
(or tentaculozooid) (Bargmannia) or a modified palpacle-less palpon termed an oleocyst (Pyrostephos)
(Dunn 2005; Pugh 1999a). This latter zooid contains an oil-filled vesicle which gives extra support to the
heavy, vermillion red stem (Totton 1965). Tentilla are also unique in pyrostephids, with a cnidoband of
mostly very small nematocysts (desmonemes and acrophores) and only a few larger stenoteles at the
proximal end (Fig. 9c). An axial gastrovascular canal penetrates the length of the terminal filament and is
probably used to extend the cnidoband during prey capture, since pyrostephid tentilla lack the paired elastic
strands found in the tentilla of most other siphonophores (Totton 1965). Pyrostephid terminal filaments
bear many small nematocysts similar to those present in the cnidoband (Mapstone 2014, Table 7).
The cormidial composition of Bargmannia elongata was revealed during an elegant SEM study by
Dunn, who also identified two growth zones: a siphosomal horn on which pro-buds develop, with
subdivision of each pro-bud into one cormidium, and a nectophoral growth zone where individual
nectophores develop (Dunn 2005, Table 2). Each cormidium was found to be completely regular and
also directionally asymmetric.
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The distribution of Bargmannia species is variable and for some species difficult to assess due to
problems of past misidentification (Pugh 1999a). Both B. elongata and B. amoena occur in the Atlantic, as
does B. lata (Pugh 1999a), while in the Pacific only B. elongata and B. lata have so far been positively
identified (Mapstone 2009). Pyrostephos vanhoeffeni, in contrast, is restricted to the southern hemisphere
(Mapstone 2014, Table 1) with an extensive distribution map published recently by Lindsay et al. (2014).
These authors found young specimens of P. vanhoeffeni concentrated close to the Antarctic coast
(nectophores previously misidentified as B. elongata), while larger and more mature nectophores were
found further north, in the open ocean. Indeed, there are records from as far north as 35 S in the Atlantic
and Pacific (Lindsay et al. 2014, map 3). Despite this, the name P. vanhoeffeni, meaning “spiral of fire,”
was originally applied to some big specimens collected not too far from the coast, near to the ice edge at
90 E. These were brightly colored, up to several meters long, and first described by Moser (1925), with
more color notes being given by Totton (1965, p. 78), who pointed out that the stem, gastrozooids, and
tentilla are all bright red, while the nectophores are pale pink with bright red ostia.
Rhodaliidae
This is one of the most species-rich “physonect” families, comprising 14 species in eight genera (Fig. 4;
Table 2; WoRMS Siphonophora List). Rhodaliids have undergone more speciation than long-stemmed
and pelagic physonects due to their semi-benthic habit, which has led to greater geographical isolation and
more restricted ranges for all species (see Mapstone 2014, Table 1, for summaries of two rhodaliid
distributions and abundances: Dromalia alexandri and Rhodalia miranda). The larvae and life cycles of
rhodaliids are unknown, and main diagnostic characters differ from those of pelagic physonects in that
nectophores are simple and lack any surface ridges and have a nectosac that is muscular throughout and a
siphosome which is coiled up into a near-spherical corm with cormidia crowded around it in whorls and
growing outward rather than being spread along a long linear stem.
Important diagnostic characters for rhodaliids include shape and surface texture of the enlarged
pneumatophore and its associated gas gland or aurophore, type of corm developed (thin walled, thick
walled, or solid throughout), type of siphosomal cormidia present (on separate stems or several on a
common stem), and type of bracts developed. In Dromalia alexandri, from off Southern California
(Fig. 9g), the pneumatophore is turreted, the aurophore is papillate, and several siphosomal cormidia
develop on common stems called cormidial units, with up to three cormidia per stem. Furthermore,
growth of cormidia continues throughout life, with units circling around the corm up to eight times in
total. D. alexandri has a unique body form, has been recently redescribed by Mapstone and Ljubenkov
(2013), and its characters compared to those of six other similar genera (Mapstone and Ljubenkov 2013,
Table 3).
Siphosomal cormidia of rhodaliids are either monogastric (each arising separately on an individual
stem), or polygastric, with several cormidia arising from a common stem and somewhat resembling a tree.
These structures are termed “cormidial units,” and mature units from the first and second whorls of
Dromalia alexandri are illustrated by Mapstone and Ljubenkov (2013, Figs. 7 and 8). In other rhodaliids,
cormidia production ceases once two whorls are formed. An important character of rhodaliids is the
presence of two types of gastrozooids instead of one: type II gastrozooids each have a long tentacle
bearing many tentilla for prey capture, while type I gastrozooids have either only a small tentacle or none,
and lack any tentilla. When a rhodaliid attaches to the substrate, it deploys many type II tentacles in a
three-dimensional array (Fig. 9g), and prey blunders into this net to be stunned and held by the tentilla.
Type I gastrozooids then extend out to these tentilla, “hoover up” the prey, and digest it. Feeding has been
described in three rhodaliid species by Hissmann (2005), who identified prey items from small copepods
and amphipods to larger amphipods and fish larvae, which are captured by the type II gastrozooids and
digested by the type I gastrozooids.
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Tentilla of rhodaliids are of typical physonect structure, with a pedicel, elongate cnidoband (sometimes
with a bilobed distal end; see Fig. 9h) of mostly larger and sometimes also smaller nematocysts, and an
elongate terminal filament of many small nematocysts, of one or two types (where known) (Mapstone
2014, Table 7). Bracts occur in the cormidia, typically arise from elongate bracteal lamellae, and are
species-specific for those species in which cormidia have been collected and studied (Mapstone and
Ljubenkov 2013).
Stephanomiidae
This family has only recently been reinstated by Pugh and Baxter (2014) for a single large species
Stephanomia amphytridis (Table 2) first figured from the siphosome only in 1807 by Lesueur and Petit,
and subsequently found with nectosomal zooids at a number of temperate and tropical locations
worldwide (except the South Pacific). The name Stephanomiidae was introduced by Huxley for a second
siphosome he found off the east coast of Australia in 1859, which was found again by Bigelow in the
tropical east Pacific in 1911, and again by Mapstone from the Flores Sea in Indonesia in 2004, this time
with the nectosome as well. All these latter specimens have since been referred to the agalmatid species
Halistemma foliacea (see below).
Stephanomia amphytridis is a large and prominent species reaching up to 5 m in length, with 25 or more
very large nectophores and a distinctive semirigid orange siphosome which is enclosed by many robust
bracts (Pugh and Baxter 2014). The orange color in the siphosome is due to pigmented gastrovascular
fluid (Dunn et al. 2005b) and not to orange pigment in the stem and zooids, as in the two Marrus species
discussed below. S. amphytridis is dioecious (with separate sexes), with a ventral nectosome, a muscle-
free zone on the nectosacs of mature nectophores, sinuous lateral radial canals, and only an ascending
pallial canal on the proximal nectophore surface (see Fig. 5). The pattern of nectophore ridges is similar to
that of Halistemma species, but the vertical-lateral ridges form a complex that is unique to S. amphytridis.
The tentilla of the long tentacles also somewhat resemble those of Halistemma species, since the
cnidoband is loosely coiled and there is only a single terminal filament; but an involucrum is absent
and the nematocysts also seem to be different. In the cnidoband nematocysts are all large, with thin ones
filling most of the cnidoband, but unidentifiable and fatter ones, which could be stenoteles, flanking it on
both sides. Similarly, two types of smaller nematocysts occur on the terminal filament, but these could not
be positively identified, although it is certain that they were not the usual acrophores and desmonemes
(Pugh and Baxter 2014, and see Mapstone 2014, Table 6, for summary of typical physonect nematocysts).
Amongst the material of Stephanomia amphytridis studied by Pugh and Baxter (2014), seven small
Nectalia postlarvae were identified and described, each about 7–8 cm in length. Their nectophores
resembled those of the mature individuals, but the siphosome was very short and had only just started
to grow. It comprised a single gastrozooid (the protozooid) with its tentacle, surrounded by some distinct
elongate larval bracts. The tentacle bore a number of unique larval tentilla unlike those found in any other
physonect, and these contained three types of nematocysts. Species of Halistemma also pass through a
Nectalia stage in their life cycle, but have quite different larval tentilla.
Unascribed Dioecious Genus

The genus Marrus was assigned to this group by Pugh (2006a, Fig. 21) for the muscle-free zone on the
nectosac (Fig. 5). Marrus is an enigmatic genus, with two well-recognized species (M. claudanielis and
M. orthocanna; see Dunn et al. 2005a), one doubtful species (M. antarcticus; see Mapstone 2009), and a
fourth which requires transfer to a different genus (M. orthocannoides; see discussion in Dunn
et al. 2005a; Mapstone 2009). Two recently studied species are striking in vivo. M. claudanielis
(Fig. 9d) was introduced and described by Dunn et al. (2005a); it has a stiff stem which never relaxes
(similar to Agalma okeni, see below) and a thick red zooid-covered siphosome that spirals around to the
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posterior end and is surrounded by a characteristic “halo” of transparent bracts (Fig. 9d). The appearance
of M. orthocanna is similar (Fig. 9e). Marrus species are very fragile and often autotomize their zooids
when illuminated or otherwise disturbed. The colony in Fig. 9d has already lost its nectophores, but in the
small M. orthocanna colony shown in Fig. 9e, the nectophores are still intact. Other images of
M. orthocanna taken under the Arctic ice (see Mapstone 2009, frontispiece A-B) show a larger individual
with at least 12 nectophores, and up to 37 nectophores or more have been recorded in other specimens
(Andersen 1981).
Nectophores of Marrus species are ridged, with straight red pigmented radial canals and a pair of
red-yellow chromatophores on each side of the ostium (possibly for disruptive coloration). The nectosac
has a large muscular-free zone, and there is only an ascending pallial canal (surface diverticulum) on the
proximal nectophore surface. Nectophore ridges are of diagnostic importance in many physonect species,
and in Marrus upper-lateral ridges divide distally in M. orthocanna, but not in M. claudanielis (Dunn
et al. 2005a). Circa 30 mature gastrozooids have been found in the largest Marrus colonies studied, and
the tentilla on their tentacles are either straight (Fig. 9f) or only loosely coiled. Gonodendra are also
numerous on the siphosome and include equal numbers of male and female gonophores in M. orthocanna
(Andersen 1981), which is monoecious, but gonophores of only one sex in M. claudanielis, which is
dioecious (Dunn et al. 2005a). Bracts are large and kite-shaped in both species, with a prominent orange
band of nematocysts and associated ectodermal cells on the upper surface. In M. orthocanna, this band is
relatively short and straight (Fig. 9e), while in M. claudanielis it is longer and arc-shaped (Fig. 9d); this
character is particularly useful for separating the two species in vivo.
Forskaliidae
This is the first monoecious physonect family listed in Tables 1 and 2 and has a ventral nectosome and a
descending pallial canal (descending surface diverticulum) on the proximal nectophore surface (Fig. 5). It
is monotypic for Forskalia and includes six valid species (WoRMS Siphonophora List). The “pallial
canals” are shorter than those shown in Fig. 6d (see Totton 1965, Figs. 57 and 58b) and the internal
pedicular canal (pi) much longer (see Pugh 2003, Figs. 2, 9, 17, 25, 34, and 40), because nectophores are
particularly flattened along the upper-lower axis and extended along the proximal-distal axis (see
Mapstone 2009, Fig. 1b for axes).
Forskaliids are fragile animals and distributed around the globe chiefly in the warmer waters of tropical
and warm-temperate latitudes. SCUBA divers have frequently observed them in the Mediterranean and
western Atlantic, although forskaliids have also been collected by manned submersibles in the Alboran Sea
(western Mediterranean) and the Bahamas (Pugh 2003). Observations on colonies show them to be mostly
snake-like and elongate, with the siphosome longer and broader than the nectosome (see WoRMS
Siphonophora List image of Forskalia edwardsi), although in some Forskalia species (Fig. 10a), the
siphosome appears to be short (F. tholoides, F. asymmetrica; Pugh 2003). One species, F. formosa has
been observed in the Mediterranean swimming powerfully and simultaneously rotating anticlockwise (Pugh
2003). However, in general, Forskalia species swim more slowly than most other physonects, because of
their long and broad siphosome, and have a small pneumatophore for greater maneuverability (Biggs 1977).
The specimen in Fig. 10a is relaxed for feeding, with the nectosome uppermost, although other forskaliids
have been observed hanging vertically in the water with the siphosome uppermost (Biggs 1977).
The family review of Pugh (2003) noted that Forskalia edwardsi could easily be distinguished from
F. contorta in the field by the presence of a small distinctive sulfur-yellow spot at the junction of the upper
radial canal and the ostial ring canal. Both species have been collected in the Mediterranean and western
North Atlantic, and also from the Indian Ocean and Far Eastern seas (Pugh 2003). Another less common
species from off India, which is also present in the Gulf of California and the North Atlantic (although not
the Mediterranean), is F. tholoides, with a distinctive bell-shaped (or fir cone-like) nectosome of
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Fig. 10 Morphology of monoecious physonects 1. Forskaliidae. (a) Forskalia sp. (# Inter-Research, revised from Luo
et al. 2014); (b) Forskalia edwardsi tentillum (# Gillian Mapstone 2014, Fig. 12H). Labels: cb cnidoband, gz gastrozooid,
n nectophores, pe pedicel of gastrozooid, pe + b gastrozooid pedicel with bracts, st stenotele nematocysts in the cnidoband,
t tentacle, tf terminal filament
nectophores that lack axial wings; this species was introduced in 1888 by Haeckel (WoRMS
Siphonophora List). A fourth species is F. formosa of which 20 specimens have been collected in the
western Mediterranean and two in the Bahamas (Pugh 2003). F. asymmetrica, a fifth species, is of similar
abundance with 15 specimens known so far from the western Mediterranean, Bahamas, and canyons off
Woods Hole in the NW Atlantic (see WoRMS Siphonophora List); the mean depth for this species is
598 m. The final forskaliid species, F. saccula, is known only from one young specimen collected close to
the surface in the Sargasso Sea and introduced by Pugh (2003).
In forskaliids, gastrozooids are held away from the siphosomal stem on long pedicels, and the tentacles
dangle down outside this cylinder as shown by Pugh (2003, Fig. 16). Bracts occur on both the gastrozooid
pedicels and the stem (Fig. 10a, Biggs 1977), and those on the pedicels provide buoyancy for the heavy
gastrozooids. Four types of bracts have been found in most species: three types on the gastrozooid
pedicels and a fourth on the stem; these are illustrated and described by Pugh (2003) for all six forskaliid
species. Also on the stem are gonodendra, comprising gonophores of both sexes (monoecious – sexes
maturing at different times), and gonopalpons, which can be species-specific (Pugh 2003).
The tentacles of forskaliids have regularly spaced tentilla, typically 15 per tentacle spaced 5 mm apart
(Fig. 10a, Biggs 1977). Each tentillum (Fig. 10b) has a short pedicel, as seen in Fig. 10a, no involucrum,
and a loosely coiled orange-red cnidoband composed mainly of homotrichous anisorhizas with some
larger lateral stenoteles (Mapstone 2014, Table 7). Beyond the cnidoband, a very long terminal filament
extends for feeding (Fig. 10a), and each such filament bears a repeating pattern of small nematocysts
including a pair of desmonemes, two pairs of acrophores, a pair of desmonemes, and so on (Mapstone
2014, Table 6). Forskaliids are known to sting fishermen and SCUBA divers badly when contact is made
with the tentacles. Copepods are the main prey, together with various other small planktonic organisms
(Purcell 1983).
Physophoridae
This family is monoecious, with a ventral nectosome, a siphosome reduced to a corm, and unique
encapsulated tentilla which may be jiggled like small copepods to act as lures. It is monogeneric for the
genus Physophora and includes two species, P. hydrostatica and P. gilmeri. P. hydrostatica is ubiquitous
with a truly cosmopolitan distribution (Mapstone 2014, Table 1), while P. gilmeri is rare (Table 2) and
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known so far only from nine specimens around the Bahamas (Pugh 2005) and one off Japan (Lindsay and
Miyake 2009).
The nectosome is typical of long-stemmed physonects and bears up to 12 nectophores arranged in two
rows (although all originate from a single ventral meridian). A ring of up to 36 prominent palpons fringes
the outer edge of the spherical corm below, each terminating in an ampulla of large microbasic
mastigophore nematocysts, which can inflict a painful sting. The ampulla of each palpon is white in
P. hydrostatica (Fig. 11a) and orange in P. gilmeri. Each mature cormidium comprises three palpons, one
smaller gastrozooid with tentacle, and a reproductive body on a single stalk which subdivides immedi-
ately into a male gonodendron branch and a female gonodendron branch (Fig. 11bb). In addition, the
cormidia of P. gilmeri each contain one or more bracts of two types (Pugh 2005). In P. hydrostatica, a
single bract develops only in the larva, for buoyancy until some large palpons develop, when the bract is
lost (Totton 1965). In P. gilmeri buoyancy provided by the palpons is apparently supplemented in mature
colonies by the large bracts; whether a single larval bract is produced in the larva of this species is
unknown.
The tentilla of physophorids differ from those of all other codonophorans in being enclosed within a
capsule on a long pedicel, and lacking any terminal filament. The cnidoband is up to 5 mm long, of many
small anisorhizas flanked by a few larger microbasic mastigophores (Mapstone 2014, Table 7); it inverts
during growth and then unwinds into a chaotic spiral and discharges through a pore near the proximal end
of the capsule (Fig. 11c).
Resomiidae
This small family, introduced in 2006 and summarized in Table 2, includes five long-stemmed species
which are very fragile, have a rigid stem, and are transparent except for buttons or arcs of nematocysts at
the tips of the bracts, faintly tinted gastrozooids, and palpons (Pugh and Haddock 2010). They also have
remarkable tentilla which transform during growth from a spirally coiled form into a (typically) zigzag
form. This has been studied in detail in all species, and the process somewhat resembles cnidoband
rearrangement in the capsulate tentilla of species in the physonect genus Physophora, although the latter
differs in having a shortened swollen siphosome and no terminal filament on the tentilla, as described
above. In Resomia, after coiling, a transparent involucrum typically grows over the entire cnidoband and
extends on to form a distal tube into which the terminal filament is withdrawn when not feeding. Once
covered, the cnidoband uncoils and rearranges itself into three zags, with the double elastic band
(employed during tentillum activation) connecting only the proximal and distal ends of the cnidoband
(see Pugh 2006a, Figs. 11 and 18). One exception to this growth pattern is R. ornicephala in which the
involucrum grows out to float above the cnidoband instead of enclose it, and is pigmented. The pigment
fluoresces under violet and blue excitation making the involucrum resemble a bird’s beak, with a central
green strip flanked by two pairs of yellow spots (Pugh and Haddock 2010). In the field the long tentacles
are repeatedly relaxed and tugged up through the water in a jiggling movement. R. ornicephala inhabits a
restricted depth range 164–298 m in Monterey Bay and must compete with the more abundant small
physonect Nanomia bijuga for food. In the dim downwelling light at this depth, the lure of R. ornicephala
may either fluoresce to attract krill prey or resemble a shoal of krill in outline when all the tentacles are
extended (Pugh and Haddock 2010).
Few resomiids have been collected worldwide, with two species found only in the Southern Ocean and
the remainder in warmer oceanic waters, mainly the NE Pacific, either off or within Monterey Bay, in the
Gulf of California, or in the Tongue of the Ocean region of the Bahamas in the Atlantic. Although a lure
has been postulated for attracting prey in Resomia ornicephala, the type of prey captured by other
resomiids is unknown. Prey must be immediately stunned by the formidable battery of nematocysts on
the long and folded cnidoband (Mapstone 2014, Tables 6 and 7), using the method described for other
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Fig. 11 Morphology of monoecious physonects 2. Physophoridae. (a) Physophora hydrostatica (# Larry Madin WHOI);
(ba) diagram of posterior surface of corm bearing ten cormidia; (bb), one cormidium exploded; (c) tentilla, a whole tentillum,
with long pedicel; b mature tentillum capsule with cnidoband reversed, uncoiled chaotically and ready for discharge; c less
mature capsule with cnidoband already reversed but still spirally coiled (# Gillian Mapstone 2014, Fig. 6Ba, b; Fig. 12Fa–c;
see these figure legends for references from which drawings were derived). Labels: ca capsule, cb cnidoband, gz gastrozooid,
gdf female gonodendron, gdm male gonodendron, p palpon, pe pedicel, pl palpacle, po pore, pn penumatophore, st stenotele
nematocysts, t tentacle, te tentillum
physonects by Mapstone (2014). So far, feeding in situ has not been observed in any resomiid, and,
indeed, in one species, R. dunni, only a single well-developed gastrozooid is present on the siphosome.
Agalmatidae Sensu Stricto

This most species-rich pelagic family of all physonects (Fig. 4) was shown to be a distinct monophyletic
clade by Dunn et al. (2005b, Fig. 3) and was delimited by Pugh (2006a) to five genera only. Diagnostic
features of the family are listed in Table 2, with the most distinctive being the twisting of the nectosome
relative to the siphosome resulting in nectophores arising from the dorsal side of the stem and siphosomal
cormidia from the ventral side. This is well illustrated in Halistemma foliacea by Pugh and Baxter (2014,
Fig. 60). The small species Nanomia bijuga is the most common physonect worldwide (Fig. 12c) between
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55 N and 59 S, with numerous records from all oceans except the North Atlantic (Mapstone 2014,
Table 1). The arrangement of zooids in one cormidium is shown in Fig. 12d, and detailed zooid
composition of the cormidia is given by Dunn and Wagner (2006). The tentillum comprises a red
cnidoband of circa three coils, with an involucrum covering the first coil, and a single distal terminal
filament (Fig. 12e).
Nanomia bijuga feeds on a range of small prey, including copepods, decapod larvae, and, in Monterey
Bay in particular, various young stages of krill (Pugh and Haddock 2010). As the physonect remains
motionless in the water with its tentacles extended for feeding, prey becomes entangled in the long
dangling terminal filament; its movements cause discharge of the cnidoband, which unwinds and slaps
Fig. 12 Morphology of monoecious physonects 3. Two common species of the family Agalmatidae sensu stricto. (a)
Agalma elegans (# Jessica Luo/Cowen Lab); (b) A. elegans tentillum (# Mapstone 2014, Fig. 13Aa); (c) Nanomia bijuga (#
Inter-Research, revised from Luo et al. 2014); (d) N. bijuga cormidium; (e) N. bijuga tentillum (# Gillian Mapstone 2014,
Figs. 6A, 13C). Labels: am ampulla, b bract, cb cnidoband, el elastic band, gdf female gonodendron, gdm male gonodendron,
gz gastrozooid, inv involucrum, mm microbasic mastigophore, n nectophore, pe pedicel, p palpon, pl palpacle, st stenotele,
t tentacle, te tentillum, tf terminal filament
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onto the prey, stunning it instantly, as described in Mapstone (2014, Fig. 15a–c). Recently, the juvenile
squid Chiroteuthis calyx has been found to mimic Nanomia bijuga in Monterey Bay, where it avoids
predators by hanging vertically among N. bijuga, a species shunned by predators because of its stinging
batteries (the tentilla) and low food value (Burford et al. 2014).
Other well-known but less abundant species in the family Agalmatidae sensu stricto include Agalma
elegans, A. okeni, and Halistemma rubrum. In the past, loose nectophores of A. elegans have sometimes
been difficult to distinguish from those of H. rubrum, but a recent and comprehensive review of the genus
Halistemma by Pugh and Baxter (2014) has resolved this problem. Halistemma nectophores are more
truncate than those of Agalma species when mature, with much shorter more truncate axial wings
separated by a prominent thrust block (see Pugh and Baxter 2014, Fig. 115). H. rubrum nectophores
display a distinctive pattern of incomplete ridges on the upper surface, whereas all those of A. elegans are
complete (see Mapstone 2009, Fig. 19a). Pugh and Baxter (2014) also introduce a new species
H. maculatum and redescribe H. rubrum, H. cupulifera, H. striata, H. foliacea, and H. transliratum
from complete specimens, most collected in exceptionally good condition by submersible vehicles. Ridge
patterns are clarified in both young and mature nectophores, bract types described (from two to five in this
genus), and tentilla compared and contrasted. The cnidoband of Halistemma species is long (sometimes
up to nine coils), with a very small cup or disk-shaped involucrum proximally and a single terminal
filament distally; the latter often, but not always, terminates in a swollen acorn-shaped sinker. Halistemma
tentilla are figured for all species by Pugh and Baxter (2014) and that of H. transliratum also shown by
Mapstone (2014, Fig. 13b).
Species in the long-stemmed genera Agalma and Nanomia have yet to be described from submersible
material, if indeed it exists, although both are well-known genera. They have differently shaped
nectophores, one or two transparent bract types on the siphosome, and distinctively different tentilla
(Fig. 12b, e). Their cnidobands tend to be shorter than those of Halistemma species and are red and in
Agalma species completely covered in a transparent sac known as the involucrum (Fig. 12b), although in
one rarer species (Agalma clausi) the involucrum is open distally and the bracts apparently bear distinctive
red spots. Agalma elegans (Fig. 12a) is a soft and flexible species, uncommon but cosmopolitan in
temperate and tropical latitudes (Mapstone 2014, Table 1). A. okeni, in contrast, is rigid, with a short stem
bearing prismatic nectophores and bracts, and is quite often collected in warmer waters worldwide (Pugh
1999b).
Two short-stemmed genera Athorybia and Melophysa are in the Agalmatidae sensu stricto family (see
Fig. 5 and Table 2) because Athorybia rosacea is sister to the three species in the genus Agalma (Fig. 3).
Athorybia rosacea is a small species without any nectophores, which resembles a floating flower
(Fig. 13a) and comprises a large pink pneumatophore surrounded by several whorls of large bracts arising
from a much reduced corm-like siphosome (Fig. 13b). Gastrozooids each bear a tentacle with tentilla,
which hangs down from the lower side of the corm for feeding, and in A. rosacea the tentilla are of two
types: dendritic, without an involucrum and with dendritic growths (Fig. 13ca), and involucrate, with a
complete involucrum when mature (Fig. 13cc) and a barely developed one when young (Fig. 13cb). Some
dendritic tentilla also have a large protruding spur, which may act as a lure (Mapstone 2014, Fig. 16b).
Melophysa melo (WoRMS Siphonophora List) differs from A. rosacea in having a short nectosome
bearing up to four functional nectophores, but has similar tricornuate tentilla (with a terminal ampulla and
two lateral terminal filaments) and a coiled cnidoband partly covered by an involucrum (Daniel 1985).
Totton (1965) concluded that Athorybia rosacea represents a young Nanomia or Agalma individual
lying on its side, and this is because species of these two genera go through an Athorybia post-larval stage
during their early development. Totton (1965) shows this stage in his Fig. 19 for A. elegans. The larva has
a ring of larval bracts around the pneumatophore (but no nectophores) and a larval tentacle with a number
of simple and different larval tentilla (shown for Agalma elegans in Mapstone 2014, Fig. 13Ab). In
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Fig. 13 Morphology of monoecious physonects 4: Athorybia rosacea. (a) Live individual floating in the sea, from above
(# Larry Madin WHOI); (b) lateral view of float with siphosomal horn (where the cormidia are formed) and attached cormidia
(# Gillian Mapstone 2014, Fig. 6D; see figure legend for reference from which the drawing derived); (c) a, b dendritic tentilla
and c involucrate tentillum (Bigelow 1911, pl. 20, Figs. 8, 9, and 10). Labels: am ampulla, b bract, bl bracteal lamella, cb
cnidoband, gdf female gonodendron, gdm male gonodendron, gz gastrozooid, inv involucrum, p palpon, pn pneumatophore, sh
siphosomal horn, t tentacle, te tentillum, tf terminal filament
Halistemma species, Pugh and Baxter (2014) have found a different developmental stage known as a
Nectalia post-larva. This comprises a few nectophores and a ring of long larval bracts, with a larval
tentacle bearing another type of larval tentillum, and has been shown so far for H. rubrum and
H. maculatum (see Pugh and Baxter 2014, Figs. 23–25, 89, and 92), although is probably also found in
the other Halistemma species. In addition, another Nectalia post-larva occurs in the dioecious physonect
Stephanomia amphytridis, but in this species the larval tentilla are very different (see Pugh and Baxter
2014, Figs. 108, 111, and 112–113).
Unassigned Monoecious Genera

Three genera with ventral nectosomes are now excluded from the Agalmatidae sensu stricto and are listed
in Fig. 5 and Table 3. Each is summarized briefly below from information given by Mapstone (2009,
2014) and references quoted therein. Cordagalma ordinata is a flexible species up to 30 cm long with a
maximum of 40 diminutive heart-shaped nectophores, distinctive kite-shaped bracts, palpons without
palpacles, and unique larval-type tentilla on the gastrozooid tentacles which are very small and lack a
cnidoband (Mapstone 2014, Fig. 13d). C. ordinata feeds only on small copepods (Purcell 1980), which
are trapped in an array of long cnidocils that project from the distal surface of the tentillum. The species
inhabits all oceans and can sometimes be abundant in deep coastal fjords. It has been collected by
submersible in the Alboran Sea in the western Mediterranean and studied in detail at Villefranche Marine
Station. These and other worldwide records are summarized in Mapstone (2009).
Frillagalma vityazi is a small physonect with larger nectophores than those of Cordagalma ordinata,
but a shorter and rigid siphosome. Nectophores bear pairs of ridges similar to those found in other
physonects (including a single pair of vertical-lateral ridges), a nectosac with simple looped lateral radial
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Table 3 Characters for calycophoran families (Derived from # Mapstone 2014, Table 5; see this paper also for additional
references omitted below; see Fig. 5 for details of fundamental siphonophore characters mentioned below)
Family Comments
14. Prayidae Probably paraphyletic and includes nested family Hippopodiidae (Dunn et al. 2005b) (see below); Praya
dubia (subfamily Prayinae) and subfamily Nectopyramidinae may be one lineage, with prayines Craseoa,
Gymnopraia, and Rosacea another (Dunn et al. 2005b), but broader taxa sampling is needed (Mapstone
2009). Prayine name Lilyopsis medusa has precedence over Lilyopsis rosea (WoRMS Siphonophora
List); new prayine species Desmophyes haematogaster, Gymnopraia lapislazula, Lilyopsis fluoracantha,
Rosacea repanda, R. limbata, and R. arabiana introduced (see WoRMS Siphonophora List); subfamily
Nectopyramidinae revised with Nectopyramis thetis and N. natans redescribed and new genus
Nectadamas introduced (for N. diomedeae and a new species N. richardi). Prayine species R. cymbiformis
also redescribed and nomenclature problems concerning R. plicata sensu Bigelow and Desmophyes
annectens resolved. Eudoxids released in amphicaryonines and nectopyramidines, but not in prayines
(Mapstone 2009). Rosacea villafrancae transferred to genus Desmophyes and Prayoides intermedia
found to be a junior synonym of Praya species (Pugh 1992, WoRMS Siphonophora List). Unique
bio-optical properties identified in G. lapislazula and L. fluoracantha, though their function is still
unknown (Haddock et al. 2005a)
15. Hippopodiidae Found nested within prayines in first siphonophore phylogeny and Hippopodius nested within Vogtia
(Dunn et al. 2005b); hippopodiid distribution correlated with feeding on various species of ostracods,
unlike other calycophorans. Family characters recently summarized and the new axes applied, together
with redescriptions given and synonomies listed for V. serrata, V. spinosa, and V. pentacantha (Mapstone
2009); V. microsticella considered a junior synonym of V. glabra and V. kuruae a junior synonym of
V. serrata (WoRMS Siphonophora List; Mapstone 2009)
16. Clausophyidae The three diphyomorph families below may have arisen from this one (Dunn et al. 2005b). New species
include Clausophyes laetmata and Cl. tropica, and two others redescribed include Cl. galeata and Cl.
moserae; a unique fuseudoxid life stage found in Crystallophyes amygdalina and a new genus Kephyes
introduced for Moser’s Cl. ovata, which, unlike Clausophyes species, has bracts with a pair of hydroecial
canals (Pugh 2006b). Four clausophyids redescribed from NE Pacific and new axes applied (Mapstone
2009)
17. Sphaeronectidae Ten species now considered valid in this family with single retained larval nectophore. Family reviewed
and history summarized (Pugh 2009); five new species introduced: Sphaeronectes christiansonae,
S. haddocki, S. tiburonae (Pugh 2009), S. pagesi, and S. pughi. An old species S. brevitruncata reinstated
(Pugh 2009) and S. bougisi concluded to be a likely calyconula of Lilyopsis medusa (WoRMS
Siphonophora List). S. gracilis relegated to a junior synonym of S. koellikeri and probably restricted to the
tropics (Pugh 2009; WoRMS Siphonophora List); specimens reported from Jervis Inlet, British Columbia
(Mapstone 2009), could be S. haddocki
18. Diphyidae Probably paraphyletic (Dunn et al. 2005b), vindicating earlier conclusions (Totton 1965), but based on
only 5 of 45 likely valid species (WoRMS Siphonophora List). Two main clades identified in the
molecular study, within one of which is nested the family Abylidae (Dunn et al. 2005b). New axes applied
to all life stages of diphyids; muscular lamellae, median gastrovascular canals, and pedicular canal
arrangements also schematically shown for two basic types of diphyids (Mapstone 2009). A new small
species added to the genus Lensia (L. quadriculata), another redescribed in detail (L. asymmetrica), and a
third (L. reticulata) transferred to a new genus Gilia within a new subfamily Giliinae, for the two
clausophyid-like canals in the bract. An enigmatic species Eudoxia macra shown, using the
mitochondrial 16S gene, to be sexual stage of a larger species L. cossack. A number of previously
described Lensia species, several Sulculeolaria species, and one Muggiaea species all reduced to junior
synonyms of various better known species (WoRMS Siphonophora List)
19. Abylidae Family nested with Diphyes dispar in one of two Diphyidae clades, based on 16S and 18S (Dunn
et al. 2005b), but only Abylopsis tetragona tested and more taxa sampling needed. Ten valid species
(WoRMS Siphonophora List), all present in the South Atlantic and summarized by Pugh (1999b); several
species also redescribed from around South Africa. Junior synonyms (including those in a confusing
abylid review by Sears) given in the WoRMS Siphonophora List
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canals and bioluminescent patches on the nectophore surface. Bracts are facetted and of three types, with
three pairs per cormidium and, as in C. ordinata, the palpons lack palpacles and the tentilla are distinctive
and diagnostic. Each tentillum comprises a very small proximal cnidosac with c. 33 nematocysts of two
types, from which project two elongate and sausage-shaped sequential ampullae (Mapstone 2014,
Fig. 13e). F. vityazi is a rare species worldwide (distribution summarized in Mapstone 2009) and has
been collected with submersibles in the Bahamas, but prey consumed is so far unknown.
The third monotypic species in this group is Lychnagalma utricularia. It is very fragile and transparent
and has only rarely been collected worldwide (WoRMS Siphonophora List), with most specimens coming
again from submersible dives in the Bahamas region (Pugh and Harbison 1986). L. utricularia shares
certain characters with Agalmatidae sensu stricto species, including nectophores with paired ridges and
the tentilla which are involucrate with a long coiled cnidoband, but differs in having a ventral nectosome.
Unusually for a physonect, it is completely non-bioluminescent, and the mature tentillum is also
exceptionally large, reaching up to 7.5 mm in length (see Mapstone 2014, Fig. 16c), with a large central
ampulla and eight terminal filaments. These pulsate like a swimming medusa and form an intriguing lure
which may perhaps attract small fish, although so far no prey has been found in any of the gastrozooids
collected from L. utricularia.
Family Prayidae
The Calycophorae is a monophyletic clade (Fig. 3) which is monoecious, has lost the pneumatophore
(Fig. 5), and retained reduced larval cormidia (Dunn and Wagner 2006). Species in the six calycophoran
families (Table 3) have only two nectophores (sometimes one and occasionally four or more) which are
alike and apposed in prayomorphs (Fig. 14b, e) and different and linearly aligned in diphyomorphs; they
also lack the axial wings and thrust block of physonect nectophores. A single larval nectophore develops
from the calyconula larva, before the first definitive nectophore appears. Prayidae is one of the largest
calycophoran families, including 27 species (Fig. 4), and systematic changes since 1987 are summarized
in Table 3. In life prayomorph nectophores attach at the anterior end of a typically very long siphosome
bearing hundreds of cormidia (Fig. 14a). When feeding a “sit and wait” strategy is employed, when the
stem relaxes and the extended tentacles hang down in a long feeding curtain.
The largest of the three subfamilies is the Prayinae, which undergo nectophore replacement, probably
throughout life. The first definitive nectophore develops inside a long proximal groove in the larval
nectophore (termed the hydroecium) and matures (Pugh 1992), and then a second definitive nectophore
may start to form before the larval nectophore is shed. Buds for third and fourth nectophores are often also
visible inside the hydroecium, and these will enlarge and replace earlier definitive nectophores over time,
as summarized by Mapstone (2009). Stem cormidia are retained throughout life in prayines and comprise
a single rounded bract (except Gymnopraia) with typically six bracteal canals enclosing a gastrozooid,
tentacle, and gonophore (Fig. 14c). Prayine tentilla are all alike (Fig. 14da) with a swollen sinker at the
distal end of the terminal filament to act as a weight (Fig. 14db). Unlike physonects, tentilla are not useful
for species or genus diagnosis in calycophorans, and prayine species are separated on nectophore and
bract characters. These include the relative size of nectosac to nectophore (small in Praya and Rosacea
species (see Fig. 14b) and large in Lilyopsis (see Fig. 14e)), presence or absence of a disjunct pedicular
canal (see Mapstone 2009, Fig. 5), the type and branching of the somatocyst when present, the branching
and courses of the lateral radial canals of the nectophore, and the number and arrangement of canals in the
bract (all summarized in Mapstone 2009).
Prayine nectophores vary in size, with some species reaching as much as 6 or 10 cm in length (Praya
species and Rosacea cymbiformis), although most are shorter (circa 3–4 cm) and others diminutive
(Mistoprayina fragosa, Prayola species, Rosacea arabiana, 3–6 mm in length (WoRMS Siphonophora
List)). Prayines with large nectosacs are very fragile and hence rarely successfully collected (Lilyopsis
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Fig. 14 Prayine prayomorph morphology. (a) Typical prayomorph Praya sp. feeding, with two rounded bells and a very
long siphosome bearing over 100 cormidia; tentacles extended for feeding, each bearing 80–90 nematocyst batteries, giving
<9,000+ batteries in all (Steven Haddock # MBARI). (b) Rosacea sp. feeding (# Jessica Luo/Cowen Lab); (c, d) Rosacea
cymbiformis; (c) cormidium enlarged; (d) a tentillum, b sinker (# Gillian Mapstone 2014, Figs. 8A and 14A); (e) Lilyopsis
rosea swimming (# Jessica Luo/Cowen Lab). Labels: ani anisorhizas, b bract, bc bracteal canal, c cormidium, dl large
desmoneme, ds small desmoneme, go gonophore, gz gastrozooid, mm microbasic mastigophore, n nectophore, ns nectosac, rh
rhopalonemes, pe pedicel, sk sinker, st stem, t tentacle, te tentilla, tf terminal filament
Fig. 14e), while others with more mesogloea are robust (Praya and Rosacea Fig. 14a, b) and collected
periodically in epi- and mesopelagic waters of most seas (Praya dubia and Rosacea plicata; see Mapstone
2014, Table 1; also R. cymbiformis Mapstone 2009).
The two much smaller prayid subfamilies Amphicaryoninae and Nectopyramidinae have only four
species apiece and are probably derived from the prayines, although only two species were included in the
molecular analysis of Dunn et al. (see Fig. 3). Amphicaryonines are small, rounded, and composed of two
unequal-sized nectophores: a larger retained larval nectophore and a smaller reduced definitive
nectophore (see summary in Mapstone 2009). The nectosac is functional in both nectophores of the
largest amphicaryonine Maresearsia praeclara (20 mm diameter), but only in the larval nectophore of the
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Fig. 15 Amphicaryonine and nectopyramidine prayomorph morphology. (a) Amphicaryon acaule colony (Bigelow
1911, pl. 4 Fig. 1); (b) Nectadamas diomedeae eudoxid (# Russ Hopcroft UAF). Labels: dn definitive nectophore, eb eudoxid
bract, go gonophore, ln larval nectophore, ns nectosac
three smaller Amphicaryon species (WoRMS Siphonophora List). The best known amphicaryonine is
A. acaule (8 mm diameter) (Fig. 15a), which inhabits epi- and mesopelagic layers of warmer waters
worldwide (see Mapstone 2014, Table 1). All amphicaryonines release small free-living eudoxids with
only two canals in the bract, for species dispersal.
Nectopyramidine prayids develop only one ridged asymmetric definitive nectophore, from a smaller
ridged larval nectophore, and also release a free-living eudoxid (Fig. 15b) (reviewed by Mapstone 2009).
Definitive nectophores vary from pyramidal to rhomboidal or bow-shaped, with a penetrating somatocyst
of one or several branches and a hydroecium of varied shape. This subfamily is rare worldwide, being
mainly mesopelagic with a greater latitudinal range than amphicaryonines (though absent from the
Mediterranean) (Mapstone 2009, 2014). Nectophores and eudoxids of three of the four species are
large, reaching up to 36 mm or more in length.
Family Hippopodiidae
Hippopodiidae is a small and unusual calycophoran family of five species each with up to 12 nectophores
(Fig. 16a–c, e) and found to be nested with the family Prayidae by Dunn et al. (2005b) (Fig. 3, Table 3).
Like prayine prayids, the first definitive nectophore develops inside the hydroecium of a small rounded
larval nectophore (see Mapstone 2009, Fig. 42), which is later shed and more definitive nectophores
formed, each from the pedicel of its predecessor. Thus, the largest nectophore, and the only one with a
functional nectosac, occurs at the base (or posterior end) of the colony (Fig. 16b). There are no bracts in
the siphosomal cormidia (Fig. 16d), and this allows the stem to be more easily withdrawn into the chamber
created by the nectophores (Fig. 16a, c, e). Buoyancy for the colony is instead provided by the thick and
typically robust nectophores, which are either rounded with two or more protuberances on the distal side
of the nectosac (Hippopodius hippopus and Vogtia glabra; see Mapstone 2009, Fig. 2f and g for
nectophore axes) or angular and pentagonal, often with ridges and cusps (Mapstone 2009). Buoyancy
in hippopodiids is likely controlled by active transport of lighter and heavier ions across the covering
epithelium (Mackie and Mackie 1967).
Although two genera are included within the family Hippopodiidae, Hippopodius and Vogtia (WoRMS
Siphonophora List), the differences between them are small. Both genus names are very old and are still
retained only to avoid confusion in the literature (Totton 1965). All five species have nectophores of
similar dimensions (<21 mm along upper-lower axis) and a widespread cosmopolitan distribution.
However, they occur at varied latitudes and depth horizons, and all feed selectively on ostracods
(Table 3), unlike all other calycophorans.
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Fig. 16 Hippopodiid prayomorph morphology. (a) Hippopodius hippopus colony (# Sonke Johnsen, Duke); (b) section
through Hippopodius (# Gillian Mapstone 2014, Fig. 8B; see figure legend for original reference); (c) Hippopodius hippopus
colony (# Russ Hopcroft UAF); (d) Hippopodius hippopus cormidium; note, no bracts (# Mapstone 2014, Fig. 8D; see figure
legend for original reference); (e) Vogtia glabra colony (# Sonke Johnsen, Duke); (f) Hippopodius hippopus tentillum (#
Gillian Mapstone 2014, Fig. 14C; see figure legend for original reference). Labels: ani anisorhizas, c bractless cormidium, gof
female gonophore, gom male gonophore, gz gastrozooid, mm microbasic mastigophore, n nectophore, nl nectophoral lamella,
ns nectosac, pe pedicel, sh siphosomal horn, st stem, t tentacle, tf terminal filament
Hippopodius hippopus is the most abundant and best known species. It has tentilla with a very long
terminal filament for feeding (Fig. 16f) and is a robust and epipelagic species, which lives in warmer
waters worldwide (Mapstone 2014), often occurring nearer the coast than the four Vogtia species. It
undergoes blanching, and this, together with nerve/epithelial conduction, has been studied in detail by
Mackie (reviewed in Mackie et al. 1987). Vogtia glabra is the only rounded Vogtia species (Fig. 16e), with
just two prominences distal of the ostium when mature (Pugh 1999b). V. glabra also prefers, like
H. hippopus, tropical and temperate waters, but differs in inhabiting mainly the mesopelagic zone, with
many fewer and sporadic records from the world’s oceans.
Of the three pentagonal Vogtia species, V. serrata is the largest and the most abundant (Mapstone 2009),
with an extensive latitudinal range in both hemispheres (Mapstone 2014, Table 1), inhabiting shallow
depths in the Antarctic, and deeper layers in temperate seas, where it is typically the dominant hippopodiid
of mesopelagic assemblages, and also the deepest living (reviewed by Mapstone 2009). V. pentacantha is
a smaller and less frequently encountered Vogtia, with cusped ridges but smooth facets on the
nectophores, and is also mainly mesopelagic. In contrast, nectophores of V. spinosa have cusps on both
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the facets and the ridges, and this species is epipelagic at lower latitudes and mesopelagic at higher
latitudes (Mapstone 2009).
Family Clausophyidae
The remaining four calycophoran families in Table 3 are all diphyomorphs, which typically have two
dissimilar angular and also often streamlined nectophores strengthened with longitudinal ridges and
containing a relatively large powerful nectosac and little mesogloea; they also contain a swollen blind-
ending diverticulum from the gastrovascular canal system termed the somatocyst (Fig. 2c) which is
mirrored in the canal system of the eudoxid bract as a swollen phyllocyst. These structures might act as
food stores and/or increase buoyancy (Mapstone 2009).
Clausophyidae is a small family of ten species which are mostly meso- and bathypelagic and hard to
sample. They were poorly understood for many years until the advent of modern sampling methods.
Clausophyids were only raised to family status in 1965, in contrast to the other calycophoran families
which are much older (Mapstone 2009, 2014; Totton 1965, WoRMS Siphonophora List). Distinctive
family characters include typically two nectophores, both containing somatocysts and with the posterior
larger than the anterior, aligned partially linearly, and partially in apposition (Mapstone 2009, Fig. 4). This
latter character suggests that they may represent the ancestors of the other diphyomorph families, as noted
in Table 3. Cormidia are released as free eudoxids in three of the five genera (Chuniphyes, Kephyes, and
Heteropyramis), but bracts are absent in Clausophyes species, and each bract is fused with a gonophore in
the monotypic genus Crystallophyes (Table 3). Few clausophyid species were sampled in the molecular
analysis of Dunn et al. (2005b) (Fig. 3), and, although the results indicate that this family might be
paraphyletic, the nodes are poorly supported and further sampling is needed.
There is considerable size variation among clausophyid genera, with anterior nectophores of
Chuniphyes growing up to 30 mm in length, while those of Heteropyramis (which does not develop a
posterior nectophore) reaching only 5 mm; nectophores of Clausophyes and Kephyes are of intermediate
size (Fig. 1; see Pugh (2006b) and Mapstone (2009) for further details and other references). The
somatocyst reaches to the anterior end of both nectophores when mature, and in the anterior nectophore
the nectosac typically extends to only half its length (Fig. 17b, c; K. ovata in Fig. 17a is an exception). The
stem attaches to the hydroecial wall of the anterior nectophore some distance anterior of the ostium
(Fig. 17b), suggesting that clausophyids might be an intermediate stage in the migration of the posterior
nectophore from the apposed position in prayids to the superimposed, or linearly aligned, position in most
Diphyidae and Abylidae (see Mapstone 2009, Fig. 4). The many nomenclatural problems among some
species of the family Clausophyidae have been resolved in recent years, as discussed by Mapstone (2009).
Clausophyids are mostly cosmopolitan worldwide (see Mapstone 2014, Table 2), but none are
common, and, indeed, species such as Clausophyes laetmata, C. galeata, and C. tropica are rare to
very rare (WoRMS Siphonophora List). Perhaps the most successful clausophyid is the large species
Chuniphyes multidentata which has a considerable latitudinal range worldwide and produces a large
number of small eudoxids when breeding (Mapstone 2009). Although it inhabits the same depth horizons
as its congener C. moserae, Mackie et al. (1987) conclude that these two species are allopatric in the North
Atlantic at least, with C. moserae being more prevalent below 40 N and C. multidentata more abundant
above this latitude; where the two species coexist, their population nuclei are spread over different depth
zones (summarized in Mapstone 2009).
Family Sphaeronectidae
Sphaeronectidae is a small and distinctive family of ten valid species and one species inquirenda
(WoRMS Siphonophora List), in which a rounded larval nectophore is retained into adulthood
(paedomorphy), producing cormidia on the elongate siphosomal stem which are released into the
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Fig. 17 Clausophyid diphyomorph morphology. (a) Kephyes ovata and (b) Clausophyes moserae (both # JMBA Plymouth,
from Pugh 2006b); (c) Chuniphyes multidentata anterior nectophore only (posterior nectophore detached during capture) (#
Casey Dunn). Labels: an anterior nectophore, ns nectosac, o ostium, pn posterior nectophore, som somatocyst, st stem
plankton as free-living eudoxids, like diphyids and abylids (Mapstone 2009). No definitive nectophores
are formed. Only one species was sequenced by Dunn et al. (2005b), appearing in their molecular
phylogeny as Sphaeronectes gracilis (Fig. 3); this species has since been referred to S. koellikeri
(Mapstone 2009; Pugh 2009). The species is firmly nested within the diphyomorph clade of
calycophorans (Fig. 3), dispelling some earlier ideas about affinities of sphaeronectids. All sphaeronectid
species are small, with a single rounded or ovoid nectophore that is very fragile and varies in size from 1.5
to 11.5 mm (Pugh 2009). As a result, most plankton nets miss these small calycophorans, and several new
species have been discovered recently by SCUBA divers and using fine-meshed nets deployed in the
surface layers of coastal waters around various continents. Despite this, two of the newly introduced
species are meso- and bathypelagic, found only so far in Monterey Bay (Pugh 2009, Fig. 18).
Axes for nectophores of Sphaeronectes koellikeri are given by Mapstone (2009, Fig. 3g), and all
species have a small hydroecial opening on the lower nectophore surface, since no second nectophore has
to be accommodated (Fig. 18a–c). All species except S. koellikeri also have a small and very short
hydroecium which originates on the lower side of the nectosac; in S. koellikeri, the hydroecium uniquely
extends over the top of the nectosac on the anterior side (Mapstone 2009, Fig. 65a). Species differ in the
ratio of nectosac to nectophore length, position of origin of the four radial canals on the nectosac, and the
courses of the lateral radial canals over the nectosac to the ostial ring canal. Size, shape, color, extent, and
position of the somatocyst are also important for species identification, with some having an elongate
tubular somatocyst, most having a pyriform one, and one species having a stalked somatocyst. In
S. tiburonae from Monterey Bay, the somatocyst is minute, and in S. christiansonae, also from this
location, the somatocyst is red (Pugh 2009). Eudoxids are so far known for only three of the ten valid
species, and all differ in phyllocyst shape and bract to gonophore ratios.
Only one sphaeronectid species, Sphaeronectes koellikeri, is well known and its distribution, together
with those of five of the other species, is summarized by Pugh (2009). Two species more recently
introduced are noted in Table 3, and their distributions are limited so far to Japanese waters and
subantarctic waters off Australia (see Mapstone 2014 for references). The identity of another small
species originally referred to the genus Sphaeronectes is now likely a calyconula of Lilyopsis medusa
(Table 3).
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Fig. 18 Sphaeronectidae diphyomorph morphology. (a, b) Sphaeronectes spp. feeding (# Jessica Luo/Cowen Lab); (c)
Sphaeronectes pagesi feeding (# D. Lindsay, R. Minemizu, JAMSTEC). Labels: c cormidium, ln larval nectophore, st stem,
t tentacle
Family Diphyidae
Diphyidae are the most successful and speciose siphonophore family (Fig. 4), currently comprising
45 species considered valid (WoRMS Siphonophora List). Diphyidae dominate surface layers in the
ocean, and their systematics is very stable because most species were introduced many years ago. Recent
changes are summarized in Table 3, and Fig. 3 shows that in the molecular phylogeny, abylids
(represented by Abylopsis tetragona) are nested within the five diphyid species tested.
Diphyids typically have two linearly aligned nectophores, led by a pointed streamlined anterior
nectophore, and followed by a smaller posterior nectophore (Fig. 19a–c). The stem is completely
withdrawn into the elongate hydroecium of the posterior nectophore for swimming, which alternates
with a motionless phase during which the stem and tentacles relax and form a fishing net for feeding
(Mapstone 2009, p. 30) (Fig. 19a, c). Nectophores are typically ridged and have a nectosac which fills the
nectophore, a mouthplate adjacent to the ostium, and, in the anterior nectophore only, a discrete
somatocyst food storage/buoyancy organ (Mapstone 2009, Fig. 3d). The stem bears numerous
cormidia which each comprise a bract, gastrozooid and tentacle (Fig. 19d), and, when mature, a
gonophore for reproduction. The structure of diphyid-type tentilla and their nematocyst compliments
are summarized by Mapstone (2014, Table 8, Fig. 14d, e), and tentillum structure is shown for three
diphyomorph species in Figs. 19e and 20c, f. The typical bract is typically helmet shaped (Fig. 20b) with a
food storage equivalent of the somatocyst, the phyllocyst, and no bracteal canals. The gonophore has a
large nectosac for propulsion, and, when released into the plankton as a free-living eudoxid from the
posterior end of the stem, can live for several months and release a large number of gonophores for sexual
reproduction.
The family includes three subfamilies: the Sulculeolariinae (five species) in which the stem is very long
because cormidia are never released and nectophores can be replaced up to four times during life, the
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Fig. 19 Diphyid diphyomorph morphology. (a) Lensia conoidea feeding (Rob Sherlock # MBARI); (b) Chelophyes
appendiculata (# Peter Schuchert MHNG); (c) Lensia multicristata feeding (# Inter-Research, revised from Luo et al. 2014);
(d) C. appendiculata cormidium; and (e) Diphyes dispar tentillum (# Gillian Mapstone 2014, Figs. 8C and 14D; see figure
legends for original references from which these figures were derived). Labels: an anterior nectophore, ani anisorhizas, b bract,
dl large desmonemes, gz gastrozooid, h hydroecium, mm microbasic mastigophores, pe pedicel, pn posterior nectophore, som
somatocyst, st stem, t tentacle, tf terminal filament
Diphyinae (39 species) with typically two ridged nectophores which cannot be replaced and a shorter stem
from which eudoxids are released when mature, and the Giliinae (summarized in Table 3).
Sulculeolariines are warm-water epipelagic and cosmopolitan species (Mapstone 2014, Table 2),
separated on the length of the somatocyst, the presence or absence of teeth around the ostium and small
swellings on the upper sides of the mouthplate (Mapstone 2009, Fig. 44). Anterior nectophores vary in
size from 8 to 26 mm, with S. quadrivalvis being the largest and probably most abundant species
worldwide (Totton 1965).
Diphyines include 39 valid species in five genera (WoRMS Siphonophora List), which are separated on
characters of the anterior nectophore, and in a few species a posterior nectophore does not develop
(Fig. 20d). The genus Chelophyes comprises two distinctive warm-water 5-ridged epipelagic species with
a claw-shaped hydroecium (Fig. 19b) and allopatric distribution. C. appendiculata is the most abundant
siphonophore species worldwide, with an anterior nectophore more than twice the size of C. contorta and
a much broader latitudinal range (Mapstone 2009, 2014; Pugh 1999b). Dimophyes arctica is monotypic
for the genus Dimophyes, has a smooth anterior nectophore with a prominent undivided mouthplate
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Fig. 20 Diphyid and abylid diphyomorph morphology. (a, b) Dimophyes arctica polygastric colony (a) and eudoxid (b)
(# Russ Hopcroft UAF); (c) D. arctica tentillum (Chun 1897, pl. 1 Fig. 9); (d) Muggiaea kochi (# Peter Schuchert MHNG);
(e) Abylopsis tetragona (# Russ Hopcroft UAF); (f) Enneagonum hyalinum tentillum (Chun 1892, pl. 12 Fig. 14). Labels: an
anterior nectophore, b bract, go gonophore, mo mouthplate, ph phyllocyst, pn posterior nectophore, som somatocyst
(Fig. 20a), a very small posterior nectophore rarely collected, and a particularly broad cosmopolitan
distribution, occupying deeper layers at lower latitudes and surface layers at higher ones (Mapstone 2009,
2014, Table 2). Diphyes anterior nectophores have a deep hydroecium, prominent teeth around the ostium,
are all epipelagic, and include the largest of all diphyid species, D. dispar (<36 mm long, Pugh 1999b).
Three of the four species are tropical, with two common worldwide (D. dispar and the much smaller
D. bojani) and the third, which lacks a posterior nectophore, inhabiting only the Indo-Pacific region
(D. chamissonis) (Totton 1965). The fourth Diphyes species, D. antarctica, is also large (<30 mm long)
and common, but only in the cold waters of the Southern Ocean (Pugh 1999b).
Two more small tropical diphyines are referable to the genus Eudoxoides and have an anterior
nectophore with five serrated ridges, a hydroecium reaching 1/3 nectophore length (from the ostium),
and a prominent mouthplate (Pugh 1999b). E. mitra is common and epipelagic in all oceans except the
Mediterranean (Mapstone 2014, Table 2), while E. spiralis, which lacks a posterior nectophore, has a
slightly more extended vertical and latitudinal distribution (Pugh 1999b). The genus Muggiaea (Fig. 20d)
includes four small species (4–10 mm long) without a posterior nectophore, so the stem is accommodated
during swimming inside a deep hydroecium in the anterior nectophore. Three species are neritic, restricted
to the shallow shelf waters fringing continents, with M. atlantica occupying temperate waters worldwide,
M. kochi replacing it in tropical Atlantic waters, and M. delsmani in tropical Indo-Pacific waters; these
temperate/tropical pairs can also coexist, for example, in the English Channel (Mapstone 2009) and
Sagami Bay (Grossmann and Lindsay 2013). The fourth species, M. bargmannae, is a bipolar species
living only at very high latitudes in epi- and mesopelagic layers (Mapstone 2014). M. delsmani from the
South China Sea was unfortunately misidentified by Lo et al. (2012) as M. kochi.
Lensia is a catch-all diphyine genus of circa 26 diverse valid species (WoRMS Siphonophora List) with
most species ridged (from five to multiridged or multistriate) and some unridged. In the anterior
nectophore, the hydroecium is typically very shallow, and the size, shape and position of the somatocyst,
and the divided mouthplate are specifically diagnostic. Many species are rare to very rare, and their
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posterior nectophores and eudoxids unknown. A few have very small anterior nectophores (3–4 mm
long), but most are intermediate (8–12 mm long), with some (L. achilles, L. conoidea, L. fowleri, L. hardy,
L. havock, L. hostile, and L. multicristata) reaching from 15 to 25 mm in length. Forty-two percent of
species inhabit epipelagic layers offshore, and several species make significant contributions to mid-water
siphonophore assemblages (L. conoidea, L. multicristata, Mackie et al. 1987). A number of small fragile
and rare multistriate species inhabit the deeper and calmer meso- and bathypelagic layers of temperate
waters (L. lelouveteau, L. quadriculata, L. grimaldii, L. exeter).
Family Abylidae
This is another well-known and successful diphyomorph family of ten species, which are most abundant
in tropical surface waters and differ from diphyids in having more prismatic and facetted nectophores,
with serrated ridges and teeth and a posterior nectophore larger than the anterior one (Fig. 20e). This large
nectophore provides the main propulsive force for abylid locomotion (Totton 1932) and also protects the
contracted stem in a long hydroecium enclosed by a serrated longitudinal flap on the inner surface of the
left hydroecial wing. Several aspects of the family are summarized in Table 3. Abylids are a stable and
long-known group, like the diphyids, with seven of the ten species already introduced by 1860 and the
remaining three by 1925 (WoRMS Siphonophora List). Species diagnoses are based on the characters of
the anterior nectophore, and are well summarized by Pugh (1999b), who also lists often used synonyms
for five of the ten valid species.
Two subfamilies are recognized. The Abylinae include six species in two genera (Abyla and
Ceratocymba), with a small facet at the anterior end of the anterior nectophore which leads the colony
during locomotion. The Abylopsinae includes four species in three genera (Abylopsis, Bassia, and
Enneagonum), with a small leading ridge at the anterior end of the anterior nectophore instead of a
facet in Abylopsis and Bassia and a very differently shaped pyramidal anterior nectophore in Enneagonum
hyalinum, which never develops a posterior nectophore.
Abyline species are all epipelagic and rare worldwide except for Ceratocymba sagittata, which is
common and mainly epipelagic, with a slightly broader latitudinal range than other abylines, although this
subfamily is absent from the Mediterranean. The anterior nectophore of most abylines is only 8–13 mm in
length, but in C. sagittata it can reach 25 mm (Pugh 1999b). This species also has a pointed anterior
extension beyond the small leading facet, and whole colonies of C. sagittata can reach 45 mm in length
(Totton 1965, Fig. 140), which is very long for a diphyomorph species. Abyla species have anterior
nectophores which are rectangular in lateral view, variable in width, and with a long hydroecium into
which fits the prominent apophysis of the posterior nectophore. The latter are larger when mature in
abylines, as noted above, and differ in width and number of teeth on the internal flap of the left hydroecial
wing (Pugh 1999b). Ceratocymba comprises three species, which, in addition to C. sagittata, include the
conspicuous but smaller opaque, rare, and sturdy species C. dentata and a third rare species C. leuckarti.
Abylopsines include three mainly epipelagic and common tropical and subtropical species: Abylopsis
eschscholtzii, A. tetragona, and Bassia bassensis, which all occur worldwide and in the Mediterranean
(Mapstone 2014 Table 2; Pugh 1999b). Anterior nectophores of Abylopsis and Bassia are similar, but can
be distinguished by their somatocysts, which in Abylopsis terminate in a thin diverticulum. A. tetragona
has a relatively longer posterior nectophore than A. eschscholtzii, although that shown in Fig. 20e has not
yet reached its maximum length (see Totton 1965, Fig. 149, for a mature colony). These three species are
often abundant in tropical siphonophore assemblages, together with certain tropical diphyid species
(Lo et al. 2012), and Bassia bassensis is distributed throughout tropical and subtropical latitudes of all
oceans and is also particularly tolerant of the varied environmental conditions found in neritic habitats
(Lo et al. 2012); the ridges of this species are also tinged blue in life. Enneagonum hyalinum, in contrast, is
a large and relatively uncommon abylopsine, from both tropical and temperate latitudes, with a pyramidal
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nectophore having nine points, a slim anteriorly directed somatocyst, and a relatively small nectosac. This
shape is unwieldy, suggesting that this species is an ineffectual swimmer with a nectosac that can do little
more than simply counteract the pull of gravity while floating in the water column (Totton 1932).

Forty three morphological siphonophore taxa have so far been successfully sampled for two genes, a
comprehensive molecular consensus generated, and some interesting new characters and relationships
revealed. More such characters and relationships might be found if further taxa are sampled (particularly
for calycophorans and also for other physonects) and wider suite of genes investigated, although CO1
should be omitted, as this gene is unsuitable for detecting interspecific differences in Siphonophora.
Investigation of mechanisms of tentillum discharge in different species and prey consumption in a wider
range of taxa might also produce interesting new findings. Sampling of siphonophores and other
zooplankton in areas not so far well studied worldwide (there are many) would also be informative,
particularly if specimens of further species with gastrozooids containing prey items are collected, and
might show further connections between siphonophores and other taxa in mid-water food webs, such as
that recently discovered by Burford et al. (2014).
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DOI 10.1007/978-94-007-6727-0_15-1
Phylogeny of Annelida
Torsten H. Struck*
Department of Research and Collections, Natural History Museum, Oslo, Norway
Abstract
Annelida are typically characterized by the presence of segmentation and can be found in all habitats on
the Earth. Traditionally regarded as being closely related to arthropods, with several very well-known
toxic or venomous species, molecular data robustly placed them within Lophotrochozoa. Besides
annelids, only one other taxon within Lophotrochozoa, Mollusca, is currently known to also contain
toxic or venomous species. The phylogeny of Annelida has been controversially discussed since the
recognition of Annelida as a taxon in the nineteenth century. However, recent phylogenomic studies have
achieved tremendous progress in this respect. Based on these results, Annelida was split into two major
clades, one clade (the Errantia) adapted to an errant mobile life and the other (the Sedentaria) which
includes earthworms and leeches, to a more sessile, sedentary one. Finally, several morphologically
aberrant annelid taxa are the first to branch off from the annelid stem lineage. Moreover, the
nonsegmented Sipuncula (peanut worms) and Echiura (spoon worms) have to be placed within Annelida.
Interestingly, the four taxa known to comprise toxic or venomous species are scattered throughout the tree.
While Amphinomidae are part of the basal radiation, Glyceridae are placed within Errantia and the
clitellate taxa Eisenia and Hirudinea are part of Sedentaria. Hence, the evolution of toxic or venomous
species within Annelida most likely occurred independently.
Keywords
Annelida; Lophotrochozoa; Articulata; Errantia; Sedentaria; Glyceridae; Amphinomidae; Hirudinea;
Eisenia
Introduction
Annelida, the ringed or segmented worms, is an ancient and ecologically important animal taxon with
over 22,000 described species occupying terrestrial, limnic, and marine habitats all around the world.
Annelids can have body length of less than 1 mm or more than 3 m. As bioturbators, scavengers, and
predators, they are important members of all ecosystems. Although some species can be found in the
plankton throughout their entire life span, annelid species usually constitute a significant part of the endo-
and epibenthos where they occupy almost every existing ecological niche in the marine environment
(Purschke et al. 2014). Moreover, they are the most abundant macrofaunal organisms in the largest habitat
on the planet, the deep sea. On the other hand, the vast majority of the limnetic and terrestrial species
belong to only one group of annelids, the Clitellata (e.g., earthworms and leeches), which exhibit specific
adaptations to the terrestrial life (e.g., Purschke 2002). Thus, annelids show a broad diversity of life
strategies including tube-dwelling, burrowing, deposit-feeding, filter-feeding, predatory, blood-feeding,
and parasitic strategies. Given this broad ecological and life history range, evolution of annelids has
*Email: torsten.struck.zfmk@uni-bonn.de
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resulted in a high morphological diversity. Typically, annelids consist of segments containing coelomic
cavities, nephridia, a pair of ganglia of the ventral nervous system, different types of muscles, and
so-called parapodia bearing different kinds of chaetae. The presence of segmentally arranged chitinous
chaetae is one of the key characters of annelids. Hence, Annelida comprises one of three major animal
groups with segmentation. Besides the segments, annelids possess a prostomium and peristomium at the
anterior end bearing different kinds of sensory organs, such as palps, antennae, and nuchal organs. In
some annelid taxa, the pharynx is equipped with elaborate jaw elements for defense, prey catching, or
blood feeding (e.g., Eunicida, Glyceridae (bloodworms), Nereididae, Gnathobdelliformes (jawed
leeches)). At the posterior end, a pygidium is present, which might also bear sensory organs in the form
of cirri. However, almost every character varies greatly among annelids, making ground pattern recon-
struction a difficult task without a solid phylogenetic framework. For example, one of the key characters,
segmentation, is virtually absent in some groups, e.g., Echiura (spoon worms), Sipuncula (peanut worms),
or Diurodrilidae, a group of small interstitial annelids (Purschke et al. 2014).
Given this diversity of different life strategies, only very few annelid taxa and species are known to be
toxic, either for defensive or predatory reasons. Amphinomidae (fireworms) bear calcareous chaetae
instead of the typical chitinous ones. Upon contact, these chaetae break and can cause serious skin
inflammation, for example, in humans (Borda et al. 2012). Upon attack, the earthworm Eisenia fetida
releases a toxin with its coelomic fluid, which is hemolytic by inserting into cell membranes (Sukumwang
and Umezawa 2013). On the other hand, parasitic leeches, Hirudinea, use different polypeptides
preventing coagulation of the blood while feeding (Kvist et al. 2014). Bloodworms (Glyceridae) are
thus far the only known annelids known to catch prey using venom (von Reumont et al. 2014b). As
mentioned above, glycerids possess jaw elements. Specifically, they have four strong clawlike jaws,
which are capable of injecting the venom due to a direct connection to the venom glands.
Phylogeny of Annelida
Phylogenetic Position of Annelida Within Bilateria
Traditionally, Annelida had been regarded as closely related to Arthropoda, which is also known as the
Articulata hypothesis. The Articulata hypothesis was substantiated by characters such as a segmented
body organization including a rope ladderlike nervous system and segment formation by a posterior
growth zone, longitudinal muscles of the body wall in distinct bundles, and presence of mushroom bodies
(Scholtz 2003). Interestingly, arthropods are among the bilaterian lineages from which several venomous
animals are known, even by non-biologists. These include insects, centipedes, remipede crustaceans,
scorpions, spiders, and ticks (Fry et al. 2009; von Reumont et al. 2014a). Given the number of toxic
annelid and arthropod taxa, the Articulata hypothesis could support the view that there is a tendency to
evolve venom in these animals.
In contrast, early molecular-phylogenetic studies based on 18S rRNA were not able to recover
Articulata, but showed a closer relationship of Annelida to Mollusca and Brachiopoda (see Struck
2012). Studies including representatives of all lophophorate lineages (i.e., Brachiopoda, Phoronida, and
Ectoprocta) confirmed these results with strong bootstrap support, and the name Lophotrochozoa was
coined for this group of taxa. Lophotrochozoa is defined as including the last common ancestor of
lophophorates, molluscs, and annelids and its descendants. The suitability of the 18S-rRNA data used in
the first molecular studies regarding bilaterian relationships has been criticized. However, since then, an
impressive array of different molecular markers had unequivocally supported a close relationship of
Annelida to lophotrochozoan taxa. These markers comprised larger 18S rRNA datasets, 28S rRNA, Hox
gene data, mitochondrial genomes, 19 nuclear protein-coding genes, microRNA data, and phylogenomic
Page 2 of 12
DOI 10.1007/978-94-007-6727-0_15-1
** Lophotrochozoa
**Annelida
*Mollusca
*
Nemertea
Ectoprocta
Platytrochozoa
Phoronida Lophophorata
Brachiopoda
Spiralia
Entoprocta
Platyhelminthes
Rouphozoa
Gastrotricha
Gnathifera
Fig. 1 Phylogeny of Spiralia based on recent phylogenomic studies (Nesnidal et al. 2013; Struck et al. 2014). Red triangles
and stars indicate group of toxic or venomous species. Higher taxonomic units are highlighted
datasets consisting of more than 50 genes mostly derived from expressed sequence taqs (EST) libraries
(see Struck 2012). Additionally, topology testing significantly rejected a close relationship of Annelida
and Arthropoda. Thus, molecular data clearly support a placement of Annelida within Lophotrochozoa,
though their position within Lophotrochozoa remains controversial (Edgecombe et al. 2011; Halanych
2004).
The term Spiralia is occasionally used as a synonym of Lophotrochozoa (Halanych 2004). However,
the definition of Lophotrochozoa as provided above does not necessarily include the same set of taxa. The
taxon Spiralia includes all animals with spiral cleavage (i.e., Annelida, Mollusca, Nemertea,
Platyhelminthes, Gnathostomulida, and Entoprocta) (Edgecombe et al. 2011). Thus, depending on the
position of the lophophorate taxa, Lophotrochozoa and Spiralia could be synonymous, Spiralia a
subgroup of Lophotrochozoa, or vice versa. One challenge in reconstructing the lophotrochozoan/
spiralian phylogeny is that many taxa (e.g., Platyhelminthes, Ectoprocta, Gastrotricha) are hampered by
misleading molecular biases such as increased substitution rates in comparison to the other taxa in the
analysis (also known as the long-branch attraction artifact) or compositional heterogeneity. Therefore,
simply increasing the number of taxa is not enough to resolve this phylogeny, but thorough analyses of the
data quality have to accompany the phylogenetic reconstruction. Recent phylogenomic studies utilizing
more genes but also more sophisticated analytical strategies have achieved some progress in this respect
(Fig. 1) (Nesnidal et al. 2013; Struck et al. 2014). These analyses showed that Lophotrochozoa is a
subgroup of Spiralia and hence not synonymous with it. The first taxon to branch off in Spiralia is
Gnathifera, which comprised among others the wheel animals “Rotifera”. The Rouphozoa comprising
Gastrotricha and Platyhelminthes are the sister of Lophotrochozoa, a clade named Platytrochozoa (Struck
et al. 2014). The relationships within Lophotrochozoa are still inconsistent, but strong evidence is
emerging that Lophophorata, comprising Ectoprocta, Brachiopoda, and Phoronida, is indeed
monophyletic. The previously recognized clades Kryptrochozoa (i.e., Brachiopoda, Phoronida,
Nemertea) and Polyzoa (i.e., Entoprocta, Cycliophora, Ectoprocta) could be attributed to misleading
biases in the dataset due base composition heterogeneity (Nesnidal et al. 2013).
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DOI 10.1007/978-94-007-6727-0_15-1
Summarizing these results, it is obvious that the molecular data unequivocally reject a close relation-
ship of Annelida to Arthropoda. In contrast, Annelida are placed in a spiralian group also comprising
Mollusca, Nemertea, and Lophophorata. Interestingly, besides annelids, molluscs are the only known
taxon within this group with venomous species (i.e., cone snails and some cephalopods) (Fry et al. 2009).
In contrast to the Articulata hypothesis, this position of Annelida does not support a view that there might
be a tendency toward evolving venom in the lineage of Spiralia and independent convergent evolution of
venomous animals seems more likely.
Monophyly and Taxon Composition of Annelida

Given the Articulata hypothesis, only very few characters like a foregut with dorsolateral folds and
capillary chaetae of b-chitin in four groups supported the monophyly of Annelida. Some regarded even
these to be present in the stem lineage of Articulata and placed arthropod taxa within Annelida (see Struck
2012). However, given the Lophotrochozoa hypothesis, several characters like segmentation with a
praepygidial proliferation zone, a dorsal brain and a ventral nerve cord, longitudinal muscle bands,
capillary chaetae of b-chitin in four groups, and nuchal organs support the monophyly of Annelida (see
Purschke 2002). In contrast, recovery of the monophyly of Annelida within Lophotrochozoa was difficult
in earlier molecular-phylogenetic studies (see McHugh 2000, 2005). However, recent studies with
increased numbers of taxa and genes and more sophisticated methods consistently recovered the mono-
phyly of Annelida even with strong nodal support (see Struck 2012). Additionally, it could be shown that
Annelida exhibit a unique mitochondrial gene order (Golombek et al. 2013). However, monophyly of
Annelida based on molecular data required the adjustment of the taxon composition of Annelida.
Traditionally, Annelida comprised Clitellata and Polychaeta (Fauchald 1977); now, other taxa, formerly
considered as separate phyla, have to be included within the annelid radiation (i.e., Myzostomida,
Siboglinidae, Echiura, Sipuncula) (Halanych et al. 2002).
For myzostomids, flat-bodied marine ectocommensals or parasites of echinoderms, a close relationship
to Annelida or placement within polychaetes had generally been assumed, even though the body plan of
Myzostomida is unique due their ectocommensalic/parasitic life history (see Struck 2012). Several
features support an inclusion of Myzostomida within Annelida: parapodia with chitinous chaetae
(aciculae and hooks) and cirri, chaetogenesis, similarities in larval development via trochophore- and
nectochaeta-like larvae, ultrastructure of the nervous system, hypertrophied axial pharynx, and serial
arrangement of protonephridia (Lanterbecq et al. 2008). However, analyses of molecular data (i.e., EF1a,
18S, and 28S rRNA) seemed to support the latter by placing Myzostomida closer to taxa like
Platyhelminthes or Ectoprocta. However, again careful analyses of the molecular data showed that the
results were due to biased data (Bleidorn et al. 2009). Molecular data of Myzostomida which is not, or is
less, affected by increased substitution rates such as myosin II heavy chain and mitochondrial proteins, as
well as mitochondrial gene order data, strongly support a close relationship of Myzostomida and
Annelida. Hox gene data also substantiate this position. Recently, phylogenomic studies based on
hundreds of genes were also able to place Myzostomida within Annelida (Hartmann et al. 2012; Weigert
et al. 2014).
Siboglinidae have had a controversial taxonomic history. Upon description of the first species, they
were compared to hemichordates. However, they were also regarded as aberrant annelids. Deuterostome
affiliations were substantiated by some by a dorsal nerve cord, radial cleavage, and tripartite body with
prosoma, mesosoma, and a modified metasoma, whereas others placed them within protostomes, specif-
ically close to Annelida, due to the possession of a ventral nerve cord, spiral cleavage, and metameric
segmentation and homology of their chaetae with uncini of polychaetes (see Struck 2012). The discovery
that the specimens investigated till then were incomplete due to the lack of the segmented ophistosoma
(Webb 1969) revealed that Siboglinidae possess a segmented body organization. Furthermore,
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ultrastructural analyses further supported the homology of the chaetae of Siboglinidae with ones of
Annelida (Bartolomaeus 1995). Finally, morphological cladistic analyses also supported a closer rela-
tionship of Annelida and Siboglinidae (Rouse and Fauchald 1997), as did different molecular data:
elongation factor 1a, cytochrome oxidase c subunit I, hemoglobin, 18S and 28S rRNA, multi-gene
analyses, mitochondrial genomes, and phylogenomic data (see Struck 2012; Weigert et al. 2014). Addi-
tionally, topology testing significantly rejected an exclusion of Siboglinidae from Annelida (Struck
et al. 2007) and analyses of combined morphological and molecular data also placed Siboglinidae within
Annelida (Zrzavy et al. 2009).
Echiura are unsegmented marine worms and were grouped together with Annelida throughout the
nineteenth century. Several features support a close affinity of Echiura with Annelida: a typical annelid-
like trochophore larva, chaetogenesis and ultrastructure of the chaetae, structure and organization of the
blood vascular system, and development and ultrastructure of spermatozoa (see Struck 2012). However,
de Quatrefages (1847) transferred them to Gephyrea, which also comprised Sipuncula, Sternapsidae, and
Priapulida, a group supposed to bridge the gap between annelids and echinoderms. As Echiura also lack
any sign of segmentation as adults as well as nuchal organs, Echiura were excluded from Annelida,
though a close relationship to Annelida or Articulata was still advocated (Clark 1969). The exclusion of
Echiura was further substantiated by morphological cladistic analyses of Annelida (Rouse and Fauchald
1997). However, the distinction between primary or secondary absence of character traits such as
segmentation in cladistic analyses based on morphological data might be problematic. Moreover, the
development of the nervous system in Echiura reveals traces of an ancestral segmentation pattern
(Hessling 2003). First support from molecular data for an inclusion of Echiura within Annelida was
derived from elongation factor 1a data (McHugh 1997). As for siboglinids, to date ample molecular data
support the inclusion of Echiura within Annelida (see Struck 2012). Topology tests significantly rejected
an exclusion of Echiura from Annelida (Struck et al. 2007).
Finally, Sipuncula are relatively large, unsegmented worms (more than 90 % are >5 mm) burrowing in
marine sediments. Sipuncula were considered to be closely related to Mollusca based on the so-called
molluscan cross during spiral cleavage (Scheltema 1993). On the other hand, on the basis of develop-
mental features, Rice (1985) suggested a close affinity to Annelida. Additionally, the early nervous system
development showed signs of a posterior growth zone similar to annelids with some metameric patterns
(Kristof et al. 2008). In contrast to Siboglinidae and Echiura, first molecular-phylogenetic studies were
inconclusive with respect to the position of Sipuncula as closer relationships to either Mollusca, Annelida,
or Mollusca plus Annelida were suggested (see Struck 2012). However, increasing the amount of
molecular data for both Sipuncula and other lophotrochozoan taxa has achieved solid placement of
Sipuncula within Annelida (see Struck 2012; Weigert et al. 2014). Moreover, topology tests significantly
rejected an exclusion of Sipuncula from Annelida (Dordel et al. 2010).
Internal Relationships of Annelida

Throughout the nineteenth century, three groups were generally recognized within Annelida: Polychaeta,
Oligochaeta, and Hirudinea. Polychaeta comprised all marine annelids, which were henceforth referred to
also as marine bristle worms. Oligochaeta comprised both earthworms and microdrilids and Hirudinea,
the leeches, acanthobdellids, and branchiobdellids. In the nineteenth century, Polychaeta had been
traditionally divided into two major groups, which however were known under different names and
with different taxon compositions. Generally, one group contained polychaetes such as Amphinomidae,
Eunicida, or Phyllodocida but also other polychaetes which were predominantly characterized by a more
or less carnivorous lifestyle. The other clade contained all remaining polychaetes like Spionidae,
Chaetopteridae, Opheliidae, or Sabellidae, which were characterized by a microphagous diet (see Struck
2012). Finally, de Quatrefages (1866) established the most influential systematic scheme, dividing
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polychaetes by their life style. Polychaeta was split into Annelidae erraticae (later named Errantia) and
Annelidae sedentariae (later named Sedentaria). The names clearly reflected their mode of living as either
being errant, more vagile, worms or sedentary, more sessile, ones, but the scheme itself was based on the
presence or absence of distinct body regions also known as tagmatization (de Quatrefages 1866). Errantia
comprised polychaetes like Amphinomidae, Eunicida, and Phyllodocida lacking any obvious body
regions. On the other hand, Sedentaria were characterized by the presence of distinct body regions.
With some modifications, the classification scheme of de Quatrefages (1866) was adopted in the
following century. In summary, Errantia comprised Eunicida, Phyllodocida and Amphinomidae, and
Sedentaria the remaining polychaetes (Struck 2012). For a detailed account of the early history of the
phylogeny of polychaetes and Annelida, please refer to Struck (2012).
Michaelsen (1928–1932) grouped Oligochaeta and Hirudinea together as Clitellata and it has been
accepted for a long time now that Hirudinea is placed within Oligochaeta (Erséus 2005). Since
Michaelsen (1928–1932), most annelid researchers have generally treated Clitellata and Polychaeta as
two separate taxa within Annelida (e.g., Dales 1963; Fauchald 1977). Support for the monophyly of
Clitellata is overwhelming based on both morphological and molecular data, but the monophyly of
Polychaeta was not similarly supported (Purschke 2002). For example, the morphological cladistic
analyses of Rouse and Fauchald (1997) strongly supported the monophyly of Clitellata, but support for
the monophyly of Polychaeta was weak, as none of the supporting characters was free of homoplasy.
In the middle of the last century, the classification scheme of polychaetes based on de Quatrefages
(1866) came under heavy criticism for showing only arbitrary groupings reflecting mode of living rather
than evolutionary history and being valuable only for practical purposes (Dales 1963, 1977). Conse-
quently this classification system of Errantia and Sedenataria was completely abandoned by the end of the
last century, but a new scheme had not yet arisen. Instead, polychaete families were grouped together into
several orders of equal rank as no morphological characters unequivocally supported any higher group-
ings (Fauchald 1977; Struck 2012). Therefore, the phylogeny of Polychaeta was regarded as unresolved.
At the end of the last century, Rouse and Fauchald (1997) tried a new approach to reconcile the different
polychaete families into higher taxonomic groupings based on morphological data. Using a cladistic
method considering all data simultaneously instead of only a single or a few characters, they employed
two coding strategies by coding either 124 morphological characters as absent/present or 55 characters as
multistate, respectively, for 80 polychaete families. Moreover, different strategies were used to analyze
the data, for example, by weighing the characters differently or by excluding symbiotic, pelagic,
interstitial, and poorly known taxa, as these taxa often show highly aberrant morphologies or the states
are not known yet in the case of poorly described species leading to instable placements of these taxa in
the reconstructed trees. Based on these analyses, Rouse and Fauchald (1997) proposed as sister group
relation of Clitellata and Polychaeta. Polychaeta was further split into Scolecida comprising Arenicolidae,
Capitellidae, Opheliidae, Orbiniidae, Paraonidae, and Cossuridae as well as Palpata consisting of the
remaining polychaetes. The scolecidan taxa are earthworm-like and hence the name Scolecida from the
Greek scolex for worm. Palpata comprised all palp-bearing polychaetes and the possession of palps was
the supporting character for this clade. Palpata was divided into Canalipalpata and Aciculata. Aciculata
consisted of Amphinomidae, Eunicida, and Phyllodocida and, hence, was in its composition similar to
Errantia. Of all clades proposed by Rouse and Fauchald (1997), Aciculata was the best supported with
respect to morphological characters, besides the presence of the name-giving aciculae, an internalized
supporting chaetae, other characters substantiated this clade (Rouse and Fauchald 1997). Moreover, it
contained both polychaete groups with toxic or venomous species. Most of the remaining polychaetes
grouped together as Canalipalpata characterized by the presence of peristomial grooved palps, but some
of the taxa listed above with aberrant morphologies were placed as incertae sedis (Rouse and Fauchald
1997). While this scheme of annelid phylogeny became generally accepted in a short time, it was also
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instantly strongly criticized for the exclusion Clitellata from polychaetes, inconsistencies in character
reconstructions, as well as their results from the different analytical strategies and lack of structural
integrity for reconstructed stem species (Bartolomaeus et al. 2005; Struck 2012; Westheide 1997).
In parallel to the analyses of Rouse and Fauchald (1997), the inclusion of Clitellata within polychaetes
was explicitly proposed in the last decade of the twentieth century based on both molecular data and
functional morphology. Nielsen (1995) suggested incorporating Clitellata within polychaetes, based on
similarities to Capitellidae. Westheide (1997) discussed the direction of evolution within polychaetes
based on the functional morphology of the coelom and coelothels and concluded that Clitellata is a highly
derived polychaete subtaxon with many of the differences to polychaetes, such as the reduction of
appendages, being related their unique reproduction. However, the polychaete subtaxon forming the
sister taxon to Clitellata could not be determined based on morphological data. In summary, based on
morphological data, it remains uncertain whether or not to place Clitellata within polychaetes. Neither
monophyly of Polychaeta nor a sister group relationship between Clitellata and a polychaete subtaxon is
convincingly supported.
The first molecular-phylogenetic studies, based on elongation factor 1a, placed Clitellata within
polychaetes. All subsequent molecular-phylogenetic studies also congruently recovered the placement
of Clitellata within polychaetes utilizing 18S rRNA, 28S rRNA, histone H3, U2 snRNA, cytochrome
oxidase subunit I, 16S rRNA mitochondrial genomes, as well as a steadily increasing number of taxa (see
Struck 2012). However, as for the morphological data, a polychaete sister group of Clitellata could not be
determined, as nodal support was weak, and the placement of Clitellata differed between the analyses.
Moreover, these molecular-phylogenetic studies also seemed to still suffer from low-resolution power as
judged by nodal support (see McHugh 2005). Nonetheless, increasing the number of both taxa and
position had, in general, a positive effect on the reconstruction on the annelid phylogeny (see Struck
2012). Due to these additional genes, monophyly of Annelida, including the taxa discussed above, was
consistently recovered, and topology testing significantly rejected monophyly of Scolecida, Palpata,
Canalipalpata and Aciculata (Struck et al. 2007). Moreover, the congruence between the results of
different studies increased, but nonetheless nodal support remained low (Struck et al. 2007, 2008; Zrzavy
et al. 2009), precluding any robust conclusions of the phylogeny with respect to polychaete families and
the position of Clitellata (Struck 2012).
Simulation studies by Struck et al. (2008) showed that annelid phylogeny could be resolved using more
data. Moreover, Dordel et al. (2010) revealed that amino acid data would be better suited to achieve this
goal than nucleotide data. Therefore, instead of using specific genes, a phylogenomic study of Annelida
was conducted using a dataset of nearly 48,000 amino acid positions and data coverage of 41.7 % per
taxon (Struck et al. 2011). This large and reasonably well-covered dataset resulted in a well-supported
phylogeny with several basal nodes showing bootstrap values of 70 or higher, even up to the maximal
value of 100. Interestingly, the results of this phylogenomic approach were congruent in many parts to the
studies of Struck et al. (2008) and Zrzavy et al. (2009), which were based on many fewer genes. One of the
major differences was the position of the Amphinomidae, which was part of Errantia in Struck
et al. (2011), instead of being part of the basal radiation. A study concentrating on the effect of paralogous
genes, which have been grouped together erroneously as orthologous genes, revealed that even in the
presence of 231 genes, a single such misplacement has the potential to mislead the placement of the
affected taxon, given certain circumstances such as low taxon coverage for the corresponding taxon
(Struck 2013). For example, due to one instance of such erroneously grouped paralogous genes,
Amphinomidae was placed within Errantia (Struck 2013). A follow-up phylogenomic study further
increasing both taxon sampling as well as number of genes (>600 genes) found results similar to the
phylogenomic analyses of Struck et al. (2011) except that Amphinomidae was part of the basal annelid
radiation and Orbiniidae of Sedentaria (Fig. 2, Weigert et al. 2014). Moreover, in this study all basal
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* Lumbricidae
Megascolecidae
* Hirudinea Clitellata
Tubificidae
Terebelliforma
Arencolidae
Sedentaria
Capitellidae
Echiura
Opheliidae
Spionidae
Sabellariidae
Pleistoannelida
Sabellidae
Serpulidae
Siboglinidae
Cirratuliformia
Orbiniidae
Annelida
Tomopteridae
* Glyceridae
Phyllodocidae
Errantia
Nephtyidae Phyllodocida
Nereididae
Aphroditiformia
Syllidae
Eunicidae
Onuphidae Eunicida
Lumbrineridae
*Amphinomidae
Sipuncula
Chaetopteridae
Oweniidae
Magelonidae
Fig. 2 Phylogeny of Annelida based on the phylogenomic study of Weigert et al. (2014). As Fig. 1 red triangles and stars
indicate group of toxic or venomous species. Higher taxonomic units are highlighted
relationships within the annelid phylogeny were supported by significant bootstrap values of 99 or
100 and misleading effects on the reconstruction by paralogy, contamination, long-branch heterogeneity,
or other biases could be excluded. Hence, the century-old debate about the phylogeny of Annelida
including the placement of Clitellata within polychaetes could finally be settled using massive amounts
of molecular data (Fig. 2, Struck et al. 2011; Weigert et al. 2014).
Oweniidae, Mageloniidae, Chaetopteridae, Amphinomidae, and Sipuncula have been found to be part
of the basal annelid radiation (Fig. 2). Hence, of the two polychaete taxa with toxic or venomous species,
Amphinomidae and Glyceridae, one was placed as part of this basal radiation closely related to Sipuncula,
which previously have been regarded as its own phylum. The remaining annelids split into two well-
supported groups. One clade comprised Eunicida and Phyllodocida and the other one the remaining
annelids including Clitellata and Echiura (Fig. 2). As the taxon compositions of these two clades were
very similar to the taxon compositions of the traditional Errantia and Sedentaria concepts, respectively,
Struck et al. (2011) resurrected these names for these two clades with some modifications. Errantia was
defined as the clade comprising all descendants of the last common ancestor of Eunicida, Phyllodocida,
and all organisms or species that share a more recent common ancestor with these two taxa than with
Clitellata and Spionidae, whereas Sedentaria was oppositely defined as the clade comprising Clitellata,
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Echiura, Spionidae, and all organisms or species that share a more recent common ancestor with these
three taxa than with Phyllodocida or Eunicida (Struck 2012). Moreover, Struck (2011) named the clade
comprising Errantia and Sedentaria Pleistoannelida as it comprises most of the recent annelid biodiversity
and defined it by the last common ancestor of Sedentaria and Errantia and all the descendants of that
ancestor. The second polychaete taxon with toxic or venomous species, Glyceridae, was placed within the
errant annelid taxon Phyllodocida (Fig. 2, Weigert et al. 2014). However, although relationships within
Phyllodocida were strongly supported in the analyses of Weigert et al. (2014), taxon representation of
Phyllodocida was still too limited to allow definite conclusions about the sister group of Glyceridae within
Phyllodocida. Based on morphological data, the most likely sister group of Glyceridae is Goniadidae
(Rouse and Fauchald 1997).
Within Sedentaria, Clitellata were placed within a clade consisting also of Arenicolidae,
Terebelliformia, Capitellidae, Echiura, and Opheliidae, as sister to Arenicolidae/Terebelliformia
(Fig. 2), with very strong support (Weigert et al. 2014). Moreover, this clade is congruent with the
placement of Clitellata in previous phylogenomic studies with a much more limited taxon sampling of
Annelida (e.g., Dordel et al. 2010). Additionally, in the analyses of Struck et al. (2008), Clitellata were
part of a congruent clade. This study was based only on 18S and 28S rRNA data, but with a much more
comprehensive taxon sampling than the phylogenomic studies (Dordel et al. 2010; Struck et al. 2011;
Weigert et al. 2014). The placement of Clitellata within polychaetes also means that Polychaeta is
synonymous with Annelida and should be therefore abandoned as a taxon name.
Two of the four groups with toxic or venomous species, Hirudinea and the genus Eisenia
(Lumbricidae), are clitellates (Fig. 2). Within Clitellata, Hirudinea is part of a clade that also comprises
the classical earthworms (Lumbricidae) as well as all other megadrilid taxa and Enchytraeidae (Erséus
2005). Hence, both clitellate groups with toxic or venomous species are part of this group. However, as in
Lumbricidae only species of the genus Eisenia are so far known to release toxins, convergent evolution of
the toxic or venomous character trait is more likely than a common origin with several losses. This is
further substantiated by the fact that there are differences in the toxins. Eisenia uses the protein lysenin,
which is hemolytic and might also play a role in the innate immune reaction, while hirudineans release a
complex mixture of anticoagulant polypeptides (von Reumont et al. 2014b).

Of the four annelid taxa with toxic or venomous species, two are placed within Clitellata as part of the
sedentary annelids, but not as sister groups to each other (Fig. 2). Glyceridae is part of Errantia and
Amphinomidae part of the basal radiation of Annelida. Hence, convergent evolution of venomous and
toxic species even within Annelida seems very likely.
With respect to the phylogeny of Annelida, and especially considering the evolution of venomous
annelids, future research should be directed to three more specific tasks. First, what are the
lophotrochozoan sister group of annelids and also the other lophotrochozoan taxon Mollusca with
venomous species? Second, while the phylogenetic position of Hirudinea, Eisenia and Amphinomida
has been settled in the last few decades, the position of Glyceridae within Phyllodocidae remains to be
determined. Third, within Amphinomidae and Glyceridae, the ingroup relationships of the genera and
species to each other are not yet resolved. This is an especially important task for Glyceridae, as glycerid
species are emerging as model systems for studying venoms in annelids (see von Reumont et al. 2014b).
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Cross-References
▶ Systematics of Cephalopods
▶ Toxicity in Cephalopods
▶ Venom Metering in Spiders and Other Animals
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Evolutionary Context of Venom in Animals

Kevin Arbuckle*
Department of Ecology, Evolution and Behaviour, University of Liverpool, Liverpool, UK
Abstract
Much of the research on venoms has understandably focused on clinical implications of human enven-
omation and detailed molecular studies of toxins. However, as with any biological trait, venom exists in an
evolutionary context and must be considered as such if we are to gain a full understanding of the biology
of animal venoms. Consequently, this chapter aims to provide an overview of the diversity of venom and
venomous animals and also a set of evolutionary principles which are particularly applicable here. There
has been substantial variation in the definition of “venom” and “venomous” in the literature, so this is
discussed first with the aim of giving a definition which encompasses a number of important features of
venoms. A survey of the functional diversity of venoms and taxonomic diversity of venomous animals is
then provided as an introduction to the evolutionary drivers of venom and how it is distributed across the
animal tree of life. The last three sections consider three principles that are important to venom evolution:
(1) the composition of venom is variable both between and within species; (2) venom evolves in the
context of antagonistic coevolutionary interactions; and (3) venom can have consequences for the ecology
and evolution of animals that possess it beyond its direct functions to their behavioral ecology.
Keywords
Diversity; Ecology; Function; Terminology; Variation
Introduction
Venomous animals have a long history of inspiring fear, fascination, interest, and dread in
humans – perhaps since the earliest evolution of our species. They have also been the subject of a great
deal of scientific study over the last several hundred years. The majority of this research has focused on
two areas. Firstly, and largely because humans are understandably self-obsessed, the medical implications
of envenomation are intensely studied, including epidemiology, toxicological effects, and clinical treat-
ment. Secondly, because venoms typically consist of a diverse set of individual toxins (Casewell
et al. 2013) with a wide range of specific and often potent physiological actions (Fry 2005), much
research on molecular aspects of venom toxins has been undertaken, including structure-function relation-
ships, toxin evolution, and “venomics” (e.g., proteomic and transcriptomic studies of venom). In fact,
these two areas of venom research have been so prolific as to form the major content of several recent
books, including Chippaux (2006), Mackessy (2009), Fry (2015), and the current Handbook of
Toxinology series.
However, despite the impressive advances that clinical and molecular toxinological studies have
achieved, much less attention has been paid to the broadscale evolutionary context of animal venoms.
The ecological relations of whole venoms are often considered little more than a sidenote to detailed
*Email: k.arbuckle@liverpool.ac.uk
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investigation of particular toxins, but these are necessary to understand the selection pressures and
therefore drivers of venom evolution. Therefore, this chapter aims to provide a review of the diversity
of, and the principles underlying the evolution of, venoms.
What is Venom?
Before a meaningful discussion of the evolution of venomous animals can be made, it is important that the
terminology is made clear. Therefore, this section begins with a broad overview of different weapons in
the chemical arsenal of animals in order to set the context within which venom falls, and then the
definition of venom (and therefore venomous) is clarified.
The Diversity of Chemical Warfare in Animals

Collectively, animals use a wide range of chemicals for several purposes (see “Venom Functions” section)
but primarily for defense against predators. Venom is only one form of chemical defense (or offense), but
it is useful to consider the additional forms that it can take because many of these fulfill the same function
from the ecologically relevant perspective of predators. This subsection refers to chemical defense
specifically because, with the notable exception of venom, all other traits considered here are used in
defensive roles as opposed to other functions. The unique characteristic of venom, that it can be
deliberately and directly transferred into the body of another organism, is likely to be an important factor
in the ability to use venom for a broader range of functions.
When considering antipredator mechanisms, it is useful to think about them in the context of Endler’s
(1986) five stages of predation: detection, identification, approach, subjugation, and consumption. In
principle, defenses can operate to disrupt predation attempts at any one of those stages. However in
practice, with one exception, chemical defenses typically act only at the later stages of predation (against
subjugation and consumption of prey), perhaps because such defenses are effective but carry high
energetic costs (e.g., Higginson et al. 2011) and so are reserved for late-stage “emergency” situations.
Indeed, many chemically defended animals use other strategies such as protective coloration (e.g.,
aposematic warning signals or camouflage) to avoid attack at earlier stages of predation. Furthermore,
the exception to late-stage chemical defenses is using a low nutrient composition of body tissues to
discourage predation (Bullard and Hay 2002): a rare and unusual strategy that carries constraints such as
an ability to store substantial energy reserves and does not involve energetically costly ways to
biosynthesize or sequester chemicals.
Avoidance of subjugation by predators can use several forms of chemical defense. Firstly, slime or
mucous production can either make the prey “slippery” and so difficult to subdue or adhere to the
predator’s jaws (which restricts their movement), depending on the properties of the mucus. Secondly,
venom is often used as a defense against subjugation by causing pain and/or actual harm to predators
during attack. Thirdly, a somewhat more cryptic form of chemical defense is the production of detoxifying
chemicals, including antibodies, as one way to confer resistance against the venom of predators. Fourthly,
there are various chemicals which are sprayed toward predators and have various effects that act from a
distance (i.e., before the prey is fully subjugated). Some of these spray defenses are well known and
include many iconic species, such as the high-temperature bombardier beetle spray, horned lizards that
squirt blood from behind their eye, and spitting cobras that spray venom with contact toxicity to eyes.
Once predators have subdued prey, limited options exist to avoid consumption, and these can generally
be considered to be the release of chemicals that are ingested by predators, hopefully before the prey
animal is mortally injured. The effects of such chemical defenses can be either (or a combination of)
simple distastefulness, irritation to the mucous membranes of the predator’s mouth (such as the blistering
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caused by some millipede secretions, pers. obs.), nausea (inducing vomiting or other sickness but not
“harmful” per se), or poisoning (causing physiological harm to the consumer). Poisons are the most
intensively studied of these, and along with venoms are the most studied chemical defenses more
generally. However, it is remarkably difficult to distinguish these effects in many cases, and so for
many such defenses we do not know, for example, whether a predator rejects prey under natural
conditions because it does not like the taste, experienced some discomfort (from irritation or sickness),
or was fully poisoned.
The different “anti-consumption” defenses highlighted in the last paragraph are often discussed
collectively as “poisons,” though this is an overly general classification. Even more broadly, the word
“poison” is sometimes used erroneously to include venoms, but these are important to distinguish for
medical, evolutionary, ecological, or behavioral studies. Therefore, following the focus of this chapter on
venoms, the following subsection attempts to provide a meaningful definition of “venom.”
In Search of a Definition
Although the questions of “what is venom” and therefore “what constitutes a venomous animal” may
seem obvious to many, a unanimously accepted definition of “venom” has been surprisingly difficult to
develop. This is perhaps also the reason that much literature, even recent work, has continued to describe
venom as “salivary secretions” and other such terms that limit the connectivity and cohesiveness of the
field (e.g., Hildebrandt and Lemke 2011). A recent review by Nelsen et al. (2014a) highlighted the many
different definitions that exist in the literature and makes a good attempt at summarizing these. In essence,
much of the disagreement exists due to the emphasis placed on different aspects of venoms. For instance,
the relative importance of the potential to inflict damage on humans may be considered an important
defining characteristic by medics but less so (or not at all) by those from molecular genetic, biochemical,
evolutionary, or ecological backgrounds.
Medical importance of envenomation to humans, although a useful distinction for clinical and public
health purposes, is of little relevance to a biologically meaningful definition of venom. If we accept that all
of biology occurs in the context of evolution, including venoms, then a meaningful definition of venom
must include evolution as a main component. Because humans have not been important in the origin of
venoms in any species, any such interactions with humans are demonstrably misguided as a part of the
definition. This is highlighted in the many species that use venom effectively to catch their prey but whose
venom is of little or no clinical importance for humans. Beyond this, purely objective arguments for what
is, and is not, important for defining “venom” are lacking. Nevertheless, we can consider those factors that
are most commonly used in the literature to generate a definition that is sensible, meaningful, and
consistent with as much of the existing literature as possible.
Nelsen et al. (2014a) found that the following attributes are frequently used in definitions of venom:
(1) the venom is produced or stored in a gland, (2) there is a specialized delivery system used to transfer
the venom to another organism, (3) the venom is transferred via an injury, (4) the venom is actively
(as opposed to passively) transferred to another organism, and (5) the venom functions in predation and/or
defense. In addition, Fry et al. (2009a) also add another characteristic that is commonly used, implicitly or
explicitly, in definitions of venom: (6) the venom contains molecules (“toxins”) which interfere with
physiological or biochemical processes in another organism. These six defining characteristics of venom
are generally sensible, but there are two points that do not capture the range of organisms traditionally
considered venomous. Firstly, attribute 1 would not consider cnidarians (such as jellyfish) to be venomous
as they do not possess a venom “gland” per se, but they do possess a sub-glandular apparatus that fulfills
the same function (specialized “nematocyst” cells). Therefore, a better attribute would be that the venom
is produced and/or stored in a specialized structure, which may include both glands and nematocysts.
Secondly, attribute 5 would not consider the platypus to be venomous because they do not use venom
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(at least primarily) in either predation or defense but in intraspecific competition. Therefore, this attribute
should be relaxed to include other potential venom functions, such that the venom need only function to
provide a benefit to the venomous animal once it is transferred to another organism, albeit this is most
commonly a predatory or defensive benefit.
A definition of venom can therefore be ascribed based on the six attributes above (with the modifica-
tions discussed) alongside the recognition that evolution is important and medical consequences to
humans are not important for this purpose. To this end, the following should provide a definition that
represents a synthesis of current use and key components of venom:
Venom is a biological substance produced by an organism that contains molecules (“toxins”) which interfere with
physiological or biochemical processes in another organism, which has evolved in the venomous organism to provide a
benefit to itself once introduced to the other organism. The venom is produced and/or stored in a specialized structure
and actively transferred to another organism through an injury by means of a specialized delivery system.
Venom Functions
As briefly touched on in the preceding section, venoms are often considered to function in predation or
defense (e.g., Edstrom 1992), with some literature giving additional mention to the use of venom in
competition (e.g., Casewell et al. 2013; Fry et al. 2015). However, the true diversity of venom functions is
broad but somewhat masked by such short and simple statements. This is not surprising because venom,
as defined above, should provide many different potential benefits, and the evolution of diverse animal
ecologies opens the door for a wide range of venom functions. Consequently, this section will highlight
the diversity of venom functions found in animals as this directly relates to the selection pressures driving
the evolution of venoms.
One key point to bear in mind is that the following functions are not mutually exclusive: venom may be
used for different functions by the same animal, although not necessarily equally (one function may still
be the main evolutionary driver). For instance, the evolution of snake venoms is primarily driven by their
use in predation (Barlow et al. 2009; Daltry et al. 1996), but that does not prevent their use as a very
effective antipredator defense often with devastating consequences for the person or other animal bitten
(e.g., Boyer et al. 2015; Chippaux 2006; Mackessy 2009). In addition to this example of co-opting venom
for another purpose than its main driver, some animals have specifically evolved a “dual-purpose” venom
systems which include separate predatory and defensive components, such as cone snails (Dutertre
et al. 2014) and scorpions (Inceoglu et al. 2003).
Predation
A predatory function for venom is arguably the most common primary driver of venom evolution (see
phylogenetic distribution of venom functions in Casewell et al. 2013). It is also the main function of
venom in many of the better known lineages of venomous animals such as snakes, spiders, and scorpions.
Nevertheless, there are various ways in which venoms can be used to aid predation.
The most direct is also the most obvious – incapacitating prey to allow the venomous predator to
consume it. Note that the function here is to incapacitate prey, not necessarily to kill it. Killing prey would
usually require more venom than incapacitation, which is unlikely to be favored by evolution since venom
is energetically expensive to produce (McCue 2006; Morgenstern and King 2013) and incapacitated prey
is just as beneficial as killed prey for consumption. That is not to say that killing prey as a standard
predatory tactic is not common, in fact many mechanisms for incapacitation may kill with more time, but
that killing is not an essential part of predatory venom functions. This is reflected in the toxicological
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effects of many predatory venoms, which typically cause two main classes of symptoms in prey:
interfering with nerve action causing paralysis (and often death later) and altering blood and blood
vessels causing blood loss and associated shock. For example, ant-specialist spiders (Zodarion) are
capable of predating prey much larger than themselves by using paralyzing venom (Pekár et al. 2014),
without the need to actually kill the prey before consumption. Furthermore, Fry et al. (2009b) demon-
strated that the Komodo monitor (Varanus komodoensis) inflicts a deep wound with recurved, serrated
teeth and uses its venom to quickly induce loss of consciousness in prey via the onset of shock. The same
authors also suggested that the extinct Varanus priscus probably used a similar strategy.
Venom has also been considered to aid in predation after prey consumption by increasing the speed of
digestion with proteolytic toxins. This is most commonly discussed in snakes because viper venoms often
have relatively high proteolytic activities and because some of the earlier studies demonstrating an effect
of venom on digestion were carried out using Crotalus atrox rattlesnakes (Thomas and Pough 1979).
Recent studies, such as McCue (2007), have failed to find an effect on digestion in Crotalus atrox, which
has been used to call into question whether increased digestive performance can be a function of venom.
However, McCue (2007) conducted experiments at higher temperatures (25–30 C) that did not produce a
large effect in Thomas and Pough (1979) – the latter authors found that venom was more important in
digestion at lower temperatures (15 C). This, combined with some evidence of improved digestive
efficiency conferred by the venom of some other species, such as Andrallus spinidens bugs (Zibaee
et al. 2012), suggests that such a function cannot be completely discounted in studied taxa and is certainly
a plausible function that has not been well studied in most venomous animals.
Finally, parasitism represents another predatory (in the broad sense) function of venoms in some
animals. Blood-feeding parasites such as ticks and vampire bats often produce anticoagulant venoms that
facilitate prolonged feeding by maintaining a constant flow of blood, alongside other actions (Cabezas-
Cruz and Valdés 2014; Low et al. 2013). In addition, parasitoids present an interesting situation wherein
the predation is not by the organism that injected the venom but by its offspring at a later date. In Asobara
parasitoid wasps, the venom acts to paralyze the host during egg laying by the wasp before killing it at a
later date (Moreau et al. 2009) – ensuring a stationary and storable food source for the larvae when they
hatch. More detailed transcriptomic work on the parasitoid wasp Nasonia vitripennis has revealed that the
venom of this species induces a variety of changes to gene expression in the host (Martinson et al. 2014).
The venom of this wasp causes the host to enter developmental arrest and also upregulates certain
antimicrobial peptides that likely help to prevent spoilage of the (live but immobile) host until the larvae
hatch out and consume the host.
Defense
Aside from predation, defense is the most common primary function for venoms, especially antipredator
defense. Furthermore, as alluded to earlier in the context of venomous snakes envenomating potential
predators, many (probably most) venomous species will use their venom in a defensive role even if the
main role is, for example, predation. However, several groups of animals, such as bees and sea urchins,
use venom primarily for defense, and many others (such as spitting cobras) regularly use venom for both
defense and another function. In spite of this variation in functional importance, there are some gener-
alities that can be made for defensive venoms when compared to predatory or other venoms.
Firstly, defensive venoms tend to be simpler in composition than predatory venoms (Casewell
et al. 2013), likely because the latter are involved in more intense arms races which generates selection
for diverse and fast-evolving venoms (see later section on “Antagonistic Coevolutionary Interactions Are
the Common Thread in Venom Evolution”). Secondly, defensive venoms are more likely to have evolved
to be effective at a distance, such as spitting cobras or spraying behavior of certain scorpions (Nisani and
Hayes 2015). This enables the venomous animal to defend itself while keeping away from the predator but
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requires chemical components which can penetrate external surfaces (mostly eyes or mucous mem-
branes). Thirdly, defensive venoms are likely to contain toxins which interfere with fast-acting physio-
logical processes such as nerve transmission – because lengthy delay in actions can give the predator
enough time to kill the animal before the venom takes effect. Consequently, many defensive venoms
contain toxins which act to cause paralysis quickly by blocking neuromuscular receptors or to target pain
receptors to cause instant and intense pain (Bohlen et al. 2011; Dutertre et al. 2014; Inceoglu et al. 2003;
Siemens et al. 2006).
Although antipredator defense is particularly well studied (and probably more important), some
venoms are also known or suspected to contribute toward immune and antiparasite defense. For instance,
in some social hymenopterans, the venom is spread over the cuticle of other individuals and the nest
combs, and appears to reduce the prevalence of infections via antimicrobial venom components (Baracchi
et al. 2011). In others, they actively apply venom to fungus-infected group-mates which helps eliminate
the fungus (Tragust et al. 2013). Similarly, Grow et al. (2015) have shown that the venom of slow loris
species (Nycticebus) is effective in killing arthropods that are similar to those which parasitize them, and
that lorises anoint themselves with the venom. However, it is difficult to know to what extent the venom is
transferred to the microbes or parasites per se, and therefore it is debatable whether such uses would be
considered as those of venom (even if the same secretions function as a typical venom in other
circumstances). The alternative is to consider the same substance both as a venom and as a contact
poison, depending on the use at any one time.
Intraspecific Competition
Few venoms seem to have a prominent function in intraspecific competitive interactions, but this is known
in a few mammal species, namely the platypus (Ornithorhynchus anatinus) and slow loris species
(Nycticebus). It is notable that both of these groups also use their venom in defense and possibly other
functions but that they nevertheless use venom to a large extent for competition. Platypus venom glands
increase in size during the breeding season in males, and scars from envenomations are usually found in
males, both of which highlight the predominance of intrasexual competition for females as a driving force
in the evolution of their venom (Whittington et al. 2009). Echidnas (Tachyglossus and Zaglossus) were
once thought to also be venomous and indeed possess similar glands to the platypus, but their “venom”
system is highly degenerate and the secretions now seem to function in scent communication during the
breeding season rather than as venom (Wong et al. 2013). Slow loris venom appears to also be used in
intraspecific competition based on the frequency, patterns, and consequences of bite wounds on wild
lorises as well as observations in captivity (Nekaris et al. 2013), though this is less well studied than in the
platypus.
Reproduction
A potentially unique venom function is found in scorpions of the genus Hadogenes. These species are
extremely reluctant to use venom in either predation (relying on their pedipalps) or defense, but during
courtship males will sting females in the side, which seems to produce sedative and perhaps aphrodisiac
effects (Leeming 2003). Other scorpions have occasionally been seen stinging during courtship, but
similar behavioral responses are not observed, and Hadogenes also possess marked sexual dimorphism
wherein males have much longer tails which facilitates this behavior. Therefore, Hadogenes represents an
interesting genus for studies of sexual selection (including sexual dimorphism of venom apparatus), toxin
evolution, and potentially a source of new pharmaceutical drugs given that the unique function may be
associated with unique toxins. However, the venom of the genus has been extremely understudied and
almost nothing is known about the details.
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Sauropods
Nonavian_theropods
Birds
Ornithischians
Crocodiles
Toxicoferans
Other_squamata
Tuatara
Turtles
Monotremes
Marsupials
Bats
Ungulates
Carnivores
Insectivores
Rodents
Primates
Caecilians
Salamanders
Frogs
Lungfish
Cod
Gobies
Scats
Stargazers
Sunfish
Surgeonfish
Bass
Rabbitfish
Weeverfish
Groupers
Scorpionfish
Stonefish
Stickleback
Perch
Gurnard_perch
Mackerel
Pufferfish
Carangoids
Toadfish
Anglerfish
Cichlids
Blennies
Sturgeons
Sharks_and_rays
Lampreys
Hagfish
Tunicates
Feather_stars
Brittle_stars
Starfish
Sea_cucumbers
Sea_urchins
Nematodes
Tardigrades
Velvet_worms
Mantis_shrimps
Decapods
Remipedes
Silverfish
Odonates
Mantids
Cockroaches
Phasmids
Orthopterans
True_bugs
Beetles
Hymenopterans
Lepidopterans
Fleas
Flies
Other_crustaceans
Centipedes
Millipedes
Horseshoe_crabs
Spiders
'Whipscorpions'
Mites
Ticks
Scorpions
Pseudoscorpions
Camel_spiders
Harvestmen
Bivalves
Gastropods
Cephalopods
Chitons
Annelids
Cnidaria
Sponges
Fig. 1 Phylogenetic tree of animals highlighting groups containing venomous species in red. Taxa were selected solely to
show the diversity of venomous animals, not to give an overview of animal diversity in general. Topology is based on the Tree
of Life Web Project (www.tolweb.org) in addition to many studies from the literature which were used to resolve uncertainty in
the ToL project phylogeny
Taxonomic Distribution of Venomous Animals

Venom is a trait which has evolved multiple times across the animal tree of life (Fig. 1), a testament to the
myriad benefits it confers and corresponding selection pressure to originate and maintain venom systems
across diverse groups of animals (Fig. 2). It should be noted that Fig. 1 is a substantial underestimate of the
number of times venom has evolved in animals as it only represents broad taxonomic groups, within
which venom may have evolved more than once. It is also likely that future research will uncover more
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Fig. 2 Examples of venomous animals. From left to right in each row: young huntsman spider (Heteropoda sp.), blue leg
centipede (Scolopendra mirabilis), adder (Vipera berus) (row 1); unidentified squid from Thailand, flat rock scorpion
(Hadogenes troglodytes) (row 2); nursery web spider (Pisaura mirabilis), unidentified tick from Uganda, assassin bug
nymph (Rhynocoris sp.) (row 3); stony coral (Favia sp.), cuttlefish (Sepia officinalis), unidentified wasp from Uganda (row
4); leaf-cutter ant (Atta cephalotes), beadlet anemone (Actinia sp.), Komodo monitor (Varanus komodoensis) (row 5). (All
photos by the author)
groups of venomous animals because many groups are understudied and research effort is directed toward
medically relevant species rather than an objective survey of animal life.
Although the focus of this chapter is on venomous animals, it is noteworthy that venom is not restricted
to animals. Other groups of organisms more commonly use other forms of chemical defenses, but venom
is used by taxa as diverse as plants and bacteria. For instance, plants in the family Urticaceae have stinging
hairs which pierce the skin of mammals and inject pain-inducing venom which deters herbivores
(Iwamoto et al. 2014). In the case of the stinging tree (Dendrocnide spp.), the pain has reported to be
excruciating and has caused deaths of domestic animals and humans (Hurley 2000). One species of
bacteria, Photorhabdus luminescens, has so far been reported to have a toxin delivery system which
would qualify it as truly venomous, although the diversity of bacteria and the lack of detailed study of
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most mean that it is likely that more examples will be revealed in the future. In P. luminescens, a “syringe
protein” penetrates the cells of an insect host and transfers a toxin molecule through the pore of the
“syringe” into the insect cells (Gatsogiannis et al. 2013).
Cnidarians
All groups of cnidarians (including jellyfish, corals, and sea anemones) use venom in either predation or
defense (Fig. 2). This is produced in specialized cellular structures, called nematocysts, and is injected
into prey or predators using a harpoon-like venom apparatus that is extremely sensitive and readily fired
upon contact (Fautin 2009). The venom contains a number of neurotoxins which can cause pain and rapid
death from respiratory paralysis (Edstrom 1992) – the latter mostly in prey, but some species are capable
of delivering lethal envenomations to humans and other predators. Of additional interest is the ability of
some specialist predators of cnidarians, especially nudibranch mollusks, to extract nematocytes intact
from eaten cnidarians (without discharging them), transport them to their own skin surface, and employ
the “stolen” venom system in their own defense (Greenwood 2009).
Annelids
Two of the three major groups of annelid worms possess venom – the exception is oligochaete worms
(such as earthworms) which are not known to have any venomous species. On the other hand, blood-
feeding leeches possess venom with similar functional characteristics to many other parasitic animals.
Specifically, the venom contains toxins which prevent blood clotting and therefore maintain sufficient
flow to enable prolonged feeding and also suppress inflammation, pain, and other immune responses in the
host to prevent detection of the leech and removal before feeding is complete (Hildebrandt and Lemke 2011).
The third major group of annelids, polychaete worms, have evolved a range of venom systems and so
presumably have also evolved venom multiple times. Among the best studied of these are bloodworms
Glycera spp., which feed on other invertebrates and inject a neurotoxic venom with their jaws which
quickly immobilizes prey and can be used to give a painful bite in defense (Edstrom 1992). Other
polychaetes, such as the various species known as “scale worms,” are considered to have a venomous bite
but are very poorly known (von Reumont et al. 2014a). In addition to species which inject venom using
mouthparts, other polychaetes (e.g., Amphinomida or bristle worms) have evolved a venom apparatus
consisting of fragile spines (actually modified chaetae) which function defensively by breaking off when
touched, causing a wound into which venom is delivered (von Reumont et al. 2014a). This group of
worms feeds on slow-moving or sessile prey, and this, combined with the position and mechanism of
venom delivery, restricts the benefits of this venom to antipredator defense.
Mollusks
Venom in mollusks is largely restricted to two of the major groups: gastropods and cephalopods (Fig. 2).
However, as mentioned above in the subsection on “Cnidarians,” nudibranch mollusks can reallocate the
nematocysts of their cnidarian prey to their own surface and use it in defense. This is a highly unusual
form of sequestration of chemical defenses, which usually entails the extraction of particular toxins or
toxin precursors from the diet but in this case involves the sequestration of the entire venom apparatus.
Many gastropods (snails) contain venom which is used primarily to aid predation and injected via a
barbed “tooth” (radula) which is thrust into prey. It is also used in defense, and some species of cone snail
(Conus spp.) are capable of lethal envenomation of humans and other predators. Although many groups of
marine gastropods are known to possess venoms (Edstrom 1992; von Reumont et al. 2014a), that of cone
snails is by far the best studied and has generated a vast quantity of literature. Conus spp. venom contains
a number of highly potent neurotoxins which serve to rapidly incapacitate (by respiratory paralysis)
fast-moving prey such as fish, which would otherwise be unavailable to a slow-moving predator such as
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the snails. However, Conus have a dual-purpose venom system which produces separate venoms for
predation and defense and so has presumably faced strong selection pressure for both functions (Dutertre
et al. 2014). It has recently been suggested that in this genus venom originally evolved in worm-eating
species for defense against predatory fish, and this subsequently allowed the shift to hunting mollusks and
fish using the venom for predation (Dutertre et al. 2014), although how or if this also applies to other
venomous gastropods is uncertain.
Cephalopods have long been known to possess venom, but recently it has been revealed that this is
likely to be far more widespread than previously thought, evolving early in the group (Fry et al. 2009c).
Although also used in defense, the venom primarily functions in predation with toxins causing paralysis in
prey organisms such as crabs (Cornet et al. 2014). There may also be some digestive function of the
venom of some species, including Octopus vulgaris, although this is based on more circumstantial
evidence (Edstrom 1992). Blue-ringed octopuses (Hapalochlaena) are famed for being one of the few
cephalopods capable of killing a human, and their venom contains tetrodotoxin, among other compo-
nents. The tetrodotoxin is contained both in the venom and within the body as a defensive poison.
In addition, female blue-ringed octopuses actively provision their offspring with tetrodotoxin to ensure
they have adequate defense early in life (Williams et al. 2011).
Centipedes
The venom system of centipedes features a unique means of injecting venom – the first pair of walking
legs evolved modifications over 400 mya to be used as venom-delivering pincers (Dugon and Arthur
2012) (Fig. 2). This means that, in essence, centipedes do not bite but instead have a venomous pinch. The
venom itself is used as an effective defense, but its evolution has likely been primarily driven by its role in
predation (Yang et al. 2012). The venom contains a complex mixture of toxins with diverse effects, but the
overarching results from a pinch occur in two phases (Undheim and King 2011). Firstly, fast-acting but
short-lived toxins produce pain and hypotension (as well as some muscle paralysis) almost immediately,
which contributes to prey being quickly driven into shock. Secondly, slower-acting toxins produce a pain
which spreads from the pinch site, some breakdown of the skeletal muscle, and cardiac arrest.
Crustaceans
Perhaps surprisingly, given the high diversity of crustaceans (even considered as a paraphyletic group
excluding insects), only one single species has (very recently) been found to have venom. Von Reumont
et al. (2014b) uncovered venom consisting of a paralyzing neurotoxin and a large number of proteinase
and other enzymes from venom glands of Xibalbanus tulumensis – a cave-dwelling remipede crustacean.
Almost nothing else is known about this venom so far, although it is plausible that the venom has evolved
to enable efficient predation in an environment highly conducive to prey escaping if not quickly subdued.
If this is the case, then venom may be genuinely rare in crustaceans for as yet unknown reasons, but von
Reumont et al. (2014a) have expressed an expectation that more venomous crustaceans will be found with
further study, especially in parasitic groups.
Arachnids
Spiders and scorpions are among the first animals that come to mind when thinking about venomous
creatures (Fig. 2). However, the literature on the venom toxinology has been slower to amass than their
cultural impact. Furthermore, at least three other groups of arachnids also possess venom: mites, ticks
(Fig. 2), and pseudoscorpions. A very limited amount of evidence exists to suggest that camel spiders
(or solifugids) may also be venomous (von Reumont et al. 2014a). This would explain the reportedly high
levels of pain from their bites, but currently enough information to confidently consider them venomous is
lacking.
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Recent investigations have provided much insight into the composition of spider venoms – though
interpretations and reviews have often been strongly focused on medical importance to humans (e.g.,
Sannaningaiah et al. 2014). Nevertheless, the primary function of all spider venoms is undoubtedly
predation. Consequently, it can enable spiders to subdue and eat much larger prey than they would
otherwise (Pekár et al. 2014). Although spiders are well known for biting defensively, only ~10–20 of the
50,000 species of spider have a venom capable of causing serious consequences to human predators, and
even infamous species such as the widow spiders (Latrodectus) are remarkably reluctant to bite in defense
(Nelsen et al. 2014b). These observations further suggest that spiders primarily use venom to catch prey
rather than in defense, and the venom often contains specific insecticidal and paralyzing neurotoxins
(Sannaningaiah et al. 2014).
Scorpion venom is not as clearly streamlined into a single function as spider venom. As highlighted
above (in the “Venom Functions” section), scorpions have a dual-function venom incorporating separate
antipredator defense and predatory components. Scorpions have one type of venom (sometimes called
prevenom) which is expelled initially and differs in appearance from the subsequently expelled
venom – in Parabuthus transvaalicus the prevenom is clear, whereas the secondary venom is milky
white (Inceoglu et al. 2003). The first venom type is particularly adapted to instill immediate and severe
pain in mammals as an antipredator deterrent, whereas the second venom type is better adapted for
causing paralysis and death in their insect prey (Inceoglu et al. 2003). This is likely to be at least in part due
to the action of different toxins which act specifically on either mammals or insects (Ochola et al. 2007).
Additionally, one genus of scorpions (Hadogenes, flat rock scorpions) appears to have co-opted its venom
primarily for use in courtship, a potentially unique venom function (see “Reproduction” subheading in
“Venom Functions” section).
All ticks and some mites are blood-feeding parasites, and consequently they use a venom rich in toxins
that interfere with blood to ensure a steady flow during feeding (Andersen 2010). Ticks are known to have
venom components that reduce the immune response by the host to avoid detection while feeding
(Cabezas-Cruz and Valdés 2014). Furthermore, both ticks and mites can induce paralysis in hosts via
their venom, which again likely acts to prevent the host from removing the parasite before it is finished
feeding (Cabezas-Cruz and Valdés 2014; Tomalski et al. 1988). Therefore, in common with other blood-
feeding parasites, ticks and parasitic mites contain venoms which function almost exclusively in promot-
ing this type of feeding ecology.
Pseudoscorpions represent the most understudied arachnids that are known to be venomous. Their
venom system is integrated into their pedipalps, which have a “venom tooth” at the ends which connects
to a duct from the venom gland. As a consequence of the lack of research, there is currently little known
about the venom of pseudoscorpions, but it seems to function in predation and may be a key factor
enabling them to prey on relatively large prey – envenomated insect prey have been observed to be
paralyzed in seconds and dead in minutes (von Reumont et al. 2014a). Furthermore, some pseudoscor-
pions engage in cooperative hunting of (relatively) large prey such as beetles and millipedes, for which the
use of venom by multiple individuals may be necessary (von Reumont et al. 2014a).
Insects
Venom is found in several groups of insect, whether functioning primarily in predation or defense: true
bugs, beetles, hymenopterans, lepidopterans, fleas, and flies. In fleas and (blood-feeding) flies, such as
mosquitoes, the venom contains toxins that interfere with blood clotting and keep a steady supply of blood
flowing, as well as reducing inflammatory and other such responses in the host (Andersen et al. 2007;
Ribeiro et al. 2004). Such effects are typical of other venoms that act to increase ease of feeding in
parasitic animals, which has led to substantial convergence in blood-feeding species (Andersen 2010).
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Predatory (and defensive) venoms in insects are exceptionally diverse, but Edstrom (1992) provides
good discussion of some of these. Venom use in predation (in addition to the blood-feeding flies and fleas)
occurs in true bugs, beetles, and hymenopterans (Fig. 2). In hymenopterans, parasitoid wasps were
highlighted earlier (“Predation” subheading in the “Venom Functions” section), but many nonparasitic
wasps and ants use venom to subdue prey. For instance, most ant families use venom (a few have
evolutionarily lost their venom), and predation is thought to be the main driver of its evolution (Aili
et al. 2014). The venom of wasps and ants quickly immobilizes and/or kills their prey with paralyzing
neurotoxins, and food is normally carried back to the nest (in both solitary and social species) to be
consumed – although some solitary ants will feed where the prey is found.
Bugs represent an interesting example of venomous insects because they are characterized by piercing
and sucking mouthparts (Fig. 2). This has the consequence that the carnivorous (or blood-feeding) species
are prime candidates for the use of venom as their mouthparts are well structured as a venom injection
apparatus. Furthermore, the necessity to feed on liquid food requires that bugs are able to liquidate their
insect (or in the case of some belostomatid bugs, vertebrate) prey before consumption. Therefore, many
bug venoms contain paralytic or otherwise immobilizing toxins alongside some digestive venom com-
ponents (Sahayaraj and Muthukumar 2011; Zibaee et al. 2012).
Although beetles are exceptionally diverse, contain a wide range of chemical defenses, and are known
to have some venomous representatives, venom appears to be either relatively rare or understudied in this
group. Nevertheless, groups such as predaceous diving beetles (Dysticidae) use potent paralytic venoms
to quickly incapacitate prey in their aquatic environment (Formanowicz 1982), which can include large
items such as fish and amphibians. Venomous beetles also contain a diversity of venom systems, which
often includes jaws but also far more unusual strategies. For instance, in the cerambycid long-horned
beetle Onychocerus albitarsis, the antennae have been modified into a sharp and flexible venom injection
system that is used primarily to subdue prey (Berkov et al. 2008).
The use of insect venom in defense is frequent, though only lepidopterans and social hymenopterans
are likely to have had their venom evolution primarily shaped by this function. In social wasps, bees, and
ants, the venom often contains numerous components which cause pain and discomfort to predators, and
in some species (e.g., bullet ants and fire ants), the pain generated can be excruciatingly intense (Aili
et al. 2014; Edstrom 1992). Furthermore, because defensive venom evolution often occurs in social
hymenopterans, multiple stings are frequent and act to increase the magnitude of the symptoms suffered
by the predator.
Although many adult Lepidoptera (butterflies and moths) contain toxic defenses, venom appears to be
restricted to the caterpillar stages. Furthermore, in this group, venom is entirely a defensive strategy as
most species are herbivorous and venom is not known from carnivorous species (see Pierce 1995 for a
review of carnivorous caterpillars). The venom system of caterpillars typically involves extremely fragile
hairs which break off easily upon contact, and venom is transferred from associated glands or specialized
secretory cells (Carrijo-Carvalho and Chudzinski-Tavassi 2007; Edstrom 1992). The venom of caterpil-
lars is relatively poorly known but causes immediate pain and intense irritation, and some species are
capable of interfering with blood systems and causing fatal envenomations in animals as large as humans
(Carrijo-Carvalho and Chudzinski-Tavassi 2007).
Echinoderms
The venom of echinoderms (starfish and sea urchins) has evolved purely for defense – sea urchins are
herbivorous grazers, and starfish feed on sessile or slow-moving organisms, and so there is little or no
additional benefit to be gained from predatory venoms. Although other starfish may be venomous, they
are an understudied group and only the crown-of-thorns starfish (Acanthaster planci) is known to use
venom from dorsal spines in defense. Envenomation by this species causes a broad range of symptoms
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ranging from intense pain, irritation, and vomiting through to more systemic effects of hemolysis and
reduction of central nervous system activity (Lee et al. 2013). In sea urchins, many species have a venom
apparatus contained within specialized structures known as pedicellaria, which are distributed over the
dorsal surface (Edstrom 1992). These are small structures which have three “claws,” each one containing
a venom gland, that are responsible for transferring venom to predators. The venom appears to act mainly
as a neurotoxin, but its function against natural predators is poorly known. The best-known species,
Toxopneustes pileolus, can cause respiratory difficulties in humans (Edstrom 1992). Furthermore, many
sea urchins possess long spines which are often easily broken in the skin of predators, though only a few of
these species seem to have venom glands attached to the spines – the others relying on irritation directly
from the wounds (O’Neal et al. 1964).
Fish
Venom systems are highly diverse in fishes and have evolved multiple times (Smith and Wheeler 2006;
Fig. 1). Representatives of venomous fish are known from both cartilaginous (in both sharks and rays) and
bony fish but across the entire group function purely for defense. A defensive role is particularly important
in species which are relatively slow moving or spend a great deal of time remaining still, and venomous
fish often exhibit such behaviors (Edstrom 1992). The venom system itself typically consists of spines,
though the precise structure varies greatly – compare, for instance, the flat and serrated spine of stingrays
to the needlelike spines of stonefish. The venom of fishes remains poorly known for most species and
varies greatly between species (Smith and Wheeler 2006) but often contains potent neurotoxins which
induce intense pain and respiratory paralysis, as is common for defensive venoms.
Amphibians
Although many amphibians are poisonous or possess other forms of chemical defense, only two genera
of salamanders and two species of frog can be considered to be venomous: the salamanders Pleurodeles
and Echinotriton, and the frog species Corythomantis greeningi and Aparasphenodon brunoi. In the
salamanders, the ribs are sharp-tipped and can be protruded through the skin, piercing poison glands and
coating the ribs in toxins before puncturing the skin of a predator (Heiss et al. 2010). The mechanism of
action remains unknown, but injection of these toxins is lethal to many potential predators including
mammals and other amphibians (Heiss et al. 2010). Recently, Jared et al. (2015) described the use of
venom in the two frog species. In both species, bony spines on the head are used to pierce the skin of
predators and inject a potent venom into a predator. The toxins present causes intense pain, edema, and
visual difficulties. Jared et al. (2015) also suggest that other frogs may possess venoms using similar spiny
delivery systems, but definitive evidence is currently lacking.
Squamate Reptiles
Reptile venoms have received more study than any other venomous animal and consequently have
generated several book-length treatments (e.g., Chippaux 2006; Fry 2015; Mackessy 2009), largely
focused on molecular toxinology and clinical implications. Until recently, venom was considered to
have evolved multiple times in snakes and once in Heloderma lizards, but recent work suggests that it has
evolved once, early in the group containing snakes, Heloderma, and Varanus monitor lizards (Fry
et al. 2006) which has subsequently been called “Toxicofera” (Fig. 2). However, it should be noted that
some authors have challenged this idea and contend that the traditional view of multiple origins of venom
in reptiles is correct (see Mulley et al.’s chapter in this volume for further discussion). Assuming a single
origin, several lineages have subsequently lost venom due to alternative adaptations that lessen the benefit
of venom (Fry et al. 2013). Because almost all squamate (snake and lizard) venoms are primarily used for
predation, the factors involved in the loss of venom tend to be related to alternative means to catch and
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subdue prey, such as constriction, or alternative diets, such as eggs or leaves, that do not require
subjugation. Because of this tight link of venom to predation, diet is often the major factor influencing
variation in squamate venoms (e.g., Daltry et al. 1996). Nevertheless, some groups have also had their
venom evolution driven substantially by defensive function, such as spitting cobras (Young et al. 2004)
and likely Heloderma (Beck 2005).
Archosaurs
The archosaurs (including crocodilians, dinosaurs, and birds) were traditionally considered a completely
non-venomous group of animals. However, recently Gong et al. (2010) proposed that the dromaeosaurid
(theropod) dinosaur Sinornithosaurus (and perhaps also other “raptors”) was probably venomous based
on the characteristics of its skull and teeth and used their venom primarily for predation. Although
absolutely conclusive evidence for venom is difficult or impossible to retrieve from the fossil record,
Gong et al. (2010) make a convincing argument that it was likely possessed in this and perhaps other
dinosaurs in the “raptor” lineage (Dromaeosauridae).
Interestingly, this dinosaur was closely related to the early ancestors of birds, and yet despite their
diversity, no birds are known to be venomous. Given the frequency of venom in other similarly diverse
groups, that birds possess a number of hard parts (such as beaks and talons) that could facilitate venom
delivery, and that some birds have used other toxic defenses such as poison, it is perhaps surprising that
none have been found. There are many potential explanations for this, although it is difficult to discount
chance as the reason. It may be that the extra weight of venom apparatus would be disfavored in flying
animals or that the additional energetic expenditure of producing venom is too costly in addition to that
needed for flight and high metabolism. Alternatively, it could be that most birds feed on small prey and
have flight as an effective defense, therefore removing selection for venom for these two functions
(although an advantage in intraspecific competition could still be beneficial for many species).
Mammals
Venom has evolved at least four times in mammals and probably more due to multiple origins within these
four groups: platypuses, vampire bats, slow lorises, and insectivores. There is also some fossil evidence
that some extinct mammals were also venomous (Fox and Scott 2005). Therefore, venom is rare but
taxonomically dispersed in mammals. Vampire bats share similar venom characteristics with other blood-
feeding animals, with toxins acting to maintain blood flow (via anticoagulant effects and vasodilation) and
avoid disturbing (in this case waking) the host by reducing pain and inflammation (Ligabue-Braun
et al. 2012; Low et al. 2013).
There are two mammals which use venom for intraspecific competition in addition to defense:
platypuses (Whittington et al. 2009) and slow lorises (Nekaris et al. 2013). Although sharing function,
these two venoms seem to act in different ways. Slow loris venom causes pain, inflammation, and
dramatic tissue destruction in conspecifics (Nekaris et al. 2013), which may give a longer-lasting
advantage to the inflicting male or may simply be a corollary of the suspected multifunctionality of
slow loris venom. The venom system is highly unusual in that the venom is formed by mixing two
nontoxic fluids, brachial gland secretion from near the elbow and saliva, which combine to form a toxic
venom which is injected by biting (Nekaris et al. 2013). In platypuses, the venom is less destructive but
still inflicts a great deal of pain and inflammation (Whittington et al. 2009). The venom is delivered via
spurs on the hind legs, though there are sex and seasonal differences in the venom system. Female
platypuses lose the spurs early in life and generally have a degenerate venom system; males keep the spurs
but the venom gland only becomes highly active during the breeding season (Ligabue-Braun et al. 2012).
These changes strongly suggest that intraspecific competition is the main driver of venom evolution in
platypuses, even if it is also occasionally used in defense.
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Several groups of insectivores have either been demonstrated or suspected of using venom to assist
predation. At least three shrews (Blarina and Neomys) are known to be venomous, as are solenodons, all
of which have venom glands in the lower jaws and transfer venom to prey via a bite, and there is some
evidence that other shrews are similar (Ligabue-Braun et al. 2012). Moles are also strongly suspected of
being venomous as they have similar glands in the lower jaw and are known to store paralyzed worms in
burrows (Ligabue-Braun et al. 2012). It seems to be generally the case for insectivores that the venom
does not usually kill prey but rather immobilizes it in a live but paralyzed state, in which it is stored for
later consumption.
Evolutionary Drivers of Variation in Venoms Between and Within Species

Venoms are highly variable both between species and within a single species. The evolutionary causes of
this variation will vary depending on the primary function(s) of the venom, and the extent of the variation
will partly depend on any constraints acting on the system. Although this section will focus on the
generation and maintenance of variation, it is worth mentioning the influence of convergent evolution in
constraining diversity of toxins. Convergence is a common theme in venom evolution (e.g., Casewell
et al. 2013; Fry 2015) and can be seen at two levels.
The first is at the level of individual toxins, wherein the same protein structures are repeatedly altered to
function as toxins across the animal kingdom (Fry et al. 2009a, c). This is likely a consequence of a
combination of similar basic protein structures being available as body proteins to many different animals,
therefore the raw materials are similar before toxin evolution, and that toxin evolution by small alterations
of particular physiologically active molecules is likely to be easier to achieve as the molecule is already
adapted to interact with physiological systems.
The second level that convergence can be seen is in whole venoms, by which the author means that
venom in some form has evolved repeatedly a large number of times across the animal kingdom (Fig. 1).
This level could be extended to the consideration that venom functions have also evolved convergently
throughout the animal kingdom, especially predatory and defensive venoms (but use in intraspecific
competition has evolved at least in monotremes and primates).
It should also be noted that venom is an energetically expensive product (McCue 2006) as so selection
would be expected to act to optimize the cost-benefit ratio of the functions for which it is used. Therefore,
venom evolution has followed a complex path of diversification and convergence which has shaped the
observed variation in animals.
Interspecific Variation
The chemical composition of venom often varies remarkably between even closely related species. For
predatory venoms, different species may have different diets, and this may drive divergence in the venom
of each as it increases the efficacy of the venom toward that species’ particular prey (Barlow et al. 2009). It
is highly likely that such dietary shifts are the main selection pressure driving variation in predatory
venoms as there are numerous observations of venom systems degenerating (presumably to save energy)
when diet changes make venom unnecessary (e.g., Fry et al. 2013).
For defensive venoms, there is little clear evidence that different predator communities drive differ-
ences in venom. However, this would be difficult to obtain for multiple reasons. Firstly, it is often
unknown what the actual predator community is in a given area for a given venomous prey species.
Secondly, predators are likely to be attacking multiple prey species, and perhaps multiple venomous prey
species, and so attributing changes to a particular predator is difficult. Thirdly, venoms (including
defensive venoms) are often effective against a wide range of predators so even if the predator community
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were fully replaced, the same venom may still be effective. In other chemical defense systems, it seems
that the defense is readily gained but difficult to lose which suggests that there are strong and general
individual benefits (Arbuckle and Speed 2015). Furthermore, natural enemies such as predators are
expected to impose strong selection on defenses, and therefore it is likely that predators do drive variation
in defensive venom, but it may be at a broader scale that is typically examined.
Competitive and reproductive venoms are so poorly understood that the drivers of their variation between
(or within) species are unknown. For venoms with a reproductive function, we may expect that sexual
conflict is strong as males chemically manipulate females and females may be selected to resist this.
Consequently, we might expect that variation in venoms between closely related species using venom in
reproductive interactions is much higher than otherwise expected, but this remains to be investigated.
Finally, environmental drivers of interspecific variation in venoms are understudied (excepting prey
choice and availability). However, we might expect, for example, that predatory venoms may be more
potent in species that hunt in environments where prey may escape out of reach unless venom takes effect
especially quickly, such as in slow-moving aquatic predators or those hunting in dense habitats or where
prey can escape to burrows.
Intraspecific Variation
The same drivers of interspecific variation may also drive intraspecific differences between populations,
but there are other considerations that are specific to the latter. However, many of these are not
evolutionary in origin. For instance, variation in venom can be a consequence of amount of energy
available to an individual for toxin manufacture, or time since last envenomation as venom supplies need
to be replenished (and different toxins may regenerate at different rates).
Other causes of intraspecific variation may be a consequence of evolution. For instance, sex differences
in venom may reflect sex differences in diet or predation risk (e.g., in Bothrops jararaca; Furtado
et al. 2006), in which niche partitioning between sexes leads to feeding on different prey types and
consequent shifts in venom, increasing variation within the species. Similarly, age-related variation may
be a consequence of diet or predation differences coupled with a smaller venom yield in smaller
individuals (e.g., in Crotalus spp.; Furtado et al. 2003; Mackessy 1988). This situation allows young,
and therefore small, individuals to possess a relatively more effective (e.g., higher toxicity) venom that
could offset the low venom yield available to secure prey or repel predators.
Finally, in predatory venoms, prey populations or communities may change over time. This could
conceivably generate selection on venomous predators to have a quick evolutionary response in their
venoms, leading to increased mutation rates in venom genes compared to other genes (either in coding or
regulatory regions controlling expression of different components). This situation would lead to high
variation in venoms within a species, and within populations, despite the selection acting on evolvability
rather than favoring the increased variation per se. Nevertheless, evidence for this scenario is currently
lacking and remains a mere possibility, though if true it could provide an additional explanation for many
toxins being part of multigene families.
Antagonistic Coevolutionary Interactions Are the Common Thread in Venom

Evolution
All venoms have evolved in the context of antagonistic coevolution. Despite the massive diversity of
venoms, venom delivery systems, venom functions, and venomous animals, this is one key point which is
applicable throughout. The idea of the “arms race” is well known in natural enemy interactions such as
predator-prey and host-parasite systems (e.g., Endler 1991) but is less well appreciated as a core concept
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in venom evolution throughout the tree of life. The antagonists may be traditional natural enemies (e.g.,
predator-prey, host-parasite) as in the case of predatory and defensive venoms or less traditional (in this
context) such as conspecifics of the same or opposite sex as in the case of competitive or reproductive
venom functions. Nevertheless, because they all represent parties with opposing interests (one side wants
to envenomate, the other wants to avoid envenomation), there are implications for considerations of the
evolution of venomous animals: it never takes place in isolation.
In essence, any evolutionary change in venom – whether gain, loss, or alteration – will impose selection
pressure on another organism to limit or remove the benefits conferred to the venomous animal. This will
both lead to diversification of the venom as the arms race forces both parties to continually adapt but also
constrain the advantages that can be gained. A common example of coevolution in the context of
venomous animals is venom resistance. Predators that eat venomous prey have often evolved resistance
to the prey’s venom to enable consumption (Drabeck et al. 2015). Similarly, prey that are eaten by
venomous predators often have a high level of resistance to the predator’s venom (Heatwole and Poran
1995; Heatwole and Powell 1998). In some cases, prey species are more resistant to the venom of their
predators than to other venomous predators that do not eat that species – a result that would be unintuitive
without an appreciation of coevolution. This concept is taken further by nudibranch mollusks which feed
on venomous cnidarians but are able to not only avoid envenomation but extract the entire venom system
from the cnidarian and transport it to the nudibranch’s surface for its own defense (Greenwood 2009).
Ecological and Evolutionary Consequences of Venom (and Other Chemical

Defenses)
Natural enemy interactions are expected to lead to phenotypic divergence and evolutionary diversification
in organisms, as a consequence of the coevolutionary arms races that they fuel (Ehrlich and Raven 1964;
Vamosi 2005). In essence, the expectation is that effective antipredator defenses should lead to a greater
freedom of movement (without having to be as cautious about potential predators) and hence occupy a
broader niche space. The broader niche space may allow more opportunities for diversification, and the
arms race itself is predicted to generate evolutionary diversification in such scenarios (Ehrlich and Raven
1964) – a phenomenon known as “escape-and-radiate” theory.
Note that these predictions stem from theories of defense but should apply well to venomous animals
since most use venom either primarily or secondarily in defense. Unfortunately, little research effort has
been focused on the evolutionary and ecological consequences of possessing venom in animals, and so
this discussion will borrow from the literature on other chemical defenses. However, because the
predictions are based on the interplay between the predators and the repellent nature of the defense, the
response should be similar in many cases across specific forms of defense.
Higher predation risk (and correlates of higher predation risk) tends to favor the evolution of effective
defenses, but the consequences to ecology and life history of the animals after the evolution of venom are
less well known. Two general points are of particular interest though. The first is that chemical defense
does seem to be associated with a broader niche space, as predicted above (Arbuckle et al. 2013).
Specifically, in musteloid mammals (a group including skunks, badgers, and otters), those that use
repugnant anal gland secretions in defense had a less constrained activity period and a broader diet.
The second point is that chemical defense is associated with slower life histories including such traits as
longer life span (Hossie et al. 2013) – although venoms used primarily for predation did not show the
same pattern of longevity, suggesting specificity to defensive venoms. This is expected based on
evolutionary theories of the life history of aging that predict slower senescence and generally slower
life histories in species with lower extrinsic mortality, such as from predation (Blanco and Sherman 2005).
Page 17 of 23
DOI 10.1007/978-94-007-6727-0_16-1
Few empirical tests of the prediction from “escape-and-radiate” theory, that chemically defended
animals should have higher diversification rates, have been conducted. Recently, Arbuckle and Speed
(2015) investigated this idea using amphibians and found that chemically defended lineages actually had
lower diversification rates, due to an increased extinction rate. The raised extinction rate was also
observed in present-day amphibians Arbuckle (2015), wherein chemically defended species are
more likely to be threatened (based on IUCN Red List conservation status) than non-defended species.
The most plausible mechanism to explain this is that because chemically defended species should have
slower life histories, they should be less resilient to population declines due to slower rates of subsequent
population increase.

Venom has evolved frequently across the tree of life and is consequently found in many disparate groups
of animals. The benefits obtained by venomous animals are most often related to enhancing prey capture
or avoiding attack by predators but can include other aspects of biology such as competition and
reproduction. These functions are not mutually exclusive, but all take place in the context of antagonistic
coevolutionary interactions – perhaps the one comprehensive rule of venom evolution. Venom displays
extensive variation both within and between species, which can be driven by various processes relating to
the functions of the particular venom. Finally, the evolution of venom, especially as a defensive trait, can
have important long-term consequences for the ecology, evolution, and conservation of venomous
animals.
Throughout this chapter, many gaps in our knowledge have been highlighted. However, perhaps the
most promising for future work falls into the following two areas. Firstly, there are many venomous
animal groups which have been given very little attention, particularly among the invertebrates, and
directed research into those groups would provide insights into the evolution and diversity of venom, as
well as uncover novel toxins which could potentially yield a multitude of new pharmaceutical products.
Secondly, the macroevolutionary consequences of venoms have been almost ignored until very recently,
yet provide an opportunity to understand how venomous animals originated and how their future is likely
to play out. These areas are likely to be extremely fruitful for further investigation.
Cross-References
▶ Evolution of Resistance to Toxins in Prey
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Page 23 of 23
Functional and Genetic Diversity of Toxins
in Sea Anemones
Marymegan Daly
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Diversity and Phylogeny of Actiniarian Sea Anemones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Diversity and Evolution of Structures and Behaviors Associated with Venom in Sea
Anemones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Diversity and Evolution of Toxins Within Sea Anemones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Voltage-Gated Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Cytolytic Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Other Venom Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Abstract
Sea anemones are benthic, sessile cnidarians that use venom for prey capture,
defense, digestion, and intraspecific competition. Lacking venom glands, sea
anemones produce venom locally in the tissue of use and deliver it via subcellular
structures called nematocysts. The majority of venoms characterized from anem-
ones are unique to the lineage. Although there are many components of venom
that are only known from particular lineages, these are generally not associated
with structures that are unique to those lineages. The few kinds of venoms that
have been explored in an evolutionary context appear to evolve under negative
selection, although positive selection may occur on select residues within the
molecule. Because there is a positive relationship between study effort and
number of toxins known from any lineage, it is likely that broader taxonomic
M. Daly (*)
Museum of Biological Diversity, The Ohio State University, Columbus, OH, USA
e-mail: marymegandaly@gmail.com; daly.66@osu.edu

2 M. Daly
representation in studies of anemone venom will increase the number of genes

and molecules reported from anemones.
Keywords
Cnidaria • Actiniaria • Cytolysins • Voltage-Gated Channel Toxins
Introduction
Cnidaria is the earliest-diverging metazoan lineage in which venoms have evolved

and is the only phylum-level lineage for which venom and venom-delivery struc-
tures are synapomorphic (Daly et al. 2007). In addition to being the oldest venomous
lineage, Cnidaria is the lineage showing greatest diversity in the known uses of
venom, which is critical to prey capture, digestion, and intraspecific aggression
(Doumenc and Van Praët 1987). Among cnidarians, venom is best characterized in
sea anemones (Cnidaria: Anthozoa: Actiniaria). Sea anemone venoms have gener-
ally been studied one species at a time, with an emphasis on the toxicology and
structure of the venom proteins. Recovering the complete venom and understanding
the genotype-to-phenotype relationship in the multigene venom phenotype is espe-
cially complicated for sea anemones and other Cnidaria because there is not a single
kind of venom or a single locus of venom production (e.g., a venom gland); venom is
produced by the nematocysts and adjacent secretory cells within tissues (Basulto
et al. 2006; Moran et al. 2011).
The evolution of venom in sea anemones can be understood through consider-
ation of the evolutionary history of the lineages in which it occurs, of the ways in
which it is used, or of the molecules themselves. These avenues are not independent
but nonetheless provide different insights into the diversity and evolution of the
complex venom phenotype in sea anemones.
Diversity and Phylogeny of Actiniarian Sea Anemones
Cnidarians are epithelial animals that produce cnidae, tiny intracellular capsules
made of protein and collagen (Daly et al. 2007). The tubule, lying introverted within
the capsule, everts upon stimulation; this action leads to a chemical or mechanical
interaction in which the tubule punctures or entangles predators, prey, or debris.
Cnidae vary across the phylum in terms of the anatomy of the capsule and tubule and
have been classified as nematocysts (double-walled capsule, tubule penetrating or
entangling, often containing venom), spirocysts (single-walled capsule, tubule
entangling), and ptychocysts (single-walled capsule, tubule entangling) (Mariscal
1974). Nematocysts are produced by all cnidarians in cells called nematocytes.
Spirocysts and ptychocysts are produced by spirocytes and ptychocytes, which are
unique to subsets of Anthozoa. Although all cnidae are inferred to have a single
origin, the relationship among the various kinds of capsules is unclear (Reft and Daly
Functional and Genetic Diversity of Toxins in Sea Anemones 3
2012). Nematocysts are assumed to be critical to the delivery of venom in cnidarians

and clearly contain some venom within the capsule (Balasubramanian et al. 2012;
Moran et al. 2013), but nematocytes are not the only kind of cell in which toxins are
made (Basulto et al. 2006; Moran et al. 2011).
Cnidaria comprises two reciprocally monophyletic groups: Anthozoa and
Medusozoa (Zapata et al. 2015). Medusozoa contains four monophyletic lineages
which have each been ranked as a class (Cubozoa, Hydrozoa, Scyphozoa, and
Staurozoa). Anthozoa contains two daughter clades, Octocorallia and Hexacorallia;
these are generally recognized as monophyletic, although the relative position of the
hexacorallian order Ceriantharia has been problematic and some analyses place it as
a sister to Hexacorallia and Octocorallia or even outside of the two primary groups
within Anthozoa (reviewed in Rodríguez et al. 2014; Zapata et al. 2015). Although
Anthozoa has historically been accorded status as a class, the diversity within it and
the hierarchical structure of the group relative to that of Medusozoa have been
offered as arguments for recognizing Hexacorallia and Octocorallia as classes rather
than subclasses (Daly et al. 2007; Zapata et al. 2015).
Octocorals and hexacorals share the anthozoan attributes of being exclusively
polypoid, having the mouth project into the gastrovascular cavity as an
actinopharynx, and having sheets of endodermal tissue (mesenteries) that project
from the body wall into the gastrovascular cavity. Octocorals generally have eight-
part symmetry, with eight pinnate tentacles and eight internal mesenteries. Despite
their name, hexacorals are varied in their symmetry, although 12 is the common
denominator for the organization of tentacles and mesenteries in the majority of its
lineages. Hexacorals are more diverse in polyp anatomy than any other lineage in
Cnidaria and so have been hard to circumscribe (Daly et al. 2007), but they can be
diagnosed by the shared production of spirocysts, the single-walled, entangling
cnida. Diversity within hexacorals has historically been organized based on differ-
ences in polyp anatomy and skeletal production (Daly et al. 2007; Rodríguez
et al. 2014). Analysis of DNA sequences has confirmed that the majority of the
ordinal distinctions are robust (Rodríguez et al. 2014), although the discovery of new
lineages remains a possibility in Hexacorallia (as for many other marine groups:
Appeltans et al. 2012).
Sea anemones belong to the hexacorallian order Actiniaria. This group contains
only solitary, sessile, benthic polyps. They are distinguished from other solitary and
soft-bodied hexacorals in lacking the signature attributes of those other orders: they
lack ptychocyst cnidae and have tentacles and mesenteries arranged unlike those of
members of Ceriantharia, lack the distinctive arrangement of mesenteries seen in
members of Zoanthidea, and have musculature and nematocysts that differentiate
them from members of Corallimorpharia. Despite the challenge of defining
Actiniaria relative to the other orders based on anatomical features, DNA sequence
data are unambiguous in finding Actiniaria to be monophyletic with respect to these
groups (see Rodríguez et al. 2014).
Sea anemones are exclusively marine, inhabiting oceans at all depths and lati-
tudes. They are among the most conspicuous inhabitants of marine ecosystems,
where they play critical roles in benthic-pelagic coupling and, through their
4 M. Daly
Fig. 1 Diagrammatic representation of sea anemone anatomy, highlighting structures relevant to

the use of venom. The polyp is shown in longitudinal section. Tissues in grey are common to all
anemones. Those in red (acrorhagi) are seen only in members of Actinioidea; those in green
(acontia, catch tentacles, cinclides) are seen only in members of Metridioidea
symbiosis with photosynthetic unicellular organisms, primary production (Shick

1991). Like all cnidarians, sea anemones have bodies that are two cell layers thick,
with no organs and no centralized nerve, sensory, circulatory, or osmoregulatory
apparatus. However, among cnidarian polyps, sea anemone bodies are complex in
having specialized structures associated with digestion, reproduction, defense, and
competition (Fig. 1). Many of these structures are defined by a specialized comple-
ment of nematocysts (Doumenc and Van Praët 1987). All of the nematocysts in sea
anemones are “stommocnidae” (Mariscal 1974), with a hollow tubule that is inferred
to penetrate the skin of the victim. The structure of the tubule within the nematocyst
capsule, and thus the way in which the nematocyst is categorized, varies across
tissue, ecology, and lineage. There is considerable species-level diversity in the
structure of the tubule and thus in the “type” of nematocyst within any tissue, but
at a broad-scale, the cnidom of each lineage within Actiniaria is similar, including
spirocysts, basitrichs, holotrichs (= atrichs), microbasic b-mastigophores, and
microbasic p-mastigophores (Rodríguez et al. 2014).
Relationships within Actiniaria as detected by DNA or modern analyses of
morphological characters do not accord with the divisions of the traditional classi-
fication. The order has been revised (Rodríguez et al. 2014) so that taxonomic
divisions correspond to phylogenetic relationships (Fig. 2). Thus, the primary
Fig. 2 Phylogenetic relationships among major lineages of sea anemones (After Rodríguez
et al. 2014) with the reported occurrence of venoms. Venoms have been associated to the most
general group possible and may not occur in all members of a lineage; for example, type I cytolysin
is known from only a subset of Actinioidea. Preliminary evidence suggests that Type II and IV
cytolysins may be common to both Actinioidea and Metridiodea (see text); these have not been fully
characterized or analyzed. Given the current state of knowledge, the localization of each of these is
subject to change; in particular, those venoms that are associated with the common ancestral lineage
for Actinioidea + Metridioidea may be shared by all Enthemonae. The occurrence of some kind of
NaTx in all anemones suggests that the venome of the common ancestor to all sea anemones
included NaTx, but this ancestral molecule likely does not fit into the existing scheme of classifi-
cation for NaTx
division within the order is between the Anenthemonae and Enthemonae.

Anenthemonae is the less species-rich of the suborders, containing members of
families Actinernidae, Edwardsiidae, and Halcuriidae; the model organism
Nematostella vectensis is the most familiar and well-studied member of this group.
6 M. Daly
Enthemonae contains the overwhelming majority of species and anatomical diver-

sity within Actiniaria and is further subdivided into three superfamilies, Actinioidea,
Actinostoloidea, and Metridiodea (Fig. 2). These correspond roughly to the tradi-
tional divisions between Endomyaria, Mesomyaria, and Acontiaria, respectively,
although the exact composition of Actinostoloidea and Metridioidea differ some-
what from the circumscriptions of Mesomyaria and Acontiaria.
Diversity and Evolution of Structures and Behaviors Associated

with Venom in Sea Anemones
Venom is fundamental to the biology of sea anemones. It is part of feeding, defense,

and intraspecific competition, a broader repertoire than reported for any other
venomous lineage (see e.g., Casewell et al. 2013). Because sea anemones do not
have a central venom gland or venom delivery surface, there is the potential for
functional specialization of venom in particular tissues based on the function of that
tissue. The functional specialization of the sea anemone body is associated with
differences in the size and distribution of nematocysts and many regions of the body
are limited in their function (Doumenc and Van Praët 1987). Although no tissue-
specific venom phenotype (as a protein profile) has been published for any species of
anemone, assessments of venom based on mucus (e.g., Rodríguez et al. 2012), gene
expression (e.g., Orts et al. 2013), and transcriptomes (e.g., Macrander et al. 2015a)
suggest that there are species- and tissue-specific differences in the venom pheno-
type. However, function does not map one-to-one with particular tissues or body
parts, and the absence of information about a toxin within a tissue should be
interpreted cautiously in light of the incompleteness of knowledge about the occur-
rence of venom in sea anemones.
Although they are flexible in the ways in which they obtain nutrition (Shick
1991), sea anemones are fundamentally predatory animals, using their tentacles to
catch prey. Because they lack true muscle tissue, have no visual capacity and lack a
centralized or coordinated nervous system, prey capture relies heavily on toxins to
subdue prey. Adhesion of prey to tentacles relies on spirocysts, cnidae which are
interpreted as adhesive rather than penetrating and which have not been shown to
contain venom (Doumenc 1971). Although spirocysts are the most abundant kind of
cnida in the tentacles of sea anemones, penetrating, venom-bearing cnidae are also
found in tentacles – generally basitrichs and often microbasic p-mastigophores. In
addition to the effect of venoms injected by nematocysts, the same or different toxins
produced by gland cells (Basulto et al. 2006; Moran et al. 2011) probably render the
copious mucus of sea anemone tentacles effective as a paralytic agent.
Venom is also inferred to have a role in the digestion of prey. Like all cnidarians,
sea anemones lack a stomach, using the gastrovascular cavity as a holding space for
prey. Food is digested within the cavity and nutrients are absorbed across the
gastrodermal epithelium. Lacking teeth, muscular gizzards, or any other means of
mechanical degradation of food, cnidarians rely on chemical digestion. Cytolytic
toxins in the tentacles may predigest food. The numerous gland cells and nemato-
cysts (primarily microbasic p-mastigophores, but also basitrichs and microbasic b-
mastigophores) of the mesenterial filaments are inferred to be important in the lysis
of food items. Cytolytic toxins that form pores in the membranes of non-anthozoan
tissues have been identified in the mesenterial filaments (Meinardi et al. 1995;
Basulto et al. 2006) and in the actinopharynx (Moran et al. 2011). Although
ubiquitous in cell membranes of eukaryotes, sphingomyelin is not present in the
membranes of sea anemones; this innovation likely protects sea anemones from self-
digestion (Meinardi et al. 1995). Whether all of these are venoms injected by
nematocysts or simply digestive toxins remains unclear.
Nematocysts and toxins in the mucus also play a role in defense. Tentacles are
used directly in agonistic interactions with potential predators; the venom contained
within or produced by nearby gland cells is inferred to be of primary importance in
these interactions (Hines and Pawlik 2012). Neurotoxins have been identified in
planula larva and juvenile polyp of Nematostella vectensis (Orts et al. 2013) and are
interpreted to have a defensive function. Nematocysts and toxins are key to
protecting photosynthetic microorganisms, fish, and crustaceans that associate with
anemones (Shick 1991; Mebs 2009). The nematocyst complement of sea anemones
that host photosynthetic organisms are not different from that of closely related
species that are aposymbiotic, bolstering the contention that capacity for harboring
photosymbionts is primitive in many lineages (Geller and Walton 2001) and perhaps
a primary function of the tentacle cnidom. For mobile animal symbionts that live on
the outside of the body rather than within the host tissue, the protection afforded by
nematocysts is a double-edged sword. The symbionts may “hide” themselves from
the triggering mechanisms of the nematocysts by behavioral and chemical means,
including coating themselves in the host’s mucus. However, instances of toxin
resistance have been reported in fish that engage in species-specific symbioses
with anemones, and the vulnerability of some fish changes over developmental
time in other cases, suggesting that coevolutionary changes in venom and receptivity
may occur (Mebs 2009).
All modern studies of the interaction between host anemones and their symbionts
to date have focused on host species belonging to Actinioidea (e.g., members of
Stichodactylidae, Entacmaea spp., Condylactis spp.). However, symbioses with
crustaceans are well characterized in several lineages within Metridioidea and in a
few species within Actinostoloidea (Ross 1974; Vader 1983). The means of
circumventing envenomation and the nature of coevolutionary changes in those
lineages are unknown. Laboratory studies of the effects of toxins from Adamsia
palliata and Calliactis parasitica, sea anemones that live in association with hermit
crabs, attest to its efficacy on crustacean targets (Mebs 2009). The toxicity of
anemone venom to crabs underpins the behavior of Lybia edmonsoni, a xanthid
crab that fight conspecifics using sea anemones (Triactis producta) held on their
chelae (Karplus et al. 1998). Although the exoskeleton clearly protects crustaceans
from the host anemones, coevolution may also play some role in the resistance of
symbiotic crustaceans to the venom of their host sea anemone. Early studies by
8 M. Daly
Cosmovici (1925) and Cantacuzene (1925) found that crude extract from the anem-
one had limited or no effects on host species and indirectly implicated crab host
hemolyph as the source of this resistance.
Defensive venom in metridiodeans is produced in the tentacles and column and
also in acontia (Fig. 1), extensions of the mesenterial filaments that can be extruded
through special pores called cinclides or ejected through the mouth. Acontia are
comprised primarily of nematocysts; they also contain secretory cells, but these are
less numerous than nematocysts and relatively less common in acontia than in
mesenterial filaments. They are interpreted to act primarily as defensive structures
but may also play a role in digestion (Shick 1991). Acontia are integral to the
defensive response of the Adamsia, Calliactis, and the other hormathiid hermit
crab anemones: when disturbed, the anemone emits these nematocyst-packed
threads through the cinclides; predators are deterred by the sting that presumably
accompanies contact with acontia (Ross 1971). Acontia emission is generally not
triggered by interaction with the symbiotic crab. Although acontia are likely central
to the protective effect of the anemone association with hermit crabs, they occur
more broadly across Metridioidea and are inferred to be ancestrally present in most
Metridioidea, arising after the split between Triactis (family Aliciidae), Paranthus
(family Actinostolidae), and all other metridiodeans (Rodríguez et al. 2014). Acontia
generally contain different types or sizes of nematocysts than the mesenterial
filament, a difference that highlights the potential for functional differentiation
between the filaments and acontia. In the evolutionary history of Metridioidea,
acontia are inferred to evolve a relatively simpler cnidom through loss of microbasic
p-mastigophores and to have been lost altogether, particularly in lineages whose
members inhabit the deep sea. Innovations in the evolution of acontia include both
loss and novel the incorporation types of cnidae into the acontia, loss of cinclides,
and the loss of acontia (Rodríguez et al. 2014).
Sea anemones are among the small number of venomous animals known to use
venom in agonistic interactions with conspecifics (Casewell et al. 2013). Intraspe-
cific agonism in anemones relies on structures that differ depending on the lineage:
actinioidean sea anemones use novel structures on the column called acrorhagi,
whereas metridioidean sea anemones use modified tentacles, referred to as catch
tentacles (Fig. 1). Both acrorhagi and catch tentacles are used only in agonistic
interactions with conspecifics. Although these structures are not homologous to one
another and each instance of intraspecific agonism is interpreted as a parallel
innovation (Rodríguez et al. 2014), in both Actinioidea and Metridioidea intraspe-
cific antagonism invokes holotrichous nematocysts (reviewed by Bigger 1988).
The anatomical distinctiveness of acrorhagi and their functional limitation to
agonism have made them relatively easy targets for studies of tissue-specific
venom. Early studies of these tissues lead to the discovery and characterization of
acrorhagin, a kind of toxin so far known only from sea anemones (Honma
et al. 2005). Investigation of the acrorhagi of Anthopleura fuscoviridis and
Anthopleura aff. xanthogrammica led to the discovery of novel Kunitz-type protease
inhibitors (Minagawa et al. 2008). A transcriptomic assay of the acrorhagi of
Anthopleura elegantissima recovered two kinds of acrorhagins, plus several genes
that were candidate cytolysins (Type II & IV), phospholipase A2s (PLA2s), voltage-
gated potassium channel toxins (Types I, II, III, IV & V), and voltage-gated sodium
channel toxins (Types I, II & III) (Macrander et al. 2015a).
The venom of catch tentacles has not been studied specifically, perhaps because
these structures are difficult to differentiate from feeding tentacles. However, among
the acrorhagi-expressed cytolysins identified by Macrander et al. (2015a) is a gene
that is highly similar to a gene retrieved from Sagartia rosea, a metridioidean
belonging to a genus whose members produce catch tentacles. Furthermore,
Macrander et al. (2015a) found a gene copy highly similar to the presumed to be
acrorhagi-specific acrorhagin in the transcriptome of Metridium senile. The function
of the acrorhagin-like product in Metridium has not been identified and no phylo-
genetic analysis has shown that this is a homolog of acrorhagin (and if so, how it is
related to the acrorhagin of Actinia, Anthopleura, etc.). These similarities in venom
highlight the potential for deeply conserved toxins that could be invoked in parallel
in agonistic interactions.
Diversity and Evolution of Toxins Within Sea Anemones
Research on venom constituents in sea anemones has focused on characterization of

the structure and function of the toxins, with much less attention to the ecology and
evolution of the molecules. Even in those areas where there has been significant
research effort, the venom of sea anemones is imperfectly known. Conclusions about
its composition, its biological role, and its evolution remain highly contingent. Many
toxins currently known from only a few species or from only a single lineage may be
more widely distributed than currently appreciated. Innovations in proteomics,
transcriptomics, and genomics promise to increase the number of species for
which toxins are characterized and thus will facilitate the comparative study of the
genes and proteins that make venom (Sunagar et al. 2016).
At a broad scale, the venom of sea anemones is quite distinct from that of
medusozoan cnidarians (Rachamim et al. 2015). Many of the key constituents of
sea anemone venom have no known ortholog outside of anemones. The functional
characterization of the venome of each major cnidarian lineage suggests that
Anthozoa and Medusozoa have contrasting strategies for their use of venom, with
anthozoans (or at least actiniarians) relying much more on neurotoxins than do
medusozoans (Rachamim et al. 2015). A similar conclusion obtains in comparisons
within Anthozoa: nematocyst-mediated defense (venom) is more critical to sea
anemones than to hexacorals of orders Corallimorpharia and Zoanthidea (which
rely on toxins that are not injected: Hines and Pawlik 2012).
Contrary to expectations of repeated recruitment of venom genes from broadly-
shared and physiologically diverse genes (e.g., Fry et al. 2009), the majority of
venom genes in sea anemones are unique to this lineage. This emphasis on taxo-
nomically restricted genes in venom seems to be more common among ancient
lineages of venomous animals (Sunagar and Moran 2015). The few elements of the
venom of sea anemones that have a counterpart in the venom of other animals (e.g.,
10 M. Daly
protease inhibitors, CRISPs) have ambiguous roles, making comparison problematic

in the absence of detailed functional study. For those toxins unique to anemones,
only the voltage-gated and cytolytic toxins have been explored in an evolutionary
framework.
Voltage-Gated Toxins
Voltage-gated sodium channel toxins (NaTx) are among the most diverse toxins
described in sea anemones. These polypeptides range in size from 3 to 5 kDa and
inhibit the inactivation of voltage-gated sodium channels (reviewed in Moran
et al. 2009). Homologs of NaTx have not been identified in other cnidarians.
These toxins or genes that are expected to encode them have been found in assays
of tentacles and acrorhagi and in whole-animal assays. They are produced in planula
larvae of Nematostella and are also present in the oocytes of Anemonia viridis
(Moran et al. 2009), suggesting that they play a role in defense. In the genome of
Nematostella, at least eight of the genes encoding NaTx are physically near one
another and potentially transcribed as a single unit (Moran et al. 2008). Although the
multiple copies presumably represent at least some ancient gene duplications,
because of concerted evolution, some gene copies within a species may be more
similar to one another than to their orthologs in other species (Moran et al. 2008,
2009). Although this is likely generally true for NaTx in anemones, not all NaTX
transcripts show concerted evolution (Macrander et al. 2015b).
All three types of NaTx are reported from Actinioidea; many fewer NaTx have
been reported for metridiodeans (Jouiaei et al. 2015) (Fig. 2). Although proposed as
different based on early studies of species of Heteractis and Anemonia, the distinc-
tion between Type I and Type II NaTx is less clear-cut when toxins from other
lineages are considered (Ishida et al. 1997; Moran and Gurevitz 2006): the NaTx of
the anenthemonaeans Nematostella vectensis and Halcurias sp. seem to be pre-
cursors to the Type I and Type II NaTx of enthemonaean anemones. This pattern
suggests that some kind of NaTx was made by the common ancestor of sea
anemones (Fig. 2).
Because they have no homolog outside of Actiniaria, interpretation of the evolution
of NaTx in sea anemones is especially reliant on comparison of the gene and
organismal phylogenies to root and contextualize the network of NaTx genes. Rooting
a tree of NaTx sequences from Nematostella (Anenethemonae), Calliactis
(Enthemonae-Metridiodea), Actinia (Enthemonae-Actiniodea), and Anemonia
(Enthemonae-Actiniodea) with Calliactis rather than Nematostella (as done by e.g.,
Moran et al. 2009) is inappropriate given the relationships among these lineages
(Fig. 2). A tree rooted on Nematostella (Macrander et al. 2015b) suggests that Type
I and Type II NaTx underwent a duplication within Actinioidea and that the “orphan
clade” of NaTx is the metridioidean sister to this large clade of NaTx gene sequences
from actinioideans.
Voltage-gated toxins that attack the potassium channel (KTx) are more diverse in
form and function than NaTX and are presumed to represent multiple independent
origins from nontoxin molecules (Orts et al. 2013). Their orthologs outside of
anemones are not known. All of these act to inhibit the activity of membrane
potassium channels, with different types having affinity for different channels. As
for NaTX, the five types of KTx are differentiated by their amino acid composition,
folding pattern, and target site. Type I KTx are reported only from enthemonaean
anemones (Frazão et al. 2012) (Fig. 2), where they may be diverse and abundant.
Macrander et al. (2015a) find that the copies from Anthopleura elegantissima are not
the result of a single radiation within this species. The gene phylogeny of type I KTx
includes mostly sequences from actinioideans, but one sequence from the
metridioidean Metridium senile is associated with these, suggesting widespread
gene loss in that lineage or many as-yet undiscovered KTx in this lineage
(Macrander et al. 2015a).
Types II, III, and IV KTx have only been reported from a subset of Enthemonae,
the actinioidean sea anemones (Frazão et al. 2012). Type IV KTx have been reported
only from Stichodactyla haddoni, although it bears some similarity to a toxin from
Heteractis aurora (as Antheopsis maculata, Honma et al. 2005), and incomplete
candidate sequences were also retrieved in the actinioidean Anthopleura
elegantissima (Macrander et al. 2015a). The narrow known distribution of type IV
KTx precludes phylogenetic analysis. Type II KTx acts both as voltage-gated
potassium channel toxins and as Kunitz-type protease inhibitors (KPI; Minagawa
et al. 2008). This dual function makes it difficult to make inferences about the
likelihood of a gene being a functional toxin based on sequence data alone but
may explain the repeated evolution of potassium channel toxins across animals (Fry
et al. 2009). A gene phylogeny of type II KTx and KPI from sea anemones suggests
that members of this gene family have undergone both ancient and recent duplica-
tions (Macrander et al. 2015a) and that there is a differentiation between those genes
that act primarily as KPI and those that act as KTx, as the genes with known function
fall into two clades. Jouiaei et al. (2015) find that Type I and Type II KTx both evolve
under strong negative selection, although some sites on the surface of each molecule
show evidence of positive selection. This evolutionary pattern may explain the
diversity of copies within a species, as the combination of functional constraint
and relaxed selection on surface sites may lead to neofunctionalization (Jouiaei
et al. 2015).
Unlike the relatively narrowly distributed types I-IV KTx, Type V KTx occur in
all major lineages of sea anemones (Orts et al. 2013). This is the only KTx reported
in Nematostella vectensis, where its expression begins early in development in cells
that appear to be nematocysts (Orts et al. 2013) (Fig. 2). It has also been reported in
Bunodosoma caissarum, Metridium senile, and Anthopleura elegantissima (Orts
et al. 2013; Macrander et al. 2015a). Because it occurs in all major lineages of sea
anemone, Orts et al. (2013) hypothesize that this toxin has a very ancient origin,
likely being part of the ancestral venome of sea anemones.
The focus on cellular target and function for the various voltage-gated toxins may
obscure evolutionary similarity. In their analysis of the voltage-gated toxins from sea
anemones, Jouiaei et al. (2015) find that Type I NaTx and Type III KTx are most
closely related to one another. Thus, from an evolutionary standpoint, type II KTx
12 M. Daly
are highly modified NaTx. This phylogenetic result contextualizes functional and
biochemical studies that had identified similarities between NaTX and type III KTx
but stands in contrast to the inferred origin and history of functionally similar toxins
in other animal lineages (e.g., Zhang et al. 2015). These toxins are also the only
toxins in sea anemones that show strong evidence of evolving under positive
selection (Jouiaei et al. 2015) and thus the only kind of toxin that conforms to the
pattern of rapid, positive selection that has been proposed as the general mode of
evolution of venom genes (reviewed by Casewell et al. 2013).
Cytolytic Toxins
Although all act to disrupt the cell membrane of target cells, cytolytic toxins in sea
anemones are heterogeneous and diverse in terms of their structure and precise mode
of action (Anderluh et al. 2011). In addition to forming pores in membranes, some
cytolysins of sea anemones may act as antihistamines (Type I) or PLA2s (Type III).
Most of the cytolysins identified to date are restricted in their phylogenetic occur-
rence and their evolutionary relationships to other cytolytic toxins in other lineages
has not been investigated. Type I cytolysins have been reported only from Heteractis
crispa (as Radianthus macrodactyla) (Monastyrnaya et al. 2002). Type III cytolysins
are similarly restricted in their phylogenetic distribution, being reported only from
members of the actinioidean genus Urticina (Anderluh et al. 2011). The type III
cytolysins UPI and UCI were isolated from Urticina piscivora and U. crassicornis,
respectively, and have cardiostimulatory properties (reviewed in Anderluh
et al. 2011). Exaiptasia pallida has a cytolysin similar in size to UPI and UCI in
the acontia (Maček 1992), but it is unclear whether this has functional or evolution-
ary similarity to the other type III cytolysins. Type IV cytolysins are the largest
cytolysins and are characterized with respect to function and structure only in
Metridium senile. Sequences similar to these were recovered in the transcriptome
of acrorhagi from Anthopleura elegantissima (Macrander et al. 2015), indicating a
broader taxonomic distribution and potentially greater functional capability than
previously recognized.
Cytolysins that resemble membrane attack complex/perforins (MAC/PF) have
been characterized in Actineria villosa and Phyllodiscus semoni (reviewed in
Anderluh et al. 2011). These species are not thought to be closely related: Actineria
is a member of Thalassianthidae, a family historically placed within Endomyaria and
thus expected to belong to Actinioidea, and Phyllodiscus belongs to Aliciidae, a
family whose members form the stem or sister group to Metridioidea (Rodríguez
et al. 2014). However, both of these species are capable of delivering powerful
stings, raising the possibility that the venoms are convergent or, given the superficial
similarity of the species, that they have been misidentified.
MAC/PF proteins have not been identified as part of the venom in other sea
anemones, but they occur in other species: at least four MAC/PF genes are present in
the genome of Nematostella (Miller et al. 2007). A broad-scale analysis of the

evolution of the complement system highlights that the MAC/PF genes in anemones
are different from those of triploblasts in several respects (Kimura et al. 2009;
Rachamim et al. 2015). The MAC/PF genes of sea anemones do not cluster with
those of other Cnidaria and lie outside of a cluster that includes those genes that play
a role in the animal immune system (Rachamim et al. 2015). The tree of MAC/PF
genes from Cnidaria is very sparse in terms of taxon sampling, but if correct, it
suggests a very ancient duplication of MAC/PF genes, with one branch contributing
to the evolution of the immune system and the other becoming critical to the venome
of sea anemones.
Type II cytolysins, or actinoporins, are the most common and best characterized
type of cytolysin (Anderluh et al. 2011). These cytolysins recognize and bind to
sphingomyelin (Bakrač and Anderluh 2010). These are only characterized for
actinioideans and metridiodeans (Fig. 2). Although Meinardi et al. (1995) did not
identify the toxin they found in the gastrovascular cavity of Phymactis clematis, the
fact that it recognized sphingomyelin suggests that it is an actinoporin and thus that
these play some role in digestion. Basulto et al. (2006) found antibodies to
actinoporins in the tentacles and filaments of Sticohodactyla helianthus, a finding
also consistent with a role in digestion. However, actinoporins were abundant in the
transcriptome of acrorhagi of Anthopleura elegantissima (Macrander et al. 2015a)
and this tissue is not used in prey capture or digestion.
Actinoporins have only been characterized from sea anemones and have no
known ortholog outside of Actiniaria. An actinoporin-like molecule has been iden-
tified in the hydrozoan Hydra, but the relationship of this molecule to actinoporins
from sea anemones is unclear (Rachamim et al. 2015). Because phylogenetic
analysis of actinoporins has included only those actinoporins for which sequence
(rather than protein or biochemical) data are available, the full diversity of toxins has
not been considered. In their analysis, Frazão et al. (2012) find that the actinoporins
from actinioidean anemones (members of Actinia, Anthopleura, Heteractis,
Oulactis, Stichodactyla, and Urticina) form a clade and that a clade of sequences
from metridiodeans (Sagartia and Phyllodiscus) plus Actineria is sister to this
actinioidean cluster. Lineage-specific duplications have occurred in actinoporins
within Actinioidea (Frazão et al. 2012; Macrander et al. 2015a). Frazão
et al. (2012) also find that a second set of sequences from Actineria and Phyllodiscus
lies outside of the clade that includes the metridioidean and actinioidean
actinoporins. These cytolysins of Actineria and Phyllodiscus have a MAC/PF
domain (based on sequence similarity) and are only distantly related to the other
actinoporins; their phylogenetic relatedness to actinoporins should not be over-
interpreted. In their study of the mode of selection acting on diverse cnidarian toxins,
Jouiaei et al. (2015) found a general pattern of negative selection for actinoporins.
Although some of their analyses support the inference of episodic diversifying
selection (Jouiaei et al. 2015), examples of positive selection that would be required
by this scenario are limited.
14 M. Daly
Other Venom Constituents
Phospholipase A2s (PLA2s) are enzymes that contribute to the venom of diverse
animals (Casewell et al. 2013). They are found in the venom of members of all
lineages of sea anemone (Nevalainen et al. 2004; Frazão et al. 2012). The PLA2s
characterized from sea anemones are diverse and thus difficult to classify in a
meaningful way. In the few cases where its activity has been assayed in discrete
tissues, PLA2 activity is higher in acontia than tentacles (Nevalainen et al. 2004),
suggesting a role in defense rather than in predation. Nonetheless, PLA2s are active
in acrorhagi (Macrander et al. 2015a), attesting to a broad role for PLA2s in
Actiniaria.
PLA2s and MAC/PF cytolysins are the only identified constituents of sea anem-
one venom that have a clear relationship to genes found in other taxa, including other
Cnidaria (Nevalainen et al. 2004). Phylogenetic analysis of the PLA2s from sea
anemones with those from other animals (Macrander et al. 2015a) indicates that the
PLA2s of anemones have multiple origins within the larger gene family, rather than
representing a single radiation. The presence of multiple copies from this gene
family within a single species and the distribution of these on the tree points to
within-species and within-lineage diversification of PLA2s in sea anemones.
Cysteine rich secretory proteins (CRiSP) play diverse roles in animals, including
a role in venom in snakes. These are part of a larger gene family (CAP) whose
members are critical to reproductive and immune function in vertebrates and critical
to the organization and development of the nematocyst capsule (Balasubramanian
et al. 2012). Genomic assays of Nematostella vectensis and studies of the nematocyst
proteome detected genes with CRiSP and CAP domains, including some that could
not be assigned to the structural proteins of nematocyst capsules (Moran et al. 2013).
Similar molecules occur in the metridioidean sea anemone Metridium senile and the
actinioidean Heteractis magnifica, in the scleractinian Porites and Acropora, and in
the hydrozoan Hydra (Moran et al. 2013). Other cysteine-rich peptides include
SCRiPs, smaller molecules that were first described from scleractinians and inferred
to play a role in stress response and skeletalization. Sequences similar to SCRiPs
occur in Metridium senile and in the actinioidean Anemonia viridis (Jouiaei
et al. 2015). The toxicity of these is unknown, as is their affinity to those molecules
in scleractinian corals.
Venoms of sea anemones differ from those of non-cnidarian animals in several

respects: the distributed nature of venom in the cnidarian body, the great age of the
lineage, and the reliance on lineage-specific genes for venoms. Perhaps unsurpris-
ingly given these differences, the mode and tempo of evolution differs in sea
anemones compared to that reported for, e.g., snakes, spiders, and cone snails.
Unlike most other lineages from which venoms have been studied, the venom
genes of sea anemones undergo concerted evolution (Moran et al. 2011) and
experience negative selection (Jouiaei et al. 2015) in addition to diversifying and

positive selection. This difference may be a general property of venom genes in
ancient lineages (e.g., Sunagar and Moran 2015) or may be an artifact of the way in
which venom genes are studied in this lineage. Those molecules shared across major
lineages conform more closely to the expectations of negative selection (see Jouiaei
et al. 2015). Because studies of venom in sea anemones have largely been directed
using the “search image” of a known molecule (Sunagar et al. 2016) and because the
taxon sample of species surveyed is sparse in number and broad in phylogenetic
diversity, it will be difficult to detect novel molecules or highly divergent ones,
reinforcing the perception that there is low diversity within and across sea anemones
in terms of their venom genes. Advancing our understanding of the evolution of
venom in sea anemones will require focused study of lineages, tissues, and mole-
cules. Evolutionary relatedness, function, and tissue of origin need to be considered.
The strong correlation between toxin diversity and research effort suggests that
taxonomic diversity will be key to the discovery of novel molecules. Nonetheless,
lineage-specific differences in venom diversity clearly obtain with the
anenthemonaean anemones generally having fewer kinds of toxins in their venom
arsenal (Fig. 2). Although genomics and transcriptomics offer much potential for
broad-based surveys of taxa and tissues, functional characterization, including
studies of the biological role in natural systems, is critical to understanding whether
and how these molecules have changed in light of sequence evolution and for
clarifying the action of multifunctional molecules.
Cross-References

▶ Independent Origins of Scorpion Toxins Affecting Potassium and Sodium
Channels
▶ Mutation, Duplication, and More in the Evolution of Venomous Animals and
Their Toxins
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Revising the Role of Defense and Predation
in Cone Snail Venom Evolution
Jutty Rajan Prashanth, Sebastien Dutertre, and Richard James Lewis
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Cone Snails and Conotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Conotoxin Transcription and the Processes Driving Diversification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Classification of Conotoxins and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
The Integrated Venomics Approach Accelerating Conotoxin Discovery . . . . . . . . . . . . . . . . . . . . . . . 7
Evolution of Cone Snails and Conotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Traditional Views on the Origins of Prey Shifts in Conidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Evolution of Distinct Predatory and Defensive Venoms: A New Hypothesis for Prey Shifts
in Conidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Abstract
Venoms are widely employed by numerous animals across disparate lineages for
predation and defense. Among them, the deadly carnivorous cone snails are
Note: This template intends to guide authors in writing a contribution for the handbook series
Toxinology. As a reference work, please avoid first-person usage. Please also define all acronyms
and abbreviations used in your contribution and avoid footnotes and endnotes. For further details, please
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J.R. Prashanth (*) • R.J. Lewis (*)
Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland,
St. Lucia/Brisbane, QLD, Australia
e-mail: p.juttyrajan@imb.uq.edu.au; r.lewis@imb.uq.edu.au
S. Dutertre (*)
Institut des Biomolécules Max Mousseron (IBMM) – UMR 5247- Equipe Sciences Analytiques des
Biomolécules et Modélisation Moléculaire, Université de Montpellier 2, Bat. 17, Montpellier,
France
e-mail: sebastien.dutertre@umontpellier.fr; Sebastien.Dutertre@univ-montp2.fr

2 J.R. Prashanth et al.
reputed for the potency of their venoms comprising small neurotoxic peptides
known as conotoxins. Though a majority of cone snails prey on worms, some
species prey on fish and other mollusks despite being slow movers. This remark-
able prey diversification contributes to their evolutionary success. The origins of
these dietary shifts have historically been explained based on the synergistic
pharmacology of toxin classes. However, the recent discovery that some mollusk-
and fish-hunting snails inject distinct defensive and predatory venoms has led to
an alternative hypothesis where defense plays a pivotal role in the evolution of
conotoxins and cone snails. This chapter provides an overview of cone snails and
highlights recent advances in our understanding of conotoxin evolution.
Keywords
Cone snails • Conotoxins • Defense • Ecological release • Evolution
Introduction
Ecological interactions between life-forms have profoundly shaped their evolution-

ary histories (Dobzhansky 1973; Valen 1973). The nature of these interactions
ranges from the mutually beneficial (mutualistic) to the competitive (competition
for resources) and adversarial (predation, parasitism, etc.). Organisms across king-
doms have evolved numerous adaptations to gain advantages in such interactions
(Dobzhansky 1973; Valen 1973). One such adaptation is the use of venom compris-
ing a mixture of proteins and peptides secreted by a specialized organ that is injected
through specialized injection mechanisms (Casewell et al. 2013; Lewis and Garcia
2003). Venoms have risen independently across many lineages for use in prey
capture and defense. Some well-known venomous animals such as snakes, spiders,
and scorpions inject venoms using their fangs or stingers into prospective prey to
impair them, while others including most venomous fish and hymenopterans use
their venoms in defense (Casewell et al. 2013).
Typically, venom peptides potently and specifically target a number of the
receptors, enzymes, and ion channels that play key roles in important physiological
processes, making these molecules highly efficient in their respective ecological
roles, as well as providing promising drug leads (Lewis and Garcia 2003). Despite
their infamy, venoms are an excellent source of novel compounds with the potential
to treat a range of diseases. One example, captopril, a highly commercially success-
ful ACE-inhibitor, was originally developed from a peptide isolated from the venom
of the snake Bothrops jararaca (Prashanth et al. 2014). In addition to their medical
potential, venom systems provide excellent opportunities to study evolutionary
mechanisms such as protein neofunctionalization, and molecular and adaptive evo-
lution. Venoms are hypothesized to be evolving in an “evolutionary arms race”
(Dawkins and Krebs 1979) where increased resistance in prey catalyzes commen-
surate gains in potency and specificity of the toxins, and necessitates variations in
overall venom composition (Casewell et al. 2013; Dawkins and Krebs 1979; Valen
Revising the Role of Defense and Predation in Cone Snail Venom Evolution 3
1973). Indeed, venom composition within a species is adaptable to local pressures,

with well-characterized intraspecific variations in cone snail venoms a leading
example (Jakubowski et al. 2005). The venoms of both snakes and cone snails
exhibit variability across geographic regions in response to changes in diet (Chang
et al. 2015; Daltry et al. 1996). Thus, selection pressure acting on venomous animals
to efficiently capture prey can lead to increased prey specificity and potency of
venoms (Pawlak et al. 2006). Several complementary genetic, transcriptional, and
peptide level mechanisms are hypothesized to collectively facilitate the adaptive
evolution of toxins and neofunctionalization to target new physiological receptors
(Casewell et al. 2013). Though venoms appear to have adaptively evolved prey
specificity, they are also used in defense, with snakebite associated with a large
number of human fatalities (Kasturiratne et al. 2008). Similarly, some cone snail
stings have also been associated with fatalities (Dutertre et al. 2014a). However,
there is scant evidence about the role of defense in venom evolution (Casewell
et al. 2013). Given defensive use of venoms against humans has prompted the
multitude of venom-based drug discovery initiatives, combining these programs
with an understanding with the evolution of venoms could better guide such efforts
and also aid antivenom discovery – an intriguing twist on Dobzhansky’s words –
“Nothing in biology makes sense except in the light of evolution” (Dobzhansky
1973).
This chapter focuses on venom evolution in cone snails, slow-moving tropical
snails whose deadly neurotoxic venoms are composed of small peptides called
conotoxins. Conotoxins have proven a valuable source of molecular probes to
dissect the pharmacology of receptors implicated in the pathophysiology of numer-
ous neuronal diseases and disorders (Lewis et al. 2012). More importantly, a
conotoxin has also been approved by the FDA to treat intractable pain and a number
of other conotoxin-based drug leads are in various stages of clinical trials (Lewis
et al. 2012; Prashanth et al. 2014). Uniquely, some cone snails inject distinct
cocktails of defensive and predatory venoms facilitated by a venom gland adaptation
allowing for localized expression and secretion of different toxins (Dutertre
et al. 2014b). This also provides an opportunity to elucidate the role of defense in
addition to predation on venom evolution. As stated above, a better understanding of
venom evolution affords the opportunity to further rationalize drug discovery efforts
for conotoxins with species-specific activity. The following sections provide a brief
overview of cone snails and conotoxins, their classification and nomenclature, the
approaches employed to study them, and aspects of their evolution.
Cone Snails and Conotoxins
Marine gastropods belonging to the family Conidae, which consists of more than
800 species, are among the most evolutionarily successful marine animals
(Puillandre et al. 2015). Conidae comprises four genera, namely, Conus,
Conasprella, Profundiconus, and Californiconus, with the genus Conus containing
~85 % of known species (Puillandre et al. 2015). Conus and Conasprella are further
divided into 57 and 11 subgenera, respectively, based on molecular phylogenetic

analysis (Puillandre et al. 2015). Cone snails have colonized tropical waters around
the world using their venom for predation and defense. The wide geographical
distribution and extended species diversity have been attributed to cone snails
adapting to new environments by rapidly evolving their venom library, allowing
them to capture newly encountered prey in novel environmental niches (Conticello
et al. 2001; Duda and Palumbi 1999; Olivera et al. 2012). Indeed, cone snails have
evolved to target a range of worm lineages and, surprisingly, mollusks and fish.
Worm-hunting species (~80 % of Conidae) exhibit a wide variety of dietary patterns
with some species employing a generalist approach, feeding on several different
worm lineages, while others have specialized prey preferences (Duda et al. 2001;
Kohn 1959; Leviten 1978). The array of biotic interactions encountered by Conidae
is biochemically reflected in its venom composition (Olivera et al. 2012). Each cone
snail species expresses a unique venom profile befitting its ecological requirements
with initial estimates of the number of peptides per species ranging between 50 and
200 (Jones et al. 1996), though more advanced mass spectrometry methods have
identified more than a thousand unique molecules (Biass et al. 2009; Dutertre
et al. 2013; Lewis et al. 2012). Such an extensive reservoir is proposed to have
evolved under natural selection pressure to maintain an efficient prey capture
strategy, driving toxin diversification through aggressive mutations, gene duplica-
tions, and recombination (Conticello et al. 2001; Duda and Palumbi 1999; Olivera
et al. 2012).
The toxins that make up cone snail venoms are called conotoxins. Conotoxins are
primarily small disulfide-rich (containing two or more disulfide bonds) neuroactive
peptides that predominantly range between 10 and 35 amino acids in length though
longer peptides and proteins have also been discovered (Lewis et al. 2012).
Conotoxins have a broad range of pharmacological targets comprising ion channels
and neuronal receptors, many of which have been implicated in pain signaling
pathways and the pathophysiology of neurodegenerative diseases such as
Parkinson’s and dementia (Lewis et al. 2012). Some prominent receptor targets
include nicotinic acetylcholine receptors (nAChRs), voltage-gated calcium and
sodium channels, G protein-coupled receptors (GPCRs), the norepinephrine trans-
porter (NET), and N-methyl D-aspartate (NMDA) receptors (Lewis et al. 2012).
Conotoxins are unusually potent, and potency is often allied with the ability to
discriminate between receptor subtypes (Lewis et al. 2012). The small size and
relative ease of synthesis combined with their exquisite pharmacological properties
have made conotoxins an invaluable source of pharmacological probes and thera-
peutic candidates (Prashanth et al. 2012). In 2004, the FDA approved the first
conotoxin-based drug, the calcium channel blocker ω-MVIIA (Prialt™) originally
isolated from the venom of Conus magus, as an intrathecal analgesic for intractable
pain, while several others are in clinical trials (Lewis et al. 2012; Olivera and Cruz
2001). Despite these advances, conotoxins remain a relatively untapped source of
therapeutic candidates given the remarkable diversity they encompass, with esti-
mates suggesting that just 0.1 % of the conotoxin pool has been studied thus far
(Lewis et al. 2012; Prashanth et al. 2012).
Conotoxin Transcription and the Processes Driving

Diversification
Conotoxins are renowned for their hyperdiverse structure and pharmacology and the
mechanisms facilitating this diversity at genetic, transcriptional, and the peptide
level (Prashanth et al. 2014). Molluscan genomes are characterized by the predom-
inance of repeat regions with preliminary genomic analysis of cone snails revealing a
similar pattern for conotoxins (Barghi et al. 2015). Numerous conotoxin genes are
seemingly punctuated by introns, some of which encompass repeat regions that
presumably facilitate recombination and influence conotoxin diversification (Barghi
et al. 2015). A number of unexpressed conotoxin genes across various superfamilies
were also discovered from preliminary genome analysis that are likely differentially
expressed to enable adaption to new environmental challenges (Barghi et al. 2015;
Chang and Duda 2014). Differential expression of conotoxin genes also appears to
facilitate the reported intraspecific diversity in venom peptide compositions (Chang
and Duda 2014).
Conotoxins are initially transcribed and translated into a prepropeptide containing
a hydrophobic signal peptide at the N-terminal end, a propeptide region, and the
mature peptide at the C-terminal. Signal and propeptide regions are enzymatically
cleaved, leaving the mature peptide to be folded into its tertiary structure by
chaperone proteins and maturation is completed with the incorporation of posttrans-
lational modifications (PTMs) (Fig. 1; Prashanth et al. 2012, 2014).
Cone snails are renowned for their combinatorial library-like strategy that diver-
sifies their venom compositions, exhibiting both inter- and intraspecific variations in
venom profiles (Abdel-Rahman et al. 2011; Davis et al. 2009). Variations in the
venom injected by a single specimen have also been observed over time in Conus
consors specimens milked in captivity. Though these injected venoms revealed an
archetypal venom fingerprint, differences were observed in minor components
between injections (Dutertre et al. 2010). Some injections comprised of an entirely
different venom profile, though these may be defensive stings (Dutertre et al. 2010,
2014b).
Several genetic mechanisms are suggested to be driving the expansion of the
conotoxin repertoire. These include gene duplication, recombination, and focal
hypermutations within the mature peptide, which has an elevated rate of
non-synonymous mutations (mutations resulting in amino acid changes) (Barghi
et al. 2015; Chang and Duda 2014; Conticello et al. 2001; Duda and Palumbi 1999;
Olivera et al. 2012). In addition to genetic mechanisms, phenotypic mutations
arising from “messy” transcriptional processing (Jin et al. 2013) and variable peptide
Proteolytic
Cleavage C C
Signal Peptide Propeptide Mature region Mature region
Translation Prepropeptide Posttranslational C C
precursor Modifications
Gene
Fig. 1 The simplified conotoxin transcription pathway

processing in the form of truncations, posttranslational modifications and differential

cleavages also greatly multiply the number of gene products, with more than 6000
peptides arising from just 105 conotoxin transcripts in the Conus marmoreus venom
gland (Dutertre et al. 2013), further expanding conotoxin diversity. However, the
majority of these variants are likely to be nonfunctional with functional versions
upregulated and fixed in the genome over evolutionary time scales (Prashanth
et al. 2016). The breadth of the conopeptide library likely provides the substrate
for selection pressures to act on to improve selectivity at some receptors and attain
activity at receptors that were not targeted previously, which in turn enables capture
of new prey (Chang and Duda 2014; Olivera et al. 2012).
Classification of Conotoxins and Nomenclature
Three schemes are used to classify conotoxins. They are classified based on the gene
superfamily, their structure and arrangement of cysteines in the mature peptide, and
their pharmacological target. The gene superfamily of a conotoxin is assigned based
on the signal peptide sequence of the conotoxin precursor and is denoted by a
capitalized alphabet. Twenty-seven superfamilies have been accepted by the
ConoServer thus far (Kaas et al. 2008) though recent transcriptome sequencing
studies have identified many more novel superfamilies than were previously thought
to exist (Prashanth et al. 2014). Whether these novel superfamilies are widespread
across a number of species remains to be ascertained (Prashanth et al. 2014). A, O1,
O2, M, and T are some of the most common conotoxin superfamilies, each with
more than a 100 different precursors from various species deposited on the
ConoServer (Kaas et al. 2008). The arrangement and connectivity of cysteines in
the mature region influence the tertiary structure of the conotoxin. Hence, the
arrangement of cysteines has been used to classify sequences into “cysteine frame-
works” that are indicated using Roman numerals. Currently, there are 26 known
frameworks, although as in the case of superfamilies, several novel frameworks have
been discovered from transcriptome sequencing experiments, their prevalence across
species is as yet unclear (Prashanth et al. 2014). Finally, based on their pharmaco-
logical targets, conotoxins are classified into families that are denoted by Greek
alphabets, with 12 pharmacological families defined so far. The general template for
naming, based on the original proposal of McIntosh et al. (1999), is illustrated with
an example below (Fig. 2).
In the above example, the first Ca2+ channel blocker (ω-conotoxins) to be
discovered and characterized from Conus geographus with the framework VI
would take the name ω-GVIA. Conotoxins that have yet to be characterized phar-
macologically are named similarly with the Roman number replaced by its equiva-
lent Arabic number, and instead of an alphabet indicating the order in which the
conotoxins were classified, the equivalent Arabic number is used with the two
numbers separated by a period. In the above example, before pharmacological
characterization, ω-GVIA would have been termed G6.1.
Fig. 2 Conotoxin nomenclature template using ω-GVIA as an example
While these classification schemes and nomenclature are applied to conotoxins,

several “conopeptides,” which are nominally defined as “cysteine-poor” peptides
with one or no disulfide bonds with similarity to neurohormones, have also been
discovered though they are less common than conotoxins and named differently
(McIntosh et al. 1999; Prashanth et al. 2012). Non-conotoxin conopeptides are
identified simply by their names, which are portmanteaus consisting of the name
of the hormone/endogenous-signaling molecule to which the peptide shares greatest
similarity, prefixed with the term “cono” and suffixed by the species name. Some
examples of conopeptides include conopressins that are related to members of the
vasopressin/oxytocin family, conopeptide Y/YY, which are similar to neuropeptide
Y/YY, and conorfamide that resembles RF-amide peptides (Lewis et al. 2012).
Based on the aforementioned nomenclature, the conopressin from C. geographus
is called Conopressin-G.
The Integrated Venomics Approach Accelerating Conotoxin

Discovery
Until recently, conotoxins were mostly isolated and characterized by a process called
bioactivity-guided fractionation where venoms were screened for activity in bio-
assays and “hits” exhibiting activity were isolated by a series of fractionations
punctuated by additional bioactivity screens (Prashanth et al. 2012, 2014). This
resource-heavy approach uses a disproportionate amount of venom sample to char-
acterize even a small number of peptides. Not only is this method taxing on venom
resources, it is also time consuming and does not permit holistic studies of venom
composition and properties which may be required to infer the ecological implica-
tions of some peptides being co-expressed/co-injected (Prashanth et al. 2012, 2014).
These drawbacks have rendered traditional bioactivity-guided methods cumber-
some, particularly when trying to extract ecological and evolutionary information
based on the composition of venoms. Rapid technological strides that have increased
the availability and affordability of next-generation sequencing, as well as the

development of advanced mass spectrometry techniques with exquisite sensitivity,
and new bioinformatic tools to combine and analyze large volumes of data have seen
the traditional approach gradually supplanted by sequence-driven approaches
(Prashanth et al. 2012).
Apart from drastically reducing the time and resources required to characterize
novel peptides, these sequence-driven approaches have greatly impacted whole
venom and venom evolution studies by increasing the scale of these studies manifold
and have also led to the discovery of several novel superfamilies (Prashanth
et al. 2012, 2014). The integration of transcriptomic and proteomic data is an
efficient way to analyze the composition of venom samples, and high-throughput
bioassays can rapidly characterize the pharmacology of these peptides (Prashanth
et al. 2012). The large volume of data generated by next-generation sequencing
combined with proteomics also allows for specific evolutionary hypotheses to be
tested.
Evolution of Cone Snails and Conotoxins
As mentioned earlier, cone snails consist of more than 800 species spread across the
world inhabiting a range of environmental niches with a multitude of dietary
preferences. Conidae are thought to have first appeared approximately 55 mya and
subsequently diverged through two radiations that were separated by a period of
extinction during the Lower Pliocene leading to the species diversity we observe
today (Duda et al. 2001). While ancestral Conidae were likely worm hunters preying
on errant polychaetes similar to the Turridae family from which they branched,
modern cone snails prey on a range of errant and sedentary worms, mollusks, and
fish (Duda et al. 2001). Each cone snail species possesses a unique mixture of venom
peptides that are used to capture prey and in defense, with venom profiles reflecting
the species’ biotic interactions (Olivera et al. 2012). Even a conservative estimate of
100 conotoxins per species gives an estimate of 80,000 conotoxins, which is a
tremendously diverse pool of toxins, though many of these conotoxins are likely
to be homologous in activity.
Phylogenetic studies suggest that fish hunting evolved between one and three
times, once or twice from worm-hunting cone snails that preyed on errant poly-
chaetes, and once from sedentary polychaete-consuming species, while mollusk
hunting evolved once from errant polychaete feeders (Duda et al. 2001). Eunicids
remain the dietary preference for a majority of worm-hunting lineages that prey on
errant polychaetes though some subgenera preferentially prey on nereids. Some
species such as Conus anemone consume both eunicids and nereids, while some
species such as Conus arenatus consume both errant and sedentary polychaetes
(Duda et al. 2001). As opposed to the more generalist feeding behaviors within
worm-hunting Conidae discussed above, C. leopardus feeds exclusively on hemi-
chordates (Duda et al. 2001) with its venom appearing to have undergone commen-
surate simplification (Remigio and Duda 2008). In keeping with this trend, increased
dietary breadth in a C. milliaris population that underwent ecological release on

Easter Island seems to have elicited a proportional expansion in its venom repertoire,
presumably to allow capture of a wider variety of prey (Duda and Lee 2009).
Furthermore, cone snail species spread across vast geographical areas with differing
prey availability in each area also exhibit intraspecific variations in venom compo-
sition (Duda et al. 2009). Thus, the diet of cone snails and venom composition and
evolution appear intertwined.
Traditional Views on the Origins of Prey Shifts in Conidae
The relationship between diet and venom leads to perhaps the most intriguing
question regarding cone snail evolution and the origins of mollusk and fish hunting.
The ability of cone snails to hunt fish is particularly surprising given snails move
slowly. Despite their lack of mobility, piscivorous species have evolved two alternate
mechanisms to capture prey. In the first, broadly called “hook-and-line,” though
variations of this strategy exist (Olivera et al. 2015), cone snails inject venom to
rapidly immobilize fast-moving piscine prey (Fig. 3). This is the predominant
feeding behavior among fish-hunting snails and presumably used by all of the
piscivorous subgenera (Olivera et al. 2015). The second type called “net feeding”
has only been reported in C. geographus and C. tulipa (subgenus Gastridium). Here,
the cone snail opens its rostrum and secretes peptides into the water eliciting a
“nirvana-like” state in the fish before engulfing them (Fig. 4; Olivera et al. 2015).
The molecular mechanisms reportedly underlying the two feeding behaviors are
characterized by synergistically acting peptides referred to as “cabals.” Hook-and-
line hunters use two complementary cabals called “lightning-strike” and “motor”
cabals. The lightning-strike cabal causes rapid tetanic paralysis, while the motor
cabal induces flaccid paralysis by deactivating neurotransmission after tetanic paral-
ysis has ensued (Olivera et al. 2015). The lightning-strike cabal is proposed to
contain a peptide to delay the inactivation of Na+ channels, usually a δ-conotoxin,
and another to block shaker K+ channels (Terlau et al. 1996). While δ-conotoxins
appear to be used broadly by most if not all piscivorous cone snails, the choice of the
K+ channel blocker varies, with Conus purpurascens (Chelyconus subgenus) using a
κ-conotoxin with the framework VII (Terlau et al. 1996), while Conus striatus
(Pionoconus subgenus) is suggested to use a kunitz domain containing conkunitzin
peptide for the same role, and C. radiatus (Phasmoconus subgenus) purportedly uses
kRIIIK, a M-superfamily peptide with the framework III that is generally associated
with Na+ channel blockers (μ-conotoxins) (Olivera et al. 2015). Thus, the use of
δ-conotoxins is widespread in the lightning-strike cabal, while many of the K+
channel blockers appear to be convergently recruited from a variety of structural
folds. Following the lightning-strike cabal, the motor cabal, which consists of
paralytic α-conotoxins (nAChR blockers), ω-conotoxins (Ca2+ channel blockers),
and μ-conotoxins (Na+ channel blockers), begins to systematically disable neuro-
transmission causing flaccid paralysis (Olivera et al. 1985; Terlau et al. 1996). The
Fig. 3 The “hook-and-line” prey capture strategy by Conus obscurus. (a) C. obscurus
approaches the fish with proboscis primed for stinging. (b) Snail stings the fish causing the fish
to experience a massive excitotoxic shock resulting in rapid uncontrolled flinching before tetanic
paralysis within seconds of injection. (c) Snail engulfs paralyzed fish
Fig. 4 The “net feeding” behavior of Conus geographus. (a) C. geographus secretes the
“nirvana” cabal into the water causing the fish to become comatose. (b) The snail slowly engulfs
the fish while it continues to remain dazed. (c) The fish is completely engulfed and is stung while
inside the rostrum, presumably inducing paralysis to prevent escape as the fish briefly regained
mobility
components of the motor cabal are independently paralytic and are widespread
across piscivorous and molluscivorous cone snails (Olivera et al. 1985).
In contrast, the net feeding C. geographus and C. tulipa appear to have replaced
the lightning-strike cabal with peptides that dampen the neuronal systems of fish
such as contulakin-G and conanotkin-G that are both associated with inducing
comatose symptoms in fish (Olivera and Cruz 2001). Recently, Conoinsulins that
induce hypoglycemic shock in fish were also isolated from C. geographus,
suggesting it may be another component of the “nirvana cabal” (Safavi-Hemami
et al. 2015). This novel feeding behavior allows these Conidae to engulf several fish
simultaneously aided by an extremely light shell and a large aperture, which in turn
is presumed to allow these species to be more mobile and prey on schools of fish
(Olivera et al. 2015). In addition, C. geographus and C. tulipa also express the motor
cabal toxins described earlier, which are suggested to be occasionally injected to
paralyze fish that have already been engulfed, likely to prevent escape (Olivera
et al. 2015).
Based on the molecular pharmacology of these toxins and their combined phys-
iological effects on fish, an evolutionary pathway to fish hunting from ancestral
worm-hunting species was hypothesized (Olivera and Cruz 2001; Olivera
et al. 2015). Briefly, it has been suggested that ancestral worm-hunting species
originally evolved δ-conotoxins for predation. However, cone snails also use their
venoms to stave off competitors and in defense. Hence, when a fish competing for
the same prey approached, the ancestral Conidae are presumed to have injected it
with a venom containing δ-conotoxins. It is suggested that δ-conotoxins that caused
pain in fish apart from potently immobilizing worms were subsequently selected for.
K+ channel blockers that were recruited later by ancestral Conidae for predatory
purposes now enabled rapid paralysis when used against competitor fish given
δ-conotoxins were already established in the venom. Immobilization of injected
fish likely provided opportunities for fish hunting before founder populations
diverged, with some species eventually evolving into specialist fish hunters (Olivera
et al. 2015). Thus, conservation in pharmacology across competitors, predators, and
prey is predicted to have led to dietary shifts to include erstwhile piscine competitors
and predators. Notably, this hypothesis does not adequately explain the evolution of
mollusk hunting even though mollusks, including other cone snails, are also likely to
act as competitors for targeted prey. No k-conotoxins have been found so far in
mollusk-hunting species though some k-conotoxins have been found in worm-
hunting snails (Lewis et al. 2012; Olivera et al. 2015), suggesting mollusk hunting
may have followed an independent evolutionary trajectory.
Evolution of Distinct Predatory and Defensive Venoms: A New

Hypothesis for Prey Shifts in Conidae
Though cone snails use their venoms primarily for prey capture, on occasion they are
used to defend themselves against predators. Indeed, the venom of C. geographus is
deadly and associated with numerous human fatalities accompanied by symptoms of
paralysis consistent with the use of the “motor cabal” toxins, i.e., α-, μ-, and
ω-conotoxins (Dutertre et al. 2014b; Fegan and Andresen 1997). Though the
venom of C. geographus has been implicated in fatalities, stings from several
other species have also been reported to cause severe symptoms suggesting that
defensive use of venom is widespread across Conidae (Olivera and Cruz 2001;
Olivera et al. 2015). The effects associated with cone snail envenomation were again
attributed to the use of venom containing peptides that had evolved to target
vertebrate prey and therefore more active on mammalian targets (Endean and
Rudkin 1963; Olivera and Cruz 2001). However, the remarkable potency of
C. geographus, which is a net feeder that rarely uses motor cabal peptides for prey
capture, appears paradoxical since the motor cabal peptides in this species are
presumably under relaxed selection pressure given their secondary role in predation.
Yet C. geographus is the only species that is widely accepted to cause human
fatalities (Dutertre et al. 2014a; Fegan and Andresen 1997).
This discrepancy maybe explained by the recent discovery that Conidae can
alternatively inject distinct venom cocktails when provoked with predatory and
defensive stimuli (Dutertre et al. 2014b). The motor cabal toxins that were originally
hypothesized to be injected for predation were found to dominate the highly complex
defensive venom of C. geographus. However, peptides purportedly part of the
nirvana cabal were found to dominate the prey-evoked venom, which was much
less paralytic than the defensive venom (Dutertre et al. 2014b). In contrast, motor
cabal toxins dominated the defensive venom from different C. geographus speci-
mens, accounting for the high lethality of the venom to humans (<0.05 mg/kg;
Dutertre et al. 2014a). Furthermore, it appears C. geographus evolved a particularly
aggressive defensive strategy, apparently trading off the protection afforded by a
heavy shell for a highly complex and potent defensive venom that is readily
deployed under duress. This stands in contrast to most Conidae that use their shells
as the primary defensive mechanism with defensive injections employed only under
exceedingly high threat (Dutertre et al. 2014b). Such a defensive envenomation
strategy presumably coevolved with the novel net feeding strategy that allows
multiple fish to be consumed simultaneously (Dutertre et al. 2014b; Olivera
et al. 2015). The ability to readily switch between predatory and defensive venoms
was also reported in Conus marmoreus, a mollusk-hunting cone snail, in which a
majority of previously isolated mammalian active peptides were found to be major
components in the defensive venom (Dutertre et al. 2014b). Defensive and predatory
milkings were also collected from a number of other piscivorous and molluscivorous
cone snails, while defensive stings were obtained from worm-hunting species,
suggesting that this ability is widespread among Conidae (Fig. 5; Dutertre
et al. 2014a, b).
The spatiotemporal stimulus-dependent injection of venom is facilitated by
compartmentalization of the venom gland (Dutertre et al. 2014b). Cone snails
express venom peptides in a long tubular organ called the venom gland or duct,
which culminates with the venom bulb at one end (proximal) and the proboscis at the
other (distal). Glands of both C. geographus and C. marmoreus were sectioned with
the “distal” section expressing predatory conotoxins and the “proximal” section of
Fig. 5 Defensive envenomation by Conus planorbis against the predatory mollusk Conus
marmoreus. (a) C. planorbis under threat from C. marmoreus extends its proboscis to defend itself
against the mollusk-hunter C. marmoreus. (b) C. planorbis stings the predator on its siphon. The
white fluid visible at the point of contact is the venom (see inset). (c) C. marmoreus withdraws its
proboscis into the shell following the unsuccessful attempt to prey on C. planorbis due to its
defensive envenomation against C. marmoreus
the gland expressing defensive venom peptides (Dutertre et al. 2014b). The target is
stung using hypodermic needles-like radula through which the venom is injected for
prey capture and/or defense (Fig. 6; Salisbury et al. 2010; Schulz et al. 2004).
Based on the above observations, Dutertre et al. have put forward an alternate
hypothesis to better explain the evolution of mollusk and fish hunting. They suggest
that the ancestral worm-hunting cone snail used a single venom cocktail, primarily
for prey capture and secondarily for defense. However, in contrast to the previous
hypothesis, some worm-hunting snails evolved a specialized venom gland that
facilitated the injection of distinct defensive and predatory venoms. This adaptation
was hypothesized to allow conotoxins to evolve separately under predatory and
defensive selection regimes, with peptides originally evolved in worm-hunting cone
snails for defense against predatory mollusks and fish (Dutertre et al. 2014b; Kohn
1959) being repurposed for mollusk and fish hunting, respectively (Dutertre
et al. 2014b; Fig. 7).
While the aforementioned hypotheses help to better explain the striking diet
diversification to mollusk and fish hunting, ~80 % of modern cone snails continue
to prey on worms. More recent studies on worm-hunting cone snails are now starting
to provide a more comprehensive understanding of the evolution of Conidae. Aman
et al. isolated a δ-conotoxin from Conus tessulatus, a worm-hunting species that is
phylogenetically closely related to fish-hunting subgenera. Behavioral observations
of this species revealed that C. tessulatus, while primarily a worm hunter, can also
opportunistically prey on fish. A homologous peptide was also isolated from a
Defence-evoked venom
PREDATORS
Bulb 0 20 40 60 80 Oesophagus
Time (min)
Salivary
Venom duct Distal gland
Proximal Proboscis
Radular sac
0 20 40 60 80 PREY
Time (min)
Predation-evoked venom
Fig. 6 The stimulus-dependent injection of venom by cone snails. (Adapted from Dutertre
et al. (2015))
Ancestral primitive cone snail Defensive evolutionary pressures Fish-hunting C. geographus

D +P Motor cabal Nirvana cabal
Fish-hunting C. consors Ligthning-

Motor cabal strike cabal
Venom duct specialization Mollusc-hunting C. marmoreus

Defensive cabal Mollusc cabal
Ancestral modern cone snail Worm-hunting

D P Defensive cabal Worm cabal
Fig. 7 An illustration of the “repurposed defense” hypothesis (Figure adapted from Dutertre
et al. (2014b))
closely related species, C. eburneus (Aman et al. 2015). A third δ-conotoxin SuVIA
was isolated from C. suturatus, another member of the Tesselliconus subgenus (Jin
et al. 2015a). Mass spectrometric studies revealed that expression of this peptide
occurred primarily in the proximal section of the venom gland, which is associated
with a defensive role, suggesting that these δ-conotoxins were likely originally
evolved for defense against vertebrate predators including fish. Further, δ-SuVIA
potently activates the human Nav1.7 channel, an important part of the pain trans-
mission pathways, thereby causing pain when defensively injected into vertebrate
predators (Jin et al. 2015a). Together, these studies indicate that defensive
δ-conotoxins from worm-hunting cone snails could have facilitated fish hunting,
providing the first direct evidence of a link between defense and piscivory in
Conidae, in a classic case of the hunter becoming the hunted (Jin et al. 2015a).
The role of defense on conotoxin evolution was also established by studying αD-
conotoxins from a number of worm-hunting subgenera, predominantly the
Rhizoconus subgenus (Prashanth et al. 2016). αD-conotoxins are dimeric
nonparalytic inhibitors of nAChRs that appear to have evolved episodically for a
defensive role in the Rhizoconus subgenus demonstrating that defense can play a role
in the evolution of a toxin class. As with the δ-conotoxins, αD-conotoxins were also
expressed mainly within the proximal venom gland of the Rhizoconus species where
they have replaced the widely used α-conotoxins from the A-superfamily found in
most if not all cone snail species surveyed to date (Prashanth et al. 2016). This
integrated venomic study also showed that the compartmentalization of the venom
gland that facilitated defensive and predatory venom injections in mollusk- and fish-
hunting cone snails originally evolved in worm-hunting species, from where the
other dietary classes arose. However, the aforementioned venom gland architecture
is not conserved in all worm-hunting species. In C. planorbis, despite large varia-
tions in peptide expression along the venom gland, defensive venom peptides were
more highly expressed in the central sections, in particular, the proximal central
section (Jin et al. 2015b). Despite this difference, the defensive venom of
C. planorbis was also highly potent at nAChRs although C. planorbis uses
A-superfamily α-conotoxins unlike the αD-conotoxin-containing Rhizoconus sub-
genus. Why the compartmentalization of the C. planorbis venom gland is different to
the other species studied is not yet clear. One possible explanation is that different
worm-hunting species have evolved different venom gland compartmentalization
with C. planorbis an example of this diversity. Another explanation is that ancestral
cone snails used a single venom for defense and predation as hypothesized by
Dutertre et al., with compartmentalization of the venom gland occurring over
evolutionary time. Based on C. planorbis’ early divergence as inferred from the
phylogenetic reconstructions of Puillandre et al., it is possible that this species
represents a transitionary venom gland from the earliest diverging species, where
differences in venom expression along the gland were minimal, to modern mollusk-
and fish-hunting species, where there are drastic differences between the different
sections of the venom gland (Dutertre et al. 2014b; Prashanth et al. 2016). Further
studies into a wider array of species across different lineages of cone snails are
required to fully uncover the evolutionary routes of Conidae, though these recent
studies have begun to make some inroads.
This chapter highlights developments in our understanding of the ecological roles

and evolution of conotoxins and cone snails. By injecting different defensive and
predatory venoms, cone snails provide a unique opportunity to study the roles of
each of these individually on the evolution of their venoms (Dutertre et al. 2014b).
Indeed, recent studies have revealed an important role for defense in the evolution of
Conidae (Prashanth et al. 2016). These discoveries have expanded our understanding
of how prey shifts may have risen in this lineage. An early hypothesis explaining the
origins of fish hunting based on the pivotal role of δ-conotoxins has been
complemented by the discovery of their defensive role in worm-hunting Conidae
that are closely related to fish-hunting species (Aman et al. 2015; Jin et al. 2015a).
However, defensive venoms are typically complex and often contain peptides
targeting Ca2+ channels and nAChRs (Dutertre et al. 2014b; Jin et al. 2015b;
Prashanth et al. 2016). The roles of these peptides, if any, in facilitating prey
diversification remain to be understood, and further research is required to unravel
the evolutionary origins of these pharmacologically important peptides. Exploring
the ancestry of these classes may also provide clues with regard to the evolution of
mollusk hunting. Finally, little is known about the origins of conopeptides and
conotoxins. The discovery of hormone-like venom peptides such as the insulin-
like peptides in C. geographus (Safavi-Hemami et al. 2015) suggests a neurohor-
monal origin for some conopeptides. Nonetheless, the vast majority of conotoxins
are disulfide-rich peptides with a rapid rate of evolution and small sizes that have
diverged significantly enough to not have left a trace of their ancestry (Prashanth
et al. 2016). Studying other cone snail organs may shed light on these fundamental
questions about ancestral conotoxins. Finally, venomic studies on cone snails are
beginning to shed further light on fundamental questions such as the origin of prey
diversity (Aman et al. 2015; Jin et al. 2015a) and the role of defense in Conidae
(Dutertre et al. 2014b; Prashanth et al. 2016). Though further study is required to
fully understand these mechanisms, answering such evolutionary questions con-
tinues to offer the promise of rationalizing the discovery of novel conotoxins.
Cross-References

Their Toxins
▶ Systematics and Evolution of the Conoidea
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Systematics and Evolution of the Conoidea
Nicolas Puillandre, Alexander E. Fedosov, and Yuri I. Kantor
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Biology and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
General Characteristics and Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Feeding Habits and Habitat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Species Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Phylogeny and Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Alpha-taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Foregut Anatomy and Venom Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Toxin Diversity and Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
What Is a Conotoxin? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Phylogeny of Conoidean Venom Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Molecular Mechanisms of Conopeptide Diversification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
State of the Art of the Conoidean Toxin Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Speciation and Diversification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Abstract
The highly diverse toxins of cone snails have been known since the 1970s;
however, the evolutionary processes that led to both the species and toxin
diversity in the group are only recently being explored. Furthermore, their closely
N. Puillandre (*)
Institut de Systématique, Evolution, Biodiversité ISYEB – UMR7205 – CNRS, MNHN, UPMC,
EPHE, Muséum National d’Histoire Naturelle, Sorbonne Universités, Paris, France
e-mail: nicolaspuillandre@gmail.com
A.E. Fedosov (*) • Y.I. Kantor (*)
A. N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia
e-mail: fedosovalexander@gmail.com; kantor.yuri1956@gmail.com

2 N. Puillandre et al.
related, also venomous but much more diversified, allies in the superfamily
Conoidea remain largely unknown, with most species still undescribed and
only few toxins characterized for a handful of conoideans other than cone snails.
This chapter is a review of the literature dealing with systematics and evolution of
the Conoidea. In particular, it will be shown how new hypotheses on the evolu-
tionary processes have emerged from interdisciplinarity between ecology, taxon-
omy, phylogeny, anatomical study, and toxinology. It is becoming increasingly
well documented that conoidean diversification is actually linked to toxin diver-
sification: recent results tend to show that the venom apparatus played a major
role in the evolution of the group by offering sets of unique molecular adaptations
for efficient interactions with other taxa of marine animals. These, in turn,
enhanced capacities of conoideans to efficiently compete for new ecological
niches, a remarkable example of which is the appearance of fish hunting in
cone snails. Speciation in conoideans was also promoted by other factors, e.g.,
episodic losses of planktotrophy that led to reduced dispersal abilities and
intensive allopatric differentiation. Testing such hypotheses remains primarily
based on the accumulation of data on the diversification patterns (in particular on
the systematics), and there is still a long road ahead to achieving a full under-
standing the evolutionary success of these remarkable mollusks.
Keywords
Conoidea • Taxonomy • phylogeny • Evolution • Conotoxins • Venom apparatus
Introduction
Although it does not come directly to mind when thinking of venomous organisms,
the superfamily Conoidea challenges snakes, scorpions, and spiders for the title of
most diverse taxon both in terms of species and toxins produced. Conoidea
(Toxoglossa) constitutes a group of highly diversified marine gastropods, found in
all oceans, from the tropics to the poles, and at all depths. It represents one of the
most abundant groups of mollusks in bathyal and abyssal environments. Among
them, cone snails (= Conidae) and the toxins they produce (“conotoxins”) have been
studied intensively since the 1970s. Focus has been on cone snails mainly because
they are bigger, easier to sample, and overall better known than other conoideans,
mainly thanks to the amateur shell-collecting community attracted by their shape and
color variation and the fascination of deadly species. However, with around 850 spe-
cies, cone snails only represent the tip of the iceberg: conoideans include 5,000
described species, but it is estimated that there may be 10–20,000 species in total.
Research on Conoidea toxins are now expanding to other families, but cone snails
remain more accessible, better known, and thus by far the most studied. Cone snails
are well established as an important model in pharmacology; the numerous toxins
they produce, mostly acting on ion channels, are characterized by a remarkable
Systematics and Evolution of the Conoidea 3
specificity to channel subtypes. Cone snails thus constitute a vast reservoir of natural
peptides with multiple potential applications in both therapeutics and neuroscience.
Initially, studies on conoideans essentially followed two routes. Toxinology
mainly focused on describing the molecular diversity of the toxins, guided by the
motivation of understanding the functional mechanisms of envenomation and ulti-
mately identifying potential therapeutic applications, while evolutionary biology
relied on the accumulation of knowledge on the diversity of venomous organisms,
including their toxins, to understand the evolutionary processes that lead to the
apparition of such diversity. These two directions remained generally weakly corre-
lated until recently, when a number of complex studies that bridged this gap through
synthesizing of interdisciplinary data started to appear. It resulted in the realization
that the diversification of the conoideans is better explained by integrating the
processes occurring at the molecular level, and vice versa, and that taking into
account phylogenetic, biological, and anatomical information enhances the under-
standing of the functional properties of the toxins. This chapter will thus be devoted
to reviewing the knowledge of the biology, ecology, systematics, anatomy, and
toxinology of the Conoidea, as well as the different evolutionary hypotheses pro-
posed to explain this astonishing diversity of species and toxins.
First, the patterns of diversification related to (i) biology and ecology,
(ii) systematics (alpha-taxonomy and phylogenetic relationships), (iii) venom appa-
ratus, and (iv) toxin diversity will be discussed. As illustrated in Fig. 1, these
different characters provide the arguments to propose hypotheses on the evolution-
ary history of the Conoidea and in particular on their diversification process. The last
section will demonstrate how these patterns can be combined to understand how this
group became one of the most successful taxa of marine animals.
Biology and Ecology
General Characteristics and Biology
Similar to most neogastropods, conoideans are carnivores, mostly, active predators.

They are characterized by a venom apparatus (although secondarily lost in several
lineages; see the section on “Venom Apparatus” below), associated with a highly
modified radula used to inject venom in their prey, and to defeat competitors and/or
predators (Dutertre et al. 2014a; Olivera et al. 2014). Several cases of human death
caused by a contact with cone snails have been reported, with Conus geographus
accounting for most of them (Dutertre et al. 2014b), but other species have also been
involved in accidents (Haddad Junior et al. 2009).
Most species of conoideans, as well as the majority of other gastropods, are
dextral, but some, including several species of Antiplanes, Pseudomelatomidae
(Kantor and Sysoev 1991a), and one species of cone snail, C. adversarius (Hen-
dricks 2005), possess a sinistral shell. Conoidea can be readily segregated into three
groups (Fig. 2) by shell shape: (1) cone snails (Conidae) with conical shells and
proportionally very low spire, high last adult whorl, and narrow slit-like aperture;
Fig. 1 Fields of research that were until recently quite independent now feed off each other to
propose hypotheses on the speciation and diversification process. The outer ring lists the different
fields and type of information for each field (non-exhaustive list); the inner ring provides an
example for the species Conus geographus
(2) family Terebridae (or so-called Auger snails) with very tall multi-whorled spire,
low aperture, and a very short siphonal canal; and (3) the rest of Conoidea that are
characterized by intermediate shell proportions, and are collectively referred to as
“turrids”. While the two former groups are rather compact and easily recognizable
and correspond to distinct phylogenetic clades, the “turrids” are extremely variable
and comprise an array of lineages that do not form a natural grouping (the polyphyly
of turrids has been convincingly demonstrated – see “Systematics” section below).
Nevertheless, many “turrids” share one characteristic feature, a so-called anal sinus
on the adapical part of the labrum (outer aperture lip), which allows their easy
recognition. However, this feature is also present in some non-conoidean gastropod
taxa (e.g., family Bursidae and subfamily Typhinae in the Muricidae) and at the same
time is absent in many groups of “turrids.”
Some lineages of Conoidea have a reduced or entirely lacking operculum. The
protoconch can be multispiral or paucispiral, corresponding to planktotrophic or
non-planktotrophic larvae, and reflecting high or low dispersal abilities, respectively
(Jablonski and Lutz 1980). Likewise in other caenogastropods, sexes are separate
and the fertilization is internal in all conoideans. One case of sexual dimorphism of
Fig. 2 Successive classification schemes proposed for the Conoidea. Top row, classification mainly
based on shell characters (Powell 1966); middle row, classification based on anatomical and shell
characters (Taylor et al. 1993); and bottom row, classification based on molecular data (Puillandre
et al. 2011a; Kantor et al. 2012a), with the corresponding phylogenetic tree. The validity of the
Strictispiridae remains to be tested. The Turridae experienced a drastic reduction of its included
diversity; the Conidae have been successfully extended and then reduced to its original concept (but
see Tucker and Tenorio (2009) for subdivisions within the Conidae); the Terebridae is the only
family that has remained unchanged
the shell has been reported in Gemmula (Kantor and Sysoev 1991b). Egg capsule
morphology is variable and well known in cone snails (Kohn 2012).
Feeding Habits and Habitat
The unprecedentedly rich radiation of the Conoidea is usually attributed to a strict

prey specialization, which is assumed to apply to most members of the superfamily
(Taylor et al. 1980). Conoidean preys are mostly polychaetes, and less frequently
nemerteans, hemichordates, and mollusks including gastropods (Miller and Morton
1990), bivalves (Fujikura et al. 2009), and even cephalopods (Marshall 1981). Cone
snails are remarkable in this respect, as they are also able to feed on fish (Kohn 1956)
and crustaceans (Biggs et al. 2010). Apart from the cone snails, preys are known
only for around 50 species, with most data coming from the examination of the gut
contents. Duda et al. (2009a) confirmed the diet of Conus ebraeus using a DNA
barcoding approach. The type of prey in cone snails can also be inferred from
indirect evidences – the morphology of the radula (Tucker and Tenorio 2009), or
using “reverse ecology” (Olivera 2002), from the composition of the venom.
Feeding strategies observed on cone snails are diverse (“hook-and-line” = “

taser-and-tether” = harpoon strategy, “net fishing,” “strike-and-stalk” (Olivera
et al. 2014, 2015)), even in fish-hunting species, to which most observations refer
(Olivera et al. 2014). These different strategies are also associated with diverse
behaviors as described for the fish-hunting cone snails, including mimetic with
brittle star arms and worms, or group attacks (Olivera et al. 2015). Scavenger
behavior has also been reported in Californiconus californicus, the cone snail
species with the widest prey spectrum, feeding on worms, mollusks, shrimps, and
fish (Biggs et al. 2010). Remarkably, some cone snails are able to engulf a prey that
is nearly equal to their own size, the ability facilitated by the reduction of the inner
shell walls (Kantor 2007a). One specimen of cone snail typically feeds on a single
prey per night (Kohn 1959), but see Olivera et al. (2015). Other specific behaviors
likely related to feeding, including ‘surfboarding’, have been described for
Terebridae (Miller 1970).
Several conoidean species, even when phylogenetically closely related, can exist
in sympatry, but probably by utilizing different niches (e.g., if the prey type is the
same, the size of the prey will differ (Kohn 1980)). Up to 30 species of cone snails
can be found in a single site, which was demonstrated for both recent (Muttenthaler
et al. 2012) and fossil species (Hendricks 2015); some unpublished data suggest that
up to 70 species can co-occur in a single locality (P. Bouchet pers. com). Typically,
closely related Conus species will feed on different prey species (Kohn 1980; Duda
et al. 2001). The same pattern was shown for terebrids: Fedosov et al. (2014) applied
stable isotope analysis (SIA) to demonstrate existence of the resource partitioning in
sympatric species of Terebridae that indirectly proves the alternate feeding special-
izations in multiple species of the family co-occurring within one habitat. In turn,
fish (Simpson et al. 2013) and turtles (Behera et al. 2015) have been reported to feed
on conoideans. In addition, the analysis by Dutertre et al. (2014a) implied that one of
the important natural enemies of cone snails are octopi. Until recently, the use of the
venom apparatus to defeat competitors or predators (and not to capture preys) was
mostly hypothetical, relying mostly on observations in aquaria (Aman et al. 2015;
Jin et al. 2015) (see “Speciation and Diversification” below).
Three species of cone snails are currently listed as critically endangered, 11 as
endangered, 27 as vulnerable, and 26 as near-threatened in the IUCN Red List
(Peters et al. 2013). Peters et al. (2015) also shown that 70 % of the red-listed
species (regardless of status) are known from areas that are presently highly
impacted by humans. In addition, over-sampling should not be neglected in consid-
ering threats to cone snails, especially by shell collectors (Duda et al. 2004).
Species Diversity
The enormous species diversity contained within Conoidea makes it one of the most
speciose groups of marine animals: the World Register of Marine Species (WoRMS,
http://www.marinespecies.org/, accessed in November 2015) lists 4,710 valid spe-
cies for which 9,492 names are available. However, there are several lines of
evidence suggesting that this number is, in fact, a great underestimation of actual
conoidean diversity, and recent estimations (Bouchet et al. 2009) suggest that there
are probably as many as 20,000 species of Conoidea. These estimates are based on
the proportion of undescribed species calculated in well-sampled local faunas, in
particular in New Caledonia (Bouchet et al. 2002, 2009). The high proportion of
undescribed species is, in part, a result of their rarity: 41 % of the turrid species in
New Caledonia were represented by only a single specimen in the data set of
Bouchet et al. (2009), and 73 % of the turrid species in New Caledonia were
recovered only as dead shells (Bouchet et al. 2002). Remarkably, only 17 % of
these species were also found in the surrounding archipelago. The other factor,
which makes the adequate documentation of conoidean fauna rather hard, is a high
proportion of very small species that are difficult to collect and study.
With 827 valid species, Conidae is currently considered the most diverse family;
however, this is probably the family with the lowest proportion of undescribed
species. If the proportion of undescribed species is taken into account, the families
Pseudomelatomidae, Raphitomidae, and Mangeliidae may well be even more
speciose. All these results tend to converge on the idea that a large part of Conoidea
diversity remains unknown.
Like all shelled mollusks, conoideans have a large fossil record (Hendricks 2005;
Hendricks 2015; Todd and Johnson 2013). The first fossil confidently identified as a
cone snail is dated to 50–55 MY, while the oldest confident record of turrid in the
fossil records is dated to the Cretaceous (Powell 1966); however, the uncertainty in
the attribution of some fossils to the Conoidea remains important, and the minimum
age of the group remains to be defined.
Systematics
Phylogeny and Classification
Conoidea (or Toxoglossa) is included in the order Neogastropoda (Mollusca,

Caenogastropoda) in a rank of superfamily, together with five other currently
recognized superfamilies, Cancellarioidea, Buccinoidea, Muricoidea, Olivoidea,
and Turbinelloidea (Fedosov et al. unpublished). Because of their distinctive shell
shape, compared to the rest of the Conoidea, the cone snails (Conidae) and the auger
snails (Terebridae) were established as separate families in the earliest classifications
of the group (e.g., Fischer 1887) (Fig. 2). The major confusion lay with the
remaining Conoidea, which was subject to numerous alterations. Sometimes, they
were included in Conidae (Thiele 1929), but most often everything besides the cones
and terebrids were placed into the very heterogeneous family Turridae
(or Pleurotomidae). Hedley (1922) correctly remarked that the family Turridae “is
more perplexing than any other molluscan family.”
The principal scheme, with three families recognized within Conoidea, persisted
until the 1990s with a few modifications concerning the subdivision of the Turridae
made by some later authors (Kilburn 1983; McLean 1971; Morrison 1965); these
modifications essentially multiplied the number of the subfamilies in the Turridae,

but their relationships remained unrevised. A first revolution occurred in 1993, when
Taylor et al. (1993) proposed a completely new classification based on the cladistic
analysis of the combined data set of shell, radular and anatomical characters (earlier
classifications were built upon the “intuitive” approach using mainly shell charac-
ters). Six families were proposed within the Conoidea, including the family Conidae
extended to include five subfamilies earlier placed in Turridae. The names Conidae,
either restricted to the cone snails or including also several turrid groups, and
Turridae, either encompassing all the conoideans except cone and auger snails or
restricted to a single group of turrids, have been thus used to designate different
taxonomic concepts in the different classifications. Tucker and Tenorio (2009)
proposed another classification of the Conoidea, based on the previous ones but
also on the published molecular phylogenies. They proposed to split the Conoidea in
two superfamilies, Conoidea and Turroidea (corresponding to the clade A and B
defined in the molecular phylogeny of Puillandre et al. (2008)), with a total of
15 families assigned to them, the traditional Conidae being split into four different
families.
The large panel of alternative classifications proposed for Conoidea attests to the
difficulty encountered by taxonomists. The differences between the classifications
are, at least partly, the consequence of the use of different characters (shell, radula,
anatomy, DNA) by the taxonomists; what is considered an essential character by one
taxonomist can be regarded as nonsignificant by another. Different schemes of
relationships resulting when different sets of characters are analyzed are mainly
explained by the fact that morphological evolution of Conoidea presents multiple
cases of homoplasy or retention of ancestral polymorphism. Thus, the phylogenetic
signal of many characters is masked to the extent that they cannot be used to infer
phylogenetic relationships. Whereas DNA characters are, by definition, genetically
determined, phenotypic traits, such as shell, radula, or anatomical features, are not
necessarily genetically determined and can vary with the environment even when the
genome remains unchanged.
The radula, in particular, although in general useful to infer phylogenetic relation-
ships (Tucker and Tenorio 2009; Kantor et al. 2008), is known to be shaped by the
diet. As phylogenetically distant conoideans can feed on similar preys, they may
acquire strikingly similar radulae (Olivera et al. 2015; Castelin et al. 2012), which
therefore would not reflect phylogenetic relationships. The same conclusion has
been reached for shell characters: molecular phylogenies revealed that highly similar
shells can actually correspond to two different families (e.g., almost identical
representatives of the former genus “Leucosyrinx” are now placed in the
Sibogasyrinx, Cochlespiridae and Leucosyrinx, Pseudomelatomidae). Even more
prominent are the cases where the counterparts not only belong to different families
but to different major clades of the Conoidea, e.g., Cochlespira (Cochlespiridae)
versus Toxicochlespira (Mangeliidae). Needless to say, misinterpretation of shell
morphology has led to erroneous placements of many conoidean species and genera;
these were resolved only when additional morphological or molecular data were
involved. Once the evolutionary lability of the shell in Conoidea was demonstrated,
other sources of phylogenetic information were examined. In particular, the high

taxonomic value of the protoconch was consistently promoted by Powell (1966) and
has been widely used in taxonomy (Bouchet and Waren 1980). Indeed, some high-
level conoidean taxa demonstrate characteristic sculpture patterns of the multispiral
protoconch. However, among the many types of sculpture found in conoideans, not
a single one is shared by all the members of a clade, and only a few of them are found
in only one lineage. In addition, all species with non-planktotrophic development
have a paucispiral protoconch, either weakly sculptured, or with no sculpture
whatsoever, and their placement therefore was troublesome. Using morphological
characters only, Bouchet (1990) discouraged the separation of species with identical
teleoconch but different types of protoconch in different genera as was suggested by
Powell (1964) and instead proposed that even sister species could have different
types of protoconch morphology (planktotrophic vs. non-planktotrophic). Molecular
analyses confirmed this hypothesis, the “planktotrophic larvae” character state being
lost regularly during the evolution of the Conoidea (Cunha et al. 2005; Duda and
Palumbi 1999; Fedosov and Puillandre 2012; Remigio and Duda 2008).
Several phylogenies have been proposed for the Conoidea that combine molec-
ular data and cladistic methods; the latest of them (Puillandre et al. 2011a) provided a
basis for the simultaneously published classification of the superfamily (Bouchet
et al. 2011). Fifteen families were recognized in this classification within the
superfamily Conoidea, with the family Conidae including all cone snails. One year
later, the sixteenth family, Bouchetispiridae, was added (Kantor et al. 2012a).
Although some previous classifications, not based on molecular data, contradicted
each other and are not in full agreement with the molecular-based classification,
some phylogenetic relationships were actually already proposed in the literature, and
in particular several features of the Taylor et al.’s classification (Taylor et al. 1993)
were actually confirmed with DNA characters. It should also be noted that the
classification by Bouchet et al. (2011), now widely accepted, is based on the
molecular phylogeny and thus does not include fossil lineages, contrary to the
shell-based classifications. Hendricks (Hendricks 2005) published the only phylog-
eny of Conoidea (more specifically, cone snails) that includes both recent and fossil
taxa, combining morphological and molecular characters. Smith and Hendricks
(Smith and Hendricks 2013) explored the possibility of including “cladistic
pseudofossils” (recent species for which the molecular information is discarded) in
a phylogeny based on both morphological and molecular characters, to test whether
morphological data alone can place the species in the same position as the molecular
data: the success rate was, however, below 50 %.
Molecular phylogenies restricted to subgroups within the Conoidea have been
published in abundance. They are especially numerous for cone snails (listed in
Puillandre et al. (2014a)) and scarcer for Terebridae (Castelin et al. 2012; Holford
et al. 2009a, b) or Turridae sensu stricto (Fedosov et al. 2011; Heralde et al. 2007,
2010; Puillandre et al. 2012a). Other phylogenies that have been published are either
limited to a few species only or reproduce already published phylogenies. Finally,
species delimitation articles (see the “Alpha-taxonomy” section) often include a
molecular phylogeny, but their scope is restricted.
Genome-based approaches have also been used in Conoidea: Bandyopadhyay

et al. (2006) published the first mitochondrial genome of any Conoidea,
Lophiotoma cerithiformis (Turridae), and several others have been published since
then (Conus textile, Conus consors, Conus borgesi, Oxymeris dimidiata, Fusiturris
similis, Conus tulipa, and Conus tribblei). Hu et al. (2011) published a preliminary
version of the nuclear genome of Conus bullatus, and Barghi et al. (2015a) published
the genome of Conus tribblei. Additionally, a karyological analysis of Conus magus
revealed a diploid chromosome number of 32 (Dalet et al. 2015). Several
transcriptomes of venom glands of Conoidea are also available (Barghi
et al. 2015b; Gonzales and Saloma 2014; Gorson et al. 2015; Prashanth
et al. 2014; Robinson et al. 2014). Even though they have not yet been used to
discuss phylogenetic relationships within the Conoidea, these data represent an
interesting source of new molecular markers that could be used to improve the
knowledge on the systematics of the Conoidea.
Alpha-taxonomy
Similar to classification, species delimitation in Conoidea is also a difficult task, for

various reasons. First, similar to most mollusks, species delimitation and descrip-
tions are mainly based on shell characters, even in recent years. Diagnoses, espe-
cially in older literature, are sometimes short, and applicable to several different
species, leading sometimes to endless nomenclatural debates (Janssen et al. 2014).
The high number of synonyms adds to the confusion. Second, it is generally difficult
to identify and formalize discrete morphological characters. The shell variability
often constitutes a continuum, and it can be very difficult to identify the limits
between the species along this continuum. Species delimitations are thus often
opinions based on an interpretation of the shell, and the absence of a clear description
and formalization of the characters prevents any formal test of species hypotheses.
Third, Conoidea diversity remains largely unknown, and each new expedition in the
field collects new species to be described, but also new forms and new occurrences
for already described species that requires taxonomists to modify their previously
proposed species hypotheses. Finally, difference in morphological characters can
reflect environmental differences (including, for example, radula convergence linked
to prey preferences as described above) and not evolutionary relationships and
species limits. These difficulties are not new: in 1884, Tryon stated “In no other
group of mollusks is it so difficult to make a satisfactory classification as in the
Pleurotomidae (= Turridae). The forms are exceedingly numerous, and known in
many species to be very variable in their characters, whilst the material for the
recognition of most of those described is generally scanty” (Tryon 1884).
As in other taxa, species delimitation tends to be more integrative, with joint
analysis of morphological, molecular, and ecological characters. Other methods and
characters have recently appeared in the toolbox, with geometric morphometry
(Smith and Hendricks 2013; Puillandre et al. 2009) and molecular analyses
(Cunha et al. 2008; Puillandre et al. 2011b), including conotoxin-coding genes
(Duda et al. 2008; Puillandre et al. 2014b). The general tendency is toward splitting
of previously recognized morphospecies in several species (e.g., Puillandre
et al. (2010a)), subtle differences in the shell being found to be correlated with
fixed genetic differences. Some exceptions can be found within cone snails, where
geographic varieties have been described as different species in some cases.
Foregut Anatomy and Venom Apparatus
The anatomy of the foregut of Conoidea is quite variable, although several characters
can be considered to be synapomorphies. The key apomorphy of the group is the
venom apparatus, consisting of normally long convoluted tubular venom gland
(often incorrectly referred to as “duct”) and terminating in uni- or multilayered
muscular bulb, serving as a propulsory organ (Fig. 3a, c). The venom gland is
presently considered homologous to midgut gland of other neogastropods, while,
based on the embryonic development of Conus lividus, the muscular bulb is a de
novo structure (Page 2012). It is the sophisticated mechanism of venom delivery
provided by radular functioning in conjunction with the venom gland, rather than the
presence of the gland itself, that is the basis of the unique feeding mechanism of
Conoidea (Kantor and Puillandre 2012).
In gastropods, the radular apparatus consists of a radular ribbon with numerous
transverse rows of teeth (radula per se) and odontophore. The latter is a massive
organ, consisting of several sub-radular cartilages and muscles, which provides the
movement. The radular apparatus is situated in the buccal cavity in close proximity
to the mouth and can be partially everted through the mouth opening. The radula
serves as an integrated organ for rasping or gripping food objects. In the vast
majority of Conoidea, the buccal cavity, together with the radular apparatus, is
situated at the proboscis base (Fig. 3a, c). Consequently, the radula cannot be
protruded through the mouth to directly contact prey. Instead, in the early stages of
conoidean evolution, a peculiar and unique mechanism appeared, whereby individ-
ual marginal radular teeth are dislodged from the membrane, transported to probos-
cis tip, where they are fixed by a sphincter, and used for penetration of the prey’s
integument (Fig. 3b). The function of the radular apparatus as integral organ in the
buccal cavity is thus very limited, since the prey is already captured and passed along
proboscis before the contact with radula is made. Nevertheless, some functions may
still be present, since in significant number of conoideans (clade B as defined in the
molecular phylogeny of Puillandre et al. (2008)) the odontophore with muscles that
provide movement of the radular membrane persists. The transformation of the
radular apparatus in representatives of clade A was much more radical, and involved
the complete disappearance of the odontophore, while the radular membrane is
vestigial or even absent in some Conus (e.g., in C. striatus).
The radula itself is rather variable in Conoidea, having from five teeth in a
transverse row (Drilliidae) to only two marginals (Powell 1966; Taylor et al. 1993;
Kantor and Puillandre 2012). Due to a particularly important role in feeding,
marginal teeth are probably under evolutionary selection, and their morphology
Fig. 3 Diagrammatic sections through the anterior foregut of Conoidea. (a) Anterior foregut of the
Conoidea with non-hypodermic marginal radular teeth and odontophore (generalized representative
of the clade B). A duplex marginal tooth detached from the sub-radular membrane is used at the
proboscis tip for stabbing and envenomating the prey. (b) Section of the tip of the proboscis with the
duplex marginal tooth held by sphincters of the buccal tube (actual specimen of Aforia kupriyanovi
Sysoev and Kantor 1988, Cochlespiridae). (c) Anterior foregut of the Terebridae with developed
rhynchostomal introvert, radula of hypodermic marginal teeth and lacking odontophore. A hypo-
dermic marginal tooth detached from the sub-radular membrane is used at the proboscis tip.
Abbreviations: bm buccal mass with radular apparatus at proboscis base; bt buccal tube leading
from buccal mass to mouth on proboscis tip; bts buccal tube sphincter holding the base of the tooth
at proboscis tip; dmt duplex (non-hypodermic) marginal tooth at the proboscis tip; intr
rhynchostomal introvert, or pseudoproboscis; mb muscular bulb of the venom gland; oe esophagus;
pr proboscis; rhs rhynchostome, or false mouth, through which the proboscis is everted; rs radular
sac without odontophore; rsod radular sac with odontophore; sg salivary gland; vg venom gland
reflects feeding specializations. In practically all groups of Conoidea of clade B, the

marginal teeth are reinforced by different elements, and they have accessory limb in
“duplex” teeth, or thickened edges, or obtain semi-enrolled form (trough shaped) and
rarely even become hypodermic (Kantor and Taylor 2000) (Fig. 4d, g, h). The use of
separate marginal teeth at the proboscis tip was demonstrated initially in clade B
conoideans by serial histological sections of the tooth gripped by special sphincter
(Kantor and Taylor 1991; Sysoev and Kantor 1987). Moreover, the teeth were
gripped by the proboscis tip sphincter in a large proportion of studied preserved
specimens. Since the studied specimens were not specially collected or treated, this
is probably an indication that the tooth is permanently or most of time “primed” for
use.
Fig. 4 Radulae of Conoidea. (a–c) Hypodermic marginal teeth of clade A of Conoidea, which
includes only the species with hypodermic teeth and without odontophore. (a) Strongly barbed
tooth of fish-hunting Conus striatus (family Conidae), and enlarged tip of the tooth. (b) Bathytoma
neocaledonica (family Borsoniidae). (c) Mangelia fieldeni (family Mangeliidae). (d–k) Radulae of
clade B of Conoidea, which includes species with odontophore. (d) Typical radula with duplex
marginal teeth and central tooth, Turridrupa jubata (Turridae). (e) Semi-enrolled, nearly hypoder-
mic marginal teeth, Toxiclionella tumida (family Clavatulidae). (f) Hypodermic marginal tooth,
Cruziturricula arcuata (family Drilliidae). (g) Radula with semi-enrolled, trough-shaped marginal
teeth, Ptychobela suturalis (family Pseudomelatomidae). (h–k) Different radulae of Terebridae. (h)
Radula with duplex marginal teeth, Clathroterebra poppei. Next three figures depict hypodermic
marginal teeth in Terebridae, originated independently in three clades, identified by molecular
phylogeny: (i) Terebra cingulifera, (j) Hastula lanceata, and (k) Myurella kilburni. Abbreviations:
ap accessory process, ct central tooth, mt marginal tooth
In Conoidea of clade A (which are better known since they include Conidae s.s.),
the marginal radular teeth normally possess very characteristic (hypodermic) mor-
phology, i.e., a hollow harpoon of syringe needle shape with holes at the base and
tip (Fig. 4a–c). In such teeth, toxins are injected through the central tooth cavity.
This, at the first glance, seems to be more efficient and may explain the broader
known diet range (including fish) compared to clade B. According to phylogenetic
analysis, the hypodermic teeth in clade A appeared only once. Another proof of
higher efficiency may be the independent origin of similar hypodermic teeth in
unrelated lineages of Conoidea in clade B – in Clavatulidae (Toxiclionella (Kilburn
1985, Kantor and Taylor 2000); Fig. 4e), in Drilliidae (Imaclava (Shimek and Kohn
1981)), and at least three times in Terebridae (Castelin et al. 2012) (Fig. 4i–k). One of
the still unexplained peculiarities of some hypodermic teeth that appeared in at least
two non-related lineages (Mangelia from Mangeliidae of clade A, and Impages from
Terebridae of clade B) is the presence of numerous lateral holes in the tooth (Fig. 4c)
(Imperial et al. 2007a). The morphology of hypodermic teeth in at least Conidae
s.s. reflects their diet (Kohn et al. 1999), and it can change in ontogeny, probably due
to diet shift (Nybakken 1990). Even within the fish-hunting cones, the radular
morphology differs in species with different hunting strategy. Species that are
known to use the “taser-and-tether” strategy have radular teeth that usually have a
long accessory process (long outgrowth directed toward the tooth base; Fig 4a),
often bearing an additional barb that can tether fish securely after a successful strike.
In species using the “net” strategy, the teeth are poorly barbed at the tip (Tucker and
Tenorio 2009; Olivera et al. 2015).
The unique envenomation mechanism of Conoidea is possible only by coordi-
nated action of separate marginal teeth and proboscis. The proboscis grips the tooth,
bringing it in contact with the prey and providing the impulse for injecting the tooth
into the body of the prey. It also channels the flow of toxins from the venom gland.
The mechanism of actual injection remains unclear, but for fish-hunting Conus catus
the tooth is propelled by a high-speed ballistic mechanism after the proboscis tip
makes contact with the fish skin (Schulz et al. 2004) and is then gripped by proboscis
tip to retain control of the stung fish prey while retracting. Thus, the sphincter at the
proboscis tip is able to maintain a strong grip on a relatively very thin object, only a
fraction of a millimeter in diameter. Since the radula of Conoidea cannot be used for
tearing or rasping prey (as it is either not an integral organ in clade A, or is situated at
the base of proboscis and cannot protrude through mouth in clade B), the
immobilized prey has to be swallowed whole. Thus, the very same sphincter of
the mouth has the ability to undergo great expansion. For mollusk-hunting Conus
textile, expansion of up to 50 times resting size was demonstrated (Kantor 2007b).
The swallowing of particularly large prey relative to the size of the predator, such as
mollusks or fish, is facilitated by a peculiar contraction of the proboscis. In Conus,
many Raphitomidae, and some Mangeliidae, this forms multiple telescopic folds,
and when in a contracted position, the proboscis occupies a small posterior part of
the rhynchocoel (proboscis sheath) (Kantor and Taylor 2002).
In addition to the venom gland, there are salivary glands and in some groups,
accessory salivary glands associated with anterior foregut. Their secretion remains
grossly unstudied and different functions were proposed for salivary glands includ-
ing cleaning the cellular debris in the radular tooth or “activating the venom”
(Shimek 1975). Biggs et al. (2008) revealed transcripts whose predicted gene
products, after posttranslational processing, strikingly resemble mature conopeptides
belonging to the A-conotoxin superfamily.
It is worth mentioning the tendency in Conoidea toward reduction and complete

loss of different foregut structures. First, radula-less turrids without proboscis,
venom, or salivary glands were found in Raphitomidae (Smith 1967); later, this
phenomenon was also recorded in several families of Conoidea other (Raphitomidae
(Kantor and Taylor 2002; Kantor and Sysoev 1989); Horaiclavidae (Fedosov and
Kantor 2008); Borsoniidae (Medinskaya and Sysoev 2003)). The loss of the venom
gland is usually (although not always) accompanied by the loss of the radula and
proboscis. A remarkable exception is the genus Strictispira (Strictispiridae), which
possess proboscis and large radulae with odontophore, but have no traces of the
venom gland (Kantor and Taylor 1994). Multiple independent losses of different
structures of the foregut were demonstrated for Terebridae (Castelin et al. 2012).
Fedosov et al. (2014) demonstrated that the isotopic trophic niches of the radula-less
terebrids are not wider than in those species, possessing radulae, which implies that
the loss of radula does not affect degree of feeding specialization.
In some Conoidea, the anterior pre-tentacular region of the head becomes greatly
expanded and forms either a large funnel (called rostrum in Conus, also present in
many Raphitomidae) or eversible tube (pseudoproboscis, or rhynchostomal intro-
vert). The latter is found in some Mangeliidae, most Raphitomidae, and all
Terebridae (Taylor et al. 1993; Kantor and Taylor 2002) (Fig. 3c). The rostrum and
pseudoproboscis are actively utilized in prey capture, as was observed for terebrids
(Miller 1975).
Toxin Diversity and Evolution
Reviews on the structural and functional diversity of the toxins produced by the
Conoidea, and in particular by the cone snails, as well as their potential or confirmed
therapeutic importance, are numerous, and this will not be the purpose here (for a
detailed review see, e.g., Olivera et al. (2014); Prashanth et al. (2014)). After a short
introduction on these topics, the evolutionary aspects related to toxin diversity will
be detailed.
What Is a Conotoxin?
Physiologically active peptides produced in the venom gland show some structural
peculiarities that, although not shared among all types of molecules, allow a general
description of conoidean toxins.
1. They are commonly short (12–40 residues) molecules with a high proportion of
cysteines, expressed in the venom gland of cone snails (typically genus Conus).
Their secondary structure is stabilized by the disulfide cross-links and achieved
through the specific folding patterns. The rather conservative arrangement of Cys
residues in the conoidean toxin peptides (Cys framework) is the major determi-
nant of their specific conformation. Many Conus venom peptides, however, are
either long, or contain few or no cysteine residues; a more general term

“conopeptides” therefore refers to the whole diversity of Conus venom peptides
(Puillandre et al. 2012b). Conopeptides are characterized by a high proportion of
posttranslationally modified residues. Of these, some identified modifications
(hydroxyproline, O-glycosylated serine, or tryptophan) are widespread in the
animal world, while others (6-bromtryptophan, γ-carboxyglutamate, and
sulfotyrosine) are exclusive for Conoidea (Bandyopadhyay et al. 2002). The
venom gland peptides in other conoidean lineages, i.e., Turridae (turripeptides),
Terebridae (teretoxins), and Pseudomelatomidae (crassipeptides), are also disul-
fide rich, but are generally longer and less posttranslationally modified than those
in Conus.
2. All studied conoidean peptides are synthesized in a same manner: the translation
of a messenger RNA produces a peptide precursor, which consists of three
conservatively arranged functional blocks. An N-terminal signal region followed
by a pro-peptide region (which may be absent in some cases), and a mature
peptide region on the C-terminal end of the precursor (Olivera 2006). The
precursor undergoes maturation with formation of a specific pattern of disulfide
cross-links, posttranslational modification of specific amino-acid residues, and
removal of the signal and pro-peptide regions.
3. One of the remarkable characteristics of conoidean venom peptides is a disparity
between extremely structurally diverse mature toxin regions (except for the Cys
framework) and highly conserved signal and pro-peptide regions. Signal regions
are used in classification of venom peptides into gene superfamilies (Puillandre
et al. 2012b), designated by uppercase Latin letters. The three most widespread
are the A-, O-, and M-superfamilies. The conotoxin families (designated with
lowercase Greek letters – α to ω) are determined by the molecular target.
4. Most conopeptides target voltage- and ligand-gated ion channels that mediate
dissemination of nerve impulses and neuromuscular transmission. Among the
best studied are the ω-conotoxins (targeting presynaptic Ca channels), μ- and
δ-conotoxins (blockers of Na channels), and α-conotoxins (antagonists of the
postsynaptic nicotinic receptors – nAChRc). Cysteine-poor venom peptides tar-
get a variety of physiological pathways, e.g., conopressin (vasopressin receptors),
conantokin (inhibitor of NMDA receptors), contulakin G (agonist of neurotensin
receptors), χ-conotoxin Mr5A (blocker of noradrenalin transporter NET). The
most recent discovery is the specialized fish-type insulin produced in the venom
gland of C. geographus (Safavi-Hemami et al. 2015).
5. An outstanding property of the conopeptides is their extreme target specificity.
Venom of one Conus species often includes multiple conotoxins of one family,
each of which is “responsible” for its own receptor’s subtype. For example,
among ω-conotoxins of Conus magus, MVIIA acts specifically on Cav2.2 and
MVIIB on Cav2.1, and neither of them shows any affinity to the other’s molecular
target (Nielsen et al. 1999). Even higher diversity and specificity was demon-
strated for the α-conotoxins reflecting structural diversity and complexity of
nAChRc (Jacobsen et al. 1997).
6. In complex conoidean venoms, individual peptides act in concert to achieve a

major physiological effect. The functional groups of the conoidean venom pep-
tides that affect multiple molecular targets within one physiological circuit, and
thus act synergistically, are known as “cabals.” “Thus, the evolution of conoidean
gastropods has produced what is in essence a highly sophisticated equivalent to
combination drug therapy” (Olivera et al. 2014).
7. The conoidean venom gland is compartmentalized, with distal and proximal
portions of the gland specialized to produce different venom components. It
was demonstrated that venoms produced in distal and proximal portions of the
Conus venom gland differ in functionality and may be discriminatively used by
the cone snail for predation or defense in response to different external stimuli
((Dutertre et al. 2014a); see below for more details).
Phylogeny of Conoidean Venom Peptides
The principles upon which the classification of conoidean toxins is based were
reassessed using a phylogenetic approach (Puillandre et al. 2012b). The traditional
definition of the gene superfamilies, based on the similarities in the signal sequences,
was generally confirmed. However, the astonishing diversity of potentially new
superfamilies (or at least new signal sequences) revealed by the numerous recent
transcriptomic analyses tends to somewhat blur the limits between superfamilies,
with the signal diversity resembling a continuum of variability rather than a partition
of clearly different signal sequences. Until recently, cysteine-rich and cysteine-poor
conotoxins were considered to be two independent groups of Conus venom peptides;
however, this distinction was not supported by the phylogenetic analysis. Several
groups of cysteine-poor conopeptides were shown to share similar signal sequences
with some well-established gene superfamilies of cysteine-rich conopeptides; there-
fore, in terms of relatedness, different groups of cysteine-poor conopeptides are not
closer to each other than they are to some cysteine-rich conotoxins, and vice versa
(Puillandre et al. 2012b). Thus, the classifications based on the Cys pattern and the
function are purely practical. A given Cys pattern or function can be found poten-
tially in several venom peptide superfamilies, and a given superfamily may include
several Cys patterns and/or functions (Prashanth et al. 2014). Phylogenetic
approaches also suggested a complex evolutionary origin of the conoidean toxins,
with different peptides found in single venom recruited from a diversity of peptide-
coding genes, with various physiological functions (Casewell et al. 2013). This
remarkable complexity of conoidean venoms is conspicuously illustrated by the
conkunitzins, structurally divergent from “traditional” conotoxins and bearing
Kunitz domains, which are conserved among many animal lineages, and by recently
discovered Conus venom insulins.
Molecular Mechanisms of Conopeptide Diversification
Several hypotheses have been proposed to explain the mechanisms underlying

diversification of conoidean toxins leading to the recruitment of novel venom
peptides. These include active hypermutational mechanisms (Olivera et al. 2014;
Espiritu et al. 2001), lack of a mismatch repair system in the mature toxin (Olivera
et al. 1999), recombination (Olivera et al. 1999), and exon shuffling (Pi et al. 2006).
These generate a pool of “trial” venom peptides that may contribute to the efficiency
of predation or defense or lead to a shift in the prey specialization; in any of these
cases, the gene undergoes positive selection (Casewell et al. 2013; Duda 2008;
Puillandre et al. 2010b), especially in the case of highly expressed genes (Chang and
Duda 2014). Additionally, expression of some toxin genes may be inactivated, and
the silenced genes retained in the genome. Toxins gene silencing and revival give
rise to the so-called lazarotoxins (Conticello et al. 2001), peptide products of some
ancestral toxin genes reemployed and intensively expressed by some derived taxon.
The aforementioned genetic mechanisms generate species-specific sets of expressed
venom toxin genes (estimated between 100 and 200 per species) in cone snails. At
the peptide level, additional mechanisms including alternative cleaving, posttrans-
lational modifications, and N- and C-terminal truncations further increase the diver-
sity of toxins in the mature venom (Dutertre et al. 2013), even if most (but not all,
including toxins confirmed by proteomic approaches) represent rare transcripts
(Prashanth et al. 2014).
It is generally accepted that each cone snail species possesses its own arsenal of
toxins (Olivera 2006), partly shaped by the distinctive species-specific gene expres-
sion profiles (Conticello et al. 2001). The crucial question is how the processes
underlying the hyper-variability of venom peptides (outlined in the previous para-
graph) are coordinated to shape individual peptide repertoires in the diverging
species. A recent study on the conopeptide expression patterns in two closely related
Conus species (Barghi et al. 2015c) shed some light on this matter. Worm-hunting
species of the subgenus Splinoconus, Conus tribblei, and Conus lenavati demon-
strated unusual diversity of gene superfamilies expressed in their venom glands –
39 and 40, respectively, with 100 and 132 unique venom peptides identified in these
species through high-throughput sequencing. Sixty-seven pairs of orthologs were
identified in these two species, and 21 of these pairs showed identical mature toxin
regions. Interestingly, highly expressed toxin gene superfamilies tend to show highly
correlated expression levels, although may differ in the number of unique peptides
expressed in each species, whereas the moderately expressed gene superfamilies
show higher between-species variation in expression. Finally, about 20 % of toxin
gene superfamilies expressed by each species (eight in C. lenavati and seven in
C. tribblei) were not detected in the other. Thus, fine-tuning of the expression pattern
and concomitant recruitment (revival) of the novel (lazaro-) toxins underlie diver-
gence of the Conus venoms ((Barghi et al. 2015c); see also Chang and Duda (2014)).
Duda and Palumbi (2004) found identical toxins in the two clades of fish-hunting
cone snails, which are supposedly not sister groups (although this remains to be
demonstrated; see “Species and Diversification” section), which could be explained
either by a convergence between the two clades, or by gene silencing. In the latter
case, some ancestral toxins become increasingly highly expressed in distantly related
clades that developed piscivory, while remaining silent in the non-fish-hunting
clades. Alternatively, these toxins might also be expressed in worm-hunting cone
snails, to be used not in predation but in defense (Dutertre et al. 2014a). Conversely,
some remarkable cases are known (Imperial et al. 2007b) when two unrelated
lineages of fish-hunting cone snails employ structurally highly divergent toxins,
that specifically interact with same molecular target in the body of prey fish.
It should be noted that the complement of toxins varies among individuals of the
same species; intraspecific and intraindividual variations of toxin diversity, either at
the genetic level or when looking at the expressed toxins, have been reported in
many studies (Prashanth et al. 2014; Jakubowski et al. 2005). Differences in toxin
composition and/or expression between different development stages in one species
have also been documented (Chang and Duda 2016; Safavi-Hemami et al. 2011).
Interestingly, these differences likely parallel the ontogenetic changes in radular
morphology supposedly reflecting prey preferences that are different in juvenile and
adult cone snails (Nybakken and Perron 1988). To add an additional degree of
complexity, some studies have proposed that toxins can be produced in organs
other than the venom gland ((Biggs et al. 2008; Jakubowski et al. 2005); but see
Dutertre et al. (2014a)).
State of the Art of the Conoidean Toxin Diversity
Knowledge about toxins from Conoidea remained limited to cone snails until
recently. A quick bibliographic search in the Web of Science (Fig. 5) suggests that
within Conoidea, there is a clear bias in the effort of the toxinologist community
toward cone snails; within cone snails, there is a bias toward Conus; and within
Conus, there is a bias toward non-worm-hunting species.
Conoidean toxins are likely the most diverse, both in terms of numbers (estimated
from the conoidean species diversity and the diversity of toxins in individual venom)
and molecular targets, compared to toxins in other groups of venomous organisms
(Prashanth et al. 2014). Within Conus (sensu (Puillandre et al. 2015)), at least one
toxin has been sequenced and eventually functionally characterized for fewer than
100 species (www.conoserver.org). A few species in other clades of cone snails
(Conasprella, Californiconus) have been studied, with generally high proportion of
new toxins that are not found in Conus (see Puillandre et al. 2012b; Kaas et al. 2012).
In particular, half of the toxin superfamilies found in C. californiconus have never
been found in Conus (Biggs et al. 2010). In the last 10 years, toxins from other
Conoidea have been sequenced, although well-documented toxins remain very
limited in number compared to the toxins of cone snails. At present, some data on
the venom gland peptides exist for eight species in Turridae (Polystira albida,
Gemmula periscelida, G. speciosa, G. sogodensis, G. diomedea, G. kieneri,
Lophiotoma olangoensis, and Unedogemmula bisaya), six in Terebridae (Hastula
hectica, Terebra subulata, T. argus, T. consorbina, T. variegata, and Oxymeris
Fig. 5 Results of the bibliographic search, with the number of species (in black) and the number of
published articles (in gray) for different groups of Conoidea and cone snails. Search was performed
in the Web of Science with the keywords “Terebrid*” AND “*toxin*” in TOPIC, “Conus” AND
“*toxin*” in TOPIC and “Turri*” AND “*toxin*” in TOPIC returned 15 articles refer to the
Terebridae, 1405 to cone snails, and 34 to the “turrids,” although they respectively represent
c.a. 9 %, 16 %, and 75 % of conoidean diversity (Fig. 5). Among 1405 articles dealing with the
cone snails, 236 cited a Conus species in the title. Of these, 77 articles were citing a fish-hunting
species, 58 a mollusk-hunting species, and 94 a worm-hunting species; in total 68 different Conus
species were cited. Although fish-hunting and mollusk-hunting species represent 17 % and 7 % of
the total number of cone snail species, they are cited in 33 % and 25 % of the articles, respectively.
The genus Conasprella represents 13 % of cone snail species but is cited in only six articles of the
236, while species of Profundiconus are never cited, and Californiconus is an exception, with seven
citations for a single species
maculata), and one each in Pseudomelatomidae (Crassispira cerithina),

Clathurellidae (“Clathurella” cincta), and Clavatulidae (Turricula javana) (Gorson
et al. 2015; Imperial et al. 2007a; Aguilar et al. 2009; Heralde et al. 2008). Most
toxin superfamilies are found in only one family of Conoidea, except one shared by
Turridae and Conidae (Olivera et al. 2014), and another shared by a turrid
Lophiotoma olangoensis, and a terebrid Hastula hectica (Imperial et al. 2007a;
Watkins et al. 2006): the latter two groups are supposedly sister clades (Puillandre
et al. 2011a). The venom peptides in Hastula hectica show extremely low frequency
of posttranslational modifications, comparing to the conotoxins (Prashanth
et al. 2014; Imperial et al. 2007a; Dutertre et al. 2010; Jakubowski et al. 2006) and
the turritoxins (Olivera et al. 2014). Outside the Conoidea, peptides with structures
and/or sequences similar to conotoxins have been found in mussels (Gerdol
et al. 2015), whereas the toxins isolated from the polychaete genus Glycera are
similar in many features to the turrid venom peptides (von Reumont et al. 2014).
Generally, toxin discovery has moved from a cDNA-based sequencing of more or
less randomly selected taxa before 2000, to a concerted discovery approach (using
systematics and transcriptomics to identify new lineages and new toxins) post-2000
(Olivera 2006; Fry et al. 2015; Puillandre and Holford 2010) and, more recently, to
venomics, which combines proteomics and next generation sequencing (NGS)-
based approaches to characterize transcriptomes (Prashanth et al. 2014; Kaas and

Craik 2015). Still, all available data on conoidean toxin diversity and evolution are
nuanced by the fact that toxin sampling of a venom gland is never exhaustive (Duda
and Remigio 2008), although this bias is notably less (but not fully resolved) when
using contemporary NGS-based approaches.
Speciation and Diversification
The knowledge reviewed in this chapter has been obtained by different research
teams working in different scientific fields: e.g., systematics and morpho-anatomical
descriptions were provided by zoologists; characterization of the toxins and their
molecular targets, as well as the physiological effects induced, by toxinologists.
These fields of research remained generally disconnected from each other, with some
notable exceptions, until the late 1990s, when more integrated approaches started to
emerge. This synthesis will be illustrated with several examples taken from the
literature, from the population level to macroevolutionary processes. Although the
analytical methods and the concepts are not necessarily the same depending on the
taxonomic level considered, the evolutionary hypotheses proposed to explain the
observed pattern always rely on the integrated analyses of various type of data (e.g.,
DNA, morphology, distribution, ecology, toxins) (Fig. 1).
Given that one of the most striking features of the Conoidea is their extreme
diversity, it is not surprising that one of the first evolutionary questions to be tackled
is why there are so many species in this group. If the allopatric model, i.e., in which
new species arise when populations become geographically disconnected, is the
canonical speciation model for terrestrial organisms, the apparent continuity of the
marine ecosystems makes this model less likely. Speciation for marine organisms is
thus generally linked either to limited dispersal abilities and/or to habitat shift that
would lead to reproductive barriers not necessarily linked to geographical isolation.
Both these hypotheses have been tested, at least partly, in the Conoidea, and in
particular, in cone snails. In most cases, these studies include the analysis of
molecular data, either to reconstruct the relationships between the studied species
and their close relatives, or to analyze the genetic differentiation between
populations within a species.
Because the dispersal ability of gastropods can be deduced from the larval shell,
several authors have tested the correlation between protoconch type and speciation
rate. This hypothesis is generally tested using dated molecular phylogenies, cali-
brated using either fossils (in particular, the oldest cone snails or the divergence
between C. quercinus and C. lividus) (Cunha et al. 2005; Duda and Kohn 2005) or
molecular substitution rates (Duda and Palumbi 1999). Cunha et al. (2005, 2008)
showed that the diversification of one of the two Cape Verde clades of cone snails is
linked to the loss of planktotrophy, limiting the dispersal abilities and favoring the
genetic differentiation of geographic populations, and ultimately resulting in speci-
ation. Similarly, Kohn (Kohn 2012) established a link between dispersal abilities,
estimated from egg size, and area of distribution.; The correlation remained valid
when the phylogenetic signal was taken into account (i.e., the species with large
distribution areas and long dispersal phases are not phylogenetically more closely
related to each other than to species with short dispersal phase and more narrow
distribution). Duda and Palumbi (1999) showed that planktotrophy has been lost
repeatedly during the evolution of cone snails and that species with limited dispersal
abilities are more numerous. This result could suggest that the speciation rate is
greater in species that lost planktotrophy, but it can also be explained by the fact that
the reverse transition (from non-planktotrophy to planktotrophy) is never observed
(or at least never convincingly demonstrated). Thus, an equal speciation rate in both
development types would lead to the same pattern. Although not correlating their
results with dispersal abilities, Duda and Kohn (2005) also interpreted their result in
an allopatric context: they delimited two main lineages in cone snails that diverged
33 MYA, one seemingly limited to the Indo-Pacific (large major clade – LMC) and
the other to the East Pacific and the Atlantic (small major clade – SMC). This
scenario has been rejected by Puillandre et al. (2014a) as a larger data set tended
to show that the distinction between the two clades is more related to the bathymetry
than to the geography.
Population genetics also helped to test whether geographic distances are corre-
lated with genetic differentiation (Duda et al. 2012; Duda and Lee 2009a). Some
cone snails in the insular regions of the Indo-Pacific are slightly divergent from the
rest of their distribution (such as Conus miliaris are slightly divergent in Easter
Island and C. sanguinolentus in Hawaii), whereas others in contrast show an absence
of genetic structure in the whole Indo-Pacific (e.g., C. chaldaeus). Thus, high
dispersal rates tend to homogenize the species with large distribution areas, this
pattern being disturbed by the stochasticity of long-distance dispersal for the periph-
eral populations. Even if a species is characterized by highly dispersing larvae, some
isolated archipelagoes may be difficult to reach, thus reducing the migration rate
between the isolated populations and the others and increasing the genetic differen-
tiation between them.
When the signal of allopatric differentiation, and thus speciation, is less clear,
ecological speciation has been evoked to explain current distribution areas (Vallejo
2005). Numerous species of ecologically similar and phylogenetically closely
related (at least for some of them) Terebridae have been found to co-occur on the
same beach in Vietnam (Kantor et al. 2012b). An efficient pattern of resource
partitioning among these species has been demonstrated, with the most abundant
or most closely related syntopic species showing minimal overlap in trophic niches
as indicated by stable isotope analysis (SIA) (Fedosov et al. 2014). Duda and Rolàn
(2005), in a study similar to Cunha et al. (2005), showed that the higher number of
species in the oldest island of the Cape Verde archipelago is not due to a longer
diversification time but to a highest variability of habitats. Cunha et al. (2014)
confirmed this hypothesis by comparing the high diversity of the cone snails in the
Cape Verde Islands with their low diversity in the Canary Islands, proposing again
that this difference is not linked to different biogeographical histories, but rather to
ecological parameters such as a lower variability of habitats in the Canary Islands.
More generally, and as for other venomous organisms, a link between toxin diversity,
prey shifts, and speciation has been proposed as a major hypothesis to explain the
evolutionary success of the Conoidea (Olivera 2006; Duda 2008). Conus conco,
limited to the Marquesas Islands, was probably a peripheral population of C. lividus,
a species present in the whole Indo-Pacific except in the Marquesas Islands. Both
species having evolved different toxins, and prey shift may also be involved in this
apparent case of peripatric speciation (Puillandre et al. 2014b). Similarly, Duda and
Lee (2009b) proposed the hypothesis of “ecological release” for Conus miliaris in
the Easter Island, where the species has supposedly less competitors and more
diversified prey than in the rest of its distribution range. Furthermore, the genetic
diversity of two toxin-coding genes is higher on this island, a pattern interpreted as
resulting from a higher selection pressure. In this case, both geographic isolation and
different environmental conditions would explain the diversification within a spe-
cies. Similarly, for Conus ebraeus, toxin-coding genes exhibit genetic differentiation
between Okinawa, Guam, and Hawaii, although the COI gene does not (Duda
et al. 2009a); the prey, identified using a metabarcoding approach, are indeed
different in Hawaii. Conus ebraeus and C. judeus, two sympatric closely related
species (but not sister species) and genetically clearly different, are actually charac-
terized by different radulae and prey (Duda et al. 2009b), suggesting again that
species occupy different ecological niches. Several authors (Barghi et al. 2015c;
Chang et al. 2015; Phuong et al. 2015) also concluded that the dietary breadth
influences the diversity in at least some toxin genes more than the type of prey, and
Chang and Duda (2016) showed that the diet and the expression pattern of toxin
genes can change throughout the life history of cone snail.
At a higher taxonomic level, Williams and Duda (2008) detected an increase in
speciation rate in Conus sensu (Puillandre et al. 2015) around 20-25 MYA, a pattern
found also in two other molluscan groups (Turbo and Echinolittorina). However,
their study includes only 100 species, and even if they correct their results by
considering that cone snails include 500 species, it still remains an underestimation
of the total number of species. Speciation rates have also been estimated using the
fossil record, and not calculated using time-calibrated phylogenies. Kohn (1990)
described the fossil records of cone snails, with successive phases of expansion and
reduction of the diversity, with a global tendency toward a less elongated shell,
which could be linked to the radiation of the cone snails. More recently, Todd and
Johnson (Todd and Johnson 2013) estimated the speciation rate in Polystira
(Turridae) between 0.2 and 0.5 species per MY, thus identical to or even higher
than the speciation rate in cone snails (Cunha et al. 2005; Kohn 1990). The absence
of apparent variation across time in species richness for Polystira could be more
linked to intrinsic (e.g., speciation favored by prey shifts and toxin diversification)
than to extrinsic factors (e.g., geological events).
However, one of the most studied evolutionary patterns is the transition from
worm-hunting cone snails (WHC) to fish-hunting cone snails (FHC) (and, to a lesser
extent, to mollusk-hunting cone snails – MHC). Originally, two hypotheses,
resulting from both phylogenetic and toxinological analyses, were proposed to
support the idea that FHC and MHC arose from a WHC ancestor. More recently,
other data have been added, perfectly illustrating the integrative approach (Fig. 1).
Fig. 6 Integration of
different patterns. Available
data on a given set of species
(a, c, d) can be used to
propose hypotheses on the
evolutionary processes that
led to the observed pattern.
Furthermore, it can also be
used to predict missing
characters states, based on a
given evolutionary
hypothesis. Similarly,
available data on some species
(a, c, d) can be used to predict
the character states for an
unknown species (b), based
on the phylogenetic
relationships
Duda et al. (2001) first proposed that the transition was from WHC to FHC, and that
this transition had occurred twice independently during the evolution of cone snails
(giving birth to two lineages of FHC: the clade including the subgenera Asprella,
Afonsoconus, Textilia, Pionoconus, Embrikena, Gastridium, and Phasmoconus on
one hand and Chelyconus on the other hand). Subsequent molecular phylogenies
also supported this result (Puillandre et al. 2014a; Espiritu et al. 2001), although it
should be noted that the node on which this hypothesis is based has never been
statistically supported. It should also be noted that not all the species in these
subgenera, and even not all the subgenera, have been confirmed to be fish hunters
by direct observations (Olivera et al. 2015); for several species, prey type has been
deduced using a comparative approach, as illustrated in Fig. 6. More generally, Duda
et al. (2001) also shown that the number of transitions from one feeding type to
another is limited in cone snails, a hypothesis supported also by the most recent
molecular phylogeny (Puillandre et al. 2014a).
Toxins provide support for this hypothesis, but have also been used to propose
more detailed scenarios of the transition from WHS to FHS and WHS species,
independently from the phylogenetic analysis. In the lightning-strike cabal, some
analogous toxins have been recruited independently in the two lineages of FHS
(Olivera et al. 2014), while others were already present in the WHS ancestor,
supporting the hypothesis that both FHS clades are independent, and that toxins
acting on fish were already present in WHS (Imperial et al. 2007b). To explain this
result, it was proposed that WHS were using toxins to deter competitors, such as fish
(Aman et al. 2015; Imperial et al. 2007b). Alternatively, it has been suggested that
WHS used these toxins to defend themselves from predators (i.e., fish), because
these toxins were found in the proximal part of the venom gland, used for the defense
(Dutertre et al. 2014a; Jin et al. 2015), although it remains to be shown that this
pattern is common to all cone snails (but see Prashanth et al. (2016)). Furthermore, in
FHS, the toxins found in the attack venom do not include the toxins supposed to be
included in the motor cabal (see chapter “▶ Evolution of Separate Predation- and
Defence-Evoked Venoms in Carnivorous Cone Snails” by Lewis et al.).
The possibilities offered by NGS have clearly enhanced, or will enhance in the near
future, our capacity to describe the pattern of diversity in three out of the four fields
of research shown in Fig. 1, i.e., systematics of the Conoidea, diversity of prey, and
diversity of toxins. Even when small geographic distances are involved, apparent
sympatry can actually correspond to bathymetrically separated species (Barghi
et al. 2015c), and allopatric speciation thus cannot to be ruled out systematically.
Nevertheless, the fact that ecological speciation occurs in conoideans is supported by
a growing amount of data, in particular for closely\ related species with different
diets that are found to co-occur in sympatry, or even in syntopy. Questions remain
about the process leading to genetic differentiation in the presence of gene flow. How
do barriers to gene flow, either pre- or postzygotic, appear, and how are they
maintained? What is the exact role of toxin and prey diversification in the speciation
process? How do these microevolutionary processes affect the general diversifica-
tion pattern of Conoidea? Is there selection for species that are more prone to evolve
new toxins (i.e., species selection for evolvability)? What is the role of prey
specialization (or, on the contrary, expansion of dietary breadth) in the evolution
of Conoidea? Other traits should not be neglected; for example, it has been shown
that dispersal ability may influence diversification process in conoideans. Many
hypotheses now bloom in the literature to explain conoideans and cone snails’
evolution, but most remain to be thoroughly tested by describing the diversity
patterns more precisely. Systematics still remains paramount to answer such ques-
tions, and as such effort should be put into describing species diversity and species
relationships: only in the light of these data can the evolutionary hypotheses be
tested.
Acknowledgments This review benefitted from the project CONOTAX, funded by the French
ANR (ANR-13-JSV7-0013-01).
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Venom Use in Mammals: Evolutionary
Aspects
Rodrigo Ligabue-Braun
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Venomous Mammals: Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Eulipotyphla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Monotremata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Chiroptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Mammalian Venom Evolution: Shadows of the Past or Rare Recurrences? . . . . . . . . . . . . . . . . . . . . 10
Anthropocentric Biases: Research Limitations, Exciting Applications . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Abstract
Mammals are recently accepted as venomous animals, with four orders having
venomous representatives. These are Eulipotyphla (solenodons and some
shrews), Monotremata (platypus), Chiroptera (vampire bats), and Primates
(slow and pygmy slow lorises). Each of them has different strategies for using
very diverse mixtures of toxic molecules. Venomous saliva is used by
eulipotyphlans to paralyze and cache prey, and by chiropterans to avoid blood
clotting in suitable prey, allowing continuous feeding. Monotremata use crural
spurs to inject a highly painful secretion as a tool in sexual selection, while
Primates lick an elbow gland, loading modified teeth with anaphylaxis-inducing
venom. There is no homology between venomous systems in these different
orders, making a common origin for all venom in Mammalia unlikely, even
R. Ligabue-Braun (*)
Graduate Program in Cellular and Molecular Biology, Center for Biotechnology, Universidade
Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
e-mail: ligabue.braun@gmail.com; rodrigobraun@cbiot.ufrgs.br

2 R. Ligabue-Braun
considering gaps in the fossil record. An emerging picture of complex interac-

tions between cost of venom producing, specialized teeth for feeding and possible
lack of benefits for venom in larger, stronger mammals may be able to justify the
rarity of venom in this group. Both basic science and biotechnology are benefited
as more knowledge accumulates about mammalian venoms.
Keywords
Mammals • Platypus • Bat • Loris • Shrew
Introduction
Mammalian venoms may be considered a novelty, but they have not been discovered
recently. For centuries, up to the 1940s, shrew bites were regarded as highly painful
(and the animal itself was taken as evil and ill intentioned). Comparisons were drawn
between shrews and venomous reptiles, including cobra and beaded lizards. How-
ever, with advances in microbiology and the advent of antibiotics, effects originally
attributed to shrew saliva were attributed to the action of microorganisms from the
animal’s mouth. It took 50 years for this topic to be rediscovered, or, more accu-
rately, rediscussed. Unfortunately, Dufton’s seminal work (Dufton 1992) remained
largely ignored by mainstream zoologists (and biologists as a whole). The emer-
gence of more solid evidence on many mammalian venoms, two decades later
prompted new investigation of the subject (Ligabue-Braun et al. 2012; Rode-
Margono and Nekaris 2015).
There are four mammalian orders with known venomous representatives, as
recognized today. These comprise solenodons and some species of shrews (Order
Eulipotyphla), platypuses (Order Monotremata), vampire bats (Order Chiroptera),
and slow lorises (Order Primates). The amount of knowledge regarding each class
varies greatly. There is also great variation in the strategies in which the venoms are
employed. These secretions are used to immobilize prey, to facilitate feeding, for
predator defense, and for sexual selection. In this chapter, historical aspects and
specifics of the venoms’ toxicity will be presented first. Subsequently, evolutionary
mechanisms that led to each of these venom-use strategies will be discussed.
Venomous Mammals: Overview
Eulipotyphla
The eulipotyphlans include the majority of venomous mammals. These are the
American short-tailed shrew (Blarina brevicauda), the Hispaniolan solenodon
(Solenodon paradoxus), the European water shrew (Neomys fodiens), and the
Mediterranean water shrew (Neomys anomalus). Some evidences support venom
in the Cuban solenodon (Solenodon cubanus) and the Canarian shrew (Crocidura
Venom Use in Mammals: Evolutionary Aspects 3
canariensis), while circumstantial evidence may point to venom in the European

mole (Talpa europaea). Despite being omnivores, the eulipotyphlans were formerly
included in the “wastebasket” taxon Insectivora (Greek for “eaters of insects”). This
was a major misnomer, since these animals prey on varied invertebrates and on
vertebrates of the same, or even larger, size as themselves. The venom asset of these
mammals is found in their saliva, produced in enlarged granular submaxillary
salivary glands. Venomous saliva is also found in vampire bats, albeit of different
composition (see below). The most extensively studied eulipotyphlan saliva is the
one from B. brevicauda, since many factors hinder studies with other species,
ranging from difficulties in keeping these animals in captivity (shrews) to their
endangered status (solenodons).
The European shrew probably was the first mammal to be historically recorded as
venomous. In 1607, Reverend Topsell’s “History of Four-footed Beasts” described
the animals as cunning and cruel, pretending to be gentle and tame, but desiring to
hurt anything with a deep, deadly, bite. In Europe, shrews have been associated with
malignancy, depravity, wickedness, and taken as signs of ill omen. The Latin name
for the European shrew, Sorex araneus, and the French common name, musaraigne,
derive from the Latin word for spider (aranea). In North America, shrews were
considered mostly harmless, and their dangerous status was relegated to folklore,
while the natives of the Caribbean Islands regarded solenodons as venomous.
Solenodons and shrews had their venomous saliva scientifically examined almost
simultaneously. In 1877, Gundlach studied bites of Cuban solenodons (S. cubanus),
comparing them to bites from venomous snakes, while Maynard, in 1889, made a
case report on the effects of an American short-tailed shrew (B. brevicauda) bite.
From 1942 to the 1960s, the saliva toxicity of various eulipotyphlans was tested on
animal models. However, this line of research seemed abandoned until 1992, when a
major review with new data (Dufton 1992) brought the subject back into the
spotlight. In the 2000s, the identification of the B. brevicauda toxin (Kita
et al. 2004) and the uncovering of fossil shrews with envenomation apparatus
(Cuenca-Bescós and Rofes 2007) once again emphasized this aspect of mammalian
physiology.
Bites from shrews are considered uncomfortable to humans, with personal per-
ceptions ranging from no detectable effect to an immediate burning sensation,
swelling, and impossibility of using the affected member for days (Ligabue-Braun
et al. 2012). One of the first observations made about the Cuban solenodon bite was
that the lower incisors caused inflammation at wound entry, while the upper incisors
had no effect, something considered common by natives. This was the first obser-
vation that the submaxillary glands were the production site of venom (Dufton
1992). Solenodons have gutter-like grooves in their inferior incisor teeth that allow
saliva flow to the bite-induced wound. Shrews lack this modification. When present
(as in the Eurasian shrew), there is only a slight concavity in the incisors (Fig. 1).
Testing Blarina, Neomys, and Solenodon submaxillary extracts on mice, rabbits,
and cats established that the venomous saliva causes general depression, breathing
disturbance, paralysis, and convulsions. Even though small vertebrate prey (espe-
cially mice and voles) are an important part of some eulipotyphlan diets,
4 R. Ligabue-Braun
Fig. 1 Venomous Eulipotyphla. (a) General aspect of a Solenodon (Solenodon paradoxus). The
detail highlights the grooved incisor teeth. (b) General aspect of a Eurasian shrew (Sorex araneus).
The detail highlights the slightly concave incisor teeth (Author’s own artwork, incorporating images
in the public domain from “Solenodon paradoxus” by GM Allen, “Règne animal” by G Cuvier, and
“Faune des vertébrés de la Suisse” by V Fatio)
invertebrates account for most of the animals’ nutrition. Based on that, tests of
B. brevicauda with experimental insect prey (such as roaches and crickets) revealed
that its saliva has immobilizing effects, with these immobilized insects being stored
for later consumption. In natural conditions, B. brevicauda caches a varied array of
preys in a comatose state. These include, besides insects, snails, earthworms, and
small mammals (Ligabue-Braun et al. 2012; Rode-Margono and Nekaris 2015).
The active compound in the saliva was thought to be a neurotoxin, based mainly
in some similarities between shrew and cobra venoms proposed in the late 1940s
(Ligabue-Braun et al. 2012). However, no resemblance was found (Lawrence 1945;
Dufton 1992). This neurotoxin-targeting search, coupled with difficulties to work
with pure B. brevicauda submaxillary gland extracts, hindered further research into
this topic until the 2000s. In 2004, it was found that the major toxic component in
B. brevicauda saliva is blarina toxin (BLTX) (Kita et al. 2004). Still, other,
unidentified synergistic components may be acting in the venom. BLTX is an
N-glycosylated kallikrein-like protease of 253 amino acids, with heterogenous
glycoforms. This toxin releases bradykinin from kininogens and would be
Fig. 2 Venomous Monotremata. General aspect of a platypus (Ornithorhynchus anatinus). The

detail highlights the crural spur (Author’s own artwork, incorporating images in the public domain
from “Genera mammalium”, by A Cabrera)
responsible for the effects observed in experimental animals (dyspnea, hypotension,

hypokinesia), since bradykinin is an inflammation mediator that acts in increasing
vascular permeability and lowering blood pressure.
Monotremata
There is only one venomous Monotremata species, the platypus (Ornithorhynchus

anatinus). This egg-laying mammal has semifossorial, semiaquatic habits, living in
rivers and streams in the eastern coast of Australia. Both males and females are born
with spurs in their hind legs, but only the former maintain them for life. These
keratinized spurs are connected to the crural glands, which produce venom, forming
the venom-injecting structure known as the crural system (Grant and Temple-Smith
1998). The crural glands are found in the dorsocaudal sides of the abdomen, and
derive from sweat glands (Whittington and Belov 2016; Fig. 2).
Platypus envenomation was first recorded in the scientific literature in 1818, and
detailed anatomical description, including venom use and tests on domestic animals,
followed. From 1935 to the 1960s, there seems to be no records on this subject. In
1968, however, a major monograph on platypus detailed the toxic properties of the
crural secretions. Some envenomation case reports have been published since but
have been normally treated more as a curiosity than as a real medical issue (Ligabue-
Braun et al. 2012). From 1995 onwards, more biochemical characterizations of the
venom became available, and the Platypus Genome Project (Warren et al. 2008)
allowed a much more detailed inspection of this secretion.
Platypuses have been hunted for their fur by Australian colonists, who sometimes
were victims of envenomation. In humans, all cases involved hands or wrists. The
venom, injected by repetitive jabbing of spurs from both hind legs pressed against
one another, causes immediate acute pain and swelling and requires anesthetic
blockade combined with intravenous narcotic infusion as regular analgesic treatment
is ineffective. The envenomation symptoms may persist for a long period, from
6 R. Ligabue-Braun
2 weeks to several months. In test animals, swelling and tenderness at the site of
injection were observed, followed by decrease in blood pressure, respiratory distress,
and death (Whittington and Belov 2007).
During the mating season, male platypuses are frequently found with punctures in
their bodies, despite attacks among platypus being rarely observed. The crural glands
show cyclic activity, becoming highly active during the mating season, producing
venom to be delivered by the channeled spur (Grant and Temple-Smith 1998). Due
to this cyclic activity and the protected status of platypuses, studies on their venom
composition have been hindered until recently. The venom is a complex mixture of
C-type natriuretic peptides, defensin-like peptides, nerve growth factors, isomerases,
hyaluronidases, proteases, and other, uncharacterized proteins (Whittington and
Belov 2007).
Four major components of the venoms have had more in-depth characterization
(Whittington and Belov 2007; Ligabue-Braun et al. 2012; Rode-Margono and
Nekaris 2015) but their exact functions have not been established (Whittington
and Belov 2016). C-type natriuretic peptides differ from A and B natriuretic pep-
tides, which act in controlling blood pressure, by lacking natriuretic activity,
suggesting other actions for these peptides. They are the most biologically active
peptides in the platypus venom, and may be responsible for envenomation signs
(such as hypotension). These peptides seem able to disrupt membranes and interact
with putative nociceptors. Defensin-like peptides are structurally similar to
β-defensins but lack sequence and functional similarity with them. These are the
most abundant peptides in the venom and could cause pain, possibly by synergistic
action with venom nerve growth factors. These factors are also devoid of their
classical function, having a putative immunogenic effect. C-type natriuretic peptides
and defensin-like peptides from platypus venom also show isoforms with either L- or
D-amino acids in specific positions. This is due to an L-to-D-amino acid-residue
isomerase. Though not confirmed, the function of these D-residues seem to be
resistance to proteases while in the crural gland.
Chiroptera
Among Chiropterans, only a subfamily of New World leaf-nosed bats

(Phyllostomidae) holds venomous representatives. These are the vampire bats
from the Desmodontinae subfamily. They are found from Mexico to southern
Argentina and comprise the common vampire bat (Desmodus rotundus), the rarer
hairy-legged vampire bat (Diphylla ecaudata), and white-winged vampire bat
(Diaemus youngi). The saliva of these bats has anticoagulant properties and is part
of many other adaptations that allow these animals to feed on blood only, including
razor-sharp teeth (Schondube et al. 2001; Tellgren-Roth et al. 2009; Fig. 3).
Venomous bats satisfy the criteria for producing venom, i.e., a secretion produced
in a specialized gland in one animal and delivered to a target animal through the
infliction of a wound, containing molecules that disrupt normal physiological pro-
cesses so as to facilitate feeding or defense by the producing animal (Brodie 2009;
Fig. 3 Venomous Chiroptera. General aspect of a common vampire bat (Desmodus rotundus). The
detail highlights the modified sharp teeth in front and side view (Author’s own artwork, incorpo-
rating images in the public domain from “Voyage dans l’Amérique Méridionale”, by AD
d’Orbigny)
Fry et al. 2009). However, the large majority of vampire preys do not perish from the
venom, which causes only a minor discomfort. In this regard, vampire bats resemble
parasites in their feeding behavior (Delpietro and Russo 2009), since the physiolog-
ical disruption facilitates feeding while keeping the prey alive, ensuring continuous
nutritional supply for the bats (Fry et al. 2009).
For centuries prior to the discovery of vampire bats, Europe had legends of
supernatural blood-sucking entities (Ligabue-Braun et al. 2012). Serendipitous
crossing of such folklore with the growing reports on hematophagous bats from
South and Central America from 1498 onwards led to the association of these
animals with the myth of the vampire, summited by the publishing of “Dracula”
(despite the fact that bats are mostly unmentioned in Bram Stoker’s book).
Vampire bats have characteristic feeding bites, which are sharply circumscribed,
crater-like, 4 mm wounds inflicted onto the attacked animal’s bare skin. The
anticoagulant saliva allows bats to ingest a continuous flow of blood for up to half
an hour, through a piston-like motion of the tongue. In vivo comparisons have shown
that a bat-inflicted wound may bleed from 180 to 480 min, while an equivalent,
blade-induced wound bleeds for about 15 min (Tellgren-Roth et al. 2009).
The three species of hematophagous bats prey upon different animals (Tellgren-
Roth et al. 2009). The common vampire bats (Desmodus rotundus) mostly feed on
farm animals, such as cattle, horses, goats, pigs, and sheep. Less commonly, they
feed on humans, poultry, and wild prey. The white-winged vampire bats (Diaemus
youngi) feed mostly on birds but are also able to feed on mammals, while the hairy-
legged vampire bats (Diphylla ecaudata) feed exclusively on birds.
The free bleeding of bat-inflicted wounds led many researchers to propose that
some kind of anticoagulant should be present in the saliva. In 1966, one such
8 R. Ligabue-Braun
compound, a plasminogen activator, was identified, followed in the 1990s by

molecular characterization of four activators and a factor Xa inhibitor. In the
2010s, yet another plasminogen activator was identified (Ma et al. 2013), along
with the molecular characterization of the fXa inhibitor (Francischetti et al. 2013;
Low et al. 2013).
Bat venomous saliva has tissue-type plasminogen activators and a lactoferrin,
while other components may still be discovered. The plasminogen activators (orig-
inally identified in 1966 but still being unfolded into different types) convert the
plasmin proenzyme to its active form, which is able to degrade blood clots. While the
rarer vampire bats have only one type of plasminogen activator in their genomes, the
common vampire bat has five (Tellgren-Roth et al. 2009; Francischetti et al. 2013;
Low et al. 2013).
The tissue-type plasminogen activator molecule has five domains: finger, epider-
mal growth factor, kringle 1, kringle 2, and serine protease. Only the D. ecaudata
plasminogen activator has all five domains, with the other two species having
smaller chains with domain deletions (Tellgren-Roth et al. 2009; Ma et al. 2013).
These chain variations, combined with variable glycosylation structures (one O- and
two N-glycosylation sites) alter binding properties of vampire bat plasminogen
activators compared to tissue-type plasminogen activators.
The second type of anticoagulant from vampire bat saliva is draculin
(Francischetti et al. 2013; Low et al. 2013). This modified lactoferrin is a
noncompetitive, tight-binding inhibitor of activated factor X from the coagulation
cascade. Factor Xa is the only enzyme that converts prothrombin into thrombin
(a key point in the blood coagulation process). Draculin action is dependent on
correct N- and O-glycosylation, and a mixture of draculin glycoforms are proposed
to modulate the degree of fXa inhibition. As with many other vampire bat studies,
draculin has been inspected only in D. rotundus so far.
Since the feeding bites from hematophagous bats are considered painless, it has
been proposed that their saliva may also have an anesthetic. However, vampire bats
are known to learn how to properly bite prey by trial and error. So far, there is no
concrete evidence for this putative anesthetic.
Primates
The nocturnal prosimians slow loris (Nycticebus coucang, N. bengalensis), Kayan

slow loris (Nycticebus kayan), and pigmy slow loris (N. pygmaeus) are the venom-
ous representatives of the Primates order (Rode-Margono and Nekaris 2015). They
inhabit trees in Southeast Asia and Western Indonesia. They are unique in their mode
of toxin delivery, since unrelated body parts produce and inject the venom. This
venom is synthesized in the brachial gland, located in the ventral, almost hairless,
side of the elbow. Then, by licking of the gland, the secretion is mixed with saliva
and loaded into the toothcomb, a specialized compression of the needle-like canines
and incisors of the loris jaw (Hagey et al. 2007; Fig. 4). Exhibition of the elbow, by
positioning of their front hands above the head, and intense spreading of the venom
Fig. 4 Venomous Primates. General aspect of a slow loris (Nycticebus coucang). The detail
highlights the modified tooth comb (Author’s own artwork, incorporating images under Creative
Commons license by Kathleen Reinhardt, and in the public domain from “A handbook to the
primates” by HO Forbes)
on the head are also taken as indications of venomousness. Most (if not all) generally
accepted definitions of a venomous animal state that the venom producing site and
the delivery (or injecting) organ must be directly connected. This is not the case with
the primates, which are only now becoming accepted as venomous (Ligabue-Braun
et al. 2012; Rode-Margono and Nekaris 2015).
Folklore in Thailand holds lorises as venomous animals capable of causing
intense pain and death. However, human envenomation is rarely reported. There
are only three records in the medical literature (a man bitten by his pet N. coucang, a
pregnant zookeeper bitten by a N. pygmaeus, and a researcher bitten by a N. kayan),
and anecdotal evidence from manuals for zookeepers and wildlife caretakers
(Madani and Nekaris 2014). The animal bite causes some effect besides the lacer-
ation itself – symptoms that do not differ from anaphylaxis. These include pulsating
pain, hypotension, extremity cyanosis, and hematuria. Researchers working in close
contact with lorises develop allergies to the venom. Despite its ability to cause
anaphylactic shock, this seems to be only an incidental effect of the venom
(Hagey et al. 2007).
There is ongoing research aiming to define the main physiological role of the loris
brachial gland secretion, which may act synergistically with the animal’s saliva
(Rode-Margono and Nekaris 2015). Venom use for prey capture is not supported,
while use in intraspecific competition seems plausible. Predator and ectoparasite
defense are the most well-supported ecological roles for the venom.
Despite being a highly complex mixture, containing dozens to hundreds of
compounds, the main toxic component of the venom has been identified as the
brachial gland exudate protein (BGE protein) (Krane et al. 2003). It is a
10 R. Ligabue-Braun
heterodimeric protein (17.6 kDa), with the α-chain (7.8 kDa) and β-chain (9.8 kDa)
linked by two disulfide bridges. All studied lorises have two BGE isoforms, due to
variable β-chains. The BGE protein is highly similar to the major cat allergen, Fel d
1. Both share the uteroglobin protein fold, are disulfide bound, and have alternate
β-chains. The uteroglobin fold is related to transport of hydrophobic molecules (such
as steroid hormones) and calcium binding. Regardless of being poorly understood,
uteroglobins are postulated to act as boxes, being able to open and close according to
physiological conditions, loading, carrying, and delivering hydrophobic cargo
(Hagey et al. 2007). The similarity to a feline allergen reinforces the possible
cross-reactivity of loris venom instead of a de facto venomous role (at least for
humans) (Ligabue-Braun et al. 2015).
Mammalian Venom Evolution: Shadows of the Past or Rare

Recurrences?
As can be observed from the mammalian phylogenetic tree (Fig. 5), orders with
venomous representatives do not cluster together, having no obvious common
origin. This observation prompts the question: Why are venomous mammals so
rare? Moreover, what is the utility of the extant venom, being so scarce among this
animal class? Since each order’s venom is different, the following segment will
describe peculiarities and strategies in which these mammalian groups employ
venoms, discussing current evolutionary hypotheses regarding their origins.
Venomous eulipotyphlans have been compared to venomous reptiles long before
the identification of BLTX. Solenodons and shrews were studied taking snakes as
reference (Dufton 1992). In 1942, when identifying that shrews had modified
salivary glands responsible for the venom production, like snakes, Pearson also
noted that in the latter the parotid glands are modified to produce venom, while in the
former, the submaxillary glands are the venom source. Despite this important
difference between the two cases, there are indeed venomous reptiles with venom-
producing submaxillary glands (including the Gila monster Heloderma suspectum
and the Mexican beaded lizard H. horridum). With the purification of BLTX, it was
confirmed that these cases were indeed linked (Kita et al. 2004). The Gila toxin
(GTX) and BLTX have similar effects on prey and are similar (34 % identical).
Horridum toxin also has high sequence similarity with BLTX (32 %). The reptilian
and mammalian kallikreins underwent convergent transitions to venomous ones,
with similar, nonhomologous residue insertions that increased the protein flexibility,
altering loop lengths, polarity, and surface charges. Both GTX and BLTX started
from independent serine proteases that had locally different alterations generating
globally similar, toxic, structures (Aminetzach et al. 2009; Fig. 6). Blarinasin,
another kallikrein-like protein from shrew saliva is not toxic on tested animals,
suggesting that small differences, including glycosylation heterogeneity, may play
key roles in their toxicity.
Regarding the venom function in Eulipotyphla, Furió et al. (2010) defined an
ongoing debate as “hunting big or hoarding small”. As part of an adaptive winter
Fig. 5 Mammalian
phylogeny highlighting orders
with venomous
representatives (in red). Please
note that there are alternate
versions for the evolutionary
history of Mammalia
(Author’s own artwork, based
on Springer et al. 2004)
profile, shrews cache various preys in a comatose state. Other adaptions include
elaborate nests, stable thermal regime for foraging, and reduced activity during
periods of cold. Within this framework, venom would be an asset to sustain a living
hoard when hunting is difficult, especially in cold winters. The high metabolic rate of
shrews would make this ability very relevant. The use of eulipotyphlan venom as a
paralyzing, conservative agent is thus taken as support for the “hoarding small”
hypotheses. The “hunting big” hypothesis proposes that venom is a tool to overcome
bigger prey. According to Dufton (1992), vertebrate food is of major importance for
eulipotyphlans, and this kind of prey is larger and more dangerous to subdue than
their power-to-weight ratio would allow, thus making venom necessary. There is no
specialized venom delivery apparatus in extant shrews, which have only a concavity
on the surface of their first incisors. However, the discovery of an extinct giant shrew
(Beremendia sp.) with an envenomation apparatus (grooved incisors similar to those
12 R. Ligabue-Braun
Fig. 6 Structural model of

blarinatoxin, BLTX, with
regulatory loops colored in
blue and insertions that
convergently evolved towards
toxicity in yellow. A substrate
is shown in orange. Model
built based on PDB ID 2ZCK,
Ménez et al. (2008) (Author’s
own artwork)
from solenodons) (Cuenca-Bescós and Rofes 2007) seems to favor Dufton’s pro-
posal. These grooves would act as a channel, directing saliva from the submaxillary
glands to wounds inflicted on the prey, as seen in solenodons. However, alternative
explanations, based on paleoenvironmental reconstructions, propose that
Beremendia lived in a highly unpredictable environment and that their prey
consisted mostly on gastropods and coleopterans (Furió et al. 2010). Most likely,
venom is used by Eulipotyphla in both ways, including combining them to hoard
larger prey. Secondarily, venom use in intraspecific competition has been observed
among captive solenodons.
In his model for venomousness in mammals, Dufton (1992) proposed that the
earliest eutherian mammals had morphologies that resemble extant hedgehogs and
shrews. These early eutherians developed during the late Cretaceous (66–144
millions of years ago), and would form a basal group for extant mammals (Rode-
Margono and Nekaris 2015). Eulipotyphlans per se are not ancestral in mammalian
phylogeny, but in this proposal, they would be the extant mammals that retained the
most from these ancient eutherians. The current distribution of venomous
eulipotyphlans, covering Asia, Europe, North and Central America, would support
this view of a more widespread occurrence of venomous mammals in their evolu-
tionary past. Since these ancestral mammals were small and not fully homeothermic,
foraging efficiency would act as a selective pressure, while the use of venom would
bestow a selective advantage on them (Rode-Margono and Nekaris 2015). Dufton
also observed that extant venomous eulipotyphlans almost exclusively co-occur with
flightless birds. This scenario would somewhat resemble their origins sharing hab-
itats with dinosaurs, suggesting that beyond egg-eating, larger flightless birds
(or dinosaurs) could be targets for venom (either predatorily or defensively). Once
their diet shifted to invertebrates, venom would become less useful, being retained in
only a few species. This is an especially cumbersome hypothesis, since extant
venomous Eulipotyphlan are very successful at using venom to prey on invertebrates

(Folinsbee 2013).
Despite the well-supported occurrence of venom in the extinct giant shrew
Beremendia (and possibly in the solenodon relative Nesophontes), other evidences
from the fossil record are still being disputed. The discovery of a Palaeocene
eutherian mammal with canine grooves, phylogenetically distant from Eulipotyphla,
prompted its classification as a venomous mammal (Fox and Scott 2005). This
animal, Bisonalveus browni, was proposed to use its grooved teeth to deliver toxic
saliva, resembling solenodons. However, the occurrence of grooved teeth alone has
been deemed insufficient and inadequate to support the occurrence of true venom
delivery apparatus in extinct mammals. Orr et al. (2007) and Folinsbee et al. (2007)
cited numerous examples of extant mammals with grooved teeth and no sign of
venom. These include suiforms, coatis, lemurs, and primates. The grooving in their
teeth seems to act as a structural reinforcement of the dentary structure, unrelated to
venom delivery. Both works conclude that the traditional comparative method alone
could not ascertain if a primitive mammal was venomous without slipping into “false
positives,” i.e., if the structure (grooved teeth) is related to function (venom deliv-
ery), all extant mammals with this anatomy would be expected to be venomous,
which is not the case. Additionally, except for solenodons, all other extant venomous
mammals lack truly grooved teeth. The mammalian masticatory apparatus, however,
is considered highly sophisticated, enabling a wide range of feeding strategies. This,
alone, could render venom use redundant (Folinsbee et al. 2007). As observed by
Rode-Margono and Nekaris (2015), this seems to be the case, since the venomous
eulipotyphlans are exceptions to the general pattern in mammalian diets. While
many orders are chiefly herbivorous or insectivorous with small prey relative to
predator body mass, the carnivorous orders are large and able to overcome their prey
by sheer strength.
In a recent phylogenetic construction of Eulipotyphla phylogeny, Folinsbee
(2013) observed that Neomys, Solenodon, and Blarina brevicauda are phylogenet-
ically distant, with the Solenodon lineage diverging around 80.5 mya and the
ancestors of Neomys and Blarina diverging around 16.5 mya. The author observed
that, if venomousness was an ancestral condition of Eulipotyphla, at least nine
convergent losses of venom would be required to explain the obtained tree. On the
other hand, if venom evolved convergently in different eulipotyphlans, only three
unique acquisitions would be required. Still, venom is rare in this group, occurring in
no more than 2 % of extant species.
Toxic saliva is not exclusive to shrews and solenodons. Vampire bats use their
venomous secretion to be able to feed on the continuous blood flow from a sharply
cut wound. Vampire bat saliva, however, is just part of a highly specialized physi-
ology, as a reflection of blood being their sole source of hydration and nutrition.
Other adaptions include a modified gastrointestinal tract, which allows the ingested
blood to enter the intestines prior to entering their tubular stomachs (due to a
T-shaped gastroesophageal-duodenal junction). When reaching around half their
weight in ingested blood, most of the water is eliminated in a process known as
instant diuresis, which leads to the highly nitrogenous blood remains being
14 R. Ligabue-Braun
processed with almost no water. Their high capacity for concentrating urea in the
urine makes vampire bats physiologically equivalent to desert mammals. Hema-
tophagous chiropterans feed almost exclusively on the proteinaceous moiety of the
ingested fluid, with the carbohydrates being almost unused, and sucrase and maltase
being absent in their gastrointestinal tracts (Schondube et al. 2001). Another phys-
iological characteristic of these animals is that they have no adipose tissue for
storage, making them dependent on daily blood meals and reliant on a highly
ordered social system, in which fed animals are able to regurgitate blood into the
mouth of individuals that are unsuccessful or unable to prey for themselves. Their
sharp teeth and anticoagulant saliva work as to facilitate the tongue-directed flow of
blood to the animals’ mouth. The tongue does not act licking up the blood, but rather
acting like a piston. For this method to work, blood cannot coagulate, as to guarantee
free flowing from the prey to the predator.
Vampire bats form a subfamily, Desmodontinae, in the Phillostomidae family.
This family is considered to have the most diverse feeding habits among all mammal
families. They include nectarivory, omnivory, frugivory, carnivory, and
hematophagy (the latter deriving from insectivory) (Schondube et al. 2001). The
three hematophagous species have different preferred preys, and this reflects the
evolution of their salivary anticoagulants. The fibrin specificity and susceptibility to
plasminogen activator inhibitor 1 of chiropteran plasminogen activators has been
altered by gene duplication, domain losses, and further sequence evolution
(Tellgren-Roth et al. 2009; Fig. 7). D. ecaudata has a single copy of plasminogen
activator, which is similar to the one found in other mammals and feeds only on
birds. D. youngi plasminogen activator lacks the kringle 2 domain, having increased
fibrin specificity. This bat is more generalist, feeding on birds and mammals. The
plasminogen activators in D. rotundus went through rounds of gene duplication and
domain loss, creating versions with decreased sensitivity to plasminogen activator
inhibitor 1 and enhanced ability to feed only on mammalian blood (Tellgren-Roth
et al. 2009). Differences in glycosylation also take part in the improved
anticoagulatory activity, since the clearance of some chiropteran plasminogen acti-
vators are up to four times slower than those observed for tissue plasminogen
activators.
Only chickens have been observed to die of hemorrhage after vampire bat attacks.
Other prey do not succumb to their bites or saliva, prompting arguments against the
inclusion of bats as venomous animals (Ligabue-Braun et al. 2012). Indeed, their
saliva facilitates feeding by disrupting regular prey physiology while ensuring its
survival for the continuous supply of nutrition for the bat. The highly specialized
saliva of Chiroptera is not homologous to Eulipotyphla venom, with the involved
teeth being different as well as the molecules involved in the toxicity. Additionally,
the emergence of hematophagy in bats is considerably more recent in evolutionary
terms than the speciation of solenodons and shrews with envenomation apparatus.
These observations highlight the emergence of venom more than once in the history
of mammals.
Male platypuses (Monotremata) employ the secretion of their crural system in a
different way to the venomous saliva from Eulipotyphla or Chiroptera, which is used
Fig. 7 Structural scheme of plasminogen activators from vampire bats. Absent domains in each
protein are color-coded, while isoforms from each species are clustered together. Depicted based on
Tellgren-Roth et al. (2009), model built based on PDB ID 4DUR, Law et al. (2012) (Author’s own
artwork)
in prey acquisition and feeding facilitation, respectively. The use of the glands and
spurs has been proposed to take part in multiple behaviors in these animals, from
helping climb riverbanks to waterproofing the fur (Grant and Temple-Smith 1998).
However, their true usage is to act as a weapon in male-male competition for
females, taking part in sexual selection. Adult male platypuses largely avoid each
other and have testicular and crural gland size increases in the mating season, in
which males become aggressive. In this season, it is common to find males with spur
punctures, especially in their tails. Grant and Temple-Smith (1998) used this evi-
dence to propose a polygynous mating system for platypuses. In this system, male
interactions direct the access to females, something that would justify the retention of
the crural system in former. Only one other Monotremata representative, the short-
beaked echidna (Tachyglossus aculeatus), had its crural apparatus examined (Krause
2009). Both genders of these animals have degenerate crural spurs, with the males
having cyclic growth of the crural gland in accordance to mating season. However,
they are unable to use their spurs aggressively or to support the structure on their
tibia during attacks. The seasonal growth cycle would suggest a role as scent gland,
but its real function is still uncertain.
Monotremata are the sole remaining mammals from the class Prototheria, the first
to diverge from other mammals (around 166 Mya). Platypus, in special, have
anatomical features that are closely related to reptiles (similar ribs and pectoral
girdle), despite being furred homeotherms. These mixed characteristics are reflected
in their genome, with a large amount of reptilian-like genes, and taken as a possible
link between reptiles and therians (Whittington and Belov 2016). Likewise, platypus
venom has many similarities with reptilian ones, via convergent evolution
(Whittington et al. 2008). In both cases, defensin-like peptides, C-type natriuretic
peptides, and nerve growth factor gene families were duplicated and then co-opted
16 R. Ligabue-Braun
Fig. 8 Structure of defensin-

like peptide 2, DLP-2, from
platypus venom (PDB ID
1ZUE, Torres et al. 2005). The
three characteristic defensin
disulfide pairs are highlighted
(Author’s own artwork)
for toxic purposes (Warren et al. 2008). Many of the proposed “venom genes” are
also expressed in non-venom tissues (i.e., outside the crural gland), while the
natriuretic peptides and nerve growth factors from venom are also expressed in
females, suggesting additional roles for these peptides. As observed in reptiles, there
is an ongoing debate on whether putative toxins expressed in multiple tissues
constitute true toxins (please see the chapter “A Critique of the Toxicoferan Hypoth-
esis” for more details). A transcriptome of platypus venom gland labeled 88 toxin
genes when filtered against transcriptomes of nonvenomous tissues (Whittington
et al. 2010). So far, only defensin-like A (Fig. 8) is considered a crural-gland
exclusive peptide (Whittington and Belov 2009).
As with eulipotyphlans, fossil evidence has been interpreted as signs that crural
spurs were widespread among primitive mammals. The proposed role for the crural
system would be in defense against larger predators, dinosaurs in particular (Hurum
et al. 2006; Kielan-Jaworowska and Hurum 2006). Once again, this proposition is
based on comparisons with extant mammals, of which only one species has a
venom-delivery apparatus involving crural glands and spurs. Still, true monotreme
fossils are rare and consist of tooth and jaw fragments, not allowing certainty in
defining ancestral monotremes as venomous. Considering the conserved (vestigial or
functional) crural systems in extant platypuses and echidnas and some shared
elements between their glandular secretions, it is possible to propose that their
ancestral species were also venomous (Whittington and Belov 2016). The fact that
spurs (functional or vestigial) are present in both sexes in extant Monotremata raises
the possibility that their original purpose was in defense, especially in confronting
large predators, from dinosaurs to large mammals (from the Jurassic to Pleistocene).
It has been proposed that, once this selective pressure was no longer present, the
crural system was co-opted to a reproductive context. This change would justify the
maintenance of energetically expensive venom production as a sexually dimorphic
trait (Whittington and Belov 2016).
There are many cross-taxa examples of convergent venom evolution (Fry
et al. 2009), and the very unusual mammalian ones are no exception. Most toxins
in mammalian venoms (with the possible exception of BGE protein) are products of
this type of process, being similar to toxins found in other animal groups. Rapid
effect, be it fast prey immobilization or quickly occurring pain, is a major require-
ment for a venom. This is proposed to be one of the main factors that limit what
kinds of protein may be co-opted for toxicity. There is only a small group of proteins
that recurrently develop into toxins once their genes are duplicated. However, this is
not the sole process acting on mammalian venoms, since mutations in regulatory or
coding regions and alternate splicing has been shown for platypus venoms, and
alternate glycosylation has been related to variable activity in shrew and vampire bat
venoms.
The order Primates displays a unique venom delivery system, unlike any other
present in venomous animals. Currently four species in the Nyctycebus genus are
recognized as being venomous. They load needle-like modified teeth with the
secretion of an elbow gland, possibly mixing it with saliva, thus establishing a
venom delivery apparatus from unrelated body parts. Many hypotheses have been
tested to ascertain the ecological role of this venom, as reviewed by Rode-Margono
and Nekaris (2015). Aiding in feeding seems unlikely, since their diet consists of
fruits, invertebrates, and small vertebrates, i.e., smaller than the animal itself. Also
unlike eulipotyphlans, there is no caching behavior observed, with all food being
consumed immediately. Testing the brachial gland exudate on arthropods did not
confirm deleterious effects on this type of prey. Predator defense is somewhat
unlikely, since the predators are diverse and the loris-predator encounters are rare.
However, when testing BGE-saliva mixes on olfactory-oriented predators (leopards,
clouded leopards, tigers, sun bears, common palm civets, and binturongs), these
were repelled by the venom. In the field, it has been observed that Javan slow lorises
move close to palm civets and leopard cats. Visually oriented predators reacted
differently when faced with loris venom. Eagles (Spizaetus, Spilornis) show incon-
clusive behavior, while orangutans actually eagerly consumed the venom-containing
swabs. These visual predators (along with pythons) are known to prey on lorises
(Hagey et al. 2007). There is growing evidence that the secretion may act as an
antiparasitic, since lorises are conspicuously low in ectoparasites, being slightly
more affected in the rainy season. Tests on leeches and tick-related models revealed
that the BGE-saliva mixture is able to kill them.
The venom may have a role in intraspecific competition (as observed for
solenodons and platypuses). Loris-on-loris bites are severe, affecting large areas
with loss of fur, prolonged edema, and are slow-healing (often life-threatening)
(Hagey et al. 2007). The chemical complexity of BGE, associated with grooming
behaviors, point to the substance being used as a signaling device. The main target,
however, may not be predators but the lorises themselves. Aside from fending off
some predators, the secretion may alert other lorises about the predator’s presence.
Since the venom shows species specificity, this may reinforce a communication role.
Taking its high similarity to Fel d 1, the major cat allergen, structural modeling
suggest that the BGE protein may act as a box (Fig. 9), being able to close its lid on
signaling molecules from saliva or BGE. This entrapping and delivery system helped
propose the physiological role of Fel d 1 in big and small cats (Ligabue-Braun
18 R. Ligabue-Braun
Fig. 9 Fel d 1, a working

model for BGE protein.
α-chain in blue, β-chain in
orange. The internal cavity,
proposed to act as a box, is
shown as a grey volume (PDB
ID 2EJN, Kaiser et al. 2007)
(Author’s own artwork)
et al. 2015). It is interesting to note that chimpanzee and human genomes harbor
remnants of the BGE protein in the form of pseudogenes, indicating a putative
venomous past for apes (Hagey et al. 2007) or a long lost redundant uteroglobin.
Nekaris et al. (2013) proposed an elegant hypothesis for the role of venom in
lorises as part of a much broader Müllerian mimicry system. In this proposal, the use
of venom to repel olfactory-orientated predators is combined with other features,
such as extra vertebra in the spine that allow serpentine movements, aggressive
snake-like vocalizations, and long dark dorsal stripes and dark ocular circles,
constituting a mimic of cobras (Naja spp.). Despite its multipurpose role (including
intraspecific competition and parasite defense), the evolution of venom may have
been an adaptive strategy against predators when combined with other features,
arising in the Miocene, when slow lorises and cobras migrated through
Southeast Asia.
Recently, Harris and Arbuckle (2016) used large datasets with comparative
phylogenetic methods to inspect patterns of venom and poison evolution in birds,
amphibians, reptiles, and mammals. They found that venom biosynthesis evolution
is much less dynamic than that of toxin sequestration from the diet. Apart from
amphibians, the remaining tetrapods show an association between the evolution of
toxins and higher diversification rates. Furthermore, the work found that mammals
and reptiles evolve under a similar regime regarding their toxicity/venomousness,
with gains and losses of toxicity sparsely and infrequently distributed across the
phylogeny. Interestingly, mammals form the only tetrapod lineage in which venom
is used for intraspecific competition. Harris and Arbuckle (2016) speculate that, due
to frequent social interactions in mammals (compared to other groups), they may be
under higher selection pressure to use venom in social situations. To support this,
the authors argue that such behavior is observed in some eusocial hymenopteran
insects.
Folinsbee (2013) summarized the main (nonmutually exclusive) hypotheses to

why venom is rare among extant mammals. Venom may not be adaptive in mammals
(i.e., there is no need for it); the production of venom may be constrained by some
biological factor; there are high costs associated with production and maintenance of
venomous secretions; and venom may be adaptive only in a narrow range of
morphologies. Regarding the need for venom, the greater size and masticatory
adaptions acquired by mammals may have supplanted the (putative) ancestral
venomousness. Still, it is unclear, at least for the diverse Eulipotyphla order
(452 extant members), what proportion is really venomous. There is an abundance
of untested mammals in this order to be evaluated prior to considering them
nonvenomous. Studying them comparatively may confirm (or disprove) multiple
origins for venom in these animals. The cost of producing venoms also needs to be
evaluated in mammals. Snakes and arachnids carefully measure the amount of
venom deployed in each attack, with pitvipers and death adders increasing signifi-
cantly their metabolic rates when synthesizing venom (Folinsbee 2013; Morgenstern
and King 2013). There is still no study regarding venom production costs in sister
mammalian taxa that would clarify the energetic costs of using venom for predation.
If it is not costly, another constraint must be in place. A likely explanation is that the
highly specialized mammalian teeth, intensively used for oral processing of food,
may hinder their modification into venom delivering tools, making venom less
adaptive. It is possible that a combination of factors makes venom adaptive only
for a specific phenotype, namely, small body size with high metabolism (Folinsbee
2013). As becomes clear when one inspects each mammalian order’s venom, its
uses, and injecting apparatus, there is no homology present (at least, none that can be
ascertained without major concessions). Venomousness seems to have indepen-
dently arisen at least four times among mammals, once in each of the four orders
presented in this chapter. Explaining the scarcity of venomous mammals may take
into account specificities of each mammalian order.
Anthropocentric Biases: Research Limitations, Exciting

Applications
The study of venoms in general suffers from a human-centered perspective. This is

understandable, since humans are the ones doing research, basic science is expen-
sive, and resources are scarce. However, this should be avoided as soon as perceived
as an obstacle to fully understand venoms and toxinology as a whole. The study of
venomous mammals also have these caveats.
Most of the available data reflects venom effects on humans, pets, farm, or
experimental animals. Venom effects, however, are context- and taxon-specific.
For this reason, using lethal dosage evaluation (LD50), for instance, is problematic,
since it is normally based on a single species, ignoring that different species respond
differently to the same compound (Brodie 2009). Only recently there has been an
effort to understand the physioecological role of venom in the animals’ life history.
20 R. Ligabue-Braun
Despite mammalian venoms reflecting such bias, there are indeed tests on animals
that are more strongly related to the mammals’ ecology. For instance, roaches and
crickets were tested with Eulipotyphla, in an attempt to mimic their invertebrate diet,
and lorises had their venom tested on spiders, maggots, ants, fleas, and caterpillars
(Grow et al. 2015).
Much information regarding venom use by mammals originated from observa-
tional studies. Still, there is room for more assessments aiming to understand venom
use by these animals in their natural habitat. Since the notion of venomousness as a
mammalian trait is just beginning to be accepted by the wider zoology community,
there are many gaps still waiting to be bridged in respect to these animals. The vast
majority of venomous mammals are difficult to maintain in captivity, and even when
this is not a limiting feature, the amount of venom is too small to allow in-depth
research without straining individual animals or requiring large numbers of individ-
uals. Genomic techniques are rising as possible answer to this conundrum.
Toxic proteins from mammals have served as models to understand mammalian
evolution, as well as providing interesting prototypes for new drugs. Anticoagulants
from vampire bat saliva have been proposed as promising treatments for myocardial
infarction, pulmonary thromboembolism, and stroke, since a key aspect of these
events is to keep blood unclotted. BGE protein from lorises may help to assess
allergy-related issues in humans, considering its high similarity to cat allergens. In its
turn, platypus venom may aid in the study of pain perception and as a model to
design novel pain relievers, particularly interesting when one targets long lasting,
treatment-unresponsive pain. Isomerases from Monotremata venom may offer tools
to develop degradation-resistant peptides with medical application. Other, less
studied, toxins from this venom may even work as scaffolds for antineoplastic
drugs (Ligabue-Braun et al. 2012, 2015; Rode-Margono and Nekaris 2015;
Whittington and Belov 2016). The field of venomics is growing together with
toxin-based drug discovery (Calvete 2009). Mammalian venoms are thus rich
sources of novel frameworks for drug development.
Mammalian venoms prompt a need to revise the definitions of venomous and

poisonous animals. In the traditional definition, venoms are produced and stored in
specialized structures (glands), associated with delivery devices, forming the enven-
omation apparatus with which the venoms is delivered directly to the recipient’s
body. Thus, venomous animals are actively toxic. Poisons may be available in
specialized structures in the toxic animal but lack any special mechanism of delivery.
The recipient animal must eat (or at least be in direct contact with) the poisonous
animals to be affected. This is considered passive toxicity. There are obvious flaws
with these definitions. For instance, the spitting cobra would be considered poison-
ous and not venomous, when venom is delivered by squirting and not directly onto
the prey. Likewise, the feeding secretions from hematophagous animals are not
universally considered as venom, even though they satisfy all requirements to
be. It is the fact that these animals depend on the survival of the food source for
continuous supply of nutrients that may divert them from traditional definitions,
despite their venoms being capable of facilitating feeding by disruption of normal
physiological processes of the prey.
Eulipotyphlans and platypuses satisfy the criteria to be taken as venomous.
Vampire bats are considered venomous only if hematophagy is enrolled along with
traditional venom uses (Fry et al. 2009). Lorises clearly do not satisfy the criteria,
since their venom-producing organ is not directly connected to the injury-inflicting
and toxin-delivery apparatus. Still, one can no longer argue that these animals cannot
act venomously.
Chemical defenses (passive toxicity) are also present in mammals. Pangolins,
skunks, the greater long-nosed armadillo (Dasypus kappleri), and the striped polecat
(Ictonyx striatus) emit noxious substances to fend off predators. This form of
chemical defense would allow these mammals to be considered poisonous. In the
most striking example of chemical defense in mammals so far, the African crested rat
(Lophiomys imhausi) is able to sequester toxic substances from plants and accumu-
late them in their manes, forming a protective mantle. These cases have been
considered “arguably venomous” (Rode-Margono and Nekaris 2015). Considering
that examples of chemical defense in other animal groups are ample (amphibians),
understudied (marine turtles) and still being elucidated (birds) (Ligabue-Braun and
Carlini 2015), it is possible that mammalian poisons may be much more widespread
than mammalian venoms. Hopefully this chapter, along with recent literature, will be
able to include mammals in the “hall of venomous animals” from the perspective of
both toxinologists and the general public.
Cross-References

▶ Evolution of Resistance to Toxins in Prey
Their Toxins
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(Toxinology) P. Gopalakrishnakone, Anita Malhotra (Eds.) - Evolution of Venomous Animals and Their Toxins-Springer Netherlands

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(Toxinology) P. Gopalakrishnakone, Anita Malhotra (Eds.) - Evolution of Venomous Animals and Their Toxins-Springer Netherlands

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Evolution of Venomous Animals and Their Toxins

Evolution, Morphology and Development of the Centipede Venom System

Phylogeny and Diversity

The Forcipular Apparatus

The Venom Gland

Location and Shape of the Venom Gland

Ultrastructure and Function of the Venom Gland

Ontogeny of Centipedes with Reference to the Forcipular Apparatus

Reproduction, Egg Laying, and Maternal Care

Development of the Venom Apparatus

Fossil Records of Centipedes with Special Reference to the Forcipular

Predatory Behavior and Prey Choice

Conclusion and Future Directions

Enghoff H. Notes on Lamyctes coeculus (Brölemann), a cosmopolitic, parthenogenetic centipede

Mrinalini and John H. Werren

# Springer Science+Business Media Dordrecht 2016 1

Parasitoid wasps (also known as parasitic wasps) (Hymenoptera) are a distinctive,

Fig. 1 Parasitic wasps are

Recently, the potential uses of parasitoid venom in pharmacology and agricul-

Evolution of Parasitic Wasps

A recent phylogeny using ~1500 protein-coding genes estimates the origin of

The Parasitic Lifestyle

Parasitoid wasps are holometabolous or metamorphosing insects, and they com-

Parasitic Wasp Venom System

The Venom Apparatus

The Ovipositor or Stinger

Parasitic wasp venom can be a complex mixture of proteins, polydnaviruses

et al. 2014a), Leptopilina boulardi and Leptopilina heterotoma (Goecks

Viruses and Virus-like Particles

whereas the venom of specialist Leptopilina boulardi interferes further downstream

Gene Regulatory Elements

How Parasitoid Venoms Function

Venom Functions in Insect Hosts

The earliest parasitoid wasps in Apocrita parasitized larvae of Coleoptera (beetles)

Ectoparasitic Host Manipulations

oxidase activity, and programmed cell death/apoptosis of hemocytes (Rivers

Endoparasitic Host Manipulations

Behavioral Manipulations of Endoparasitoid Hosts

Parasitoid Venoms and Arachnid Hosts

Parasitoid-host interaction can be an arms race between successful parasitization

Models of Venom Evolution

Gene Duplication and Neo-functionalization

Co-option of Non-venom Genes for Venom Function

Evolution of Alternative Splicing of Venom Transcripts

Evolution from Noncoding DNA

Venom Gene Evolution Following Lateral Gene Transfer (LGT) from

Questions to Explore in Parasitic Venom Evolution

has mostly focussed on functions of individual toxins, usually of high abundance,

Technological advances in recent years have made venom analysis in parasitoid

Rapid advancement of parasitic venom research is needed to establish parasitoid

Venom as a Component of External Immune Defense in Hymenoptera

Immune Defenses in Solitary and Social Hymenoptera

Hymenoptera Venoms: A Complex Multifunctional Secretion

The Evolutionary History of Venom in Hymenoptera

Venom Use in Solitary and Parasitic Hymenoptera

Rise of Sociality and the Threat of Predators and Pathogens

Venom as Externalized Immune Defense in Social Hymenoptera

Venom on the Cuticle

Venom on the Nest Surface

Proportion surviving 0.7