BiochemicalSystematicsand Ecology,Vol. 12, No. 4, pp. 395--406,1984. 0305-1978/84 $3.00+0.

00
Printed in GreatBritain. PergamonPressLtd.

Chemical Defences in Leaf Beetles and their Larvae:

The Ecological, Evolutionary and Taxonomic Significance

JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER,* JEAN-CLAUDE BRAEKMAN

and DESIRE DALOZE
Collectif de Bio-ecologla, Facult6 des Sciences, Universit~ Libre de Bruxelles, 1050 Bruxellas, Belgium; and
*Zoologisches Institut der Universit~t Basel, Rheinsprung 9, Basel 4051, Swilzerland

Key Word I n d e x - Chwsomelinae; defensive secretions; host plant influence; predator spectrum; methylcyclopentanoid
monoterpenes; salicylaldehyde; cardenolides; isoxazolin-5-one glucosides.
Abatmct- The chemical defences of the Chrysomelinae ere reviewed. Defensive glandular secretions have evolved
independently in larvae end adults, and faster than the morphology of the glands. Both characters are used in a phylogenetic
study of the Chrysornelini, disclosing suprageneric affinities. First, a close relationship between the Chrysomelina and
Phratora is proposed. Secondly, Leptinotarsa and Gonioctena are probably more closely related to the Chrysolinina than to
the Chrysomelina and Phratora. The qualitative and quantitative composition of larval and adult secretions sometimes differs
between geographically isolated populations of the same species, which are therefore probably genetically distinct. The
host plant shows an influence which explains discrepancies between the current classification and the chemical nature of the
secretions. Some beetles sequester secondary plant metabolites or use them as precursors for their own defences. The original
biosynthetic pathway has been lost in these species. Other factors which could affect the chemical diversity of the secretions are
discussed: random events such as neutral mutations and genetic drift, conditioning or adaptation of predators to specific defen-
sive mixtures, and the diversity of potential predators. The latter could help to explain the divergence in the chemical nature of the
larval and adult secretions. The volatile irritants produced by the larvae are well suited to repel small arthropods, such as ants,
whereas poisons associated with aposematic coloration in the adults could be directed against small vertebrates, such as birds.

Introduc~on based on more restricted faunae [5-9] are however

Chrysomelidae, or leaf beetles, are phytophagous also mentioned (summary in Table 1).
insects, often forming large aggregations on their In addition to phylogenetic aspects, we attempt
food-plants. This makes them particularly to relate the nature of the defensive secretions to
apparent to predators and parasitd(ds, and spec- ecological factors such as the host plants and the
tacular chemical, mechanical and behavioral probable predator spectrum. In temperate regions,
defensive mechanisms have evolved in this family Chrysomelinae are often found in damp, open,
[1]. Defensive glandular secretions of both larvae relatively undisturbed habitats such as riversides,
and adults are particularly prominent in the brightly marshes and meadows. There, the favored food
colored Chrysomelini (Chrysomelinae). plants include both herbs [docks (Polygonaceae),
The classification of the Chrysomelinae into buttercups (Ranunculaceae) or mints (Labiateae)]
tribes and groups of genera is controversial. Data and also some trees and shrubs [willows and
on the chemical ecology of the chrysomeline larvae poplars (Salicaceae) or alders (Betulaceee)]. The
and adults might bring new taxonomical criteria. In Chrysomelinae are either monophagous or oligo-
this paper, new and previously published (refer- phagous on these plants. There is evidence that
ences in [2]) chemical data on the defensive secre- evolution within this habitat proceeds, at least, in
tions of the Chrysomelini are summarized, and part through sympatric speciation via shifts of host
their ecological and possible evolutionary implica- plants [10].
tions are discussed (see also [3] for a first account
of these aspects). We follow the new pan-global Glandular secretions of larvae
classification of Daccordi [4]; other classifications The larvae of the subtribe Chrysomelina and of
the genus Phratora all possess 9 pairs of glands
(Received 17 December 1983) on the meso- and metathorax and on the first 7
395
396 JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER, JEAN-CLAUDE BRAEKMAN AND DESIRE DALOZE

TABLE 1. CLASSIFICATIONS OF THE CHRYSOMELINE GENERA CITED IN THIS PAPER, MODIFIED FROM DACCORDI 14], COX 15],
HENNIG [6], KIMOTO [7] AND WEISE [9] GIVEN IN CHRONOLOGICAL ORDER OF APPEARANCE

Weise Hennig Kimoto Cox Daccordi

on world species* on German species'i" on Japanese species1 on British species1 on world species* 1"

Chyrsornelini Group 1 Generic group Chrysolina Chrysometini Chrysomelini

Chrysolina Chrysolina Chrysolina Chrysolina Doryphorina
Oreina Oreina Oreina Leptinotarsa Leptinotarsa St~l
Leptinotarsa Lep#notarsa Leptinorarsa Gonioctena Chrysolinina
Chrysolina Motschulsky
Oreina Chevrolat
Phaedonini Group 3 Generic group Phaedon Phaedonini Chrvsomelina
Phaedon Phaedon Phaedon Phaedon Phaedoj~ Latreille
Gastrophysa Gastrophysa Gastrophysa Gastrophysa Gastrophysa Chevrolat
Chrysomela Chrysomela Phratora Phratora Chrysomela L.
(incl. Linaeidea) (incl. Linaeidea) Chrysomela Linaeidea Motschulsky
Plegiodera Hydrothassa Generic group Chrysomela Plagiodera Plagiodera Chevrotat
Gastrolina Pfagiodera Chrysomela Prasocuri$ Gastrolina Baly
PrasocurJs Prasocutis Linaeid~ Hydrothassa Prasocu/is Latreille
/-lydrothassa Phratora Plagiodera Hydrothassa Thomson
Phaedonia Gastrolina Phaedonia Weise

Generic group Prasocuris

Prasocuris
Hydrothassa

Ph~lodectini Group 2 Generic group Gonioctena Gonioctenina

Phratora Gonioctena Gonioctena Gonioctena Chevrolat
Gonioctena Paropsina
Paropsis Olivier
Chrysophtharta Weise
Phyllodectina
Phratora Chevrolat

*Chiefly on adult criteria.

1.Chiefly on larval criteria.
To avoid confusion due to synonymy we have adopted the generic nomenclature of Daccordi (Column 5) in all classifications.

f.o c.o c.o

H
Chrysomelidial Epich rysomelidial Plagiodial
0

H ;
Plagiolactone Epiplagiolactone Gastrolactone

FIG. 1. METHYLCYCLOPENTANOID MONOTERPENES IDENTIFIED IN THE LARVAL SECRETIONS OF CHRYSOMELINI,

CHEMICAL DEFENCES IN LEAF BEETLES AND THEIR LARVAE: THE ECOLOGICAL EVOLUTIONARY AND TAXONOMIC SIGNIFICANCE 397

abdominal segments. The glands are eversible, very reduced, is also present in other groups (e.g.
and when the larvae are disturbed, a drop of Chrysolina and Gonioctena species) but have not
secretion appears at the tip of the everted yet been studied in detail. Takizawa [8] put these
reservoirs. Most of this secretion can be species with only one pair of glands or without
withdrawn back into the reservoir during the glands in a "'non-glanduliferous group".
retraction of the latter into the body [11, 12]. The principal classes of compounds found so far
According to the descriptions of Cu6not [13], in the larval secretions of Chrysomelini are listed in
Hollande [12], Garb [11], Berti [14] and Renner Table 2 together with the host plant of each
[15] there seems to be little morphological species. The structures of these compounds are
difference between the glands of the different given in Rgs 1 and 2. Table 3 gives the composition
genera. For Takizawa [8] the genera with such of the mixtures of methylcyclopentanoid mono-
glands belong to the "glanduliferous group". terpenes secreted by species of the subtribe
The Paropsina have only one single pair of well Chrysomelina and the genus Phratora, including
developed glands between the 8th and 9th data for geographically isolated populations of
abdominal tergites [16]. One pair of glands, often some species.
TABLE 2. PRINCIPAL CLASSES OF COMPOUNDS FOUND IN THE GLANDULAR SECRETIONS OF LARVAL CHRYSOMELINI*

Host plants Referenoes

Chrysomelina
ChPfsome~ populi + Populus 45
C. tremulae + Populus 2
C. saliceb" + + Salix t
C. 20-punc=a= costelta + + Salix 32
C. 20-punctata + +~ Salix 2
C. scripta + Sa/ix 46
C eenicollis + SMi~ Populus
C. interrupta Alnus 31
Gastrolina depressa Juglans 32
Gastrophysaatrocyanea + + Rumex 36, 47
G. cyanea + Rumex 35
G. viridula + Rumex 2
Hydmtha=~ marginella + Ranunculus 2
Linaeidee eenee + + Alnus 37
Phaedon brasdcae + Brassica 35
P. coch/eariae + Nasturtium 2
Plagiodera versicolora + + ~ Salix 49
Prasocuris phellandrii + Ranunculus 2

Ph~dec~'na
Phratora vitNlinee + Salix, Popu/us 49
P. taticollis + Populus
P. ~'bialis + Se/ix
P. vulgatissima + Salix

Peropsina
Paro/xcis atumetia + + Eucalyptus 16
Chrysophtharta varfico//is d" + Eucalyptus 16
C. amoena + + Eucalyptus 16

*Seefigures.
I"This paper.
Present in only one of the four populations studied.
3S8 JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER, JEAN-CLAUDE BRAEKMAN AND DESIRE DALOZE

OH

~IHO ~'~CFIO
Salieyl~dehyde Benzaldehyde -Phenylethy!- isobutyrate

OH 0

Juglone 8 " Phenylethyl - 2 - methylbatyrate

FIG. 2. AROMATIC COMPOUNDS IDENTIFIED IN THE LARVAL SECRETIONS OF CHRYSOMELINI.

Chemical defence in adults unique food plant (Eucalyptus species) and the
In adults, the defensive apparatus consists of defensive secretion of the larvae. The presence of
clusters of glands opening into grooves on the benzaldehyde together with hydrocyanic acid in
surface of the pronotum and the elytra [1]. These this secretion suggests a biosynthetic pathway
glands are found not only in the Chrysomelinae starting from phenylalanine, and with
but also in the Criocerinae, Alticinae and Galeru- mandelonitrile as an intermediate (review [21]). In
cinae, forming one phyletic line [17]. They have the secretion of Chrysomela 20-punctata and C.
been briefly described by Cu~not [13], Tower saliceti, benzaldehyde is associated with
[18], Hollande [12] and Patay [19]. Deroe and salicylaldehyde rather than hydrocyanic acid. This
Pasteels [20] made a detailed histological study of implies a different metabolic pathway and does
the glands of Leptinotarsa decemlineata. not indicate a close taxonomic relationship
When the beetles are disturbed the secretion between the Paropsina and those Chrysomela.
oozes from the openings of the glands, covers No defensive secretion has been reported as yet
the integument and accumulates in the marginal in adult Paropsina.
grooves. The different classes of compounds
recognized so far in the secretion of adult Chrysolinina
Chrysomelini are listed in Table 4. The structures The adults possess secretions characterized by
of the cardenolides and the isoxazolin-5-one the presence of cardenolides, which so far have
glucosides identified in the secretions are given in not been detected in other chrysomelid taxa,
Figs 3 and 4. either within or without the Chrysomelini [22, 23].
Since cardenolides were not detected in the food
Taxonomic implications plants (e.g. Mentha and Galeopsis) they are
As can be seen from Tables 1, 2 and 4 the probably synthesized by the insects, presumably
taxonomic grouping of Daccordi [4] accords well from a plant sterol precursor [22].
with the distribution of the different classes of Five species of Chrysolina ( C. brunsviscensis, C.
compounds found in the defensive secretions of varians, C. hyperici, C. gerninata and C. carnifex)
both larvae and adults. do not produce cardenolides. This could
represent an adaptation to a particular host plant
Paropsina (see next section).
The Paropsina (Paropsis and Chrysophtharta) form Another characteristic of the Crysolinina is the
a distinct group. This is consistent with their presence in the secretions of the adults of amino
single pair of defensive glands in the larvae, their acid derivatives and of small quantities of lipids.
CHEMICAL DEFENCES IN LEAF BEETLESAND THEIR LARVAE: THE ECOLOGICALEVOLUTIONARYAND TAXONOMIC SIGNIFICANCE 399

No larval secretion is known in this group, but the 8 genera studied (Table 2). The exceptions
cardenolides were detected in extracts from whole can be explained by a secondary adaptation to
individuals of all immature stages, in increasing particular host plants (see next section).
quantity with age [23]. The secretion of the adults is characterized by
the presence of isoxazolin-5-one glucosides and a
Doryphorina large quantity of lipids, unlike the Chrysolinina,
In the Doryphorina, the adult secretion of only the Doryphodna and Gonioctena.
one species has been studied. It does not contain
any cardenolides but amino acid derivatives and Phyllodectina
relatively small quantities of saturated hydro- In the Phyllodectina, defensive compounds have
carbons are presem. This suggests a closer rela- been fully identified only in Phratora. The larvae,
tionship between the Doryphorina and the like those of the Chrysornelina, secrete methyl-
Chrysolinina than between the Doryphorina and cyclopentanoid monoterpenes (R vitellinae
the Chrysomelina. Indeed, Lep~'notarsa and being an exception) and the adults produce
Chrysolina have been put in the same taxon by isoxazolin-5-one glucosides.
many authors [5-7, 9; Table 1), on morphological The taxonomic affinities of Phratora are
criteria. controversial (Table 1). Weisa [9], using morpho-
No larval secretion has yet been described or logical characteristics of adults, grouped Phratora
analyzed. with Gonioctena (=Phytodecta) in a distinct
tribe, the Phytlodectini. This subdivision has been
Chrysomelina followed by Chen [24], Derenne [25] and Jolivet
The Chrysomelina seem also to be a natural and Petitplerre [26]. However, on the basis of
category on the basis of their defensive larval characteristics Cox [5], Hennig [6], Kimoto
secretions. Methylcylo;Jentanoid monoterpenes [7] and Takizawa [8] recognized that the Phyllo-
have been found in the larval secretions of 6 of dectini of Weise do not seem to be a natural tribe,

TABLE 3. PROPORTIONS (%) OF THE DIFFERENT IDENTIFIED METHYLCYCLOPENTANOID MONOTERPENES IN THE SECRETIONS OF
LARVAL CHRYSOMELINI*

Origin (reference)

Gastrophysa cyanea 50 50 U.S.A. [35]

G. atrocyanea 100 - - Japan [36]
G. vindula 90 10 - - Belgium 121
Hydrothassa marginella 9O 9O - 10 Belgium [2]
Linaeidea aenea 35 15 50 Japan (371
L. aenea 85 15 Belgium 12]
Phaedon brassicae 100 Japan 135l
P. cochlearme 100 100 Belgium 12]
Rag~odera versicolora 68 66 - 33 U.S.A. 1481
P. versicolora 70 3O Belgium [21
P. versicolora distincta 3 9O 3 Japan 1371
P. versicolora ~ § 2 2 4 17 Germany1.
Prasocuris phe#andm" 100 Belgium 121
Phratora dbidis§ 7 7 91 tr Belgium 1"
P. la~'collis§ 3 3 79 1 Bqdgium 1"
P. vulga~ssima§ 68 68 tr - Switzerland 1.

*For formulae see Fig. 1.

1.This paler.
:~68% of salicylaldehydealso presem.
§Minor comgounOs remain unidentified.
- The compound has not been detected in the secretion.
400 JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER, JEAN-CLAUDE BRAEKMAN AND DESIRE DALOZE

Phratora being more closely related to the chemistry neither supports nor contradicts this
Chrysomelina than to Gonioctena. This is association. It is, however, interesting to note
consistent with the presence of 9 pairs of that in captivity P. polaris accepts as food plant
defensive glands in the larvae of Phratora but not Polygonum viviparum, a member of the same
in those of Gonioctena. The composition of the family (Polygonaceae) as the food plants of
defensive secretions of both larvae and adults Gastrophysa [27]. As already suggested by Cox
confirms this opinion. The precise nature of the [5] (Table 1), Gonioctena might have some affini-
secretion of adult Gonioctena is unknown. The ties with both Doryphorina and Chrysolinina.
isoxazolin-5-one glucosides characteristic of
Phratora and of the Chrysomelina are not Secondarily derived secretions and the
present, but amino acid derivatives were detected influence of the host plant
as well as small amount of lipids. On the basis of The wide distribution of methylcyclopentanoid
chemical data from both larvae and adults, we monoterpenes in the secretions of the Chrysome-
favor the idea that Phratora should be included lina and Phratora larvae suggests that these
within the Chrysomelina. Kimoto [7] and compounds represent their primitive defensive
Takizawa [8] put Phratora in the same generic secretion. These monoterpenes are most likely
group as Phaedon and Gastrophysa. Defensive synthesized de novo, since they have no obvious

0 o ~,

HO. HO..Oo

OH
OH H

H
H H
Sarmentogenin O Sarmentogenin - 33- xyloside 0
~. O o

HO. HO.,..

OH HO...-T,~ Z H

HO
OH
Bipindogenin 0 Bipuldogenin - 33 • x~,'loside
O O

OH
OH HO HO OH
H
OH
Periplogenin Periplogenin-3~- xyloside

FIG. 3. CARDENOLIDES IDENTIFIED IN THE SECRETIONS OF ADULT CHRYSOL/NA COERULANSAND C. HERBACEA.

CHEMICALDEFENCESIN LEAFBEETLESAND THEIR LARVAE: THE ECOLOGICALEVOLUTIONARYANDTAXONOMICSIGNIFICANCE 401

0 various reasons. First, they are one of the most

abundant woody plants in the biotopes favored
O/!~CH= CH= NO= by the beetles. Secondly, the leaves of some
Salix species, in contrast to those of most woody
plants, do not contain proanthocyanidins [30] and
their secondary chemistry might therefore be
more favorable for a herbivore adapted to digest-
OH ing herbs [28]. That the secretion of salicylalde-
hyde is advantageous to the larvae feeding on
Salicaceae is supported by the fact that it has
occurred in at least three genera: Chrysomela,
Phratora and in one German Upper-Rhein popu-
lition of Plagiodera versicolora. The litter is per-
OH ticulady interesting because it secretes a mixture
of salicylaldehyde (as major constituent) and
FIG. 4. ISOXAZOLIN-~-ONE GLUCOSIDES IDENTIFIED IN THE
SECRETIONS OF ADULT CHRYSOMELINA AND PHRATORA. monoterpenes, thus differing from the previously
studied American, Japanese and Belgian popu-
litions (Table 1) which secrete only monoter-
precursors in the food plants of the larvae and penes. This mixture of salicylaldehyde and
their presence is independent of the food plant; monoterpenes in the secretion of the larvae of
e.g. chrysomelidial is secreted by the larvae of only one of the populations studied could
different species feeding on Alnus, Rumex, represent an intermediary step in the evolution of
Ranunculus, Brassica, Nasturtium and Salix, res- the secretion of aromatic compounds. Hollande
pectively. [12], working in Grenoble (France), also reported
The larvae of some species of Chrysomelina the presence of salicylildehyde in the secretions
and Phratora, however, secrete not monoter- of P. versicolora larvae; unfortunately he does not
penes but instead aromatic compounds; most of give precise information on the chemical methods
them feed on trees belonging to the Salicaceae or used nor on the exact origin of the insects
Betulaceae. For both C. tremulae and P. vitellinae studied. The change from the secretion of
the salicylaldehyde secreted by the larvae has monoterpenes to that of salicylaldehyde has
been shown to be derived from salicin, a phenyl- probably occurred early in the evolution of the
glucoside present in the leaves of their salicaceous genus Chrysomela, since none of its present
food plants [28, 29]. Thus in contrast to the situa- studied species are still producing monoterpenes.
tion described for the species producing monoter- The change must have occurred more recently in
penes, in at least some of the species producing the genus Phratora, because only one of its four
aromatic compounds the defensive secretion is studied species secretes salicylildehyde; and even
dependent upon the host plant. more recently and not completely in Plagiodera.
The secretion of aromatic compounds by The Alnus-feeding C. interrupta also produces
chrysomeline larvae can be best explained as a aromatic compounds, but these are phenylethyl-
consequence of a shift of food plant, associated esters, not salicylaldehyde. It has already been
with speciation, from herbs to Salicaceae or shown that C. interrupta belongs to a group of
Batulaceae. The secretion of salicylaldehyde sibling species which have apparently evolved
would be an adaptation to the secondary through ecological isolation, some feeding on
chemistry of the Salicaceae, enabling the larvae Alnus and others on Populus or Salix [10]. Brown
not only to produce cheap defensive compounds successfully fed the larvae of the Populus-feeder
largely presynthesized by the food plant, but also on Alnus and obtained hybrids between the
to mobilize an otherwise unexploited energy Alnus- and the Populus-feeders.We suggest that
source, the glucose moiety of selicin [29]. C. interrupta originated through a secondary
Salicaceous bushes and trees might have been ecological speciation from a Salicaceae-feeder
particularly likely objects of host plant switches of which had lost the ability to synthesize monoter-
primitively herb feeding Chrysomelinae for penes for defence but had acquired the capacity
402 JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER, JEAN-CLAUDE BRAEKMAN AND DESIRE DALOZE

to incorporate aromatic compounds in its The sporadic occurrence in the secretions of

defensive secretion. Alnus being devoid of salicin, compounds derived from plant precursors
it is not salicylaldehyde but phenylethylesters supports the idea that their production is a secon-
which are produced. These phenylethylesters are dary development within the lineage.
not themselves present in Alnus leaves [31] and In adults, the host plant may also influence the
their origin remains unknown. nature of the chemical defence. The five species
Another example of the influence of host plant of Chrysolina not producing cardenolides (Table
chemistry on the composition of the defensive 4) feed on chemically protected plants such as
secretion of the larvae is that afforded by Hypericum and Artemisia. They could rely on
Gastrolina depressa [32] which secretes juglone, plant toxins for their defence [23] rather than
a naphthoquinone that could be easily derived synthesize costly cardenolides. Indeed, C. brunsvi-
from a 1,4,5-trihydroxynaphtalene glucoside censis sequesters hypericin from Hypericum [33]
present in the walnut tree (Juglans) on which it but does not incorporate it in an exocrine secre-
feeds. tion. C. didymata, another species feeding on
TABLE 4. CLASSES OF DEFENSIVE COMPOUNDS IN THE GLANDULAR SECRETIONS OF ADULT CHRYSOMELINI*

Amino-acid Isoxazolin-5-one
Cardenolides derivatives Lipids glucosides References

Dotyphorina
Leptinotarsa decernlineata - + + 2, 22
Chrysolinina
Chrysolina americana + + 2,22
banksi + + 2, 2 3
brunsvicensis 2,22
carnffex - 2, 23
cereali$ + + 2, 22
coerulans + + + + 2, 22, 23, 51
diversipes + + 2, 23
didimata + 2, 23
fas~osa + + 2, 22
geminata + "r
grossa + + 2, 22
haemoptera + + - 2, 23
herbacea + + + 2, 22, 51
hyperici - + - 2, 22
polita + + + ~ 2 , 2 2 , 23
sanguinolenta + + 2, 23
staphylaea + + 2, 23
vatians + ~ 2, 22
Oreina cacaliae + + + ~ 2, 22
tristis + + 2, 22
Chrysome/ina
Chrysomela populi - + + + + 2
tremulae - + + + + 2, 23
20-punctata - + + + + 2, 23
saliceti - + + + + ~r
Gastrophysa viridula - + + + + 2, 22
cyanea - + + 50
Hydrothassa margine/la - + + 2, 22
P h a e d o n brassicae - - + + 2, 22
Phaedonia circumcincta - (23) - + + ~r
Plagiodera versicolora - [22] - + + "t
Phy#odectina
P h r a t o r a vitellinae - + + 2
laticollis - + + 2, 22
tibialis - + + 2
Gonioctenina
Gonioctena olivacea - 121] + -

*Sea figures.
This paper.
- The compound has not been detected in the secretion.
CHEMICALDEFENCESIN LEAF BEETLESAND THEIR LARVAE: THE ECOLOGICALEVOLUTIONARYAND TAXONOMICSIGNIFICANCE 403

Hypeffcurn secretes only one cardenolide, sar- The significance of the diversity of chemical
mentogenin, whereas a mixture of 4-12 different defence between different sympatric species or
cardenolides is usually present in the secretion of between allopatric races of the same species is
other Chrysolina species. C. didyrnata could not clear. In most cases, no pheromonal function
represent an intermediate stage in the secondary is known which could explain the specificity of
loss of cardenolide production by insects feeding the blends. Tim larvae are often gregarious, but
on toxic plants [23]. groups of mixed species are easy to obtain in the
laboratory (e.g. R versicolora with R vitellinae).
Inter- and i,;~,=apedfic diversity Larvae of R vitellinae experimentally made
As seen above, the chemical defence of both devoid of defens'Ne secretion remain grouped. It
larvae and adults provides good taxonomic has been suggested that the secretion of the
characters at the supragenic level for the larvae of Gastrophysa viHdula may lower intra-
Chrysomelini. When the precise composition of specific competition. Indeed, the larvae evart
the defensive mixtures is considered, however, their glands at the approach of adults of the same
great variation is observed between genera and species which are strongly repelled [15].
species. Defens'Ne secretions of the larvae may also
It is clear from Table 3 that the larvae of most reduce interspecific competition. In the spring,
species produce a different blend of methylcyclo- the same Salix bushes are colonized first by
pentanoid monoterpenes. The secretions of adults of C. 20-punctata coming out of
different species within a genus are in some cases hibernation and a couple of weeks later by P.
more different than the secretions from species vitellinae. The larvae of the new generation of C.
belonging to different genera. Quantitative and 20-punctata develop rapidly and smell strongly of
qualitative differences exist between geographic- salicylaldehyde when they reach the third larval
ally isolated populations, e.g. Linaeida aenea in instar or the pupal stage. A decrease in the
Japan and Belgium, and P. versicolora in North population of P. vitellinae adults on the initially
America, Japan, Belgium and Germany. The favored trees is positively correlated with the
Japanese P. versicolora is usually considered a number of larvae and pupae of C. 20-punctata
distinct subspecies, but both the American and (RowelI-Rahler, unpublished observations).
the European forms belong to the same The diversity of defensive secretions could he
subspecies, the American having probably origin- due to genetic drift in the absence of selective
ated from insects introduced from Europe. These pressure for uniformity. Tschinkel [34], in his
differences are not due to sampling error: most of study of the defensive secretions of tenebrionids,
the proportions were calculated from the pooled suggested that their precise composition was not
secretions of hundreds of larvae. At least in P. critical within a given range of compounds as
versicolora, individual or year to year variations long as the secretion meets certain physical
are smaller than the differences observed requirements (e.g. lipophilicity, volatility, melting
between species or distant populations of the point, irritant value). Variation on the theme
same species [2]. would then be possible without any particular
Similarly, in the secretions of adult Chrysolina, ecological significance. On the other hand, it is
the spectrum of cardenolides varies quantitatively also possible that intraspecific variation between
and qualitatively from species to species. The geographically isolated populations reflects
mixture of cardenolides can also differ between adaptation to the different communities in which
isolated populations of the same species, e~g. the beetles are living. No data supporting this
between Belgian and Greek populations of both hypothesis are presently available.
C. po/h'a and C. herbacea. As judged by thin Selection for diversity would be another
layer chromatography, however, there is little possible explanation of the specificity of the
variation in the proportion of the different defensive secretions in sympatric species. In
cardenolides between individuals of one popula- nature, larvae of Hydrothassa marginella and
tion, from one generation to the next, or between Prasocuris phe/landri~ for example, are often
individuals fed on different food plant species found together on Ranunculus. They are probably
[23]. exposed to the same set of potential predators and
4O4 JACQUES M. PASTEELS, MARTINE ROWELL-RAHIER, JEAN-CLAUDE BRAEKMAN AND DESIRE DALOZE

parasitd='ds but they produce different mixtures of noticing that the relatively aposematic adults of P.
compounds. So far, the activity of the defensive vitellinae feed on the upper surface of the poplar
compounds has only been demonstrated with and willow leaves whereas the larvae which are
"standard laboratory predators" such as ants for more cryptic feed on the lower surface.
which at least chrysomelidial and salicylaldehyde The adults, on the other hand, are mechanic-
are deterrent [29, 35-37]. Using adult sawflies ally protected by their hard cuticle against small
(Tenthredo olivacea), a predator observed to feed predators like ants. Further, many species fall to
on chrysomeline larvae in nature, Pasteels and the ground on the slightest mechanical stimulus;
GrL~goire (in preparation) demonstrated that the this behavior pattern is probably effective against
sawflies can be conditioned and show a preference birds. The adults which are mostly brightly
for the larvae producing the secretion they have colored tend to clump together. The association
already experienced. Such behavior of predators of highly toxic compounds and aposematic color-
favors the maintenance or evolution of diversity of ation has been shown to be a good defence
defensive secretions in sympatric species. Especially against birds [41-43]. The toxicity of cardenolides
for species feeding next to each other on a plant, a for vertebrates is well known. The physiological
high diversity of defensive blends may be a good effect of the isoxazolin-5-one glucosides secreted
strategy to prevent adaptation of the predator. by the adults of the Chrysomelina and Phratora
Similarly, interspecific and intraspecific diversity of remains to be investigated./3-Nitropropionic acid
plant secondary metabolites is thought to limit the (Fig. 4), present as an ester in one of the major
adaptation of herbivores to these plants [38, 39]. constituents of the secretions, is known to be
very toxic for both vertebrates and insects [44]. In
Differences between the chemical nature of some legumes it is also present as glucosides and
the defensive secretion of the larvae and of responsible for their toxicity to mammals.
theadults We do not claim that larval defence is
The glands of the Chrysomelina larvae are not invariably inefficient against vertebrates, or adult
morphologically homologous to those of the defence against ants, but rather that one group of
adults. Moreover, the chemical nature of their defensive chemicals could be more adequate
secretion is very different. Although they live against one group of predators. This might be
together on the same plant, the chemical defence sufficient to explain the divergence in the
of the larvae and of the adults might be a chemical nature of the secretions of larvae and
response to different selective pressures or, adults.
alternatively, there might be no selection for them
to be identical. Conclusions
The small soft larvae of the Chrysomelinae are Enough data are presently available to allow some
easy prey for predators such as ants. A small taxonomic and evolutionary deductions from
wound can be fatal to a larva and it is therefore comparative studies of chemical defence of the
important for them to repel the potential predator Chrysomelini, especially when they are analyzed
at a distance. The secretion of a small volatile in their ecological context.
molecule is appropriate in this case [40]. Volatile Chemical defence has evolved separately in
monoterpenes appear to be very effective against larvae and adults. In both, the evolution of the
arthropods, acting as irritants at a distance. chemical nature of the secretions is not strictly
Salicylaldehyde, more volatile than its precursors linked to the evolution of the glandular structures.
saligenin and salicin, has been shown to be an Independent morphological and chemical infor-
effective deterrent against ants, even at low con- mation can thus be used to assess taxonomical
centration [29]..On the other hand, volatile com- affinities.
pounds may not provide an efficient defence So far, the existing information on chemical
against larger predators such as birds. The small defence is in agreement with Deccordi's
size of most larvae, their cryptic coloration and classification of the Chrysomelini from temperate
their feeding habit on the lower side of the leaves regions at the suprageneric level. The defence
are probably more effective in protecting the mechanism of Phratora shows more similarities
larvae against predation by birds. It is worth with that of the Chrysomelina than with
CHEMICAL DEFENCESIN LEAFBEETLESANDTHEIR LARVAE:THE ECOLOGICALEVOLUTIONARYANDTAXONOMIC SIGNIFICANCE 405

Gonioctena; and we consider the grouping of 9. Weise, J.'(1916) Coleoptm Cat. 88, 1.
Phratora and Gonioctena in a distinct subtribe to 10. Brown, W. J. (1956) Can. Ent. suppl. III I~, 1.
11. Garb, G. (1915) J. Ent. Zoo/. 8, 88.
be unnatural. Unfortunately, there is not enough 12. Hollande, A. Ch. (1909) Ann/s Univ. Grenoble U, 459.
information on the chemical ecology of tropical 13. Cu6not, L. (1896) ArchsZool. Exp. 4, 655.
species to suggest a rearrangement of the 14. Berti, N. (1968) Bull. Soc. Ent. Ft. 73, 114.
Chrysomelini on a world-wide basis. 15. Renner, K. (1970) Beltr. Ent. 20, 527.
16. Moore, B. P. (1967) J. AusL EnL Soc. 6, 36.
Some discrepancy between classification and
17. Jolivet, P. (1978)ActaZool. Path. Antverp. 70, 167.
the chemical nature of defensive secretions can !8. Tower, W. L. (1906) Carnegie Inst. Wash. 42, 1.
be interpreted as secondary adaptation to new 19. Patay, R. (1937) Bull. Soc. scient. Bretagne 14, 94.
host plants; such close adaptation to ecological 20. Deroe, C. and Pasteels, J. M. (1977) ArchsBiol., Bruxelies
conditions may thus mask taxonomical affinity. 88,289.
21. Blum, M. S. (1981) Chemical Defense of Arthropods.
The precise composition of the secretions Academic Press, New York.
offers no reliable taxonomical characters at the 22. Pasteels, J. M. and Daloze, D. (1977) Science 197, 70.
generic or specific levels. Differences are as great 23. Daloze, D. and Pasteels, J. M. (1979) J. chem. Ecol. 5, 63.
between species of different genera as between 24. Chen, S. H. (1936) Notes Ent. Chin. 3, 63.
congeneric species, or even between distinct 25. Derenne, E. (1963) Bull. Ann. Soc. R. Ent. Belgique94, 1.
25. Jolivet, P. and Petitpierre, E. (1976) Acta Zoo/. Path.
populations of the same species. This high Antverp. 88, 59.
diversity can be interpreted as the result of several 27. Morris, M. G. (1980) Entomologist's Mon. Mag. 106, 48.
factors: first, the absence of selective pressure for 28. RowelI-Rahier, M. and Pasteels, J. M. (1982) Proc. Vth
uniformity, leading to a certain degree of genetic Int. Syrup. "Insect-Rant Relationships" Wageningen
drift; secondly, a selective pressure for diversity in (Visser, J. H. and Minks, A. K., eds), p. 73. Pudoc,
Wageningen.
syrnpatric species, to take advantage of the 29. Pasteels, J. M., RowelI-Rahier, M., Braekman, J. C. and
establishment of preferences in the predators. Dupont, A. (1983) Physiol. Entomol. 8, 307.
The dissimilarity of the chemical defences of 30. Hegnauer, R. (1973) Chemotaxonomie der Pflanzen.
the larvae and adults living in the same habitat Birkhauser Vedag, Basel.
31. Blum, M. S., Brand, J. M., Wallace, J. B. and Fales, H. F.
could be explained by differences in the type of (1972) Life Sci. 11,525.
predators to which they are most vulnerable, e.go 32. Matsuda, K. and Sugawara, F. (1980) Appl. Ent. Zoo/.
ants or other small arthropods for the larvae and 15, 316.
birds or other vertebrates for the adults. This last 33. Rees, J. C. (1969) Proc. 2ndlnt. Syrup. "'lnsect and Host
hypothesis remains to be supported by further Plant" Wageningen, The Netherlands (De Wilde, J. and
Schoonhoven, L. M., eds), p. 565. North-Holland,
ecological and experimental data. Amsterdam.
34. Tschinkel, W. R. (1975) J. Inst. Physiol. 21,763.
Acknowledgments - We are grateful to the "National Fund 35. Blum, M. S., Wallace, J. B., Duffield, R. M., Brand, J. M:;
for Scientific Research" (Belgium) and the "Geigy Jubil~um Fales, H. M. and Sokolski, E. A. (1978) J. Chem. Ecol. 4, 47.
Stiftung" (Swilzedand) for financial support. Many thanks 36. Sugawara, F., Matsuda, K., Kobayashi, A. and Yama-
are also due to H. F. Rowell and D. B. McKey for valuable shita, K. (1979) J. Chem. Ecol. 5, 635.
discussions and critical reading of the manuscript. The 37. Sugawara, F., Matsuda, K., Kobayashi, A. and Yama-
sample of larval secretion of Chrysomela aenicollis was kindly shim, K. (1979) J. Chem. Ecol. 5, 929.
provided by J. Smiley and C. Wisdom. 38. Dolinger, P. M., Ehdich, P. R., Fitch, W. L. and Breed-
lose, D. E. (1973) Oecologia 13, 191.
39. Atsett, P. R. and O'Dowd, D. J. (1976) Science 193, 24.
40. Pasteels, J. M., Gregoire, J. C. and Rowell-Rahier, M.
(1963)A. Rev. Ent. 28,263.
41. Rothschild, M. (1971) Ecological Genetics and Evolution
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