ORIGINAL RESEARCH
published: 12 February 2019
doi: 10.3389/fevo.2019.00026
Male Sex Pheromone of the
Parasitoid Wasp Urolepis rufipes
Demonstrates Biosynthetic Switch
Between Fatty Acid and Isoprenoid
Metabolism Within the Nasonia
Group
Joachim Ruther 1*, Tyler Wittman 2 , Christopher Grimm 3 , Florian S. Feichtner 1 ,
Sonja Fleischmann 1 , Josef Kiermaier 4 , Bethia H. King 2 , Werner Kremer 5 ,
Hans Robert Kalbitzer 5 and Stefan Schulz 3
1
Edited by:
Astrid T. Groot,
University of Amsterdam, Netherlands
Reviewed by:
Marjorie A. Lienard,
Broad Institute, United States
Angel Guerrero,
Instituto de Química Avanzada de
Cataluña (IQAC), Spain
*Correspondence:
Joachim Ruther
joachim.ruther@ur.de
Specialty section:
This article was submitted to
Chemical Ecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 04 December 2018
Accepted: 23 January 2019
Published: 12 February 2019
Citation:
Ruther J, Wittman T, Grimm C,
Feichtner FS, Fleischmann S,
Kiermaier J, King BH, Kremer W,
Kalbitzer HR and Schulz S (2019)
Male Sex Pheromone of the Parasitoid
Wasp Urolepis rufipes Demonstrates
Biosynthetic Switch Between Fatty
Acid and Isoprenoid Metabolism
Within the Nasonia Group.
Front. Ecol. Evol. 7:26.
doi: 10.3389/fevo.2019.00026
Institute of Zoology, University of Regensburg, Regensburg, Germany, 2 Department of Biological Sciences, Northern Illinois
University, DeKalb, IL, United States, 3 Institute of Organic Chemistry, Technische Universität Braunschweig, Braunschweig,
Germany, 4 Faculty of Chemistry and Pharmacy, University of Regensburg, Regensburg, Germany, 5 Institute of Biophysics
and Physical Biochemistry, University of Regensburg, Regensburg, Germany
Sex pheromones play a crucial role in the mate finding process of insects. The information
has to be encoded in a species specific manner to avoid fitness costs due to courtship
by or even mismating with closely related species. Hence, sex pheromones normally
diversify when new species evolve. Pheromones of closely related species typically vary
in their chemical composition, but the compounds they use are often biosynthetically
related. The biosynthetic pathways of insect pheromones are variations of conserved
pathways that lead to primary and secondary metabolites. A switch from one pathway
to another, particularly in species that use the same type of pheromone glands, is the
exception. Here we use chemical analyses and behavioral bioassays to examine the
male sex pheromone of the parasitoid wasp Urolepis rufipes. The genus Urolepis is most
closely related to Nasonia and Trichomalopsis, forming the so-called “Nasonia group.”
All Nasonia species and T. sarcophagae, the only Trichomalopsis species studied so
far, produce fatty acid-derived hydroxylactones in their rectal vesicle. The pheromones
are deposited on the ground and other substrates and attract virgin females. We show
that males of U. rufipes produce a sex pheromone in the same gland and use it in
the same manner as the other species of the Nasonia group. Structure elucidation and
stable isotope labeling experiments revealed, however, that the U. rufipes pheromone is
2,6-dimethyl-7-octene-1,6-diol, a monoterpenoid derived from the mevalonate pathway.
This suggests a biosynthetic switch between the fatty acid and isoprenoid metabolism
in the Nasonia group.
Keywords: 2,6-dimethyl-7-octene-1,6-diol, biosynthesis, mate finding, evolution, monoterpenoids, parasitoid, sex
pheromone, Urolepis rufipes
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Ruther et al.
Male Sex Pheromone in Urolepis rufipes
INTRODUCTION
America and with Nl in western North America (Darling and
Werren, 1990; Raychoudhury et al., 2010; Werren et al., 2010).
Nasonia males deposit volatile sex pheromones on substrates,
such as host pupae from which females are about to emerge or
the ground in the vicinity of these hosts (Mair and Ruther, 2018).
A male does this by dragging the tip of his abdomen, leaving
behind short, white marks. Nv males show increased marking
behavior after copulation and after mere contact with a female
during courtship (Barrass, 1969; Van Den Assem et al., 1980;
Steiner and Ruther, 2009). Marked areas are highly attractive to
virgin females (Ruther et al., 2007), and males are arrested by
their own marks (Van Den Assem et al., 1980; Ruther et al., 2008).
Studies on Nv have shown that the female’s response is plastic,
with her becoming unresponsive to the pheromone after mating
(Ruther et al., 2007, 2014). This switch in responsiveness occurs
quickly and is independent of sperm transfer. Instead, the switch
correlates with the female’s receptivity signal (lowering of the
antennae and simultaneous opening of the genital orifice), which
she exhibits during courtship by a mounted male (Ruther et al.,
2010; Ruther and Hammerl, 2014; Lenschow et al., 2018).
The pheromones of all Nasonia species contain (4R,5S)-5hydroxy-4-decanolide (RS) as a major component, whereas only
Nv males produce significant amounts of its epimer, (4R,5R)5-hydroxy-4-decanolide (RR). RR is produced enzymatically
from RS by short chain dehydrogenases/reductases (Niehuis
et al., 2013; Ruther et al., 2016). Nv males emerge without
the pheromone in the gland, but titers increase within the
first 2 days after emergence (Ruther et al., 2007). Traces of 4methylquinazoline (MQ) also occur in the pheromone glands
of all Nasonia species, synergizing the female response to
the hydroxylactones and mediating site fidelity of males at
marked areas (Ruther et al., 2008, 2011). Stable isotope labeling
experiments with Nv have revealed that both RS and RR are
derived from fatty acid metabolism, with oleic and linoleic acid
functioning as precursors (Abdel-Latief et al., 2008; Blaul and
Ruther, 2011; Blaul et al., 2014).
The genus Nasonia is closely related to the genera
Trichomalopsis (Crawford, 1913) and Urolepis (Walker, 1846),
and all three genera have been proposed to form a monophyletic
taxon, the so-called “Nasonia group” (Burks, 2009). As for the
57 Trichomalopsis species described so far (Noyes, 2017), only
T. sarcophagae has been studied with respect to its pheromone
communication (Niehuis et al., 2013). The male sex pheromone
components of T. sarcophagae match those of Nl, Ng and No,
i.e., consist of RS and MQ. In contrast, sex pheromones of the
three described Urolepis species, U. maritima, U. singularis, and
U. rufipes (Ur) (Gibson, 2000; Noyes, 2017), have not yet been
identified, although recent studies suggest that Ur males produce
a sex pheromone (Cooper and King, 2015). Like the Nasonia
species, Ur is a pupal parasitoid of several Diptera such as house
flies and stable flies (Stenseng et al., 2003) and has been found
together with Nv in North American dairies (Smith and Rutz,
1991; Gibson and Floate, 2004) as well as in European pig and
cattle farms (Skovgard and Jespersen, 2000). Ur males exhibit
substrate-borne marking similar to what has been described
for Nasonia males (Cooper and King, 2015). Ur females spend
more time near these markings, and males show site fidelity at
The mate finding process of animals typically includes sexual
communication via visual, auditory, or olfactory signals (Smith
and Harper, 2003). Most insects produce sex pheromones
to attract potential mates from a distance and to exchange
information at close range about their species, sex, or
individual quality (Johansson and Jones, 2007; Wyatt, 2014). Sex
pheromones have to encode information in a species-specific
manner to prevent costly sexual interactions with unsuitable
mates or even mismating with closely related species. As a
consequence, sex pheromones typically diversify when new
species evolve (Smadja and Butlin, 2009). Insect pheromones
are mostly blends of several compounds which are synthesized
in specialized glands or tissues and often occur in speciesspecific ratios (Schulz and Francke, 2010). The biosynthetic
pathways by which insects produce sex pheromones are often
variations of the conserved pathways leading to common primary
and secondary metabolites, such as fatty acids, amino acids,
aromatics, polyketides or isoprenoids (Tillman et al., 1999;
Morgan, 2004; Jurenka et al., 2017).
Novel pheromone phenotypes can evolve either quantitatively
by shifts of the relative proportions of the same components
or qualitatively by the gain or loss of single components. This
process can occur gradually by subtle changes over evolutionary
time or by saltational shifts generating phenotypes that are greatly
or completely different from the antecedent ones (Symonds
and Wertheim, 2005; Symonds and Elgar, 2008; Symonds
et al., 2009). New pheromone components can derive from
simple chemical modifications of existing ones, for instance
by enzymatic oxidation, reduction, acetylation, decarboxylation
or epimerization (Symonds and Elgar, 2008; Ruther et al.,
2016). Even when pheromones of closely related species differ
considerably in their chemical composition, the compounds
they use are often biosynthetically related (Tillman et al., 1999;
Symonds and Elgar, 2008; Wyatt, 2014). Much less common is
a switch from one pathway to another, particularly in species
that use the same type of pheromone glands. For example, in
moths virtually all known female sex pheromones are fatty acid
derivatives which are either synthesized de novo from acetylCoA units (Type I pheromones) or produced by modification of
dietary polyunsaturated fatty acids (Type II) (Ando et al., 2004).
In bark beetles, the vast majority of aggregation pheromones
are isoprenoids synthesized de novo via the mevalonate
pathway (Blomquist et al., 2012) and many compounds are
shared by numerous bark beetle species from different genera
(El-Sayed, 2018).
Parasitoid wasps are excellent models to study pheromone
communication in insects (Niehuis et al., 2013; Ruther, 2013;
Weiss et al., 2013). In particular, the genus Nasonia (Ashmead,
1904), which includes the genetic model organism N. vitripennis
(Nv), has allowed significant advances in our understanding of
the behavioral, biochemical, genetic, and evolutionary aspects of
pheromone communication. Apart from Nv, the genus consists
of the species N. longicornis (Nl), N. giraulti (Ng), and N. oneida
(No), all of which parasitize the pupae of numerous fly species. Nv
is cosmopolitan and sympatric with Ng and No in eastern North
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Male Sex Pheromone in Urolepis rufipes
revealed that male extracts contained a compound later identified
as 2,6-dimethyl-7-octene-1,6-diol (DMOD, 6 see section Results).
To investigate whether the rectal vesicle is the site of DMOD
biosynthesis, 2-day-old unmated males (n = 8) were dissected
with fine forceps (Dumont 5A) under a stereo microscope.
Freeze-killed males were transferred to a drop of distilled water in
a clean glass dish. Males were dissected by pulling the abdominal
tip and spreading the internal genitalia and the intestinal tract
in the water. Genitalia and gut were carefully separated using a
fine needle and the yellowish-white spherical rectal vesicle was
transferred to the glass vial for extraction.
Because the rectal vesicle is the production site of DMOD
(see section Results), we extracted only dissected abdomens
for quantitative analyses. To study the age-dependency of the
DMOD titers in Ur males, we extracted the abdomens of males
of different age (<1 h, 1 day, 2 days, n = 10 per age) in 30
µl dichloromethane containing 10 ng µl−1 methyl undecanoate
(Sigma-Aldrich, Deisenhofen, Germany) as an internal standard.
For DMOD quantification, peak areas of DMOD were related to
the peak area of the internal standard.
To investigate the influence of male mating frequency on
their DMOD titers, we extracted the abdomens of 2-day-old
males that were virgin or had mated 1, 3 or 5 times prior
to pheromone extraction. Ur males exhibit increased marking
activity after mating (Cooper and King, 2015), and comparable
tests with N. vitripennis revealed decreasing pheromone titers in
multiply mated males (Steiner and Ruther, 2009). Each Ur male
was exposed in a glass Petri dish (5 cm diameter) to a virgin
female until mating was observed. After each mating, the female
was removed and the male was kept alone for 10 min in the
same glass dish to enable pheromone deposition. A clean glass
Petri dish was used for each mating and the respective postmating period. Each control male was transferred successively
to 5 clean glass dishes without exposing him to females. DMOD
extraction was done as described above for the age dependency of
DMOD titers.
To test bioactivity of natural pheromone extracts and fractions
thereof, we extracted batches of 30–50 1- to 3-day-old males
for 30 min with dichloromethane (25 µl/wasp). These raw
extracts were concentrated under a gentle stream of nitrogen
to a concentration of 0.5 male equivalents per µl and used
for the bioassay. Additionally, we isolated DMOD from the
natural extracts either by size exclusion high performance
liquid chromatography (SE-HPLC) (Sperling et al., 2015) or
by solid phase extraction (SPE) on silica gel. For SE-HPLC,
raw extracts of 30–50 males (see section Pheromone Extraction
and Quantification From Wasps) were concentrated to 20 µl
and separated on a 300 × 7.5 mm PLgel SE-HPLC column
(particle size 5 µm, pore size 100 Å) (Agilent Technologies,
Waldbronn, Germany) with DCM as mobile phase as described
previously (Sperling et al., 2015). The DMOD fraction eluted
between 7.40 and 8.00 min, and purity was controlled by
GC/MS (Supplementary Figure S1). A total of 45 µg pheromone
was purified. Dichloromethane was evaporated under a gentle
stream of nitrogen, and the residue was re-dissolved in
deuterated dichloromethane and used for nuclear magnetic
resonance (NMR) spectroscopy experiments (see section Nuclear
their own marks and defend them aggressively against other
males. Furthermore, Ur males increase marking activity in the
presence of females and when fed honey (Cooper and King,
2015). Virgin females prefer areas where multiple males have
marked over areas where a single male has marked (Wittman
et al., 2016). Taken together, these results suggest that Ur males
use a substrate-borne sex pheromone in a similar manner as
described for Nasonia males.
In the present study we report the identification of the
male sex pheromone in Ur and compare several aspects of
its pheromone communication with the results previously
reported for Nasonia species. In particular, we investigate the
production site, the influence of age and mating frequency on
male pheromone titers, as well as the influence of previous
mating on the females’ pheromone response. Furthermore, we
perform stable isotope labeling experiments to identify the
biosynthetic pathway by which the pheromone is synthesized.
Our results demonstrate striking similarities between Ur and
the closely related Nasonia species with respect to production
site, release behavior and plasticity of the female pheromone
response. However, the sex pheromone of Ur males is an
isoprenoid, suggesting a biosynthetic switch between fatty acid
and isoprenoid metabolism in the Nasonia group.
MATERIALS AND METHODS
Insects
The Ur strain used in this study was kindly provided by K.
Floate and originated from cattle feedlots in southern Alberta,
Canada. Ur can be reared conveniently in the lab on freezekilled host pupae (Floate, 2002). We used freeze-killed pupae of
the green bottle fly Lucilia caesar, except the initial comparative
analysis and the bioassays with male extract and fractionated
pheromone that used Ur that had been reared on the house fly,
Musca domestica. Batches of approximately 100 host pupae were
transferred to Petri dishes and thawed for 30 min at 24◦ C in
an incubator. Subsequently, about 20 female Urolepis wasps per
Petri dish were exposed to the hosts in an incubator at a 16:8 h
light dark cycle, 25◦ C and 60% RH. After 13 days, parasitized fly
pupae were kept singly in tubes to ensure virginity of emerging
wasps. Mated females were produced by placing a virgin female
and a virgin male together and observing them until mating was
complete. Except where noted, females used were 3-day-old and
allowed to acclimate to the laboratory conditions for at least
30 min before trials. To obtain enough males for the pheromone
isolation, all-male broods were produced by using virgin females
for parasitization. Because wasps are haplodiploid, virgin females
produce only sons.
Pheromone Extraction and Quantification
From Wasps
For an initial comparative analysis, 2-day-old virgin males and
females (n = 5) were killed by freezing, transferred to a 1.5 ml
glass vial equipped with a 200 µl micro insert, and extracted
individually for 30 min with 30 µl of dichloromethane. After
removal of the wasps, the extracts were analyzed by coupled gas
chromatography-mass spectrometry (GC/MS). These analyses
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Male Sex Pheromone in Urolepis rufipes
GC/MS Analysis
Magnetic Resonance (NMR) Experiments). Subsequently, the
same pheromone fraction was diluted with dichloromethane to
a DMOD concentration of 100 ng µl−1 and used for the bioassay
(see section Bioassays). For SPE, male-derived raw extracts were
applied to conditioned 100 mg silica gel cartridges and rinsed
twice with 500 µl dichloromethane each. Subsequently, DMOD
was eluted twice with 500 µl methanol each. The methanol
was carefully evaporated under a gentle stream of nitrogen and
the residue was dissolved in dichloromethane, resulting in a
final concentration of 0.5 male equivalents per µl. This DMOD
fraction was used for the bioassay.
Aliquots (1 µl) of extracts and fractions were analyzed on a
Shimadzu QP2010 Plus GC/MS system equipped with a nonpolar BPX5 or a polar BP-20 capillary column (both columns
30 m x 0.25 mm inner diameter, 0.25 µm film thickness) (SGE
Analytical Science Europe, Milton Keynes, UK). Samples were
injected at 280◦ C (BP-20: 240◦ C) in splitless mode using an AOC
20i auto sampler. Helium was used as the carrier gas at a linear
velocity of 40 cm s−1 . The initial oven temperature of 80◦ C was
increased at 5◦ C min−1 to 280◦ C (BP-20: 240◦ C) and held for
30 min. Enantioselective GC/MS was performed on chiral ß-Dex
225 column (=25% 2,3-di-O-acetyl-6-O-tert-butyldimethylsilylß-cyclodextrin in polydimethylsiloxane, 30 m, 0.25 mm inner
diameter, 0.25 µm film thickness) (Supelco, Bellefonte, PA).
Injector temperature for enantioselective GC was 220◦ C; the
linear velocity of the carrier gas was 30 cm s−1 . The temperature
program started at 100◦ C and was increased by 0.25◦ C min−1
to the final temperature of 220◦ C. For all analyses, the mass
spectrometer was operated in the electron ionization (EI) mode
at 70 eV, and the mass range was m/z 35–600. Given that the
EI mass spectrum did not show a reasonable molecular ion (see
section Results), we analyzed a pheromone extract also by GC
(parameter as given above for the non-polar column) coupled
to atmospheric pressure chemical ionization mass spectrometry
(APCI-MS) using an Agilent high resolution Q-TOF 6540 UHD
mass spectrometer (conditions: mass range m/z 60–600; scan
rate: 5 spectra s−1 ; drying gas: 6 L min−1 at 300◦ C; corona
discharge current: 1 µA).
Quantification of Deposited Pheromone
DMOD deposited by individual males was quantified by GC/MS
using thermal desorption (TD) sampling as described by Blaul
and Ruther (2011). For this purpose, empty 89 × 5 mm inner
diameter TD glass tubes were filled at one end with 50 mg
of Tenax TA (Supelco, Bellefonte, PA). The adsorbent layer
of 25 mm was fixed using fine mesh metal screens (Supelco).
One microliter of an internal standard solution containing
100 ng µl−1 methyl undecanoate (Sigma-Aldrich, Deisenhofen,
Germany) in methanol was applied to the adsorbent, and the
tube was purged for 5 min with a nitrogen flow of 100 ml
min−1 to remove the solvent. Subsequently, 1-day-old males
were released into the empty side of the adsorption tubes and
kept there for 24 h. Males were either virgin (n = 12) or
newly mated (n = 10). TD tubes were purged every 6 h for
30 min with charcoal filtered air at a flow rate of 60 ml min−1
(adsorbent upwind) to trap the volatilized DMOD proportion.
By this means, losses of DMOD during sampling were kept to a
minimum and both the pheromone adsorbed to the substrate and
the volatilized proportion could be detected when the tube was
thermally desorbed. TD sampling was done using an automated
Shimadzu TD20 thermal desorption unit coupled to the QP2010
Plus GC/MS system (Shimadzu GmbH, Duisburg, Germany) as
described previously (Blaul and Ruther, 2011).
Nuclear Magnetic Resonance (NMR)
Experiments
NMR spectra were recorded with Bruker Avance 600 and
800 NMR spectrometers operating at a 1 H frequency of
600.03 and 800.2 MHz, respectively. Measurements were
performed in a 5 mm TXI (Avance 600) or TCI (Avance 800)
cryoprobe. Approximately 45 µg of the sample was dissolved in
deuterated dichloromethane (see section Pheromone Extraction
and Quantification From Wasps). For the assignment of the
chemical shifts standard 1D-NOESY, 2D-COSY, 2D-TOCSY
(80 ms mixing time), 2D-ROESY (250 ms mixing time), 2D[1 H,13 C] HSQC [1.725 ms mixing time (1 JC−H = 145 Hz)], and
2D-[1 H,13 C] HMBC [50 ms mixing time (10 Hz coupling)], and
1D 13 C spectra (including DEPT45 and DEPT135) were recorded
at 278 K and carbon frequencies of 150.88 and 201.21 MHz,
respectively. The chemical shifts were indirectly referenced to
TMS using the residual 1 H signal at 5.32 ppm and 13 C signal at
53.84 ppm of methylene chloride (Fulmer et al., 2010).
Stable Isotope Labeling Experiment
To investigate whether mevalonic acid is a precursor for
the biosynthesis of DMOD in Ur males, we performed
labeling experiments using racemic 3-fold deuterated mevalonic
acid lactone (MAL-d3 , 4-hydroxy-4-(methyl-d3 )tetrahydro-2Hpyran-2-one) (Santa Cruz Biotechnology, Heidelberg, Germany).
MAL-d3 is hydrolyzed under physiological conditions to
mevalonic acid (Figure 6B) which can be incorporated into
products of isoprenoid metabolism (Dewick, 2002). We used
3-day-old parasitoid males on the assumption that they were
widely depleted of intrinsically synthesized mevalonic acid and
thus would have a higher tendency to use the externally applied
precursor. Prior to application, males were cold-sedated on an
ice bath, and 0.2 µl of a solution containing 10 µg µl−1 MALd3 dissolved in acetone was applied to the abdominal tip (n = 8)
as described previously (Blaul et al., 2014). The pure solvent was
applied to control wasps (n = 8). Applications were done between
2 and 3 pm, and males were freeze-killed the next morning and
extracted for GC/MS analysis as described above.
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Chemical Synthesis
Synthesis of racemic 2,6-dimethyl-7-octene-1,6-diol started with
a catalytic Riley oxidation of racemic linalyl acetate 1 in 50
% yield (Umbreit and Sharpless, 1977). Subsequent ParikhDoering oxidation furnished aldehyde 3 in 93 % yield (Tojo and
Fernandez, 2006; Figure 1). Reduction of unsaturated aldehyde
3 was accomplished by a biomimetic approach. Hantzsch
ester 7 was used as an NADH analog in a 1,4-conjugate
4
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Ruther et al.
Male Sex Pheromone in Urolepis rufipes
FIGURE 2 | Static two-choice olfactometer used for bioassays. (1) Bottom of
a plastic Petri dish (9 cm diameter, 0.8 cm) with a hole (1 cm diameter) in the
center for female entrance; (2) rubber stopper; (3) glass Petri dish (9 cm
diameter, 1.5 cm height) divided into four quarters of equal size; (4) odor fields
with filter paper (1 cm diameter, each disk 5 cm apart, attached with double
sided tape) for application of pheromone extract (test) and pure solvent
(control). A 2 cm diameter circle was drawn around each filter disk. After
application of the test substances, evaporation of solvent and an equilibration
time of 5 min, the stopper was removed and the arena was flipped so that the
opening was over a female thus allowing the female to enter the olfactometer.
FIGURE 1 | Synthesis of racemic 2,6-dimethyl-7-octene-1,6-diol (6).
In experiments 1 and 2, we tested whether crude extracts
and purified DMOD are arresting (residence time, experiment 1)
and attractive (first choice, experiment 2) toward virgin females
and whether mating triggers a switch in responsiveness as has
been shown for females of Nv (Ruther et al., 2007, 2010). In
experiment 3 we asked whether the pheromone response of
females is switched off after receptivity signaling, like in Nv
(Ruther et al., 2010), or whether copulation and sperm transfer
are essential for the switch. Copulation was prevented by allowing
the male to court the female, but then brushing the male off the
female with a fine paint brush just as the male began to back up
for copulation. In experiment 4 we tested the response of virgin
females to synthetic racemic DMOD.
addition, catalyzed by the trifluoroacetic acid salt of tertbutyl valinate 8 (Martin and List, 2006). The catalyst was
prepared by anion exchange of the corresponding commercially
available hydrochloride salt, whereas Hantzsch ester 7 was
prepared as reported previously (Eey and Lear, 2010). Sodium
borohydride reduction gave alcohol 5 in 20% yield. Finally,
acetate cleavage by saponification with K2 CO3 in methanol/water
furnished the target compound 2,6-dimethyl-7-octene-1,6-diol
(6) in 63% yield. Full details of the procedures are given in the
Electronic Supplementary Material.
Bioassays
Statistical Analysis
Clean arenas were used for each trial (Figure 2). Females
were each presented with two filter paper disks simultaneously.
Depending on the experiment (see Table 1), one filter paper
disk received 2 µl of a male extract (representing one male
equivalent), 300 ng of purified natural DMOD (representing
approximately the mean pheromone titer of a 1-day-old male),
or 300 and 1,000 ng of synthetic racemic DMOD. The other disk
was treated with the respective amount of pure dichloromethane
(solvent control). The pheromone and solvent sides were
switched between trials to minimize any side effects.
Prior to testing, the female had been tapped out of the glass vial
in which she had emerged, onto the clean desk. Depending on the
experiment, we tested virgin or mated females and we recorded
either the circle around the filter disks entered first by the female
(first choice), or we recorded for 5 min the time females spent in
the circles around each filter disks (residence time). The residence
time was recorded using the scientific software The Observer XT
(Noldus, Wageningen, The Netherlands).
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Pheromone titers of males of different age and mating history
were compared by a Kruskal-Wallis H-test followed by pairwise
Mann-Whitney U-tests with sequential Bonferroni correction.
Deposited DMOD amounts of virgin and mated males were
compared by a Mann-Whitney-U-test. Residence time in the test
and control fields of the olfactometer was analyzed by a Wilcoxon
matched pairs test. First choice between the test and control fields
was analyzed by a two-sided binominal test.
RESULTS
The comparative GC/MS analysis of whole body extracts from
male and female Ur revealed the presence of a male-specific
compound with the linear retention indices 1,350 and 2,155 on
non-polar BPX-5 and polar BP-20 stationary phases, respectively
(Figure 3). The EI mass spectrum of the compound (Figure 4A)
showed a base peak at m/z 71 and the highest mass at m/z 157. A
5
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Male Sex Pheromone in Urolepis rufipes
TABLE 1 | Outline of the bioassays performed.
Exp.
Pheromone source
Dose
Parameter
Female status
n
P
1a
Crude abdomen extract
300 ng DMODc
Residence time
Virgin
20
0.0072a
1b
DMOD fraction (SE-HPLC)
300 ng DMOD
Residence time
Virgin
20
0.0015a
1c
DMOD fraction (SE-HPLC)
300 ng DMOD
Residence time
Mated
20
0.5461a
2a
Crude abdomen extract
1 male eq.
First choice
Virgin
22
<0.0001b
2b
DMOD fraction (SPE)
1 male eq.
First choice
Virgin
17
0.0023b
2c
DMOD fraction (SPE)
1 male eq.
First choice
Mated
15
1.0000b
3a
Crude abdomen extract
300 ng DMOD
Residence time
Virgin
20
0.0089a
3b
Crude abdomen extract
300 ng DMOD
Residence time
Mated
20
0.1930a
3c
Crude abdomen extract
300 ng DMOD
Residence time
Receptivity signal
20
0.1570a
4a
rac. synthetic DMOD
300 ng DMOD
Residence time
Virgin
22
0.0018a
4b
rac. synthetic DMOD
1,000 ng DMOD
Residence time
Virgin
22
0.0004a
Given are the pheromone source, tested pheromone dose, recorded parameter, mating status of the females, number of replicates, and results of the statistical analyses of experiments
1–4 (details given in section Bioassays).
a Wilcoxon matched pairs test.
b two-sided binominal test.
c 2,6-dimethyl-7-octene-1,6-diol.
mass difference of only 12 mass units to the next lighter fragment
(m/z 145) indicated, however, that m/z 157 does not represent
the molecular ion of the compound. Therefore, we analyzed the
extract by GC/APCI-MS. This analysis revealed an exact mass
of 190.1801 for the quasi molecular ion ([MNH+
4 ], Figure 4B)
matching the empirical formula C10 H24 O2 N. Two subsequent
losses of H2 O from the ion [MH+ ] furthermore suggested the
presence of two hydroxyl groups.
[1 H, 13 C] HSQC with a mixing time of 1.725 ms (1 JC−H =
145 Hz) allowed the detection of all carbon resonances except
the tertiary carbon in position 6 as well as the resonances of
the protons directly coupled to the respective carbon atoms. In
the [1 H, 13 C] HMBC optimized for long range couplings all
expected couplings to neighboring carbons as well as the carbon
in position 6 could be observed (Supplementary Figure S2).
1 H-TOCSY-spectroscopy was used to confirm the assignments
leading to the chemical structure of DMOD. The chemical shifts
are listed in Table 2. Within the limits of error they are identical
to spectra obtained from synthetic DMOD.
With the suggested structure in hand, synthetic material
was prepared according to Figure 1. GC/MS analysis of the
synthetic product 6 revealed that all four stereoisomers eluted
as a single peak on both the non-polar and the polar
stationary phase. Mass spectra, 13 C- and 1 H-NMR-spectra as
well as linear retention indices matched the natural product.
Enantioselective GC/MS analysis of the racemate on the chiral βDEX 225 column separated all four DMOD isomers and revealed
that only the stereoisomer eluting first is produced by the
males (Supplementary Figure S3). The absolute configuration
of this compound, however, remains to be established by
enantioselective synthesis of the four DMOD stereoisomers.
GC/MS analysis of dissected tissues revealed that Ur males
produce DMOD in the rectal vesicle (Supplementary Figure S1).
Male pheromone titers were almost zero in newly emerged
males (mean ± SEM: 4.6 ± 2.5 ng) but increased within the first
2 days after emergence (day one: 333 ± 57 ng; day two: 510 ±
Frontiers in Ecology and Evolution | www.frontiersin.org
FIGURE 3 | Total ion current chromatograms obtained by GC/MS analysis of
dichloromethane extracts from male and female U. rufipes on a non-polar
BPX5 capillary column (CHCs = cuticular hydrocarbons).
81 ng). Although the variability of pheromone titers increased in
mated males compared to virgin males, the mating history had
no significant effect on the pheromone titers of Ur males and the
presence of a female did not influence the deposited pheromone
amounts (Figures 5A–C).
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Ruther et al.
Male Sex Pheromone in Urolepis rufipes
FIGURE 4 | (A) EI mass spectrum of the male-specific compound extracted from U. rufipes; (B) high resolution APCI mass spectrum; suggested empirical formulas
are given above the accurate masses of selected diagnostic ions.
chromatography, respectively (Figures 7A,B, 8). Likewise, virgin
females were attracted by the racemic mixture of all four DMOD
stereoisomers (Figures 7C,D) demonstrating that DMOD is
indeed used by Ur males as a sex pheromone to attract virgin
females. Mated females, however, were no longer attracted to or
arrested by the male sex pheromone (Figure 7E). More detailed
observations revealed that this switch in responsiveness of mated
Ur females is independent of sperm transfer. Females that had
signaled receptivity during courtship but were prevented from
copulating with the male became nonetheless unresponsive to the
male sex pheromone (Supplementary Figure S4).
The mass spectrum of DMOD from MAL-d3 treated males
showed an additional ion at m/z 74 indicative of a mass shift
of the base peak m/z 71 by three mass units. This shift can be
explained by the deuterated methyl group at C6 of the DMOD
molecule (Figure 6C). The incorporation rate determined using
the ion pair m/z 71/74 was 1.7 ± 0.2 % (mean ± SEM). A
second ion indicating the incorporation of the precursor was
m/z 46 resulting from a mass shift of the ion m/z 43 by three
mass units. Deuterium labeled compounds are characterized
by decreased retention times in comparison to the respective
fully hydrogenated derivatives (Matucha et al., 1991). Hence, the
presence of the deuterium label in the ions m/z 74 and 46 was
supported by a retention time decreased by approximately 3 s
in comparison to the unlabeled ions m/z 71 and 43, respectively
(Figures 6D,E). Due to the relatively low incorporation rates, no
diagnostic ions were detected in the mass spectrum of labeled
DMOD that would have indicated the incorporation of two
deuterated isoprene units.
In our behavioral bioassays, virgin females were attracted
to and arrested by filter paper disks treated with both
crude abdomen extracts and natural DMOD purified by solid
phase extraction on a polar adsorbent or size exclusion
Frontiers in Ecology and Evolution | www.frontiersin.org
DISCUSSION
The present study demonstrates that Ur males use DMOD as
a volatile sex pheromone to attract virgin females, functionally
similar to the pheromone used by males of the Nasonia species
and T. sarcophagae (Ruther et al., 2007; Niehuis et al., 2013).
Thus, the use of male sex pheromones has been shown in all
three genera of the “Nasonia group.” Our data and previous
studies reveal numerous parallels between the Nasonia and Ur
pheromone communication in that (a) the male sex pheromone
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Ruther et al.
Male Sex Pheromone in Urolepis rufipes
TABLE 2 | 1 H and 13 C NMR shifts (δ) of 2,6-dimethyl-7-octene-1,6-diol.
Position
2,6-dimethyl-7-octene-1,6-diol
δ(1 H) [ppm], J-couplings and
δ(13 C) [ppm]
multiplicities
1
3.42 (m, 1H) [3.45–3.33 (m, 2H)]
3.35 (m, 1H)
68.22 (CH2 ) [68.23/68.22 (CH2 )]
2
1.559 (m, 1H) [1.59–1.52 (m,
1H)]
35.97 (CH) [35.68/35.67 (CH)]
3
1.340 (m, 1H) [1.36–1.31 (m,
1H)]
1.050 (m, 1H) [1.08–1.02 (m,
1H)]
33.63 (CH2 ) [33.45/33.43 (CH2 )]
4
1.319 (m, 1H) [1.35–1.30, m,
1H]
1.248 (m, 1H) [1.26–1.20, m,
1H]
21.47 (CH2 ) [21.23 (CH2 )]
5
1.479 (m, 1H) [1.47–1.39 (m,
2H)]
1.433 (m, 1H)
42.73 (CH2 ) [42.46/42.45 (CH2 )]
6
FIGURE 5 | Quantification of the pheromone 2,6-dimethyl-7-octene-1,6-diol
(DMOD) from male U. rufipes wasps (A,B) and from male deposits (C).
Pheromone titres of 0, 1, and 2-d old males (A) and 2-d old males with 0, 1, 3,
and 5 previous matings (B). Amounts of pheromone deposited within 24 h by
single 1-d old virgin or newly mated males (C). Box-and-whisker plots show
median (horizontal line), 25–75% quartiles (box), maximum/minimum range
(whiskers) and outliers (◦ >1.5 and * >3 x above box height). Different
lowercase letters indicate significant differences at p < 0.001; data analysis by
Kruskal-Wallis test followed by multiple pairwise Mann-Whitney U-tests with
sequential Bonferroni correction.
73.30 (C) [73.29/73.28 (C)]
7
5.893 (dd, 3 J = 10.7 Hz, 3 J =
17.3 Hz, 1H)
[5.84 (dd, J = 17.3, 10.8 Hz, 1H)]
145.71 (CH) [145.16/145.11
(CH)]
8
5.153 (dd, 3 J = 17.4 Hz
(pro-Z), 2 J = 1.4 Hz, 1H)
[5.13 (d, J = 17.3 Hz, 1H)]
5.01 (dd, 3 J = 10.8 Hz (pro-E),
2 J = 1.4 Hz, 1H)
[4.98 (d, J = 10.7 Hz, 1H)]
111.28 (CH2 ) [111.60/111.58
(CH2 )]
9
0.862 (d, 3 J2,9 = 6.8 Hz, 3H)
[0.85, d, J = 6.6 Hz, 3H)]
16.58 (CH3 ) [16.57/16.55 (CH3 )]
10
1.218 (s, 3H) [1.21, s, 3H]
27.75 (CH3 ) [27.83/27.72 (CH3 )]
species studied so far, and this is the biosynthetic pathway
by which the male sex pheromone is synthesized. The
incorporation of deuterated mevalonic acid lactone into DMOD
by Ur males clearly indicates that they synthesize it via
the mevalonate pathway. Hence, a switch between two basic
biosynthetic pathways (fatty acid and isoprenoid metabolism)
has occurred during pheromone evolution in the Nasonia group,
while parameters such as production site and release mode
remained unchanged.
Which of the two pathways is ancestral and which is derived
will remain inconclusive until the phylogenetic relationships
among the genera of the Nasonia group are better understood.
Phylogenetic analyses of the Nasonia group based on 28S rDNA,
cytochrome b and cytochrome oxidase II sequences suggest that
the species rich genus Trichomalopsis is rendered paraphyletic
by the genera Nasonia and Urolepis, and that T. sarcophagae is
the sister group of Nasonia (Burks, 2009). Close relationship
between T. sarcophagae and Nasonia is consistent with the
qualitative pheromone composition of T. sarcophagae males
being the same as that of the Nasonia species, except for Nv
having evolved RR as an additional pheromone component. The
biosynthetic switch found in the present study might question
that Urolepis is a member of the Nasonia group. However,
that facts that Ur produces a male-derived sex pheromone and
uses it in a similar way like the Nasonia species support the
close relationship given that preliminary chemical analyses of
other species from the tribus Pteromalini such as Pteromalus
puparum, Muscidifurax raptorellus, M. uniraptor, and Dibrachis
Shifts (bold: wildtype DMOD, values in squared brackets, synthetic DMOD) are referenced
indirectly to TMS using the 1 H and 13 C signal of CD2 Cl2 . Temperature 278 K. For definition
of position numbers (see Figure 6A). Stereospecific assignment from J-coupling pattern.
is produced in the rectal vesicle (Abdel-Latief et al., 2008) and
released through the anal orifice by abdomen dipping (Steiner
and Ruther, 2009; Cooper and King, 2015); (b) males emerge
without the pheromone but titers increase within the first 2 days
after emergence (Ruther et al., 2007); (c) pheromone markings
are attractive only for virgin females; mated females do not
respond to the male pheromone (Ruther et al., 2007); (d) unlike
in tephritid fruit flies (Jang, 1995, 2002), this virgin to mated
switch in responsiveness is independent of sperm transfer but
correlates with receptivity signaling during courtship (Ruther
et al., 2010). In Nv, the switch in responsiveness is mediated by
oral fatty acid ethyl esters which are released by the male to the
female antennae during courtship (Ruther and Hammerl, 2014).
Dopamine release in the female brain is the neuromodulator
underlying the switch (Lenschow et al., 2018). Further studies
are necessary to elucidate whether the same mechanism is
involved in Ur’s virgin to mated female switch in responsiveness
and in previously reported post-mating modifications of mating
behavior (King and Miller, 2018).
Despite the many parallels outlined above, Ur diverges
in a decisive feature from the Nasonia and Trichomalopsis
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Male Sex Pheromone in Urolepis rufipes
FIGURE 6 | Mevalonic acid lactone is a precursor of the male sex pheromone in U. rufipes. (A) Structure of 2,6-dimethyl-7-octene-1,6-diol (DMOD). The two isoprene
units indicating the isoprenoid origin are indicated by differently colored dots. Numbers refer to the NMR-data given in Table 2. (B) Hydrolysis of racemic MAL-d3 to
threefold deuterated (±)-mevalonic acid-d3 . (C) EI mass spectrum of DMOD extracted from a male U. rufipes after treatment with MAL-d3 . Red arrows indicate
diagnostic ions resulting from the incorporation of MAL-d3 . Insert shows putative structures of the diagnostic ions m/z 74 and m/z 46. (D,E) Total ion chromatograms
(TIC) and diagnostic ion chromatograms (43/46 and m/z 71/74, magnification factors given in parentheses) of pheromone extracts from males treated with an acetone
solution of MAL-d3 or the pure solvent (Control).
the biosynthetic switch occurred gradually or saltationally
(Symonds and Elgar, 2008).
A second feature by which the pheromone communication
in Ur differs from Nasonia concerns the quantitative dynamics
of pheromone release. Nv males show increased pheromone
marking after contact with a female and after copulation (Barrass,
cavus revealed that these species do not use male-derived sex
pheromones (J.R., unpublished data). Further phylogenetic and
pheromone analyses of the hitherto unstudied Trichomalopsis
and Urolepis species are needed to improve our understanding
of the exact species relationships and the pheromone
evolution in the Nasonia group and to conclude whether
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Male Sex Pheromone in Urolepis rufipes
FIGURE 7 | Arrestment of females to the male-derived sex pheromone. Given are the residence times of (A–D) virgin and (E) mated U. rufipes females in the two odor
fields of a static two-choice olfactometer when given the choice between a solvent control (Con) and (A) a male-derived pheromone extract and (B,E) natural
2,6-dimethyl-7-octene-1,6-diol (DMOD) purified by SE-HPLC, or a synthetic DMOD racemate (C,D). Box-and-whisker plots show median (horizontal line), 25–75%
quartiles (box), maximum/minimum range (whiskers) and outliers [◦ >1.5 × above box height; * >3 × above box height; data analysis by Wilcoxon matched pairs
tests (n = 20)].
are less attractive than those of virgins (Ruther et al., 2009).
Like Nv, Ur males make more marks when held with a female
and after copulation (Cooper and King, 2015). Unlike Nv, mated
and multiply mated Ur males did not have lower pheromone
titers than virgins. Mated Ur males also did not deposit a
greater amount of pheromone through marking than virgin
males did (Figures 5B,C). Thus, mated Ur males make more
marks without depositing a greater amount of pheromone. One
possibility is that males make shorter marks after mating, despite
making more marks. The length of marks varies in Ur, but
whether it differs between virgin and mated males has not
been examined.
That Nv males, but not Ur males, deposit more pheromone
after interaction with a female may result from their differing
life histories, which may select for the deposition of lower
pheromone amounts per site in Ur. Nv is a gregarious species
with typically 15–30 wasps emerging from the same host
(Whiting, 1967), and sib-mating is common (Shuker et al., 2004).
Females emerge in rapid succession; thus, the presence of one
virgin female reliably predicts the very nearby occurrence of
others, which will be attracted to the male sex pheromone.
Nv males are territorial and defend the exit holes of hosts
aggressively against their competitors (Mair and Ruther, 2018)
and Nv males do not fly (Weston et al., 1999). In contrast,
Ur males have full wings and fly, and Ur generally produces
only one wasp per host. However, Ur can be described as
quasigregarious (Cooper et al., 2013) where the distribution
of their hosts is highly clumped, as can also be the case for
Nv hosts (Werren, 1983; King, 1990). A group of hosts may
even consist of thousands of hosts in contact with each other.
However, hosts also sometimes occur farther apart. As a result,
the presence of one virgin female is probably a less reliable
predictor of the presence of other nearby virgin females for Ur
FIGURE 8 | Attraction of females to a male-derived pheromone extract or a
natural 2,6-dimethyl-7-octene-1,6-diol (DMOD) fraction purified by solid phase
extraction on silica gel. Given are the first choices of virgin or mated U. rufipes
females for the two odor fields of a static two-choice olfactometer. Females
were given the choice between a solvent control (Con) and one male
equivalent of pheromone extract or DMOD fraction; data analysis by a
two-sided binominal test (number of replicates in parentheses).
1969; Van Den Assem et al., 1980; Steiner and Ruther, 2009).
Consequently, multiply mated males suffer both sperm and
pheromone depletion, and pheromone markings of those males
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Ruther et al.
Male Sex Pheromone in Urolepis rufipes
than for Nv. In addition, Ur males may benefit by conserving
pheromone to later mark sites that they fly to in search of
additional females.
DMOD is a minor compound in several plant-derived
materials, such as rose petals (Knapp and Winterhalter, 2000),
birch leaves (Schulz et al., 1997), and wine (Oliveira et al.,
2004). Enzymatic conversion of (±)-linalool by the cytochrome
P450 CYP76C1 from Arabidopsis thaliana also results in the
formation of DMOD (Boachon et al., 2015). In insects, DMOD
has only been reported once as a minor compound, in the
male “hairpencils” of the butterfly Danaus genutia (Francke
et al., 1989), but whether DMOD is involved in the pheromone
communication of this species remains unknown. Hence the
present study is the first report of DMOD as an insect
pheromone. Insect pheromones are typically blends of several
compounds enhancing each other additively or synergistically.
In Ur, however, there is no evidence for minor compounds
being involved in the pheromone function. Our enantioselective
GC/MS analysis revealed that only one of the four possible
stereoisomers occurs in Ur males. The fact that the racemic
DMOD mixture attracted females at both tested doses suggests
that the non-natural stereoisomers do not inhibit the females’
response to the natural product. As a next step, enantioselective
organic syntheses are needed to clarify the absolute configuration
of the natural product.
Interspecific sexual interactions are not uncommon in
sympatric parasitoid wasp species that parasitize fly pupae. Males
of all Nasonia species exhibit some level of courtship to chemical
cues present on the cuticle of both conspecific and congeneric
females (Buellesbach et al., 2013, 2018; Mair et al., 2017). Males of
Nv and T. sarcophagae even respond reciprocally to the cuticular
lipids of heterogeneric females (Buellesbach et al., 2018). Ur is
partially sympatric with Nv and T. sarcophagae. It remains to be
investigated whether there is similar overlap of courtship-related
communication with these species and whether the male-derived
abdominal sex attractant DMOD has evolved as a prezygotic
isolation mechanism to avoid the costs of interspecific mating.
ETHICS STATEMENT
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AUTHOR CONTRIBUTIONS
JR initiated the study, coordinated the structure elucidation,
performed the stable isotope labeling, and enantioselective
GC/MS analyses, analyzed data, and wrote the first draft of the
manuscript. TW and BK developed and performed the behavioral
bioassays. FF and SF performed behavioral bioassays and the
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NMR spectroscopy. JK performed the GC/APCI-MS analysis.
CG and SS synthesized the synthetic racemate of 2,6-dimethyl7-octene-1,6-diol. All authors read and revised the manuscript.
FUNDING
This
research
was
funded
by
the
Deutsche
Forschungsgemeinschaft (DFG) Grant Ru717-10/2 to JR
and by the Verband der Chemischen Industrie (VCI) to HK.
ACKNOWLEDGMENTS
We thank K. Floate for providing a starter culture of U. rufipes
and Melanie Schlossberger for rearing the insects and Derdar
Dilek for technical assistance during synthesis.
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Conflict of Interest Statement: The authors declare that the research was
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be construed as a potential conflict of interest.
Copyright © 2019 Ruther, Wittman, Grimm, Feichtner, Fleischmann, Kiermaier,
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