Male Sex Pheromone Research Articles

Partial role of volatile organic compounds in behavioural responses of mice to bedding from cancer-affected congeners.

Tumours induce changes in body odours. We compared volatile organic compounds (VOCs) in soiled bedding of a lung adenocarcinoma male mouse model in which cancer had (CC) versus had not (NC) been induced by doxycycline at three conditions: before (T0), after 2weeks (T2; early tumour development), after 12 weeks (T12; late tumour development) of the induction. In an earlier study, wild-derived mice behaviourally discriminated between CC and NC soiled bedding at T2 and T12. Here, we sought to identify VOCs present in the same soiled bedding that could have triggered the behavioural discrimination. Solid phase micro-extraction was performed to extract VOCs from 3 g-sample stimuli. While wild-derived mice could discriminate the odour of cancerous mice at a very early stage of tumour development (T2), the present study did not identify VOCs that could explain this behaviour. However, consistent with the earlier behavioural study, four VOCs, including two well-known male mouse sex pheromones, were found to be present in significantly different proportions in soiled bedding of CC as compared to NC at T12. We discuss the potential involvement of non-volatile molecules such as proteins and peptides in behavioural discrimination of early tumour development (T2), and point-out VOCs that could help diagnose cancer.

Complexity of Chemical Emissions Increases Concurrently with Sexual Maturity in Heliconius Butterflies

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown.

Sensory trap leads to reliable communication without a shift in nonsexual responses to the model cue.

The sensory trap model of signal evolution suggests that males manipulate females into mating using traits that mimic cues used in a nonsexual context. Despite much empirical support for sensory traps, little is known about how females evolve in response to these deceptive signals. Female sea lamprey (Petromyzon marinus) evolved to discriminate a male sex pheromone from the larval odor it mimics and orient only toward males during mate search. Larvae and males release the attractant 3-keto petromyzonol sulfate (3kPZS), but spawning females avoid larval odor using the pheromone antagonist, petromyzonol sulfate (PZS), which larvae but not males, release at higher rates than 3kPZS. We tested the hypothesis that migratory females also discriminate between larval odor and the male pheromone and orient only to larval odor during anadromous migration, when they navigate within spawning streams using larval odor before they begin mate search. In-stream behavioral assays revealed that, unlike spawning females, migratory females do not discriminate between mixtures of 3kPZS and PZS applied at ratios typical of larval versus male odorants. Our results indicate females discriminate between the sexual and nonsexual sources of 3kPZS during but not outside of mating and show sensory traps can lead to reliable sexual communication without females shifting their responses in the original context.

Courtship and mating behavior of Endoclita signifer (Hepialidae: Lepidoptera) and the male sex pheromones in hairbrushes.

Endoclita signifer Walker is the most destructive wood-boring pest of Eucalyptus in China, causing significant economic and ecological damage. As an insect of the primitive Lepidoptera family Hepialidae, E. signifer fly and mat for only 10-20 min at dusk. The courtship and mating behavior of E. signifer adults and whether male moths release sex pheromones are still unknown, especially since transitory flight survival strategies in primitive moths differ from advanced moths like noctuids. In this study, we first observed the courtship and mating behavior of E. signifer by considering the effects of space and then analyzed extracts of male hairbrushes using gas chromatography-electroantennogram detection. Our results indicated that during the courtship period, flying males form courtship fields, lekking, and chase flying females before mating with them; E. signifer were more successful in mating in larger spaces (Length × Width × Height = 9.6 × 7 × 4 m); 5 compounds in the hairbrushes of the male moths which elicited antennal responses of 2 sexes, despite at high concentrations. Combined with it, indicating that communication between male and female may rely on male sex pheromones. These findings can serve as a basis for studying the mechanisms of sex communication in E. signifer and developing sex pheromone-based trapping techniques.

The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster.

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility, though functional evidence in any species is lacking. Here, we used functional genetics to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes are dispensable. We found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage, likely due to its expression in the male ejaculatory bulb. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Diet-derived male sex pheromone compounds affect female choice in a noctuid moth.

Sexual signals often function in species recognition and may also guide mate choice within a species. In noctuid moths, both males and females may exercise mate choice. Females of the tobacco budworm Chloridea virescens prefer to mate with larger males, but the signal(s) underlying female choice remain unknown. Male hairpencil volatiles are emitted during close range courtship displays. However, previously identified male hairpencil volatiles, namely acetate esters, aldehydes, alcohols, and fatty acids, are not associated with female choice. Recently, two new hairpencil compounds were identified that elicit strong electrophysiological responses in female antennae: methyl salicylate (MeSA) and δ-decalactone. In this study, we investigated the effect of larval diet and adult feeding on MeSA and δ-decalactone content in hairpencils and determined whether these compounds are involved in female choice. We found that larval diet affected MeSA content in hairpencils, but not δ-decalactone. Conversely, adult feeding affected the level of δ-decalactone, but not MeSA: sugar-water feeding increased δ-decalactone content compared to plain water. In two-choice assays, females mated more with males that had higher amounts of δ-decalactone, and less with males with higher amounts of MeSA.

Synergistic behavioral antagonists of a sex pheromone reduce reproduction of invasive sea lamprey

Sex pheromones impart maximal attraction when their components are present at optimal ratios that confer balanced olfactory inputs in potential mates. Altering ratios or adding pheromone analogs to optimal mixtures may disrupt balanced olfactory antagonism and result in reduced attraction, however, tests in natural populations are lacking. We tested this hypothesis in sea lamprey (Petromyzon marinus), a fish whose male sex pheromone attracts females when two critical components, 3-keto petromyzonol sulfate (3kPZS) and petromyzonol sulfate (PZS), are present at certain ratios. Here, we report a pheromone analog, petromyzonol tetrasulfate (3sPZS), reduced female attraction to 3kPZS but not to PZS. 3sPZS mixed with additional PZS synergistically disrupted female attraction to the male pheromone and reduced spawning by 97% in a high-density population. Our results provide evidence of balanced olfactory antagonism in a vertebrate and establish a tactic to disrupt spawning of sea lamprey, a destructive invader of the Laurentian Great Lakes.

Cross-mating between the alien bumblebee Bombus terrestris and two native Japanese bumblebees, B. hypocrita sapporensis and B. cryptarum florilegus, in the Nemuro Peninsula, Japan

The rapid naturalization of Bombus terrestris across the Nemuro Peninsula has led to a decline in two closely related native Japanese species, namely Bombus hypocrita sapporensis and Bombus cryptarum florilegus, both belonging to the common subgenus Bombus. Although it is widely believed that cross-mating of native and non-native species is influenced by the common male sex pheromone in this region, no study has been conducted to substantiate this claim. Thus, we investigated the cross-activities of male sex pheromones between native and non-native bumblebees, as well as the frequencies of cross-mating, using chemical and DNA assays. Our gas chromatography–electroantennographic detector analyses and behavioral tests revealed the presence of sex pheromonal cross-activities between B. terrestris and the two Japanese bumblebees species. Furthermore, DNA analyses revealed the occurrence of cross-mating between native and non-native species in the Nemuro Peninsula. Overall, these results indicate the immediate need for conservation measures to safeguard Japanese bumblebee populations in the Nemuro Peninsula.

Behavioural and Electrophysiological Response of Anastrepha fraterculus (Diptera: Tephritidae) to a γ-Lactone Synthetic Semiochemical.

Attractants are a powerful tool for pest management. The lack of specific attractants for the South American fruit fly, Anastrepha fraterculus, a complex of cryptic species of great economic importance in South America, makes it difficult to monitor the pest in the field. The γ-lactone male sex and aggregation pheromones of several Anastrepha species, naturally released in a 7:3 epianastrephin to anastrephin ratio, and a structurally related naturally occurring γ-lactone ((±)-trans-tetrahydroactinidiolide) with gem-dimethyl groups (dimethyl) at C(4), were evaluated as potential attractants of this species. Different age and mating conditions of A. fraterculus males and females were evaluated during electroantennography (EAG) and field cage experiments in which polymeric lures were deployed to contain 100 mg of attractant. Epianastrephin and dimethyl were EAG+ for all fly conditions, with epianastrephin eliciting the highest response for both sexes and immature flies showing greater responsiveness than mature flies. In the field cage experiments, immature flies were only attracted to leks; virgin females were attracted to leks, dimethyl, and both epianastrephin-anastrephin formulations (95 and 70 wt.% epianastrephin); mature-mated males were attracted to leks, dimethyl and 70 wt.% epianastrephin; and mature-mated females were only attracted to leks. Our bioassays showed a promising performance of the analog dimethyl since it elicited the same response as epianastrephin, requires fewer steps to synthesize, and contains one less chiral center than the natural pheromones. The attraction to leks was recorded for all mating conditions and ages of flies and suggests that air-borne volatiles of calling males contain cues that could act as sensory traps. The addition of any of these compounds in the synthetic attractants may result in a greater attraction and thus deserves further evaluation. Dose-response experiments will provide additional information to move a step forward and validate the results obtained in open-field conditions.

Male sex pheromone in the parasitoid wasp Nasonia longicornis: Chemical and behavioral analyses

The use of sex pheromones for the attraction of potential mating partners is widespread in insects. Species-specificity of these chemical signals is essential, particularly in closely related species with overlapping habitats. In parasitoid wasps of the genus Nasonia, it is the males that produce sex pheromones in their rectal vesicles. The genus consists of four species: N. vitripennis (Nv), N. giraulti (Ng), N. oneida (No), and N. longicornis (Nl). The cosmopolitan species Nv is sympatric with Ng and No in eastern North America and with Nl in the west. Interspecific courtship is common in Nasonia although hybridization is prevented in most combinations by Wolbachia-mediated cytoplasmic incompatibility. The pheromone of Nv males differs from all Nasonia congeners by an additional component that is believed to ensure precopulatory isolation from the sympatric congeners. Detailed investigations on these interactions, however, exist only for the species combination Nv/Ng. Here, we report the results of chemical and behavioral investigations on the sex pheromone of Nl males. The pheromone consists of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as a minor component which are produced only after eclosion. Pheromone titers peaked 2 days after eclosion and remained constant on day three. The pheromone is deposited by abdomen dipping which males increasingly exhibited after mating or brief contact with a female. The presence of hosts containing females about to emerge did not increase marking behavior. Site fidelity of males at their own pheromone markings is mediated by MQ. Both natural and synthetic pheromone attracted virgin but not mated females and both RS and MQ are required for female attraction. Females collected during emergence from the host responded likewise to the pheromone and 84% of them produced mixed-sex offspring showing that most of them emerge unmated. Nl females preferred pheromone extract from conspecific males to extract from Nv males, and the addition of the Nv-specific component (4R,5R)-5-hydroxy-4-decanolide (RR) to the Nl pheromone made them unresponsive. The present study demonstrates that Nl uses the male sex pheromone in a similar way as previously shown for Nv and Ng. Furthermore, it shows that Nl females use the Nv-specific pheromone component RR to avoid costly sexual interactions with Nv males in regions of sympatry.

No evidence for long-range male sex pheromones in two malaria mosquitoes

Cues involved in mate seeking and recognition prevent hybridization and can be involved in speciation processes. In malaria mosquitoes, females of the two sibling species Anopheles gambiae s.s. and An. coluzzii mate in monospecific male swarms and hybrids are rare. Long-range sex pheromones driving this behaviour have been debated in literature but so far, no study has proven their existence or their absence. Here, we attempted to bring to light their existence. To put all the odds in our favour, we used different chemical ecology methods such as behavioural and electrophysiological assays as well chemical analyses, and we worked with mosquitoes at their optimal physiological mating state that is with swarming males during their natural swarming windows. Despite all our efforts, our results support the absence of long-range sex pheromones involved in swarm detection and recognition by females. We briefly discuss the implications of this finding in ecology, evolution and for control strategies.

Exploitation of an ancestral pheromone biosynthetic pathway contributes to diversification in Heliconius butterflies.

During courtship, male butterflies of many species produce androconial secretions containing male sex pheromones (MSPs) that communicate species identity and affect female choice. MSPs are thus likely candidates as reproductive barriers, yet their role in speciation remains poorly studied. Although Heliconius butterflies are a model system in speciation, their MSPs have not been investigated from a macroevolutionary perspective. We use GC/MS to characterize male androconial secretions in 33 of the 69 species in the Heliconiini tribe. We found these blends to be species-specific, consistent with a role in reproductive isolation. We detected a burst in blend diversification rate at the most speciose genus, Heliconius; a consequence of Heliconius and Eueides species using a fatty acid (FA) metabolic pathway to unlock more complex blends than basal Heliconiini species, whose secretions are dominated by plant-like metabolites. A comparison of 10 sister species pairs demonstrates a striking positive correlation between blend dissimilarity and range overlap, consistent with character displacement or reinforcement in sympatry. These results demonstrate for the first time that MSP diversification can promote reproductive isolation across this group of butterflies, showcasing how implementation of an ancestral trait, the co-option of the FA metabolic pathway for pheromone production, can facilitate rapid speciation.

The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster

Specific mate communication and recognition underlies reproduction and hence speciation. Our study provides new insights in Drosophila melanogaster premating olfactory communication. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Female-produced, species-specific volatile pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone (Z)-11-octadecenyl acetate (cVA) interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Two isoforms of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. However, only Z4-11Al mediates courtship in experienced males, not (R)-linalool. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in contrast to Z4-11Al. A blend of the food odour vinegar and cVA attracted females, while a blend of vinegar and female pheromone Z4-11Al attracted males, instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.

Pest categorisation of Xylotrechus chinensis.

The EFSA Panel on Plant Health performed a pest categorisation of Xylotrechus chinensis (Coleoptera: Cerambycidae) for the EU territory. This species is not included in the EU Commission Implementing Regulation 2019/2072. X. chinensis is native to China, Japan, the Korean peninsula and Taiwan. It has recently been reported from Spain (Catalonia; Region of Valencia), Greece (Athens; Crete) and France (Hérault; Gironde). X. chinensis attacks and kills Morus spp. in Europe and is also a pest of Malus domestica, Pyrus sp. and Vitis vinifera in Asia. This last species, however, was not confirmed as a host in an experimental study in Spain. The pest is univoltine. The adults are 1.5–2.5 cm long; they emerge between May and August. Each female produces approximately 80 eggs which are laid on the bark. The larvae live in the phloem and tunnel into the xylem where they pupate. Infested trees show injuries including longitudinal slits in the bark, caused by larval activity next to the surface and round exit holes from which frass emerges. The females respond to a male sex pheromone, which has not been developed into a detection method. The adults spread by flight as suggested by the local expansion of damage in Europe. However, wood packaging material and wooden objects can also be a pathway as suggested by interceptions in Germany and the USA. In Greece and Spain, hundreds of Morus trees have already been attacked within a few years, and often killed. The infested area has been observed to expand from 44 to 380 km2 within 2 years in Spain (Catalonia). Phytosanitary measures are available to inhibit further introductions and slow the spread within the EU. X. chinensis satisfies all the criteria that are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest.

Mating, but Not Male Accessory Gland Products, Changes Female Response to Olfactory Cues in Anastrepha Fruit Flies.

Copulation and/or ejaculate components can alter female physiological state and female post-mating behavior. The objective of the present study was to determine if copulation and male reproductive accessory gland products (MAGs) modify the behavior of female Anastrepha ludens (Loew) and Anastrepha obliqua (Macquart; Diptera: Tephritidae) in response to two stimuli: male-emitted pheromone and oviposition host volatiles. Olfactometry studies revealed that mated females of both A. ludens and A. obliqua have a stronger response for host volatiles compared to unmated females, which have a stronger response for male pheromone. We also examined olfactory responses of females mated to testectomized males who could transfer MAGs but not sperm. In both species, MAGs alone did not cause the change in the olfactory response observed after copulation, unlike what has been found in Ceratitis capitata (Wiedemann). Females mated to testectomized males responded equally to the male sex pheromone or to host volatiles, thus suggesting that the whole ejaculate is needed to elicit the complete behavioral switch in olfactory response. The function of MAGs is still unknown in these two pests of economic importance. The response for host volatiles by mated females has implications for the development of baits and traps that should preferably attract and target this population.

Hormonal Prostaglandin F2α Mediates Behavioral Responsiveness to a Species-Specific Multi-component Male Hormonal Sex Pheromone in a Female Fish.

Although hormonally-derived female sex pheromones have been well described in approximately a dozen species of teleost fish, only a few male sex pheromones have been characterized and the neuroendocrine underpinnings of behavioral responsiveness to them is not understood. Herein, we describe a study that addresses this question using the goldfish, Carassius auratus, an important model species of how hormones drive behavior in egg-laying teleost fishes. Our study had four components. First, we examined behavioral responsiveness of female goldfish and found that when injected with prostaglandin F2α (PGF2α), a treatment that drives female sexual receptivity, and found that they became strongly and uniquely attracted to the odor of conspecific mature males, while non-PGF2α-treated goldfish did not discern males from females. Next, we characterized the complexity and specificity of the male pheromone by examining the responsiveness of PGF2α-treated females to the odor of either mature male conspecifics or male common carp odor, as well as their nonpolar and polar fractions. We found that the odor of male goldfish was more attractive than that of male common carp, and that its activity was attributable to both its nonpolar and polar fractions with the later conveying information on species-identity. Third, we hypothesized that androstenedione (AD), a 19-carbon sex steroid produced by all male fish might be the nonpolar fraction and tested whether PGF2α-treated goldfish were attracted to either AD alone or as part of a mixture in conspecific water. We found that while AD was inactive on its own, it became highly attractive when added to previously unattractive female conspecific water. Lastly, in a test of whether nonhormonal conspecific odor might determine species-specificity, we added AD to water of three species of fish and found that while AD made goldfish water strongly attractive, its effects on other species holding water were small. We conclude that circulating PGF2α produced at the time of ovulation induces behavioral sensitivity to a male sex pheromone in female goldfish and that this male pheromone is comprised of AD and a mixture of body metabolites. Because PGF2α commonly mediates ovulation and female sexual behavior in egg-laying fishes, and AD is universally produced by male fishes as a precursor to testosterone, we suggest that these two hormones may have similar roles mediating male-female behavior and communication in many species of fish.

Identification and functional characterization of odorant-binding proteins 69a and 76a of Drosophila suzukii

The fruit fly Drosophila suzukii is a fruit crop pest that causes a severe economic threat to soft summer fruit worldwide. The male sex pheromone, cis-vaccenyl acetate (cVA) has multiple functions in intra-species communication in Drosophila melanogaster, which is required in male to suppress male-male courtship. D. suzukii males do not produce cVA; however, the odorant receptor for cVA (Or67d) is still functional. The lack of cVA in D. suzukii casts the question of whether this pheromone might have been replaced by another compound similar to cVA that disrupts mating in D. suzukii. In order to address this question, we cloned two D. suzukii adult antenna-specific odorant-binding proteins (OBPs) DsOBP69a and DsOBP76a and aligned with their D. melanogaster orthologues. Moreover, we examined the binding properties of the newly identified recombinant proteins against 26 potential ligands including cVA, using the fluorescence-based ligand binding assay. The alignment showed that DsOBP69a and DsOBP76a, have six conserved cysteines and belong to the classic OBP family. Furthermore, our results revealed that cVA did not bind to DsOBP69a or DsOBP76a proteins. Interestingly, the floral odorant β-ionone and the bitter substance berberine chloride and coumarin displayed high binding ability. It is also worth noting that DsOBP69a and DsOBP76a have different affinities to (Z)-7-Tricosene that may reflect different functional roles. These findings suggest that DsOBP69a and DsOBP76a are potentially involved in olfaction and gustation of D. suzukii.

Mating-induced changes in responses of female Queensland fruit fly to male pheromones and fruit: A mechanism for mating-induced sexual inhibition

In order to reproduce, female tephritid fruit flies need both mates for fertilization and fruit for oviposition. Virgin females are prone to mating and approach males, attracted by their pheromones. Mated females, however, may experience an abrupt reduction of mating propensity and prioritise the search for suitable fruit rather than additional mates. Accordingly, mating in fruit flies may induce a switch in olfactory preferences of females from pheromones to fruit stimuli, and this switch may also be an important mediator of mating-induced sexual inhibition. To test for mating-induced switches in olfactory preference of female Queensland fruit fly, Bactrocera tryoni, we used wind tunnel assays to assess attraction of mated and virgin females to (1) male sex pheromone delivered through a perforated glass sphere or (2) an entire fruit. Electroantennogram (EAG) responses were also used to test for mating-induced changes in olfactory sensitivity to pheromones and fruit odours. Pheromones elicited quicker upwind responses in virgin females than in mated females; during the first six minutes of trials more virgin females than mated females were observed in the upwind end of the wind tunnel where pheromone odours were released. Fruit cues, in contrast, elicited stronger association with the upwind end of the wind tunnel in mated females than in virgin females from the fifth minute onwards. Also, mated females were observed on the fruit for longer periods than virgin females. EAG responses to pheromones and fruit odours were similar in virgin and mated females, indicating that changes in preferences are not a consequence of changes in peripheral sensitivity of antennae to odours but instead appear to be mediated by post-receptor processing. Our results show that mating reduces attraction to male-produced pheromones and increases attraction to fruit stimuli in B. tryoni females. We propose that this behavioural switch from mating stimuli to oviposition stimuli is an important mediator of mating-induced sexual inhibition in this species.

Acetone application for administration of bioactive substances has no negative effects on longevity, fitness, and sexual communication in a parasitic wasp.

Administration of defined amounts of bioactive substances is a perseverative problem in physiological studies on insects. Apart from feeding and injection, topical application of solutions of the chemicals is most commonly used for this purpose. The solvents used should be non-toxic and have least possible effects on the studied parameters. Acetone is widely used for administration of chemical substances to insects, but possible side-effects of acetone application on fitness and behavioral parameters have been rarely investigated. Here we study the effects of acetone application (207 nl) on fitness and sexual communication in the parasitic wasp Nasonia giraulti Darling. Application of acetone had neither negative effects on longevity nor on offspring number and offspring sex ratio of treated wasps. Treatment of females hampered courtship and mating of N. giraulti couples neither directly after application nor one day after. Male sex pheromone titers were not influenced by acetone treatment. Three application examples demonstrate that topical acetone application is capable of bringing active amounts of insect hormones, neuromodulators, and biosynthetic precursors even in tiny insects. We advocate the use of acetone as a convenient, conservative, and broadly applicable vehicle for studying the effects of bioactive substances in insects.

Clustering of loci controlling species differences in male chemical bouquets of sympatric Heliconius butterflies.

The degree to which loci promoting reproductive isolation cluster in the genome—that is, the genetic architecture of reproductive isolation—can influence the tempo and mode of speciation. Tight linkage between these loci can facilitate speciation in the face of gene flow. Pheromones play a role in reproductive isolation in many Lepidoptera species, and the role of endogenously produced compounds as secondary metabolites decreases the likelihood of pleiotropy associated with many barrier loci. Heliconius butterflies use male sex pheromones to both court females (aphrodisiac wing pheromones) and ward off male courtship (male‐transferred antiaphrodisiac genital pheromones), and it is likely that these compounds play a role in reproductive isolation between Heliconius species. Using a set of backcross hybrids between H. melpomene and H. cydno, we investigated the genetic architecture of putative male pheromone compound production. We found a set of 40 significant quantitative trait loci (QTL) representing 33 potential pheromone compounds. QTL clustered significantly on two chromosomes, chromosome 8 for genital compounds and chromosome 20 for wing compounds, and chromosome 20 was enriched for potential pheromone biosynthesis genes. There was minimal overlap between pheromone QTL and known QTL for mate choice and color pattern. Nonetheless, we did detect linkage between a QTL for wing androconial area and optix, a color pattern locus known to play a role in reproductive isolation in these species. This tight clustering of putative pheromone loci might contribute to coincident reproductive isolating barriers, facilitating speciation despite ongoing gene flow.