Female Cuticular Hydrocarbons Research Articles

Antennal sensilla of Cyclocephala barrerai (Coleoptera: Melolonthidae): morphology, sexual dimorphism, allometric relationships, and function.

Scarab beetles use pheromones and volatiles to search for their partners and host plants. The perception of these compounds occurs in the beetle antennae, particularly in the sensilla. Relatively few studies have morphologically and physiologically characterized the sensilla of scarab beetles. Cyclocephala barrerai Martínez is a beetle distributed in Mexico. Male beetles have larger antennal lamellae than females, and in both cases, the size of this structure is related to their weight. Previous studies have reported that both sexes are attracted to bacterial volatiles isolated from the female genital chambers. Female cuticular hydrocarbons may act as a sexual contact pheromone. However, antennal sensilla and their electrophysiological responses to behaviorally relevant compounds remain to be investigated. Here, we describe and report the types, allometric relationships, and functions of sensilla found in the lamellae of both C. barrerai sexes. Sensilla were identified, classified, measured, and counted to identify intra and intersexual relationships. The single sensillum recordings showed that plant volatiles, hydrocarbons, and heat stimulated receptor neurons. We identified 2 new types of sensilla basiconica in the scape. Males have more and larger antennal sensilla placodea IV, which specializes in detecting pheromones, plant volatiles, and heat. Females have a greater diversity of sensilla.

Behavioural consequences of intraspecific variability in a mate recognition signal.

Mate recognition is paramount for sexually reproducing animals, and many insects rely on cuticular hydrocarbons (CHCs) for close-range sexual communication. To ensure reliable mate recognition, intraspecific sex pheromone variability should be low. However, CHCs can be influenced by several factors, with the resulting variability potentially impacting sexual communication. While intraspecific CHC variability is a common phenomenon, the consequences thereof for mate recognition remain largely unknown. We investigated the effect of CHC variability on male responses in a parasitoid wasp showing a clear-cut within-population CHC polymorphism (three distinct female chemotypes, one thereof similar to male profiles). Males clearly discriminated between female and male CHCs, but not between female chemotypes in no-choice assays. When given a choice, a preference hierarchy emerged. Interestingly, the most attractive chemotype was the one most similar to male profiles. Mixtures of female CHCs were as attractive as chemotype-pure ones, while a female-male mixture negatively impacted male responses, indicating assessment of the entire, complex CHC profile composition. Our study reveals that the evaluation of CHC profiles can be strict towards 'undesirable' features, but simultaneously tolerant enough to cover a range of variants. This reconciles reliable mate recognition with naturally occurring variability.

Cuticular Hydrocarbon Polymorphism in a Parasitoid Wasp

Cuticular hydrocarbons (CHCs) are major constituents of the cuticular lipid layer of insects. They serve not only as a barrier to desiccation, but often additionally mediate communication at close range. The compositions of the CHC profiles, i.e., the specific compounds and their relative amounts, usually differ between species. Additional intraspecific variability can be found between different populations, between colonies and castes of social insects, and between the sexes. Thus, such groups can often be distinguished based on distinctive compounds and/or specific compound ratios. The CHC profile may further be influenced by biotic and abiotic factors, which therefore can impact, e.g., nestmate recognition or mate choice. However, consistent intrasexual variation seems to be rare. Here, we investigated a case of intrasexual CHC variability within a single population of a parasitoid wasp. While wasps of both sexes produced the same set of compounds, the relative amounts of specific compound classes revealed the presence of intrasexual chemical phenotypes. This is, to our knowledge, the first report of three distinct female CHC profile patterns within a population of a solitary insect that uses CHCs for mate recognition. Additionally, male CHC profiles, while overall very similar, could be separated into two chemotypes by multivariate analysis. The study of species exhibiting such intraspecific and intrasexual CHC variation will advance our understanding of the effects of CHC variability on both, desiccation resistance and intraspecific communication.

Biologically active cuticular compounds of female Sirex noctilio

AbstractThe Eurasian woodwasp Sirex noctilio Fabricius (Hymenoptera: Siricidae) is a major invasive pest in pine plantations of the Southern Hemisphere. In this study, we aimed to identify pheromone compounds in S. noctilio females, for potential use in lures for pest monitoring and control. An examination of S. noctilio body washes resulted in the identification of a behaviorally active compound, 10‐oxo‐decanoic acid (10‐ODA), that is derived from cuticular hydrocarbons of females. This oxocarboxylic acid is likely produced through oxidative reactions at the cleavage of double bonds of certain female‐produced cuticular dienes. Gas chromatography‐electroantennogram detection (GC‐EAD) analyses showed that only male antennae consistently responded to natural and synthetic 10‐ODA. In olfactometer assays, 10‐ODA alone was attractive to S. noctilio males, although males marginally preferred the combination of 10‐ODA and nonanal, compared with 10‐ODA alone. This suggests pheromonal activity of this compound. However, no wasps were captured in initial field trapping experiments with 10‐ODA and further work is required.

Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males.

Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge of its role in regulating sexual behaviour is primarily limited to Drosophila. Here, we show with the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced (NvDsx-i) underwent a three-level pheromonal feminization: (i) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titres of the long-range sex pheromone; (ii) NvDsx-i males were courted by wild-type males as though they were females, which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males, to NvDsx-i males interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in N. vitripennis; and (iii) Nvdsx-i males were hampered in eliciting female receptivity and thus experienced severely reduced mating success, suggesting that they are unable to produce the to-date unidentified oral aphrodisiac pheromone reported in N. vitripennis males. We conclude that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis.

A Sexually Dimorphic Olfactory Neuron Mediates Fixed Action Transition during Courtship Ritual in Drosophila melanogaster.

Animals perform a series of actions in a fixed order during ritualistic innate behaviors. Although command neurons and sensory pathways responding to external stimuli that trigger these behaviors have been identified, how each action is induced in a fixed order in response to multimodal sensory stimuli remains unclear. Here, the sexually dimorphic lateral antennal lobe tract projection neuron 4 (lPN4) in male Drosophila melanogaster mediates the expression of a fixed behavioral action pattern at the beginning of the courtship ritual, in which a male taps a female body and then extends a wing unilaterally to produce a courtship song. We found that blocking the synaptic output of lPN4 caused an increase in the ratio of male flies that extended a wing unilaterally without tapping the female body, whereas excitation of lPN4 suppressed the transition from the tapping phase to the unilateral wing extension phase. Real-time calcium imaging showed that lPN4 is activated by a volatile pheromone, palmitoleic acid, whose responses were inhibited by simultaneous gustatory stimulation with female cuticular hydrocarbons, showing the existence of an "AND-gate" for multimodal sensory inputs during male courtship behaviors. These results suggest that the function of lPN4 is to suppress unilateral wing extension while responding to a female smell, which is released by appropriate contact chemosensory inputs received when tapping a female. As the female smell also promotes male courtship behaviors, the olfactory system is ready to simultaneously promote and suppress the progress of courtship actions while responding to a female smell.SIGNIFICANCE STATEMENT Although it has been 80 years since Konrad Lorenz and Niko Tinbergen introduced how multiple acts comprising separate innate behaviors are released in a fixed order according to external stimuli, the neural circuits responsible for such fixed action patterns remain largely unknown. The male courtship behavior of Drosophila melanogaster is a good model to investigate how such a fixed behavioral sequence is determined in the brain. Here, we show that lateral antennal lobe tract projection neuron 4 (lPN4) in D. melanogaster functions as an "AND-gate" for volatile and contact chemosensory inputs, mediating the expression of tapping behaviors before unilateral wing extension during male courtship rituals.

Micro‐climate correlations and conserved sexual dimorphism of cuticular hydrocarbons in European populations of the jewel wasp Nasonia vitripennis

1. Protection against desiccation and chemical communication are two fundamental functions of cuticular hydrocarbons (CHCs) in insects. In the parasitoid jewel wasp Nasonia vitripennis (Walker), characterised by a cosmopolitan distribution through largely different environments, CHCs function as universally recognised female sex pheromones. However, CHC uniformity as basis for sexual recognition may conflict with the desiccation protection function, expected to display considerable flexibility through adaptation to different environmental conditions.2. We compared male and female CHC profiles of N. vitripennis across a wide latitudinal gradient in Europe and correlated their CHC variation with climatic factors associated with desiccation. Additionally, we tested male mate discrimination behaviour between populations to detect potential variations in female sexual attractiveness.3. Results did not conform to the general expectation that longer, straight‐chain CHCs occur in higher proportions in warmer and drier climates. Instead, unexpected environmental correlations of intermediate chain‐length CHCs (C31) were found exclusively in females, potentially reflecting the different life histories of the sexes in N. vitripennis.4. Furthermore, we found no indication of population‐specific male mate preference, confirming the stability of female sexual attractiveness, likely conveyed through their CHC profiles. C31 mono‐ and C33 di‐methyl‐branched alkanes were consistently and most strongly associated with sexual dimorphism, suggesting their potential role in encoding the female‐specific sexual signalling function.5. Our study sheds light on how both adaptive flexibility and conserved sexual attractiveness can potentially be integrated and encoded in CHC profiles of N. vitripennis females across a wide distribution range in Europe.

Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness.

Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.

3D-printed insect models offer a feasible method for mating studies of chrysomelid beetles

A variety of models have been used in mating bioassays of insects to assess the contribution of chemical and visual signals to mate location and mate selection. Although the use of such ‘dummies’ has had varying degrees of success, some insect species refuse to accept simplistic models. In the present study, we developed a 3D-printed model to explore whether more realistic models will be more successful than simplistic models in mating assays of difficult to manipulate species such as the flea beetle Altica fragariae. We ran five experiments to test (1) whether males could discriminate between males and females solely based on differences in cuticular hydrocarbons (CHCs), (2) whether males use shape or (3) color to choose mates, and (4) whether males can discriminate between 3D-printed models and freshly killed beetles either with or (5) without legs and antennae. The results of these experiments confirmed that male A. fragariae preferred models coated with CHCs of females over that of male CHCs, providing strong support for the role of CHCs in mate choice in Altica. We also showed that males use both shape and color in mate selection, and that males are capable of discriminating between the models and real beetle specimens. Together, the results indicate that 3D-printed models can provide a feasible and cost-effective method for mating studies of insects.

Genes underlying species differences in cuticular hydrocarbon production between Drosophila melanogaster and D. simulans.

Surface chemical compounds are key components of survival and reproduction in many species. Cuticular hydrocarbons (CHCs) are chemical compounds produced by all insects that are used for both desiccation resistance and chemical communication, including communication related to mating. In the species pair of Drosophila melanogaster and D. simulans, female CHCs stimulate conspecific males to mate and repel heterospecific males. While CHCs are a critical contributor to both reproductive success within a species and isolation between species, few genes underlying species variation in CHC profiles are known. Here, we use genetic mapping of the 3rd chromosome to test a suite of candidate genes for interspecies variation in CHCs. Candidate gene CG5946 was found to be involved in species differences in the production of 7,11-heptacosadiene and 7-tricosene between D. melanogaster and D. simulans. This is therefore a new candidate locus contributing to species-specific variation in the CHC profile. In the process of mapping genes for CHCs, we also identified 29 candidate genes for the reduced survival or inviability of interspecies hybrids.

Seasonal changes in cuticular hydrocarbons in response to polyphenism in the host‐alternating aphid Prociphilus oriens

AbstractIn most terrestrial arthropods, cuticular hydrocarbons (CHCs) function to assist in desiccation tolerance and chemical communications. However, few studies have clarified whether CHC profiles change among developmental stages or among different morphs in non‐social insects. In the present study, we evaluated how CHC profiles change in accordance with polyphenism in the host‐alternating aphid Prociphilus oriens, which exhibits a complex life cycle and five distinct morphs. These morphs are sexual or asexual and adapt to different host plants. We found that all generations of P. oriens shared high proportions of n‐alkanes, but its composition varied among morphs. Three morphs that are attended by ants were characterized by relatively high proportions of n‐C25 to n‐C27, whereas two morphs that are not attended by ants had higher proportions of longer‐chain n‐alkanes, such as n‐C27 and n‐C29. The CHC profiles of sexual females were largely different from those of males. Considering that sexual females of Prociphilus spp. lack organs that secrete sex pheromones (scent plaques), the CHCs of sexual females are likely to function as a sex attractant. High proportions of methyl‐branched alkanes were detected in the long and flocculent waxy substances of autumnal migrants. These methyl‐branched alkanes are considered a cue to recognize conspecifics. We concluded that the functions and components of CHCs differ among morphs, and that those of sexual females differ from those of males and asexual generations because of their function in sexual communication.

Sexual selection and population divergence III: Interspecific and intraspecific variation in mating signals

A major challenge for studying the role of sexual selection in divergence and speciation is understanding the relative influence of different sexually selected signals on those processes in both intra- and interspecific contexts. Different signals may be more or less susceptible to co-option for species identification depending on the balance of sexual and ecological selection acting upon them. To examine this, we tested three predictions to explain geographic variation in long- versus short-range sexual signals across a 3,500+km transect of two related Australian field cricket species (Teleogryllus spp.): (a) selection for species recognition, (b) environmental adaptation and (c) stochastic divergence. We measured male calling song and male and female cuticular hydrocarbons (CHCs) in offspring derived from wild populations, reared under common garden conditions. Song clearly differentiated the species, and no hybrids were observed suggesting that hybridization is rare or absent. Spatial variation in song was not predicted by geography, genetics or climatic factors in either species. In contrast, CHC divergence was strongly associated with an environmental gradient supporting the idea that the climatic environment selects more directly upon these chemical signals. In light of recently advocated models of diversification via ecological selection on secondary sexual traits, the different environmental associations we found for song and CHCs suggest that the impact of ecological selection on population divergence, and how that influences speciation, might be different for acoustic versus chemical signals.

Mating has opposite effects on male and female sexually selected cuticular hydrocarbons

In Drosophila serrata flies, there is female choice for male cuticular hydrocarbon (CHC) profiles and male choice for female CHC profiles. Furthermore, both males and females can alter their CHCs: when there is more opportunity for mating, males express combinations of CHCs preferred by females; however, females appear to change CHC profiles to avoid male harassment. In this study, I investigate the effect of number of matings (0–4) on male and female sexually selected CHCs. Mating caused males to express CHCs associated with higher male mating success. Thus, successfully mating males are likely to have increased future mating success. Conversely, females that mated more times expressed CHC profiles that were associated with lower female mating success. Females maximized their offspring production by mating more than once, but additional matings did not provide additional benefits. Furthermore, number of matings did not affect female survival. In total, these results suggest that females alter CHC expression to discourage male courtship when additional matings are not beneficial. In conclusion, plasticity in male and female CHC expression can both increase variance in male mating success and decrease variance in female mating success, driving the evolution of sexually selected chemical signals.

Influence of female cuticular hydrocarbon (CHC) profile on male courtship behavior in two hybridizing field crickets Gryllus firmus and Gryllus pennsylvanicus

BackgroundThe hybridizing field crickets, Gryllus firmus and Gryllus pennsylvanicus have several barriers that prevent gene flow between species. The behavioral pre-zygotic mating barrier, where males court conspecifics more intensely than heterospecifics, is important because by acting earlier in the life cycle it has the potential to prevent a larger fraction of hybridization. The mechanism behind such male mate preference is unknown. Here we investigate if the female cuticular hydrocarbon (CHC) profile could be the signal behind male courtship.ResultsWhile males of the two species display nearly identical CHC profiles, females have different, albeit overlapping profiles and some females (between 15 and 45%) of both species display a male-like profile distinct from profiles of typical females. We classified CHC females profile into three categories: G. firmus-like (F; including mainly G. firmus females), G. pennsylvanicus-like (P; including mainly G. pennsylvanicus females), and male-like (ML; including females of both species). Gryllus firmus males courted ML and F females more often and faster than they courted P females (p < 0.05). Gryllus pennsylvanicus males were slower to court than G. firmus males, but courted ML females more often (p < 0.05) than their own conspecific P females (no difference between P and F). Both males courted heterospecific ML females more often than other heterospecific females (p < 0.05, significant only for G. firmus males).ConclusionsOur results suggest that male mate preference is at least partially informed by female CHC profile and that ML females elicit high courtship behavior in both species. Since ML females exist in both species and are preferred over other heterospecific females, it is likely that this female type is responsible for most hybrid offspring production.

Experimental evidence for the role of sexual selection in the evolution of cuticular hydrocarbons in the dung beetle, Onthophagus taurus.

A role for sexual selection in the evolution of insect cuticular hydrocarbons (CHCs) is suggested by observations of selection acting on male CHCs during female mate choice. However, evidence that CHCs evolve in response to sexual selection is generally lacking, and there is a need to extend our understanding beyond well-studied taxa. Experimental evolution offers a powerful approach to investigate the effect of sexual selection on the evolution of insect CHCs. We conducted such an experiment using the dung beetle, Onthophagus taurus. After six, 12 and 21 generations of experimental evolution, we measured the CHCs of beetles from three populations subject to sexual selection and three populations within which sexual selection had been removed via enforced monogamy. We found that the male CHC profile responded to the experimental removal of sexual selection. Conversely, the CHC profile of females responded to the presence of sexual selection but not to its removal. These results show that sexual selection can be an important mechanism affecting the evolution of insect CHCs and that male and female CHCs can evolve independently.

Female cuticular hydrocarbons can signal indirect fecundity benefits in an insect.

Male choosiness of prospective female mating partners provides an increasingly recognized pathway through which males can increase their fitness. For example, males may increase their number of offspring by targeting more fecund females as mating partners. If fecundity is heritable, males that mate with more fecund females can also receive the indirect benefit of more fecund daughters. In species where female fecundity is not directly assessable, female ornaments may act as signals of fecundity. However, whether female ornaments reliably signal the indirect benefit of more fecund daughters is not well understood. We investigated this question using the field cricket, Teleogryllus oceanicus. Previous work had identified the cuticular hydrocarbon (CHC) profile as a female sexual display trait in T. oceanicus. To examine whether CHCs can provide a reliable signal of fecundity, we tested whether individual CHC compounds and the first principal axis of CHC variation (PC1) are genetically correlated with ovary mass, a reliable proxy for fecundity in this species. We found significant genetic correlations between ovary mass and three individual CHC compounds, as well as PC1. This result indicates that by targeting females as mating partners based on their CHC profile, males can sire more fecund daughters.

A costly chemical trait: phenotypic condition dependence of cuticular hydrocarbons in a dung beetle.

Chemical traits are increasingly recognized as important cues used in mate choice. For example, the cuticular hydrocarbons (CHCs) of insects have been shown to influence mating success in a range of taxa. Less is known, however, about how CHCs are expressed in proportion to an individual's condition, and consequently whether CHCs can function as condition-dependent signals of quality. We investigated this question using the dung beetle, Onthophagus taurus. CHCs are subject to sexual selection in this species through mate choice. A dietary manipulation revealed condition dependence of CHC expression for both sexes: dietary restriction decreased overall CHC production and altered the composition of CHCs. Furthermore, CHC production was associated with a measure of condition in beetles fed a limited diet but not those fed adlibitum. These results implicate a resource cost to CHC production that is likely to result in trade-offs with other fitness components in this species, as these respond similarly to a dietary restriction. The CHC profiles showed sexual dimorphism: males produced more CHCs and the sexes differed in the blend of compounds they produced. There was evidence for a male dimorphism in the CHC profile, in line with the presence of the alternative reproductive tactics (minor sneaks and major fighters) in this species. However, rather than mimicking a female CHC profile, minor males differed more from females than major males. Our results suggest that CHCs are a costly trait in O.taurus that has the potential to act as a condition-dependent signal of quality.

Cuticular hydrocarbons determine sex, caste, and nest membership in each of four species of yellowjackets (Hymenoptera: Vespidae)

Cuticular hydrocarbons (CHCs) of social insects have typically been studied for their roles in reproductive signaling (i.e., fertility) rather than sexual signaling (i.e., interest in mating), resulting in little information about CHCs of males and virgin females. This dearth of information applies particularly to social wasps. We tested the hypothesis that CHCs differentiate sex, caste, and nest membership in each of four yellowjacket species (baldfaced hornets, Dolichovespula maculata; southern yellowjackets, Vespula squamosa; western yellowjackets, V. pensylvanica; V. alascensis). Cold-euthanized queens (21), gynes (81), workers (125), and males (77) from 35 nests were extracted with pentane, and each of the resulting 304 extracts was analyzed by gas chromatography (GC) and GC–mass spectrometry to identify and quantify CHC constituents (aliphatic alkanes and alkenes; mono-, di-, and tri-methyl-branched alkanes). To determine whether caste and sex differ in CHC profiles of wasps, linear discriminant analyses were performed, using Z-transformed relative CHC peak areas as predictor variables and sex and caste, or nest, as grouping variables. When caste and sex were used as a grouping variable, plots of the first two discriminant functions revealed that wasps from each of the four species clustered into their respective groups (queens, gynes, workers, males), with significant differences in group centroids, as measured by Wilks’ lambda. When nest was used as a grouping variable, plots of the first two discriminant functions revealed that workers from each of the four species and males from each of three species (insufficient sample size for V. pensylvanica) clustered according to nest. Diagnostic power calculations show greater inter-caste than inter-nest variation. Our data support the above hypothesis and inspire future studies to determine the definitive role(s) that gyne- and male-specific CHCs play in the context of sexual communication, from the perspective of both males and females.

Interference of chemical defence and sexual communication can shape the evolution of chemical signals

According to current evolutionary theory, insect pheromones can originate from extant precursor compounds being selected for information transfer. This is exemplified by females of the parasitoid wasp Leptopilina heterotoma whose defensive secretion consisting mainly of (−)-iridomyrmecin has evolved secondary functions as cue to avoid other females during host search and as female sex pheromone. To promote our understanding of pheromone evolution from defensive secretions we studied the chemical ecology of Leptopilina clavipes. We show here that L. clavipes also produces a defensive secretion that contains (−)-iridomyrmecin as major component and that females use it to detect and avoid host patches occupied by other females. However, the female sex pheromone of L. clavipes consists solely of cuticular hydrocarbons (CHCs) and males did not respond to female CHCs if presented in combination with the defensive secretion containing (−)-iridomyrmecin. This is in contrast to other species of Leptopilina, in which the iridoid compounds have no inhibiting effect or even function as sex pheromone triggering courtship behaviour. This indicates that Leptopilina species differ in the cost-benefit ratio for males searching for females, which might explain the strong divergence in the composition of the sex pheromone in the genus.

Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species.

Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, we have little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. In the present study, we estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and show that male mate discrimination accounts for the majority of the current isolation between these species. We further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. Our findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process.