Male Cuticular Hydrocarbons Research Articles

The transfer of male cuticular hydrocarbons provides a reliable cue of the risk and intensity of sperm competition in decorated crickets.

Theoretically, males should increase their ejaculate expenditure when the probability of sperm competition occurring (or risk) is high but decrease ejaculate expenditure as the number of competing ejaculates (or intensity) increases. Here we examine whether male decorated crickets (Gryllodes sigillatus) use cuticular hydrocarbons (CHCs) transferred to females by rival males at mating to assess the risk and intensity of sperm competition and adjust their ejaculate accordingly. Unmated females and those perfumed with CHCs extracted from one, three, or five males could be distinguished chemically, providing a reliable cue of the risk and intensity of sperm competition. In agreement with theory, males mating with these females increased sperm number with the risk of sperm competition and decreased sperm number with the intensity of sperm competition. Similarly, as the risk of sperm competition increased, males produced a larger and more attractive spermatophylax (an important non-sperm component of the ejaculate) but these traits did not vary with the intensity of sperm competition. Our results therefore demonstrate that both sperm and non-sperm components of the male ejaculate respond to the risk and intensity of sperm competition in different ways and that CHCs provide males with an important cue to strategically tailor their ejaculate.

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.

An experimental test of the evolutionary consequences of sympatry in Drosophila subquinaria.

Prezygotic isolation is often stronger between sympatric as opposed to allopatric taxa, but the underlying cause can be difficult to infer from comparative studies alone. Experimental evolution, where evolutionary responses to treatments manipulating the presence/absence of heterospecific individuals are tracked, can provide a powerful complementary approach. We used experimental evolution to investigate a naturally occurring pattern of reproductive character displacement in the mushroom-feeding fly, Drosophila subquinaria. In nature, female D. subquinaria from populations sympatric with the closely related Drosophila recens discriminate more strongly against heterospecific males than do females from allopatric populations. Starting with 16 replicate allopatric populations of D. subquinaria, we manipulated the presence/absence of D. recens during mating (experimental sympatry vs. control) and, when present, we allowed hybrids to live or kill them each generation. Across 12 generations, heterospecific offspring production from no-choice mating trials between D. subquinaria females and D. recens males declined in both experimental sympatry treatments relative to the control, suggesting increased sexual isolation. Male cuticular hydrocarbon profiles also evolved, but only in the hybrids killed treatment. Our results strongly imply that the existing reproductive character displacement in wild D. subquinaria populations was an evolutionary response to selection arising from secondary contact with D. recens.

A supergene in seaweed flies modulates male traits and female perception.

Supergenes, tightly linked sets of alleles, offer some of the most spectacular examples of polymorphism persisting under long-term balancing selection. However, we still do not understand their evolution and persistence, especially in the face of accumulation of deleterious elements. Here, we show that an overdominant supergene in seaweed flies, Coelopa frigida, modulates male traits, potentially facilitating disassortative mating and promoting intraspecific polymorphism. Across two continents, the Cf-Inv(1) supergene strongly affected the composition of male cuticular hydrocarbons (CHCs) but only weakly affected CHC composition in females. Using gas chromatography-electroantennographic detection, we show that females can sense male CHCs and that there may be differential perception between genotypes. Combining our phenotypic results with RNA-seq data, we show that candidate genes for CHC biosynthesis primarily show differential expression for Cf-Inv(1) in males but not females. Conversely, candidate genes for odorant detection were differentially expressed in both sexes but showed high levels of divergence between supergene haplotypes. We suggest that the reduced recombination between supergene haplotypes may have led to rapid divergence in mate preferences as well as increasing linkage between male traits, and overdominant loci. Together this probably helped to maintain the polymorphism despite deleterious effects in homozygotes.

Beauty or function? The opposing effects of natural and sexual selection on cuticular hydrocarbons in male black field crickets.

Although many theoretical models of male sexual trait evolution assume that sexual selection is countered by natural selection, direct empirical tests of this assumption are relatively uncommon. Cuticular hydrocarbons (CHCs) are known to play an important role not only in restricting evaporative water loss but also in sexual signalling in most terrestrial arthropods. Insects adjusting their CHC layer for optimal desiccation resistance is often thought to come at the expense of successful sexual attraction, suggesting that natural and sexual selection are in opposition for this trait. In this study, we sampled the CHCs of male black field crickets (Teleogryllus commodus) using solid-phase microextraction and then either measured their evaporative water loss or mating success. We then used multivariate selection analysis to quantify the strength and form of natural and sexual selection targeting male CHCs. Both natural and sexual selection imposed significant linear and stabilizing selection on male CHCs, although for very different combinations. Natural selection largely favoured an increase in the total abundance of CHCs, especially those with a longer chain length. In contrast, mating success peaked at a lower total abundance of CHCs and declined as CHC abundance increased. However, mating success did improve with an increase in a number of specific CHC components that also increased evaporative water loss. Importantly, this resulted in the combination of male CHCs favoured by natural selection and sexual selection being strongly opposing. Our findings suggest that the balance between natural and sexual selection is likely to play an important role in the evolution of male CHCs in T. commodus and may help explain why CHCs are so divergent across populations and species.

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.

Integrating genomics and multivariate evolutionary quantitative genetics: a case study of constraints on sexual selection in Drosophila serrata.

In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, β, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and β have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. Here, we integrate multivariate genome-wide association study (GWAS) with G and β estimation in a well-studied system of multivariate constraint: sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived re-sequenced lines, we augment genome-based restricted maximum likelihood to estimate G alongside multivariate single nucleotide polymorphism (SNP) effects, detecting 532 significant associations from 1 652 276 SNPs. Constraint was evident, with β lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on β. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. We discuss potential factors leading to these varied results including multivariate stabilizing selection and mutational bias. Our framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations.

A shift to shorter cuticular hydrocarbons accompanies sexual isolation among Drosophila americana group populations.

Because sensory signals often evolve rapidly, they could be instrumental in the emergence of reproductive isolation between species. However, pinpointing their specific contribution to isolating barriers, and the mechanisms underlying their divergence, remains challenging. Here, we demonstrate sexual isolation due to divergence in chemical signals between two populations of Drosophila americana (SC and NE) and one population of D. novamexicana, and dissect its underlying phenotypic and genetic mechanisms. Mating trials revealed strong sexual isolation between Drosophila novamexicana males and SC Drosophila americana females, as well as more moderate bi‐directional isolation between D. americana populations. Mating behavior data indicate SC D. americana males have the highest courtship efficiency and, unlike males of the other populations, are accepted by females of all species. Quantification of cuticular hydrocarbon (CHC) profiles—chemosensory signals that are used for species recognition and mate finding in Drosophila—shows that the SC D. americana population differs from the other populations primarily on the basis of compound carbon chain‐length. Moreover, manipulation of male CHC composition via heterospecific perfuming—specifically perfuming D. novamexicana males with SC D. americana males—abolishes their sexual isolation from these D. americana females. Of a set of candidates, a single gene—elongase CG17821—had patterns of gene expression consistent with a role in CHC differences between species. Sequence comparisons indicate D. novamexicana and our Nebraska (NE) D. americana population share a derived CG17821 truncation mutation that could also contribute to their shared “short” CHC phenotype. Together, these data suggest an evolutionary model for the origin and spread of this allele and its consequences for CHC divergence and sexual isolation in this group.

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.

Similar Is Not the Same – Mate Recognition in a Parasitoid Wasp

Finding and recognizing a suitable mate is a key prerequisite to reproductive success. Insects often recognize prospective mates using chemical cues and signals. Among these, cuticular lipids commonly serve for mate recognition at close range. The lipid layer on the surface of insects is comprised predominantly of cuticular hydrocarbons (CHCs), though more polar compounds may also be present. While the composition of the cuticular profile is typically species specific, many species additionally show differentiation between the sexes by sex specific compounds and/or compound ratios. It is often assumed that a clear sexual dimorphism of cuticular lipid profiles is a prerequisite for a potential function as sex pheromones. Both, sex specific single compounds or the profiles as a whole have been shown to serve as sex pheromones in parasitoid wasps. Here, we studied parasitoid wasps of the speciesTachinaephagus zealandicus(Encyrtidae, Hymenoptera). Chemical analyses revealed that this species presents a case where males and females produce the same set of CHCs in similar relative amounts. To test whether these wasps nonetheless can use the cuticular lipids for close range mate recognition, we tested the reaction of males toward freeze-killed conspecifics. Males showed copulation behavior exclusively toward dead females, but not toward dead males. Dead females from which the cuticular lipids had been removed did not elicit copulation behavior by tested males. Reapplication of female whole body extracts restored bioactivity, and males reacted with copulation attempts as often as toward the freeze-killed females. Bioassays with lipid fractions revealed that only the CHC fraction was bioactive on its own. Here, again, males reacted to female, but not to male CHCs. Our results indicate that these wasps are capable of using CHCs for close range sex recognition despite the similarity of male and female profiles.

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.

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.

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.

The complex interplay between macronutrient intake, cuticular hydrocarbon expression and mating success in male decorated crickets.

The condition dependence of male sexual traits plays a central role in sexual selection theory. Relatively little, however, is known about the condition dependence of chemical signals used in mate choice and their subsequent effects on male mating success. Furthermore, few studies have isolated the specific nutrients responsible for condition-dependent variation in male sexual traits. Here, we used nutritional geometry to determine the effect of protein (P) and carbohydrate (C) intake on male cuticular hydrocarbon (CHC) expression and mating success in male decorated crickets (Gryllodes sigillatus). We show that both traits are maximized at a moderate-to-high intake of nutrients in a P:C ratio of 1:1.5. We also show that female precopulatory mate choice exerts a complex pattern of linear and quadratic sexual selection on this condition-dependent variation in male CHC expression. Structural equationmodelling revealed that although the effect of nutrient intake on mating success is mediated through condition-dependent CHC expression, it is not exclusively so, suggesting that other traits must also play an important role. Collectively, our results suggest that the complex interplay between nutrient intake, CHC expression and mating success plays an important role in the operation of sexual selection in G.sigillatus.

Crowd control: sex ratio affects sexually selected cuticular hydrocarbons in male Drosophila serrata.

Although it is advantageous for males to express costly sexually selected signals when females are present, they may also benefit from suppressing these signals to avoid costly interactions with rival males. Cuticular chemical profiles frequently function as insect sexual signals; however, few studies have asked whether males alter these signals in response to their social environment. In Drosophila serrata, an Australian fly, there is sexual selection for a multivariate combination of male cuticular hydrocarbons (CHCs). Here, we show that the ratio of females to males that an adult male experiences has a strong effect on his CHC expression, with female-biased adult sex ratios eliciting greater expression of CHC profiles associated with higher male mating success. Classical models predict that male reproductive investment should be highest when there is a small but nonzero number of rivals, but we found that males expressed the most attractive combination of CHCs when there were no rivals. We found that male CHCs were highly sensitive to adult sex ratio, with males expressing higher values of CHC profiles associated with greater mating success as the ratio of females to males increased. Moreover, sex ratio has a stronger effect on male CHC expression than adult density. Finally, we explore whether sex ratio affects the variance among a group of males in their CHC expression, as might be expected if individuals respond differently to a given social environment, but find little effect. Our results reveal that subtle differences in social environment can induce plasticity in male chemical signal expression.

Sexual Selection on male cuticular hydrocarbons via male-male competition and female choice.

Traditional views of sexual selection assumed that male–male competition and female mate choice work in harmony, selecting upon the same traits in the same direction. However, we now know that this is not always the case and that these two mechanisms often impose conflicting selection on male sexual traits. Cuticular hydrocarbons (CHCs) have been shown to be linked to both social dominance and male attractiveness in several insect species. However, although several studies have estimated the strength and form of sexual selection imposed on male CHCs by female mate choice, none have established whether these chemical traits are also subject to sexual selection via male–male competition. Using a multivariate selection analysis, we estimate and compare sexual selection exerted by male–male competition and female mate choice on male CHC composition in the broad‐horned flour beetle Gnatocerus cornutus. We show that male–male competition exerts strong linear selection on both overall CHC abundance and body size in males, while female mate choice exerts a mixture of linear and nonlinear selection, targeting not just the overall amount of CHCs expressed but the relative abundance of specific hydrocarbons as well. We discuss the potential implications of this antagonistic selection with regard to male reproductive success.

Female choice for male cuticular hydrocarbon profile in decorated crickets is not based on similarity to their own profile.

Indirect genetic benefits derived from female mate choice comprise additive (good genes) and nonadditive genetic benefits (genetic compatibility). Although good genes can be revealed by condition-dependent display traits, the mechanism by which compatibility alleles are detected is unclear because evaluation of the genetic similarity of a prospective mate requires the female to assess the genotype of the male and compare it to her own. Cuticular hydrocarbons (CHCs), lipids coating the exoskeleton of most insects, influence female mate choice in a number of species and offer a way for females to assess genetic similarity of prospective mates. Here, we determine whether female mate choice in decorated crickets is based on male CHCs and whether it is influenced by females' own CHC profiles. We used multivariate selection analysis to estimate the strength and form of selection acting on male CHCs through female mate choice, and employed different measures of multivariate dissimilarity to determine whether a female's preference for male CHCs is based on similarity to her own CHC profile. Female mating preferences were significantly influenced by CHC profiles of males. Male CHC attractiveness was not, however, contingent on the CHC profile of the choosing female, as certain male CHC phenotypes were equally attractive to most females, evidenced by significant linear and stabilizing selection gradients. These results suggest that additive genetic benefits, rather than nonadditive genetic benefits, accrue to female mate choice, in support of earlier work showing that CHC expression of males, but not females, is condition dependent.

Reproductive character displacement of female mate preferences for male cuticular hydrocarbons in Drosophila subquinaria.

Several lines of evidence implicate sexual isolation in both initiating and completing the speciation process. Although its existence is straightforward to demonstrate, understanding the evolution of sexual isolation requires identifying the underlying phenotypes responsible so that we can determine how these have diverged. Here, we study geographic variation in female mate preferences for male sexual displays in the fly Drosophila subquinaria. Female D. subquinaria that are sympatric with its sister species D. recens discriminate strongly against both D. recens and allopatric conspecific males, whereas females from allopatric populations do not. Furthermore, female mate preferences target at least in part a suite of cuticular hydrocarbons (CHCs) in males and geographic variation in CHCs mirrors the pattern of mate discrimination. In this study, we quantify female mate preferences for male CHCs from populations that span the geographic range of D. subquinaria. We find that the direction of linear sexual selection varies significantly between populations that are sympatric versus allopatric with D. recens in a pattern of reproductive character displacement. Differences in preference partially align with existing differences in CHCs and patterns of sexual isolation, although discrepancies remain that suggest the involvement of additional traits and/or more complex, nonlinear preference functions.

Rival male chemical cues evoke changes in male pre- and post-copulatory investment in a flour beetle.

Males can gather information on the risk and intensity of sperm competition from their social environment. Recent studies have implicated chemosensory cues, for instance cuticular hydrocarbons (CHCs) in insects, as a key source of this information. Here, using the broad-horned flour beetle (Gnatocerus cornutus), we investigated the importance of contact-derived rival male CHCs in informing male perception of sperm competition risk and intensity. We experimentally perfumed virgin females with male CHCs via direct intersexual contact and measured male pre- and post-copulatory investment in response to this manipulation. Using chemical analysis, we verified that this treatment engendered changes to perfumed female CHC profiles, but did not make perfumed females "smell" mated. Despite this, males responded to these chemical changes. Males increased courtship effort under low levels of perceived competition (from 1-3 rivals), but significantly decreased courtship effort as perceived competition rose (from 3-5 rivals). Furthermore, our measurement of ejaculate investment showed that males allocated significantly more sperm to perfumed females than to control females. Together, these results suggest that changes in female chemical profile elicited by contact with rival males do not provide males with information on female mating status, but rather inform males of the presence of rivals within the population and thus provide a means for males to indirectly assess the risk of sperm competition.

Do the benefits of polyandry scale with outbreeding?

There have been many potential explanations put forward as to why polyandry often persists despite the multiple costs it can inflict on females. One such explanation is avoidance of costs associated with mating with genetically incompatible males. Genetic incompatibility can be thought of as a spectrum from individuals that are genetically too similar (inbreeding) to those that are too dissimilar (outbreeding or hybridization). Here we look for evidence that the level of outbreeding influences the benefits of polyandry in the seed bug Lygaeus equestris. Our system allows us to test for benefits of polyandry at levels of genetic similarity ranging from full siblings to heterospecifics, both in terms of egg production and hatching success. We found that while outbreeding level appeared to have no effect on fitness for intraspecific matings, and polyandry did not appear to result in any increase in fertility or fecundity, hybridization with a closely related species, Lygaeus simulans, carried considerable fitness costs. However, these costs could be rescued with a single mating to a conspecific. Thus, polyandry may be beneficial in populations that co-occur with closely related species and where there is reproductive interference. However, within-species genetic incompatibility is unlikely to be the driving force behind polyandry in this species. Furthermore, the mechanism underlying this rescue of fertility remains unclear as manipulation of male cuticular hydrocarbon profile, a possible mechanism by which females can assess male identity, had no effect on female offspring production.