Natural Variation in the Strength and Direction of Male Mating Preferences for Female Pheromones in Drosophila melanogaster

Many animal species communicate using chemical signals. In Drosophila, cuticular hydrocarbons (CHCs) are involved in species and sexual identification, and have long been thought to act as stimulatory pheromones as well. However, a previous study reported that D. melanogaster males were more attracted to females that were lacking CHCs. This surprising result is consistent with several evolutionary hypotheses but is at odds with other work demonstrating that female CHCs are attractive to males. Here, we investigated natural variation in male preferences for female pheromones using transgenic flies that cannot produce CHCs. By perfuming females with CHCs and performing mate choice tests, we found that some male genotypes prefer females with pheromones, some have no apparent preference, and at least one male genotype prefers females without pheromones. This variation provides an excellent opportunity to further investigate the mechanistic causes and evolutionary implications of divergent pheromone preferences in D. melanogaster males.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection

Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within‐population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair‐mated adults from no‐choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population‐specific plasticity in CHCs. Different groups of CHCs mediated female choice‐based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. X. Age‐specific dynamics of adult epicuticular hydrocarbon expression in response to different host plants

Analysis of sexual selection and sexual isolation in Drosophila mojavensis and its relatives has revealed a pervasive role of rearing substrates on adult courtship behavior when flies were reared on fermenting cactus in preadult stages. Here, we assessed expression of contact pheromones comprised of epicuticular hydrocarbons (CHCs) from eclosion to 28 days of age in adults from two populations reared on fermenting tissues of two host cacti over the entire life cycle. Flies were never exposed to laboratory food and showed significant reductions in average CHC amounts consistent with CHCs of wild‐caught flies. Overall, total hydrocarbon amounts increased from eclosion to 14–18 days, well past age at sexual maturity, and then declined in older flies. Most flies did not survive past 4 weeks. Baja California and mainland populations showed significantly different age‐specific CHC profiles where Baja adults showed far less age‐specific changes in CHC expression. Adults from populations reared on the host cactus typically used in nature expressed more CHCs than on the alternate host. MANCOVA with age as the covariate for the first six CHC principal components showed extensive differences in CHC composition due to age, population, cactus, sex, and age × population, age × sex, and age × cactus interactions. Thus, understanding variation in CHC composition as adult D. mojavensis age requires information about population and host plant differences, with potential influences on patterns of mate choice, sexual selection, and sexual isolation, and ultimately how these pheromones are expressed in natural populations. Studies of drosophilid aging in the wild are badly needed.     

Flexible origin of hydrocarbon/pheromone precursors in Drosophila melanogaster

In terrestrial insects, cuticular hydrocarbons (CHCs) provide protection from desiccation. Specific CHCs can also act as pheromones, which are important for successful mating. Oenocytes are abdominal cells thought to act as specialized units for CHC biogenesis that consists of long-chain fatty acid (LCFA) synthesis, optional desaturation(s), elongation to very long-chain fatty acids (VLCFAs), and removal of the carboxyl group. By investigating CHC biogenesis in Drosophila melanogaster, we showed that VLCFA synthesis takes place only within the oenocytes. Conversely, several pathways, which may compensate for one another, can feed the oenocyte pool of LCFAs, suggesting that this step is a critical node for regulating CHC synthesis. Importantly, flies deficient in LCFA synthesis sacrificed their triacylglycerol stores while maintaining some CHC production. Moreover, pheromone production was lower in adult flies that emerged from larvae that were fed excess dietary lipids, and their mating success was lower. Further, we showed that pheromone production in the oenocytes depends on lipid metabolism in the fat tissue and that fatty acid transport protein, a bipartite acyl-CoA synthase (ACS)/FA transporter, likely acts through its ACS domain in the oenocyte pathway of CHC biogenesis. Our study highlights the importance of environmental and physiological inputs in regulating LCFA synthesis to eventually control sexual communication in a polyphagous animal.     

Time flies: time of day and social environment affect cuticular hydrocarbon sexual displays in Drosophila serrata

Recent work on Drosophila cuticular hydrocarbons (CHCs) challenges a historical assumption that CHCs in flies are largely invariant. Here, we examine the effect of time of day and social environment on a suite of sexually selected CHCs in Drosophila serrata. We demonstrate that males become more attractive to females during the time of day that flies are most active and when most matings occur, but females become less attractive to males during the same time of day. These opposing temporal changes may reflect differences in selection among the sexes. To evaluate the effect of social environment on male CHC attractiveness, we manipulated male opportunity for mating: male flies were housed either alone, with five females, with five males or with five males and five females. We found that males had the most attractive CHCs when with females, and less attractive CHCs when with competitor males. Social environment mediated how male CHC attractiveness cycled: males housed with females and/or other males showed temporal changes in CHC attractiveness, whereas males housed alone did not. In total, our results demonstrate temporal patterning of male CHCs that is dependent on social environment, and suggest that such changes may be beneficial to males.     

‘Does my Diet Affect my Perfume?’ Identification and Quantification of Cuticular Compounds in Five Drosophila melanogaster Strains Maintained over 300 Generations on Different Diets

Cuticular hydrocarbons (CHCs) in Drosophila melanogaster represent the basis of chemical communication being involved in many important biological functions. The aim of this study was to characterize chemical composition and variation of cuticular profiles in five D. melanogaster strains. These strains were reared for approximately 300 generations on five diets: standard cornmeal medium and substrates prepared with apple, banana, tomato, and carrot. Differences in quantity and/or quality in CHCs were assumed as a result of activation of different metabolic pathways involved in food digestion and adaptations to the particular diet type. In total, independently of sex and strain, 66 chemical compounds were identified. In females of all strains, 60 compounds were identified, while, in males, 47 compounds were extracted. Certain new chemical compounds for D. melanogaster were found. MANOVA confirmed that CHC amounts significantly depend on sex and substrates, as well as on their interactions. Discriminant analysis revealed that flies belonging to ‘apple’ and ‘carrot’ strains exhibited the most noticeable differences in CHC repertoires. A non‐hydrocarbon pheromone, cis‐vaccenyl acetate (cVA) also contributed to the variation in the pheromone bouquet among the strains. Variability detected in CHCs and cVA may be used in the explanation of differences in mating behaviour previously determined in analyzed fly strains.     

Dietary Effects on Cuticular Hydrocarbons and Sexual Attractiveness in Drosophila

Dietary composition is known to have profound effects on many aspects of animal physiology, including lifespan, general health, and reproductive potential. We have previously shown that aging and insulin signaling significantly influence the composition and sexual attractiveness of Drosophila melanogaster female cuticular hydrocarbons (CHCs), some of which are known to be sex pheromones. Because diet is intimately linked to aging and to the activity of nutrient-sensing pathways, we asked how diet affects female CHCs and attractiveness. Here we report consistent and significant effects of diet composition on female CHC profiles across ages, with dietary yeast and sugar driving CHC changes in opposite directions. Surprisingly, however, we found no evidence that these changes affect female attractiveness. Multivariate comparisons among responses of CHC profiles to diet, aging, and insulin signaling suggest that diet may alter the levels of some CHCs in a way that results in profiles that are more attractive while simultaneously altering other CHCs in a way that makes them less attractive. For example, changes in short-chain CHCs induced by a high-yeast diet phenocopy changes caused by aging and by decreased insulin signaling, both of which result in less attractive females. On the other hand, changes in long-chain CHCs in response to the same diet result in levels that are comparable to those observed in attractive young females and females with increased insulin signaling. The effects of a high-sugar diet tend in the opposite direction, as levels of short-chain CHCs resemble those in attractive females with increased insulin signaling and changes in long-chain CHCs are similar to those caused by decreased insulin signaling. Together, these data suggest that diet-dependent changes in female CHCs may be sending conflicting messages to males.     

Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: Combined effects of octopamine and juvenile hormone

Male moths use sex pheromones to find their mating partners. In the moth, Agrotis ipsilon, the behavioral response and the neuron sensitivity within the primary olfactory centre, the antennal lobe (AL), to sex pheromone increase with age and juvenile hormone (JH) biosynthesis. By manipulating the JH level, we previously showed that JH controls this age-dependent neuronal plasticity, and that its effects are slow (within 2 days). We hypothesized that the hormonal effect might be indirect, and one neuromodulator candidate, which might serve as a mediator, is octopamine (OA). Here, we studied the effects of OA and an OA receptor antagonist, mianserin, on behavioral and AL neuron responses of mature and immature males during stimulation with sex pheromone. Our results indicate that, although OA injections enhanced the behavioral pheromone response in mature males, OA had no significant effect on behavior in immature males. However, mianserin injections decreased the behavioral response in mature males. AL neuron sensitivity increased after OA treatment in immature males, and decreased after mianserin treatment in mature males. Determination of OA levels in ALs of immature and mature males did not reveal any difference. To study the possible interactive effects of JH and OA, the behavioral pheromone response was analyzed in JH-deprived mature males injected with OA, and in immature males injected with fenoxycarb, a JH agonist, and mianserin. Results show that both JH and OA are necessary to elicit a behavioral response of A. ipsilon males to sex pheromone.     

Cuticular hydrocarbons as contact sex pheromone in the parasitoid Dibrachys cavus

Cuticular hydrocarbons (CHCs) cover the cuticle of virtually any insect and do not only protect them from desiccation, but also possess various communicative functions. Previous studies suggested a function as contact sex pheromones in the parasitic wasp family Pteromalidae, but further species need to be studied before more general conclusions are possible. Here, we demonstrate a contact sex pheromone in females of Dibrachys cavus (Walker) (Hymenoptera: Pteromalidae). Males of this parasitoid were arrested on cadavers of 1‐ to 2‐day‐old females, but neither on newly emerged females nor on males. Extracts from 1‐ to 2‐day‐old females and non‐polar fractions were also behaviorally active suggesting a non‐polar contact sex pheromone. Chemical analyses of bioactive and non‐active CHC profiles by gas chromatography–mass spectrometry and subsequent principal component analysis (PCA) revealed that the bioactive profiles from 1‐ to 2‐day‐old females were clearly distinguishable from all other profiles. Among the chemicals showing the strongest impact in the PCA were the two 3‐methylalkanes 3‐methylnonacosane and 3‐methylhentriacontane, which were absent or trace components in the inactive profiles and whose relative amounts increased in females when they became sexually attractive. The putative function of 3‐methylalkanes as key components of the contact sex pheromones in Pteromalidae is discussed.     

Pheromone production in bark beetles

The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids. The structural similarity between the major pheromone components of bark beetles and the monoterpenes found in the host trees, along with the association of monoterpenoid production with plant tissue, led to the paradigm that most if not all bark beetle pheromone components were derived from host tree precursors, often with a simple hydroxylation producing the pheromone. In the 1990s there was a paradigm shift as evidence for de novo biosynthesis of pheromone components began to accumulate, and it is now recognized that most bark beetle monoterpenoid aggregation pheromone components are biosynthesized de novo. The bark beetle aggregation pheromones are released from the frass, which is consistent with the isoprenoid aggregation pheromones, including ipsdienol, ipsenol and frontalin, being produced in midgut tissue. It appears that exo-brevocomin is produced de novo in fat body tissue, and that verbenol, verbenone and verbenene are produced from dietary α-pinene in fat body tissue. Combined biochemical, molecular and functional genomics studies in Ips pini yielded the discovery and characterization of the enzymes that convert mevalonate pathway intermediates to pheromone components, including a novel bifunctional geranyl diphosphate synthase/myrcene synthase, a cytochrome P450 that hydroxylates myrcene to ipsdienol, and an oxidoreductase that interconverts ipsdienol and ipsdienone to achieve the appropriate stereochemistry of ipsdienol for pheromonal activity. Furthermore, the regulation of these genes and their corresponding enzymes proved complex and diverse in different species. Mevalonate pathway genes in pheromone producing male I. pini have much higher basal levels than in females, and feeding induces their expression. In I. duplicatus and I. pini, juvenile hormone III (JH III) induces pheromone production in the absence of feeding, whereas in I. paraconfusus and I. confusus, topically applied JH III does not induce pheromone production. In all four species, feeding induces pheromone production. While many of the details of pheromone production, including the site of synthesis, pathways and knowledge of the enzymes involved are known for Ips, less is known about pheromone production in Dendroctonus. Functional genomics studies are under way in D. ponderosae, which should rapidly increase our understanding of pheromone production in this genus. This chapter presents a historical development of what is known about pheromone production in bark beetles, emphasizes the genomic and post-genomic work in I. pini and points out areas where research is needed to obtain a more complete understanding of pheromone production.     

Quantitative genetic analysis suggests causal association between cuticular hydrocarbon composition and desiccation survival in Drosophila melanogaster

Survival to low relative humidity is a complex adaptation, and many repeated instances of evolution to desiccation have been observed among Drosophila populations and species. One general mechanism for desiccation resistance is Cuticular Hydrocarbon (CHC) melting point. We performed the first Quantitative Trait Locus (QTL) map of population level genetic variation in desiccation resistance in D. melanogaster. Using a panel of Recombinant Inbred Lines (RILs) derived from a single natural population, we mapped QTL in both sexes throughout the genome. We found that in both sexes, CHCs correlated strongly with desiccation resistance. At most desiccation resistance loci there was a significant association between CHCs and desiccation resistance of the sort predicted from clinal patterns of CHC variation and biochemical properties of lipids. This association was much stronger in females than males, perhaps because of greater overall abundance of CHCs in females, or due to correlations between CHCs used for waterproofing and sexual signalling in males. CHC evolution may be a common mechanism for desiccation resistance in D. melanogaster. It will be interesting to compare patterns of CHC variation and desiccation resistance in species which adapt to desiccation, and rainforest restricted species which cannot.     

Conspecific and heterospecific pheromone effects on female receptivity

Courtship pheromones play an important role in salamander reproductive behaviour. In salamanders of the family Plethodontidae, males deliver specialized pheromones to females during courtship interactions. These courtship pheromones increase female receptivity and may be involved in mate discrimination. In order to test hypotheses related to mate discrimination, we staged courtship encounters between male-female Plethodon shermani pairs in which the female received pheromones obtained from either conspecific (P. shermani) or heterospecific (P. yonahlossee orP. montanus ) males. Both conspecific and heterospecific pheromones increased female receptivity. Moreover, pheromones from both heterospecific species were as effective as the conspecific pheromone in increasing female receptivity inP. shermani females. Our results suggest that the courtship pheromone signal and function may be conserved across related species, with mate discrimination occurring before pheromone delivery. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.     

Evolution of sexually dimorphic pheromone profiles coincides with increased number of male‐specific chemosensory organs in Drosophila prolongata

Binary communication systems that involve sex‐specific signaling and sex‐specific signal perception play a key role in sexual selection and in the evolution of sexually dimorphic traits. The driving forces and genetic changes underlying such traits can be investigated in systems where sex‐specific signaling and perception have emerged recently and show evidence of potential coevolution. A promising model is found in Drosophila prolongata, which exhibits a species‐specific increase in the number of male chemosensory bristles. We show that this transition coincides with recent evolutionary changes in cuticular hydrocarbon (CHC) profiles. Long‐chain CHCs that are sexually monomorphic in the closest relatives of D. prolongata (D. rhopaloa, D. carrolli, D. kurseongensis, and D. fuyamai) are strongly male‐biased in this species. We also identify an intraspecific female‐limited polymorphism, where some females have male‐like CHC profiles. Both the origin of sexually dimorphic CHC profiles and the female‐limited polymorphism in D. prolongata involve changes in the relative amounts of three mono‐alkene homologs, 9‐tricosene, 9‐pentacosene, and 9‐heptacosene, all of which share a common biosynthetic origin and point to a potentially simple genetic change underlying these traits. Our results suggest that pheromone synthesis may have coevolved with chemosensory perception and open the way for reconstructing the origin of sexual dimorphism in this communication system.     

Juvenile hormones: Their role in the regulation of the pheromonal communication system of the armyworm moth,Pseudaletia unipuncta

Recently, much effort has been devoted to the elucidation of the neuro-endocrine mechanisms regulating the biosynthesis and emission of sex pheromones in the Lepidoptera. The available data indicate that the hormonal mechanisms involved vary considerably among species. For example, compelling evidence that juvenile hormones (JH) play a role in the control of sex pheromone production has been presented only for the armyworm moth, Pseudaletia unipuncta. In this species, females that are allatectomized at emergence neither produce nor release pheromone, but both activities are restored following replacement therapy with synthetic JH. However, injection of synthetic JH into neck-ligated females does not induce pheromone biosynthesis, whereas treatment with either a brain homogenate or synthetic PBAN results in a rise in the pheromone titer. These results indicate that the role played by JH is an indirect one and that the tropic factor is a PBAN-like substance. Studies on in vitro JH biosynthesis by isolated corpora allata of P. unipuncta have shown that the low JH output observed early in the life of adult females coincides with the absence of both calling behavior and pheromone production. The subsequent increase in the rates of JH biosynthesis correlates with the onset of pheromone production and release. We have therefore proposed that JH titers must pass a threshold level before the circadian release of PBAN and calling behavior can begin. Furthermore, recent experiments suggest that the continuous presence of JH is necessary for calling behavior to be maintained once initiated. Lastly, we present data suggesting a role for JH or JH acids in the receptivity of P. unipuncta males to the female sex pheromone. © 1994 Wiley-Liss, Inc.     

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

In 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.     

Identification of a Male-Produced Pheromone Component of the Citrus Longhorned Beetle,Anoplophora chinensis

The Asian wood-boring beetle Anoplophora chinensis (Forster) (Coleoptera: Cerambycidae) is an important pest of hardwood trees in its native range, and has serious potential to invade other areas of the world through worldwide commerce in woody plants and wood products. This species already has been intercepted in North America, and is the subject of ongoing eradication efforts in several countries in Europe. Attractants such as pheromones would be immediately useful as baits in traps for its detection. Because long-range pheromones are frequently conserved among closely related species of cerambycids, we evaluated two components of the volatile pheromone produced by males of the congener A. glabripennis (Motschulsky), 4-(n-heptyloxy)butan-1-ol and 4-(n-heptyloxy)butanal, as potential pheromones of A. chinensis. Both compounds subsequently were detected in headspace volatiles from male A. chinensis, but not in volatiles from females. Only 4-(n-heptyloxy)butanol elicited responses from beetle antennae in coupled gas chromatography-electroantennogram analyses, and this compound attracted adult A. chinensis of both sexes in field bioassays. These data suggest that 4-(n-heptyloxy)butan-1-ol is an important component of the male-produced attractant pheromone of A. chinensis, which should find immediate use in quarantine monitoring for this pest.     

Hormonal modulation of reproduction and fertility signaling in polistine wasps

In social insects, it has been suggested that reproduction and the production of particular fertility-linked cuticular hydrocarbons (CHC) may be under shared juvenile hormone (JH) control, and this could have been key in predisposing such cues to later evolve into full-fledged queen pheromone signals. However, to date, only few studies have experimentally tested this “hormonal pleiotropy” hypothesis. Here, we formally test this hypothesis using data from four species of Polistine wasps, Polistes dominula, Polistes satan, Mischocyttarus metathoracicus, and Mischocyttarus cassununga, and experimental treatments with JH using the JH analogue methoprene and the anti-JH precocene. In line with reproduction being under JH control, our results show that across these four species, precocene significantly decreased ovary development when compared with both the acetone solvent-only control and the methoprene treatment. Consistent with the hormonal pleiotropy hypothesis, these effects on reproduction were further matched by subtle shifts in the CHC profiles, with univariate analyses showing that in P. dominula and P. satan the abundance of particular linear alkanes and mono-methylated alkanes were affected by ovary development and our hormonal treatments. The results indicate that in primitively eusocial wasps, and particularly in Polistes, reproduction and the production of some CHC cues are under joint JH control. We suggest that pleiotropic links between reproduction and the production of such hydrocarbon cues have been key enablers for the origin of true fertility and queen signals in more derived, advanced eusocial insects.     

The role of cuticular hydrocarbons in male attraction and repulsion by female Dawson's burrowing bee, Amegilla dawsoni

Cuticular hydrocarbons (CHCs) are long-chain fatty acids and their derivatives that protect insects from desiccation. They can also be important semiochemicals in insect reproduction. We used behavioural and chemical assays to examine the potential role of CHCs in sexual communication in a solitary burrowing bee, Amegilla dawsoni. Washing CHC blends from the cuticle of emerging virgin females made them unattractive to mate-searching males. Returning the CHC blends restored their attractiveness. Nesting females were unattractive to mate-searching males, whether they were washed or not. Chemical analysis identified significant differences between male and female CHC blends and between virgin female and nesting female blends. Some of these differences were due to specific compounds. Loss of attractiveness is unlikely to be due to antiaphrodisiac compounds delivered by males, because male-specific compounds were not found on nesting females, and because recently mated females with intact CHC blends were attractive to searching males. Nesting females could not be made attractive to searching males by removing their CHC blends. Adding virgin female CHC blends tended to improve attractiveness but the effect was weak, suggesting that some form of volatile compound may also be involved in signalling unreceptivity.