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.     

GENETICS OF INCIPIENT SPECIATION IN DROSOPHILA MOJAVENSIS. III. LIFE‐HISTORY DIVERGENCE IN ALLOPATRY AND REPRODUCTIVE ISOLATION

We carried out a three‐tiered genetic analysis of egg‐to‐adult development time and viability in ancestral and derived populations of cactophilic Drosophila mojavensis to test the hypothesis that evolution of these life‐history characters has shaped premating reproductive isolation in this species. First, a common garden experiment with 11 populations from Baja California and mainland Mexico and Arizona reared on two host species revealed significant host plant X region and population interactions for viability and development time, evidence for host plant adaptation. Second, replicated line crosses with flies reared on both hosts revealed autosomal, X chromosome, cytoplasmic, and autosome X cactus influences on development time. Viability differences were influenced by host plants, autosomal dominance, and X chromosomal effects. Many of the F₁, F₂, and backcross generations showed evidence of heterosis for viability. Third, a QTL analysis of male courtship song and epicuticular hydrocarbon variation based on 1688 Baja × mainland F₂ males also revealed eight QTL influencing development time differences. Mainland alleles at six of these loci were associated with longer development times, consistent with population‐level differences. Eight G × E interactions were also detected caused by longer development times of mainland alleles expressed on a mainland host with smaller differences among Baja genotypes reared on the Baja host plant. Four QTL influenced both development time and epicuticular hydrocarbon differences associated with courtship success, and there was a significant QTL‐based correlation between development time and cuticular hydrocarbon variation. Thus, the regional shifts in life histories that evolved once D. mojavensis invaded mainland Mexico from Baja California by shifting host plants were genetically correlated with variation in cuticular hydrocarbon‐based mate preferences.     

Interaction between temperature and male pheromone in sexual isolation in Drosophila melanogaster

In Drosophila, female hydrocarbons are known to be involved in premating isolation between different species and pheromonal races. The role of male‐specific hydrocarbon polymorphism is not as well documented. The dominant cuticular hydrocarbon (CHC) in male D. melanogaster is usually 7‐tricosene (7‐T), with the exception of African populations, in which 7‐pentacosene (7‐P) is dominant. Here, we took advantage of a population from the Comoro Islands (Com), in which males fell on a continuum of low to high levels of 7‐T, to perform temperature selection and selection on CHCs' profiles. We conducted several experiments on the selected Com males to study the plasticity of their CHCs in response to temperature shift, their role in resistance to desiccation and in sexual selection. We then compared the results obtained for selected lines to those from three common laboratory strains with different and homogenous hydrocarbon profiles: CS, Cot and Tai. Temperature selection modified the CHC profiles of the Com males in few generations of selection. We showed that the 7‐P/7‐T ratio depends on temperature with generally more 7‐P at higher temperatures and observed a relationship between chain length and resistance to desiccation in both temperature‐ and phenotypically selected Com lines. There was partial sexual isolation between the flies with clear‐cut phenotypes within the phenotypically selected lines and the laboratory strains. These results indicate that the dominant male pheromones are under environmental selection and may have played a role in reproductive isolation.     

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle. Lipophorin loads and transports lipids, including hydrocarbons, from one tissue to another. In some insects, the lipophorin receptor (LpR), which binds to lipophorin and accepts its lipid cargo, is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes. However, it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown. We herein present the results of our molecular characterization, phylogenetic analysis, and functional annotation of the pea aphid (Acyrthosiphon pisum) LpR gene (ApLpR). This gene was transcribed throughout the A. pisum life cycle, but especially during the embryonic stage and in the abdominal cuticle. Furthermore, we optimized the RHA interference (RNAi) parameters by determining the ideal dose and duration for gene silencing in the pea aphid. We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity. Additionally, a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress, with decreased survival rates under simulated drought conditions. Moreover, ApLpR expression levels significantly increased in response to the desiccation treatment. These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress. Furthermore, this gene is vital for aphid reproduction. Therefore, the ApLpR gene of A. pisum may be a novel RNAi target relevant for insect pest management.     

Polymorphisms in a desaturase 2 ortholog associate with cuticular hydrocarbon and male mating success variation in a natural population of Drosophila serrata

Elucidating the nature of genetic variation underlying both sexually selected traits and the fitness components of sexual selection is essential to understanding the broader consequences of sexual selection as an evolutionary process. To date, there have been relatively few attempts to connect the genetic variance in sexually selected traits with segregating DNA sequence polymorphisms. We set out to address this in a well‐characterized sexual selection system – the cuticular hydrocarbons (CHCs) of Drosophila serrata – using an indirect association study design that allowed simultaneous estimation of the genetic variance in CHCs, sexual fitness and single nucleotide polymorphism (SNP) effects in an outbred population. We cloned and sequenced an ortholog of the D. melanogaster desaturase 2 gene, previously shown to affect CHC biosynthesis in D. melanogaster, and associated 36 SNPs with minor allele frequencies > 0.02 with variance in CHCs and sexual fitness. Three SNPs had significant multivariate associations with CHC phenotype (q‐value < 0.05). At these loci, minor alleles had multivariate effects on CHCs that were weakly associated with the multivariate direction of sexual selection operating on these traits. Two of these SNPs had pleiotropic associations with male mating success, suggesting these variants may underlie responses to sexual selection due to this locus. There were 15 significant male mating success associations (q‐value < 0.1), and interestingly, we detected a nonrandom pattern in the relationship between allele frequency and direction of effect on male mating success. The minor‐frequency allele usually reduced male mating success, suggesting a positive association between male mating success and total fitness at this locus.     

Replicated evolutionary divergence in the cuticular hydrocarbon profile of male crickets associated with the loss of song in the Hawaiian archipelago

Female choice based on male secondary sexual traits is well documented, although the extent to which this selection can drive an evolutionary divergence in male traits among populations is less clear. Male field crickets Teleogryllus oceanicus attract females using a calling song and once contacted switch to courtship song to persuade them to mate. These crickets also secrete onto their cuticle a cocktail of long‐chained fatty acids or cuticular hydrocarbons (CHCs). Females choose among potential mates based on the structure of male acoustic signals and on the composition of male CHC profiles. Here, we utilize two naturally occurring mutations that have arisen independently on two Hawaiian islands and render the male silent to ask whether the evolutionary loss of acoustic signalling can drive an evolutionary divergence in the alternative signalling modality, male CHC profiles. Q_ST‐F_ST comparisons revealed strong patterns of CHC divergence among three populations of crickets on the islands of Hawaii, Oahu and Kauai. Contrasts between wild‐type and flatwing males on the islands of Oahu and Kauai indicated that variation in male CHC profiles within populations is associated with the loss of acoustic signalling; flatwing males had a relatively low abundance of long‐chained CHCs relative to the short‐chained CHCs that females find attractive. Given their dual functions in desiccation resistance and sexual signalling, insect CHCs may be particularly important traits for reproductive isolation and ultimately speciation.     

Cuticular hydrocarbon profiles of codling moth larvae,Cydia pomonella (Lepidoptera: Tortricidae), reflect those of their host plant species

The cuticular hydrocarbons (CHCs) of codling moth larvae collected in the field from their host plant species, apple and walnut, were analyzed and compared with the CHCs of fruits from these two phylogenetically distant hosts. The CHC profiles of the larvae consisted solely of n‐alkanes (C₂₃–C₃₁) and differed quantitatively between host populations. Amounts of the CHCs from the walnut‐collected larvae were shifted towards longer‐chain alkanes compared to those from apple‐collected larvae. A similar shift was observed for the CHC profiles of walnut and apple fruits. Analysis of the CHCs of larvae reared on artificial diet, in comparison with hydrocarbons from the diet, confirmed that larval CHCs scarcely reflect hydrocarbons from the food source. This finding indicates that the larval hydrocarbons must be biosynthesized to a large degree by the insect, rather than being gained directly from its diet. Hence, codling moth populations from apple and walnut each synthesize their own CHC profiles, which largely resemble those of their respective host plant, yielding a potential tool of chemical camouflage from certain natural antagonists of the larvae. The findings of the present study, together with recent molecular population analyses, provides evidence for a process that might ultimately lead to sympatric speciation of this herbivore species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 376–384.     

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.     

CLINES IN CUTICULAR HYDROCARBONS IN TWO DROSOPHILA SPECIES WITH INDEPENDENT POPULATION HISTORIES

We took a comparative approach utilizing clines to investigate the extent to which natural selection may have shaped population divergence in cuticular hydrocarbons (CHCs) that are also under sexual selection in Drosophila. We detected the presence of CHC clines along a latitudinal gradient on the east coast of Australia in two fly species with independent phylogenetic and population histories, suggesting adaptation to shared abiotic factors. For both species, significant associations were detected between clinal variation in CHCs and temperature variation along the gradient, suggesting temperature maxima as a candidate abiotic factor shaping CHC variation among populations. However, rainfall and humidity correlated with CHC variation to differing extents in the two species, suggesting that response to these abiotic factors may vary in a species‐specific manner. Our results suggest that natural selection, in addition to sexual selection, plays a significant role in structuring among‐population variation in sexually selected traits in Drosophila.     

Sensitivity to larval density in populations of Drosophila mojavensis: Influences of host plant variation on components of fitness

Summary Chromosomally polymorphic populations of Drosophila mojavensis from Baja California feed and breed on agria cactus, Stenocereus gummosus; whereas, monomorphic Arizona populations are associated exclusively with organ pipe cactus, S. thurberi. The effects of this host plant shift in expanding the kinds of feeding and breeding sites were assessed by manipulating larval density and recording differences in egg to adult development time and viability, and adult thorax size in both populations on artificially rotted substrates of both cactus species. Older agria rots increased development time but had no effect on viability. Organ pipe rots were qualitatively poorer substrates than agria rots for both monomorphic and polymorphic populations of D. mojavensis, especially at higher larval densities causing longer egg to adult development times, lower viabilities, and smaller thorax sizes than agria.The Baja population expressed shorter development times, higher viabilities, and smaller thorax sizes than the Arizona population on both cactus substrates. No evidence for cactus host race formation was found. The Baja population was less sensitive to increasing larval densities for all fitness characters studied on both cactus substrates indicating greater developmental homeostasis than in the monomorphic Arizona population. These data support the hypothesized central-marginal population structure within this species coincident with the distribution of host plants and lend insight into the process of adaptive divergence at different life history stages caused by host plant shifts.     

The taste of origin in a lady beetle: do males discriminate between females based on cuticular hydrocarbons?

The Asian lady beetle Harmonia axyridis originates from Asia and has established invasive populations worldwide. Recent population genetic studies trace their invasion routes and demonstrate that bottlenecks in population size have reduced their genetic diversity. Consequently, phenotypical differences are highlighted between native and invasive populations. Among phenotypical traits, cuticular hydrocarbons (CHCs) might reflect the geographical origin of a lady beetle, especially because of their genetic basis. The present study investigates whether (i) the CHC profiles qualitatively and quantitatively differ between females of H. axyridis from native and invasive populations and (ii) males discriminate females from native and invasive populations using CHC profiles. CHCs are solvent‐extracted before being quantified and identified by gas chromatography and mass spectrometry. In total, 17 CHCs are detected from female elytra, including six alkanes, three polyunsaturated and eight monounsaturated alkenes. The total quantity of CHCs differs among the populations, with lady beetles from Tai'an (China) displaying higher CHC concentrations than lady beetles from Gembloux (Belgium) and from Beijing (China) populations. Multivariate analyses detect differences in CHC qualitative profiles, with females from Tai'an being different from the two other populations. Finally, behavioural assays show that females originating from the native Tai'an population are less preferred by males, whereas females from the invasive population are mounted more often. The behavioural assays suggest that CHCs are not involved in discrimination of mating partners based on their origin.