Evolutionary genomics of host plant adaptation: insights from Drosophila.

Variation in gene expression in response to the use of alternate host plants can reveal genetic and physiological mechanisms explaining why insect-host relationships vary from host specialism to generalism. Interpreting transcriptome variation relies on well-annotated genomes, making drosophilids valuable model systems, particularly those species with tractable ecological associations. Patterns of whole genome expression and alternate gene splicing in response to growth on different hosts have revealed expression of gene networks of known detoxification genes as well as novel functionally enriched genes of diverse metabolic and structural functions. Integrating trancriptomic responses with fitness differences and levels of phenotypic plasticity in response to alternate hosts will help to reveal the general nature of genotype-phenotype relationships.

Effects of temperature on transcriptome and cuticular hydrocarbon expression in ecologically differentiated populations of desert Drosophila.

We assessed the effects of temperature differences on gene expression using whole-transcriptome microarrays and cuticular hydrocarbon variation in populations of cactophilic Drosophila mojavensis. Four populations from Baja California and mainland Mexico and Arizona were each reared on two different host cacti, reared to sexual maturity on laboratory media, and adults were exposed for 12 hr to 15, 25, or 35°C. Temperature differences influenced the expression of 3,294 genes, while population differences and host plants affected >2,400 each in adult flies. Enriched, functionally related groups of genes whose expression changed at high temperatures included heat response genes, as well as genes affecting chromatin structure. Gene expression differences between mainland and peninsular populations included genes involved in metabolism of secondary compounds, mitochondrial activity, and tRNA synthases. Flies reared on the ancestral host plant, pitaya agria cactus, showed upregulation of genes involved in metabolism, while flies reared on organ pipe cactus had higher expression of DNA repair and chromatin remodeling genes. Population × environment (G × E) interactions had widespread effects on the transcriptome where population × temperature interactions affected the expression of >5,000 orthologs, and there were >4,000 orthologs that showed temperature × host plant interactions. Adults exposed to 35°C had lower amounts of most cuticular hydrocarbons than those exposed to 15 or 25°C, including abundant unsaturated alkadienes. For insects adapted to different host plants and climatic regimes, our results suggest that temperature shifts associated with climate change have large and significant effects on transcriptomes of genetically differentiated natural populations.

Preadult life history variation determines adult transcriptome expression.

Preadult determinants of adult fitness and behaviour have been documented in a variety of organisms with complex life cycles, but little is known about expression patterns of genes underlying these adult traits. We explored the effects of differences in egg-to-adult development time on adult transcriptome and cuticular hydrocarbon variation in order to understand the nature of the genetic correlation between preadult development time and premating isolation between populations of Drosophila mojavensis reared in different host cactus environments. Transcriptome variation was analysed separately in flies reared on each host and revealed that hundreds of genes in adults were differentially expressed (FDR P < 0.05) due to development time differences. For flies reared on pitaya agria cactus, longer preadult development times caused increased expression of genes in adults enriched for ribosome production, protein metabolism, chromatin remodelling and regulation of alternate splicing and transcription. Baja California flies reared on organ pipe cactus showed fewer differentially expressed genes in adults due to longer preadult development time, but these were enriched for ATP synthesis and the TCA cycle. Mainland flies reared on organ pipe cactus with shorter development times showed increased transcription of genes enriched for mitochondria and energy production, protein synthesis and glucose metabolism: adults with longer development times had increased expression of genes enriched for adult life span, cuticle proteins and ion binding, although most differentially expressed genes were unannotated. Differences due to population, sex, mating status and their interactions were also assessed. Adult cuticular hydrocarbon profiles also showed shifts due to egg-to-adult development time and were influenced by population and mating status. These results help to explain why preadult life history variation determines subsequent expression of the adult transcriptome along with traits involved with reproductive isolation and revealed previously undocumented connections between genetic and environmental influences over the entire life cycle in this desert insect.

Genome-wide tests for introgression between cactophilic Drosophila implicate a role of inversions during speciation.

Models of speciation-with-gene-flow have shown that the reduction in recombination between alternative chromosome arrangements can facilitate the fixation of locally adaptive genes in the face of gene flow and contribute to speciation. However, it has proven frustratingly difficult to show empirically that inversions have reduced gene flow and arose during or shortly after the onset of species divergence rather than represent ancestral polymorphisms. Here, we present an analysis of whole genome data from a pair of cactophilic fruit flies, Drosophila mojavensis and D. arizonae, which are reproductively isolated in the wild and differ by several large inversions on three chromosomes. We found an increase in divergence at rearranged compared to colinear chromosomes. Using the density of divergent sites in short sequence blocks we fit a series of explicit models of species divergence in which gene flow is restricted to an initial period after divergence and may differ between colinear and rearranged parts of the genome. These analyses show that D. mojavensis and D. arizonae have experienced postdivergence gene flow that ceased around 270 KY ago and was significantly reduced in chromosomes with fixed inversions. Moreover, we show that these inversions most likely originated around the time of species divergence which is compatible with theoretical models that posit a role of inversions in speciation with gene flow.

Deciphering life history transcriptomes in different environments.

We compared whole transcriptome variation in six pre-adult stages and seven adult female ages in two populations of cactophilic Drosophila mojavensis reared on two host plants to understand how differences in gene expression influence standing life history variation. We used singular value decomposition (SVD) to identify dominant trajectories of life cycle gene expression variation, performed pairwise comparisons of stage and age differences in gene expression across the life cycle, identified when genes exhibited maximum levels of life cycle gene expression, and assessed population and host cactus effects on gene expression. Life cycle SVD analysis returned four significant components of transcriptional variation, revealing functional enrichment of genes responsible for growth, metabolic function, sensory perception, neural function, translation and ageing. Host cactus effects on female gene expression revealed population- and stage-specific differences, including significant host plant effects on larval metabolism and development, as well as adult neurotransmitter binding and courtship behaviour gene expression levels. In 3- to 6-day-old virgin females, significant upregulation of genes associated with meiosis and oogenesis was accompanied by downregulation of genes associated with somatic maintenance, evidence for a life history trade-off. The transcriptome of D. mojavensis reared in natural environments throughout its life cycle revealed core developmental transitions and genome-wide influences on life history variation in natural populations.

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.

No boundaries: genomes, organisms, and ecological interactions responsible for divergence and reproductive isolation.

Revealing the genetic basis of traits that cause reproductive isolation, particularly premating or sexual isolation, usually involves the same challenges as most attempts at genotype-phenotype mapping and so requires knowledge of how these traits are expressed in different individuals, populations, and environments, particularly under natural conditions. Genetic dissection of speciation phenotypes thus requires understanding of the internal and external contexts in which underlying genetic elements are expressed. Gene expression is a product of complex interacting factors internal and external to the organism including developmental programs, the genetic background including nuclear-cytotype interactions, epistatic relationships, interactions among individuals or social effects, stochasticity, and prevailing variation in ecological conditions. Understanding of genomic divergence associated with reproductive isolation will be facilitated by functional expression analysis of annotated genomes in organisms with well-studied evolutionary histories, phylogenetic affinities, and known patterns of ecological variation throughout their life cycles. I review progress and prospects for understanding the pervasive role of host plant use on genetic and phenotypic expression of reproductive isolating mechanisms in cactophilic Drosophila mojavensis and suggest how this system can be used as a model for revealing the genetic basis for species formation in organisms where speciation phenotypes are under the joint influences of genetic and environmental factors.

Sex-specific triacylglycerides are widely conserved in Drosophila and mediate mating behavior.

Pheromones play an important role in the behavior, ecology, and evolution of many organisms. The structure of many insect pheromones typically consists of a hydrocarbon backbone, occasionally modified with various functional oxygen groups. Here we show that sex-specific triacylclyerides (TAGs) are broadly conserved across the subgenus Drosophila in 11 species and represent a novel class of pheromones that has been largely overlooked. In desert-adapted drosophilids, 13 different TAGs are secreted exclusively by males from the ejaculatory bulb, transferred to females during mating, and function synergistically to inhibit courtship from other males. Sex-specific TAGs are comprised of at least one short branched tiglic acid and a long linear fatty acyl component, an unusual structural motif that has not been reported before in other natural products. The diversification of chemical cues used by desert-adapted Drosophila as pheromones may be related to their specialized diet of fermenting cacti.

Cuticular hydrocarbons of Drosophila montana: geographic variation, sexual dimorphism and potential roles as pheromones.

Sexual selection within populations can play an important role in speciation when divergence in mating signals and their corresponding preferences occur along different coevolutionary trajectories in different populations. In insects, one potential target of sexual selection is the blend of cuticular hydrocarbons (CHCs), which often show intra- and interspecific variation, sexual dimorphism and may act as pheromones. In Drosophila montana, a cold-adapted, circumboreal member of the Drosophila virilis species group, flies from different populations have been found to show significant premating isolation as well as variation in male mating signal (song) and female preference. While the role of male courtship song in mate choice has been studied extensively, CHCs in this species have received little attention. In this study, we identified most of the CHCs found on the cuticle of D. montana and characterized population divergence and sexual dimorphism of CHC profiles among flies established from three natural populations - one European and two North American. We also studied their potential role as pheromones by analyzing CHCs of flies used in female-choice mating experiments. We report significant population×sex effects on CHC profiles, as well as significant relationships between some CHC principal components and particular mating behaviours, such as female attractiveness and male mating success, providing evidence that CHCs may play a role in mate choice in this species. The study also provides evidence for variation in the degree to which CHCs play a role in chemical communication among these populations, which may have an influence on the speciation process itself, and could be due to variation in interactions with other closely-related species that occur sympatrically with D. montana in some, but not other, parts of its distribution.

Transcriptome-wide expression variation associated with environmental plasticity and mating success in cactophilic Drosophila mojavensis.

Ecological speciation occurs with the adaptation of populations to different environments and concurrent evolution of reproductive isolation. Phenotypic plasticity might influence both ecological adaptation and reproductive traits. We examined environment-specific gene expression and male mating success in cactophilic Drosophila mojavensis using transcriptome sequencing. This species exhibits cactus-dependent mating success across different species of host plants, with genotype-by-environment interactions for numerous traits. We cultured flies from egg to eclosion on two natural cactus hosts and surveyed gene expression in adult males that were either successful or unsuccessful in achieving copulation in courtship trials. We identified gene expression differences that included functions involved with metabolism, most likely related to chemical differences between host cactus species. Several epigenetic-related functions were identified that might play a role in modulating gene expression in adults due to host cactus effects on larvae, and mating success. Cactus-dependent mating success involved expression differences of genes implicated in translation, transcription, and nervous system development. This suggests a role of neurological function genes in the mating success of D. mojavensis males. Together, these results suggest that the influence of environmental variation on mating success via regulation of gene expression might be an important aspect of ecological speciation.

Functional genomic and phenotypic responses to desiccation in natural populations of a desert drosophilid.

We used whole-transcriptome microarrays to assess changes in gene expression and monitored mortality rates and epicuticular hydrocarbons (CHCs) in response to desiccation stress in four natural populations of Drosophila mojavensis from Baja California and mainland Mexico. Desiccation had the greatest effect on gene expression, followed by biogeographical variation at regional and population levels. Genes involved in environmental sensing and cuticular structure were up-regulated in dry conditions, while genes involved in transcription itself were down-regulated. Flies from Baja California had higher expression of reproductive and mitochondrial genes, suggesting that these populations have greater fecundity and higher metabolic rates. Host plant differences had a surprisingly minor effect on the transcriptome. In most cases, desiccation-caused mortality was greater in flies reared on fermenting cactus tissues than that on laboratory media. Water content of adult females and males was significantly different and was lower in Baja California males. Different groups of CHCs simultaneously increased and decreased in amounts due to desiccation exposure of 9 and 18 h and were population-specific and dependent on larval rearing substrates. Overall, we observed that changes in gene expression involved a coordinated response of behavioural, cuticular and metabolic genes. Together with differential expression of cuticular hydrocarbons, this study revealed some of the mechanisms that have allowed D. mojavensis to exploit its harsh desert conditions. Certainly, for D. mojavensis that uses different host plants, population-level understanding of responses to stressors associated with future climate change in desert regions must be evaluated across geographical and local ecological scales.

Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group.

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group.

Model-based comparisons of phylogeographic scenarios resolve the intraspecific divergence of cactophilic Drosophila mojavensis.

The cactophilic fly Drosophila mojavensis exhibits considerable intraspecific genetic structure across allopatric geographic regions and shows associations with different host cactus species across its range. The divergence between these populations has been studied for more than 60years, yet their exact historical relationships have not been resolved. We analysed sequence data from 15 intronic X-linked loci across populations from Baja California, mainland Sonora-Arizona and Mojave Desert regions under an isolation-with-migration model to assess multiple scenarios of divergence. We also compared the results with a pre-existing sequence data set of eight autosomal loci. We derived a population tree with Baja California placed at its base and link their isolation to Pleistocene climatic oscillations. Our estimates suggest the Baja California population diverged from an ancestral Mojave Desert/mainland Sonora-Arizona group around 230,000-270,000years ago, while the split between the Mojave Desert and mainland Sonora-Arizona populations occurred one glacial cycle later, 117,000-135,000years ago. Although we found these three populations to be effectively allopatric, model ranking could not rule out the possibility of a low level of gene flow between two of them. Finally, the Mojave Desert population showed a small effective population size, consistent with a historical population bottleneck. We show that model-based inference from multiple loci can provide accurate information on the historical relationships of closely related groups allowing us to set into historical context a classic system of incipient ecological speciation.

Variations on a theme: diversification of cuticular hydrocarbons in a clade of cactophilic Drosophila.

BACKGROUND: We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the Drosophila buzzatii cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the D. buzzatii species cluster in order to assess the concordance of CHC differentiation with species divergence. RESULTS: Thirty-six CHC components were scored in single fly extracts with carbon chain lengths ranging from C29 to C39, including methyl-branched alkanes, n-alkenes, and alkadienes. Multivariate analysis of variance revealed that CHC amounts were significantly different among all species and canonical discriminant function (CDF) analysis resolved all species into distinct, non-overlapping groups. Significant intraspecific variation was found in different populations of D. serido suggesting that this taxon is comprised of at least two species. We summarized CHC variation using CDF analysis and mapped the first five CHC canonical variates (CVs) onto an independently derived period (per) gene + chromosome inversion + mtDNA COI gene for each sex. We found that the COI sequences were not phylogenetically informative due to introgression between some species, so only per + inversion data were used. Positive phylogenetic signal was observed mainly for CV1 when parsimony methods and the test for serial independence (TFSI) were used. These results changed when no outgroup species were included in the analysis and phylogenetic signal was then observed for female CV3 and/or CV4 and male CV4 and CV5. Finally, removal of divergent populations of D. serido significantly increased the amount of phylogenetic signal as up to four out of five CVs then displayed positive phylogenetic signal. CONCLUSIONS: CHCs were conserved among species while quantitative differences in CHC profiles between populations and species were statistically significant. Most CHCs were species-, population-, and sex-specific. Mapping CHCs onto an independently derived phylogeny revealed that a significant portion of CHC variation was explained by species' systematic affinities indicating phylogenetic conservatism in the evolution of these hydrocarbon arrays, presumptive waterproofing compounds and courtship signals as in many other drosophilid species.

Male-specific transfer and fine scale spatial differences of newly identified cuticular hydrocarbons and triacylglycerides in a Drosophila species pair.

We analyzed epicuticular hydrocarbon variation in geographically isolated populations of D. mojavensis cultured on different rearing substrates and a sibling species, D. arizonae, with ultraviolet laser desorption/ionization mass spectrometry (UV-LDI MS). Different body parts, i.e. legs, proboscis, and abdomens, of both species showed qualitatively similar hydrocarbon profiles consisting mainly of long-chain monoenes, dienes, trienes, and tetraenes. However, D. arizonae had higher amounts of most hydrocarbons than D. mojavensis and females of both species exhibited greater hydrocarbon amounts than males. Hydrocarbon profiles of D. mojavensis populations were significantly influenced by sex and rearing substrates, and differed between body parts. Lab food-reared flies had lower amounts of most hydrocarbons than flies reared on fermenting cactus substrates. We discovered 48 male- and species-specific hydrocarbons ranging in size from C(22) to C(50) in the male anogenital region of both species, most not described before. These included several oxygen-containing hydrocarbons in addition to high intensity signals corresponding to putative triacylglycerides, amounts of which were influenced by larval rearing substrates. Some of these compounds were transferred to female cuticles in high amounts during copulation. This is the first study showing that triacylglycerides may be a separate class of courtship-related signaling molecules in drosophilids. This study also extends the kind and number of epicuticular hydrocarbons in these species and emphasizes the role of larval ecology in influencing amounts of these compounds, many of which mediate courtship success within and between species.

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 F(1) , F(2) , 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 F(2) 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.

Species hybrids in the laboratory but not in nature: a reanalysis of premating isolation between Drosophila arizonae and D. mojavensis.

Understanding speciation relies critically on the identification of mechanisms responsible for maintaining species integrity (i.e., reproductive isolation) especially when closely related species are sympatric in nature. Studies of reproductive isolation in Drosophila often involve laboratory mating experiments that assume that patterns of mate choice in the laboratory are similar to those in the wild. Two sibling species, Drosophila arizonae and D. mojavensis, known to exhibit low levels of interspecific hybridization in the laboratory, but not in nature, were used in multiple-choice mating trials using various mating chamber designs as well as synthetic and natural media for developing larvae and courting adults. Sympatric populations were more sexually isolated than allopatric ones, consistent with past studies, and all experimental variables tested (chamber size, host plant presence and rearing substrates) had significant effects on levels of premating isolation between these species. Flies reared on cactus showed increased premating isolation versus those reared on synthetic laboratory food as did providing fermenting host plant tissue during mating trials. Also, surprisingly, smaller mating chambers led to an increase in premating isolation versus larger containers. The design of these types of mating trials is thus critical to understanding how mating behaviors in the laboratory are related to those in natural populations.

Rapid response to perturbation of chromosome frequencies in natural populations of Drosophila robusta.

Perturbation of gene or chromosome frequencies in natural populations is one of the most powerful ways of demonstrating whether natural selection maintains genetic polymorphism or if other evolutionary forces are at work. Gene arrangement frequencies in two natural populations of Drosophila robusta were perturbed multiple times by releasing adult flies with contrasting karyotypes and carefully monitoring post-perturbation presence of hybrids and chromosome frequencies. In all cases, frequencies quickly returned to pre-perturbation levels, and in the following sampling periods, no evidence of the introduced chromosomes was apparent. Analysis of post-perturbation frequency changes included tests for heterogeneity among chromosome arrangements in rates of return to equilibrium values using population admixture analysis. In several cases, significant heterogeneity was detected indicating some form of natural selection was operating. Technical challenges to carrying out perturbation experiments in the wild are also discussed.

Genetics of incipient speciation in Drosophila mojavensis: II. Host plants and mating status influence cuticular hydrocarbon QTL expression and G x E interactions.

We performed a quantitative trait locus (QTL) analysis of epicuticular hydrocarbon variation in 1650 F(2) males from crosses of Baja California and mainland Mexico populations of Drosophila mojavensis cultured on two major host cacti. Principal component (PC) analysis revealed five PCs that accounted for 82% of the total epicuticular hydrocarbon variation. Courtship trials with mainland females were used to characterize hydrocarbon profiles of mated and unmated F(2) males, and logistic regression analysis showed that cactus substrates, two PCs, and a PC by cactus interaction were associated with mating success. Multiple QTLs were detected for each hydrocarbon PC and seven G x E (cactus) interactions were uncovered for the X, second, and fourth chromosomes. Males from the courtship trials and virgins were used, so "exposure to females" was included as a factor in QTL analyses. "Exposed" males expressed significantly different hydrocarbon profiles than virgins for most QTLs, particularly for the two PCs associated with mating success. Ten QTLs showed G x E (exposure) interactions with most resulting from mainland genotypes expressing altered hydrocarbon amounts when exposed to females compared to Baja genotypes. Many cactus x exposure interaction terms detected across QTL and all PCs confirmed that organ pipe-reared males expressed significantly lower hydrocarbon amounts when exposed to females than when reared on agria cactus. Epicuticular hydrocarbon variation in D. mojavensis is therefore a multigenic trait with some epistasis, multiple QTLs exhibited pleiotropy, correlated groups of hydrocarbons and cactus substrates determined courtship success, and males altered their hydrocarbon profiles in response to females.