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Mate choice is a crucial decision in any animal. In terms of fitness, the best mate is the one that leads to the most abundant and productive offspring. Pairing with a low-quality mate would reduce fitness, generating selection for accurate and subtle mate choice in all animal species. Hence, mate choice is expected to be highly context dependent, and should depend on other potential options. For instance, a medium-quality male can constitute the best option when all other males are in poorer condition, but not when there are better-quality males available. Therefore, animals are predicted to gather information about their social context and adapt their mate choice to it. Here, we report on experiments in which we manipulated the social environment of females of Drosophila melanogaster and found that after encountering a high or a low-quality male, they take more or less time to accept copulation with another male, suggesting that females adapt their mating strategy to their social context. We also report on a similar effect in D. biarmiceps. Thus, male attractiveness appears to depend on the quality of recently met males, suggesting that male attractiveness is subjective, indicating plastic and context dependent mate choice., (© 2024. The Author(s).)

Although the environment is three-dimensional (3-D), humans are able to extract subtle information from two-dimensional (2-D) images, particularly in the domain of sex. However, whether animals with simpler nervous systems are capable of such information extraction remains to be demonstrated, as this ability would suggest a functional generalisation capacity. Here, we performed mate-copying experiments in Drosophila melanogaster using 2-D artificial stimuli. Mate copying occurs when naïve females observe the mating success of potential mates and use that social information to build their own mating preference. By replacing live demonstrations with (i) photos or (ii) simplified images of copulating pairs, we found that even crudely simplified images of sexual intercourse still elicit mate copying, suggesting that Drosophila is able to extract sex-related information even from a degraded image. This new method constitutes a powerful tool to further investigate mate copying in that species and sexual preferences in general., (© 2022. The Author(s).)

Insects hold considerable ecological and agricultural importance making it vital to understand the factors impacting their reproductive output. Environmental stressors are examples of such factors which have a substantial and significant influence on insect reproductive fitness. Insects are also ectothermic and small in size which makes them even more susceptible to environmental stresses. The present study assesses the consequence of desiccation on the mating latency and copulations duration in tropical Drosophila melanogaster. We tested flies for these reproductive behavioral parameters at varying body water levels and with whole metabolome analysis in order to gain a further understanding of the physiological response to desiccation. Our results showed that the duration of desiccation is positively correlated with mating latency and mating failure, while having no influence on the copulation duration. The metabolomic analysis revealed three biological pathways highly affected by desiccation: starch and sucrose metabolism, galactose metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results are consistent with carbohydrate metabolism providing an energy source in desiccated flies and also suggests that the phenylalanine biosynthesis pathway plays a role in the reproductive fitness of the flies. Desiccation is a common issue with smaller insects, like Drosophila and other tropical insects, and our findings indicate that this lack of ambient water can immediately and drastically affect the insect reproductive behaviour, which becomes more crucial because of unpredictable and dynamic weather conditions., Competing Interests: The authors have declared that no competing interests exist.

In Drosophila, long sperm are favoured in sperm competition based on the length of the female's primary sperm storage organ, the seminal receptacle (SR). This sperm-SR interaction, together with a genetic correlation between the traits, suggests that the coevolution of exaggerated sperm and SR lengths may be driven by Fisherian runaway selection. Here, we explore the costs and benefits of long sperm and SR genotypes, both in the sex that carries them and in the sex that does not. We measured male and female fitness in inbred lines of Drosophila melanogaster derived from four populations previously selected for long sperm, short sperm, long SRs or short SRs. We specifically asked: What are the costs and benefits of long sperm in males and long SRs in females? Furthermore, do genotypes that generate long sperm in males or long SRs in females impose a fitness cost on the opposite sex? Answers to these questions will address whether long sperm are an honest indicator of male fitness, male post-copulatory success is associated with male precopulatory success, female choice benefits females or is costly, and intragenomic conflict could influence evolution of these traits. We found that both sexes have increased longevity in long sperm and long SR genotypes. Males, but not females, from long SR lines had higher fecundity. Our results suggest that sperm-SR coevolution is facilitated by both increased viability and indirect benefits of long sperm and SRs in both sexes., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

Drosophila melanogaster males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the yellow gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that yellow influences mating behavior through effects on wing extension, song, and/or courtship vigor. Here, we rule out these explanations, as well as effects on the nervous system more generally, and find instead that the effects of yellow on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of yellow expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior., Competing Interests: JM, DC, IS, DS No competing interests declared, PW Senior editor, eLife, (© 2019, Massey et al.)

The emergence of behavioural isolation between populations under divergent selection can be crucial for ecological speciation, but the mechanisms underlying such isolation are poorly understood. Several experimental evolution studies have shown that positive assortative mating (preference for similar mates) can arise rapidly in Drosophila laboratory populations reared in different stressful conditions, while other studies failed to confirm this effect. Here, we present the results of an evolution experiment in which outbred lines of Drosophila melanogaster were reared for 1-2 years on one of the three different diets (standard, starch based or high salt). We show that nonrandom mating arose in some, but not all lines, and that the manifestations and possible interpretations of this nonrandomness depend strongly on the type of tests used to assess mating preferences. More specifically, multiple-choice four-fly tests revealed positive assortative mating (prevalence of homogamic matings) in some starch-adapted and salt-adapted lines when paired with a control line reared on the standard diet, but competitive three-fly tests rather revealed competitive advantage of control males and females over the flies reared on stressful diets. The results imply that divergent adaptation can result in differences in mating propensity or competitive ability, which, in turn, may either facilitate or hamper speciation depending on the relative frequency of high- vs. low-competition settings in natural habitats of the diverging populations. The results also emphasize the importance of using diverse tests for assessing mating structure in natural and laboratory populations., (© 2018 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2018 European Society For Evolutionary Biology.)

In many animal species, females choose potential mating partners according to their own preferences. Thus, female preference-based mate choice affects intraspecific mating success and prevents interspecific mating. To clarify the neuronal basis of female mate choice, it is essential to identify the important relevant sensory cues. In the fruitfly Drosophila melanogaster, the courtship song of males promotes female sexual receptivity. When wild-type virgin females can freely choose one of two types of courting males (winged or wingless males), they prefer to mate with winged males. Here, we report a crucial sensory cue relevant to this female mate choice. In a female choice test, female receptivity toward winged and wingless males was markedly reduced when females had auditory impairments, although females with visual or olfactory impairments showed normal receptivity similar to wild-type females. However, females with visual impairments did not show clear mate preference toward winged males. Thus, these findings suggest that females utilize visual cues in mate choice between winged and wingless males in Drosophila.

Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

Mutual mate choice occurs when males and females base mating decisions on shared traits. Despite increased awareness, the extent to which mutual choice drives phenotypic change remains poorly understood. When preferences in both sexes target the same traits, it is unclear how evolution will proceed and whether responses to sexual selection from male choice will match or oppose responses to female choice. Answering this question is challenging, as it requires understanding, genetic relationships between the traits targeted by choice, mating success, and, ultimately, fitness for both sexes. Addressing this, we applied artificial selection to the cuticular hydrocarbons of the fly Drosophila serrata that are targeted by mutual choice and tracked evolutionary changes in males and females alongside changes in mating success. After 10 generations, significant trait evolution occurred in both sexes, but intriguingly there were major sex differences in the associated fitness consequences. Sexually selected trait evolution in males led to a genetically based increase in male mating success. By contrast, although trait evolution also occurred in females, there was no change in mating success. Our results suggest that phenotypic sexual selection on females from male choice is environmentally, rather than genetically, generated. Thus, compared with female choice, male choice is at best a weak driver of signal trait evolution in this species. Instead, the evolution of apparent female ornamentation seems more likely due to a correlated response to sexual selection on males and possibly other forms of natural selection., Competing Interests: The authors declare no conflict of interest.

Sexual traits convey information about individual quality to potential mates. Environmental and genetic factors affect sexual trait expression and perception via effects on animal condition and health. High fat diet (HFD) is one environmental factor that adversely affects Drosophila melanogaster health, and its effects on animal health are mediated through conserved metabolic signaling pathways. HFD decreases female attractiveness, resulting in reduced male mating behaviors toward HFD females. HFD also affects the ability of males to judge mate attractiveness and likely alters fly condition and sexual traits to impact mating behavior. Here we show that HFD affects both visual (body size) and non-visual (pheromone profiles) sexual traits, which likely contribute to decreased fly attractiveness. We also demonstrate that adult-specific HFD effects on male mate preference can be rescued by changing metabolic signaling. These results demonstrate that HFD alters Drosophila sexual cues to reflect concurrent effects on condition and that less severe behavioral defects can be reversed by genetic manipulations that rescue fly health. This work expands on current knowledge of the role that metabolic signaling pathways play in linking animal health, sexual traits, and mating behavior, and provides a robust assay in a genetically tractable system to continue examining these processes.

Experimental studies of the evolution of reproductive isolation (RI) in real time are a powerful way in which to reveal fundamental, early processes that initiate divergence. In a classic speciation experiment, populations of Drosophila pseudoobscura were subjected to divergent dietary selection and evolved significant positive assortative mating by diet. More recently, a direct role for the gut microbiome in determining this type of RI in Drosophila melanogaster has been proposed. Manipulation of the diet, and hence the gut microbiome, was reported to result in immediate assortative mating by diet, which could be eliminated by reducing gut microbes using antibiotics and recreated by adding back Lactobacillus plantarum We suggest that the evolutionary significance of this result is unclear. For example, in D. melanogaster , the microbiome is reported as flexible and largely environmentally determined. Therefore, microbiome-mediated RI would be transient and would break down under dietary variation. In the absence of evolutionary coassociation or recurrent exposure between host and microbiome, there are no advantages for the gut bacteria or host in effecting RI. To explore these puzzling effects and their mechanisms further, we repeated the tests for RI associated with diet-specific gut microbiomes in D. melanogaster Despite observing replicable differences in the gut microbiomes of flies maintained on different diets, we found no evidence for diet-associated RI, for any role of gut bacteria, or for L. plantarum specifically. The results suggest that there is no general role for gut bacteria in driving the evolution of RI in this species and resolve an evolutionary riddle., Competing Interests: The authors declare no conflict of interest.

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male-male competition and female choice as discrete processes.

The developmental environment can potentially alter the adult social environment and influence traits targeted by sexual selection such as body size. In this study, we manipulated larval density in male and female Drosophila melanogaster, which results in distinct adult size phenotypes-high (low) densities for small (large) adults-and measured sexual selection in experimental groups consisting of adult males and females from high, low, or a mixture of low and high larval densities. Overall, large adult females (those reared at low larval density) had more matings, more mates and produced more offspring than small females (those reared at high larval density). The number of offspring produced by females was positively associated with their number of mates (i.e. there was a positive female Bateman gradient) in social groups where female size was experimentally varied, likely due to the covariance between female productivity and mating rate. For males, we found evidence that the larval environment affected the relative importance of sexual selection via mate number (Bateman gradients), mate productivity, paternity share, and their covariances. Mate number and mate productivity were significantly reduced for small males in social environments where males were of mixed sizes, versus social environments where all males were small, suggesting that social heterogeneity altered selection on this subset of males. Males are commonly assumed to benefit from mating with large females, but in contrast to expectations we found that in groups where both the male and female size varied, males did not gain more offspring per mating with large females. Collectively, our results indicate sex-specific effects of the developmental environment on the operation of sexual selection, via both the phenotype of individuals, and the phenotype of their competitors and mates.

Symbionts and parasites can manipulate their hosts' reproduction to their own benefit, profoundly influencing patterns of mate choice and evolution of the host population. Wolbachia is one of the most widespread symbionts among arthropods, and one that alters its hosts' reproduction in diverse and dramatic ways. While we are beginning to appreciate how Wolbachia's extreme manipulations of host reproduction can influence species diversification and reproductive isolation, we understand little about how symbionts and Wolbachia, in particular, may affect intrapopulation processes of mate choice. We hypothesized that the maternally transmitted Wolbachia would increase the attractiveness of its female hosts to further its own spread. We therefore tested the effects of Wolbachia removal and microbiome disruption on female attractiveness and male mate choice among ten isofemale lines of Drosophila melanogaster. We found variable effects of general microbiome disruption on female attractiveness, with indications that bacteria interact with hosts in a line-specific manner to affect female attractiveness. However, we found no evidence that Wolbachia influence female attractiveness or male mate choice among these lines. Although the endosymbiont Wolbachia can greatly alter the reproduction of their hosts in many species, there is no indication that they alter mate choice behaviours in D. melanogaster., (© 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.)

The mating success of larger male Drosophila melanogaster in the laboratory and the wild has been traditionally been explained by female choice, even though the reasons are generally hard to reconcile. Female choice can explain this success by virtue of females taking less time to mate with preferred males, but so can the more aggressive or persistent courtships efforts of large males. Since mating is a negotiation between the two sexes, the behaviors of both are likely to interact and influence mating outcomes. Using a series of assays, we explored these negotiations by testing for the relative influence of male behaviors and its effect on influencing female courtship arousal threshold, which is the time taken for females to accept copulation. Our results show that large males indeed have higher copulation success compared to smaller males. Competition between two males or an increasing number of males had no influence on female sexual arousal threshold;-females therefore may have a relatively fixed 'arousal threshold' that must be reached before they are ready to mate, and larger males appear to be able to manipulate this threshold sooner. On the other hand, the females' physiological and behavioral state drastically influences mating; once females have crossed the courtship arousal threshold they take less time to mate and mate indiscriminately with large and small males. Mating quicker with larger males may be misconstrued to be due to female choice; our results suggest that the mating advantage of larger males may be more a result of heightened male activity and relatively less of female choice. Body size per se may not be a trait under selection by female choice, but size likely amplifies male activity and signal outputs in courtship, allowing them to influence female arousal threshold faster.

Oviposition is a female-specific behavior that directly affects fecundity, and therefore fitness. If a fertilized female encounters another male that she has evaluated to be of better quality than her previous mate, it would be beneficial for her to remate with this male rather than depositing her eggs. Females who decided not to remate exhibited rejection behavior toward a courting male and engaged in oviposition. Although recent studies of Drosophila melanogaster identified sensory neurons and putative second-order ascending interneurons that mediate uterine afferents affecting female reproductive behavior, little is known about the brain circuitry that selectively activates rejection versus oviposition behaviors. We identified the sexually dimorphic pC2l and female-specific pMN2 neurons, two distinct classes of doublesex (dsx)-expressing neurons that can initiate ovipositor extension associated with rejection and oviposition behavior, respectively. pC2l interneurons, which induce ovipositor extrusion for rejection in females, have homologues that control courtship behavior in males. Activation of these two classes of neurons appears to be mutually exclusive and each governs hierarchical control of the motor program in the VNC either for rejection or oviposition, contributing centrally to the switching on or off of the alternative motor programs.

Drosophila melanogaster females commonly mate with multiple males establishing the opportunity for pre- and postcopulatory sexual selection. Traits impacting sexual selection can be affected by a complex interplay of the genotypes of the competing males, the genotype of the female, and compatibilities between the males and females. We scored males from 96 2nd and 94 3rd chromosome substitution lines for traits affecting reproductive success when mated with females from 3 different genetic backgrounds. The traits included male-induced female refractoriness, male remating ability, the proportion of offspring sired under competitive conditions and male-induced female fecundity. We observed significant effects of male line, female genetic background, and strong male by female interactions. Some males appeared to be "generalists" and performed consistently across the different females; other males appeared to be "specialists" and performed very well with a particular female and poorly with others. "Specialist" males did not, however, prefer to court those females with whom they had the highest reproductive fitness. Using 143 polymorphisms in male reproductive genes, we mapped several genes that had consistent effects across the different females including a derived, high fitness allele in Acp26Aa that may be the target of adaptive evolution. We also identified a polymorphism upstream of PebII that may interact with the female genetic background to affect male-induced refractoriness to remating. These results suggest that natural variation in PebII might contribute to the observed male-female interactions., (© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sexual selection is responsible for the evolution of male ornaments and armaments, but its role in the evolution of cognition--the ability to process, retain and use information--is largely unexplored. Because successful courtship is likely to involve processing information in complex, competitive sexual environments, we hypothesized that sexual selection contributes to the evolution and maintenance of cognitive abilities in males. To test this, we removed mate choice and mate competition from experimental populations of Drosophila melanogaster by enforcing monogamy for over 100 generations. Males evolved under monogamy became less proficient than polygamous control males at relatively complex cognitive tasks. When faced with one receptive and several unreceptive females, polygamous males quickly focused on receptive females, whereas monogamous males continued to direct substantial courtship effort towards unreceptive females. As a result, monogamous males were less successful in this complex setting, despite being as quick to mate as their polygamous counterparts with only one receptive female. This diminished ability to use past information was not limited to the courtship context: monogamous males (but not females) also showed reduced aversive olfactory learning ability. Our results provide direct experimental evidence that the intensity of sexual selection is an important factor in the evolution of male cognitive ability.

How mating preferences evolve remains one of the major unsolved mysteries in evolutionary biology. One major impediment to the study of ornament-preference coevolution is that many aspects of the theoretical literature remain loosely connected to empirical data. Theoretical models typically streamline mating preferences by describing preference functions with a single parameter, a modeling convenience that may veil important aspects of preference evolution. Here, we use a high-throughput behavioral assay in Drosophila melanogaster to quantify attractiveness and multiple components of preferences in both males and females. Females varied genetically with respect to how they ranked males in terms of attractiveness as well as the extent to which they discriminated among different males. Conversely, males showed consistent preferences for females, suggesting that D. melanogaster males tend to rank different female phenotypes in the same order in terms of attractiveness. Moreover, we reveal a heretofore undocumented positive genetic correlation between male attractiveness and female choosiness, which is a measure of the variability in a female's response to different male phenotypes. This genetic correlation sets the stage for female choosiness to evolve via a correlated response to selection on male traits and potentially adds a new dimension to the Fisherian sexual selection process.

Indirect genetic effects (IGEs) describe how an individual's behaviour-which is influenced by his or her genotype-can affect the behaviours of interacting individuals. IGE research has focused on dyads. However, insights from social networks research, and other studies of group behaviour, suggest that dyadic interactions are affected by the behaviour of other individuals in the group. To extend IGE inferences to groups of three or more, IGEs must be considered from a group perspective. Here, I introduce the 'focal interaction' approach to study IGEs in groups. I illustrate the utility of this approach by studying aggression among natural genotypes of Drosophila melanogaster. I chose two natural genotypes as 'focal interactants': the behavioural interaction between them was the 'focal interaction'. One male from each focal interactant genotype was present in every group, and I varied the genotype of the third male-the 'treatment male'. Genetic variation in the treatment male's aggressive behaviour influenced the focal interaction, demonstrating that IGEs in groups are not a straightforward extension of IGEs measured in dyads. Further, the focal interaction influenced male mating success, illustrating the role of IGEs in behavioural evolution. These results represent the first manipulative evidence for IGEs at the group level.

Studies of mating preferences have largely neglected the potential effects of individuals encountering their previous mates ('directly sexually familiar'), or new mates that share similarities to previous mates, e.g. from the same family and/or environment ('phenotypically sexually familiar'). Here, we show that male and female Drosophila melanogaster respond to the direct and phenotypic sexual familiarity of potential mates in fundamentally different ways. We exposed a single focal male or female to two potential partners. In the first experiment, one potential partner was novel (not previously encountered) and one was directly familiar (their previous mate); in the second experiment, one potential partner was novel (unrelated, and from a different environment from the previous mate) and one was phenotypically familiar (from the same family and rearing environment as the previous mate). We found that males preferentially courted novel females over directly or phenotypically familiar females. By contrast, females displayed a weak preference for directly and phenotypically familiar males over novel males. Sex-specific responses to the familiarity of potential mates were significantly weaker or absent in Orco(1) mutants, which lack a co-receptor essential for olfaction, indicating a role for olfactory cues in mate choice over novelty. Collectively, our results show that direct and phenotypic sexual familiarity is detected through olfactory cues and play an important role in sex-specific sexual behaviour.

How females store and use sperm after remating can generate postcopulatory sexual selection on male ejaculate traits. Variation in ejaculate performance traits generally is thought to be intrinsic to males but is likely to interact with the environment in which sperm compete (e.g., the female reproductive tract). Our understanding of female contributions to competitive fertilization success is limited, however, in part because of the challenges involved in observing events within the reproductive tract of internally fertilizing species while discriminating among sperm from competing males. Here, we used females from crosses among isogenic lines of Drosophila melanogaster, each mated to two genetically standardized males (the first with green- and the second with red-tagged sperm heads) to demonstrate heritable variation in female remating interval, progeny production rate, sperm-storage organ morphology, and a number of sperm performance, storage, and handling traits. We then used multivariate analyses to examine relationships between this female-mediated variation and competitive paternity. In particular, the timing of female ejection of excess second-male and displaced first-male sperm was genetically variable and, by terminating the process of sperm displacement, significantly influenced the relative numbers of sperm from each male competing for fertilization, and consequently biased paternity. Our results demonstrate that females do not simply provide a static arena for sperm competition but rather play an active and pivotal role in postcopulatory processes. Resolving the adaptive significance of genetic variation in female-mediated mechanisms of sperm handling is critical for understanding sexual selection, sexual conflict, and the coevolution of male and female reproductive traits.

Male mate choice has been reported in the fruit fly, Drosophila melanogaster, even though males of this species were previously thought to maximise their fitness by mating with all available females. To understand the evolution of male mate choice it is important to understand variation in male mating preferences. Two studies, using different stock populations and different methods, have reported contrasting patterns of variation in male mate choice in D. melanogaster. Two possible explanations are that there are evolved differences in each stock population or that the methods used to measure choice could have biased the results. We investigated these hypotheses here by repeating the methods used in one study in which variable male mate choice was found, using the stock population from the other study in which choice was not variable. The results showed a significant resource-independent male preference for less fecund, smaller females, which contrasts with previous observations of male mate choice. This indicates that different selection pressures between populations have resulted in evolved differences in the expression of male mate choice. It also reveals phenotypic plasticity in male mate choice in response to cues encountered in each choice environment. The results highlight the importance of variation in male mate choice, and of identifying mechanisms in order to understand the evolution of mate choice under varying ecological conditions.

We are unique in reporting a repetition of Bateman [Bateman AJ (1948) Heredity (Edinb) 2:349-368] using his methods of parentage assignment, which linked sex differences in variance of reproductive success and variance in number of mates in small populations of Drosophila melanogaster. Using offspring phenotypes, we inferred who mated with whom and assigned offspring to parents. Like Bateman, we cultured adults expressing dramatic phenotypes, so that each adult was heterozygous-dominant at its unique marker locus but had only wild-type alleles at all other subjects' marker loci. Assuming no viability effects of parental markers on offspring, the frequencies of parental phenotypes in offspring follow mendelian expectations: one-quarter will be double-mutants who inherit the dominant gene from each parent, the offspring from which Bateman counted the number of mates per breeder; half of the offspring must be single mutants inheriting the dominant gene of one parent and the wild-type allele of the other parent; and one-quarter would inherit neither of their parent's marker mutations. Here we show that inviability of double-mutant offspring biased inferences of mate number and number of offspring on which rest inferences of sex differences in fitness variances. Bateman's method overestimated subjects with zero mates, underestimated subjects with one or more mates, and produced systematically biased estimates of offspring number by sex. Bateman's methodology mismeasured fitness variances that are the key variables of sexual selection.

Courtship behaviour involves a complex exchange of signals and responses. These are usually studied at the phenotypic level, and genetic or transcriptional responses to courtship are still poorly understood. Here, we examine the gene-expression changes in Drosophila melanogaster females in response to one of the key male courtship signals in mate recognition, song produced by male wing vibration. Using long oligonucleotide microarrays, we identified several genes that responded differentially to the presence or absence of acoustic courtship stimulus. These changes were modest in both the number of genes involved and fold-changes, but notably dominated by antennal signalling genes involved in olfaction as well as neuropeptides and immune response genes. Second, we compared the expression patterns of females stimulated with synthetic song typical of either conspecific or heterospecific (Drosophila simulans) males. In this case, antennal olfactory signalling and innate immunity genes were also enriched among the differentially expressed genes. We confirmed and investigated the time course of expression differences of two identified immunity genes using real-time quantitative PCR. Our results provide novel insight into specific molecular changes in females in response to courtship song stimulation. These may be involved in both signal perception and interpretation and some may anticipate molecular interactions that occur between the sexes after mating.

When the cost of reproduction for males and variance in female quality are high, males are predicted to show adaptive mate choice. Using Drosophila melanogaster, we test this prediction and show that sperm limited males preferentially mated with young and/or well fed females. The preferred females had higher reproductive output--direct evidence of adaptive precopulatory male mate choice. Our most striking finding is the strong positive correlation between the degree of mating bias showed by the males and the variance in the fitness of the females. We discuss the possible mechanism for such adaptive male mate choice and propose that such choice has important consequences with respect to the existing understanding of the mating system and the evolution of aging.

Stronger selection on males has the potential to lower the deleterious mutation load of females, reducing the cost of sex. However, few studies have directly quantified the strength of selection for both sexes. As the magnitude of inbreeding depression (ID) is related to the strength of selection, we measured the cost of inbreeding for both males and females in a laboratory population of Drosophila melanogaster. Using a novel technique for inbreeding, we found significant ID for both juvenile viability and adult fitness in both sexes. The genetic variation responsible for this depression in fitness appeared to be recessive for adult fitness (h=0.11) and partially additive for juvenile viability (h=0.29). ID was identical across the sexes in terms of juvenile viability but was significantly more deleterious for males than females as adults, even though female X-chromosome homogamety should predispose them to a higher inbreeding load. We estimated the strength of selection on adult males to be 1.24 greater than on adult females, and this appears to be a consequence of selection arising from competition for mates. Combined with the generally positive intersexual genetic correlation for inbred lines, our results suggest that the mutation load of sexual females could be meaningfully reduced by stronger selection acting on males.

Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.

Development of mating preference is considered to be an early event in speciation. In this study, mating preference was achieved by dividing a population of Drosophila melanogaster and rearing one part on a molasses medium and the other on a starch medium. When the isolated populations were mixed, "molasses flies" preferred to mate with other molasses flies and "starch flies" preferred to mate with other starch flies. The mating preference appeared after only one generation and was maintained for at least 37 generations. Antibiotic treatment abolished mating preference, suggesting that the fly microbiota was responsible for the phenomenon. This was confirmed by infection experiments with microbiota obtained from the fly media (before antibiotic treatment) as well as with a mixed culture of Lactobacillus species and a pure culture of Lactobacillus plantarum isolated from starch flies. Analytical data suggest that symbiotic bacteria can influence mating preference by changing the levels of cuticular hydrocarbon sex pheromones. The results are discussed within the framework of the hologenome theory of evolution.

In animal populations, sib mating is often the primary source of inbreeding depression (ID). We used recently wild-caught Drosophila melanogaster to test whether such ID is amplified by environmental stress and, in males, by sexual selection. We also investigated whether increased ID because of stress (increased larval competition) persisted beyond the stressed stage and whether the effects of stress and sexual selection interacted. Sib mating resulted in substantial cumulative fitness losses (egg to adult reproduction) of 50% (benign) and 73% (stressed). Stress increased ID during the larval period (23% vs. 63%), but not during post-stress reproductive stages (36% vs. 31%), indicating larval stress may have purged some adult genetic load (although ID was uncorrelated across stages). Sexual selection exacerbated inbreeding depression, with inbred male offspring suffering a higher reproductive cost than females, independent of stress (57% vs. 14% benign, 49% vs. 11% stress)., (© 2010 The Authors. Journal Compilation © 2010 European Society For Evolutionary Biology.)

Variation in female choice for mates has implications for the maintenance of genetic variation and the evolution of male traits. Yet, estimates of population-level variation in male mating success owing to female genotype are rare. Here, we used a panel of recombinant inbred lines to estimate the strength of selection at many genetic loci in a single generation and attempt to assess differences between females with respect to the males they mated with. We performed selection assays in a complex environment to allow differences in habitat or social group preference to be expressed. We detected directional selection at loci across the genome, but are unable to provide support for differential male success because of variation in female genotype.

Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila melanogaster, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. Here we provide direct evidence that a single compound is used to communicate female identity among D. melanogaster, and to define a reproductive isolation barrier between D. melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes, specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe(-)) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe(-) males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe(-) females compared to wild-type females. This difference was eliminated when oe(-) females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe(-) D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe(-) females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons.

Explanations for the maintenance of variation in reproductive traits influenced by seminal fluid accessory gland proteins (Acps) in male Drosophila melanogaster include nontransitivity in the outcome of sperm competition and/or condition dependence of the traits involved. We investigated the effects of adult male nutrition (five diets) on the expression of Acp- and sperm- mediated traits. We found novel, nonlinear effects, with females showing lower levels of refractoriness to remating after mating with males held on the lowest and highest yeast diets. There were no significant effects of adult male nutrition on male paternity share, but there was a striking, nonlinear effect on second male progeny production, with males kept on intermediate yeast diets fathering the highest number of offspring. Such "bell shaped" responses of life-history traits to nutrition have only previously been reported for longevity. Consistent with previous reports, males maintained on low protein diets had lower premating success and gained fewer rematings with nonvirgins. We show novel and body size independent effects of adult male nutrition on traits influenced by Acps and sperm, which do not fit current condition-dependent handicap models and can affect the strength of sexual selection acting upon such fitness-related traits.