Adult male nutrition and reproductive success in Drosophila melanogaster

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.     

The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila

During mating, males transfer seminal proteins and peptides, along with sperm, to their mates. In Drosophila melanogaster, seminal proteins made in the male's accessory gland stimulate females' egg production and ovulation, reduce their receptivity to mating, mediate sperm storage, cause part of the survival cost of mating to females, and may protect reproductive tracts or gametes from microbial attack. The physiological functions of these proteins indicate that males provide their mates with molecules that initiate important reproductive responses in females. A new comprehensive EST screen, in conjunction with earlier screens, has identified approximately 90% of the predicted secreted accessory gland proteins (Acps). Most Acps are novel proteins and many appear to be secreted peptides or prohormones. Acps also include modification enzymes such as proteases and their inhibitors, and lipases. An apparent prohormonal Acp, ovulin (Acp26Aa) stimulates ovulation in mated Drosophila females. Another male-derived protein, the large glycoprotein Acp36DE, is needed for sperm storage in the mated female and through this action can also affect sperm precedence, indirectly. A third seminal protein, the protease inhibitor Acp62F, is a candidate for contributing to the survival cost of mating, given its toxicity in ectopic expression assays. That male-derived molecules manipulate females in these ways can result in a molecular conflict between the sexes that can drive the rapid evolution of Acps. Supporting this hypothesis, an unusually high fraction of Acps show signs consistent with their being targets of positive Darwinian selection.     

Associations between sperm competition and natural variation in male reproductive genes on the third chromosome of Drosophila melanogaster

We applied association analysis to elucidate the genetic basis for variation in phenotypes affecting postcopulatory sexual selection in a natural population of Drosophila melanogaster. We scored 96 third chromosome substitution lines for nine phenotypes affecting sperm competitive ability and genotyped them at 72 polymorphisms in 13 male reproductive genes. Significant heterogeneity among lines (P < 0.01) was detected for all phenotypes except male-induced refractoriness (P = 0.053). We identified 24 associations (8 single-marker associations, 12 three-marker haplotype associations, and 4 cases of epistasis revealed by single-marker interactions). Fewer than 9 of these associations are likely to be false positives. Several associations were consistent with previous findings [Acp70A with the male's influence on the female's refractoriness to remating (refractory), Esterase-6 with a male's remating probability (remating) and a measure of female offspring production (fecundity)], but many are novel associations with uncharacterized seminal fluid proteins. Four genes showed evidence for pleiotropic effects [CG6168 with a measure of sperm competition (P2') and refractory, CG14560 with a defensive measure of sperm competition (P1') and a measure of female fecundity, Acp62F with P2' and a measure of female fecundity, and Esterase-6 with remating and a measure of female fecundity]. Our findings provide evidence that pleiotropy and epistasis are important factors in the genetic architecture of male reproductive success and show that haplotype analyses can identify associations missed in the single-marker approach.     

The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster

Drosophila melanogaster male accessory gland proteins (Acps) that are transferred in the ejaculate with sperm mediate post-mating competition for fertilizations between males. The actions of Acps include effects on oviposition and ovulation, receptivity and sperm storage. Two Acps that modulate egg production are Acp26Aa (ovulin) and Acp70A (the sex peptide). Acp26Aa acts specifically on the process of ovulation (the release of mature eggs from the ovaries), which is initiated 1.5 h after mating. In contrast, sperm storage can take as long as 6-9 h to complete. Initial ovulations after matings by virgin females will therefore occur before all sperm are fully stored and the extra eggs initially laid as a result of Acp26Aa transfer are expected to be inefficiently fertilized. Acp26Aa-mediated release of existing eggs should not cause a significant energetic cost or lead to a decrease in female lifespan assuming, as seems likely, that the energetic cost of egg laying comes from de novo egg synthesis (oogenesis) rather than from ovulation. We tested these predictions using Acp26Aa(1) mutant males that lack Acp26Aa but are normal for other Acps and Acp26Aa(2) males that transfer a truncated but fully functional Acp26Aa protein. Females mating with Acp26Aa(2) (truncation) males that received functional Acp26Aa produced significantly more eggs following their first matings than did mates of Acp26Aa(1) (null) males. However, as predicted above, these extra eggs, which were laid as a result of Acp26Aa transfer to virgin females, showed significantly lower egg hatchability. Control experiments indicated that this lower hatchability was due to lower rates of fertilization at early post-mating times. There was no drop in egg hatchability in subsequent non-virgin matings. In addition, as predicted above, females that did or did not receive Acp26Aa did not differ in survival, lifetime fecundity or lifetime progeny, indicating that Acp26Aa transfer does not represent a significant energetic cost for females and does not contribute to the survival cost of mating. Acp26Aa appears to remove a block to oogenesis by causing the clearing out of existing mature eggs and, thus, indirectly allowing oogenesis to be initiated immediately after mating. The results show that subtle processes coordinate the stimulation of egg production and sperm storage in mating pairs.     

Effects of different male remating intervals on the reproductive success of Choristoneura rosaceana males and females

The mass of the spermatophore transferred by a previously mated Choristoneura rosaceana male increases with time elapsed since the last mating but, even after 4 days, it never reaches the mass of the spermatophore of a virgin male. However, spermatophore mass is clearly not a good indicator of the male reproductive investment as the quantity of sperm in the second ejaculate of a previously mated male is the same as that of his first, if he is allowed a 2 (eupyrene sperm) to 3 day (apyrene sperm) recovery period. The interval between the first two matings had no influence on female fecundity or longevity but significantly affected fertility if the male had only 1 day to recover. The length of the post-copulatory refractory period was also shorter in females mated with previously mated males than in those mated with virgins, regardless of the male's remating interval. Furthermore, a significant variation in the eupyrene sperm content of the spermatophore transferred by virgin males had no influence on the length of the female refractory period. Globally, these results support the hypothesis that a factor, other than sperm numbers in the spermatheca, is responsible for maintaining the inhibition of pheromone production in this species.     

Tactic-dependent plasticity in ejaculate traits in the swordtail Xiphophorus nigrensis

In species with alternative reproductive tactics, males that sneak copulations often have larger, higher quality ejaculates relative to males that defend females or nest sites. Ejaculate traits can, however, exhibit substantial phenotypic plasticity depending on a male's mating role in sperm competition, which may depend on the tactic of his competitor. We tested whether exposure to males of different tactics affected sperm number and quality in the swordtail Xipophorus nigrensis, a species with small males that sneak copulations and large males that court females. Sperm swimming speed was higher when the perceived competitor was small than when the competitor was large. Plasticity, however, was only exhibited by small males. Sperm number and viability were invariant between social environments. Our results suggest sperm quality is role-dependent and that plastic responses to the social environment can differ between male reproductive tactics.     

Coevolution of male and female reproductive traits in a simultaneously hermaphroditic land snail

Inter- and intraspecific studies in gonochoristic animals reveal a covariation between sperm characteristics and the size of the female reproductive tract, indicating a rapid evolutionary divergence, which is consistent with the theory of post-copulatory sexual selection. Simultaneous hermaphrodites differ from species with separate sexes (gonochorists) in that they possess both functional male and female reproductive organs at the same time. We investigated whether in hermaphroditic animals intraspecific variation in reproductive traits results from divergent coevolution, by quantifying the variation in male and female traits among six natural populations of the snail Arianta arbustorum and examining the covariation in interacting traits. There was a significant among-population variation in spermatophore volume, number of sperm transferred and sperm length, as well as in volume of the sperm storage organ (spermatheca) and number of tubules, but not in spermatheca length. We found a positive association between sperm number transferred and spermatheca volume. This result suggests that the same post-copulatory mechanisms as in gonochorists drive the correlated evolution of reproductive characters in hermaphrodites.     

Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.     

Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes.     

Context-dependent expression of sperm quality in the fruitfly

In most species, females mate multiply within a reproductive cycle, invoking post-copulatory selection on ejaculatory components. Much research has focused on disentangling the key traits important in deciding the outcomes of sperm competition and investigating patterns of covariance among these traits. Less attention has focused on the degree to which such patterns might be context-dependent. Here, we examine whether the expression of sperm viability—a widely used measure of sperm quality—and patterns of covariance between this trait and male reproductive morphologies, change across distinct age classes and across naturally occurring genotypes, when expressed in both heterozygotic (extreme outbred) and homozygotic (extreme inbred) states in the fruitfly Drosophila melanogaster. Older males, and heterozygous males, generally exhibited higher sperm viability. The male age effect seems at least partly explained by a positive association between sperm numbers and viability. First, old males possessed more stored sperm than young males, and second, sperm numbers and viability were also positively associated within each age class. Furthermore, we found a positive association between sperm viability and testis size, but only among heterozygous, old males. These results suggest that sperm quality is a labile trait, with expression levels that are context-dependent and shaped by multiple, potentially interacting, factors.     

The consequences of genetic variation in sex peptide expression levels for egg laying and retention in females

The accessory gland proteins (Acps) that male Drosophila melanogaster produce and transfer to females during copulation are key to male and female fitness. One Acp, the sex peptide (SP), is largely responsible for a dramatic increase in female egg laying and decrease in female receptivity after copulation. While genetic variation in male SP expression levels correlate with refractory period duration in females, it is unknown whether male SP expression influences female egg laying or if any effect of SP is mediated by SP retention in the female reproductive tract. Here we measured the amount of SP retained in the female reproductive tract after mating and female egg laying after copulating with virgin males. We found no correlation between male SP expression levels and egg laying, or the amount of SP in the female reproductive tract after mating. Additionally, the amount of SP retained in the female did not influence egg laying. These finding suggests that additional factors, such as variation in other Acps, are important for the retention of SP in females and its quantitative effects on egg laying. It also shows that egg laying and refractory period response to SP is at least partially uncoupled.     

The benefits of male ejaculate sex peptide transfer in Drosophila melanogaster

The accessory gland protein (Acp) ejaculate molecules of male Drosophila melanogaster mediate sexual selection and sexual conflict at the molecular level. However, to date no studies have comprehensively measured the timing and magnitude of fitness benefits to males of transferring specific Acps. This is an important omission because without this information it is not possible to fully understand the strength and form of selection acting on adaptations such as Acps. Here, we measured the fitness benefits to males of ejaculate sex peptide (SP) transfer. SP is of interest because it is a candidate for mediating sexual conflict: its frequent receipt reduces female fitness. In single matings with virgin females SP is known to increase egg laying and decrease receptivity. Hence, we predicted that SP could: (i) boost a male’s absolute paternity by increasing offspring production and delaying female remating and/or (ii) boost relative paternity share. We tested these predictions using two different lines of SP‐lacking males, in both two‐mating and free‐mating assay conditions. SP transfer conferred higher absolute, but not relative, male reproductive success. In matings with virgin females, SP transfer increased mating productivity and delayed remating and hence the onset of sperm competition. In already mated females, SP transfer did not elevate absolute progeny production, but did increase intermating intervals and hence the period over which a male could gain paternity. Consistent with this, under free‐mating conditions over an extended period, we detected a ‘per‐mating’ fitness benefit for males transferring SP. These benefits are consistent with a role for SP in mediating conflict, with SP acting to maximize short‐term fitness benefits for males.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster

A crucial factor determining sperm fertilization success in multiply mated Drosophila melanogaster females is the efficiency with which sperm are stored. This process is modulated by the accessory gland protein Acp36DE. In this study, we show that the effect of Acp36DE on sperm storage itself alters the outcome of sperm competition. As second-mating males, Acp36DE1 (null) males had significantly lower P2-values than Acp36DE2 (truncation) or Acp36DE+ (control) males, as might be expected as the null males' sperm are poorly stored. We used spermless males, which are null for Acp36DE, to show that, in the absence of sperm co-transfer, Acp36DE itself could not displace first-male sperm. The results therefore suggest that males null for Acp36DE suffer in sperm displacement because fewer sperm are stored or retained, not because Acp36DE itself displaces sperm. Acp36DE1 (null) males also gained significantly fewer fertilizations than controls when they were the first males to mate. Using spermless males, we also showed that significantly more second-male offspring were produced following the transfer of Acp36DE by spermless first-mating males. This implies that the transfer of Acp36DE itself by the first male facilitated the storage or use of the second male's sperm and that co-transfer with sperm is not necessary for Acp36DE effects on second-male sperm storage. Acp36DE may persist in the reproductive tract and aid the storage of any sperm including those of later-mating males or prime the female for future efficient sperm storage. Our results indicate that mutations in genes that affect sperm storage can drastically affect the outcome of sperm competition.     

Molecular evolution of X-linked accessory gland proteins in Drosophila pseudoobscura

In Drosophila melanogaster and Drosophila simulans, positive Darwinian selection drives high rates of evolution of male reproductive genes, and accessory gland proteins (Acps) in particular. Here, we tested whether 13 X-linked male-specific genes, 4 Acps and 9 non-Acps, are under selective forces in the Drosophila pseudoobscura species group, much as those in the D. melanogaster group. We observed a statistically significant correlation in relative rates of nonsynonymous evolution between the two species groups tested. One Acp examined had a higher rate of nonsynonymous substitution than predicted by a neutral model in both species groups, suggesting its divergence was driven by positive Darwinian selection. To further test for the signature of selection, we examined polymorphism of three Acps within D. pseudoobscura. From this test, no Acp individually bore the signature of positive selection, but the 3 Acps together possessed an excess of nonsynonymous differences between species, relative to polymorphism within species. We conclude that faster evolution of Acps in the D. pseudoobscura group appears to be driven by positive selection, as previously suggested in the D. melanogaster group.     

Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.     

An integrative view of sexual selection in Tribolium flour beetles

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.