An assessment of sperm survival in Drosophila melanogaster

Recently published evidence based on cytological staining indicates that sperm die rapidly after being stored in female Drosophila melanogaster. However, measuring sperm death in this way has a potential artifact: the death of sperm owing to the extraction, mounting, and staining of sperm. Here we use a protocol that bypasses all of these potential extraneous mortality factors to test the hypothesis that there is high mortality of stored sperm in D. melanogaster. Contrary to the findings from cytological staining, our data indicates that mortality of stored sperm is quite low.     

Patterns of sperm precedence are not affected by female mating history in Drosophila melanogaster

In promiscuously mating species, there is strong selection on males to maximize their share of paternity through both defensive and offensive means. This has been most extensively examined using the Drosophila melanogaster model system. In these studies, sperm competition has been examined by mating a virgin female to two consecutive males and then determining the fertilization success of both the first male (defending, P1) and the second male (offending, P2). Recent evidence suggests that male defense may be influenced by female mating history (i.e., virgin versus nonvirgin). Here, by mating females to males with three different genotypes, we show that female mating history does not affect male defensive or offensive abilities in sperm competition. We also show that, although female lifetime fecundity was not correlated with the number of times that she mated, it was reduced by increased exposure to males. These data indicate that measures of P1 and P2 previously reported in D. melanogaster may be robust to the specific mating history of the females used in these studies.     

Male genotype affects female longevity in Drosophila melanogaster

Several recent studies suggest that interactions with conspecific males can reduce the longevity of female Drosophila melanogaster or support the idea that male and female fitness components are involved in antagonistic interactions. Here we report that males from third-chromosome isogenic lines demonstrated significant genetic variation in male reproductive performance and in the longevity of their mates. Increased male performance was marginally significantly associated with one measure of increased female survival rate. However, there was no indication of tradeoffs or negative correlations between male reproductive success and female survival. We discuss alternative hypotheses for the cause of the induced variation in female longevity.     

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.     

Postmating reproductive barriers contribute to the incipient sexual isolation of the United States and Caribbean Drosophila melanogaster

The nascent stages of speciation start with the emergence of sexual isolation. Understanding the influence of reproductive barriers in this evolutionary process is an ongoing effort. We present a study of Drosophila melanogaster admixed populations from the southeast United States and the Caribbean islands known to be a secondary contact zone of European‐ and African‐derived populations undergoing incipient sexual isolation. The existence of premating reproductive barriers has been previously established, but these types of barriers are not the only source shaping sexual isolation. To assess the influence of postmating barriers, we investigated putative postmating barriers of female remating and egg‐laying behavior, as well as hatchability of eggs laid and female longevity after mating. In the central region of our putative hybrid zone of American and Caribbean populations, we observed lower hatchability of eggs laid accompanied by increased resistance to harm after mating to less‐related males. These results illustrate that postmating reproductive barriers act alongside premating barriers and genetic admixture such as hybrid incompatibilities and influence early phases of sexual isolation.     

Sexual conflict is not counterbalanced by good genes in the laboratory Drosophila melanogaster model system

Sexual conflict theory is based on the observation that females of many species are harmed through their interactions with males. Direct harm to females, however, can potentially be counterbalanced by indirect genetic benefits, where females make up for a reduction in offspring quantity by an increase in offspring quality through a generic increase in offspring fitness (good genes) and/or one restricted to the context of sexual selection (sexy sons). Here, we quantify the magnitude of the good genes mechanism of indirect benefits in a laboratory-adapted population of Drosophila melanogaster. We find that despite high-standing genetic variance for fitness, females gain at most only a modest benefit through the good genes form of indirect benefits--far too little to counterbalance the direct cost of male-induced harm.     

Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster

Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

Costs and benefits of giant sperm and sperm storage organs in Drosophila melanogaster

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.     

Gene expression changes in male accessory glands during ageing are accompanied by reproductive decline in Drosophila melanogaster

Senescence is accompanied by loss of reproductive functions. Here, we studied reproductive ageing in Drosophila melanogaster males and asked whether the expected decline in male reproductive success is due to diminished functionality of the male accessory gland (AG). The male AG produces the majority of seminal fluid proteins (SFPs) transferred to the female at mating. SFPs induce female postmating changes and are key to male reproductive success. We measured age‐dependent gene expression changes for five representative SFP genes in males from four different age groups ranging from 1 to 6 weeks after eclosion. Simultaneously, we also measured male reproductive success in postmating traits mediated by transfer of these five SFPs. We found a decreased in male SFP gene expression with advancing age and an accompanying decline in male postmating success. Hence, male reproductive senescence is associated with a decline in functionality of the male AG. While overall individual SFP genes decreased in expression, our results point towards the idea that the composition of an ejaculate might change with male age as the rate of change was variable for those five genes.     

Mechanisms underlying the sperm quality advantage in Drosophila melanogaster

Contrary to early predictions of sperm competition theory, postcopulatory sexual selection favoring increased investment per sperm (e.g., sperm size, sperm quality) has been demonstrated in numerous organisms. We empirically demonstrate for Drosophila melanogaster that both sperm quality and sperm quantity independently contribute to competitive male fertilization success. In addition to these independent effects, there was a significant interaction between sperm quality and quantity that suggests an internal positive reinforcement on selection for sperm quality, with selection predicted to intensify as investment per sperm increases and the number of sperm competing declines. The mechanism underlying the sperm quality advantage is elucidated through examination of the relationship between female sperm-storage organ morphology and the differential organization of different length sperm within the organ. Our results exemplify that primary sex cells can bear secondary sexual straits.     

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.     

Selection on the Drosophila seminal fluid protein Acp62F

Sperm competition and sexual conflict are thought to underlie the rapid evolution of reproductive proteins in many taxa. While comparative data are generally consistent with these hypotheses, few manipulative tests have been conducted and those that have provided contradictory results in some cases. Here, we use both comparative and experimental techniques to investigate the evolution of the Drosophila melanogaster seminal fluid protein Acp62F, a protease inhibitor for which extensive functional tests have yielded ambiguous results. Using between‐species sequence comparisons, we show that Acp62F has been subject to recurrent positive selection. In addition, we experimentally evolved populations polymorphic for an Acp62F null allele over eight generations, manipulating the opportunities for natural and sexual selection. We found that the Acp62F null allele increased in frequency in the presence of natural selection, with no effect of sexual selection.     

Paternity costs from polyandry compensated by increased fecundity in the hide beetle

Polyandry-induced sperm competition is assumed to impose costson males through reduced per capita paternity success. In contrast,studies focusing on the consequences of polyandry for femalesreport increased oviposition rates and fertility. For thesespecies, there is potential for the increased female fecundityassociated with polyandry to offset the costs to males of sharedpaternity. We tested this hypothesis by comparing the proportionand number of offspring sired by males mated with monandrousand polyandrous females in the hide beetle, Dermestes maculates,both for males mating with different females and for males rematingwith the same female. In 4 mating treatments, monandrous femalesmated either once or twice with the same male and polyandrousfemales mated either twice with 2 different males or thricewith 2 males (where 1 male mated twice). Polyandrous and twice-matingmonandrous females displayed greater fecundity and fertilitythan singly mating monandrous females. Moreover, males rematedto the same female had greater paternity regardless of whetherthat female mated with another male. In both polyandrous treatments,male mating order did not affect paternity success. Finally,although the proportion of eggs sired decreased if a male matedwith a polyandrous female, multiply mating females or femalesthat remated with a previous mate laid significantly more eggsand thus the actual number of eggs sired was comparable. Thus,males do not necessarily accrue a net fitness loss when matingwith polyandrous females. This may explain the absence of anyobvious defensive paternity-protection traits in hide beetlesand other species.     

Influence of developmental environment on male- and female-mediated sperm precedence in Drosophila melanogaster

Length of the sperm flagellum and of the female's primary sperm-storage organ, the seminal receptacle (SR), exhibit a pattern of rapid correlated evolution in Drosophila and other lineages. Experimental evolution studies with Drosophila melanogaster indicate that these traits have coevolved through sexual selection, with length of the SR representing the proximal basis of female sire discrimination, biasing paternity according to sperm length. Here, we examine the impact of experimentally varying the developmental environment, including larval density and larval and adult nutrition, on sperm length, SR length and on the pattern of sperm precedence. Expression of SR length was far more sensitive to variation among developmental environments than was sperm length. Nevertheless, there was striking co-variation in sperm and SR length. The developmental environment of both females and second males, but not first males, significantly contributed to variation in male competitive fertilization success.     

No evidence that polyandry benefits females in Drosophila melanogaster

Understanding the evolution of polyandry (mating with multiple males) is a major issue in the study of animal breeding systems. We examined the adaptive significance of polyandry in Drosophila melanogaster, a species with well-documented costs of mating in which males generally cannot force copulations. We found no direct fitness advantages of polyandry. Females that mated with multiple males had no greater mean fitness and no different variance in fitness than females that mated repeatedly with the same male. Subcomponents of reproductive success, including fecundity, egg hatch rate, larval viability, and larval development time, also did not differ between polyandrous and monogamous females. Polyandry had no affect on progeny sex ratios, suggesting that polyandry does not function against costly sex-ratio distorters. We also found no evidence that polyandry functions to favor the paternity of males successful in precopulatory sexual selection. Experimentally controlled opportunities for precopulatory sexual selection had no effect on postcopulatory sperm precedence. Although these results were generally negative, they are supported with substantial statistical power and they help narrow the list of evolutionary explanations for polyandry in an important model species.     

No evidence for postcopulatory inbreeding avoidance in Drosophila melanogaster

Selection to avoid inbreeding is predicted to vary across species due to differences in population structure and reproductive biology. Over the past decade, there have been numerous investigations of postcopulatory inbreeding avoidance, a phenomenon that first requires discrimination of mate (or sperm) relatedness and then requires mechanisms of male ejaculate tailoring and/or cryptic female choice to avoid kin. The number of studies that have found a negative association between male-female genetic relatedness and competitive fertilization success is roughly equal to the number of studies that have not found such a relationship. In the former case, the underlying mechanisms are largely unknown. The present study was undertaken to verify and expand upon a previous report of postcopulatory inbreeding avoidance in D. melanogaster, as well as to resolve underlying mechanisms of inbreeding avoidance using transgenic flies that express a sperm head-specific fluorescent tag. However, siblings did not have a lower fertilization success as compared to unrelated males in either the first (P(1) ) or second (P(2) ) mate role in sperm competition with a standard unrelated competitor male in our study population of D. melanogaster. Analyses of mating latency, copulation duration, egg production rate, and remating interval further revealed no evidence for inbreeding avoidance.     

Covariance among premating, post-copulatory and viability fitness components in Drosophila melanogaster and their influence on paternity measurement

In polyandrous mating systems, male fitness depends on success in premating, post-copulatory and offspring viability episodes of selection. We tracked male success across all of these episodes simultaneously, using transgenic Drosophila melanogaster with ubiquitously expressed green fluorescent protein (i.e. GFP) in a series of competitive and noncompetitive matings. This approach permitted us to track paternity-specific viability over all life stages and to distinguish true competitive fertilization success from differential early offspring viability. Relationships between episodes of selection were generally not present when paternity was measured in eggs; however, positive correlations between sperm competitive success and offspring viability became significant when paternity was measured in adult offspring. Additionally, we found a significant male × female interaction on hatching success and a lack of repeatability of offspring viability across a focal male's matings, which may underlay the limited number of correlations found between episodes of selection.     

A species‐specific multigene family mediates differential sperm displacement in Drosophila melanogaster

Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. Here, we compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first‐to‐mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.