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.     

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.     

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.     

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.     

Natural genetic variation in male reproductive genes contributes to nontransitivity of sperm competitive ability in Drosophila melanogaster

Female Drosophila melanogaster frequently mate with multiple males, and the success of a given male depends not only on his genotype but also on the genotype of his competitor. Here, we assess how natural genetic variation affects male–male interactions for traits influencing pre‐ and postcopulatory sexual selection. Males from a set of 66 chromosome substitution lines were competed against each other in a ‘round‐robin’ design, and paternity was scored using bulk genotyping. We observed significant effects of the genotype of the first male to mate, the second male to mate and an interaction between the males for measures of male mating rate and sperm utilization. We also identified specific combinations of males who show nontransitive patterns of reproductive success and engage in ‘rock‐paper‐scissors’ games. We then tested for associations between 245 polymorphisms in 32 candidate male reproductive genes and male reproductive success. We identified eight polymorphisms in six reproductive genes that associate with male reproductive success independent of the competitor (experimentwise P < 0.05). We also identified four SNPs in four different genes where the relative reproductive success of the alternative alleles changes depending on the competing males' genetic background (experimentwise P < 0.05); two of these associations include premature stop codons. This may be the first study that identifies the genes contributing to nontransitivity among males and further highlights that ‘rock‐paper‐scissors’ games could be an important evolutionary force maintaining genetic variation in natural populations.     

Female influence on pre- and post-copulatory sexual selection and its genetic basis in Drosophila melanogaster

Genetic variation among females is likely to influence the outcome of both pre- and post-copulatory sexual selection in Drosophila melanogaster. Here we use association testing to survey natural variation in 10 candidate female genes for their effects on female reproduction. Females from 91 chromosome two substitution lines were scored for phenotypes affecting pre- and post-copulatory sexual selection such as mating and remating rate, propensity to use sperm from the second male to mate, and measures of fertility. There were significant genetic contributions to phenotypic variation for all the traits measured. Resequencing of the 10 candidate genes in the 91 lines yielded 68 non-synonymous polymorphisms which were tested for associations with the measured phenotypes. Twelve significant associations (markerwise P<0.01) were identified. Polymorphisms in the putative serine protease homolog CG9897 and the putative odorant binding protein CG11797 associated with female propensity to remate and met an experimentwise significance of P<0.05. Several other associations, including those impacting both fertility and female remating rate suggest that sperm storage might be an important factor mitigating female influence on sexual selection.     

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.     

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.     

Autosomal variation for male body size and sperm competition phenotypes is uncorrelated in Drosophila melanogaster

Correlations between male body size and phenotypes impacting post-copulatory sexual selection are commonly observed during the manipulation of male body size by environmental rearing conditions. Here, we control for environmental influences and test for genetic correlations between natural variation in male body size and phenotypes affecting post-copulatory sexual selection in Drosophila melanogaster. Dry weights of virgin males from 90 second-chromosome and 88 third-chromosome substitution lines were measured. Highly significant line effects (p<0.001) documented a genetic basis to variation in male body size. No significant correlations were identified between male body size and the components of sperm competitive ability. These results suggest that natural autosomal variation for male body size has little impact on post-copulatory sexual selection. If genetic correlations exist between male body size and post-copulatory sexual selection then variation in the sex chromosomes are likely candidates, as might be expected if sexually antagonistic coevolution was responsible.     

The evolution of sperm and non-sperm producing organs in male Drosophila

The male ejaculate is made up of two components: sperm and non-sperm. There has been little consideration of how these two basic compartments evolve. If they are subject to trade-offs, theory predicts that when the sperm competition raffle is unfair, when seminal fluid proteins stimulate fecundity and/or when ejaculate components alter fertilization success, there will be differential selection on sperm versus non-sperm ejaculate characteristics. However, the fundamental assumption that there are trade-offs between sperm and non-sperm ejaculate compartments in Drosophila has not yet been tested. To address this, we examined testis (sperm producing) and accessory gland (non-sperm producing) size across 22 species of Drosophila . We also examined how these characters varied with copulation duration, which may represent an additional target for sperm competition. The results showed no evidence of a trade-off between testis length and accessory gland length. Copulation duration correlated negatively with accessory gland length and there was a positive correlation with testis length, but only after correcting for body size. Overall, the results suggest no evidence for gross trade-offs in sperm versus non-sperm compartments across these Drosophila species, and motivate more detailed examination of ejaculate investment patterns.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 505–512.     

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.     

Larval competition in Drosophila melanogaster: frequency-dependence of viability

Summary The application of the overfeeding technique (interruption of the competition during larval development) to the study of larval competition in two-strain cultures of Drosophila melanogaster demonstrates the following points: (1) viability is a function of competition time; (2) viability becomes more frequency-dependent as competition time increases; (3) the dynamics of the inner subpopulation (adults that have passed all their development in a crowded condition) and outer subpopulation (adults coming from larvae recovered by interruption of competition) vary with time as regards frequency-dependence; and (4) the wild type strain Oregon is the active agent in competition with the strain cardinal.     

Intraspecific variation in ejaculate traits of the northern watersnake (Nerodia sipedon)

Sperm competition occurs when the sperm of more than one male compete to fertilize the eggs of a female. In reptiles, sperm competition is particularly prevalent and is an important agent of sexual selection in males. Spermatogenesis in reptiles can be energetically expensive, suggesting that there may be costs to producing high-quality ejaculates. The northern watersnake Nerodia sipedon has a mating system characterized by aggregations in which a single female mates with multiple males, resulting in high levels of multiple paternities. Under these circumstances sperm competition is likely important, and selection should favour sperm and ejaculate traits that enhance a male's reproductive success. In this study, we examined intraspecific variation in ejaculate quality (sperm length, motility, sperm density, spermactocrit) in male watersnakes and determined whether ejaculate traits varied with body size and condition, using both size-corrected mass and haematocrit as indices of condition. We found large variation among males in all these traits, except for sperm length. Although there was significant variation in sperm length among males, the majority of variation in sperm length occurred within rather than among individuals. Males with high haematocrit had sperm that were less variable with respect to length, and large males produced ejaculates that were less concentrated with respect to sperm than small males. The lack of condition dependence of most ejaculate traits is consistent with previous studies that indicate that male reproductive effort in this species is generally not energy limited, perhaps because of opportunistic foraging during the mating season.     

Trade-off between pre and post-copulatory traits depends on locomotor activity in male Tribolium castaneum beetles

Locomotor performance is an indicator of dynamic exercise; thus, it is a central trait in many animal behaviours. Although higher locomotor endurance may increase male reproductive success (e.g., in mate searching and male–male contests), investment in other male reproductive traits (e.g., male attractiveness and sperm competition) may be decreased through energy consumption due to higher activity levels. Here, I investigated male attractiveness, mating success, and paternity success using males of the red flour beetle Tribolium castaneum selected for higher (H) and lower (L) locomotor endurance. Although there was no difference in male attractiveness between the selection regimes, H males had significantly higher mating success than L males. Conversely, L males had significantly higher paternity success than H males. Therefore, there was a trade-off between mating success and paternity success among the selection regimes, suggesting that locomotor endurance affects male reproduction in T. castaneum, and individual variation of locomotor endurance may be maintained within a population.     

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.     

Body size of virtual rivals affects ejaculate size in sticklebacks

Sperm competition occurs when sperm of two or more males competeto fertilize a given set of eggs. Theories on sperm competitionexpect males under high risk of sperm competition to increaseejaculate size. Here we confirm this prediction experimentallyin the three-spined stickleback (Gasterosteus aculeatus). Inthis species, sneaking (i.e., stealing of fertilizations byneighboring males) can lead to sperm competition. Sneaking malesinvade foreign nests, and the owners vigorously try to preventthis intrusion. In such fights, male body size is assumed tobe an important predictor of success. Consequently, the riskof sperm competition may depend on the size of a potential competitor.We experimentally confronted males before spawning with eithera large or a small computer-animated rival. We show that malesejaculated significantly more sperm after the presentation ofthe larger virtual rival than after the small stimulus. In addition,the time between the initiation of courting and the spawningwas shorter in the large virtual male treatment. The resultssuggest that stickleback males tailor ejaculate size relativeto the risk of sperm competition perceived by the size of apotential competitor.     

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.     

Fitness consequences of redundant cues of competition in male Drosophila melanogaster

Phenotypic plasticity can allow animals to adapt their behavior, such as their mating effort, to their social and sexual environment. However, this relies on the individual receiving accurate and reliable cues of the environmental conditions. This can be achieved via the receipt of multimodal cues, which may provide redundancy and robustness. Male Drosophila melanogaster detect presence of rivals via combinations of any two or more redundant cue components (sound, smell, and touch) and respond by extending their subsequent mating duration, which is associated with higher reproductive success. Although alternative combinations of cues of rival presence have previously been found to elicit equivalent increases in mating duration and offspring production, their redundancy in securing success under sperm competition has not previously been tested. Here, we explicitly test this by exposing male D. melanogaster to alternative combinations of rival cues, and examine reproductive success in both the presence and absence of sperm competition. The results supported previous findings of redundancy of cues in terms of behavioral responses. However, there was no evidence of reproductive benefits accrued by extending mating duration in response to rivals. The lack of identifiable fitness benefits of longer mating under these conditions, both in the presence and absence of sperm competition, contrasted with some previous results, but could be explained by (a) damage sustained from aggressive interactions with rivals leading to reduced ability to increase ejaculate investment, (b) presence of features of the social environment, such as male and female mating status, that obscured the fitness benefits of longer mating, and (c) decoupling of behavioral investment with fitness benefits.