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.     

Sperm competition and the evolution of male reproductive anatomy in rodents

Sperm competition is a pervasive selective force in evolution, shaping reproductive anatomy, physiology and behaviour. Here, we present comparative evidence that varying sperm competition levels account for variation in the male reproductive anatomy of rodents, the largest and most diverse mammalian order. We focus on the sperm-producing testes and the accessory reproductive glands, which produce the seminal fluid fraction of the ejaculate. We demonstrate a positive association between relative testis size and the prevalence of within-litter multiple paternity, consistent with previous analyses in which relative testis size has been found to correlate with sperm competition levels inferred from social organization and mating systems. We further demonstrate an association between sperm competition level and the relative size of at least two accessory reproductive glands: the seminal vesicles and anterior prostate. The size of the major product of these glands-the copulatory plug-is also found to vary with sperm competition level. Our findings thus suggest that selection for larger plugs under sperm competition may explain variation in accessory gland size, and highlight the need to consider both sperm and non-sperm components of the male ejaculate in the context of post-copulatory sexual selection.     

Males' evolutionary responses to experimental removal of sexual selection

We evaluated the influence of pre- and post-copulatory sexual selection upon male reproductive traits in a naturally promiscuous species, Drosophila melanogaster. Sexual selection was removed in two replicate populations through enforced monogamous mating with random mate assignment or retained in polyandrous controls. Monogamous mating eliminates all opportunities for mate competition, mate discrimination, sperm competition, cryptic female choice and, hence, sexual conflict. Levels of divergence between lines in sperm production and male fitness traits were quantified after 38-81 generations of selection. Three a priori predictions were tested: (i) male investment in spermatogenesis will be lower in monogamy-line males due to the absence of sperm competition selection, (ii) due to the evolution of increased male benevolence, the fitness of females paired with monogamy-line males will be higher than that of females paired with control-line males, and (iii) monogamy-line males will exhibit decreased competitive reproductive success relative to control-line males. The first two predictions were supported, whereas the third prediction was not. Monogamy males evolved a smaller body size and the size of their testes and the number of sperm within the testes were disproportionately further reduced. In contrast, the fitness of monogamous males (and their mates) was greater when reproducing in a non-competitive context: females mated once with monogamous males produced offspring at a faster rate and produced a greater total number of surviving progeny than did females mated to control males. The results indicate that sexual selection favours the production of increased numbers of sperm in D. melanogaster and that sexual selection favours some male traits conferring a direct cost to the fecundity of females.     

Adaptation to experimental alterations of the operational sex ratio in populations of Drosophila melanogaster

Theory predicts that males adapt to sperm competition by increasing their investment in testis mass to transfer larger ejaculates. Experimental and comparative data support this prediction. Nevertheless, the relative importance of sperm competition in testis size evolution remains elusive, because experiments vary only sperm competition whereas comparative approaches confound it with other variables, in particular male mating rate. We addressed the relative importance of sperm competition and male mating rate by taking an experimental evolution approach. We subjected populations of Drosophila melanogaster to sex ratios of 1:1, 4:1, and 10:1 (female:male). Female bias decreased sperm competition but increased male mating rate and sperm depletion. After 28 generations of evolution, males from the 10:1 treatment had larger testes than males from other treatments. Thus, testis size evolved in response to mating rate and sperm depletion, not sperm competition. Furthermore, our experiment demonstrated that drift associated with sex ratio distortion limits adaptation; testis size only evolved in populations in which the effect of sex ratio bias on the effective population size had been compensated by increasing the numerical size. We discuss these results with respect to reproductive evolution, genetic drift in natural and experimental populations, and consequences of natural sex ratio distortion.     

Female social preference for males that have evolved via monogamy: evidence of a trade-off between pre- and post-copulatory sexually selected traits?

When females mate with multiple males both pre- and post-copulatory sexual selections occur. It has been suggested that females benefit from polyandry when better-quality males are successful in sperm competition and sire high-quality offspring. Indeed, studies of experimental evolution have confirmed that sperm competition selects for both increased ejaculate quality and elevated offspring viability. Fewer investigations have explored whether these fitness benefits are evident beyond early life-history stages. Here, I used house mice (Mus domesticus) from selection lines that had been evolving for 25 generations under either polygamy or monogamy to test whether females preferred males from lines that had evolved with sperm competition. Males from the polygamous lines had previously been shown to achieve a fitness advantage under semi-natural conditions, deeming them to be of high genetic quality and leading to the a priori expectation that females would prefer males that had evolved with sperm competition compared with males that had not. Contrary to expectation, the data showed that sexually receptive females spent more time associating with males from the monogamous lines. This unexpected but interesting result is discussed in relation to sperm competition theory that predicts a trade-off between male investment in pre- and post-copulatory sexually selected traits.     

Sperm competition and the coevolution of pre‐ and postcopulatory traits: Weapons evolve faster than testes among onthophagine dung beetles

Reproductive competition generates episodes of both pre‐ and postcopulatory sexual selection. Theoretical models of sperm competition predict that as the fitness gains from expenditure on the weapons of male combat increase, males should increase their expenditure on weapons and decrease their expenditure on traits that contribute to competitive fertilization success. Although traits subject to sexual selection are known to have accelerated evolutionary rates of phenotypic divergence, it is not known whether the competing demands of investment into pre‐ and postcopulatory traits affect their relative rates of evolutionary divergence. We use a comparative approach to estimate the rates of divergence in pre‐ and postcopulatory traits among onthophagine dung beetles. Weapons evolved faster than body size while testes mass and sperm length evolved more slowly than body size, suggesting that precopulatory competition is the stronger episode of sexual selection acting on these beetles. Although horns evolved faster than testes, evolutionary increases in horn length were not associated with evolutionary reductions in testes mass. Our data for onthophagines support the notion that in taxa where males are unable to monopolize paternity, expenditure on both weapons and testes should both be favored.     

Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles

Sexual selection is thought to favor the evolution of secondary sexual traits in males that contribute to mating success. In species where females mate with more than one male, sexual selection also continues after copulation in the form of sperm competition and cryptic female choice. Theory suggests that sperm competition should favor traits such as testes size and sperm production that increase a male's competitive fertilization success. Studies of experimental evolution offer a powerful approach for assessing evolutionary responses to variation in sexual selection pressures. Here we removed sexual selection by enforcing monogamy on replicate lines of a naturally polygamous horned beetle, Onthophagus taurus, and monitoring male investment in their testes for 21 generations. Testes size decreased in monogamous lines relative to lines in which sexual selection was allowed to continue. Differences in testes size were dependent on selection history and not breeding regime. Males from polygamous lines also had a competitive fertilization advantage when in sperm competition with males from monogamous lines. Females from polygamous lines produced sons in better condition, and those from monogamous lines increased their sons condition by mating polygamously. Rather than being costly for females, multiple mating appears to provide females with direct and/or indirect benefits. Neither body size nor horn size diverged between our monogamous and polygamous lines. Our data show that sperm competition does drive the evolution of testes size in onthophagine beetles, and provide general support for sperm competition theory.     

EXPERIMENTAL EVOLUTION OF SPERM QUALITY VIA POSTCOPULATORY SEXUAL SELECTION IN HOUSE MICE

Individuals of many species copulate with multiple mates (polygamy). Multiple mating by females (polyandry) promotes sperm competition, which has broad implications for the evolution of the ejaculate. Multigenerational studies of polygamous insects have shown that the removal of sexual selection has profound fitness consequences for females, and can lead to an evolutionary divergence in ejaculate traits. However, the evolutionary implications of polygamous mating across successive generations have not before been demonstrated in a vertebrate. By manipulating the mating system we were able to reinstate postcopulatory sexual selection in a house mouse population that had a long history of enforced monogamy. Following eight generations of selection, we performed sperm quality assays on males from both the polygamous and monogamous selection lines. We applied a principal component analysis to summarize the variation among 12 correlated sperm traits, and found that males evolving under sperm competition had significantly larger scores on the first axis of variation, reflecting greater numbers of epididymal sperm and increased sperm motility, compared to males from lines under relaxed selection. Moreover, we found a correlated response in the size of litters born to females in lines subject to sperm competition. Thus, we present significant evidence that sperm competition has profound fitness consequences for both male and female house mice.     

Postmating sexual selection: the effects of male body size and recovery period on paternity and egg production rate in a water strider

The role of male body size in postmating sexual selection wasexplored in a semiaquatic insect, the water strider Gerris lateralis.To separate effects of male size per se from those due to numericsperm competition, male recovery period (shown here to be proportionalto ejaculate size) was manipulated independently of body sizein a factorial experiment where virgin females were mated firstwith sterile males and then with focal males. Both relativemale fertilization success and female reproductive rate were measured.The number of sperm transferred increased with male recoveryperiod, an effect that was mediated by longer copulation duration,but there were no effects of body size on ejaculate size. Neithermale size nor recovery period had any significant direct effectson male fertilization success. However, copulation durationinfluenced relative fertilization success, suggesting that malesable to transfer more sperm also achieved higher fertilizationsuccess. Females exercised cryptic female choice by modulatingtheir reproductive rate in a manner favoring large males andmales that were successful in terms of achieving high relativefertilization success. Thus, successful males gained a twofoldadvantage in postmating sexual selection. This study has important implicationsfor previous estimates of sexual selection in this group of insectsbecause pre- and postmating sexual selection will be antagonisticdue to limitations in male sperm production: males mating frequently(high mating success) will on average transfer fewer sperm ineach mating and will hence tend to fertilize fewer eggs permating (low fertilization success).     

Correlated responses to selection on female egg size in male reproductive traits in a butterfly

Genetic correlations between male and female traits can act as evolutionary constraints and, if involving reproductive traits, potentially influence sexual selection. Artificial selection on egg size in the tropical butterfly Bicyclus anynana has yielded highly divergent lines. Here we report evidence for correlated evolution in male traits. Males from the large-egg selected lines produced significantly heavier spermatophores independent of body size and tended to have more fertile sperm stored in their reproductive tracts than those from the small-egg selected lines. This may be due to an underlying genetic correlation in reproductive effort between the sexes. However, non-fertile sperm number and testis size remained unaffected by selection on egg size. Phenotypic correlations within an unselected population revealed that spermatophore mass and fertile sperm number, but not testis size and non-fertile sperm number, were positively related to male body size, and that larger spermatophores contained more fertile, but not non-fertile sperm. In addition, males provided larger females with bigger spermatophores and more fertile sperm, indicating males may be exercising mate choice during copulation.     

Bigger testes increase paternity in a simultaneous hermaphrodite,independently of the sperm competition level

Hermaphroditic animals face the fundamental evolutionary optimization problem of allocating their resources to their male vs. female reproductive function (e.g. testes and sperm vs. ovaries and eggs), and this optimal sex allocation can be affected by both pre‐ and post‐copulatory sexual selection. For example, local sperm competition (LSC) – the competition between related sperm for the fertilization of a partner's ova – occurs in small mating groups and can favour a female‐biased sex allocation, because, under LSC, investment into sperm production is predicted to show diminishing fitness returns. Here, we test whether higher testis investment increases an individual's paternity success under sperm competition, and whether the strength of this effect diminishes when LSC is stronger, as predicted by sex allocation theory. We created two subsets of individuals of the simultaneously hermaphroditic flatworm Macrostomum lignano – by sampling worms from either the highest or lowest quartile of the testis investment distribution – and estimated their paternity success in group sizes of either three (strong LSC) or eight individuals (weak LSC). Specifically, using transgenic focal individuals expressing a dominant green‐fluorescent protein marker, we showed that worms with high testis investment sired 22% more offspring relative to those with low investment, corroborating previous findings in M. lignano and other species. However, the strength of this effect was not significantly modulated by the experienced group size, contrasting theoretical expectations of more strongly diminishing fitness returns under strong LSC. We discuss the possible implications for the evolutionary maintenance of hermaphroditism in M. lignano.     

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.     

The influence of mating system on seminal vesicle variability among gobies (Teleostei, Gobiidae)

A variety of sexual selection mechanisms have been implicated to drive the variability of the male reproductive tract in internal fertilizers, while studies on external fertilizers have been largely limited to exploring the influence of sperm competition on testis size and sperm number. Males in the Gobiidae, a speciose teleost family of demersal spawners with external fertilization, are known to be characterized by accessory structures to the sperm duct called seminal vesicles. These seminal vesicles secrete a mucus-enriched seminal fluid. Seminal vesicle size and function have been demonstrated to be influenced by sperm competition at the intraspecific level. With the aim to test the factors influencing the development of these male organs at the interspecific level, an independent contrast analysis was performed on 12 species, differing in mating system type, sperm competition risk, and duration of egg deposition. The type of mating system appears to be the main factor significantly affecting development of seminal vesicles, with males of monogamous species completely lacking or having extremely reduced organs.     

Intrasexual selection and testis size in strepsirhine primates

The main objective of this study was to investigate two predictionsof sexual selection theory concerning interspecific variationin testis size among strepsirhine primates (Lemuriformes andLorisiformes). First, the unique evolutionary history of lemursprovides an opportunity for an independent test of the predictionsof sperm competition theory regarding the relationship betweenmating system and relative testis size. Second, I examined theevolutionary relationship between the morphological correlatesof pre- and postcopulatory competition (i.e., between sexualdimorphism and testis size) because polygamous lemurs, in contrastto other polygamous primates, lack sexual dimorphism. Basedon measurements from 174 captive strepsirhines from 24 species,I found that multi-male species had significantly larger testesthan pair-living ones, but that they did not differ significantlyfrom solitary species. This result deviates from theoreticalexpectations, but may be the result of yet-unknown heterogeneityin mechanisms of male-male competition in both multi-male andsolitary species. There was no difference in relative testissize between nonmonogamous lemurs and lorises, indicating thatpresumably lower levels of precopulatory competition are notnecessarily compensated by more intense sperm competition. Bodysize and phylogenetic effects were also found to considerablyaffect interspecific variability in testis size. Analyses ofindependent contrasts revealed that evolutionary changes inmating system, testis size, sexual size, and canine dimorphismwere not, or only weakly, associated in this monophyletic groupof primates. Additional comprehensive comparative studies ofsexual dimorphism, testis size, mating system, and copulatorybehavior in these and other taxa are indicated to illuminategeneral patterns and causes of covariation among these traits     

Accessory gland size influences time to sexual maturity and mating frequency in the stalk-eyed fly, Cyrtodiopsis dalmanni

Age at first reproduction is an extremely important life-historytrait. Several factors such as nutritional state and age-specificfecundity have been shown to influence time to sexual maturity;however, little work has been done in insects. We addressedthis in a stalk-eyed fly (Cyrtodiopsis dalmanni), by testingthe hypothesis that time to sexual maturity is associated withthe development of male internal reproductive structures. Wefound that sexual maturity was attained after an increased rateof growth in the accessory glands, several days after maturesperm bundles, and motile sperm were observed in the testes.Although testis development is essential, the results suggestthat accessory gland growth is more closely associated withthe time taken to reach sexual maturity than is testis growth.When we manipulated the growth of testes and accessory glandsvia a dietary manipulation, we found that delayed growth ratesincreased the time taken to reach sexual maturity. Among thedelayed individuals, sexually mature males had larger accessoryglands, but not testes, than did immature males. In adult males,mating frequency was significantly positively correlated withaccessory gland size, but not with testis length or body size.We conclude that accessory gland size is a critical determinantof sexual maturity and male mating frequency in this species.     

Larval social cues influence testicular investment in an insect

Socio-sexual environment can have critical impacts on reproduction and survival of animals. Consequently, they need to prepare themselves by allocating more resources to competitive traits that give them advantages in the particular social setting they have been perceiving. Evidence shows that a male usually raises his investment in sperm after he detects the current or future increase of sperm competition because relative sperm numbers can determine his paternity share. This leads to the wide use of testis size as an index of the sperm competition level, yet testis size does not always reflect sperm production. To date, it is not clear whether male animals fine-tune their resource allocation to sperm production and other traits as a response to social cues during their growth and development. Using a polygamous insect Ephestia kuehniella, we tested whether and how larval social environment affected sperm production, testis size, and body weight. We exposed the male larvae to different juvenile socio-sexual cues and measured these traits. We demonstrate that regardless of sex ratio, group-reared males produced more eupyrenes (fertile and nucleate sperm) but smaller testes than singly reared ones, and that body weight and apyrene (infertile and anucleate sperm) numbers remained the same across treatments. We conclude that the presence of larval social, but not sexual cues is responsible for the increase of eupyrene production and decrease of testis size. We suggest that male larvae increase investment in fertile sperm cells and reduce investment in other testicular tissues in the presence of conspecific juvenile cues.     

Rapid evolution of testis size relative to sperm morphology suggests that post‐copulatory selection targets sperm number in Anolis lizards

Post‐copulatory sexual selection is thought to be responsible for much of the extraordinary diversity in sperm morphology across metazoans. However, the extent to which post‐copulatory selection targets sperm morphology versus sperm production is generally unknown. To address this issue, we simultaneously characterized the evolution of sperm morphology (length of the sperm head, midpiece and flagellum) and testis size (a proxy for sperm production) across 26 species of Anolis lizards, a group in which sperm competition is likely. We found that the length of the sperm midpiece has evolved 2–3 times faster than that of the sperm head or flagellum, suggesting that midpiece size may be the most important aspect of sperm morphology with respect to post‐copulatory sexual selection. However, testis size has evolved faster than any aspect of sperm morphology or body size, supporting the hypothesis that post‐copulatory sexual selection acts more strongly upon sperm production than upon sperm morphology. Likewise, evolutionary increases in testis size, which typically indicate increased sperm competition, are not associated with predictable changes in sperm morphology, suggesting that any effects of post‐copulatory selection on sperm morphology are either weak or variable in direction across anoles. Collectively, our results suggest that sperm production is the primary target of post‐copulatory sexual selection in this lineage.     

Male courtship attractiveness and paternity success in Photinus greeni fireflies

Although female mate choice and male sperm competition have separately attracted much attention, few studies have addressed how precopulatory and postcopulatory episodes of sexual selection might interact to drive the evolution of male traits. In Photinus fireflies, females preferentially respond to males based on their bioluminescent courtship signals, and females gain direct benefits through male nuptial gifts acquired during multiple matings over several nights. We experimentally manipulated matings of P. greeni fireflies to test the hypothesis that postcopulatory paternity success might be biased toward males that are more attractive during courtship interactions. We first measured male courtship attractiveness to individual females using field behavioral assays. Females were then assigned to two double-mating treatments: (1) least attractive second male-females were first mated with their most attractive male, followed by their least attractive male, or (2) most attractive second male-females mated with males in reverse order. Larval offspring produced by each female following these double matings were genotyped using random amplified polymorphic DNA (RAPD) markers, and male paternity was determined. Contrary to prediction, firefly males that were more attractive to females based on their bioluminescent courtship displays subsequently showed significantly lower paternity, reflecting possible male trade-offs or sexual conflict. Differences in male paternity were not related to male body condition, testes or accessory gland mass, or to variation in female spermathecal size. Additionally, this study suggests that changes in phenotypic selection gradients may occur during different reproductive stages. These results indicate that it is crucial for future studies on sexual selection in polyandrous species to integrate both precopulatory and postcopulatory episodes to fully understand the evolution of male traits.     

No evidence for a trade‐off between sperm length and male premating weaponry

Male ornaments and armaments that mediate success in mate acquisition and ejaculate traits influencing competitive fertilization success are under intense sexual selection. However, relative investment in these pre‐ and post‐copulatory traits depends on the relative importance of either selection episode and on the energetic costs and fitness gains of investing in these traits. Theoretical and empirical work has improved our understanding of how precopulatory sexual traits and investments in sperm production covary in this context. It has recently also been suggested that male weapon size may trade off with sperm length as another post‐copulatory sexual trait, but the theoretical framework for this suggestion remains unclear. We evaluated the relationship between precopulatory armaments and sperm length, previously reported in ungulates, in five taxa as well as meta‐analytically. Within and between taxa, we found no evidence for a negative or positive relationship between sperm length and male traits that are important in male–male contest competition. It is important to consider pre‐ and post‐copulatory sexual selection together to understand fitness, and to study investments in different reproductive traits jointly rather than separately. A trade‐off between pre‐ and post‐copulatory sexual traits may not manifest itself in sperm length but rather in sperm number or function. Particularly in large‐bodied taxa such as ungulates, sperm number is more variable interspecifically and likely to be under more intense selection than sperm length. We discuss our and the previous results in this context.     

The evolution of sperm morphometry in pheasants

Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.