Sexual selection and sperm quantity: meta‐analyses of strategic ejaculation

Multiple mating or group spawning leads to post‐copulatory sexual selection, which generally favours ejaculates that are more competitive under sperm competition. In four meta‐analyses we quantify the evidence that sperm competition (SC) favours greater sperm number using data from studies of strategic ejaculation. Differential investment into each ejaculate emerges at the individual level if males exhibit phenotypic plasticity in ejaculate properties in response to the likely risk and/or intensity of sperm competition after a given mating. Over the last twenty years, a series of theoretical models have been developed that predict how ejaculate size will be strategically adjusted in relation to: (a) the number of immediate rival males, with a distinction made between 0 versus 1 rival (‘risk’ of SC) and 1 versus several rivals (‘intensity’ of SC); (b) female mating status (virgin or previously mated); and (c) female phenotypic quality (e.g. female size or condition). Some well‐known studies have reported large adjustments in ejaculate size depending on the relevant social context and this has led to widespread acceptance of the claim that strategic sperm allocation occurs in response to each of these factors. It is necessary, however, to test each claim separately because it is easy to overlook studies with weak or negative findings. Compiling information on the variation in outcomes among species is potentially informative about the relevance of these assumptions in different taxa or mating systems. We found strong evidence that, on average, males transfer larger ejaculates to higher quality females. The effect of female mating status was less straightforward and depended on how ejaculate size was measured (i.e. use of proxy or direct measure). There is strong evidence that ejaculate size increased when males were exposed to a single rival, which is often described as a response to the risk of SC. There is, however, no evidence for the general prediction that ejaculate size decreases as the number of rivals increases from one to several males (i.e. in response to a higher intensity of SC which lowers the rate of return per sperm released). Our results highlight how meta‐analysis can reveal unintentional biases in narrative literature reviews. We note that several assumptions of theoretical models can alter an outcome's predicted direction in a given species (e.g. the effect of female mating status depends on whether there is first‐ or last‐male sperm priority). Many studies do not provide this background information and fail to make strong a priori predictions about the expected response of ejaculate size to manipulation of the mating context. Researchers should be explicit about which model they are testing to ensure that future meta‐analyses can better partition studies into different categories, or control for continuous moderator variables.     

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.     

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.     

Indirect genetic effects and sexual conflicts: Partner genotype influences multiple morphological and behavioral reproductive traits in a flatworm

The expression of an individual's phenotypic traits can be influenced by genes expressed in its social partners. Theoretical models predict that such indirect genetic effects (IGEs) on reproductive traits should play an important role in determining the evolutionary outcome of sexual conflict. However, empirical tests of (i) whether reproductive IGEs exist, (ii) how they vary among genotypes, and (iii) whether they are uniform for different types of reproductive traits are largely lacking. We addressed this in a series of experiments in the simultaneously hermaphroditic flatworm Macrostomum lignano. We found strong evidence for IGEs on both morphological and behavioral reproductive traits. Partner genotype had a significant impact on the testis size of focal individuals—varying up to 2.4‐fold—suggesting that IGEs could mediate sexual conflicts that target the male sex function. We also found that time to first copulation was affected by a genotype × genotype interaction between mating partners, and that partner genotype affected the propensity to copulate and perform the postcopulatory suck behavior, which may mediate conflicts over the fate of received ejaculate components. These findings provide clear empirical evidence for IGEs on multiple behavioral and morphological reproductive traits, which suggests that the evolutionary dynamics of these traits could be altered by genes contained in the social environment.     

Ejaculate traits in relation to male body size in the eastern mosquitofish Gambusia holbrooki

In the present study, the correlation between sperm number, sperm quality (speed, viability, longevity and length), sperm bundles quality (size and dissolving rate) and male body size has been tested in the eastern mosquitofish Gambusia holbrooki a poecilid species characterized by coercive mating tactics where males do not possess obvious ornaments, and the body size is the key determinant of pre‐copulatory male mating success. The results do not tally with theoretical predictions. Indeed, no correlation between male body size and either sperm or sperm‐bundle traits has been found, evidencing the lack of the theoretically expected trade‐off between the investment in characters involved in mate acquisition and the investment in ejaculate quality. An explanation for the observed pattern comes from the extremely dynamic mating system of G. holbrooki, characterized by variable size‐related male mating success and strong post‐copulatory selective pressure, with all males facing a similar high level of sperm competition. In this situation, a higher investment in growth and maintenance at the expense of ejaculate quality is not expected. These results underscore the necessity to comprehend detailed information on species’ reproductive biology and reproductive environment to understand both the evolution of ejaculate characteristics and possible deviations from theoretical predictions.     

Impaired sperm quality,delayed mating but no costs for offspring fitness in crickets winning a fight

The outcome of male–male contest competition is known to affect male mating success and is believed to confer fitness benefits to females through preference for dominant males. However, by mating with contest winners, females can incur significant costs spanning from decreased fecundity to negative effects on offspring. Hence, identifying costs and benefits of male dominance on female fitness is crucial to unravel the potential for a conflict of interests between the sexes. Here, we investigated males' pre‐ and post‐copulatory reproductive investment and its effect on female fitness after a single contest a using the field cricket Gryllus bimaculatus. We allowed males to fight and immediately measured their mating behaviour, sperm quality and offspring viability. We found that males experiencing a fight, independently of the outcome, delayed matings, but their courtship effort was not affected. However, winners produced sperm of lower quality (viability) compared to losers and to males that did not experience fighting. Results suggest a trade‐off in resource allocation between pre‐ and post‐mating episodes of sexual selection. Despite lower ejaculate quality, we found no fitness costs (fecundity and viability of offspring) for females mated to winners. Overall, our findings highlight the importance of considering fighting ability when assessing male reproductive success, as winners may be impaired in their competitiveness at a post‐mating level.     

Random mating with respect to mating status in the simultaneously hermaphroditic land snail Arianta arbustorum

In promiscuous species with sperm storage, males are expected to show a preference for mating with virgin and young females to reduce the risk of sperm competition. In the simultaneous hermaphrodite land snail Arianta arbustorum, sperm production precedes egg production by 2–4 weeks, resulting in a short period of protandric hermaphroditism before shell growth is completed. In natural populations, copulating pairs involving individuals which have not yet completed shell growth (virgins) have been observed. We ran a series of mate-choice experiments to examine whether virgin and nonvirgin (experienced) individuals of A. arbustorum discriminate between virgin and nonvirgin mating partners. We also assessed the number of sperm delivered to partners with different mating status. Neither virgin nor nonvirgin snails showed any preference for mating with a virgin partner. In all test situations mating was random and the number of sperm delivered was not adjusted to the mating status of the partner. Mating success was mainly determined by the activity of the individual. The random mating pattern does not imply random fertilization of eggs because the presence of a sperm-digesting organ and the morphology of the sperm storage organ allow a selective storage and use of sperm in A. arbustorum.     

No Plastic Responses to Experimental Manipulation of Sperm Competition per se in a Free‐Living Flatworm

In the absence of sperm competition evolutionary theory predicts low mating rates and low ejaculate expenditure per mating, and sex allocation theory for simultaneous hermaphrodites predicts a strongly female‐biased sex allocation. In the presence of sperm competition a shift towards a more male‐biased sex allocation and a higher ejaculate expenditure are predicted. The free‐living flatworm Macrostomum lignano has been shown to respond plastically in mating rate, testis size, and sperm transfer to manipulation of the social group size, a proxy of the strength of sperm competition. However, manipulation of social group size may manipulate not only sperm competition, but also other factors, such as food supply and metabolite concentration. In this study we therefore manipulated sperm competition per se by repeatedly exposing individuals to partners that have either mated with rivals or not, while keeping the social group size constant. Our results suggest that M. lignano does not have the ability to detect sperm competition per se, as worms experimentally exposed to the presence or absence of sperm competition did not differ in sex allocation, sperm transfer or mating behavior. A response to our manipulation would have required individual recognition, the ability to detect self‐referencing tags, or tags or traces left by rivals on or in the mating partners. We first discuss the possibility that highly efficient sperm displacement may have decreased the difference between the treatment groups and then propose three alternative cues that may allow M. lignano to respond plastically to the social group size manipulation used in earlier studies: assessment of the mating rate, chemical cues, or tactile cues.     

Don't pull the plug! the Drosophila mating plug preserves fertility

ABSTRACT

Mating plugs are hardened structures—typically a coagulation of seminal fluid components—that are transferred to, or formed within, the female reproductive tract of numerous animal species (both mammals and insects). Analysis of the role(s) of the mating plug in reproduction has been conducted in a wide array of diverse species. These structures have been proposed to have a multitude of functions, which include altering female re-mating rate, acting as a barrier to re-mating and being required for sperm storage or sperm movement to occur in mated females. A recent analysis of the Drosophila melanogaster mating plug has shown that proper formation of the structure is required for optimal fertility in flies: the Drosophila mating plug is required to retain the ejaculate within the female reproductive tract once mating has terminated. Here, we discuss the possible implications of the Drosophila mating plug in the fertility of this species in light of these new results.     

Differential sperm expenditure reveals a possible role for post‐copulatory sexual selection in a lekking moth

Male reproductive success in the lesser wax moth Achroia grisella is strongly determined by pre‐copulatory mate choice, during which females choose among males aggregated in small leks based on the attractiveness of ultrasonic songs. Nothing is known about the potential of post‐copulatory mechanisms to affect male reproductive success. However, there is evidence that females at least occasionally remate with a second male and that males are unable to produce ejaculates quickly after a previous copulation. Here we investigated the effects of mating history on ejaculate size and demonstrate that the number of transferred sperm significantly decreased from first (i.e., virgin) to second (i.e., nonvirgin) copulation within individual males. For males of identical age, the number of sperm transferred was higher in virgin than in nonvirgin copulations, too, demonstrating that mating history, is responsible for the decrease in sperm numbers transferred and not the concomitant age difference. Furthermore, the number of transferred sperm was significantly repeatable within males. The demonstrated variation in ejaculate size both between subsequent copulations as well as among individuals suggests that there is allocation of a possibly limited amount of sperm. Because female fecundity is not limited by sperm availability in this system, post‐copulatory mechanisms, in particular sperm competition, may play a previously underappreciated role in the lesser wax moth mating system.     

Condition‐dependent ejaculate production affects male mating behavior in the common bedbug Cimex lectularius

Food availability in the environment is often low and variable, constraining organisms in their resource allocation to different life‐history traits. For example, variation in food availability is likely to induce condition‐dependent investment in reproduction. Further, diet has been shown to affect ejaculate size, composition and quality. How these effects translate into male reproductive success or change male mating behavior is still largely unknown. Here, we concentrated on the effect of meal size on ejaculate production, male reproductive success and mating behavior in the common bedbug Cimex lectularius. We analyzed the production of sperm and seminal fluid within three different feeding regimes in six different populations. Males receiving large meals produced significantly more sperm and seminal fluid than males receiving small meals or no meals at all. While such condition‐dependent ejaculate production did not affect the number of offspring produced after a single mating, food‐restricted males could perform significantly fewer matings than fully fed males. Therefore, in a multiple mating context food‐restricted males paid a fitness cost and might have to adjust their mating strategy according to the ejaculate available to them. Our results indicate that meal size has no direct effect on ejaculate quality, but food availability forces a condition‐dependent mating rate on males. Environmental variation translating into variation in male reproductive traits reveals that natural selection can interact with sexual selection and shape reproductive traits. As males can modulate their ejaculate size depending on the mating situation, future studies are needed to elucidate whether environmental variation affecting the amount of ejaculate available might induce different mating strategies.     

Determinants of male ejaculate investment in the cotton bollworm Helicoverpa armigera: mating history,female body size and male age

Many studies demonstrate that ejaculate size may be influenced by male condition, female quality and the risk or intensity of sperm competition. In the present study, the effect of male and female conditions, male mating history and female mating status on ejaculate sperm numbers in the polyandrous moth Helicoverpa armigera is examined. A large variation in ejaculate size is found and, although female body size and male age influence ejaculate size, female age and copula duration do not. Both male and female mating histories have significant effects on ejaculate sperm numbers. Males reduce ejaculate expenditure in successive matings but deliver significantly more apyrene and eupyrene sperm to nonvirgin than to virgin females.     

Sperm trading and sex roles in the hermaphroditic opisthobranch sea slug Navanax inermis : eager females or opportunistic males?

The sea slug Navanax inermis (Gastropoda, Opisthobranchia) is a well‐known example of a simultaneous hermaphrodite in which mating partners trade sperm. According to previous work by others, sperm trading follows from a general preference for the female role when the expected variance in reproductive success in the male role is higher and the risk of failure therefore larger. However, this view contradicts theoretical and empirical studies of other systems, which predict a general preference for the male role; sperm trading is assumed to follow from the fact that individuals benefit from sperm receipt as a nutritional compensation for sperm investment. In this study, we investigate the behaviour of N. inermis in more detail. In addition to observations of regular pairs, we also paired individuals with a partner that had been isolated for 33 days in order to induce changes in sex‐role preference in the non‐isolated partner. We also collected all clutches produced throughout the study to check for signs of infertility as a consequence of allosperm depletion. Fertility of field‐collected and isolated individuals suggested that sperm depletion occurs under natural conditions and may be caused by a lack of partners. Although this argues in favour of female preference, low mating rates both reduce variance in male reproductive success and remove the intent to use sperm for nutritional purposes, thus eliminating the conditions under which both hypotheses are intended to operate. The observational data indicate that animals are eager to mate as males, particularly at the beginning of a mating session. Intromissions lasted longer when a simultaneous intromission was received from the partner. Increases in intromission were recorded in non‐isolated individuals with partners which were previously isolated and therefore more attractive as females. This response would not have been expected were the female role the preferred one. A summary of the arguments concerning preference for either gender suggests some preference for the male role, but indicates that sexual preferences may actually change or become neutral within an individual, even in the course of a mating session. Overall, our results clearly confirm previously published observations of sperm trading in N. inermis . © 2003 The Linnean Society of London. Biological Journal of the Linnean Society, 2003, 78 , 105?116.     

A double role of sperm in scorpions: The mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel‐like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel‐like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3‐5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Resource availability,mating opportunity and sexual selection intensity influence the expression of male alternative reproductive tactics

The expression of alternative reproductive tactics can be plastic and occur simultaneously depending on cues that vary spatially or temporally. For example, variation in resources and sexual selection intensity is expected to influence the pay‐off of each tactic and shape the decision of which tactic to employ. Males of the nuptial gift‐giving spider Pisaura mirabilis can adopt three tactics: offering a genuine prey gift, a ‘worthless’ non‐nutritious gift or no gift. We hypothesized that resources and/or male body condition, and mating opportunity and sexual selection intensity, vary over the course of the mating season to shape the co‐existence of alternative traits. We measured these variables in the field over two seasons, to investigate the predictions that as the mating season progresses, (i) males become more likely to employ a gift‐giving tactic, and (ii) the likelihood of switching from worthless to genuine gifts increases. Prey availability increased over the season and co‐varied with the propensity of males to employ the gift‐giving tactic, but we found no support for condition‐dependent gift giving. Males responded to an increase in female availability by increasing their mating effort (gift production). Furthermore, the frequency of genuine gift use increased with sexual selection intensity, consistent with the assumption that sperm competition intensity increases with time. Our results suggest that the frequency of alternative tactics is shaped by seasonal changes in ecological factors and sexual selection. This leads to relaxed selection for the gift‐giving tactic early in the season when females are less choosy and resources more scarce, and increased selection for genuine gifts later in the season driven by mating opportunity and risk of sperm competition.     

Opening Pandora's box: comparative studies of genetic mating systems reveal reproductive complexity

Genetic analyses of realized reproductive success have fundamentally changed our understanding of mating behaviour in natural systems. While behavioural ecologists have long been interested in what factors influence mating behaviour, early studies were limited to direct observations of matings and thus provided an incomplete picture of reproductive activity. Genetic assessments of parentage have revolutionized the study of reproductive behaviour, revealing that many individuals engage in extra‐pair copulations ( Griffith et al. 2002 ) and that social mating partners frequently invest substantial resources into raising offspring that are unrelated to one or both of them ( Avise et al. 2002 ). While these findings have changed the way we think about reproductive behaviour, most investigations of genetic parentage have been restricted to single populations at a single point in time, obscuring spatial and/or temporal variation in mating behaviour and limiting our ability to determine how environmental changes can lead to shifts in mating strategies. In this issue of Molecular Ecology, Mobley & Jones (2009) compare genetic mating behaviour across five populations of Syngnathus floridae ( Fig. 1 ), a widespread species of pipefish distributed along the Gulf‐ and Atlantic Coasts of North America. The authors document how genetic mating behaviour varies across space in S. floridae and identify correlations between reproductive variation and particular ecological characteristics. Mobley & Jones’ paper is one of an increasing number of studies which address the question of how ecological variables influence mating behaviour, and highlights how our understanding of mating system variation and evolution is likely to expand through the wider application of high‐throughput parentage assessment in a comparative context.

Figure 1 Open in figure viewer PowerPoint A pregnant male dusky pipefish (Syngnathus floridae) in its natural habitat. Photo credit: Joe O’Hop.     

Male Mating History Influences Female Mate Choice in the Trinidadian Guppy (Poecilia reticulata)

Based on the phenotype‐linked fertility hypothesis, sexual selection should favour females that can accurately assess the recent mating history of available sexual partners and preferentially avoid mating with recently‐mated males [who may be sperm depleted (SD)] so as to minimize the risk of their eggs not being fertilized. This hypothesis has received to date only limited attention and empirical support. Therefore, in the current study, we investigated experimentally whether females of a vertebrate species, the Trinidadian guppy (Poecilia reticulata), are able to assess the recent mating history of males, and thus potentially their functional fertility, and choose to avoid mating with males that appear to have recently mated and who may be sperm limited. Individual virgin females were first given a dichotomous choice between a male that had not been recently observed to interact sexually with another female (i.e. not sperm‐depleted) and another male that had been observed to interact sexually with a female (i.e. potentially sperm‐depleted) as sexual partners. Paired males were matched for body length and coloration. Immediately following this test, the focal females were subjected to a free‐swimming mate‐choice test using the same paired stimulus males. As predicted, on average, female guppies avoided the apparently recently‐mated (and potentially sperm‐depleted) male and exhibited a significant preference for the other male not recently observed mating (and thus not likely sperm limited) during both tests. We do not yet fully understand the underlying mechanisms of this preference. Therefore, further research on the particular cues that females use to assess the recent mating history and fertility status of males is required.     

Multiple mating increases cocoon hatching success in the earthworm Eisenia andrei (Oligochaeta: Lumbricidae)

In simultaneous hermaphrodites with reciprocal mating, multiple mating may be a male strategy that conflicts with female interests, and therefore an intra‐individual sexual conflict regarding the number of matings may be expected. The evolutionary outcome of this sexual conflict will depend on the costs and benefits that extra mating entails for each sexual function. In the present study, we investigated the costs and benefits of multiple mating on cocoon number, cocoon mass, and cocoon hatching success in the redworm Eisenia andrei, a simultaneous hermaphrodite with reciprocal insemination, by manipulating the number of matings with different partners. We did not detect any reduction in the female reproductive output (number and mass of cocoons) with increasing number of mating partners. However, we found that multiple mating showed benefits for female reproduction that increased the hatching success of the cocoons. This effect may be a result of increased quantity and/or diversity of sperm in the spermathecae of multiple mated earthworms. Further studies are required to clarify the mechanism underlying the increased cocoon hatching success when redworms engage in multiple matings. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.