Influence of female reproductive anatomy on the outcome of sperm competition in Drosophila melanogaster

Females as well as males can influence the outcome of sperm competition, and may do so through the anatomy of their reproductive tracts. Female Drosophila melanogaster store sperm in two morphologically distinct organs: a single seminal receptacle and, normally, two spermathecae. These organs have different temporal roles in sperm storage. To examine the association between sperm storage organ morphology and sperm competition, we used a mutant type of female with three spermathecae. Although the common measure of sperm competition, P(2), did not differ between females with two and three spermathecae, the pattern of sperm use over time indicated that female morphology did affect male reproductive success. The rate of offspring production by females with three spermathecae rose and fell more rapidly than by females with two spermathecae. If females remate or die before using up second male sperm, then second male reproductive success will be higher when they mate with females with three spermathecae. The results indicate that temporal patterns of sperm use as well as P(2) should be taken into account when measuring the outcome of sperm competition.     

Broken Copulatory Organs are Low-Cost Adaptations to Sperm Competition in Redback Spiders

In some spiders, a discrete portion of the male's copulatory organ (the apical sclerite) breaks off during copulation and remains in the female's reproductive tract. Apical sclerites may prevent insemination by rivals (sperm competition), stimulate females to favourably bias paternity (cryptic choice) or breakage may reflect sexual conflict over copulation duration with little direct effect on paternity. It has been assumed that any benefits of organ breakage are balanced by a large cost (male sterility) in species where males could otherwise mate multiply, but this has never been experimentally tested. We examined these ideas in the Australian redback spider (Latrodectus hasselti Thorell 1870, Araneae: Theridiidae), a species where males are functionally sterile after one normal mating. We experimentally removed sclerites and found males were able to mate, had similar copulation durations and transferred similar numbers of sperm as males with intact sclerites. Benefits of organ breakage were examined by forcing intact, rival males to inseminate the same or opposite reproductive tracts (female have paired, independent tracts in this taxon) and assessing paternity as a function of sclerite location. As predicted, apical sclerites were typically deposited at the entrance to the female's sperm storage organ, where they could physically block insemination by rivals. First male precedence was common when males inseminated the same tract and deposited sclerites at the entrance to the spermatheca, but not when sclerites were found elsewhere in the tract, or when rivals inseminated opposite tracts (where physically blocking rivals was impossible). Our data show that, in redbacks, copulatory organ breakage is not a side‐effect of sexual conflict, is unlikely to be a cue for cryptic female choice, but allows males to avoid sperm competition. Moreover, copulatory organ damage can have minimal reproductive cost for males, so assumptions of sterility after organ breakage are unjustified without supporting data.     

Copulation patterns in the golden orb-web spider Nephila madagascariensis

A consequence of multiple mating by females can be that the sperm of two or more males directly compete for the fertilisation of ova inside the female reproductive tract. Selection through sperm-competition favours males that protect their sperm against that of rivals and strategically allocate their sperm, e.g., according to the mating status of the female and the morphology of the spermatheca. In the majority of spiders, we encounter the otherwise unusual situation that females possess two independent insemination ducts, both ending in their own sperm storage organ, the spermatheca. Males have paired mating organs, but generally can only fill one spermatheca at a time. We investigated whether males of the African golden orb-web spider Nephila madagascariensis can prevent rival males from mating into the same spermatheca and whether the mating status of the female and/or the spermatheca causes differences in male mating behaviour. There was no significant difference in the duration of copulations into unused spermathecae of virgin and mated females. We found that copulations into previously inseminated spermathecae were generally possible, but shorter than copulations into the unused side of mated females or with virgins. Thus, male N. madagascariensis may have an advantage when they mate with virgins, but cannot prevent future males from mating. However, in rare instances, parts of the male genitals can completely obstruct a female genital opening.     

Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Cryptic female choice in the yellow dung fly Scathophaga stercoraria (L.)

Both female choice and male-male competition may take place during reproduction in many species. Female choice tends to be less obvious than male-male competition and consequently has received less attention from researchers. The opportunity for cryptic female choice arises after multiple insemination. Through postcopulatory processes, a female could alter the pattern of paternity among her offspring so that it does not directly reflect the different contributions of sperm made by her mates. To be able to determine if a female alters the relative sperm contributions of her mates, the behaviors and influences of the males must therefore be first taken into account. The interest of each male is to father all the offspring, and the interest of each female is to maximize paternal quality. Female yellow dung flies have complex internal reproductive tracts that may give them considerable control over the fertilization success of stored sperm from different males. In laboratory trials to date, the last male to mate has usually been most successful. In the present study, cryptic choice occurred in Scathophaga stercoraria and the pattern of choice was consistent with previously reported results. The fertilization success of a female's second mate (P2) was substantially larger if a female was kept at constant temperature and if the second male was genetically similar to her at the phosphoglucomutase (Pgm) locus. Females from the field normally have three spermathecae, but some have four. Lines were bred to have either three or four spermathecae. Flies from the different lines were crossed to generate females with similar genetic backgrounds that had either three or four spermathecae. P2 was significantly lower for high-quality females, that is, those that laid larger-than-average-clutches, with four spermathecae than for low-quality females with four spermathecae; female quality had no influence on P2 for females with three spermathecae. The results suggest that only large females may benefit from increased spermathecae number by being able to act against male interests. Females may only have three spermathecae, even though genetic variation for more is present, because selection for more spermathecae is weak.     

Male insemination decisions and sperm quality influence paternity in the golden orb-weaving spider

In polyandrous species, paternity may be influenced by the timingand frequency of mating. Female spiders possess 2 genital openingsthat lead to separate sperm-storage structures. Thus, even whenmating with a previously mated female, a male may reduce directsperm competition by inseminating the opposite opening to herfirst mate. Such morphology may provide females with greatercontrol over paternity. We examined simultaneously whether malesavoided already inseminated female genital openings and whetherthis behavior varied with the time between successive matings.To explore these questions, we mated female golden orb weaverspiders, Nephila edulis, each to 2 males and manipulated thetiming of their second mating. We documented male inseminationpatterns and explored the influence of male mating decisionson paternity success using the irradiated male technique. Wefound that 60% of males avoided sperm competition by discriminatingagainst inseminated genital openings. Moreover, male matingbehavior had a dramatic impact on the paternity success of irradiatedmales. When males inseminated the same genital opening, thecompetitive ability of the irradiated male's sperm was dramaticallyreduced resulting in lower paternity success. In contrast, whenthe 2 males inseminated opposite genital openings both malessired equal proportions of offspring regardless of their radiationstatus. There was no evidence that the timing of the secondmating affected patterns of paternity. Our data suggest thatdifferences in sperm quality may influence paternity successof N. edulis males under a sperm-competitive scenario. In contrast,females appear to have limited postmating control over paternity.     

Effects of broken male intromittent organs on the sperm storage capacity of female earwigs, <Emphasis Type="Italic">Euborellia plebeja</Emphasis>

In earwigs of the family Anisolabididae, male intromittent organs (virgae) sometimes break off inside female sperm-storage organs (spermathecae) during mating. I examined the effects of this genital breakage on the sperm storage capacity of females using Euborellia plebeja as a representative species. When genital breakage was artificially induced in virgin females, subsequent males successfully inseminated these females. However the sperm-storage capacity of these females was limited by the presence of broken virgae in their spermathecae. In another experiment, genital breakage was experimentally induced in the spermathecae of inseminated females, and their reproductive performance was then monitored for 60 days. In all of four cases where the entire piece of the broken virga remained inside the spermatheca, females deposited fertile eggs (more than 60% hatchability). The average number of clutches, that of eggs laid, and that of hatchlings were similar to those of controls. On the other hand, females laid no eggs in the other two cases where the broken virgae protruded from the spermathecal opening. I discuss the relevance of the results to the mating system and possible removal of rival sperm, which has been reported for E. plebeja. Electronic Publication     

Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae)

Sperm storage organs allow females to temporally separate insemination from fertilization, manipulate ejaculates and control fertilization. In the reproductive tract of female fruit flies (Diptera: Tephritidae), sperm are found in two different organs--a pair or triplet of spermathecae, and a "fertilization chamber". In order to understand the specific function of each of these organs, we tested the following hypotheses: (1) Sperm are distributed equally amongst the various sperm storage organs; (2) Both organ types maintain sperm viability; and (3) Sperm used in fertilization come from the fertilization chamber. We counted sperm in spermathecae and fertilization chamber of Mediterranean fruit flies (Ceratitis capitata) every 3 days for 18 days following insemination, and used a live/dead staining technique to determine the viability of sperm in these organs. Finally, by extirpating spermathecae from inseminated females and allowing them to oviposit, we were able to identify the fertilization chamber as the source of fertilizing sperm. Numbers of sperm in the spermathecae declined from an average of 3575 on the day of copulation to 649, 18 days later. Conversely, the fertilization chamber maintained a fairly constant level of sperms, ranging between an average of 207 cells on day 3 to 115 sperms on day 18. Throughout the period we monitored, we found high levels of sperm viability in both organs (> 80%). Sperm viability was similarly high in the fertilization chambers of females without spermathecae. However, fertility of eggs laid by these females declined rapidly, as did the number of sperm in the fertilization chamber. We conclude that both the spermathecae and the fertilization chamber are active sperm storage organs, with separate functions: the spermathecae for long-term storage and the fertilization chamber, periodically filled by the spermathecae, a staging point for fertilizing sperm. We suggest that the use of both organs by females results in sperm economy, which adaptively prolongs the intervals between copulations.     

Molecular anatomy of an onychophoran: compartmentalized sperm storage and heterogeneous paternity

Onychophorans (peripatus or velvet worms) show extraordinarily high local endemism, and cryptic species are common. As part of a programme addressing issues of endemicity at hierarchical spatial scales, we investigated reproduction in Euperipatoides rowelli (Onychophora: Peripatopsidae) using microsatellite analysis. This species is ovoviviparous, and females have up to 70 embryos in their uteri simultaneously. Batches of undeveloped and well-developed embryos may be present in the uteri of a female. Paired ovaries lead via a common oviduct into paired uteri, each of which has a spermatheca (sperm storage organ). Insemination in E. rowelli is dermal-haemocoelic: spermatophores are placed on the skin of the female, the body wall is breeched, and sperm are released into the haemocoel through which they migrate to the spermathecae. There is no obvious mechanism to prevent sperm mixing, yet microsatellite analysis indicated that offspring in a female's paired reproductive tracts can be sired by different males, and that the paired spermathecae can contain sperm from different males. More than 70% of females had broods with multiple paternity. The data are consistent with the potential for female postcopulatory influence over fertilizations: in particular, compartmentalization of sperm from different males into different spermathecae. Female control of fertilizations could lead to benefits including increased diversity of offspring, minimization of maternal-paternal genetic incompatibility, and influence on offspring genotypes. Multiple mating alone may increase the genetic diversity of offspring: this could be of importance in E. rowelli, which has very small genetic neighbourhoods and low genetic marker diversity.     

Female mate choice predicts paternity success in the absence of additive genetic variance for other female paternity bias mechanisms in Drosophila serrata

After choosing a first mate, polyandrous females have access to a range of opportunities to bias paternity, such as repeating matings with the preferred male, facilitating fertilization from the best sperm or differentially investing in offspring according to their sire. Female ability to bias paternity after a first mating has been demonstrated in a few species, but unambiguous evidence remains limited by the access to complex behaviours, sperm storage organs and fertilization processes within females. Even when found at the phenotypic level, the potential evolution of any mechanism allowing females to bias paternity other than mate choice remains little explored. Using a large population of pedigreed females, we developed a simple test to determine whether there is additive genetic variation in female ability to bias paternity after a first, chosen, mating. We applied this method in the highly polyandrous Drosophila serrata, giving females the opportunity to successively mate with two males ad libitum. We found that despite high levels of polyandry (females mated more than once per day), the first mate choice was a significant predictor of male total reproductive success. Importantly, there was no detectable genetic variance in female ability to bias paternity beyond mate choice. Therefore, whether or not females can bias paternity before or after copulation, their role on the evolution of sexual male traits is likely to be limited to their first mate choice in D. serrata.     

A light and electron microscopical study of the spermathecae and ventral receptacle of Anastrepha suspensa (Diptera: Tephritidae) and implications in female influence of sperm storage

Female insects with multiple sperm storage organs may potentially influence patterns of paternity by differential storage of sperm from competing males. The Caribbean Fruit Fly, Anastrepha suspensa, stores sperm differentially with respect to its three spermathecae. To understand the mechanisms and processes responsible for patterns of sperm storage and use in A. suspensa, details of the fine structure of female sperm storage organs were resolved by UV-light microscopy, confocal microscopy, tissue sectioning, and scanning and transmission electron microscopy. Structures not previously described for this species include a ventral receptacle for sperm storage and osmoregulation, a conical-shaped valve at the junction between the spermathecal capsules and their ducts, laminar and granular secretions, secretions from the signum, hemocytes surrounding the spermathecae, and spermathecae with sclerotized, hollow projections that terminate in single glandular cells. The independent organization of sperm storage organs, spermathecal ducts, associated musculature, gland cells, and innervation offer possible mechanisms by which sperm movement may be influenced by females. The implications of these structures for insemination and fertilization events are discussed.     

Sperm storage and utilization in female Queensland fruit flies (Bactrocera tryoni)

Abstract.  Female animals that use sperm from a single mating to fertilize eggs over an extended period require efficient mechanisms for sperm storage and use. There have been few studies of these mechanisms in tephritid flies. Mating, copula duration, sperm storage and sperm usage patterns are assessed in an Australian tephritid, the Queensland fruit fly ( Bactrocera tryoni ; a.k.a. 'Q-fly'). In particular, the present study investigates whether each of these aspects of mating varies in relation to female size or male size, whether sperm storage patterns change over time after mating (1, 5, 10 and 15 days), and the relative roles of the ventral receptacle and the two spermathecae as sperm storage organs. Large females are more likely to mate than are small females, and are also more fecund in the first 5 days after mating. Females are more likely to store some sperm and, among those that store some sperm, store more sperm if their mate is large. Most sperm are stored in the spermathecae (median = 97%), often with high levels of asymmetry between the two spermathecae. Asymmetry of sperm storage is related to number of sperm stored, but not to male or female size. Total number of stored sperm declines over the 15 days after mating, but this decrease in sperm numbers only reflects changes in the spermathecae; numbers of sperm in the ventral receptacle remain unchanged over this period. As a consequence, the proportion of total sperm stored in the spermathecae declines relative to the ventral receptacle. These results are consistent with a system in which small numbers of sperm are maintained in the ventral receptacle for fertilizations, and are replenished by sperm from the spermathecae as required. Sperm distribution and usage patterns in Q-flies are comparable with recent findings in medflies, Ceratitis capitata , but differ markedly from patterns found in several Anastrepha species.     

Sperm transport after insemination in the Alpine newt (Triturus alpestris, Caudata, Salamandridae)

The goal of this study was to test if sperm transport to the spermathecae in the Alpine newt (Triturus alpestris) requires active co-operation of the female. Artificial insemination of anaesthetised female newts was conducted using spermatophores collected from courting males and with sperm duct contents collected from sacrificed males. Sperm was present in the spermathecae of 9 out of 10 females inseminated with the spermatophores but in only 1 out of 8 females inseminated with sperm duct contents. The females of both groups laid some eggs after insemination, and a portion of these eggs in group of females inseminated with spermatophores were fertilized. However, the number of eggs produced by the females was much lower than typical egg-production in newts. The presence of sperm in the spermathecae of females inseminated with spermatophores and lack of sperm in the spermathecae of females inseminated with sperm duct contents suggests that sperm transport is either induced by the substances present in spermatophores and/or that sperm from the sperm duct is not fully mobile in comparison with sperm from the spermatophores.     

Offspring and sex ratio are independent of sperm management in Eupelmus orientalis females

Sperm stocks in both males and females of the parthenogenetic wasp Eupelmus orientalis were investigated at various points during reproduction and compared to the progeny of females in controlled conditions. One day-old virgin males had approximately 5500 sperm, and from a total of about 1697 sperm transferred per copulation, 21% are stored in the spermathecae by females 24 hours after mating. At the end of the egg-laying period (at least 42 days), 2/5 of the initial amount of sperm remained in this storage organ. This decrease (from approximately 350 to 150) occurred essentially during the first 21 days of egg-laying activity, indicating that the majority of sperm stored were used during this period. Between 21 days and the end of fertile life, the number of sperm remained constant. The mean offspring production throughout reproductive life after one mating was 153, with 56.5% of the daughters laid at the beginning of the laying activity. Sex ratio was entirely female biased during the first 15 days (mean=0.65), then it decreased and became nearly equal after 20 days. Present results propose that females maximize the production of daughters i.e. of inseminated eggs until the 20th day and after this time lay as many daughters and sons despite their still having stored sperm. Physiological constraints due to ageing are proposed to explain this phenomenon.     

Sperm Storage in the Yellow Dung Fly Scathophaga stercoraria: Identifying the Sperm of Competing Males in Separate Female Spermathecae

We examined the effects of male and female behaviour and morphology on the process of sperm storage in the yellow dung fly Scathophaga (Scatophaga) stercoraria. The larger of two males was more successful in transferring sperm to females' spermathecae the greater the difference in weight to his smaller competitor, as expected from previous studies by other authors. Sperm length, which is not correlated to body size, affected sperm access to the spermathecae, the female storage organs; longer sperm were more likely to be found in the spermathecae. A female typically had a singlet spermatheca and two spermathecae arranged as a pair, a doublet. However, there was variation from this pattern, which influenced the pattern of sperm storage. We measured the proportion of sperm from two competing males in females' singlet and doublet spermathecae. When the larger male's sperm were longer than his competitor's, they were more often in a female's singlet when he was her first mate and equally likely to be in the singlet or doublet when he was her second mate. When the larger male's sperm were shorter than his competitor's, the pattern was more complicated, principally because his sperm were not as successful at entering the female's doublet when he was her second mate. Counts of sperm, made using the same experimental procedure, showed that these effects were due to greater numbers of sperm entering the females' doublets when the larger male mated second. Sperm length was thus the factor with the largest single influence on the pattern of sperm storage. However, our most important result is that it was the interactions between male and female characters that were significant. Males mostly determine the early, especially precopulatory, events and females strongly influence the later ones.     

The assessment of insemination success in yellow dung flies using competitive PCR

In spite of considerable interest in postcopulatory sexual selection, separating the effects of sperm competition from cryptic female choice remains difficult because mechanisms underlying postcopulatory processes are poorly understood. One methodological challenge is to quantify insemination success for individual males within the sperm stores of multiply mated females to discover how insemination translates into eventual paternity. Any proposed method must be applicable in organisms without extensive DNA sequence information (which include the majority of model species for sexual selection). Here, we describe the development and application of microsatellite competitive-multiplex-PCR for quantifying relative contributions to a small number of sperm in storage. We studied how DNA template characteristics affect PCR amplification of known concentrations of mixed DNA and generated regressions for correcting observations of allelic signal strength based on such characteristics. We used these methods to examine patterns of sperm storage in twice-mated female yellow dung flies, Scathophaga stercoraria. We confirm previous findings supporting sperm displacement and demonstrate that average paternity for the last mate accords with the mean proportion of sperm stored. We further find consistent skew in storage across spermathecae, with more last male sperm stored in the singlet spermatheca on one side of the body than in the doublet on the opposite side. We also show that the time between copulations may be important for effectively sorting sperm. Finally, we demonstrate that male size may influence the opportunity for sperm choice, suggesting future work to disentangle the roles of male competition and cryptic female choice.     

Sperm storage and viability in Photinus fireflies

In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.     

Sperm mobility: mechanisms of fertilizing efficiency, genetic variation and phenotypic relationship with male status in the domestic fowl, Gallus gallus domesticus

When females are sexually promiscuous, sexual selection continues after insemination through sperm competition and cryptic female choice, and male traits conveying an advantage in competitive fertilization are selected for. Although individual male and ejaculate traits are known to influence paternity in a competitive scenario, multiple mechanisms co-occur and interact to determine paternity. The way in which different traits interact with each other and the mechanisms through which their heritability is maintained despite selection remain unresolved. In the promiscuous fowl, paternity is determined by the number of sperm inseminated into a female, which is mediated by male social dominance, and by the quality of the sperm inseminated, measured as sperm mobility. Here we show that: (i) the number of sperm inseminated determines how many sperm reach the female sperm-storage sites, and that sperm mobility mediates the fertilizing efficiency of inseminated sperm, mainly by determining the rate at which sperm are released from the female storage sites, (ii) like social status, sperm mobility is heritable, and (iii) subdominant males are significantly more likely to have higher sperm mobility than dominant males. This study indicates that although the functions of social status and sperm mobility are highly interdependent, the lack of phenotypic integration of these traits may maintain the variability of male fitness and heritability of fertilizing efficiency.     

Hold on: Females modulate sperm depletion from storage sites in the fly Drosophila melanogaster

Among many species of insects, females gain fitness benefits by producing numerous offspring. Yet actions related to producing numerous offspring such as mating with multiple males, producing oocytes and placing offspring in sub-optimal environments incur costs. Females can decrease the magnitude of these costs by retaining gametes when suitable oviposition sites are absent. We used the pomace fly, Drosophila melanogaster, to explore how the availability of fresh feeding/oviposition medium influenced female fitness via changes in offspring survivorship and the modulation of gamete release. Availability of fresh medium affected the absolute number and temporal production of offspring. This outcome was attributable to both decreased larval survival under crowded conditions and to female modulation of gamete release. Direct examination of the number of sperm retained among the different female storage organs revealed that females ‘hold on’ to sperm, retaining more sperm in storage, disproportionately within the spermathecae, when exposed infrequently to fresh medium. Despite this retention, females with lower rates of storage depletion exhibited decreased sperm use efficiency shortly after mating. This study provides direct evidence that females influence the rate of sperm depletion from specific storage sites in a way that can affect both female and male fitness. The possible adaptive significance of selective gamete utilization by female Drosophila includes lowering costs associated with frequent remating and larval overcrowding when oviposition sites are limiting, as well as potentially influencing paternity when females store sperm from multiple males.     

Multiple mating and repeated copulations: effects on male reproductive success in red flour beetles

In many insects, both sexes mate multiple times and females use stored sperm for fertilizations. While males frequently engage in two distinct behaviours, multiple mating (with different females) and repeated copulations (with the same female), the reproductive consequences of these behaviours for males have been quantified for only a few species. In this study, males of the red flour beetle, Tribolium castaneum, were found to be capable of mating with as many as seven different virgin females within 15 min. Across sequential copulations with virgin females, there was no decline in either male insemination success or average female progeny production over 48 h. However, when males copulated with previously mated females, there was a significant decline in male paternity success across sequential copulations, possibly due to male sperm depletion. In separate experiments, T. castaneum males were found to engage in two to six repeated copulations with the same, individually marked female. These repeated copulations did not increase male insemination success, short-term female fecundity, or male paternity success. Repeated copulations may possibly play a role in sperm defence. This study indicates that males may frequently engage in multiple matings, but these additional matings may lead to diminishing male reproductive returns.