Butterflies tailor their ejaculate in response to sperm competition risk and intensity

Males of many insects eclose with their entire lifetime sperm supply and have to allocate their ejaculates at mating prudently. In polyandrous species, ejaculates of rival males overlap, creating sperm competition. Recent models suggest that males should increase their ejaculate expenditure when experiencing a high risk of sperm competition. Ejaculate expenditure is also predicted to vary in relation to sperm competition intensity. During high intensity, where several ejaculates compete for fertilization of the female''s eggs, ejaculate expenditure is expected to be reduced. This is because there are diminishing returns of providing more sperm. Additionally, sperm numbers will depend on males'' ability to assess female mating status. We investigate ejaculate allocation in the polyandrous small white butterfly Pieris rapae (Lepidoptera). Males have previously been found to ejaculate more sperm on their second mating when experiencing increased risk of sperm competition. Here we show that males also adjust the number of sperm ejaculated in relation to direct sperm competition. Mated males provide more sperm to females previously mated with mated males (i.e. when competing with many sperm) than to females previously mated to virgin males (competing with few sperm). Virgin males, on the other hand, do not adjust their ejaculate in relation to female mating history, but provide heavier females with more sperm. Although virgin males induce longer non-receptive periods in females than mated males, heavier females remate sooner. Virgin males may be responding to the higher risk of sperm competition by providing more sperm to heavier females. It is clear from this study that males are sensitive to factors affecting sperm competition risk, tailoring their ejaculates as predicted by recent theoretical models.     

Regulation of sexual receptivity of female Mediterranean fruit flies: old hypotheses revisited and a new synthesis proposed

The objective of this study was to examine the relative contributions of copula duration and sperm transfer to the inhibition of sexual receptivity of female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae). Females choosing to remate had significantly fewer sperm in their spermathecae than females who chose not to remate. Duration of a female's first copulation did not affect her subsequent receptivity. Furthermore, on the first day following copulation significantly more females whose first mate was sterile and from a laboratory strain (sterile males transfer fewer sperm than wild males) chose to copulate than did females whose mate was fertile and recently derived from wild stock. Finally, we offer a synthesis of the available information on remating in this species, and suggest that while females are facultatively polyandrous, copula duration, sperm transfer and male accessory gland secretions act in succession to inhibit female receptivity.     

Team swimming in ant spermatozoa

In species where females mate promiscuously, competition between ejaculates from different males to fertilize the ova is an important selective force shaping many aspects of male reproductive traits, such as sperm number, sperm length and sperm–sperm interactions. In eusocial Hymenoptera (bees, wasps and ants), males die shortly after mating and their reproductive success is ultimately limited by the amount of sperm stored in the queen''s spermatheca. Multiple mating by queens is expected to impose intense selective pressure on males to optimize the transfer of sperm to the storage organ. Here, we report a remarkable case of cooperation between spermatozoa in the desert ant Cataglyphis savignyi. Males ejaculate bundles of 50–100 spermatozoa. Sperm bundles swim on average 51% faster than solitary sperm cells. Team swimming is expected to increase the amount of sperm stored in the queen spermatheca and, ultimately, enhance male posthumous fitness.     

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.     

Strategic ejaculation in the common dung fly Sepsis cynipsea

Sperm competition has been a major selective force acting on male and female behaviour. Theory predicts that when sperm compete numerically, selection will favour males that vary the number of sperm they transfer with sperm competition risk. This often leads to increased copula duration when sperm competition risk is high, the selective advantage of which is increased paternity. We investigated the copulatory behaviour of the common dung fly Sepsis cynipsea in relation to male and female size, female mating status, age, and presence or absence of dung. This fly is unusual in that males mate-guard before copula while females use the sperm of previous males for their current clutch. Body size had no effect on copula duration, but duration of first copulations depended on female age, with older females having longer copulations. For females that copulated twice, there was an interaction between female age and mating status influencing copula duration: old females had longer copulations than young females, but second copulas were longer for young females. Residual testis size of nonvirgin males was smaller than for virgins, and testis shrinkage was significantly associated with copula duration, which indicates that males transfer more ejaculate with longer copulations. We therefore conclude that copulation duration and ejaculate transfer vary in accordance with sperm competition theory.     

Male-derived cuticular hydrocarbons signal sperm competition intensity and affect ejaculate expenditure in crickets

Female sexual promiscuity can have significant effects on male mating decisions because it increases the intensity of competition between ejaculates for fertilization. Because sperm production is costly, males that can detect multiple matings by females and allocate sperm strategically will have an obvious fitness advantage. The presence of rival males is widely recognized as a cue used by males to assess sperm competition. However, for species in which males neither congregate around nor guard females, other more cryptic cues might be involved. Here, we demonstrate unprecedented levels of sperm competition assessment by males, which is mediated via the use of chemical cues. Using the cricket Teleogryllus oceanicus, we manipulated male perception of sperm competition by experimentally coating live unmated females with cuticular compounds extracted from males. We found that males adjusted their ejaculate allocation in response to these compounds: the viability of sperm contained within a male's ejaculate decreased as the number of male extracts applied to his virgin female partner was increased. We further show that males do not respond to the relative concentration of male compounds present on females, but rather to the number of distinct signature odours of individual males. Our results conform to sperm competition theory, and show for the first time, to our knowledge, that males can detect different intensities of sperm competition by using distinct chemical cues of individual males present on females.     

Condition-dependent ejaculate size and composition in a ladybird beetle

Sexually selected male ejaculate traits are expected to depend on the resource state of males. Theory predicts that males in good condition will produce larger ejaculates, but that ejaculate composition will depend on the relative production costs of ejaculate components and the risk of sperm competition experienced by low- and high-condition males. Under some conditions, when low condition leads to poorer performance in sperm competition, males in low condition may produce ejaculates with higher sperm content relative to their total ejaculate and may even transfer more sperm than high-condition males in an absolute sense. Previous studies in insects have shown that males in good condition transfer larger ejaculates or more sperm, but it has not been clear whether increased sperm content represents a shift in allocation or simply a larger ejaculate, and thus the condition dependence of ejaculate composition has been largely untested. We examined condition dependence in ejaculate by manipulating adult male condition in a ladybird beetle (Adalia bipunctata) in which males transfer three distinct ejaculate components during mating: sperm, non-sperm ejaculate retained within the female reproductive tract, and a spermatophore capsule that females eject and ingest following mating. We found that high condition males indeed transferred larger ejaculates, potentially achieved by an increased rate of ejaculate transfer, and allocated less to sperm compared with low-condition males. Low-condition males transferred ejaculates with absolutely and proportionally more sperm. This study provides the first experimental evidence for a condition-dependent shift in ejaculate composition.     

Sperm storage and use in polyandrous females of the globally invasive fruitfly, Ceratitis capitata

The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.     

Sperm viability and sperm competition in insects

Sperm quality plays an important role in vertebrates in determining which male has the advantage when two or more males compete to fertilize a female's ova. In insects, however, the importance of sperm quality has never been considered, despite sperm competition being widespread and well studied in this group. We tested the hypothesis that sperm viability, measured as the proportion of live sperm, covaried with the intensity of sperm competition in insects. In a pairwise comparison of seven closely related species pairs, each comprising a monandrous and a polyandrous species (i.e., with and without sperm competition, respectively), we found that in all cases the polyandrous species had a higher proportion of live sperm in their sperm stores. The distribution of the percentage of live sperm showed considerable inter- and intraspecific variation, suggesting that, all else being equal, males will vary in their ability to fertilize ova on the basis of sperm viability alone. Our results suggest that sperm viability is one of a suite of male adaptations to sperm competition in insects.     

Adult Responses to Larval Population Size in the Almond Moth, Cadra cautella

Juvenile population size may affect the potential for future mating opportunities and therefore potentially sperm competition; this may favour ontogenetic adjustments in sperm production. Theory predicts that males should optimize their ejaculatory investment in accordance with the risk of sperm competition. Evidence for these theories is typically revealed in males of highly polyandrous species. Whether such responses to environmental cues exist for females, or are maintained in mildly polyandrous species in which most females do not re-mate, is unknown. Male lepidopterans produce normal, fertilizing sperm (eupyrene) and non-fertilizing (apyrene) sperm. Apyrene sperm are associated with reduced female receptivity, suggesting a role in sperm competition. We tested the effect of juvenile population size on life-history parameters and reproductive investment in the mildly polyandrous almond moth, Cadra cautella , a species in which current male ejaculate traits suggest previous selection for paternity protection consistent with a sperm-competitive environment. Larvae were reared at high (H) or low population sizes (L). We recorded larval development time, adult longevity and male gametic investment. Our results show a response by adults to signals in the juvenile environment. H males transferred more apyrene, but not eupyrene sperm. We also examined potential trade-offs between somatic characters and reproductive behaviours. Larval duration was longer for H individuals, females and heavier individuals. Further, H females and L males lived longer than L females. Our data are consistent with the theory that males should adjust their reproductive investment in accordance with sperm competition risk.     

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.     

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.     

Age-dependent insemination success of sterile Mediterranean fruit flies

The Sterile Insect Technique (SIT) is used in many regions worldwide to manage wild populations of the Mediterranean fruit fly (`medfly'), an important pest species. This technique relies on released sterile males outcompeting their wild counterparts in fertilizing ova of wild females. Numerous studies have investigated the ability of sterile males to secure copulations, an essential step toward overall success. Here we progress further along the mating sequence by studying reproductive barriers that may remain ahead of sterile males that manage to secure copulations in field cage experiments and whether ability to pass these barriers is influenced by a male's age, diet and size, or the size of his mate. Amongst those virgin males that succeeded in copulating, both the number of sperm stored by mates and the chances of having any sperm stored at all decreased with age. Sperm tended to be stored asymmetrically between the females' two spermathecae, and this tendency was more apparent when few sperm were stored. In accord with effects of male age on number of sperm stored, sperm of older males were stored more asymmetrically than that of young males. We found no evidence that male size, male diet or female size influenced copula duration, number of sperm stored or allocation of sperm between the female's two spermathecae. The decline in number of sperm stored as males aged was not accompanied by age-dependent changes in copula duration, indicating that copula duration and insemination success are not deterministically linked. We discuss these results in light of their relevance to SIT and the medfly mating system.     

Female sperm storage mediates post‐copulatory costs and benefits of ejaculate anticipatory plasticity in the guppy

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade‐offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live‐bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready‐to‐use sperm (an anticipatory response called ‘sperm priming’). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm‐primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female‐stored sperm, weeks after insemination. By suggesting a plastic trade‐off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.     

Sperm allocation and cost of mating in a tropical tephritid fruit fly

Males that copulate repeatedly may suffer from reduced sperm stores. However, few studies have addressed sperm depletion from both the female and male perspective. Here, we show that male Anastrepha obliqua (Diptera: Tephritidae) do not ejaculate all available sperm and are left with mature sperm in the seminal vesicles even after copulating as often as three times in half a day. Ejaculate size was not related to male mating history; time elapsed since the last mating, copulation duration, female thorax length or head width. Larval host origin did not affect the number of sperm stored by females. More sperm was found in the ventral receptacle compared to sperm stored in the three spermathecae. Males apparently do not suffer a cost of mating in terms of longevity, although we cannot rule out other fitness costs. Sperm production in this species may not be as costly as it is for other species. Results suggest that males strategically allocate similar numbers of sperm among successive mates without exhausting sperm reserves for future encounters. We discuss the role that differential sperm storage may have in mediating sperm competition and tie our results to the unique natural history of A. obliqua.     

Determinants of Sperm Transfer by Males of the Noctuid Moth Heliothis virescens

Ejaculate composition can be an important determinant of male reproductive success in the face of sperm competition, which varies with the mating history of the female. Here we examine the effect of various male and female mating histories and morphological traits on ejaculate sperm numbers in the polyandrous moth Heliothis virescens. We show that when mating with nonvirgin females, males passed larger sperm packages (spermatophores) but did not alter either the sperm count or the ratio of nucleated-to-nonnucleated sperm. Males also passed fewer sperm in their second ejaculates. Finally, older males passed more sperm than did younger males. Earlier research found that females store more sperm from older males and that older males are more likely to gain sperm precedence over younger rivals. These earlier results, taken together with the present results, indicate that the advantage enjoyed by older males is due to an increased sperm count.     

Sperm selection and genetic incompatibility: does relatedness of mates affect male success in sperm competition?

Sperm selection may be said to occur if females influence the relative success of ejaculates competing to fertilize their ova. Most evidence that female animals or their ova are capable of sperm selection relates to male genetic incompatibility, although relatively few studies focus on competition between conspecific males. Here I look for evidence of sperm selection with respect to relatedness of mates. Reduced fitness or inbreeding effects in offspring resulting from copulations between close relatives are well documented. If females are capable of sperm selection, they might therefore be expected to discriminate against the sperm of sibling males during sperm competition. I describe an experimental protocol designed to test for evidence of sperm selection while controlling for inbreeding effects. Using decorated field crickets (Gryllodes supplicans), I found that sibling males achieved lower fertilization success in competition with a male unrelated to the female than in competition with another sibling more frequently than expected by chance, although the mean paternity values did not differ significantly between treatments. The tendancy for sibling males to achieve relatively lower fertilization success in competition with males unrelated to the female could not be explained by the effects of increased ejaculate allocation, female control of sperm transfer or inbreeding. This study therefore provides some evidence in support of the idea that female insects (or their ova) may be capable of selection against sperm on the basis of genetic similarity of conspecific males.     

Effects of different male remating intervals on the reproductive success of Choristoneura rosaceana males and females

The mass of the spermatophore transferred by a previously mated Choristoneura rosaceana male increases with time elapsed since the last mating but, even after 4 days, it never reaches the mass of the spermatophore of a virgin male. However, spermatophore mass is clearly not a good indicator of the male reproductive investment as the quantity of sperm in the second ejaculate of a previously mated male is the same as that of his first, if he is allowed a 2 (eupyrene sperm) to 3 day (apyrene sperm) recovery period. The interval between the first two matings had no influence on female fecundity or longevity but significantly affected fertility if the male had only 1 day to recover. The length of the post-copulatory refractory period was also shorter in females mated with previously mated males than in those mated with virgins, regardless of the male's remating interval. Furthermore, a significant variation in the eupyrene sperm content of the spermatophore transferred by virgin males had no influence on the length of the female refractory period. Globally, these results support the hypothesis that a factor, other than sperm numbers in the spermatheca, is responsible for maintaining the inhibition of pheromone production in this species.     

Influences of sex, size, and symmetry on ejaculate expenditure in a moth

Although sperm fundamentally function to fertilize eggs, forcesarising from both sexes select for optimal ejaculate composition.Sperm competition is one recognized agent in the evolution ofsperm and ejaculate structure. Few studies, however, have examinedhow female factors influence ejaculate structure, despite somebehavioral evidence for male mate choice. Male Plodia interpunctella(Lepidoptera, Pyralidae) accrue all resources for reproductionas larvae. Adults emerge with a limited sperm complement andare therefore under intense selection to optimize gamete allocation.I detected no effect of male body weight on ejaculate size.However, female reproductive potential (ovary masses) was dictatedby body weight In addition, heavier females had greater spermathecalvolumes, but there was no such relationship with bursal size.Finally, heavier females showed a higher mating frequency. Ifound that mating males were sensitive to female size and producedlarger ejaculates when mating with heavier females. Males mayejaculate more sperm into larger females either because it paysthem to "spend" more reproductive resources on matings thatprovide greater reproductive potential, or because heavier (longerlived and more attractive) females mate more frequently andhave larger spermathecal volumes. Alternatively, females maycontrol spermatophore formation and "accept" an appropriateejaculate to maximize fertility. Males may therefore be alsoselected to ejaculate more sperm into larger females to counteractgreater risks of sperm competition associated with heavier females.There was no association between male or female femur asymmetryand ejaculate size. P.interpunctella may be selected to exercisemodulation of ejaculate size because males invest paternally,sperm for the single reproductive episode are limited, and femalefecundity and mating pattern vary between individuals and areassociated with body weight. More obvious variability in malereproductive behavior and choice may therefore be paralleledat the cryptic gametic level by plasticity in ejaculate allocation.