Sperm numbers,their storage and usage in the fly Dryomyza anilis

In the fly Dryomyza anilis females have two kinds of sperm storage organs: one bursa copulatrix and three spermathecae (two spermathecae with a common duct form the doublet, and the third is a singlet spermathecal unit). At the beginning of a mating the male deposits his sperm in the bursa copulatrix. After sperm transfer the male taps the female''s abdomen with his claspers. This behaviour has been shown to increase the male''s fertilization success. After mating, the female discharges large quantities of sperm before oviposition. To find out where the sperm remaining in the female are stored, I counted the number of sperm in the droplet and in the female''s sperm storage organs after different types of mating. I carried out three mating experiments. In experiment 1, virgin females were mated with one male and the matings were interrupted either immediately after sperm transfer or after several tapping sequences. The results show that during male tapping more sperm moved into the singlet spermatheca. In addition, the total number of sperm correlated with sperm numbers in all sperm storage organs, and male size was positively related to the number of sperm remaining in the bursa. In experiment 2, females mated with several males. The number of sperm increased with increasing number of matings only in the doublet spermatheca. No increase in the number of sperm in the singlet spermatheca during consecutive matings suggests that sperm were replaced or did not reach this sperm storage organ. In experiment 3, virgin females were mated with a single male and half of them were allowed to lay eggs. The experiment showed that during egglaying, females primarily used sperm from their singlet spermatheca. The results from the three experiments suggest that sperm stored in the singlet spermatheca is central for male fertilization success and male tapping is related to sperm storage in the singlet spermatheca. The different female''s sperm storage organs in D. anilis may have separate functions during sperm storage as well as during sperm usage.     

Ejaculate esterase 6 and initial sperm use by female Drosophila melanogaster

The period of initial sperm storage and use by Drosophila melanogaster females is examined for effects of the seminal fluid enzyme esterase 6. Females mated to males differing in their level of esterase 6 activity were dissected from 5 min to 50 hr after the start of copulation and numbers of sperm contained in the uterus, ventral receptacle and paired spermathecae were counted. Of the 4000–6000 sperm transferred at copulation, about 700 are stored in the receptacle by 4 hr post mating and 400 in the spermathecae by 7 hr. However, sperm are released rapidly from storage organs following these peaks and may be found again in the uterus in numbers up to 100 or more. The rate of sperm release is closely related to the level of esterase 6 activity, suggesting that this seminal fluid enzyme is involved in sperm motility.     

Copula duration and sperm storage in Mediterranean fruit flies from a wild population

In the Mediterranean fruit fly (Ceratitis capitata Weidemann, ‘medfly’), a lekking tephritid, evidence from laboratory studies of flies from laboratory strains suggests that copulation is shorter, and sperm storage more abundant, if males are large or protein‐fed, and that copulation is longer when females are large. In addition, sperm tend to be stored asymmetrically between the female’s two spermathecae and this asymmetry declines with abundance of stored sperm. The primary objective of this study was to investigate whether these trends persist in other experimental contexts that bear closer resemblance to nature. Accordingly, we carried out experiments in a field‐cage using males derived as adults from a wild population and virgin females reared from naturally infested fruit. The results of this study were consistent with laboratory studies in that copula duration increased with female size, that sperm were stored asymmetrically between the females’ spermathecae, and that this asymmetry declined with number of sperm stored. However, we also found some previously unreported effects of female size; large females stored more sperm and stored sperm more asymmetrically between their two spermathecae than did small females. Unlike the laboratory studies, copula duration and sperm storage patterns were unaffected by male size and diet. This may be due to overwhelming variation from other sources in the wild‐collected males used, as well as environmental variability in the semi‐natural setting.     

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.     

Female control of sperm ejection and retention in the cornsilk fly Euxesta eluta (Diptera: Ulidiidae)

Promiscuous mating systems provide the opportunity for females to bias fertilization toward particular males. However, distinguishing between male sperm competition and active female sperm choice is difficult for species with internal fertilization. Nevertheless, species that store and use sperm of different males in different storing structures and species where females are able to expel all or part of the ejaculates after copulation may be able to bias fertilization. We report a series of experiments aimed at providing evidence of female sperm choice in Euxesta eluta (Hendel), a species of ulidiid fly that expels and consumes ejaculates after copulation. We found no evidence of greater reproductive success for females mated singly, multiply with the same male, or mated multiply with different males. Female E. eluta possesses two spherical spermathecae and a bursa copulatrix for sperm storage, with a ventral receptacle. There was no significant difference in storing more sperm in spermathecae 24 h after copulation than immediately after copulation. Females mated with protein-fed males had greater reproductive success than similar females mated to protein-deprived males. Protein-fed females prevented to consume the ejaculate, retained more sperm when mated to protein-fed males than when mated to protein-deprived males. Our results suggest that female E. eluta can exert control of sperm retention of higher quality males through ejaculate ejection.     

Sperm of the wasted mutant are wasted when females utilize the stored sperm in Drosophila melanogaster

Females of many animal species store sperm after copulation for use in fertilization, but the mechanisms controlling sperm storage and utilization are largely unknown. Here we describe a novel male sterile mutation of Drosophila melanogaster, wasted (wst), which shows defects in various processes of sperm utilization. The sperm of wst mutant males are stored like those of wild-type males in the female sperm storage organs, the spermathecae and seminal receptacles, after copulation and are released at each ovulation. However, an average of thirteen times more wst sperm than wild type sperm are released at each ovulation, resulting in rapid loss of sperm stored in seminal receptacles within a few days after copulation. wst sperm can enter eggs efficiently at 5 hr after copulation, but the efficiency of sperm entry decreases significantly by 24 hr after copulation, suggesting that wst sperm lose their ability to enter eggs during storage. Furthermore, wst sperm fail to undergo nuclear decondensation, which prevents the process of fertilization even when sperm enter eggs. Our results indicate that the wst gene is essential for independent processes in the utilization of stored sperm; namely, regulation of sperm release from female storage organs, maintenance of sperm efficiency for entry into eggs, and formation of the male pronucleus in the egg at fertilization.     

Isometric patterns for male genital allometry in four damselfly species

Recent studies have found that insect genitalic traits show negative allometry, i.e., are relatively small in relation to body size. One interpretation of this is that males use their genitalia to stimulate females. Thus, given the nature of damselfly copulation in which males physically reach the rival sperm that females have stored from previous matings, male genitalic traits are not expected to show negative allometry. To test this idea, we assessed (a) the rival sperm displacement function by the mating male and (b) allometry of aedeagal length of four damselfly species (Argia anceps, Argia tezpi, Argia extranea, and Enallagma praevarum). Sperm displacement was assessed by inspecting whether the aedeagus reached the rival sperm during copulation in mating pairs for the four species. To have a standard for comparing allometric patterns, allometry of aedeagal was compared to that of two non-genital traits, tibial, and fourth abdominal segment length. In all cases, the aedeagus was found to reach the rival sperm which supports the idea that stimulation is not the mechanism for sperm displacement but physical displacement. Aedeagal length was isometric, and its slope was lower in general compared to that of tibial length and fourth abdominal segment. Given that this isometric pattern is not common for other odonate species, our interpretation of these varying aedeagal scaling patterns in this insect order is that males’ and females’ sexual interests are in conflict (males are evolving an elongated aedeagus to reach rival sperm while females are evolving unreachable sperm storage organs to prevent displacement of stored sperm). This sexual conflict scenario would favor varying scaling patterns for aedeagal length in odonates. A final interpretation is that the risk of interspecific matings in damselflies, may also explain different species-specific, aedeagal allometries.     

Multiple sperm storage organs facilitate female control of paternity

It has been proposed that multiple sperm storage organs (spermathecae) could allow polyandrous females to control paternity. There is little conclusive evidence for this since insemination of individual spermathecae is generally not experimentally manipulable. Here, we examined sperm use patterns in the Australian redback spider (Latrodectus hasselti), which has paired, independent spermathecae. We assessed paternity when two rivals were forced to inseminate a single storage organ or opposite storage organs. When males inseminated a single spermatheca, mean paternity of the female's first mate was 79.8% (median 89.4%), and 38% of first mates achieved 100% paternity. In contrast, when males inseminated opposite organs, the mean paternity of the first mate was 49.3% (median 49.9%), only 10% of males achieved complete precedence, and paternity was normally distributed, suggesting sperm mixing. Males responded to this difference by avoiding previously inseminated female reproductive tracts. Complete sperm precedence can only be achieved if females permit males to copulate with both reproductive tracts. Females often cannibalize smaller males during their first copulation, thus limiting their paternity to 50%. These data show that multiple sperm storage organs can increase female control of paternity.     

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.     

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 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.     

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 mediated by cryptic female choice for nuptial gifts

Polyandrous females are expected to discriminate among males through postcopulatory cryptic mate choice. Yet, there is surprisingly little unequivocal evidence for female-mediated cryptic sperm choice. In species in which nuptial gifts facilitate mating, females may gain indirect benefits through preferential storage of sperm from gift-giving males if the gift signals male quality. We tested this hypothesis in the spider Pisaura mirabilis by quantifying the number of sperm stored in response to copulation with males with or without a nuptial gift, while experimentally controlling copulation duration. We further assessed the effect of gift presence and copulation duration on egg-hatching success in matings with uninterrupted copulations with gift-giving males. We show that females mated to gift-giving males stored more sperm and experienced 17% higher egg-hatching success, compared with those mated to no-gift males, despite matched copulation durations. Uninterrupted copulations resulted in both increased sperm storage and egg-hatching success. Our study confirms the prediction that the nuptial gift as a male signal is under positive sexual selection by females through cryptic sperm storage. In addition, the gift facilitates longer copulations and increased sperm transfer providing two different types of advantage to gift-giving in males.     

Sperm storage and copulation duration in a sexually cannibalistic spider

Female St Andrew’s Cross spiders control copulation duration by timing sexual cannibalism and may thereby control paternity if cannibalism affects sperm transfer. We have investigated the effect of copulation duration on sperm transfer and documented sperm storage patterns when we experimentally reduced the ability of females to attack and cannibalise the male. Virgin males and females were paired and randomly allocated either to a control treatment, where females were allowed to attack and cannibalise the male during copulation, or to an experimental treatment, where females were unable to cannibalise the male. The latter was achieved by placing a paintbrush against her chelicerae during copulation. Our experimental manipulation did not affect copulation duration or sperm storage. However, the number of sperm stored by the female increased with copulation duration only if the male was cannibalised, suggesting that cannibalism increases relative paternity not only through prolonged copulation duration following a fair raffle model but also through the cannibalism act itself. Future studies should explore whether cannibalised males ejaculate more sperm or whether females selectively store the sperm of cannibalised males.     

Long-term sperm storage in eusocial Hymenoptera

In internally fertilizing species, sperm transfer is not always immediately followed by egg fertilization, and female sperm storage (FSS) may occur. FSS is a phenomenon in which females store sperm in a specialized organ for periods lasting from a few hours to several years, depending on the species. Eusocial hymenopterans (ants, social bees, and social wasps) hold the record for FSS duration. In these species, mating takes place during a single nuptial flight that occurs early in adult life for both sexes; they never mate again. Males die quickly after copulation but survive posthumously as sperm stored in their mates' spermathecae. Reproductive females, also known as queens, have a much longer life expectancy, up to 20 years in some species. Here, we review what is currently known about the molecular adaptations underlying the remarkable FSS capacities in eusocial hymenopterans. Because sperm quality is crucial to the reproductive success of both sexes, we also discuss the mechanisms involved in sperm storage and preservation in the male seminal vesicles prior to ejaculation. Finally, we propose future research directions that should broaden our understanding of this unique biological phenomenon.     

Factors Affecting Sperm Quality Before and After Mating of Calopterygid Damselflies

Damselflies (Odonata: Zygoptera) have a more complex sperm transfer system than other internally ejaculating insects. Males translocate sperm from the internal reproductive organs to the specific sperm vesicles, a small cavity on the body surface, and then transfer them into the female. To examine how the additional steps of sperm transfer contribute to decreases in sperm quality, we assessed sperm viability (the proportion of live sperm) at each stage of mating and after different storage times in male and female reproductive organs in two damselfly species, Mnais pruinosa and Calopteryx cornelia. Viability of stored sperm in females was lower than that of male stores even just after copulation. Male sperm vesicles were not equipped to maintain sperm quality for longer periods than the internal reproductive organs. However, the sperm vesicles were only used for short-term storage; therefore, this process appeared unlikely to reduce sperm viability when transferred to the female. Males remove rival sperm prior to transfer of their own ejaculate using a peculiar-shaped aedeagus, but sperm removal by males is not always complete. Thus, dilution occurs between newly received sperm and aged sperm already stored in the female, causing lower viability of sperm inside the female than that of sperm transferred by males. If females do not remate, sperm viability gradually decreases with the duration of storage. Frequent mating of females may therefore contribute to the maintenance of high sperm quality.     

Live for the moment—Adaptations in the male genital system of a sexually cannibalistic spider (Theridiidae, Araneae)

Monogyny in spiders culminates in extreme traits, like dramatic male self-sacrifice and emasculation of the male by the female during copulation. Here we show that monogynous males can be highly adapted for this fatal sexual behaviour. Dwarf males of the one-palped theridiid spider Tidarren argo, which are cannibalised immediately after the insertion of their single copulatory organ, stop spermiogenesis when reaching adulthood. Their testes atrophy, which might economise the energy expenditures of these males. We also found that the amount of seminal fluid produced is stored in an enlarged seminal vesicle until the single sperm induction takes place. The volume of the seminal vesicle is similar to the sperm droplet taken up into the copulatory organ (palpal organ). Sperm uptake takes much longer than in related species most likely due to the large amount of seminal fluid. As shown by histological observations males are able to fill one of the paired female sperm storage organs during copulation thereby presumably impeding subsequent charging by rival males.     

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.     

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.     

The prediapause copulation and its significance in the butterflyEurema hecabe

In the pierid butterflyEurema hecabe, which shows seasonal polymorphism (summer and autumn morphs) and overwinters at adult stage, whether or not the prediapause copulation may be of usual occurrence and reproductively functional was examined. From the counts of the spermatophores carried by the females, it is clear that the prediapause copulation characteristically occurs in reproductively inactive females of the autumn corph. From behavioral observations in the field, mating partners of those females are mostly males of the summer morph rather than from the autumn morph. In autumn, males of the summer morph remained abundant and searched for females on the larval food plants. Furthermore, they mated frequently with autumn morph females. Autumn morph males seemed to be sexually less active or inactive before hibernation. Microscopic examinations of the spermathecae were made in mated autumn-morph females collected before and after hibernation. The results indicate that sperm is passed by the males at autumnal copulation. The sperm may be stored in the female reproductive tract and utilized for fertilization in spring. This supposition is strongly supported by field data; that is, once-mated autumn-morph females laid fertilized eggs in spring. Finally, physiological basis of the prediapause copulation, its adaptive signficance and the behavioral advantage inE. hecabe are discussed from the viewpoint of seasonal adaptation.