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.     

Prior oviposition, female receptivity and last-male sperm precedence in the cosmopolitan pest Callosobruchus maculatus (Coleoptera: Bruchidae)

Sperm competition studies have shown that P₂ (the proportion of ova fertilized by the last male to mate) increases as the interval between inseminations is experimentally increased. Variation in the number of sperm in storage is associated with sperm use (or loss) from the female's sperm stores between copulations (fewer sperm from previous mates at the time of the last copulation) and with the extent of prior oviposition and female receptivity to further copulation: females that lay many eggs tend to have few remaining sperm in storage and to be more receptive to further copulation. Using the bruchid beetle Callosobruchus maculatus, we examined the effect of prior oviposition and female receptivity to further copulation on the extent of last-male sperm precedence (measured as P₂). Extent of prior oviposition was experimentally manipulated independently of the intermating interval by altering the availability of oviposition sites between inseminations. Females given few or no oviposition sites laid fewer eggs, were less receptive and had a lower P₂ than females given abundant oviposition sites. To examine the effect of female receptivity on P₂ independently of prior oviposition, we examined the outcome of sperm competition experiments using (1) females from lines that had been selected for different latencies to copulation and (2) natural variation in female latency to receptivity. Female receptivity to further copulation had no detectable effect on P₂. When oviposition resource is abundant, female receptivity may be a poor predictor of current sperm load.     

Remating inhibition in the melon fly,Bactrocera (=Dacus) cucurbitae (Diptera: Tephritidae): Copulation with spermless males inhibits female remating

Eight hour copulation of the melon fly,Bactrocera cucurbitae, which usually mates at dusk and finishes copulation at dawn, inhibited female remating, while 3 h copulation did not. Copulation of females with either normal or virgin sterile males inhibited female remating. Sperm-depleted sterile males inhibited female remating at the same rate as normal males when the copulation duration was 8 h, indicating that existence or amount of sperm in females' spermathecae is not important in remating inhibition. Females of a wild strain remated later than females of a mass-reared strain, irrespective of strains of 1st and 2nd males. This suggests that the females may control their own remating, or that there is a difference between wild and mass-reared strains in their sensitivity to a male substance that inhibits females' receptivity.     

Female-mediated differential sperm storage in a fly with complex spermathecae, Scatophaga stercoraria

Multiple spermathecae potentially allow selective sperm use, provided that sperm from rival males are stored differentially, that is, in different proportions across storage compartments. In the yellow dung fly, Scatophaga stercoraria, females have three spermathecae arranged as a doublet and singlet. To test whether females store the sperm of rival males actively and differentially, we mated fixed male pairs to three females. After copulation, females were (1) dissected immediately before they could start laying a clutch of eggs, (2) left awake for 30 min but prevented from oviposition, or (3) anaesthetized with carbon dioxide for 30 min to interfere with the muscular control presumably required for sperm transport from the site of insemination to the spermathecae. For each female, we estimated the proportion of the second male's sperm stored in her spermathecae (S(2)value), using sperm length as a male marker. After copulation, the S(2)values in the singlet and doublet spermathecae differed significantly, indicating differential sperm storage during copulation. Postcopulatory treatment affected differential sperm storage significantly. Females dissected immediately had lower S(2)values in the doublet than in the singlet spermatheca, while females left awake showed the reverse pattern for the same two males. This reversal did not occur when females were treated with carbon dioxide. The results indicate differential storage of sperm from different males during copulation and that female muscular activity can affect storage and separation of competing ejaculates beyond copulation. Copyright 2000 The Association for the Study of Animal Behaviour.     

Females of the grasshopper Chorthippus parallelus (Zett.) do not remate for fresh sperm

The evolution of female multiple mating is still a largely debated field. Among the benefits that have been proposed to explain this risky behaviour is the replenishment of sperm reserves. Apart from an increase in total sperm number, it can be an expression of post-copulatory mate choice or can be directed towards the uptake of fresh sperm. Using fresh sperm for fertilization instead of sperm aged by storage in the female genital tract may avoid a lowered fertilization capacity, an increase in deleterious effects or a skewed offspring sex ratio. We investigated the influence of sperm age on female fitness in the grasshopper Chorthippus parallelus, a species where females mate multiply. After copulation, females store sperm over the course of weeks until fertilization. An average ejaculate of 250 000 spermatozoa exponentially declined with time within the female''s spermatheca. The number of days since copulation better explained the variation in actual sperm number than the number of pods or eggs laid. We investigated differences in female fitness parameters in two treatments. In the first, females were mated only once, while in the second, females always had freshly ejaculated sperm available. Although in our experiment, multiply mated females had heavier offspring than singly mated females, egg number per pod, hatching and fertilization success, their composite effects and offspring sex ratio did not vary with respect to season or sperm age. We therefore reject the hypothesis that the reason for remating in females of this species is the uptake of fresh sperm.     

Function of multiple sperm-storage organs in female damselflies (Ischnura senegalensis): difference in amount of ejaculate stored, sperm loss, and priority in fertilization

We studied changes in the number of sperm within two kinds of female sperm-storage organ in the damselfly Ischnura senegalensis (Odonata: Coenagrionidae): the bursa copulatrix and the spermatheca. We counted the number of sperm within each storage organ and tested their viability after a single copulation in female damselflies kept for seven days with and without oviposition. We also counted sperm and tested their viability in females that underwent an interrupted second copulation after the sperm-removal stage, and after subsequent oviposition. Our results showed that the bursa copulatrix and spermatheca have different sperm storage roles. Immediately after copulation, most eggs appear to have been fertilized with bursal sperm, which were positioned near the fertilization point. By seven days after copulation, a greater proportion of spermathecal sperm were used for fertilization, as the number of bursal sperm had decreased. We hypothesize that female damselflies use the spermatheca for long-term storage and the bursa copulatrix for short-term storage: bursal sperm are more likely to be used for fertilization but may have a higher risk of mortality due to sperm removal by a competing male and/or sperm expelling by the female, whereas spermathecal sperm are safer but will be used for fertilization only after their release from the spermatheca.     

Expandable spermatheca influences sperm storage in the simultaneously hermaphroditic snail Arianta arbustorum

Summary

In many simultaneously hermaphroditic land snail species, the sperm storage organ (spermatheca) is highly structured, suggesting that the female function might be able to influence offspring paternity. Physical properties of the sperm storage organ, including its initial size and sperm storage capacity, may also affect fertilization patterns in multiply mated snails. We examined the structure, volume and tubule length of empty spermathecae in the land snail, Arianta arbustorum, and assessed differences in spermatheca size following a single copulation. The number of spermathecal tubules ranged from 2–7, but was not correlated with the volume of empty spermathecae. The volume of sperm stored in the spermatheca after a copulation was correlated with neither the number of spermathecal tubules nor copulation duration. Mean spermathecal volume more than doubled between two and thirty-six hours after sperm uptake, but the length of the spermathecal tubules did not change. Interestingly, the volume of sperm stored in the spermatheca seems not to be related to the size of the spermatophore and thus not to the number of sperm received (= allosperm). The amount of allosperm digested in the bursa copulatrix was highly variable and no significant relationship with the size of the spermatophore received was found. These findings suggest that numerical aspects of sperm transfer are less important in influencing fertilization success of sperm in A. arbustorum than properties of the female reproductive tract of the sperm receiver.     

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     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.     

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.     

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.     

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.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

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.     

Short-and long-term sperm precedence in the beetle Tenebrio molitor: a test of the 'adaptive sperm removal' hypothesis

Abstract. Sperm removal in Tenebrio molitor L. (Coleoptera: Tenebrionidae) has been proposed as an adaptation to sperm competition and has been documented when the remating interval between successive copulations is short, but not when it is long (Gage, 1992). If sperm removal is adaptive, it follows that there should be different fertilization outcomes from double matings with different remating intervals.
Sperm precedence patterns were assessed using reciprocal double matings of normal and γ-irradiated (sterile) virgin males of controlled size and age with virgin females of controlled size and age.
Immediate last male sperm precedence was high whether the remating interval was short (<10 min) (P_2,= 0.89) or long (24h) (P₂= 0.92).
Sperm precedence in eggs laid in a 16-day period after the last copulation showed no difference in the pattern of change between females with short and long remating intervals.
By examining the aedeagus of males we show that sperm are removed at the end of copulation by the first and the second male to mate with a virgin female regardless of whether the remating interval is short or long.
We conclude that sperm removal is unlikely to be the primary mechanism by which males gain such high levels of last male sperm precedence.     

Material Benefits from Multiple Mating in Female Mealworm Beetles (Tenebrio molitor L.)

In yellow mealworm beetles (Tenebrio molitor), females are sexually receptive throughout their adult lives. We examined how access to mates affected female fecundity by varying the number of matings per female and quantifying cumulative egg production. Also, we dissected females at successive intervals after a single mating to assess the relationship among time since mating, sperm supplies, egg load, and oviposition rate. Females that mated at intervals greater than 2 days did not produce as many eggs as females that mated every 2 days or were allowed to mate ad libitum. Dissections showed that the amount of sperm remaining in a female spermatheca was correlated with the number of eggs she had laid recently, which suggests sperm replenishment as the material benefit gained through multiple mating. However, females mate more frequently than necessary for sperm replenishment, and therefore material benefits alone may not fully explain the continuous receptivity of T. molitor females.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

The male ejaculate as inhibitor of female remating in two tephritid flies

The inhibition of female receptivity after copulation is usually related to the quality of the first mating. Males are able to modulate female receptivity through various mechanisms. Among these is the transfer of the ejaculate composed mainly by sperm and accessory gland proteins (AGPs). Here we used the South American fruit fly Anastrepha fraterculus (where AGP injections inhibit female receptivity) and the Mexican fruit fly Anastrepha ludens (where injection of AGPs failed to inhibit receptivity) as study organisms to test which mechanisms are used by males to prevent remating. In both species, neither the act of copulation without ejaculate transfer nor sperm stored inhibited female receptivity. Moreover, using multiply mated sterile and wild males in Mex flies we showed that the number of sperm stored by females varied according to male fertility status and number of previous matings, while female remating did not. We suggest female receptivity in both flies is inhibited by the mechanical and/or physiological effect of the full ejaculate. This finding brings us closer to understanding the mechanisms through which female receptivity can be modulated.     

Adaptive control of copulation duration by males under sperm competition in the mite,Macrocheles muscaedomesticae

In a manure-inhabiting predatory mite, Macrocheles muscaedomesticae (Gamasida, Macrochelidae), when the female mates with two males, the first male takes nearly perfect fertilization priority (Yasui, 1988). The present study examined whether the first-male's sperm precedence is influenced by the copula-duration of the first and second males mating with the same female, and whether males control their copulation duration by assessing the probability that the mate has been inseminated by other males. Results of the artificial interruption of copulation showed that sperm precedence value, P₂ (the proportion of the offspring fathered by the second male), was negatively correlated with the copulation duration of the first male but positively correlated with that of the second male. There was a threshold (ca. 180–300 seconds) in the first-male's copulation duration beyond which P₂ decreased drastically; when length of the first copulation exceeded this threshold, the second males did not fertilize eggs, whereas they fertilized more than half of the eggs when the first-copulation duration was shorter than the threshold. Almost all males copulated for a longer period (average 509.8 seconds) than this threshold if the copulation duration of the previous male had not exceeded the threshold, but if it was longer than the threshold, second males had shortened their copulation (67.6 seconds). These results suggest that males are able to assess the insemination status of their mates and to adjust their copulation duration depending on the probability of fertilizing eggs by their own sperm. A mechanistic explanation for sperm precedence (i.e., plug-formation within sperm receptive organ of the females) is proposed.