Females suffer a reduction in the viability of stored sperm following an immune challenge

Despite the ubiquitous nature of sperm storage in invertebrates, relatively little is known about its costs, or the impact that immune activation can have on a female's ability to maintain viable sperm stores. We explored the effects of an immune challenge on sperm storage under food‐limited and ad libitum conditions in the field cricket, Teleogryllus oceanicus, by injecting mated adult females with either a LD₅ dose of live bacteria or a nonpathogenic immune elicitor [lipopolysaccharide (LPS)] and then scoring the viability of their stored sperm. Females that were infected with bacteria showed a reduction in the viability of stored sperm 48 h after infection; interestingly, this pattern was not evident when females were injected with LPS. Reduction in sperm viability post‐infection may reflect a reproductive trade‐off between immune function and sperm store maintenance, as only females injected with bacteria showed an elevated antibacterial immune (lytic) response. Alternatively, bacteria may act directly on sperm quality. Dietary manipulations showed that lytic activity in females is condition dependent, irrespective of their immune challenge treatment. Diet affected the ability of females to maintain the viability of stored sperm, suggesting that sperm storage is condition dependent. That bacterial infection associated with a reduction in stored sperm quality has potentially important implications for the outcomes of sperm competition in T. oceanicus and in other species in which females store sperm between matings.     

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.     

Male scorpionflies assess the amount of rival sperm transferred by females' previous mates

Theory predicts that when sperm compete numerically, selection will favor males who vary the number of sperm they transfer with the immediate level of sperm competition. In this study, I measured male mating investment in response to both female mating status (virgin vs. mated) and the number of foreign sperm stored by females in a previous mating in the scorpionfly Panorpa cognata. Female sperm storage was manipulated by interrupting copulations at different time points. Female mating status did not significantly influence male mating investment, but resource-limited males invested strategically in relation to the amount of sperm stored by females in a previous mating. I found continuously decreasing male investment in response to increasing amounts of competing sperm. These results demonstrate an unprecedented male ability to assess the number of sperm stored by females. As a result, males are capable of an extraordinarily fine-tuned reaction to the intensity of sperm competition.     

Multiple paternity in a natural population of a salamander with long-term sperm storage

Sperm competition appears to be an important aspect of any mating system in which individual female organisms mate with multiple males and store sperm. Post-copulatory sexual selection may be particularly important in species that store sperm throughout long breeding seasons, because the lengthy storage period may permit extensive interactions among rival sperm. Few studies have addressed the potential for sperm competition in species exhibiting prolonged sperm storage. We used microsatellite markers to examine offspring paternity in field-collected clutches of the Ocoee salamander (Desmognathus ocoee), a species in which female organisms store sperm for up to 9 months prior to fertilization. We found that 96% of clutches were sired by multiple males, but that the majority of females used sperm from only two or three males to fertilize their eggs. The high rate of multiple mating by females suggests that sperm competition is an important aspect of this mating system. Comparison of our data with those of other parentage studies in salamanders and newts reveals that multiple mating may be common in urodele amphibians. Nevertheless, the number of males siring offspring per clutch in D. ocoee did not differ appreciably from that in other species of urodeles with shorter storage periods.     

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

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

The evolution of polyandry: an examination of the genetic incompatibility and good‐sperm hypotheses

I have examined the adaptive significance of polyandry using the Australian field cricket Teleogryllus oceanicus. Previous studies of polyandry have examined differences in offspring production by females mated multiply to a single male or females mated multiply to different males. Here I combine this approach with a study of parentage of offspring produced in the later group. Females mated to two different males had a higher proportion of their eggs hatching than did females mating twice with a single male. Offspring fitness parameters were not effected. There was little evidence to suggest that females elevate their hatching success via fertilizing their eggs with sperm from genetically compatible males. Although the average paternity points towards random sperm mixing, there was considerable individual variation in sperm competition success. Patterns of parentage were consistent across females mating twice or four times. Sperm competition success was not related to offspring viability or performance. Thus, the notion that competitively superior sperm produce competitively superior offspring is not supported either. The mechanism underlying increased hatching success with polyandry requires further study.     

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.     

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.     

Paternity analysis of wild‐caught females shows that sperm package size and placement influence fertilization success in the bushcricket Pholidoptera griseoaptera

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a ‘fair raffle’. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1–6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.     

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.     

Strategic sperm allocation in the Small White butterfly Pieris rapae (Lepidoptera: Pieridae)

1. In species where females mate multiply, it is important for males to recuperate quickly in order to maximize their fertilization success. Butterflies produce a spermatophore at mating containing accessory secretions and sperm of two types: a large number of non-fertile 'apyrene' sperm and fewer fertile 'eupyrene' sperm. Many butterfly species eclose with most nutrients for reproduction already present. Males must therefore decide how to allocate resources to the various spermatophore components at any given mating.
2. Recovery rates of apyrene and eupyrene sperm number and spermatophore size was studied in the polyandrous Small White butterfly Pieris rapae . The mass of the first spermatophore increases with time since eclosion, as does the number of both types of sperm. Similarly, on a male's second mating, both the mass of the spermatophore and the number of sperm increases with time since the first mating.
3. However, the rate of increase in eupyrene sperm numbers is higher after the first mating. The difference in rate of increase may be the result of different probabilities of virgin and non-virgin males obtaining future matings.
4. Males have a sperm storage organ, the duplex, in which they retain sperm after their first mating. This ensures that high sperm numbers are available for their second mating, even when remating only 1 h later. Thus, males do not ejaculate all available sperm on any given mating, and seem to have different strategies on their first and second matings.
5. It can be argued that Small White butterfly males allocate sperm strategically according to the probability of obtaining subsequent matings, and the level of sperm competition.     

Sperm length influences fertilization success during sperm competition in the snail Viviparus ater

Sperm form and size is tremendously variable within and across species. However, a general explanation for this variation is lacking. It has been suggested that sperm size may influence sperm competition, and there is evidence for this in some taxa but not others. In addition to normal fertilizing sperm, a number of molluscs and insects produce nonfertile sperm that are also extremely morphologically variable, and distinct from fertilizing forms. There is evidence that nonfertile sperm play an indirect role in sperm competition by decreasing female remating propensity in Lepidopterans, but in most taxa the function of parasperm is unknown. We investigated the role of nonfertile (oligopyrene) sperm during sperm competition in the fresh water snail Viviparus ater. Previous studies found that the proportion of oligopyrene sperm increased with the risk of sperm competition, and hence it seems likely that these sperm influence fertilization success during competitive matings. In mating experiments in which females were sequentially housed with males, we examined a range of male characteristics which potentially influence fertilization success. We found that the size of oligopyrene sperm was the best predictor of fertilization success, with males having the longer sperm siring the highest proportion of offspring. Furthermore, we found a positive shell size and sperm concentration effect on paternity, and females with multiply sired families produced more offspring than females mating with only one male. This result suggests polyandry is beneficial for female snails.     

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.     

Male sperm donation consequences in single and double matings in Anisopteromalus calandrae

Abstract. The number of spermatozoa that a male transfers to the female during copulation is a main component of its individual fitness, especially under the pressure of sperm competition. This paper presents experimental results on the direct relationship between the male's sperm investment and its paternity in the offspring of dual-mated females. An eye colour mutant (red-eyed) is used to study the differences in the mating and fertilization abilities of males through observation of single and dual matings of females in Anisopteromalus calandrae (Hymenoptera, Chalcidoidea, Pteromalidae). Experimentally, females accept dual matings only in the simultaneous presence of two males. Counts of spermatozoa in the seminal vesicles of virgin males show that red-eyed males have more sperm than wild-eyed ones (approximately 1.46-fold greater). Red- and wild-eyed males do not differ in their mating behaviour and females mate indifferently with both phenotypes. Compared with once-mated females, double-mated females increase neither sperm storage nor lifetime fecundity, and the offspring sex ratio is female-biased. Females mated with two males of different phenotypes produce offspring of both phenotypes throughout their reproductive life, whatever the order of males in the copulation sequence. Any mating pattern appears to produce more red- than wild-eyed offspring (between 1.45- and 1.88-fold greater). Thus, proportions of offspring of each male match the proportions of their sperm potential. With no preference of female for red-eye or wild-eye males being demonstrated at either behavioural or physiological levels, a male's investment in sperm quantity appears to determine its individual reproductive success, at least in these experimental conditions.     

Mating strategy in Aleochara bilineata: sperm storage and allocation

Abstract By contrast to females that can maximize reproductive success with only one or a few copulations, males generally increase their fitness with frequency of mating. Sperm storage and allocation is therefore crucial for both male and female fitness. Sperm storage in Aleochara bilineata (Coleoptera; Staphylinidae) is investigated by measuring the number of spermatozoa stored in the female spermatheca after single, double or triple successive copulations with different males. The potential advantages of polyandry are studied in terms of the number of sperm stored by females mated twice with the same male (i.e. repeated copulation), compared with females mated twice with two different virgin males (i.e. polyandry). Level of polygyny is also estimated by measuring sperm allocation when ten successive mates are offered to a virgin male. Aleochara bilineata females store the sperm of the same or different males additively, suggesting no advantage for polyandry in terms of the number of sperm stored. A virgin male is able to inseminate ten different females but the number of sperm transferred decreases linearly. Finally, the latencies and durations of copulations are measured in all experiments to estimate changes according to the male or female status (i.e. virgin or mated). The latency before mating is higher when females are virgin than when females have already mated.     

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.     

Male mating behavior and ejaculate expenditure under sperm competition risk in the eastern mosquitofish

Theory predicts that males should tailor the size of their ejaculatesaccording to temporal changes in the risk of sperm competition.Specifically, males are predicted to allocate more sperm toeach mating event with increasing risk (i.e., the probabilitythat the sperm from two males will compete for fertilization).We tested this hypothesis by using the eastern mosquitofish,a freshwater species of fish exhibiting a coercive mating systemand internal fertilization. We manipulated the perception ofsperm competition risk by adjusting the sex ratio under whichmales were maintained over 8 days. Males were housed eitherwith three females and one male (simulating high sperm competitionrisk) or with four females (low risk). After the treatment,we presented each test male individually to an unfamiliar male-deprivedfemale for 30 minutes and observed his mating behavior. We thenartificially stripped the test males of sperm and recoveredthe ejaculates from the females. Our results revealed that malesin the high-risk group performed higher levels of mating activityand sperm expenditure (i.e., used up more of their sperm reserves)than did low-risk males. A control experiment, in which testmales were treated but did not participate in the mating trials,revealed no significant difference in the number of sperm strippedfrom high- and low-risk males, indicating that sperm productionwas not affected by the treatment. We did not detect a differencein the number of sperm retrieved from females among the groups,raising the possibility that some sperm are lost during matingactivity, either through ejaculation with incomplete or interruptedpenetration, or via female ejection.     

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.     

Female remating in butterflies: interaction between female genotype and nonfertile sperm

Female mating rate is fundamental to evolutionary biology as it determines the pattern of sexual selection and sexual conflict. Despite its importance, the genetic basis for female remating rate is largely unknown and has only been demonstrated in one species. In paternally investing species there is often a conflict between the sexes over female mating rate, as females remate to obtain male nutrient donations and males try to prevent female remating to ensure high fertilization success. Butterflies produce two types of sperm: fertilizing, eupyrene sperm, and large numbers of nonfertile, apyrene sperm. The function of apyrene sperm in the polyandrous, paternally investing green‐veined white butterfly, Pieris napi, is to fill the female’s sperm storage organ thereby reducing her receptivity. However, there is large variation in number of apyrene sperm stored. Here, I examine the genetic basis to this variation, and if variation in number of apyrene sperm stored is related to females’ remating rate. The number of apyrene sperm stored at the time of remating has a genetic component and is correlated with female remating tendency, whereas no such relationship is found for fertilizing sperm. The duration of the nonreceptivity period in P. napi also has a genetic component and is inversely related to the degree of polyandry. Sexual conflict over female remating rate appears to be present in this species, with males using their apyrene sperm to exploit a female system designed to monitor sperm in storage. Ejaculates with a high proportion of nonfertile sperm may have evolved to induce females to store more of these sperm, thereby reducing remating. As a counter‐adaptation, females have evolved a better detection system to regain control over their remating rate. Sexually antagonistic co‐evolution of apyrene sperm number and female sperm storage may be responsible for ejaculates with predominantly nonfertile sperm in this butterfly.     

Mating tactics in external fertilizers when sperm is limited

Among externally fertilizing animals in aquatic habitats, theproportion of a female's egg clutch that is successfully fertilizedoften falls below 100%. In many such species, particularly incoral reef fishes, males spawn daily at high frequencies, oftenwith little or no sperm competition. A major evolutionary problemfor such males is how best to allocate sperm over successivespawns. Females face the problem of ensuring complete fertilizationof their egg clutch. Here we model male and female mating tacticswhen daily sperm production is limited and with various assumptionsconcerning how differences in the number of sperm released duringa mating influence the number of eggs fertilized. The modelsreveal conditions under which males can maximize daily reproductivesuccess, either by releasing a fixed number of sperm duringall successive spawns or by matching sperm numbers to the clutchsize of their mates. These patterns of sperm allocation exertdifferent pressures on females, which may respond evolutionarilyby developing various mating tactics of their own.