Reproductive success by large, closely related males facilitated by sperm storage in an aggregate breeding amphibian

The outcome of sexual selection on males may depend on female mate choice and male–male competition as well as postcopulatory processes such as cryptic female choice and sperm competition. We studied the outcome of sexual selection in the spotted salamander ( Ambystoma maculatum ), specifically examining the role of body size and relatedness on male reproductive success. Using controlled mating experiments in the field, we gave females access to three males of different sizes. We used seven microsatellite loci to determine paternity in the resulting larvae, estimate relatedness ( r ) between females and their mates, and calculate md ² (a measure of within-individual genomic divergence), heterozygosity, and standardized heterozygosity in the larvae. Both body size and relatedness to the female were significant predictors of male reproductive success. The relatedness of the males available to a female did not influence the amount of stored sperm she used to sire her larvae. Nonetheless, computer simulations showed that the average md ², heterozygosity, and standardized heterozygosity of the offspring were lower than expected by random mating. These differences are due to the use of stored sperm to fertilize some eggs; md ², heterozygosity, and standardized heterozygosity of larvae sired by stored sperm were significantly lower than those of larvae sired by the experimental males. These results suggest that relatedness may further influence a male's long-term reproductive success by determining whether his sperm is stored for later breeding seasons. Sexual selection in this salamander likely involves a complex interaction among many factors and may act over many seasons.     

SPERM COMPETITION AND THE EVOLUTION OF NUPTIAL FEEDING BEHAVIOR IN THE CRICKET,GRYLLODES SUPPLICANS (WALKER)

The pattern of sperm predominance in doubly mated female crickets, Gryllodes supplicans, was investigated using a radiation-sterility technique. Female G. supplicans made significant use of sperm from both males in fertilizing eggs; overall, first males to mate enjoyed a small advantage, fertilizing about 60% of the offspring produced subsequent to the second mating. The combined use of the sperm of both males in fertilizing eggs occurred soon after the second mating; evidently, mixing of ejaculates within a female's spermatheca does occur. Male G. supplicans provide females with a nuptial gift, the spermatophylax, which influences the time at which a female removes the externally attached sperm-ampulla; this in turn determines the quantity of sperm that is transferred. Moreover, the degree of sperm precedence achieved by a male may be positively related to the time at which the female removes his sperm ampulla. Thus males, by feeding females, ensure not only that a sufficient number of sperm are transferred to fertilize all of a female's eggs, but also may increase the certainty of their paternity. In mating systems in which females control sperm transfer and paternity is influenced by numbers of sperm (i.e., numerical sperm competition), an increase in prezygotic investment in females may be an adaptive male response.     

Determinants of sperm precedence in a noctuid moth Heliothis virescens: a role for male age

1. Females of the noctuid moth Heliothis virescens F. mate more than once. Thus, sperm from two or more males normally compete for fertilisations within the female reproductive tract. The eggs are typically fertilised by sperm from only one male, either the female's last mate or an earlier mate. Twice‐mated females store only one ejaculate's worth of fertilising sperm (eupyrene) but nearly two ejaculates' worth of a nonfertilising sperm morph (apyrene), which is thought to play a role in sperm competition. 2. The mechanism of sperm use in H. virescens was investigated by examining factors that vary with paternity, which was assigned based on allozyme variation. The factors included male and female body masses and ages, male genital characters, the size of the sperm package, and the number of sperm stored by the female. 3. One male typically gained sperm precedence; this was nearly twice as likely to be the second male as it was to be the first. Two factors were found to vary significantly with paternity: female mass and male age. The second male to mate was more likely to gain sperm precedence if the female was larger and if the male was older than the female's first mate. 4. The significance of male age and female mass to several hypothetical models of the mechanism of sperm use is discussed.     

Indirect parental effects on offspring viability by egg-derived fluids in an external fertilizer

The capacity for parents to influence offspring phenotypes via nongenetic inheritance is currently a major area of focus in evolutionary biology. Intriguing recent evidence suggests that sexual interactions among males and females, both before and during mating, are important mediators of such effects. Sexual interactions typically extend beyond gamete release, involving both sperm and eggs, and their associated fluids. However, the potential for gamete-level interactions to induce nongenetic parental effects remains under-investigated. Here, we test for such effects using an emerging model system for studying gamete interactions, the external fertilizer Mytilus galloprovincialis. We employed a split-ejaculate design to test whether exposing sperm to egg-derived chemicals (ECs) from a female would affect fertilization rate and offspring viability when those sperm were used to fertilize a different female''s eggs. We found separate, significant effects of ECs from non-fertilizing females on both fertilization rate and offspring viability. The offspring viability effect indicates that EC-driven interactions can have nongenetic implications for offspring fitness independent of the genotypes inherited by those offspring. These findings provide a rare test of indirect parental effects driven exclusively by gamete-level interactions, and to our knowledge the first evidence that such effects occur via the gametic fluids of females.     

SPERM MIXING IN THE COCKROACH DIPLOPTERA PUNCTATA

Females of the viviparous cockroach Diploptera punctata store sperm from their first mating, and do not remate until after giving birth to their first batch of young. The irradiated male technique was used to determine the outcome of sperm competition in the second batch of eggs of females mated sequentially to normal and irradiated males. It is estimated that the second male to mate with a female fertilizes approximately two thirds of the eggs in a female's second batch of eggs. Direct evidence for sperm mixing was obtained. Undeveloped eggs (fertilized by irradiated sperm) and developing embryos (fertilized by normal sperm) were found interspersed throughout oothecae that were extruded from females, demonstrating that normal and irradiated sperm were released from the spermathecae at oviposition and that they competed for fertilizations.     

A female nervous system is necessary for normal sperm storage in Drosophila melanogaster: a masculinized nervous system is as good as none

A male Drosophila melanogaster deposits many more sperm in a female''s bursa copulatrix than are stored in her ventral receptacle or paired spermathecae soon after copula has ended. The remaining sperm are expelled by the female. These observations suggest a sexual conflict over the processes involved in sperm storage. We used genetically manipulated flies to study the role of the central nervous system in sperm storage. Flies with female bodies but masculinized nervous systems, or isolated female abdomens, stored significantly fewer sperm than did control females. Furthermore, compared with control flies, there were relatively more sperm in the ventral receptacle and relatively fewer in the spermathecae. These results suggest that the female nervous input counteracts the male''s attempts to force sperm into the ventral receptacle during copula and promotes active transport of sperm to the spermathecae during and after copula. The female is clearly a very active partner in influencing processes involved in sperm competition, especially as only stored sperm can be used later to fertilize eggs. To our knowledge, this is the first study to show directly the involvement of the female nervous system in sperm storage.     

Breeding Strategies in Females of the Parasitoid Wasp Spalangia endius: Effects of Mating Status and Size

Does the mating status or body size of a female parasitoid wasp affect her host size choice or propensity to burrow? In Spalangia endius, using smaller hosts appears to reduce a female's cost of parasitization but not her son's fitness. However, virgin females, which produce only sons, did not preferentially parasitize smaller hosts. Mated females also showed no host size preference. Mated females burrowed more than virgins in the presence of hosts, although not in their absence. Burrowing may reduce a mated female's harassment from males, and not burrowing may increase a virgin female's chance of mating because males avoid burrowing. Mating did not increase female longevity. Greater female size increased the offspring production of mated females burrowing for hosts but not in the absence of burrowing and not in virgin females. A female's size had no significant effect on whether her first drill attempt was on a large or a small host or on the duration of her successful drills.     

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more,but females influence insemination success by removing spermatangia

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.     

Sperm Swimming Velocity Predicts Competitive Fertilization Success in the Green Swordtail Xiphophorus helleri

Sperm competition is expected to favour the evolution of traits that influence the performance of sperm when they compete to fertilize a female''s eggs. While there is considerable evidence that selection favours increases in sperm numbers, much less is known about how sperm quality contributes towards competitive fertilization success. Here, we determine whether variation in sperm quality influences competitive fertilization success in the green swordtail Xiphophorus helleri, a highly promiscuous livebearing fish. We use artificial insemination as a method of controlled sperm delivery and show that sperm swimming velocity is the primary determinant of fertilization success when ejaculates from two males compete to fertilize a female''s eggs. By contrast, we found no evidence that sperm length had any effect on siring success. We also found no evidence that pre- and postcopulatory sexual traits were phenotypically integrated in this species, suggesting that the previous observation that reproductive skew favours males with high mating rates is unlikely to be due to any direct association between sperm quality and male sexual ornamentation.     

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.     

Field experiments on the distributions of eggs of different phosphoglucomutase (PGM) genotypes in the yellow dung fly Scathophaga stercoraria (L.)

Female yellow dung flies can, in the laboratory, influence the probability that stored sperm from different males are used to fertilize eggs. This matches offspring phosphoglucomutase genotypes to the environmental conditions in which the larvae will grow, increasing larval growth success. We conducted field experiments in which dung topology or shading conditions were controlled. The proportions of the five common phosphoglucomutase alleles in eggs laid in north-facing slopes or in shaded conditions was related to their electrophoretic mobility. We suggest that females lay eggs of different genotypes, by appropriately choosing their fathers, in different places.     

Wild yellow dung fly females may not select sperm based on dung pat microclimate but could nevertheless benefit from polyandry

Molecular techniques have substantially improved our knowledge of postcopulatory sexual selection. Nevertheless, studies examining sperm utilization in natural populations of nonsocial insects are rare, support for sperm selection (biased use of stored sperm, e.g. to match offspring genotypes to prevailing environmental conditions) is elusive, and its relevance within natural populations unknown. We performed an oviposition site choice experiment in the field where female yellow dung flies Scathophaga stercoraria could deposit eggs into three different microenvironments on a dung pat (the east–west ridge, north- or south-exposed side), and genotyped the offspring and sperm remaining in storage after oviposition. Females exhibited plasticity in the number of eggs deposited according to pat age. Additionally, temperature strongly influenced egg placement: the warmer the temperature, the higher the proportion of eggs laid into the north-exposed side of dung. The number of ejaculates in storage differed amongst spermathecae, and females stored sperm from more males than fathered their offspring (2.11 sires vs. 2.84 males within sperm stores). Mean last male paternity was 83.4%, roughly matching previous laboratory estimates. Importantly, we found no evidence that females selectively lay eggs of different genotypes, by biasing paternity towards certain males, depending on offspring’s microclimate. Thus, while we show female choice over number of eggs and where these are deposited, there was no evidence for sperm selection. We further revealed positive effects of multiple mating on total number of offspring and proportion of offspring emerging from the dung. We argue that the integration of field studies and laboratory experiments is essential to promote our understanding of polyandry and cryptic female choice.     

The more the better – polyandry and genetic similarity are positively linked to reproductive success in a natural population of terrestrial salamanders (Salamandra salamandra)

Although classically thought to be rare, female polyandry is widespread and may entail significant fitness benefits. If females store sperm over extended periods of time, the consequences of polyandry will depend on the pattern of sperm storage, and some of the potential benefits of polyandry can only be realized if sperm from different males is mixed. Our study aimed to determine patterns and consequences of polyandry in an amphibian species, the fire salamander, under fully natural conditions. Fire salamanders are ideal study objects, because mating, fertilization and larval deposition are temporally decoupled, females store sperm for several months, and larvae are deposited in the order of fertilization. Based on 18 microsatellite loci, we conducted paternity analysis of 24 female‐offspring arrays with, in total, over 600 larvae fertilized under complete natural conditions. More than one‐third of females were polyandrous and up to four males were found as sires. Our data clearly show that sperm from multiple males is mixed in the female's spermatheca. Nevertheless, paternity is biased, and the most successful male sires on average 70% of the larvae, suggesting a ‘topping off’ mechanism with first‐male precedence. Female reproductive success increased with the number of sires, most probably because multiple mating ensured high fertilization success. In contrast, offspring number was unaffected by female condition and genetic characteristics, but surprisingly, it increased with the degree of genetic relatedness between females and their sires. Sires of polyandrous females tended to be genetically similar to each other, indicating a role for active female choice.     

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.     

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.     

Female genotypes affect sperm displacement in Drosophila.

Differential success of sperm is likely to be an important component of fitness. Extensive variation among male genotypes in competitive success of sperm in multiply mated females has been documented for Drosophila melanogaster. However, virtually all previous studies considered the female to be a passive vessel. Nevertheless, under certain conditions female fitness could be determined by her role in mediating use of sperm from multiple males. Here we ask whether females differ among genotypes in their tendency to exhibit last-male precedence. Competition of sperm from two tester male genotypes (bwD and B3-09, a third-chromosome isogenic line from Beltsville, MD) was quantified by doubly mating female lines that had been rendered homozygous for X, second, or third chromosomes isolated from natural populations. The composite sperm displacement parameter, P2'', was highly heterogeneous among lines, whether or not viability effects were compensated, implying the presence of polymorphic genes affecting access of sperm to eggs. Genetic variation of this type is completely neutral in the absence of pleiotropy or interaction between variation in the two sexes.     

Quantity or quality? Determinants of maternal reproductive success in tropical pythons (Liasis fuscus)

A female''s reproductive output (size and number of offspring) may say little about her reproductive success; the ''quality'' of her progeny and the time that they are produced may be more important in this respect. We marked and released 1224 hatchling water pythons (Liasis fuscus) from 116 clutches of laboratory-incubated eggs, in a study site in tropical Australia. Clutches varied widely in the number of eggs, mean offspring sizes, the proportion of eggs that hatched, and recapture rates of offspring. Clutch size was not significantly correlated with the number of recaptured hatchlings in either year, nor was mean offspring size related to recapture probability. Instead, offspring recapture rates varied as a function of the time of hatching. Recapture rates were higher for hatchlings released in a year with high prey availability than in a year with few prey. In addition, recapture rates were higher for earlier-hatching clutches in the year with few prey. The other significant effect on recapture rates involved offspring viability: some clutches had a much higher proportion of surviving offspring than did others, and recapture rates were higher from clutches with higher hatching success (i.e. proportion of eggs hatching). Hence, the usual measures of reproductive output (clutch size and offspring size) offer only a poor index of a female''s reproductive success in this system.     

Asymmetric larval competition between two species of solitary parasitoid wasps: the influence of superparasitism

Abstract.

• 1 We test the hypothesis that a solitary parasitoid wasp may gain in fitness if she lays more eggs in a host.
• 2 Using heterospecific superparasitism (=multiparasitism) between the solitary aphid parasitoids, Aphidius smithi Sharma & Subba Rao and Ephedrus californicus Baker, we show that (i) a superparasitizing female's chance that her offspring will survive competition is an increasing function of egg density, and (ii) survival among same-aged larvae is independent of the oviposition sequence.
• 3 These findings on asymmetric larval competition provide indirect evidence that supports two fundamental, but untested, assumptions underlying models of adaptive superparasitism between conspecific wasps.

     

Sperm competition and the significance of female multiple mating in the predatory miteParasitus fimetorum

Females of the predatory miteParasitus fimetorum (Gamasida; Parasitina) inhabiting animal manure indiscriminately copulate with many mates. The sperm competition between the males was estimated by electrophoresis of allozymes and the effects of multiple mating on female reproduction were investigated. When females were forced to mate only once, their fecundity decreased drastically compared to the case of multiple mating (but longevity was unaffected). When one female mated with two males, the outcome of sperm competition depended greatly upon the mating interval. When the second mating occurred immediately after the first, the female fecundity decreased as in the case of single mating and the second male fertilized only a few eggs. However, when there was an interval of 1 day between the two matings, the females achieved normal fecundity and the second male fertilized approximately half the eggs. This suggests that the spermatophore deposited by the first male may act as a short-term copulatory ‘plug’ in the female's genital opening. When one female mated with several males with 1 day intervals, three or more males shared fertilization of the eggs. This study suggests that the multiple mating of females is a necessary stimulus to continue oogenesis and some physiological factors for this stimulation may exist in spermatophores.     

Coevolution of non-fertile sperm and female receptivity in a butterfly

Sexual conflict can promote rapid evolution of male and female reproductive traits. Males of many polyandrous butterflies transfer nutrients at mating that enhances female fecundity, but generates sexual conflict over female remating due to sperm competition. Butterflies produce both normal fertilizing sperm and large numbers of non-fertile sperm. In the green-veined white butterfly, Pieris napi, non-fertile sperm fill the females'' sperm storage organ, switching off receptivity and thereby reducing female remating. There is genetic variation in the number of non-fertile sperm stored, which directly relates to the female''s refractory period. There is also genetic variation in males'' sperm production. Here, we show that females'' refractory period and males'' sperm production are genetically correlated using quantitative genetic and selection experiments. Thus selection on male manipulation may increase the frequency of susceptible females to such manipulations as a correlated response and vice versa.