Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Does long-term storage of spermatozoa occur in the adder (Vipera berus)?

In the adder ( Vipera berus) multiple inseminations during the spring mating season have been demonstrated, with fertilization taking place several weeks after mating activities have ceased. Subsequent sperm competition in the female reproductive tract can result in within-season mixed paternity. Prolonged sperm storage between mating seasons has been suggested as another mechanism of sperm competition in the adder. Competition between new and old spermatozoa would obstruct efforts to reveal reproductive strategies that correlate observed behaviour to paternity in natural populations. In the present study, controlled breeding experiments with adders were performed and DNA fingerprinting was used to determine paternity among all offspring of a total of 15 litters. The offspring of 11 large females, that had most probably also mated in a previous season, were sired exclusively by enclosure males. This result and earlier empirical data support the hypothesis that long-term sperm storage, over one or several reproductive cycles, does not occur in the adder.     

Effects of age and repeated mating on male sperm supply and paternity in a parasitoid wasp

Post-copulatory paternity biases after female multiple mating are major constraints on both male and female reproductive systems. The outcome of paternity in certain situations is only controlled directly by male sperm stock. This was tested experimentally in the parasitoid wasp Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), in which sperm stocks are small (several hundred) and the fertilizing efficiency of stored sperm is high (the ratio of sperm stored/fertilized eggs is about 0.75). Sperm in seminal vesicles and paternity of males of different status (virgin young, virgin old, or young previously mated) were measured after female single and double mating. The amount of sperm in the seminal vesicle differed according to male status (increasing from previously mated males to old males), but there was no difference in sperm stored by females after a single mating. In double mating experiments with two males of different status, paternity increased linearly with the relative amount of sperm in seminal vesicles. Paternity distribution conforms to 'a fair raffle' of sperm from both donors following complete mixing of sperm prior to fertilization. Thus, in a female multiple mating context, male fitness depends principally on their sperm stock, which in turn depends on life history parameters, such as age and previous mating.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Female influence over offspring paternity in the red flour beetle Tribolium castaneum

In animals having internal fertilization, both sexes can potentially influence the post-copulatory processes of sperm transfer, sperm storage and sperm use for fertilization. In this experiment, we investigated whether Tribolium castaneum females can influence male paternity success following consecutive matings with two different males. We compared second male paternity success (P2) between females exposed to carbon dioxide (CO2) and control females kept in air, in both cases for 30 min between two matings. CO2 exposure inhibits muscular activity and has previously been shown to decrease sperm storage by T. castaneum females. Females exposed to CO2 after their first mating showed significantly higher P2 than control females during the later portion of a one-month oviposition period. These results are consistent with reduced storage of first male sperm by CO2-exposed females. Also, T. castaneum females showed considerable variation in spermathecal morphology, and P2 decreased with increasing spermathecal tubule volume. These results demonstrate that T. castaneum females can influence male paternity success, and suggest that differential sperm storage may be an important mechanism of post-copulatory female choice.     

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.     

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.     

Sperm precedence in a novel context: mating in a sessile marine invertebrate with dispersing sperm

The compound ascidian Diplosoma listerianum releases aquatic sperm which are dispersed passively to potential mates as individual gametes prior to storage of sperm, internal fertilization and brooding of embryos. The storage of exogenous sperm enables D. listerianum to produce a lengthy series of progeny following a brief period of mating. Molecular paternity analysis following sequential mating of colonies in laboratory culture revealed a consistent pattern with a clear initial bias in paternity towards the first of two acting males. The sites of sperm storage and fertilization and the morphology of the ovary in D. listerianum suggest that this bias reflects first-in-first-out use of individual stored gametes. The proportion of second-male paternity subsequently increased with time within the progeny arrays. This may have reflected the ageing or passive loss of first-male sperm. It is also possible that the modular nature of the organism contributed to this temporal trend: any recently budded colony modules maturing in the interval between matings would have been available exclusively to second-male sperm as virgin zooids. Two sets of mating trials were run. In the first, the collection of progeny suffered an interruption of 13 days and each male gained a larger proportion of recorded paternity within the progeny analysed when mating first rather than when mating second. In one mating combination, the first male obtained almost 100% of recorded paternity. In the second set of trials, with different clonal combinations, the complete sequence of progeny was collected and the estimated overall proportion of second-male paternity (P2) was consistently > 0.5. Taken as a whole, the results suggest that the overall P2-value can vary widely within the population studied. Proposed mechanisms of mating-order effects in species with copulatory mating include several which can have no counterpart in indirect aquatic mating since they involve the active removal, sealing off, volumetric displacement or incapacitation of first-male ejaculates. It is nevertheless clear that mating-order effects can be pronounced during the type of non-copulatory mating examined here, which is widespread in marine invertebrates.     

Multiple sperm storage organs facilitate female control of paternity

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

The outcome of sperm competition is affected by behavioural and anatomical reproductive traits in a simultaneously hermaphroditic land snail

Sperm competition is important in species with reproductive strategies that involve multiple mating and prolonged sperm storage such as the simultaneously hermaphroditic land snail Cornu aspersum. Double mating trials in this species have revealed that mating order and courtship behaviour affect paternity success. We investigated the effect of behavioural and anatomical reproductive traits on paternity success from triple mating trials. Triple mating resulted in triple fertilization in 58% of the cases whereas zero paternity was observed in 16% of sperm donors. Third sperm donors achieved higher paternity followed by first and second sperm donors. Snails with a longer epiphallus, the spermatophore forming organ, sired more offspring regardless of their mating order. Genetic compatibility between sperm donor and recipient did not influence paternity success. The results of the present study identified mating order and epiphallus length, as traits affecting the outcome of sperm competition in this species.     

An advantage for young sperm in the house cricket Acheta domesticus

We show that males of the house cricket Acheta domesticus regularly expel sperm packages (spermatophores) independently of copulation and at a rate that is not affected by the presence of females. We then show for the first time that the age of sperm affects their likelihood of being stored by females after copulation; younger sperm were overrepresented in the female sperm storage organ and therefore in the sperm population used for fertilization. Our results suggest that the reproductive success of males may increase if they deliver ejaculates with young sperm, and the results may explain why the males of several species are regularly observed to discard ejaculates. Our results also suggest that phenomena such as female multiple mating, paternity bias, and/or exaggerated ejaculate sizes may be related to the advantage both genders gain by using young sperm.     

Reconstructing paternal genotypes to infer patterns of sperm storage and sexual selection in the hawksbill turtle

Postcopulatory sperm storage can serve a range of functions, including ensuring fertility, allowing delayed fertilization and facilitating sexual selection. Sperm storage is likely to be particularly important in wide‐ranging animals with low population densities, but its prevalence and importance in such taxa, and its role in promoting sexual selection, are poorly known. Here, we use a powerful microsatellite array and paternal genotype reconstruction to assess the prevalence of sperm storage and test sexual selection hypotheses of genetic biases to paternity in one such species, the critically endangered hawksbill turtle, Eretmochelys imbricata. In the majority of females (90.7%, N = 43), all offspring were sired by a single male. In the few cases of multiple paternity (9.3%), two males fertilized each female. Importantly, the identity and proportional fertilization success of males were consistent across all sequential nests laid by individual females over the breeding season (up to five nests over 75 days). No males were identified as having fertilized more than one female, suggesting that a large number of males are available to females. No evidence for biases to paternity based on heterozygosity or relatedness was found. These results indicate that female hawksbill turtles are predominantly monogamous within a season, store sperm for the duration of the nesting season and do not re‐mate between nests. Furthermore, females do not appear to be using sperm storage to facilitate sexual selection. Consequently, the primary value of storing sperm in marine turtles may be to uncouple mating and fertilization in time and avoid costly re‐mating.     

Emasculation to plug up females: the significance of pedipalp damage in Nephila fenestrata

Female multiple mating selects for male adaptations that maximizefertilization success in a context of sperm competition. Whilemale mating strategies usually reflect a trade-off between presentand future reproduction, this trade-off is largely removed insystems where the maximum number of matings for males is verysmall. Selection may then favor extreme mechanisms of paternityprotection that amount to a maximal investment in a single mating.Males in several arthropod taxa break off parts of their copulatoryorgans during mating, and it has frequently been suggested thatmutilated males can thus secure their paternity. Nevertheless,such a mechanism has rarely been confirmed directly. Here westudy the golden orb spider Nephila fenestrata, which has amating system with potentially cannibalistic, polyandrous females,and males that are often functionally sterile after mating withone female only. We show that males in this species can indeedprotect their paternity by obstructing the female's genitalopenings with fragments of their copulatory organs.     

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Frass clearing by male pine engraver beetles (Ips pini; Scolytidae): paternal care or paternity assurance

Male parental care and paternity assurance are often associatedwith long-duration pair bonds. The mating system of the pineengraver beetle, Ips pini, includes an association between themale and female that persists for most of the prolonged oppositionperiod. The male beetles remove frass that arnmmlatn as thefemales lay their eggs in die phloem tissue of the host tree.Experiments and field observations were done to test possiblebenefits to males that stay in the galleries removing frasswhile die females are ovipositing. Two hypotheses were thatclearing frass (1) provides some form of care that results inmore offspring being produced and (2) is part of a paternityassurance mechanism. Male removal experiments in the field producedno evidence that male presence significantly influenced anyof five measures of offspring production. Laboratory experimentsin which virgin females were bred reciprocally to sterile andfertile males showed that, while there is no strong patternof last-male pr, last-male paternity does increase over time.Field observations revealed that female pine engravers oftencarry sperm from previous maringi when they solicit entry toa male's breeding gallery. The pattern of paternity and thefemale's sperm storage capacity suggest that males must maintainprolonged mating access to females in order to ensure high paternity.Hence, frass clearing is necessary to maximize paternity     

HOW WELL DO MATING FREQUENCY AND DURATION PREDICT PATERNITY SUCCESS IN THE POLYGYNANDROUS WATER STRIDER AQUARIUS REMIGIS?

Abstract.— The relationship between mating success and paternity success is a key component of sexual selection but has seldom been estimated for species in which both sexes mate with many partners (polygynandry). We used a modification of Parker's sterile male technique to measure this relationship for the water strider Aquarius remigis in 47 laboratory populations simulating natural conditions of polygynandry. We also tested the hypothesis that prolonged copulation, a characteristic of this species, enhances paternity success. Mating behavior and paternity success were assayed for four days while males and females freely interacted. Paternity success was also assayed for an additional 7 days when females were isolated from males. Mating success significantly predicted paternity success and accounted for ≤ 36% of the variance. Copulation duration was negatively related to both mating success and paternity success and did not explain any of the residual variance in paternity success. Thus, we found no evidence that prolonged copulation functions as a paternity assurance strategy in this species. Comparisons of sterile and fertile males suggested that paternity success is directly influenced by the quantity of sperm transferred. Our results support previous studies that have used mating success to estimate sexual selection, but also highlight the potential importance of sperm competition and other postinsemination processes.     

Female remating and sperm competition patterns in a terrestrial crustacean

In most species, both sexes may mate with more than one partner during their life. In terrestrial isopods (woodlice) female remating can occur within a reproductive season (immediate remating) or after a period of sexual rest and sperm storage, that is in a subsequent reproductive season (delayed remating). The pattern of sperm precedence is unknown in both cases. These two female remating patterns may shape male-male competition in different ways. To elucidate both patterns of female remating and sperm precedence, we used an albinism mutation in Armadillidium vulgare to track paternity in laboratory experiments. Males had low remating success after immediate remating attempts, mainly because of the female's refractory behaviour. However, refractory behaviour seemed to be lost after female sexual rest: delayed remating attempts were as successful as first mating attempts with virgin females. In both immediate and delayed remating, competing males had similar fertilization success, but varied in sperm precedence. We hypothesize that males might induce the refractory mating behaviour in females to ensure their paternity. This could be a strategy that evolved in woodlice after the loss of precopulatory mate guarding during adaptation to the terrestrial environment. We discuss the consequences of these findings for woodlice optimal mating strategies. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Male insemination decisions and sperm quality influence paternity in the golden orb-weaving spider

In polyandrous species, paternity may be influenced by the timingand frequency of mating. Female spiders possess 2 genital openingsthat lead to separate sperm-storage structures. Thus, even whenmating with a previously mated female, a male may reduce directsperm competition by inseminating the opposite opening to herfirst mate. Such morphology may provide females with greatercontrol over paternity. We examined simultaneously whether malesavoided already inseminated female genital openings and whetherthis behavior varied with the time between successive matings.To explore these questions, we mated female golden orb weaverspiders, Nephila edulis, each to 2 males and manipulated thetiming of their second mating. We documented male inseminationpatterns and explored the influence of male mating decisionson paternity success using the irradiated male technique. Wefound that 60% of males avoided sperm competition by discriminatingagainst inseminated genital openings. Moreover, male matingbehavior had a dramatic impact on the paternity success of irradiatedmales. When males inseminated the same genital opening, thecompetitive ability of the irradiated male's sperm was dramaticallyreduced resulting in lower paternity success. In contrast, whenthe 2 males inseminated opposite genital openings both malessired equal proportions of offspring regardless of their radiationstatus. There was no evidence that the timing of the secondmating affected patterns of paternity. Our data suggest thatdifferences in sperm quality may influence paternity successof N. edulis males under a sperm-competitive scenario. In contrast,females appear to have limited postmating control over paternity.     

Relative fitness components measured with competitive PCR

In mating systems with sperm competition, paternity is frequently established with modern DNA techniques. These methods are often expensive and cumbersome, and can be especially difficult for highly fecund species. An additional objective of many paternity studies is to discover the relationship between sperm number and paternity. We present here a competitive polymerase chain reaction (PCR) protocol, coupled with the use of an automated sequencer, that has two functions: (i) to measure directly relative sperm output of males in sperm competition; and (ii) to estimate paternity distributions of large numbers of offspring simultaneously. Our technique was calibrated using a microsatellite locus of the bluehead wrasse, Thalassoma bifasciatum, with the result that product ratio after competitive PCR accurately reflected the initial template proportions of known mixtures of DNA. When we applied our technique to multiple larvae of separate mating events we found that paternity distributions estimated with the competitive PCR technique closely matched the estimates derived from the traditional method of pooling paternity data from individual larvae. Finally, we compared paternity of these spawns with relative sperm contribution estimates. This comparison suggests that ejaculate size alone does not predict a male's proportion of paternity within the group.     

Female mate choice predicts paternity success in the absence of additive genetic variance for other female paternity bias mechanisms in Drosophila serrata

After choosing a first mate, polyandrous females have access to a range of opportunities to bias paternity, such as repeating matings with the preferred male, facilitating fertilization from the best sperm or differentially investing in offspring according to their sire. Female ability to bias paternity after a first mating has been demonstrated in a few species, but unambiguous evidence remains limited by the access to complex behaviours, sperm storage organs and fertilization processes within females. Even when found at the phenotypic level, the potential evolution of any mechanism allowing females to bias paternity other than mate choice remains little explored. Using a large population of pedigreed females, we developed a simple test to determine whether there is additive genetic variation in female ability to bias paternity after a first, chosen, mating. We applied this method in the highly polyandrous Drosophila serrata, giving females the opportunity to successively mate with two males ad libitum. We found that despite high levels of polyandry (females mated more than once per day), the first mate choice was a significant predictor of male total reproductive success. Importantly, there was no detectable genetic variance in female ability to bias paternity beyond mate choice. Therefore, whether or not females can bias paternity before or after copulation, their role on the evolution of sexual male traits is likely to be limited to their first mate choice in D. serrata.