Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Remating, sperm transfer, and sperm displacement in the cactophilic species Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae)

The mating system of Drosophila buzzatii is characterized by short copulation duration, frequent remating in both males and females, and male ejaculate partitioning. Additional features of the system are strong sperm displacement and a high frequency of sterile matings. Remating frequencies and the effects of remating on various mating parameters were studied. In order to characterize variation, five isofemale lines from geographically distant localities in Australia (three localities), Brazil and the Canary Islands were used. Mating parameters studied were: premating time, copulation duration, interval between successive matings, and progeny number as a measure of sperm transfer. Variation for sperm displacement was studied in crosses between laboratory stocks and a number of isofemale lines from Australia. There were significant between‐line differences in female remating frequencies, premating time, copulation duration, interval between successive matings, and progeny numbers, indicating genetic variation for these traits. Females from the five lines mated on average 1.6 to 3.1 times in 4 h, with a maximum of eight matings for one female. The males were given a maximum of ten virgin females in sequence and more than one‐third of the males mated all ten females in the 2 h observation period. Copulation duration decreased and interval between matings increased with copulation number in multiply mated males. Mean copulation duration was c. 2 min. Sperm transfer, measured as the average number of progeny from a single mating, was low (c. 25) and multiply mated females gave more progeny than single mated females, although with much lower progeny numbers than observed in wild‐caught non‐virgin females. A surprisingly high proportion of observed matings gave no progeny, i.e. they were sterile matings. Sperm displacement was strong in most crosses and remained strong in multiply mated females. The results are discussed in relation to the evolution of mating patterns in Drosophila.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

Reproductive costs of mating with a sibling male: sperm depletion and multiple mating in Cephalonomia hyalinipennis

Polygynous parasitoid males may be limited by the amount of sperm they can transmit to females, which in turn may become sperm limited. In this study, I tested the effect of male mating history on copula duration, female fecundity, and offspring sex ratio, and the likelihood that females will have multiple mates, in the gregarious parasitoid Cephalonomia hyalinipennis Ashmead (Hymenoptera: Bethylidae: Epyrinae), a likely candidate for sperm depletion due to its local mate competition system. Males were eager to mate with the seven females presented in rapid succession. Copula duration did not differ with male mating history, but latency before a first mating was significantly longer than before consecutive matings. Male mating history had no bearing on female fecundity (number of offspring), but significantly influenced offspring sex ratio. The last female to mate with a given male produced significantly more male offspring than the first one, and eventually became sperm depleted. In contrast, the offspring sex ratio of first‐mated females was female biased, denoting a high degree of sex allocation control. Once‐mated females, whether sperm‐depleted or not, accepted a second mating after a period of oviposition. Sperm‐depleted females resumed production of fertilized eggs after a second mating. Young, recently mated females also accepted a second mating, but extended in‐copula courtship was observed. Carrying out multiple matings in this species thus seems to reduce the cost of being constrained to produce only haploid males after accepting copulation with a sperm‐depleted male. I discuss the reproductive fitness costs that females experience when mating solely with their sibling males and the reproductive fitness gain of males that persist in mating, even when almost sperm‐depleted. Behavioural observations support the hypothesis that females monitor their sperm stock. It is concluded that C. hyalinipennis is a species with a partial local mating system.     

Last male sperm precedence in a polygamous squid

Differential sperm usage from consecutive matings, or sperm precedence, is vital in determining male reproductive success and the outcome of sperm competition for many organisms. Sperm precedence also has significant consequences for mating system dynamics, including both male and female adaptations for increasing reproductive success and avoiding the costs of mating. Despite sexual selection being a strong driver of reproductive behaviour and morphology in cephalopods, surprisingly few studies have investigated sperm dynamics in this group. To redress this gap, we experimentally quantified sperm precedence patterns in the dumpling squid, Euprymna tasmanica, controlling for recent male mating history (first vs. second mating), mating position, and mating frequency. We found that the last male to mate gains an advantage in this system, with the second mating male siring up to 75% of offspring at the beginning of the laying period. The proportion of offspring attributable to the second mating male decreases to 54% by the end of the laying period, potentially as a result of changes in the velocity or number of sperm released from spermatangia over time. There is also significant variation among females in patterns of sperm precedence. This variation was not associated with whether it was the male's first or second mating, male mass, the duration of copulation or the number of pumps (sperm removal behaviour) by the second male. If widespread in cephalopods, last male sperm precedence could help to explain the evolution of mate guarding (or long copulation duration) and sperm removal behaviour in this group.     

Strong cryptic prezygotic isolation despite lack of behavioral isolation between sympatric host races of the leaf beetle Lochmaea capreae

One of the major goals in speciation research is to understand which isolation mechanisms form the first barriers to gene flow. This requires examining lineages that are still in the process of divergence or incipient species. Here, we investigate the presence of behavioral and several cryptic barriers between the sympatric willow and birch host races of Lochmaea capreae. Behavioral isolation did not have any profound effect on preventing gene flow. Yet despite pairs mating indiscriminately, no offspring were produced from the heterospecific matings between birch females and willow males due to the inability of males to transfer sperm to females. We found evidence for differences in genital morphology that may contribute to failed insemination attempts during copulation. The heterospecific matings between willow females and birch males resulted in viable offspring. Yet fecundity and hatchability was remarkably reduced, which is likely the result of lower efficiency in sperm transportation and storage and lower survival of sperm in the foreign reproductive tract. Our results provide evidence for the contribution of several postmating‐prezygotic barriers that predate behavioral isolation and act as primary inhibitors of gene flow in this system. This is a surprising, yet perhaps often overlooked feature of barriers acting early in sympatric speciation process.     

A nonsemen copulatory fluid influences the outcome of sperm competition in Japanese quail

Sperm competition is a powerful and widespread evolutionary force that drives the divergence of behavioural, physiological and morphological traits. Elucidating the mechanisms governing differential fertilization success is a fundamental question of sperm competition. Both sperm and nonsperm ejaculate components can influence sperm competition outcomes. Here, we investigate the role of a nonsemen copulatory fluid in sperm competition. Male Japanese quail possess a gland that makes meringue‐like foam. Males produce and store foam independent of sperm and seminal fluid, yet transfer foam to females during copulation. We tested whether foam influenced the outcome of sperm competition by varying foam state and mating order in competitive matings. We found that the presence of foam from one male decreased the relative fertilization success of a rival, and that foam from a given male increased the probability he obtained any fertilizations. Mating order also affected competitive success. Males mated first fertilized proportionally more eggs in a clutch and had more matings with any fertilizations than subsequent males. We conclude that the function of foam in sperm competition is mediated through the positive interaction of foam with a male's sperm, and we speculate whether the benefit is achieved through improving sperm storage, fertilizing efficiency or retention. Our results suggest males can evolve complex strategies to gain fertilizations at the expense of rivals as foam, a copulatory fluid not required for fertilization, nevertheless, has important effects on reproductive performance under competition.     

Determinants of sperm transfer in the scorpionfly Panorpa cognate: male variation, female condition and copulation duration

Recent studies suggest that sperm production and transfer may have significant costs to males. Male sperm investment into a current copulation may therefore influence resources available for future matings, which selects for male strategic mating investment. In addition, females may also benefit from actively or passively altering the number of sperm transferred by males. In the scorpionfly Panorpa cognata, the number of sperm transferred during copulation depended on copulation duration and males in good condition (residual weight) copulated longer and also transferred more sperm. Moreover, sperm transferred and stored per unit time was higher in copulations with females in good condition than in copulations with females in poor condition. Males varied greatly and consistently in their sperm transfer rate, indicative of costs associated with this trait. The duration of the pairing prelude also varied between males and correlated negatively with the male's sperm transfer rate, but no other male character correlated significantly with male sperm transfer rate. The results are consistent with strategic mating effort but sperm transfer could also be facilitated by the physical size of females and/or females in good condition may be more cooperative during sperm transfer.     

Fertilization by proxy: rival sperm removal and translocation in a beetle

Competition between different males'' sperm for the fertilization of ova has led to the evolution of a diversity of characters in male reproductive behaviour, physiology and morphology. Males may increase sperm competition success either by enhancing the success of their own sperm or by negating or eliminating the success of rival sperm. Here, we find that in the flour beetle Tribolium castaneum, the second male to mate gains fertilization precedence over previous males'' sperm and fertilizes approximately two-thirds of the eggs. It is not known what mechanism underlies this pattern of last-male sperm precedence; however, the elongate tubules of the female sperm storage organ may encourage a ''last-in, first-out'' sperm use sequence. Here we present an additional or alternative mechanism of sperm precedence whereby previously deposited sperm are removed from the female tract by the mating male''s genitalia. In addition to providing evidence for sperm removal in T. castaneum, we also show that removed, non-self sperm may be translocated back into the reproductive tracts of new, previously unmated females, where the translocated sperm go on to gain significant fertilization success. We found that, in 45 out of 204 crosses, sperm translocation occurred and in these 45 crosses over half of the offspring were sired by spermatozoa which had been translocated between females on the male genitalia. In the natural environment of stored food, reproductively active T. castaneum adults aggregate in dense mating populations where copulation is frequent (we show in three naturally occurring population densities that copula duration and intermating intervals across three subsequent matings average 1 to 2 min). Selection upon males to remove rival sperm may have resulted in counter-selection upon spermatozoa to survive removal and be translocated into new females where they go on to fertilize in significant numbers.     

Sperm transfer and paternity in the scorpionfly <Emphasis Type="Italic">Panorpa cognata</Emphasis>: large variance in traits favoured by post-copulatory episodes of sexual selection

Post-copulatory episodes of sexual selection can be a powerful selective force influencing the reproductive success of males. In order to understand variation in male fertilisation success, we first need to consider the pattern of sperm utilisation by females following matings with more than one male. Second, we need to study those traits responsible for male success in sperm competition. Here we study both male sperm transfer characteristics as well as offspring paternity of females mated to two males in the scorpionfly Panorpa cognata. By repeatedly mating males to virgin females and interrupting copulation at defined time points, we found for all males that sperm transfer set off after approximately 40 min. During the remaining copulation, sperm transfer of individual males was continuous and with constant rate. Yet the rate of sperm transfer differed between individual males from about one sperm per minute to more than eight sperm per minute for the most successful males. In addition, we measured the fertilisation success in sperm competition of males with known sperm transfer capability. The relative number of sperm transferred by males during copulation, estimated from copulation duration and the males’ individual sperm transfer rate, explained a large proportion of variation in offspring paternity. The mode of sperm competition in this species, thus, conforms largely to a fair raffle following complete mixing of sperm prior to fertilisation. Hence, male differences in both the ability to copulate for long and of rapid sperm transfer will translate directly into differences in reproductive success.     

Heritability of sperm competition success in the bulb mite,Rhizoglyphus robini

Multiple mating by females has been proposed to function as a form of mate-choice, which implies that males should show heritable variation in sperm-competitive abilities. In this study, repeatability and heritability of sperm competition success was estimated in the bulb mite, Rhizoglyphus robini. Fertilization success of males was estimated in competition with sperm of two other males. Males differed consistently in their sperm competition success, with repeatability estimated at 0.22. The heritability of sperm competition success was estimated using parent-offspring regression, with the mean fertilization success from two matings used as a measure of each male's competitive ability. There was a significant association between the sperm competition success of fathers and sons. Narrow sense heritability (h²) was 0.284. This result supports the hypotheses proposing the multiple mating is selectively maintained in females by enhancing the reproductive success of their progeny.     

Immune investment and sperm competition in a beetle

Individuals that invest more in immunity may not be able to invest as much in o\ther life history traits. The overall effects on fitness depend on the balance of investment in life history traits and unnecessary investment in immunity may lower fitness. Adult mealworm beetles (Tenebrio molitor L.) modulate their investment according to the perceived risk of infection as larvae; the amount of investment can be assessed by body coloration. This prophylactic investment in immunity can be used to assess the costs of investment when no immune challenge is present. Whether investment in immunity is traded off against sperm competitive ability, another important fitness trait in insects, was investigated. Males that had invested more in immunity (dark males) competed against males that had invested less (light males) for fertilization of offspring. Dark males did lose sperm precedence over time, whereas light males did not. However, this decrease in sperm offensive ability may not result in decreased fitness for darker males under normal female mating frequencies; the decrease in offspring did not occur for 1 week, but females that have constant access to males mate once a day, which would negate any long‐term effects of male mating order. Thus, prophylactic investment in immunity does not produce immediate reductions in a male's ability to gain fertilizations. The costs to immune investment may be born by other fitness traits in T. molitor.     

Strategic male mating effort and cryptic male choice in a scorpionfly

In animal species with high male mating effort, males often find themselves in a dilemma: by increasing their mating effort, the gain from each copulation increases but simultaneously reduces available resources and, thus, the opportunity for future copulations. Therefore, we expect males to spend less reproductive resources on matings that provide low reproductive potential, thereby saving resources for future copulations, possibly with high-quality females, a sort of cryptic male choice. However, the strength of the trade-off between investment in a current mating and resources available for future matings must not be the same for all males. Males with relatively high mating costs should allocate their limited resources more cautiously than males with more plentiful resources. Here, we examine this prediction in the scorpionfly Panorpa cognata. Prior to copulation, males produce a large salivary mass on which females feed during copulation. We show that the production of larger salivary masses leads to longer copulations. Moreover, the size of the salivary gland and salivary mass increases with increasing male condition. However, males in poor condition make a relatively higher mating investment than males in good condition. We therefore expect male condition to influence cryptic male choice. In accordance with our hypothesis, only males in poor condition choose cryptically, producing larger salivary masses in copulations with females of high fecundity.     

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.     

SPERM DISPLACEMENT WITHOUT SPERM TRANSFER IN DROSOPHILA MELANOGASTER

In this paper we show that when Drosophila melanogaster females are mated twice, the semen of the second male causes a reduction of the effective number of resident sperm from the previous mating. This is demonstrated by two different kinds of experiments. In one set of experiments, mated females were remated to two different kinds of sterile males, one with normal semen and the other with deficient semen. The effect on the resident sperm was determined from the number of remaining progeny after mating to the sterile male, with the result that the normal semen reduced the amount of resident sperm in comparison with matings to the males with deficient semen. The second set of experiments employed interrupted matings. These experiments were based on the observation that semen is delivered before sperm during the first 5 min of copulation. The second matings were interrupted instantly, 2 min, and 4 min after the initiation of copulation. Compared to the instant interruptions, the two later interruptions had the effect of reducing the amount of resident sperm. The results of these two experiments clearly indicate that a sperm-incapacitation process plays a role in the well-documented phenomenon of sperm displacement (last-male advantage) in this species. Such a process could play a role in sperm displacement in the many cases where the mechanism is unknown.     

Genetic variation but weak genetic covariation between pre‐ and post‐copulatory episodes of sexual selection in Drosophila melanogaster

When females mate polyandrously, male reproductive success depends both on the male's ability to attain matings and on his ability to outcompete rival males in the fertilization of ova post‐copulation. Increased investment in  ejaculate components may trade off with investment in precopulatory traits due to resource allocation. Alternatively, pre‐ and post‐copulatory traits could be positively related if individuals can afford to invest heavily in traits advantageous at both episodes of selection. There is empirical evidence for both positive and negative associations between pre‐ and post‐copulatory episodes, but little is known about the genetic basis of these correlations. In this study, we measured morphological, chemical and behavioural precopulatory male traits and investigated their relationship with measures of male fitness (male mating success, remating inhibition and offensive sperm competitiveness) across 40 isofemale lines of Drosophila melanogaster. We found significant variation among isofemale lines, indicating a genetic basis for most of the traits investigated. However, we found weak evidence for genetic correlations between precopulatory traits and our indices of male fitness. Moreover, pre‐ and post‐copulatory episodes of selection were uncorrelated, suggesting selection may act independently at the different episodes to maximize male reproductive success.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

Male’s copulation investment, including spermatophore and sperm investment were very high in the Chinese bushcricket Gampsocleis gratiosa. The effects of mating status of both males and females on male’s copulation investment were examined in this study. The fresh weight of spermatophylax increased positively with the weight of males’ body. This indicated that the nutritional investment during copulation depended on male’s quality. Spermatophore investment showed insignificant differences in every copulation protocols. This finding supported the paternal investment hypothesis, that is, males contributed to their offspring with little attention to their partners. Sperm releasing per ejaculation varied significantly among the trials. Males decreased 54.19% sperm in second mating than in its first mating, demonsrated that males regarded the first mating highly, and were more prudent in subsequent mating. These males’ strategies may contribute to the viability of the offspring.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

Male’s copulation investment, including spermatophore and sperm investment were very high in the Chinese bushcricket Gampsocleis gratiosa. The effects of mating status of both males and females on male’s copulation investment were examined in this study. The fresh weight of sper-matophylax increased positively with the weight of males’ body. This indicated that the nutritional in-vestment during copulation depended on male’s quality. Spermatophore investment showed insig-nificant differences in every copula...~     

Mating with sperm-depleted males does not increase female mating frequency in the parasitoid Lariophagus distinguendus

Sexual conflicts due to divergent male and female interests in reproduction are common in parasitic Hymenoptera. The majority of parasitoid females are monandrous, whereas males are able to mate repeatedly. Thus, accepting only a single mate might be costly when females mate with a sperm‐depleted male, which may not transfer a sufficient amount of sperm. In the present study, we investigated the reproductive performance in the parasitoid Lariophagus distinguendus Först. (Hymenoptera: Pteromalidae) and studied whether mating with experimentally sperm‐depleted males increases the tendency of females to remate. Males were able to mate with up to 17 females offered in rapid succession within a 10‐h test period. The resulting female offspring, as an indirect measure of sperm transfer, remained constant during the first six matings and then decreased successively with increasing number of copulations by the males. Experimentally sperm‐depleted males continued to mate even if they transferred only small amounts or no sperm at all. Unlike males, the majority of females mated only once during a 192‐h test period. A second copulation was observed only in a few cases (maximum 16%). The frequency of remating was not influenced by the mating status of the first male the females had copulated with, suggesting that these events are not controlled by sperm deficiency of the females. Furthermore, we investigated male courtship behaviour towards mated females. Male courtship intensity towards mated females decreased with increasing time. However, females that had mated with an experimentally sperm‐depleted male did not elicit stronger or longer‐lasting behavioural responses in courting males than those that had mated with a virgin male. As the observed behaviours in L. distinguendus are known to be elicited by a courtship pheromone, these results suggest that females no longer invest in pheromone biosynthesis after mating (as indicated by ceasing behavioural responses of courting males), irrespective of whether they have received a sufficient amount of sperm or not. We discuss the results with respect to a possible mating strategy of sperm‐depleted males.     

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.