High variation in sperm precedence and last male advantage in the scorpionfly Panorpa germanica L. (Mecoptera, Panorpidae): possible causes and consequences

In the last decades, many insect species have been studied in terms of sperm competition. Patterns of sperm use are often inferred from the mean species value of P(2), defined as the mean proportion of offspring sired by the second male in double-mating trials. In Panorpa germanica (Mecoptera, Panorpidae), P(2) largely depends on relative copulation durations of both males, but with the second male on average having some advantage over the first male. Estimating the presence of fertile sperm inside the female's reproductive tract in relation to time after copulation we conclude this partial last male sperm precedence not to be caused by natural death, loss, or depletion of first male sperm. Estimating sperm transfer rates of both mates of a female we, furthermore, found that the high intraspecific variance in P(2) that can be observed cannot solely be explained by variances in sperm transfer rates among P. germanica males. Other factors possibly causing the observed patterns of paternity success are discussed.     

Mating duration and sperm precedence in the spider <Emphasis Type="Italic">Linyphia triangularis</Emphasis>

In many animal species, mating behaviour is highly ritualised, which may allow us to relate some of its consequences, e.g. male paternity and female receptivity, to the progression of phases in the mating sequence; at the same time, ritualisation raises the question of to what extent the partners, especially the males, are able to influence the outcome of mating for their own benefit. We studied the linyphiid spider Linyphia triangularis in which mating follows a strict sequence during which the male inducts two droplets of sperm and transfers them to the female. We performed sperm competition experiments (sterile-male technique) including four treatments, in which the copulation of the first male was interrupted at prescribed phases of the mating sequence, while the second male was allowed a complete mating. Second males spent a shorter time than first males on the behaviours prior to sperm transfer, but the amount of sperm (2 droplets) and the time spent in sperm transfer were independent of the females’ mating status. The proportion of females accepting the second male depended on the mating duration of the first male, i.e. whether the first male had transferred one or two sperm droplets. After a complete first mating, most females accepted no further males. A last-male sperm precedence was apparent if only half of the first sperm droplet had been transferred by the first male, but this switched to a first male precedence if one full sperm droplet had been transferred. Thus, even in the face of sperm competition, it is sufficient for the first male to transfer one sperm droplet. The second sperm droplet and the extended copulatory courtship associated with its transfer may serve to induce a lack of receptivity in the female, but the males seem unable to enhance their reproductive success through variable copulatory tactics.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

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 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.     

A potential mechanism for cryptic female choice in a flour beetle

In polyandrous mating systems, events occurring during copulation can alter the fate of the mating male's sperm. These events may exert selective pressures resulting in the evolution of diverse reproductive behaviours, morphologies and physiologies. This study investigates the role of male and female copulatory behaviours on sperm fate in the red flour beetle, Tribolium castaneum. I describe and quantify copulatory behaviours for mating pairs, and examine sperm fate by quantifying sperm transfer, sperm storage and second male sperm precedence. Whereas female quiescence during copulation and male leg rubbing during copulation together account for significant variation (26%) in sperm precedence, female copulatory quiescence alone provides information about the timing and numbers of sperm transferred. These experiments show that a female copulatory behaviour predicts sperm fate, and emphasize the value of studying variation in both male and female copulatory behaviours for understanding factors affecting sperm use.     

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.     

'Sloppy' sperm mixing and intraspecific variation in sperm precedence (P2) patterns

Sperm precedence patterns are typically highly variable within (and between) species. Intraspecific variation in sperm precedence (measured as P2, the proportion of progeny fathered by the last male to mate' is frequently seen as a candidate for adaptive interpretation through either male effects (e.g. body size), female effects (e.g. cryptic female choice) or an interaction between the two. Here we show, using computer simulation, that if ejaculates divide into a number of 'packets' and packets from two males mix randomly, then a variety of patterns of sperm precedence may result. We term this process 'sloppy' mixing. If ejaculates break into a small number of packets, bimodal P2 distributions are predicted. As the number of packets is increased, then a complex series of changes through multimodal and flat to unimodal distributions results. Sloppy mixing can thus result in many of the observed P2 distributions. Sloppy mixing is unlikely to change the predictions of adaptive models of sperm competition.     

Sperm survival in female stalk-eyed flies depends on seminal fluid and meiotic drive

Sperm competition is common in many insect species; however, the mechanisms underlying differences in sperm precedence are not well understood. In the stalk-eyed fly, Cyrtodiopsis whitei (Diptera, Diopsidae), sperm precedence is influenced by the presence of sex chromosome meiotic drive. When drive-carrying males compete with non-driving males for fertilizations within a female, the number of progeny sired by drive males is significantly fewer than predicted by sperm mixing alone. Thus, drive males apparently suffer not only a reduction in the number of viable sperm produced, but also a reduction in sperm competitive ability. In this study, we manipulated the amount and source of seminal fluid and sperm received by females by interrupting copulations before sperm, but after seminal fluid, was transferred. We find that seminal fluid from another male influences the number of progeny sired by a drive-carrying male when both males mate with the same female. Sperm viability staining reveals that sperm from drive males are incapacitated by seminal fluid from other males within the female reproductive tract. These results suggest that multiple mating by females enables seminal fluid products to interact differentially with sperm and may reduce the transmission advantage of the drive chromosome.     

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.     

Sperm competition promotes the exploitation of rival ejaculates

In many polyandrous species, the second male to mate with a female has fertilization priority. Strategic mechanisms generating this pattern tend to involve facultative increases in ejaculate size by informed males. Here we suggest a novel mechanism to generate second male precedence in internal fertilizers, based on the established fact that the female site of insemination is hostile to sperm, and that non-sperm components of the ejaculate frequently act to ameliorate these hostile conditions. We suggest that in species where female remating is frequent and rapid, second male sperm precedence may be due to the prior buffering of the female tract by previous males' ejaculates. In this scenario, second male costs are lower due to reduced sperm mortality and/or the redundancy of seminal fluid. Contrary to many classical predictions, second males may gain a paternity advantage despite investing less in an ejaculate.     

FEMALE CONTROL OF SPERM TRANSFER AND INTRASPECIFIC VARIATION IN SPERM PRECEDENCE: ANTECEDENTS TO THE EVOLUTION OF A COURTSHIP FOOD GIFT

Manipulation of ejaculates is believed to be an important avenue of female choice throughout the animal kingdom, but evidence of its importance to sexual selection remains scarce. In crickets, such manipulation is manifest in the premature removal of the externally attached spermatophore, which may afford females an important means of postcopulatory mate choice. We tested the hypothesis that premature spermatophore removal contributes significantly to intraspecific variation in sperm precedence by (1) experimentally manipulating spermatophore attachment durations of competing male Gryllodes sigillatus and (2) employing protein electrophoresis to determine the paternity of doubly mated females. The relative spermatophore attachment durations of competing males had a significant influence on male paternity, but the pattern of sperm precedence deviated significantly from the predictions of an ideal lottery. Instead, paternity data and morphological evidence accorded best with a model of partial sperm displacement derived here. Our model is similar to a displacement model of Parker et al. in that sperm of the second male mixes instantaneously with that of the first throughout the displacement process, but the novel feature of our model is that the number of sperm displaced is only a fraction of the number of sperm transferred by the second male. Regardless of the underlying mechanism, female G. sigillatus can clearly alter the paternity of their offspring through their spermatophore-removal behavior, and employ such cryptic choice in favoring larger males and those providing larger courtship food gifts. We discuss how female control of sperm transfer and intraspecific variation in sperm precedence may be important precursors to the evolution of gift giving in insects.     

Precedence of the first male sperm in successive matings in the HymenopteraDiadromus pulchellus

In most insect species where double matings occur, sperm from the second male preferentially fertilize subsequent eggs. However, we demonstrate here that, as already shown for some other hymenopteran species, this is not the case in the ichneumonid waspDiadromus pulchellus (Wesmeal): sperm from the first male usually father all the female progeny. This precedence of the first male sperm is also observed in double matings involving an haploid male and a diploid sterile male, whichever is the first mating male. We discuss the consequences of this phenomenon from an evolutionary point of view.     

Multiple paternity in wild-caught Drosophila mojavensis

Female remating frequency and sperm allocation patterns can strongly influence levels of sperm competition and reproductive success in natural populations. In the laboratory, Drosophila mojavensis males transfer very few sperm per copulation and females remate often, suggesting multiple paternity should be common in nature. Here, we examine female sperm loads, incidence of multiple paternity, and sperm utilization by genotyping progeny from 20 wild-caught females at four highly polymorphic microsatellite loci. Based on indirect paternity analyses of 814 flies, we found evidence for high levels of multiple paternity coupled with relatively low reproductive output, consistent with the high levels of female remating predicted in this sperm-limited species. Overall, we found little evidence for last -- male sperm precedence though some temporal variation in sperm utilization was observed, consistent with laboratory findings.     

Biases in sperm use in the mallard: no evidence for selection by females based on sperm genotype

If we are to understand fully the factors influencing fertilization success, it is essential to untangle male and female effects on sperm use. In many species, differences in fertilizing ability have been found between males or male genotypes, but the impact of female effects is less clear and may vary between taxa. Here, we examine sperm use in the mallard (Anas platyrhynchos), a species of bird in which forced copulation forms a major component of the mating system, to investigate whether there is any evidence for post-insemination female choice or rejection of particular sperm genotypes. Current models of sperm use in birds suggest observed patterns of paternity are a result of passive sperm loss from the reproductive tract and the relative timing of inseminations. Although this type of model successfully predicted average values of last male precedence observed in this species, there was considerable variation between females in their pattern of sperm use, with a tendency for females to use sperm of a single genotype. However, females did not consistently prefer one genotype over another in repeated inseminations with identical sperm mixtures, suggesting that post-insemination female preference based on sperm genotype did not account for this variation.     

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

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

Mechanisms of conspecific sperm precedence in Drosophila

The postmating, prezygotic isolating mechanism known as conspecific sperm precedence (CSP) may play an important role in speciation, and understanding the mechanism of CSP is important in reconstructing its evolution. When a Drosophila simulans female mates with both a D. simulans male and a D. mauritiana male, the vast majority of her progeny are fathered by D. simulans, regardless of the order of mating. The dearth of hybrid progeny does not result from inviability of eggs fertilized by heterospecific sperm or from the relative inviability of heterospecific larvae. Instead, CSP apparently results from a prefertilization obstacle to heterospecific sperm. We identified two independent barriers to heterospecific fertilization, sperm displacement and incapacitation, whose action depends on the order of mating. When a D. simulans female mates first with a conspecific male, the seminal fluid from this mating incapacitates heterospecific sperm transferred two days later. This sperm incapacitation occurs with no change in the retention of stored sperm over time, but does not occur when the conspecific mating lasts for only 5 min. When the order of matings is reversed, the seminal fluid from the second mating physically displaces heterospecific sperm from storage, even if the conspecific copulation lasts only 5 min. Conspecific sperm are not susceptible to displacement by a second conspecific copulation, but are susceptible to interference by heterospecific sperm if the conspecific copulation is interrupted after 12 min. Curing the D. mauritiana males of their infection with the endosymbiont Wolbachia had no effect on CSP. Sperm displacement and incapacitation involve the same basic mechanisms seen in second-male sperm precedence within species, supporting the hypothesis that CSP is an evolutionary by-product of adaptations affecting sperm competition within species.     

Age-related sperm transfer and sperm competitive ability in the male hide beetle

The influence of male age on reproductive success after a singlemating has been explored widely; however, few studies have investigatedwhether quantitative or qualitative differences in male spermare responsible for the observed patterns. Moreover, the roleof male age on sperm competitive ability has been largely ignored.We examined the importance of male age on the probability andamount of sperm transferred during a single mating and exploredwhether sperm competitive ability varies with male age in thehide beetle Dermestes maculatus, a species where sperm viabilitydoes not vary with male age. We also investigated whether spermtransfer rates varied with female age. We found that the probabilityof sperm transfer and the amount of sperm transferred variedwith male, but not female, age. All males performed behaviorallysuccessful copulations, but intermediate-age and old males weremore likely to transfer sperm successfully and also transferreda greater quantity of sperm than young males. Old males wereless likely to transfer sperm than intermediate-age males, butif they did transfer sperm successfully, they transferred comparableamounts. Sperm competitive ability varied with male age andreflected the quantity of sperm transferred. On average, intermediate-agemales achieved greater fertilization success when competingagainst young or old males than when competing against otherintermediate-age males. Old males were poor competitors againstintermediate-age males, but they achieved significantly higherrates of fertilization when competing against young males. Ourfindings suggest that quantitative differences in the amountof sperm transferred determine male success in sperm competitionin the hide beetle.     

Infertile matings and sperm competition: the effect of "nonsperm representation" on intraspecific variation in sperm precedence patterns

In theoretical and experimental approaches to the study of sperm competition, it is often assumed that ejaculates always contain enough sperm of good quality and that they are successfully transferred and used for fertilization. However, this view neglects the potential effects of infertility and sperm limitation. Permanent or temporal male infertility due to male sterility, insemination failures, or failures to fertilize the ova implies that some males do not achieve sperm representation in the female reproductive tract after mating. A review of the literature suggests that rates of nonsperm representation may be high; values for the proportion of infertile matings across 30 insect species vary between 0% and 63%, with the median being 22%. I simulated P2 (the proportion of offspring fathered by the second male to copulate with a female in a double-mating trial) distributions under a mechanism of random sperm mixing when sample sizes and rates of male infertility varied. The results show that nonsperm representation can be responsible for high intraspecific variance in sperm precedence patterns and that it can generate misleading interpretations about the mechanism of sperm competition. Nonsperm representation might be a common obstacle in the studies of sperm competition and postcopulatory female choice.     

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.