Intraspecific variation in sperm precedence in the bruchid beetle Callosobruchus maculatus

Abstract.

• 1 By examining potential sources of intraspecific variation in sperm precedence, the underlying mechanisms of sperm competition in Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) were investigated.
• 2 The extent of sperm precedence was not related to either copulatory behaviour or body size (male and female).
• 3 The extent of sperm precedence increased during the egg-laying period, suggesting that the stratification of sperm within the spermatheca is not the mechanism of sperm precedence.
• 4 Direct removal of sperm from the female's reproductive tract was not observed.
• 5 Four other mechanisms (not mutually exclusive) are proposed to account for last-male sperm precedence in this species.

     

Number of mating males and mating interval affect last-male sperm precedence in Tenebrio molitor L.

Polyandrous females often mate with more than two males, and yet most studies of postcopulatory sexual selection involve only two males. In insects, second-male sperm precedence is usually taken as evidence of overall last-male sperm precedence despite some studies to the contrary. Furthermore, the processes or mechanisms causing the patterns are often unknown and yet are important when estimating how postcopulatory sexual selection might act on males. Whether the patterns and processes change in normal sperm competitive situations and the effects of other factors besides mating order need to be examined to better assess the evolutionary potential of postcopulatory sexual selection. In this study, I assessed the effects of mating interval and number of mating males on sperm precedence patterns and their causal mechanisms in the mealworm beetle, Tenebrio molitor. Last-male sperm precedence was the same when two or three males mated, but also depended on mating intervals and hence mechanisms of paternity bias. However, when females mated with many males, one of the mechanisms no longer created last-male sperm precedence. This example illustrates the importance of knowing both the patterns and mechanisms of paternity bias and whether they change depending on female mating frequency to make reasonable inferences about the potential for postcopulatory sexual selection on males. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Conspecific sperm precedence in Callosobruchus subinnotatus (Coleoptera: Bruchidae): mechanisms and consequences

Conspecific sperm precedence (CSP) has been identified as an important post-copulatory, pre-zygotic mechanism that can act to reduce gene flow between populations. The evolution of CSP is thought to have arisen as a by-product of male and female coevolution in response to intraspecific post-copulatory sexual selection. However, little is known about the mechanisms that generate CSP. When Callosobruchus subinnotatus females copulate with both C. subinnotatus and Callosobruchus maculatus males, regardless of mating order, the majority of eggs are fertilized by conspecific sperm. The low number of heterospecific fertilizations does not result from general differences in the viability of sperm in the female reproductive tract, as heterospecific sperm fertilized equivalent numbers of eggs as conspecific sperm in the absence of sperm competition. Instead, CSP results from disadvantages to heterospecific sperm that are manifest only when in competition with conspecific sperm. CSP in C. subinnotatus appears to result from two, not mutually exclusive, mechanisms. First, conspecific sperm are better able to displace heterospecific sperm from female storage. Second, conspecific sperm achieve disproportionately higher numbers of fertilizations relative to their proportional representation in the fertilization set. Thus, we provide evidence of differential sperm use from the female spermatheca.     

Nuptial gift size prolongs copulation duration in the dance fly Empis borealis

Abstract. 1. The time that pairs of the dance fly Empis borealis (L.) (Diptera: Empididae) spent in copula was positively correlated with the volume of the nuptial gift.
2. In some cases the copulation duration was short although prey volume was large. Females engaged in these matings had more worn wings, were therefore older, and most probably had sperm already stored in the spermatheca. It is suggested that these copulations were interrupted because either the spermatheca was full of sperm and the male was not able to transmit any sperm, or because prey content was depleted (males may have fed on the prey or the prey had been used in a previous mating). The latter explanation seemed more probable.
3. A male providing a large nuptial gift may transfer larger quantities of sperm. Quantitative sperm competition is more likely than last-male sperm predominance.     

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.     

Longer exaggerated male genitalia confer defensive sperm-competitive benefits in an earwig

As evidence mounts that male genitalia can affect relative fertilisation success, the role that sexual selection has played in the rapid and divergent evolution of genitalia is becoming increasingly recognized. Unfortunately, the limited functional understanding of these complex structures and their interactions with the female reproductive tract often limit interpretation regarding their evolution. Here, we address this issue using the earwig Euborellia brunneri, where both the male intromittent organ and the female spermatheca are highly exaggerated in length yet structurally simple. In a double mating design, we use the sterile male technique to study how sperm precedence patterns are affected by male genital length, male age, and the size of the male sperm storage organ, the seminal vesicle. Relative fertilisation success exhibited considerable variation around modest last-male paternity. Only an interaction between first and second male genital length affected paternity, where males gained reduced paternity when preceded by rivals with longer genitalia. Longer genitalia confer defensive benefits in sperm competition by apparently depositing ejaculate deeper in the tubular spermatheca, safe from removal by rivals. Paternity similarly depended on an interaction between the ages of both males, likely mediated by sperm traits as testes size decreased with age. Seminal vesicle size showed positive allometry but did not affect paternity; instead, greater seminal vesicle size in last males expedited oviposition. The exaggerated yet relatively simple genitalia of E. brunneri facilitate an unusually clear example of post-copulatory selection on phenotypic variation in multiple reproductive traits.     

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.     

Sperm competition in the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae): Effects of sperm “longevity” on sperm precedence

Sperm competition inBactrocera cucurbitae was studied by double matings of one female with normal and sterile males, with different intervals between the first and the second matings and with or without allowing oviposition after the first or the second mating. When the interval was less than 4 days, the last-male sperm precedence,P ₂ , was not different from 0.5, but as the interval was prolonged,P ₂ was higher than 0.5. There was no significant difference between treatments in which females were allowed to oviposit after the first mating and only after the second mating. The reason for the higherP ₂ when the interval was long was therefore attributed not to sperm usage for egg fertilization during the two matings but, possibly, to sperm mortality. ThatP ₂ was 0.5 for shorter intervals suggests that particular sperm replacement mechanisms such as removal and inactivation are absent in B. cucurbitae. Our study is the first to demonstrate a significant effect of short sperm longevity on the last-male sperm precedence.     

Age-Related Sperm Production,Transfer, and Storage in the Sweet Potato Weevil, <Emphasis Type="Italic">Cylas formicarius</Emphasis> (Fabricius) (Coleoptera: Curculionidae)

The relationship between sperm production, insemination rate, and sperm transfer were studied in the sweet potato weevil, Cylas formicarius. Older adult males retained more sperm in the testes-seminal vesicle complex (TSC) and thus more was ejaculated into females at first mating. Number of matings per day for males was relatively constant across different ages, and frequent mating resulted in a reduced amount of sperm transferred to females, especially in young males. Young virgin males had a relatively small ejaculate, and almost all sperm transferred to females was stored in the spermatheca, whereas older virgin males transferred a larger amount of sperm to females, in whom sperm was found in both the spermatheca and post-spermathecal organs (PSO) after mating. The number of sperm in the PSO decreased markedly within 24 h after mating, but amounts in the spermatheca remained the same. Just where the sperm in the PSO went is a point that remained undetermined. The amount of sperm in the spermatheca was reduced more rapidly in females that laid eggs than in females that did not, although sperm reduction occurred even in the latter. Insemination of this weevil corresponded with the volume of the spermatheca, and the amount of sperm stored in the TSC was determined by the age and mating history of the males.     

Strategic Investment in Sperm Removal Behaviour in a Bushcricket

Multiple mating by females is widespread and generates sperm competition among the ejaculates of rival males over fertilization. One way in which males can avoid or reduce sperm competition is by displacing or removing previous males’ sperm from female sperm stores. An apparent example of this occurs in the bushcricket Metaplastes ornatus. Males perform a specialised sperm removal behaviour (SRB), using their highly-derived subgenital plate, with which they remove sperm from the female’s spermatheca during the early phases of mating before transferring a spermatophore of their own. Here we investigated whether males strategically invest in SRB according to the amount of previously stored sperm present in females. Each male was tested twice, once with a female containing sperm (‘filled’ condition) and once with a female from whom most previously deposited sperm had recently been removed by another male (‘emptied’ condition). For comparison, a separate group of males was paired with virgin females. Males did not discriminate between non-virgin females in the ‘emptied’ or ‘filled’ conditions in terms of their investment in SRB, suggesting they may not able to perceive the amount of sperm present in the female’s spermatheca. By contrast, male investment in SRB was significantly reduced in pairings with virgin females, indicating that males are sensitive to some aspect of a female’s mating status. Our results thus suggest that males modulate SRB in response to female-mediated cues, possibly chemical cues left by previous males, which would not be present on virgin but would be on non-virgin females.     

Size, operational sex ratio, and mate-guarding success of the carrion beetle, Necrophila americana

When male insects guard females until oviposition, the benefitsfrom last-male sperm precedence must outweigh the costs of relinquishingadditional fertilizations. The profitability of guarding isincreased when males guard large, fecund females and when femalesare scarce because fewer fertilizations are sacrificed. However,the male reproductive success is not only determined by theprofitability of guarding but also by his ability to maintainguarding. In this study, we used male carrion beetles (Necrophilaamericana) to examine the effects of sex ratio, male relativesize, and female quality on the ability to guard. First, wepresent a model of mate guarding that explores factors, suchas sperm precedence, sex ratio, male size, and female quality,that influence the profitability of postcopulatory riding. Ourmodel predicts that large N. americana males should preferentiallyguard the largest female only when the sex ratio is male biasedand sperm precedence is above 80%. In contrast, small malesgain little from guarding because they are not likely to maintainit and be the last male to mate. Then, we tested these predictionsby manipulating sex ratio, relative male size, and female quality.All males in equal sex ratio and large males in male-biasedsex ratio guarded females significantly longer than did malesin female-biased sex ratio. In male-biased sex ratio, largemales guarded significantly longer and achieved more takeoversthan small males. Large females were guarded longer. The successof guarding males in this beetle depends on their size relativeto other males and the operational sex ratio.     

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

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

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.     

Sexual selection in flour beetles: the relationship between sperm precedence and male olfactory attractiveness

Sperm precedence, defined as nonrandom differential fertilizationsuccess among mating males, is an important postmating componentof sexual selection. This study examined the relationship betweenpremating and postmating components of sexual selection in malesof the flour beetle (Tribolium castanewn). Male olfactory attractivenessto females was positively correlated with a male's subsequentfertilization success: more attractive males achieved highersecond-male sperm precedence when allowed to mate with previouslyinseminated females. Attractive males may achieve compoundedgains in their reproductive success through enhanced matingopportunities as well as through greater fertilization success.Thus, the relationship between these reproductive fitness componentsmay augment differences in reproductive success among males.Female fecundity, estimated as the number of adult progeny produced,increased significantly with multiple malings. This result supportsincreased female reproductive success as a direct benefit ofmultiple mating in T. caslaneum and suggests that progeny productionis partially limited by sperm availability. Total progeny productionby doubly mated females remained constant at all levels of second-malesperm precedence. However, higher sperm precedence was associatedwith a decline in firstmale progeny and a concomitant increasein second-male progeny. This pattern of progeny production suggeststhat more attractive males may achieve higher fertilizationsuccess through a combination of displacement of previouslystored sperm, transfer of greater sperm quantities, or females'preferential use of sperm of attractive males for fertilizations.     

Female age and sperm competition: last-male precedence declines as female age increases

Until very recently, most studies of sperm competition have focused on variation in male competitive ability. However, we now know that a number of reproductive traits, including oviposition rate, use of stored sperm and receptivity to mating, vary with female condition. Because females can play an active part in the movement of sperm within their reproductive tract, sperm competition may be influenced by female condition. Existing studies of sperm competition in fruitflies ignore the effects of female condition, using females that are 3-4 days old and in their reproductive prime. But condition will decline as a female senesces. Here, we examine the effect of female age on the outcome of sperm competition in three strains of the fruitfly, Drosophila melanogaster. Previous studies have shown that female age influences preference for mates and male ejaculation strategies. In this study, we find that when males are mated to females that are older than 17 days, last-male sperm precedence decreases significantly. These results could lead to a greater understanding of the physiological mechanisms that regulate the outcome of sperm competition.     

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

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

Effect of male body size on sperm precedence in the polyandrous butterfly Pieris napi L. (Lepidoptera: Pieridae)

Male Lepidoptera produce an ejaculate during copulation thatcontains both sperm and accessory gland nutrients and may functionas paternal investment and/or male mating effort Several studieshave examined how ejaculates function as paternal investment,but few have determined the influence of sperm competition onmale investment This study examines the effect of male bodysize on sperm precedence in the polyandrous butterfly Pierisnapi L. We used male body mass as an indicator of the size ofejaculate transferred and found that relative male size hada significant effect on paternity. The offspring of twice-matedfemales showed a low incidence of mixed paternity. Larger malesobtained the majority of fertilizations, and the degree of second-malesperm precedence was influenced by relative body size of matingmales. In general, second mates obtained fewer fertilizationsthe larger the size of the first mate. The interval betweenthe first and second mating was influenced by the size of thefirst male mate Females first mated to small males remated soonerthan females first mated to larger males Our results suggestthat large males may have a selective advantage over small maleswhen both a male's fertilization success and a female's refractoryperiod are influenced by the size of ejaculate transferred.Furthermore, the effect of male body size on the proportionof offspring sired lends support to the hypothesis that spermcompetition has played a major role in the evolution of ejaculatesize.     

The effects of exposure to seaweed on willingness to mate, oviposition, and longevity in seaweed flies

Abstract  1. Large male seaweed flies (Diptera: Coelopidae) are more likely to mate than smaller males. This is due to sexual conflict over mating, by which females physically resist male attempts to copulate. In some species, large males are simply more efficient at overpowering female resistance.
2. Female reluctance to mate is likely to have evolved due to the costs of mating to females. In many dipterans, males manipulate female behaviour through seminal proteins that have evolved through sperm competition. This behavioural manipulation can be costly to females, for example forcing females to oviposit in sub-optimal conditions and increasing their mortality.
3. Previous work has failed to identify any ubiquitous costs of mating to female coelopids. The work reported here was designed to investigate the effects of exposure to oviposition sites ( Fucus algae) on the reproductive behaviour of four species of coelopid. Algae deposition in nature is stochastic and females mate with multiple males in and around oviposition sites. Spermatogenesis is restricted to the pupal stage and there is last-male sperm precedence. It was predicted that males would avoid wasting sperm and would be more willing to mate, and to remain paired with females for longer, when exposed to oviposition material compared with control males. Females were predicted to incur longevity costs of mating if mating increased their rate of oviposition, especially in the presence of algae.
4. The behaviour of males of all four species concurred with the predictions; however mating did not affect female receptivity, oviposition behaviour, or longevity. Exposure to algae induced oviposition and increased female mortality in all species independently of mating and egg production. The evolutionary ecology of potential costs of mating to female coelopids are discussed in the light of these findings.     

Mechanisms of sperm competition: testing the fair raffle

Sperm competition is a major force of sexual selection, but its implications for mating system and life-history evolution are just beginning to be understood. Of particular importance is understanding the mechanisms of sperm competition. Models have been developed to determine if sperm competition operates in a fair raffle process, whereby each sperm from competing males has an equal chance of fertilizing a female's ova, or if it operates in a loaded raffle process, whereby one male's sperm has a fertilization advantage. These models require data on relative sperm and offspring (paternity) numbers of competing males. Here we develop a model based on maximum-likelihood methods for differentiating between the fair and loaded raffle processes. The model calculates the relative competitiveness of two males' sperm (loadings) as well as the economy of scale (nonlinear returns to sperm number). Previous models implicitly assumed that there is no economy of scale, which may not be the case when there is cooperation or interference among sperm from a given male. We demonstrate that our model has superior power-in some instances more than double-than previous models. We apply our model to an example of sperm competition in the guppy (Poecilia reticulata) and show that the system may be characterized by a loaded raffle attributable to effects of second male precedence.     

Multiple paternity and postcopulatory sexual selection in a hermaphrodite: what influences sperm precedence in the garden snail Helix aspersa?

Sperm competition has been studied in many gonochoric animals but little is known about its occurrence in simultaneous hermaphrodites, especially in land snails. The reproductive behaviour of the land snail Helix aspersa involves several features, like multiple mating, long-term sperm storage and dart-shooting behaviour, which may promote sperm competition. Cryptic female choice may also occur through a spermatheca subdivided into tubules, which potentially allows compartmentalized sperm storage of successive mates. In order to determine the outcome of postcopulatory sexual selection in this species, we designed a cross-breeding experiment where a recipient ('female') mated with two sperm donors ('males'). Mates came from either the same population as the recipient or from a distinct one. To test for the influence a recipient can have on the paternity of its offspring, we excluded the effects of dart shooting by using only virgin snails as sperm donors because they do not shoot any dart before their first copulation. We measured the effects of size of mates as well as time to first and second mating on second mate sperm precedence (P2; established using microsatellite markers). Multiple paternity was detected in 62.5% of clutches and overall there was first-mate sperm precedence with a mean P2 of 0.24. Generalized linear modelling revealed that the best predictors of paternity were the time between matings and the time before first mating. Overall, both first and second mates that copulated quickly got greater parentage, which may suggest that postcopulatory events influence patterns of sperm precedence in the garden snail.