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

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

Sperm length influences fertilization success during sperm competition in the snail Viviparus ater

Sperm form and size is tremendously variable within and across species. However, a general explanation for this variation is lacking. It has been suggested that sperm size may influence sperm competition, and there is evidence for this in some taxa but not others. In addition to normal fertilizing sperm, a number of molluscs and insects produce nonfertile sperm that are also extremely morphologically variable, and distinct from fertilizing forms. There is evidence that nonfertile sperm play an indirect role in sperm competition by decreasing female remating propensity in Lepidopterans, but in most taxa the function of parasperm is unknown. We investigated the role of nonfertile (oligopyrene) sperm during sperm competition in the fresh water snail Viviparus ater. Previous studies found that the proportion of oligopyrene sperm increased with the risk of sperm competition, and hence it seems likely that these sperm influence fertilization success during competitive matings. In mating experiments in which females were sequentially housed with males, we examined a range of male characteristics which potentially influence fertilization success. We found that the size of oligopyrene sperm was the best predictor of fertilization success, with males having the longer sperm siring the highest proportion of offspring. Furthermore, we found a positive shell size and sperm concentration effect on paternity, and females with multiply sired families produced more offspring than females mating with only one male. This result suggests polyandry is beneficial for female snails.     

No Effect of Male Courtship Intensity on Female Remating in the Butterfly <Emphasis Type="Italic">Pieris napi</Emphasis>

In Pieris napi, female fitness increases with number of matings, but wild females mate at an unexpectedly low rate. From a sexual conflict perspective this could be because males manipulate female remating, or alternatively, because wild females experience costs associated with remating which are not applicable under laboratory conditions. To get an indication which sex controls remating and/or the different sexes’ relative costs and benefits of remating, we here test whether female mating frequency is affected by male courtship intensity. We found no effect on female mating frequency or lifespan. This indicates that (i) females control remating and their optimal mating frequency is lower compared to males, or (ii) males can manipulate female remating. We argue that both these alternatives may apply simultaneously to P. napiand that they are inseparable.     

Strategic sperm allocation in the Small White butterfly Pieris rapae (Lepidoptera: Pieridae)

1. In species where females mate multiply, it is important for males to recuperate quickly in order to maximize their fertilization success. Butterflies produce a spermatophore at mating containing accessory secretions and sperm of two types: a large number of non-fertile 'apyrene' sperm and fewer fertile 'eupyrene' sperm. Many butterfly species eclose with most nutrients for reproduction already present. Males must therefore decide how to allocate resources to the various spermatophore components at any given mating.
2. Recovery rates of apyrene and eupyrene sperm number and spermatophore size was studied in the polyandrous Small White butterfly Pieris rapae . The mass of the first spermatophore increases with time since eclosion, as does the number of both types of sperm. Similarly, on a male's second mating, both the mass of the spermatophore and the number of sperm increases with time since the first mating.
3. However, the rate of increase in eupyrene sperm numbers is higher after the first mating. The difference in rate of increase may be the result of different probabilities of virgin and non-virgin males obtaining future matings.
4. Males have a sperm storage organ, the duplex, in which they retain sperm after their first mating. This ensures that high sperm numbers are available for their second mating, even when remating only 1 h later. Thus, males do not ejaculate all available sperm on any given mating, and seem to have different strategies on their first and second matings.
5. It can be argued that Small White butterfly males allocate sperm strategically according to the probability of obtaining subsequent matings, and the level of sperm competition.     

Sperm storage by females of the polyandrous noctuid moth Heliothis virescens

Female tobacco budworm moths, Heliothis virescens, generally mate with more than one male, receiving from each mate both fertilizing sperm (eupyrene) and nonfertilizing anucleate sperm (apyrene), which is thought to play a role in sperm competition. One male typically gains sperm precedence, but it is not consistently the last or the first male to mate. I investigated the mechanism of this variable pattern of paternity by examining the patterns of storage of both types of sperm in the female's spermatheca as a function of multiple mating and male phenotype. The number of stored apyrene sperm varied with mating history, being greatest in twice-mated females and least in females mated to one nonvirgin male. In contrast, only one ejaculate's worth of eupyrene sperm was stored regardless of female mating history (once or twice mated). Thus, while they store two complements of apyrene sperm, twice-mated females apparently store only one ejaculate's worth of eupyrene sperm. This biased pattern of sperm storage may contribute to the variable pattern of paternity observed in this species. Eupyrene sperm storage also correlated positively with female size, male age and spermatophore size. Finally, a new sperm storage site was identified and described. It is a bulged region in the seminal duct. Copyright 2000 The Association for the Study of Animal Behaviour.     

SEX RATIO DRIVE PROMOTES SEXUAL CONFLICT AND SEXUAL COEVOLUTION IN THE FLY DROSOPHILA PSEUDOOBSCURA

Selfish genetic elements occur in all living organisms and often cause reduced fertility and sperm competitive ability in males. In the fruit fly Drosophila pseudoobscura, the presence of a sex‐ratio distorting X‐chromosome meiotic driver Sex Ratio (SR) has been shown to promote the evolution of increased female remating rates in laboratory populations. This is favored because it promotes sperm competition, which decreases the risk to females of producing highly female‐biased broods and to their offspring of inheriting the selfish gene. Here, we show that non‐SR males in these SR populations evolved an increased ability to suppress female remating in response to the higher female remating rates, indicating male–female coevolution. This occurred even though SR was rare in the populations. This was further supported by a correlation between females’ remating propensity and males’ ability to suppress female remating across populations. Thus SR can generate sexual conflict over female remating rate between females and the noncarrier males that make up the majority of the males, promoting evolution of increased ability of males to suppress female remating.     

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.     

Fertilization competence and sperm size variation in sperm-heteromorphic insects

Between species, variation in sperm size has been related to male–female coevolution and male–male competition. In contrast, variation within species is poorly understood. A particular case of intraspecific sperm-size variation occurs in sperm-heteromorphic species, where males produce distinct sperm morphotypes, usually only one of which is fertile. This allows to investigate sperm size variation under different selection regimes. Nonfertile morphotypes, whose role is aside from fertilization, may have other functions, and this may be reflected by changes in developmental processes and a different phenotype compared to fertile sperm. We show that the intraspecific coefficient of variation in sperm length is up to four times lower for fertile than nonfertile morphotypes across 150 sperm-heteromorphic species (70 butterfly, 71 moth, 9 diopsid fly species). This is in agreement with a previous study on 11 species in the Drosophila obscura group. Significantly lower variation in fertile than nonfertile sperm morphometry may result from fertilization-related selection for optimal sperm size, novel functions of nonfertile sperm, or from tighter control of fertile sperm development. More data are needed to clarify the consequences and adaptive significance of within-morph variation, and its consistent pattern across sperm-heteromorphic insects.Co-ordinating editor: Hurst     

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.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Female influence on pre- and post-copulatory sexual selection and its genetic basis in Drosophila melanogaster

Genetic variation among females is likely to influence the outcome of both pre- and post-copulatory sexual selection in Drosophila melanogaster. Here we use association testing to survey natural variation in 10 candidate female genes for their effects on female reproduction. Females from 91 chromosome two substitution lines were scored for phenotypes affecting pre- and post-copulatory sexual selection such as mating and remating rate, propensity to use sperm from the second male to mate, and measures of fertility. There were significant genetic contributions to phenotypic variation for all the traits measured. Resequencing of the 10 candidate genes in the 91 lines yielded 68 non-synonymous polymorphisms which were tested for associations with the measured phenotypes. Twelve significant associations (markerwise P<0.01) were identified. Polymorphisms in the putative serine protease homolog CG9897 and the putative odorant binding protein CG11797 associated with female propensity to remate and met an experimentwise significance of P<0.05. Several other associations, including those impacting both fertility and female remating rate suggest that sperm storage might be an important factor mitigating female influence on sexual selection.     

Female remating, sperm competition and sexual selection in Drosophila

Female remating is fundamental to evolutionary biology as it determines the pattern of sexual selection and sexual conflict. Remating in females is an important component of Drosophila mating systems because it affects sperm usage patterns and sexual selection. Remating is common in females of many species of Drosophila in both natural and laboratory populations. It has been reported in many insect species and also in vertebrates. Female remating is a prerequisite for sperm competition between males, and the consequences of this competition, such as sperm precedence or sperm displacement, have been reported for many species of Drosophila. Female remating is dependent on the amount of sperm stored, the male seminal fluid components, nutrition, the quantity of eggs laid, experimental design and density of flies in laboratory. Remating by a female is an insurance against male sterility and sub-fertility and increases genetic heterogeneity of female offspring. Remating gives greater female productivity in many species of Drosophila. We examined female remating with respect to sperm competition and sexual selection in Drosophila and addressed the possible benefits for females. We also reviewed the role of accessory gland fluid in remating, costs associated with remating, the genetic basis of female remating and some possible mechanisms of sperm competition in the light of last male sperm priority and paternity assurance in Drosophila and other insects. We also suggest future areas of research.     

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated males.     

Functional significance of seminal receptacle length in Drosophila melanogaster

Despite its central role in post-copulatory sexual selection, the female reproductive tract is poorly understood. Here we provide the first experimental study of the adaptive significance of variation in female sperm-storage organ morphology. Using populations of Drosophila melanogaster artificially selected for longer or shorter seminal receptacles, we identify relationships between the length of this primary sperm-storage organ and the number of sperm stored, pattern of progeny production, rate of egg fertilization, remating interval, and pattern of sperm precedence. Costs and benefits of relatively short or long organs were identified. Benefits of longer receptacles include increased sperm-storage capacity and thus progeny production from a single insemination. Results suggest that longer receptacles have not naturally evolved because of developmental time costs and a correlated reduction in longevity of mated females. This latter cost may be a consequence of sexual conflict mediated by ejaculate toxicity. Receptacle length did not alter the pattern of sperm precedence, which is consistent with data on the co-evolution of sperm and female receptacle length, and a pattern of differential male fertilization success being principally determined by the interaction between these male and female traits.     

Effects of male nutrition on sperm storage and remating behavior in wild and laboratory Anastrepha fraterculus (Diptera: Tephritidae) females

Male physiological condition can affect his ability to modulate female sexual receptivity. Thus, studying this aspect can have biological and practical implications. Here, we examine how male nutritional status affected the amount of sperm stored, remating rate and refractory period of the tephritid fruit fly Anastrepha fraterculus (Wiedemann) females. Both wild and laboratory flies were evaluated. We also examine female sperm storage patterns. Experiments were carried out by manipulating male adult diet and exposing these males to virgin females. Females mated with differently treated males were either dissected to count the amount of sperm stored or exposed to virgin males to determine remating rate and the length of the refractory period. We found that male nutritional status affected the amount of the sperm stored and the renewal of sexual receptivity in wild flies. For laboratory flies, male nutritional status affected the length of the refractory period but not the amount of sperm stored by females. In addition, we report that the ventral receptacle is not an important organ of sperm storage in this species. We conclude that male nutritional condition influences the ability to modulate female sexual receptivity, possibly through a combination of the quantity and quality of the ejaculate. From an applied perspective, providing males with an enriched diet will likely result in increased efficacy of the sterile insect technique.     

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.     

Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.     

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.     

Female wolf spiders exert cryptic control drastically reducing ejaculate size

Sexes' roles in post‐copulatory processes have important effects on individual fitness and are promising to study in species showing complex mating behaviours. In the spider Schizocosa malitiosa, males perform two different copulatory patterns, pattern 1 includes 80% of total pedipalp insertions and pattern 2 includes 20%. Both patterns produce similar number of offspring, but pattern 1 induces higher female reluctance to remating than pattern 2. We hypothesised that the complex copulatory patterns are linked to post‐copulatory sexual selection, affecting males' sperm transfer and the resulting sperm storage by females. First, we examined amounts of sperm in males and live females from uninterrupted (pattern 1 + 2) and interrupted matings (pattern 1, pattern 2). Second, in order to disentangle male and female actions, we induced males to mate with dead females and examined amounts of sperm. Males transfer in total 71% of the sperm available in their pedipalps, being higher but not significant in pattern 1 than in pattern 2. Females drastically reduced the amount of sperm stored in their spermathecae and such control is stronger in pattern 1 compared to pattern 2 matings. We propose that cryptic female control is a main factor driving males to strengthen sperm transfer. Active female reduction in ejaculate most probably diminished her reluctance to remate.     

Sexual conflict and cooperation under naturally occurring male enforced monogamy

An evolutionary conflict often exists between the sexes in regard to female mating patterns. Females can benefit from polyandry, whereas males mating with polyandrous females lose reproductive opportunities because of sperm competition. Where this conflict occurs, the evolution of mechanisms whereby males can control female remating, often at a fitness cost to the female, are expected to evolve. The fitness cost to the female will be increased in systems where a few high status males monopolise mating opportunities and thus have limited sperm supplies. Here we show that in the cockroach Nauphoeta cinerea, a species where males enforce female monogamy in the first reproductive cycle, males that have become sperm depleted continue to be able to manipulate female remating behaviour. Although the manipulation severely decreases fecundity in females mated to sperm-depleted males, males benefit, increasing their relative fitness by preventing other males from reproducing. Our results suggest that there is selection on maintaining the mechanism of manipulation rather than maintaining sperm numbers. Taken with previous research on sexual conflict in N. cinerea, this study suggests that the causes and consequences of sexual conflict are complex and can change across the life history of an individual.