Co-evolution of male and female reproductive characters across the Scathophagidae (Diptera)

Sperm morphometry is extremely variable across species, but a general adaptive explanation for this diversity is lacking. As sperm must function within the female, variation in sperm form may be associated with variation in female reproductive tract morphology. We investigated this and other potential evolutionary associations between male and female reproductive characters across the Scathophagidae. Sperm length was positively associated with the length of the spermathecal (sperm store) ducts, indicating correlated evolution between the two. No association was found between sperm length and spermathecal size. However, the size of the spermathecae was positively associated with testis size indicating co-evolution between male investment in sperm production and female sperm storage capacity. Furthermore, species with a higher degree of polyandry (larger testes) had longer spermathecal ducts. However, no associations between sperm length or length variation and testis size were found which suggests greater sperm competition sensu stricto does not select for longer sperm.     

Selection on sperm size in response to promiscuity and variation in female sperm storage organs

Sperm cells are exceptionally morphologically diverse across taxa. However, morphology can be quite uniform within species, particularly for species where females copulate with many males per reproductive bout. Strong sexual selection in these promiscuous species is widely hypothesized to reduce intraspecific sperm variation. Conversely, we hypothesize that intraspecific sperm size variation may be maintained by high among-female variation in the size of sperm storage organs, assuming that paternity success improves when sperm are compatible in size with the sperm storage organ. We use individual-based simulations and an analytical model to evaluate how selection on sperm size depends on promiscuity level and variation in sperm storage organ size (hereafter, female preference variation). Simulations of high promiscuity (10 mates per female) showed stabilizing selection on sperm when female preference variation was low, and disruptive selection when female preference variation was high, consistent with the analytical model results. With low promiscuity (2–3 mates per female), selection on sperm was stabilizing for all levels of female preference variation in the simulations, contrasting with the analytical model. Promiscuity level, or mate sampling, thus has a strong impact on the selection resulting from female preferences. Furthermore, when promiscuity is low, disruptive selection on male traits will occur under much more limited circumstances (i.e. only with higher among-female variation) than many previous models suggest. Variation in female sperm storage organs likely has strong implications for intraspecific sperm variation in highly promiscuous species, but likely does not explain differences in intraspecific sperm variation for less promiscuous taxa.     

Sperm release and use at fertilization by yellow dung fly females (Scathophaga stercoraria)

Females of many species mate multiple times and store transferred sperm in storage organs. The mechanisms underlying sperm release from the stores at fertilization remain poorly understood, although they are central to an understanding of the female influence on post-copulatory male competition. Using double-mated females of the yellow dung fly, we counted the sperm sticking to the surface of deposited eggs of two successive clutches to obtain insight into the physiological processes associated with fertilization. The number of sperm released to fertilize an egg decreased between the first and second clutches, as well as within clutches from early to late eggs. These results indicate that: (1) sperm are lost from the stores over time independent of egg laying and (2) the number of sperm released depends on the amount of sperm stored. The lower number of sperm on eggs of the second clutches was accompanied by a strong increase of the proportion of sperm adhering to the micropyle region, suggesting that sperm use is more efficient and sperm release better controlled when sperm supply is substantially reduced. Finally, our approach indicates that sperm storage capacity of the female is higher than assumed from counts of spermathecal sperm.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 511–518.     

Female sperm storage mediates post‐copulatory costs and benefits of ejaculate anticipatory plasticity in the guppy

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade‐offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live‐bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready‐to‐use sperm (an anticipatory response called ‘sperm priming’). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm‐primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female‐stored sperm, weeks after insemination. By suggesting a plastic trade‐off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.     

Paternity in mallards: effects of sperm quality and female sperm selection for inbreeding avoidance

Postcopulatory processes might play an important role in sexualselection. In theory, fertilization success could be controlledby females via selection of particular sperm within their reproductivetract, or it could be determined by sperm competition per se.In practice, these two mechanisms are difficult to disentangle.To assess the relative importance of both mechanisms we usedartificial insemination in combination with measurements ofsperm quality (swimming speed and motility) in mallards. Inthis species, females often lack behavioral control over copulationsand hence may use postcopulatory mechanisms to optimize theirreproductive output. One important factor affecting female fitnessmay be selection of genetically compatible males. To investigatethe influence of sperm quality and parental relatedness on paternitywe inseminated 12 groups of related females with a sperm mixturecontaining equal numbers of sperm from a brother and from anunrelated male. Paternity was independent of the relatednessof the siring male to the female but was significantly affectedby long-term sperm swimming speed and motility. No interactionbetween relatedness and sperm quality on paternity was observed.These results suggest that female mallards are not able to selectsperm on a purely genetic basis and emphasize the importanceof sperm quality in gaining paternity.     

A panel of microsatellite markers to study sperm precedence patterns in the emperor dragonfly Anax imperator (Odonata: Anisoptera)

Odonates were the first group of organisms where sperm competition and last male sperm precedence have been identified. With the development of 10 microsatellites for the emperor dragonfly Anax imperator, the function and priority patterns of the multiple sperm storage organs of females can be studied and compared between species in natural populations. In addition, two microsatellite loci developed for the sister species Anax parthenope, are also highly polymorphic in A. imperator. For the presented 12 microsatellite loci, the number of alleles per locus ranged from two to 24. Observed heterozygosity ranged from 0.07 to 0.88.     

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

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

Female genital system of the folding-trapdoor spider Antrodiaetus unicolor (Hentz, 1842) (Antrodiaetidae, Araneae): ultrastructural study of form and function with notes on reproductive biology of spiders

The genitalia of the female folding-trapdoor spider Antrodiaetus unicolor are characterized by two pairs of spermathecae that are arranged in a single row and connected to the roof of the bursa copulatrix. Each single spermatheca is divided into three main parts: stalk, bowl, and bulb, which are surrounded by the spermathecal gland. The epithelium of the spermathecal gland is underlain by a muscle meshwork and consists of different types of cells partly belonging to glandular cell units (Class 3 gland cells) that extend into pores in the cuticle of the stalk and bowl. Interestingly, the bulb lacks glandular pores and is characterized by a weakly sclerotized cuticle. This peculiarly structured bulb probably plays an important role in the discharge of the sperm mass. It is suggested that by contraction of the muscle layer the sperm mass may be squeezed out, when the bulb invaginates and expands into the spermathecal lumen, pushing the sperm to the uterus lumen. Each glandular unit consists of usually one or two central secretory cells that are for the most part surrounded by a connecting cell that again is surrounded by a canal cell. The canal cell, finally, is separated from the other epithelial cells (intercalary cells) located between the glandular units by several thin sheath cells that form the outer enveloping layer of the unit. The secretions are released through a cuticular duct that originates proximally between the apical part of the connecting cell and the apical microvilli of the secretory cells and runs into a pore of the spermathecal cuticle. The glandular products of the Class 3 gland cells likely contribute to the conditions allowing long-term storage of the spermatozoa in this species. Details regarding the ovary, the uterus internus, and the uterus externus are reported. Most of the secretion that composes the chorion of the egg is produced in the ovary. Glandular cell units observed in the uterus externus differ structurally from those in the spermathecae and likely play a different role. Finally, we briefly discuss our results on the female genitalia of A. unicolor in the light of knowledge about the reproductive biology of spiders.     

Geographic variation in sperm traits reflects predation risk and natural rates of multiple paternity in the guppy

Guppies (Poecilia reticulata) are models for understanding the interplay between natural and sexual selection. In particular, predation has been implicated as a major force affecting female sexual preferences, male mating tactics and the level of sperm competition. When predation is high, females typically reduce their preferences for showy males and engage more in antipredator behaviours, whereas males exploit these changes by switching from sexual displays to forced matings. These patterns are thought to account for the relatively high levels of multiple paternity in high‐predation populations compared to low‐predation populations. Here, we assess the possible evolutionary consequences of these patterns by asking whether variation in sperm traits reflect differences in predation intensity among four pairs of Trinidadian populations: four that experience relatively low levels of predation from a gape‐limited predator and four that experience relatively high levels of predation from a variety of piscivores. We found that males in high‐predation populations had faster swimming sperm with longer midpieces compared to males in low‐predation populations. However, we found no differences among males in high‐ and low‐predation populations with respect to sperm number, sperm head length, flagellum length and total sperm length.     

Female mate choice and mating costs in the polyandrous butterflyPieris napi (Lepidoptera: Pieridae)

Males of the green-veined white butterfly (Pieris napi L.) transfer large ejaculates that represent on average 15% of their body mass when mating for a first time. Shortly after mating a male is able to transfer only a small ejaculate when mating a second time. Male ejaculate production plays a crucial role in the mating system ofP. napi because females use male-derived nutrients for egg production and somatic maintenance. Here we study how timing of female rematings and copulation duration are influenced by the mating history of their mates and, also, study if females exert mate choice to minimize their mating costs. Mating with a recently mated male increased female mating costs by increasing time in copula and mating frequency. Virgin females that mated with virgin males remated after an average of 6 days, whereas virgin females that mated with recently mated males remated after an average of 2 days. Moreover, copulations involving recently mated males lasted on average almost 7 h, whereas copulations involving virgin males lasted on average 2 h. Recently mated males were eager to remate, in spite of the fact that the size of the ejaculate they transfer is small and that they remain in copula for a long time. Hence it seems that males are more successful in the sexual conflict over mating decisions and that females do not minimize mating costs by choosing to mate preferentially with virgin males.     

Functional morphology of the copulatory organs of a reed beetle and a shining leaf beetle (Coleoptera: Chrysomelidae: Donaciinae,Criocerinae) using X-ray micro-computed tomography

For more than 100 years it has been known that the sclerotised median lobe of beetles harbours a membranous structure (the "internal sac" or "endophallus") which is everted during copula inside the female genital tract. In order to explore the functional role of this structure and those associated with it, we cryofixed copulating pairs of Donacia semicuprea and Lilioceris lilii and studied the relative position of the elements of the copulatory apparatus of males and females by micro-computer-tomography.We found that the everted endophallus fills the lumen of the bursa copulatrix completely. Our data suggest that in Lilioceris lilii the tip of the sclerotised distal part of the ejaculatory duct, the flagellum, is positioned exactly over the opening of the spermathecal duct inside the bursa copulatrix. The mouth of the bursa copulatrix in Donacia semicuprea is armed with a strong muscle ring, and the whole wall of the bursa is covered externally with a layer of muscle fibres. These morphological differences correspond with differences in mating behaviour: In reed beetles (Donaciinae), females seemingly can control mating to a higher degree than in lily beetles (Lilioceris spp.).     

Colourful male guppies produce faster and more viable sperm

In guppies (Poecilia reticulata) precopulatory sexual selection (via female choice) and post-copulatory selection (via sperm competition) both favour males with relatively high levels of carotenoid (orange) pigmentation, suggesting that colourful males produce more competitive ejaculates. Here we test whether there is a positive association between male orange pigmentation and sperm quality. Our analysis of sperm quality focused on sperm swimming speeds (using CASA: computer-assisted sperm analysis to estimate three parameters of sperm velocity in vitro), sperm viability (proportion of live sperm per stripped ejaculate) and sperm lengths. We found that males with relatively large areas of orange pigmentation had significantly faster and more viable sperm than their less ornamented counterparts, suggesting a possible link between dietary carotenoid intake and sperm quality. By contrast, we found no relationship between sperm length (head length and total sperm length) and male phenotype. These findings, in conjunction with previous work showing that highly ornamented male guppies sire higher quality offspring, suggest that female preference for colourful males and sperm competition work in concert to favour intrinsically higher quality males.     

Dynamics of eupyrene and apyrene sperm storage in ovipositing females of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

A male swallowtail butterfly, Papilio xuthus, transfers both eupyrene and apyrene sperm during copulation, both of which migrate to the spermatheca via the spermatophore in the bursa copulatrix of the female. Because the spermatheca seems to remain constant in size during the female lifespan, the excess sperm migration may cause the spermatheca to overflow. Approximately 9000 eupyrene and 265 000 apyrene spermatozoa were transferred during a single copulation, and approximately 1000 eupyrene and 1100 apyrene spermatozoa successfully arrived in the spermatheca. The number of both types of spermatozoon decreased in the spermatheca after the onset of oviposition, and no eupyrene spermatozoa were found by 7 days after copulation, partly due to insemination. The spermathecal gland leading from the distal end of the spermatheca was gradually filled by eupyrene spermatozoa. Although the function of the gland remains unclear, the final destination of the sperm is likely to be the gland.     

The spermatheca in the land snail, Arianta arbustorum (Pulmonata: Stylommatophora): muscle system and potential role in sexual selection

Abstract. Previous studies have shown variable patterns of paternity after multiple mating, and also variation in sperm storage among individuals of Arianta arbustorum , which suggests that the spermatheca may influence paternity in this promiscuous land snail. To identify possible morphological correlates of sperm manipulation, we investigated arrangement and ultrastructure of the muscles of the spermatheca. The musculature surrounding the 2–9 spermathecal tubules is arranged in a complex three dimensional network. In addition, each tubule has a thin sheath in which longitudinally oriented cells make up the innermost layer. Usually, the smooth muscle cells are enclosed by connective tissue. Only occasionally is direct muscle-muscle contact established through dense plaques. The short thick filaments, their small diameter, the relatively weak development of the tubular system and sarcoplasmic reticulum, and the low density of mitochondria indicate that the muscle cells contract relatively fast but with little strength, that they recover slowly, and have low endurance. A single muscle cell may be innervated by several axons and one axon may contact several muscle cells. Combining evidence of the present paper and a foregoing investigation on the spermathecal epithelium, we suggest that the main function of the spermathecal muscles is to expel sperm stored for fertilization, while the ciliation of the common duct is probably responsible for the distribution of sperm among the tubules.     

Spermicide, cryptic female choice and the evolution of sperm form and function

Sperm competition and cryptic female choice profoundly affect sperm morphology, producing diversity within both species and individuals. One type of within-individual sperm variation is sperm heteromorphism, in which each male produces two or more distinct types of sperm simultaneously, only one of which is typically fertile (the "eusperm"). The adaptive significance of nonfertile "parasperm" types is poorly understood, although numerous sperm-heteromorphic species are known from many disparate taxa. This paper examines in detail two female-centred hypotheses for the evolution and maintenance of this unconventional sperm production strategy. First, we use game theoretical models to establish that parasperm may function to protect eusperm from female-generated spermicide, and to elucidate the predictions of this idea. Second, we expand on the relatively undeveloped idea that parasperm are used by females as a criterion for cryptic female choice, and discuss the predictions generated by this idea compared to other hypotheses proposed to explain sperm heteromorphism. We critically evaluate both hypotheses, suggest ways in which they could be tested, and propose taxa in which they could be important.     

Variation in sperm precedence in Chorthippus grasshoppers (Caelifera: Gomphocerinae)

Variation in the proportions of offspring fathered by a second male to mate (the P₂ value) has been studied in two species of grasshoppers, Chorthippus parallelus (Zetterstedt) and Ch. biguttulus (Linnaeus), by means of the sterile‐male technique. In both species the P₂‐values of the first egg pods laid were in the range of 50–100%, and the temporal variation of P₂‐values appeared to follow two modes. In one, the P₂‐value steadily declined with time, in the other it remained constant at a high level. It is concluded that sperm is passively lost between two matings. The remaining sperm then either mixes within the spermatheca or is stratified. Further variability in sperm precedence can possibly be explained by spermatophore ejection or differential use of stored sperm.     

Trinucleotide microsatellite loci in the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae)

We describe six polymorphic, trinucleotide microsatellite loci that were isolated from the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), a model system for examining the mechanisms behind sperm competition in species with internal fertilization. These microsatellites yielded between 3 and 11 alleles per locus in a sample of 20–48 dung flies collected from several sites in Cheshire, UK. Observed and expected heterozygosities varied between 0.195 and 0.900 and 0.335–0.842, respectively. These markers should allow us to investigate postcopulatory sexual selection processes in this fly much more fully than has been possible in the past.     

Sperm ejection as a possible cryptic female choice mechanism in Odonata (Insecta)

Abstract.  The few odonate studies of sperm use suggest that females spend apparently more sperm than appears necessary during oviposition (sometimes females may have their sperm stores reduced to 50% after a single oviposition episode). Furthermore, some studies document that females eject sperm during and after copulation. This raises the question of whether sperm reduction may be interpreted as a cryptic female choice mechanism. Using two zygopterans, Ischnura denticollis Burmeister and Enallagma praevarum Hagen, and one anisopteran, Pantala flavescens Fabricius, it is shown that females mate more than once, show a marked reduction in stored sperm, and that this is by ejection of sperm before to oviposition. The extent of sperm reduction is inversely related to the number of eggs laid. When mated to the same male, females show similar reductions in sperm stores and egg load and only rarely does the vaginal duct contain sperm. This suggests that marked sperm reduction is common in this insect order and is not explained by an excess of sperm obstructing the egg passage. It is suggested that female's sperm shortage is better explained as a cryptic female choice mechanism aimed at favouring the sperm of some males. This study provides exciting research avenues for future studies of female choice in an animal taxa whose sexual biology is otherwise regarded as controlled by males.