Sexual conflict and cryptic female choice in the black field cricket, Teleogryllus commodus

The prevalence and evolutionary consequences of cryptic female choice (CFC) remain highly controversial, not least because the processes underlying its expression are often concealed within the female reproductive tract. However, even when female discrimination is relatively easy to observe, as in numerous insect species with externally attached spermatophores, it is often difficult to demonstrate directional CFC for certain male phenotypes over others. Using a biological assay to separate male crickets into attractive or unattractive categories, we demonstrate that females strongly discriminate against unattractive males by removing their spermatophores before insemination can be completed. This results in significantly more sperm being transferred by attractive males than unattractive males. Males respond to CFC by mate guarding females after copulation, which increases the spermatophore retention of both attractive and unattractive males. Interestingly, unattractive males who suffered earlier interruption of sperm transfer benefited more from mate guarding, and they guarded females more vigilantly than attractive males. Our results suggest that postcopulatory mate guarding has evolved via sexual conflict over insemination times rather than through genetic benefits of biasing paternity toward vigorous males, as has been previously suggested.     

Effects of male mating interval on spermatophore formation,transfer, and subsequent female receptivity and fecundity in the sorghum plant bug,Stenotus rubrovittatus

Males of the sorghum plant bug, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), transfer a spermatophore to females during copulation. After a 1‐day interval between the first and second copulation, males transferred both sperm and a spermatophore to females during the second copulation. However, when male mating interval was <1 h, they transferred sperm but no spermatophores to females during the second copulation. Therefore, the male mating interval probably produces two types of mated females, those with and those without a spermatophore. Mated females of S. rubrovittatus do not remate for at least 3 days after mating, even when courted, and lay more eggs than virgin females at the beginning of the oviposition period. The effects of spermatophores on female sexual receptivity and fecundity were examined using mated females with or without a spermatophore. Only one of the 40 (2.5%) mated females with a spermatophore remated, whereas 10 of the 26 (38.5%) without a spermatophore remated. Furthermore, mated females with a spermatophore laid more eggs than those without a spermatophore. These results suggest that spermatophores participate in reducing female sexual receptivity and enhancing female fecundity in S. rubrovittatus.     

Paternity Analysis in a Hexapod (Orchesella cincta; Collembola) with Indirect Sperm Transfer

In species where males and females interact during mating, the role of females in sexual selection cannot always be demonstrated unambiguously. Here we present a model system to study female choice for mates. Orchesella cinca is a soil-dwelling hexapod with indirect sperm transfer. Females and males do not interact physically for reproduction. We gave females the choice between spermatophores produced by two different males. Paternity analysis based on microsatellite variation revealed that offspring in one clutch were sired by one male only. Direct observations showed that after a female has taken up a spermatophore, the female's receptivity to further spermatophore uptake seem to end. Our results imply that the female is in full control of paternity.     

The production and transfer of spermatophores in three Asian species of Luciola fireflies

During mating, many male insects transfer sperm packaged within a spermatophore that is produced by reproductive accessory glands. While spermatophores have been documented in some North American fireflies (Coleoptera: Lampyridae), little is known concerning either production or transfer of spermatophores in the aquatic Luciola fireflies widespread throughout Asia. We investigated this process in Japanese Luciola lateralis and L. cruciata by feeding males rhodamine B, a fluorescent dye known to stain spermatophore precursors. We then mated males with virgin females, and dissected pairs at various timepoints after mating. In both of these Luciola species, spermatophores were produced by three pairs of male accessory glands and were transferred to females during the second stage of copulation. Male spermatophores were highly fluorescent, and were covered by a thin outer sheath; a narrow tube leading from an internal sperm-containing sac fit precisely into the female spermathecal duct, presumably for sperm delivery. Both L. lateralis and L. cruciata females have a spherical spermatheca as well as a highly extensible gland where spermatophore breakdown commences by 24h post-mating. Similar reproductive anatomy was observed for both sexes in Luciola ficta from Taiwan. These results suggest that nuptial gifts may play an important role in many firefly-mating systems.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Male Condition, Female Choice, and Extreme Variation in Repeated Mating in a Scaly Cricket, Ornebius aperta (Orthoptera: Gryllidae: Mogoplistinae)

Mating in the scaly cricket Ornebius aperta often includes the transfer of many spermatophores to individual females during extended copulatory interactions. We manipulated male condition in staged matings to determine whether this could explain variation in the number of repeated copulations seen across pairs. Males on a high nutrient diet were in good condition, were more likely to mate repeatedly, and transferred more spermatophores on average than low-diet males (in poor condition). High-diet males were more likely to produce a vibratory signal that increased female receptivity to repeated mating attempts. Courtship and copulatory interactions were always terminated by females, and in every case males had already formed a spermatophore when deserted by females. We conclude that variation in male repeated mating success may be due to female choice rather than an inability or unwillingness of low-diet males to produce spermatophores.     

No Coolidge Effect in Decorated Crickets

Female decorated crickets, Gryllodes sigillatus , obtain genetic benefits by mating with different males and, when given a choice, prefer novel males over previous mates. It is unknown, however, whether males exhibit a similar preference for novel females. Although female crickets control copulation, there are at least two ways in which males can exercise choice: (1) the amount of courtship directed towards prospective mates and (2) the size of the spermatophore transferred to the female at mating. To determine whether males devote more courtship effort to novel females while controlling for female behavioral cues, male courtship effort toward two dead females, one, a previous mate and the other, a novel female, was measured. To determine whether males manufacture larger spermatophores when paired with novel females, males were mated to novel or previous mates, and the different components of the spermatophore weighed. Males did not spend more time courting dead novel females than previous mates. There was no difference in the latency to remating of males confined with novel females and those paired with previous mates, and there was no difference in the mass of spermatophores transferred to novel and familiar females. Contrary to previous studies in other taxa, this study suggests that male crickets do not prefer novel mates and thus, are not subject to the Coolidge effect. Although mating with novel females may be beneficial to males, selection on males to identify and discriminate against previous mates may be relaxed because of a strong female preference for novel males.     

Competitive PCR reveals the complexity of postcopulatory sexual selection in Teleogryllus commodus

The outcome of mate choice depends on complex interactions between males and females both before and after copulation. Although the competition between males for access to mates and premating choice by females are relatively well understood, the nature of interactions between cryptic female choice and male sperm competition within the female reproductive tract is less clear. Understanding the complexity of postcopulatory sexual selection requires an understanding of how anatomy, physiology and behaviour mediate sperm transfer and storage within multiply mated females. Here we use a newly developed molecular technique to directly quantify mixed sperm stores in multiple mating females of the black field cricket, Teleogryllus commodus. In this species, female postcopulatory choice is easily observed and manipulated as females delay the removal of the spermatophore in favour of preferred males. Using twice‐mated females, we find that the proportion of sperm in the spermatheca attributed to the second male to mate with a female (S₂) increases linearly with the time of spermatophore attachment. Moreover, we show that the insemination success of a male increases with its attractiveness and decreases with the size of the female. The effect of male attractiveness in this context suggests a previously unknown episode of mate choice in this species that reinforces the sexual selection imposed by premating choice and conflicts with the outcome of postmating male harassment. Our results provide some of the clearest evidence yet for how sperm transfer and displacement in multiply mated females can lead directly to cryptic female choice, and that three distinct periods of sexual selection operate in black field crickets.     

When do the Costs of Spermatogenesis Constrain Sperm Expenditure? Remarks on the Pattern of the Spermatogenic Cycle

The costs of spermatogenesis constrain sperm expenditure when sperm production per day is limited. Thus, males are challenged to allocate available resources to sperm production and other life history functions. However, this prevailing assumption is not applicable to species in which spermatogenesis becomes quiescent during the breeding season. Males of these species prepare large quantities of sperm before the breeding season. Among these species, constraints on ejaculates have been intensively investigated in salamanders that deposit spermatophores. Although it is predicted that sperm expenditure should not be limited because of abundantly prepared sperm, spermatophore deposition is often limited during the breeding season when vas deferens are full of sperm. We tested a hypothesis regarding limited spermatophore deposition by measuring sperm quantity and volume of spermatophores sequentially deposited by male eastern newts Notophthalmus viridescens. A male newt rarely deposits more than three spermatophores per mating. If depletion of non‐sperm components of spermatophores limits spermatophore deposition, we predicted that spermatophore volume decreases while sperm quantity remains constant as a male deposits more spermatophores. Alternatively, some regulative mechanisms allow a limited portion of available sperm to be expended per mating, in which sperm quantity is predicted to decrease while the spermatophore volume remains constant. Finally, depletion of non‐sperm components may regulate sperm expenditure, which predicted that both spermatophore volume and sperm quantity decrease. We found that both sperm quantity and the spermatophore volume decreased as a male deposited more spermatophores during a single mating. Sperm expenditure was constrained without the costs involved in active spermatogenesis, and depletion of non‐sperm components likely regulate sperm quantity loaded in spermatophores. In dissociated spermatogenesis, constrained sperm expenditure do not mean that costly spermatogenesis is directly limiting male mating capacity but rather suggest that the evolution of physiological mechanisms regulating sperm expenditure per mating maximizes male reproductive success.     

Spermatophore and Sperm Allocation in Males of the Monandrous Butterfly Pararge aegeria: the Female’s Perspective

In insects, spermatophore production represents a non‐trivial cost to a male. Non‐virgin males have been shown to produce small spermatophores at subsequent matings. Particularly in monandrous species, it may be an issue to receive a sufficiently large spermatophore at the first and typically only mating. Females of the monandrous Speckled wood butterfly Pararge aegeria (L.) produce fewer offspring after mating with a non‐virgin male. After mating, females spend all their active time selecting oviposition sites and typically ignore other males. Here, we show that females did not discriminate between a virgin male and a recently mated male in our laboratory experiments. We demonstrate that the number of eupyrene sperm bundles relative to spermatophore mass differed with subsequent male matings. Males transferred a significantly smaller spermatophore after the first copulation, but the spermatophore mass did not decrease further with subsequent matings. However, the number of eupyrene sperm bundles decreased linearly. Therefore, there was proportionally more eupyrene sperm in the male’s second spermatophore compared with the first and the later spermatophores. Such a pattern has been shown in polyandrous species. Hence, it suggests that differences in sperm allocation strategy between polyandrous and monandrous butterflies may be quantitative rather than qualitative. There was also a tendency for females that had mated with a recently mated male to have higher propensity to remate than did females that had mated with a virgin male. We discuss the results relative to the mating system in P. aegeria, including female remating opportunities in the field and male mate‐locating behaviour.     

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.     

Regulation of long-term oviposition in the house cricket,Acheta domesticus: Roles of prostaglandin and factors associated with sperm

The transfer of spermatophore contents derived from testes during mating greatly stimulates ovipositional activity for long periods of time in the house cricket, Acheta domesticus (L.). Since prostaglandins appear to play a role in reproduction in several insect species, and since prostaglandin synthesis enzymes occur in cricket testes and spermatophores, we investigated the role of prostaglandins in the regulation of long-term oviposition. Inactivation of prostaglandin synthesis enzymes in males or females using specific inhibitors failed to block mating-induced increases in egg laying. However, males lacking sperm because of X-irradiation were unable to induce oviposition even though they mated, transferred spermatophores, and had high levels of prostaglandins in both testes and spermatophores. X-irradiation was also used to generate males with nonfunctional sperm. Females mated to these animals readily laid eggs, which failed to develop. It appeared that sperm or a factor associated with sperm induced long-term oviposition in female house crickets. Prostaglandin synthesis enzymes transferred from the male to females may have other roles in the female, for example, in sperm maintenance in the spermatheca. Previous observations strongly suggest that prostaglandins induce egg laying behavior and activity; they may be synthesized by female enzymes that are regulated by male-derived factors.     

Pairing and insemination patterns in a giant weta (Deinacrida rugosa: Orthoptera; Anostostomatidae)

Positive size assortative mating can arise if either one or both sexes prefer bigger mates or if the success of larger males in contests for larger females leaves smaller males to mate with smaller females. Moreover, body size could not only influence pairing patterns before copulation but also the covariance between female size and size of ejaculate (number of spermatophores) transferred to a mate. In this field study, we examine the pre-copulatory mate choice, as well as insemination, patterns in the Cook Strait giant weta (Deinacrida rugosa). D. rugosa is a large orthopteran insect that exhibits strong female-biased sexual dimorphism, with females being nearly twice as heavy as males. Contrary to the general expectation of male preference for large females in insects with female-biased size dimorphism, we found only weak support for positive size assortative mating based on size (tibia length). Interestingly, although there was no correlation between male body size and the number of spermatophores transferred, we did find that males pass more spermatophores to lighter females. This pattern of sperm transfer does not appear to be a consequence of those males that mate heavier females being sperm depleted. Instead, males may provide lighter females with more spermatophores perhaps because these females pose less of a sperm competition risk to mates.     

Mating behavior,insemination and sperm transfer in the ground beetle Carabus insulicola

Mating behavior and the processes of insemination and sperm transfer in the ground beetle Carabus insulicola were analyzed. C. insulicola has elaborate genitalia, in which the strongly sclerotized male copulatory piece is inserted into the female vaginal appendix in copula. During mating, I observed pre-copulatory struggles of males and females, as well as delays in ejaculation, suggesting the presence of intersexual conflicts. Insemination was achieved with a spermatophore, which strongly adhered to the openings of the spermatheca, common oviduct, and vaginal appendix. The spermatophore dissolved after copulation, and sperm were transferred into the spermatheca within three hours after copulation. Sperm bundles were contained within the testes and spermatophores, but free spermatozoa were found in the spermatheca.     

Condition-dependent spermatophore size is correlated with male's age in a bushcricket (Orthoptera: Phaneropteridae)

During mating, male bushcrickets transfer a spermatophore that consists of a sperm-containing ampulla and a product of the accessory glands, the spermatophylax, which females directly ingest. In the present study, we demonstrate male spermatophore allocation in the bushcricket Poecilimon zimmeri . Males of this species show condition-dependent spermatophore investment. This investment depended upon the age at first mating of males, with older individuals transferring larger spermatophores than younger ones of the same body mass. Independently of age, heavier males transfer larger spermatophores, but the size of males (as measured by femur length) was not a good predictor. Heavier males allocate a lower proportion of their mass to spermatophores and reach their maximal investment point earlier than less heavy males. Spermatophylax production levelled off to a species specific maximum earlier than that of sperm investment (measured as ampulla mass), suggesting that males face high levels of sperm competition.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 354–360.     

Ejaculate expenditure by malebush crickets decreases with sperm competition intensity

Male bushcrickets transfer a spermatophore at mating that consists of a sperm-containing ampulla and a sperm-free mass, the spermatophylax, that is consumed by the female during insemination. The costs of spermatophore production for males and benefits of consumption for females result in reversals in courtship roles in nutrient limited populations that increase both the risk and intensity of sperm competition. Here we show that under conditions characteristic of courtship role reversal, male expenditure on the spermatophore is dependent on female size. When mating with small females, males increase the amount of spermatophylax material and sperm, as expected from the increased sperm competition risk associated with courtship role reversal. However, males reduce the amount of spermatophylax material and sperm transferred to larger females. Since larger females have a higher mating success when competing for nurturant males, the intensity of sperm competition covaries with female size. Reduced ejaculate expenditure under increased sperm competition intensity is in accord with theoretical expectation.     

An integrative view of sexual selection in Tribolium flour beetles

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.     

Extreme Costs of Mating for Male Two-Spot Ladybird Beetles

Male costs of mating are now thought to be widespread. The two-spot ladybird beetle (Adalia bipunctata) has been the focus of many studies of mating and sexual selection, yet the costs of mating for males are unknown. The mating system of A. bipunctata involves a spermatophore nuptial gift ingested by females after copulation. In this study, we investigate the cost to males of mating and of transferring spermatophores in terms of lifespan, ejaculate production and depletion of nutritional reserves. We found that males faced a strong trade-off between mating and survival, with males that were randomly assigned to mate a single time experiencing a 53% reduction in post-mating lifespan compared to non-mating males. This is among the most severe survival costs of a single mating yet reported. However, spermatophore transfer did not impact male survival. Instead, the costs associated with spermatophores appeared as a reduced ability to transfer spermatophores in successive matings. Furthermore, males ingested more food following spermatophore transfer than after matings without spermatophores, suggesting that spermatophore transfer depletes male nutritional reserves. This is to our knowledge the first report of an effect of variation in copulatory behaviour on male foraging behaviour. Overall, our study highlights the advantages of assessing mating costs using multiple currencies, and suggests that male A. bipunctata should exhibit mate choice.     

Daily production of spermatophores, sperm number and spermatophore size in two eriophyoid mite species

Under dissociated sperm transfer, (non-pairing) males deposit spermatophores on a substrate, while females seek spermatophores and pick up sperm on their own. Spermatophore expenditures of non-pairing males should be high, due to the increased uncertainty of sperm uptake by a female. In this study I examined spermatophore expenditures in two eriophyoid species that differed in the degree of dissociation between sexes: (1) Aculus fockeui (Nalepa and Trouessart) males rarely visit quiescent female nymphs (QFNs), and mostly deposit spermatophores all over the leaves, whereas (2) Aculops allotrichus (Nalepa) males guard QFNs for many hours and deposit several spermatophores beside them. Males of both species were collected from the field and tested in solitude. Aculus fockeui males deposited on average 19.1 spermatophores per day, whereas A. allotrichus deposited only 3.6 spermatophores per day, and had a very large coefficient of variation. Males and spermatophores of A. allotrichus were significantly smaller and contained less sperm than those of A. fockeui. In both eriophyoids, spermatophore size was fitted to the size of female genitalia and the height of females. The ratio between the diameter of spermatophore head and the width of a female genital coverflap was 0.6, whereas the ratio between the female leg and the length of spermatophore stalk was 0.5. Several factors could be responsible for the discrepancy in spermatophore expenditures between species. Among other factors, the effects of male size, male reproductive strategy and female genitalia size on spermatophore output and size of spermatophores are discussed.     

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.