Sexual selection on bushcricket genitalia operates in a mosaic pattern

In most species with internal fertilization, male genitalia evolve faster than other morphological structures. This holds true for genital titillators, which are used exclusively during mating in several bushcricket subfamilies. Several theories have been proposed for the sexual selection forces driving the evolution of internal genitalia, especially sperm competition, sexually antagonistic coevolution (SAC), and cryptic female choice (CFC). However, it is unclear whether the evolution of genitalia can be described with a single hypothesis or a combination of them. The study of species‐specific genitalia action could contribute to the controversial debate about the underlying selective evolutionary forces. We studied female mating behaviors in response to experimentally modified titillators in a phylogenetically nested set of four bushcricket species: Roeseliana roeselii, Pholidoptera littoralis littoralis, Tettigonia viridissima (of the subfamily Tettigoniinae), and Letana inflata (Phaneropterinae). Bushcricket titillators have several potential functions; they stimulate females and suppress female resistance, ensure proper ampulla or spermatophore attachment, and facilitate male fixation. In R. roeselii, titillators stimulate females to accept copulations, supporting sexual selection by CFC. Conversely, titillator modification had no observable effect on the female's behavior in T. viridissima. The titillators of Ph. l. littoralis mechanically support the mating position and the spermatophore transfer, pointing to sexual selection by SAC. Mixed support was found in L. inflata, where manipulation resulted in increased female resistance (evidence for CFC) and mating failures by reduced spermatophore transfer success (evidence for SAC). Sexual selection is highly species‐specific with a mosaic support for either cryptic female choice or sexually antagonistic coevolution or a combination of both in the four species.     

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.     

Copulatory courtship by bushcricket genital titillators revealed by functional morphology, μCT scanning for 3D reconstruction and female sense structures

Genitalia are rapidly evolving morphological structures most likely under sexual selection. Due to their internal nature they are often hidden inside the body, thus morpho-functional studies of animal genitalia are broadly lacking. Males of some bushcricket taxa bear paired genital appendices called titillators, the exact function of which is unknown since they are obscured inside the female body during pairing. To investigate titillator morphology and possible function during copulation, we studied the bushcricket Metrioptera roeselii (Hagenbach, 1822) using a novel combination of independent, yet complementary, techniques. Copulating pairs were snap-frozen and scanned by X-ray micro-computed tomography (μCT) to visualize the coupling of male and female genitalia in situ. Video recordings of copulating pairs also showed rhythmical insertion of male titillators into the female's genital chamber, where they percuss a softened structure on the female's subgenital plate. Movements did not induce damage to the female's structure, which lacks any sclerotized genital counterparts. Instead, scanning electron microscopy and histological sections show the female subgenital plate to be covered with two different types of sensory receptors at the contact zone between the male's titillator and the female genital chamber. We interpret the non-harmful function of the titillator processes, the lack of a genital counter-structure and the presence of sensory cells on the female's subgenital plate as indicators of a copulatory courtship function of titillators, subject to sexual selection by female choice.     

Operational Sex Ratio and Alternative Reproductive Behaviours in Chinese Bushcricket, Gampsocleis gratiosa

The effects of operational sex ratio (OSR) on male mating tactics in the Chinese bushcricket Gampsocleis gratiosa were investigated in male‐ and female‐biased environments. We measured fresh and dry spermatophore contents and copulation duration, and counted sperm numbers of each copulation. The fresh weight of spermatophore and spermatophylax was positively correlated with male body weight. The males in a strongly male‐biased environment produced significantly heavier fresh ampulla and more sperm per ejaculation, which were likely tactics for successful matings under the competition of rivals. The spermatophore might function as a structure to protect the fertilization potential of the ejaculate from rival males.     

Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Increased copulation duration before ejaculate transfer is associated with larger spermatophores, and male genital titillators, across bushcricket taxa

Copulation duration varies considerably across species, but few comparative studies have examined factors that might underlie such variation. We examined the relationship between copulation duration (prior to spermatophore transfer), the complexity of titillators (sclerotized male genital contact structures), spermatophore mass and male body mass across 54 species of bushcricket. Using phylogenetic comparative analyses, we found that copulation duration was much longer in species with titillators than those without, but it was not longer in species with complex compared with simple titillators. A positive relationship was found between spermatophore size and copulation duration prior to ejaculate transfer, which supports the hypothesis that this represents a period of mate assessment. The slope of this relationship was steeper in species with simple rather than complex titillators. Although the data suggest that the presence of titillators is necessary to maintain long copulation prior to ejaculate transfer, mechanisms underlying this association remain unclear.     

Discriminating Males and Unpredictable Females: Males Bias Sperm Allocation in Favor of Virgin Females

When both sexes mate with multiple partners, theory predicts that males should adjust their investment in ejaculates in response to the risk and/or intensity of sperm competition. Here, we demonstrate that, in the harlequin beetle riding pseudoscorpion, Cordylochernes scorpioides, males use cues deposited on females by previous males to distinguish between virgin, once‐mated, and multiply‐mated females and adjust sperm allocation accordingly. Sperm number declined in direct proportion to the number of previous males, with virgin females receiving nearly three times more sperm than females exposed to three previous males. Given the lack of first‐male sperm precedence in C. scorpioides, this pattern is not consistent with current sperm competition models and appears best explained by a significant risk of wasting ejaculates on deceitful, mated females. In C. scorpioides, males transfer sperm indirectly to females via a stalked spermatophore deposited on the substrate. Mated females often feign sexual receptivity and cooperate throughout mating, only to reject the sperm packet produced by the male. While indirect sperm transfer facilitates a high level of female deceit and control, females of many species are able to influence the number and fate of sperm transferred during copulation and are likely to conceal their sexual unreceptivity to minimize male retaliation. If males cannot accurately assess female receptivity, increased risk of sperm rejection by mated females could outweigh the risk of sperm competition and favor greater sperm allocation to virgin females.     

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.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

Male’s copulation investment, including spermatophore and sperm investment were very high in the Chinese bushcricket Gampsocleis gratiosa. The effects of mating status of both males and females on male’s copulation investment were examined in this study. The fresh weight of spermatophylax increased positively with the weight of males’ body. This indicated that the nutritional investment during copulation depended on male’s quality. Spermatophore investment showed insignificant differences in every copulation protocols. This finding supported the paternal investment hypothesis, that is, males contributed to their offspring with little attention to their partners. Sperm releasing per ejaculation varied significantly among the trials. Males decreased 54.19% sperm in second mating than in its first mating, demonsrated that males regarded the first mating highly, and were more prudent in subsequent mating. These males’ strategies may contribute to the viability of the offspring.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

Male’s copulation investment, including spermatophore and sperm investment were very high in the Chinese bushcricket Gampsocleis gratiosa. The effects of mating status of both males and females on male’s copulation investment were examined in this study. The fresh weight of sper-matophylax increased positively with the weight of males’ body. This indicated that the nutritional in-vestment during copulation depended on male’s quality. Spermatophore investment showed insig-nificant differences in every copula...~     

Males prefer virgin females,even if parasitized,in the terrestrial isopod Armadillidium vulgare

In many species, males increase their reproductive success by choosing high‐quality females. In natural populations, they interact with both virgin and mated females, which can store sperm in their spermatheca. Therefore, males elaborate strategies to avoid sperm competition. In the terrestrial isopod Armadillidium vulgare, females can store sperm and produce several clutches. Moreover, this species can be parasitized by Wolbachia, which feminizes genetic males, transforming them into functional females. Our study compared attractiveness and mate choice when a male is exposed to both virgin and experienced females (i.e., females who have produced offspring and rested for 6 months), with or without Wolbachia. Our results revealed that males are more attracted to virgin females than experienced females, even if these virgin females are parasitized. Moreover, the chemical analysis highlighted different odors in females according to their reproductive and infection (Wolbachia‐free or vertically Wolbachia‐infected) status. Males attempted copulation more frequently and for longer with virgin females, even if Wolbachia‐infected, while experienced females refused further copulation. The evolutionary consequences of both male choice and female resistance on their fitness are discussed in this study.     

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 ejection and retention in the cornsilk fly Euxesta eluta (Diptera: Ulidiidae)

Promiscuous mating systems provide the opportunity for females to bias fertilization toward particular males. However, distinguishing between male sperm competition and active female sperm choice is difficult for species with internal fertilization. Nevertheless, species that store and use sperm of different males in different storing structures and species where females are able to expel all or part of the ejaculates after copulation may be able to bias fertilization. We report a series of experiments aimed at providing evidence of female sperm choice in Euxesta eluta (Hendel), a species of ulidiid fly that expels and consumes ejaculates after copulation. We found no evidence of greater reproductive success for females mated singly, multiply with the same male, or mated multiply with different males. Female E. eluta possesses two spherical spermathecae and a bursa copulatrix for sperm storage, with a ventral receptacle. There was no significant difference in storing more sperm in spermathecae 24 h after copulation than immediately after copulation. Females mated with protein-fed males had greater reproductive success than similar females mated to protein-deprived males. Protein-fed females prevented to consume the ejaculate, retained more sperm when mated to protein-fed males than when mated to protein-deprived males. Our results suggest that female E. eluta can exert control of sperm retention of higher quality males through ejaculate ejection.     

Mated Redback Spider Females Re-Advertise Receptivity Months after Mating

In many species, selection acts on males to recognize female reproductive status at a distance using pheromones. Unmated females may actively seek to attract males; however, mated females may become cryptic to avoid attracting additional males if multiple matings are costly. Although females of many species cease pheromone production after mating, it is often unclear whether this is a strategic part of a female reproductive strategy, or whether this is because of chemical manipulation by males. If variation in pheromone production is part of the female’s strategy, then we predicted mated females should eventually re‐advertise receptivity if the benefits of multiple mating increase with time since copulation (e.g. because of sperm depletion). Here, we tested this prediction in Australian redback spiders (Latrodectus hasselti). First, we replicated earlier results by showing that virgin males discriminate female maturity and mating status based exclusively on web‐borne chemicals. Our results show this difference must arise from a change in chemical deposition in the web as we controlled for web volume differences between mated and virgin females. Male activity on extracts from webs of virgin females exceeded activity on a solvent control and on extracts of webs of just‐mated females—confirming that female redbacks cease pheromone production immediately after mating. Second, we tested a new prediction that mated females might re‐advertise receptivity near the end of their normal breeding season to replenish diminished sperm stores prior to overwintering. Consistent with the prediction of strategic advertisement, we show that male activity on extracts from females’ webs increased significantly 3 mo after the female first mated (typical length of the breeding season). Thus, these females had begun to add pheromone to their web again. At this time, 26% of these females re‐mated with a second male. If females re‐advertise receptivity to ensure adequate sperm stores, then we predicted a positive relationship between female reproductive output during the 3‐mo interval after copulation and the subsequent intensity of male response to web extracts. However, differences in male activity time were not related to the total number of spiderlings or the number of egg sacs a female had produced during the 3‐mo interval after the first copulation. This result could arise if male chemical manipulation of female receptivity decreases with time after copulation, or if the testing interval used in our study was too long to reveal variation in sperm depletion in females. Thus, although our results are consistent with the idea that females strategically alter pheromonal advertisement, we cannot distinguish this from the hypothesis that female receptivity arises from chemical manipulation by males.     

Spermatophore expulsion in the carrion beetle Silpha perforata (Coleoptera: Silphidae)

Silphinae (Coleoptera: Silphidae) is an abundant decomposer that plays important roles in the ecosystem. However, there is little information about the life history of this taxon. We found sperm displacement behavior in carrion beetle Silpha perforata. Copulating males bit the female's antenna strongly and inserted the penis into the partner's genital organ more than once. We found a white substance on the tip of penis during copulation. We examined whether this white substance is a previous male's spermatophore, which was removed from the mating partner. When females were dissected just after mating, the same substance that often presents on the penis of mating males was found in the bursa copulatrix of females, although the bursa copulatrix of virgin females was empty. Male behavior during copulation with females of different mating history was also observed to confirm that the removal of spermatophores was observed only in copulation with females that have the spermatophores of previous males. Consequently, we estimated that S. perforata males removed spermatophores of previous males from mating partners. In addition, we dissected the males frozen during copulation, and inspected the penis morphology. This observation revealed that the apical part of the penis was usually hidden in the basal part of penis, but expanded and appeared during insertion. This apical part had many spines, which play an important role in sperm displacement and sexual conflict in some species. These results indicate that there is the sperm competition in S. perforata. This is the first report on sperm competition in Silphinae.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Multiple mating and lifetime reproductive output in female dobsonflies that receive nuptial gifts

In mating of the dobsonfly, Protohermes grandis (Thunberg), the male attaches the spermatophore externally to the female genitalia. The spermatophore includes a large gelatinous mass which the female detaches and feeds on after mating. While the female consumes this nuptial food gift, sperm is evacuated from the remaining portion of the spermatophore (sperm package) into her reproductive tract. Under laboratory conditions, mated females maintained receptivity throughout their lifetime, and they remated even on the day following copulation. A single insemination may supply enough sperm, as females mated only once deposited fertile eggs throughout life and, when dissected after death, all females had sperm in the spermatheca. There was a positive correlation between longevity and the number of matings. Lifetime fecundity also increased as mating multiplied. However, the size of eggs and hatchlings was not influenced by the number of matings. It seems that large spermatophore consumption by female P. grandis provides nutrients that increase fitness not in offspring quality, but in their quantity.     

Mating with sperm-depleted males does not increase female mating frequency in the parasitoid Lariophagus distinguendus

Sexual conflicts due to divergent male and female interests in reproduction are common in parasitic Hymenoptera. The majority of parasitoid females are monandrous, whereas males are able to mate repeatedly. Thus, accepting only a single mate might be costly when females mate with a sperm‐depleted male, which may not transfer a sufficient amount of sperm. In the present study, we investigated the reproductive performance in the parasitoid Lariophagus distinguendus Först. (Hymenoptera: Pteromalidae) and studied whether mating with experimentally sperm‐depleted males increases the tendency of females to remate. Males were able to mate with up to 17 females offered in rapid succession within a 10‐h test period. The resulting female offspring, as an indirect measure of sperm transfer, remained constant during the first six matings and then decreased successively with increasing number of copulations by the males. Experimentally sperm‐depleted males continued to mate even if they transferred only small amounts or no sperm at all. Unlike males, the majority of females mated only once during a 192‐h test period. A second copulation was observed only in a few cases (maximum 16%). The frequency of remating was not influenced by the mating status of the first male the females had copulated with, suggesting that these events are not controlled by sperm deficiency of the females. Furthermore, we investigated male courtship behaviour towards mated females. Male courtship intensity towards mated females decreased with increasing time. However, females that had mated with an experimentally sperm‐depleted male did not elicit stronger or longer‐lasting behavioural responses in courting males than those that had mated with a virgin male. As the observed behaviours in L. distinguendus are known to be elicited by a courtship pheromone, these results suggest that females no longer invest in pheromone biosynthesis after mating (as indicated by ceasing behavioural responses of courting males), irrespective of whether they have received a sufficient amount of sperm or not. We discuss the results with respect to a possible mating strategy of sperm‐depleted males.     

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.     

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.