The relationship between mating system and penis morphology in ischnuran damselflies (Odonata: Goenagrionidae)

Zygopterans belonging to the genus Ischnura are unusual amongst damselflies because of the variety of mate guarding techniques employed by males of different species. The lack of post-copulatory guarding combined with lengthy copulations in one group of ischnuran species suggest that these males guard females in copula. An examination of the accessory penes of species in this group indicates that all but one species have considerable microspination on the distal end (the flagella) of their penes that can function in sperm displacement. The flagella of these species are long and thin compared to those of other ischnurans. This is likely an adaptation to gain access to the spermatheca of the female. Two species tandem guard their mates during ovipositing. These species are the only ischnurans missing a stout pair of basal spines on the penultimate segments of their penes. They have considerable microspination over much of their penes but their flagella are of only moderate length and stout. Ischnurans that do not mate guard have short, stout flagella and most species examined from this group (5 of 7) have litde microspination on their flagella tips. It is proposed that females of these species mate only once and therefore their males do not displace sperm.     

Evidence for widespread sperm displacement ability among Zygoptera (Odonata) and the means for predicting its presence

Males of the coenagrionid damselflies Argia moesta, A. sedula and hchnura ramburii use similar penis morphology to remove and/or reposition sperm of previous males from the storage organs of females prior to inseminating them. Although the species vary in the degree to which sperm is removed from or packed into the spermatheca, in all three species, sperm is removed from the bursa copulatrix. Since sperm in the bursa probably has priority in fertilizing eggs in at least the first oviposition after mating, sperm precedence can be estimated as the percentage of sperm (by volume) in the bursa belonging to the last male to mate. Estimated sperm precedence for these species is approximately 71% for Argia sedula, 82% for I. ramburii and 93% for A. moesta. These results, combined with similar ones for other damselflies clearly indicate that the ability to displace sperm may be widespread among temperate-zone Zygoptera. Species with each of the four major variations in damselfly penis structure have now been shown to displace sperm using this morphology. The systematic distribution of these major variants suggests several origins of sperm displacement ability within the Zygoptera. Whether or not all damselflies are capable of sperm displacement depends on both the presence of micro-structures used in sperm removal or repositioning and on the presence of sperm of previous males in mating females. It is possible, therefore, to predict that sperm displacement occurs in a damselfly if (1) females mate more than once, (2) mating females store sperm in organs accessible to penis morphology, (3) the distal segment of the male penis has structures similar to those known to be involved in sperm removal or repositioning, and (4) oviposition occurs in tandem or with the male non-contact guarding his mate.     

EVOLUTION OF MULTIPLE KINDS OF FEMALE SPERM-STORAGE ORGANS IN DROSOPHILA

Females of all species belonging to the family Drosophilidae have two kinds of sperm-storage organs: paired spherical spermathecae and a single elongate tubular seminal receptacle. We examined 113 species belonging to the genus Drosophila and closely allied genera and describe variation in female sperm-storage organ use and morphology. The macroevolutionary pattern of organ dysfunction and morphological divergence suggests that ancestrally both kinds of organs stored sperm. Loss of use of the spermathecae has evolved at least 13 times; evolutionary regain of spermathecal function has rarely if ever occurred. Loss of use of the seminal receptacle has likely occurred only once; in this case, all descendant species possess unusually elaborate spermathecae. Data further indicate that the seminal receptacle is the primary sperm-storage organ in Drosophila. This organ exhibits a pattern of strong correlated evolution with the length of sperm. The evolution of multiple kinds of female sperm-storage organs and the rapidly divergent and correlated evolution of sperm and female reproductive tract morphology are discussed.     

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.     

Evidence for biased use of sperm sources in wild female giant cuttlefish (Sepia apama)

In species where females store sperm from their mates prior to fertilization, sperm competition is particularly probable. Female Sepia apama are polyandrous and have access to sperm from packages (spermatangia) deposited by males onto their buccal area during mating and to sperm stored in internal sperm-storage organs (receptacles) located below the beak. Here, we describe the structure of the sperm stores in the female's buccal area, use microsatellite DNA analyses to determine the genetic diversity of stored sperm and combine these data with offspring genotypes to determine the storage location of paternal sperm. The number of male genotypes represented in the sperm receptacles was significantly lower than that found among the spermatangia. Estimation of the volumes of sperm contained in the receptacles and the spermatangia were statistically comparable; however, paternal sperm were more likely to have come from spermatangia than from the sperm receptacles. These results confirm a genetic polyandrous mating system in this species and suggest that fertilization pattern with respect to the sperm stores used is not random.     

Evolution of female sperm-storage organs in the carrefour of stylommatophoran gastropods

The presence of specialized female sperm-storage organs has been recognized as an important factor influencing postcopulatory sexual selection via sperm competition and cryptic female choice in internally fertilizing species. We morphologically examined the complexity of sperm-storage organs in the carrefour (spermatheca and fertilization pouch) in 47 species of stylommatophoran gastropods. We used partial 28S rDNA sequences to construct a molecular phylogeny, and applied maximum likelihood (ML) and Bayesian methods to investigate the history of spermatheca diversification and to test different hypotheses of sperm-storage organ evolution. The phylogenetic reconstruction supported several gains and losses of spermathecae. Moreover, a complex spermatheca was associated with the occurrence of love darts or other kinds of auxiliary copulatory organs, the presence of a long penial flagellum, and cross-fertilization as the predominant mating system. However, our results also suggest associations of carrefour complexity with body size, reproductive strategy (semelparity versus iteroparity), reproductive mode (oviparity versus ovoviviparity), and habitat type. Carrefour length in 17 snail species possessing a spermatheca was positively correlated with sperm length. Our results indicate that postcopulatory sexual selection as well as life history and habitat specificity may have influenced the evolution of female sperm-storage organs in hermaphroditic gastropods.     

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.     

Sperm competition and male ejaculate investment in Nauphoeta cinerea: effects of social environment during development

Selective pressure arising from sperm competition has been predicted to influence evolutionary and behavioural adjustment of ejaculate investment, but also may influence developmental adjustment of ejaculate investment. Immature males able to target resources strategically based on the competitive environment they will experience when they become sexually mature should be at a selective advantage. In our study we investigated how the presence of potential competitors or mates affects ejaculate and testes investment during development in the cockroach Nauphoeta cinerea, a species where males control female remating via their ejaculate size (large spermatophores prevent females from remating and therefore function to avoid sperm competition for males) and females store sperm. Our aim was to determine whether the social environment influences developmental adjustment of ejaculate investment and the relative importance of ejaculate components with different functions; avoidance of or engagement in sperm competition. We conclude that the social environment can influence developmental and behavioural flexibility in specific ejaculate components that may function to avoid or engage in sperm competition.     

Effects of broken male intromittent organs on the sperm storage capacity of female earwigs, <Emphasis Type="Italic">Euborellia plebeja</Emphasis>

In earwigs of the family Anisolabididae, male intromittent organs (virgae) sometimes break off inside female sperm-storage organs (spermathecae) during mating. I examined the effects of this genital breakage on the sperm storage capacity of females using Euborellia plebeja as a representative species. When genital breakage was artificially induced in virgin females, subsequent males successfully inseminated these females. However the sperm-storage capacity of these females was limited by the presence of broken virgae in their spermathecae. In another experiment, genital breakage was experimentally induced in the spermathecae of inseminated females, and their reproductive performance was then monitored for 60 days. In all of four cases where the entire piece of the broken virga remained inside the spermatheca, females deposited fertile eggs (more than 60% hatchability). The average number of clutches, that of eggs laid, and that of hatchlings were similar to those of controls. On the other hand, females laid no eggs in the other two cases where the broken virgae protruded from the spermathecal opening. I discuss the relevance of the results to the mating system and possible removal of rival sperm, which has been reported for E. plebeja. Electronic Publication     

Morphological structures for potential sperm storage in poeciliid fishes. Does superfetation matter?

Sperm storage within the female reproductive tract has been reported as a reproductive strategy in several species of vertebrates and invertebrates. However, the morphological structures that allow for sperm to be stored and kept viable for long periods are relatively unknown in osteichthyes. We use histological and stereological tools to identify and quantify sperm storage structures (spermathecae) in 12 species of viviparous Poeciliidae. We found spermathecae in nine species, six of which exhibit superfetation (the ability of females to simultaneously carry within the ovary two or more broods of embryos at different stages of development). These spermathecae are folds of ovarian tissue that close around spermatozoa. We compared the number and size (volume) of spermathecae between species with and without superfetation. Species that exhibit superfetation had a significantly higher number of spermathecae than species that do not exhibit this reproductive strategy. In addition, we found that the mean volume of spermathecae and total volume of spermathecae present in the ovary are marginally higher in species with superfetation. Our results contribute to the understanding of the morphological structures that allow for sperm storage in viviparous osteichthyes and suggest a positive relationship between superfetation and the capacity of females to store sperm.     

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.     

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.     

The spider Harpactea sadistica: co-evolution of traumatic insemination and complex female genital morphology in spiders

The males of invertebrates from a few phyla, including arthropods, have been reported to practise traumatic insemination (TI; i.e. injecting sperm by using the copulatory organ to penetrate the female''s body wall). As all previously reported arthropod examples have been insects, there is considerable interest in whether TI might have evolved independently in other arthropods. The research reported here demonstrates the first case of TI in the arthropod subphylum Chelicerata, in particular how the genital morphology and mating behaviour of Harpactea sadistica (Řezáč 2008), a spider from Israel, has become adapted specifically for reproduction based on TI. Males have needle-like intromittent organs and females have atrophied spermathecae. In other spiders, eggs are fertilized simultaneously with oviposition, but the eggs of H. sadistica are fertilized in the ovaries (internal fertilization) and develop as embryos before being laid. Sperm-storage organs of phylogenetically basal groups to H. sadistica provide males with last male sperm priority and allow removal of sperm by males that mate later, suggesting that TI might have evolved as an adaptive strategy to circumvent an unfavourable structure of the sperm-storage organs, allowing the first male to mate with paternity advantage. Understanding the functional significance of TI gives us insight into factors underlying the evolution of the genital and sperm-storage morphology in spiders.     

Mating strategy in Aleochara bilineata: sperm storage and allocation

Abstract By contrast to females that can maximize reproductive success with only one or a few copulations, males generally increase their fitness with frequency of mating. Sperm storage and allocation is therefore crucial for both male and female fitness. Sperm storage in Aleochara bilineata (Coleoptera; Staphylinidae) is investigated by measuring the number of spermatozoa stored in the female spermatheca after single, double or triple successive copulations with different males. The potential advantages of polyandry are studied in terms of the number of sperm stored by females mated twice with the same male (i.e. repeated copulation), compared with females mated twice with two different virgin males (i.e. polyandry). Level of polygyny is also estimated by measuring sperm allocation when ten successive mates are offered to a virgin male. Aleochara bilineata females store the sperm of the same or different males additively, suggesting no advantage for polyandry in terms of the number of sperm stored. A virgin male is able to inseminate ten different females but the number of sperm transferred decreases linearly. Finally, the latencies and durations of copulations are measured in all experiments to estimate changes according to the male or female status (i.e. virgin or mated). The latency before mating is higher when females are virgin than when females have already mated.     

Sperm length evolution in the fungus-growing ants

Eusocial insects offer special opportunities for the comparativestudy of sperm traits because sperm competition is absent (inspecies with obligatory monandry) or constrained (in lineageswhere queens mate multiply but never remate later in life).We measured sperm length in 19 species of fungus-growing ants,representing 9 of the 12 recognized genera, and mapped theseonto the ant phylogeny. We show that average sperm length acrossspecies is highly variable and decreases with mature colonysize in basal genera with singly mated queens, suggesting thatsperm production or storage constraints affect the evolutionof sperm length. Sperm length does not decrease further in multiplymating leaf-cutting ants, despite substantial further increasesin colony size. In a combined analysis, sexual dimorphism explained63.1% of the variance in sperm length between species. As colonysize was not a significant predictor in this analysis, we concludethat sperm production trade-offs in males have been the majorselective force affecting sperm length across the fungus-growingants, rather than storage constraints in females. The relationshipbetween sperm length and sexual dimorphism remained robust inphylogenetically independent contrasts. Some of the remainingvariation was explained by the relative size of the sperm-storageorgan, but only in the multiply mating leaf-cutting ants, suggestingthat sperm-storage constraints become important for the evolutionof sperm length in this derived group. Mate number affectedsperm length to a minor extent, and only in interaction withother predictor variables, suggesting that sperm competitionhas not been a major selective force for sperm length evolutionin these ants.     

Mechanisms of sperm removal and sperm transfer in Orthetrum coerulescens (Fabricius) (Odonata: Libellulidae)

ABSTRACT An account of the structure of the secondary genitalia of male Orthetrum coerulescens (Fabricius) is given together with an outline of the internal genitalia of the female. Observations on the experimental inflation of the penis are described. No sperm was released during fast inflations and deflations of the penis, but sustained inflation of the penis led to the release of a clear fluid followed by the slow discharge of sperm. Examination of natural copulations in the field has shown that rapid rhythmic movements at up to 5 Hz take place in the male's third abdominal segment throughout most of copulation, but towards the end they cease though inflation is maintained. It is suggested that the rapid movements coincide with the removal of rival sperm from the female and that sperm is transferred to the female only during the maintained inflation. Possible mechanisms of sperm translocation, sperm removal and sperm transfer are discussed with reference to male and female genital structures and the action of relevant muscles.     

Effects of Sexual Selection Upon Sperm Morphology and Sexual Skin Morphology in Primates

We review possible effects of sexual selection upon sperm morphology, and sexual skin morphology, in primates. Comparative morphometric studies, involving 31 species representing 21 primate genera, revealed a positive relationship between volume of the sperm midpiece, occurrences of multiple partner matings by females, and large relative testes sizes, which indicate sperm competition. The midpiece houses the mitochondria required to power sperm motility. Hence, sperm competition may have influenced the evolution of increased mitochondrial loading in species where females mate with multiple partners during the fertile period. Females of some Old World monkey species and female chimpanzees exhibit large estrogen-dependent sexual skin swellings during the follicular phase of the menstrual cycle. Studies of mandrills support the conclusion that swellings act primarily as sexually attractive, graded signals and that swelling size may indicate current reproductive quality. Measurements of the genitalia in chimpanzees indicate a secondary function for female swellings. The swelling increases the operating depth of the female's vagina by 50% during the fertile phase of her cycle. Males have evolved long, filiform penes capable of placing sperm close to the os cervix during competitive multipartner matings. This may exemplify how morphologic specializations in females can influence the coevolution of advantageous genitalic specializations in males: the phenomenon that Eberhard (1985) dubbed cryptic female choice.     

Multiple paternity in a natural population of a salamander with long-term sperm storage

Sperm competition appears to be an important aspect of any mating system in which individual female organisms mate with multiple males and store sperm. Post-copulatory sexual selection may be particularly important in species that store sperm throughout long breeding seasons, because the lengthy storage period may permit extensive interactions among rival sperm. Few studies have addressed the potential for sperm competition in species exhibiting prolonged sperm storage. We used microsatellite markers to examine offspring paternity in field-collected clutches of the Ocoee salamander (Desmognathus ocoee), a species in which female organisms store sperm for up to 9 months prior to fertilization. We found that 96% of clutches were sired by multiple males, but that the majority of females used sperm from only two or three males to fertilize their eggs. The high rate of multiple mating by females suggests that sperm competition is an important aspect of this mating system. Comparison of our data with those of other parentage studies in salamanders and newts reveals that multiple mating may be common in urodele amphibians. Nevertheless, the number of males siring offspring per clutch in D. ocoee did not differ appreciably from that in other species of urodeles with shorter storage periods.     

Sperm storage and utilization in female Queensland fruit flies (Bactrocera tryoni)

Abstract.  Female animals that use sperm from a single mating to fertilize eggs over an extended period require efficient mechanisms for sperm storage and use. There have been few studies of these mechanisms in tephritid flies. Mating, copula duration, sperm storage and sperm usage patterns are assessed in an Australian tephritid, the Queensland fruit fly ( Bactrocera tryoni ; a.k.a. 'Q-fly'). In particular, the present study investigates whether each of these aspects of mating varies in relation to female size or male size, whether sperm storage patterns change over time after mating (1, 5, 10 and 15 days), and the relative roles of the ventral receptacle and the two spermathecae as sperm storage organs. Large females are more likely to mate than are small females, and are also more fecund in the first 5 days after mating. Females are more likely to store some sperm and, among those that store some sperm, store more sperm if their mate is large. Most sperm are stored in the spermathecae (median = 97%), often with high levels of asymmetry between the two spermathecae. Asymmetry of sperm storage is related to number of sperm stored, but not to male or female size. Total number of stored sperm declines over the 15 days after mating, but this decrease in sperm numbers only reflects changes in the spermathecae; numbers of sperm in the ventral receptacle remain unchanged over this period. As a consequence, the proportion of total sperm stored in the spermathecae declines relative to the ventral receptacle. These results are consistent with a system in which small numbers of sperm are maintained in the ventral receptacle for fertilizations, and are replenished by sperm from the spermathecae as required. Sperm distribution and usage patterns in Q-flies are comparable with recent findings in medflies, Ceratitis capitata , but differ markedly from patterns found in several Anastrepha species.