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.     

X-irradiation of theDrosophila male and its effect on the number of sperm transferred to the female

Summary Experiments were carried out to determine the number of sperm transferred by the maleDrosophila after X-irradiation. Acute doses (1000 r/min) of 5,000 r, 25,000 r and 50,000 r delivered to the male results in reductions of some 20–25%, 60–65%, and 85–90%, respectively, in the number of sperm scored in the ventral receptable of inseminated females. It appears, further, that sperm in ejaculates of relatively small size (50 or fewer) from irradiated as well as unirradiated males distribute themselves such that 20–50% or more found in the spermathecae. The bearing of this observation on the apparent 1:1 sperm: progeny ratio reported byPeacock andErickson (1965) from ejaculates with low sperm number is noted.Supported by National Science Foundation Grant GB-3117.     

A female nervous system is necessary for normal sperm storage in Drosophila melanogaster: a masculinized nervous system is as good as none

A male Drosophila melanogaster deposits many more sperm in a female''s bursa copulatrix than are stored in her ventral receptacle or paired spermathecae soon after copula has ended. The remaining sperm are expelled by the female. These observations suggest a sexual conflict over the processes involved in sperm storage. We used genetically manipulated flies to study the role of the central nervous system in sperm storage. Flies with female bodies but masculinized nervous systems, or isolated female abdomens, stored significantly fewer sperm than did control females. Furthermore, compared with control flies, there were relatively more sperm in the ventral receptacle and relatively fewer in the spermathecae. These results suggest that the female nervous input counteracts the male''s attempts to force sperm into the ventral receptacle during copula and promotes active transport of sperm to the spermathecae during and after copula. The female is clearly a very active partner in influencing processes involved in sperm competition, especially as only stored sperm can be used later to fertilize eggs. To our knowledge, this is the first study to show directly the involvement of the female nervous system in sperm storage.     

Ultrastructure of the Spermathecae of Parafabricia ventricingulata and Three Species of Oriopsis (Polychaeta: Sabellidae)

Parafabricia ventricingulata females have a pair of spermathecae located in the radiolar crown anterio-dorsal to the buccal opening. The spermathecae have three regions; an entrance, 7 μm across, leading into a ciliated ‘atrium’ that is approximately 50 μm long; a connecting piece, 2–5 μm across and 25 μm long, leading from the ‘atrium’ to the sperm receptacle. The sperm receptacle is heavily pigmented and spherical. The sperm lie in a large mass in the receptacle with no particular orientation. Oriopsis bicoloris females have a pair of unpigmented spermathecae in the collar behind the radiolar crown. Each spermatheca is a simple blind duct 100 μm long, with a lumen 8 μm in diameter. Between 30 and 40 sperm lie in the lumen of each spermatheca. Oriopsis brevicollaris females have a pair of spermathecae located in the radiolar crown above the buccal opening. From the opening, 10 μm across, a blind duct runs for 90 μm. Sperm are stored in the distal region of the duct. Sperm lie along the margins of the duct in close contact with microvilli. Up to 10 sperm were found in each spermatheca. Oriopsis mobilis females have a pair of spermathecae located in the radiolar crown above the buccal opening. The opening, 3 μm across, leads into a blind duct that runs for 30 μm. Sperm are stored in the distal region of the spermathecae where they are embedded in spermathecal cells. Between 10 and 20 sperm were found in each spermatheca. Oriopsis dentata was found not to have spermathecae. The homologies of the spermathecae found within the Sabellinae and Fabriciinae (Sabellidae) and the Spirorbinae (Serpulidae) are discussed, but cannot be resolved on present evidence.     

Ejaculate esterase 6 and initial sperm use by female Drosophila melanogaster

The period of initial sperm storage and use by Drosophila melanogaster females is examined for effects of the seminal fluid enzyme esterase 6. Females mated to males differing in their level of esterase 6 activity were dissected from 5 min to 50 hr after the start of copulation and numbers of sperm contained in the uterus, ventral receptacle and paired spermathecae were counted. Of the 4000–6000 sperm transferred at copulation, about 700 are stored in the receptacle by 4 hr post mating and 400 in the spermathecae by 7 hr. However, sperm are released rapidly from storage organs following these peaks and may be found again in the uterus in numbers up to 100 or more. The rate of sperm release is closely related to the level of esterase 6 activity, suggesting that this seminal fluid enzyme is involved in sperm motility.     

Coevolution of sperm and female reproductive tract morphology in stalk-eyed flies

Sperm and female reproductive tract morphology are among the most rapidly evolving characters known in insects. To investigate whether interspecific variation in these traits results from divergent coevolution we examined testis size, sperm length and female reproductive tract morphology for evidence of correlated evolution using 13 species of diopsid stalk-eyed flies. We found that sperm dimorphism (the simultaneous production of two size classes of sperm by individual males) is ancestral and occurs in four genera while sperm monomorphism evolved once and persists in one genus. The length of ''long-sperm'' types, though unrelated to male body or testis size, exhibits correlated evolution with two regions of the female reproductive tract, the spermathecae and ventral receptacle, where sperm are typically stored and used for fertilization, respectively. Two lines of evidence indicate that ''short sperm'', which are probably incapable of fertilization, coevolve with spermathecae. First, loss of sperm dimorphism coincides phylogenetically with reduction or loss of spermathecae. Second, evolutionary change in short-sperm length correlates with change in spermathecal size but not spermathecal duct length or ventral receptacle length. Morphological coevolution between sperm and female reproductive tracts is consistent with a history of female-mediated selection on sperm length.     

A light and electron microscopical study of the spermathecae and ventral receptacle of Anastrepha suspensa (Diptera: Tephritidae) and implications in female influence of sperm storage

Female insects with multiple sperm storage organs may potentially influence patterns of paternity by differential storage of sperm from competing males. The Caribbean Fruit Fly, Anastrepha suspensa, stores sperm differentially with respect to its three spermathecae. To understand the mechanisms and processes responsible for patterns of sperm storage and use in A. suspensa, details of the fine structure of female sperm storage organs were resolved by UV-light microscopy, confocal microscopy, tissue sectioning, and scanning and transmission electron microscopy. Structures not previously described for this species include a ventral receptacle for sperm storage and osmoregulation, a conical-shaped valve at the junction between the spermathecal capsules and their ducts, laminar and granular secretions, secretions from the signum, hemocytes surrounding the spermathecae, and spermathecae with sclerotized, hollow projections that terminate in single glandular cells. The independent organization of sperm storage organs, spermathecal ducts, associated musculature, gland cells, and innervation offer possible mechanisms by which sperm movement may be influenced by females. The implications of these structures for insemination and fertilization events are discussed.     

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.     

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.     

A hypothesis about the origin of sperm storage in the Eubrachyura, the effects of seminal receptacle structure on mating strategies and the evolution of crab diversity: How did a race to be first become a race to be last?

The origins and evolution of sperm storage in Brachyura are enigmatic: sperm is either stored in seminal receptacles, accessible via the vulvae on the sixth thoracic sternite, or in spermathecae at the border between the seventh and eighth sternites. Crabs with spermathecae are collectively referred to as “podotremes” while crabs with seminal receptacles belong to the Eubrachyura. The position of gonopores is the primary basis for subdividing the Eurachyura into the Heterotremata (female vulvae + males with coxal gonopores) and Thoracotremata (female vulvae + males with sternal gonopores). We present a hypothesis about the evolution of seminal receptacles in eubrachyuran female crabs and argue that the sternal gonopore has been internalized into chitin-lined seminal receptacles and the vulva is in fact a secondary aperture. The loss of some or all of the ancestral chitinous seminal receptacle lining was linked to ventral migration of the oviduct connection. Male and female strategies are to maximize gamete fertilization. The most important variable for females is sperm supply, enhanced by long-term storage made possible by the seminal receptacle. To maximize their fertilization rates males must adapt to the structure of the seminal receptacle to ensure that their sperm are close to the oviduct entrance. The major evolutionary impetus for female mating strategies was derived from the consequences of better sperm conservation and the structure of the seminal receptacle. The advantages were all to the females because their promiscuity and sperm storage allowed them to produce more genetically variable offspring, thereby enhancing variation upon which natural selection could act. We extend our arguments to Brachyura as a whole and offer a unifying explanation of the evolution of seminal receptacles, comparing them with the spermathecae found in “Podotremata”: they were independent solutions to the same problem: maintaining sperm supply during evolutionary carcinization.Explanation of eubrachyuran mating strategies requires analysis of the mating–moulting link, indeterminate vs. determinate growth format and seminal receptacle structure. Two alternatives for each of these characters means that there are eight possible outcomes. Six of these outcomes have been realized, which we term Portunoid, Majoid, Eriphoid, Xanthoid, Cancroid, and Grapsoid–Ocypodoid strategies, respectively. Mapping these characters on to a workable phylogeny (wherein some changes to the seminal receptacle + moulting–mating links are assumed to have occurred more than once) produces the following relationships: Portunoids + Majoids are a sister group to the rest of the Eubrachyura, which fall into two sister groups, Eriphoids + Xanthoids and Cancroids + Grapsoid–Ocypodoids and the “Podotremata” is sister group to all the Eubrachyura. We conclude that what began as a race to be the first to mate was turned on its head to become a race to be last, by the evolutionary changes to the seminal receptacle. Eubrachyuran females were advantaged by greater reproductive autonomy, more opportunity to mate with other males, resulting in more genetically variable progeny and leading to the evolution of much greater taxonomic diversity compared to “podotremes”.     

Age‐specific changes in sperm levels among female tsetse (Glossina spp.) with a model for the time course of insemination

The age, insemination and ovulation status of tsetse flies Glossina pallidipes Austen (n = 154369) and Glossina morsitans morsitans Westwood (n = 19659), captured over 11 years in Zimbabwe, are assessed by ovarian dissection. Instantaneous rates of insemination increase exponentially with age in both species; 90% insemination levels are reached after 5 days post‐emergence in G. m. morsitans and 7 days in G. pallidipes, varying little with season. More than 95% of both species have ovulated by the age of 8 days and 99% by 12 days. Older flies that have not ovulated are > 100‐fold more likely to be caught in October and November than in other months. A 500‐fold decrease in trap catches did not result in any detectible decrease in the probability of females being inseminated. The proportion of partially filled spermathecae rises for approximately 6 days then declines, consistent with some flies having mated more than once. For flies caught on electric nets, with wings undamaged during capture, wing‐fray data are used to extend ovarian age estimates up to 11 ovulations. Among these flies, the volume of sperm in the spermathecae declines little in flies that have ovulated up to seven times; thereafter, it declines by approximately 1% per ovulation. The time course of insemination and the mating frequency of females are important considerations in modelling tsetse fly populations, as well as for the dynamics of interventions involving the release of genetically‐modified insects, which should not be seriously compromised by the limited levels of polyandry currently observed.     

Control of copula duration and sperm storage by female Queensland fruit flies

Copula duration and sperm storage patterns can directly or indirectly affect fitness of male and female insects. Although both sexes have an interest in the outcome, research has tended to focus on males. To investigate female influences, we compared copula duration and sperm storage of Queensland fruit fly females that were intact, or had been incapacitated through decapitation or abdomen isolation. We found that copulations were far longer when females had been incapacitated, indicating that constraints imposed on copula duration by intact females had been relaxed. Repeatability of copula duration for males was very low regardless of female treatment, and this is also consistent with strong female influence. Number of sperm in the spermathecae was not influenced by female treatment, suggesting that female abdominal ganglia control the transport of sperm to these long-term storage organs. However, more sperm were found in the ventral receptacles of incapacitated females compared to intact females. Overall, results implicate cephalic ganglia in regulation of copula duration and short-term sperm storage in the ventral receptacle and abdominal ganglia in regulation of long-term sperm storage in the spermathecae.     

Sperm allocation and cost of mating in a tropical tephritid fruit fly

Males that copulate repeatedly may suffer from reduced sperm stores. However, few studies have addressed sperm depletion from both the female and male perspective. Here, we show that male Anastrepha obliqua (Diptera: Tephritidae) do not ejaculate all available sperm and are left with mature sperm in the seminal vesicles even after copulating as often as three times in half a day. Ejaculate size was not related to male mating history; time elapsed since the last mating, copulation duration, female thorax length or head width. Larval host origin did not affect the number of sperm stored by females. More sperm was found in the ventral receptacle compared to sperm stored in the three spermathecae. Males apparently do not suffer a cost of mating in terms of longevity, although we cannot rule out other fitness costs. Sperm production in this species may not be as costly as it is for other species. Results suggest that males strategically allocate similar numbers of sperm among successive mates without exhausting sperm reserves for future encounters. We discuss the role that differential sperm storage may have in mediating sperm competition and tie our results to the unique natural history of A. obliqua.     

Copula duration and sperm storage in Mediterranean fruit flies from a wild population

In the Mediterranean fruit fly (Ceratitis capitata Weidemann, ‘medfly’), a lekking tephritid, evidence from laboratory studies of flies from laboratory strains suggests that copulation is shorter, and sperm storage more abundant, if males are large or protein‐fed, and that copulation is longer when females are large. In addition, sperm tend to be stored asymmetrically between the female’s two spermathecae and this asymmetry declines with abundance of stored sperm. The primary objective of this study was to investigate whether these trends persist in other experimental contexts that bear closer resemblance to nature. Accordingly, we carried out experiments in a field‐cage using males derived as adults from a wild population and virgin females reared from naturally infested fruit. The results of this study were consistent with laboratory studies in that copula duration increased with female size, that sperm were stored asymmetrically between the females’ spermathecae, and that this asymmetry declined with number of sperm stored. However, we also found some previously unreported effects of female size; large females stored more sperm and stored sperm more asymmetrically between their two spermathecae than did small females. Unlike the laboratory studies, copula duration and sperm storage patterns were unaffected by male size and diet. This may be due to overwhelming variation from other sources in the wild‐collected males used, as well as environmental variability in the semi‐natural setting.     

The anatomy of fertilization in the yellow dung fly Scathophaga stercoraria

Female yellow dung flies, Scathophaga stercoraria, can influence the traffic of sperm stored in their spermathecae to the site of fertilization in the bursa copulatrix. However, the anatomical mechanisms employed are largely unknown. We investigated the anatomy of the female genital tract, seeking structures involved in sperm transfer and egg fertilization. We found a membranous structure descending from the ends of the spermathecal and accessory gland ducts into the bursa copulatrix. We call this the prolatus. Sperm accumulate in the prolatus during oviposition. When an egg is in the bursa the egg micropyle, rather than being aligned towards the dorsal openings of the spermathecal ducts, lies on the opposite, ventral side. We also confirm the presence, and suggest a function for, a cuticularized pouch on the ventral wall of the anterior bursa copulatrix. This pouch, plus a previously undescribed chamber, may be homologous to the ventral receptacle/fertilization chamber found in other dipterans. Further, we describe a translucent cap, apparently transversed by channels, covering the micropyle. Sperm were observed to aggregate on and in the micropyle cap, which appears to attract and hold sperm. We interpret the prolatus as a structure that allows an ovipositing female to transfer a few sperm onto the ventral bursal wall and thus, indirectly, onto the micropyle cap. Such anatomy potentially gives the female a large degree of control over sperm traffic from storage to the site of fertilization.     

Function of multiple sperm storage organs in female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae)

Sperm storage organs allow females to temporally separate insemination from fertilization, manipulate ejaculates and control fertilization. In the reproductive tract of female fruit flies (Diptera: Tephritidae), sperm are found in two different organs--a pair or triplet of spermathecae, and a "fertilization chamber". In order to understand the specific function of each of these organs, we tested the following hypotheses: (1) Sperm are distributed equally amongst the various sperm storage organs; (2) Both organ types maintain sperm viability; and (3) Sperm used in fertilization come from the fertilization chamber. We counted sperm in spermathecae and fertilization chamber of Mediterranean fruit flies (Ceratitis capitata) every 3 days for 18 days following insemination, and used a live/dead staining technique to determine the viability of sperm in these organs. Finally, by extirpating spermathecae from inseminated females and allowing them to oviposit, we were able to identify the fertilization chamber as the source of fertilizing sperm. Numbers of sperm in the spermathecae declined from an average of 3575 on the day of copulation to 649, 18 days later. Conversely, the fertilization chamber maintained a fairly constant level of sperms, ranging between an average of 207 cells on day 3 to 115 sperms on day 18. Throughout the period we monitored, we found high levels of sperm viability in both organs (> 80%). Sperm viability was similarly high in the fertilization chambers of females without spermathecae. However, fertility of eggs laid by these females declined rapidly, as did the number of sperm in the fertilization chamber. We conclude that both the spermathecae and the fertilization chamber are active sperm storage organs, with separate functions: the spermathecae for long-term storage and the fertilization chamber, periodically filled by the spermathecae, a staging point for fertilizing sperm. We suggest that the use of both organs by females results in sperm economy, which adaptively prolongs the intervals between copulations.     

Receptivity of female remating and sperm number in the sperm storage organ in the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

This study examines the relationship between the number of sperm in the seminal receptacle (spermatheca) and the receptivity of female remating in the bean bugRiptortus clavatus Thunberg. On the 21 st day after the first mating when receptivity to remating was > 70%, females receptive to remating had significantly fewer sperm ( < 40 on average) in the spermathecae than females reluctant to do (about 150 on average). However, averages of the number of eggs laid by receptive and reluctant females within 21 days were almost same. The proportion of fertilized eggs for receptive females at 15–21 days after copulation was significantly lower than that for reluctant females. Spermatozoa transferred from a male to a female’s spermatheca were detected 5 min after copulation and then increased continuously to about 500 with the first hour. When copulation durations were manipulated artificially, the shorter the copulation period (=females had less sperm in their spermathecae), the higher the remating rate became. Females may perceive the number of sperm in their seminal receptacles and then determine whether they copulate or not. These results support the hypothesis that females mate multiply in order to replenish inadequate sperm supplies to fertilize all eggs produced.     

Morphology and function of the reproductive tract of the spider crab Libinia spinosa (Crustacea, Brachyura, Majoidea): pattern of sperm storage

Morphology and function of the male reproductive tract, female spermatheca and patterns of sperm storage were assessed in the crab Libinia spinosa using histological methods. Testes are characterized by the presence of peripheral spermatogonia and different sequences of sperm maturity. Spermatophores begin to be packed in the last portion. The vas deferens consists of three sections: anterior, with undeveloped spermatophores and free sperm; median, with well-developed spermatophores; and posterior with granular secretions. Female spermathecae are of the ventral type, with a velum separating dorsal and ventral chambers. Live individuals were kept in the laboratory and arranged in pairs. An experiment was conducted toward the end of the reproductive season, in which males with the right gonopod excised were placed with receptive females. After mating, females were killed and the spermathecae dissected for histological study and observation of the pattern of sperm storage. Spermatozoa were found forming discrete sperm packages. New ejaculates can fill the entire spermatheca or be restricted to the ventral chamber; sperm are rounded, with a distinguishable acrosomal core. Old ejaculates are restricted to the dorsal chamber and are of irregular shape and larger size; an acrosomal core was not distinguishable. The secretions produced by the glandular epithelium of the dorsal chamber of the spermathecae are likely to have a role in the removal of dead sperm.     

Structural organization of the copulation site in the medfly Ceratitis capitata (Diptera: Tephritidae) and observations on sperm transfer and storage

The copulation site of the medfly Ceratitis capitata was investigated at anatomical and ultrastructural levels. It consists of the anterior vagina, with a ventral fertilization chamber and a dorsal insemination pocket into which the two spermathecal ducts open. The fertilization chamber is an organ comprised of a number of alveoli that in virgin females are filled with a filamentous secretion, whereas in mated females contain sperm bundles. Through study of the internal morphology of the aedeagus, its position in the anterior vagina, and the direct observation of sperm transfer and storage, we confirmed that sperm are ejaculated through two gonopores at the top of the distiphallus and another at the base of the genital rod. The sperm flow dorsally into the insemination pocket and ventrally into the fertilization chamber. During copulation, the two spermathecae and the fertilization chamber are progressively filled with spermatozoa.     

Influence of female reproductive anatomy on the outcome of sperm competition in Drosophila melanogaster

Females as well as males can influence the outcome of sperm competition, and may do so through the anatomy of their reproductive tracts. Female Drosophila melanogaster store sperm in two morphologically distinct organs: a single seminal receptacle and, normally, two spermathecae. These organs have different temporal roles in sperm storage. To examine the association between sperm storage organ morphology and sperm competition, we used a mutant type of female with three spermathecae. Although the common measure of sperm competition, P(2), did not differ between females with two and three spermathecae, the pattern of sperm use over time indicated that female morphology did affect male reproductive success. The rate of offspring production by females with three spermathecae rose and fell more rapidly than by females with two spermathecae. If females remate or die before using up second male sperm, then second male reproductive success will be higher when they mate with females with three spermathecae. The results indicate that temporal patterns of sperm use as well as P(2) should be taken into account when measuring the outcome of sperm competition.