Paternity analysis of wild‐caught females shows that sperm package size and placement influence fertilization success in the bushcricket Pholidoptera griseoaptera

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a ‘fair raffle’. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1–6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.     

Sperm storage by spermatodoses in the spermatheca of Trioza alacris (Flor, 1861) hemiptera,psylloidea, triozidae: A structural and ultrastructural study

Female insects generally store sperm received during mating in specific organs of their reproductive tract, i.e., the spermathecae, which keep the sperm alive for a long time until fertilization occurs. We investigated spermatheca morphology and ultrastructure in the psylloidean insect Trioza alacris (Flor, 1861 ) in which spheroidal sperm packets that we refer to as ‘spermatodoses’ are found after mating. The ectoderm‐derived epithelium of the sac‐shaped spermatheca that has a proximal neck, consists of large secretory and flat cuticle‐forming cells. Secretory cells are characterized by a wide extracellular cavity, bordered by microvilli, in which electron‐dense secretion accumulates before discharge into the spermathecal lumen. The cuticle‐forming cells produce the cuticular intima of the organ and a peculiar specialized apical structure, through which secretion flows into the lumen. At mating, the male transfers bundles of sperm cells embedded in seminal fluid into the spermathecal neck. Sperm cells proceed towards the spermathecal sac lumen, where they are progressively compacted and surrounded with an envelope that also encloses secretions of both male and female origin. We describe the formation of these sperm containing structures and document the contribution of the female secretion to spermatodose or female‐determined spermatophore construction. We also discuss the choice of the term ‘spermatodose’ for T. alacris and suggest it be used to refer to sperm masses constructed in the female reproductive organs, at least when they involve the contribution of female secretion. © 2011 Wiley Periodicals, Inc.     

Female Bush-Crickets,Pholidoptera griseoaptera,that have Received Smaller Ejaculates Show a Higher Mating Rate in the Field

Females of numerous insect species are known to be polyandrous, but there have been relatively few studies of factors associated with the degree of polyandry in females in the field. Number of copulations by females is negatively associated with ejaculate size across bush-cricket species. Assessing intraspecific variability is important when looking for and interpreting trait evolution. Therefore the aim of this study was to test the association between ejaculate size (i.e. volume of spermatodose–spermatophore-like structure formed within the spermatheca) and mating rate (i.e. number of spermatodoses) of females of Pholidoptera griseoaptera, while accounting for female body size (pronotum length) and age (number of hind leg’s cuticular bands). The results based on field-caught individuals suggested that there were statistically significant negative association between smallest and mean spermatodose volume, respectively, and number of copulations in this nuptial gift-giving bush-cricket species. This is in accordance with interspecific associations between ejaculate size and polyandry. However, lower slope of the intraspecific relationship may suggest lower importance of the ejaculate size in explaining females’ mating rate variability in this nuptial gift-giving species.     

Mode of transfer of spermatozoa in Orthoptera Tettigoniidae

A morphological and ultrastructural study was carried out on the spermatophore and spermatodoses of some species of Orthoptera Tettigoniidae. From the results concerning the spermatophore it emerged that this structure has a morphological and ultrastructural organization represented by a dilated ampulla and a peduncle or neck. From the examination of freshly deposited spermatophores and those at various time intervals thereafter, it was seen that these structures other than allowing gamete transfer, represent the site where spermatodesms, organized in the male genital tracts, undergo reorganization to acquire their definitive morphological and structural characteristics as found in the female genital tracts. The spermatodoses, in the same way as the spermatophore, represent capsules containing spermatodesms, which are originated in the spermatheca, their specific morphology seems to diversify according to the species considered. As regards their role, it is hypothesized that these structures represent a long-term conservation mechanism for spermatozoa inside the seminal receptacle.     

优雅蝈螽与暗褐蝈螽精子束的显微观察

本文应用微分干涉相衬法对优雅蝈螽Ｇampsocleis gratiosa Brunner von Wattenwyl和暗褐蝈螽G. sedakovii （Fischer von Waldheim） 雄性精巢管基部、输精管、贮精囊和精包,及雌性受精囊中精子束的形态变化进行了观察,对探讨螽斯近缘种的生殖隔离机制和生殖生物学具有重要意义.结果表明：这两种蝈螽的精子束通过精包转移到雌性受精囊后,精子束的形态发生了显著变化.精巢管基部的精子为游离的单个精子;输精管、贮精囊和精包中精子成束排列形成较分散的精子束,精子束头部包裹有粘液帽;雌性受精囊中的精子束的精子呈羽状排列,精子的头部汇集在中央轴上.两种蝈螽精子束形态差异不显著.     

Spermatodesm reorganization in the spermatophore and in the spermatheca of the bushcricket Tylopsis liliifolia (Fabricius) (Orthoptera,Tettigoniidae)

Spermatozoa of Tettigoniidae are usually transferred to the female by means of a spermatophore which is also the site of feather-shaped spermatodesm formation. These spermatodesms are then transferred to a spermatheca, composed of a spermathecal duct and of a seminal receptacle, involved in storing spermatozoa. In order to extend the knowledge about sperm transfer and spermatodesms reorganization in the Tettigoniidae, a morpho-structural investigation was carried out on spermatophore and spermatheca of Tylopsis liliifolia and on the reorganization of the gametes from the spermatophore. Our results show that the spermatodesms undergo disorganization in the spermatophore; unlike other Tettigoniidae, however, feather-shaped spermatodesms are never found. The epithelium of the spermatheca consists of two cell types, the cuticle-forming and the gland cells, with secretory features. The gland cells, absent in the distal tract of the seminal receptacle, release their secretion in a “reservoir” where an efferent duct opens. In the distal tract of the spermathecal duct, adjacent epithelial cells show diversified ultrastructural characteristics whose probable role is discussed. A particular feature of T. liliifolia is the genesis of the feather-shaped spermatodesms in the seminal receptacle. This feature and the peculiar organization of the feather-shaped spermatodesm are a possible autapomorphy of T. liliifolia.     

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

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

Histochemical characteristics of spermatophore layers of Scylla serrata (Forskal) (Decapoda: Portunidae)

The spermatophores of S. serrata are protected by an outer thick chitinous layer and an inner thin non-chitinous one. Both layers are rich in acid mucopolysaccharides containing sulphated (outer layer) and carboxylic groups (inner layer). The proteins of the two layers show much tryptophan, but lack tyrosyl, sulfhydryl and disulphide groups. No phenols or phenol oxidases could be detected histochemically in either layer, suggesting the absence of phenolic tanning in the spermatophore. The physical properties, as revealed from treatment with acids and alkali, indicate the resistant nature of the outer layer; the inner layer easily shrinks or disrupts under such treatment. The outer layer, though resistant, is readily permeable to low molecular weight dye substances employed in permeability experiments. The mechanism of sperm release is recorded and discussed. It is suggested that, in S. serrata, the dehiscence of spermatophore may be caused by imbibing of low molecular weight substances by the sperm mass substances of the spermatophore while the latter is inside the spermatheca.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Sperm co-operation in the Fishfly, Parachauliodes japonicus

1. Males of the Fishfly Parachauliodes japonicus (McLachlan) produce sperm in bundles. Each bundle consists of hundreds of sperm with their heads agglutinated. At copulation, on average 500 bundles are packed in a single spermatophore which is attached externally to the female genitalia. The bundles swim forward by synchronous flagellate movements in viscous seminal fluids and finally enter a spermatheca of the female. Females detach the spermatophore without guarding by the male; males guard the spermatophore for 5 h.
2. In this study, the effects of sperm-bundle size and medium viscosity on their swimming velocities were examined. As sperm-bundle mass increased, the amount of time it took to move a unit length decreased. As viscosity increased, those sperm-bundles with greater mass moved faster than sperm-bundles with less mass.
3. The distance sperm move seems to be longer in P. japonicus than in most other insects that ejaculate directly or place the spermatophore internally in the female storage organs. Thus, large sperm-bundles may be an advantage for sperm of P.japonicus who move over a long distance in a viscous environment.     

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

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

The sperm-egg fusion in the nematode Parascaris equorum

Summary

The process of fertilization and the sperm storage in the female apparatus in Parascaris equorum is described in this paper. The sperm approaches the egg by means of pseudopodia containing bundles of microfilaments. The sperm and egg membranes fuse and the sperm penetrates progressively into the ovum. The egg and sperm plasma membranes and glycocalyces disappear at the point of fusion. At the end of fertilization, they are reformed at the egg's surface, while the egg and sperm chromatin begins to decondense. Spermatozoa are stored in the female apparatus prior to fertilization; here they come into contact with the epithelial cells of the spermatheca, protruding pseudopodia rich in microfilaments into the cellular body.     

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.     

Impact of male mating history on the postmating resumption of sexual receptivity and lifetime reproductive success in Choristoneura rosaceana females

Abstract.  The first objective of the present study is to test the hypothesis that the decrease in the number of eupyrene spermatozoa in the spermatheca is directly associated with the resumption of sexual receptivity in female moths, an aspect that has not been examined in previous studies. The obliquebanded leafroller, Choristoneura rosaceana , is used and females mated with previously mated males have a shorter refractory period than those mated with virgins. This difference is associated with a faster rate of movement of sperm from the spermatheca. Overall, the length of the female refractory period coincides with the mean time required for the number of eupyrene sperm in the spermatheca to drop to approximately 3000, regardless of male mating history. Although such a decline in sperm numbers may be a factor responsible for the resumption of sexual receptivity, this is clearly not the only one because more than 40% of females remate even though sperm numbers in the spermatheca are well above this threshold. Virgin males do not vary the mass or the content of their ejaculate as a function of the female's reproductive status and this may increase the risk of sperm competition if the female is previously mated. The second objective of this study is to examine the effect of previous male mating history on female reproductive potential. Females mated with previously mated males have a significantly lower fecundity than those mated with virgin males. However, in all treatments, remating increases both female longevity and lifetime fecundity. There is also a significant effect of female mass on the length of the refractory period and on lifetime fecundity, with large females resuming sexual receptivity sooner and laying more eggs than small ones, regardless of male mating history.     

Strategic Investment in Sperm Removal Behaviour in a Bushcricket

Multiple mating by females is widespread and generates sperm competition among the ejaculates of rival males over fertilization. One way in which males can avoid or reduce sperm competition is by displacing or removing previous males’ sperm from female sperm stores. An apparent example of this occurs in the bushcricket Metaplastes ornatus. Males perform a specialised sperm removal behaviour (SRB), using their highly-derived subgenital plate, with which they remove sperm from the female’s spermatheca during the early phases of mating before transferring a spermatophore of their own. Here we investigated whether males strategically invest in SRB according to the amount of previously stored sperm present in females. Each male was tested twice, once with a female containing sperm (‘filled’ condition) and once with a female from whom most previously deposited sperm had recently been removed by another male (‘emptied’ condition). For comparison, a separate group of males was paired with virgin females. Males did not discriminate between non-virgin females in the ‘emptied’ or ‘filled’ conditions in terms of their investment in SRB, suggesting they may not able to perceive the amount of sperm present in the female’s spermatheca. By contrast, male investment in SRB was significantly reduced in pairings with virgin females, indicating that males are sensitive to some aspect of a female’s mating status. Our results thus suggest that males modulate SRB in response to female-mediated cues, possibly chemical cues left by previous males, which would not be present on virgin but would be on non-virgin females.     

Genesis of spermatodesms in Tylopsis liliifolia (Orthoptera: Phaneropterinae) and their transit in the male genital tract

The spermatodesms of Tylopsis liliifolia form in the most proximal follicular cysts and are composed of a large number of sperm held together by a cap located in the anterior region of the acrosome. The cap is formed by short thin fibrils, loosely arranged at random, probably derived from secretory activity of cells of the cyst wall. Compared to other Tettigoniidae, a peculiar feature is acrosomal wings that twist gradually around the anterior region of the nucleus; at the end of the twisting process, the region of the sperm acrosome, observed in cross section, shows a typical spiral form. Spermatodesms do not undergo any substantial changes in the spermiduct. The epithelial cells of the wall have secretory activity and many show marked spermiophagic activity, which is conducted by epithelial cell protrusions that envelop the gametes, taking them into the cytoplasm. When removed from seminal vesicles and observed in vivo, spermatodesms show accentuated corkscrew movement, and when observed by SEM, slight torsion. Thus organized, spermatodesms are transferred to the spermatophore during mating, where they are transformed before reaching the seminal receptacle.     

Ultrastructure and function of long and short sperm in Cicadidae (Hemiptera)

The cicada, Graptopsaltria nigrofuscata, produces two distinct sizes of sperm, as determined by either nuclear volume of early spermatids or nuclear length of mature sperm. Between both sperm, there is no difference in location of the acrosome and flagellum during spermiogenesis. The acrosome is covered by an anteacrosomal bleb, which is inserted in a common mass, spermatodesm, derived from cyst cells. Both kinds of sperm linked to the spermatodesm form sperm bundles, respectively. During copulation, the sperm bundles are transported from the vesicula seminalis of the male to the bursa copulatrix of the female. Morphometric analyses of the nuclear length revealed that the two kinds of sperm reach the bursa copulatrix in the same condition as that found in the vesicula seminalis. Once transferred inside the latter, the sperm bundles disintegrated to individual sperm within a few hours, and the tail components, such as the axoneme and mitochondrial derivatives, become separated from each other over time. The tail completely splits from the sperm nucleus 24 h after copulation. Fertile sperm accumulate in the spermatheca, the final storage organ, where only long sperm survived for any length of time. Fertilized eggs examined by vital staining contain only sperm with long nuclei.     

Pheromonostasis is not directly associated with post-mating sperm dynamics in Choristoneura fumiferana and C. rosaceana females

In Lepidoptera, a number of humoral and neural cues are involved in post-mating pheromonostasis, including the presence of sperm in the spermatheca. However, as there are two types of sperm, apyrene and eupyrene, they may play different roles in pheromonostasis, an aspect not considered in previous studies. As a first step to examine this possibility, we determined the quantity of sperm transferred by the male at the time of mating and the temporal migration of both sperm types from the bursa copulatrix to the spermatheca in the spruce budworm, Choristoneura fumiferana, and the obliquebanded leafroller, C. rosaceana. While the mass of the ejaculate was positively correlated to male body mass, there was no relation between ejaculate mass and sperm numbers. In both species, the migration of the two sperm types was asynchronous, with the apyrene sperm migrating before the eupyrene type. There were, however, some interspecific temporal differences in the migration of both sperm types. Eupyrene sperm would not serve as a direct signal for pheromonostasis in either species as it does not reach the spermatheca for at least 7 h while the neural message for pheromonostasis in both tortricids occurs within 3 h of mating. Given the time apyrene sperm arrives in the spermatheca (between 3 and 5 h post-mating), it could serve as a direct cue for pheromonostasis in C. fumiferana but not in C. rosaceana. However, considering that these two Choristoneura species have similar pheromone physiologies, it seems somewhat unlikely that apyrene sperm would be involved in one species and not the other.     

Ultrastructural analysis of the morphology and function of the spermatheca of the pulmonate snail,Sonorella santaritana

The ultrastructure of the spermatheca of the reproductive tract in the pulmonate snail, Sonorella santaritana, was investigated. This organ has a debris-filled lumen and an outer wall which can be divided into three distinct layers. The cell layer adjacent to the lumen is comprised of two cell types, tall columnar epithelial cells with microvilli and cells lacking microvilli. The next layer also has two cell types, muscle cells and apparent pigment cells. The most distant layer is an adventitia of large glycogen-containing cells. The lumen of the spermatheca contains a core of partially digested sperm and related materials. The luminal contents and the cellular morphology of this organ suggest that the spermathecal functions are both digestive and absorptive. It is proposed that excess sperm and related materials are transported to the spermatheca, digested, and the usable products are reabsorbed.     

Beyond the mouse model: Using Drosophila as a model for sperm interaction with the female reproductive tract

Although the fruit fly, Drosophila melanogaster, has emerged as a model system for human disease, its potential as a model for mammalian reproductive biology has not been fully exploited. Here we describe how Drosophila can be used to study the interactions between sperm and the female reproductive tract. Like many insects, Drosophila has two types of sperm storage organs, the spermatheca and seminal receptacle, whose ducts arise from the uterine wall. The spermatheca duct ends in a capsule-like structure surrounded by a layer of gland cells. In contrast, the seminal receptacle is a slender, blind-ended tubule. Recent studies suggest that the spermatheca is specialized for long-term storage, as well as sperm maturation, whereas the receptacle functions in short-term sperm storage. Here we discuss recent molecular and morphological analyses that highlight possible themes of gamete interaction with the female reproductive tract and draw comparison of sperm storage organ design in Drosophila and other animals, particularly mammals. Furthermore, we discuss how the study of multiple sperm storage organ types in Drosophila may help us identify factors essential for sperm viability and, moreover, factors that promote long-term sperm survivorship.