Sperm competition and reproductive success in the decapod Inachus phalangium (Majidae): a male ghost spider crab that seals off rivals' sperm

Parker's (1970a) hypothesis that the overlap of multiple mating sperm in the female's storage organs promotes sperm competition is tested here for the first time in Crustacea: specifically, the mechanisms and consequences of sperm competition are detailed for the spider crab Inachus phalangium . Females of this species store ejaculates from successive copulations with different males discretely and consecutively in sac-like twin seminal receptacles. During copulation males transfer a large quantity of a sperm-free seminal plasma, followed by the sperm which is stored in small spermatophores and forms a densely-packed sperm packet. It was shown, using ³H-thymidine-labelled ejaculate, that the last male to mate displaces the ejaculate of his predecessors dorsally into the apex of the receptacle. Sperm of previous matings are sealed in with the hardening seminal plasma (sperm gel) and are thus prevented from being used to fertilize eggs, while the last male to mate places his sperm closest to the oviduct and vaginal openings. In experiments using the 'sterile-male' method, sperm from the last male to mate gained all fertilizations in subsequent broods. The seminal plasma forms the sperm gel in ghost spider crabs which is used for displacement of previously stored sperm, whereas various other brachyuran taxa use seminal plasma to produce the sperm plug, which prevents a male's sperm from being displaced.     

Male reproductive system of the arrow crab Stenorhynchus seticornis (Inachoididae)

Our aim was to describe the reproductive system of males and the formation of sperm packages in the seminal receptacle (SR) of recently mated females of the arrow crab Stenorhynchus seticornis. The male reproductive system was analyzed, and was described using light microscopy and histological and histochemical methods. The first pair of gonopods was described by means of scanning electron microscopy. Additionally, the dehiscence of spermatophores was tested using samples obtained from the vas deferens of males and from the seminal receptacle of recently mated females. Testes were tubular type, and each vas deferens consisted of three regions: the anterior vas deferens (AVD), including a proximal portion that was filled with free spermatozoa and a distal portion contained developing spermatophores; the median vas deferens (MVD) that contained completely formed spermatophores; and the posterior vas deferens (PVD), which contained only granular secretions. The accessory gland, which was filled with secretions, was located in the transition region between the MVD and the PVD. The spermatophores from the MVD were of different sizes, and none of them showed dehiscence in seawater, whereas those spermatophores in contact with the seminal receptacle were immediately broken. The ultrastructure of the gonopods revealed the presence of denticles at the distal portion, which contribute to the mechanical rupture of the spermatophore wall during the transfer of sperm. The contents of the PVD and accessory gland of males are transferred together with the spermatophores, and are responsible for the secretions observed among the sperm packets in the SR of the female. We suggest that these secretions formed the layers found in the SR of recently mated females, and may play a role in sperm competition in arrow crabs.     

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

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

Costs and benefits of giant sperm and sperm storage organs in Drosophila melanogaster

In Drosophila, long sperm are favoured in sperm competition based on the length of the female's primary sperm storage organ, the seminal receptacle (SR). This sperm–SR interaction, together with a genetic correlation between the traits, suggests that the coevolution of exaggerated sperm and SR lengths may be driven by Fisherian runaway selection. Here, we explore the costs and benefits of long sperm and SR genotypes, both in the sex that carries them and in the sex that does not. We measured male and female fitness in inbred lines of Drosophila melanogaster derived from four populations previously selected for long sperm, short sperm, long SRs or short SRs. We specifically asked: What are the costs and benefits of long sperm in males and long SRs in females? Furthermore, do genotypes that generate long sperm in males or long SRs in females impose a fitness cost on the opposite sex? Answers to these questions will address whether long sperm are an honest indicator of male fitness, male post‐copulatory success is associated with male precopulatory success, female choice benefits females or is costly, and intragenomic conflict could influence evolution of these traits. We found that both sexes have increased longevity in long sperm and long SR genotypes. Males, but not females, from long SR lines had higher fecundity. Our results suggest that sperm–SR coevolution is facilitated by both increased viability and indirect benefits of long sperm and SRs in both sexes.     

Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female.

Biochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm-specific fraction, LDHx, the M-type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm-agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constituents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.     

Repeated visitations of spermatophores and polyandry in females of eriophyoid mites

Eriophyoid females store sperm either asymmetrically in one spermatheca, or symmetrically in both spermathecae. Previous studies have suggested that species in which females store sperm asymmetrically pick up sperm from only one spermatophore, while those with symmetrical sperm storage pick up sperm from two or more spermatophores during their lifetime. The aim of this study was to examine spermatophore visitation behaviour and symmetry of sperm storage in Aculops allotrichus from the black locust tree and Cecidophyopsis hendersoni from the yucca. This would indicate monandry or polyandry in these species. In both eriophyoids, the spermatophore visitation consisted of three phases: mounting, lying on the spermatophore and dismounting. Aculops allotrichus stored sperm asymmetrically. However, nearly one-third of the observed females visited two spermatophores, rather than only one in their lives. When A. allotrichus females visited two spermatophores they spent a similar amount of time at the first and at the second visitation. Also, the times of visitation of the first of the two spermatophores and the single spermatophore in a female lifetime did not differ significantly. This would suggest that apart from monandry, double insemination also occurs in this species. By contrast, C. hendersoni females were polyandrous. They stored sperm symmetrically and visited several spermatophores, on average 1.54 (max 6) per day, and up to 33 spermatophores in their lives. The benefits of repeated spermatophore visitation and the possible mechanisms of sperm storage in both species are discussed.     

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

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

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

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

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

Parasperm: morphological and functional studies on nonfertile sperm

Sperm polymorphism, a phenomenon in which more than one type of sperm is produced within a species, occurs widely in animals from invertebrates to vertebrates. Sperm in this phenomenon can be categorized into fertile sperm and nonfertile sperm on the basis of fertilization ability. Nonfertile sperm can be further classified into parasperm and aberrant deformed sperm. Parasperm are sperm produced through a constant developmental process along with normal fertile eusperm, and they are readily distinguished from deformed sperm, which are irregularly crippled by unpredictable errors at certain stages during spermatogenesis. Sperm identified as parasperm occur widely in invertebrates but are presently quite limited in vertebrates. This may be the result of the deficiency both of studies on parasperm and of clear criteria to identify parasperm in vertebrates. Some vertebrates show unique spermatogenesis, such as symplastic spermatid and semicystic spermatogenesis. Thus, parasperm must be identified by comparing cells in each cyst and in semen, because irregularly shaped cells in the seminal duct could be either parasperm or normal spermatids. Although parasperm are identified by clear criteria in vertebrates, only in cottoid fishes to date, it is possible for parasperm to be discovered in other vertebrates. Recently, roles related to sperm competition have been reported in several species (e.g., the marine cottoid fish Hemilepidotus gilberti), and, with some of them, parasperm production is influenced by an intraspecific factor such as a sex ratio or the density of a population. Sperm competition is one of the important candidates for influencing evolution of parasperm functions, but not all parasperm seem to have a relation to sperm competition. Parasperm function may relate to the ecological conditions of each species that produces parasperm. Studies on parasperm function will be advanced by an ecological approach concerning male fertilization success as well as cytological investigation for parasperm. An erratum to this article is available at .     

Courtship and sperm transfer in Charinus neocaledonicus Kraepelin, 1895 and Charinus australianus (L. Koch, 1867) (Arachnida, Amblypygi, Charinidae)

The Charinus australianus group is a well-defined species group characterised by rounded, cushion-like female gonopods. Before the present study, the morphology of the gonopods and their function have not been understood. This paper describes courtship behaviour, spermatophore morphology, and the morphology of the female genitalia of Charinus neocaledonicus Kraepelin, 1895 and C. australianus (L. Koch, 1867). Courtship behaviour, though different in details, is similar to that of many other species. The spermatophores are large and soft and carry very small sperm packages, each with a short stalk. After sperm transfer, the spermatophore may be eaten by the female. The spermatophore thus transfers not only spermatozoa but also nutritious paternal investment to the female. Each female gonopod is equipped with a seminal receptacle consisting of an atrium and a spacious inner receptacle. The cover of the atrium can be elevated by high blood pressure and pulled back by a group of muscles attached to the inner part of the receptacle. The female probably picks up the sperm packages with the atria of her receptacles. The observations are compared to those on other amblypygids, and the evolution of different types of spermatophores and of gonopods with seminal receptacles is discussed.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Determinants of paternity in a butterfly

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

Reproductive investment patterns,sperm characteristics,and seminal plasma physiology in alternative reproductive tactics of Chinook salmon (Oncorhynchus tshawytscha)

Although alternative reproductive tactics (ARTs) are common across a range of taxa, little is known about whether the different tactics have adapted to sperm competition risk. Chinook salmon, Oncorhynchus tshawytscha, have two ARTs: large males that participate in dominance‐based hierarchies for access to spawning females, known as hooknoses, and small males that attempt to sneak fertilizations during spawning events from peripheral positions, known as jacks. Jacks continually face sperm competition risk because they always spawn in the presence of another male, whereas hooknoses face relatively low sperm competition risk because other males are not always present during spawning events. Based on the sneak‐guard model of sperm competition this asymmetry in sperm competition risk predicts that jacks ought to invest significantly more into sperm‐related traits important for sperm competition success relative to hooknoses. In the present study we report on reproductive investment patterns, sperm characteristics, and seminal plasma physiology of males that exhibit ARTs in Chinook salmon. We found that jacks invest significantly more of their somatic tissue into gonads compared with hooknoses. Sperm velocity also varied significantly between the ARTs, with jacks having significantly faster sperm than hooknoses. No significant differences in seminal plasma physiology metrics related to sperm quality were detected between the ARTs. We interpret these sperm investment patterns in light of the sneak‐guard model of sperm competition that is based on differences in sperm competition risk and alternative investment possibilities among ARTs. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae)

Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism.     

Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster

In Drosophila melanogaster, sperm and accessory gland proteins ("Acps," a major component of seminal fluid) transferred by males during mating trigger many physiological and behavioral changes in females (reviewed in ). Determining the genetic changes triggered in females by male-derived molecules and cells is a crucial first step in understanding female responses to mating and the female's role in postcopulatory processes such as sperm competition, cryptic female choice, and sexually antagonistic coevolution. We used oligonucleotide microarrays to compare gene expression in D. melanogaster females that were either virgin, mated to normal males, mated to males lacking sperm, or mated to males lacking both sperm and Acps. Expression of up to 1783 genes changed as a result of mating, most less than 2-fold. Of these, 549 genes were regulated by the receipt of sperm and 160 as a result of Acps that females received from their mates. The remaining genes whose expression levels changed were modulated by nonsperm/non-Acp aspects of mating. The mating-dependent genes that we have identified contribute to many biological processes including metabolism, immune defense, and protein modification.     

Paternity in mallards: effects of sperm quality and female sperm selection for inbreeding avoidance

Postcopulatory processes might play an important role in sexualselection. In theory, fertilization success could be controlledby females via selection of particular sperm within their reproductivetract, or it could be determined by sperm competition per se.In practice, these two mechanisms are difficult to disentangle.To assess the relative importance of both mechanisms we usedartificial insemination in combination with measurements ofsperm quality (swimming speed and motility) in mallards. Inthis species, females often lack behavioral control over copulationsand hence may use postcopulatory mechanisms to optimize theirreproductive output. One important factor affecting female fitnessmay be selection of genetically compatible males. To investigatethe influence of sperm quality and parental relatedness on paternitywe inseminated 12 groups of related females with a sperm mixturecontaining equal numbers of sperm from a brother and from anunrelated male. Paternity was independent of the relatednessof the siring male to the female but was significantly affectedby long-term sperm swimming speed and motility. No interactionbetween relatedness and sperm quality on paternity was observed.These results suggest that female mallards are not able to selectsperm on a purely genetic basis and emphasize the importanceof sperm quality in gaining paternity.     

Sperm transport after insemination in the Alpine newt (Triturus alpestris, Caudata, Salamandridae)

The goal of this study was to test if sperm transport to the spermathecae in the Alpine newt (Triturus alpestris) requires active co-operation of the female. Artificial insemination of anaesthetised female newts was conducted using spermatophores collected from courting males and with sperm duct contents collected from sacrificed males. Sperm was present in the spermathecae of 9 out of 10 females inseminated with the spermatophores but in only 1 out of 8 females inseminated with sperm duct contents. The females of both groups laid some eggs after insemination, and a portion of these eggs in group of females inseminated with spermatophores were fertilized. However, the number of eggs produced by the females was much lower than typical egg-production in newts. The presence of sperm in the spermathecae of females inseminated with spermatophores and lack of sperm in the spermathecae of females inseminated with sperm duct contents suggests that sperm transport is either induced by the substances present in spermatophores and/or that sperm from the sperm duct is not fully mobile in comparison with sperm from the spermatophores.     

Functional morphology of the female reproductive system of a crab with highly extensible seminal receptacles and extreme sperm storage capacity

We studied the functional morphology of the female reproductive system of the purple stone crab Danielethus crenulatus . The most remarkable feature is the relative storage capacity and extensibility of the seminal receptacles. These receptacles are a pair of simple sacs that lack internal structures dividing the internal lumen. Differences in seminal receptacle size and contents are accompanied by conspicuous changes in receptacle lining at a tissue level. Full seminal receptacles contain discrete sperm masses formed by hardened fluid and densely packed spermatophores. Different sperm masses are likely from different mates and their stratified disposition within the seminal receptacles is compatible with rival sperm displacement and last sperm precedence. Additionally, the anatomical structure of the vulva and vagina suggest active female control over copula. We discuss our results in the general context of sperm storage in brachyurans and the implications for the mating system of this species.