Ultrastructure of spermatozoa of orsolobidae (Haplogynae,Araneae) with implications on the evolution of sperm transfer forms in Dysderoidea

Haplogynae are highly diverse with respect to the primary male genital system and sperm characteristics. Additionally, all sperm transfer forms (STF) known for spiders are present. Besides individually transferred sperm (cleistospermia), sperm are transferred as conjugates, both primary (synspermia) and secondary sperm conjugates (coenospermia, rouleaux) occur. Nevertheless, the ultrastructure of spermatozoa and STF are described for few Haplogynae and often only one representative species was studied, resulting in a superficial insight in the evolution of these traits. To elucidate the evolution of STF within Haplogynae we investigated representatives of four genera of the dysderoid family Orsolobidae. Our data show the presence of synspermia (Orsolobus, Osornolobus, Hickmanolobus, and Tasmanoonops) and also cleistospermia (Osornolobus). The occurrence of different STF within one family or even genus has not been described for any other spider taxon so far. Moreover, the synspermia of species of Tasmanoonops and Hickmanolobus were not covered by a secretion sheath suggesting a previously unknown strategy of transferring sperm that is possibly related to sperm residency time or female triggered processes after copulation. Based on serial ultrathin sectioning and subsequent 3D‐reconstruction, we obtained detailed measurements revealing remarkable size differences of STF. To evaluate the previously suggested correlation with the most distal region of the spermophor inside the embolus (intromittent part of the copulatory organ) we measured the diameter of the spermophor using micro‐computed X‐ray tomography data to obtain corresponding morphometric parameters. Based on these data only two species show similarity in STF and spermophor diameter. J. Morphol. 275:1238–1257, 2014. © 2014 Wiley Periodicals, Inc.     

The ultrastructure of the peculiar synspermia of some Dysderidae (Araneae, Arachnida)

The present study reports on the ultrastructure features of spermatozoa and spermatogenesis of several species of Dysderidae (Dysdera crocata, Dysdera erythrina, Dysdera ninnii, Harpactea arguta, Harpactea piligera, Dasumia taeniifera). Dysderid spiders are known to possess a peculiar sperm transfer form known as synspermia, characterized by fused spermatozoa surrounded by a secreted sheath. Until now the exact mode of formation of the synspermia is unknown. The present study demonstrates that the spermatids are connected via narrow cell bridges during the entire spermiogenesis as is usual, although in Dysderidae they do not separate at end of the spermiogenesis. Instead, they fuse completely within the testes shortly after the spermatid has coiled to get a spherical shape. The number of fusing sperm cells is different in the different observed species. The species of the genus Harpactea thus have synspermia consisting of two fused spermatozoa; whereas in the species of the genus Dysdera four sperm cells are fused and in D. taeniifera at least three spermatozoa are fused. In contrast with other known families with this peculiar form transfer of sperm, the synspermia in Dysderidae are mainly characterized by a conspicuous vesicular area which extends through the entire synspermium surrounding the cell organelles. Thus, all main cell components (e.g., nucleus, acrosomal vacuole, and axoneme) are covered by the vesicular membrane. The vesicular area seems to be functional and probably it is important during sperm activation in female genital system. Simultaneously to the extension of the vesicular area, the synspermium accumulates large amounts of glycogen. The glycogen is mainly located around the centriolar adjunct and along the axoneme accompanying the postcentriolar elongation of the nucleus. A further peculiar feature is the extremely elongated acrosomal vacuole, which seems to be synapomorphic trait for sperm cells of dysderids. Interestingly, spermatogenesis, including the fusion, exclusively occurs within the testes (in contrast to the formation of coenospermia). In the vas deferens only synspermia were found. The secreted sheath surrounding the spermatozoa is finally synthesized in the parts of the vasa deferentia, which are close to the genital opening where numerous vacuoles and microvilli are seen in the epithelial cells.     

Evolutionary morphology of the male reproductive system,spermatozoa and seminal fluid of spiders (Araneae,Arachnida) – Current knowledge and future directions

The male reproductive system and spermatozoa of spiders are known for their high structural diversity. Spider spermatozoa are flagellate and males transfer them to females in a coiled and encapsulated state using their modified pedipalps. Here, we provide a detailed overview of the present state of knowledge of the primary male reproductive system, sperm morphology and the structural diversity of seminal fluids with a focus on functional and evolutionary implications. Secondly, we conceptualized characters for the male genital system, spermiogenesis and spermatozoa for the first time based on published and new data. In total, we scored 40 characters for 129 species from 56 families representing all main spider clades. We obtained synapomorphies for several taxa including Opisthothelae, Araneomorphae, Dysderoidea, Scytodoidea, Telemidae, Linyphioidea, Mimetidae, Synotaxidae and the Divided Cribellum Clade. Furthermore, we recovered synspermia as a synapomorphy for ecribellate Haplogynae and thus propose Synspermiata as new name for this clade. We hope that these data will not only contribute to future phylogenetic studies but will also stimulate much needed evolutionary studies of reproductive systems in spiders.     

Effects of sperm conjugation and dissociation on sperm viability in vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability.     

Ultrastructural aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia (Araneae: Sicariidae)

This study reports ultrastructural and cytochemical aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia. The roundish early spermatids are initially interconnected by cytoplasmic bridges, forming groups of four cells. During spermiogenesis, these cells pass through a series of modifications: (1) progressive nuclear condensation brings chromatin into a fibrillar arrangement; (2) the nucleus becomes long and asymmetric, with a short post-centriolar elongation; (3) formation of the long, cone-shaped acrosome and the F-actin acrosomal filament; (4) establishment of the implantation fossa and the 9x2+3 pattern flagellum, which extends away from the sperm cell body. Eventually, the entire cell undergoes twisting and folding resulting in a synspermium, containing four sperm cells in which the flagellum and nucleus are delimitated by the plasma membrane, as individualized structures, but remain involved by the fused remaining cytoplasm and plasma membrane. Reaching the vas deferens, the synspermia are surrounded by a basic glycoproteic secretion. Synspermia are considered a derivative character, probably developed in this Sicariidae species, as well as in other Haplogynae, as an adaptation to improve the reproductive strategy.     

Molecular phylogeny of araneomorph spiders

The reconstruction of the phylogeny of spiders based on morphological and ethological characters is hampered by frequent homoplasies. Therefore, fragments of the 28S rRNA gene have been sequenced to elucidate some crucial points in the evolution of spiders. The following results were obtained. (1) The 28S rRNA gene data support the monophyly of the Entelegynae. Thus, the number of different subunits and the number of hexamers which form a haemocyanin complex must have been reduced at least twice within the spiders. The character state pattern within the spiders and within the Arachnida suggests that these reductions are due to a decrease in the selection on the oxygen binding properties of the haemocyanins as a result of the evolution of tracheae. (2) The semientelegyne spiders are not a transition stage between the Haplogynae and the Entelegynae. The lack of separate fertilization ducts in these species is probably due to secondary reduction. This reduction has decisive consequences for the flow of the sperm and for sperm competition. (3) Furthermore, the 28S rRNA gene data tentatively support the hypothesis that the orb web is either a plesiomorphy or has evolved at least twice.     

Spermiogenesis in Psilochorus simoni (Berland, 1911) (Pholcidae, Araneae): evidence for considerable within-family variation in sperm structure and development

A large number of characters and considerable variation among taxa make animal sperm cells promising objects for phylogenetic studies. However, our knowledge about sperm structure and development in spiders is still rudimentary. In pholcids, previous studies of two species representing different subfamily level taxa have revealed conspicuous differences. Here, we report on a representative of a third subfamily level taxon, confirming substantial variation in sperm structure and development within the family. The male genital system in Psilochorus simoni (Berland, 1911) consists of paired testes and deferent ducts which lead into a common ejaculatory duct. The somatic cells of the testes show a high secretory activity, and produce at least two different kinds of secretion. The spermatozoa show features already known from other Pholcidae as well as unique characters. The acrosomal vacuole is tube-like with a narrow subacrosomal space. The axoneme migrates deep into the nucleus and is finally located near the acrosomal vacuole. Thus, the postcentriolar elongation of the nucleus is very long. A centriolar adjunct is not present and after the coiling process the implantation fossa is completely filled with glycogen which is also found in larger amounts within the cytoplasm of the sperm cell. After the coiling process, a vesicular area is present that becomes most prominent in the periphery of the sperm cell and surrounds the axoneme and parts of the nucleus. The secretion sheath surrounding the mature spermatozoon is already formed in the lumen of the testis, possibly by a secretion present in the testis but absent in the deferent duct. Sperm are transferred as cleistospermia. Results are compared with previous studies on pholcid spermiogenesis and sperm structure.     

The oviduct as a complex mediator of mammalian sperm function and selection

The fallopian tube, or oviduct, is no longer considered merely a conduit that joins the uterine horns and the ovaries, being recognised as a venue for the capacitation of spermatozoa and fertilisation. However, recent evidence has implicated the oviduct in the stringent selection of spermatozoa prior to fertilisation, sperm storage prior to fertilisation, the regulation of sperm motility and possibly the guidance of spermatozoa towards the egg. Moreover, the arrival of spermatozoa within the oviduct is now known to regulate gene expression in oviductal epithelial cells with the consequent up- and downregulation of various proteins. In this review, we examine the emerging significance of sperm-oviduct interactions, as they relate to both physiological functions and the likelihood that the oviduct has a role in post-copulatory sperm selection by females (cryptic female choice) under conditions of sperm competition. The mechanisms by which sperm selection might operate still remain a mystery, especially when the underlying rationale for such mechanism appears to require the recognition by the female tract of sperm qualities related to the intrinsic integrity and information content of the sperm DNA. The oviduct not only selects against spermatozoa containing fragmented DNA but also imposes selection related to the fitness or quality of individual males. This implies the existence of, as yet unrecognised, mechanisms for the detection and interpretation of sperm-surface markers that link phenotypic and genotypic qualities of each individual cell.     

Fine structure of spermiogenesis, spermatozoa and spermatophore of Saxidromus delamarei (Saxidromidae, Actinotrichida, Acari)

Ultrastructural details of spermiogenesis, spermatozoa and the spermatophore of the early derived actinedid mite Saxidromus delamarei are described. Spermatids and mature sperm cells are provided with up to four acrosomal complexes and nuclei derivatives (chromatin bodies). Due to this reason, the sperm cells may be classified as synspermia, a sperm type found only in some spiders until now. The acrosomal complex is composed of a remarkably complicated vacuole and filament. Other peculiarities of sperm structure correspond to those found in prostigmatic mites, i.e. penetration of the chromatin body by the acrosomal filament and the presence of peripheral invaginations of the plasmalemma. The sperm cells are covered by a thin secretion layer of probably proteinaceous material. Stalked spermatophores are rather large, but simply structured and contain relatively few sperm cells. The results are discussed taking systematical and behavioural aspects into account. In particular, it is suggested that the peculiar mating behaviour of these mites secures both sperm transfer and first male's sperm priority and that this allowed reduction of sperm numbers.     

Morphological evidence for limited sperm production in the enigmatic Tasmanian cave spider Hickmania troglodytes (Austrochilidae,Araneae)

The male reproductive systems of spiders are highly diverse in structure across all major spider taxa. Little is known about this organ system in basal araneomorph spiders, especially Austrochiloidea; such knowledge is necessary for a more complete understanding of the evolutionary morphology of the male reproductive system in spiders. In the present study, we describe the male reproductive system of an austrochilid spider, the enigmatic troglophilic Tasmanian cave spider Hickmania troglodytes, using light and electron microscopic techniques. The male reproductive system consists of tubular testes leading into convoluted deferent ducts, which are fused close to the genital opening to an unpaired ejaculatory duct. Spermatogenesis occurs only in the subadult testes, whereas adult testes showed neither spermatogenic stages nor any generative tissue in all investigated specimens. The testes of adult males are drastically reduced in size compared with those of subadult males, but the deferent ducts are filled with large numbers of mature spermatozoa. Thus, our data suggest that males of H. troglodytes are sperm‐limited, but not necessarily sperm‐depleted as described for certain orb‐weaving spiders. Due to the absence of generative tissue, limited sperm production is permanent (PSL) and probably has an influence on the reproductive strategies in this species. As nearly no data are available on the life history of H. troglodytes, and in particular information on the phenology of males is lacking, implications of the evolution of PSL in this species are unclear. Nonetheless, our data on other representatives of Austrochilidae (Austrochilus forsteri, Thaida chepu) and Gradungulidae (Progradungula otwayensis) suggest that PSL evolved within Austrochiliodea only in H. troglodytes and might be an adaptation to its troglophilic lifestyle.     

Evolution of intra‐ejaculate sperm interactions: do sperm cooperate?

Sperm are often considered to be individuals, in part because of their unique genetic identities produced as a result of synapsis during meiosis, and in part due to their unique ecology, being ejected away from the soma to continue their existence in a foreign environment. Selection at the level of individual sperm has been suggested to explain the evolution of two enigmatic sperm phenotypes: sperm heteromorphism, where more than one type of sperm is produced by a male, and sperm conjugation, where multiple sperm join together for motility and transport through the female reproductive tract before dissociation prior to fertilization. In sperm heteromorphic species, only one of the sperm morphs typically participates in fertilization, with the non‐fertilizing “parasperm” being interpreted as reproductive altruists. Likewise, in species with sperm conjugation, high levels of sperm mortality have been suggested to be required for conjugate break‐up and this has been considered evidence of kin‐selected altruism. However, it is unclear if sperm possess the heritable variation in fitness (i.e. are individuals) required for the evolution of cooperation. We investigate the question of sperm individuality by focusing on how sperm morphology is determined and how sperm conjugates are formed. Concentrating on sperm conjugation, we discuss functional hypotheses for the evolutionary maintenance of this remarkable trait. Additionally, we speculate on the potential origins of sperm heteromorphism and conjugation, and explore the diversification and losses of these traits once they have arisen in a lineage. We find current evidence insufficient to support the concept of sperm control over their form or function. Thus, without additional evidence of haploid selection (i.e. sperm phenotypes that reflect their haploid genome and result in heritable differences in fitness), sperm heteromorphism and conjugation should be interpreted not as cooperation but rather as traits selected at the level of the male, much like other ejaculatory traits such as accessory gland proteins and ejaculate size.     

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.     

The long and short of sperm polymorphisms in insects

Production of more than one morphological type of sperm in a common testis has been documented for a variety of invertebrates, including gastropods, spiders, centipedes, and insects. This unusual phenomenon is difficult to explain by current theory, particularly since available evidence indicates that one sperm type is often incapable of effecting fertilization. In this review we critically examine evidence on the distribution and development of sperm heteromorphisms among insects in light of competing hypotheses for the evolutionary origin, maintenance, and function of a non-fertilizing class of sperm. To date, no single hypothesis, including alternatives which assume non-fertilizing sperm are non-adaptive, or that they provision, facilitate, or compete with fertilizing sperm, has received strong empirical support by any group of insects. The diversity of sperm heteromorphisms suggests that non-fertilizing sperm may have different functions in different clades or even serve multiple functions within a clade. We suggest that insight could be gained from (1) new models for the evolution of sperm polymorphism, (2) comparative studies that focus on multiple traits simultaneously (e.g. sperm number, proportion, length, and remating rate) and utilize clades in which more than one gain or loss of sperm heteromorphism has been documented (e.g. Pentatomidae, Carabidae, or Diopsidae), and (3) experimental studies that exploit individual variation or directly manipulate the composition of the male ejaculate.     

Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity

Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.     

Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay

Defective mammalian spermatozoa become ubiquitinated during epididymal passage, a mechanism that may mark the abnormal spermatozoa for proteolytic destruction (Sutovsky et al., 2001a: J Cell Sci 114:1665-1675). It is not known how such spermatozoa are recognized by the epididymal ubiquitination pathway and whether there is a selection against certain types of sperm defects. We examined the relationship between sperm ubiqutination, lifelong sperm morphology and sperm DNA defects using a single chanel, ubiquitin-activated flow cytometric assay, and a dual, ubiquitin-TUNEL assay. Semen samples from nine service sires of good-to-average fertility were screened. A positive correlation was found between sperm ubiquitination and the average frequency of morphological semen abnormalities from field evaluations performed throughout the reproductive life of individual sires. Sample correlation coefficients were r=0.65 for primary (head and tail) and r=0.60 for total semen abnormalities in the single channel assay. In a dual assay, we found a high, positive correlation (r=0.93) between the ubiquitin-positive sperm and the TUNEL positive sperm. Substantial correlations (r=0.47-0.64) were observed when the measurements from these two respective assays were compared for individual sires. While anti-ubiquitin antibodies recognized most of the TUNEL-positive sperm cells, the TUNEL-positive spermatozoa represented only a subset (approximately 20-40%) of all ubiquitin-positive cells. It appears that the ubiquitin-dependent sperm quality control, residing in the epididymal epithelium, has the ability to detect spermatozoa with apoptotic or necrotic DNA, while spermatozoa with defects other than DNA fragmentation are also recognized and ubiquitinated.     

Light‐microscopic examinations of sperm in several lineages of Carabidae (Insecta: Coleoptera): Implications for the evolution of sperm conjugation

Sperm show marked morphological diversity, but the processes and mechanisms driving this diversity have not been fully elucidated. The beetle family Carabidae represents a potential model system for studying sperm trait evolution. In this study, sperm traits (mainly conjugation and sperm conjugate gross morphology) of 42 species from nine subfamilies of Carabidae were examined using light microscopy. Except in Harpalinae, the type of conjugation was shared by all members of a particular subfamily: in Carabinae, Elaphrinae, Patrobinae and Brachinae, sperm conjugates were observed in which variable numbers of sperm clumped together; in Nebriinae, Cicindelinae and Trechinae, sperm were not organized as conjugates but were present individually; and in Broscinae, both individual sperm and sperm conjugates were observed. In the remaining subfamily, Harpalinae, sperm conjugates were formed in most species, but a loss of conjugation was observed in some species. Mapping the observed sperm traits onto within‐family molecular phylogenetic trees suggested that sperm conjugation was ancestral, with loss of conjugation evolving in several lineages. In sperm conjugates, a short spermatostyle (the axis of sperm conjugates) was the ancestral state, while a long spermatostyle evolved in subsequent lineages. In the long spermatostyle trait, the flexible type without a conspicuous 3D structure was ancestral, while the type with a conspicuous 3D structure, such as the spiral structure, evolved in derived lineages.     

Embryonic expression patterns of Wnt genes in the RTA-clade spider Cupiennius salei

Spiders represent widely used model organisms for chelicerate and even arthropod development and evolution. Wnt genes are important and evolutionary conserved factors that control and regulate numerous developmental processes. Recent studies comprehensively investigated the complement and expression of spider Wnt genes revealing conserved as well as diverged aspects of their expression and thus (likely) function among different groups of spiders representing Mygalomorphae (tarantulas), and both main groups of Araneae (true spiders) (Haplogynae/Synspermiata and Entelegynae). The allegedly most modern/derived group of entelegyne spiders is represented by the RTA-clade of which no comprehensive data on Wnt expression were available prior to this study. Here, we investigated the embryonic expression of all Wnt genes of the RTA-clade spider Cupiennius salei. We found that most of the Wnt expression patterns are conserved between Cupiennius and other spiders, especially more basally branching species. Surprisingly, most differences in Wnt gene expression are seen in the common model spider Parasteatoda tepidariorum (a non-RTA clade entelegyne species). These results show that data and conclusions drawn from research on one member of a group of animals (or any other organism) cannot necessarily be extrapolated to the group as a whole, and instead highlight the need for comprehensive taxon sampling.     

A comparative analysis of the morphology and evolution of permanent sperm depletion in spiders

Once thought to be energetically cheap and easy to produce, empirical work has shown that sperm is a costly and limited resource for males. In some spider species, there is behavioral evidence that sperm are permanently depleted after a single mating. This extreme degree of mating investment appears to co-occur with other reproductive strategies common to spiders, e.g. genital mutilation and sexual cannibalism. Here we corroborate that sperm depletion in the golden orb-web spider Nephila clavipes is permanent by uncovering its mechanistic basis using light and electron microscopy. In addition, we use a phylogeny-based statistical analysis to test the evolutionary relationships between permanent sperm depletion (PSD) and other reproductive strategies in spiders. Male testes do not produce sperm during adulthood, which is unusual in spiders. Instead, spermatogenesis is nearly synchronous and ends before the maturation molt. Testis size decreases as males approach their maturation molt and reaches its lowest point after sperm is transferred into the male copulatory organs (pedipalps). As a consequence, the amount of sperm available to males for mating is limited to the sperm contained in the pedipalps, and once it is used, males lose their ability to fertilize eggs. Our data suggest that PSD has evolved independently at least three times within web-building spiders and is significantly correlated with the evolution of other mating strategies that limit males to monogamy, including genital mutilation and sexual cannibalism. We conclude that PSD may be an energy-saving adaptation in species where males are limited to monogamy. This could be particularly important in web-building spiders where extreme sexual size dimorphism results in large, sedentary females and small, searching males who rarely feed as adults and are vulnerable to starvation. Future work will explore possible energetic benefits and the evolutionary lability of PSD relative to other mate-limiting reproductive behaviors.