The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster.

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility, though functional evidence in any species is lacking. Here, we used functional genetics to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes are dispensable. We found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage, likely due to its expression in the male ejaculatory bulb. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Fine structure of the female genital system of diving beetle Stictonectes optatus (Seidlitz, 1887) (Dytiscidae-Hydroporinae) and evidence of mating plug formation.

The general organization of the female genital system of the diving beetle Stictonectes optatus was studied, clarifying the complex structure of the spermatheca and spermathecal gland. The two structures adhere closely to each other, sharing a small area of their cuticular epithelium. A long duct connects the bursa copulatrix to the spermatheca, where the sperm are stored. The sperm reach the common oviduct, where egg fertilization occurs, via a fertilization duct. The spermathecal gland cells have extracellular cisterns where secretions are stored. Thin ducts composed of duct-forming cells transport these secretions to the apical gland region and into the spermathecal lumen. Soon after mating, the bursa copulatrix is almost completely occupied by a plug secreted by the male accessory glands. The secretions of the bursa epithelium seem to contribute to plug formation. Later this plug becomes large and spherical, obstructing the bursa copulatrix.

Drosophila female reproductive glands contribute to mating plug composition and the timing of sperm ejection.

Reproductive traits that influence female remating and competitive fertilization rapidly evolve in response to sexual selection and sexual conflict. One such trait, observed across diverse animal taxa, is the formation of a structural plug inside the female reproductive tract (FRT), either during or shortly after mating. In Drosophila melanogaster, male seminal fluid forms a mating plug inside the female bursa, which has been demonstrated to influence sperm entry into storage and latency of female remating. Processing of the plug, including its eventual ejection from the female's reproductive tract, influences the competitive fertilization success of her mates and is mediated by female × male genotypic interactions. However, female contributions to plug formation and processing have received limited attention. Using developmental mutants that lack glandular FRT tissues, we reveal that these tissues are essential for mating plug ejection. We further use proteomics to demonstrate that female glandular proteins, and especially proteolytic enzymes, contribute to mating plug composition and have a widespread impact on plug formation and composition. Together, these phenotypic and molecular data identify female contributions to intersexual interactions that are a potential mechanism of post-copulatory sexual selection.

Functional morphology of immature mating in a widow spider.

BACKGROUND: Mating generally occurs after individuals reach adulthood. In many arthropods including spiders, the adult stage is marked by a final moult after which the genitalia are fully developed and functional. In several widow spider species (genus Latrodectus), however, immature females may mate a few days before they moult to adulthood, i.e. in their late-subadult stage. While the "adult" mating typically results in cannibalism, males survive the "immature" mating. During both "immature" and "adult" matings, males leave parts of their paired copulatory organs within female genitalia, which may act as mating plugs. To study potential costs and benefits of the two mating tactics, we investigated female genital morphology of the brown widow spider, L. geometricus. Light microscopy, histology and micro-computed tomography of early-subadult, late-subadult and adult females were conducted to determine the overall pattern of genital maturation. We compared genitalia of mated late-subadult and adult females to reveal potential differences in the genitalic details that might indicate differential success in sperm transfer and different environments for sperm storage and sperm competition. RESULTS: We found that the paired sperm storage organs (spermathecae) and copulatory ducts are developed already in late-subadult females and host sperm after immature mating. However, the thickness of the spermathecal cuticle and the staining of the secretions inside differ significantly between the late-subadult and adult females. In late-subadult females mating plugs were found with higher probability in both spermathecae compared to adult females. CONCLUSIONS: Sperm transfer in matings with late-subadult females follows the same route as in matings with adult females. The observed differences in the secretions inside the spermathecae of adult and late-subadult females likely reflect different storage conditions for the transferred sperm which may lead to a disadvantage under sperm competition if the subadult female later re-mates with another male. However, since males mating with late-subadult females typically transfer sperm to both spermathecae they might benefit from numerical sperm competition as well as from monopolizing access to the female sperm storage organs. The assessment of re-mating probability and relative paternity will clarify the costs and benefits of the two mating tactics in light of these findings.

Control of mating plug expelling and sperm storage in Drosophila: A gynandromorph- and mutation-based dissection.

INTRODUCTION: In this study, we analyzed gynandromorphs with female terminalia, to dissect mating-related female behaviors in Drosophila. MATERIALS AND METHODS: We used gynandromorphs, experimentally modified wild-type (Oregon-R) females, and mutant females that lacked different components of the female reproductive apparatus. RESULTS: Many of the gynandromorphs mated but did not expel the mating plug (MP). Some of these - with thousands of sperm in the uterus - failed to take up sperm into the storage organs. There were gynandromorphs that stored plenty of sperm but failed to release them to fertilize eggs. Expelling the MP, sperm uptake into the storage organs, and the release of stored sperm along egg production are separate steps occurring during Drosophila female fertility. Cuticle landmarks of the gynandromorphs revealed that while the nerve foci that control MP expelling and also those that control sperm uptake reside in the abdominal, the sperm release foci derive from the thoracic region of the blastoderm. DISCUSSION AND CONCLUSION: The gynandromorph study is confirmed by analyses of (a) mutations that cause female sterility: Fs(3)Avar (preventing egg deposition), Tm2gs (removing germline cells), and iab-4DB (eliminating gonad formation) and (b) by experimentally manipulated wild-type females: decapitated or cut through ventral nerve cord.

A review of the occurrence and diversity of the sphragis in butterflies (Lepidoptera, Papilionoidea).

Males of many butterfly species secrete long-lasting mating plugs to prevent their mates from copulating with other males, thus ensuring their sperm will fertilize all future eggs laid. Certain species have further developed a greatly enlarged, often spectacular, externalized plug, termed a sphragis. This distinctive structure results from complex adaptations in both male and female genitalia and is qualitatively distinct from the amorphous, internal mating plugs of other species. Intermediate conditions between internal plug and external sphragis are rare. The term sphragis has often been misunderstood in recent years, hence we provide a formal definition based on accepted usage throughout most of the last century. Despite it being a highly apparent trait, neither the incidence nor diversity of the sphragis has been systematically documented. We record a sphragis or related structure in 273 butterfly species, representing 72 species of Papilionidae in 13 genera, and 201 species of Nymphalidae in 9 genera. These figures represent respectively, 13% of Papilionidae, 3% of Nymphalidae, and 1% of known butterfly species. A well-formed sphragis evolved independently in at least five butterfly subfamilies, with a rudimentary structure also occurring in an additional subfamily. The sphragis is probably the plesiomorphic condition in groups such as Parnassius (Papilionidae: Parnassiinae) and many Acraeini (Nymphalidae: Heliconiinae). Some butterflies, such as those belonging to the Parnassius simo group, have apparently lost the structure secondarily. The material cost of producing the sphragis is considerable. It is typically offset by production of a smaller spermatophore, thus reducing the amount of male-derived nutrients donated to the female during mating for use in oogenesis and/or somatic maintenance. The sphragis potentially represents one of the clearest examples of mate conflict known. Investigating its biology should yield testable hypotheses to further our understanding of the selective processes at play in an 'arms race' between the sexes. This paper provides an overview, which will inform future study.

On the new monotypic wolf spider genus Ovia gen. nov. (Araneae: Lycosidae, Lycosinae).

A new monotypic wolf spider genus, Ovia gen. nov. is proposed to accommodate a misplaced species: Pardosa procurva Yu & Song, 1988. Ovia procurva comb. nov. is redescribed, illustrated and designated as the type species for the genus. The subfamily placement of the new genus is discussed and it is considered as a member of Lycosinae Sundevall, 1833 and possibly closely related to Alopecosa Simon, 1885. The presence of an apical process (spur) on the median apophysis is proposed as the putative synapomorphy of Ovia gen. nov. The possible sister-taxon relationship of Ovia gen. nov. with Alopecosa is discussed and evidence on the occurrence of sexual dimorphism and mating plug within the genus are presented. Ovia gen. nov. is assumed to be of Holarctic origin, from which it has migrated to the Indomalayan region. Additionally, a current distribution map for the genus is provided.

Don't pull the plug! the Drosophila mating plug preserves fertility.

Mating plugs are hardened structures--typically a coagulation of seminal fluid components--that are transferred to, or formed within, the female reproductive tract of numerous animal species (both mammals and insects). Analysis of the role(s) of the mating plug in reproduction has been conducted in a wide array of diverse species. These structures have been proposed to have a multitude of functions, which include altering female re-mating rate, acting as a barrier to re-mating and being required for sperm storage or sperm movement to occur in mated females. A recent analysis of the Drosophila melanogaster mating plug has shown that proper formation of the structure is required for optimal fertility in flies: the Drosophila mating plug is required to retain the ejaculate within the female reproductive tract once mating has terminated. Here, we discuss the possible implications of the Drosophila mating plug in the fertility of this species in light of these new results.

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme.

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.

Description of new species of Stenaelurillus Simon, 1886 from the Western Ghats of India with the redescription of Stenaelurilluslesserti Reimoser, 1934 and notes on mating plug in the genus (Arachnida, Araneae, Salticidae).

A new species of the jumping spider genus Stenaelurillus Simon, 1886, Stenaelurillusalbus sp. n., is described from the Western Ghats of India, one of the biodiversity hotspots of the world. Detailed morphological descriptions, diagnostic features and illustrations of copulatory organs of both sexes are given. Detailed redescription, diagnosis and illustration of Stenaelurilluslesserti Reimoser, 1934 are provided. The occurrence of a mating plug in the genus is reported.