Male accessory gland proteins in Drosophila: a multifaceted field [corrected

Male accessory gland in Drosophila is a secretory tissue of the reproductive system. The proteins synthesized in the accessory gland are tissue specific, stage specific-seen only during the adult stage and sex specific in the sense of male limited expression. These secretions that form a component of the seminal fluid are transferred to the female at the time of copulation and play an important role in reproduction. In conjunction with sperm, these secretory proteins assure reproductive success by reducing the female's receptivity to mating and escalating the rate of egg laying. Some of these proteins are antibacterial in nature with a likely function of protecting the female's genital tract against microbial infection during/after mating. Most of the genes involved in the synthesis of accessory gland proteins are autosomal but a few are still X-linked. Their male specific expression is achieved at the time of sex determination. The level of expression of these genes is dose dependent and they follow Mendelian pattern of segregation. Further, majority of these proteins are rapidly evolving with high rates of non-synonymous substitutions. In this review, by considering the work carried out in different fields, we have tried to generate a comprehensive picture about the male accessory gland and the role of its proteins in the reproduction of Drosophila.     

Female Drosophila melanogaster suffer reduced defense against infection due to seminal fluid components

Reduced defense against infection is commonly observed as a consequence of reproductive activity, but little is known about how post-mating immunosuppression occurs. In this work, we use Drosophila melanogaster as a model to test the role of seminal fluid components and egg production in suppressing post-mating immune defense. We also evaluate whether systemic immune system activity is altered during infection in mated females. We find that post-mating reduction in female defense depends critically on male transfer of sperm and seminal fluid proteins, including the accessory gland protein known as "sex peptide." However, the effect of these male factors is dependent on the presence of the female germline. We find that mated females have lower antimicrobial peptide gene expression than virgin females in response to systemic infection, and that this lower expression correlates with higher systemic bacterial loads. We conclude that, upon receipt of sperm and seminal fluid proteins, females experience a germline-dependent physiological shift that directly or indirectly reduces their overall ability to defend against infection, at least in part through alteration of humoral immune system activity.     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.     

昆虫雄性附腺功能蛋白研究进展

昆虫雄性附腺蛋白是精液蛋白的主要来源,对雌雄虫生殖过程具有重要生理功能,按功能可分为精包结构蛋白和功能蛋白两类。精包结构蛋白参与精包的形成;功能蛋白在交配过程中随精子一起转移到雌虫体内,导致雌虫行为和生理的深刻变化,如降低雌虫再交配率、提高产卵量、促进精子转移、储存和竞争等。随着对昆虫雄性附腺功能蛋白研究的深入,特别对果蝇附腺功能蛋白的详细研究,从分子水平上阐述蛋白质序列与功能的关系,明确其作用机制,可为进一步阐明昆虫生殖和进化机制等提供新依据。     

Molecular dissection of nuptial gifts in divergent strains of Ostrinia moths

Seminal fluid proteins (SFPs) produced in the male accessory glands and ejaculatory duct are subject to strong sexual selection, often evolve rapidly and therefore may play a key role in reproductive isolation and species formation. However, little is known about reproductive proteins for species in which males transfer ejaculate to females using a spermatophore package. By combining RNA sequencing and proteomics, we characterize putative SFPs, identify proteins transferred in the male spermatophore and identify candidate genes contributing to a one‐way gametic incompatibility between Z and E strains of the European corn borer moth Ostrinia nubilalis. We find that the accessory glands and ejaculatory duct secrete over 200 highly expressed gene products, including peptidases, peptidase regulators and odourant‐binding proteins. A comparison between Ostrinia strains reveals that accessory gland and ejaculatory duct sequences with hormone degradation and peptidase activity are among the most extremely differentially expressed. However, most spermatophore peptides lack reproductive tissue bias or canonical secretory signal motifs and aproximately one‐quarter may be produced elsewhere before being sequestered by the male accessory glands during spermatophore production. In addition, most potential gene candidates for postmating reproductive isolation do not meet standard criteria for predicted SFPs and almost three‐quarters are novel, suggesting that both postmating sexual interactions and gametic isolation likely involve molecular products beyond traditionally recognized SFPs.     

Identity and transfer of male reproductive gland proteins of the dengue vector mosquito, Aedes aegypti: potential tools for control of female feeding and reproduction

Male reproductive gland proteins (mRGPs) impact the physiology and/or behavior of mated females in a broad range of organisms. We sought to identify mRGPs of the yellow fever mosquito, Aedes aegypti, the primary vector of dengue and yellow fever viruses. Earlier studies with Ae. aegypti demonstrated that "matrone" (a partially purified male reproductive accessory gland substance) or male accessory gland fluid injected into virgin female Ae. aegypti affect female sexual refractoriness, blood feeding and digestion, flight, ovarian development, and oviposition. Using bioinformatic comparisons to Drosophila melanogaster accessory gland proteins and mass spectrometry of proteins from Ae. aegypti male accessory glands and ejaculatory ducts (AG/ED) and female reproductive tracts, we identified 63 new putative Ae. aegypti mRGPs. Twenty-one of these proteins were found in the reproductive tract of mated females but not of virgin females suggesting that they are transferred from males to females during mating. Most of the putative mRGPs fall into the same protein classes as mRGPs in other organisms, although some appear to be evolving rapidly and lack identifiable homologs in Culex pipiens, Anopheles gambiae, and D. melanogaster. Our results identify candidate male-derived molecules that may have an important influence on behavior, survival, and reproduction of female mosquitoes.     

Microsatellite analysis of sperm-use patterns in the bushcricket Requena verticalis

Abstract.— The proportion of offspring sired by the second male to mate with a doubly mated female, P2, is a ubiquitously measured statistic in the study of insect sperm competition. Nevertheless, the underlying mechanisms of sperm transfer, storage, and use that determine P2 are poorly understood. Typically the second male to mate gains moderate to high paternity. More rarely, the first male to mate gains the majority of fertilizations. Here we examine the transfer, storage, and use of sperm in the bushcricket Requena verticalis , a species with male parental investment and almost complete first male paternity. Sperm drain from an externally attached spermatophore into the female's reproductive tract, where they are transported to the sperm store or spermatheca. We find that only sperm from the first male to mate are transported to the spermatheca. We provide some data that address a number of different mechanisms that might account for the lack of storage of second-male sperm. DNA microsatellite markers are developed to assign paternity. By manipulating the numbers of sperm transferred by first and second males, we show that the size of the ejaculate transferred by the first male has a major impact on paternity; increasing ejaculate size of the first male assures his paternity. Paternity assurance in R. verticalis holds significant implications for the evolution of paternal investment via the male's nuptial gift.     

The genetic architecture of immune defense and reproduction in male Bombus terrestris bumblebees

Understanding the architecture of genetic variation, that is the number, effect, location, and interaction, of genes responsible for phenotypic variability in nature is important for the understanding of microevolutionary processes. In this study, we have used a quantitative trait loci (QTL) approach to uncover the genetic architecture of fitness-relevant traits associated with reproduction and immune defense in male Bombus terrestris bumblebees. Three male reproductive investment traits, the number and length of the produced sperm and the size of the accessory glands, were studied. Two branches of the insect innate immune system, the activation of the Phenoloxidase-cascade and the hemolymph's antibacterial activity, were investigated. We found that variation in most of the studied traits is based on a network of minor QTLs and epistatic interactions. Unexpectedly, there was no evidence for phenotypic or genetic trade-offs between the presumably costly investment in immune defense and reproductive effort in this population for the measured traits. In fact, we found a positive correlation, both, in phenotype and genetic architecture for the number of produced sperm and antibacterial activity against an insect pathogen. A major finding for all traits analyzed was that the epistatic interactions accounted for a major proportion of the explained phenotypic variance. Especially for traits involved in immune defense, this pattern highlights the possible role of parasites in the evolution and maintenance of recombination and sexual reproduction.     

A comparative study on the arginine degradation cascade for sperm maturation ofBombyx mori andDrosophila melanogaster

Summary The spermatophore of the silkmoth,Bombyx mori, is a reactor with a specific energy-yielding system for sperm maturation, the arginine degradation cascade. On mating, the highly viscous secretions from various glands in the male reproductive tract, which contain many enzymes and their substrates, are transferred to the female bursa (b.) copulatrix to form the spermatophore. In the spermatophore, transferred arginine-rich proteins are digested by initiatorin, an Arg-C endopeptidase of serine-protease type, and a carboxypeptidase. The produced free arginine is then hydrolyzed to urea and ornithine by arginase. Ornithine is metabolized to glutamate, follwed by forming alanine and 2-oxoglutarate. The latter, as a member of TCA-cycle, is a preferred respiratory substrate for spermatozoa and accelerates the post-testicular sperm maturation.In contrast toBombyx mori, Drosophila melanogaster produces only eupyrene spermatozoa and does not form the spermatophore. The sperm of this dipteran insect acquire motility in the v. seminalis of males. As reported forDrosophila, a high glutamate-pyruvate aminotransferase activity was found in the spermatophore as well as the v. seminalis of the silkmoth. The value in the latter organ reaches 58.3% of the whole male reproductive tract that participates in transfer of the seminal fluid.In the male reproductive system ofDrosophila, the concentration of arginine is low, whereas those of urea and ammonia are high. The accessory gland secretion contains much phosphoserine. Theses substances are transferred to female uterus with spermatozoa during mating. Most amino acids increase distinctly at 30 min after the termination of mating (ATM) and then decline, suggesting active degradation of transferred proteins in the uterus. As found inBombyx, urea increases at the post-mating period, while ornithine shows a rather low concentration. Ornithine must be converted to glutamate. In this connection, it is notable that alanine rises markedly at 30 min following mating. As in the silkmoth, the energy metabolism of the fruit fly spermatozoa involves also arginine, ornithine, urea, and proline. These findings suggest that the occurrence of the arginine degradation cascade or related metabolic pathway in this insect.Abbreviations ATM after the termination of mating - Arg-C arginine-carbon - b. bursa - d. ductus - g. glandula - GPA l-glutamate-pyruvate aminotransferase - NADH₂ reduced nicotinamide-adenine dinucleotide - TCA tricarbonic acid - v. vesicula     

Genomic organization, tissue distribution and functional characterization of the rat Pate gene cluster

The cysteine rich prostate and testis expressed (Pate) proteins identified till date are thought to resemble the three fingered protein/urokinase-type plasminogen activator receptor proteins. In this study, for the first time, we report the identification, cloning and characterization of rat Pate gene cluster and also determine the expression pattern. The rat Pate genes are clustered on chromosome 8 and their predicted proteins retained the ten cysteine signature characteristic to TFP/Ly-6 protein family. PATE and PATE-F three dimensional protein structure was found to be similar to that of the toxin bucandin. Though Pate gene expression is thought to be prostate and testis specific, we observed that rat Pate genes are also expressed in seminal vesicle and epididymis and in tissues beyond the male reproductive tract. In the developing rats (20-60 day old), expression of Pate genes seem to be androgen dependent in the epididymis and testis. In the adult rat, androgen ablation resulted in down regulation of the majority of Pate genes in the epididymides. PATE and PATE-F proteins were found to be expressed abundantly in the male reproductive tract of rats and on the sperm. Recombinant PATE protein exhibited potent antibacterial activity, whereas PATE-F did not exhibit any antibacterial activity. Pate expression was induced in the epididymides when challenged with LPS. Based on our results, we conclude that rat PATE proteins may contribute to the reproductive and defense functions.     

Seminal fluids mediate sexual inhibition and short copula duration in mated female Queensland fruit flies

Molecules in male seminal fluid transferred to female insects during mating can have potent effects on their subsequent sexual and reproductive behaviour. Like many other tephritids, female Queensland fruit flies (Bactrocera tryoni) typically have diminished sexual receptivity after their first mating. Also, copulations of females that do remate tend to be shorter than those of virgins. We here find that virgin females injected with small doses (0.1, 0.2 or 0.5 male equivalents) of extracts from the male reproductive tract accessory tissues, which consist of male accessory glands, ejaculatory apodeme and ejaculatory duct (AG/EA/ED), have diminished receptivity and short copula duration very similar to naturally mated females. In contrast, virgin females injected with saline or with high doses of AG/EA/ED (1 or 2 male equivalents) that likely exceed the range of natural variation retain the higher levels of sexual receptivity and longer copulations of un-injected virgins. We conclude that reduced sexual receptivity and shorter copulations of mated female Q-flies are mediated by products in the male seminal fluid derived from the male reproductive tract accessory tissues.     

Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.     

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.     

Sexual ornamentation reflects antibacterial activity of ejaculates in mallards

Bacteria present in ejaculates can impair sperm function and reduce male reproductive success. Thus, selection should favour the evolution of antimicrobial defences to limit the detrimental effects of sperm-associated bacteria. Additionally, current hypotheses suggest that ornamental traits may signal information about the infection status of an individual or the ability of an individual to resist bacterial-induced sperm damage. However, despite the evolutionary implications of ejaculate antimicrobials, and the putative importance of pathogens for the evolution of male ornamentation, tests of these hypotheses are lacking. We examined the antibacterial activity of semen from mallard ducks (Anas platyrhynchos) and tested whether the bactericidal capacity of semen was associated with bill coloration, a sexually selected trait. We show that mallard semen exhibits significant antibacterial activity, as measured by the in vitro capacity to kill Escherichia coli and Staphylococcus aureus. Furthermore, we demonstrate that males with more colourful bills have semen with superior bacterial-killing ability. These results suggest that females could use male phenotypic traits to avoid sexually transmitted pathogens and acquire partners whose sperm suffer less bacteria-induced damage.