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.     

Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction

Successful reproduction requires contributions from both the male and the female. In Drosophila, contributions from the male include accessory gland proteins (Acps) that are components of the seminal fluid. Upon their transfer to the female, Acps affect the female's physiology and behavior. Although primary sequences of Acp genes exhibit variation among species and genera, the conservation of protein biochemical classes in the seminal fluid suggests a conservation of functions. Bioinformatics coupled with molecular and genetic tools available for Drosophila melanogaster has expanded the functional analysis of Acps in recent years to the genomic/proteomic scale. Molecular interplay between Acps and the female enhances her egg production, reduces her receptivity to remating, alters her immune response and feeding behavior, facilitates storage and utilization of sperm in the female and affects her longevity. Here, we provide an overview of the D. melanogaster Acps and integrate the results from several studies that bring the current number of known D. melanogaster Acps to 112. We then discuss several examples of how the female's physiological processes and behaviors are mediated by interactions between Acps and the female. Understanding how Acps elicit particular female responses will provide insights into reproductive biology and chemical communication, tools for analyzing models of sexual cooperation and/or sexual conflict, and information potentially useful for strategies for managing insect pests.     

Proteomic analysis of Drosophila mojavensis male accessory glands suggests novel classes of seminal fluid proteins

Fruit-flies of the genus Drosophila are characterized by overwhelming variation in fertilization traits such as copulatory plug formation, sperm storage organ use, and nutritional ejaculatory donation. Despite extensive research on the genetic model Drosophila melanogaster, little is known about the molecular underpinnings of these interspecific differences. This study employs a proteomic approach to pin-point candidate seminal fluid proteins in Drosophila mojavensis, a cactophilic fruit-fly that exhibits divergent reproductive biology when compared to D. melanogaster. We identify several classes of candidate seminal fluid proteins not previously documented in the D. melanogaster male ejaculate, including metabolic enzymes, nutrient transport proteins, and clotting factors. Conversely, we also define 29 SFPs that are conserved despite >40 million years of Drosophila evolution. We discuss our results in terms of universal processes in insect reproduction, as well as the specialized reproductive biology of D. mojavensis.     

Perceived sperm competition intensity influences seminal fluid protein production prior to courtship and mating

Sperm competition is a potent postcopulatory selective force where sperm from rival males compete to fertilize a limited set of ova. Considering that sperm production is costly, we expect males to strategically allocate sperm in accordance with the level of competition. Accordingly, previous work has examined a male's strategic allocation in terms of sperm number. However, the seminal fluid proteins (Sfps) transferred along with sperm may also play a crucial role in competition. Surprisingly, the strategic allocation of Sfps has remained largely unexplored. Using Drosophila melanogaster, we examined the expression of three seminal fluid and four spermatogenesis genes in response to perceived sperm competition intensity by manipulating male density in a pre-mating and courtship environment. In the pre-mating environment, we found that males modified Sfp ratios by reducing the production of two spfs when potential rivals were present, while one Sfp and all spermatogenesis genes remained unaltered. In the courtship environment, males did not modify spermatogenesis or Sfp production in response to either rival males or female presence. Our data suggest that perceived competition in the pre-mating environment places a significant influence on Sfp allocation, which may be a general trend in promiscuous animal systems with internal fertilization.     

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.     

Drosophila males transfer antibacterial proteins from their accessory gland and ejaculatory duct to their mates

The male fruitfly, Drosophila melanogaster, transfers to his mate proteins that increase his reproductive success by causing changes in her behavior and physiology. Here we show that among the transferred proteins are ones with antibacterial activity. We performed Escherichia coli overlay assays of native PAGE or renatured SDS-PAGE of reproductive tissue extracts of wild-type or transgenic males deficient in accessory gland function. We detected a 28 kDa male accessory gland-derived protein and two ejaculatory duct-derived proteins all with antibacterial activity. Based on its gel mobility and tissue of synthesis, one of the ejaculatory duct proteins is likely to be andropin, a previously-reported 6 kDa antibacterial peptide. All three proteins are transferred to females during mating. Therefore, they could assist in protecting the male's reproductive tract and, after transfer to the female, the female's reproductive tract or eggs against bacterial infection. Since seminal fluid proteins are transferred before the sperm, these antibacterial proteins may also protect sperm from bacterial infection.     

Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies

The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred together with sperm by mating males. The functions of many of these substances are not yet known. However, those that have been characterized have marked effects on the reproductive success of males and females. For example, seminal fluid proteins and peptides can decrease female receptivity, can increase egg production and can increase sperm storage, and are necessary for sperm transfer and success in sperm competition. In this review we focus on the currently known functions of seminal fluid molecules and on new technologies and approaches that are enabling novel questions about their form and function to be addressed. We discuss how techniques for disrupting the production of seminal fluid proteins, such as homologous recombination and RNA interference, along with the use of microarrays and yeast two hybrid systems, should allow us to address ever more sophisticated questions about seminal fluid protein function. These and similar techniques promise to reveal the function of naturally-occurring variants of these proteins and hence the evolutionary significance of genetic variation for them.     

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.     

Adaptive evolution of recently duplicated accessory gland protein genes in desert Drosophila

The relationship between animal mating system variation and patterns of protein polymorphism and divergence is poorly understood. Drosophila provides an excellent system for addressing this issue, as there is abundant interspecific mating system variation. For example, compared to D. melanogaster subgroup species, repleta group species have higher remating rates, delayed sexual maturity, and several other interesting differences. We previously showed that accessory gland protein genes (Acp's) of Drosophila mojavensis and D. arizonae evolve more rapidly than Acp's in the D. melanogaster subgroup and that adaptive Acp protein evolution is likely more common in D. mojavensis/D. arizonae than in D. melanogaster/D. simulans. These findings are consistent with the idea that greater postcopulatory selection results in more adaptive evolution of seminal fluid proteins in the repleta group flies. Here we report another interesting evolutionary difference between the repleta group and the D. melanogaster subgroup Acp's. Acp gene duplications are present in D. melanogaster, but their high sequence divergence indicates that the fixation rate of duplicated Acp's has been low in this lineage. Here we report that D. mojavensis and D. arizonae genomes contain several very young duplicated Acp's and that these Acp's have experienced very rapid, adaptive protein divergence. We propose that rapid remating of female desert Drosophila generates selection for continuous diversification of the male Acp complement to improve male fertilization potential. Thus, mating system variation may be associated with adaptive protein divergence as well as with duplication of Acp's in Drosophila.     

Pervasive adaptive evolution in primate seminal proteins

Seminal fluid proteins show striking effects on reproduction, involving manipulation of female behavior and physiology, mechanisms of sperm competition, and pathogen defense. Strong adaptive pressures are expected for such manifestations of sexual selection and host defense, but the extent of positive selection in seminal fluid proteins from divergent taxa is unknown. We identified adaptive evolution in primate seminal proteins using genomic resources in a tissue-specific study. We found extensive signatures of positive selection when comparing 161 human seminal fluid proteins and 2,858 prostate-expressed genes to those in chimpanzee. Seven of eight outstanding genes yielded statistically significant evidence of positive selection when analyzed in divergent primates. Functional clues were gained through divergent analysis, including several cases of species-specific loss of function in copulatory plug genes, and statistically significant spatial clustering of positively selected sites near the active site of kallikrein 2. This study reveals previously unidentified positive selection in seven primate seminal proteins, and when considered with findings in Drosophila, indicates that extensive positive selection is found in seminal fluid across divergent taxonomic groups.     

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being "movers" to "fertilizers." Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization.     

Transfer and fate of seminal fluid molecules in the beetle, Diaprepes abbreviatus: implications for the reproductive biology of a pest species

Molecules transferred from males to females via seminal fluids are important to the study of insect reproduction because they affect female physiology, reproductive behavior, and longevity. These molecules (seminal fluid molecules or SFMs) interest applied entomologists because of their potential use in insect control. SFMs are also interesting because of their relatively rapid evolution and important role in post-mating sexual selection. We studied SFMs in Diaprepes abbreviatus, a major pest of numerous plant species of economic importance. Using radiolabeled-methionine (35S), we found that D. abbreviatus males synthesized proteins de novo in their reproductive tissues after mating. Males that were fed radiolabeled methionine transferred radioactivity to females beginning within the first 10 min of mating. Male-derived substances are absorbed from the female's reproductive tract into the hemolymph and circulated throughout the body, but are found primarily in the eggs and ovaries. As a result, SFMs may be a useful means of both horizontal (to mates) and vertical transfer (to offspring) of control agents between conspecifics.     

Female resistance to male harm evolves in response to manipulation of sexual conflict

The interests of males and females over reproduction rarely coincide and conflicts between the sexes over mate choice, mating frequency, reproductive investment, and parental care are common in many taxa. In Drosophila melanogaster, the optimum mating frequency is higher for males than it is for females. Furthermore, females that mate at high frequencies suffer significant mating costs due to the actions of male seminal fluid proteins. Sexual conflict is predicted to lead to sexually antagonistic coevolution, in which selection for adaptations that benefit males but harm females is balanced by counterselection in females to minimize the extent of male-induced harm. We tested the prediction that elevated sexual conflict should select for increased female resistance to male-induced harm and vice versa. We manipulated the intensity of sexual conflict by experimentally altering adult sex ratio. We created replicated lines of D. melanogaster in which the adult sex ratio was male biased (high conflict lines), equal (intermediate conflict lines), or female biased (low conflict lines). As predicted, females from high sexual conflict lines lived significantly longer in the presence of males than did females from low conflict lines. Our conclusion that the evolutionary response in females was to the level of male-induced harm is supported by the finding that there were no female longevity differences in the absence of males. Differences between males in female harming ability were not detected. This suggests that the response in females was to differences between selection treatments in mating frequency, and not to differences in male harmfulness.     

Evolutionary expressed sequence tag analysis of Drosophila female reproductive tracts identifies genes subjected to positive selection

Genes whose products are involved in reproduction include some of the fastest-evolving genes found within the genomes of several organisms. Drosophila has long been used to study the function and evolutionary dynamics of genes thought to be involved in sperm competition and sexual conflict, two processes that have been hypothesized to drive the adaptive evolution of reproductive molecules. Several seminal fluid proteins (Acps) made in the Drosophila male reproductive tract show evidence of rapid adaptive evolution. To identify candidate genes in the female reproductive tract that may be involved in female-male interactions and that may thus have been subjected to adaptive evolution, we used an evolutionary bioinformatics approach to analyze sequences from a cDNA library that we have generated from Drosophila female reproductive tracts. We further demonstrate that several of these genes have been subjected to positive selection. Their expression in female reproductive tracts, presence of signal sequences/transmembrane domains, and rapid adaptive evolution indicate that they are prime candidates to encode female reproductive molecules that interact with rapidly evolving male Acps.     

Mating and hormonal triggers regulate accessory gland gene expression in male Drosophila

Drosophila melanogaster males transfer accessory gland proteins, as part of their seminal fluid, to females during each mating. Since accessory gland proteins are important for male reproductive success, it is important that the male replenish the proteins he transferred during mating. Previous studies had shown that mating induces the resynthesis of accessory gland proteins, but since mating includes a set of stereotyped behavior patterns as well as the act of copulation, it was not known which aspect of the mating process induces accessory gland protein synthesis. By exposing males to females whose ovipositors had been sealed shut, we have shown that resynthesis of accessory gland proteins occurs only when seminal fluid is transferred to females. By applying juvenile hormone or 20-hydroxyecdysone topically to the cuticle of male flies, we showed that these hormones can act in vivo to stimulate the synthesis of accessory gland proteins to levels similar to those observed after mating.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

Cross-species comparison of Drosophila male accessory gland protein genes

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.     

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.