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.     

The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila

During mating, males transfer seminal proteins and peptides, along with sperm, to their mates. In Drosophila melanogaster, seminal proteins made in the male's accessory gland stimulate females' egg production and ovulation, reduce their receptivity to mating, mediate sperm storage, cause part of the survival cost of mating to females, and may protect reproductive tracts or gametes from microbial attack. The physiological functions of these proteins indicate that males provide their mates with molecules that initiate important reproductive responses in females. A new comprehensive EST screen, in conjunction with earlier screens, has identified approximately 90% of the predicted secreted accessory gland proteins (Acps). Most Acps are novel proteins and many appear to be secreted peptides or prohormones. Acps also include modification enzymes such as proteases and their inhibitors, and lipases. An apparent prohormonal Acp, ovulin (Acp26Aa) stimulates ovulation in mated Drosophila females. Another male-derived protein, the large glycoprotein Acp36DE, is needed for sperm storage in the mated female and through this action can also affect sperm precedence, indirectly. A third seminal protein, the protease inhibitor Acp62F, is a candidate for contributing to the survival cost of mating, given its toxicity in ectopic expression assays. That male-derived molecules manipulate females in these ways can result in a molecular conflict between the sexes that can drive the rapid evolution of Acps. Supporting this hypothesis, an unusually high fraction of Acps show signs consistent with their being targets of positive Darwinian selection.     

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.     

Male contributions to egg production: the role of accessory gland products and sperm in Drosophila melanogaster

Drosophila melanogatser seminal fluid components, accessory gland proteins (Acps) and sperm, induce females to deposit high numbers of fertilized eggs for about 11 days. This high and sustained level of egg deposition requires that oogenesis be stimulated to provide the necessary mature oocytes. To investigate the relative timing and contributions of Acps and sperm in the egg-production process, we examined the rates of oogenic progression and egg deposition in females mated to genetically altered males that have seminal fluid deficient in Acps and/or sperm, and subjected these data to path analysis. We found that Acps and sperm are complementary stimuli necessary for inducing high rates of oogenic progression and rapid egg deposition. While egg deposition and oogenic progression can be induced by Acps alone, both Acps and sperm are required for maximum stimulation of oogenic progression and egg deposition immediately after mating.     

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.     

Fates and targets of male accessory gland proteins in mated female Drosophila melanogaster

Male accessory gland proteins (Acps) in Drosophila are components of the seminal fluid and are transferred to females during copulation. In mated females, Acps enhance egg production, augment sperm storage, induce refractory mating behaviors, and affect the female's longevity. To address the functions of eight previously uncharacterized Acps and further analyze five others, we determined the tissues to which they target after transfer to females. Each Acp has multiple targets and is unique in its pattern of localization. Within the reproductive tract, Acps target to the uterus, oviduct, sperm storage organs, ovary and oocytes. Some Acps also leave the reproductive tract, to enter the hemolymph. Some Acps are detected on the surface of eggs laid by mated females but were not detectable within those eggs. Our results can help to identify the likely functions of these Acps as well as to create models for the mechanism of action of Acps.     

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.     

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.     

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.     

A Novel Function for the Hox Gene Abd-B in the Male Accessory Gland Regulates the Long-Term Female Post-Mating Response in Drosophila

In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland''s other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), we discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer from the iab-6 regulatory domain that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. We determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of sex peptide in female mates of iab-6 mutant males. We also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. Our results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR.     

The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster

Drosophila melanogaster male accessory gland proteins (Acps) that are transferred in the ejaculate with sperm mediate post-mating competition for fertilizations between males. The actions of Acps include effects on oviposition and ovulation, receptivity and sperm storage. Two Acps that modulate egg production are Acp26Aa (ovulin) and Acp70A (the sex peptide). Acp26Aa acts specifically on the process of ovulation (the release of mature eggs from the ovaries), which is initiated 1.5 h after mating. In contrast, sperm storage can take as long as 6-9 h to complete. Initial ovulations after matings by virgin females will therefore occur before all sperm are fully stored and the extra eggs initially laid as a result of Acp26Aa transfer are expected to be inefficiently fertilized. Acp26Aa-mediated release of existing eggs should not cause a significant energetic cost or lead to a decrease in female lifespan assuming, as seems likely, that the energetic cost of egg laying comes from de novo egg synthesis (oogenesis) rather than from ovulation. We tested these predictions using Acp26Aa(1) mutant males that lack Acp26Aa but are normal for other Acps and Acp26Aa(2) males that transfer a truncated but fully functional Acp26Aa protein. Females mating with Acp26Aa(2) (truncation) males that received functional Acp26Aa produced significantly more eggs following their first matings than did mates of Acp26Aa(1) (null) males. However, as predicted above, these extra eggs, which were laid as a result of Acp26Aa transfer to virgin females, showed significantly lower egg hatchability. Control experiments indicated that this lower hatchability was due to lower rates of fertilization at early post-mating times. There was no drop in egg hatchability in subsequent non-virgin matings. In addition, as predicted above, females that did or did not receive Acp26Aa did not differ in survival, lifetime fecundity or lifetime progeny, indicating that Acp26Aa transfer does not represent a significant energetic cost for females and does not contribute to the survival cost of mating. Acp26Aa appears to remove a block to oogenesis by causing the clearing out of existing mature eggs and, thus, indirectly allowing oogenesis to be initiated immediately after mating. The results show that subtle processes coordinate the stimulation of egg production and sperm storage in mating pairs.     

Seminal fluid causes temporarily reduced egg hatch in previously mated females

In Drosophila, male accessory gland fluid (seminal fluid) has multiple effects on the female's reproductive efficiency. Here, we show the effect of seminal fluid on rate of egg hatch immediately following mating. Singly mated females were remated to two classes of sterile males, one with seminal fluid and one without seminal fluid. Transfer of seminal fluid results in a strong reduction in egg hatch shortly after the mating. Also, it is shown that remating with normal males causes an immediate reduction of egg hatch followed by recovery to normal egg hatch. In all cases, unhatched eggs contained no sperm. These results are consistent with a role for seminal fluid in sperm competition, mediated by incapacitation or inefficient use of resident sperm.     

The consequences of genetic variation in sex peptide expression levels for egg laying and retention in females

The accessory gland proteins (Acps) that male Drosophila melanogaster produce and transfer to females during copulation are key to male and female fitness. One Acp, the sex peptide (SP), is largely responsible for a dramatic increase in female egg laying and decrease in female receptivity after copulation. While genetic variation in male SP expression levels correlate with refractory period duration in females, it is unknown whether male SP expression influences female egg laying or if any effect of SP is mediated by SP retention in the female reproductive tract. Here we measured the amount of SP retained in the female reproductive tract after mating and female egg laying after copulating with virgin males. We found no correlation between male SP expression levels and egg laying, or the amount of SP in the female reproductive tract after mating. Additionally, the amount of SP retained in the female did not influence egg laying. These finding suggests that additional factors, such as variation in other Acps, are important for the retention of SP in females and its quantitative effects on egg laying. It also shows that egg laying and refractory period response to SP is at least partially uncoupled.     

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。     

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 remating, sperm competition and sexual selection in Drosophila

Female remating is fundamental to evolutionary biology as it determines the pattern of sexual selection and sexual conflict. Remating in females is an important component of Drosophila mating systems because it affects sperm usage patterns and sexual selection. Remating is common in females of many species of Drosophila in both natural and laboratory populations. It has been reported in many insect species and also in vertebrates. Female remating is a prerequisite for sperm competition between males, and the consequences of this competition, such as sperm precedence or sperm displacement, have been reported for many species of Drosophila. Female remating is dependent on the amount of sperm stored, the male seminal fluid components, nutrition, the quantity of eggs laid, experimental design and density of flies in laboratory. Remating by a female is an insurance against male sterility and sub-fertility and increases genetic heterogeneity of female offspring. Remating gives greater female productivity in many species of Drosophila. We examined female remating with respect to sperm competition and sexual selection in Drosophila and addressed the possible benefits for females. We also reviewed the role of accessory gland fluid in remating, costs associated with remating, the genetic basis of female remating and some possible mechanisms of sperm competition in the light of last male sperm priority and paternity assurance in Drosophila and other insects. We also suggest future areas of research.     

Evidence for positive selection on Drosophila melanogaster seminal fluid protease homologs

Proteins present in the seminal fluid of Drosophila melanogaster (accessory gland proteins Acps) contribute to female postmating behavioral changes, sperm storage, sperm competition, and immunity. Consequently, male-female coevolution and host-pathogen interactions are thought to underlie the rapid, adaptive evolution that characterizes several Acp-encoding genes. We propose that seminal fluid proteases are likely targets of selection due to their demonstrated or potential roles in between-sex interactions and immune processes. We use within- and between-species sequence data for 5 predicted protease-encoding Acp loci to test this hypothesis. Our polymorphism-based analyses find evidence for positive selection at 2 genes, both of which encode predicted serine protease homologs. One of these genes, CG6069, also shows evidence for consistent selection on a subset of codons over a deeper evolutionary time scale. The second gene, CG9997, was previously shown to be essential for normal sperm usage, suggesting that sexual selection may underlie its history of adaptation.     

Adult male nutrition and reproductive success in Drosophila melanogaster

Explanations for the maintenance of variation in reproductive traits influenced by seminal fluid accessory gland proteins (Acps) in male Drosophila melanogaster include nontransitivity in the outcome of sperm competition and/or condition dependence of the traits involved. We investigated the effects of adult male nutrition (five diets) on the expression of Acp- and sperm- mediated traits. We found novel, nonlinear effects, with females showing lower levels of refractoriness to remating after mating with males held on the lowest and highest yeast diets. There were no significant effects of adult male nutrition on male paternity share, but there was a striking, nonlinear effect on second male progeny production, with males kept on intermediate yeast diets fathering the highest number of offspring. Such "bell shaped" responses of life-history traits to nutrition have only previously been reported for longevity. Consistent with previous reports, males maintained on low protein diets had lower premating success and gained fewer rematings with nonvirgins. We show novel and body size independent effects of adult male nutrition on traits influenced by Acps and sperm, which do not fit current condition-dependent handicap models and can affect the strength of sexual selection acting upon such fitness-related traits.     

Sex peptide causes mating costs in female Drosophila melanogaster

Conflicts between females and males over reproductive decisions are common . In Drosophila, as in many other organisms, there is often a conflict over how often to mate. The mating frequency that maximizes male reproductive success is higher than that which maximizes female reproductive success . In addition, frequent mating reduces female lifespan and reproductive success , a cost that is mediated by male ejaculate accessory gland proteins (Acps) . We demonstrate here that a single Acp, the sex peptide (SP or Acp70A), which decreases female receptivity and stimulates egg production in the first matings of virgin females , is a major contributor to Acp-mediated mating costs in females. Females continuously exposed to SP-deficient males (which produce no detectable SP ) had significantly higher fitness and higher lifetime reproductive success than control females. Hence, rather than benefiting both sexes, receipt of SP decreases female fitness, making SP the first identified gene that is likely to play a central role in sexual conflict.     

Cross-generational fitness benefits of mating and male seminal fluid

In many species, the physical act of mating and exposure to accessory gland proteins (Acps) in male seminal fluid reduces female survival and offspring production. It is not clear what males gain from harming their sexual partners or why females mate frequently despite being harmed. Using sterile strains of Drosophila melanogaster that differ in their production of Acps, we found that both the physical act of mating and exposure to male seminal fluid in mothers increase the fitness of daughters. We show that the changes in daughter fitness are mediated by parental effects, not by sexual selection involving good genes or owing to variation in maternal egg production. These results support the idea that male harm of females might partly evolve through cross-generational fitness benefits.