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There is extensive variation in males for sperm competitive abilities, and in females for the ability to distinguish among sperm from different males. But it is still not known how females distinguish males... In many species, sperm can remain viable in the reproductive tract of a female well beyond the typical interval to remating. This creates an opportunity for sperm from different males to compete for oocyte fertilization inside the female’s reproductive tract. In Drosophila melanogaster, sperm characteristics and seminal fluid content affect male success in sperm competition. On the other hand, although genome-wide association studies (GWAS) have demonstrated that female genotype plays a role in sperm competition outcome as well, the biochemical, sensory, and physiological processes by which females detect and selectively use sperm from different males remain elusive. Here, we functionally tested 26 candidate genes implicated via a GWAS for their contribution to the female’s role in sperm competition, measured as changes in the relative success of the first male to mate (P1). Of these 26 candidates, we identified eight genes that affect P1 when knocked down in females, and showed that five of them do so when knocked down in the female nervous system. In particular, Rim knockdown in sensory pickpocket (ppk)+ neurons lowered P1, confirming previously published results, and a novel candidate, caup, lowered P1 when knocked down in octopaminergic Tdc2+ neurons. These results demonstrate that specific neurons in the female’s nervous system play a functional role in sperm competition and expand our understanding of the genetic, neuronal, and mechanistic basis of female responses to multiple matings. We propose that these neurons in females are used to sense, and integrate, signals from courtship or ejaculates, to modulate sperm competition outcome accordingly.

The yellow fever mosquito, Aedes aegypti,, transmits several viruses causative of serious diseases, including dengue, Zika, and chikungunya. Some proposed efforts to control this vector involve manipulating reproduction to suppress wild populations or to replace them with disease-resistant mosquitoes. The design of such strategies requires an intimate knowledge of reproductive processes, yet our basic understanding of reproductive genetics in this vector remains largely incomplete. To accelerate future investigations, we have comprehensively catalogued sperm and seminal fluid proteins (SFPs) transferred to females in the ejaculate using tandem mass spectrometry. By excluding female-derived proteins using an isotopic labeling approach, we identified 870 sperm proteins and 280 SFPs. Functional composition analysis revealed parallels with known aspects of sperm biology and SFP function in other insects. To corroborate our proteome characterization, we also generated transcriptomes for testes and the male accessory glands-the primary contributors to Ae. aegypti, sperm and seminal fluid, respectively. Differential gene expression of accessory glands from virgin and mated males suggests that transcripts encoding proteins involved in protein translation are upregulated post-mating. Several SFP transcripts were also modulated after mating, but >90% remained unchanged. Finally, a significant enrichment of SFPs was observed on chromosome 1, which harbors the male sex determining locus in this species. Our study provides a comprehensive proteomic and transcriptomic characterization of ejaculate production and composition and thus provides a foundation for future investigations of Ae. aegypti, reproductive biology, from functional analysis of individual proteins to broader examination of reproductive processes.

In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca2+ in the oocyte's cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster,. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.

The biogenic monoamine octopamine (OA) is a crucial regulator of invertebrate physiology and behavior. Since its discovery in the 1950s in octopus salivary glands, OA has been implicated in many biological processes among diverse invertebrate lineages. It can act as a neurotransmitter, neuromodulator and neurohormone in a variety of biological contexts, and can mediate processes including feeding, sleep, locomotion, flight, learning, memory, and aggression. Here, we focus on the roles of OA in female reproduction in insects. OA is produced in the octopaminergic neurons that innervate the female reproductive tract (RT). It exerts its effects by binding to receptors throughout the RT to generate tissue- and region-specific outcomes. OA signaling regulates oogenesis, ovulation, sperm storage, and reproductive behaviors in response to the female's internal state and external conditions. Mating profoundly changes a female's physiology and behavior. The female's OA signaling system interacts with, and is modified by, male molecules transferred during mating to elicit a subset of the post-mating changes. Since the role of OA in female reproduction is best characterized in the fruit fly Drosophila melanogaster, we focus our discussion on this species but include discussion of OA in other insect species whenever relevant. We conclude by proposing areas for future research to further the understanding of OA's involvement in female reproduction in insects.

Significance In many species, mating induces physiological changes in the female that increase the reproductive success of the mating pair. The postmating response (PMR) is caused by male seminal fluid proteins interacting with the female reproductive system. Because of the importance of the PMR in many insect species relevant to human health and agriculture, we examined the gene expression profile of one important cell type of the gland that produces most of the seminal fluid proteins in Drosophila. Interestingly, among proteins necessary for the PMR in this species, we discovered that a micropeptide encoded by a supposedly noncoding transcript is produced and carries out important reproductive functions. Such micropeptides were previously unrecognized but are emerging as important actors in complex biological processes., Even in well-characterized genomes, many transcripts are considered noncoding RNAs (ncRNAs) simply due to the absence of large open reading frames (ORFs). However, it is now becoming clear that many small ORFs (smORFs) produce peptides with important biological functions. In the process of characterizing the ribosome-bound transcriptome of an important cell type of the seminal fluid-producing accessory gland of Drosophila melanogaster, we detected an RNA, previously thought to be noncoding, called male-specific abdominal (msa). Notably, msa is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. We find that this RNA encodes a “micropeptide” (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. In addition to bringing insights into the biology of a rare cell type, this work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes.

Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. Here, we demonstrate that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, we report a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state.

Summary X-ray fluorescence microscopy (XFM) is a powerful tool for mapping and quantifying the spatial distribution of elemental composition of biological samples. Recently, it was reported that transition metal fluctuations occur during Drosophila reproduction, analogous to what is seen in mammals and nematodes, and may contribute to Drosophila female fertility. To further support XFM studies on Drosophila reproduction, we describe procedures for isolating oocytes and activated eggs and examining their elemental composition by XFM scanning and analysis. For complete details on the use and execution of this protocol, please refer to Hu et al. (2020)., Graphical abstract, Highlights • Preparing Drosophila oocytes and activated eggs for X-ray fluorescence microscopy scans • XFM scanning to reveal the elemental contents and distribution of samples • Analysis and interpretation of XFM data on Drosophila oocyte and egg samples, X-ray fluorescence microscopy (XFM) is a powerful tool for mapping and quantifying the spatial distribution of elemental composition of biological samples. Recently, it was reported that transition metal fluctuations occur during Drosophila reproduction, analogous to what is seen in mammals and nematodes, and may contribute to Drosophila female fertility. To further support XFM studies on Drosophila reproduction, we describe procedures for isolating oocytes and activated eggs and examining their elemental composition by XFM scanning and analysis.

Drosophila melanogaster females undergo a variety of post-mating changes that influence their activity, feeding behavior, metabolism, egg production and gene expression. These changes are induced either by mating itself or by sperm or seminal fluid proteins. In addition, studies have shown that axenic females—those lacking a microbiome—have altered fecundity compared to females with a microbiome, and that the microbiome of the female’s mate can influence reproductive success. However, the extent to which post-mating changes in transcript abundance are affected by microbiome state is not well-characterized. Here we investigated fecundity and the post-mating transcript abundance profile of axenic or control females after mating with either axenic or control males. We observed interactions between the female’s microbiome and her mating status: transcripts of genes involved in reproduction and genes with neuronal functions were differentially abundant depending on the females’ microbiome status, but only in mated females. In addition, immunity genes showed varied responses to either the microbiome, mating, or a combination of those two factors. We further observed that the male’s microbiome status influences the fecundity of both control and axenic females, while only influencing the transcriptional profile of axenic females. Our results indicate that the microbiome plays a vital role in the post-mating switch of the female’s transcriptome.

Postcopulatory sexual selection (PCSS), comprised of sperm competition and cryptic female choice, has emerged as a widespread evolutionary force among polyandrous animals. There is abundant evidence that PCSS can shape the evolution of sperm. However, sperm are not the whole story: they are accompanied by seminal fluid substances that play many roles, including influencing PCSS. Foremost among seminal fluid models isDrosophila melanogaster, which displays ubiquitous polyandry, and exhibits intraspecific variation in a number of seminal fluid proteins (Sfps) that appear to modulate paternity share. Here, we first consolidate current information on the identities ofD. melanogasterSfps. Comparing betweenD. melanogasterand human seminal proteomes, we find evidence of similarities between many protein classes and individual proteins, including someD. melanogasterSfp genes linked to PCSS, suggesting evolutionary conservation of broad-scale functions. We then review experimental evidence for the functions ofD. melanogasterSfps in PCSS and sexual conflict. We identify gaps in our current knowledge and areas for future research, including an enhanced identification of PCSS-related Sfps, their interactions with rival sperm and with females, the role of qualitative changes in Sfps and mechanisms of ejaculate tailoring.This article is part of the theme issue ‘Fifty years of sperm competition’.

Aedes aegypti mosquitoes are the primary vectors of numerous viruses that impact human health. As manipulation of reproduction has been proposed to suppress mosquito populations, elucidation of biological processes that enable males and females to successfully reproduce is necessary. One essential process is female sperm storage in specialized structures called spermathecae. Aedes aegypti females typically mate once, requiring them to maintain sperm viably to fertilize eggs they lay over their lifetime. Spermathecal gene products are required for Drosophila sperm storage and sperm viability, and a spermathecal-derived heme peroxidase is required for long-term Anopheles gambiae fertility. Products of the Ae. aegypti spermathecae, and their response to mating, are largely unknown. Further, although female blood-feeding is essential for anautogenous mosquito reproduction, the transcriptional response to blood-ingestion remains undefined in any reproductive tissue. We conducted an RNAseq analysis of spermathecae from unfed virgins, mated only, and mated and blood-fed females at 6, 24, and 72 h post-mating and identified significant differentially expressed genes in each group at each timepoint. A blood-meal following mating induced a greater transcriptional response in the spermathecae than mating alone. This study provides the first view of elicited mRNA changes in the spermathecae by a blood-meal in mated females.

When females mate with more than one male, the males’ paternity share is affected by biases in sperm use. These competitive interactions occur while female and male molecules and cells work interdependently to optimize fertility, including modifying the female’s physiology through interactions with male seminal fluid proteins (SFPs). Some modifications persist, indirectly benefiting later males. Indeed, rival males tailor their ejaculates accordingly. Here, we show that SFPs from one male can directly benefit a rival’s sperm. We report that Sex Peptide (SP) that a female Drosophila receives from a male can bind sperm that she had stored from a previous male, and rescue the sperm utilization and fertility defects of an SP-deficient first-male. Other seminal proteins received in the first mating ‘primed’ the sperm (or the female) for this binding. Thus, SP from one male can directly benefit another, making SP a key molecule in inter-ejaculate interaction., eLife digest When fruit flies and other animals reproduce, a compatible male and a female mate, allowing sperm from the male to swim to and fuse with the female’s egg cells. The males also produce proteins known as seminal proteins that travel with the sperm. These proteins increase the likelihood of sperm meeting an egg and induce changes in the female that increase the number, or quality, of offspring produced. Some seminal proteins help a male to compete against its rivals by decreasing their chances to fertilize eggs. However, since many of the changes seminal proteins induce in females are long-lasting, it is possible that a subsequent male may actually benefit indirectly from the effects of a prior male’s seminal proteins. It remains unclear whether the seminal proteins of one male are also able to directly interact with and help the sperm of another male. Male fruit flies make a seminal protein known as sex peptide. Normally, a sex peptide binds to the sperm it accompanies into the female, increasing the female’s fertility and preventing her from mating again with a different male. To test whether the sex peptide from one male can bind to and help a rival male’s sperm, Misra and Wolfner mated female fruit flies with different combinations of males that did, or did not, produce the sex peptide. The experiments found that female flies that only mated with mutant males lacking the sex peptide produced fewer offspring than if they had mated with a ‘normal’ male. However, in females that mated with a mutant male followed by another male who provided the sex peptide, the second male’s sex peptide was able to bind to the mutant male’s sperm (as well as to his own). This in turn allowed the mutant male’s sperm to be efficiently used to sire offspring, at levels comparable to a normal male providing the sex peptide. These findings demonstrate that the ways individual male fruit flies interact during reproduction are more complex than just simple rivalry. Since humans and other animals also produce seminal proteins comparable to those of fruit flies, this work may aid future advances in human fertility treatments and strategies to control the fertility of livestock and pests, including mosquitoes that transmit diseases.

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed inDrosophila melanogasterbut are less common for other laboratory organisms. Here, we reportmosaicanalysis bygRNA-inducedcrossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline ofDrosophila. Further, by developing a MAGIC toolkit for one chromosome arm, we demonstrate the method’s application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derivedDrosophilastrains. Eliminating the need to introduce recombinase-recognition sites in the genome, this simple and versatile system simplifies mosaic analysis inDrosophilaand can be applied in any organism that is compatible with CRISPR/Cas9.

In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins, and are critical for fertilization success. Unfortunately, our understanding of the molecular interplay between female and male reproductive proteins remains superficial. Here we analyze the post-mating response in aDrosophilaspecies that has evolved strong gametic incompatibility with its sister species;D. novamexicanafemales produce only 1% fertilized eggs in crosses withD. americanamales, compared to ~98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study we use short-read RNA sequencing to examine the evolutionary dynamics of female reproductive genes and the post-mating transcriptome response in crosses within and between species. First, we found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, post-mating responses in con- and heterospecific matings are largely congruent, but heterospecific mating induces expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. We also identify several genes in the JAK/STAT signaling pathway that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, we also detect it in the female head and ovaries. Our results show that the female’s post-mating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.

Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating “junk”. Yet multiple lines of evidence indicate these regions may be an important source of variation that could drive adaptation and species divergence, particularly through regulation of fertility. The ∼40 Mb Y chromosome ofDrosophila melanogastercontains only 16 known protein-coding genes and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. We devised a genetic strategy for recovering and retaining stocks with sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three Y-linked genes. Two genes,PRYandFDY, had no previously identified functions. We found thatPRYmutant males are sub-fertile, butFDYmutant males had no detectable fertility defects.FDY, the newest known gene on the Y chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene,CCY, had been predicted but never formally shown to be required for male fertility. CRISPR-targeting and RNAi ofCCYcaused male sterility. Surprisingly, however, ourCCYmutants were sterile even in the presence of an extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to dominant sterility. Our approach provides an important step toward understanding the complex functions of the Y chromosome and parsing which functions are accomplished by genes versus repeat elements.

Temporal fluctuations in zinc concentration are emerging as essential signaling events in the regulation of meiosis during oogenesis and early embryogenesis. For example, accumulation and release of zinc in mammalian gametes are required for oocyte maturation and egg activation, respectively. Here, we report that significant zinc fluctuations occur in Drosophila melanogaster oocytes and activated eggs, and that zinc is required for female fertility. Using synchrotron-based X-ray fluorescence microscopy (XFM), we mapped the subcellular localization of essential transition metals during fly oogenesis and egg activation. Quantitative analysis of the XFM data reveals that zinc is the most abundant transition metal in oocytes and that its levels increase during oocyte maturation. This increase is accompanied by appearance of zinc-enriched intracellular granules in the oocyte. The zinc transporter Znt35C is required for maintenance of these zinc-enriched granules in mature oocytes. During egg activation, the process that follows oocyte maturation and mediates the transition of the egg to an embryo, oocyte zinc levels decrease significantly, accompanied by a decrease in the number of zinc-enriched granules. This essential pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, rising during oogenesis and decreasing during egg activation. This further extends the parallels in female gamete processes between Drosophila and mammals, suggesting that the former may provide an important genetically tractable model for studying the role of zinc in female reproduction.

Chemicals released into the environment by food, predators and conspecifics play critical roles in Drosophila reproduction. Females and males live in an environment full of smells, whose molecules communicate to them the availability of food, potential mates, competitors or predators. Volatile chemicals derived from fruit, yeast growing on the fruit, and flies already present on the fruit attract Drosophila, concentrating flies at food sites, where they will also mate. Species-specific cuticular hydrocarbons displayed on female Drosophila as they mature are sensed by males and act as pheromones to stimulate mating by conspecific males and inhibit heterospecific mating. The pheromonal profile of a female is also responsive to her nutritional environment, providing an honest signal of her fertility potential. After mating, cuticular and semen hydrocarbons transferred by the male change the female's chemical profile. These molecules make the female less attractive to other males, thus protecting her mate's sperm investment. Females have evolved the capacity to counteract this inhibition by ejecting the semen hydrocarbon (along with the rest of the remaining ejaculate) a few hours after mating. Although this ejection can temporarily restore the female's attractiveness, shortly thereafter another male pheromone, a seminal peptide, decreases the female's propensity to re-mate, thus continuing to protect the male's investment. Females use olfaction and taste sensing to select optimal egg-laying sites, integrating cues for the availability of food for her offspring, and the presence of other flies and of harmful species. We argue that taking into account evolutionary considerations such as sexual conflict, and the ecological conditions in which flies live, is helpful in understanding the role of highly species-specific pheromones and blends thereof, as well as an individual's response to the chemical cues in its environment.

Aedes aegypti is a vector of medically important viruses including those causing Zika, dengue, and chikungunya. During mating, males transfer a number of proteins and other molecules to the female and these components of the male ejaculate are essential in shifting female post-mating behaviors in a number of insect species. Because these molecules are highly variable by species, and female post-mating behavior by species is also varied, behavioral assays testing the function of the ejaculate are necessary before we can develop control strategies targeting the mating system to reduce mosquito populations. Because increased survival in mosquitoes strongly increases vectorial capacity and can influence population sizes and potential risk we tested the effect of mating on female survival. The ejaculate can either promote or reduce female survival, as both have been shown in multiple insect species, yet this effect has not been directly assessed in mosquitoes. We compared survival of females in four treatment groups: mated females, virgin females, and virgin females injected with either an extract from the male reproductive glands or a saline control. Survival, blood feeding frequency, fecundity and cumulative net reproductive rate (R(0)) were determined after multiple feedings from a human host. Our results confirm that male reproductive gland substances increase female fecundity and blood feeding frequency, resulting in dramatic increases in fitness (R(0)). We also demonstrate, for the first time, an effect of male reproductive gland extracts alone on female survival, regardless of whether or not the female ingested a vertebrate blood meal. Thus, the effects of MAG extract on survival are not secondary effects from altered blood feeding. Collectively, we demonstrate a direct role for Ae. aegypti male-derived molecules on increasing female fitness, reproductive success and, ultimately, transmission potential for vector borne pathogens.

The ejaculatory bulb of Drosophila melanogaster males produces proteins and pheromones that play important roles in reproduction. This tissue is also the final mixing site for the ejaculate before transfer to the female. The ejaculatory bulb's dynamics remain largely unstudied. By microscopy of the ejaculatory bulb in maturing adult males, we observed that the ejaculatory bulb expands in size as males age. Moreover, we document that when males mate, their ejaculatory bulb expands further as ejaculate transfer begins, and then contracts halfway through the course of mating as ejaculate transfer finishes. Although there is some male-to-male variation in the timing of these changes, ultimately the tissue changes in a predictable pattern that gives insight into the active mating process in Drosophila.

Background Aedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results. Methodology/Principal findings To resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection. Conclusions/Significance Mating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory., Author summary Controlling mosquitoes and the disease agents they transmit during blood feeding remains a global public heath priority. Some vector control tools in development target the mosquito reproductive system as a means of control. The transfer of semen to a female during mating profoundly impacts her biology by inhibiting re-mating and stimulating egg production and laying. Previous studies in Aedes aegypti that examined potential effects of mating on blood feeding yielded contradictory results. We examined the potential of mating and seminal fluids to modulate female blood feeding and controlled for numerous experimental factors, such as ingestion of sugar prior to blood feeding, that may have led to conflicting results in prior studies. In laboratory studies with field-derived Thai mosquitoes, we show that mating and seminal fluids do not impact blood feeding during a mosquito’s first meal, but prior sugar feeding does. Furthermore, we show that mating, seminal fluids, and sugar feeding can impact blood feeding across multiple consecutive meals. Furthermore, field studies in Colombia show that virgin and mated females were equally likely to contain blood. Our research clarifies the impact of mating on blood feeding and suggests ways to improve our understanding of these behaviors in nature.

Declining ejaculate performance with male age is taxonomically widespread and has broad ramifications for fertility and fitness. However, we have a poor understanding of age-related changes to specific ejaculate components, how they cause reduced performance, and whether the decline is ameliorable. Here, we show that, in Drosophila, sperm production chronologically declines with age, invariant to mating activity, while repeated mating causes infertility via reduced sperm stores and viability. However, changes to sperm do not fully explain ejaculate deterioration: impacts on seminal fluid contribute to aspects of reduced ejaculate performance, associated with shifts in proteome abundance and quality. We show that ablation of insulin-like peptide-producing cells in males ameliorates aspects of ejaculate performance loss, suggesting that anti-ageing interventions can be coopted to benefit male reproductive health.One Sentence SummaryEjaculate performance declines with male age via mating-dependent sperm and seminal protein deterioration, but it can be ameliorated.

Aedes aegypti mosquitoes transmit pathogens such as yellow fever, dengue, Zika, and chikungunya viruses to millions of human hosts annually [1]. As such, understanding Ae. aegypti courtship and mating biology could prove crucial to the success of pest and disease control efforts that target reproduction. Potentially to communicate reproductive fitness [2,3], mosquito males and females harmonize their flight tones prior to mating in a behavior known as harmonic convergence (HC) [4]. Furthermore, after mating or treatment with male accessory gland (MAG) extracts, female Ae. aegypti become resistant, or refractory, to re-mating [5]. To test the hypothesis that mating and MAG fluids inhibit a female’s ability to induce HC in males, we recorded audio of pre-copulatory flight interactions between virgin males and either virgin, mated, or MAG extract-injected females and analyzed these recordings for the presence or absence of HC. We further assayed HC with heat-treated MAG- and bovine serum albumin (BSA)-injected females to test if potential MAG effects are due specifically to heat-labile molecules. We found that mating and MAG extract, but not heat-treated MAG or BSA, lower HC occurrence by 53% compared to all other controls. Because MAG-injected females formed only nonproductive pseudocopulas and HC was most often accomplished by male tone modulations, these results suggest that mating may inhibit HC indirectly via the broader range of MAG-induced female refractory mating behaviors. Together, our results demonstrate an important new role for MAG molecules in mediating female post-mating behavior.

Summary Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc., Graphical Abstract, Highlights • Dietary zinc is required for Drosophila female fertility • Oocyte zinc levels increase over oogenesis and decrease during egg activation • Zinc is enriched in the oocyte and forms aggregated granules in mature oocytes • Transporters are required for zinc retention and zinc-enriched granules in oocytes, Evolutionary Developmental Biology; Biotechnology; Embryology

[][1] By the early 1920s, the existence of mutations was well established, but how they could be generated remained a topic of lively speculation. One interesting case was the Drosophila Bar mutation ([Tice 1914][2]). While normal flies have round eyes, the X-linked mutation Bar ( B

The yellow fever mosquito, Aedes aegypti, transmits several viruses, including dengue, Zika, and chikungunya. Some proposed efforts to control this vector involve manipulating reproduction to suppress wild populations or replacing them with disease-resistant mosquitoes. The design of such strategies requires an intimate knowledge of reproductive processes, yet our basic understanding of reproductive genetics in this vector remains largely incomplete. To accelerate future investigations, we have comprehensively catalogued sperm and seminal fluid proteins (SFPs) transferred to females in the ejaculate using tandem mass spectrometry. By excluding female-derived proteins using an isotopic labelling approach, we identified 870 sperm proteins and 280 seminal fluid proteins. Functional composition analysis revealed parallels with known aspects of sperm biology and SFP function in other insects. To corroborate our proteome characterization, we also generated transcriptomes for testes and the male accessory glands—the primary contributors to Ae. aegypti sperm and seminal fluid, respectively. Differential gene expression of accessory glands from virgin and mated males suggests that protein translation is upregulated post-mating. Several SFP transcripts were also modulated after mating, but >90% remained unchanged. Finally, a significant enrichment of SFPs was observed on chromosome 1, which harbors the male sex determining locus in this species. Our study provides a comprehensive proteomic and transcriptomic characterization of ejaculate production and composition and thus provides a foundation for future investigations of Ae. aegypti reproductive biology, from functional analysis of individual proteins to broader examination of reproductive processes.

Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. Here, we show that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues., Author summary In many animals, the male seminal fluid induces physiology changes in the mated female that increase a male’s reproductive success. These changes are often referred to as the post-mating response (PMR). In Drosophila, the seminal fluid proteins responsible for generating the PMR are made in a specialized gland, analogous to the mammalian seminal vesicle and prostate, called the accessory gland (AG). In this work, we show that a male-specific, long, non-coding RNA (lncRNA), called msa, plays a critical role in the development and function of this gland, primarily through a microRNA (miRNA) encoded within its sequence. This same miRNA had previously been shown to be expressed in the central nervous system (CNS) via an alternative promoter, where its ability to repress homeotic genes is required for both male and female fertility. Here, we present evidence that the targets of this miRNA in the AG are likely different from those found in the CNS. Thus, the same miRNA seems to have been selected to affect Drosophila fertility through two different mechanisms. Although many non-coding RNAs have now been identified, very few can be shown to have function. Our work highlights a lncRNA that has multiple biological functions, affecting cellular morphology and fertility.

Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SP's sperm binding is mediated by a network of at least eight seminal proteins. We show here that some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2 h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. We also observed that the same network proteins are detectable at 2 h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. Our results suggest that these network proteins act transiently to facilitate the conditions for SP's binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.

Male reproductive ageing has been mainly explained by a reduction in sperm quality with negative effects on offspring development and quality. In addition to sperm, males transfer seminal fluid proteins (Sfps) at mating; Sfps are important determinants of male reproductive success. Receipt of Sfps leads to female post-mating changes including physiological changes, and affects sperm competition dynamics. Using the fruit fly Drosophila melanogaster we studied ageing males' ability to induce female post-mating responses and determined the consequences of male ageing on their reproductive success. We aged males for up to 7 weeks and assayed their ability to: i) gain a mating, ii) induce egg-laying and produce offspring, iii) prevent females from remating and iv) transfer sperm and elicit storage after a single mating. We found that with increasing age, males were less able to induce post-mating responses in their mates; moreover ageing had negative consequences for male success in competitive situations. Our findings indicate that with advancing age male flies transferred less effective ejaculates and that Sfp composition might change over a male's lifetime in quantity and/or quality, significantly affecting his reproductive success.

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This drastic change of cell state is accompanied by large scale alteration of the phospho-proteome of the cell. Despite the importance of this transition in cell and developmental state, it has been difficult to find many of its regulators. We hypothesize that phosphorylation state changes between oocyte and early embryo regulate the activities of proteins that are necessary during or after this transition, and thus that the set of phospho-regulated proteins would be an enriched source for finding critical players in the egg-to-embryo transition. To test this, we used germline-specific RNAi to examine the function of 189 maternal proteins that are phospho-regulated during egg activation inDrosophila melanogaster. We identified 53 genes whose knockdown reduced or abolished egg production, as well as 50 genes for which maternal knockdown led to significant impairment or abolishment of the eggs’ ability to hatch (hatchability). We observed different stages of developmental arrest in the embryos with impaired hatchability and several distinct categories of abnormalities in the maternal knockdown embryos that arrest early in development, suggesting potential roles of the candidates in egg activation and early embryogenesis. Our results, validated by our detection of multiple genes with previously-documented maternal-effect phenotypes among the proteins we tested, revealed 15 genes with new roles in egg activation and early embryogenesis. Given that protein phospho-regulation also occurs during this transition in other organisms, we suggest that the phospho-regulated proteins may provide an enriched dataset for identifying important players in the egg-to-embryo transition.

Physiological changes in females during and after mating are triggered by seminal fluid components in conjunction with female-derived molecules. In insects, these changes include increased egg production, storage of sperm, and changes in muscle contraction within the reproductive tract (RT). Such postmating changes have been studied in dissected RT tissues, but understanding their coordination in vivo requires a holistic view of the tissues and their interrelationships. Here, we used high-resolution, multiscale micro-computed tomography (CT) scans to visualize and measure postmating changes in situ in the Drosophila female RT before, during, and after mating. These studies reveal previously unidentified dynamic changes in the conformation of the female RT that occur after mating. Our results also reveal how the reproductive organs temporally shift in concert within the confines of the abdomen. For example, we observed chiral loops in the uterus and in the upper common oviduct that relax and constrict throughout sperm storage and egg movement. We found that specific seminal fluid proteins or female secretions mediate some of the postmating changes in morphology. The morphological movements, in turn, can cause further changes due to the connections among organs. In addition, we observed apparent copulatory damage to the female intima, suggesting a mechanism for entry of seminal proteins, or other exogenous components, into the female’s circulatory system. The 3D reconstructions provided by high-resolution micro-CT scans reveal how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology.

The storage of sperm in mated females is important for efficient reproduction. After sperm are transferred to females during mating, they need to reach and enter into the site(s) of storage, be maintained viably within storage, and ultimately be released from storage to fertilize eggs. Perturbation of these events can have drastic consequences on fertility. In Drosophila melanogaster, females store sperm for up to 2 weeks after a single mating. For sperm to be released normally from storage, Drosophila females need to receive the seminal fluid protein (SFP) sex peptide (SP) during mating. SP, which binds to sperm in storage, signals through the sex peptide receptor (SPR) to elicit two other effects on mated females: the persistence of egg laying and a reduction in sexual receptivity. However, it is not known whether SPR is also needed to mediate SP's effect on sperm release. By phenotypic analysis of flies deleted for SPR, and of flies knocked down for SPR, ubiquitously or in specific tissues, we show that SPR is required to mediate SP's effects on sperm release from storage. We show that SPR expression in ppk(+) neurons is needed for proper sperm release; these neurons include those that mediate SP's effect on receptivity and egg laying. However, we find that SPR is also needed in the spermathecal secretory cells of the female reproductive tract for efficient sperm release. Thus, SPR expression is necessary in both the nervous system and in female reproductive tract cells to mediate the release of stored sperm.

Little is known about the genetic basis of naturally occurring variation for sexually selected behavioral traits. Drosophila melanogaster, with its rich repertoire of courtship behavior and genomic and genetic resources, is an excellent model organism for addressing this question. We assayed a genetically diverse panel of lines with full genome sequences, the Drosophila Genetic Reference Panel, to assess the heritability of variation in courtship behavior and mating progression. We subsequently used these data to quantify natural variation in transition probabilities between courtship behaviors. We found heritable variation along the expected trajectory for courtship behaviors, including the tendency to initiate courtship and rate of progression through courtship, suggesting a genetic basis to male modulation of courtship behavior based on feedback from unrelated, outbred, and genetically identical females. We assessed the genetic basis of variation of the transition with the greatest heritability—from copulation to no engagement with the female—and identified variants in Serrate and Furin 1 as well as many other polymorphisms on the chromosome 3R associated with this transition. Our findings suggest that courtship is a highly dynamic behavior with both social and genetic inputs, and that males may play an important role in courtship initiation and duration.

[Figure][1] Lindsley and Sandler et al. is, arguably, the first Drosophila functional genomics article; it even has “genome” in its title. The authors generated flies duplicated or deleted for specific, mapped regions across the major autosomes by making and combining Y:autosome

Female aggression is enhanced after mating. Genetic manipulation and behavioural observation show that the receipt of sperm, and a seminal fluid protein, enhances female Drosophila aggression towards other females.

Sperm storage in the mated female reproductive tract (RT) is required for optimal fertility in numerous species with internal fertilization. In Drosophila melanogaster, sperm storage is dependent on female receipt of seminal fluid proteins (SFPs) during mating. The seminal fluid protein Acp36DE is necessary for the accumulation of sperm into storage. In the female RT, Acp36DE localizes to the anterior mating plug and also to a site in the common oviduct, potentially "corralling" sperm near the entry sites into the storage organs. Genetic studies showed that Acp36DE is also required for a series of conformational changes of the uterus that begin at the onset of mating and are hypothesized to move sperm towards the entry sites of the sperm storage organs. After Acp36DE is transferred to the female RT, the protein is cleaved by the astacin-metalloprotease Semp1. However, the effect of this cleavage on Acp36DE's function in sperm accumulation into storage is unknown. We used mass spectrometry to identify the single cleavage site in Acp36DE. We then mutated this site and tested the effects on sperm storage. Mutations of Acp36DE's cleavage site that slowed or prevented cleavage of the protein slowed the accumulation of sperm into storage, although they did not affect uterine conformational changes in mated females. Moreover, the N-terminal cleavage product of Acp36DE was sufficient to mediate sperm accumulation in storage, and it did so faster than versions of Acp36DE that could not be cleaved or were only cleaved slowly. These results suggest that cleavage of Acp36E may increase the number of bioactive molecules within the female RT, a mechanism similar to that hypothesized for Semp1's other substrate, the seminal fluid protein ovulin.

Females and males of sexually reproducing animals must cooperate at the molecular and cellular level for fertilization to succeed, even though some aspects of reproductive molecular biology appear to involve antagonistic interactions. We previously reported the existence of a proteolytic cascade in Drosophila melanogaster seminal fluid that is initiated in the male and ends in the female. This proteolytic cascade, which processes at least two seminal fluid proteins (Sfps), is a useful model for understanding the regulation of Sfp activities, including proteolysis cascades in mammals. Here, we investigated the activation mechanism of the downstream protease in the cascade, the astacin-family metalloprotease Seminal metalloprotease-1 (Semp1, CG11864), focusing on the relative contribution of the male and female to its activation. We identified a naturally occurring semp1 null mutation within the Drosophila Genetic Reference Panel. By expressing mutant forms of Semp1 in males homozygous for the null mutation, we discovered that cleavage is required for the complete activation of Semp1, and we defined at least two sites that are essential for this activational cleavage. These amino acid residues suggest a two-step mechanism for Semp1 activation, involving the action of at least two male-derived proteases. Although the cascade’s substrates potentially influence both fertility and sperm competition within the mated female, the role of female factors in the activation or activity of Semp1 is unknown. We show here that Semp1 can undergo its activational cleavage in male ejaculates, without female contributions, but that cleavage of Semp1’s substrates does not proceed to completion in ejaculates, indicating an essential role for female factors in Semp1’s full activity. In addition, we find that expression of Semp1 in virgin females demonstrates that females can activate this protease on their own, resulting in activity that is complete but substantially delayed.

Gene duplication is an important mechanism for the evolution of new reproductive proteins. However, in most cases, each resulting paralog continues to function within the same sex. To investigate the possibility that seminal fluid proteins arise through duplicates of female reproductive genes that become “co-opted” by males, we screened female reproductive genes in Drosophila melanogaster for cases of duplication in which one of the resulting paralogs produces a protein in males that is transferred to females during mating. We identified a set of three tandemly duplicated genes that encode secreted serine-type endopeptidase homologs, two of which are expressed primarily in the female reproductive tract (RT), whereas the third is expressed specifically in the male RT and encodes a seminal fluid protein. Evolutionary and gene expression analyses across Drosophila species suggest that this family arose from a single-copy gene that was female-specific; after duplication, one paralog evolved male-specific expression. Functional tests of knockdowns of each gene in D. melanogaster show that one female-expressed gene is essential for full fecundity, and both female-expressed genes contribute singly or in combination to a female’s propensity to remate. In contrast, knockdown of the male-expressed paralog had no significant effect on female fecundity or remating. These data are consistent with a model in which members of this gene family exert effects on females by acting on a common, female-expressed target. After duplication and male co-option of one paralog, the evolution of the interacting proteins could have resulted in differential strengths or effects of each paralog.

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.

Drosophila seminal fluid proteins elicit physiological and behavioral changes in the female after mating. For example, the seminal protein sex peptide (SP) causes females to lay more eggs, reduce receptivity to re-mating, consume more food and produce more concentrated excreta upon mating. It has been reported that SP indirectly increases food consumption as a result of its stimulation of egg production, but its role in producing more concentrated excreta in the mated female was reported to be independent of egg production. Additionally, it has been shown that SP's effect on food consumption persists for several days after mating, while it is unknown whether this is true for its effect on excretion. SP can have both transient and long-term effects on mated females; the latter occur because of the peptide's binding to, and slow release from, sperm in the female. Here we used timed measures of excretion by female flies that had mated to males mutant in SP or in its regulators, to test the duration of SP's effect on excretion. We found that SP's effect on excretion persists for at least ~1 week after mating, and that this persistence requires that SP bind to and be released from sperm. Although these binding/release requirements of SP are similar to those for increased egg production (and consequent increased food intake) following mating, we find that the long-term change in excretion phenotype is only partially dependent on the presence of eggs in the female. Our data indicate that a change in intestinal transit is part of the long-term post-mating response elicited by the gradual release of sperm-bound SP in the female after mating, even though it is not fully dependent on other long-term responses elicited by SP.

Proteins in the seminal fluid of animals with internal fertilization effect numerous responses in mated females that impact both male and female fertility. Among these proteins is the highly represented class of proteolysis regulators (proteases and their inhibitors). Though proteolysis regulators have now been identified in the seminal fluid of all animals in which proteomic studies of the seminal fluid have been conducted (as well as several other species in which they have not), a unified understanding of the importance of proteolysis to male fertilization success and other reproductive processes has not yet been achieved. In this review, we provide an overview of the identification of proteolysis regulators in the seminal fluid of humans and Drosophila melanogaster, the two species with the most comprehensively known seminal fluid proteomes. We also highlight reports demonstrating the functional significance of specific proteolysis regulators in reproductive and post-mating processes. Finally, we make broad suggestions for the direction of future research into the roles of both active seminal fluid proteolysis regulators and their inactive homologs, another significant class of seminal fluid proteins. We hope that this review aids researchers in pursuing a coordinated study of the functional significance of proteolysis regulators in semen.

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.

The Aedes aegypti mosquito is a significant public health threat, as it is the main vector of dengue and chikungunya viruses. Disease control efforts could be enhanced through reproductive manipulation of these vectors. Previous work has revealed a relationship between male seminal fluid proteins transferred to females during mating and female post-mating physiology and behavior. To better understand this interplay, we used short-read RNA sequencing to identify gene expression changes in the lower reproductive tract of females in response to mating. We characterized mRNA expression in virgin and mated females at 0, 6 and 24 hours post-mating (hpm) and identified 364 differentially abundant transcripts between mating status groups. Surprisingly, 60 transcripts were more abundant at 0hpm compared to virgin females, suggesting transfer from males. Twenty of these encode known Ae. aegypti seminal fluid proteins. Transfer and detection of male accessory gland-derived mRNA in females at 0hpm was confirmed by measurement of eGFP mRNA in females mated to eGFP-expressing males. In addition, 150 transcripts were up-regulated at 6hpm and 24hpm, while 130 transcripts were down-regulated at 6hpm and 24hpm. Gene Ontology (GO) enrichment analysis revealed that proteases, a protein class broadly known to play important roles in reproduction, were among the most enriched protein classes. RNAs associated with immune system and antimicrobial function were also up-regulated at 24hpm. Collectively, our results suggest that copulation initiates broad transcriptome changes across the mosquito female reproductive tract, “priming” her for important subsequent processes of blood feeding, egg development and immune defense. Our transcriptome analysis provides a vital foundation for future studies of the consequences of mating on female biology and will aid studies seeking to identify specific gene families, molecules and pathways that support key reproductive processes in the female mosquito., Author Summary Female post-mating behavior has important consequences for mosquito populations and their ability to transmit diseases. Male Aedes aegypti seminal fluid substances transferred during mating cause many important changes to female behavior and physiology, including blood feeding behavior, egg development, and oviposition. In an effort to understand how males induce these responses in Ae. aegypti females, we characterized the transcriptome changes that occur in the female reproductive tract at different time points after mating. We found several RNAs that are apparently transferred by the male, and 280 genes whose mRNA abundance in the female is affected by mating. The nature of the predicted products of many of these genes suggests roles in priming the reproductive tract for egg development, protecting the female against bacterial infections or processing the blood meal. This identification of mating-responsive genes provides information potentially useful for developing tools aimed at preventing disease transmission by manipulating female mosquitoes’ post-mating responses.

Successful reproduction requires interactions between males and females at many levels: the organisms, their cells (the gametes), and their molecules. Among the latter, secreted products of male and female reproductive glands are especially important. These molecules are particularly well understood in Drosophila melanogaster, because of this insect’s excellent molecular genetic tools. Here, we discuss the biology of Drosophila reproductive glands, including their development, structure, and secreted products. These glands include important secretory centers, tissues that play roles in gamete maintenance and perhaps in modification, and organs that mediate dynamic transfer of gametes and molecules, and gamete support and/or discharge. Components of seminal fluid produced by male reproductive glands enter the female during mating. There, they interact with female proteins, neurons, and pathways to convert the female from a “poised” pre-mated state to an active post-mating state. This mated state is characterized by high levels of egg production, by sperm storage, and by post-mating behaviors related to re-mating, activity, and feeding. Female reproductive gland secretions include additional molecules important for sperm survival or egg transit. The interplay and coordination between male- and female-derived molecules is an area of intense study. Its conclusions are relevant to understanding reproduction in insects and, more broadly, in all animals, and as well as to questions about chemical communication, hormone biology and evolution.

Male mosquitoes transfer seminal fluid proteins (hereafter 'SFPs') during mating. These proteins can have profound effects on female behavior in the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. SFPs are thought to be responsible for female refractoriness to mating in both species. However, only limited information is available about the duration of induced refractoriness or the quantity of SFPs required to be effective in Ae. albopictus. Here, we tested the duration of the effect of SFPs on female refractory behavior for both Aedes species. Additionally, we determined the lowest SFP dose required to induce female refractory behavior in Ae. aegypti. Virgin females were injected intra-thoracically with doses ranging from 0.25 to 0.008 equivalents of one male's SFP amount. Our results demonstrate high sensitivity of female Ae. aegypti and Ae. albopictus to SFPs of their own species, with the majority of females becoming refractory at doses ≥ 0.031 male-equivalents after injection into the hemocoel. This effect was long-lasting in both species; none of the injected females were inseminated when presented with males of their own species 30 to 34 days post-injection, whereas most saline-injected control females mated at this time point. These results will aid future work to characterize individual SFPs involved in post-mating refractoriness in these two species. Moreover, they show that as is the situation in the mosquito Anopheles gambiae, and unlike Drosophila melanogaster, sperm are not required for the maintenance of a sexual refractoriness response in Ae. aegypti and Ae. albopictus.

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 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.

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.

Dimerization is an important feature of the function of some proteins, including prohormones. For proteins whose amino acid sequences evolve rapidly, it is unclear how such structural characteristics are retained biochemically. Here we address this question by focusing on ovulin, a prohormone that induces ovulation in Drosophila melanogaster females after mating. Ovulin is known to dimerize, and is one of the most rapidly evolving proteins encoded by the Drosophila genome. We show that residues within a previously hypothesized conserved dimerization domain (a coiled-coil) and a newly identified conserved dimerization domain (YxxxY) within ovulin are necessary for the formation of ovulin dimers. Moreover, dimerization is conserved in ovulin proteins from non-melanogaster species of Drosophila despite up to 80% sequence divergence. We show that heterospecific ovulin dimers can be formed in interspecies hybrid animals and in two-hybrid assays between ovulin proteins that are 15% diverged, indicating conservation of tertiary structure amidst a background of rapid sequence evolution. Our results suggest that because ovulin's self-interaction requires only small conserved domains, the rest of the molecule can be relatively tolerant to mutations. Consistent with this view, in comparisons of 8510 proteins across 6 species of Drosophila we find that rates of amino acid divergence are higher for proteins with coiled-coil protein-interaction domains than for non-coiled-coil proteins.