Sex peptide receptor is not required for refractoriness to remating or induction of egg laying in Aedes aegypti.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating-processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract.

Unique patterns of inheritance and selection on Y chromosomes have led to the evolution of specialized gene functions. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as those of wild-type and that mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract. Therefore, although mature sperm are produced by WDY mutant males, and are transferred to females, those sperm fail to enter the female sperm storage organs. We report genotype-dependent and regional differences in sperm motility that appear to break the correlation between sperm tail beating and propulsion. Furthermore, we identify a significant change in hydrophobicity at a residue at a putative calcium-binding site in WDY orthologs at the split between the melanogaster and obscura species groups, when WDY first became Y-linked. This suggests that a major functional change in WDY coincided with its appearance on the Y chromosome. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

Reproductive biology: A genetic recipe for parthenogenesis.

New work reveals differences in oogenic gene expression between parthenogenetic and sexually reproducing Drosophila mercatorum strains. Recapitulating those changes in D. melanogaster oocytes induced parthenogenesis in this normally sexually reproducing species, providing molecular insight into how these reproductive modes arise.

Transcriptional programs are activated and microRNAs are repressed within minutes after mating in the Drosophila melanogaster female reproductive tract.

BACKGROUND: The female reproductive tract is exposed directly to the male's ejaculate, making it a hotspot for mating-induced responses. In Drosophila melanogaster, changes in the reproductive tract are essential to optimize fertility. Many changes occur within minutes after mating, but such early timepoints are absent from published RNA-seq studies. We measured transcript abundances using RNA-seq and microRNA-seq of reproductive tracts of unmated and mated females collected at 10-15 min post-mating. We further investigated whether early transcriptome changes in the female reproductive tract are influenced by inhibiting BMPs in secondary cells, a condition that depletes exosomes from the male's ejaculate. RESULTS: We identified 327 differentially expressed genes. These were mostly upregulated post-mating and have roles in tissue morphogenesis, wound healing, and metabolism. Differentially abundant microRNAs were mostly downregulated post-mating. We identified 130 predicted targets of these microRNAs among the differentially expressed genes. We saw no detectable effect of BMP inhibition in secondary cells on transcript levels in the female reproductive tract. CONCLUSIONS: Our results indicate that mating induces early changes in the female reproductive tract primarily through upregulation of target genes, rather than repression. The upregulation of certain target genes might be mediated by the mating-induced downregulation of microRNAs. Male-derived exosomes and other BMP-dependent products were not uniquely essential for this process. Differentially expressed genes and microRNAs provide candidates that can be further examined for their participation in the earliest alterations of the reproductive tract microenvironment.

The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract.

Unique patterns of inheritance and selection on Y chromosomes lead to the evolution of specialized gene functions. Yet characterizing the function of genes on Y chromosomes is notoriously difficult. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. WDY mutants produce mature sperm with beating tails that can be transferred to females but fail to enter the female sperm storage organs. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as wild-type sperm's and that the mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely cause the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggest that sperm tail beating and propulsion do not always correlate. Furthermore, we find significant differences in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs of WDY that are Y-linked and those that are autosomal. Given that WDY appears to be evolving under positive selection, our results suggest that WDY's functional evolution coincides with its transition from autosomal to Y-linked in Drosophila melanogaster and its most closely related species. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. In contrast to WDY, PRY mutants do swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

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

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Previous work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility in the ejaculate, though functional evidence in any species is lacking. Here, we used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the male's ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. We found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Time series transcriptome analysis implicates the circadian clock in the Drosophila melanogaster female's response to sex peptide.

Sex peptide (SP), a seminal fluid protein of Drosophila melanogaster males, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses including increased egg laying, activity, and food intake and a decreased remating rate. While it is known that SP achieves this, at least in part, by altering neuronal signaling in females, the genetic architecture and temporal dynamics of the female's response to SP remain elusive. We used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 h after mating to learn about the genetic architecture, at the gene and exon levels, of the female's response to SP. We find that SP is not essential to trigger early aspects of a virgin-to-mated transcriptional switch, which includes changes in a metabolic gene regulatory network. However, SP is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that SP alters rhythmic gene expression in females and suggests that SP's disruption of the female's circadian rhythm might be key to its widespread effects.

Suspension of hostility: Positive interactions between spermatozoa and female reproductive tracts.

Interactions between spermatozoa and the female reproductive tract (FRT) are complex, in many cases poorly understood, and likely to contribute to the mechanistic basis of idiopathic infertility. As such, it is not surprising that the FRT was often viewed historically as a "hostile" environment for spermatozoa. The FRT has also been touted as a selective environment to ensure that only the highest quality spermatozoa progress to the oocyte for the opportunity to participate in fertilization. Recent advances, however, are giving rise to a far more nuanced view in which supportive spermatozoa × FRT interactions-in both directions-contribute to beneficial, even essential, effects on fertility. In this perspective article, we discuss several examples of positive spermatozoa × FRT interactions. We believe that these examples, arising in part from studies of taxonomically diverse nonmammalian systems, are useful to efforts to study mammalian spermatozoa × FRT interactions and their relevance to fertility and the advancement of assisted reproductive technologies.

Female factors modulate Sex Peptide's association with sperm in Drosophila melanogaster.

BACKGROUND: Male-derived seminal fluid proteins (SFPs) that enter female fruitflies during mating induce a myriad of physiological and behavioral changes, optimizing fertility of the mating pair. Some post-mating changes in female Drosophila melanogaster persist for ~10-14 days. Their long-term persistence is because the seminal protein that induces these particular changes, the Sex Peptide (SP), is retained long term in females by binding to sperm, with gradual release of its active domain from sperm. Several other "long-term response SFPs" (LTR-SFPs) "prime" the binding of SP to sperm. Whether female factors play a role in this process is unknown, though it is important to study both sexes for a comprehensive physiological understanding of SFP/sperm interactions and for consideration in models of sexual conflict. RESULTS: We report here that sperm in male ejaculates bind SP more weakly than sperm that have entered females. Moreover, we show that the amount of SP, and other SFPs, bound to sperm increases with time and transit of individual seminal proteins within the female reproductive tract (FRT). Thus, female contributions are needed for maximal and appropriate binding of SP, and other SFPs, to sperm. Towards understanding the source of female molecular contributions, we ablated spermathecal secretory cells (SSCs) and/or parovaria (female accessory glands), which contribute secretory proteins to the FRT. We found no dramatic change in the initial levels of SP bound to sperm stored in mated females with ablated or defective SSCs and/or parovaria, indicating that female molecules that facilitate the binding of SP to sperm are not uniquely derived from SSCs and parovaria. However, we observed higher levels of SP (and sperm) retention long term in females whose SSCs and parovaria had been ablated, indicating secretions from these female tissues are necessary for the gradual release of Sex Peptide's active region from stored sperm. CONCLUSION: This study reveals that the SP-sperm binding pathway is not entirely male-derived and that female contributions are needed to regulate the levels of SP associated with sperm stored in their storage sites.

On how to identify a seminal fluid protein: A commentary on Hurtado et al.

Seminal fluid proteins (Sfps) have striking effects on the behaviour and physiology of females in many insects. Some Drosophila melanogaster Sfps are not highly or exclusively expressed in the accessory glands, but derive from, or are additionally expressed in other male reproductive tissues. The full suite of Sfps includes transferred proteins from all male reproductive tissues, regardless of expression level or presence of a signal peptide.

Octopaminergic/tyraminergic Tdc2 neurons regulate biased sperm usage in female Drosophila melanogaster.

In polyandrous internally fertilizing species, a multiply-mated female can use stored sperm from different males in a biased manner to fertilize her eggs. The female's ability to assess sperm quality and compatibility is essential for her reproductive success, and represents an important aspect of postcopulatory sexual selection. In Drosophila melanogaster, previous studies demonstrated that the female nervous system plays an active role in influencing progeny paternity proportion, and suggested a role for octopaminergic/tyraminergic Tdc2 neurons in this process. Here, we report that inhibiting Tdc2 neuronal activity causes females to produce a higher-than-normal proportion of first-male progeny. This difference is not due to differences in sperm storage or release, but instead is attributable to the suppression of second-male sperm usage bias that normally occurs in control females. We further show that a subset of Tdc2 neurons innervating the female reproductive tract is largely responsible for the progeny proportion phenotype that is observed when Tdc2 neurons are inhibited globally. On the contrary, overactivation of Tdc2 neurons does not further affect sperm storage, release or progeny proportion. These results suggest that octopaminergic/tyraminergic signaling allows a multiply-mated female to bias sperm usage, and identify a new role for the female nervous system in postcopulatory sexual selection.

The Effects of Male Seminal Fluid Proteins on Gut/Gonad Interactions in Drosophila.

Mating initiates broad physiological changes encompassing multiple organ systems in females. Elucidating the complex inter- and intra-organ signaling events that coordinate these physiological changes is an important goal in the field of reproductive biology. Further characterization of these complex molecular and physiological interactions is key to understanding how females meet the energetic demands of offspring production. Many recent studies of the fruit fly, Drosophila melanogaster, have described the mechanisms of post-mating changes within the female reproductive tract and digestive system. Additionally, other studies have described post-mating signaling crosstalk between these systems. Interestingly, male seminal fluid proteins have been linked to post-mating responses within the female reproductive tract and gut, and to signaling events between the two organ systems. However, information about the hormonal and neuronal signaling pathways underlying the post-mating signaling events within and between the reproductive tract and digestive systems that are triggered by seminal fluid proteins has yet to be combined into a single view. In this article, we summarize and integrate these studies into a single "network schematic" of the known signaling events within and between the reproductive and digestive systems downstream of male seminal fluid proteins. This synthesis also draws attention to the incomplete parts of these pathways, so that outstanding questions may be addressed in future studies.

A single mating is sufficient to induce persistent reduction of immune defense in mated female Drosophila melanogaster.

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. We compared the immune defense of mated females when infected at 2, 4, 7, or 10 days after mating to that of unmated females and saw no recovery of immune capacity regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, we additionally tested whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance. We found that females mated either once or twice before infection survived at equal proportions, both with significantly lower probability than unmated females. We conclude that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, we observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform our understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.

A standardized nomenclature and atlas of the female terminalia of Drosophila melanogaster.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.

The life history of Drosophila sperm involves molecular continuity between male and female reproductive tracts.

SignificanceIn species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, we used semiquantitative proteomics and sex-specific isotopic labeling in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. Our data reveal a molecular "hand-off" from males to females, which we postulate to be an important component of sperm-female interactions.

Seminal fluid proteins induce transcriptome changes in the Aedes aegypti female lower reproductive tract.

BACKGROUND: Mating induces behavioral and physiological changes in the arbovirus vector Aedes aegypti, including stimulation of egg development and oviposition, increased survival, and reluctance to re-mate with subsequent males. Transferred seminal fluid proteins and peptides derived from the male accessory glands induce these changes, though the mechanism by which they do this is not known. RESULTS: To determine transcriptome changes induced by seminal proteins, we injected extract from male accessory glands and seminal vesicles (MAG extract) into females and examined female lower reproductive tract (LRT) transcriptomes 24 h later, relative to non-injected controls. MAG extract induced 87 transcript-level changes, 31 of which were also seen in a previous study of the LRT 24 h after a natural mating, including 15 genes with transcript-level changes similarly observed in the spermathecae of mated females. The differentially-regulated genes are involved in diverse molecular processes, including immunity, proteolysis, neuronal function, transcription control, or contain predicted small-molecule binding and transport domains. CONCLUSIONS: Our results reveal that seminal fluid proteins, specifically, can induce gene expression responses after mating and identify gene targets to further investigate for roles in post-mating responses and potential use in vector control.

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti.

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.

Identification and bioinformatic analysis of neprilysin and neprilysin-like metalloendopeptidases in Drosophila melanogaster.

The neprilysin (M13) family of metalloendopeptidases comprises highly conserved ectoenzymes that cleave and thereby inactivate many physiologically relevant peptides in the extracellular space. Impaired neprilysin activity is associated with numerous human diseases. Here, we present a comprehensive list and classification of M13 family members in Drosophila melanogaster. Seven Neprilysin (Nep) genes encode active peptidases, while 21 Neprilysin-like (Nepl) genes encode proteins predicted to be catalytically inactive. RNAseq data demonstrate that all 28 genes are expressed during development, often in a tissue-specific pattern. Most Nep proteins possess a transmembrane domain, whereas almost all Nepl proteins are predicted to be secreted.

Identification of a micropeptide and multiple secondary cell genes that modulate Drosophila male reproductive success.

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.