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Transfer and receipt of seminal fluid proteins crucially affect reproductive processes in animals. Evolution in these male ejaculatory proteins is explained with post-mating sexual selection, but we lack a good understanding of the evolution of female post-mating responses (PMRs) to these proteins. Some of these proteins are expected to mediate sexually antagonistic coevolution generating the expectation that females evolve resistance. One candidate in

Anopheles gambiae and An. coluzzii, the two most important malaria vectors in sub-Saharan Africa, are recently radiated sibling species that are reproductively isolated even in areas of sympatry. In females from these species, sexual transfer of male accessory gland products, including the steroid hormone 20-hydroxyecdysone (20E), induces vast behavioral, physiological, and transcriptional changes that profoundly shape their post-mating ecology, and that may have contributed to the insurgence of post-mating, prezygotic reproductive barriers. As these barriers can be detected by studying transcriptional changes induced by mating, we set out to analyze the post-mating response of An. gambiae and An. coluzzii females captured in natural mating swarms in Burkina Faso. While the molecular pathways shaping short- and long-term mating-induced changes are largely conserved in females from the two species, we unravel significant inter-specific differences that suggest divergent regulation of key reproductive processes such as egg development, processing of seminal secretion, and mating behavior, that may have played a role in reproductive isolation. Interestingly, a number of these changes occur in genes previously shown to be regulated by the sexual transfer of 20E and may be due to divergent utilization of this steroid hormone in the two species.

In Drosophila melanogaster, seminal fluid proteins influence several components of female physiology and behavior, including re-mating rates, ovulation and oviposition, and sperm use. It is well-known that female flies are not simply passive vessels and that female-mediated interactions with male products are important to female (and thus male) reproductive success. While the population genetics, molecular evolution and physiological effects of seminal fluid proteins have been examined, the genetics and evolution of the female side of these post-mating interactions is unexplored in spite of work showing that female genotype and female-by-male genotype interactions are important determinants of sperm competition outcomes. Here we use microarrays to identify candidate genes involved in the female side of post-mating sexual interactions. We report the results of a whole-genome oligonucleotide chip experiment that reveals 23 genes differentially expressed between virgin females exposed and unexposed to courting males, and 38 genes differentially expressed between virgin and recently mated females. Immune related genes are overrepresented among the mating-influenced candidates. We use quantitative reverse-transcriptase PCR to independently assess gene expression changes for roughly half of the mating-affected candidate genes.Key words: reproduction, gene expression, Drosophila immune related genes, serine proteases, accessory gland proteins.

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks., Author Summary Reproduction requires more than a sperm and an egg. In animals with internal fertilization, other proteins in the seminal fluid and the female are essential for full fertility. Although hundreds of such reproductive proteins are known, our ability to understand how they interact remains limited. In this study, we investigated whether shared patterns of protein sequence evolution were predictive of functional interactions by focusing on a small network of proteins that control fertility and female post-mating behavior in the fruit fly, Drosophila melanogaster. We first showed that the six proteins already known to act in this network display correlated patterns of evolution across the Drosophila phylogeny. We then screened hundreds of otherwise uncharacterized male and female reproductive proteins and identified those with patterns of evolution most similar to those of the known network proteins. We tested each of these candidate genes and found six new network members that are each required for long-term fertility. Using molecular genetics, we also observed that the steps in the network at which these new proteins act are consistent with their strongest evolutionary correlations. Our results suggest that patterns of coevolution may be broadly useful for predicting protein interactions in a variety of biological processes.

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.

Six groups of adult beagles were tested for the display of masculine mating responses to sexually receptive bitches. Treatment and constitution of the several groups were as follows: females whose mothers were injected with TP for 10 days during the second trimester, females implanted with a testosterone pellet 1–3 days postpartum, females whose mothers were injected with TP during pregnancy and who received a testosterone pellet 1–3 days postpartum, females ovariohysterectomized in adulthood but given no androgen during development, males castrated within 48 hr postpartum, and males castrated in adulthood. All groups received periodic injections of TP as adults before and during mating tests. Prenatal androgenic stimulation prevented development of an external vagina and induced development of a penis in all cases. Treatment starting at birth resulted in clitoral hypertrophy but the vagina was patent. Females untreated during development and ovariectomized as adults showed relatively little masculine behavior when injected with TP in adulthood. Females untreated before birth but given testosterone implants during the neonatal period were not appreciably “masculinized.” In contrast, females treated in utero but receiving no treatment in infancy displayed significantly more masculine sexual activity than either of the two preceding groups. The most frequent and complete masculine behavior shown by any of the females was exhibited by those which had been exposed to androgen both before and immediately after birth. Most of the males castrated in adulthood and injected with TP 15–19 months later displayed the full complement of coital responses characteristic of the normal male. Males castrated at birth and given TP as adults showed much more masculine sexual behavior than any group of females, but on most measures were noticeably inferior to male subjects castrated after attainment of sexual maturity.

Seminal fluid proteins (Sfps) alter female behaviour and physiology and can mediate sexual conflict. In Drosophila melanogaster , a single Sfp, the sex peptide (SP), triggers remarkable post-mating responses in females, including altered fecundity, feeding, immunity and sexual receptivity. These effects can favour the evolutionary interests of males while generating costs in females. We tested the hypothesis that SP is an upstream master-regulator able to induce diverse phenotypes through efficient induction of widespread transcriptional changes in females. We profiled mRNA responses to SP in adult female abdomen (Abd) and head+thorax (HT) tissues using microarrays at 3 and 6 h following mating. SP elicited a rich, subtle signature of temporally and spatially controlled mRNAs. There were significant alterations to genes linked to egg development, early embryogenesis, immunity, nutrient sensing, behaviour and, unexpectedly, phototransduction. There was substantially more variation in the direction of differential expression across time points in the HT versus Abd. The results support the idea that SP is an important regulator of gene expression in females. The expression of many genes in one sex can therefore be under the influence of a regulator expressed in the other. This could influence the extent of sexual conflict both within and between loci.

Alphitobius diaperinus is one of the most significant pests in the poultry industry. Identifying the role of self-produced chemical signals can help control it. Here, we exposed adults to the olfactory signals of other adults of similar and different genders (either males or females) and sexual experiences (i.e., virgin and experienced) to assess their long-range attractiveness and, at short-range, their mating behavior responses (i.e., touching, mounting, and copulation). In olfactometric experiments, our results indicate that adults are attracted to the olfactory signals of other male adults, independently of gender, or sexual condition, indicating the presence of generalized long-range attractive signals, in contrast to female signals, can be both factor-dependent. However, in mating experiments, virgin males developed more robust mating responses (i.e., they mount and copulate longer with females) compared to sexually experienced males, even though they both have similar precopulatory behavioral responses (i.e., time of antennal and leg touching). These results address the importance of short-range chemical signals in eliciting copulation. Furthermore, when virgins of both genders were tested, their mating responses were significantly longer than any other pair combination, indicating that sexual experience also affects mating behavior. Chemical analyses of adult extracts showed that sexual experience, but not gender, is linked to differences in chemical profiles of adults, primarily involved in short-range signaling. These findings provide new insights into the attractiveness and mating responses of A. diaperinus and the role of sexual experience in shaping the behavior and chemical profile of insects that mate multiple times during their lifetime.

Mating causes considerable alterations in female physiology and behaviour, and immune gene expression, partly due to proteins transferred from males to females during copulation. The magnitude of these phenotypic changes could be driven by the genotypes of males and females, as well as their interaction. To test this, we carried out a series of genotype-by-genotype (G × G) experiments using Drosophila melanogaster populations from two distant geographical locations. We expected lines to have diverged in male reproductive traits and females to differ in their responses to these traits. We examined female physiological and behavioural post-mating responses to male mating traits, that is behaviour and ejaculate composition, in the short to mid-term (48 hr) following mating. We then explored whether a sexually transferred molecule, sex peptide (SP), is the mechanism behind our observed female post-mating responses. Our results show that the genotypes of both sexes as well as the interaction between male and female genotypes affect mating and post-mating reproductive traits. Immune gene expression of three candidate genes increased in response to mating and was genotype-dependent but did not show a G × G signature. Males showed genotype-dependent SP expression in the 7 days following eclosion, but female genotypes showed no differential sensitivity to the receipt of SP. The two genotypes demonstrated clear divergence in physiological traits in short- to mid-term responses to mating, but the longer-term consequences of these initial dynamics remain to be uncovered.

Ageing is nearly ubiquitous and encompasses all biological functions. We here focus on age-dependent changes in male reproductive capacity across a broad range of animal taxa. While there has been a long-standing focus on mating ability and overall reproductive success, we here highlight the underlying mechanisms that explain loss in fertilisation capacity in ageing males. Fertilisation is mediated by not only the presence of sperm, but also the cocktail of seminal fluid proteins that ensure sperm survival, capacitation and interaction with female physiology. Sperm ageing has received much attention in studies of male reproductive senescence; however, post-mating processes include a number of interlocked steps that together cumulate in successful fertilisation. As such we consider male ability to elicit female post mating responses such as uterine conformational changes, sperm storage and ovulation and the components within the ejaculate that mediate these post-mating processes. For the latter seminal fluid proteins are key and hence we reflect on age-dependent changes in quality of the entire ejaculate and its consequences for male reproductive capacity. While first studies accrue and highlight that changes in the non-sperm fraction can explain substantial variation in senescent male reproductive success and male ability to induce post-mating responses necessary for fertilisation many open questions still remain that warrant further investigations. One being what the potential age-dependent changes in composition are or whether there is a general decline and how this interacts with sperm to affect fertilisation success. Further, the impact females might have to ameliorate these changes will be an area of interest.

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.

Sexually dimorphic phenotypes arise largely from sex-specific gene expression, which has mainly been characterized in sexually naïve adults. However, we expect sexual dimorphism in transcription to be dynamic and dependent on factors such as reproductive status. Mating induces many behavioral and physiological changes distinct to each sex and is therefore expected to activate regulatory changes in many sex-biased genes. Here, we first characterized sexual dimorphism in gene expression in Callosobruchus maculatus seed beetles. We then examined how females and males respond to mating and how it affects sex-biased expression, both in sex-limited (abdomen) and sex-shared (head and thorax) tissues. Mating responses were largely sex-specific and, as expected, females showed more genes responding compared with males (∼2,000 vs. ∼300 genes in the abdomen, ∼500 vs. ∼400 in the head and thorax, respectively). Of the sex-biased genes present in virgins, 16% (1,041 genes) in the abdomen and 17% (243 genes) in the head and thorax altered their relative expression between the sexes as a result of mating. Sex-bias status changed in 2% of the genes in the abdomen and 4% in the head and thorax following mating. Mating responses involved de-feminization of females and, to a lesser extent, de-masculinization of males relative to their virgin state: mating decreased rather than increased dimorphic expression of sex-biased genes. The fact that regulatory changes of both types of sex-biased genes occurred in both sexes suggests that male- and female-specific selection is not restricted to male- and female-biased genes, respectively, as is sometimes assumed.

Mating affects the reproductive behaviour of insect females: the egg-laying rate increases and courting males are rejected. These post-mating responses are induced mainly by seminal fluid. In Drosophila melanogaster, males transfer two peptides (sex-peptides, = Sps) that reduce receptivity and elicit increased egg laying in their mating partners. Similarities in the open reading frames of the genes suggest that they have arisen by gene duplication. In females, Sps bind to specific sites in the central and peripheral nervous system, and to the genital tract. The binding proteins of the nervous system and genital tract are membrane proteins, but they differ molecularly. The former protein is proposed to be a receptor located at the top of a signalling cascade leading to the two post-mating responses, whereas the latter is a carrier protein moving Sps from the genital tract into the haemolymph. Sps bind to sperm. Together with sperm they are responsible for the persistence of the two post-mating responses. But Sps are the molecular basis of the sperm effect; sperm is merely the carrier.

Several signal transduction pathways play important roles in the sexual life cycle of Chlamydomonas. Nitrogen deprivation, perhaps sensed as a drop in intracellular [NH4+], triggers a signal transduction pathway that results in altered gene expression and the induction of the gametogenic pathway. Blue light triggers a second signalling cascade which also culminates in gene induction and completion of gametogenesis. New screens have uncovered several mutants in these pathways, but so far we know little about the biochemical events that transduce the environmental signals of nitrogen deprivation and blue light into the changes in gene transcription that produce gametes. Cell-cell contact of mature, complementary gametes elicits a number of responses that prepare the cells for fusion. Contact is sensed by the agglutinin-mediated cross-linking of flagellar membrane proteins. An increase in [cAMP] couples protein cross-linking to the mating responses. In C. reinhardtii the cAMP signal appears to be generated by the sequential stimulation of as many as 3 distinct adenylyl cyclase activities. Although the molecular mechanisms of adenylyl cyclase activations are poorly understood, Ca2+ may play a role. Most of the mating responses appear to be triggered by a cAMP-dependent protein kinase, but here too, Ca2+ may play a role. Numerous mutants are facilitating studies of the signalling pathways that trigger the mating responses. Cell fusion triggers another series of events that culminate in the expression of zygote specific genes. The mature zygote is sensitive to a light signal which stimulates the expression of genes whose products are essential for germination. The signal transduction pathways that trigger zygospore formation and germination are ripe for investigation in this experimentally powerful system.

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females., Author Summary Proteases can destroy, activate, or otherwise modulate the function of other proteins. In seminal fluid, many proteins have to be activated or degraded after mating; proteolysis is an effective way to accomplish this because seminal fluid proteins act outside of the cell, where most other regulatory processes cannot be used. Despite the presence of proteases in the seminal fluid of many animals, nearly nothing is known about the kinds of processes they regulate. Here, we present evidence of a protease cascade in the seminal fluid of the fruit fly Drosophila melanogaster. This cascade involves two proteases that are activated during mating. Once in the female, the downstream protease acts on two other proteins that are important for ovulation and sperm storage. Interestingly, the protease at the top of the cascade, CG10586, is also required for other female post-mating responses, including egg laying and sperm usage, independent of the second protease. Thus, CG10586 might be a general regulatory switch used by the male to quickly activate many female responses after mating.

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.

In the Mediterranean fruit fly, Ceratitis capitata (Wiedemann)(Diptera: Tephritidae), mating has a strong impact on female biology, leading to a decrease in sexual receptivity and increased oviposition and fecundity. Previous studies suggest that sperm transfer may play a role in inducing these behavioural changes. Here we report the identification of a medfly innexin gene, Cc-inx5, whose expression is limited to the germ-line of both sexes. Through RNA interference of this gene, we generated males without testes and, consequently, sperm, but apparently retaining all the other reproductive organs intact. These sperm-less males were able to mate and, like their wild-type counterparts, to induce in their partners increased oviposition rates and refractoriness to remating. Interestingly, matings to sperm-less males results in oviposition rates higher than those induced by copulation with control males. In addition, the observed female post-mating behavioural changes were congruent with changes in transcript abundance of genes known to be regulated by mating in this species. Our results suggest that sperm transfer is not necessary to reduce female sexual receptivity and to increase oviposition and fecundity. These data pave the way to a better understanding of the role/s of seminal components in modulating female post-mating responses. In the long term, this knowledge will be the basis for the development of novel approaches for the manipulation of female fertility, and, consequently, innovative tools to be applied to medfly control strategies in the field.

The Drosophila sex-peptide (SP) has been identified as a seminal fluid component that induces post-mating responses (PMRs) in the inseminated females, such as inhibition of remating and stimulation of egg-laying. SP has been thought to play a central role in sexual conflict and sexually antagonistic co-evolution. Most of the sequenced Drosophila genomes contain SP orthologs, but their functions have been poorly characterized. Recently, we have investigated cross-species activity of D. melanogaster SP by means of injection into virgin females of other species. Among 11 species examined, SP response was observed in 6 species belonging to the D. melanogaster species group only. These species females express SP receptor (SPR) in their oviducts at relatively high levels, which was visualized by using a GFP-tagged SP. Furthermore, females of this species group responded to their own SP orthologs. However, females of the species outside the group did not respond to their own SP orthologs, even though all of them were potent inducers of SP-response in D. melanogaster. Our results suggested that the SP/SPR-mediated PMR was established in the lineage of the D. melanogaster species group.

The Drosophila melanogaster sex peptide receptor (DrmSPR), which is a G protein-coupled receptor (GPCR), is known as the specific receptor for sex peptide (SP). It is responsible for the reproductive behavior in the Drosophila model system; in particular, it is involved in the post-mating responses such as the increase in egg-laying ability and decrease in receptivity in females. In a previous study, we discovered a small molecule agonist of DrmSPR for the first time, which could not, however, activate Aedes aegypti SPR (AedesSPR). To investigate the binding mechanism of the small molecule agonist of DrmSPR, the ensemble structures of low-lying packing structures of DrmSPR and AedesSPR were assembled using the GEnSeMBLE (GPCR Ensemble of Structures in Membrane BiLayer Environment) method. The generated homology models exhibited the typical pattern of inter-helical interactions of the class A GPCRs. The docking experiments of the small molecule agonist suggest that Tyr(5.35) and Phe(2.67) residues may be involved in a hydrophobic interaction and that Ser(3.25) forms a hydrogen bond with the agonist. Additionally, we found that the docking results were consistent with the experimental data of the reference compounds with variable agonistic activities. Moreover, a potential distinction of the putative binding sites in two GPCR models of DrmSPR and AedesSPR, which was determined in this study, can explain the selective action of the agonist for DrmSPR but not for AedesSPR.

Although the majority of flowering plants achieve pollination by exploiting the food‐seeking behavior of animals, some use alternative ploys that exploit their mate‐seeking behavior. Sexual deception is currently known only from the Orchidaceae and almost always involves pollination by male hymenoptera. An outstanding problem has been to identify the selective factors in plants that favor exploitation of mating versus feeding behaviors in pollinators. Here we show that the insectlike petal ornaments on inflorescences of the daisy Gorteria diffusa elicit copulation attempts from male bombyliid flies and that the intensity of the mating response varies across geographical floral morphotypes, suggesting a continuum in reliance on feeding through mating responses for pollination. Using pollen analogues applied to a morphotype with prominent insectlike petal ornaments, we demonstrate that mate‐seeking male flies are several‐fold more active and export significantly more pollen than females. These results suggest that selection for traits that exploit insect mating behavior can occur through the male component of plant fitness and conclusively demonstrates pollination by sexual deception in Gorteria, making this the first confirmed report of sexual deception outside of the Orchidaceae.

The roles of visual and/or olfactory stimuli in eliciting mating responses from male Glossina morsitans morsitans Westwood were examined, using a system for automatically recording the number and duration of mating strikes made towards decoys, under controlled conditions. The results confirm that there is no olfactory component of the female sex recognition pheromone sensed by the male antennae, and the attraction of males to females appears to be visual. The absence of male-male mating strikes was the result of the absence of female sex-pheromone, rather than the presence of a repellent mating deterrent in the male cuticle. Experiments with coloured, artificial, sex-pheromone-dosed, cotton decoys showed that colour had only weak effects on attractiveness and number of encounters with decoys, and that no colour caused significant enhancement of mating responses over those shown to decoy females.

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.

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.

As hubs for eukaryotic cell signaling, scaffold proteins are attractive targets for engineering and manipulating signaling circuits. We designed synthetic scaffolds with a repeated PDZ domain that interacted with engineered kinases of the mitogen-activated protein kinase (MAPK) cascade involved in yeast mating to investigate how modular interactions mediate kinase cascades. The synthetic scaffolds functioned as logic gates of signaling circuits. We replaced the endogenous yeast scaffold Ste5 with designer scaffolds with a variable numbers of a PDZ domain that bound kinases or phosphatases engineered with a PDZ-binding motif. Although association with the membrane was necessary for pathway activity, surprisingly, mating responses occurred when the circuit contained a scaffold with only two PDZ domains, which could only bind two of the three kinases simultaneously. Additionally, the three tiers of the MAPK pathway exhibited decreasing positional plasticity from the top [MAPK kinase kinase (MAPKKK)] to the bottom (MAPK) tier such that binding of a MAPKKK, but not a MAPK, from the osmoregulatory pathway or protein kinase C pathway to the synthetic scaffold activated a reporter of the mating response. We also showed that the output duration and intensity could be altered by recruiting phosphatases or varying the affinity of the recruited proteins for the scaffold and that a designer MAPK scaffold functioned in mammalian cells. Thus, this synthetic approach with designer scaffolds should enable the rational manipulation or engineering of signaling pathways and provide insight into the functional roles of scaffold proteins.

6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.

A male fruit fly influences the behavior and physiology of his mate via molecules that he transmits to her'in his semen. The mated female fly has an elevated rate of egg laying, a decreased receptivity to mating and a shorter life span; she also stores sperm from the mating. Molecular genetic analyses possible in this insect model system permit the dissection of seminal fluid components that cause these mating responses in the female. Studies with transgenic and mutant flies have shown that products of the male's accessory gland cause short-term changes in the female's behavior and physiology; persistence of these changes requires the storage of sperm. Further dissection of accessory gland function has defined several molecules that cause these effects. A “sex peptide” and a prohormone-like molecule (Accessory gland protein 26Aa) stimulate the female's egg-laying rate; the sex peptide also depresses her receptivity to mating. A large glycoprotein (Acp36DE) appears to function in “corralling” sperm for storage. Studies of accessory gland products and the regulation of the genes that encode them will be important in understanding insect reproduction, behavior, and speciation and ultimately in designing ways to control the impressive fertility of unwanted insects. These studies also provide excellent models to address basic questions in cell biology such as the control of genes in response to sex-specific, mating-regulated and cell type-specific cues and the function and targeting of peptide hormones.

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992) J. Cell Sci. 101, 303–313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, ‘LAGSE’ and, HIPYRESKLT, ‘HIPYR’) reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, approximately 110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the ‘97 kDa’ proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotide-dependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation., Author Summary Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised individuals, killing over 600,000 people per year worldwide. A major factor in the ability of this microbe to cause disease is an extensive polysaccharide capsule that surrounds the cell and interferes with the host immune response to infection. This capsule expands dramatically in certain growth conditions, including those found in the mammalian host. We grew cells in multiple conditions and assessed gene expression and capsule size. This allowed us to identify a ‘transcriptional signature’ of genes whose expression correlates with capsule size; we speculated that a subset of these genes acts in capsule regulation. To test this hypothesis, we characterized one previously unstudied gene in this signature and found it to be a novel regulator of capsule expansion, fungal virulence, and mating. This gene encodes cryptococcal Ada2, a well-conserved protein that regulates genes involved in stress response and development. We used phenotypic analysis, RNA sequencing, and chromatin-immunoprecipitation sequencing (ChIP-Seq) to situate Ada2 in the complex network of genes that regulate capsule and other cryptococcal virulence factors. This approach, which yielded insights into the regulation of a critical fungal virulence factor, is applicable to similar questions in other pathogens.

Background Genes involved in post-mating processes of multiple mating organisms are known to evolve rapidly due to coevolution driven by sexual conflict among male-female interacting proteins. In the malaria mosquito Anopheles gambiae - a monandrous species in which sexual conflict is expected to be absent or minimal - recent data strongly suggest that proteolytic enzymes specifically expressed in the female lower reproductive tissues are involved in the processing of male products transferred to females during mating. In order to better understand the role of selective forces underlying the evolution of proteins involved in post-mating responses, we analysed a cluster of genes encoding for three serine proteases that are down-regulated after mating, two of which specifically expressed in the atrium and one in the spermatheca of A. gambiae females. Results The analysis of polymorphisms and divergence of these female-expressed proteases in closely related species of the A. gambiae complex revealed a high level of replacement polymorphisms consistent with relaxed evolutionary constraints of duplicated genes, allowing to rapidly fix novel replacements to perform new or more specific functions. Adaptive evolution was detected in several codons of the 3 genes and hints of episodic selection were also found. In addition, the structural modelling of these proteases highlighted some important differences in their substrate specificity, and provided evidence that a number of sites evolving under selective pressures lie relatively close to the catalytic triad and/or on the edge of the specificity pocket, known to be involved in substrate recognition or binding. The observed patterns suggest that these proteases may interact with factors transferred by males during mating (e.g. substrates, inhibitors or pathogens) and that they may have differently evolved in independent A. gambiae lineages. Conclusions Our results - also examined in light of constraints in the application of selection-inference methods to the closely related species of the A. gambiae complex - reveal an unexpectedly intricate evolutionary scenario. Further experimental analyses are needed to investigate the biological functions of these genes in order to better interpret their molecular evolution and to assess whether they represent possible targets for limiting the fertility of Anopheles mosquitoes in malaria vector control strategies.

Background During copulation, the major Afro-tropical malaria vector Anopheles gambiae s.s. transfers male accessory gland (MAG) proteins to females as a solid mass (i.e. the "mating plug"). These proteins are postulated to function as important modulators of female post-mating responses. To understand the role of selective forces underlying the evolution of these proteins in the A. gambiae complex, we carried out an evolutionary analysis of gene sequence and expression divergence on a pair of paralog genes called AgAcp34A-1 and AgAcp34A-2. These encode MAG-specific proteins which, based on homology with Drosophila, have been hypothesized to play a role in sperm viability and function. Results Genetic analysis of 6 species of the A. gambiae complex revealed the existence of a third paralog (68-78% of identity), that we named AgAcp34A-3. FISH assays showed that this gene maps in the same division (34A) of chromosome-3R as the other two paralogs. In particular, immuno-fluorescence assays targeting the C-terminals of AgAcp34A-2 and AgAcp34A-3 revealed that these two proteins are localized in the posterior part of the MAG and concentrated at the apical portion of the mating plug. When transferred to females, this part of the plug lies in proximity to the duct connecting the spermatheca to the uterus, suggesting a potential role for these proteins in regulating sperm motility. AgAcp34A-3 is more polymorphic than the other two paralogs, possibly because of relaxation of purifying selection. Since both unequal crossing-over and gene conversion likely homogenized the members of this gene family, the interpretation of the evolutionary patterns is not straightforward. Although several haplotypes of the three paralogs are shared by most A. gambiae s.l. species, some fixed species-specific replacements (mainly placed in the N- and C-terminal portions of the secreted peptides) were also observed, suggesting some lineage-specific adaptation. Conclusions Progress in understanding the signaling cascade in the A. gambiae reproductive pathway will elucidate the interaction of this MAG-specific protein family with their female counterparts. This knowledge will allow a better evaluation of the relative importance of genes involved in the reproductive isolation and fertility of A. gambiae species and could help the interpretation of the observed evolutionary patterns.

Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd2+. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40 degrees C, first described by Zhang et al. (1991), is insensitive to Cd2+ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40 degrees C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type flagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of flagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different flagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell-body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.

The Drosophila seminal fluid protein (SFP) sex peptide (SP) elicits numerous post-mating responses, including increased egg laying and decreased sexual receptivity, in the mated female. Unlike other SFPs, which are detectable in mated females for only a few hours post mating, SP is maintained—and its effects are sustained—for several days. The persistence of SP in the mated female's reproductive tract is thought to be a consequence of its binding to, and gradual release from, sperm in storage, which maintains SP's ability to act within the female reproductive tract. Recent studies have shown that several other SFPs, acting in a network, are needed for SP's localization to sperm and are necessary for the efficient release of sperm from storage. This result suggested an additional new role for SP modulating the release of sperm from storage. We tested for this possibility by examining sperm storage parameters in mated females that did not receive SP. We found that while sperm accumulation into storage was unaffected, sperm depletion from storage sites was significantly decreased (or impaired) in the absence of SP. Mates of males expressing a modified SP that is unable to be released from sperm showed a similar phenotype, indicating that release of sperm-bound SP is a necessary component of normal sperm depletion. Additionally, SP null males were more successful in a sperm competitive environment when they were first to mate, which is likely a consequence of higher retention of their sperm due to defective sperm release. Our findings illustrate a direct role for SP in the release of sperm from storage.

Ejaculate chemicals transferred from males to females during mating cause significant changes in female behaviour and physiology, but the causes of phenotypic variation in these responses is little understood. We tested here the effect of adult female nutrition on the response of female Drosophila melanogaster to a specific ejaculate component, the sex peptide (SP), which is of interest because of its effects on female egg laying, sexual receptivity, feeding rate, immune responses and potential role in mediating sexual conflict. We exposed adult females to five different diets and kept them continuously with males that did or did not transfer SP. Diet altered the presence, magnitude and sign of the effects of SP on different phenotypic traits (egg laying, receptivity and lifespan) and different traits responded in different ways. This showed that the set of responses to mating can be uncoupled and can vary independently in different environments. Importantly, diet also significantly affected whether exposure to SP transferring males was beneficial or costly to females, with beneficial effects occurring more often than expected. Hence, the food environment can also shape significantly the strength and direction of selection on mating responses.

In Drosophila melanogaster, mating radically transforms female physiology and behaviour. Post-mating responses include an increase in the oviposition rate, a reduction in female receptivity and an activation of the immune system. The fitness consequences of mating are similarly dramatic – females must mate once in order to produce fertile eggs, but additional matings have a clear negative effect. Previously, microarrays have been used to examine gene expression of females differing in their reproductive status with the aim of identifying genes influenced by mating. However, as only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and male-induced effects (possibly under sexually antagonistic selection) cannot be disentangled. We partitioned these fundamentally different effects by instead examining the expression profiles of virgin, single mated and double mated females. We found substantial effects relating to reproduction and further effects that are only attributable to mating itself. Immune response genes dominate this male-induced effect indicating that the cost of mating may be due partly to this system’s activation. We propose that both sexually antagonistic and natural selection have been important in the evolution of the innate immunity genes, thereby contributing to the sexual dimorphism and rapid evolution at these loci.