Project description:Insects, unlike vertebrates, are generally believed to lack steroid hormones with functions predominantly associated with adult male biology. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to both control egg development in females and induce mating refractoriness and oviposition when sexually transferred by males. Here we show that these sex-specific functions are instead carried out by distinct steroids. We identify a male-specific oxidized form of 20E (3D20E) that upon sexual transfer switches off female mating receptivity, ensuring male paternity. Endogenous female 20E does not induce mating refractoriness, while it triggers oviposition in mated females when expression of a 20E-inhibiting kinase is repressed. 3D20E and 20E have different downstream targets, with 3D20E inducing expression of a tolerance factor that preserves female fitness during Plasmodium infection. The evolution of this male steroid has therefore not only shaped the mating biology of An. gambiae, but also impacted malaria transmission.
Project description:Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behaviour. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterised protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term post-mating responses including increased ovulation, elevated feeding and reduced receptivity to remating, primarily signalling through the SP receptor (SPR). We demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating, where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing non-functional SP mutant proteins that affect SP binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data reveal a novel role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signalling functions seen in D. melanogaster. In this experiment we assessed the effect of SP loss-of-function on the transferred seminal proteome.
Project description:Male Anophele gambiae mosquitoes transfer the steroid hormone 20-hydroxyecdysone (20E) to the female lower reproductive tract during mating. We have shown that this sexually transferred hormone is a key regulator of monandry and oviposition in An. gambiae females. In order to identify possible molecular pathways underlying the effects induced by sexually transferred 20E we investigated the transcriptional response in the atrium and spermatheca at 24 hours after 20E injection in the thorax of virgin females, which delivers to these reproductive tissues 20E levels comparable to those detected after mating. By comparing these results to those of microarrays assessing the post-mating transcriptional response in the female lower reproductive tract (LRT) at 24 hours post copulation, we were able to identify mating responsive genes likely regulated by male transferred 20E. For each experiment atrium and spermatheca were dissected from females 24 hours after 20E or ethanol (10%) injection and transcriptional profiles compared across 4 biological replicates using Agilent two-color microarrays
Project description:In insects, male accessory gland proteins (ACPs) are important reproductive proteins secreted by male accessory glands (MAGs) of the internal male reproductive system. During mating, ACPs were transferred along with sperms inside female bodies and have a significant impact on the physiology of female reproduction. Under sexual selection pressures, the ACPs exhibit remarkably rapid and divergent evolution and varies from species to species. The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a major insect pest of cruciferous vegetables worldwide. The reproductive physiology on post-mating state of this species is still largely unknown, which is important for management of this pest. In this study, the ACPs transferred into females during mating were identified by using a tandem mass tags quantitative proteomic analysis. The MAGs were compared before and after mating immediately. In total, we identified 123 putative secreted ACPs, including most important physiological: regulators of proteolysis, transporters and protein export machinery, signal transduction and immunity. Comparing P. xylostella with other four insect ACPs, trypsins were the only ACPs detected in all insect species. This was the first time to identify and analyze ACPs in P. xylostella. Our results have provided an important list of putative secreted ACPs, and have set the stage for further explore functions of these putative proteins in P. xylostella reproduction.
Project description:In insects, male accessory gland proteins (ACPs) are important reproductive proteins secreted by male accessory glands (MAGs) of the internal male reproductive system. During mating, ACPs were transferred along with sperms inside female bodies and have a significant impact on the physiology of female reproduction. Under sexual selection pressures, the ACPs exhibit remarkably rapid and divergent evolution and varies from species to species. The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a major insect pest of cruciferous vegetables worldwide. The reproductive physiology on post-mating state of this species is still largely unknown, which is important for management of this pest. In this study, the ACPs transferred into females during mating were identified by using a tandem mass tags quantitative proteomic analysis. The MAGs were compared before and after mating immediately. The proteomes of copulatory bursas (CB) in mated females shortly after mating were also analyzed by shotgun LC-MS/MS technique. In total, we identified 123 putative secreted ACPs, including most important physiological: regulators of proteolysis, transporters and protein export machinery, signal transduction and immunity. Comparing P. xylostella with other four insect ACPs, trypsins were the only ACPs detected in all insect species. We also identified some new insect ACPs, including protein with chitin binding Peritrophin-A domain, PMP-22/EMP/MP20/Claudin tight junction domain-containing protein, netrin-1, type II inositol 1,4,5-trisphosphate 5-phosphatase, two spaetzles, allatostatin-CC and cuticular protein. This was the first time to identify and analyze ACPs in P. xylostella. Our results have provided an important list of putative secreted ACPs, and have set the stage for further explore functions of these putative proteins in P. xylostella reproduction.
Project description:We custom-built a bioinformatics pipeline to search for 20E-modifying enzymes in the accessory glands of Anopheles gambiae males, searching for ecdysteroid kinases (EcK), ecdysone oxidases (EO), and ecdysteroid-phosphate phosphatases (EPP). To this end, we generated RNAseq datasets of different An. gambiae tissues dissected from virgin and mated females and males, and produced similar datasets for Anopheles albimanus, a South American species that does not synthetize and transfer ecdysteroids during mating. These analyses led to the identification of one candidate EPP and two potential EcKs (EcK1 and EcK2), which we demonstrated are involved in the activity of a male-specific oxidized ecdysteroid (3D20E). We further determined that 3D20E is specifically produced by the An. gambiae male accessory glands and is transferred to females during copulation, where it triggers a series of post-mating responses.
Project description:Globally invasive Aedes aegypti mosquitoes disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with the intracellular bacterium Wolbachia pipientis, a symbiont that naturally infects ~40-52% of insects but is normally absent from Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allowing infected individuals to rapidly invade native populations. Further, wMel Wolbachia-infected females display refractoriness to medically relevant arboviruses. Thus, wMel Wolbachia-infected Ae. aegypti are being released in several areas to replace native populations, thereby suppressing disease transmission by this species. Wolbachia is reported to have minimal effects on Ae. aegypti fertility, but its influence on other reproductive processes is unknown. Female insects undergo several post-mating physiological and behavioral changes required for optimal fertility. Post-mating responses (PMRs) in female insects are typically elicited by receipt of male seminal fluid proteins (SFPs) transferred with sperm during mating, but can be modified by other factors, such as adult age, nutritional status, and microbiome composition. To assess how Wolbachia infection influences Ae. aegypti female PMRs, we collected wMel Wolbachia-infected Ae. aegypti from the field in Medellín, Colombia and introduced the bacterium into our laboratory strain. We found that Wolbachia influences female fecundity, fertility, and re-mating incidence. Further, we observed that Wolbachia significantly extends longevity of virgin females. Changes in female PMRs are not due to defects in sperm transfer by infected males, or sperm storage by infected females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia infection has a moderate effect on SFP composition. However, we identified 125 Wolbachia proteins that are paternally transferred to females by infected males. Surprisingly, the CI factor proteins (Cifs), were not detected in the ejaculates of Wolbachia-infected males. Our findings indicate that Wolbachia infection of Ae. aegypti alters female post-mating responses, potentially influencing control programs that utilize Wolbachia-infected individuals.
Project description:Male-derived accessory gland proteins (Acps) that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in Acps may be driven by sperm competition and sexual conflict, processes which may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 hrs postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 hrs after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 hrs postmating, including proteins which appeared to undergo post-translational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization. Experiment Overall Design: Three-day-old mated and unmated females were dissected to remove the lower reproductive tract (upper uterus, sperm-storage organs, and accessory glands). Mated females were dissected either immediately following mating (0 hr) or at 3, 6, or 24 hrs following the termination of mating. Tracts of 12-40 females of like category were pooled and total RNA extracted via a TRIzol-based protocol. Processing and labeling of transcript was performed by the Molecular Biology Core Facility at the Medical College of Georgia. Arrays from mated females at the different timepoints were compared to unmated females.
Project description:Male-derived accessory gland proteins (Acps) that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in Acps may be driven by sperm competition and sexual conflict, processes which may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 hrs postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 hrs after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 hrs postmating, including proteins which appeared to undergo post-translational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization. Keywords: keywords: reproduction, reproductive tract, accessory gland proteins, sperm, timecourse
Project description:In Drosophila melanogaster, mating radically transforms female physiology and behavior. 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, since only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and the effects of male-induced harm (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 a second mating. Immune response genes dominate this male-induced harm 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 dimorphismand rapid evolution at these loci. Keywords: Female response to mating