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

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.

Background No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating. Methodology and Principal Findings Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera. Conclusion and Significance This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti., Author Summary Dengue is a potentially lethal infection that impacts millions of humans annually. This disease is caused by viruses transmitted by infected female Aedes aegypti mosquitoes during blood feeding. No commercial vaccine or treatment is available for dengue infection. One way to break the disease transmission cycle is to develop new tools to reduce dengue vector populations. Seminal fluid proteins (Sfps) produced in the reproductive glands of male mosquitoes and transferred to females in the ejaculate during mating could be the target of such a tool. In related insects, Sfps modulate female reproduction and feeding patterns. Here we report 145 proteins that are transferred to females in the Ae. aegypti ejaculate. The proteins, which include Sfps and sperm proteins, fall into biochemical classes that suggest important potential roles in mated females. Of particular interest are proteins that could play roles in fertility and hormonal activity (including pathways involved in egg development and utilization of the blood meal). Our results lay important groundwork for new control strategies by identifying candidate proteins that may alter the reproductive biology or blood-feeding patterns of female Ae. aegypti and ultimately reduce the global burden of dengue.

The Asian tiger mosquito (Aedes albopictus) is an important vector for pathogens that affect human health, including the viruses that cause dengue and Chikungunya fevers. It is also one of the world's fastest-spreading invasive species. For these reasons, it is crucial to identify strategies for controlling the reproduction and spread of this mosquito. During mating, seminal fluid proteins (Sfps) are transferred from male mosquitoes to females, and these Sfps modulate female behavior and physiology in ways that influence reproduction. Despite the importance of Sfps on female reproductive behavior in mosquitoes and other insects, the identity of Sfps in Ae. albopictus has not previously been reported. We used transcriptomics and proteomics to identify 198 Sfps in Ae. albopictus. We discuss possible functions of these Sfps in relation to Ae. albopictus reproduction-related biology. We additionally compare the sequences of these Sfps with proteins (including reported Sfps) in several other species, including Ae. aegypti. While only 72 (36.4%) of Ae. albopictus Sfps have putative orthologs in Ae. aegypti, suggesting low conservation of the complement of Sfps in these species, we find no evidence for an elevated rate of evolution or positive selection in the Sfps that are shared between the two Aedes species, suggesting high sequence conservation of those shared Sfps. Our results provide a foundation for future studies to investigate the roles of individual Sfps on feeding and reproduction in this mosquito. Functional analysis of these Sfps could inform strategies for managing the rate of pathogen transmission by Ae. albopictus., Author Summary The highly invasive Asian tiger mosquito (Aedes albopictus) transmits several pathogens that cause disease in humans and other animals. Therefore, Ae. albopictus poses a large and growing threat to public health across the world. One step toward managing the reproduction and threat of this species is to determine factors that influence its reproductive biology. Seminal fluid proteins (Sfps) are transferred from male mosquitoes to females during mating, and receipt of Sfps changes female reproductive behavior and physiology. Here we report the identity of 198 Ae. albopictus Sfps. We discuss the potential roles and impacts of these Sfps on reproduction. In addition, we compare Ae. albopictus Sfps with proteins (including reported Sfps) from other species, including two other important mosquito vectors of pathogens that cause human diseases. Our results provide a foundation for future studies to investigate the roles of individual Sfps on Ae. albopictus reproduction.