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.

Cryo-Electron Microscopy Reveals That Sperm Modification Coincides with Female Fertility in the Mosquito Aedes aegypti.

Manipulating mosquito reproduction is a promising approach to reducing mosquito populations and the burden of diseases they carry. A thorough understanding of reproductive processes is necessary to develop such strategies, but little is known about how sperm are processed and prepared for fertilization within female mosquitoes. By employing cryo-electron microscopy for the first time to study sperm of the mosquito Aedes aegypti, we reveal that sperm shed their entire outer coat, the glycocalyx, within 24 hours of being stored in the female. Motility assays demonstrate that as their glycocalyx is shed in the female's sperm storage organs, sperm transition from a period of dormancy to rapid motility-a critical prerequisite for sperm to reach the egg. We also show that females gradually become fertile as sperm become motile, and that oviposition behavior increases sharply after females reach peak fertility. Together, these experiments demonstrate a striking coincidence of the timelines of several reproductive events in Ae. aegypti, suggesting a direct relationship between sperm modification and female reproductive capacity.

Proteins, Transcripts, and Genetic Architecture of Seminal Fluid and Sperm in the Mosquito Aedes aegypti.

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

A mosquito sperm's journey from male ejaculate to egg: Mechanisms, molecules, and methods for exploration.

The fate of mosquito sperm in the female reproductive tract has been addressed sporadically and incompletely, resulting in significant gaps in our understanding of sperm-female interactions that ultimately lead to fertilization. As with other Diptera, mosquito sperm have a complex journey to their ultimate destination, the egg. After copulation, sperm spend a short time at the site of insemination where they are hyperactivated and quickly congregate near the entrance of the spermathecal ducts. Within minutes, they travel up the narrow ducts to the spermathecae, likely through the combined efforts of female transport and sperm locomotion. The female nourishes sperm and maintains them in these permanent storage organs for her entire life. When she is ready, the female coordinates the release of sperm with ovulation, and the descending egg is fertilized. Although this process has been well studied via microscopy, many questions remain regarding the molecular processes that coordinate sperm motility, movement through the reproductive tract, maintenance, and usage. In this review, we describe the current understanding of a mosquito sperm's journey to the egg, highlighting gaps in our knowledge of mosquito reproductive biology. Where insufficient information is available in mosquitoes, we describe analogous processes in other organisms, such as Drosophila melanogaster, as a basis for comparison, and we suggest future areas of research that will illuminate how sperm successfully traverse the female reproductive tract. Such studies may yield molecular targets that could be manipulated to control populations of vector species. Mol. Reprod. Dev. 83: 897-911, 2016 © 2016 Wiley Periodicals, Inc.

Polyandry Depends on Postmating Time Interval in the Dengue Vector Aedes aegypti.

Aedes aegypti is the primary vector of the dengue and chikungunya viruses. After mating, male seminal fluid molecules cause females to become unreceptive to a subsequent mating. This response is often assumed to be immediate and complete, but a growing body of evidence suggests that some females do mate more than once. It is unknown how quickly a female becomes unreceptive to a second mating. Furthermore, the degree to which she remains monandrous after laying several batches of eggs has not been rigorously tested. Therefore, we assessed the rates of polyandry in two sets of experiments using wild-type males and those with fluorescent sperm. The first experiment tested the likelihood of polyandry after postmating intervals of various durations. Most females became refractory to a second mating within 2 hours after mating, and rates of polyandry ranged from 24% immediately after mating to 3% at 20 hours after mating. The second experiment tested whether females were polyandrous after cycles of blood meals and oviposition. No re-insemination was found after one, three, or five such cycles. This study is the first to demonstrate that polyandrous behavior depends on the postmating interval. Our results will inform future applications that depend on an accurate knowledge of Ae. aegypti mating behavior, including models of gene flow, investigations of molecules that drive female mating behavior, and control strategies that deploy genetically modified mosquitoes into the field.

Mating-Induced Transcriptome Changes in the Reproductive Tract of Female Aedes aegypti.

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 0 hpm 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 0 hpm was confirmed by measurement of eGFP mRNA in females mated to eGFP-expressing males. In addition, 150 transcripts were up-regulated at 6 hpm and 24 hpm, while 130 transcripts were down-regulated at 6 hpm and 24 hpm. 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 24 hpm. 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.