The Timing of Reproductive Events in the Mosquito Aedes aegypti

Abstract

The mosquito Aedes aegypti is the primary vector of dengue, yellow fever, chikungunya, and Zika viruses. Despite decades of effort to curb transmission of these diseases, this mosquito’s continued status as the most proficient arbovirus vector highlights the need for new and improved vector control methods. Manipulating entire mosquito populations in the wild is a promising avenue by which mosquitoes may be controlled. Typically, such strategies aim either to suppress a wild population by limiting reproduction or to replace a population with mosquitoes with a desirable trait (such as refractoriness to disease). Regardless of the type of manipulation, these strategies require that altered individuals (usually reared in laboratories or factories) are deployed into the wild to compete with and mate with wild individuals. While mosquito reproduction has been studied for decades, many questions still remain to be answered that could help to improve mosquito release strategies. The purpose of this dissertation was to answer fundamental questions surrounding Ae. aegypti reproduction that will contribute to the development of new or enhanced mosquito control strategies. Specifically, it addresses the timeline with which several reproductive events occur and lays a foundation for future investigations to describe the cellular and molecular underpinnings of important reproductive events. First, I addressed the question of when Ae. aegypti females mate more than once. While consensus in the literature suggested that they do occasionally mate more than once, some studies suggest that re-mating occurs soon after a female’s first mating, and others claim that females only mate after they have undergone several gonotrophic cycles. Using males with fluorescently labeled sperm, I demonstrate that females are most likely to mate a second time within 2 h of their first mating, that almost no re-insemination occurs after 24 h post-mating (hpm), and that no females re-mate after they have begun laying eggs. Given this 24 hpm period in which behavior drastically shifts, I wondered if other aspects of reproductive physiology also follow similar timelines. Studies of other mosquitoes’ sperm suggested that sperm are initially equipped with a thick glycocalyx, but that this outermost layer is removed some time after mating. To understand whether this occurs in Ae. aegypti, when it occurs, and if it is typical of all sperm, I used cryo-electron microscopy to image sperm from the male prior to insemination and at different times post-insemination from the female. Within 24 hpm, sperm indeed shed a thick outer glycocalyx. Motility assays over the same 24 h period indicated that within 8 h, sperm had achieved their maximum motility. Finally, I tested whether oviposition is initiated and fertility is achieved during this period. Both reached their peak at ~16 hpm, with fertility gradually setting in beginning at 4 hpm and oviposition experiencing a sharp increase after 12 hpm. After investigating Ae. aegypti reproduction in the laboratory, I aimed to describe aspects of its reproductive biology in an epidemiologically relevant field setting. I intensively sampled Ae. aegypti in Medellín, Colombia, by aspirating resting adults from homes. By recording data on each female’s mating status, parity status, and whether she had taken a blood meal and developed eggs, I compared virgin and mated females’ behavior and physiology. Females frequently took blood meals prior to mating. However, blood fed virgins’ abdominal distension was significantly lower than that of mated females, suggesting that virgins may take smaller blood meals. Some virgins also produced eggs, and those that did produced a number of eggs similar to their mated counterparts. These observations provide the first comprehensive comparison of mated and virgin Ae. aegypti feeding behavior and reproductive physiology in a field setting. Knowing when critical reproductive events occur is a critical first step to understanding how they occur. To enable future investigations of the cellular and molecular mechanisms that govern reproductive events, I developed a comprehensive proteome for both Ae. aegypti sperm and seminal fluid. Building on previous work by Sirot et al. (2011) and using state-of-the-art mass spectrometry technology, I was able to identify over 870 putative sperm proteins and 280 putative seminal fluid proteins. These findings were largely corroborated and supported by transcriptomes that I generated from the testes and male accessory glands. The findings in this dissertation will support future investigations of crucial reproductive processes that may be manipulated for the purposes of vector control.