The eggs of the dengue fever vector Aedes aegypti possess the ability to undergo an extended quiescence period hosting a fully developed first instar larvae within its chorion. As a result of this life history stage, pharate larvae can withstand months of dormancy inside the egg where they depend on stored reserves of maternal origin. This adaptation known as pharate first instar quiescence, allows A. aegypti to cope with fluctuations in water availability. An examination of this fundamental adaptation has shown that there are trade-offs associated with it. Aedes aegypti mosquitoes are frequently associated with urban habitats that may contain metal pollution. My research has demonstrated that the duration of this quiescence and the extent of nutritional depletion associated with it affects the physiology and survival of larvae that hatch in a suboptimal habitat; nutrient reserves decrease during pharate first instar quiescence and alter subsequent larval and adult fitness. The duration of quiescence compromises metal tolerance physiology and is coupled to a decrease in metallothionein mRNA levels. My findings also indicate that even low levels of environmentally relevant larval metal stress alter the parameters that determine vector capacity. My research has also demonstrated that extended pharate first instar quiescence can elicit a plastic response resulting in an adult phenotype distinct from adults reared from short quiescence eggs. Extended pharate first instar quiescence affects the performance and reproductive fitness of the adult female mosquito as well as the nutritional status of its progeny via maternal effects in an adaptive manner, i.e., anticipatory phenotypic plasticity results as a consequence of the duration of pharate first instar quiescence and alternative phenotypes may exist for this mosquito with quiescence serving as a cue possibly signaling the environmental conditions that follow a dry period. M findings may explain, in part, A. aegypti’s success as a vector and its geographic distribution and have implications for its vector capacity and control.

Master of Science / Department of Biology / Rollie Clem / Apoptosis, or programmed cell death, is a crucial conserved process among organisms for deleting damaged unwanted cells, as well as for development and viral defense, and plays an important role in multiple diseases. Too much apoptosis may lead to Alzheimer’s disease, and too little may result in cancer. Therefore, the ability to understand this process is essential for improved medical knowledge today. Apoptosis has been explored in a number of species and pathways seem relatively conserved among most, with unique aspects contained in each, but little is known about apoptosis in mosquitoes. Improved knowledge and growing interest concerning apoptosis in mosquitoes is necessary considering the vast health effects seen across the globe as a result of diseases transferred by the mosquito vector. The Dengue virus mosquito vector Aedes aegypti was the focus here. A new player named defense repressor 1 was discovered in Drosophila melanogaster (DmDnr1), shown to play a role in apoptosis, and the homolog discovered in A. aegypti (AeDnr1). Silencing Dmdnr1 resulted in cells sensitized to apoptosis but was not enough to induce spontaneous apoptosis. In contrast, silencing Aednr1 in the A. aegypti cell line, Aag2, led to spontaneously induced apoptosis. This showed the importance of AeDnr1 as a member of the apoptotic pathway in this species. Epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the initiator caspase Dronc and the effector caspase CASPS8, whereas apoptosis induced by silencing the inhibitor of apoptosis, Aeiap1, also requires Dronc but acts through the effector caspase CASPS7. Further epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the IAP antagonist Mx, but not IMP. This showed for the first time a gene regulating upstream of an IAP antagonist. Biochemical studies showed that AeDnr1 regulates active CASPS8 but not CASPS7, and interacts with Mx and CASPS8 but not AeDronc, CASPS7 nor AeIAP1. Studies also showed Mx competes effectively with CASPS8 but not CASPS7 for AeIAP1 binding, and IMP competes effectively with CASPS7 but not CASPS8 for AeIAP1 binding. An improved apoptosis pathway for the mosquito A. aegypti emerged involving a potential feedback loop with explanations for the upstream IAP antagonist preference as well as the downstream effector caspase preference resulting from apoptosis induced by Aednr1 silencing. Through the discussed research, multiple unique findings resulted. Studying the mosquito model will allow us to find certain gene relations that are more difficult to uncover in the Drosophila model. Because Dnr1 is found in most systems, this improved pathway may shed light not only on a potential role of Dnr1 in apoptosis in insects but higher organisms as well.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Each year, over 500 million people are infected with mosquito-borne diseases, including malaria, yellow fever and dengue fever, which cause several million deaths, and long-term disability and suffering. This dissertation focused on the mosquito Aedes aegypti, a vector for dengue virus and yellow fever virus. Since Sindbis virus (SINV) is an arthropod-borne virus (arbovirus) that is vectored by A. aegypti and is well characterized at the molecular level, the SINV - A. aegypti model was used to determine whether apoptosis plays a role in the control of vector competency. In Chapter 2, the effects of inducing or inhibiting apoptosis on SINV replication were tested in mosquito cells. It was observed that recombinant SINVs expressing pro-apoptotic genes caused extensive apoptosis in mosquito cells, with decreased virus production after the cells underwent apoptosis. Infection of mosquito cells with SINV expressing the caspase inhibitor P35 inhibited actinomycin D-induced apoptosis, but had no observable effects on virus replication. This study was the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells. Chapter 3 examined the effects of silencing apoptosis regulatory genes on SINV replication and dissemination in A. aegypti. Genes which either positively or negatively regulate apoptosis were silenced by RNA interference in mosquitoes, which were then infected with a recombinant SINV expressing green fluorescent protein (GFP). Reciprocal effects were observed on both the occurrence and intensity of expression of GFP in various tissues. These results suggest that systemic apoptosis positively influences SINV replication in A. aegypti. This was the first direct study to explore the role of apoptosis in determining mosquito vector competence for arboviruses. Finally, in Chapter 4, the mechanisms of apoptosis were explored in A. aegypti. Overexpression of IAP antagonists caused extensive cell death in mosquito cells, while silencing the expression of IAP antagonists attenuated apoptosis. The results showed that the IAP binding motif (IBM) of IAP antagonists was critical for their binding to AeIAP1. The IAP antagonists released initiator and effector caspases from AeIAP1 by competing for the binding sites and caused caspase-dependent apoptosis. These findings imply that the mechanisms of IAP antagonists regulating apoptosis are conserved between mosquitoes and the model insect where apoptosis has been mainly studied, Drosophila melanogaster.

Mosquitoes spread diseases that shorten and worsen the lives of many people, chiefly children in poor countries, around the world. Since Ronald Ross' discovery at the end of the nineteenth century that mosquitoes transmit malaria, field entomologists have collected a great deal of information about mosquito ecology. Despite this tremendous effort, there still remain significant gaps in our knowledge of mosquito ecology, in part, reflecting the significant variation in mosquito ecology across species and geographies. The main aim of this thesis is an attempt to synthesise the substantial information that field entomologists have collected on mosquito lifespan. In Chapters 2 and 3, I conduct meta-analyses of the two predominant approaches used to estimate mosquito lifespan: mark-release-recapture experiments and female mosquito dissection-based studies, respectively. These analyses produce estimates of mosquito lifespan by species and genus, and more broadly, allow for an appraisal of these two experimental approaches. In Chapter 4, I describe a recently developed approach, known as near-infrared spectroscopy, which enables estimation of the age of individual mosquitoes, and then perform an in silico analysis to explore the use of this technology for estimating the average lifespan of wild populations of mosquitoes. The emergence of mosquito resistance to the main insecticides used in vector control, along with the concerning recent discovery that the malaria parasites in Asia are becoming resistant to arteminisin - an important drug used to treat malaria - highlight the need for novel approaches to control disease transmission. Some recently-proposed approaches involve genetic modification of the mosquito vectors, for example, to render them incapable of acting as hosts for disease or to reduce their fecundity. In Chapter 5, I model the impact of a release of mosquitoes carrying a genetic construct known as a homing endonuclease, which has been constructed to bias the sex of mosquito offspring towards males, in computational environments that capture some facets of the real life landscapes where mosquito borne disease is rife. About a century ago, the famous Italian Malariologist Giovanni Grassi declared that malaria was a "giant with clay feet"; reflecting the optimism, in some academic circles at the time, that eradication of this terrible disease would soon occur. Unfortunately, a century of often unsuccessful attempts to control and eradicate malaria, and other mosquito borne diseases, would follow Grassi's statement, meaning that this fight is likely to continue throughout the twenty-first century. We now know much more about mosquitoes and mosquito borne disease than we did a generation ago, but there is still crucial information that we do not. In this thesis, I argue that in order to make significant inroads to disease eradication, further research on mosquito ecology is crucial. Only when we better understand our unwitting mosquito foe, can we design and implement effective disease control measures that are so desperately needed in those most desperate parts of the world.

Bed bugs (Hemiptera:Cimicidae) are a common, hematophagous ectoparasite of humans and other animals, and are experiencing an international resurgence. Cimicids have been implicated in the transmission many disease agents, including various Bartonella species, however disease transmission has not yet been confirmed. Bartonella spp. are transmitted by a variety of arthropods, including fleas, lice and sand flies, and it is speculated that bed bugs may also serve as a potential vector. In this study, we used an artificial membrane to feed two groups of adult Cimex lectularius rabbit blood, either infected or uninfected with Bartonella henselae. After two weeks, the presence of Bartonella henselae was assessed via PCR, and salivary glands from infected and uninfected bed bugs were dissected and processed for transmission electron microscopy. We were unable to visually identify Bartonella henselae in the images, and therefore unable to confirm the role of bed bugs in B. henselae transmission.