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The state of Florida is the third most populous state in the United States of America, with six (6) of its metropolitan areas dubbed as the fastest growing in the entire country. A mosquito bite may mean the transmission of a virus or disease which might be fatal. Hence, there is a need for the state to control mosquitoes through the various Departments of Mosquito Control in each of its sixty-seven (67) counties. Six locally acquired mosquito-borne viruses which affect humans and animals in the state of Florida were considered. This thesis used statistical methods to examine data for rainfall, population estimate, as well as, the data on six (6) arboviruses, over the course of thirteen (13) years, namely 2002 to 2014. The first hypothesis that was tested, was that greater precipitation increased the likelihood of a greater number of arbovirus cases. It was important to also examine the relationship that this growing human population had with mosquito-borne diseases, and so the second hypothesis that was tested, was that, an increase in the human population would increase the likelihood of a greater number of arbovirus cases. Subsequently, an analysis was done for eleven (11) of Florida*s 67 counties with the greatest cumulative occurrence of human and animal arbovirus cases combined. Of the eleven counties, seven exhibited a weak associated between the size of the human population and the spread of animal and human arbovirus cases; three exhibited a somewhat moderate association; and one – Osceola County – had a strong negative association. This indicated that, as the size of the human population increased in Osceola County, the combined number of human and animal arbovirus cases decreased, which refuted the second hypothesis of this thesis. A linear regression model for the data for Osceola County was derived and that model was used to simulate what will occur in future years with the use of population projection data. In each simulated year, the number of combined human and arbovirus cases was negative. This prediction meant that, as the projected population increased from year to year, then the number of cases should be zero in each year. The reliability of these predictions are questionable, since Osceola County does not exist in a vacuum and it cannot be isolated from the surrounding counties which may be experiencing an outbreak of arboviruses.

MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases. / Ph. D.

Many insects rely on olfaction as their primary method of interaction with their environment. One of the best examples of this is the olfactory driven host-seeking behavior displayed by female mosquitoes. Although mosquitoes are capable of extracting blood from a variety of hosts many mosquito species show marked preferences for particular host species. Mosquitoes displaying preference for humans above bovines are more likely to be disease vectors. Therefore understanding the molecular basis of this preference is important for public health. These differences may be the result of genetic variations in olfactory signaling components such as mosquito odorant receptors. This hypothesis is supported by several lines of evidence including the highly divergent and lineage-specific nature of this receptor family. Likely these differences are subtle and will be identified in highly focused studies. Even closely related sibling species of mosquitoes can display large behavioral differences. In our current study I have studied several aspects of both Anopheles and Aedes genus odorant receptors with emphasis on comparing receptors in species that are part of the Anopheles genus. The first goal of this project was to study the insect odorant receptor family for potential sites of heterodimer formation. Numerous studies have shown that insect odorant receptors are involved in detection of odorants. More recent studies have demonstrated that odorant receptors are also involved in protein trafficking and in forming cation channels. Both of these activities involve heterodimer formation between odorant receptors that bind odorants and those that are part of the Or83b subfamily. There is little informaiton on how heterodimers are formed and where within the protein heterodimer sites exist. The C-terminal region has been implicated as sites for such heterodimer formation. A hidden markov model based program, Multiple em for motif elicitation (MEME), was used to uncover three motifs in the C-terminus of the odorant receptor peptides from Anopheles gambiae, D. melanogaster, and Apis mellifera. Previous studies have shown that insect odorant receptors are highly divergent between different insect lineages suggesting conservation of these motifs is functionally important. I propose that these motifs are involved in receptor-receptor protein interactions, contributing to the heterodimer formation between Or83b subfamily members and other odorant receptors.The next goal was to identify odorant receptors in closely related mosquito species and compare and contrast them. This was accomplished by using public sequence data of An. gambiae and BAC library screening to identify orthologous gene clusters in An. stephensi and An. quadriannulatus. Although I have identified many different odorant receptor genes the chapter in this dissertation discusses my work with the Or2 gene cluster. Multi-species comparison of these orthologous regions in An. gambiae, An. quadriannulatus, and An. stephensi revealed highly conserved gene structure among the OR genes and the discovery of the An. stephensi Or10x gene (AsOr10x), which is present only in An. stephensi. AsOr10x showed a different expression pattern than AsOr2 and AsOr10, the other members of this gene subfamily in An. stephensi. Therefore AsOr10x might be adapting or has adapted a new function. Analysis of the phylogeny and physical location of all known members of the Or2/Or10 gene subfamily in Anopheles, Aedes, and Culex mosquitoes suggest that a few events of gene duplication and loss resulted in the current gene distribution. The final focus of this project was to develop a method to study the function of mosquito odorant receptors. There is currently no in vivo system to study mosquito odorant receptors, and experimental systems pioneered in D. melanogaster are not transferable to mosquitoes. I decided to employ a reverse genetics strategy involving the silencing of three Aedes aegypti odorant and gustatory receptors of known or suspected function. These gustatory receptors are members of a small subfamily that encode olfactory and not taste receptors. As a preliminary step the expression profiles of these three genes and an additional gustatory receptor were determined using non-quantitative and quantitative RT-PCR. We found that the putative CO₂-detecting gustatory receptors are expressed in Ae. aegypti larvae, and hence these larvae may respond to CO₂, an observation that has not been reported previously. The purpose of silencing these receptors is to generate a loss-of-function behavior phenotype that will allow for inference of receptor function. Recombinant Sindbis viruses were used to knockdown mRNA levels of these receptors. GFP-expressing recombinant Sindbis viruses were shown to infect chemosensory tissue. Additional viruses containing fragments of receptor genes were found capable of lowering odorant and gustatory receptor mRNA levels. Infected mosquitoes displayed varying levels of gene knockdown with one virus generating supression of mRNA levels to 15.0% of normal. These mRNA levels may not be low enough to generate an unambiguous phenotype. Future experimentation is focused on developing more effective recombinant viruses and identifying characteristics of viruses more effective in receptor gene knockdown. A safe and effective behavior assay setup is needed to test the behavioral responses of these infected mosquitoes. In this study I outline a preliminary behavior assay that is being developed and optimized. When established it will provide a powerful tool in the study of both basic mosquito behavior and phenotype screening of recombinant Sindbis virus-infected mosquitoes. / Ph. D.

Mycobacterium ulcerans is an acid-fast bacillus that is the causative agent of Buruli ulcer, a necrotizing skin disease. The transmission route for M. ulcerans is unknown, but many insects have been posited as part of the web, including Belostomatids, Naucorids, and Culicids. Aedes albopictus was selected for use in a set of experiments where the first-generation larvae were inoculated with M. ulcerans, and mosquitoes were reared throughout the third generation to interrogate presence and quantity of the bacteria. Using qPCR, second and third generations displayed positivity (22% and 5.6% respectively). 16S V4 sequencing was used to obtain microbiota for all life stages as well as environmental samples, and many relationships between generations, life stages, and treatments displayed statistical significance in alpha diversity, beta diversity, and relative abundance of microbiomes. This study opens multiple avenues of further investigation into the transmission web of Buruli ulcer.

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Vector-borne diseases (VBDs) cause significant harm to humans, plants, and animals worldwide. For instance, VBDs are very difficult to manage, as they are governed by complex interactions. VBD transmission depends on the pathogen itself, vector-host movement, and environmental conditions. Mosquito-borne diseases are a perfect example of how all these factors contribute to changes in VBD dynamics. Although vectors are highly sensitive to climate, modeling studies tend to ignore climate effects. Here, I am interested in the arthropod small vectors that are sensitive to climate factors such as temperature, precipitation, and drought. In particular, I am looking at the effect of temperature on vector traits for two VBDs, namely, dengue, caused by a virus that infects humans and bluetongue disease, caused by a virus that infects ruminants. First, I collect data on mosquito traits' response to temperature changes, this includes adult traits as well as juvenile traits. Next, I use these traits to model mosquito density, and then I incorporate the density into our mathematical models to investigate the effect it has on the basic reproductive ratio R0, a measure of how contagious the disease is. I use R0 to determine disease risk. For dengue, my results show that using mosquito life stage traits response to temperature improves our vector density approximation and disease risk estimates. For bluetongue, I use midge traits response to temperature to show that the suitable temperature for bluetongue risk is between 21.5 °C and 30.7 °C. These results can inform future control and prevention strategies. / Doctor of Philosophy / Infectious diseases are a type of illness that occurs when microorganisms spread between hosts. Some infectious diseases are directly transmitted and some require indirect transmission such as vector-borne diseases (VBDs). Each VBD requires the presence of a vector for the disease to be transmitted. For example, dengue that puts 40% of the world population at risk, requires mosquitoes to transmit the disease between humans. My research aims to investigate how the main climate factor, temperature, influences the spread of VBDs. I develop mathematical and statistical models that explain the effect of temperature on vector traits of a mosquito-borne disease (dengue) and a midge-borne disease (bluetongue) and investigate modeling formulas to improve our estimates for dengue mosquito densities. My results can be used to inform future prevention and control strategies.

Mosquito reproduction is tightly regulated by the endocrine system. The sesquiterpenoid insect hormone, Juvenile hormone (JH), plays a crucial role in mosquito reproductive maturation. JH signaling pathways consist of a hierarchy of transcriptional regulators that modulate the transcriptional responses to this hormone. Genomic action of JH is mediated through the intracellular receptor Methoprene tolerant (Met) and Krüppel homolog 1 (Kr-h1), an intermediate effector that acts downstream of Met. Kr-h1 is an essential transcription factor for proper oogenesis and egg production in several insects. However, the regulatory mechanism of Kr-h1 in mosquito reproduction has not been well studied. In the current study, we performed global analyses of the Kr-h1 binding sites at multiple time points before and after a blood meal. In addition to known JH-regulated genes, we identified the binding of Kr-h1 to several genes that are controlled by the insect steroid hormone 20-hydroxyecdysone (20E). Kr-h1 seemed to have different roles in regulating the 20E-responsive genes before and after the blood-feeding. RNAi mediated Kr-h1 silencing demonstrated the activator role of Kr-h1 on several 20E-regulated genes in the previtellogenic stage, while Kr-h1 mostly repressed those genes after blood feeding. On the genes that were activated by Kr-h1 in the previtellogenic stage, the binding of Kr-h1 was associated with an increase of the histone marker H3K27ac. For the first time, we demonstrated that the regulatory action of Kr-h1 involves histone modification on the 20E-responsive genes. This study significantly extends our understanding of the regulatory mechanism of Kr-h1, and the cross-talk of JH and 20E in coordinating mosquito reproduction. JH analogs are commonly used as mosquito larvicides. Recent studies reported that the application of a JH analog, pyriproxyfen (PPF), on adult female mosquitoes substantially reduces their reproduction. A big knowledge gap was the poor understanding of the mechanism underlying this sterilizing effect of PPF. Here, with our customized laboratory setup that mimics the bed net intervention, we established a dose-dependent effect of PPF in compromising mosquito fecundity and fertility. We carefully assessed the effects of PPF exposure on mosquito physiology and follicular development. PPF induced excessive growth of primary follicles during the previtellogenic stage. However, the follicular development in the PPF-treated mosquitoes was severely impaired after blood feeding. The primary follicles were much smaller than their counterparts in the control groups and their development stopped at Christopher's stage III. Moreover, PPF triggered the atypical premature growth of secondary follicles at ~36 h PBM. In addition to the follicular developmental reprogramming, PPF also altered the levels of storage metabolites, enhancing the accumulation of glycogen and triglyceride (TAG) before a blood meal and speeding their depletion after blood-feeding. Consistent with the observed phenotypical changes and relevant metabolic genes, several 20E-responsive genes were significantly altered in their expression as a result of PPF exposure. Furthermore, RNAi experiments demonstrated that the JH receptor Met is required in the PPF-induced sterilization. In summary, we evaluated the sterilizing effects of PPF on mosquito reproduction, investigated the molecular action of PPF in regulating mosquito gene expression, and determined the signaling pathway involved in the PPF-induced sterilization of female mosquitoes. / Doctor of Philosophy / Among different insect-borne diseases, mosquito causes the highest disease burden with almost 700 million infections and over a million deaths every year. Aedes aegypti mosquitoes are the major vehicle to transmit several viral diseases including dengue, yellow fever, chikungunya, and Zika fever. They pose a global threat to public health and economic sectors. Different mosquito control strategies are used, and a very quick, powerful, and popular strategy is using chemical insecticides to decrease mosquito populations. However, insecticide resistance in mosquitoes and non-specific toxicity to other animals are great challenges associated with the commonly used insecticides. To resolve this problem, new insecticides are urgently needed. If we can broaden our understanding of mosquito reproductive biology, new targets will be identified and can be exploited to develop new insecticides. In our study, we investigated an insect-specific hormone, Juvenile hormone (JH), to understand its regulatory action in mosquito reproduction. Also, this study improved our knowledge of the molecular understanding of the insecticide (synthetic JH-like compound) in decreasing mosquito egg numbers and reducing the hatching rate. Overall, we gained a significant understanding of the hormonal regulation of mosquito reproduction. This knowledge can be used in the future to develop new insecticides with better efficiency to decrease the mosquito population and mosquito-borne disease burden.