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The Culicinae is the most extensive subfamily among the Culicidae family of mosquitoes. Two genera, Culex and Aedes, from this subfamily have world-wide distribution and are responsible for transmitting of several deadly diseases including Zika, West Nile fevers, chikungunya, dengue, and Rift Valley fevers. Developing high-quality genome assembly for mosquitoes, studying their population structure, and evolution can help to facilitate the development of new strategies for vector control. Studies on Aedes albopitcus as well as on species from the Culex pipiens complex, which are widely spread in the United States, provide excellent models on these topics. Ae. albopictus is one of the most dangerous invasive mosquito species in the world that transmits more than 20 arboviruses. This species has highly repetitive genome that is the largest among mosquito genomes sequenced so far. Thus, sequencing and assembling of such genome is extremally challenging. As a result, the lack of high-quality Ae. albopictus genome assembly has delayed the progress in understanding its biology. To produce a high-quality genome assembly, it was important to anchor genomic scaffolds to the cytogenetic map creating a physical map of the genome assembly. We first developed a new gene-based approach for the physical mapping of repeat-rich mosquito genomes. The approach utilized PCR amplification of the DNA probes based on complementary DNA (cDNA) that does not include repetitive DNA sequences. This method was then used for the development of a physical map for Ae. albopictus based on the in situ hybridization of fifty cDNA fragments or gene exons from twenty-four scaffolds to the mitotic chromosomes from imaginal discs. This study resulted in the construction of a first physical map of the Ae. albopictus genome as well as mapping viral integration and polyphenol oxidase genes. Moreover, comparing our present Ae. albopictus physical map to the current Ae. aegypti assembly indicated the presence of multiple chromosomal inversions between them. To better understand population structure and chromosome evolution in Culicinae mosquitoes, especially in the Culex pipiens complex, we studied genomic and chromosomal differentiation between two subspecies Cx. pipiens pipiens and Cx. pipiens molestus. For the species responsible for the spread of human diseases, understanding the population dynamics and processes of taxa diversification is important for an effective mosquito control . Two vectors of West Nile virus, Cx. p. pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The first goal of this study was to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations, but not in Cx. p. molestus populations. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. The second goal was to study differences at the chromosome level between these two organisms. We first measured whole chromosome and chromosome arm length differences between Cx. p. molestus and Cx. p. pipiens as a basic cytogenetic approach. In addition, we used the novel Hi-C approach to detect chromosomal rearrangements between them since Hi-C was successful in detecting a known inversion in Cx. quinquefasciatus. Cx. p. molestus and Cx. p. pipiens embryos were used to perform the Hi-C technique. Analysis of the Hi-C data showed the presence of two different inversions in Cx. p. pipiens and Cx. p. molestus heatmap, which could explain their different physiology and adaptation in nature. Developing modern genomic and cytogenetic tools is important to enhance the quality of genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics of mosquitoes; also it is the foundation for the development of novel genome-based approaches for vector control. / Doctor of Philosophy / Mosquitoes are medically important insects because they vector a range of diseases that infect humans. The subfamily Culicinae is responsible for transmitting such diseases as Zika, dengue, and West Nile fevers, which have triggered fatal infections and epidemics in multiple parts of the world. Since 2010-2016, studies have reported exceeding levels of insecticide resistance that slows the disease elimination process. Novel transgenic techniques have a tremendous potential for more efficiently minimizing mosquito-borne diseases and transmission. Availability of high-quality genome assemblies for mosquitoes may help to better understand their population structure and to develop effective and safe vector-control approaches that we urgently need. For the development of high-quality genome assemblies, we need to construct a physical genome map, that shows the physical locations of genes or other DNA sequences of interest along the chromosomes. For this reason, we developed a new gene-based approach for the physical mapping of the mosquito genomes. This method was then used for the development of a physical map for Ae. albopictus. This study resulted in the generation of the first physical map of the Ae. albopictus genome. To understand population structure in Culicinae mosquitoes, we used mosquitoes from the Culex pipiens complex. Species in this complex transmit different arthropod-borne viruses or arboviruses. Notable is the West Nile Virus, which has triggered fatal infections and epidemics in Eastern and Central Europe, North America and is also known in Asia, Australia, Africa, and the Caribbean. We specifically focused on two subspecies in this complex, Cx. pipiens pipiens and Cx. pipiens molestus that are morphologically identical, but are different physiologically and behaviorally. Although they are spread globally in temperate regions, their population structure and taxonomic status remains unclear. The first goal of this study was to better understand the level of genomic differentiation of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units that are currently undergoing ecological speciation. The second goal was to study differences at the chromosome level between them. Using the Hi-C approach we detected presence of two different inversions in Cx. p. pipiens and Cx. p. molestus, which could potentially explain their different physiology and adaptation.

Mosquitoes belonging to the genera Anopheles transmit malaria parasites, attributing the highest mortality of any vector-borne disease worldwide. Mosquitoes belonging to the genera Aedes transmit arboviruses including dengue, which has become the most important vector-borne virus due to a drastic surge in disease incidence. The scope of the studies in this dissertation is broad, with investigations bringing together elements of classical genetics, recent advances in sequencing and genome-editing technologies, and the use of modern forward genetics approaches. Chapter 2 of this dissertation explores the use of the Oxford Nanopore Sequencing Technology for the first time in mosquitoes. This new technology provides long reads that were used to piece together the AabS3 chromosomal assembly for Anopheles albimanus. The utility of this genomic resource is demonstrated by the discovery of novel telomeric repeats at the ends of the chromosomes that could have important implications in mosquito biology and control. Chapter 3 describes a forward genetics strategy called 'Marker-Assisted Mapping' (MAM) that enables high-resolution mapping of the causal gene locus of a mutant phenotype. The principle and effectiveness of MAM is first demonstrated by mapping a known transgene insertion. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. Genetic crosses between the re mutant and cardinal knocking out individuals generated using CRISPR/Cas9 confirmed that cardinal indeed is the causal gene for re mutation. Chapter 4 explores three innovative strategies for mosquito sex separation by exploiting several sex-linked marker lines. We show that by linking a transgenic marker to the male-determining locus (M locus), or by linking the male-determining Nix gene to a marker, males can be precisely separated from females. We also produce a two-marker transgenic line that allows for both non-transgenic male separation and for efficient line maintenance. Finally, we discuss further applications of the resources generated and future directions stemming from these findings. Altogether, the studies described in this dissertation contribute to the overall goal of understanding mosquito biology and of controlling mosquito-borne infectious diseases. / Doctor of Philosophy / Female mosquitoes bite and transmit deadly pathogens including the malaria parasite, and viruses such as dengue, Zika, and West Nile. Control programs that attempt to limit the spread of these deadly diseases rely on the control of mosquitoes themselves. These mosquito control methods have relied heavily on indoor and outdoor insecticidal spraying. However, the efficacy of these methods has been jeopardized by the increasing prevalence of insecticide resistance. Thus, it is necessary to implement other methods for effective mosquito control. Genetic control strategies such as the Sterile Insect Technique (SIT) and Wolbachia-based Incompatible Insect Technique (IIT) are excellent solutions to overcome the limitations of current control strategies. As female mosquitoes bite and transmit disease-causing pathogens, only males are released, which necessitate the separation of the non-biting males from females before release. The aim of this work was to use recent technological advancements to better understand the genome and basic genetics of vector mosquito species, and to identify possible approaches to improve current sex separation practices. To develop a deep understanding of mosquito biology and genetics, it is crucial that a high-quality and accurate genome assembly is available. However, many mosquito genome assemblies remain fragmented. To address this limitation, we used recent advances in sequencing technologies to produce a high-quality genome assembly for the New World malaria mosquito, Anopheles albimanus. These sequencing and assembly efforts led to the discovery of novel telomere sequences at the ends of chromosomes, which could have implications for mosquito control. Forward genetics, which identifies the gene(s) responsible for a given phenotype, has been hindered by the low recombination rate in the yellow and dengue fever mosquito, Aedes aegypti. We develop a Marker-Assisted Mapping (MAM) strategy to address this problem. We first demonstrate this method by mapping the known insertion of a transgene. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. MAM identification of the Cardinal gene was then verified by knocking out Cardinal, which represents the first successful gene mapping in Aedes aegypti using forward genetics. The MAM strategy has broad implications as it could enable the discovery of genes involved in important traits such as insecticide resistance. To improve sex separation methods, we took advantage of several sex-linked transgenic lines to develop three novel strategies. First, we demonstrate that screening for a genetic marker that is tightly linked to the male-determining locus (M locus) is an effective approach to reduce female contamination. Second, we demonstrate that instead of linking a marker to the M locus, we can link the male-determining factor, Nix, to a genetic marker. When a Nix transgene is located adjacent to the red-eye locus with extremely tight linkage, the red-eye phenotype becomes a faithful marker for separation of males and females. Finally, we developed a two-marker genetic sexing strain that produces non-transgenic males that could be used for release, and transgenic marked males and females for efficient line maintenance.

Blood-feeding mosquitoes, by transmitting parasites and viruses to their hosts, kill several hundred thousand people every year. Mosquito populations are currently developing raising levels of insecticide resistance, and there is a need for a better understanding of their behavior so that new control solutions can be imagined, and existing ones can be improved. There has been a vast number of studies examining the host seeking behavior of mosquitoes, however there is a lack of knowledge concerning how mosquitoes are evading the threats their hosts pose via their defensive behaviors. Female mosquitoes are indeed in this unique position where their fitness and reproduction depend on them being able to locate hosts as well as evade them. In order to do this, they rely on sensory cues that they must be able to continuously re-evaluate during host tracking to potentially decide to quickly escape at any point during these interactions. Host seeking is mediated by multiple sensory modalities such as vision, olfaction, and thermosensation. However, it is not clear whether mosquitoes may also be using some of these same cues to identify that their host is turning into a threat. Focusing solely on visual cues in the context of escape behavior, we used a looming stimulus to elicit escape responses from the Yellow Fever mosquito, Aedes aegypti. A virtual reality environment was adapted from previous work, to display the looming stimulus while the mosquito is in a variety of conditions (i.e., landed, in free or tethered flight). Results from these experiments allowed us to characterize the escape responses of mosquitoes, by determining the angles and distances to the stimuli that will most likely trigger an avoidance response. / Master of Science in Life Sciences / Mosquitoes are the deadliest animals in the world because of the several diseases they can transmit. Females are the only ones that bite, since they require a blood meal in order to produce offspring. The task of obtaining this blood meal from a mobile, and defensive host can be extremely dangerous. The females must find a host, approach it, land, feed, and flee without being killed so they can reproduce. At any point, the host might detect the mosquito and try to kill it, typically by swatting at it. For this reason, mosquitoes have evolved the ability to navigate in close proximity to the host, while assessing their level of defensiveness and avoiding these threats. Insects, in general, are well known to display escape behaviors in response to visual, predator-like, looming stimuli. However, in spite of great epidemiological importance, very little is known about the mechanisms that allow mosquitoes to evade their predators, as well as swatting from their hosts. Mosquitoes use visual, olfactory, and thermal cues to track their hosts but what kinds of sensory cues are being used to avoid threats? As a first step towards bridging this knowledge gap, we analyzed the behavioral responses of the Yellow Fever mosquito, Aedes aegypti, to looming visual stimuli. For this, we used a virtual-reality environment designed for mosquitoes, where we displayed looming squares to mosquitoes in a variety of conditions (for example: landed, in flight). Results from these experiments allowed us to characterize the escape responses of mosquitoes, by determining the angles and distances to the stimuli that will most likely trigger an avoidance response. Ultimately, better understanding mosquito vision in the context of their escape behavior, can help us improve the design of control tools, such as traps, to increase their efficiency.

Virginia's temperate climate is suitable for several mosquito species capable of transmitting pathogens to humans. In southwest Virginia, La Crosse encephalitis and West Nile fever are most prominent. The objective of this research, which uses the Health Belief Model (HBM) as a theoretical framework, is to assess knowledge of mosquito-borne disease in southwest Virginia, as well as perceptions and practices of mosquito prevention. Given that several cases of La Crosse encephalitis have been reported in Wise and Tazewell counties, they were selected as study sites to conduct surveys. Five demographic and socioeconomic variables (gender, age, income, education level and length of time one has lived in the county) were used as predictor variables in logistic regression analyses. Gender, age, and length of residence time in the county were found to be statistically significant predictors of specific health-related behaviors. Within the framework of the HBM, barriers to removing standing water around the home and wearing insect repellent were highlighted. Knowledge of mosquito-borne diseases within the area was generally low, with only one individual correctly identifying La Crosse encephalitis as a threat in the region. Higher numbers (6%) were aware of West Nile virus, while 4% reported malaria in the region, demonstrating a disconnect between actual and perceived risk. These results can enhance existing public health programs by increasing knowledge, addressing public uncertainty about insect repellent safety, and addressing ways to make recommended practices more effective with the knowledge of how different aspects are perceived by varying groups within the community. / Master of Science