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Leopard frogs (Rana pipiens) rely on vision to recognize behaviorally meaningful aspects of their environment. The optic tectum has been shown to mediate the frog's ability to recognize and respond to moving prey and looming objects. Nonetheless, atectal frogs are still able to appropriately respond to non-moving aspects of their environment. There appears to be independent visual systems operating in the frog: one system for recognizing moving objects; and another system for recognizing stationary objects. Little is known about the neural mechanisms mediating the recognition of stationary objects in frogs. Our laboratory showed that a retino-recipient area in the anterior lateral thalamus--the NB/CG zone--is involved in processing visual information concerning stationary aspects of the environment. This thesis aims to characterize the frog's responses to a range of stationary stimuli, and to elucidate the thalamic visual system that mediates those responses. I tested leopard frogs' responses to different stationary stimuli and found they respond in stereotypical ways. I discovered that leopard frogs are attracted to dark, stationary, opaque objects; and tested the extent of this attraction under different conditions. I found that frogs' preference to move toward a dark area versus a light source depends on the intensity of the light source relative to the intensity of ambient light. Unilateral lesions applied to the NB/CG zone of the anterior lateral thalamus resulted in temporary deficits in frogs' responses to stationary stimuli presented in the contralateral visual field. Deficits were observed in response to: dark objects, entrances to dark areas, light sources, and gaps between stationary barriers. However, responses to moving prey and looming stimuli were unaffected. Interestingly, these deficits tended to recover after about 6 days in most cases. Recovery time ranged from 2 - 28 days. The NB/CG zone is anatomically and functionally connected to a structure in the posterior thalamus called the "PMDT." The PMDT has no other connections in the brain. Thus, I have discovered a "satellite" of the NB/CG zone. Preliminary evidence suggests that the PMDT is another component of the visual system mediating stationary object recognition in the frog. / Biology

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.

Understanding chromosome structure and genome organization of Culicine mosquitoes can potentially contribute to the development of novel approaches to vector control. However, because of highly repetitive nature of the Aedes and Culex genomes, the structure of their polytene chromosomes is damaged by ectopic contacts that make the analysis difficult. Mitotic chromosomes from imaginal discs of 4th instar larvae of Aedes aegypti were tested as a source for the physical genome mapping for this mosquito. Chromosomes in imaginal discs are 10 times more abundant than chromosomes in nervous ganglia, and they do not accumulate chromosomal mutation as cell line chromosomes do. Prometaphase chromosomes in imaginal discs of Ae. aegypti are 4-5 times longer than metaphase chromosomes and can provide higher resolution for physical mapping. Cold temperature (+16°C) was proven to increase the number of the chromosomes. Hypotonic solution treatment of live larvae was proven to elongate chromosomes and improve banding patterns. We differentially stained these mitotic chromosomes with Giemsa and YOYO-1 to revile the banding pattern. We applied fluorescent in situ hybridization (FISH) procedure developed for human chromosomes to Ae. aegypti chromosomes. A strain from Culex pipiens, Cx. quinquefasciatus and their hybrids from the natural population in Virginia was successfully colonized in the laboratory. This strain can be used as a reliable source for cytogenetic studies. / Master of Science in Life Sciences

Field studies were conducted in southwestern Virginia to determine the bionomics and ovipositional preferences of Culex restuans Theobald and Culex pipiens Linnaeus using ovitraps and gravid traps. Both species have been implicated as enzootic and epizootic vectors of West Nile virus (WNV) and these studies provide information on the relative abundance of gravid mosquitoes. Ovitraps were used in the summers of 2002 and 2003 to measure the oviposition activity of Culex mosquitoes, mainly Cx. restuans and Cx. pipiens. In 2002, 1,345 egg rafts were collected from 5 traps set at different locations in the New River Valley (NRV). Cx. restuans constituted 93.2% of the catch; the remainder was Cx. pipiens (6.7%) and Cx. salinarius (<1%). In 2003, 4 ovitraps were placed at each of 6 locations in the NRV. Of 9,794 egg rafts collected, Cx. restuans comprised 92.8%, Cx. pipiens 6.5%, and Cx. salinarius <1%. Oviposition patterns were similar in both years. Cx. restuans oviposition was detected about mid-May, and raft numbers were highest in late June and the middle of July and then showed a steady decline throughout the remainder of each season. Cx. pipiens oviposition activity began later in the season and gradually increased, reaching its peak in August. Although the number of egg rafts of Cx. restuans decreased in August and September while the number of Cx. pipiens egg rafts increased, a crossover in the relative abundance of the two species never occurred. In 2003, the attractiveness of four infusions (cow manure, straw, grass, and rabbit chow) were compared in oviposition traps. For Cx. restuans, the manure infusions collected more egg rafts than the other three infusions for the first four weeks, with two of the weeks showing significance. During week 1, the manure infusions collected significantly more egg rafts than straw (P<0.01), grass (P<0.01), and rabbit (P<0.001). During week 2, manure collected more than grass (P<0.05) and rabbit (P<0.01). The straw and grass infusions yielded the most egg rafts after week 2, and only three weeks showed any significance. When traps began to collect Cx. pipiens, the majority were collected in the straw and grass infusions. During week 6, the hay infusions had significantly more egg rafts when compared to manure (P<0.001) and rabbit infusions (P<0.001) and the grass infusions had significantly more when compared to manure (P<0.05) and rabbit (P<0.01). Week 9 also showed significance when the hay infusions collected significantly more egg rafts than manure and rabbit (P<0.01 and P<0.01, respectively). The attractiveness of the cow manure and straw infusions were also compared in gravid traps. Because it is difficult to accurately discriminate between Cx. pipiens and Cx. restuans that have been collected as adults in gravid traps, these collections were combined into Culex. More Culex mosquitoes were collected in the manure infusions for the first two weeks (P<0.05). No significant differences were found between the numbers of mosquitoes captured in the traps baited with the different infusions after the second week. The shift observed in oviposition preference for both types of traps may have been due to cooler temperatures in the early part of the season. The straw infusions were aged outdoors for 3 days prior to use, and a sufficient incubation temperature to generate the bacteria producing the attractant chemicals may not have been attained. In addition, the manure lagoon had been drawn down and the consistency of the remaining manure became much drier by this time. No chemicals were ever added to the lagoon, but the draw down may have affected the attractiveness of the manure. / Master of Science

Avian malaria is a devastating disease that has decimated numerous bird species. This study sought to identify the vectors of avian malaria at four central Virginia Prothonotary warbler breeding sites. Twenty one thousand mosquitoes were collected and Culex salinarius, Cx. erraticus, and Cx. pipiens/restuans were found to be the dominant species at these sites. Geographic factors, such as crop land and forest type, were determined to be potential indicators for species abundance variation between sites. Of the mosquitoes collected, ninety one (0.4%) were identified as blood fed. The blood fed mosquitoes were found to have fed on avian, mammalian, amphibian, and reptilian hosts and a 12.1% Plasmodium infection rate. Of the non-blood fed mosquito pools tested, Deep Bottom had the highest rate of infection (10.5%). Of the species tested, Cx. salinarius, Cx. erraticus, and Cx. pipiens/restuans were determined to be the most probable vectors of avian malaria the four sites.

Despite a thorough understanding of the proximate mechanisms that drive genetic diversity, we are still very poor at predicting the genetic diversity of natural populations. Understanding patterns of genetic diversity is important for many reasons, including predicting species' adaptation to climate change and predicting the spread of invasive species, but it is particularly important for species that are declining. This dissertation attempts to explain patterns in genetic diversity at multiple spatial scales across the range of the Northern Leopard Frog, Rana pipiens, which is declining across large portions of its range. Genetic diversity is often lower in edge populations than in central populations. Genetic diversity may be reduced in edge populations per se, or populations that occur at the edge of the species' range may have low diversity because they have recently expanded into new habitat and thus show signs of founder effects. In Chapter 2, we tested several alternative hypotheses to explain genetic diversity across the species' range, and to explain why some edge populations may not show reduced genetic diversity. We found that genetic diversity was reduced in edge populations relative to central populations, but was not reduced in populations in previously glaciated areas relative to previously unglaciated areas; therefore position at range edge had a stronger effect in reducing diversity than recent colonization of new habitat. We found that genetic diversity declined linearly towards the range edge in one of two transects from range center to range edge. We concluded that genetic diversity in this species is generally reduced by position at the range edge, but that this effect may differ among edges. In Chapter 3, we tested the hypothesis that eastern and western populations were genetically distinct. We found two distinct clades that introgress in some markers but are distinct and defined by narrow boundaries in the eastern Great Lakes region in others. We concluded that genetic diversity in the Mississippi River region was elevated by the introgression of descendants from two Pleistocene refugia. In Chapter 4, we analyzed genetic diversity within populations throughout Arizona to assess potential source populations for reintroductions. We also analyzed mitochondrial DNA to determine whether any populations contained genetic material not native to the region. Populations in one area had high genetic diversity and high gene flow among populations, but also contained evidence of introduction of eastern frogs. We conclude that supplementing genetic diversity in other populations with translocations from this area is not recommended.

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Dirofilaria immitis Leidy (dog heartworm) is a life-threatening parasite transmitted by mosquitoes to domestic dogs. Endemic in the eastern United States, cases have become more prevalent over the last few decades. While prevalence in California is generally low, Lake and San Joaquin Counties have reported rates comparable to the East Coast at 3.73% and 0.71%(CAPC 2017), respectively. Aedes sierrensis is thought to be responsible for transmission in California, but in some cases, it exists in inadequate quantities and temporal ranges to explain parasite activity. Based on Huang et al. (2013) and Tran (2016), bloodfeeding patterns, and other vector criteria, Culex pipiens complex and Culiseta incidens were chosen to evaluate for vector competence. Female field-caught mosquitoes were reared, infected (2.5-5 mff/μl), and decapitated at 15, 18, or 21 days post infection (dpi). Cs. incidens was reluctant to feed using an artificial feeding system and will require additional trials. On the contrary, trials on Ae. sierrensis and Cx. pipiens complex were both completed successfully. Both species were determined to be competent vectors of D. immitis. Based on our findings, more than half of Ae. sierrensis females produced emerging L3s by 21 dpi, while Cx. pipiens complex never produced L3s in more than 5% of females. In conjunction with other factors such as the detection of D. immitis in wild mosquitoes, host-seeking preferences for domestic dogs, and appropriate temporal overlap, this suggests that both Ae. sierrensis and Cx. pipiens complex may play central roles in Lake or San Joaquin Counties, CA when abundant. Targeted control efforts are necessary to reduce the incidence of canine heartworm in these areas. While Lake and San Joaquin Counties, CA were the focus of this study, our results may be applicable to the western United States when these species are relevant.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.