Le Virus du Nil Occidental (VNO) et le Virus de la Fièvre de la Vallée du Rift (VFVR) sont deux arbovirus transmis par le moustique Culex pipiens comprenant deux biotypes: pipiens et molestus. Au cours de ce projet, nous avons évalué la circulation du VNO au Liban dans des populations de moustiques, des humains, des chevaux et des poulets. Nous avons aussi évalué la compétence vectorielle des populations locales de Cx. pipiens à transmettre le VNO et le VFVR.Des moustiques ont été récoltés et testés pour la présence d’un gène spécifique du VNO. En plus, des sérums humains, de chevaux et de poulets ont été analysés pour rechercher des anticorps spécifiques par ELISA puis confirmés par neutralisation. En outre, des spécimens de Cx. pipiens ont été infectés avec la lignée 1 du VNO ou la souche de VFVR Clone 13. Ensuite, les taux d’infection, de dissémination et de transmission ont été déterminés à différents jours après infection des moustiques. La compétence vectorielle a été comparée entre les différents biotypes.Les résultats entomologiques ont révélé que Cx. pipiens est dominant (87.2%). Tous les moustiques analysés étaient négatifs pour le VNO. Les taux de séroprévalence étaient de 1.01% et 1.98% parmi les humains et les chevaux respectivement. De plus, Cx. pipiens s’est révélé bien plus compétent pour transmettre le VNO que le VFVR. Le biotype molestus est capable de transmettre le VNO plus tôt que celui de pipiens. Cette étude présente des preuves sur une faible circulation du VNO au Liban. Cx. pipiens s’est révélé compétent pour assurer cette transmission. Ainsi, il est essentiel d'établir des programmes de surveillance pour prévenir les éventuelles épidémies. / West Nile virus (WNV) and Rift Valley Fever virus (RVFV) are two emerging arboviruses that have never been reported in Lebanon. They can be transmitted by Culex pipiens mosquito species including two biotypes: pipiens and molestus. During this project, we assessed the circulation of WNV among mosquitoes, human, horse and chicken populations in Lebanon. Moreover, we evaluated, under experimental conditions, the capacity of local Cx. pipiens biotypes to transmit both viruses.Adult mosquitoes were collected, identified and tested to detect WNV RNA. Besides, human, horse and chicken blood samples were collected and screened for WNV antibodies using an in-house ELISA and then confirmed by neutralization assay. Moreover, local Cx. pipiens specimens were experimentally infected with WNV lineage 1 or RVFV Clone 13 strain. The viral infection, dissemination and transmission were then estimated at different days post infection.The vector competence was compared between Cx. pipiens biotypes.Entomological results revealed that 87.2% of collected adult mosquitoes were Cx. pipiens. Screened mosquitoes were negative for WNV. Seroprevalence rates were 1.01% and 1.98% among humans and horses respectively. Besides, local Cx. pipiens were highly competent for WNV transmission and to a lesser extent to RVFV. The molestus biotype was able to transmit WNV earlier than pipiens biotype.The present study provides new evidence of a low circulation of WNV among human and horses in Lebanon. Cx. pipiens is the suspected vector and is experimentally competent to ensure transmission. Therefore, there is a need to establish surveillance program to predict and prevent potential outbreaks.

Mosquito species of the Culex genus are the enzootic vectors for several bird-associated viruses that cause disease in humans. In Europe, these viruses include Sindbis (SINV), West Nile and Usutu viruses. The morphologically similar females of Cx. torrentium and Cx. pipiens are potential vectors of these viruses, but difficulties in correctly identifying the mosquito species have caused confusion regarding their respective distribution, abundance, ecology, and consequently their importance as vectors. Species-specific knowledge from correctly identified field material is however of crucial importance since previous research shows that the relatively unknown Cx. torrentium is a far more efficient SINV vector than the widely recognized Cx. pipiens. The latter is involved in the transmission of several other viruses, but its potential importance for SINV transmission is debated. In this thesis I describe the development of a molecular method for species identification, based on reliably identified males of Cx. torrentium and Cx. pipiens. This identification method was then used in consecutive studies on the distribution and relative abundance of the two species in Sweden and 12 other European countries, SINV field infection rates in mosquitoes identified to species level, and evaluation of potential trap bias associated with common sampling techniques. The results showed that Cx. torrentium is a far more common species in Europe than previously assumed. In Sweden and Finland, it is the dominant species, accounting for 89% of the sampled Culex population. In central Europe, it is equally common to Cx. pipiens, while Cx. pipiens dominates south of the Alps Mountain range. Larvae of both species are often found together in both artificial containers (e.g. car tires) and natural sites. Also, a trapping bias against Cx. torrentium was revealed for CDC-traps. For the first time, SINV was isolated from species-identified Cx. torrentium and Cx. pipiens mosquitoes caught in the field, with Cx. torrentium being superior in infection rates (36/1,000 vs. 8.2/1,000). Future studies on SINV, as well as other mosquito-borne bird viruses in Europe, can hopefully gain from the baseline information provided here, and from principles of vector discrimination discussed in the thesis.

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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.

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.

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