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West Nile virus (WNV; family Flaviviridae) has been an annual public health concern in the continental United States since its introduction in 1999. It is particularly difficult to determine when and where human infections will occur because WNV is highly focal and amplifies in bird-mosquito cycles with incidental spillover to humans. WNV is transmitted by female mosquitoes in the genus Culex, most commonly Cx. tarsalis, Cx. pipiens, and Cx. quinquefasciatus. The mosquitoes’ capacity to transmit WNV depends on the environment, particularly temperature, resulting in seasonal cycles with most human cases occurring between July and September. WNV prevention is predominantly through vector management, consisting of a combination of larval and adult control. Integrated vector management programs in California conduct surveillance to monitor mosquito abundance and mosquito infection prevalence as a means for targeting control strategies, but these estimates are not adjusted for biases that could be induced by short-term variation in weather. This dissertation investigates the complicated relationship between environmental factors, entomological surveillance observations, and human WNV disease.Chapter 1 focused on the relationship between entomological surveillance indicators and risk for human WNV disease. In particular, we evaluated the ability of the vector index (VI), the product of mosquito abundance and infection prevalence, to predict periods of above-average WNV incidence. We used receiver operating characteristic (ROC) curves to identify the VI threshold that maximized sensitivity and specificity of these predictions and found that these thresholds were highly dependent on the dominant vector species in an area and the trap type used for targeting surveillance. We also used statistical models with observed entomological surveillance and human disease data aggregated to different spatial scales to examine the effect of spatial scale on the ability of the VI to predict human WNV disease incidence. These results found cities to be the best balance between being small enough to be operationally relevant but large enough to have adequate predictive accuracy during high-risk periods. Chapter 2 considered short-term weather variability as a potential source of bias in entomological surveillance that may affect estimates of WNV disease risk. We collected mosquitoes and gathered weather data from 10 field sites equipped with devices to record mosquito counts, temperature, and wind speed every 15 minutes in the rice-growing region of northern California. We used the 15-minute mosquito data to estimate four outcomes for the primary WNV vector in the study area, Cx. tarsalis: the total overnight count, the onset time of evening host-seeking activity, the median time of nightly host-seeking activity, and the hour of peak host-seeking activity. We related each of these outcomes to the wind speed and temperature recorded at a range of times in the afternoon leading to the host-seeking night through statistical models and found both wind speed and temperature at 20:00, or just prior to the onset of host-seeking, to be the best predictors of all four outcomes. These predictable factors will help guide the timing of vector control applications to maximize their effects on the local vector population, thus reducing the overall risk of WNV transmission. Chapter 3 applied spatio-temporal predictive models that accounted for ecological factors and were capable of highlighting gaps in surveillance coverage for estimating risk of WNV transmission. We used a generalized additive model (GAM) to model the nonlinear weekly seasonal trends of Cx. tarsalis abundance in a range of land use types using trap count data for the years 2008-2020 collected by 20 Central Valley mosquito and vector control districts in California. Overall, the model captured strong seasonal patterns in abundance, modified by local land use and ephemeral spatio-temporal anomalies. Models maintained a similar predictive accuracy for out-of-sample data compared to that for the training data set. To determine the ability of the model to extrapolate from known surveillance locations, we then used the GAM to predict weekly Cx. tarsalis abundance at unmeasured locations across a 2.5-km grid of the Central Valley. We found that these predictions were most accurate when the nearest observed trap was within 2 km and one week prior. Taken together, these three chapters provide a basis for improving WNV risk estimation through entomological surveillance. These chapters will inform efforts to prevent human WNV disease through a more complete understanding of the link between WNV vector dynamics and the risk of human disease, improving the ability to predict risk and target mosquito control.

Culex tarsalis is a major vector of West Nile Virus (WNV) in North America. Although the genome for this species was recently sequenced, the physical genome map has not developed. Unlike other Culex species, that have sex-determination locus on chromosome 1, the sex locus in Cx. tarsalis is located on chromosome 3, the longest chromosome. It is currently unknown if this difference is associated with chromosomal rearrangements. The objectives of this study were to develop a high-resolution map for the precise physical genome mapping in Cx. tarsalis and to compare mitotic chromosomes between three species of Culicinae mosquitoes. Using mitotic chromosomes from imaginal discs of 4th instar larvae of Cx. tarsalis, we developed idiograms based on morphology and proportions of the mitotic chromosomes. In addition, the physical mapping of ribosomal genes using fluorescence in situ hybridization was performed. The comparative analysis of Cx. tarsalis to Cx. pipiens and Cx. quinquefasciatus chromosomes showed that the total chromosome length in Cx. tarsalis is longer than the other two species suggesting the bigger genome size in this mosquito. A comparison of the relative chromosome length between the species indicated no significant differences suggesting that no large chromosomal translocation occurred between the species. Comparisons of the centromeric indexes demonstrated a significant difference in chromosome 1 between Cx. pipiens and Cx. quinquefasciatus. This difference suggests the presence of pericentric inversion between the species or amplification of ribosomal genes in Cx. pipiens. Studying mosquito chromosomes advances our understanding of Culex cytogenetics. Further comparative physical mapping of the three major mosquito genera will help us to understand the evolution of genus Culex better and to develop genome-based strategies for the vector control.

Culex tarsalis, the western encephalitis mosquito, predominantly uses large water bodies as oviposition sites. These water bodies are home not only to macroinvertebrate predators, but larger predators such as fish. The western mosquitofish, Gambusia affinis, has been used worldwide for the control of larval mosquito larvae for several decades.. My research demonstrates that Cx. tarsalis can detect the presence of semiochemicals associated with the western mosquitofish, Gambusia affinis, without the fish being physically present, leading to a decrease in oviposition rate. Oviposition onto fish-conditioned water was reduced relative to that in control treatments in bioassays using different densities of fish to condition water, in no-choice assays comparing mosquito oviposition on fish-conditioned water with aged tap water controls, and in bioassays using aged tap as well as pond water against Gambusia-associated semiochemicals at different concentrations to simulate “real-world” conditions. Assays with individual females provided responses to fish-conditioned water that were congruous with the results from binary choice experiments using multiple gravid females in replicate cages. Mosquito oviposition onto fish-conditioned water that had been filter-sterilized was reduced relative to filter-sterilized control water suggesting that the semiochemical was still present after bacteria were removed. Female Cx. tarsalis were deterred from ovipositing in the majority of trials conducted, and the response appears to be correlated with the concentration of Gambusia-associated semiochemicals present in oviposition sites.I identified two non-volatile and three volatile chemical compounds in water in which G. affinis had been held using a variety of chemistry techniques. Oviposition bioassays were conducted using standards of the three volatile compounds identified: dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) and S-methyl methanethiosulphonate to determine their role in mosquito oviposition behavior. The non-volatile compounds isolated from the Gambusia-treated water could not be fully characterized, but one appears to contain a ring structure similar to that of cholesterol. Female Cx. tarsalis were deterred from ovipositing into sites containing solid phase extracts of Gambusia-treated water. This may indicate that it is the non-volatile compounds that the females are detecting, leading to a decrease in oviposition rate.