International malaria control initiatives such as the Roll Back Malaria Initiative (RBM) and the Medicines for Malaria Venture (MMV) mobilize resources and spur research aimed at vector control as well as the treatment and eventual eradication of the disease. These efforts have managed to reduce incidence of malaria by an estimated 37% worldwide since 2000. However, despite the promising success of control efforts such as these, the World Health Organization reports a staggering 438,000 deaths from malaria in 2015. The continuing high death toll of malaria as well as emerging insecticide and antimalarial drug resistance suggests that while encouraging, success in reducing malaria incidence may be tenuous. Current vector control strategies are often complicated by ecological and behavioral heterogeneity of vector mosquito populations. As an additional obstruction, mosquito genomes are highly plastic as evidenced by the wealth or chromosomal inversions that have occurred in this genus. Chromosomal inversions have been correlated with differences in adaptation to aridity, insecticide resistance, and differences in resting behavior. However, a good understanding of the molecular mechanisms for inversion generation is still lacking. One possible contributor to inversion formation in Anopheles mosquitoes includes repetitive DNA such as transposable elements (TEs), tandem repeats (TRs) and inverted repeats (IRs). This dissertation provides physical maps for two important malaria vectors, An. stephensi and An. albimanus (Ch.2 and Ch. 3) and then applies those maps to the identification of inversion breakpoints in malaria mosquitoes. Repeat content of each chromosomal arm and the molecular characterization of lineage specific breakpoints is also investigated (Ch. 2 and Ch.4). Our study reveals differences in patterns of chromosomal evolution of Anopheles mosquitoes vs. Drosophila. First, mosquito chromosomes tend to shuffle as intact elements via whole arm translocations and do not under fissions or fusions as seen in fruitflies. Second, the mosquito sex chromosome is changing at a much higher rate relative to the autosomes in malaria mosquitoes than in fruit flies. Third, our molecular characterization of inversion breakpoints indicates that TEs and TRs may participate in inversion genesis in an arm specific manner. / Ph. D. / Malaria is a complex and devastating disease vectored by the bite of a female Anopheles mosquito. This disease claimed an estimated 438,000 lives in 2015. The mobilization of funding and resources as part of global malaria eradication initiatives have reduced the global incidence of malaria by 37% in the last 15 years. Deaths from malaria are also 60% lower vs. the year 2000. These promising gains are threatened by the ability of Anopheles mosquitoes to adapt in the face of malaria control efforts. Anopheles mosquito chromosomes are known to be highly plastic, as evidenced by numerous chromosomal inversions. Recent years have seen increases in insecticide resistance, and behavioral change in mosquito populations that allow them to avoid insecticides and remain prolific vectors of disease. This ability of mosquito vectors to adapt threatens to unravel recent progress towards a malaria free world. The projects presented in this dissertation explore mechanisms of chromosomal evolution, specifically the potential role of repetitive DNA in the generation of chromosomal inversions. The exploration of chromosomal inversions was facilitated by the creation of physical maps for Anopheles species. Prominent malaria vectors An. stephensi andAn. albimanus were physically mapped in Chapter 2 and Chapter 3 respectively. In chapter 1 and chapter 3 physical maps are utilized for the identification of chromosomal inversion breakpoints using 2 species (Ch. 2) and many species (Ch. 4). Repeat content was quantified along each chromosomal arm (Ch 2,4) and in inversion breakpoint regions (Ch 3). This dissertation presents physical maps for two important malaria species that have been applied to the study of chromosomal evolution and will also serve as community tools for further study of malaria mosquitoes. Our work on chromosomal evolution has revealed the Anopheles chromosomes tend to undergo translocations as intact elements and do not under fissions and fusions as seen in fruitflies. We also find that the malaria mosquito sex chromosome changes much more rapidly relative to the autosomes than in fruitflies. Additionally, repetitive DNA including transposable elements (TEs) and tandem repeats (TRs) may be encouraging chromosomal inversions but with differing roles on different chromosomal arms.

Every year three million deaths are attributed to malaria, of which one-third are of children. Malaria is a vector-borne disease, where a mosquito acts as the vector that transmits the disease. In the last few years, computer simulation based models have been used effectively to study the vector population dynamics and control strategies of vector-borne diseases. Typically, these models use ordinary differential equations to simulate the spread of malaria. Although these models provide a powerful mechanism to study the spread of malaria, they have several shortcomings. The research in this thesis focuses on creating a simulation model based on the framework of cellular automata, which addresses many shortcomings of previous models. Cellular automata are dynamical systems, which are discrete in time and space. The implementation of the model proposed can easily be integrated with EpiSims/TRANSIMS. EpiSims is an epidemiological modeling tool for studying the spread of infectious diseases; it uses social contact network from TRANSIMS (A Transport Analysis and Simulation System). Simulation results from the prototype implementation showed qualitatively correct results for vector densities, diffusion and epidemiological curves. / Master of Science

Eastern Equine Encephalitis virus is a vector-borne virus amplified by the Culiseta melanura mosquito in an enzootic avian cycle, causing high morbidity and mortality to horses and humans when contracted as incidental hosts. The virus is distributed across most of the eastern United States, Canada, and Gulf coast, and has been expanding in geographic range and season of activity over time. Spatial-temporal trends in Cs. melanura abundance were correlated with available meteorological (temperature and precipitation) and remotely sensed environmental data for the period of 1997-2012 in Connecticut. The effects of inter-annual changes in precipitation, temperature, and groundwater levels on Cs. melanura abundances using time-series linear regression and cross-correlation analyses were inconclusive. Habitat modeling using logistic regression and landscape-based predictive variables demonstrated strong efficiency (46.2%) and acceptable sensitivity and specificity (65.6 and 78.6%, respectively) using NDVI difference and distance from palustrine areas as predictive factors. Remotely sensed data can improve the understanding of vector abundance patterns, helping to forecast future outbreaks and regional expansions by guiding surveillance efforts.

We develop and evaluate a novel host-vector model of West Nile virus (WNV) transmission that incorporates multiple avian host species and host stage-structure (juvenile and adult stages), with both species-specific and stage-specific biting rates of vectors on hosts. We use this model to explore WNV transmission dynamics that occur between vectors and multiple structured host populations as a result of heterogeneous biting rates. Our analysis shows that increased exposure of juvenile hosts results in earlier, more intense WNV transmission when compared to the effects of differential host species exposure, regardless of other parameter values. We also find that, in addition to competence, increased juvenile exposure is an important mechanism for determining the effect of species diversity on the disease risk of a community.

Abstract The age of an adult female mosquito is a critical determinant of its ability to transmit a range of human pathogens such as the dengue viruses. Since most pathogens require a relatively long period of development in their mosquito vector before they can be transmitted to a new host, only older insects are of epidemiological importance. It has been proposed that life-shortening strains of the inherited bacterial symbiont Wolbachia may be potentially useful to shift mosquito population age structure towards younger individuals, thereby reducing pathogen transmission without eradicating the mosquito population. However, life-shortening Wolbachia strains do not occur in mosquitoes naturally. To facilitate the evaluation of this potential strategy, the major aim of this project was to transfer a life-shortening Wolbachia strain, wMelPop, from the fruit fly, Drosophila melanogaster, to the dengue vector, Aedes aegypti. After successful transinfection, I then characterized the wMelPop infection in this mosquito species, and the associated phenotypic effects of this Wolbachia strain on several aspects of Ae. aegypti life-history. The horizontal transfer of the obligate intracellular bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. Previous attempts to directly transfer wMelPop from D. melanogaster to Ae. aegypti have been unsuccessful, presumably due to mal-adaptation of wMelPop to this novel mosquito host. In an attempt to adapt wMelPop to the mosquito intracellular environment to facilitate transfer, we serially passaged this infection for 3 years in Aedes cell culture. After long-term serial passage in mosquito cell lines, wMelPop was then initially reintroduced into its native host, D. melanogaster, by embryonic microinjection to examine the phenotypic outcome of this process. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into Drosophila, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility (CI), when compared to the original wMelPop strain. These results were suggestive of adaptation of this Wolbachia strain to the mosquito intracellular environment. Subsequently, we successfully established wMelPop in Ae. aegypti using embryonic microinjection. Two independently transinfected Ae. aegypti lines, PGYP1 and PGYP2, were generated. Analogous to infection in Drosophila, the presence of wMelPop in both of these lines halved adult mosquito lifespan under a range of laboratory conditions. The new association is stable, and wMelPop is maternally inherited with high fidelity across Ae. aegypti life span. It is capable of inducing near perfect CI that does not diminish as male Ae. aegypti age, which should facilitate its invasion into natural field populations and persistence over time. Fecundity assays using young PGYP1 females revealed that wMelPop infection induced minimal costs for reproductive fitness for females during their first gonotrophic cycle. The phenotypic effects of wMelPop on several aspects of Ae. aegypti life-history were additionally characterized. Overall, minor costs of wMelPop infection for pre-imaginal survivorship, development and adult size were observed. However, wMelPop infection decreased the viability of quiescent Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-infected Ae. aegypti females declined with age. These results revealed a general pattern associated with wMelPop pathogenesis in Ae. aegypti, where host fitness costs incurred by this infection increase during aging of both immature and adult life-history stages. We also discovered that wMelPop-infected Ae. aegypti have a reduced ability to utilise blood from non-human hosts for egg development. Blood feeding by wMelPop-infected females on mouse, guinea pig or chicken hosts resulted in a near complete abolishment of reproductive output associated with both a decline in the numbers of eggs oviposited as well as the hatching rate of successfully laid eggs. In contrast, the reproductive output of wMelPop-infected females fed human blood was normal, with intermediate effects observed with other vertebrate blood sources. Removal of Wolbachia from mosquitoes using antibiotic treatment restored egg development to standard levels on all blood sources. Further blood feeding assays over two reproductive cycles definitively illustrated a nutritional interaction between host blood source and egg development in wMelPop-infected Ae. aegypti. This dramatic phenotype may provide new insights into the nutritional basis of mosquito anthropophily. The results presented in this thesis highlight several important parameters required to theoretically model the infection dynamics of wMelPop, and its potential impacts on Ae. aegypti populations. Moreover, the successful establishment of wMelPop in Ae. aegypti forms the primary basis for further field-based evaluations, which will ultimately determine the viability of this Wolbachia-based strategy as an applied tool to reduce dengue transmission.

Rodents have been reported to play a significant role as reservoirs of over 22 rodent-associated Bartonella species. In this study, we contrast prevalence and diversity of Bartonella infections in 377 small mammals, representative of three terrestrial rodent genera, namely Aethomys, Gerbilliscus and Rhabdomys and one subterranean mole-rat species (Bathyergus suillus). The latter was sampled in close proximity to an informal human settlement, whereas the afore-mentioned murid rodent genera were sampled across a range of landscapes inclusive of natural, agricultural, urban, peri-urban and rural settings, from three provinces (Free State, Gauteng and Western Cape) in South Africa. Molecular estimates of Bartonella infection rates were determined through multi-gene screening of DNA extracted from clinical samples, primarily heart and spleen. PCR assays targeting the citrate synthase (gltA) and NADH dehydrogenase gamma subunit (nuoG) and/or beta subunit of bacterial RNA polymerase (rpoB) genes were used to ensure enhanced molecular estimates of Bartonella prevalence. Aethomys had the highest infection rate (86.7%), whereas Rhabdomys had the lowest (15%). Nucleotide sequencing and phylogenetic analyses revealed that the different primers sets used for Bartonella screening have different affinities to the different strains present in rodents from South Africa. Furthermore, the presence of Bartonella co-infections, confirmed through the presence of multiple peaks at 15% of the nucleotide sequences sites, ranged from 33.8% (in Aethomys) to 42.9% (in Gerbilliscus species) for the gltA gene region. For Aethomys ineptus, of the discrete Bartonella lineages recovered, one was closely related to zoonotic B. elizabethae. The latter species, which is associated with Rattus hosts worldwide and has been linked to cases of human endocarditis, suggests spillover from invasive to indigenous rodents. This is supported by previous studies indicating that indigenous Micaelamys namaquensis, a highly adaptable species, which like Aethomys is capable of utilizing natural and modified landscapes also hosts B. elizabethae-related lineages. Of potential public health importance, Bathyergus suillus were shown to be infected with a zoonotic Bartonella species, B. rochalimae. Our results further indicate that the level of anthropogenic transformation is significantly correlated with Bartonella prevalence, with Rhabdomys sampled from rural settings in the Western Cape Province having infection rates of 36% versus 0% in a nearby urban setting. This study also uncovered high levels of strain diversity in members of the Gerbilliscus cryptic species complex, sampled from an agricultural setting. The overall Bartonella PCR-positivity rate was 67.5 % and the gltA gene phylogeny confirmed the presence of six discrete Bartonella gerbil-specific lineages (I-VI). Lineages I and II clustered with Bartonella strains identified previously in G. leucogaster sampled from Sandveld nature reserves in the Free State Province South Africa, whereas lineages III-VI comprised of lineages that were restricted to either G. leucogaster or to G. brantsii, indicative of host-specificity. From the findings of this study, it is clear that the public health importance of the Bartonella species present in indigenous rodents warrants further investigation as at least two species, with known zoonotic potential (B. elizabethae and B. rochalimae) were shown to be present in rodents sampled in close proximity to human settlements. / Thesis (PhD (Zoology))--University of Pretoria, 2019. / This research was made possible through the financial support from the (AZD-IRT), CDC Co-Ag 5 NU2GGH001874-02-00 and through the NRF incentive and SARChI PI funding awarded to ADSB. / Zoology and Entomology / PhD (Zoology) / Unrestricted

Aedes aegypti mosquitoes, vectors for many human diseases, begin life as larvae developing in water, potentially exposed to runoff with herbicides and pesticides. This study serves as a novel investigation into the transstadial effects of exposure to Roundup on A. aegypti life history, immunity, and stress response and aims to account for these effects in an R0 model for vectorborne disease transmission. Prior work has shown that Roundup negatively affects mosquito life history. I hypothesized that larval exposure to the maximum sublethal dose of Roundup (7189µg/L) would negatively impact A. aegypti life history, immunity (candidate gene approach), and stress response (heat shock protein expression and fluctuating asymmetry). No significant differences were found for survival from the larval to adult stages, body size, size or shape fluctuating asymmetry, or sex ratio. However, the Roundup treatment group developed significantly slower for both time to pupation and to adult eclosion (both p < 0.0001). Adult immune gene expression showed no difference between groups, but the larval immune genes Dome (JAK-STAT pathway) and Spatzle (TOLL pathway) were downregulated in the Roundup treatment (p=0.0383 and p=0.0035, respectively), suggesting the larvae have reduced immunity. This study suggests that Roundup may have off-target effects on A. aegypti mosquitoes that are unaccounted for by current models, and these effects may potentially alter disease transmission to human hosts.

The recent discovery of Amblyomma maculatum sensu lato (s. l.) ticks in southern Arizona has renewed discussions around species designations for members of the Amblyomma maculatum tick group. Amblyomma maculatum s. l. from Arizona appear to be morphologically intermediate between A. maculatum sensu stricto (s. s.) and A. triste s. s. At present there is no conclusive species designation for the ticks from Arizona. My research focused on analyzing the systematics of both A. maculatum s. l. and Rickettsia parkeri, a common bacterial pathogen transmitted by these ticks. In the laboratory, A. maculatum s. l. from Arizona and A. maculatum s. s. from Georgia readily mated on experimental animals to produce F1 hybrid ticks; there was no difference in fertility with these two populations when compared with homologous populations. However, the F1 hybrids produced during these experiments exhibited diminished fitness and did not produce a viable F2 generation. These results suggest that A. maculatum s. l. and A. maculatum s. s. represent separate biological species. Results of the crossbreeding experiment conflict with recent genetic analyses of A. maculatum s. l. and A. maculatum s. s. suggesting they are a single species. Thus, I developed and optimized 14 microsatellite loci that amplify both A. maculatum s. s. and A. maculatum s. l. These novel microsatellite markers can be used in future analyses of A. maculatum s. l. and A. maculatum s. s. to further test for conspecificity between the two. I also investigated the genetic relationships within geographically distinct R. parkeri strains through development and implementation of a multi-locus sequence typing analysis. I showed that while there is no consistent genetic delineation of strains isolated from A. maculatum s. l. versus A. maculatum s. s., there is a subset of R. parkeri strains from A. maculatum s. l. that appear to represent an intermediate genotype between the North and South American strains. While the biological causes for these results are not immediately clear, coevolution of R. parkeri and A. maculatum s. l. may account for the detection of the intermediate genotype only found in association with A. maculatum s. l.

This study evaluates the roles of temperature variability and disease awareness in the emergence of Chagas disease (American trypanosomiasis). Chagas disease is endemic in Latin America and primarily spreads to humans directly via the triatomine vector. Hosts for most triatomine species are mainly rodents and occasionally dogs. The disease itself is caused by a parasitic protozoan, Trypanosoma cruzi (T. cruzi) which is found in the triatomine's feces and is often spread while the triatomine is consuming a blood meal. T. cruzi from feces enters the body via an abrasion on the skin, the mucous membranes, conjunctivae, or through consumption. To determine the risk of Chagas disease transmission one must define qualities that make the triatomine an effective disease vector as well as investigate the level of disease awareness among physicians and the population within the vector's range. This thesis maps triatomine species within the U.S. that harbor T. cruzi naturally and that exhibit qualities of domesticity. These qualities are defined by whether the species bites humans and dogs as well as reports that the species has been found in the domestic setting. Ranges illustrating temperature thresholds for increased triatomine activity for 2000 and 2030 are also depicted. Additionally, outcomes of a physician survey are presented to gauge the status of Chagas disease awareness in areas at higher risk for disease transmission. Results reveal limited consideration of Chagas disease in physician diagnosis despite the higher risk range which extends through the southern U.S. and is predicted to expand significantly by 2030. / Master of Science

A large epizootic of the vector-borne disease bluetongue occurred in northern Europe from 2006-2009, costing the economies of the infected countries several hundreds of millions of euros. During this time, the United Kingdom (UK) was exposed to the risk of bluetongue by windborne incursions of infected Culicoides biting midges from the northern coast of mainland Europe. The first outbreaks which occurred in the UK in 2007 were attributed to this cause. Although bluetongue virus (BTV) no longer appears to be circulating in northern Europe, it is widely suggested that it and other midge-borne diseases may emerge again in the future, particularly under a changing climate. Spread of BTV is strongly influenced by the weather and climate however limited use has been made of meteorologically based models to generate predictions of its spread to the UK. The extent to which windborne BTV spread can be modelled at timescales from days to decades ahead, to inform tactical and strategic decisions taken to limit its transmission, is therefore examined here. An early warning system has been developed to predict possible incursion events on a daily timescale, based on an atmospheric dispersion model adapted to incorporate flight characteristics of the Culicoides vectors. The system’s warning of the first UK outbreak in September 2007 was found to be greatly beneficial to the UK livestock industry. The dispersion model is also shown to be a useful post-outbreak epidemiological analysis tool. A novel approach has been developed to predict BTV spread into the UK on climate-change timescales as dispersion modelling is not practical over extended periods of time. Using a combination of principal component and cluster analyses the synoptic scale atmospheric circulations which control when local weather conditions are suitable for midge incursions were determined. Changes in the frequency and timing of these large scale circulations over the period 2000 to 2050 were then examined using an ensemble of regional climate model simulations. The results suggest areas of UK under the influence of easterly winds may face a slight increase in risk and the length of the season where temperatures are suitable for BTV replication is likely to increase by around 20 days by 2050. However a high level of uncertainty is associated with these predictions so a flexible decision making approach should be adopted to accommodate better information as it becomes available in the future.