Flaviviruses (genus Flavivirus, family Flaviviridae) includes a number of medically and veterinary important arboviruses. Most of them are transmitted by mosquitoes, such as West Nile virus, yellow fever virus, dengue virus or Zika virus. In the last decade, there has been discovered number of flaviviruses which have not known vertebrate host. They are called insect-specific flaviviruses (ISFs). These viruses have been detected in dipteran insects (mostly in mosquitoes) from all over the world. It seems they induce no pathogenic changes even in insect and they are not able to infect any vertebrate cells. However in some recent studies has been shown that some ISFs could influence the replication of other medically important flaviviruses in mosquito cells. In addition, they probably represent an ancestrial lineage of the family Flaviviridae and further studies focused on them could help to clarify which characteristics of flaviviruses enable them to infect vertebrates. There is not a lot of information about the ecology of ISFs and it is suggested that most ISFs have not been discovered yet. Main goal of this thesis was to detect ISFs in mosquitoes in the Czech Republic, where have not been made any exstensive research of these viruses yet. In case of discover some undiscribed ISFs we wanted to characterize...

Culex quinquefasciatus has been identified as one of the most prominent vectors of West Nile virus (WNV) in Southern California. WNV is a zoonotic disease that is endemic in North America and is known to primarily cause flu-like symptoms in humans, and in rare cases, life-threatening conditions. The goal of this study was to identify which animal species are most frequently fed upon by these mosquitoes in this region. To examine the relationship between blood-feeding patterns and West Nile virus activity in San Bernardino County, the feeding patterns of Cx. quinquefasciatus are determined in a variety of habitat types, which was the primary focus of this study. Furthermore, potential shifts in seasonal blood-feeding patterns of this population of Cx. quinquefasciatus towards increased mammalian feeding was examined. The WNV activity in the county during 2011 was also analyzed. Over 740 Cx. quinquefasciatus samples were collected by West Valley Mosquito and Vector Control District in San Bernardino County during 2011 from 34 different sites. DNA from the bloodmeals was extracted and purified, and a 658-base pair region of DNA located in the mitochondrial gene cytochrome c-oxidase I (COI) was amplified. This was followed by DNA sequencing of the PCR product, and identification of the individual sequences using the Bar Code of Life Data Systems. A total of 683 bloodmeals were successfully identified. These bloodmeals belong to 29 vertebrate species across four different habitats. It was found that species richness was not significantly different between habitats, even though the sample sizes for each habitat varied. Across habitats, the highest percentage of avian bloodmeals were taken from House Sparrows and House Finches. Bloodmeals were identified from five mammalian species which included Humans. A seasonal shift towards increased mammalian bloodmeal prevalence was observed in urban habitats. It was found that WNV activity during 2011 in San Bernardino County was relatively low when compared to the following six years.

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Malaria is a serious and life-threatening mosquito-borne disease that every year affects over 200 million individuals and causes 400,00 deaths. An additional 0.5 billion people globally are at risk of malaria infection. The unique role of climate in influencing malaria transmission outcomes across individual communities by acting on multiple dimensions of the malaria vector and parasite ecology has been long recognized. This recognition has led to the development of explicit and implicit climate-driven models of malaria transmission designed to better understand and predict patterns of population vulnerability and uncover potential challenges to malaria control. However, existing implicitly-forced process-based models of malaria have relied on indirectly correlated predictors of malaria transmission, instead of direct relationships among climate, vector entomology and parasite ecology. The lack of biologically-motivated modulation of malaria transmission compromises meaningful interpretation of the ecological role played by climate in malaria transmission. Similarly, the specific influence of climate on vector and parasite dynamics is obscured, limiting the utility of these simple and powerful model forms. This dissertation focuses on elaborating the direct ecological relationships between climate, the malaria vector and parasite to enhance the ecological utility of lower dimensional mathematical models of malaria transmission. In the 2nd chapter of this thesis, a climate-driven entomological modeling framework is developed, consisting of a simple dynamic model that explicitly tracks malaria transmission in human populations and implicitly represents the malaria force of infection through climate-regulation of multiple aspects of the Entomological Inoculation Rate (EIR). The EIR-model construct is found to accurately capture seasonal malaria dynamics under free-simulation, when coupled to local rainfall and temperature climatology across multiple local regions in Rwanda. Furthermore, local rainfall modulation of sub-adult survivorship is found to be a more critical driver of seasonal malaria dynamics than other environmentally-regulated components of EIR. In chapter 3, the model framework is paired with data assimilation methods to dynamically simulate interannual malaria incidence in Rwanda, infer parameters of malaria transmission and validate the malaria model. Results indicate that the implicitly-forced transmission model is able to reproduce interannual and seasonal malaria incidence at regional and local scales. However, accuracy of model description of malaria incidence is more varied at the more resolved local level. Intensified malaria control efforts during the later years of the study are suspected to increase the discrepancy between the vector and parasite dynamics dictated by climate and the observed widespread decline in malaria activity in the region. Nonetheless, the parameters of transmission identified across populations in Rwanda were comparable to existing estimates of malaria, further validating the transmission model and data assimilation approach. For the 4th chapter, a state-of-the-art Bayesian inference forecasting system for the EIR-model framework is developed, as well as a multi-model forecasting system consisting of weighted-average predictions from the dynamic malaria model and historical expectance predictions. Retrospective forecasts of four years of malaria data from 42 regions in Rwanda indicate that the model-inference forecasting system predicts malaria incidence more accurately than historical expectance alone, particularly for predictions with 1-6 weeks lead times. Although slightly less skillful, the multi-model system was found to substantively enhance forecast reliability of the EIR-model system, bolstering the utility of the malaria model as a robust forecaster of malaria in the region. The concluding chapter describes areas for improving the specification of the parsimonious model construct. The need to include malaria control coverage data as exogenous forces of transmission, non-climate drivers and alternate sources of climate exposure that support transmission are highlighted. Future works on forecast calibration needed to improve model performance for real-time prediction are also detailed. In addition, areas for application within information systems for evaluating malaria risk and for advising malaria control efforts, specifically relating to local variability in malaria burden and characterization of entomological drivers of local malaria, are identified and further discussed. The model systems developed in this thesis advance the capabilities of lower dimension dynamic models to connect the ecological drivers of malaria transmission to climate variation. Such process-based formulations could provide better climate-driven descriptions of malaria, while limiting model complexity, without compromising representation of entomological relationships that are potentially valuable for improved understanding and control of malaria transmission.

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The rise of vector-borne diseases transmitted by the Aedes spp. mosquitoes is attributed, in part, to the dramatic rates of contemporary urbanization. Over the past 30 years, scientists have developed a wealth of knowledge around the drivers of heterogeneity in Aedes-borne disease risk within and between cities. However, in current Aedes-borne disease research, characterizations of “urban” are oversimplified, with the built environment and social institutions of the city often relegated to a background context. To mitigate the spread of Aedes-borne diseases, under the dual global pressures of urbanization and climate change, there is an urgent need to incorporate the multi-dimensionality of urban systems in driving Aedes-borne disease risk. This dissertation is anchored in socio-ecological sciences, and tailored to the complexities of urban eco-epidemiological dynamics. Herein, theory and methods from ecology, epidemiology, geography, and urban science are synthesized to develop and implement a novel urban systems approach for Aedes-borne diseases. T he first chapter establishes the theoretical foundation for this approach, integrating concepts from three bodies of knowledge: “cities as complex adaptive systems”, hierarchical patch systems theory, and relational geography. In the framework, cities are conceptualized as hierarchically-structured patches of different land uses and characteristics. Patch composition determines localized disease risk, while patch configuration and connectivity contribute to emergent patterns of disease risk and spread. Complexity is added to the system by considering the cross-scale and dynamical processes occurring within a city. Furthermore, the framework establishes how individual and collective social structures interact with the biophysical landscape to generate risk. The empirical research for this dissertation uses a range of data sets, from open source remotely-sensed environmental data and census-derived socio-economic data to fine-scale household survey and entomological data. Chapter 2 is carried out at the scale of the city, and examines how extreme climate and weather conditions in Colombia differentially affects the onset of peak dengue incidence for urban settlements with varying landscape and socio-economic properties. Using Bayesian spatio-temporal hierarchical models we discovered that extreme temperature anomalies (10–12°C) result in an earlier onset of dengue risk for high-elevation compared to low-elevation settlements, which experience increases in dengue risk two to four months after extreme temperature anomalies. Furthermore, the risk of dengue after extremely dry conditions is higher and extends for a longer duration in highly urban areas compared to areas with a low proportion of the population living in urban settlements. These findings indicate the potential for landscape-specific dengue early warning and forecasting frameworks. Chapter 3 is based in a mid-sized, rapidly growing city (Ibagué) embedded within the Andes Mountains of Colombia, and establishes homogenous urban typologies of dengue risk. Measuring dengue incidence across census block and higher order urban sections, we show that distinctive signatures of incidence can emerge from interactions between heterogeneous socio-environmental composition and configuration. Finally, Chapter 4 is carried out at the household and neighborhood scale in Ibagué, and examines how water governance and neighborhood-based social processes drive household-level dengue risk. We documented the role of collective societal memory of water scarcity in fostering a culture of water storage. We determined that neighborhood-based metrics of social cohesion do not necessarily translate to dengue household preventative practices and that to scale dengue prevention strategies, public health agencies may consider interventions rooted in “place-making” to foster linkages between perceived neighborhood-level versus household-level risk. This dissertation demonstrates how trans-disciplinary research bridges urban science, ecology, and public health research communities, and provides a pathway for mosquito-borne disease interventions to be incorporated into national-level early warning systems as well as community-based initiatives that collectively, set cities on more healthy and sustainable trajectories for the 21st century.

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Aedes aegypti mosquitoes are found globally and also act as the primary vector of Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. The use of insecticides as the main control strategy against diseases transmitted by this mosquito, is increasingly challenged by emerging resistance. Thus, there is a dire need for the development of novel approaches informed by an improved understanding of mosquito biology, to control mosquito populations and, ultimately, disease transmission. Rhythmic biological processes in mosquitoes help optimize resource exploitation by coordinating behaviors and physiology with fluctuating environmental conditions. Such synchronization enables organisms to adjust their physiology, metabolism, and behavior to predictable external cycles. In mosquitoes, circadian rhythmicity has been demonstrated in their biting and oviposition behavior, as well as their locomotor activity. However, little is known regarding how responses to long-range host cues are modulated by the circadian system. Here we show that both antennal sensitivity and olfactory behavior are time-of-day and odor-specific in Ae. aegypti females. Global transcriptomic analysis in whole heads of Ae. aegypti females reveal chemosensory genes differentially expressed throughout the day, providing insight into the molecular mechanisms behind daily variations in olfactory sensitivity and behaviors. We additionally show an odor-induced activation of mosquito behavior. Mosquito locomotion and behavior are also mediated by physiological state, and activity decreases after blood-feeding. Since the central clock components have been shown in other organisms to be redox-sensitive, we explored the role that diet heme plays in mediating behavioral changes following blood ingestion using artificial blood diets. We found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a meal containing hemoglobin, but peripheral period transcription is reduced throughout the course of digestion following ingestion of a protein meal independent of hemoglobin inclusion. Overall, our results show that Ae. aegypti behavioral rhythms mediated by rhythmic gene expression are plastic and susceptible to external host cues and host blood digestion. This work can be leveraged for future studies investigating mosquito host-seeking and blood digestion to identify novel targets for vector control. / Doctor of Philosophy / Female mosquitoes rely on blood-feeding in order to produce eggs, but can unfortunately act as vectors of disease if they transmit pathogens when biting. Insecticides are currently our strongest main tool for controlling mosquito disease vectors such as Aedes aegypti, the yellow fever mosquito. However, increasing cases of insecticide resistance present new challenges in vector control, and new strategies to prevent vector-borne disease are needed. The Ae. aegypti mosquito is found globally and transmits Zika, dengue, and Chikungunya viruses, for which there are limited treatment options and no vaccines available. Mosquitoes exhibit rhythms in their gene expression and behaviors such as biting and activity patterns, in order to optimize energy efficiency and coordinate their biology and behaviors with daily fluctuations in the environment. However, it is unknown how their responses to human host odor cues are modulated by their central timekeeping system in the brain. Mosquitoes primarily find a human host via their sense of smell, or olfaction. Odor molecules in the air, emitted by humans, can be detected by mosquitoes' antennae. Here we show that both antennal sensitivity and behavioral responses to odors are time of day and odor-specific in Ae. aegypti females. We quantified gene transcripts in whole heads of Ae. aegypti females as a measure of gene expression, which revealed that genes involved in odor detection are expressed differently throughout the day, providing insight into the molecular mechanisms behind behavioral observations. We also show that mosquito behavior can be activated by odor exposure, and that their behavioral patterns can be influenced for multiple days following exposure. Mosquito behavior is also influenced by blood-feeding, which reduces mosquito activity and flight. Time-keeping genes in the fly brain have been shown to be sensitive to oxidative stress. Blood contains the protein hemoglobin, which can lead to oxidative stress when digested. Using artificial blood diets that allowed us to include or exclude hemoglobin in the meal, we found that the transcription of the timekeeping gene period is reduced in the head immediately after feeding on a diet containing hemoglobin, but is reduced in the rest of the body throughout the course of digestion following ingestion of a protein meal, whether hemoglobin was included or not. This work can be leveraged for future studies investigating mosquitoes' rhythms in host-seeking and blood digestion to identify new effective targets for vector control.

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.