This study was conducted to elucidate and to compare some factors which influence the host-seeking behaviour of adult female simuliids in two geographically isolated regions. Various observations and experiments were performed with the knowledge of a mechanism inherent in the feeding behaviour of simuliids. This approach has helped to clarify the orientation of simuliids to their hosts and has enhanced the understanding of the transmission of parasites by these flies. Further, it was found valuable to determine whether the behaviour state of simuliids influenced their reaction to visual stimuli. / Thesis / Master of Science (MSc)

Mosquitoes, by transmitting vector-borne diseases through their saliva, impact nearly half of the world's population. Mosquito survival is dependent on their sense of smell, or olfaction, which allows a mosquito to differentiate between plant nectar, required for metabolic processes, and host odors, which will help them navigate towards hosts, source of the blood required for producing eggs. Mosquitoes interpret and respond to chemical volatiles very differently depending on their environment (temperature, humidity, time of day, etc.) and species-specific host preferences (humans, mammals, etc.). However, the impact of ecological factors on mosquito prevalence, sugar-feeding, and host seeking is relatively unknown. In an attempt to address this knowledge gap, we first investigate how several ecological factors (i.e., temporal, seasonal, and topographical) affect mosquito sugar feeding and population dynamics. Second, we focused on the anthropophilic mosquito species Aedes aegypti to investigate the role of plant associated compounds in host-seeking behavior, such as those which are commonly found in personal care products (i.e., body washes). Our results demonstrate that several ecological factors alter mosquito prevalence and behavior, including both sugar and host seeking behaviors. We anticipate these results to be a starting point for mosquito control strategies that depend not only on olfactory perception of plant odors, but also on the ecological and species-specific characteristics which shape the dynamics of vector-borne diseases. / Master of Science in Life Sciences / Most female mosquitoes must feed on a blood source in order to produce eggs, but in doing this they can transmit viruses and pathogens between hosts through their saliva, which are contracted by 700,000 people per year. In order to survive and fly to locate these host sources, both female and male mosquitoes require sugar and carbohydrates present in plant nectar. Mosquito identification of nectar and blood sources is primarily dependent on their sense of smell, or olfaction, which allows a mosquito to identify and locate chemical odors. The interpretation of these chemical odors is context dependent on both the mosquitoes' hunger status, ideal host (human, mammal, bird, etc.) and environmental cues (temperature, humidity, time of day, etc.). It is relatively unknown how the combination of these biological and environmental factors influences mosquito survival and biting frequency. In order to better understand this relationship, we first investigated mosquito species' habitat preferences (i.e., urban and forested) and ecological factors (i.e., season, weather). Second, we focused on Aedes aegypti mosquitoes, which almost exclusively feed on humans in urban habitats, to investigate mosquito behavior in the presence of combined plant and human odors caused by the usage of fragrant personal care products that are scented with plant odors. Our results demonstrate that mosquito prevalence, host-seeking, and plant-seeking are highly dependent on chemical and ecological factors. We anticipate that this research has avenues for both improving mosquito control strategies and better understanding the ecological dynamics of vector-borne diseases.

The association of olfactory information with a resource is broadly known as olfactory-based associative learning. From an ecological perspective, associative learning can reduce search time for resources and fine tune responses to changing biotic and abiotic factors in a variable environment, which in mosquitoes has implications for pathogen transmission and vector control strategies. The purpose of this dissertation was to examine the ability for olfactory-based associative learning across the major life history domains of mosquitoes. Six different experiments comprise this dissertation. The first was to evaluate the response of mosquitoes following conditioning to 5, 10 or 50% sucrose concentrations with individual level mosquito conditioning and testing and introduction of statistical analysis with binary logistic regression. Mosquitoes did not respond in a dose dependent manner with respect to positive response to target odors following conditioning. This effect appears to be related to the mosquitoes' prior exposure to sugar as those exposed to 10% sucrose before conditioning did not prefer 50% sucrose but significantly fewer chose 5% sucrose. In an evaluation of host associated odors and second blood meal choice by females using a dual-choice olfactometer no significant effects were observed. The lack of significance may have been due to insufficient sample sizes, problems with odor collection or physiological state of mosquitoes. Effects of predatory mosquitofish on larval development and female oviposition choice were evaluated by rearing in separated habitats under three different treatments followed by an oviposition choice assay. Females did not prefer their natal habitat or avoid predators but chose substrate that had contained mosquitofish fed conspecific larvae. Mosquitofish affected larval development with acceleration in treatments with mosquitofish fed Tetramin® and delayed pupation in treatments with mosquitofish fed conspecific larvae. Mosquito memory length was evaluated by conditioning and testing at six time intervals from colony and field populations at two ages. Younger mosquitoes showed higher levels of positive response after conditioning at all time intervals except the longest (24h). Finally the olfactory-based associative learning ability of Anopheles cracens was evaluated. Significant evidence for learning was observed in males but not females at a memory length interval of 24h.

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.

La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population. / Ph. D.