Benjamin Matthews

Aquatic larvae of the genus Chaoborus (Diptera: Chaoboridae) are the only truly pelagic insects in the world because they can precisely regulate their buoyancy. By controlling the volumes of two internal pairs of air-filled sacs Chaoborus larvae are able to float, sink or remain neutrally buoyant in the water column, and they can also compensate for the changes in air-sac volume that result from changes in hydrostatic pressure with depth. Varying the air-sac volume changes the overall density of the larvae: more volume of air allows the larvae to ascend, and less allows them to descend. This is due to the unique mechanochemical mechanism of the air-sac wall, comprised of alternating transverse bands of cuticle and resilin and surrounded by a thin epithelial layer. Resilin is an elastic protein which responds to changes in pH, expanding when in an alkaline environment and contracting in an acidic environment. Exactly how the pH of the air-sac wall was controlled was previously unknown. Using an RNAseq approach, I uncovered a suite of pathways found in the air-sac that are potentially involved in pH regulation. I propose a model in which apical VHA secretes H⁺ into the lumen, generating an electrical potential that drives the efflux of Cl⁻ via pHCl-2 to reduce the transmembrane potential and acidify the air-sac’s resilin bands. To alkalinize the resilin, I propose a model in which an apical NHA1 likely acts as either a symporter or exchanger, moving Cl⁻ and H⁺ back into the epithelium or exchanging Cl⁻ for OH⁻. These proposed models arise from a transcriptomic exploration into a previously unknown regulatory mechanism, uncovering potential pathways that can be verified in future studies. By comparing these expression pattern of Chaoborus air-sacs to the basal tracheae of the closely related Eucorethra underwoodi, I uncovered the potential evolutionary trajectory of the air-sac.

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The yellow fever mosquito, Aedes aegypti, is a prevalent vector that spreads transmissible diseases in human populations. In the past few decades, increasing effort have been made to study these fascinating animals. The rising research in CRISPR/Cas9 technology has allowed the potential for gene editing to be done in Ae. aegypti. This thesis focuses on three novel applications of CRISPR/Cas9 in Ae. aegypti that focus on improving gene editing efficiency and expanding the mosquito genetic toolbox.In the first chapter, germline gene promoters (zpg and nanos) are used to promote the expression of Cas9 proteins. We hypothesized that germline gene promoters can bias Cas9 expression in space and time to favor Homology Directed Repair (HDR). We discovered that transient tail expression of fluorescence markers in injected mosquitoes successfully predicted integration of transgene into the germline. This transgene was able to be passed on to the next generation. The use of germline gene promoters can reduce efforts in creating transgenic mosquitoes, as it increases mosquito survival rate and reduce time needed to screen fluorescence.In the second chapter, a sgRNA and donor template cassette was inserted into the mosquito genome through piggyBac transposon integration to create a split CRISPR system. We hypothesized that the endogenous expression of CRISPR components in a split system can favor HDR. We found no offspring that had a stable integration of transgene through HDR, while most strains exhibited Mendelian inheritance offluorescence genes. Our results show that split systems in gene editing should test multiple candidates to ensure Cas9 activity.In the third chapter, we aimed to create a novel balancer chromosome in Ae. aegypti through utilizing CRISPR/Cas9 to generate a large chromosomal inversion. We applied methods of CRISPR gene editing at two target sites simultaneously, which has the potential for HDR repair to invert the chromosome segment between these sites. Future work will validate the sequences of putative G0 founders. These are the first steps towards creating a novel balancer chromosome.

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Insects possess gustatory receptor neurons that respond to different taste modalities. Theseneurons express receptor proteins that are tuned to specific taste molecules to elicit a response inthe neuron. Taste processing has been extensively studied in Drosophila melanogaster, whichmakes it an ideal model to identify molecular mechanisms conserved in other insects. In thisstudy, we explore the neural basis of taste processing in the yellow fever mosquito, Aedesaegypti which utilizes its gustatory system to detect non-volatile chemosensory cues duringbehaviours such as blood feeding, nectar feeding, and egg laying. We hypothesize that tasteprocessing mechanisms in A. aegypti are broadly conserved with D. melanogaster, but thatmosquito-specific adaptations have evolved to support mosquito-specific behaviours. To test this,I produced a comprehensive anatomical and functional characterization of gustatory receptorneurons expressing taste receptors in the tarsi of mosquitoes. I used genetic driver lines to labelsensory neurons, characterizing their anatomy and neural activity in response to taste stimuli. Toinvestigate the anatomical map, I examined four different populations of neurons across all threelegs in males and females. I found that specific populations of neurons are distributed throughoutthe legs, and across sexes in accordance with the behaviours they are associated with. Toinvestigate the functional characterization of the neurons, I developed a novel protocol for livecell imaging in the tarsus during liquid tastant delivery. With this setup, I explored whether,similarly to D. melanogaster, neurons in the mosquito are broadly tuned to appetitive tastants,with overlapping populations of neurons responding to various appetitive taste molecules. Ispecifically tested sucrose, associated with mosquito nectar feeding behaviour, and lactic acid orlow concentrations of sodium chloride, associated with blood feeding behaviour. My resultsshow that non-overlapping populations of neurons in the mosquito respond to the appetitivetastants tested. These results are not consistent with observations in D. melanogaster but supportthe duality of the feeding systems in mosquitoes. Overall, this work aims to provide a betterunderstanding of the neural coding of mosquito gustation for better understanding of themolecular basis of various key behaviour in mosquitoes.

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There are over 3,500 species of mosquito, occupying a huge range of environments including inland and coastal habitats. Salinity tolerance plays a crucial role in the ecology of mosquitoes as it can determine whether a particular habitat is suitable for larvae. Understanding how aquatic larvae physiologically cope with salt and how adult mosquitoes detect salt is of critical importance to understanding the biology of important vector species. In this study, the larval salinity tolerance, oviposition preference and breeding habitat ecology of a widespread (Ae. aegypti) and restricted (Ae. togoi) mosquito species was investigated to try to understand key physiological and behavioral differences. Ae. aegypti breed and develop in freshwater containers associated with urban environments while Ae. togoi develop in high fluctuating salt-water rockpools. By conducting larval survival studies, I found that, although Ae. togoi larvae are capable of surviving in extremely high concentrations of seawater, Ae. aegypti larvae can tolerate a wider range of ions at intermediate concentrations. This ability to survive in a wide variety of water types could partially explain how Ae. aegypti have been able to spread globally while Ae. togoi remains restricted to coastal areas. Oviposition bioassays found that salinity may not be a key driver of oviposition for Ae. togoi, as it is for Ae. aegypti. Finally, a field study of Ae. togoi in metro Vancouver uncovered that temperature, precipitation, salinity and the interaction of these factors are important predictors of larval abundance. This study has highlighted the importance of investigating field populations as well as conducting laboratory experiments when investigating the biology of a species. In reality, a suite of interacting factors modulates the ability of species to survive in their habitat and these factors can only be uncovered by examining field populations. In addition, a two-species comparison can aid into providing preliminary insight about how evolution has shaped osmoregulatory strategies as well as the chemosensory system in the face of exploiting new habitats. Investigating mosquito physiology and behavior can aid in predicting distribution of mosquito populations and eventually this can aid in developing targeted control strategies for vector species.

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