Investigating Odorant Receptor Function in Aedes aegypti Mosquitoes: Response to Human Odorants and Chemical Repellents
Type of DegreePhD Dissertation
Entomology and Plant Pathology
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As a vector of some diseases affecting humans and certain animals, the yellow fever mosquito (Aedes aegypti) has been increasingly widespread worldwide, drawing attention from governments and the general public due to its biting nuisance and disease transmission. The mosquito's host-seeking behavior is mediated by its olfactory system, which detects attractive odors or chemical repellents. While the composition of human emanations and chemical insect repellents has been thoroughly elucidated, there remain numerous unresolved mysteries regarding which components are active and how they are recognized by insects. To elucidate the mechanisms of human odorants reception and chemical repellents reception in mosquito olfactory receptors, this study investigated the responses of human odorants and chemical insect repellents by mosquito odorant receptors and peripheral phase olfactory system, particularly focusing on the response to DEET. I have optimized a protocol for the functional characterization of mosquito olfactory receptors in response to human odors. While there is a previously published protocol that shares similarities with our procedure, we have made important modifications and enhancements. These include improvements in cRNA aliquoting and storage to maintain high quality, as well as advancements in frog surgery techniques. Our protocol utilizes a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a reliable and effective method for studying the function of mosquito olfactory receptors in the presence of human odors or chemical repellents. With the help of Xenopus oocytes expression system, we have identified three specific odorant receptors (ORs), namely AaegOR13, AaegOR15, and AaegOR55, in Ae. aegypti that specifically respond to prominent human odorants, such as alcohols, aldehydes, and aromatics. These three ORs are believed to play crucial roles in the mosquito's host-seeking behavior. Our research also investigated the inhibitory effects of two botanical compounds, α-terpinene and citronellal, on the reception of these human odorants by the AaegORs and the peripheral olfactory system. Furthermore, we focused on elucidating the mechanism underlying the repellency of EPA-approved repellents in AaegOR13 and AaegOR15. While these synthetic repellents do not directly stimulate AaegOR13 and AaegOR15, they significantly reduce the reception of human odorants by these receptors and diminish the detection of human odorants in the mosquito's peripheral olfactory system. These inhibitory effects have the potential to impede the mosquito's ability to seek hosts, presenting a promising approach to protect humans from mosquito bites and reduce the transmission of mosquito-borne diseases. DEET (N,N-diethyl-meta-toluamide) is one of the most successful synthetic mosquito repellents, known for its significant mosquito-repellent effects. We have successfully identified two DEET receptors, AaegOR54 and AaegOR55, in Ae. aegypti. This marks the first discovery of the "true" DEET receptors in this mosquito species. AaegOR54 is a DEET-specific receptor that exhibits a strong response to DEET and plays a specialized role in DEET detection. On the other hand, AaegOR55 is a versatile receptor involved in detecting human odors but also demonstrates a moderate response to DEET. By performing gene knockout studies on AaegOR54, we observed a significant reduction in the mosquito's response to DEET perception. These findings reveal the true DEET receptors in Ae. aegypti and highlight the presence of specialized DEET-detection receptors alongside overlapping olfactory pathways. This knowledge deepens our understanding of the molecular mechanisms underlying DEET reception in mosquitoes and significantly contributes to the understanding of the molecular basis of DEET reception. This knowledge holds important implications for the development of enhanced mosquito repellents and the implementation of more effective strategies for mosquito-borne disease prevention and control. In conclusion, this dissertation extensively examined how receptors in Ae. aegypti respond to human odors and repellents, and explored the mechanisms involved in the interaction between repellents and human odors. These findings enhance our understanding of the molecular processes underlying mosquito host-seeking behavior and the effectiveness of repellents. They offer valuable insights for developing strategies to prevent or hinder mosquito responses to host-seeking behavior, ultimately aiming to protect humans from mosquito bites and reduce the spread of mosquito-borne diseases.