Department of Entomology and Plant Pathology, Auburn University.
Department of Entomology and Plant Pathology, Auburn University; Departments of Biochemistry & Biophysics, and Cellular & Molecular Pharmacology, University of California, San Francisco.
J Vis Exp. 2021 Jun 8(172). doi: 10.3791/61813.
The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti's olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor (Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.
埃及伊蚊（Linnaeus）是许多重要人类疾病的传播媒介，包括黄热病、登革热和寨卡热，它对人类宿主有强烈的偏好，而不是其他温血动物。嗅觉线索在蚊子探索环境并寻找人类宿主以获取血液时起着至关重要的作用，从而传播人类疾病。已知在嗅觉感觉神经元中表达的气味受体（OR）负责蚊子与人类气味的相互作用。为了更深入地了解埃及伊蚊的嗅觉生理学，并在分子水平上研究它们与人类的相互作用，我们使用了优化的非洲爪蟾卵母细胞异源表达方案，以功能分析埃及伊蚊气味受体对人类气味的反应。本文呈现了三个示例实验：1）从 4 至 6 天大的埃及伊蚊触角克隆和合成 ORs 和气味受体共受体（Orco）的 cRNAs；2）在非洲爪蟾卵母细胞中进行 ORs 和 Orco 的微注射和表达；3）用双电极电压钳记录表达蚊子 ORs/Orco 的非洲爪蟾卵母细胞的全细胞电流。这些优化的程序为研究人员提供了一种新的方法来研究埃及伊蚊对人类气味的感知，并揭示了它们在分子水平上寻找宿主活动的潜在机制。
