A New Genetic Expression System to Determine the Odor Tuning of Insect Vector Ionotropic Receptors Sensitive to Human-Derived Odorants

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Project Abstract/Summary Mosquitoes are a major threat to human health throughout the world. From an infected bite, mosquitoes can transmit a variety of pathogens that lead to such diseases as malaria, Zika, and West Nile. The deadliest of all mosquito species is Anopheles gambiae, the predominant vector for Plasmodium parasites that lead to malaria; in 2020, over 600,000 people died as a result of bites from this insect. To identify and navigate towards humans, mosquitoes use their sense of smell to recognize human-derived odorants. Carboxylic acids on human skin, such as lactic acid, propanoic acid, butanoic acid, heptanoic acid, octanoic acid, and tetradecanoic acid, play a critical role in both attracting host-seeking mosquitoes as well as in host-preference. The family of olfactory receptors that detect such human-derived acid odorants are likely olfactory ionotropic receptors (IRs). However, previous attempts to use expression systems to characterize the odor-response profiles of mosquito IRs met with limited success. Here, we propose the development of a genetic method in Drosophila melanogaster flies that will facilitate the functional characterization of Anopheles mosquito IRs. To accomplish this goal, we will generate a “IR Decoder System” fly which allows Anopheles mosquito IrXs to be heterologously expressed in Drosophila neurons lacking their endogenous odor-binding IrX. The specific aims are: 1) to develop a genetic method in Drosophila for expressing an Anopheles tuning IrX receptor in a sensillum devoid of endogenous odorant responses, and 2) to identify the Anopheles gambiae IrXs that detect carboxylic acid odorants, particularly those known to attract mosquitoes. We will first generate an “Empty Sensillum” fly in which the three Drosophila IrXs (Ir41a, Ir75a, Ir75d) natively expressed in the three olfactory neurons in ac2 sensilla are mutated in the background of a Ir41a-GAL4 transgene and Phi-C31 attP2 docking site. We will generate attB-UAS-AgIrX constructs for all AgIrXs abundantly expressed in the Anopheles antenna. Insertion of the attB-UAS-AgIrX into the attP2 site of a Empty Sensillum fly will generate an IR Decoder System fly in which the AgIrX is ectopically expressed in a single ac2 neuron. The odor-response properties of the AgIrX- expressing neuron will be quantitatively analyzed by single sensillum recordings to a panel of 60 odorants, focused on those that are human-derived carboxylic acids and/or mosquito attractants. These experiments will identify the AgIrXs that are the most sensitive to attractive carboxylic acid odorants such as lactic acid, and could suggest suitable molecular targets for mosquito control. This study will generate a valuable new genetic tool for the investigation of olfactory responses of mosquito IrXs, and could further serve as the foundation to investigate the function of olfactory IrXs from a variety of other insect disease vectors.