Abstract Opsins are classical G-protein coupled receptors, which for more than a century have been thought to function exclusively as light detectors. However, using Drosophila, we have uncovered multiple, light-independent roles for opsins including detection of bitter tastants. This discovery raises exciting questions, which we propose to address here. Do opsins only respond to bitter compounds, or do they function more broadly in taste sensation? Of particular interest, do opsins serve as taste receptors in other animals, including the mosquito, Aedes (Ae.) aegypti? This vector spreads viruses that cause dengue, yellow fever, and other diseases that afflict millions of people each year. Both male and female Aedes use taste to identify nectar and avoid toxic chemicals, while females use many senses to identify people for blood meals. However, the receptors required for mosquito taste are largely unknown. Another fascinating question is whether opsins serve as taste receptors in mammals. To address these questions, we propose to employ an extensive suite of approaches including electrophysiology, Ca2+ imaging, behavioral assays, cell biology, an in vitro receptor activation assay, and state-of-the art molecular genetics. We will also bring to bear our expertise in Drosophila taste, opsins, and our recent experience performing molecular genetics and behavior in Ae. aegypti and in the mouse. Aim 1 will define the role of a Drosophila opsin in amino acid taste, which in flies is a poorly understood taste modality. Attraction to amino acids increases if the flies are kept on an amino acid deficient diet. We outline experiments to define the contribution of the opsin to this dynamic change in amino acid appeal. We will also test the proposal that the opsin is activated directly by amino acids. Aim 2 will build on our preliminary data that two Ae. aegypti opsins are expressed in the major taste organ, and are required for sensing a flavonoid. The goals of this aim are to reveal the chemical specificity of these opsins in vitro and in vivo, determine whether they act independently or together, and identify the gustatory receptor neurons in which these opsins function. Aim 3 will characterize the roles of opsins in mammalian taste. This aim is supported by preliminary data showing that multiple mouse opsins are expressed in the mouse tongue, and are activated in vitro by bitter compounds, including a flavonoid for which there is no known receptor. In addition, we present preliminary data that the response to this flavonoid is impaired in the opsin mutant. We propose to test the hypothesis that this opsin functions broadly in sensing flavonoids, identify the taste receptor cells that express this and the other “gustatory opsins,” and perform behavioral tests with mutants to characterize the requirements for each opsin for sensing bitter compounds. The results from this project would establish opsins as a new class of taste receptor conserved from insects...