The objective of the proposed R01 project is to build on our recent findings that attractive odors from sources of nectar activate conserved olfactory channels in mosquitoes, and the odorant ratios within the scent, along with the inhibition in the antennal lobe (AL), are critical for Aedes aegypti behavior. Feeding on fruits and flowers is a vital behavior for mosquitoes, and mosquito attraction to these sources of nectar is mediated by the ratio of key odorants in the bouquet. Bait-and-kill traps that use fruit syrups have effectively controlled local mosquito populations, but variation in the fruit odor can strongly impact its attractiveness. We still have not identified the odor constituents and ratios that are attractive in the nectar odors – enabling the development of synthetic lures –, nor how this information is detected and processed by the mosquito's olfactory system. Nectar-feeding by female mosquitoes increases their life-span and decreases the gonotrophic cycle, thereby increasing their vectorial capacity, and for adult males, it is the only source of nutrients. Our recent work allowed us to identify the odor constituents that mediate nectar-feeding behaviors in Ae. aegypti and understand how the odor is processed in the brain. Now in this application, we propose to use a combination of behavioral assays, chemical methods combined with calcium imaging in tethered flying mosquitoes, and genetic approaches to study the olfactory basis of nectar-seeking behaviors. Aim 1 will allow us to identify attractive odorants in the scents of diverse plant nectar sources and use heterologous expression systems to de-orphan the cognate odorant receptors (Ors). We also take advantage of the Q-system for genetically characterizing three Ors (Or15, Or49, and Or2) that are essential for representing the proper ratios in nectar odors. In Aim 2, we leverage our existing and new GCaMP expression lines to examine how the ratios of these key odorants are processed in the AL, and how GABAergic inhibition shapes these responses. In Aim 3, we will use our identified odor lures, and artificial lures that vary in their natural ratios of key odorants, to determine their efficacy in bait-and-kill systems. Together, these experiments will test the working hypothesis that nectar odors and their specific odorant ratios activate conserved olfactory channels to be processed similarly by AL circuits to mediate feeding behaviors. While there has been an extensive study of mosquito attraction to blood hosts, we know comparatively less about nectar feeding. Our experiments will identify new odors that can be immediately deployed as attractant lures. Additionally, GABAergic systems are involved in diverse physiological processes in insect vectors, including olfaction, immune response, and arbovirus replication, as well as being potent targets for insecticides. Generating mutants that target the mosquito's olfactory responses or GABAergic pathways could provide additional insight into these diverse processes. Sugar feeding plays an essential role in the vectorial capacity of mosquitoes and the spread of diseases that afflict over a billion people annually. Therefore, unraveling the neural bases of nectar-feeding will enable new gene-targets and tools for their control.

PROJECT NARRATIVE Plant nectar-feeding is essential for survival and reproduction for many mosquito species, and mosquitoes locate sugar sources by using their sensitive olfactory system. Trap-and-kill systems that exploit the nectar-feeding behaviors have shown to be an effective control intervention, but we don't know which odorants in the plant scent that mosquitoes find attractive, or how they process this information in their olfactory system. Using Aedes aegypti mosquitoes, we will characterize the genes involved in the odorant reception of nectar source odors and determine the mechanisms by which this olfactory input is processed in the brain, thereby providing new attractant lures and tools for mosquito control.