Project Summary During infection and transmission, the larvae of mosquito-borne filarial nematode parasites perform patterned migrations, likely using a variety of sensory modalities to navigate the hostile environment of host barriers and immune attack. Migrations are coordinated with developmental time points in the mosquito vector and definitive mammalian host, and aberrant migrations result in transmission failure. Control of diseases caused by filarial worm infections, such as lymphatic filariasis (LF) in humans or heartworm disease in companion animals, has been hampered by a dearth of available antifilarials, and current control strategies through mass preventative chemotherapy are susceptible to loss of drug efficacy. Parasite migrations may be vulnerable to therapeutic inter- vention or strategies for vector-based control, but advances are limited by a lack of knowledge of the molecular receptors and pathways that mediate intra-host sensory processes. Filarial worms such as Brugia malayi, an eti- ological agent of LF, and Dirofilaria immitis, the canine heartworm, express nematode-specific G protein-coupled receptors (chemoreceptors) in amphid sensory organs, and expression is patterned throughout the life cycle in accordance with non-uniform sensory needs. However, a lack of scalable behavioral assays has prevented the investigation of filarial worm chemosensory behaviors and the functional annotation of putative chemosen- sory receptors. To fill this knowledge gap, this project will (1) develop new higher throughput behavioral assays using advanced microdevices, microfluidics, and high-content imaging, (2) identify host-derived cues that elicit chemosensory behaviors, and (3) functionally characterize chemoreceptors expressed during migratory time points. While other parasitic nematodes have large datasets of behavioral responses to host-derived cues, filar- ial worms lack this comparative data due to the difficulty of performing chemotaxis experiments in vitro. Aim 1 of this project will develop innovative higher throughput behavioral assays that are optimized for multiple larval stages. These will be used to screen host-derived cues for attractive or repulsive effects. Aim 2 will functionally annotate putative chemoreceptors by first employing RNA-seq to identify chemoreceptors that are upregulated during migratory time points. These will then be prioritized for annotation with in vivo functional genomics (RNA interference) and deorphanization leveraging a novel approach that uses expression of parasite chemoreceptors in the amphid neurons of C. elegans paired with behavioral assays and assessment of receptor activation by live calcium imaging of amphid neurons in response to activating cues. The result of this proposal will be (1) new behavioral assays that are capable of assessing a variety of sensory phenotypes of multiple stages and species of filarial worms, (2) a vastly expanded set of host-derived cues that induce chemotaxis of larval filaria...