PROJECT SUMMARY Infections with the parasitic nematode Ascaris lumbricoides affect up to an estimated 1.2 billion people worldwide, causing significant disease and accounting for the loss of 749,000 Disability Adjusted Life Years (DALYs) annually. Treatment of these infections in most endemic regions depends on mass drug administration (MDA) programs primarily comprising benzimidazoles (BZ), such as albendazole and mebendazole. Large-scale and prolonged usage of BZs selects for resistance in parasitic nematodes, as observed in veterinary parasites (e.g., Haemonchus contortus, a parasite of small ruminants). Although no confirmed cases of BZ resistance in human ascariasis have been described, BZ resistance has been confirmed in ascarids of veterinary importance. Importantly, these veterinary parasites also appear to lack canonical alleles associated with BZ resistance in other parasite species, suggesting that resistance mechanisms in ascarids likely differ. Everything currently known about BZ resistance comes from studies of the model nematode Caenorhabditis elegans and closely related parasites such as H. contortus. However, 350 million years of evolution separate C. elegans and ascarids, limiting its use as a model for ascarid research. Therefore, it is necessary to establish a new, more closely related, model to study BZ resistance in ascarids. Ascaridia dissimilis offers a powerful model system to study BZ resistance in human ascarids because established resistant and susceptible isolates exist, labor costs are lower than other veterinary parasites, and importantly no human infections are required. Human parasites cannot be readily studied because of the ethical issues associated with controlled infections, as well as difficulties in manipulating such a model. This project will identify genetic variants associated with BZ resistance in ascarids, using poultry ascarids as a new model system, allowing for diagnostics to be developed and improving treatment programs to control human infections. Aim 1 will create high-quality reference genomes for sensitive and resistant A. dissimilis isolates. Reference genomes will allow known resistance associated genes to be analyzed for high-impact variants, and enable future studies, including genome-wide mapping. Aim 2 will use genetic crosses and BZ selection to create recombinant isolates with BZ resistance associated loci in an otherwise susceptible background. After initial crossing of the resistant and susceptible isolates, progeny will be backcrossed with the susceptible parental line and selected for BZ resistance and used in a bulk-segregant analysis approach to identify BZ resistance loci. This innovative poultry ascarid system enables discoveries of conserved BZ resistance loci in a tractable model. Results can then be translated to human ascarids to improve the quality of care and quality of life for infected individuals across the developing world.