Intersecting experimental evolution and CRISPR screens to identify novel toxin resistance loci

Understanding toxin resistance in insects is key to appreciate niche adaptations, but remains challenging due to its often complex genetic basis. A well-known example is the specialized association of Drosophila sechellia with noni fruit ( Morinda citrifolia ), which is toxic to most other insects, including the closely-related drosophilids D. simulans and D. melanogaster . Noni toxicity is due to its high concentration of octanoic acid (OA), but the mechanisms that determine sensitivity or resistance to OA remain poorly understood. Here, we experimentally-evolved D. simulans with increased OA resistance, identifying multiple loci under selection. Cross-referencing these with a genome-wide, OA-resistance CRISPR screen in a D. melanogaster cell line highlighted two proteins: Kraken, a putative detoxification enzyme expressed in digestive and renal tissues, and Alkbh7, a mitochondrial protein linked to fatty acid metabolism. Both genes show elevated expression in D. sechellia and OA-resistant D. simulans . In D. melanogaster , kraken mutants are more OA-sensitive, while Alkbh7 overexpression increased OA resistance. Importantly, mutation of these genes in D. sechellia reduced OA tolerance. Our identification of genes underlying OA resistance in laboratory and natural contexts demonstrates how complementary, cross-species selection approaches can provide insights into toxin susceptibility and adaptation mechanisms, with practical applications in the characterization of novel insecticides.