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

Understanding the evolution of insect resistance to natural and artificial poisons is important both to appreciate the adaptation of species to toxic ecological niches ¹ and manage pest insects ² . While resistance to chemicals that target neuronal ion channels is well-appreciated ^3,4 , dissecting toxin resistance mechanisms is hampered by its complex genetic basis. Here, we investigated how Drosophila sechellia has evolved resistance to octanoic acid (OA), a historically-important paradigm of adaptation to a natural toxin ^5,6 . OA is an abundant component of Morinda citrifolia noni fruit – the exclusive niche of D. sechellia – and is toxic to many other insects, including its close relatives Drosophila simulans and Drosophila melanogaster ^5,6 . We experimentally evolved D. simulans populations with increased OA resistance, identifying hundreds of loci displaying signatures of selection. Cross-referencing of these loci with the results of a genome-wide, CRISPR knockout screen for OA tolerance genes in D. melanogaster S2R+ cells highlighted two proteins of interest: Kraken, a putative detoxification enzyme of the serine hydrolase family expressed most strongly in the digestive and renal systems ^7,8 , and Alkbh7, a ubiquitously-expressed, mitochondrial-localised protein, whose mammalian orthologue functions in fatty acid metabolism ^9,10 . In D. sechellia , kraken and Alkbh7 display both signs of selection and elevated expression, mirrored by their higher transcription in artificially-evolved, OA-resistant D. simulans . In D. melanogaster , loss-of-function of kraken , but not Alkbh7 , led to increased susceptibility to OA; conversely, overexpression of Alkbh7 , but not kraken , increased OA resistance. Importantly, mutation of these genes in D. sechellia demonstrated a contribution of these proteins to the natural resistance of this species, identifying the first specific loci underlying this multigenic trait. Our results suggest an adaptive role of these genes in shaping OA resistance both under laboratory selection and during D. sechellia ’s evolutionary history. More generally, this work emphasises the experimental power of drosophilids to dissect multilayered molecular mechanisms of toxin resistance, with applications in the development of novel insecticides.