CRISPR/Cas9 knock-out of nAChR α6 confers resistance to spinosyns in Frankliniella occidentalis and is associated with a higher fitness cost than target-site mutation G275E

Thrips are major agricultural pests globally and spinosyn insecticides like spinosad and spinetoram are commonly used for their control. However, numerous cases of resistance have emerged, often linked to mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit, the main molecular target of spinosyns. In this study, toxicological data for spinosad and spinetoram were obtained from a susceptible strain of Frankliniella occidentalis, as well as two field-collected resistant strains carrying the G275E resistance mutation. Notably, a new candidate resistance mutation never reported before, T202A, was identified in one of the field collected populations and its possible role in resistance is discussed. Further, CRISPR/Cas9-mediated knockout (KO) of α6 was performed in the susceptible strain to shed light on the phenotypic strength of this resistance mechanism previously observed in the field. The KO conferred complete insensitivity to spinosad and significant resistance to spinetoram, although higher doses of spinetoram remained lethal, suggesting potential interaction with a secondary target. Finally, in an experimental evolution approach, the α6 KO allele rapidly disappeared, indicating a substantial fitness cost. In contrast, G275E alleles persisted.