Field resistance monitoring and nAChR α6 target validation support spinetoram as a candidate insecticide for rotation in Nilaparvata lugens resistance management.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a major pest threatening rice production in Asia. Severe resistance to commonly used insecticides has become a major challenge for its effective control, highlighting the need for alternative insecticides. In this study, we evaluated the susceptibility of field populations of N. lugens to spinetoram and functionally validated the nicotinic acetylcholine receptor (nAChR) α6 subunit, Nlα6, as a molecular determinant of spinosyn susceptibility. Resistance monitoring showed that field populations remained highly susceptible to spinetoram despite exhibiting moderate to high resistance to several commonly used insecticides. A homozygous Nlα6 knockout strain generated using CRISPR/Cas9 developed high-level resistance to spinosad and spinetoram, with resistance ratios of 554.1 and 357.1, respectively, but did not show broad cross-resistance to nine other nAChR-targeting insecticides. Co-segregation analysis further confirmed tight linkage between Nlα6 loss of function and spinosyn resistance. Inheritance analysis showed that spinetoram resistance mediated by Nlα6 knockout was autosomal and completely recessive. Transcriptome analysis revealed that Nlα6 deficiency was associated with reduced expression of immune defense genes, enrichment of functions related to axonemes and neural processes, and coordinated activation of energy metabolism pathways, suggesting that Nlα6 disruption may alter neural homeostasis and energy demand. These results demonstrate that Nlα6 is a key subunit mediating spinosyn susceptibility in N. lugens and support spinetoram as a candidate rotation insecticide for resistance management.