miRNAs modulate altered expression of cytochrome P450s and nicotinic acetylcholine receptor subunits conferring both metabolic and target resistance to sulfoxaflor in Nilaparvata lugens (Stål).

Understanding the insecticide resistance mechanisms and their underlying regulatory pathways is essential for pest management. Previous findings indicated that the overexpression of P450 gene, CYP6ER1, was a key mechanism for sulfoxaflor metabolic resistance in Nilaparvata lugens. However, it remains unclear whether quantitative changes in the target nicotinic acetylcholine receptors (nAChRs) contribute to sulfoxaflor resistance and the underlying regulatory mechanisms involved. Here, qRT-PCR, pairwise correlation analyses and RNAi confirmed that the down-regulation of Nlα4, along with the up-regulation of Nlα10 and Nlβ1, were linked to sulfoxaflor resistance in N. lugens. Four microRNAs, novel-m0262-5p, novel-m0071-3p and novel-m0196-3p, and miR-10471-x were found to target CYP6ER1, Nlα4 and Nlβ1, respectively. Subsequently, the binding activity between these miRNAs and their target genes was verified by dual fluorescence in vitro. Over-supplementation of novel-m0262-5p and miR-10471-x via miRNA agomir injections suppressed the expression of CYP6ER1 and Nlβ1, and decreased nymph resistance to sulfoxaflor. Conversely, novel-m0262-5p and miR-10471-x antagomirs treatment induced the expression of CYP6ER1 and Nlβ1, thereby enhancing sulfoxaflor resistance. Additionally, overexpression of novel-m0071-3p and novel-m0196-3p inhibited Nlα4 expression and increased sulfoxaflor resistance. These findings indicate that miRNAs regulate the differential expression of P450s and nAChRs, mediating both metabolic and target resistance to sulfoxaflor in N. lugens.