miR-210–5p Regulates <i>CYP6ER1</i> Expression To Confer Clothianidin Resistance in <i>Nilaparvata lugens</i>

Insecticide resistance poses a critical challenge to crop protection, and the brown planthopper has developed substantial resistance to neonicotinoids, including clothianidin. To investigate the role of miRNAs in clothianidin resistance, we performed small RNA sequencing on resistant (CLR) and susceptible (CLS) strains of <i>Nilaparvata lugens</i>. A total of 206 differentially expressed miRNAs (DEmiRNAs) were identified, including 73 upregulated and 133 downregulated miRNAs in the CLR strain. Bioinformatic analysis predicted that miR-993-3p, novel_919, and miR-210-5p, could target the detoxification gene <i>CYP6ER1</i>. Dual-luciferase reporter assays confirmed the direct binding of these miRNAs to the coding sequence of <i>CYP6ER1</i>. Functional validation showed that miR-210-5p agomir significantly reduced <i>CYP6ER1</i> expression and increased clothianidin susceptibility, whereas antagomir treatment produced the opposite effect. These results indicate that miR-210-5p was involved in the post-transcriptional regulation of <i>CYP6ER1</i> and contribute to clothianidin resistance. This study provides insights into the mechanisms underlying insecticide resistance and identifies potential miRNA targets for improved pest management strategies.