RNA-seq-based identification of imidacloprid resistance mechanisms in Nilaparvata lugens and development of diagnostic tools for the CYP6ER1 variant

Neonicotinoid insecticides, such as imidacloprid, have been used for over three decades to control major agricultural pests, including brown planthopper (Nilaparvata lugens), aphids, and other sap-feeding insects. However, the extensive use of imidacloprid has led to widespread resistance across Asia in many pest populations. Bioassay results demonstrated significant resistance in N. lugens, with a resistance ratio of 32. RNA-seq analysis revealed that the overexpression of detoxification enzyme genes plays a central role in resistance, with CYP6ER1 being the most significantly upregulated gene, showing a 28-fold increase in resistant strain compared to susceptible strain. Among detoxification enzymes, carboxylesterases and glutathione-S-transferases exhibited slightly resistance-specific overexpression, while others remained unchanged. To date, six main variants of CYP6ER1 have been reported. In the current study, CYP6ER1vA has been identified as the predominant variant across all resistant strains, field populations collected in 2024, and museum samples from 1975. This suggests that the vA variant predated the widespread use of imidacloprid and that its expression level, rather than its mere presence, is the key factor driving resistance. A LAMP-PCR diagnostic method was developed to detect CYP6ER1vA, offering a rapid and reliable tool for field-based resistance monitoring. These findings support systematic imidacloprid resistance management in N. lugens, integrating molecular diagnostic with sustainable pest control strategies.