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BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)‐mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens. RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1‐KO) with a 5‐bp deletion and 1‐bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl‐R), the susceptibility of knockout strain Nl6CS1‐KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3‐, 3.4‐, 7.0‐, 4.2‐ and 3.9‐fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1‐KO strain resembled the Nl‐R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl‐R strain in the survival rate of eggs and nymphs, reproduction, and body weight. CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance. [Display omitted] • A knock-in strain (Nl-G932C) of Nilaparvata lugens was created using CRISPR/Cas9. • Nl-G932C showed a 94.9-fold resistance to buprofezin compared to susceptible strain. • Nl-G932C showed fitness disadvantage with low survivorship, emergence and fecundity. • The low fitness may help manage buprofezin resistance of N. lugens in the field. [ABSTRACT FROM AUTHOR]