Cloning and Functional Verification of CYP408A3 and CYP6CS3 Related to Chlorpyrifos Resistance in the Sogatella furcifera (Horváth) (Hemiptera: Delphacidae)

SIMPLE SUMMARY: As an important enzyme system in organisms, P450 multi-functional oxidase not only participates in the metabolism and synthesis of substances in organisms but can also maintain the normal physiological functions of organisms under stress. As one of the important rice pests, the harm caused by white-backed planthoppers has been increasing in recent years. Although the application of chemical pesticides as one of the prevention and control measures can slow down the harm of white-backed planthoppers, its resistance is also rising rapidly. Among them, the generation of metabolic resistance dominated by the P450 enzyme is more common. In this study, we measured the expression of ten P450 gene in vivo situations with a background of chlorpyrifos resistance in white-backed planthoppers. After selecting the two genes with the highest expression, the function of these two genes in the pesticide resistance process was verified by RNA interference and provided a theoretical basis for a follow-up study of the molecular mechanism of the P450 gene mediated by pesticide resistance formation. ABSTRACT: The white-back planthopper (WBPH), Sogatella furcifera, mainly harms rice and occurs in most rice regions in China and Asia. With the use of chemical pesticides, S. furcifera has developed varying degrees of resistance to a variety of pesticides. In our study, a chlorpyrifos-resistant population (44.25-fold) was built through six generations of screening with a sublethal dose of chlorpyrifos (LD(50)) from a field population. The expression levels of ten selected resistance-related P450 genes were analyzed by RT-qPCR and found that CYP408A3 and CYP6CS3 were significantly more expressed in the third instar nymphs of the XY17-G5 and XY17-G6 populations, about 25-fold more than the Sus-Lab strain, respectively (p < 0.01). To elucidate their molecular function in the development of resistance towards chlorpyrifos, we cloned two P450 full lengths and predicted their tertiary protein structures. CYP408A3 and CYP6CS3 were also downregulated after injecting dsCYP408A3, dsCYP6CS3, or their mixture compared to the control group. Moreover, the mortality rates of the dsCYP6CS3 (91.7%) and the mixture injection treatment (93.3%) treated by the LC(50) concentration of chlorpyrifos were significantly higher than the blank control group (51.7%) and dsCYP408A3 injection treatment (69.3%) at 72 h (p < 0.01). Meanwhile, the P450 enzyme activities in the dsRNA treatments were lower than that in the control (XY17-G6) (p < 0.01). Therefore, the P450 gene CYP6CS3 may be one of the main genes in the development of chlorpyrifos resistance in S. furcifera.