Two point mutations in CYP4CE1 promoter contributed to the differential regulation of CYP4CE1 expression by FoxO between susceptible and nitenpyram resistant Nilaparvata lugens

Four P450s were reported to be important for imidacloprid resistance in Nilaparvata lugens , a major insect pest on rice, which was confirmed in this study in an imidacloprid resistant strain (ImiR). Here we found that only two ( CYP4CE1 and CYP6ER1 ) from these four P450 genes were over-expressed in a nitenpyram resistant strain (NitR) when compared to a susceptible strain SUS. CYP4CE1 RNAi reduced nitenpyram and imidacloprid resistance in NitR and ImiR strains, with a more reduction in nitenpyram resistance. The transcription factor FoxO mediated nitenpyram resistance in NitR and ImiR strains, but it was not differentially expressed among strains. The potential reason for the differential regulation of FoxO on CYP4CE1 expression was mainly from sequence differences in CYP4CE1 promoter between susceptible and resistant insects. In six FoxO response elements predicted in CYP4CE1 promoter, the single-nucleotide polymorphisms were frequently detected in over 50% of NitR and ImiR individuals. The luciferase reporter assays showed that two mutations, -650T/G and -2205T/A in two response elements at the position of -648 bp and -2200 bp, mainly contributed to the enhanced regulation on CYP4CE1 expression by FoxO in resistant insects. The frequency was over 69% for both -650T/G and -2205T/A detected in NitR and ImiR individuals, but less than 20% in SUS insects. In conclusion, CYP4CE1 over-expression importantly contributed to nitenpyram resistance in N. lugens , and two mutations in CYP4CE1 promoter of resistant insects led to an enhanced regulation on CYP4CE1 expression by FoxO.