*4.7. Function Analysis of CYP6FD1 and CYP4FD2 via RNAi*

Third-instar nymphs were used for dsRNA injection experiments. First, the tested insects were anesthetized with CO2 for approximately 30 s, and each insect received 120 ng (approximately 30 μL) of the dsRNA for each target gene using an UMP3/Nanoliter2010 microinjection device (World Precision Instruments, Sarasota, Florida), with dsGFP used as a negative control, and 300 3rd-instar nymphs were prepared to check the RNAi efficiency and bioassay for each gene. The relative expression of *CYP6FD1* and *CYP4FD2* was detected at 24, 48, 72 and 96 h after injection. For insecticide bioassays after RNAi, thirty 3rd-instar nymphs were collected 24 h after injection and sixty 3rd-instar nymphs for each treatment were transferred to rice seedlings that had been treated with the LC50 of sulfoxaflor in solution. Mortality was calculated at 72 h and 96 h after insecticide treatment. Three biological replicates were performed.
