*2.12. Functional Analysis of CYP6FD1 and CYP4FD2 via RNAi*

The results indicated that the relative normalized expression of *CYP6FD1* and *CYP4FD2* was significantly lower than that of dsGFP (*p* < 0.05) at 24 h after treatment, only reaching 0.72- and 0.68-fold, respectively. With prolongation of the interference time (48–72 h), the RNAi efficacy of *CYP6FD1* and *CYP4FD2* dsRNA became greater, with relative normalized expression of 0.42- and 0.55-fold for *CYP6FD1* and *CYP4FD2,* respectively, at 48 h to 72 h after injection, while that of *dsCYP6FD1* was higher than that of *dsCYP4FD2* (*p* < 0.05). However, the RNAi efficacy of *CYP6FD1* and *CYP4FD2* dsRNA gradually decreased, with the relative normalized expression of 0.61- and 0.71-fold for *CYP6FD1* and *CYP4FD2*, respectively, at 96 h after injection (Figure 7A).

The results of the bioassay showed that after 72 h of treatment with sulfoxaflor at the LC50, the mortality of the SF-Sel strain treated with *CYP6FD1* dsRNA was 65.89% ± 5.38, which was significantly higher than the mortality of the same strain treated with *CYP4FD2* dsRNA (48.14% ± 4.33) and that in the control treatment (dsGFP, 38.95% ± 3.07) (*p* < 0.05). With prolongation of the interference time (96 h), the mortality of the *CYP6FD1* and *CYP4FD2* dsRNA treatment groups increased, reaching 62.01% ± 2.12~77.94% ± 5.30, respectively; this difference was also significant (*p* < 0.05), and mortality in these groups was significantly higher than under treatment with dsGFP (48.14% ± 0.93) (Figure 7B).

**Figure 7.** RNA interference and biological activity of two major P450 genes. (**A**)—RNA interference efficiency; (**B**)—Activity of sulfoxaflor against post-RNAi insects. Different letters (a, b, c) above bars indicate significant differences (*p* < 0.05) according to Duncan's multiple range test. The relative normalized expression of *dsGFP*, *dsCYP6FD1* and *dsCYP4FD2* in 3rd-instar nymph of *S furcifera,* and the mortalities for each treatment are presented as the mean of three replications ± *SE*. Means followed by the same letters did not differ significantly (*p* > 0.05) according to the ANOVA test. The F2, 6 values of relative normalized expressions at 24 h, 48 h, 72 h, 96 h and mortlities at 72 h, 96 h for different treatments were 158.933, 230.377, 164.477, 51.036 and 9.949, 19.901, respectively. and the corresponding *p* values were < 0.0001, < 0.0001, < 0.0001, < 0.0001, and = 0.012 < 0.05, = 0.02 < 0.05, respectively.
