Chemosensory proteins (CSPs) can bind and transport odorant molecules and play important roles in insect chemoreception. In this study, we focused on the roles of a chemosensory protein (BodoCSP1) in perception of host plant volatiles in Bradysia odoriphaga. The expression of BodoCSP1 was significantly higher in adults than in larvae and pupae, without a significant difference between male and female adults. Recombinant protein BodoCSP1 exhibited relatively high binding affinities to 9 out of 10 tested ligands (K i < 10 μM). Behavioral assays revealed that adults of B. odoriphaga showed a significant preference for five compounds. The predicted three-dimensional (3D) structure of BodoCSP1 has the typical six α-helices that form the hydrophobic ligand-binding pocket. Molecular docking and site-directed mutagenesis combined with ligand-binding assays indicated that Val48 and Thr66 may be the key binding site in BodoCSP1 for host plant volatiles. RNAi results indicated that dsBodoCSP1-treated adults showed significant reductions in response to diallyl disulfide, dipropyl disulfide, and allyl methyl disulfide. These results indicated that BodoCSP1 plays essential functions in the perception of host plant volatiles in B. odoriphaga.

BACKGROUND Insect cytochrome P450 monooxygenases (P450s) play a key role in the detoxification metabolism of insecticides and their overexpression is often associated with insecticide resistance. Our previous research showed that the overexpression of four P450 genes is responsible for clothianidin resistance in B. odoriphaga. In this study, we characterized another P450 gene, CYP6FV21, associated with clothianidin resistance. However, the molecular basis for the overexpression of P450 genes in clothianidin‐resistant strain remains obscure in B. odoriphaga. RESULTS In this study, the CYP6FV21 gene was significantly overexpressed in the clothianidin‐resistant (CL‐R) strain. Clothianidin exposure significantly increased the expression level of CYP6FV21. Knockdown of CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to clothianidin. The transcription factor Cap ‘n’ Collar isoform‐C (CncC) was highly expressed in the midgut of larvae in B. odoriphaga. The expression level of CncC was higher in the CL‐R strain compared with the susceptible (SS) strain. Clothianidin exposure caused reactive oxygen species (ROS) accumulation and significantly increased the expression level of CncC. Knockdown of CncC caused a significant decrease in the expression of CYP3828A1 and CYP6FV21, and P450 enzyme activity, and led to a significant increase in mortality after exposure to lethal concentration at 30% (LC30) of clothianidin. After treatment with CncC agonist curcumin, the P450 activity and the expression levels of CYP3828A1 and CYP6FV21 significantly increased, and larval sensitivity to clothianidin decreased. The ROS scavenger N‐acetylcysteine (NAC) treatment significantly inhibited the expression levels of CncC, CYP3828A1 and CYP6FV21 in response to clothianidin exposure and increased larval sensitivity to clothianidin. CONCLUSION Taken together, these results indicate that activation of the CncC pathway by the ROS burst plays a critical role in clothianidin resistance by regulating the expression of CYP3828A1 and CYP6FV21 genes in B. odoriphaga. This study provides more insight into the mechanisms underlying B. odoriphaga larval resistance to clothianidin. © 2023 Society of Chemical Industry.