**Background:** Bemisia tabaci MED (Mediterranean cryptic species) is a globally invasive agricultural pest that causes direct damage by feeding on plant sap and indirect damage by transmitting plant viruses. It has developed high-level resistance to neonicotinoid insecticides, particularly imidacloprid, which targets nicotinic acetylcholine receptors. Cytochrome P450 monooxygenases are major detoxification enzymes involved in insecticide metabolism, but no systematic genome-wide analysis of the P450 gene family had been conducted in B. tabaci MED prior to this study.
**Methods:** The B. tabaci MED Lingshui population (collected from Hainan Province, China in January 2017) was reared on tobacco without pesticide exposure. Whole genome sequencing was performed on the PacBio platform, yielding 105.92 Gb of clean data at ~166.16× coverage (read N50 = 23.42 Kb, average read length = 14.74 Kb). Gene prediction used three strategies: de novo prediction (Genscan, Augustus, GlimmerHMM, GeneID, SNAP), homology-based prediction (GeMoMa, Hisat, Stringtie, TransDecoder, GeneMarkS-T), and transcriptome-based prediction (PASA). Predicted genes were annotated against NR, KOG, GO, KEGG, and TrEMBL databases by BLAST (e-value 1e-5). P450 candidates were confirmed by NCBI CDD analysis. Phylogenetic trees were constructed using MEGA7 (neighbor-joining method, 1000 bootstrap replicates) and conserved motifs were analyzed by MEME. For expression analysis, whitefly adults were fed 50 ppm imidacloprid for two days. RNA was extracted from 100 adults using TRIzol, cDNA was synthesized, and RT-qPCR was performed using the 2^−ΔΔCT method. For RNAi, dsRNA targeting five selected P450 genes (CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, CYP4CS6) and EGFP (control) was synthesized using T7 RiboMAX Express RNAi kit and fed to adults for 48 hours. Bioassays used 100 ppm imidacloprid, with mortality recorded after 24 hours. Statistical analysis used Student's t-test and ANOVA in SPSS v.21 (p < 0.05).
**Key Results:** Twenty-four P450 genes were identified in the B. tabaci MED genome, classified into four families: CYP4, CYP6, CYP9, CYP301-318, and CYP18a, belonging to four clans (Clan 2, 3, 4, and M). The CYP4 and CYP6 families were the largest. After 50 ppm imidacloprid treatment, nine genes were significantly upregulated: CYP4CS2, CYP4CS5, CYP4CS6, CYP4CS8, CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP6EN1. Five of these (CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP4CS6) were selected for RNAi. qPCR confirmed significant suppression of these genes after two days of dsRNA feeding. In bioassays with 100 ppm imidacloprid, silencing each of these five genes significantly increased mortality compared to dsEGFP controls (p < 0.05).
**Clinical Implications:** This study provides the first genome-wide identification of the P450 gene family in B. tabaci MED, establishing a genomic database of 24 P450 genes. The confirmation that CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP4CS6 directly contribute to imidacloprid resistance identifies these genes as potential targets for RNAi-based pest control strategies or for designing new insecticides that circumvent P450-mediated detoxification. The findings also support the conserved role of CYP4 and CYP6 family genes in xenobiotic metabolism across insect species. Further research is needed to understand the molecular interactions between imidacloprid-related compounds and these P450 proteins to guide rational pesticide design.