Expression and Functional Analysis of Two Cytochrome P450 Monooxygenase Genes and a UDP-Glycosyltransferase Gene Linked with Thiamethoxam Resistance in the Colorado Potato Beetle

Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are involved in the evolution of insecticide resistance. Leptinotarsa decemlineata (Say), the Colorado potato beetle (CPB), is a notorious insect that has developed resistance to various insecticides including neonicotinoids. This study investigated whether the differentially expressed P450 genes CYP9Z140 and CYP9AY1 and UGT gene UGT321AP1, found in our transcriptome results, conferred resistance to thiamethoxam in L. decemlineata. Resistance monitoring showed that the sampled field populations of L. decemlineata adults collected from Urumqi City and Qapqal, Jimsar, and Mulei Counties of Xinjiang in 2021-2023 developed low levels of resistance to thiamethoxam with resistance ratios ranging from 6.66- to 9.52-fold. Expression analyses indicated that CYP9Z140, CYP9AY1, and UGT321AP1 were significantly upregulated in thiamethoxam-resistant populations compared with susceptible populations. The expression of all three genes also increased significantly after thiamethoxam treatment compared with the control. Spatiotemporal expression patterns showed that the highest expression of CYP9Z140 and CYP9AY1 occurred in pupae and the midgut, whereas UGT321AP1 was highly expressed in adults and Malpighian tubules. Knocking down all three genes individually or simultaneously using RNA interference increased the sensitivity of adult L. decemlineata to thiamethoxam. These results suggest that overexpression of CYP9Z140, CYP9AY1, and UGT321AP1 contributes to the development of thiamethoxam resistance in L. decemlineata and provides a scientific basis for improving new resistance management of CPB.