Tuta absoluta has developed resistance to many biological insecticides, causing substantial agricultural and economic losses annually. P450s have been the most extensively studied enzymes in the context of insecticide metabolism in insect pests, and the detoxification metabolism of P450s in T. absoluta against biological insecticides remains poorly understood. In T. absoluta, CYP304F1 was screened from the comparative transcriptome of 2 regional populations in Xinjiang, China. The objective of the present study was to characterize and analyze CYP304F1 of T. absoluta and explore its role in detoxification of spinetoram as well as the growth and development of T. absoluta. Following cloning and sequence analysis of the target gene, it was named CYP304F1. Expression levels of CYP304F1 were then determined after spinetoram exposure and across various developmental instars and tissues. Finally, dsCYP304F1 was synthesized and utilized to assess the effects of post-RNAi on larval spinetoram susceptibility, growth, and development. Sequence analysis revealed that CYP304F1 harbors conserved domains characteristic of P450 proteins, exhibiting high conservation within the Lepidoptera clade. Treatment with an LC₅₀ dose of spinetoram significantly upregulated CYP304F1 expression in T. absoluta larvae. Silencing CYP304F1 significantly enhanced larval susceptibility to spinetoram and prolonged leaf-mining duration and developmental time from the 2nd instar to 4th instar by 40% and 17.6%, respectively, compared to controls. And feeding on dsCYP304F1-treated leaves for 6 days resulted in 71% larval mortality. These results suggested that CYP304F1 played a crucial role in detoxification of spinetoram as well as in the growth and development of T. absoluta larvae.

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