Cytochrome P450 monooxygenase (CYP) is one of the three detoxification metabolic enzymes in insects, and is involved in the metabolism and transformation of endogenous substances as well as the activation and degradation of exogenous compounds. This study aims to reveal the molecular mechanism of CYP9E2 in Tribolium castaneum in adapting to high-CO₂ stress. By predicting the sequence function of CYP9E2, analyzing the temporal and spatial expression profile of TcCYP9E2, and using RNAi to silence TcCYP9E2 combined with a high-CO₂ stress treatment, we measured the carbohydrate content, trehalase activity, and gene expression levels in trehalose metabolism of T. castaneum. A bioinformatics analysis showed that the predicted molecular weight of the protein encoded by TcCYP9E2 is 60.15, the theoretical isoelectric point is 8.63, there is no signal peptide, and the protein is hydrophilic. An evolutionary tree analysis showed that TcCYP9E2 belongs to the CYP6 family and belongs to the CYP3 group; and the spatiotemporal expression profile results showed that TcCYP9E2 was highly expressed in the larvae midgut 48 h after injection of dsCYP9E2, with survival rates decreasing with the increase in CO₂ concentration. Under the condition of 75% CO₂, the contents of glycogen, glucose, ATP, and membrane-bound trehalase decreased significantly after the injection of dsCYP9E2. The expression of TRE-1, TRE-2, and GP in trehalose metabolism and energy pathways was significantly downregulated.

Spodoptera frugiperda was used to investigate its role as a trehalase inhibitor of three new thiothiazolidone compounds—6d, 6e, and 6f. The development and reproduction of S. frugiperda were investigated to evaluate the potential of these compounds as a novel pesticide for pest control. Compounds 6d, 6e, and 6f were injected into larvae and female pupae. Molecular changes in trehalose and chitin metabolism genes were monitored. Pupal trehalase activity, the female pupal emergence rate, ovarian development, and SfVg and SfVgR gene expression levels were assessed. The results revealed that all three compounds significantly inhibited trehalase activity in the larvae. The expression of TRE was significantly downregulated, and compounds 6d and 6f significantly downregulated the expression of TRE2. Treated larvae exhibited significantly decreased survival rates and a higher incidence of abnormalities. The egg production and hatching rates were markedly diminished by the inhibitors, and the ovaries displayed blackening and clumping. These compounds exhibit promise as eco-friendly insecticides, but further experiments are required to test their multifaceted capabilities.