Integrated metabolomic and cuticular hydrocarbon analyses reveal physiological reprogramming associated with...

Tribolium castaneum is a major stored-product pest with strong phosphine (PH₃) resistance. Here, PH₃-resistant (PH₃-R) and PH₃-susceptible (PH₃-S) strains were compared to elucidate transcriptional, biochemical, metabolic, cuticular, physiological, and behavioral changes associated with PH₃ resistance. In the PH₃-R strain, strongly upregulated detoxification-related genes included one Phase I gene (Cyp6bq9) and three Phase II genes (Gst, Ugt5-2, and Ugt5-3), with 8.20-, 4.13-, 2.92-, and 5.33-fold increases, respectively. Total AMPKα abundance was reduced in the PH₃-R strain, while RNAi-mediated 98% knockdown of Cyp6bq9 did not significantly alter PH₃-induced mortality. Reactive oxygen species production was significantly reduced under PH₃ pressure. Enzyme activities such as carboxylesterase (2.63-fold), glutathione S-transferase (1.83-fold), and catalase (1.88-fold) were substantially reduced in the PH₃-R strain compared to the PH₃-S strain. Cytochrome c oxidase (COX) activity, which has been implicated in PH₃ toxicity, was also lower in the PH₃-R strain. Metabolomics analysis revealed that pathways related to glycolysis and the TCA cycle were enriched in the PH₃-R strain. Levels of non-methylated cuticular hydrocarbons (C25-C35) were elevated, suggesting changes in cuticular hydrocarbon composition. The PH₃-R strain had 15.4% lower body water content than the PH₃-S strain. Interestingly, both strains showed similar responses under desiccation stress. Preliminary DeepLabCut analysis suggested differences in locomotor responses between the two strains under PH₃ exposure, with PH₃-R individuals showing higher angular velocity at later exposure and post-ventilation periods. Collectively, these findings characterize multiple resistance-associated phenotypes in T. castaneum and support a multifactorial interpretation rather than a single newly identified resistance mechanism.