Metabolic Remodeling of the Tricarboxylic Acid Cycle and Glycolysis Reveals Cold-Induced Respiratory Adaptations in Streltzoviella insularis (Staudinger) (Lepidoptera: Cossidae) Larvae.

Global climate change is pushing insects into colder regions. Understanding their cold tolerance is important for predicting population dynamics. During overwintering, Streltzoviella insularis larvae activate the AMPK signaling pathway. This suggests that energy metabolism plays a key role under cold stress. In this study, we used enzyme activity assays, LC-MS-based targeted metabolomics, and transcriptome sequencing. We focused on six key enzymes in glycolysis and the TCA cycle. We also measured related metabolites and regulatory genes. Hexokinase (HK) and citrate synthase (CS) activities were highly sensitive to temperature. HK increased then markedly decreased; CS was significantly downregulated. Pyruvate kinase (PK), isocitrate dehydrogenase (IDH), and α-ketoglutarate dehydrogenase (KGD) showed trends that matched changes in larval cold tolerance, exhibiting an up-down-up expression trend. Glycolytic metabolites (glucose-6-phosphate, fructose-6-phosphate, 1,6-fructose-diphosphate, phosphoenolpyruvic acid) peaked at -10 °C. TCA intermediates (citrate, acetyl-CoA, α-ketoglutaric acid, and isocitrate) were more abundant at 0-4 °C. Pyruvate increased significantly. PYR content showed a significant increase followed by a decrease, peaking at 0 °C. It was converted into lactate and acetyl-CoA. ATP levels dropped and then increased, reaching their lowest level at 0 °C. These results suggest a shift from aerobic to mixed aerobic-anaerobic metabolism. Transcriptome data showed differential expression of key metabolic genes such as phosphoenolpyruvate carboxykinase, phosphoglycerate kinase, and ATP synthase subunit beta. These gene changes supported the trends in enzymes and metabolites. Our findings reveal a coordinated metabolic and transcriptional response to cold. This provides a basis for understanding the cold adaptation and potential range expansion of S. insularis.