Agonistic Behavior and Energy Metabolic Responses of Eriocheir sinensis Under Thermal Stress with Insights from Untargeted Metabolomics

The Chinese mitten crab (Eriocheir sinensis) is an important freshwater aquaculture species in China, but high summer temperatures may disrupt its behavioral patterns and energy metabolism. In this study, male E. sinensis were maintained at 25 °C or 35 °C, and behavioral tracking, histological observation, biochemical assays, RT-qPCR, and untargeted metabolomics were combined to investigate the effects of thermal stress on swimming and aggressive behaviors and their associated metabolic responses. For swimming behavior, compared with 25 °C, crabs at 35 °C showed decreases in total moving distance, average speed, and the proportions of slow, medium, and fast-speed movement, together with a significant increase in inactive time (p < 0.05). For agonistic behavior, compared with 25 °C, both the overall proportion of agonistic intensity and the proportion of strong fighting decreased significantly at 35 °C (p < 0.05). According to agonistic behavior data, the crabs were classified as winners or losers, generating four groups: T25W, T25L, T35W, and T35L. Non-contact behaviors decreased significantly in both winners and losers, whereas contact behaviors also decreased significantly in winners (p < 0.05). Histopathological analysis revealed that, at 35 °C, E. sinensis exhibited disorganized muscle fibers and structural damage to the gills, including swelling of the distal lamellar tips and morphological abnormalities in epithelial and pillar cells. With respect to energy metabolism, muscle glycogen in winners was significantly higher than that in losers at both temperatures, whereas haemolymph lactate concentrations were significantly higher in winners only at 25 °C. Haemolymph glucose concentrations were significantly higher in winners at 25 °C but in losers at 35 °C (p < 0.05). Compared with 25 °C, exposure to 35 °C significantly decreased muscle succinate dehydrogenase (SDH) activity in both winners and losers and increased gill citrate synthase (CS) activity in winners. In losers, muscle pyruvate kinase (PK) activity decreased nonsignificantly; lactate dehydrogenase (LDH) activity increased (p < 0.05). Under 35 °C conditions, significant upregulation of AMPK, Sestrin, and SIRT1 expression and significant downregulation of mTOR expression (except in losers’ muscle) were exhibited in both muscle and gill tissues (p < 0.05). Untargeted metabolomics identified 297, 301, 484, and 393 differential metabolites in comparisons in T25W vs. T25L, T35W vs. T35L, T35W vs. T25W, and T35L vs. T25L, respectively. These differential metabolites were mainly associated with amino acid, nucleotide, and lipid metabolism, aminoacyl-tRNA biosynthesis, ABC transporters, and neuroactive ligand–receptor interaction. Collectively, acute exposure to 35 °C suppressed swimming and agonistic behavior, with this behavioral inhibition being associated with tissue damage and regulation of energy metabolism. This study provides candidate indicators and a scientific basis for the early assessment and monitoring of thermal stress in E. sinensis aquaculture.