Integrated untargeted metabolomics and physiological analysis reveal the response mechanisms of Procambarus clarkii larvae to carbonate alkalinity exposure

High alkalinity stress represents a critical yet understudied threat to aquatic organisms in saline-alkaline ecosystems. This study provides the first comprehensive investigation of carbonate alkalinity tolerance mechanisms in larval Procambarus clarkii, employing an innovative dual-approach methodology. Two experiments were conducted: the first aimed to determine the semi-lethal concentration (LC₅₀) and safe concentration (SC) of alkalinity for larval P. clarkii, and the second to evaluate the physiological and biochemical enzyme activities and metabolic level under 96 h acute alkalinity stress. The LC₅₀ alkalinity values of larval P. clarkii at 24, 48, 72, and 96 h were 25.57, 21.43, 18.40 and 16.03 mmol/L, respectively. The results of biochemical and physiological analyses showed that alkalinity stress disrupted oxidative immune balance, and physiological metabolism leading to increased superoxide dismutase (SOD), alkaline phosphatase (AKP), acid phosphatase (ACP), Na⁺-K⁺-ATPase, succinate dehydrogenase (SDH), glutamine synthetase (GS), glutamate dehydrogenase (GLDH), glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activities and malondialdehyde (MDA) contents and decreased catalase (CAT) and glycogen activities. Metabolomics analysis showed that in comparison with the control group, 414 (70 up- and 344 downregulated) and 675 (67 up- and 608 downregulated) significantly differential metabolites were identified in the A6 and A12 experimental groups, respectively. Our findings suggest that P. clarkii possess a certain level of tolerance to alkalinity stress; however, exposure to high alkalinity stress can adversely affect the antioxidant defense system, energy metabolism and immune response of crayfish. Our integrated approach provides a new framework for assessing crustacean resilience to saline-alkaline stress, with direct implications for aquaculture management under climate change scenarios.