Carbonate Alkalinity Stress Induces Hepatopancreas Injury and Activates TLR2-MyD88-NF-κB-Related Responses in Chinese Mitten Crab

Global freshwater salinization endangers aquatic species, yet its impacts on crustaceans remain poorly understood. This study investigated the hepatopancreatic response of Eriocheir sinensis to carbonate alkalinity stress (0, 4.375, 8.75, 17.5, and 35 mmol/L) over 24, 48, and 96 h, integrating histology, ultrastructure, gene expression (RT-qPCR), and non-specific immune enzyme assays. Histopathological and ultrastructural analyses revealed concentration- and time-dependent damage, including vacuolization, hepatic tubule disintegration, nuclear condensation, mitochondrial reduction, and loss of cellular integrity. Molecular analysis demonstrated upregulation of genes associated with the TLR2-MyD88-NF-κB pathway and inflammatory genes (LITAF, IL-16), alongside increased HSP70 expression, confirming severe inflammation and cellular stress. Furthermore, apoptosis was induced via upregulated Bax and Caspase-3, downregulated Bcl-2, and DNA fragmentation. Non-specific immune responses in the hepatopancreas exhibited dynamic changes: acid phosphatase (ACP) was initially activated at low alkalinity but inhibited at high concentrations, while alkaline phosphatase (AKP) activity increased at 96 h. Notably, the hepatopancreas proved more sensitive to this stress than the hemolymph. Collectively, carbonate alkalinity causes multidimensional hepatopancreatic injury in E. sinensis through structural disruption, inflammation mediated by TLR2-MyD88-NF-κB signaling pathway-related genes, apoptosis induction, and immune enzyme dysregulation, posing a significant threat to crab health in salinized waters.