Immune-physiological responses of hemocytes to NH4Cl stress in Litopenaeus vannamei: cell death, proliferation and inflammation

Ammonia-N pollution has become a worldwide concern in aquatic environments, posing threats to organisms via the effects of immunotoxicity and inflammation induction. In our study, the mechanisms of the adverse effect of ammonia-N on immune-physiological responses were investigated. Litopenaeus vannamei was exposed to multi-gradient NH₄Cl (0, 5, 10, 20 mg/L) for 0, 1, 3, 6, and 10 d. Under NH₄Cl stress, hemolymph ammonia was elevated, and plasma neuroendocrine factors acetylcholine and norepinephrine increased at 1–3 d and 6–10 d, respectively, inducing the upregulation of endoplasmic reticulum (ER) and mitochondrial stress-related genes, and causing increased reactive oxygen species (ROS) content. Moreover, apoptosis, ferroptosis, and cuprotosis-related biomarkers were enhanced in hemocytes under NH₄Cl stress. The expression of proliferation, differentiation, and migration-related genes in hemopoietic tissues was inhibited by ACh and NE during 6–10 d, leading to a decline in the total hemocyte count. The antibacterial activity and bacteriolytic activity were inhibited by PLC/NF-κB-relish pathway and TLR2/MyD88/NF-κB-relish pathway under NH₄Cl stress, while the aseptic inflammatory response was activated by ASK1/JNK/AP-1-c-Jun pathway and TLR9/NF-κB-dorsal pathway. Overall, prolonged exposure to NH₄Cl led to the intensification of hemocyte death, suppression of hematopoietic functions, and induction of inflammation in crustaceans. Our study enriched the knowledge of the immune physiological homeostasis of invertebrates under the global nitrogen pollution crisis in the aquatic environment.