The mechanism of reactive oxygen species generation, DNA damage and apoptosis in hemocytes of Litopenaeus vannamei under ammonia nitrogen exposure

Ammonia-N poses a significant threat to aquatic animals. However, the mechanism of ROS production leading to DNA damage in hemocytes of crustaceans is still unclear. Additionally, the mechanism that cells respond to DNA damage by activating complex signaling networks has not been well studied. Therefore, we exposed shrimp to 0, 2, 10, and 20 mg/L NH4Cl for 0, 3, 6, 12, 24, 48, and 72 h, and explored the alterations in endoplasmic reticulum stress and mitochondrial fission, DNA damage, repair, autophagy and apoptosis. The findings revealed that ammonia exposure led to an increase in plasma ammonia content and neurotransmitter content (DA, 5-HT, ACh), and significant changes in gene expression of PLC and Ca2+ levels. The expression of disulfide bond formationrelated genes (PDI, ERO1) and mitochondrial fission-related genes (Drp1, FIS1) were significantly increased, and the unfolded protein response was initiated. Simultaneously, ammonia-N exposure leads to an increase in ROS levels in hemocytes, resulting in DNA damage. DNA repair and autophagy were considerably influenced by ammonia-N exposure, as evidenced by changes in DNA repair and autophagy-related genes in hemocytes. Subsequently, apoptosis was induced by ammonia-N exposure, and this activation was associated with a caspasedependent pathway and caspase-independent pathway, ultimately leading to a decrease in total hemocytes count. Overall, we hypothesized that neurotransmitters in the plasma of shrimp after ammonia-N exposure bind to receptors on hemocytes membrane, causing endoplasmic reticulum stress through the PLC-IP3R-Ca2+ signaling pathway and leading to mitochondrial fission. Consequently, this process resulted in increased ROS levels, hindered DNA repair, suppressed autophagy, and activated apoptosis. These cascading effects ultimately led to a reduction in total hemocytes count. The present study provides a molecular support for the understanding of the detrimental toxicity of ammonia-N exposure to crustaceans.