The immunotoxicity mechanism of NH4Cl exposure to Litopenaeus vannamei based on the cerebral ganglion-eyestalk-haemocytes axis

Ammonia nitrogen, as a water environmental toxin, poses a potential threat to aquatic animals. Although NH4Cl stress is known to cause immunotoxicity, mechanistic pathways linking stress networks in the neuroendocrine system to immunotoxicity remain poorly understood. In this study, firstly, using transcriptome analysis of cerebral ganglion and eyestalk in shrimp, we identified significant changes in genes related to biogenic amines, acetylcholine, crustacean hyperglycemic hormones, and neuropeptide F. Additionally, expression patterns of neuroendocrine factors in different tissues of shrimp were evaluated to explore the sources of these factors. Here, we showed that NH4Cl exposure activates acetylcholine (ACh) neurons in cerebral ganglion of shrimp and dramatically upregulates high affinity choline transporter 1 (ChT1) gene expression. The knockdown of ChT1 gene enhanced the immunity of haemocytes in shrimp compared with saline and GFP dsRNA groups. And after eyestalk ablation, the levels of neuroendocrine factors in the cerebral ganglion and thoracic ganglion were disturbed, and haemocytes parameters induced by NH4Cl were significantly decreased. Combined with different doses of NH4Cl exposure experiments, we demonstrated that: (1) In a short period of NH4Cl exposure, the neuroendocrine factors CRH-ACTH-cortisol and 5-HT-DA in the cerebral ganglion-eyestalk axis of shrimp play a major role in regulating haemocytes immunity; (2) With the prolongation of exposure, the immunotoxicity induced by NH4Cl was mainly due to the release of more ACh in the cerebral ganglion, which promoted the release of NPF in the thoracic ganglion, and CHH and NPF in the eyestalk, as well as weakened the effect of biogenic amines. Subsequently, these neuroendocrine factors regulate immunity through intracellular signaling pathways. Collectively, these results established a new mechanism that NH4Cl might directly regulate haemocytes immunotoxicity through the cerebral ganglion and thoracic ganglion; or through the cerebral ganglion-eyestalk axis or cerebral ganglion-thoracic ganglion axis cause haemocytes immunotoxicity.