Cloning and expression of CRH genes in Epinephelus coioides and its potential application in stress resistance evaluation

Corticotropin-releasing hormone (CRH) is a hypothalamic regulatory peptide that modulates the hypothalamic-pituitary-interrenal (HPI) axis and mediates stress responses in fish. Fluctuations in environmental factors such as temperature, ammonia nitrogen, and dissolved oxygen are among the most common triggers of fish stress, which can disrupt homeostasis, increase disease susceptibility and mortality, and thereby constrain the sustainable development of grouper aquaculture. To identify more suitable stress tolerance indicators for timely assessment of fish stress status in grouper farming, this study focused on CRH to evaluate its response characteristics under environmental stress and its feasibility as a candidate indicator. To this end, we cloned and characterized the CRH gene from orange spotted grouper (Epinephelus coioides) and examined its temporal expression patterns alongside serum physiological and biochemical parameters under 48 h acute challenges of high temperature, high ammonia, and low oxygen. The results showed that the open reading frame (ORF) of CRH is 516 bp in length, encoding 171 amino acids with a conserved CRF domain. Following acute stress, CRH expression in the gill, liver, and brain tissues exhibited significant stress responsive changes, but with distinct tissue specific patterns. Under high temperature and high ammonia stress, gill expression showed a bimodal fluctuation, liver expression rose initially then declined before stabilizing, and brain expression increased, then decreased, and rose again or continued to rise; under hypoxia, all three tissues displayed a unimodal pattern of initial increase followed by a decrease, with markedly smaller fluctuation amplitudes. The magnitude of stress response was positively correlated with stress intensity, and fish showed strong tolerance and adaptation to hypoxic conditions. This study clarifies the gene sequence and evolutionary conservation of CRH in E. coioides, reveals its response patterns to environmental stress, and preliminarily demonstrates that CRH has potential and feasibility as a stress tolerance evaluation indicator for grouper.