Chronic stress is a serious threat to aquaculture as it lowers fish growth performance and compromises fish welfare. The exact mechanism by which growth is retarded is, however, not clearly understood. This study sought to elucidate the gene expression profiles associated with chronic stress in cultured Nile tilapia (Oreochromis niloticus) reared for 70 days at different ammonia concentrations and stocking densities. Fish in the treatment groups showed negative growth, while the controls showed positive allometric growth. The specific condition factor (K_n) ranged from 1.17 for the controls to 0.93 for the ammonia and 0.91 for the stocking density treatments. RNA was extracted from muscle tissue using TRIzol followed by library construction and Illumina sequencing. Comparative transcriptome analysis revealed 209 differentially expressed genes (DEGs) (156 up- and 53 down-regulated) in the ammonia and 252 DEGs (175 up- and 77 down-regulated) in the stocking density treatment. In both treatments, 24 and 17 common DEGs were up- and down-regulated, respectively. DEGs were significantly enriched in six pathways associated with muscle activity, energy mobilization and immunity. The heightened muscular activity consumes energy which would otherwise have been utilized for growth. These results bring to fore the molecular mechanisms underlying chronic stress’ suppression of growth in cultured Nile tilapia.

Chronic stress poses a significant challenge to aquaculture production, and cortisol and glucose have traditionally served as stress markers. However, recent doubts have emerged about the reliability of scale cortisol as a chronic stress determinant due to its involvement in calcium homeostasis. While cortisol influences gene expression in the stress response cascade, few studies explore its impact on metabolic pathways in cultured Nile tilapia. This study delves into the relationship between cortisol signatures of chronic stress in Nile tilapia and the regulation of the Mitogen Activated Protein Kinase (MAPK) signalling pathway, crucial for cell processes. Juvenile Nile tilapia were subjected to different ammonia concentrations and stocking densities for 70 days, with assessments of growth performance, stress levels, RNA sequencing, and differential gene expression. The results revealed a positive correlation between stressors and blood glucose, plasma cortisol, and scale cortisol concentrations. Fish in water with 0.8 mg/L ammonia exhibited elevated plasma glucose and cortisol, while those in 1.2 mg/L showed increased scale cortisol. Higher stocking densities also correlated with elevated stress markers. Notably, cortisol levels rose with ammonia concentration and stocking density, negatively impacting growth performance. The MAPK signalling pathway, linked to cell processes, showed significant downregulation with increasing ammonia concentrations, suggesting its sensitivity to stress. Taken together, six genes in this pathway were significantly enriched following ammonia treatment (Padj less than or equal to 0.05) i.e. Dual Specific Protein Phosphatase 1 (DUSP1), Nuclear Hormone Receptor 38 (NHR38), Heat Shock Protein 1 (HSP 72 KDa), Myelocytomatosis oncogene homologue (Mych), Growth arrest and DNA damage inducible alpha a (GADD 45aa) and Mitogen Activated Protein Kinase 4 (MAP2K4). This study contributes to unravelling the intricate relationship between chronic stress, cortisol, and the MAPK pathway in aquaculture, providing insights crucial for optimizing fish welfare and production.