Growth-physiology divergence, metabolic adjustment, and ribosome-related gene-expression suppression characterize distinct stocking-density states in juvenile Acrossocheilus fasciatus: An integrated transcriptomic and metabolomic analysis.

Abstract

Although the adverse effects of excessive stocking density are well documented, physiological and muscle molecular differences between low and moderate densities, and their changes at higher densities, remain poorly characterized. Juvenile Acrossocheilus fasciatus were reared for 61 days at 15 (LD), 30 (MD), 60 (HD), or 120 fish m-2 (EHD). Growth, plasma cortisol, and hepatic antioxidant and immune-related indices [total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), alkaline phosphatase (AKP), and acid phosphatase (ACP)] were assessed in all groups, while muscle transcriptomic and metabolomic analyses were integrated for LD, MD, and EHD. Individual growth declined with increasing density, with LD showing the highest final body weight and specific growth rate (SGR). At MD, growth was reduced relative to LD, whereas cortisol and MDA remained comparable. Gene set enrichment analysis (GSEA) showed positive enrichment of amino-acid and carbon-substrate metabolic pathways and the ribosome pathway at MD. Cross-omics integration further identified arginine and proline metabolism, with sms significantly associated with a metabolite feature annotated as D-Proline (ρ = 0.883, FDR = 0.038). At EHD, cortisol and malondialdehyde increased and alkaline phosphatase activity declined; the major metabolic pathways enriched at MD were no longer significantly enriched, while the ribosome pathway showed strong negative enrichment (NES = -2.30, FDR < 0.001). These findings distinguish low, moderate, and extremely high densities as biologically distinct states: LD supported maximal individual growth, MD combined reduced growth with coordinated metabolic adjustment without marked physiological stress, whereas EHD was characterized by overt stress and ribosome-related transcriptional suppression.