Effect of dietary protein/carbohydrate ratio on growth, digestion, and intestinal development in Apostichopus japonicus: Underlying molecular mechanisms

The dietary protein/carbohydrate (P/CH) ratio represents a critical nutritional determinant influencing growth performance, intestinal health, and feed utilization efficiency in aquatic animals, but its impact on sea cucumber Apostichopus japonicus has not been evaluated. This study formulated three isoenergetic diets (P40/CH25, P25/CH40, and P15/CH50) for an 8-week feeding trial. The effects of P/CH ratio on growth performance, digestive enzyme activity, intestinal histomorphology and gut microbiota of A. japonicus were systematically evaluated, while integrated transcriptomic and proteomic analyses were employed to elucidate the underlying molecular mechanisms. The results demonstrated that the P25/CH40 (MP/MC) group exhibited significantly higher specific growth rate (SGR), feed efficiency (FE), and intestinal protease, amylase, alginate lyase, and cellulase activities compared to the other two groups ( P < 0.05). Additionally, this group showed significant increases in intestinal villus length and fold width ( P < 0.05). Microbiota analysis revealed that the MP/MC diet with high microbial diversity and stability enriched beneficial bacterial genera (e.g., Muribaculaceae , Bacillus ), while reducing potential opportunistic taxa, indicating improved microbial homeostasis. Proteomic and transcriptomic analysis revealed that the MP/MC diet synergistically regulated the following pathways to promote growth and intestinal development in A. japonicus : (1) significant upregulation of genes related to amino acid transport and anabolic metabolism; (2) activation of the AMPK-PGC1α and PI3K-AKT-mTOR pathways to boost energy metabolism and protein synthesis; (3) upregulation of cell cycle-related genes, driving cellular proliferation; and (4) moderate activation of the LGR5/WNT signaling axis, regulating intestinal stem cell proliferation and differentiation. In conclusion, the MP/MC diet optimally promoted the growth, digestive capacity, and intestinal development of A. japonicus by synergistically activating protein synthesis, cell cycle progression, and intestinal stem cell-related signaling pathways (mTOR, AKT, WNT), thereby achieving efficient nutrient utilization and tissue renewal. This study provides an important molecular theoretical foundation for precise nutritional regulation and high-efficiency feeds for A. japonicus . • Optimal dietary protein/carbohydrate (P/CH) ratio improved growth performance and digestive enzyme activities in Apostichopus japonicus . • Optimal P/CH ratio enhanced intestinal development and ameliorated microbial homeostasis. • Integrated transcriptomic and proteomic analyses elucidated the core underlying molecular regulatory mechanisms. • Optimal P/CH ratio synergistically activated protein synthesis, cell cycle progression and intestinal stem cell-associated signaling pathways.