From multi-omics insights to single-strain proof: How traditional agricultural system enhances fish flavor via the microbiome-gut-muscle axis.

Intensive aquaculture has significantly boosted aquatic product yields, but it often compromises sensory quality and remains constrained by reliance on unsustainable fishmeal. The Mulberry-dyke and Fish-pond system, used in China for millennia, suggests a circular approach, yet the underlying mechanism remains poorly understood. In this study, silkworm excrement (SE) from traditional sericulture (TSE) and modern insect factories (ISE) was evaluated as a functional aquafeed. Body weight, gut histology, and immunohistochemistry were employed to assess host health. Gut microbiome, electronic tongue analysis, and muscle metabolomics were conducted to assess fish flavor and identify flavor-related microorganisms. A single-strain feeding experiment further validated the microbiome-gut-muscle axis using electronic tongue analysis, gut transcriptomics, and determination of free amino acids and nucleotides. SE supplementation maintained fish yield while improving intestinal structure. Compared with commercial feed (CF), both SE treatments increased gut microbial diversity and community stability, and more than half of the significantly different ASVs were shared between the TSE and ISE groups, mainly involving immune regulation, nutrient metabolism, and flavor formation. SE, particularly TSE, enhanced antioxidant capacity and reduced lipid peroxidation, possibly through microbial regulation of lysophosphatidylcholines. As consistently indicated by electronic tongue and muscle metabolomics analyses, SE significantly improved fish flavor, with increased umami and reduced bitterness. Network analysis and single-strain feeding further suggested that Methylorubrum populi, Gemmobacter aquatilis, and Rhodobacter sphaeroides contributed to flavor improvement by regulating host amino acid and nucleotide metabolism along the microbiome-gut-muscle axis. These findings highlight SE as a promising sustainable bioresource for aquaculture.