Gut Microbiota Alterations and Intestinal Pathology in Healthy, Moribund, and Recently Dead Mastacembelus Armatus from a Pond Aquaculture System

Spiny eel (Mastacembelus armatus) pond aquaculture suffers from recurrent nonspecific mortality, yet the associations between gut-microbiota shifts, host histological alterations, and fish physiological status, as well as the potential roles of sediment microbiota, remain unclear. In this study, we collected healthy (H), moribund (M), and recently dead (D) fish, as well as pond sediment (S), and performed histopathological examination and 16S rRNA amplicon sequencing combined with FEAST source-tracking analysis. Histology revealed severe intestinal villus atrophy, epithelial shedding, and extensive hepatic vacuolation in M fish. Results of 16S rRNA sequencing revealed a significant decrease in the alpha diversity (Chao1) of gut microbial richness in M and D groups, compared with healthy (H) (p < 0.05). Healthy guts were dominated by Proteobacteria and the keystone genus Cetobacterium (about 40% relative abundance). In M/D, Cetobacterium nearly disappeared, while Firmicutes and Fusobacteriota became predominant, with potentially opportunistic taxa (Fusobacterium, Clostridiaceae) showing markedly higher relative abundance. Beta-diversity and LEfSe analyses showed overlapping microbial communities between M and D fish, which were distinct from those of healthy (H) fish. FEAST modeling output showed that the conditionally estimated sediment-derived proportion increased from approximately 28% in healthy (H) to ~99% in M fish. Functional profiles predicted by PICRUSt2 shifted from oxidative phosphorylation toward peptidoglycan biosynthesis and peptidase-related functional potentials. Collectively, our results demonstrate strong associations between gut-microbiota disruption, intestinal barrier impairment, and fish physiological decline. We observed associations between damaged intestinal barrier status and increased sediment-attributed fractions of potentially opportunistic microbes within the gut microbiota.