Tetrodotoxin (TTX) reshapes the functional potential of the gut microbiome in juvenile tiger pufferfish (Takifugu rubripes) across salinity gradients

BACKGROUND: The gut microbiota of aquatic organisms responds dynamically to environmental stressors such as salinity fluctuations. However, how microbial communities respond to combined environmental and dietary stressors, and how these interactions influence functional potential, remains incompletely understood. Here, we investigated whether dietary administration of tetrodotoxin (TTX), a neurotoxin naturally accumulated by juvenile tiger pufferfish (Takifugu rubripes), alters gut bacterial community composition and functional potential across salinity gradients. RESULTS: Juvenile T. rubripes were reared under four salinity conditions (34.0, 17.0, 8.5, and 2.1 ppt) and fed either a control or TTX-containing diet (1.22 MU/g). Integrated 16S rRNA gene amplicon and shotgun metagenomic analyses revealed that salinity was the primary driver of gut microbiota structure, with only 5.1% of amplicon sequence variants (ASVs) shared across salinity levels. In contrast, TTX ingestion induced salinity-dependent shifts in specific bacterial taxa rather than broad community restructuring. Core taxa, including Arcobacteraceae, Mycoplasma, Brevinema, and Vibrio, were consistently detected across treatments but exhibited pronounced changes in relative abundance and functional potential under salinity and toxin stress. Metagenomic profiling indicated that Arcobacteraceae encode genetic modules for amino acid and B vitamin biosynthesis that are absent or incomplete in the host genome, suggesting metabolic complementarity. TTX ingestion reduced the genetic representation of these biosynthetic pathways at specific salinities, particularly those associated with Arcobacteraceae. Conversely, phenylalanine biosynthesis potential enriched in TTX-fed fish, primarily associated with Vibrio spp., indicating a possible microbial functional adaptation to toxin administration. Despite these microbiome and functional shifts, TTX ingestion did not affect host growth. CONCLUSIONS: Dietary neurotoxin administration reshaped gut microbiome functional profiles in a salinity-dependent manner, highlighting microbiome plasticity and improving our understanding of host-microbiota-environment interactions relevant to aquaculture health management.