Vibrio anguillarum is a major pathogenic bacterium causing vibriosis in aquatic animals, leading to substantial economic losses in the global aquaculture industry. Previous studies have indicated that L-arginine modulates the virulence of the pathogen, but the underlying molecular mechanisms remain elusive. The present study aimed to clarify the regulatory role of L-arginine metabolism in V. anguillarum virulence. We first evaluated the effects of L-arginine and its major metabolites (agmatine, putrescine, spermine) on the hemolytic activity of V. anguillarum. Results showed that L-arginine and its metabolites regulated hemolytic activity in a concentration-dependent biphasic manner, with agmatine exerting the most potent promoting effect. To identify the critical metabolic branch involved, four isogenic mutants were constructed targeting key genes in arginine metabolism (adc, astA, astD). Phenotypic analysis revealed that only the adc deletion mutant (Δadc) exhibited near-complete loss of hemolytic activity, which was dose-dependently restored by supplementation with agmatine, putrescine, or spermine. Transcriptomic analysis identified 704 significantly differentially expressed genes (DEGs) between Δadc and WT strains, with downregulated DEGs enriched in virulence-associated pathways. Key hemolysin and secretion system genes were validated to be downregulated in ∆adc by quantitative real-time PCR (qRT-PCR). Additionally, Δadc displayed attenuated anti-phagocytic ability in Tetrahymena co-culture assays, impaired biofilm formation, and increased susceptibility to multiple classes of antibiotics. Collectively, our findings demonstrate that L-arginine modulates V. anguillarum hemolysis and overall virulence through the adc-mediated agmatine biosynthesis branch. This study fills the knowledge gap in the regulatory mechanism of L-arginine on V. anguillarum virulence and provides a potential target for the control of vibriosis in aquaculture.

Ayu (Plecoglossus altivelis) is an East Asian amphidromous river fish, yet seasonal microbiota dynamics remain unclear. We investigated ayu in the Dajing Stream (Zhejiang Province, China) by synchronously sampling water microbiota (H), gut content microbiota (N), and gut tissue-associated microbiota (C) across four seasons. Each season, four fish were collected, and an overlapping pooling strategy (abc/abd/bcd) generated three composite replicates for C and N (n = 3 composites/season); water was collected as three field replicates (n = 3/season), yielding 36 samples (12 per niche). Using 16S rRNA amplicon sequencing and COI barcoding of stomach contents, we observed the clearest seasonal differentiation in H and seasonal variation in N consistent with diet shifts, whereas C was comparatively stable. COI signals indicated a diet dominated by aquatic insects in spring/summer, which shifted toward smaller prey (e.g., rotifers) in winter. Together, these results highlight strong niche partitioning and season-linked shifts in water and gut content communities relative to the more stable tissue-associated microbiota. These findings should be interpreted as exploratory and require validation in larger individual-level studies.

The development of intermuscular bones (IBs) in teleost fish is regulated by osteogenic genes, but the upstream signals that initiate ossification remain elusive. Given the spatiotemporal correlation between IB formation and swimming behavior, mechanical stimuli from muscle activity have been hypothesized as potential regulators. In this study, we investigated the role of the muscle-specific microRNA miR499 in this process. Using CRISPR/Cas9-generated miR499 knockout zebrafish, we demonstrated that the mutant of miR499 induces a fast-twitch muscle phenotype through the upregulation of sox6, accompanied by increased fast-myosin gene expression and fast-twitch fiber proliferation. This shift led to distinct locomotor alterations, characterized by reduced tail-beat frequency and increased stride length. Crucially, these changes resulted in delayed IB ossification onset, slower ossification rates, and simplified morphology. The total ossification area decreased by 27%. Molecular analysis revealed a downregulation of osteogenic genes in IB tissue. Our findings establish miR499 as a key regulator of a muscle–bone functional axis, where miRNA-mediated muscle fiber specification determines mechanical output that subsequently guides IB development. This work provides new insights into the mechanobiological regulation of skeletal development and identifies a promising genetic target for aquaculture breeding programs aimed at attenuated IBs.