Sex-Stratified Gut Microbiota Dysbiosis in Cultured Big-Belly Seahorses During a Mass Mortality Outbreak

Mass mortality outbreaks threaten cultured big-belly seahorses (Hippocampus abdominalis), but gut microbial changes associated with disease and host sex remain unclear. We analyzed 36 seahorses from a single aquaculture system, classified as apparently normal males (NMHA), diseased males (EMHA), apparently normal females (NFMHA), and diseased females (EFMHA). Intestinal contents from three individuals were pooled per biological replicate, yielding three pooled replicates per group and 12 microbiome samples in total (effective n = 3 per group). The V3–V4 region of the 16S rRNA gene was sequenced, and ASVs were inferred using DADA2 in QIIME 2. α-diversity showed marginal overall group differences, whereas exploratory two-way ANOVA indicated effects of health status and Health × Sex interactions on observed ASVs, Shannon, and Simpson indices. Bray–Curtis PERMANOVA also detected significant effects of health status and the Health × Sex interaction on community structure. Pseudomonadota dominated all groups, while Vibrio was strongly enriched in diseased females, reaching 86.94 ± 13.03%. LEfSe identified Vibrio/Vibrionaceae as biomarkers of diseased females, whereas Arcobacteraceae and Litoreibacter characterized diseased males. PICRUSt2-based prediction suggested higher predicted representation of pathways related to signal transduction, membrane transport, biofilm formation, chemotaxis, and flagellar assembly in diseased groups. Overall, gut microbiota dysbiosis during the outbreak showed sex-stratified patterns, with pronounced Vibrio enrichment representing a prominent disease-associated microbial feature, particularly in females.