Prevalence-Based Classification of Gut Microbiota in the Medicinal Leech Hirudinaria manillensis Reveals Differential Roles of Persistent and Intermittent Taxa in Disease Development

Abstract The gut microbiota is increasingly recognized as a key determinant of host health, but its compositional dynamics in diseased medicinal leeches ( Hirudinaria manillensis ) remain unexplored. Using 16S rRNA amplicon sequencing, we profiled gut microbial communities in healthy leeches and those with characteristic disease symptoms. Across 528 amplicon sequence variants (ASVs), 232 were unique to healthy and 173 to diseased individuals. Alpha diversity showed no significant differences between groups, and beta diversity revealed no overall separation (ANOSIM, R = -0.0019, p = 0.442), though healthy individuals clustered more tightly. A Gut Microbiome Health Index (GMHI), based on health- and disease-associated bacterial signatures, was significantly higher in healthy leeches and outperformed Shannon diversity in discriminatory power. Prevalence-based classification showed persistent taxa comprised 57.9% of the healthy community versus 46.4% in diseased ones. Intermittent taxa like Thomasclavelia and Desulfovibrio showed nominal trends toward higher abundance in healthy leeches (uncorrected p = 0.034 and p = 0.020), but these did not survive FDR correction, and other dominant taxa showed no significant differences. Co-occurrence network analysis revealed destabilized interspecies interactions in diseased leeches, indicating disrupted community robustness. Collectively, disease in H. manillensis is associated with functional and ecological shifts—particularly reflected by GMHI and network destabilization—rather than simple diversity loss. This descriptive study provides a foundational dataset and underscores the need for functional validation and longitudinal monitoring to establish causative links in medicinal leech aquaculture.