Phylogenetically distinct Vibrio mediterranei lineages confer robust protection under thermal stress against oyster pathogens

Introduction Marine bivalve mortality events cause substantial economic losses in aquaculture and threaten global food security. While pathogenic Vibrio species are frequently implicated, growing evidence suggests that loss of beneficial microbes can increase host susceptibility to disease. We previously observed that Vibrio mediterranei was consistently isolated from healthy oysters but systematically disappeared prior to mortality events, coinciding with proliferation of pathogenic Vibrio species. Here, we test whether this pattern reflects a protective functional role. Methods Screening eleven V. mediterranei strains against eastern oyster ( Crassostrea virginica ) larvae revealed three distinct virulence phenotypes – avirulent, pathogenic, and intermediate – that correspond to monophyletic phylogenetic clades. Results Pre-colonization with the avirulent strain Vm02 increased larval survival from 10–19% (pathogen-only controls) to 94–97% when challenged with V. harveyi or V. coralliilyticus , representing near-complete protection maintained at both ambient (28 °C) and thermal stress (32 °C) temperatures. Protection was rapid, effective even under simultaneous co-inoculation with the pathogen, and durable through >96 hours post-exposure. Fluorescence microscopy confirmed persistent association of fluorescently tagged Vm02 with larval digestive tissues. Phylogenomic analysis of 33 V. mediterranei genomes placed the three phenotypic groups within well-supported clades separated by 97.1–97.8% average nucleotide identity, approaching species-level thresholds. Pangenome analysis revealed that protective-clade strains harbor 230 unique orthogroups encoding regulatory systems, stress tolerance, metabolic versatility, and a conserved bacteriocin biosynthetic operon. These same strains do not encode the Type I secretion system, Type VI secretion system variants, and TCP pathogenicity island found in pathogenic lineages. Discussion Together, these findings demonstrate that beneficial and pathogenic phenotypes are phylogenetically constrained within distinct V. mediterranei lineages, providing a framework for probiotic development and disease forecasting in shellfish aquaculture.