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

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. Pre-colonization with V. mediterranei strain Vm02 increased Crassostrea virginica oyster larval survival from 10-19% (pathogen-only controls) to 94-97% when challenged with V. harveyi or V. coralliilyticus, representing near-complete protection from pathogen-induced mortality. Protection was maintained at both ambient (28{degrees}C) and thermal stress (32{degrees}C) temperatures where pathogen virulence is enhanced, rapid (effective from co-inoculation), and durable (maintained for >96 hours). Fluorescence microscopy confirmed stable colonization of larval digestive tissues by fluorescently-tagged Vm02. Larval colonization by eleven V. mediterranei strains revealed three distinct phenotypes - protective, pathogenic, and intermediate - corresponding to monophyletic clades with 97.1-97.8% average nucleotide identity between protective and pathogenic lineages. Pangenome analysis identified 230 protective-specific versus 80 pathogenic-specific orthogroups. Protective strains encode unique regulatory systems, stress tolerance mechanisms, and metabolic versatility while lacking Type I and Type VI secretion system variants associated with pathogenicity. Together, these findings demonstrate that beneficial versus pathogenic phenotypes are phylogenetically constrained within distinct V. mediterranei lineages. This supports reports that V. mediterranei acts as both a pathogen and potential symbiont in marine hosts and reveals a clade that provides robust protection against oyster pathogens. ImportanceAquaculture disease management has traditionally emphasized either prophylactic treatment using antibiotics to avoid disease and dysbiosis or has focused entirely on pathogen detection. Both of these approaches have overlooked the potential contributions of beneficial microbes to host defense and grow-out performance. Developing beneficial probiotic tools for disease prevention represents an emerging opportunity for sustainable aquaculture management. This study demonstrates that specific lineages of Vibrio mediterranei function as protective symbionts capable of rescuing oyster larvae from near-complete pathogen-induced mortality. By integrating field observations of microbial succession during mortality events with experimental validation and comparative genomics, we show that protective versus pathogenic phenotypes are phylogenetically constrained within V. mediterranei clades separated by 97.1-97.8% average nucleotide identity. This resolution of strain-level functional variation provides fundamental insights into how host-microbe mutualisms evolve within species complexes that also harbor pathogens. The unique genomic markers identified here enable reliable screening for protective symbionts, while the temperature-stable and durable protection demonstrated in this study highlights the potential for biological control strategies in shellfish hatcheries increasingly affected by warming oceans and Vibrio-driven mortality events.