Abstract

Infectious diseases are a major threat to sustainable aquaculture, yet the biosafety risks associated with emerging pathogens in farming sites are often overlooked, particularly when no mass mortality events are observed. In this study, we investigated the occurrence and virulence potential of Vibrio spp. within the microbiota of Pacific Oysters (Magallana gigas) farmed in Tongoy Bay, Chile. Using in vitro cytotoxicity assays, comparative genomics, and fluorescence microscopy, we characterized the cytotoxicity of Vibrio strains on oyster hemocytes and identified key virulence determinants. We found a positive correlation between virulence and cytotoxicity, with strains such as V. lentus TO6_3 and V. lentus TO5_9 causing significant hemocyte lysis. Whole-genome sequencing revealed the presence of diverse macromolecular systems, including Type VI secretion systems (T6SS) and a multifunctional auto-processing repeats-in-toxin (MARTX) cluster exclusive to the highly virulent but moderately cytotoxic V. lentus TO6_11. This strain induced hemocyte rounding, a phenotype consistent with the presence of the actin crosslinking domain (ACD) and the makes caterpillar floppy (MCF) effector domain, probably linked to a mechanism of pathogenicity that does not completely rely on hemocyte lysis for the establishment of infection. The seasonal monitoring of virulence factors revealed a winter increase in T6SS frequency, while MARTX prevalence remained low. Our findings expose the silent circulation of virulence factors in oyster-associated Vibrio communities and highlight the need for targeted monitoring to prevent future disease outbreaks in emerging aquaculture systems.