Sergio Rodriguez

Vibrio europaeus is an important pathogen in shellfish aquaculture, yet its genomic diversity remains poorly understood. Here, we present the first comprehensive analysis of the V. europaeus pangenome, integrating genomic data from all strains available to date ( n =39) sequenced specifically for this study. These were isolated from different aquaculture facilities (shellfish hatcheries) associated with mass mollusc mortalities from different geographical locations, years and host species. Our findings revealed an open pangenome with 61% of the genes associated with the accessory genome that contribute to environmental and host adaptations. Phylogenomic analyses of the core-genome (39% of the pangenome size) allowed us to evaluate the evolutionary history and intraspecific diversity of V. europaeus and revealed that Spanish strains displayed a much lower genetic variability than French, Chilean or American strains, probably due to a monophyletic radiation event. Functional annotation of core and accessory genes revealed the key virulence factors of the species, while it also disclosed that these are located mainly in the core genes. The high number of anti-phage defence systems encoded in the accessory genome explained almost all the variability of the species. The results provide important insights into the evolutionary history and ecological versatility of V. europaeus , with potential implications for diagnostics, epidemiological surveillance and disease management strategies in aquaculture.

Vibrio europaeus has emerged as a significant pathogen in shellfish aquaculture, causing mass mortality outbreaks in key bivalve species and leading to severe economic losses for the industry. Studies on the structure and characteristics of the accessory genome in aquaculture pathogens remain scarce, despite its crucial role in evolutionary and ecological adaptation. The accessory genome provides indeed genetic variability that enables rapid responses to environmental challenges, host adaptation and selective pressures such as antibiotics or phage predation. Here, we present the first comprehensive comparative genomic analysis of the V. europaeus pangenome to investigate the structural organization and functional content of its accessory genome. The soft mobilome of V. europaeus comprises 73% of accessory genes and 44% of the total pangenome, including non-chromosomic (plasmids) and chromosomic genetic elements such as prophages, integrative and conjugative/mobilizable elements, phage satellites and other mobile genetic elements (MGEs) designated as unclassified chromosomic regions of genomic plasticity (unclassified chromosomic RGPs). Among accessory elements, unclassified chromosomic RGPs were the primary drivers of evolutionary dynamics in V. europaeus , acting as the main genetic reservoir of anti-phage defence systems and antimicrobial resistance genes. Notably, the identification of abundant insertion hotspots in chromosomic genetic elements facilitates the rapid acquisition of anti-phage defence systems, thereby enabling rapid turnover of these systems and enhancing host fitness. In addition, novel pVE1-like plasmids (>300 kb) – only found in this species and its closest relative Vibrio tubiashii – emerged as the largest and most ubiquitous MGEs in V. europaeus . These plasmids encode the highest number of virulence genes and secondary metabolite biosynthetic genes, as well as a remarkable diversity of anti-phage defence systems among closely related strains. Although the genome dataset analysed here is limited to strains isolated from moribund/dead animals in aquaculture environments, this study provides new insights into the role of accessory genetic elements in the evolution, adaptation and diversification of the shellfish pathogen V. europaeus . The findings reveal the complexity and plasticity of its pangenome and highlight the importance of RGPs and plasmids in bacterial fitness.

Two Vibrio strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop Argopecten purpuratus during vibriosis outbreaks that occurred in two commercial scallop larvae hatcheries located in the Inglesa and Tongoy bays in Northern Chile. The strains were identified as Vibrio chagasii using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and β-hemolysis, whereas only VPAP36 produced phospholipase and only VPAP40 produced caseinase. The whole genome analysis showed that the strains harbored genes encoding for the virulence factors, the EPS type II secretion system, and Quorum Sensing (auto-inductor 1 and auto-inductor 2), whereas genes encoding a metalloproteinase and a capsular polysaccharide were detected only in the VPAP40 genome. When challenge bioassays using healthy 11-day-old scallop larvae were performed, the V. chagasii VPAP36 and VPAP40 strains exhibited significant (p < 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 ± 4.40% and 28.56 ± 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 ± 2.40% and 18.37 ± 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of V. chagasii from the massive larval mortalities of the farmed scallop (Argopecten purpuratus) in Chile, and demonstrates the pathogenic activity of V. chagasii towards the Chilean scallop, the second most important species for Chilean mariculture.

Vibrio europaeus is an emergent pathogen affecting clams, oysters and scallops produced in the most important countries for bivalve aquaculture. Studies concerning virulence factors involved in the virulence of V. europaeus are very scarce despite its global significance for aquaculture. Zinc-metalloproteases have been described as a major virulence factor in some Vibrio spp., although their contribution and role in the virulence of V. europaeus is not clear. To address this, we have studied an extracellular zinc-metalloprotease (VemA) encoded by V. europaeus, which was identified as a vibriolysin, highly conserved in this species and homologous in other pathogenic and non-pathogenic species. Virulence challenge experiments demonstrated that infection processes were faster when Manila clam larvae and juveniles were infected with the wildtype rather than with a mutant defective in the vemA gene (ΔvemA). V. europaeus was able to resist the bactericidal action of mucus and displayed a chemotaxis ability favoured by VemA to colonize the body mucus of clams and form a biofilm. The overall results suggest that VemA, although it is not a major virulence factor, plays a role in the colonization of the Manila clam mucus, and thus boosts the infection process as we observed in virulence challenge experiments.