Javier Dubert

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.

Invertebrates lack adaptive immunity and rely exclusively on innate defenses to combat pathogens. Recent studies have shown that invertebrates exhibit immune priming, a form of innate immune memory in which prior pathogen exposure enhances protection against reinfections. Despite its ecological and applied relevance, immune priming remains poorly explored in marine invertebrates. Most studies adopt a host-centric view, focusing on the immune response while overlooking that immunity occurs within a complex microbial context in which the host, its microbiota, and pathogens interact. This gap limits our understanding of how microbiota and pathogens behave under priming pressure. Here, we investigated the effects of immune priming on host survival, pathogen dynamics, and microbiota composition in the Manila clam ( Ruditapes philippinarum ), a key species for global aquaculture, challenged with the bacterial pathogen Vibrio europaeus . Immune priming was induced orally using live V. europaeus at a sublethal dose, followed by a lethal secondary challenge. Primed clams exhibited a significant increase in survival after the second challenge (87% survival vs. 0% in non-primed clams) and rapidly reduced the pathogen concentration within 48 h post-challenge below the mortality threshold observed in non-primed clams (~10 ⁵ copies mg ⁻¹ ). Notably, the pathogen was detected at low but non-harmful concentrations (~10 ² copies mg ⁻¹ ) in primed clams during both challenges. Full-length 16S rRNA amplicon sequencing analyses revealed that immune priming reshaped the host microbiota. Beta diversity analyses suggest the establishment of a specific bacterial community in primed clams. Clustering analyses identified a priming-associated microbiota dominated by Acinetobacter, Brevundimonas, Sphingobium , and Psychrobacter , which persisted at the end of the priming and the secondary challenge but was absent or depleted in non-primed clams. In contrast, members of Arcobacteraceae emerged only during the second challenge, decreasing in primed clams but increasing in non-primed clams in association with high mortality. This study provides the first phenotypic evidence of oral immune priming in the Manila clam, identifies a specific microbiota and detects the pathogen despite effective host protection. This offers a new perspective to address immune priming as an emergent property of dynamic interactions between the host, its microbiota, and the pathogen.

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.

Immunological memory was long considered an exclusive feature of vertebrates. However, extensive evidence now shows that invertebrates possess forms of innate immune memory, known as immune priming, where previous exposure to a pathogen enhances subsequent immune responses and host protection. Immune priming has been proposed as a promising strategy for disease prevention in shellfish aquaculture. However, immune priming remains largely unexplored in marine bivalves, particularly in the Manila clam (Ruditapes philippinarum), a top-ten global aquaculture species. Here, we investigated for the first time the effects of oral immune priming on host survival, pathogen dynamics, and microbiota composition in R. philippinarum against the emergent bivalve pathogen V. europaeus. Priming was induced using the live bacterial pathogen at a sublethal dose, followed by a lethal secondary exposure. Primed clams exhibited a significant survival following the second challenge (87% survival vs. 0% in non-primed clams), demonstrating robust protection against reinfection. Quantitative PCR (qPCR) revealed that primed clams rapidly reduced pathogen loads after 48 h during the second challenge, reaching concentrations below the mortality threshold observed in non-primed clams (~10 ⁵ copies/mg). Interestingly, the pathogen was able to persist at low and non-harmful concentration (~10 ² copies/mg) in primed clams along both challenges. Full-length 16S rRNA metabarcoding analyses showed that immune priming shifts the host microbiota. Alpha and beta diversity indicated a progressive reduction in diversity and the establishment of a specific and resilient bacterial community in primed clams. Clustering analyses identified a priming-associated microbiota dominated by Acinetobacter, Brevundimonas, Sphingobium, and Psychrobacter, which persisted through the secondary challenge but was absent or depleted in non-primed clams. Conversely, members of the Arcobacteraceae (e.g., Arcobacter, Poseidonibacter) were absent after priming and emerged only during second infection, decreasing in primed clams but increasing in non-primed clams coinciding with high mortalities. Our findings provide the first phenotypic and microbiome-level evidence of oral immune priming in Manila clam. Here we demonstrate that priming enhances pathogen control and promotes the establishment of a protective microbiota that may interact with the host immune system to confer resistance against bacterial infection. These results open new avenues for immune-priming and microbiota-based strategies to improve disease resistance in bivalve aquaculture.

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.

Wild phage evolution: Bacteria possess a diversity of highly specific phage defense mechanisms that evolve rapidly and that account for a large proportion of bacterial genomes. These dynamics must be understood if phage therapy is to be seriously considered for clinical use. Hussain et al . studied a set of nearly clonal wild-caught Vibrio lentus hosts and their phage and found that the hosts segregated into two groups infected by different viruses (see the Perspective by Meaden and Fineran). Puzzlingly, both host groups have the same surface phage receptors. This apparent paradox was resolved by sequencing of the hosts, which revealed distinct suites of endogenous but mobile phage defense elements (PDEs) in the two host phenotypes. Indeed, the PDEs constitute a large fraction of the flexible noncore genome of the bacteria. This means the PDEs can evolve and transfer from cell to cell without interfering with metabolic processes required to synthesize essential cell surface molecules. —CA