Rey-Varela, D. | Search bioRxiv

bioRxiv2025

Oral immune priming modulates microbiota composition and supports pathogen control in the Manila clam (Ruditapes philippinarum)

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 ([~]105 copies mg-{superscript 1}). Interestingly, the pathogen was able to persist at low and non-harmful concentration ([~]102 copies mg-{superscript 1}) 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.

microbiology↗

bioRxiv2025

Deciphering the pangenome of the shellfish pathogen Vibrio europaeus: Evolutionary history and functional impact of core and accessory genes in aquaculture.

Vibrio europaeus is an important pathogen in shellfish aquaculture, yet its genomic diversity and adaptive potential remain poorly understood. Here, we present the first comprehensive analysis of the V. europaeus pangenome, integrating genomic data from all strains available to the date sequenced specifically for this study. Those were isolated from different aquaculture facilities (shellfish hatcheries) associated to mass mollusks mortalities from different geographical locations, years and host species. Our findings revealed an open pangenome with the 61% of the genes associated to the accessory genome that contributes to environmental and host adaptations. Phylogenomic analyses of the core-genome (39% of the pangenome size) allowed 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 those are located mainly into the core genes. The high number of anti-phage defense 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. Impact statementThis study presents the first comprehensive pangenome analysis of Vibrio europaeus, an emergent pathogen responsible for severe economic losses in shellfish aquaculture, the second most important sector of global aquaculture. Here, we characterized for the first time the V. europaeus pangenome, integrating genomic data from all strains isolated to date, sequenced specifically for this study using NGS and/or third-generation (PacBio) technologies. This work achieved the most complete species pangenome to date and is among the first studies on aquaculture-related bacterial pathogens. Beyond a descriptive framework, the pangenome was critically examined to identify key traits, including virulence factors, secondary metabolite biosynthesis, and antimicrobial resistance genes, essential for host infection and adaptation. Moreover, the study of anti-phage defense systems was shown to account for much of the species genomic variability. The genomic resources and insights generated here substantially expand our understanding of V. europaeus biology and provide valuable information that can be applied for diagnostics, epidemiological surveillance, and sustainable management of this pathogen in aquaculture industry. Data summaryAll genome assemblies have been uploaded to the National Center for Biotechnology Information. The GenBank accession numbers for each of the 39 strains used in this study and detailed information can be found in Table S1. All bioinformatics tools used for comparative genomics have been listed in the Methods section including references, associated databases and analysis parameters.

microbiology↗