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.