Generating gnotobiotic bivalves: a new method on Manila clam (Ruditapes philippinarum)

Understanding how microbiomes influence the physiology of animal hosts requires detailed mechanistic insights, often obtained through gnotobiological approaches. Model organisms are central to this research, offering key advantages such as experimental tractability, reproducibility, and ease of manipulation. However, there is a lack of established gnotobiotic models for the marine environment-especially for bivalves, which play a critical role in ecosystem functioning. This gap is particularly important in the context of climate change, where harnessing microbiome resilience could mitigate environmental challenges and enhance host responses. In this study, we present a protocol to generate microbiome-depleted and gnotobiotic clams (Ruditapes philippinarum), one of the most widely farmed molluscs in the world and a key sentinel species for environmental and climate change impacts. Our microbiome depletion protocol effectively eliminated all detectable bacterial genera in the clams, with the exception of Endozoicomonas elysicola, which was identified solely by 16S rRNA amplicon sequencing and not by cultivation methods. In addition, we developed a microbiome transplantation protocol using inoculation of a mock bacterial community that successfully colonized the recipient clams within 1 h of transplantation. By extending gnotobiotic methods to marine invertebrates, this work opens new avenues for investigating microbial influences on ecologically and economically important species, particularly under the pressure of a changing climate.IMPORTANCEThe extensive diversity of host-microbe symbioses across ecosystems requires the use of different models to identify conserved and specific processes underlying such relationships. The need for novel models is particularly relevant in the context of the rapid environmental modifications due to climate change. Bivalve molluscs play a crucial role in the functioning of marine ecosystems. In this study, we present the first experimental protocol for the generation of gnotobiotic clams of the species Ruditapes philippinarum, one of the most widely farmed molluscs in the world, and a sentinel organism for environmental pollution. Our work extends the current technical understanding of the establishment of gnotobiotic animals, providing an important method for testing research hypotheses on a key taxonomic group in animal ecology. This study will also open new avenues for investigating the influence of microorganisms on animal health and elucidate the transferability of mechanisms studied predominantly in vertebrates to marine invertebrates.