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

Understanding how microbiomes influence influence the physiology of animal hosts requires detailed mechanistic insights, often obtained through gnotobiological approaches. Model organisms are central to this research, offering 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.