To cohabit or inoculate? Testing the limits of microbiota transplantation in Manila clams

Microbiota transplantation has emerged as a powerful tool to investigate host-microbe interactions across a wide range of organisms, yet its applicability and ecological limits in marine bivalves remain poorly understood. Here, we experimentally test whether microbiota associated with chronic environmental contamination can be transferred and persist in adult Manila clams (Ruditapes philippinarum). We compared two water-mediated approaches: (i) inoculation of a donor tissue homogenate into the water and (ii) cohabitation with live donor clams through shared seawater flow. Donors originated from a chronically contaminated site (Porto Marghera, Italy) while recipients were naïve clams (France), never previously exposed. Each approach was performed with and without antibiotic pretreatment to evaluate whether transient microbiota disruption could enhance donor signal acquisition. Using RNA-based 16S rRNA profiling, we tracked both overall community similarity to the donor prokaryotic community and the presence of donor-exclusive amplicon sequence variants (ASVs) through a Transfer Index. Although similarity-based metrics suggested modest convergence, donor-exclusive ASVs were detected at low levels and did not persist, indicating weak establishment rather than stable colonization. Antibiotic pretreatment did not significantly reduce prokaryotic diversity or total bacterial load, nor did it improve transfer efficiency or promote convergence toward the donor prokaryotic community. Finally, predicted functional profiles further revealed that pathways associated with xenobiotic metabolism in donor clams were not transmitted under short-term experimental conditions. Together, our results demonstrate that prokaryotic community transplantation in adult bivalves is experimentally achievable but results in limited and transient donor community establishment, providing new insight into the host- and environment-driven ecological constraints governing this process.