Biofilm presence and substrate type influence oyster larval settlement and survival.

Marine invertebrates with a bipartite life cycle typically undergo a developmental transition from pelagic larvae to benthic juveniles. The success of this transition largely depends on appropriate habitat and substrate selection, which is crucial for completing metamorphosis, subsequent development, and survival. While larvae are known to select microhabitats based on substrate-associated biofilms, whether the influence of these biofilms persists beyond post-metamorphosis remains poorly understood. Here, we investigated how microhabitat and biofilm types influence larval settlement choice and how these choices, in turn, affect settlement success and post-metamorphosis survival in the ecologically and economically important Hong Kong oyster (Crassostrea hongkongensis). The results indicate that biofilm presence positively influenced larval settlement, irrespective of substrate type. Following larval addition, biofilm profiles on substrates converged between control and treatment groups. In contrast, pre-existing biofilms had no significant effect on post-metamorphic survival, which instead depended primarily on substrate type. High-throughput sequencing of pre-existing biofilms on oyster shell substrates revealed six bacterial families enriched in association with higher larval settlement, including Cryomorphaceae, Rs-E47_termite_group, Bacteriovoracaceae, Rhodothermaceae, Vampirovibrionaceae, and Rubritaleaceae. Together, these findings suggest that substrate-associated biofilms primarily influence early settlement dynamics, whereas substrate type governs post-metamorphic survival. This decoupling highlights the distinct roles of microbial biofilm and substrate characteristics across different life-history stages, offering insights with potential implications for restoration and conservation strategies for marine invertebrates.IMPORTANCEThe transition from free-swimming larvae to bottom-dwelling juveniles represents a critical bottleneck for marine invertebrates, with direct implications for the recovery and resilience of coastal ecosystems. Using the Hong Kong oyster, an ecologically and economically important reef-building species, this study demonstrates that larvae preferentially settle on biofilm-covered shells and that this preference enhances settlement success. However, we show that the benefits of biofilm selection do not extend to post-metamorphic survival, which is instead governed by substrate type, revealing a previously unrecognized decoupling between the cues driving settlement and those determining subsequent survival. By characterizing the bacterial communities associated with higher larval settlement, this work identifies candidate microbial taxa that could inform the development of improved settlement substrates. Together, these findings provide a mechanistic foundation for optimizing restoration strategies for oyster reefs and offer a framework applicable to the conservation of other marine invertebrate species.