Settlement Induction in Mytilus coruscus Is Driven by Cue Diversity: Evidence from Natural Biofilms and Bacterial Isolates

Mytilus coruscus, a commercially important mariculture mussel in China, has shown a marked decline in larval settlement and metamorphosis over the past decade, a trend often linked to environmental degradation and resource depletion. Numerous studies have identified bacterial biofilms as key modulators of mussel larval settlement. To investigate this, we deployed PVC plates in situ within aquaculture zones near Shengsi (Zhoushan, Zhejiang) and Lianjiang (Fuzhou, Fujian). After natural biofilm colonization on the plates, juvenile M. coruscus were introduced to assess settlement rates. The attached juveniles were homogenized, leading to the isolation of four dominant bacterial strains: Pseudomonas sp. LJBF001, Vibrio sp. LJBF002, Pseudomonas sp. LJBF003 and Bacillus sp. LJBF004. Compared to control PVC plates, natural biofilms significantly promoted juvenile settlement, with the Lianjiang (LJ) group reaching up to >29% under our assay conditions. In contrast, monospecific biofilms prepared from these isolates did not significantly increase larval metamorphosis; the numerically highest response (LJBF004) reached ~9% and was not significant versus the control. These contrasting outcomes are consistent with a threshold–multi-cue synergy mechanism, whereby cue diversity and partial redundancy in natural biofilms favour threshold crossing, while restricted cue sets in single-strain films often fall short. Guided by this framework, priority next steps include testing c-di-GMP delivery (soluble and via OMVs), probing EPS structure–function and EPS–OMV/LPS–free-fatty-acid blends alongside minimal multi-strain consortia, and adopting stage-gated assays with time-to-event endpoints and effect-size/CI reporting.