Metagenomic insights into mechanisms of coral larval settlement induction and inhibition by marine biofilms

Background. Biofilms are essential to larval settlement in many marine invertebrates, yet the mechanisms driving settlement induction or inhibition in corals remain poorly resolved. This challenge lies in the vast taxonomic and functional diversity of marine biofilms, making it difficult to identify cues associated with settlement. To address this, we analysed the metagenomes of biofilms used to induce settlement of four broadcast-spawning non-acroporid coral species: Dipsastrea favus , Platygyra sinensis , Echinophyllia aspera and Porites lobata. Biofilms were developed for one or two months, under light or dark treatments, with light biofilms inducing significantly higher settlement than dark biofilms. Results. Gene composition varied strongly among treatments, with light biofilms enriched in genes encoding carotenoid biosynthesis and nitrate reduction, while dark biofilms encoded more genes for denitrification and nitric oxide production. Modelling revealed the abundance of genes encoding GABA biosynthesis and the type III secretion system (SS) were positively associated with settlement, while genes encoding the type II secretion system, flagellar and lipopolysaccharides were negatively associated. Genes predicted to promote settlement were concentrated in metagenome assembled genomes (MAGs) assigned to Flavobacteriaceae , Rhodobacteraceae and Pirellulaceae , consistent with previous research identifying these lineages as potential inducers. Additionally, we detected homologues of cycloprodigiosin and tetrabromopyrrole biosynthesis genes in MAGs classified as Sphingomonadaceae and Cellvibrionaceae , suggesting these settlement-inducing compounds may be synthesised by previously unrecognised taxa. Conclusions. These findings link biofilm metagenomics to coral larval settlement for the first time, suggesting carotenoids may attract larvae to biofilm surfaces, while GABA may promote searching and attachment. Compounds such as cycloprodigiosin, tetrabromopyrrole or effector proteins may be required to complete metamorphosis. Simultaneously, elevated levels of nitric oxide, type II SS exudates or an abundance of flagellar potentially inhibit the settlement process. This study advances our understanding of the complex microbial processes underpinning coral larval settlement.