Untargeted metabolomics reveals a selectively enriched surface metabolome underpinning constitutive chemical defence in the red alga Halymenia floresii (Clemente) C.Agardh, 1817 ((Rhodophyta, Halymeniales)

Abstract: Marine macroalgae rely on chemically mediated surface defences to prevent microbial colonization and fouling, yet the composition of surface-associated metabolites and their ecological roles remain poorly understood. In this study, untargeted liquid chromatography/mass spectrometry (LC/MS) based metabolomics was used to characterize the surface-associated metabolome of the tropical red macroalga Halymenia floresii and identify metabolites potentially involved in chemical defence. Selective dipping extraction (DIP) was used to recover surface-associated metabolites and compared with whole-cell metabolome (WCM) extracts. Across both datasets, 22,455 LC/MS features were detected, including 10,505 features in the surface extracts and 11,495 additional features detected only in the WCM extracts. Statistical filtering identified 722 LC/MS features consistently enriched by at least four-fold in surface extracts relative to WCM extracts. These features yielded 928 putative metabolite annotations using xMSannotator, with biological interpretation supported by KEGG, LIPID MAPS, and MassBank. Pathway reconstruction using the Lilikoi R package identified 44 metabolite pathway associations, predominantly involving lipid-associated metabolism, including steroidogenesis, arachidonic acid metabolism, plasmalogen synthesis, fatty acid biosynthesis, sphingolipid metabolism and sterol biosynthesis. Manual curation further indicated enrichment of fatty acids, oxylipin-like metabolites, sphingolipids, phospholipid precursors and isoprenoid intermediates at the algal surface. Rather than indicating the presence of metabolites unique to the surface, the findings suggest that H. floresii selectively enriches lipid-associated metabolites at the thallus seawater interface. Spatial localization of relatively hydrophobic metabolites may provide an energetically efficient chemical defence strategy by retaining bioactive compounds at the site where microbial attachment and biofilm formation are initiated while minimizing their loss to the surrounding seawater. Overall, this study demonstrates that chemical defence in H. floresii is associated with the selective surface enrichment of lipid-associated metabolites rather than the occurrence of unique surface-specific compounds, providing new insights into the spatial organization of macroalgal chemical defences and the mechanisms underlying resistance to microbial colonization and fouling.