Proteomic insights into the antifouling molecular responses of nature-inspired gallic and bile acid derivatives in Mytilus galloprovincialis

Nature-inspired compounds are increasingly recognized as promising sources of effective and environmentally safer antifouling (AF) agents. However, the development of new AF solutions under current regulatory frameworks requires a better understanding of the biological responses associated with their activity. Previous studies identified two synthetic derivatives of gallic and bile acids, 2-(3,4,5-trihydroxybenzamido)ethan-1-aminium bromide ( 1 ) and methyl deoxycholate ( 2 ), as potent inhibitors of Mytilus galloprovincialis larval settlement, a prominent biofouling species. Building on these findings, here we used an untargeted shotgun proteomics approach to explore the molecular responses of mussel plantigrades following exposure to compounds 1 and 2 . Proteomic analysis identified 3,214 and 2,091 proteins in plantigrades exposed to compounds 1 and 2 , respectively. Based on a predefined two-fold abundance-change threshold, 999 and 393 proteins, respectively, were identified as candidate abundance-shifted proteins. Exposure to both compounds was associated with abundance shifts in proteins containing von Willebrand factor (vWF) and epidermal growth factor (EGF)-like domains, which are also found in some mussel adhesive proteins. Functional enrichment analysis identified candidate abundance shifts associated with ciliary motility, vesicle transport pathways, and protein-processing pathways, while exposure to gallic acid derivative 1 was additionally associated with proteins involved in ATP-related metabolic processes. These patterns highlight cellular processes that may be associated with the response to compound exposure and provide potential hypotheses regarding processes linked to settlement inhibition. Although these findings require further validation through complementary targeted approaches, they provide valuable insights into the molecular processes associated with exposure to compounds 1 and 2 and reveal candidate pathways that may contribute to their settlement-inhibitory activity. Overall, this study expands the molecular understanding of AF responses in mussel plantigrades and strengthens the rationale for further developing these nature-inspired derivatives as promising candidates for safer and more environmentally compatible biofouling control.