All surfaces and interfaces in the marine environment are susceptible to biological colonisation, a process known as biofouling. This phenomenon poses significant ecological and economic challenges, affecting marine industries and infrastructure. Conventional solutions to biofouling rely predominantly on chemical bioactive coatings, many of which cause harmful environmental effects, such as toxicity to non-target organisms and disruption of marine ecosystems. In contrast, nature has developed sophisticated strategies to combat biofouling. Certain marine organisms—such as sea squirts, corals, and algae—effectively prevent colonisation (epibiosis) through mechanisms that may involve chemical defences, producing bioactive compounds that repel fouling species. Understanding these natural mechanisms offers a promising avenue for developing ecologically relevant anti-fouling technologies that mitigate biofouling while preserving marine ecosystems.

Through monitoring biofouling in the Port of Leixões, where CIIMAR is based, we observed that individuals of the ascidian species Ciona sp. and Clavelina sp. exhibit tunics free of epibiosis, while those of the species Molgula sp. and Styela sp. have colonised surfaces. These observations formed the basis for FOULPROOF, an exploratory project that aims to discover the biological and chemical mechanisms that allow certain ascidians to resist fouling. We hypothesise that the control of epibiosis in some ascidian species is mediated by small molecules produced by specialised symbiotic microbial communities that exist in their tunics.
Recent omics-based studies suggest that some species enrich their microbial communities with symbionts capable of synthesising bioactive compounds. However, the link between the composition of the microbiome and the metabolomic profiles of these communities remains unexplored, especially in the context of fouling resistance. FOULPROOF seeks to fill this gap by elucidating the interactions between ascidian microbiota, metabolite production, and natural anti-fouling strategies.

This project aims to generate and integrate independent high-resolution datasets at different biological levels to discover the microbial composition and metabolite diversity in ascidian tunics. Specifically, it seeks to: i) characterise the microbial communities inhabiting colonised and non-colonised ascidian tunics through metagenomic sequencing; ii) identify and analyse the expression of secondary metabolite biosynthetic gene clusters (BGCs) in the tunic microbiota using metagenomic and metatranscriptomic analyses; iii) compare metabolite diversity between ascidian species using metabolomic techniques, revealing potential anti-fouling compounds.

FOULPROOF employs a metagenomic, metatranscriptomic and metabolomic strategy to correlate microbial taxa and BGCs linked to metabolite production, revealing their ecological roles and biotechnological potential.
The results of FOULPROOF will advance the understanding of microbiome-mediated anti-fouling mechanisms in ascidians, with direct implications for marine ecology, biotechnology and the development of anti-fouling technologies. The knowledge gained will: i) unravel the role of symbiotic microbiota in modulating resistance to biofouling; ii) identify natural antifouling compounds with potential applications in eco-friendly marine coatings; iii) provide a functional framework for studying the control of epibiosis, facilitating future research in chemical ecology and microbial interactions.
The project will be conducted by an interdisciplinary team in marine biofouling and antifouling ecology (JRA), natural product chemistry (PNL, AR), metagenomics and bioinformatics (AR), and molecular ecology of ascidians (AM). Their combined expertise ensures a robust approach to achieving the objectives of FOULPROOF.

The impact of the project will be maximised through open access data management and dissemination. The datasets generated will be deposited in public repositories, and findings will be communicated through peer-reviewed publications, international conferences, and outreach activities. In short, by investigating the role of microbial symbionts and their metabolites in biofouling resistance, FOULPROOF aims to contribute to research in marine chemical ecology and pave the way for the development of sustainable anti-fouling technologies. At the end of this exploratory project, we will be in a position to determine whether ascidian epibiosis can be studied from a small molecule perspective or whether alternative mechanisms should be considered.