Ciona intestinalis in the spotlight of metabolomics and microbiomics : New insights into its invasiveness and the biotechnological potential of its associated microbiota

The tunicate Ciona intestinalis is one of the most notorious invasive ascidian species. In Prince Edward Island (PEI, Canada), C. intestinalis causes heavy fouling on farmed mussels leading to significant economic losses. Except for general beneficial eco-physiological characteristics of invasive ascidians, reasons underlying C. intestinalis’ invasiveness remain obscure. This study aimed to shed light on two additional factors potentially promoting its invasion success, i.e., bioactive secondary metabolites and associated microbiota, which reportedly contribute to the invasiveness of other marine species. Therefore, microbiomes and metabolomes of invasive (PEI) and native (Helgoland and Kiel, Germany) C. intestinalis populations were comparatively studied, a novelty in invasive ascidian research. Apart from being problematic invasive species, ascidians and their associated microbiota are a rich source for bioactive marine natural products (MNPs) relevant for human health. However, the biodiscovery potential of C. intestinalis-associated microorganisms remains largely unknown. Accordingly, this doctoral research project targeted to explore bioactivities and the chemical repertoire of culturable bacteria and fungi associated with C. intestinalis. Amplicon sequencing-based bacterial community analysis of gut, tunic, and seawater (control) samples revealed species-specificity and a diverse microbiota (39 phyla). The UPLC-MS/MS-based untargeted metabolomics approach revealed a diverse chemical inventory dominated by alkaloids and lipids. In addition to core bacteria and metabolites present in all samples, also tissue- and location-specific bacteria and metabolites were observed. Notably, highest microbial and chemical diversity were detected in the invasive C. intestinalis population (PEI). In combination, these results suggest a high adaptive capacity of C. intestinalis. In addition, several detected bacteria and secondary metabolites reportedly have antimicrobial, antifouling, and other relevant bioactivities, potentially promoting its overall health, fitness, and competitiveness. In conjunction with microbiome data, this first global metabolome study on C. intestinalis indicated microbial associates and chemical weapons as additional relevant factors promoting its invasion success. Therefore, this work contributes important basic knowledge for future projects scrutinizing the invasiveness of C. intestinalis. To investigate the potential of microorganisms associated with C. intestinalis in marine biodiscovery, isolates were obtained from tunics (T) and guts (G) due to their pivotal functions for the ascidian’s defense against, e.g., pathogens, and their reportedly different bacterial communities. In total, 89 (T) and 61 (G) bacteria as well as 22 (T) and 40 (G) fungi were isolated and identified from Helgoland and Kiel specimens. Many extracts showed antibacterial (T: 42%, G: 64%), antifungal (T: 10%, G: 11%), and/or anticancer (T: 6%, G: 22%) activities. A 2-step selection procedure considering bioactivity and metabolite profiles was applied to prioritize the most promising MNPs producers. This led to the selection of seven tunic- and nine gut-derived microbial extracts affiliated to the fungal group of ascomycetes (69%) and the bacterial taxa Actinobacteria (25%) and Bacillus sp. (6%). Through an UPLC-MS/MS-based dereplication workflow including molecular networking, in-silico approaches and manual database comparison, 170 compounds belonging to >40 different chemical families were putatively annotated, displaying a vast chemical diversity. Although this represents a significant increase in annotation rates compared to previous studies, still many compounds even from well-studied organisms (e.g., Penicillium and Streptomyces spp.) remained unknown. In summary, this study demonstrated a huge pharmaceutical potential of the culturable microbiota associated with C. intestinalis, including discovery of various putatively novel compounds. Application of novel selection and integrated dereplication procedures proved successful for strain prioritization and compound annotation. Furthermore, this strategy highlighted particularly fungi as so far uncharted and exceptionally promising resource for putatively novel anticancer and antimicrobial lead compounds of high interest.


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