Culture-Dependent Microbiome of the Ciona intestinalis Tunic: Isolation, Bioactivity Profiling and Untargeted Metabolomics

Ascidians and their associated microbiota are prolific producers of bioactive marine natural products. Recent culture-independent studies have revealed that the tunic of the solitary ascidian Cionaintestinalis (sea vase) is colonized by a diverse bacterial community, however, the biotechnological potential of this community has remained largely unexplored. In this study, we aimed at isolating the culturable microbiota associated with the tunic of C.intestinalis collected from the North and Baltic Seas, to investigate their antimicrobial and anticancer activities, and to gain first insights into their metabolite repertoire. The tunic of the sea vase was found to harbor a rich microbial community, from which 89 bacterial and 22 fungal strains were isolated. The diversity of the tunic-associated microbiota differed from that of the ambient seawater samples, but also between sampling sites. Fungi were isolated for the first time from the tunic of Ciona. The proportion of bioactive extracts was high, since 45% of the microbial extracts inhibited the growth of human pathogenic bacteria, fungi or cancer cell lines. In a subsequent bioactivity- and metabolite profiling-based approach, seven microbial extracts were prioritized for in-depth chemical investigations. Untargeted metabolomics analyses of the selected extracts by a UPLC-MS/MS-based molecular networking approach revealed a vast chemical diversity with compounds assigned to 22 natural product families, plus many metabolites that remained unidentified. This initial study indicates that bacteria and fungi associated with the tunic of C.intestinalis represent an untapped source of putatively new marine natural products with pharmacological relevance.