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

Abstract

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.

Diversity, Bioactivity Profiling and Untargeted Metabolomics of the Cultivable Gut Microbiota of Ciona intestinalis

“…Bacterial isolates derived mainly from Marine Broth (MB; 26%) and tryptic soy broth (TSB; 31%) media, while gut-associated fungal strains were mainly obtained from WSP medium (20 isolates; other media: 1–7 fungal isolates). Similar to our previous study that reported the tunic-associated microbiota of C. intestinalis [ 31 ], we used media resembling the natural habitat of the isolates, i.e., media adjusted to Baltic (CB) or North Sea (CN) salinity containing C. intestinalis powder. They yielded many microbial strains ( n = 20), and the microbial genera Acrostalagmus , Arthopyrenia , Cordyceps, and Sporosarcina were exclusively isolated from these media.…”

“…[ 29 ]. We have recently described the cultivable microbiota of the tunic of C. intestinalis [ 31 ] that differed strikingly from the gut-associated microbial community isolated herein. Both tissues shared only few microbial genera (e.g., bacteria: Bacillus and Vibrio ; fungi: Fusarium and Penicillium ), which is in accordance with culture-independent microbiome studies on the gut and tunic of Ciona spp., indicating tissue-specific microbial communities [ 29 , 30 ].…”

“…Both tissues shared only few microbial genera (e.g., bacteria: Bacillus and Vibrio ; fungi: Fusarium and Penicillium ), which is in accordance with culture-independent microbiome studies on the gut and tunic of Ciona spp., indicating tissue-specific microbial communities [ 29 , 30 ]. Moreover, the diversity of culture-dependent fungi was higher in the gut (gut: 40 isolates assigned to 20 genera; tunic: 22 isolates, 15 genera), while bacteria were more prominent in the tunic (89 isolates assigned 37 genera; gut: 61 isolates, 13 genera) [ 31 ]. In line with our previous study on the tunic-associated microbiota [ 31 ], the gut-associated microbiota, especially the bacterial community, was more diverse in Kiel than in Helgoland samples ( Figure 1 and Figure 2 ).…”

“…Moreover, the diversity of culture-dependent fungi was higher in the gut (gut: 40 isolates assigned to 20 genera; tunic: 22 isolates, 15 genera), while bacteria were more prominent in the tunic (89 isolates assigned 37 genera; gut: 61 isolates, 13 genera) [ 31 ]. In line with our previous study on the tunic-associated microbiota [ 31 ], the gut-associated microbiota, especially the bacterial community, was more diverse in Kiel than in Helgoland samples ( Figure 1 and Figure 2 ). This may be attributed to different salinity levels (Kiel: brackish, Helgoland: oceanic salinity) and the comparably higher anthropogenic input, i.e., more eutrophic conditions, at the sampling site in Kiel Fjord [ 31 ].…”

“…In line with this, amplicon sequencing of the bacterial community associated with C. intestinalis and C. robusta described a specific and diverse gut community dominated by Gammaproteobacteria [ 29 ], while culture-dependent and -independent studies on the tunic-associated microbiota of Ciona spp. revealed comparably high abundance of Alphaproteobacteria [ 30 , 31 ]. However, no information exists on chemical composition or biological activities of the gut-associated microbial community of C. intestinalis .…”