Bacterial Diversity Associated With Two Species Of Ascidians And Screeningof Cytotoxic Activity Of The Strains

Ascidians are one of the major marine animals from which many kinds of marine natural products were isolated. By now, over 1000 metabolites with significant bioactivities were isolated from ascidians, including metabolites with antibacterial, antifungal, cytotoxic and anti-inflammation activities. Direct and indirect evidences showed that most of the bioactive metabolites isolated from ascidians were actually produced by symbiotic bacteria. There are abundant ascidian species in China, including of some unique species. Microbial communities associated with ascidian are significant different between host species. The aim of this study was to exploit cultivable bacteria associated with ascidians from the coast of Weihai,to study the bacterial diversity and to investigate their potential antitumor activities. It’s good for broadening our knowledge about ascidian-microbial symbiotic relationship, and providing the research foundation for the development of marine microbial resource and the discovery of novel marine natural products.Culture-dependent method was used to isolate bacteria associated with ascidians of Styela clava and Aplidium sp. collected from Xiaoshi Dao and Poyu Zhen in Weihai of Shandong pronivce. The identification and phylogenetic analysis of the isolated bacterial strains were determined by 16 S r DNA sequencing. Microbial diversity of two ascidian species from different locations were compared and analyzed to study the host and location specificity of the microbial communities. MTT analysis was performed to determine the potential cytotoxicity of the crude extract of the isolated bacterial strains.With several culture media and isolation methods, a total of 453 bacterial strains were isolated from two species of ascidians. 124 bacterial strains were isolated from Styela clava of Xiaoshi Dao, 144 bacterial strains were isolated from Styela clava of Poyu Zhen, 96 bacterial strains were isolated from Aplidium sp. of Xiaoshi Dao, and 89 bacterial strains were isolated from Aplidium sp. of Poyu Zhen. 282 bacterial strains were selected and sequenced for their 16 S r DNA to analyze their phylogenetic positions. Our results showed a high microbial diversity associated with two ascidian species from both locations. The bacteria associated with Styela clava of Xiaoshi Dao belonged to 51 species of 18 genera of 13 families in 3 phyla. The bacteria associated with Styela clava of Poyu Zhen belonged to 48 species of 16 genera of 15 families in 3 phyla. The bacteria associated with Aplidium sp. of Xiaoshi Dao belonged to 35 species of 14 genera of 10 families in 3 phyla. And the bacteria associated with Aplidium sp. of Poyu Zhen belonged to 31 species of 15 genera of 12 families in 3 phyla. Bacillus was the dominant genus in Firmicutes and Streptomyces was the dominant genus in Actinobacteria. The dominant genus in Proteobacteria varies between ascidian samples.Of the 453 bacterial strains,the extracts from 80 bacterial strains(17.6% strains) showed good cytotoxicity against human hepatocellular cell line Bel-7402, the IC50 of 22 strains among them are below 500 μg?m L-1. The extracts from 107 bacterial strains(23.6% strains) exhibited good cytotoxicity against human cervical cancer cell line He La, the IC50 of 11 strains among them are below 500 μg?m L-1. The active bacterial strains mainly belonged to the genera of Bacillus and Streptomyces.Our results suggest that the bacterial diversity associated with ascidian is abundant and may be developed as promising sources for the discovery of novel bioactive substances.