Determination of sponge microbiome by metagenomic approaches

It has been known that most of the microorganisms that constitutes microbial diversity in marine environments has symbiotic relationship with sponges. The metagenomic approaches in which the genetic material of the microorganism is used due to the inadequacy of conventional cultivation techniques exposing this diversity took an important place in the recent years. In this thesis, microbial diversity of Agelas oroides, Ciocalypta carballoi, Petrosia ficiformis and Sarcotragus foetidus sponges collected from Mediterranean sea has been analyzed by metagenomic methods. Metagenomic DNA was isolated and amplified by PCR with 16S rDNA-V3 and ITS1 primers. 0-50% and 40-70% denaturant concentrations were used for Denaturing Gradient Gel Electrophoresis of 16S rDNA-V3 amplicons and 20 and 14 bacterial bands were isolated respectively. 0-50% denaturant concentration was used for Denaturing Gradient Gel Electrophoresis of ITS1 amplicons and 10 fungal bands were isolated. Totally 44 bands were isolated and sequenced. BLAST analysis were performed to define the nearest sequence match and phylogenetic trees were constructed by using MEGA6 software. Sequences have been registered to GenBank with accession numbers; KY379824-KY379849 for the 16S rDNA-V3 sequences, KY363543-KY363552 for the ITS1 sequences. It was determined that bacterial diversity of A. oroides, C. carballoi, P. ficiformis and S. foetidus was higher than their fungal diversity. Most common Proteobacteria phylum members were identified in all sponges. In this thesis, uncultured Roseospirillum sp. was determined in A. oroides for the first time. Uncultured terrestrial Cellulomonas sp. and Actinobacterium was also determined in C. carballoi. Fungal diversity was determined only in A. oroides and P. ficiformis. Fungal sequences obtained from A. oroides, was found highly similar to uncultured fungal sequences obtained from marine sediments. Uncultured lichen forming terrestrial Trichopezizella relicina from P. ficiformis was identified in this study for the first time.