Diversity Of The Culturable Aerobic And Facultative Anaerobic Bacteria In The Gut Of Reticulitermes Chinensis Snyder & Phylogenetic Analysis Of The Symbiotic Protists In The Gut Of Reticulitermes Chinensis Snyder

Wood-feeding termites are one of the most important insects that can digest lignocellulose effectively. Although it was assumed that symbiotic microbiota play important roles in the process of cellulose degradation, so far, little was known about the physiology and metabolic activities of most of the gut symbionts. It has been estimated that termites harbor a dense and diverse population of bacteria (between 109-1011 cells per gut), which may involve in the digestion of wood cellulose, nitrogen fixation and uric acid recycling. To obtain a better understanding of the microbiota inhabiting the intestinal tract of wood-feeding termites, the subterranean termite, Reticulitermes chinensis (Snyder) widely distributed in the area of Yangtze River, was used as a model. The population structure and distribution of the aerobic and facultative anaerobic bacteria in the gut of this termite was evaluated using culture-dependent method. In total 104 bacterial strains were isolated from the gut of R. chinensis with three types of media. In addition, the symbiotic flagellates were phylogenetically characterized using molecular techniques such as clonal analysis,RFLP and fluorescence in situ hybridization. All of the results form the basis for futher studies on the metabolic mechanisms of the gut microbiota, the discovery and use of new microbial resources. The main results of this study are as follows:The quatity and community structure of the aerobic and facultative anaerobic bacteria were studied by spread plate method combined with 16S rDNA sequence-based phylogenetic analysis. In total 104 bacterial strains, which were classfied into 55 ribotypes by ARDRA patterns, were isolated from three different media. Blast search based on the 16S rRNA gene sequnces of the representatives of the 55 ribotypes showed that all of the isolates belong to 20 bacterial genera. Quatitative analysis showed that 30.7% of the isolates belong to Bacillus, representing an absolute dominant population, whereas bacteria affiliated with Lactococcus account for 20.2% in the whole community. Enterobacter bacterial isolates have been found in all three kinds of media.Based on the 16S rRNA gene sequences of the isolates,6 newly isolated strains could be novel bacterial genus or species since their sequnces similarity are lower than 97% compared with the other bactera retrieved from Genbank. One of the isolates, Strain TM-1T exhibited maximum 16S rRNA gene sequence similarity (94%) with Deniococcus gobiensis DSM 21396T. On the basis of the phylogenetic, chemotaxonomic and phenotypic data, strain TM-1T was supposed to a novel species of the genus Deinococcus spp.Real-time quantitative PCR method was used for measuring the abundance of bacteria and strain Lactococcus sp.TSB-13 in termite gut. PCR primers targeting the strain Lactococcus sp. TSB-13-specific region of the 16S rRNA gene were tested.The termite gut sample was analyzed by RTQ-PCR to determine the relative abundance of strain Lactococcus sp.TSB-13 16S rRNA gene compared to the total abundance of eubacterial rDNA.The rDNA fraction of strain Lactococcus sp.TSB-13 compared to all Eubacteria were 0.05%.Phylogenetic characterization of the symbiotic flagellates in the gut of R.chinensis was also reported for the first time. With the combination of phylogentic analysis of small subunit rRNA gene sequences and fluorescence in situ hybridization techniques, three oxymonads and four parabasalids were identified. The three oxymonad protists in the intestinal gut were P. grandis, D. exilis and D. parva, whereas the identified parabasalids belonged to the order Spirotrichonymphade, Trichonymphidae and Trichomonadidae.