A Study Of The Effect Of Hydrogen-consuming Microorganisms On The Metabolisms Of Reticulitermes Chinensis Snyder

Termite is a kind of social insect that can efficiently degrade lignocellulose.It is known that various of intestinal symbiotic microorganisms in termite gut playing important roles in lignocellulose-degradation.In the gut of lower termites,the symbiotic protists first can digest lignocellulose through anaerobic fermentation to produce acetic acid,CO2 and H2.Primary studies showed that two kinds of H2-consuming microorganisms,namely homoacetogens and methanogens,can further remove H2 by reduce CO2 to produce acetic acid and methane,respectively.How these two kinds of H2-consuming microorganisms can affect the host metabolism in the gut of lower termites and if the population changes of these microorganisms will greatly affect termite life cycle remains unknown.In this thesis,we studied the effect of the two H2-consuming microorganisms on the host metabolisms of Reticulitermes chinensis with the use of inhibitors combined with different molecular techniques,which is significant for the further studies of the symbiotic relationship between intestinal microbes and wood-feeding termites.The main results of this study are as follows:1.The effects of inhibitors to the metabolisms products of R.chinensisBES(2-Bromoethanesulfonic Acid Sodium Salt)and metronidazole were used to inhibit the activities of two types of H2-consuming microorganisms,namely,homoacetogens and methanogens,respectively.In order to explore their effects on the metabolisms products of R.chinensis.The results showed that with the treatment of increasing concentrations of BES,methane emission gradually decrease and eventually disappear.However,this will not cause the death of host.When R.chinensis were treated with high concentrations of metronidazole,methane emission are also showed a trend of decline.While the concentration of metronidazole is 800mg/l,methanogens are significantly inhibited,almost no methane release.2.The effects of inhibitors on the microbial community structure and composition of R.chinensis16S rRNA Pyrosequencing was used to study the effects on microbial community structure and composition of R.chinensis with the treatment of two inhibitors.The results showed that the microbial community structure and composition of R.chinensis changed obviously with the treatment of different inhibitors.With the increasing of concentration and treatment time of metronidazole,the number of major homoacetogens,Spirochaeta decreased,and disappeared.While the number of bacteria affiliated with Proteobacteria,Firmicutes and Bacteroides increased.With the treatment of BES,the number of methanogens decreased.When the concentration of BES achieved 800mg/l,nearly no methanogens were detected from the gut of R.chinensis.Meantime,the number of Elusimicrobia increased,whereas the number of several other groups of bacteria such as Spirochaetes and Firmicutes presented little changes.R.chinensis died within three weeks of treatment of metronidazole(1000mg/l),whereas they kept active with the treatment of BES.The results showed that,Spirochaetes were major groups of symbiotic microbes in the intestinal tract of R.chinensis and played important roles in the metabolisms of homoacetogenesis.Although methanogens are one of the H2-consuming microorganisms,the absence of these microbes do not affect the activities of host termites.3.The effects of inhibitors on gene of key enzymes involved in H2-consuming metabolismsMolecular techniques such as qRT-PCR and T-RFLP were used to detect the changes of number of gene copies and types of key enzymes such as FTHFS and mcrA that involved in homoacetogenesis and methanogenesis with the treatment of inhibitors.Compared with normal termites,most of the T-RFs of FTHFS genes corresponding to homoacetogenic spirochaetes cannot be detected after treatment with high concentrations of metronidazole for one week.Only quite a few number of gene copies of FTHFS can be detected with qRT-PCR.The termites died gradually within three weeks,which indicated that the inhibitions to the homoacetogenesis associated with Spirochaetes will cause the death of host termites.The genes encoded key enzymes of methanogenesis(mcrA)cannot nearly detected with qRT-PCR after treatment of the termites with one week,indicating that BES can efficiently inhibit the activity of methanogens.However,this inhibition won't affect the life of termite,which can live for two months.In summary,homoacetogens and methanogens are two kinds of H2-consuming microorganisms in the gut of R.chinensis.But the ability of homoacetogenesis using hydrogen significantly higher than that of methanogens.Spirochaetes are the most important homoacetogens and play important roles in the reductive acetogenesis in host termites.Mainly to keep normal physiological activities of termites.