| Zokor(Myospalacinae)is one of subterranean rodents who feeds on the roots of plants,and could cause serious damage to crops and forests.The special feeding sites and living environment of zokor are significantly different from that of other pest harmful rodents,and may lead to the formation of unique internal microenvironment.At present,few studies have been done on the intestinal microbes of wild zokors,and it is unclear how the zokors adapt to special underground environment by regulating the composition and function of its intestinal microbes,especially the food source of plant roots.In addition,the relationship between zokor intestinal microbes and soil microbes of its habitat was still unclear.To answer above problems,Eospalax cansus and Eospalax rothschildi,which are endemic to China,closely related but different in habitat,were selected as the research objects.This dissertation focused on the composition and function of intestinal microbes between two zokor species and its correlation to soil microbes of its habitat.The main conclusions are as follows:(1)The composition and function of intestinal microbes between two zokor species were studied and compared by 16 S rRNA and metagenomic high-throughput sequencing.The results showed that the intestinal microbes of these two zokor species were similar in composition but distinct in proportion,and the structure of intestinal microbes between two zokor species showed significant differentiation.Firmicutes and Bacteroidota were dominant at phylum level,and E.cansus have a significantly high proportion of Firmicutes/Bacteroidota than that of E.rothschildi.The dominant genera were unclassified＿f＿lachnospiraceae,Ruminococcus and Clostridium.Some bacterium associated with cellulose degradation such as unclassified＿f＿Lachnospiraceae,Ruminococcus and Clostridium were significantly enriched in E.cansus.In addition,E.cansus harbored higher alpha diversity than E.rothschildi.Furthermore,the results based on egg NOG and KEGG database annotation showed that the relative abundance of genes annotated for carbohydrate metabolism was the highest in all samples,and these genes were significantly enriched in E.cansus.Through further analysis of KEGG pathway of carbohydrate transport and metabolism,it was found that the relative abundance of genes annotated for starch and sucrose metabolism was the highest in all samples,and they were significantly enriched in E.cansus.In the pathway of starch and sucrose metabolism,β-glucosidase(EC 3.2.1.21),the enzyme with the highest gene abundance in all samples,was related to cellulose degradation,and the genes encoding this enzyme were also significantly enriched in E.cansus.Above results demonstrated that the carbohydrate degradation is the most important function of intestinal microbes of two zokor species.Compared with E.rothschildi,E.cansus harbored a stronger carbohydrate degradation ability,mainly in starch and sucrose metabolism,and further in cellulose degradation.(2)The differences of abundance of glycoside hydrolase encoding cellulase/hemicellulase genes were compared between two zokor species based on annotated information of CAZy database,and the corresponding enzyme activities were determined.The results showed that there were three cellulases(β-glucosidase,endoglucanase and exoglucanase)and one hemicellulase(xylanase)in both species.The abundance of cellulase and cellulase activities of E.cansus were significantly higher than that of E.rothschildi,indicating that E.cansus had stronger ability of cellulose decomposition.The results of xylanase activity determination showed that there was no difference in hemicellulose degradation ability between two zokor species.In addition,the dominant microbes encoding cellulase/hemicellulose genes between two zokor species were similar in composition but distinct in proportion.Firmicutes and Bacteroidota were dominant at phylum level.The dominant genera were Clostridium,Ruminococcus,Prevotella and Bacteroides.Compared with E.rothschildi,the stronger ability of cellulose decomposition of E.cansus was mainly realized by increasing the abundance of intestinal microbes that could efficiently degrade cellulose,such as Clostridium and Ruminococcus.Based on the α diversity of cellulase gene encoded by different microbes,the results showed that those microbes could encode more diverse and numerous cellulase genes,have the ability to make more efficient use of plant fibers.(3)Cellulose-decomposing bacteria and hemicellulose-decomposing bacteria in caecum of E.cansus and E.rothschildi were isolated,cultured and identified,respectively.In addition,the characteristics of growth and enzyme production of cellulolytic bacteria with strong cellulose-degradation ability were studied.The results showed that 9,5 bacteria species with cellulose decomposing ability,and 8,5 bacteria species with hemicellulose decomposing ability were isolated from E.cansus and E.rothschildi,and the dominant bacteria all belonged to the genus Bacillus.B.wiedmanni,B.proteolyticus,B.cereus and B.thuringiensis in E.cansus and B.thuringiensis,B.toyonensis in E.rothschildi could degrade both cellulose and hemicellulose.Compared with E.rothschildi,more cellulose/hemicellulose-decomposing bacteria were isolated from E.cansus,and the corresponding enzyme activities were also higher,indicating that the cultural intestinal microbes of E.cansus had stronger cellulose/hemicellulose-decomposing ability.The strain with stronger cellulose-degradation ability in E.cansus was identified as B.cereus,a gram-positive bacterium.The cellulase produced by this strain had a wide range of tolerance to p H and temperature.Further research showed that the adaptive characteristics of this cellulase were consistent with the characteristics of intestinal environment of zokor.(4)The composition of intestinal bacteria of E.cansus and E.rothschildi and related soil bacteria(outside and inside the cave)at each site were studied based on 16 S rRNA sequences.The results showed that the composition and structure between zokor intestinal bacteria and related soil bacteria were significantly distinct.Compared with zokor intestinal bacteria,soil bacteria harbored more OTUs and higher alpha diversity,and PICRUSt metagenome predictions showed that they had more functions.The aggregation of zokor samples and their significantly separation from soil samples in β diversity results indicated that host factors played a more important role than environment factors in shaping intestinal microbes of zokor.Although the shared OTUs between zokors and related soils were present in the soil at relatively low abundance(<0.5%),these shared OTUs were rich in composition and function,belonging to diverse bacterial taxa,and composition and function of them were similar to the intestinal microbes of zokor.In conclusion,by exploring the composition,gene function,and enzyme activity of zokor intestinal microbes,it was proved that E.cansus had stronger ability of cellulose decomposition than that of E.rothschildi.The stronger ability of fermentation and degradation of cellulose of intestinal microbes of E.cansus may be a long-time adaptation to food resources underground.In addition,in interaction with soil bacteria,zokor’s gut tends to select soil bacteria with low abundance but diverse species and functions when zokor’s gut ingested soil of its habitat.This mechanism is usually a host-specific choice for zokor to meet its own dietary needs.In this study,the adaptive survival strategy of two zokor species in the face of low food quality were explained from the perspective of intestinal microbes.This result will help to deepen the understanding of the choice strategies of habitats and diet habits,and provide theoretical basis for ecological control of zokors. |
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