The Research On Microbial Community In The Gut Of Giant Panda(Ailuropoda Melanoleuca) And Its Influencing Factors

Phylogenetically,giant panda belongs to family Ursidae,which includes both carnivorous and omnivorous members.It possesses a typical carnivore-like short gastrointestinal tract.However,it became an exclusively bamboo-eating animal nearly 2million years ago(by the late Pliocene).It is surprising and a matter of debate how giant panda is surviving on low nutrition bamboo diet,while its carnivorous digestive system does not support the digestion of such highly fibrous food.Whereas,the giant panda lacks genes involved in digesting cellulose in its genome.Therefore,panda’s gut microbiota is considered to play an essential role in digesting bamboo diet.To reveal the adaptability of panda’s gut microbiota to its specialized diet and the main factors affecting the structure and function of gut microbiota of giant panda,we used the methods of 16 S r DNA and metagenome to carry out a systematic study on the structure and function in the intestinal microflora of giant panda.This study included four parts:Part one: High throughput sequencing of 16 S rRNA genes was performed on 8 giant panda cubs(from 4 to 17 months old).Based on bioinformatics analysis,we revealed the dynamic changes in the gut microbiota of giant panda during the growth period.Moreover,the source of the giant panda’s gut microbes was traced through the analysis of microorganisms in the diet(breast milk,formula milk,carrot,bamboo and bamboo shoots)and the environment(water,soil,mother’s vagina,milk,nipplen and oral).A previous study on the gut microbiota of giant panda cubs showed that the composition of gut microbiomes of giant pandas at different ages is significantly different.At the genus level,Bacteroidetes＿unclassified was the predominant genus in the cubs Ⅰ(4-7 months,The main diet is milk),cubs Ⅱ(8-13 months,The diet mainly is milk and all kinds of supplementary food),and cubs Ⅲ(14-17 months,The main diet is bamboo)were dominated by Escherichia/Shigella whereas Streptococcus and Escherichia/Shigella were the most abundant genus in the adult giant panda.In addition,cubs Ⅲ and the adult giant pandas have shown similar alpha diversities.Furthermore,the alpha diversity was significantly lower in cubs Ⅲ(14-17 months)and adults than in those of the cubsⅠ(4-7months)and cubs Ⅱ(8-13 months).The results of the beta diversity analysis demonstrated that overall gut microbiota structures of cubsⅠ(4-7 months),cubs Ⅱ(8-13 months),and cubs Ⅲ(14-17 months)were distinct from each other,and cubs Ⅲ(14-17 months)clustered closer to those of the adult.Our study on the source of the gut microbiomes of the giant pandas confirmed that mother’s vagina,milk,and nipple are the primary sources of giant panda’s gut microbes.Part two: We examined and compared the composition of gut microbiotas in giant pandas,red pandas and black bears by sequencing the V1-V3 region of 16 S rRNA genes.The result of beta diversity analysis of gut microbiotas indicated that the gut microbiotas of giant pandas are significantly distinct from those of red pandas and similar with the black bears.Gaint pandas were dominated by Streptococcus whereas Sarcina was the most abundant genus in the red panda,and their relative abundances were significantly different in the two species of pandas.Our data suggests that the evolution of gut microbiotas in the three carnivores is consistent with the phylogeny of host.Part three: The fecal DNA of 6 captive giant pandas,4 captive black bears,and 4 captive bamboo rats were used for shotgun metagenomic sequencing.To reveal the adaptability of gut microbiomes of giant pandas to its bamboo diet,we compared our data with similar data from other animal species representing 25 herbivores,19 carnivores,and12 omnivores from the current and the earlier studies.The comparison of metabolic pathways of the intestinal KEGG of animals with different diets showed that the giant panda hosts a bear and carnivore-like gut microbiota distinct from those of herbivores indicated by the predicted metagenomic functions.In addition,the relative abundances of genes involved in cellulose-and hemicellulose-digestion in the giant pandas and carnivores were significantly lower than those of herbivores.Similarly with carnivores,the gut microbiotas of giant pandas harbored the enrichment of genes associated with amino acid degradation pathways,but lacked those genes involved in amino acid biosynthetic and fermentation pathways.These phenomenons were contrary to typical herbivores.Finally,an in-vitro experimental assay of enzyme activity for cellulose and hemicellulose-degradation of the feces of animals with different diets also supported the above results.The findings of this enzyme assay demonstrated that the capacity of digesting cellulose and hemicellulose of gut microbiomes in giant pandas is poor.Part four: We examined and compared the composition,functional metabolism,resistance genes and virulence factors of gut microbiomes in 49 captive pandas and 98 wild giant pandas.The diversity,structure and functional potential in the fecal microbiomes of wild and captive giant pandas differ significantly.For example,in wild pandas,Pseudomonas was the most abundant genus,while Streptococcus and Escherichia/Shigella were the most abundant genera in captive pandas.The gut microbiota in the captive giant pandas had significantly lower diversity than those in the wild ones.Captive pandas had reduced functional potential for cellulose degradation but enriched metabolic pathways for starch metabolism.Also,antibiotic resistance genes and virulence factors,as well as heavy metal tolerance genes were enriched in the microbiomes of captive pandas.These results indicated that the management and environment of artificial breeding have a negative impact on the gut microbiomes of the giant pandas.Taken together,this study revealed that carnivorous digestive system and limited diet were the major factor to lead to the lower diversity of gut microbiotas in giant pandas;Meanwhile,gut microbiotas of giant panda is not adapted to its highly specialized diet;The structure and function of gut microbiotas of giant panda were significantly altered by artificial feeding,and brought a series of negative effects.