Functional Coupling Of Nutritional Adaptation Of Microbiota For Bamboo Diet Of The Giant Panda In The Diet Transition Period

Gut microbiome is important to the host with nutrition,metabolism and other aspects.For the endangered mammal,giant panda(Ailuropoda melanoleura),studying the gut microbiota is particularly critical.The giant panda has successfully evolved that bamboo as its exclusive diet.However,the genome sequence of this mammal illustrated that the carnivorous digesting system lacks gene encoding enzymes for cellulose and hemicellulose metabolism.Therefore,intestinal microbiota likely plays the major function for bamboo digestion.Recently,with the development of next generation sequencing technology,there were several studies using metagenomics which focus on the giant panda microbiota communities and potential functions.However,it remains the unclear that gut microbiome transcript activities and transition.An important key in understanding the function of panda gut microbiota is the diet transition period.In this stage,the main diet changes from milk to bamboo that will contribute significantly to the development of gut microbiota.In this study,we collected fecal samples from six captive panda cubs during summer(May)and winter(November)of 2013,at their ages of 0.5-and 1-year old,or 1.5-and 2-year old.In these pandas,the diet changed from ~600 mL of milk for the 0.5-year old cubs to exclusive 7-kg bamboo for the 2-year old pandas per day.Our study was covered both summer and winter,which allowed us to analyze how the microbiotas of pandas responded to seasonal switch.We extracted genomic DNA and total RNA from the same fecal samples used for library preparation and applied both metagenomic and metaRNA-seq approaches to capture the microbiota dynamics.1.Metagenomics technology illustrated the gut microbiome structural and potential functional shifts of the giant panda in the transition periodAfter metagenomic sequencing,we obtained a total of ~404 million high-quality reads from all 12 samples,that assembled 582,082 contigs.We used different bioinformatics databases to analyze the microbiome communities(SILVA,MetaPhlAn2,MBGD)and potential functions(COG-eggNOG,Pfam).A total of 457,013 contigs(78.5%)were annotated by at least one of the five databases.Statistics of the rDNA abundance show that phylum Firmicutes were the most dominant in panda microbiota,followed by Proteobacteria.The giant panda intestinal environment seems to evolve a good preparation for cellulose digestion,that we detected almost all the gut cellulolytic bacterial species pervious reported in each panda,even in 0.5-year fresh milk ones.However,their abundances have a great change with the age growth.For example,Lactobacillus is the most abundant genus in youngest group but difficult to find in any older ones.Interest,both of cluster analysis and principal component analysis shows that season is the most important factor affecting intestinal microflora of giant pandas,instead of age element.The most abundant functional categories in COG include amino acid transport and metabolism,carbohydrate transport and metabolism,as well as energy production and conversion,underlining the importance of panda gut microbiota in nutrient supply.These genes at least include 55 kinds of carbohydrate enzymes,of which 44%(2,325/5,302)copies encoded GH enzymes targeting plant structural polysaccharides.Most copies of the GH gene can encode oligosaccharide-degrading enzymes,whereas the less copies encoding cellulases and debranching enzymes.Among all the bacteria,Escherichia coli,Clostridium sp.JCC,and Clostridium baratii keep the most abundant GH gene copies.2.Meta-transcriptomics technology investigated the activities and carbohydrate functional expression of the giant panda intestinal microbiomeIn the meta-RNAseq approach,we obtained totally ~527 million reads of the cDNA sequence from all rRNA,tRNA and mRNA.Since the currently premature meta-transcriptomics algorithm combined our obtained low level of mRNA,we could not further analyze much for every general expressed function but that gives us opportunities to explore bacterial activates and carbohydrate performance.No surprise,we found mainly functional bacterial species,especially for the digesting microorganisms show transcriptional pronounced since their larger rRNA proportions than rDNA.For example,the amount of two carbohydrates digesting related bacterial phylum Firmicutes and Proteobacteria keep in ~90% proportion among all samples,both far higher than their rDNA.In addition,the activity of intestinal bacterial transcription has important links with food composition,e.g.,Lactobacillus has an active transcription(rRNA/rDNA>1)in breast milk-based food for the 0.5-year-old panda intestinal,but keeps a state of inhibition(rRNA/rDNA<1)in the other the groups with the increasing bamboo diet.Because of the activation of Clostridium and Escherichia-Shigella in almost all panda individuals,we consider these two bacteria genera should be considering as the major functional bacteria in the intestinal tract of giant pandas during this period.Also,the amount number of activated bacterial species(rRNA/rDNA>1)in summer were much more than that in winter,which further supports the seasonal effects for the intestinal microbiome.GH genes were active in most of samples,playing important of GH genes for the digestion function of pandas.Among them,high expression GH genes were mostly related to oligosaccharide degrading enzymes,but less to cellulose.The high expression of oligosaccharide degrading enzymes may be responsible for the digestion of downstream products which are degraded by cellulose and hemicellulose in the up-stream intestinal microflora.In addition,the expression of GH gene was significantly correlated with that of food,the expression of GH gene in the intestinal tract of the giant panda was lower than that of the giant panda,and the expression of GH gene increased significantly with the increase of the content of bamboo.3.Transition trends of intestinal microbiome of the giant panda during diet transformationSince the analysis of metagenomics and meta-transcriptome indicated the individual differences in dietary pandas decreased with age,and it seems that gut microflora development transited in a specific trend.Therefore,we further use a variety of statistical methods to analyze the development trend and the reasons for the change.We at first sorted the intestinal flora of each group by abundance and found that many moderate abundance bacteria accounted for most of the intestinal microflora in the 0.5-year-old giant panda intestinal tract.While in the 2-year-old panda,a small number of high abundance of bacteria accounted for a major proportion.This situation indicated that the intestinal microflora of giant pandas experienced a pressure selection during the dietary transition period: most bacteria in the intestinal tract decreased or disappeared,while a small number of bacteria changed to dominate in the microbiome.This selection pressure also presented at the genetic level,with more favor with GH gene.We detected 88.86% of total GH genes(71.83% of non-GH genes)in the 0.5-year-old giant panda,but only detected 44.76% of the total GH genes(35.40% of non-GH genes)in the 2-year-old giant panda.We further mapped the cumulative abundance patterns of GH and non-GH species,and found that there was a significant pressure selection in the non-GH species in the giant panda gut during the transition period,of which loss abundance was much greater than that of GH-containing species.In contrast,the abundance of GH species was relatively stable or increased during the period(except for 0.5 years).This phenomenon indicated that GH gene plays an important role in regulating the selection pressure of giant panda.At the transcriptional level,the activity of the flora and the gene expression were like that of DNA,but with greater individual differences and less group differences,that indicated transcriptional regulation plays important role for the adaptation for the pressure.To verify the reliability of our sequencing data,we also randomly selected 26 GH genes for qPCR or RT-qPCR validation.The results showed that their relative abundance(and expression)was consistent with the sequencing results.ConclusionIn summary,the combination of comprehensive metagenomic and meta-expression study of gut microbiota of panda cubs during their diet transition period has reached several important findings.First,the intestinal flora of the giant panda was mainly composed by Firmicutes and Proteobacteria.Genus Lactobacillus(0.5 years),Clostridium and Escherichia were the main functional bacteria.Second,we confirmed the widespread presence of the GH gene in the intestinal microbiome of the giant panda.The transcriptional regulation of these genes played an important role in the adaptation of bacteria to the increase in bamboo of diet.Third,season is one of the major factors affecting the intestinal structure of the giant panda,which may be due to the different nutrients in bamboo and other food,as well as the eating preference in different seasons.Fourth,there is a trend that the intestinal microflora transited a more stable but less diversified structure during the dietary transition period.GH genes make a significant effect for the adaptation of the select pressure.In summary,our results suggest that the giant panda and its intestinal flora seem to have a very effective mechanism of cooperation in the digestion of bamboo.