The ISME Journal (2018) 12:1319–1328 https://doi.org/10.1038/s41396-018-0051-y ARTICLE Age-associated microbiome shows the giant panda lives on hemicelluloses, not on cellulose 1 2 1 1 3 2 1 Wenping Zhang ● Wenbin Liu ● Rong Hou ● Liang Zhang ● Stephan Schmitz-Esser ● Huaibo Sun ● Junjin Xie ● 4 1 2 5 1 1 1 Yunfei Zhang ● Chengdong Wang ● Lifeng Li ● Bisong Yue ● He Huang ● Hairui Wang ● Fujun Shen ● Zhihe Zhang1 Received: 12 June 2017 / Revised: 30 August 2017 / Accepted: 10 January 2018 / Published online: 1 February 2018 © The Author(s) 2018. This article is published with open access Abstract The giant panda feeds almost exclusively on bamboo, a diet highly enriched in lignin and cellulose, but is characterized by a digestive tract similar to carnivores. It is still large unknown if and how the giant panda gut microbiota contributes to lignin and cellulose degradation. Here we show the giant pandas’ gut microbiota does not significantly contribute to cellulose and lignin degradation. We found that no operational taxonomic unit had a nearest neighbor identified as a cellulolytic species or strain with a significant higher abundance in juvenile than cubs, a very low abundance of putative lignin and cellulose genes fi 1234567890();,: existed in part of analyzing samples but a signi cant higher abundance of genes involved in starch and hemicellulose degradation in juveniles than cubs. Moreover, a significant lower abundance of putative cellulolytic genes and a significant higher abundance of putative α-amylase and hemicellulase gene families were present in giant pandas than in omnivores or herbivores. Introduction [1]. Unlike other species in Arctoidea, >99% of the food consumed by the giant panda consists of highly fibrous The giant panda (Ailuropoda melanoleuca) is an endemic plant—bamboo. In order to process and utilize highly lig- flagship species in China. It is well known that the giant nified bamboo, the giant panda exhibits several specialized panda belong to the Arctoidea (a superfamily of Carnivora) morphological adaptations, such as panda’s thumb, large skull, wide flaring zygomatic arches, a prominent sagittal crest, and flat cheek teeth with elaborate crown patterns ’ Electronic supplementary material The online version of this article [2, 3]. However, the giant panda s gastrointestinal (GI) tract (https://doi.org/10.1038/s41396-018-0051-y) contains supplementary is largely characteristic of carnivores [2]. The giant panda’s material, which is available to authorized users. cecum is absent and its large intestine may not have any function for digesta fermentation [4] and its genome lacks * Wenping Zhang [email protected] homologs of the enzymes needed for cellulose degradation * Zhihe Zhang [5]. It has thus been hypothesized that the degradation of [email protected] cellulose, a key component of its bamboo diet, should be dependent on its gut microbiota. 1 Chengdu Research Base of Giant Panda Breeding, Some researchers have found some cellulose-degrading Chengdu 611081 Sichuan, China bacteria in the feces from giant pandas (such as, [6]). Based 2 Novogene Bioinformatics Institute, Beijing 100083, PR China on metagenomic technology to detect bacteria diversity in gut microbiome of the giant panda, Zhu et al. [7] detected 3 Department of Animal Science, Iowa State University, Ames 50011 IA, USA 13 operational taxonomic units closely related to Clos- tridium groups I and XIVa, both of which contain taxa 4 Biogas Institute of Ministry of Agriculture, Chengdu 610064 Sichuan, China known to digest cellulose, and putative genes coding for two cellulose-digesting enzymes. In contrast, other studies 5 Key Laboratory of Bioresources and Ecoenvironment (Ministry of Education), College of Life Sciences, Sichuan University, suggested that the giant panda gut microbiome does not Chengdu 610064 Sichuan, China have significant cellulose degradation potential [4, 8, 9], and 1320 W. Zhang et al. their overall microbiome composition converged with those resuspended in 1.2 ml of lysis buffer of the kit and trans- of carnivorous and omnivorous bears and not with herbi- ferred to a 2-ml screw-cap tube containing 0.3 g silica bead vores [9]. The giant panda has a very low daily energy mixtures (0.2 g, 0.1 mm, and 0.1 g 1.0 mm silica beads) expenditure, which can partially explain why the giant with a bead beater (BioSpec, Bartlesville, OK) set on high panda can survive and adapt to the low-quality bamboo for 2 min. After agitation by the bead beater, DNA was with low-energy digestive efficiency [10]. The functional extracted by following the manufacturer’s instructions. For role of the giant panda gut microbiota, particularly in cel- samples with bamboo, a pretreatment protocol prior to DNA lulose degradation, is still largely unknown. extraction according to the procedure described previously Diet is one key factor influencing the composition of GI by Xue et al. [9] was added. Then, the cell suspension tract microbial communities [11–13]. Thus, if the giant obtained from the pretreatment was used to extract DNA panda has the capacity to break-down cellulose, its gut same as the samples without bamboos. The DNA con- microbiota should adapt to the dietary change from breast centrations of each sample were adjusted to 50 ng/μl for milk to bamboo during growth. Thus, the abundance of subsequent 16 S rRNA genes, first internal transcribed putative cellulolytic genes and/or bacteria should increase spacer (ITS-1), and metagenomic DNA libraries. Details on from birth to juveniles. Here, we perform the first large the 16 S, ITS-1, and metagenome data processing are scale metagenome sequencing study of giant panda gut available in the Supplementary Information data. microbiota following animals from nursing to juvenile stages switching from milk to a bamboo diet. Comparison of carbohydrate-active genes between giant pandas and other mammalian metagenomes Materials and methods To maximize the breadth of possible comparisons of carbohydrate-active genes, our data of S3 and S4 were Samples combined with previously published mammalian metagen- ome data [11, 14] available on the MG-RAST database To investigate the microbiota development of the giant [15]. To minimize the potential effect of differences in panda from birth to juvenile, 14 captive-born giant pandas sequence size among the different data sets, short reads in housed in the Chengdu Research Base of Giant Panda our study were mapped to the contig sequences of the pan- Breeding and born in 2008 or 2011 were enrolled in this metagenome, then the mapping contigs were cut into 300 bp study (Supplementary Table S1) following the method bins starting from the first mapping base for each single pipeline (Supplementary Figure S1–S2). Following their short read as a mimic data set. Then these data sets of S3 diet and birth days, giant pandas’ feces were divided into and S4 samples with other mammalian microbiome data four groups: S1 (panda breast milk as diet and <2 months were used to annotate against the CAZy database following old), S2 (between 3 and 12 months old and no bamboo previously published methods [11]. The Pearson’s correla- found in their feces, commercial milk as dietary supple- tion coefficient index between the mimic data set and cor- ment), S3 (>6 months old and bamboo stems or leaves as responding paired-end data set was 0.75–0.802, which diet), and S4 (>6 months old and bamboo shoots as diet) showed that our mimic data could represent the total data (Supplementary Table S1). The ingestion and health status set. of each giant panda were monitored daily by veterinarians and giant panda keepers and no samples were included in Amylase copy number analysis in giant pandas host our study if giant pandas were fed antibiotics in the genomes 2 months prior to sampling. Fresh fecal samples were col- lected immediately after defecation and were snap frozen on Primers for qPCR were designed to be specific to pancreatic dry ice and shipped to the laboratory on dry ice, and were amylase based on the giant panda, polar bear, cat, cheetah, stored at −80 °C for no > 6 months before DNA and tiger reference genome sequences to compare copy extractions. numbers of amylase genes in Felidae (strict carnivore), Ursidae (omnivore), and the giant panda. In addition, pri- DNA extraction and data processing mers for qPCR targeting the housekeeping gene TP53 were designed based on the five reference genome sequences and DNA was isolated by bead-beating procedure described by were used to adjust for DNA dilution quantity variation of Xue et al. [9] with the Qiagen QIAamp DNA Stool Mini Kit different samples. DNA from blood samples of 41 giant (Qiagen, Germany) according to the protocol for isolation pandas, 2 black bears, 2 brown bears, 22 tigers, 3 lions, 2 of DNA for pathogen detection with slight improvements. leopards was used as templates for qPCR. Experiments For fecal samples without bamboo, ~1 g of each sample was were performed and analyzed as described by Perry et al. Age-associated microbiome shows the giant panda lives on hemicelluloses 1321 Fig. 1 16S rRNA gene-based comparison of gut microbiota structure metagenomic survey samples. d Heatmap of the 51 OTU-level phy- of giant pandas from birth to juvenile. a Relationship between alpha lotypes identified as key variables for differentiation between S1 and diversity and increasing age. Principal coordinate analysis (PCoA) S3/S4 gut microbiota structure of the giant pandas. The OTUs are using unweighted b and weighted c UniFrac distances of 16 S rRNA arranged according to their Spearman correlation coefficient (SCC) data. Each point corresponds to a community colored by collection index between the relative abundance of OTUs and age of giant date and diet (Table S1) and the black outer ring indicates pandas.