Seasonal variation in nutrient utilization shapes gut microbiome structure and function in wild giant pandas.
ABSTRACT: Wild giant pandas use different parts of bamboo (shoots, leaves and stems) and different bamboo species at different times of the year. Their usage of bamboo can be classified temporally into a distinct leaf stage, shoot stage and transition stage. An association between this usage pattern and variation in the giant panda gut microbiome remains unknown. Here, we found associations using a gut metagenomic approach and nutritional analyses whereby diversity of the gut microbial community in the leaf and shoot stages was significantly different. Functional metagenomic analysis showed that in the leaf stage, bacteria species over-represented genes involved in raw fibre utilization and cell cycle control. Thus, raw fibre utilization by the gut microbiome was guaranteed during the nutrient-deficient leaf stage by reinforcing gut microbiome robustness. During the protein-abundant shoot stage, the functional capacity of the gut microbiome expanded to include prokaryotic secretion and signal transduction activity, suggesting active interactions between the gut microbiome and host. These results illustrate that seasonal nutrient variation in wild giant pandas substantially influences gut microbiome composition and function. Nutritional interactions between gut microbiomes and hosts appear to be complex and further work is needed.
Project description:Bamboo-eating giant panda (Ailuropoda melanoleuca) is an enigmatic species, which possesses a carnivore-like short and simple gastrointestinal tract (GIT). Despite the remarkable studies on giant panda, its diet adaptability status continues to be a matter of debate. To resolve this puzzle, we investigated the functional potential of the giant panda gut microbiome using shotgun metagenomic sequencing of fecal samples. We also compared our data with similar data from other animal species representing herbivores, carnivores, and omnivores from current and earlier studies. We found that the giant panda hosts a bear-like gut microbiota distinct from those of herbivores indicated by the metabolic potential of the microbiome in the gut of giant pandas and other mammals. Furthermore, the relative abundance of genes involved in cellulose- and hemicellulose-digestion, and enrichment of enzymes associated with pathways of amino acid degradation and biosynthetic reactions in giant pandas echoed a carnivore-like microbiome. Most significantly, the enzyme assay of the giant panda's feces indicated the lowest cellulase and xylanase activity among major herbivores, shown by an in-vitro experimental assay of enzyme activity for cellulose and hemicellulose-degradation. All of our results consistently indicate that the giant panda is not specialized to digest cellulose and hemicellulose from its bamboo diet, making the giant panda a good mammalian model to study the unusual link between the gut microbiome and diet. The increased food intake of the giant pandas might be a strategy to compensate for the gut microbiome functions, highlighting a strong need of conservation of the native bamboo forest both in high- and low-altitude ranges to meet the great demand of bamboo diet of giant pandas.
Project description:The gut microbiota diversity of eight panda cubs was assessed during a dietary switch.Gut microbiota diversity of panda cubs significantly decreased after bamboo consumption.Carnivorous species living on a plant-based diet possess low microbial diversity.Mice were fed a bamboo diet but did not display low gut microbiota diversity.Giant pandas have an exclusive diet of bamboo; however, their gut microbiotas are more similar to carnivores than herbivores in terms of bacterial composition and their functional potential. This is inconsistent with observations that typical herbivores possess highly diverse gut microbiotas. It is unclear why the gut bacterial diversity of giant pandas is so low. Herein, the dynamic variations in the gut microbiota of eight giant panda cubs were measured using 16S rRNA gene paired-end sequencing during a dietary switch. Similar data from red panda (an herbivorous carnivore) and carnivorous species were compared with that of giant pandas. In addition, mice were fed a high-bamboo diet (80% bamboo and 20% rat feed) to determine whether a bamboo diet could lower the gut bacterial diversity in a non-carnivorous digestive tract. The diversity of giant panda gut microbiotas decreased significantly after switching from milk and complementary food to bamboo diet. Carnivorous species living on a plant-based diet, including giant and red pandas, possess a lower microbial diversity than other carnivore species. Mouse gut microbiota diversity significantly increased after adding high-fibre bamboo to their diet. Findings suggest that a very restricted diet (bamboo) within a carnivorous digestive system might be critical for shaping a low gut bacterial diversity in giant pandas.
Project description:The giant panda is known worldwide for having successfully moved to a diet almost exclusively based on bamboo. Provided that no lignocellulose-degrading enzyme was detected in panda's genome, bamboo digestion is believed to depend on its gut microbiome. However, pandas retain the digestive system of a carnivore, with retention times of maximum 12 h. Cultivation of their unique gut microbiome under controlled laboratory conditions may be a valid tool to understand giant pandas' dietary habits, and provide valuable insights about what component of lignocellulose may be metabolized. Here, we collected gut microbiomes from fresh fecal samples of a giant panda (either entirely green or yellow stools) and supplied them with green leaves or yellow pith (i.e., the peeled stem). Microbial community composition was substrate dependent, and resulted in markedly different fermentation profiles, with yellow pith fermented to lactate and green leaves to lactate, acetate and ethanol, the latter to strikingly high concentrations (?3%, v:v, within 3.5 h). Microbial metaproteins pointed to hemicellulose rather than cellulose degradation. The alpha-amylase from the giant panda (E.C. 188.8.131.52) was the predominant identified metaprotein, particularly in reactors inoculated with pellets derived from fecal samples (up to 60%). Gut microbiomes assemblage was most prominently impacted by the change in substrate (either leaf or pith). Removal of soluble organics from inocula to force lignocellulose degradation significantly enriched Bacteroides (in green leaf) and Escherichia/Shigella (in yellow pith). Overall, different substrates (either leaf or pith) markedly shaped gut microbiome assemblies and fermentation profiles. The biochemical profile of fermentation products may be an underestimated factor contributing to explain the peculiar dietary behavior of giant pandas, and should be implemented in large scale studies together with short-term lab-scale cultivation of gut microbiomes.
Project description:Adaptation to a bamboo diet is an essential process for giant panda growth, and gut microbes play an important role in the digestion of the polysaccharides in bamboo. The dietary transition in giant panda cubs is particularly complex, but it is an ideal period in which to study the effects of gut microbes on polysaccharide use because their main food changes from milk to bamboo (together with some bamboo shoot and coarse pastry). Here, we used 16S rDNA and internal transcribed spacer 1 (ITS1) DNA sequencing and metagenomic sequencing analysis to investigate the succession of the gut microbial structure in feces sampled from twin giant panda cubs during the completely dietary transition and determine the abundances of polysaccharide-metabolizing genes and their corresponding microbes to better understand the degradation of bamboo polysaccharides. Successive changes in the gut microbial diversity and structure were apparent in the growth of pandas during dietary shift process. Microbial diversity increased after the introduction of supplementary foods and then varied in a complex way for 1.5-2 years as bamboo and complex food components were introduced. They then stabilized after 2 years, when the cubs consumed a specialized bamboo diet. The microbes had more potential to metabolize the cellulose in bamboo than the hemicellulose, providing genes encoding cellulase systems corresponding to glycoside hydrolases (GHs; such as GH1, GH3, GH5, GH8, GH9, GH74, and GH94). The cellulose-metabolizing species (or genes) of gut bacteria was more abundant than that of gut fungi. Although cellulose-metabolizing species did not predominate in the gut bacterial community, microbial interactions allowed the giant pandas to achieve the necessary dietary shift and ultimately adapt to a bamboo diet.
Project description:Gut microbes can enhance the ability of hosts to consume secondary plant compounds and, therefore, expand the dietary niche breadth of mammalian herbivores. The giant and red pandas are bamboo-eating specialists within the mammalian order Carnivora. Bamboo contains abundant plant secondary metabolites (e.g., cyanide-containing compounds). However, Carnivora species, including the giant panda, have deficient levels of rhodanese (one of the essential cyanide detoxification enzymes) in their tissues compared with the same tissues of herbivores. Here, we make a comparative analysis of 94 gut metagenomes, including 25 from bamboo-eating pandas (19 from giant pandas and 6 from red pandas), 30 from Père David's deer, and 39 from published data for other mammals. The bamboo-eating pandas' gut microbiomes had some common features, such as high proportions of Pseudomonas bacteria. The results revealed that bamboo-eating pandas' gut microbiomes were significantly enriched in putative genes coding for enzymes related to cyanide degradation (e.g., rhodanese) compared with the gut microbiomes of typical herbivorous mammals, which might have coevolved with their special bamboo diets. The enrichment of putative cyanide-digesting gut microbes, in combination with adaptations related to morphology (e.g., pseudothumbs) and genomic signatures, show that the giant panda and red panda have evolved some common traits to adapt to their bamboo diet.IMPORTANCE The giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens), two obligate bamboo feeders, have distinct phylogenetic positions in the order Carnivora. Bamboo is extraordinarily rich in plant secondary metabolites, such as allied phenolic and polyphenolic compounds and even toxic cyanide compounds. Here, the enrichment of putative cyanide-digesting gut microbes, in combination with adaptations related to morphology (e.g., pseudothumbs) and genomic signatures, show that the giant panda and red panda have evolved some common traits to adapt to their bamboo diet. Thus, here is another story of diet-driven gut microbiota in nature.
Project description:The giant panda genome codes for all necessary enzymes associated with a carnivorous digestive system but lacks genes for enzymes needed to digest cellulose, the principal component of their bamboo diet. It has been posited that this iconic species must therefore possess microbial symbionts capable of metabolizing cellulose, but these symbionts have remained undetected. Here we examined 5,522 prokaryotic ribosomal RNA gene sequences in wild and captive giant panda fecal samples. We found lower species richness of the panda microbiome than of mammalian microbiomes for herbivores and nonherbivorous carnivores. We detected 13 operational taxonomic units closely related to Clostridium groups I and XIVa, both of which contain taxa known to digest cellulose. Seven of these 13 operational taxonomic units were unique to pandas compared with other mammals. Metagenomic analysis using ~37-Mbp contig sequences from gut microbes recovered putative genes coding two cellulose-digesting enzymes and one hemicellulose-digesting enzyme, cellulase, ?-glucosidase, and xylan 1,4-?-xylosidase, in Clostridium group I. Comparing glycoside hydrolase profiles of pandas with those of herbivores and omnivores, we found a moderate abundance of oligosaccharide-degrading enzymes for pandas (36%), close to that for humans (37%), and the lowest abundance of cellulases and endohemicellulases (2%), which may reflect low digestibility of cellulose and hemicellulose in the panda's unique bamboo diet. The presence of putative cellulose-digesting microbes, in combination with adaptations related to feeding, physiology, and morphology, show that giant pandas have evolved a number of traits to overcome the anatomical and physiological challenge of digesting a diet high in fibrous matter.
Project description:Adult giant pandas (Ailuropoda melanoleuca) express transitional characteristics in that they consume bamboos, despite their carnivore-like digestive tracts. Their genome contains no cellulolytic enzymes; therefore, understanding the development of the giant panda gut microbiome, especially in early life, is important for decoding the rules underlying gut microbial formation, inheritance and dietary transitions. With deep metagenomic sequencing, we investigated the gut microbiomes of two newborn giant panda brothers and their parents living in Macao, China, from 2016 to 2017. Both giant panda cubs exhibited progressive increases in gut microbial richness during growth, particularly from the 6th month after birth. Enterobacteriaceae dominated the gut microbial compositions in both adult giant pandas and cubs. A total of 583 co-abundance genes (CAGs) and about 79 metagenomic species (MGS) from bacteria or viruses displayed significant changes with age. Seven genera (Shewanella, Oblitimonas, Helicobacter, Haemophilus, Aeromonas, Listeria, and Fusobacterium) showed great importance with respect to gut microbial structural determination in the nursing stage of giant panda cubs. Furthermore, 10 orthologous gene functions and 44 pathways showed significant changes with age. Of the significant pathways, 16 from Escherichia, Klebsiella, Propionibacterium, Lactobacillus, and Lactococcus displayed marked differences between parents and their cubs at birth, while 29 pathways from Escherichia, Campylobacter and Lactobacillus exhibited significant increase in cubs from 6 to 9 months of age. In addition, oxidoreductases, transferases, and hydrolases dominated the significantly changed gut microbial enzymes during the growth of giant panda cubs, while few of them were involved in cellulose degradation. The findings indicated diet-stimulated gut microbiome transitions and the important role of Enterobacteriaceae in the guts of giant panda in early life.
Project description:Captive breeding has been used as an effective approach to protecting endangered animals but its effect on the gut microbiome and the conservation status of these species is largely unknown. The giant panda is a flagship species for the conservation of wildlife. With integrated efforts including captive breeding, this species has been recently upgraded from "endangered" to "vulnerable" (IUCN 2016). Since a large proportion (21.8%) of their global population is still captive, it is critical to understand how captivity changes the gut microbiome of these pandas and how such alterations to the microbiome might affect their future fitness and potential impact on the ecosystem after release into the wild. Here, we use 16S rRNA (ribosomal RNA) marker gene sequencing and shotgun metagenomics sequencing to demonstrate that the fecal microbiomes differ substantially between wild and captive giant pandas. Fecal microbiome diversity was significantly lower in captive pandas, as was the diversity of functional genes. Additionally, captive pandas have reduced functional potential for cellulose degradation but enriched metabolic pathways for starch metabolism, indicating that they may not adapt to a wild diet after being released into the wild since a major component of their diet in the wild will be bamboo. Most significantly, we observed a significantly higher level of amylase activity but a lower level of cellulase activity in captive giant panda feces than those of wild giant pandas, shown by an in vitro experimental assay. Furthermore, antibiotic resistance genes and virulence factors, as well as heavy metal tolerance genes were enriched in the microbiomes of captive pandas, which raises a great concern of spreading these genes to other wild animals and ecosystems when they are released into a wild environment. Our results clearly show that captivity has altered the giant panda microbiome, which could have unintended negative consequences on their adaptability and the ecosystem during the reintroduction of giant pandas into the wild.
Project description:The red panda is the only living species of the genus Ailurus. Like giant pandas, red pandas are also highly specialized to feed mainly on highly fibrous bamboo. Although several studies have focused on the gut microbiota in the giant panda, little is known about the gut microbiota of the red panda. In this study, we characterized the fecal microbiota from both wild (n?=?16) and captive (n?=?6) red pandas using a pyrosequecing based approach targeting the V1-V3 hypervariable regions of the 16S rRNA gene. Distinct bacterial communities were observed between the two groups based on both membership and structure. Wild red pandas maintained significantly higher community diversity, richness and evenness than captive red pandas, the communities of which were skewed and dominated by taxa associated with Firmicutes. Phylogenetic analysis of the top 50 OTUs revealed that 10 of them were related to known cellulose degraders. To the best of our knowledge, this is the first study of the gut microbiota of the red panda. Our data suggest that, similar to the giant panda, the gut microbiota in the red panda might also play important roles in the digestion of bamboo.
Project description:The digestion of lignin and lignin-related phenolic compounds from bamboo by giant pandas has puzzled scientists because of the lack of lignin-degrading genes in the genome of the bamboo-feeding animals. We constructed a 16S rRNA gene library from the microorganisms derived from the giant panda feces to identify the possibility for the presence of potential lignin-degrading bacteria. Phylogenetic analysis showed that the phylotypes of the intestinal bacteria were affiliated with the phyla Proteobacteria (53%) and Firmicutes (47%). Two phylotypes were affiliated with the known lignin-degrading bacterium Pseudomonas putida and the mangrove forest bacteria. To test the hypothesis that microbes in the giant panda gut help degrade lignin, a metagenomic library of the intestinal bacteria was constructed and screened for clones that contained genes encoding laccase, a lignin-degrading related enzyme. A multicopper oxidase gene, designated as lac51, was identified from a metagenomic clone. Sequence analysis and copper content determination indicated that Lac51 is a laccase rather than a metallo-oxidase and may work outside its original host cell because it has a TAT-type signal peptide and a transmembrane segment at its N-terminus. Lac51 oxidizes a variety of lignin-related phenolic compounds, including syringaldazine, 2,6-dimethoxyphenol, ferulic acid, veratryl alcohol, guaiacol, and sinapinic acid at conditions that simulate the physiologic environment in giant panda intestines. Furthermore, in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringic acid, or ferulic acid as mediators, the oxidative ability of Lac51 on lignin was promoted. The absorbance of lignin at 445 nm decreased to 36% for ABTS, 51% for syringic acid, and 51% for ferulic acid after incubation for 10 h. Our findings demonstrate that the intestinal bacteria of giant pandas may facilitate the oxidation of lignin moieties, thereby clarifying the digestion of bamboo lignin by the animal.