Diet drives convergent evolution of gut microbiomes in bamboo-eating species.

Gut microbiota plays a critical role in host physiology and health. The coevolution between the host and its gut microbes facilitates animal adaptation to its specific ecological niche. Multiple factors such as host diet and phylogeny modulate the structure and function of gut microbiota. However, the relative contribution of each factor in shaping the structure of gut microbiota remains unclear. The giant (Ailuropoda melanoleuca) and red (Ailurus styani) pandas belong to different families of order Carnivora. They have evolved as obligate bamboo-feeders and can be used as a model system for studying the gut microbiome convergent evolution. Here, we compare the structure and function of gut microbiota of the two pandas with their carnivorous relatives using 16S rRNA and metagenome sequencing. We found that both panda species share more similarities in their gut microbiota structure with each other than each species shares with its carnivorous relatives. This indicates that the specialized herbivorous diet rather than host phylogeny is the dominant driver of gut microbiome convergence within Arctoidea. Metagenomic analysis revealed that the symbiotic gut microbiota of both pandas possesses a high level of starch and sucrose metabolism and vitamin B12 biosynthesis. These findings suggest a diet-driven convergence of gut microbiomes and provide new insight into host-microbiota coevolution of these endangered species.

Potential Mechanism of Detoxification of Cyanide Compounds by Gut Microbiomes of Bamboo-Eating Pandas.

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.

Bamboo Specialists from Two Mammalian Orders (Primates, Carnivora) Share a High Number of Low-Abundance Gut Microbes.

Bamboo specialization is one of the most extreme examples of convergent herbivory, yet it is unclear how this specific high-fiber diet might selectively shape the composition of the gut microbiome compared to host phylogeny. To address these questions, we used deep sequencing to investigate the nature and comparative impact of phylogenetic and dietary selection for specific gut microbial membership in three bamboo specialists-the bamboo lemur (Hapalemur griseus, Primates: Lemuridae), giant panda (Ailuropoda melanoleuca, Carnivora: Ursidae), and red panda (Ailurus fulgens, Carnivora: Musteloideadae), as well as two phylogenetic controls-the ringtail lemur (Lemur catta) and the Asian black bear (Ursus thibetanus). We detected significantly higher Shannon diversity in the bamboo lemur (10.029) compared to both the giant panda (8.256; p = 0.0001936) and the red panda (6.484; p = 0.0000029). We also detected significantly enriched bacterial taxa that distinguished each species. Our results complement previous work in finding that phylogeny predominantly governs high-level microbiome community structure. However, we also find that 48 low-abundance OTUs are shared among bamboo specialists, compared to only 8 OTUs shared by the bamboo lemur and its sister species, the ringtail lemur (Lemur catta, a generalist). Our results suggest that deep sequencing is necessary to detect low-abundance bacterial OTUs, which may be specifically adapted to a high-fiber diet. These findings provide a more comprehensive framework for understanding the evolution and ecology of the microbiome as well as the host.

Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens).

A 9-year-old male red panda (Ailurus fulgens) became emaciated and died. Necropsy examination revealed systemic lymphadenomegaly. The liver, lungs and left kidney contained multifocal yellow nodules. Microscopical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli. Sequencing of genetic material isolated from the tissues classified the pathogen as Mycobacterium gastri. Lymphoma was found in the liver, lungs, kidney and lymph nodes. The neoplastic cells were strongly labelled for expression of CD3, Ki67 and proliferating cell nuclear antigen by immunohistochemistry. This is the first report of M. gastri infection with T-cell lymphoma in a red panda.

First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China.

Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n=4) and genotype EbpC (n=5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China.

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens).

Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of illness; the other had a brief history of laboured breathing. Post-mortem examination revealed disseminated protozoal infection. To further characterize the causative agent, transmission electron microscopy (TEM), immunohistochemistry (IHC), polymerase chain reaction (PCR) and amplification and nucleic acid sequencing were performed. IHC was negative for Toxoplasma gondii and Neospora caninum, but was positive for a Sarcocystis spp. TEM of cardiac muscle and lung revealed numerous intracellular apicomplexan protozoa within parasitophorous vacuoles. PCR and nucleic acid sequencing of partial 18S rRNA and the internal transcribed spacer (ITS)-1 region confirmed a Sarcocystis spp. that shared 99% sequence homology to Sarcocystis neurona and Sarcocystis dasypi. This represents the first report of sarcocystosis in red pandas. The histopathological, immunohistochemical, molecular and ultrastructural findings are supportive of vertical transmission resulting in fatal disseminated disease.

Characterization of the gut microbiota in the red panda (Ailurus fulgens).

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.