Effects of climate warming on soil nitrogen cycles and bamboo growth in core giant panda habitat.

Climate change can pose a significant threat to terrestrial ecosystems by disrupting the circulation of soil nitrogen. However, experimental analyses on the effect of climate change on soil nitrogen cycles and the implications for the conservation of key wildlife species (i.e., the giant panda, Ailuropoda melanoleuca) remain understudied. We investigated the effects of a 1.5 °C, 3 °C, and 4.5 °C temperature increase on nitrogen distribution in different soil layers of bamboo forest via an in-situ experiment and assessed the implications for the growth and survival of arrow bamboo (Bashania faberi), a critical food resource for giant pandas. Our results showed that warming treatments generally increased soil N content, while effects differed between surface soil and subsurface soil and at different warming treatments. Particularly an increase of 1.5 °C raised the subsurface soil NO3-N content, as well as the content of N in bamboo leaves. We found a significant positive correlation between the subsurface soil NO3-N content and the N content of arrow bamboo. An increase of 3-4.5 °C raised the content of total N and NO3-N in the surface soil and led to a reduction in the total aboveground biomass and survival rate of arrow bamboo. Limited warming (e.g., the increase of 0-1.5 °C) may promote the soil N cycle, raise the N-acetylglucosaminidase (NAG) enzyme activity, increase NO3-N in subsurface soil, increase the N content of bamboo, and boost the biomass of bamboo - all of which could be beneficial to giant panda survival. However, higher warming (e.g., an increase of 3-4.5 °C) resulted in mass death of bamboo and a large reduction in aboveground biomass. Our findings provide a cautiously optimistic scenario for bamboo forest ecosystems under low levels of warming over a short period of time, but risks from higher levels of warming may be serious, especially considering the unpredictability of global climatic change.

Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda.

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

Effects of environmental disinfection on microbial population and resistance genes: A case study of the microecology within a panda enclosure.

Widespread use of disinfectants raises concerns over their involvement in altering microbial communities and promoting antimicrobial resistance. This study explores the influence of disinfection protocols on microbial populations and resistance genes within an isolated enclosure environment and in the gut of giant pandas (GPs) held within. Samples of panda feces, air conditioning ducts, soil and bamboo were collected before and after disinfection. High-throughput sequencing characterized the microbial flora of GP gut and environmental microbes inside the artificial habitat. Microbial cultures showed that Escherichia coli (34.6%), Enterococcus (15.4%) and other pathogenic bacteria deposited in feces and the enclosure. Isolates exhibit a consistent resistance to disinfectant, with the greatest resistance shown to cyanuric acid, and the lowest to glutaraldehyde-dodecyl dimethyl ammonium bromide (GD-DDAB) and dodecyl dimethyl ammonium bromide (DDAB). The total number of the culturable bacteria in soil and bamboo were significantly diminished after disinfection but increased in the gut. After disinfection, the richness (Chao1 index) of environment samples increased significantly (P < 0.05), while the richness in gut decreased significantly (P < 0.05). Ten genera showed significant change in feces after disinfection. Metagenome sequencing showed that 126 types of virulence genes were present in feces before disinfection and 37 in soil. After disinfection, 110 virulence genes localized in feces and 53 in soil. Eleven virulence genes including ECP and T2SS increased in feces. A total of 182 antibiotic resistance genes (ARGs) subtypes, potentially conferring resistance to 20 classes of drugs, were detected in the soils and feces, with most belonging to efflux pump protein pathways. After disinfection, the number of resistance genes increased both in gut and soil, which suggests disinfection protocols increase the number of resistance pathways. Our study shows that the use of disinfectants helps to shape the microbial community of GPs and their habitat, and increases populations of resistant strain bacteria.

Lipidomics for Determining Giant Panda Responses in Serum and Feces Following Exposure to Different Amount of Bamboo Shoot Consumption: A First Step towards Lipidomic Atlas of Bamboo, Giant Panda Serum and Feces by Means of GC-MS and UHPLC-HRMS/MS.

Lipidic metabolites play essential roles in host physiological health and growth performance, serving as the major structural and signaling components of membranes, energy storage molecules, and steroid hormones. Bamboo, as wild giant pandas' exclusive diet, is the main determinant of giant pandas' lipidome, both as a direct source and through microbiota activity. Interestingly, the consumption of bamboo has attracted little attention from a lipidomic perspective. In the current study, we outline the lipidomic atlas of different parts of bamboo. By gas chromatography-mass spectrometry (GC-MS), we have been able to obtain the absolute quantification of 35 fatty acids pertaining to short chain fatty acids (8), medium chain fatty acids (6), long chain fatty acids (17), and very long chain fatty acids (4), while liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) allowed us to obtain the relative quantification of another 1638 lipids. Among the fatty acids quantified in absolute terms, eight showed significantly distinct concentrations among different bamboo parts. Subsequently, we investigated how the giant panda's serum and fecal lipidome adapt to the most important annual change in their diet, represented by the consumption of high amounts of bamboo shoots, typical of spring, the weight-gaining season. Five fatty acids were significantly altered in feces and two in serum, respectively, due to the different levels of bamboo shoot consumption. Furthermore, significant differences of the main bacteria strains were observed in feces between the two groups at the genus level, pertaining to Streptococcus, Leuconostoc, and Vagococcus. Correlations between giant panda fecal microbiome and lipidome were evaluated by Pearson correlation analysis. These findings suggest that a balanced diet, important for the overall lipidomic function and giant panda health, could be reached even in this remarkable case of a single food-based diet, by administering to the giant panda's combinations of different parts of bamboo, with specific lipidome profiles.

Antimicrobial resistance of Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp. isolated from the feces of giant panda.

BACKGROUND: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp., common gut bacteria in giant pandas, include opportunistic pathogens. The giant panda is an endangered species, classified as vulnerable by the World Wildlife Foundation. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from giant pandas is vital not only for their protection but also for public health. RESULTS: A total of 166 E. coli, 68 Enterobacter spp., 116 K. pneumoniae and 117 Enterococcus spp. isolates were collected from fecal samples of 166 giant pandas. In the antimicrobial susceptibility tests, 144 E. coli isolates, 66 Enterobacter spp. isolates, 110 K. pneumoniae isolates and 43 Enterococcus spp. isolates were resistant to at least one antimicrobial. The resistant isolates carried antimicrobial resistance genes (ARGs), including sul3, blaTEM, blaSHV and tetA. The differences in the prevalence of the bla types implied that the genetic basis for ß-lactam resistance among the E. coli, Enterobacter spp. and K. pneumoniae isolates was different. The strain K. pneumoniae K85 that was resistant to sixteen antimicrobials was selected for whole genome sequencing. The genome contained Col440I, IncFIBK and IncFIIK plasmids and altogether 258 ARGs were predicted in the genome; 179 of the predicted ARGs were efflux pump genes. The genetic environment of the ß-lactamase genes blaCTX-M-3 and blaTEM-1 in the K. pneumoniae K85 genome was relatively similar to those in other sequenced K. pneumoniae genomes. In comparing the giant panda age groups, the differences in the resistance rates among E. coli, K. pneumoniae and Enterobacter spp. isolates suggested that the infections in giant pandas of different age should be treated differently. CONCLUSIONS: Antimicrobial resistance was prevalent in the bacterial isolates from the giant pandas, implying that the gut bacteria may pose serious health risks for captive giant pandas. The resistance genes in the genome of K. pneumoniae K85 were associated with insertion sequences and integron-integrase genes, implying a potential for the further spread of the antimicrobial resistance.

The Fecal and Serum Metabolomics of Giant Pandas Based on Untargeted Metabolomics.

Little is comprehensively known or understood about giant panda fecal and serum metabolites, which could serve as important indicators of the physiological metabolism of giant pandas. Therefore, we determined the contents of fecal and serum metabolites of giant pandas based on an untargeted metabolome. Four hundred and 955 metabolites were detected in the feces and serum of giant panda, respectively. Glycerophospholipid and choline metabolism were the main metabolic pathways in feces and serum. A significant correlation between the gut microbiota and fecal metabolites was found (P < 0.01). Fecal metabolites were not greatly affected by the age or gender of giant pandas, but serum metabolites were significantly affected by age and gender. The majority of different metabolites caused by age were higher in serum of younger giant pandas, including fatty acids, lipids, metabolites of bile acids, and intermediate products of vitamin D3. The majority of different metabolites caused by gender included fatty acids, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). A separate feeding diet should be considered according to different ages and genders of giant panda. Therefore, our results could provide helpful suggestions to further protect captive giant pandas.

Diet, habitat environment and lifestyle conversion affect the gut microbiomes of giant pandas.

Gut microbiota (GM) are important for the health of giant pandas (GPs), in addition to the utilization of bamboo in their diets. However, it is not fully understood how diet, habitat environment and lifestyle contribute to the composition of GM in GP. Consequently, we evaluated how dietary changes, habitat environment conversions and lifestyle shifts influence the GM of GPs using high-throughput sequencing and genome-resolved metagenomics. The GM of GPs were more similar when their hosts exhibited the same diet. High fiber diets significantly increased the diversity and decreased the richness of gut bacterial communities alone or interacted with the age factor (p < 0.05). The abundances of Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium significantly increased during diet conversion process (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA > 4). Reconstruction of 60 metagenome-assembled-genomes (MAGs) indicated that these bacteria were likely responsible for bamboo digestion via gene complements involved in cellulose, hemicellulose, and lignin degradation. While habitat environment may play a more important role in shaping the GM of GP, lifestyle can also greatly affect bacterial communities. The GM structure in reintroduced GPs notably converged to that of wild pandas. Importantly, the main bacterial genera of wild GPs could aid in lignin degradation, while those of reintroduced GPs were related to cellulose and hemicellulose digestion. Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium may contribute to lignocellulose digestion in GP. The results revealed that diet conversion, habitat environment and lifestyle could remarkably influence the GM of GP. In addition, results suggested that increasing the ability of lignin degradation with GM may aid to change the GM of reintroduced pandas to resemble those of wild pandas.

Chromosome-scale genomes provide new insights into subspecies divergence and evolutionary characteristics of the giant panda.

Extant giant pandas are divided into Sichuan and Qinling subspecies. The giant panda has many species-specific characteristics, including comparatively small organs for body size, small genitalia of male individuals, and low reproduction. Here, we report the most contiguous, high-quality chromosome-level genomes of two extant giant panda subspecies to date, with the first genome assembly of the Qinling subspecies. Compared with the previously assembled giant panda genomes based on short reads, our two assembled genomes increased contiguity over 200-fold at the contig level. Additional sequencing of 25 individuals dated the divergence of the Sichuan and Qinling subspecies into two distinct clusters from 10,000 to 12,000 years ago. Comparative genomic analyses identified the loss of regulatory elements in the dachshund family transcription factor 2 (DACH2) gene and specific changes in the synaptotagmin 6 (SYT6) gene, which may be responsible for the reduced fertility of the giant panda. Positive selection analysis between the two subspecies indicated that the reproduction-associated IQ motif containing D (IQCD) gene may at least partly explain the different reproduction rates of the two subspecies. Furthermore, several genes in the Hippo pathway exhibited signs of rapid evolution with giant panda-specific variants and divergent regulatory elements, which may contribute to the reduced inner organ sizes of the giant panda.

First Steps toward the Giant Panda Metabolome Database: Untargeted Metabolomics of Feces, Urine, Serum, and Saliva by 1H NMR.

Differences in the concentration of metabolites in the biofluids of animals closely reflect their physiological diversities. In order to set the basis for a metabolomic atlas for giant panda (Ailuropoda melanoleuca), we characterized the metabolome of healthy giant panda feces (23), urine (16), serum (6), and saliva (4) samples by means of 1H NMR. A total of 107 metabolites and a core metabolome of 12 metabolites was quantified across the four biological matrices. Through univariate analysis followed by robust principal component analysis, we were able to describe how the molecular profile observed in giant panda urine and feces was affected by gender and age. Among the molecules modified by age in feces, fucose plays a peculiar role because it is related to the digestion of bamboo's hemicellulose, which is considered as the main source of energy for giant panda. A metagenomic investigation directed toward this molecule showed that its concentration was indeed positively related to the two-component system pathway and negatively related to the amino sugar and nucleotide sugar metabolism pathway. Such work is meant to provide a robust framework for further -omics research studies on giant panda to accelerate our understanding of the interaction of giant panda with its natural environment.

Acoustic recordings provide detailed information regarding the behavior of cryptic wildlife to support conservation translocations.

For translocated animals, behavioral competence may be key to post-release survival. However, monitoring behavior is typically limited to tracking movements or inferring behavior at a gross scale via collar-mounted sensors. Animal-bourne acoustic monitoring may provide a unique opportunity to monitor behavior at a finer scale. The giant panda is an elusive species of Ursid that is vulnerable to extinction. Translocation is an important aspect of the species' recovery, and survival and recruitment for pandas likely hinge on behavioral competence. Here we tested the efficacy of a collar-mounted acoustic recording unit (ARU) to remotely monitor the behavior of panda mothers and their dependent young. We found that trained human listeners could reliably identify 10 behaviors from acoustic recordings. Through visual inspection of spectrograms we further identified 5 behavioral categories that may be detectable by automated pattern recognition, an approach that is essential for the practical application of ARU. These results suggest that ARU are a viable method for remotely observing behaviors, including feeding. With targeted effort directed towards instrumentation and computing advances, ARU could be used to document how behavioral competence supports or challenges post-release survival and recruitment, and allow for research findings to be adaptively integrated into future translocation efforts.

Comparative Analysis of Gut Microbiota Among the Male, Female and Pregnant Giant Pandas (Ailuropoda Melanoleuca).

The giant panda (GP) was the most endangered species in China, and gut microbiota plays a vital role in host health. To determine the differences of the gut microbiota among the male, female and pregnant GPs, a comparative analysis of gut microbiota in GPs was carried out by 16S rRNA and ITS high-throughput sequencing. In 16S rRNA sequencing, 435 OTUs, 17 phyla and 182 genera were totally detected. Firmicutes (53.6%) was the predominant phylum followed by Proteobacteria (37.8%) and Fusobacteria (7.1%). Escherichia/Shigella (35.9%) was the most prevalent genus followed by Streptococcus (25.9%) and Clostridium (11.1%). In ITS sequencing, 920 OTUs, 6 phyla and 322 genera were also detected. Ascomycota (71.3%) was the predominant phylum followed by Basidiomycota (28.4%) and Zygomycota (0.15%). Purpureocillium (4.4%) was the most prevalent genus followed by Cladosporium (2.5%) and Pezicula (2.4%). Comparative analysis indicated that the male GPs harbor a higher abundance of phylum Firmicutes than female GPs with the contribution from genus Streptococcus. Meanwhile, the female GPs harbor a higher abundance of phylum Proteobacteria than male GPs with the contribution from genus Escherichia/ Shigella. In addition, the shift in bacteria from female to pregnant GPs indicated that phylum Firmicutes increased significantly with the contribution from Clostridium in the gut, which may provide an opportunity to study possible associations with low reproduction of the GPs.

Metagenomic Analysis of Bacteria, Fungi, Bacteriophages, and Helminths in the Gut of Giant Pandas.

To obtain full details of gut microbiota, including bacteria, fungi, bacteriophages, and helminths, in giant pandas (GPs), we created a comprehensive microbial genome database and used metagenomic sequences to align against the database. We delineated a detailed and different gut microbiota structures of GPs. A total of 680 species of bacteria, 198 fungi, 185 bacteriophages, and 45 helminths were found. Compared with 16S rRNA sequencing, the dominant bacterium phyla not only included Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria but also Cyanobacteria and other eight phyla. Aside from Ascomycota, Basidiomycota, and Glomeromycota, Mucoromycota, and Microsporidia were the dominant fungi phyla. The bacteriophages were predominantly dsDNA Myoviridae, Siphoviridae, Podoviridae, ssDNA Inoviridae, and Microviridae. For helminths, phylum Nematoda was the dominant. In addition to previously described parasites, another 44 species of helminths were found in GPs. Also, differences in abundance of microbiota were found between the captive, semiwild, and wild GPs. A total of 1,739 genes encoding cellulase, ß-glucosidase, and cellulose ß-1,4-cellobiosidase were responsible for the metabolism of cellulose, and 128,707 putative glycoside hydrolase genes were found in bacteria/fungi. Taken together, the results indicated not only bacteria but also fungi, bacteriophages, and helminths were diverse in gut of giant pandas, which provided basis for the further identification of role of gut microbiota. Besides, metagenomics revealed that the bacteria/fungi in gut of GPs harbor the ability of cellulose and hemicellulose degradation.