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The giant panda (Ailuropoda melanoleuca) is recognized worldwide as an icon for wildlife conservation. The Qinling subspecies (Ailuropoda melanoleuca qinlingensis) inhabiting the Qinling Mountains is more endangered. Previous studies have indicated that Qinling pandas are potentially at risk from environmental pollutants, which they are exposed to via food and drinking water. However, there is little information about the surface water quality in the Qinling Mountains, and it is unknown whether drinking water is an important pollutant source for pandas. Water samples were collected from five different nature reserves in Qinling, each of which is home to a population of pandas. The samples were analyzed for five essential metals of copper (Cu), zinc (Zn), manganese (Mn), chromium (Cr), and nickel (Ni) and four non-essential metals of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg). The concentrations of all heavy metals (HMs) were higher in buffer zones than in the core areas and were highest in Foping and Niuweihe nature reserves. The concentrations of mercury exceeded the water quality standard in the core zone of three reserves, suggesting that NWH (Niuweihe), THS (Tianhuashan), and PHL (Pingheliang) giant panda populations were at risk from mercury toxicity. The accumulation of other elements over time could also pose a serious risk to pandas. Three main sources of pollution were identified: coal combustion, waste incineration, and fertilizer use; traffic-related activities; and metal mining. Environmental pollution is compromising the efforts to conserve the giant panda, and measures need to be put in place to control pollution sources.

IntroductionMicrobial community composition is closely associated with host disease onset and progression, underscoring the importance of understanding host–microbiota dynamics in various health contexts.MethodsIn this study, we utilized full-length 16S rRNA gene sequencing to conduct species-level identification of the microorganisms in the oral cavity of a giant panda (Ailuropoda melanoleuca) with oral malignant fibroma.ResultsWe observed a significant difference between the microbial community of the tumor side and non-tumor side of the oral cavity of the giant panda, with the latter exhibiting higher microbial diversity. The tumor side was dominated by specific microorganisms, such as Fusobacterium simiae, Porphyromonas sp. feline oral taxon 110, Campylobacter sp. feline oral taxon 100, and Neisseria sp. feline oral taxon 078, that have been reported to be associated with tumorigenic processes and periodontal diseases in other organisms. According to the linear discriminant analysis effect size analysis, more than 9 distinct biomarkers were obtained between the tumor side and non-tumor side samples. Furthermore, the Kyoto Encyclopedia of Genes and Genomes analysis revealed that the oral microbiota of the giant panda was significantly associated with genetic information processing and metabolism, particularly cofactor and vitamin, amino acid, and carbohydrate metabolism. Furthermore, a significant bacterial invasion of epithelial cells was predicted in the tumor side.DiscussionThis study provides crucial insights into the association between oral microbiota and oral tumors in giant pandas and offers potential biomarkers that may guide future health assessments and preventive strategies for captive and aging giant pandas.

Jiajin Mountain, where the giant pandas reside, is an essential nature reserve in China. To comprehend the land use classification of the habitat, this article proposes a remote sensing interpretation algorithm based on spatial case reasoning, known as spatial case-based reasoning (SCBR). The algorithm incorporates specific spatial factors into its framework and does not require an extensive amount of domain knowledge and eliminates the need for a complex model training process, making it capable of completing land use classification in the study area. SCBR comprises a spatial case expression model and a spatial case similarity reasoning model. The paper conducted comparative experiments between the proposed algorithm and support vector machine (SVM), U-Net, vision transformer (ViT), and Trans-Unet, and the results demonstrate that spatial case-based reasoning produces superior classification outcomes. The land use classification experiment based on spatial case-based reasoning at the Jiajinshan giant panda habitat produced satisfactory experimental results. In the comparative experiments, the overall accuracy of SCBR classification reached 95%, and the Kappa coefficient reached 90%. The paper further analyzed the changes in land use classification from 2018 to 2022, and the average accuracy consistently exceeds 80%. We discovered that the ecological environment in the region where the giant pandas reside has experienced significant improvement, particularly in forest protection and restoration. This study provides a theoretical basis for the ecological environment protection of the area.

Escherichia coli is a major bacterial pathogen which causes diarrhea in the giant panda. This study investigated the biological characteristics of 100 E. coli strains isolated from fecal samples collected from 100 captive giant pandas of different age groups and sexes. A standard Kirby-Bauer disk diffusion antimicrobial susceptibility test was performed with the isolates and we then further evaluated the antibiotic resistance genes (ARGs) by high-throughput quantitative PCR. Additionally, we then analyzed O serogroups through a slide agglutination test, virulence genes and the multi-locus sequence typing (MLST) by PCR. Antimicrobial susceptibility testing demonstrated that the 100 E. coli strains were mainly resistant to ENR (68%), AM (56%), IPM (55%), AMX (54%) and CA (52%), but were susceptible to MEM and FOX. The resistance to TZP, AK, FEP, CAZ, AMS, AZM, AT and IPM was significantly related to age (p

IntroductionThe giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus.MethodsThis study combined with high-throughput sequencing and laboratory testing to compare the diversity of the vaginal mycobiota in giant pandas during estrus and non-estrus, and to investigate the presence of potentially pathogenic fungi. Potentially pathogenic fungi were studied in mice to explore their pathogenicity.Results and discussionThe results revealed that during estrus, the vaginal secretions of giant pandas play a crucial role in fungal colonization. Moreover, the diversity of the vaginal mycobiota is reduced and specificity is enhanced. The abundance of Trichosporon and Cutaneotrichosporon in the vaginal mycobiota of giant pandas during estrus was significantly higher than that during non-estrus periods. Apiotrichum and Cutaneotrichosporon were considered the most important genera, and they primarily originate from the environment owing to marking behavior exhibited during the estrous period of giant pandas. Trichosporon is considered a resident mycobiota of the vagina and is an important pathogen that causes infection when immune system is suppressed. Potentially pathogenic fungi were further isolated and identified from the vaginal secretions of giant pandas during estrus, and seven strains of Apiotrichum (A. brassicae), one strain of Cutaneotrichosporon (C. moniliiforme), and nine strains of Trichosporon (two strains of T. asteroides, one strain of T. inkin, one strain of T. insectorum, and five strains of T. japonicum) were identified. Pathogenicity results showed that T. asteroides was the most pathogenic strain, as it is associated with extensive connective tissue replacement and inflammatory cell infiltration in both liver and kidney tissues. The results of this study improve our understanding of the diversity of the vaginal fungi present in giant pandas and will significantly contribute to improving the reproductive health of giant pandas in the future.

Represented by feline panleukopenia virus (FPV) and canine parvovirus (CPV), the species carnivore protoparvovirus 1 has a worldwide distribution through continuous ci13rculation in companion animals such as cats and dogs. Subsequently, both FPV and CPV had engaged in host-to-host transfer to other wild animal hosts of the order Carnivora. In the present study, we emphasized the significance of cross-species transmission of parvoviruses with the isolation and characterization of an FPV from giant panda displaying severe and fatal symptoms. The isolated virus, designated pFPV-sc, displayed similar morphology as FPV, while phylogenetic analysis indicated that the nucleotide sequence of pFPV-sc clades with Chinese FPV isolates. Despite pFPV-sc is seemingly an outcome of a spillover infection event from domestic cats to giant pandas, our study also provided serological evidence that FPV or other parvoviruses closely related to FPV could be already prevalent in giant pandas in 2011. Initiation of host transfer of pFPV-sc is likely with association to giant panda transferrin receptor (TfR), as TfR of giant panda shares high homology with feline TfR. Strikingly, our data also indicate that pFPV-sc can infect cell lines of other mammal species, including humans. To sum up, observations from this study shall promote future research of cross-host transmission and antiviral intervention of Carnivore protoparvovirus 1, and necessitate surveillance studies in thus far unacknowledged potential reservoirs.

ObjectiveProbiotics are beneficial to the intestinal barrier, but few studies have investigated probiotics from giant pandas. This study aims to explore the preventive effects of giant panda-derived Clostridium butyricum on dextran sodium sulfate (DSS)-induced colitis in mice.MethodsClostridium butyricum was administered to mice 14 days before administering DSS treatment to induce enteritis.ResultsClostridium butyricum B14 could more effectively prevent colitis in mice than C. butyricum B13. C. butyricum B14 protected the mouse colon by decreasing the histology index and serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels, which improved intestinal inflammation-related symptoms. In addition, the treatment led to the regulation of the expression of Tifa, Igkv12-89, and Nr1d1, which in turn inhibited immune pathways. The expression of Muc4, Lama3, Cldn4, Cldn3, Ocln, Zo1, Zo2, and Snai is related the intestinal mucosal barrier. 16S sequencing shows that the C. butyricum B14 significantly increased the abundance of certain intestinal probiotics. Overall, C. butyricum B14 exerted a preventive effect on colitis in mice by inhibiting immune responses, enhancing the intestinal barrier and increasing the abundance of probiotic species. Thus, C. butyricum B14 administration helps regulate the balance of the intestinal microecology. It can suppress immune pathways and enhance barrier-protective proteins.

IntroductionTick-borne viruses (TBVs) pose a significant risk to the health of humans and other vertebrates. A class of multisegmented flavi-like viruses, Jingmen tick virus (JMTV) was first discovered in Rhipicephalus microplus ticks collected from Jingmen of Hubei Province, China in 2010. JMTV has been confirmed to have a relatively wide distribution in vectors and hosts and is associated with human diseases.MethodsParasitic and host-seeking ticks were collected in Wolong Nature Reserve, Sichuan Province. Total RNA was extracted and then enriched the viral RNA. The DNA library was constructed and then were sequenced with MGI High-throughput Sequencing Set (PE150). After the adaptor sequences,low-quality bases and host genome were removed, resulting reads classified as a virus were subsequently de novo assembled into contigs, which were then compared to the NT database. Those annotated under the kingdom virus were initially identified as potential virus-associated sequences. Phylogenetic and Reassortment analysis of sequences were performed using MEGA and SimPlot software, respectively.Results and discussionTwo host-seeking ticks and 17 ticks that fed on giant pandas and goats were collected. Through high-throughput sequencing, whole virus genomes were attained from four tick samples (PC-13, PC-16, PC-18, and PC-19) that shared 88.7–96.3% similarity with known JMTV. Phylogenetic tree showed that it was a novel JMTV-like virus, referred to as Sichuan tick virus, which also had the signals of reassortment with other JMTV strains, suggesting a cross-species transmission and co-infection of segmented flavi-like viruses among multiple tick hosts.ConclusionWe discovered and confirmed one new Jingmen tick virus, Sichuan tick virus. Further investigation is required to determine the pathogenicity of Sichuan tick virus to humans and animals, as well as its epidemiological characteristics in nature.

Canine parvovirus (CPV) and Canine distemper virus (CDV) can cause fatal diseases in giant panda (Ailuropoda melanoleuca). The main capsid protein of CPV VP2 can be self-assembled to form virus-like particles (VLPs) in vitro, which is of great significance for potential vaccine development. In the present study, we remodeled the VP2 protein of a giant panda-derived CPV, where the major CDV F and N epitopes were incorporated in the N-terminal and loop2 region in two combinations to form chimeric VLPs. The reactivity ability and morphology of the recombinant proteins were confirmed by Western blot, hemagglutination (HA) test and electron microscopy. Subsequently, the immunogenicity of the VLPs was examined in vivo. Antigen-specific antibodies and neutralizing activity were measured by ELISA, hemagglutination inhibition (HI) test and serum neutralization test (SNT), respectively. In addition, antigen specific T cell activation were determined in splenic lymphocytes. The results indicated that the VLPs displayed good reaction with CDV/CPV antibodies, and the heterologous epitopes do not hamper solubility or activity. The VLPs showed decent HA activity, and resembled round-shaped particles with a diameter of 22–26 nm, which is identical to natural virions. VLPs could induce high levels of specific antibodies to CPV and CDV, shown by the indication of neutralizing antibodies in both VP2N and VP2L VLPs group. In addition, splenic lymphocytes of mice immunized with VLPs could proliferate rapidly after stimulation by specific antigen. Taken together, the CPV VP2 VLPs or chimeric VLPs are highly immunogenic, and henceforth could function as CPV/CDV vaccine candidates for giant pandas.

Gram-negative bacteria are major pathogens that can cause illnesses in giant pandas. Lipopolysaccharides (LPS), components of Gram-negative bacteria, can activate immune responses in mammals (i.e., humans and mice) through recognition by toll-like receptors (TLRs). However, the giant pandas’ immune response to LPS stimulation and the differences between the giant panda and other mammals are not fully known. In this study, we administrated peripheral blood mononuclear cells (PBMCs) from giant pandas, humans, C57BL/6 mice, and rhesus monkeys by LPS treatment at 6 h followed by RNA sequencing (RNA-seq), respectively, with control of non-stimulation. KEGG analyses of differentially expressed genes (DEGs) pathways indicated that LPS could activate the classic signaling pathway of NF-κB in PBMCs from those four tested species. Thus, similar to the other three species, NF-κB is an LPS-responsive regulator of innate immune responses in giant pandas. Furthermore, the expression patterns of adapter genes, inflammatory cytokine genes, chemokines, interferon genes, cytokine genes related to cell growth and development, costimulatory molecules, Th1/Th2 cytokine genes, Th17 cytokine genes, Th9, and Th22 cytokine genes were compared among giant pandas and three other species. Our data indicated that in addition to the similar expression patterns of certain genes among giant pandas and other species, the unique expression pattern response to LPS in giant pandas was also discovered. Furthermore, Th9, Th17, and Th22 cells might be involved in the response to LPS in giant pandas at this tested time point. This study reveals that LPS-induced immune responses have different sensitivities and response timelines in giant pandas compared with other mammals. This study facilitates further understanding of the role of the TLR signaling pathway and the immune system in giant pandas, which might be helpful for disease prevention and protection.

To contribute to the conservation of endangered animals, the utilization of model systems is critical to analyze the function of their gut microbiota. In this study, the results of a fecal microbial transplantation (FMT) experiment with germ-free (GF) mice receiving giant panda or horse fecal microbiota showed a clear clustering by donor microbial communities in GF mice, which was consistent with the results of blood metabolites from these mice. At the genus level, FMT re-established approximately 9% of the giant panda donor microbiota in GF mice compared to about 32% for the horse donor microbiota. In line with this, the difference between the panda donor microbiota and panda-mice microbiota on whole-community level was significantly larger than that between the horse donor microbiota and the horse-mice microbiota. These results were consistent with source tracking analysis that found a significantly higher retention rate of the horse donor microbiota (30.9%) than the giant panda donor microbiota (4.0%) in GF mice where the microbiota remained stable after FMT. Further analyzes indicated that the possible reason for the low retention rate of the panda donor microbiota in GF mice was a low relative abundance of Clostridiaceae in the panda donor microbiota. Our results indicate that the donor microbiota has a large effect on GF mice microbiota after FMT.

IntroductionToxoplasma gondii, a globally zoonotic protozoan parasite, infects most warm-blooded animals including the giant panda, and poses a serious threat to the giant panda conservation. However, the seroprevalence and the risk factors for toxoplasmosis in giant pandas are unknown. Here we aimed to determine the seroprevalence of T. gondii in the captive population of giant pandas and analyze the factors associated with the increased risk of infection.MethodsA total of 203 serum samples were collected from 157 (95 females and 62 males) captive giant pandas from 2007 to 2022, antibodies against T. gondii were screened using commercial ELISA and MAT kits.ResultsThe results showed 56 (35.67%) giant pandas were seropositive, age and transfer history between institutions were identifified as risk factors for T. gondii infection. It is suggested that age-related seroprevalence was the main factor, and housing multiple species in the same environment may increase the chance of cross-infection of T. gondii.DiscussionThis study can provide research data for developing policies for the prevention and control of T. gondii and protecting the health of captive giant pandas and other wildlife.

The giant panda has been considered to maximize nutritional intake including protein and soluble carbohydrates in bamboo, but it has spent almost entire life with the high-cellulose diet. Whether giant panda is still helpless about digesting bamboo cellulose or not is always contentious among many researchers around the world. The work has systematically clarified this issue from the perspectives of digestive enzymes, functional genes, and microbial structures in giant panda gut. The intestinal cellulase activities of panda increase with bamboo consumption, performing that the endoglucanase activity of adults reaches 10-fold that of pandas first consuming bamboo. More abundance and types of microbial endoglucanase genes occur in bamboo-diet giant panda gut, and the corresponding GH5 gene cluster is still efficiently transcribed. Gut microbes possessing cellulose-degrading genes, belong to the phylum Firmicutes and some Bacteroidetes, but their structural and functional configurations are insufficient to completely degrade cellulose. Therefore, giant panda is striving to digest cellulose in bamboo, but this adaptation is incomplete. This is probably related to the short straight carnivore-like gut structure of the giant panda, preventing the colonization of some efficient functional but anaerobic-preferred flora.

The Giant pandas (Ailuropoda melanoleuca) are mammals belonging to the bear family, order Carnivora, and their characteristic hair color and distribution has been in the spotlight. In recent years, the gradual prevalence of skin diseases in giant pandas and even the discovery of albino individuals have made the study of the substrate of their skin hair distribution more and more urgent. In this study, by comparing the skin histology and transcriptomes for hairs of different color of giant pandas, we found that the melanin contents of hair follicles at the bases of black and white hairs differed, but the hair follicles at the base of white hairs also contained some amount of melanin. The transcriptome sequencing results showed that there were great differences in the expression of the transcriptome of the skin under different hair color blocks, in which the number of differentially expressed genes in the white skin was much smaller than that in the black skin. Transcriptomes for skin tissue samples for different hair colors revealed several enriched Kyoto encyclopedia of genes (KEGG) pathways that include tumor, cell adhesion and melanocyte growth-related signaling pathways. This study provides a theoretical basis for subsequent studies on hair color distribution and skin diseases in giant pandas.

Dental caries severely hinders efficient access to adequate energy in wildlife. Different food supplies will develop characteristic plaque, and the microorganisms of these plaque are closely related to dental health. Here, plaque samples from panda cubs with caries and caries-free were collected for 16S rRNA high-throughput sequencing. All sequences clustered into 337 operational taxonomic units (OTUs; 97% identity), representing 268 independent species belonging to 189 genera, 98 families, 51 orders, 24 classes, and 13 phyla. Two groups shared 218 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in plaques with caries exceeded that of caries-free. The dominant phyla of plaque microbiota included Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. The dominant genera included unclassified Neisseriaceae, Actinobacillus, Lautropia, Neisseria, Porhyromonas, unclassified Pasteurellaceae, Moraxella, Streptococcus, Bergeywlla and Capnocytophaga. β diversity analysis showed that the plaque microbial community structure was different between two groups. Using LEfSe analysis, 19 differentially abundant taxa were identified as potential biomarkers. Finally, function predictions analysis showed All the energy related metabolic pathways on KEGG level 2 were enriched in caries-active group. Consistent with the mainstream caries-causing narrative, our results illuminate the lack of information regarding the oral microflora composition and function within giant panda cubs.

Social equity is essential in the governance of protected areas (PAs), as ignoring such consideration can lead to resistance and jeopardize conservation objectives. However, more research is required to understand the spatial heterogeneity of perceived social equity and its underlying spatial factors. Using a survey of 361 respondents, we presented spatial distribution patterns of perceived equity by kernel density estimation (KDE) in Giant Panda National Park, China. The regression analysis showed that local residents who live closer to the PA boundary are more likely to develop negative responses and those who with easy access to tourism spots have more positive procedural and distributional perceptions. Notably, the proximity to the PA authority decreases locals’ perceptions of fairness in all aspects, which is potentially due to the opaque participative channels provided by the PA authority. We argue that those spatial differentials in fairness perceptions are driven by the intrinsic discrepancy of biodiversity protection requirements and the unevenly distributed consequences of management policies. Key steps to advance social equity considerations include multi-industry guidance, extending participative channels, and co-producing better compensation plans. Herein, this study appeals to a greater focus on the spatial aspect of social equity issues in PAs.

Dermatomycosis is the second major cause of morbidity in giant pandas (Ailuropoda melanoleuca), and seriously endangers its health. Previous observations indicated that the occurrence of dermatomycosis in the giant panda varies in different seasons. The skin microbiota is a complex ecosystem, but knowledge on the community structure and the pathogenic potentials of fungi on the skin of the giant panda remains limited. In this study, samples from the giant panda skin in different seasons were collected, and the mycobiota were profiled by 18S rRNA gene sequencing. In total, 375 genera in 38 phyla were detected, with Ascomycota, Basidiomycota, Streptophyta, and Chlorophyta as the predominant phyla and Trichosporon, Guehomyces, Davidiella, Chlorella, Asterotremella, and Klebsormidium as the predominant genera. The skin mycobiota of the giant panda changed in the seasons, and the diversity and abundance of the skin fungi were significantly higher in spring, autumn, and summer than in the winter. Several dermatomycosis-associated fungi were detected as opportunists in the skin mycobiota of healthy giant pandas. Clinical dermatomycosis in the giant panda is observed more in summer and autumn. In this study, the results indicated that the high diversity and abundance of the skin fungi may have enhanced the occurrence of dermatomycosis in autumn and summer, and that dermatomycosis-associated fungi are the normal components of the skin mycobiota.

The giant panda is one of the rarest animals in the world. Skin diseases seriously endanger the health of giant panda and are considered the second major cause of its morbidity. Skin microbiota is a complex ecosystem, and the community structure and the pathogenic potential of bacteria on giant panda skin remain largely unclear. In order to understand the skin bacterial flora of captive giant pandas, the microbiota in giant panda skin samples collected during different seasons was profiled via 16S rRNA gene sequencing. In total, 522 genera from 53 bacterial phyla were detected, with Proteobacteria (40.5%), Actinobacteria (23.1%), Firmicutes (21.1%), Bacteroidetes (9.5%), Cyanobacteria (2.1%), and Thermi (1.2%) as the predominant phyla and Streptococcus (13.9%), Acinetobacter (9.2%), Staphylococcus (2.9%), Pseudomonas (5.9%), Dermacoccus (4.8%), Brachybacterium (2.9%), Escherichia (2.7%), Chryseobacterium (2.1%), Arthrobacter (1.6%), Kocuria (1.5%), Psychrobacter (1.2%), Deinococcus (1.1%), and Flavobacterium (1.1%) as the predominant genera. The results indicated that the diversity was lower in winter than in other seasons and higher in autumn than in other seasons, and the abundance in spring was significantly higher than that in other seasons. Several skin disease-associated bacteria were detected as opportunists in the skin microbiota of healthy giant pandas. In this study, the results indicated that the high diversity and abundance of the skin bacteria may have enhanced the occurrence of skin disease in autumn and spring and that skin disease-associated bacteria are the normal components of the skin microbiota.

Cryopreservation induces sperm cryoinjuries, including physiological and functional changes. However, the molecular mechanisms of sperm cryoinjury and cryoresistance are still unknown. Cryoresistance or the freeze tolerance of sperm varies across species, and boar sperm is more susceptible to cold stress. Contrary to boar sperm, giant panda sperm appears to be strongly freeze-tolerant and is capable of surviving repeated cycles of freeze-thawing. In this study, differentially expressed (DE) PIWI-interacting RNAs (piRNAs) of fresh and frozen-thawed sperm with different freeze tolerance capacity from giant panda and boar were evaluated. The results showed that 1,160 (22 downregulated and 1,138 upregulated) and 384 (110 upregulated and 274 downregulated) DE piRNAs were identified in giant panda and boar sperm, respectively. Gene ontology (GO) enrichment analysis revealed that the target DE messenger RNAs (mRNAs) of DE piRNAs were mainly enriched in biological regulation, cellular, and metabolic processes in giant panda and boar sperm. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target DE mRNAs of DE piRNAs were only distributed in DNA replication and the cyclic adenosine monophosphate (cAMP) signaling pathway in giant panda, but the cAMP, cyclic guanosine monophosphate (cGMP), and mitogen-activated protein kinase (MAPK) signaling pathways in boar sperm were considered as part of the olfactory transduction pathway. In conclusion, we speculated that the difference in the piRNA profiles and the DE piRNAs involved in the cAMP signaling pathway in boar and giant panda may have contributed to the different freeze tolerance capacities between giant panda and boar sperm, which helps to elucidate the molecular mechanism behind sperm cryoinjury and cryoresistance.

The giant panda (Ailuropoda melanoleuca) is a vulnerable species and a charismatic member of zoological collections worldwide. Despite its importance as a representative species for global wildlife conservation efforts, no studies to date have described normal cell morphology or cytoplasmic constituents by traditional techniques such as cytochemical staining and evaluation of ultrastructural features. The objective of this study was to accurately identify and characterize the leukocytes and platelets of clinically healthy giant pandas using routine Wright-Giemsa stain, eight cytochemical stains, immunocytochemistry (CD3), and transmission electron microscopy (TEM) to further the collective understanding of normal cellular morphological features, cytochemical reactivity, and cytoplasmic contents found in health. Voluntary venipuncture was performed on four healthy individual animals (two adults and two juveniles), as part of routine preventive health evaluation. Blood was collected for routine and cytochemical stains, and into 2.5% glutaraldehyde for TEM. On Wright-Giemsa-stained blood films, leukocytes were differentiated into granulocytes (neutrophils, eosinophils, basophils) and mononuclear cells (lymphocytes, monocytes). Cytochemical staining revealed similar leukocyte and platelet staining patterns to those reported in other mammals, with some notable differences. By TEM, leukocytes with nuclear and cytoplasmic features of mononuclear cells were readily differentiated from granulocytes, and platelets had similar ultrastructural features to those reported in other mammals. Neutrophils were the predominant cell type followed by lymphocytes, while basophils were rare. Rare large or reactive lymphocytes, rare reactive monocytes, and rare large platelets were noted in apparently healthy giant pandas of this study. A unique mononuclear cell, with a moderately indented nucleus and shared cytochemical and ultrastructural characteristics of lymphocytes and monocytes, was discovered in this species. The combined cytochemical, immunocytochemical (CD3), and ultrastructural features of these unique cells more closely resemble those of monocytes, but the definitive cell lineage remains unknown at this time. This study provides novel information on giant panda leukocyte morphology and cellular constituents in health, shows the importance of manual blood film review, has important implications for hemogram interpretation in future clinical cases and research, and provides a baseline for future characterization and understanding of hemogram changes in response to disease.

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. 3.2.1.1) 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.

Giant pandas are the flagship species in world conservation. Due to bamboo being the primary food source for giant pandas, dental wear is common owing to the extreme toughness of the bamboo fiber. Even though research on tooth enamel wear in humans and domestic animals is well-established, research on tooth enamel wear in giant pandas is scarce. The purpose of this study is to evaluate tooth enamel wear resistance in giant pandas to provide a basis for a better understanding of their evolutionary process. From microscopic and macroscopic perspectives, the abrasion resistance of dental enamel in giant pandas is compared with that of herbivorous cattle and carnivorous dogs in this study. This involves the use of micro-scratch and frictional wear tests. The results show that the boundary between the enamel prism and the enamel prism stroma is well-defined in panda and canine teeth, while bovine tooth enamel appears denser. Under constant load, the tribological properties of giant panda enamel are similar to those of canines and significantly different from those of bovines. Test results show that the depth of micro scratches in giant panda and canine enamel was greater than in cattle, with greater elastic recovery occurring in dogs. Scratch morphology indicates that the enamel substantive damage critical value is greater in pandas than in both dogs and cattle. The analysis suggests that giant panda enamel consists of a neatly arranged special structure that may disperse extrusion stress and absorb impact energy through a series of inelastic deformation mechanisms to cope with the wear caused by eating bamboo. In this study, the excellent wear resistance of giant panda's tooth enamel is verified by wear tests. A possible theoretical explanation of how the special structure of giant panda tooth enamel may improve its wear resistance is provided. This provides a direction for subsequent theoretical and experimental studies on giant panda tooth enamel and its biomaterials.

Effectively prediction of the tourism demand is of great significance to rationally allocate resources, improve service quality, and maintain the sustainable development of scenic spots. Since tourism demand is affected by the factors of climate, holidays, and weekdays, it is a challenge to design an accurate forecasting model obtaining complex features in tourism demand data. To overcome these problems, we specially consider the influence of environmental factors and devise a forecasting model based on ensemble learning. The model first generates several sub-models, and each sub-model learns the features of time series by selecting informative sequences for reconstructing the forecasting input. A novel technique is devised to aggregate the outputs of these sub-models to make the forecasting more robust to the non-linear and seasonal features. Tourism demand data of Chengdu Research Base of Giant Panda Breeding in recent 5 years is used as a case to validate the effectiveness of our scheme. Experimental results show that the proposed scheme can accurately forecasting tourism demand, which can help Chengdu Research Base of Giant Panda Breeding to improve the quality of tourism management and achieve sustainable development. Therefore, the proposed scheme has good potential to be applied to accurately forecast time series with non-linear and seasonal features.

A feline panleukopenia virus (FPV), Giant panda/CD/2018, was isolated from a captive giant panda with mild diarrhea in 2018 in Chengdu, China, and further identified via indirect immunofluorescence assay (IFA), transmission electron microscopy (TEM) observation, and genetic analysis. Phylogenetic analysis based on the complete VP2 nucleotide sequences showed that it shared high homology with Chinese FPV isolates and grouped within FPV cluster 1. One unique substitution Gly(G)299Glu(E) in the capsid protein VP2 was first identified with Giant panda/CD/2018. The presence of the G299E substitution is notable as it is located on the top region of the interconnecting surface loop 3, which may be involved in controlling the host range and antigenicity of FPV. These findings first demonstrate that FPV with natural point mutation G299E in the VP2 gene is prevalent in giant panda and suggest that etiological surveillance and vaccination among all giant pandas are urgently needed to protect this endangered species against FPV infection.

By analyzing the voiceprint characteristics of giant panda’s voice, this study proposes a giant panda individual recognition method based on the characteristics of the composite Mel composite frequency cepstral coefficient (CMFCC) and proves that the characteristic sequence of the CMFCC has long-range dependent characteristics. First, the MFCC (Mel composite frequency cepstral coefficient) with a low frequency resolution is obtained by the Mel filter bank; then, the inverse Mel frequency cepstral coefficient (IMFCC) features of giant panda calls are extracted. The CMFCC characteristic sequence of giant panda voice composed of the MFCC and IMFCC improves the resolution of high- and low-frequency resolution characteristics of giant panda voice. Finally, the first-order difference characteristic parameters of the MFCC are integrated to obtain the difference characteristics between frames. Through experiments, the improvement of the system recognition effect is verified, and the recognition accuracy meets the theoretical expectation.

Giant panda 3D reconstruction technology plays an important role in the research of giant panda protection. Through the analysis of giant panda video image sequence (GPVS), we prove that it has the long-range–dependent characteristics. This article proposes an algorithm to accurately reconstruct the giant panda 3D model by using the long-range–dependent characteristics of GPVS. First, the algorithm uses a skinned multi-animal linear model (SMAL) to obtain the initial 3D model of giant panda, and the 3D model of the single-frame giant panda image is reconstructed by controlling shape parameters and attitude parameters; then, we use the coherence information contained in the long-range–dependent characteristics between video sequence images to construct a smooth energy function to correct the error of the 3D model. Through this error, we can judge whether the 3D reconstruction result of the giant panda is consistent with the real structural characteristics of the giant panda. The algorithm solves the problem of low 3D reconstruction accuracy and the problem that 3D reconstruction is easily affected by occlusion or interference. Finally, we realize the accurate reconstruction of the giant panda 3D model.

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.

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.

A feline panleukopenia virus (FPV), Giant panda/CD/2018, was isolated from a captive giant panda with mild diarrhea in 2018 in Chengdu, China, and further identified via indirect immunofluorescence assay (IFA), transmission electron microscopy (TEM) observation, and genetic analysis. Phylogenetic analysis based on the complete VP2 nucleotide sequences showed that it shared high homology with Chinese FPV isolates and grouped within FPV cluster 1. One unique substitution Gly(G)299Glu(E) in the capsid protein VP2 was first identified with Giant panda/CD/2018. The presence of the G299E substitution is notable as it is located on the top region of the interconnecting surface loop 3, which may be involved in controlling the host range and antigenicity of FPV. These findings first demonstrate that FPV with natural point mutation G299E in the VP2 gene is prevalent in giant panda and suggest that etiological surveillance and vaccination among all giant pandas are urgently needed to protect this endangered species against FPV infection. [ABSTRACT FROM AUTHOR]

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) is regarded as one of the most important priority pathogens within the One Health interface. However, few studies have investigated the occurrence of ESBL-EC in giant pandas, along with their antibiotic-resistant characteristics and horizontal gene transfer abilities. In this study, we successfully identified 12 ESBL-EC strains (8.33%, 12/144) out of 144 E. coli strains which isolated from giant pandas. We further detected antibiotic resistance genes (ARGs), virulence-associated genes (VAGs) and mobile genetic elements (MGEs) among the 12 ESBL-EC strains, and the results showed that 13 ARGs and 11 VAGs were detected, of which blaCTX-M (100.00%, 12/12, with 5 variants observed) and papA (83.33%, 10/12) were the most prevalent, respectively. And ISEcp1 (66.67%, 8/12) and IS26 (66.67%, 8/12) were the predominant MGEs. Furthermore, horizontal gene transfer ability analysis of the 12 ESBL-EC showed that all blaCTX-M genes could be transferred by conjugative plasmids, indicating high horizontal gene transfer ability. In addition, ARGs of rmtB and sul2, VAGs of papA, fimC and ompT, MGEs of ISEcp1 and IS26 were all found to be co-transferred with blaCTX-M. Phylogenetic analysis clustered these ESBL-EC strains into group B2 (75.00%, 9/12), D (16.67%, 2/12), and B1 (8.33%, 1/12), and 10 sequence types (STs) were identified among 12 ESBL-EC (including ST48, ST127, ST206, ST354, ST648, ST1706, and four new STs). Our present study showed that ESBL-EC strains from captive giant pandas are reservoirs of ARGs, VAGs and MGEs that can co-transfer with blaCTX-M via plasmids. Transmissible ESBL-EC strains with high diversity of resistance and virulence elements are a potential threat to humans, animals and surrounding environment. [ABSTRACT FROM AUTHOR]

Dental caries severely hinders efficient access to adequate energy in wildlife. Different food supplies will develop characteristic plaque, and the microorganisms of these plaque are closely related to dental health. Here, plaque samples from panda cubs with caries and caries-free were collected for 16S rRNA high-throughput sequencing. All sequences clustered into 337 operational taxonomic units (OTUs; 97% identity), representing 268 independent species belonging to 189 genera, 98 families, 51 orders, 24 classes, and 13 phyla. Two groups shared 218 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in plaques with caries exceeded that of caries-free. The dominant phyla of plaque microbiota included Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. The dominant genera included unclassified Neisseriaceae , Actinobacillus , Lautropia , Neisseria , Porhyromonas , unclassified Pasteurellaceae , Moraxella , Streptococcus , Bergeywlla and Capnocytophaga. β diversity analysis showed that the plaque microbial community structure was different between two groups. Using LEfSe analysis, 19 differentially abundant taxa were identified as potential biomarkers. Finally, function predictions analysis showed All the energy related metabolic pathways on KEGG level 2 were enriched in caries-active group. Consistent with the mainstream caries-causing narrative, our results illuminate the lack of information regarding the oral microflora composition and function within giant panda cubs. [ABSTRACT FROM AUTHOR]

Intestinal diseases caused by opportunistic pathogens seriously threaten the health and survival of giant pandas. However, our understanding of gut pathogens in different populations of giant pandas, especially in the wild populations, is still limited. Here, we conducted a study based on 52 giant panda metagenomes to investigate the composition and distribution of gut pathogens and virulence factors (VFs) in five geographic populations (captive: GPCD and GPYA; wild: GPQIN, GPQIO, and GPXXL). The results of the beta-diversity analyzes revealed a close relationship and high similarity in pathogen and VF compositions within the two captive groups. Among all groups, Proteobacteria, Firmicutes, and Bacteroidetes emerged as the top three abundant phyla. By using the linear discriminant analysis effect size method, we identified pathogenic bacteria unique to different populations, such as Klebsiella in GPCD, Salmonella in GPYA, Hafnia in GPQIO, Pedobacter in GPXXL, and Lactococcus in GPQIN. In addition, we identified 12 VFs that play a role in the intestinal diseases of giant pandas, including flagella, CsrA, enterobactin, type IV pili, alginate, AcrAB, capsule, T6SS, urease, type 1 fimbriae, polar flagella, allantoin utilization, and ClpP. These VFs influence pathogen motility, adhesion, iron uptake, acid resistance, and protein regulation, thereby contributing to pathogen infection and pathogenicity. Notably, we also found a difference in virulence of Pseudomonas aeruginosa between GPQIN and non-GPQIN wild populations, in which the relative abundance of VFs (0.42%) of P. aeruginosa was the lowest in GPQIN and the highest in non-GPQIN wild populations (GPXXL: 23.55% and GPQIO: 10.47%). In addition to enhancing our understanding of gut pathogens and VFs in different geographic populations of giant pandas, the results of this study provide a specific theoretical basis and data support for the development of effective conservation measures for giant pandas. [ABSTRACT FROM AUTHOR]

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. 3.2.1.1) 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. [ABSTRACT FROM AUTHOR]

In this work, we searched for an effective probiotic that can help control intestinal infection, particularly enterotoxigenic Escherichia coli K88 (ETEC) invasion, in giant panda (Ailuropoda melanoleuca). As a potential probiotic strain, Lactobacillus plantarum BSGP201683 (L. plantarum G83) was isolated from the feces of giant panda and proven beneficial in vitro. This study was aimed to evaluate the protective effect of L. plantarum G83 in mice challenged with ETEC. The mice were orally administered with 0.2 mL of PBS containing L. plantarum G83 at 0 colony-forming units (cfu) mL−1 (control; negative control, ETEC group), 5.0 × 108 cfu mL−1 (LDLP), 5.0 × 109 cfu mL−1 (MDLP), and 5.0 × 1010 cfu mL−1 (HDLP) for 14 consecutive days. At day 15, the mice (LDLP, MDLP, HDLP, and ETEC groups) were challenged with ETEC and assessed at 0, 24, and 144 h. Animal health status; chemical and biological intestinal barriers; and body weight were measured. Results showed that L. plantarum G83 supplementation protected the mouse gut mainly by attenuating inflammation and improving the gut microflora. Most indices significantly changed at 24 h after challenge compared to those at 0 and 144 h. All treatment groups showed inhibited plasma diamine oxidase activity and D-lactate concentration. Tight-junction protein expression was down-regulated, and interleukin (IL)-1β, IL-6, IL-8, TLR4, and MyD88 levels were up-regulated in the jejunum in the LDLP and MDLP groups. The number of the Enterobacteriaceae family and the heat-labile enterotoxin (LT) gene decreased (P < 0.05) in the colons in the LDLP and MDLP groups. All data indicated that L. plantarum G83 could attenuate acute intestinal inflammation caused by ETEC infection, and the low and intermediate doses were superior to the high dose. These findings suggested that L. plantarum G83 may serve as a protective probiotic for intestinal disease and merits further investigation.

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 gutmicrobiome 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. [ABSTRACT FROM AUTHOR]

The changes in the expression of genes related to digestion and metabolism may be various in different dietary mammals from juvenile to adult, especially, the giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens), which were once carnivores but have shifted to being specialized bamboo eaters, are unique features of their changes are more unclear. To elucidate the changing patterns of gene expression related to digestion and metabolism from juvenile to adult in different dietary mammals, we performed transcriptome analysis of the liver or pancreas in giant and red pandas, herbivorous rabbits (Oryctolagus cuniculus) and macaques (Macaca mulatta), carnivorous ferrets (Mustela putorius furo), and omnivorous mice (Mus musculus) from juvenile to adult. During the transition from juvenile to adulthood, giant and red pandas, as well as rabbits and macaques, show significant upregulation of key genes for carbohydrate metabolism, such as starch hydrolysis and sucrose metabolism, and unsaturated fatty acid metabolism, such as linoleic acid, while there is no significant difference in the expression of key genes for fatty acid ß-oxidation. A large number of amino acid metabolism related genes were upregulated in adult rabbits and macaques compared to juveniles. While adult giant and red pandas mainly showed upregulation of key genes for arginine synthesis and downregulation of key genes for arginine and lysine degradation. In adult stages, mouse had significantly higher expression patterns in key genes for starch hydrolysis and sucrose metabolism, as well as lipid and protein metabolism. In contrast to general expectations, genes related to lipid, amino acid and protein metabolism were significantly higher expressed in adult group of ferrets, which may be related to their high metabolic levels. Our study elucidates the pattern of changes in the expression of genes related to digestion and metabolism from juvenile to adult in different dietary mammals, with giant and red pandas showing adaptations associated with specific nutritional limitations of bamboo. [ABSTRACT FROM AUTHOR]

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) is regarded as one of the most important priority pathogens within the One Health interface. However, few studies have investigated the occurrence of ESBL-EC in giant pandas, along with their antibiotic-resistant characteristics and horizontal gene transfer abilities. In this study, we successfully identified 12 ESBL-EC strains (8.33%, 12/144) out of 144 E. coli strains which isolated from giant pandas. We further detected antibiotic resistance genes (ARGs), virulence-associated genes (VAGs) and mobile genetic elements (MGEs) among the 12 ESBL-EC strains, and the results showed that 13 ARGs and 11 VAGs were detected, of which blaCTX-M (100.00%, 12/12, with 5 variants observed) and papA (83.33%, 10/12) were the most prevalent, respectively. And ISEcp1 (66.67%, 8/12) and IS26 (66.67%, 8/12) were the predominant MGEs. Furthermore, horizontal gene transfer ability analysis of the 12 ESBL-EC showed that all blaCTX-M genes could be transferred by conjugative plasmids, indicating high horizontal gene transfer ability. In addition, ARGs of rmtB and sul2, VAGs of papA, fimC and ompT, MGEs of ISEcp1 and IS26 were all found to be co-transferred with blaCTX-M. Phylogenetic analysis clustered these ESBL-EC strains into group B2 (75.00%, 9/12), D (16.67%, 2/12), and B1 (8.33%, 1/12), and 10 sequence types (STs) were identified among 12 ESBL-EC (including ST48, ST127, ST206, ST354, ST648, ST1706, and four new STs). Our present study showed that ESBL-EC strains from captive giant pandas are reservoirs of ARGs, VAGs and MGEs that can co-transfer with blaCTX-M via plasmids. Transmissible ESBL-EC strains with high diversity of resistance and virulence elements are a potential threat to humans, animals and surrounding environment.

DNA methylation modification can regulate gene expression without changing the genome sequence, which helps organisms to rapidly adapt to new environments. However, few studies have been reported in non-model mammals. Giant panda (Ailuropoda melanoleuca) is a flagship species for global biodiversity conservation. Wildness and reintroduction of giant pandas are the important content of giant pandas' protection. However, it is unclear how wildness training affects the epigenetics of giant pandas, and we lack the means to assess the adaptive capacity of wildness training giant pandas. We comparatively analyzed genome-level methylation differences in captive giant pandas with and without wildness training to determine whether methylation modification played a role in the adaptive response of wildness training pandas. The whole genome DNA methylation sequencing results showed that genomic cytosine methylation ratio of all samples was 5.35%--5.49%, and the methylation ratio of the CpG site was the highest. Differential methylation analysis identified 544 differentially methylated genes (DMGs). The results of KEGG pathway enrichment of DMGs showed that VAV3, PLCG2, TEC and PTPRC participated in multiple immune-related pathways, and may participate in the immune response of wildness training giant pandas by regulating adaptive immune cells. A large number of DMGs enriched in GO terms may also be related to the regulation of immune activation during wildness training of giant pandas. Promoter differentially methylation analysis identified 1,199 genes with differential methylation at promoter regions. Genes with low methylation level at promoter regions and high expression such as, CCL5, P2Y13, GZMA, ANP32A, VWF, MYOZ1, NME7, MRPS31 and TPM1 were important in environmental adaptation for wildness training giant pandas. The methylation and expression patterns of these genes indicated that wildness training giant pandas have strong immunity, blood coagulation, athletic abilities and disease resistance. The adaptive response of giant pandas undergoing wildness training may be regulated by their negatively related promoter methylation. We are the first to describe the DNA methylation profile of giant panda blood tissue and our results indicated methylation modification is involved in the adaptation of captive giant pandas when undergoing wildness training. Our study also provided potential monitoring indicators for the successful reintroduction of valuable and threatened animals to the wild. [ABSTRACT FROM AUTHOR]

Bacterial infection and imbalance of bacterial community in the genitourinary system of giant panda could affect the reproductive health. In severe cases, it can also lead to abortion. In this study, 13 of vaginal secretions in the estrue (E) group and seven of vaginal secretions in the non-estrue (NE) group were used to study the composition and diversity of vaginal bacterial communities between estrus and non-estrus by 16S rRNA gene sequencing analysis. The results showed that the vaginal microbiome in giant pandas shared the same top five abundant species between estrus and non-estrus at the phylum level. However, the vaginal microbiome changed significantly during estrus at the genus level. In top 10 genera, the abundance of Escherichia , Streptococcus , and Bacteroides in the E group was significantly higher than that in the NE group (p <0.05); Azomonas , Porphyromonas , Prevotella , Campylobacter , and Peptoniphilus in the NE group was significantly higher than that in the E group (p <0.05). The richness and diversity of vaginal microbiome in giant panda on estrus were significantly lower than those on non-estrus (p <0.05). It is noteworthy that the abundance of Streptococcus , Escherichia , and Bacteroides of vagina in giant pandas maintained low abundance in the daily. Whereas, they increased significantly during estrus period, which may play an important role in female giant pandas during estrus period. It was hypothesized that hormones may be responsible for the changes in the vaginal microbiome of giant pandas between estrus and no-estrus stages. [ABSTRACT FROM AUTHOR]

Introduction: The circadian clock influences many aspects of animal welfare including metabolism, breeding, and behavior. In most species, circadian clocks are internal clocks regulated by external environmental cues called zeitgebers. The most common zeitgebers are light/dark cycles, food, and temperature. However, within captive environments, animals can be housed at latitudes with different light/dark cycles than their natural habitat and most other zeitgebers are controlled by humans. The effects that modified zeitgebers have on captive animals' circadian and circannual rhythmicity is largely unknown. To explore this and potential welfare implications, we measured and analyzed observational behavioral data of zoo-housed giant pandas for one year utilizing live camera footage from six zoos across the world. The worldwide distribution of the zoos gives us the unique opportunity to investigate how housing giant pandas within and outside of their natural latitudinal range can affect circadian rhythmicity and behavior. Methods: Focal sampling was completed for 11 giant pandas each month for 12 consecutive months to gain an estimate of one circannual cycle. Within each month, we estimated one daylight or 24 h cycle of activity/behavior by conducting 10-min observation sessions systemically each hour the pandas were visible. Results: Zero-inflated negative binomial mixture models found that latitude is associated with activity levels, with pandas housed outside of their natural latitudinal range displaying less activity than those within their latitudinal range. Amount of daylight, temperature minimum, and temperature range were also associated with activity cycles, potentially acting as zeitgebers. An association between sexual-related and stereotypic behavioral cycles was found, with the circannual cycles fluctuating in synchrony throughout several points in a year. Discussion: These results indicate that changes to common zeitgebers and environmental conditions can influence circadian and circannual cycles. The widespread evolution of circadian rhythms suggests an adaptive advantage to possessing one in an environment with cyclical changes, allowing species to anticipate changes in their environment and respond accordingly. Therefore, although animals are highly adaptive, creating a captive environment that mimics the environmental conditions for which the animal has evolved can encourage naturalistic cycles that ultimately aid in promoting positive welfare states and increasing chances of successful breeding and conservation. [ABSTRACT FROM AUTHOR]

Despite lacking clear historical significance, the appeal pandas have to the people of China has played an integral role in the emergence of the country's cultural identity and ideals. Few studies have explored the giant panda due to the ongoing dialogue between the West and China, which, according to Edward Said, is permeated with imperialist, colonial, and orientalist flavors. In 1869, the French missionary Armand David encountered a dead specimen of a giant panda in Baoxing, Sichuan, which sparked the beginning of this dialogue. David shipped the skin to Paris, where the animal was named and aesthetically recreated for the first time for Western audiences. In this paper, we approach the giant panda as a dark tourism attraction embodying a process of making and remaking Chinese national identities over the past two centuries. Using “virtual curating” to study the Giant Panda Museum located at the Chengdu Research Base of Giant Panda Breeding, we demonstrate that the giant panda, which has achieved iconic status in China, represents a national history that is dark, backward, and based on suffering and death. We argue that understanding the giant panda's history as a dark tourism attraction provides an ethical vantage point from which to perceive tourist-panda relationships.

The decline in natural mating behavior is the primary reason underlying in the poor population growth of captive giant pandas. However, the influencing factors and underlyingmechanisms remain unclear to data. It is speculated that the decline in natural mating behavior could be related to the psychological stress caused by captivity, which restricts their free choice of mates. In order to test this hypothesis, we performed urinary metabolomics analysis using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC/-MS) combined with 16S rDNA sequencing for exploring the physiological mechanism underlying the decline in the natural mating behavior of captive giant panda. The results demonstrated that the decline in mating ability could be related to abnormalities in arginine biosynthesis and neurotransmitter synthesis. Additionally, the relative abundance of bacteria from the Firmicutes, Proteobacteria, and Actinobacteria phyla and the Acinetobacter, Weissella, and Pseudomonas genus was significantly reduced in the group with low natural mating behavior. These findings imply that the inhibition of arginine synthesis induced by environmental changes could be related to the poor libido and failure of mate selection in captive giant pandas during the breeding period. The results also demonstrate the relationship between the altered urinary microbes and metabolites related to arginine and neurotransmitter synthesis. These findings may aid in understanding the mechanism underlying environment-induced mate selection in captive giant pandas and propose a novel strategy for determining the sexual desire of giant pandas based on urinarymicrobes. Themethod would be of great significance in improving the natural reproductive success rate of captive giant pandas. [ABSTRACT FROM AUTHOR]

Intestinal diseases are one of the main causes of captive giant panda death. Their special dietary habits and gastrointestinal tract structure often lead to intestinal epithelium damage and secondary intestinal infection. The captive giant panda is predisposed to suffer from microbiota dysbiosis due to long-term artificial feeding and antibiotic misuse. However, there are few reported probiotics to treat giant panda enteritis and the associated dysbiosis. This study aims to elucidate the mechanism by which Lactobacillus plantarum G201683 (L. plantarum G83), a promising panda-derived probiotic, exerts a protective effect on intestinal inflammation in the dextran sulfate sodium- (DSS) induced panda microbiota-associated (DPMA) mouse model. The DPMA mouse was generated by antibiotic treatment and 5% DSS drinking water administration to assess the effect of L. plantarum G83 on intestinal inflammation and microbiota in vivo. Our results demonstrated the successful generation of a DPMA mouse model with Enterobacteriaceae enrichment, consistent with the giant panda intestinal microbiota. L. plantarum G83 decreased clinical and histological severity of intestinal inflammation, enhanced intestinal tight junction protein expression (ZO-1, Occludin) and alleviated inflammatory cytokine production (TNF-) in the colon of DPMA mice. The administration of L. plantarum G83 altered the microbiota composition by decreasing pathogen associated taxa such as E. coli and increasing abundance of beneficial bacteria including Bifidobacterium spp. These changes in microbiota composition were associated with an increased concentration of short chain fatty acids (SCFA), reduced NF-κB signaling, and an altered balance of T helper cell subsets. Our findings support L. plantarum G83 as a promising probiotic to treat intestinal inflammation in the giant panda. [ABSTRACT FROM AUTHOR]

The giant panda has developed a series of evolutionary strategies to adapt to a bamboo diet. The abundance and diversity of the phyllosphere microbiome change dramatically depending on the season, host species, location, etc., which may, in turn, affect the growth and health of host plants. However, few studies have investigated the factors that influence phyllosphere bacteria in bamboo, a staple food source of the giant panda. Amplicon sequencing of the 16S rRNA gene was used to explore the abundance and diversity of phyllosphere bacteria in three bamboo species (Arundinaria spanostachya , Yushania lineolate , and Fargesia ferax) over different seasons (spring vs. autumn), elevation, distance from water, etc., in Liziping National Nature Reserve (Liziping NR), China. And whole-genome shotgun sequencing uncovered the differences in biological functions (KEGG and Carbohydrate-Active enzymes functions) of A. spanostachya phyllosphere bacteria between spring and autumn. The results showed that the abundance and diversity of F. ferax phyllosphere bacteria were greater than that of the other two bamboo species in both seasons. And three kinds of bamboo phyllosphere bacteria in autumn were significantly higher than in spring. The season was a more important factor than host bamboo species in determining the community structure of phyllosphere bacteria based on the (un)weighted UniFrac distance matrix. The composition, diversity, and community structure of phyllosphere bacteria in bamboo were primarily affected by the season, species, altitude, tree layer, and shrub layer. Different bacterial communities perform different functions in different bamboo species, and long-term low temperatures may shape more varied and complex KEGG and Carbohydrate-Active enzymes functions in spring. Our study presented a deeper understanding of factors influencing the bacterial community in the bamboo phyllosphere. These integrated results offer an original insight into bamboo, which can provide a reference for the restoration and management of giant panda bamboo food resources in the Xiaoxiangling mountains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To achieve reproduction, male solitary mammals need to locate females using chemical communication with high levels of precision. In the case of giant pandas, the total estrus period of females was usually 15 days each year, however, successful mating activity is finished within 3 days from respective home range. The mating pattern of giant pandas, where multiple males compete for each female requires females employ efficient systems to communicate their estrus phases. To verifying whether the scent secretions of giant pandas changes by gender and estrus progression, the microbiota and compounds in 29 anogenital gland samples from 14 individuals during estrus were analyzed by 16S rRNA sequencing and GC-MS. We show that the microbiota communities covary by gender with 4 particular compounds of scent secretions. Among 597 genera, 34 were identified as biomarkers that could be used to distinguish between different estrus phases. By bacterialcompounds co-analysis, 3 fatty ester acids and squalene compounds covaried with the development of estrus in the bacterial communities of female giant pandas. This study helps clarify how a large, solitary mammal expresses accurate information to improve the likelihood of successful reproduction by changing the composition of microbiota and odor compounds of anogenital glands during estrus. [ABSTRACT FROM AUTHOR]

Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a "green enzyme," laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda's gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants. [ABSTRACT FROM AUTHOR]

Human activities are one of the main factors threatening the long-term survival of wild giant pandas. Long-term and large-scale human activities can be quickly assessed by night light remote sensing data, which has been more and more widely used. In this paper, we analyzed the spatial patterns of nighttime light intensity and their dynamic changes from 2000 to 2020 in the entire giant panda distribution area using long-term nighttime light remote sensing data, and evaluated the dynamic threats of nighttime light-represented human activities to giant panda populations by proximity of light source and its intensity. We also assessed the relationships between the nighttime light dynamics and the trends of habitat fragmentation. Results showed that the intensity and range of the nighttime light around panda habitat had increased significantly from 2000 to 2020. The nighttime light intensity inside the natural reserves is significantly lower than outside, and it becomes more obvious over time. The intensity and range of nighttime light inside the natural reserves first increased and then decreased during the two decades, indicating that the protection inside the natural reserves effectively reversed the rapid increase in human activities in the previous decade. From 2000 to 2020, nighttime light sources became closer to local panda populations. In particular, they approached the pandas in the first decade, and stayed away in the second decade. This is mainly reflected in the weakened threats of nighttime light-represented human activities on the core large populations of the Qinling, Minshan and Qionglai Mountains from 2010 to 2020, but the threats on the peripheral populations continued to strengthen, where nighttime light became not only closer but also more intensified. The increase in nighttime light intensity and range were also significantly positively correlated with the increase in habitat fragmentation, especially during 2000–2010. Our study reveals the dynamic changes of nighttime light-represented human activities’ threats to wild giant panda and its habitat, gives advices for effectively protecting giant pandas and their habitats from human activities, and has implications for the assessment of human activities’ threats to other species in the world.