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Introduction: Over 25% of the world’s population has non-obese or lean non-alcoholic fatty liver disease (NAFLD), and the prevalence is higher than average in Asia. The present study focused on the relationship between body mass index (BMI) and non-obese NAFLD in non-overweight people in China, particularly the influence of triglycerides (TG) in the pathogenesis of non-obese NAFLD. The findings suggest new treatments for NAFLD patients with normal BMI, as well as provide an early warning system for the understanding and prevention of NAFLD in non-obese patients. Methods: This cross-sectional study enrolled 159,959 Chinese subjects with BMI

Abstract Background Energy homeostasis is essential for the adaptation of animals to their environment and some wild animals keep low metabolism adaptive to their low-nutrient dietary supply. Giant panda is such a typical low-metabolic mammal exhibiting species specialization of extremely low daily energy expenditure. It has low levels of basal metabolic rate, thyroid hormone, and physical activities, whereas the cellular bases of its low metabolic adaptation remain rarely explored. Results In this study, we generate a single-nucleus transcriptome atlas of 21 organs/tissues from a female giant panda. We focused on the central metabolic organ (liver) and dissected cellular metabolic status by cross-species comparison. Adaptive expression mode (i.e., AMPK related) was prominently displayed in the hepatocyte of giant panda. In the highest energy-consuming organ, the heart, we found a possibly optimized utilization of fatty acid. Detailed cell subtype annotation of endothelial cells showed the uterine-specific deficiency of blood vascular subclasses, indicating a potential adaptation for a low reproductive energy expenditure. Conclusions Our findings shed light on the possible cellular basis and transcriptomic regulatory clues for the low metabolism in giant pandas and helped to understand physiological adaptation response to nutrient stress.

ABSTRACT Mycobacterium tuberculosis (Mtb) continues to pose a significant threat to global health because it causes granulomas and systemic inflammatory responses during active tuberculosis (TB). Mtb can induce macrophage pyroptosis, which results in the release of IL-1β and causes tissue damage, thereby promoting its spread. In the absence of anti-TB drugs, host-directed therapy (HDT) has been demonstrated to be an effective strategy against TB. In this study, we used an in vitro Mtb-infected macrophage model to assess the effect of baicalein, derived from Scutellariae radix, on pyroptosis induced in Mtb-infected macrophages. Further, we investigated the molecular mechanisms underlying the actions of baicalein. The results of the study suggest that baicalein inhibits pyroptosis in Mtb-infected macrophages by downregulating the assembly of AIM2 and NLRP3 inflammasome and promoting autophagy. Further research has also shown that the mechanism by which baicalein promotes autophagy may involve the inhibition of the activation of the Akt/mTOR pathway and the inhibition of the AIM2 protein, which affects the levels of CHMP2A protein required to promote autophagy. Thus, our data show that baicalein can inhibit Mtb infection-induced macrophage pyroptosis and has the potential to be a new adjunctive HDT drug. IMPORTANCE Current strategies for treating drug-resistant tuberculosis have limited efficacy and undesirable side effects; hence, research on new treatments, including innovative medications, is required. Host-directed therapy (HDT) has emerged as a viable strategy for modulating host cell responses in order to enhance protective immunity against infections. Baicalein, extracted from Scutellariae radix, was shown to inhibit pyroptosis caused by Mycobacterium tuberculosis-infected macrophages and was associated with autophagy. Our findings reveal that baicalein can be used as an adjunctive treatment for tuberculosis or other inflammatory diseases by regulating immune function and enhancing the antibacterial ability of the host. It also provides a new idea for exploring the anti-inflammatory mechanism of baicalein.

Abstract 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, bla TEM, bla SHV and tetA. The differences in the prevalence of the bla types implied that the genetic basis for β-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 β-lactamase genes bla CTX-M-3 and bla TEM-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.

Abstract Mode-localized sensors have attracted attention because of their high parametric sensitivity and first-order common-mode rejection to temperature drift. The high-fidelity detection of resonator amplitude is critical to determining the resolution of mode-localized sensors where the measured amplitude ratio in a system of coupled resonators represents the output metric. Operation at specific bifurcation points in a nonlinear regime can potentially improve the amplitude bias stability; however, the amplitude ratio scale factor to the input measurand in a nonlinear regime has not been fully investigated. This paper theoretically and experimentally elucidates the operation of mode-localized sensors with respect to stiffness perturbations (or an external acceleration field) in a nonlinear Duffing regime. The operation of a mode-localized accelerometer is optimized with the benefit of the insights gained from theoretical analysis with operation in the nonlinear regime close to the top critical bifurcation point. The phase portraits of the amplitudes of the two resonators under different drive forces are recorded to support the experimentally observed improvements for velocity random walk. Employing temperature control to suppress the phase and amplitude variations induced by the temperature drift, 1/f noise at the operation frequency is significantly reduced. A prototype accelerometer device demonstrates a noise floor of 95 ng/√Hz and a bias instability of 75 ng, establishing a new benchmark for accelerometers employing vibration mode localization as a sensing paradigm. A mode-localized accelerometer is first employed to record microseismic noise in a university laboratory environment.

Abstract This paper describes a novel electrostatically actuated microgripper with freeform geometries designed by a genetic algorithm. This new semiautomated design methodology is capable of designing near-optimal MEMS devices that are robust to fabrication tolerances. The use of freeform geometries designed by a genetic algorithm significantly improves the performance of the microgripper. An experiment shows that the designed microgripper has a large displacement (91.5 μm) with a low actuation voltage (47.5 V), which agrees well with the theory. The microgripper has a large actuation displacement and can handle micro-objects with a size from 10 to 100 μm. A grasping experiment on human hair with a diameter of 77 μm was performed to prove the functionality of the gripper. The result confirmed the superior performance of the new design methodology enabling freeform geometries. This design method can also be extended to the design of many other MEMS devices.

Abstract Here, we present new evidence that evolutionary adaptation of the Ailuripodinae lineage to bamboo diet has taken place by morphological adaptations in the masticatory system. The giant panda in the wild and in captivity removes without an exception the outer skin of all bamboo shoots, rich in abrasive and toxic compounds, by the highly adapted premolars P3 and P4. The temporomandibular joint (TMJ) allows sidewise movement of the jaw and the premolars can, in a cusp-to-cusp position, remove the poorly digestible outer skin of the bamboo before crushing the bamboo with molars. Based on the evidence presented here, we suggest that adaptation of TMJ to lateral movement for enabling cusp-to-cusp contact of premolars is the crucial evolutionary factor as which we consider the key to understand the Ailuropodinae lineage adaptive pathway to utilize the bamboo resource.

Abstract Background The gut microbiome is essential for the host’s health and serves as an essential reservoir of antibiotic resistance genes (ARGs). We investigated the effects of different factors, including the dietary shifts and age, on the functional characteristics of the giant panda’s gut microbiome (GPs) through shotgun metagenome sequencing. We explored the association between gut bacterial genera and ARGs within the gut based on network analysis. Results Fecal samples (n=60) from captive juvenile, adult, and geriatric GPs were processed, and variations were identified in the gut microbiome according to different ages, the abundance of novel ARGs and the biosynthesis of antibiotics. Among 667 ARGs identified, nine from the top ten ARGs had a higher abundance in juveniles. For 102 ARGs against bacteria, a co-occurrence pattern revealed a positive association for predominant ARGs with Streptococcus. A comparative KEGG pathways analysis revealed an abundant biosynthesis of antibiotics among three different groups of GPs, where it was more significantly observed in the juvenile group. A co-occurrence pattern further revealed a positive association for the top ten ARGs, biosynthesis of antibiotics, and metabolic pathways. Conclusion Gut of GPs serve as a reservoir for novel ARGs and biosynthesis of antibiotics. Dietary changes and age may influence the gut microbiome’s functional characteristics; however, it needs further studies to ascertain the study outcomes.

Solar-to-fuel conversion represents a renewable means to harvest sunlight, but the most efficient materials are often expensive or rare. Here, authors demonstrate gradient tantalum-doped hematite homojunctions as a method to improve photoelectrochemical water splitting performances.

Reintroducing captive giant pandas (Ailuropoda melanoleuca) to the wild is the ultimate goal of their ex situ conservation. Choosing higher fitness candidates to train prior to release is the first step in the giant panda reintroduction program. Disease resistance is one important index of individual fitness and presumed to be related to variation at major histocompatibility complex genes (MHC). Here, we used seven polymorphic functional MHC genes (Aime-C, Aime-I, Aime-L, Aime-DQA1, Aime-DQA2, Aime-DQB1 and Aime-DRB3) and estimate their relationship with Baylisascaris schroederi (Ascarididae) infection in giant panda. We found that DQA1 heterozygous pandas were less frequently infected than homozygotes. The presence of one MHC genotype and one MHC allele were also associated with B. schroederi infection: Aime-C*0203 and Aime-L*08 were both associated with B. schroederi resistance. Our results indicate that both heterozygosity and certain MHC variants are important for panda disease resistance, and should therefore be considered in future reintroduction programs for this species alongside conventional selection criteria (such as physical condition and pedigree-based information).

Conditioning efficiently high-voltage triboelectric nanogenerators for low-voltage applications remains a challenge. Here, the authors demonstrate two orders of magnitude improvement of the energy harvesting efficiency by applying a conditioning circuit with self-sustained and automatic hysteresis MEMS micro-plasma switches.

Abstract Reintroduction is a key approach in the conservation of endangered species. In recent decades, many reintroduction projects have been conducted for conservation purposes, but the rate of success has been low. Given the important role of gut microbiota in health and diseases, we questioned whether gut microbiota would play a crucial role in giant panda's wild‐training process. The wild procedure is when captive‐born babies live with their mothers in a wilderness enclosure and learn wilderness survival skills from their mothers. During the wild‐training process, the baby pandas undergo wilderness survival tests and regular physical examinations. Based on their performance through these tests, the top subjects (age 2–3 years old) are released into the wild while the others are translocated to captivity. After release, we tracked one released panda (Zhangxiang) and collected its fecal samples for 5 months (January 16, 2013 to March 29 2014). Here, we analyzed the Illumina HiSeq sequencing data (V4 region of 16S rRNA gene) from captive pandas (n = 24), wild‐training baby pandas (n = 8) of which 6 were released and 2 were unreleased, wild‐training mother pandas (n = 8), one released panda (Zhangxiang), and wild giant pandas (n = 18). Our results showed that the gut microbiota of wild‐training pandas is significantly different from that of wild pandas but similar to that of captive ones. The gut microbiota of the released panda Zhangxiang gradually changed to become similar to those of wild pandas after release. In addition, we identified several bacteria that were enriched in the released baby pandas before release, compared with the unreleased baby pandas. These bacteria include several known gut‐health related beneficial taxa such as Roseburia, Coprococcus, Sutterella, Dorea, and Ruminococcus. Therefore, our results suggest that certain members of the gut microbiota may be important in panda reintroduction.

Haemaphysalis longicornis (Ixodida: Ixodidae) is a common blood-feeding ectoparasite of the giant panda and poses significant health burden to wild and captive populations. In the present study, the complete mitogenome of the giant panda tick H. longicornis was sequenced using Illumina sequencing technology. The entire mitogenome was 14,706 bp in length and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. Phylogeny showed that four isolates of H. longicornis, regardless of host origins or locations, clustered together and had a closer relationship with Haemaphysalis hystricis than other Haemaphysalis species among the subfamily Haemaphysalinae of Ixodidae. The cumulative mitochondrial DNA resources provide insights into genetic and phylogenetic studies of Haemaphysalis ticks.

The blood-sucking tick Haemaphysalis hystricis is a common ectoparasite of the giant panda and represents a significant threat to both wild and captive populations. Herein, the complete mitogenome of H. hystricis was sequenced using Illumina sequencing technology. The complete mitogenome sequence was 14,715 bp in size and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogeny revealed that two isolates of H. hystricis, regardless of host origins and locations, grouped together and had a closer relationship with Haemaphysalis longicornis than other tick species among the genus Haemaphysalis. The cumulative mitochondrial DNA data provides novel resources for genetic and phylogenetic studies of Haemaphysalis ticks.

The tick Haemaphysalis flava (Acari, Ixodidae) is an obligatory blood-feeding ectoparasite of the giant panda and is also a vector for transmission of pathogenic microorganisms. In this study, the complete mitochondrial genome of this tick was sequenced through Illumina sequencing technology. The genome was 14,699 bp in length and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. Phylogeny revealed that three isolates of H. flava, regardless of host origins and locations, clustered together and formed a monophyletic relationship with Haemaphysalis japonica, supporting their species validity among the genus Haemaphysalis. These cumulative mitochondrial DNA data provides insights into phylogenetic studies among Haemaphysalis ticks.

As an assisted breeding technique, artificial insemination has become the main effective practical approach in the captive breeding programs of giant panda worldwide. The composition of seminal plasma plays an important role in the success of breeding. The present work is the first attempt to characterize, by proton magnetic resonance spectroscopy (1H-NMR), the metabolome of healthy giant panda seminal plasma. A total of 35 molecules were quantified, with the concentration of 2,3-butanediol being significantly different between individuals younger than 8 years and older than 13 years, and other distinct age-related trends were highlighted by a multivariate analysis. Isopropanol’s concentration was significantly linked to estrus stages. Besides, the variations in the metabolome’s profile during storage were also evaluated. This study may serve as a reference for further research wishing to shed light on the biological mechanisms affecting giant panda sperm’s overall quality and may ultimately lead to novel approaches to giant panda artificial insemination.

Giant pandas feed almost exclusively on bamboo but miss lignocellulose-degrading genes. Their gut microbiome may contribute to their nutrition; however, the limited access to pandas makes experimentation difficult. In vitro incubation of dung samples is used to infer gut microbiome activity. In pandas, such tests indicated that green leaves are largely fermented to ethanol at neutral pH and yellow pith to lactate at acidic pH. Pandas may feed on either green leaves or yellow pith within the same day, and it is unclear how pH, dung sample, fermentation products and supplied bamboo relate to one another. Additionally, the gut microbiome contribution to solid bamboo digestion must be appropriately assessed. Here, gut microbiomes derived from dung samples with mixed colors were used to ferment green leaves, also by artificially adjusting the initial pH. Gut microbiomes digestion of solid lignocellulose accounted for 30–40% of the detected final fermentation products. At pH 6.5, mixed-color dung samples had the same fermentation profile as green dung samples (mainly alcohols), while adjusting the initial pH to 4.5 resulted in the profile of yellow dung samples (mainly lactate). Metaproteomics confirmed that gut microbiomes attacked hemicellulose, and that the panda’s alpha amylase was the predominant enzyme (up to 75%).

With the introduction of the working principle of coupled resonators, the coupled bulk acoustic wave (BAW) Micro-Electro-Mechanical System (MEMS) resonators have been attracting much attention. In this paper, coupled BAW MEMS resonators are discussed, including the coupling theory, the actuation and sensing theory, the transduction mechanism, and the applications. BAW MEMS resonators normally exhibit two types of vibration modes: lateral (in-plane) modes and flexural (out-of-plane) modes. Compared to flexural modes, lateral modes exhibit a higher stiffness with a higher operating frequency, resulting in a lower internal loss. Also, the lateral mode has a higher Q factor, as the fluid damping imposes less influence on the in-plane motion. The coupled BAW MEMS resonators in these two vibration modes are investigated in this work and their applications for sensing, timing, and frequency reference are also presented.

Environment, diet, and host phylogeny are the main factors that shape the gut microbiota of humans and animals. Gut microbial communities of captive animals are often drastically altered by disease or anthropogenic activities. These changes in the microbial community profoundly influence animal fitness. Fecal microbiota transplantation (FMT) offers enormous potential to mend a disturbed gut microbiota. In addition, differences generally exist between the gut microbiotas of wild and captive animals. FMT can be used to improve the gut bacterial community structure of candidate endangered animals before releasing them into the wild. However, there are few reviews on the application of FMT in wildlife conservation. This review discusses the application of FMT for the conservation of endangered wildlife. First, the necessity for the application of FMT to conservation biology is introduced; second, the potential benefits of FMT techniques for endangered species conservation are discussed; third, successful cases where FMT was applied for the protection of endangered animals are listed; finally, future research directions and concerns with regard to FMT for the conservation of endangered wildlife are discussed. Although the attempts to perform FMT for conservation biology still remain preliminary, FMT has already indicated an enormous potential for the improvement of both the management and conservation of wildlife. We hope this review can promote future studies focused on the application of FMT for the preservation of vulnerable species.

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.

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 Background Ovarian cancer is diagnosed clinically by detecting ovarian cancer-related factors and markers. Here, we report a case of giant panda ovarian tumor metastasis with a combination of clinical and histopathological diagnosis. Case presentation Histopathological studies revealed severe lesions and tumor cells in the ovaries, lungs, spleen, kidneys and perianal tissue. Immunohistochemistry staining showed that the ovarian cancer markers B7-H4, CA125, and HE4 were highly expressed in the lungs, kidneys, spleen, ovaries and perianal tissue. Tumor marker tests detected significantly high levels of AFP in serum. Conclusion Clinical biomarkers combined with histopathology can provide a more accurate diagnosis of ovarian cancer metastasis and identification of ovarian cancer types than either method alone. The giant panda’s death may be due to granulosa cell tumor and theca cell tumor metastasis causing multiple organ dysfunction or even failure.

Abstract The physiological role of miRNAs is widely understood to include fine-tuning the post-transcriptional regulation of a wide array of biological processes. Extensive studies have indicated that exosomal miRNAs in the bodily fluids of various organisms can be transferred between living cells for the delivery of gene silencing signals. Here, we illustrated the expression characteristics of exosomal miRNAs in giant panda breast milk during distinct lactation periods and highlighted the enrichment of immune- and development-related endogenous miRNAs in colostral and mature giant panda milk. These miRNAs are stable, even under certain harsh conditions, via the protection of extracellular vesicles. These findings indicate that breast milk may facilitate the dietary intake of maternal miRNAs by infants for the regulation of postnatal development. We also detected exogenous plant miRNAs from the primary food source of the giant panda (bamboo) in the exosomes of giant panda breast milk that were associated with regulatory roles in basic metabolism and neuron development. This result suggested that dietary plant miRNAs are absorbed by host cells and subsequently secreted into bodily fluids as potential cross-kingdom regulators. In conclusion, exosomal miRNAs in giant panda breast milk may be crucial maternal regulators for the development of intrinsic ‘slink’ newborn cubs.

Documenting the different social and behavioural contexts that vocalisations are produced in remains an important step towards understanding the functional relevance of specific call types in a given species' vocal repertoire. In this study we investigated whether seasonal differences and the presence or absence of male and female conspecifics influence the production of male giant panda vocal signals. To this end, captive male giant pandas were observed during and outside of the breeding season in three social contexts: only male conspecific neighbours, only female conspecific neighbours, and a context with no neighbours. We found that males were more likely to bleat, chirp, honk and moan during the breeding season, and showed a tendency to growl more outside of the reproductive period. The contextual analysis revealed that bleats were more likely to be produced by males when opposite-sexed conspecifics are in close attendance during the breeding season. Conversely, males were more likely to chirp when neighboured by males than females or no neighbours. In addition, males were more likely to honk in the absence of neighbouring conspecifics during the breeding season, raising the possibility that these calls function to signal location and gain the attention of potential mates. Moans were produced more often when male giant pandas had male than female neighbours during the breeding season, which may reflect mild aggression towards these same-sexed rivals, whereas the production of barks and growls did not vary according to season or the sex of conspecific neighbours. Our findings underscore the importance of male giant panda bleats for coordinating reproduction and promoting contact with potential mating partners in this non-gregarious species, and yield fresh insights into the function of male honks that warrant further investigation. They also provide a basis for comparison with free-ranging giant panda vocal behaviour that could potentially inform conservation efforts.

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, β-glucosidase, and cellulose β-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.

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.

The giant pandas' (Ailuropoda melanoleuca) reproductive strategy is unique among mammals. Yet there are characteristics of giant panda behaviour we do not fully understand. Probably one of the least understood is the assumption that in captivity virtually all giant panda females rear only one cub when twins are born and abandon the other if given the chance. So far, only two females have raised twins simultaneously, but just with intensive human assistance. This case-study marks the first successful rearing of giant panda twins in captivity entirely by the mother. Using video data for detailed behavioural observations, we provide the first behavioural assessment of a giant panda female raising two cubs simultaneously without direct human assistance or disturbance. We compared the maternal behaviour during the denning period of twin cubs raised in 2016 with two singleton cubs born 2007 and 2010. YANG YANG, the dam, rested less and interacted more with the twins than with the singletons in the first month postpartum and invested a greater part of her daily time budget on rearing the twins. We discuss potential favourable factors for the autonomous twin-rearing of a female giant panda, which could serve as a model for similar efforts elsewhere.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Surprisingly little is known about how mammal vocal signals are used to achieve behavioural synchrony in the lead up to copulation. The ability to signal short-term fluctuations in arousal levels and behavioural intention is likely to be particularly important for synchronizing mating behaviour in asocial species, which must overcome their natural avoidance and aggressive tendencies to mate. Here, we examined vocal behaviour during breeding encounters in captive giant pandas (Ailuropoda melanoleuca) to gain a greater understanding of how close-range vocal signalling mediates reproduction in this asocial, and conservation-dependent species. Our results revealed that the occurrence of different giant panda vocalizations and acoustic variation within these calls is predictive of successful encounters leading to copulation, as opposed to unsuccessful encounters that do not. In addition, key differences were detected between vocalizations produced during and just prior to copulation. These findings illustrate that vocal exchanges are crucial for achieving behavioural synchrony and signalling intention to mate in giant pandas, and could also provide a valuable tool for breeding programmes, helping conservation managers to assess the likelihood of breeding introductions leading to copulation or potentially injurious failure.

To explore the effects of the mutations of ATP6 and ATP8 genes on energy metabolism and genetic structure, we sequenced the ATP6 and ATP8 genes of Ailuropoda melanoleuca. Our results showed that ATP8 is a conserved gene and ATP6 gene is positively selected during the evolution of the giant panda population with a low genetic diversity. Population expansion was observed in the giant panda group. The T179C mutation on Haplotype7 made the production of a potential phosphorylation site. This non-synonymous mutation may occur at the post-translational modification site that have a potential effect on the function of ATP synthase, related to the maintenance of body temperature of pandas at low metabolic rates.

Animals make choices about where to spend their time in complex and dynamic landscapes, choices that reveal information about their biology that in turn can be used to guide their conservation. Using GPS collars, we conducted a novel individual-based analysis of habitat use and selection by the elusive and endangered giant pandas (Ailuropoda melanoleuca). We constructed spatial autoregressive resource utilization functions (RUF) to model the relationship between the pandas' utilization distributions and various habitat characteristics over a continuous space across seasons. Results reveal several new insights, including use of a broader range of habitat characteristics than previously understood for the species, particularly steep slopes and non-forest areas. We also used compositional analysis to analyze habitat selection (use with respect to availability of habitat types) at two selection levels. Pandas selected against low terrain position and against the highest clumped forest at the at-home range level, but no significant factors were identified at the within-home range level. Our results have implications for modeling and managing the habitat of this endangered species by illustrating how individual pandas relate to habitat and make choices that differ from assumptions made in broad scale models. Our study also highlights the value of using a spatial autoregressive RUF approach on animal species for which a complete picture of individual-level habitat use and selection across space is otherwise lacking.

In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the panda's vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous study, we determined that several strains of B. subtilis isolated from pandas exhibited good cellulose decomposition ability, and we hypothesized that this bacterial species can survive in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed RNA-Seq technology to analyze the differentially expressed genes of the selected strain B. subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon sources (cellulose and glucose). In addition, we used bioinformatics software and resources to analyze the functions and pathways of differentially expressed genes. Interestingly, comparison of the cellulose and glucose groups revealed that the up-regulated genes were involved in amino acid and lipid metabolism or transmembrane transport, both of which are involved in cellulose utilization. Conversely, the down-regulated genes were involved in non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 triggered a series of adaptive mechanisms at the transcriptional level, which suggests that this bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellulose is not a very suitable carbon source for this bacterial species. In this study, we present a model to understand the dynamic organization of and interactions between various functional and regulatory networks for unicellular organisms in a high-fiber environment.

Increasingly, the world is becoming socioeconomically and environmentally connected, but many studies have focused on human-environment interactions within a particular area. Although some studies have considered the impacts of external factors, there is little research on multiple reciprocal socioeconomic and environmental interactions between a focal area and other areas. Here we address this important knowledge gap by applying the new integrated framework of telecouplings (socioeconomic and environmental interactions between two or more areas over distances). Results show that even a protected area - i.e., the Wolong Nature Reserve for giant pandas in southwest China - has multiple telecoupling processes with the rest of the world; these include panda loans, tourism, information dissemination, conservation subsidies, and trade of agricultural and industrial products. The telecoupling processes exhibit nonlinear patterns, they change over time, and they have varying socioeconomic and environmental effects across the world. We also find complex relationships among different telecouplings - e.g., amplification, offsetting, spatial overlaps - which cannot be detected by traditional separate studies. Such an integrated study leads to a more comprehensive understanding of distant human-environment interactions and has significant implications for global sustainability and human well-being.