Using non-destructive sampling to evaluate the population genomic status of captive Brown Eared Pheasants

Evaluating the genetic status of threatened species is an essential task in conservation genetics.However,the genetic status of threatened species has been mostly evaluated through techniques that fail to estimate genetic diversity at the whole genomic level.Next generation sequencing can meet this demand,but high quality samples such as blood or muscle tissues are required.However,it is difficult to collect such samples from threatened species because sampling work may impact their health.Therefore,it is essential to design a workflow to evaluate the whole genomic status of threatened species using non-destructive sampling.Even though non-destructive sampling has been used in traditional barcoding technique,the barcoding technique cannot evaluate the whole genomic status.Brown Eared Pheasant(Crossoptilon mantchuricum)is an endangered species,with captive populations maintained in Taiyuan Zoo,China,and Europe.However,the genetic diversity,inbreeding pattern,and mutation load of these two populations are unclear.To uncover the genetic status of these two captive populations,we applied 2b-RAD technology to evaluate the genomic status of these populations using feathers as samples.The feathers could be collected by non-destructive sampling.The results indicate that the Taiyuan Zoo population has a lower genetic diversity and higher inbreeding coefficient than the European population.The Taiyuan Zoo population has lethal mutations when homozygous.The current project uses a non-destructive sampling technique to evaluate the whole genomic status of the two captive populations,providing a paradigm for conservation genetics,which will facilitate the development of conservation biology.

A rare case of giant panda cancer:Pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant cancer of the digestive system common among humans.However,it is rarely reported in wild animals.In 2018,a giant panda died in the Beijing Zoo.During subsequent histological observation of the pancreas,it was discovered that the glandular epithelial cells had lost the pancreatic acinar structure,tubular areas with obvious structure in the pancreas,and the ductal epithelium was substituted by high columnar mucus cells.Masson staining showed that there were several fibrous tissue proliferative reactions around the ductal adenocarcinoma and immunohistochemical staining revealed that CK7 and CK19 were positively expressed in the pancreatic tissue.Therefore,the pathological diagnosis indicated that the panda had PDAC.In this paper,the panda’s living conditions and pathological diagnosis results are examined,with the aim of providing a reference point for the future diagnosis of wild animal tumors.

Friend or foe? Using eye-tracking technology to investigate the visual discrimination ability of giant pandas

The role that visual discriminative ability plays among giant pandas in social communication and individual discrimination has received less attention than olfactory and auditory modalities.Here,we used an eye-tracker technology to investigate pupil fixation patterns for 8 captive male giant pandas Ailuropoda melanoleuca.We paired images(N=26)of conspecifics against:1)sympatric predators(gray wolves and tigers),and non-threatening sympatric species(golden pheasant,golden snub-nosed monkey,takin,and red panda),2)conspecifics with atypical fur colora-tion(albino and brown),and 3)zookeepers/non-zookeepers wearing either work uniform or plain clothing.For each session,we tracked the pan-da's pupil movements and measured pupil first fixation point(FFP),fixation latency,total fixation count(TFC),and duration(TFD)of attention to each image.Overall,pandas exhibited similar attention(FFPs and TFCs)to images of predators and non-threatening sympatric species.Images of golden pheasant,snub-nosed monkey,and tiger received less attention(TFD)than images of conspecifics,whereas images of takin and red panda received more attention,suggesting a greater alertness to habitat or food competitors than to potential predators.Pandas'TFCs were greater for images of black-white conspecifics than for albino or brown phenotypes,implying that familiar color elicited more interest.Pandas reacted differently to images of men versus women.For images of women only,pandas gave more attention(TFC)to familiar combinations(uniformed zookeepers and plain-clothed non-zookeepers),consistent with the familiarity hypothesis.That pandas can use visual perception to discriminate intra-specifically and inter-specifically,including details of human appearance,has applications for panda conservation and captive husbandry.

Multi-omic Analyses Shed Light on The Genetic Control of High-altitude Adaptation in Sheep

Sheep were domesticated in the Fertile Crescent and then spread globally,where they have been encountering various environmental conditions.The Tibetan sheep has adapted to high altitudes on the Qinghai-Tibet Plateau over the past 3000 years.To explore genomic variants associated with high-altitude adaptation in Tibetan sheep,we analyzed Illumina short-reads of 994 whole genomes representing∼60 sheep breeds/populations at varied altitudes,PacBio High fidelity(HiFi)reads of 13 breeds,and 96 transcriptomes from 12 sheep organs.Association testing between the inhabited altitudes and 34,298,967 variants was conducted to investigate the genetic architecture of altitude adaptation.Highly accurate HiFi reads were used to complement the current ovine reference assembly at the most significantly associatedβ-globin locus and to validate the presence of two haplotypes A and B among 13 sheep breeds.The haplotype A carried two homologous gene clusters:(1)HBE1,HBE2,HBB-like,and HBBC,and(2)HBE1-like,HBE2-like,HBB-like,and HBB;while the haplotype B lacked the first cluster.The high-altitude sheep showed highly frequent or nearly fixed haplotype A,while the low-altitude sheep dominated by haplotype B.We further demonstrated that sheep with haplotype A had an increased hemoglobin–O_(2) affinity compared with those carrying haplotype B.Another highly associated genomic region contained the EGLN1 gene which showed varied expression between high-altitude and low-altitude sheep.Our results provide evidence that the rapid adaptive evolution of advantageous alleles play an important role in facilitating the environmental adaptation of Tibetan sheep.

Sequence variation and genetic diversity in the giant panda

About 336-444 bp mitochondrial D-loop region and tRNA gene were sequenced for 40 individuals of the giant panda which were collected from Mabian, Meigu, Yuexi, Baoxing, Pingwu, Qingchuan, Nanping and Baishuijiang, respectively. 9 haplotypes were found in 21 founders. The results showed that the giant panda has low genetic variations, and that there is no notable genetic isolation among geographical populations. The ancestor of the living giant panda population perhaps appeared in the late Pleistocene, and unfortunately, might have suffered bottle-neck attacks. Afterwards, its genetic diversity seemed to recover to some extent.

Climate warming shifts the time interval between flowering and leaf unfolding depending on the warming period

The timing of flowering(FL)and leaf unfolding(LU)determine plants’reproduction and vegetative growth.Global warming has substantially advanced FL and LU of temperate and boreal plants,but their responses to warming differ,which may influence the time interval between FL and LU(ΔLU-FL),thereby impacting plant fitness and intraspecific physiological processes.Based on twigs collected from two flowering-first tree species,Populus tomentosa and Amygdalus triloba,we conducted a manipulative experiment to investigate the effects of winter chilling,spring warming and photoperiod on theΔLU-FL.We found that photoperiod did not affect theΔLU-FL of Amygdalus triloba,but shortenedΔLU-FL by 5.1 d of Populus tomentosa.Interestingly,spring warming and winter chilling oppositely affected theΔLU-FL of both species.Specifically,low chilling accumulation extended theΔLU-FL by 3.8 and 9.4 d for Populus tomentosa and Amygdalus triloba,but spring warming shortened theΔLU-FL by 4.1 and 0.2 d℃^(-1).Our results indicate that climate warming will decrease or increase theΔLU-FL depending on the warming periods,i.e.,spring or winter.The shifted time interval between flowering and leaf unfolding may have ecological effects including affecting pollen transfer efficiency and alter the structure and functioning of terrestrial ecosystem.

Characterization of intestinal microbiota and fecal cortisol,T3,and IgA in forest musk deer(Moschus berezovskii)from birth to weaning

Analysis of the intestinal microbiota and physiological parameters in mammalian infancy can reveal health status.In this study,we used a combination of molecular and immunochemical approaches to assess fecal microbiota as well as Cortisol(Cor),Triiodothyronine(T3),and immunoglobulin A(IgA)levels of young forest musk deer(FMD),from birth to one month after weaning(7 days of age–110 days of age).During development as the diet of FMD changes from consuming milk to eating plants,the richness and diversity of intestinal microbiota of young FMD increased significantly.Cor levels remained unchanged throughout early development while significantly increased after weaning,T3 and IgA initially were derived from milk during lactation,significantly decreased after weaning.Correlation network analysis showed that the community of food-oriented microbes were highly structured and that many genera were correlated.Overall,this study provides scientific insights into effective management strategies for the protection of FMD population.

Effects of pentachlorophenol on the detoxification system in white-rumped munia(Lonchura striata)

Pentachlorophenol（PCP）, a priority pollutant due to its persistence and high toxicity, has been used worldwide as a pesticide and biocide. To understand the adverse effects of PCP, adult male white-rumped munias（Lonchura striata） were orally administrated commercial PCP mixed with corn oil at dosages of 0, 0.05, 0.5, and 5 mg/（kg·day） for 42 day. Gas chromatography–mass spectrometry（GC–MS） analysis found that PCP was preferentially accumulated in the kidney rather than in the liver and muscle in all exposure groups. To examine the function of CYP1A in pollutant metabolism, we isolated two full-length cDNA fragments（designated as CYP1A4 and CYP1A5） from L. striata liver using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. PCP induced the expression of CYP1A5, although no obvious change was observed in CYP1A4 expression. Furthermore, PCP significantly elevated the activities of ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase and decreased the activity of benzyloxy-trifluoromethyl-coumarin, with no significant responses observed in benzyloxyresorufin O-debenzylase. PCP induced significant changes in antioxidant enzyme（superoxide dismutase and catalase） activities and malondialdehyde content, but decreased glutathione peroxidase（GSH-Px） and glutathione S-transferase activities and GSH content in the liver of L. striata. The present study demonstrated that PCP had hepatic toxic effects by affecting CYP1As and anti-oxidative status.

Multidrug-resistant Proteus mirabilis isolates carrying blaOXA-1 and blaNDM-1 from wildlife in China:increasing public health risk

The emergence of multidrug resistance(MDR)in Proteus mirabilis clinical isolates is a growing public health concern and has serious implications for wildlife.What is the role of wildlife has been become one of the hot issues in disseminating antimicrobial resistance.Here,54 P.mirabilis isolates from 12 different species were identified.Among them,25 isolates were determined to be MDR by profile of antimicrobial susceptibility;10 MDR P.mirabilis isolates were subjected to comparative genomic analysis by whole genome sequencing.Comprehensive analysis showed that chromosome of P.mirabilis isolates mainly carries multidrug-resistance complex elements harboring resistance to carbapenem genes blaOXA-1,blaNDM-1,and blaTEM-1.Class I integron is the insertion hotspot of IS26;it can be inserted into type I integron at different sites,thus forming a variety of multiple drug resistance decision sites.At the same time,Tn21,Tn7,and SXT/R391 mobile elements cause widespread spread of these drug resistance genes.In conclusion,P.mirabilis isolates from wildlife showed higher resistance to commonly used clinic drugs comparing to those from human.Therefore,wild animals carrying MDR clinical isolates should be paid attention to by the public health.

Hand-Rearing of Newborn Giant Panda

The mortality of captive newborn giant pandas (Ailuropoda melanoleuca) is ashigh as 71.3%, but it can be greatly reduced by hand-rearing of newborn cubs, whosemother animals are unable to feed them. This is of tremendous significance to thepopulation breeding and the species conservation.Over the past ten years, many newborn cubs that did not receive any milk fromthe mother animals were hand-reared both at home and abroad, but they all diedin the early stage due to medical treatment failure.

MOLECULAR CLONING OF HIGHLY REPETITIVE DNA SEQUENCES FROM GIANT PANDA, Ailuropoda melanoleuca

The taxonomy and phylogeny of one of the endangered species found only in China, the giant panda, have been disputed for 120 years. It has been questioned whether it should be classified as the bear family(Ursidae), racoon family(Procyonidae)or giant panda family(Ailuropodidae). Recently, based on the modern biological analyses of(1)single-

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD)analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA(RAPD)was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifications showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father(No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.

Analysis of nutrient components of food for Asian Elephants in the wild and in captivity

Thirty-seven wild plants as food for Asian elephants in the field in Simao,Yunnan province,China and five cultivated plants as food for captive elephants in the Beijing Zoo were collected and analyzed for their main nutrient components.Protein,fat,fiber,dry material,ash as well as major microelements:calcium,kalium,zincum,sodium in the food were analyzed by standard methodology.No significant differences were found between the wild plants taken in the field and forage provided in captivity.However,the calcium content in the forage is significantly less than the average of those in the wild plants.It is suggested that the increase in calcium intake may contribute to the relief of low plasma calcium diseases of elephants in captivity.

Sexual interference and allomaternal behavior as predictors of rank recognition in female golden snub-nosed monkeys

Rank recognition allows social animals to adapt to complex and changeable environments and to cope with hierarchical relationships within their societies(Crone 2017).Rank recognition can improve the distribution of advantageous resources,individual adaptation,and social cohesion among group-living animals(Marmolejo-Ramos and Angiulli 2014).Empirical evidence suggests that rank recognition is a basic behavioral manifestation of social cognition adopted by a wide range of insects,birds,and mammals,including nonhuman primates(Schmitt and Fischer 2011;Smith et al.2017).Unlike most other vertebrates,primates have unusually large brains and form complex social groups.

Characteristics of the multiple replicon plasmid IncX1-X1 in multidrug-resistant Escherichia coli from Malayan pangolin(Manis javanica)

Potential zoonotic pathogens may be transmitted from wildlife to humans through the illegal wild meat trade,which has become a pressing issue.However,research on the antimicrobial resistance genes(ARGs)of Malayan pangolin(Manis javanica)intestinal bacteria is limited.Here,multidrug-resistant Escherichia coli M172-1(ST354)isolated from Malayan pangolin feces in 2019 was found to be resistant to 13 antibiotics.BGWAS analysis revealed 4 plas-mids,namely,pM172-1.1,pM172-1.2,pM172-1.3,and pM172-1.4,in the isolate.The pM172-1.2,pM172-1.3,and pM172-1.4 plasmids carried ARGs,namely,IncHI2-HI2A,IncX1-X1,and IncX1,respectively.pM172-1.3 and pM172-1.4 contained intact IntI1 integrons(Is26/IntI1/arr2/cmlA5/blaOXA-10/ant(3′′)-IIA/dfrA14/Is26).No-tably,pM172-1.3 resulted from the fusion of 2 pM172-1.4 copies and carried many more ARGs.In addition to pM172-1.3 from the same host,other drug-resistant bacteria(E.coli M159-1(ST48),E.coli S171-1(ST206),and Klebsiella pneumoniae S174-1(ST2354))in the same Malayan pangolin fecal samples also carried 3 plasmids with 100%gene coverage of pM172-1.4 and 99.98%identity.Therefore,ARGs in IncX1 might spread in the intestinalflora of Malayan pangolin and between species via the illegal food chain,posing a potential threat to public health and safety.