Zoological Research

477-494

Authors: ZengLi;LiWei;ChenCe-Shi;

Breast cancer is the most common malignancy in women. Basic and translational breast cancer research relies heavily on experimental animal models. Ideally, such models for breast cancer should have commonality with human breast cancer in terms of tumor etiology, biological behavior, pathology, and response to therapeutics. This review introduces current progress in different breast cancer experimental animal models and analyzes their characteristics, advantages, disadvantages, and potential applications. Finally, we propose future research directions for breast cancer animal models.

503-516

Authors: SongTian-Zhang;ZhengHong-Yi;HanJian-Bao;JinLin;YangXiang;LiuFeng-Liang;LuoRong-Hua;TianRen-Rong;FengXiao-Li;LiuChao;LiMing-Hua;ZhengYong-Tang;

As of June 2020, Coronavirus Disease 2019 (COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years, making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To examine the effect of age on death, we established a SARS-CoV-2 infection model in Chinese rhesus macaques (Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b+ and CD8+ cells in lungs at one-week post infection (wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b+ cells, and persistent infiltration of CD8+ cells in the lungs at 2 wpi. In addition, peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

527-538

Authors: Rong-Nan Li;Zhen-Dong Zhu;Yi Zheng; Ying-Hua Lv;Xiu-E Tian;De Wu; Yong-Jun Wang;Wen-Xian Zeng;

Sperm are specialized cells that require adenosine triphosphate (ATP) to support their function. Maintaining sperm energy homeostasis in vitro is vitally important to improve the efficacy of boar sperm preservation. Metformin can activate 5′-AMP-activated protein kinase (AMPK) to improve metabolic flexibility and maintain energy homeostasis. Thus, the aim of the present study was to investigate whether metformin can improve boar sperm quality through AMPK mediation of energy metabolism. Sperm motility parameters, membrane integrity, acrosome integrity, mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake, and lactate efflux were analyzed. Localization and expression levels of AMPK and phospho-Thr172-AMPK (p-AMPK) were also detected by immunofluorescence and western blotting. We found that metformin treatment significantly increased sperm motility parameters, ΔΨm, and ATP content during storage at 17 °C. Moreover, results showed that AMPK was localized at the acrosomal region, connecting piece, and midpiece of sperm and p-AMPK was distributed at the post-acrosomal region, connecting piece, and midpiece. When sperm were incubated with metformin for 4 h at 37 °C, sperm motility parameters, ΔΨm, ATP content, p-AMPK, glucose uptake, and lactate efflux all significantly increased, whereas the addition of Compound C treatment, an inhibitor of AMPK, counteracted these positive effects. Together, our results suggest that metformin promotes AMPK activation, which contributes to the maintenance of energy hemostasis and mitochondrial activity, thereby maintaining boar sperm functionality and improving the efficacy of semen preservation.

552-556

Authors: Zia Ur Rahman;Yu-Chun Li;Jiao-Yang Tian;Qing-Peng Kong;

With a population of around 4 000 individuals, the Kalash people have been living in the Hindu-Kush mountain valleys of present-day northern Pakistan for centuries. Due to their mysterious origin and fairer European complexion, the genetic history of this ethnic group has been investigated previously using different markers. To date, however, the maternal genetic architecture has not been systematically dissected based on high-resolution complete mitochondrial genomes (mitogenomes), making their maternal genetic history, especially their genetic connection with Europeans from a matrilineal perspective, unclear. To unravel this issue, we analyzed mitogenome data of 34 Kalash samples together with 6 075 individuals from across Eurasia. Our results indicated exclusive western Eurasian origin of the Kalash people, represented by eight haplogroups. Among these haplogroups, J2b1a7a and R0a5a (accounting for ~50% of the Kalash gene pool) displayed in situ differentiations in the Kalash and could be traced to the Mediterranean region. Age estimations suggested these haplogroups arose in the Kalash population ~2.26 and 3.01 thousand years ago (kya), a time frame consistent with the invasion of Alexander III of Macedon to the region. One possible explanation for the maternal genetic contribution from Europeans to the Kalash people would be the involvement of women in foreign campaigns of ancient Greek warfare, followed by a founder effect. Our study thus sheds important light on the genetic origin of the Kalash community of Pakistan.

564-568

Authors: Qi Chen;Zhao-Xia Ma;Li-Bin Xia;Zhen-Ni Ye;Bao- Ling Liu;Tie-Kun Ma;Peng-Fei Bao; Xing-Fei Wu;Cong-Tao Yu;Dai-Ping Ma;Yuan-Yuan Han;Wen- Guang Wang;De-Xuan Kuang; Jie-Jie Dai; Rong- Ping Zhang;Min Hu;Hong Shi; Wen-Lin Wang;Yan-Jiao Li;

Osteonecrosis is a common human disease in orthopedics. It is difficult to treat, and half of patients may need artificial joint replacement, resulting in a considerable economic burden and a reduction in quality of life. Hormones are one of the major causes of osteonecrosis and high doses of corticosteroids are considered the most dangerous factor. Because of the complexity of treatment, we still need a better animal model that can be widely used in drug development and testing. Tree shrews are more closely related to primates than rodents. As such, we constructed a successful tree shrew model to establish and evaluate steroid-associated osteonecrosis (SAON). We found that low-dose lipopolysaccharide (LPS) combined with high-dose methylprednisolone (MPS) over 12 weeks could be used to establish a tree shrew model with femoral head necrosis. Serum biochemical and histological analyses showed that an ideal model was obtained. Thus, this work provides a useful animal model for the study of SAON and for the optimization of treatment methods.

576-580

Authors: Li-Na Du; Shuo Liu;Mian Hou;Guo-Hua Yu;

Theloderma pyaukkya is recorded for the first time in China based on a specimen collected from western Yunnan. Morphologically, the specimen shows good agreement with the original description of T. pyaukkya, and phylogenetically is clustered with the type specimens and holotype of T. pyaukkya from Kachin State (northern Myanmar) with strong support. The taxonomic status of T. pyaukkya from Chin State (western Myanmar) needs further examination. In addition, Theloderma moloch is also recorded in Yunnan for the first time. This brings the number of Theloderma species recorded in Yunnan, China, to seven, namely, T. albopunctatum, T. baibungense, T. bicolor, T. gordoni, T. moloch, T. pyaukkya, and T. rhododiscus.

589-594

Authors: Jian-Li Xiong;Wei Luo;Xiao-Mao Zeng;

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

715 - 720

Authors: Ying Geng; Jia-Fei He; Ying-Chun Li; Zhen-Hua Guan1; Xiao-Yang He; Jun Sun; Zhi-Pang Huang; Yan-Peng Li; Fan Yong; Wen Xiao; Liang-Wei Cui

Understanding dietary selection and feeding strategies is important for the conservation and management of endangered primate species. Here, we conducted a preliminary study on the diet and feeding behavior of endangered Shortridge’s langurs (Trachypithecus shortridgei) within the Drung River Valley (Dulongjiang) in southwestern China. The study site lies at a high latitude (N27°47.5′) and elevation (1 900 m a.s.l.) and is characterized by substantial annual rainfall (2 745.1 mm). From August 2012 to September 2013, we observed five groups of langurs and analyzed their overall food composition and dietary variation in spring and autumn. To understand their dietary adaptations to the distinctive habitat of the Drung River Valley, we also compared the diet of Shortridge’s langurs to that of other Trachypithecus species inhabiting different environments. Results indicated that T. shortridgei fed on 52 plant species, 23 of which each accounted for ≥1% of their annual feeding time. Their primary dietary components included leaves (46.2%, young, mature, and petioles), fruits (28.7%, unripe 17.6%, ripe 11.1%), and mosses (10.2%). The langurs mainly consumed mature (34.2%) and young leaves (27.5%) in spring and ripe fruits (39.4%) and mature leaves (24.7%) in autumn. Two species of moss (Macrothamnium macrocarpum and Scapania verrucosa, 21.2% of annual feeding time), which are usually found growing together on cliffs, played a relatively important role in the diet of T. shortridgei. The langurs mainly consumed ripe fruits of Saurauia napaulensis (7.1%) and Dendropanax burmanicus (7.1%), which were abundant at lower elevations. Trachypithecus species in temperate forests consumed more fruits and seeds but fewer leaves (similar mature leaves but fewer young leaves) than those species in tropical forests, which may be related to their availability and abundance. Compared to Trachypithecus species in temperate forests, the higher proportion of mosses and mature leaves but fewer young leaves in the annual diet of T. shortridgei are likely a response to the distinctive Drung River Valley habitat. Therefore, conservation of the main food plants of this threatened species could be vital for its survival and conservation management.

351-364

Authors: Sun Yan-Bo , Zhang Yi , Wang Kai

Understanding the genetic mechanisms underlying particular adaptations/phenotypes of organisms is one of the core issues of evolutionary biology. The use of genomic data has greatly advanced our understandings on this issue, as well as other aspects of evolutionary biology, including molecular adaptation, speciation, and even conservation of endangered species. Despite the well-recognized advantages, usages of genomic data are still limited to non-mammal vertebrate groups, partly due to the difficulties in assembling large or highly heterozygous genomes. Although this is particularly the case for amphibians, nonetheless, several comparative and population genomic analyses have shed lights into the speciation and adaptation processes of amphibians in a complex landscape, giving a promising hope for a wider application of genomics in the previously believed challenging groups of organisms. At the same time, these pioneer studies also allow us to realize numerous challenges in studying the molecular adaptations and/or phenotypic evolutionary mechanisms of amphibians. In this review, we first summarize the recent progresses in the study of adaptive evolution of amphibians based on genomic data, and then we give perspectives regarding how to effectively identify key pathways underlying the evolution of complex traits in the genomic era, as well as directions for future research.

373-380

Authors: Ma Chi , Xiong Wei-Guo , Yang Li , Zhang Lu , Tomlin Peter Robert , Chen Wu , Fan Peng-Fei

Studies on behavioral flexibility in response to habitat differences  and  degradation  are  crucial  for developing  conservation  strategies  for  endangered species. Trachypithecus species  inhabit  various habitats and display different patterns of strata use; however, the effect of habitat structure on strata use remains poorly studied. Here, we investigated strata use  patterns  of  Indo-Chinese  gray  langurs (Trachypithecus crepusculus) in a primary evergreen forest  in  Mt.  Wuliang,  southwest  China,  from  June 2012 to January 2016. In addition, we compared T. crepusculus strata  use  and  terrestriality  with  five other Trachypithecus species from previous studies. Unlike langurs living in karst forests, our study group was typically arboreal and spent only 2.9% of time on the ground. The group showed a preference for higher strata when resting and lower strata (<20 m) when moving. The langurs primarily used time on the ground  for  geophagy,  but  otherwise  avoided  the ground during feeding. These strata use patterns are similar  to  those  of  limestone  langurs  (T. francoisi) when  using  continuous  forests.  At  the  genus  level (n=6  species),  we  found  a  negative  relationship between habitat forest cover and terrestriality. This negative  relationship  was  also  true  for  the  five limestone langur species, implying limestone langurs increase territoriality in response to decreased forest cover. Our results document behavioral flexibility in strata  use  of Trachypithecus langurs  and  highlight the  importance  of  the  protection  of  continuous forests to promote langur conservation.

395-409

Authors: Liu Lei , Song Da-Wei , Liu Guang-Lu , Shan Li-Peng , Qiu Tian-Xiu , Chen Jiong

Spring  viremia  of  carp  virus  (SVCV)  causes devastating losses in aquaculture. Coumarin has an advantageous  structure  for  the  design  of  novel antiviral  agents  with  high  affinity  and  specificity.  In this  study,  we  evaluated  a  hydroxycoumarin medicine,  i.e.,  7-(6-benzimidazole)  coumarin  (C10), regarding its anti-SVCV effects in vitro and in vivo. Results  showed  that  up  to  12.5  mg/L  C10 significantly  inhibited  SVCV  replication  in  the epithelioma papulosum cyprini (EPC) cell line, with a maximum inhibitory rate of >97%. Furthermore, C10 significantly reduced cell death and relieved cellular morphological  damage  in  SVCV-infected  cells. Decreased mitochondrial membrane potential (ΔΨm) also  suggested  that  C10  not  only  protected mitochondria, but also reduced apoptosis in SVCV- infected  cells.  For in vivo studies,  intraperitoneal injection of C10 resulted in an anti-SVCV effect and substantially  enhanced  the  survival  rate  of  virus- infected  zebrafish.  Furthermore,  C10  significantly enhanced  antioxidant  enzyme  activities  and decreased  reactive  oxygen  species  (ROS)  to maintain antioxidant-oxidant balance within the host, thereby contributing to inhibition of SVCV replication. The  up-regulation  of  six  interferon  (IFN)-related genes  also  demonstrated  that  C10  indirectly activated  IFNs  for  the  clearance  of  SVCV  in zebrafish.  This  was  beneficial  for  the  continuous maintenance of antiviral effects because of the low viral  loads  in  fish.  Thus,  C10  is  suggested  as  a therapeutic agent with great potential against SVCV infection in aquaculture.

423-430

Authors: Shen Zhong-Yuan , Yu Dan , Gao Xin , Zhang Fu-Tie , Liu Huan-Zhang

The  Chinese  sturgeon  (Acipenser sinensis Gray, 1835) is a large anadromous fish species, which is under considerable threat due to dramatic declines in population numbers. In the current study, population genetic diversity and individual reproductive success were assessed using nuclear microsatellite markers (simple  sequence  repeat,  SSR)  and  complete mitochondrial (mtDNA) genome analysis of juveniles born in 2 014. Results showed the existence of size polymorphism  in  the  mtDNA  genome  of  Chinese sturgeon,  which  was  caused  by  a  repeat  motif. Population genetic diversity was high based on both SSR  (Ho:  0.728±0.211; He:  0.779±0.122)  and mtDNA  genome  analyses  (H:  0.876±0.003 5; Pi: 0.001 1±0.001 0).  A  positive  inbreeding  coefficient (FIS:  0.066±0.143)  was  also  found,  indicating  the occurrence  of  inbreeding.  Reconstruction  of  sibling groups identified 11 mothers and 11 fathers involved in  reproduction  of  Chinese  sturgeons  in 2 014. Variance in individual reproductive success was not significant, with reproductive success of parent fish instead shown to be relatively even (P=0.997>0.05), thus  suggesting  the  absence  of  sweepstakes reproductive success (SRS). These results indicate that,  in  regard  to  conservation,  loss  of  genetic diversity due to the effects of SRS is not of particular concern.  However,  we  must  focus  on  having  an adequate  number  of  adults  and  suitable environmental  conditions  to  ensure  that  fish  can reproduce.

437-443

Authors: Qin Dong-Dong , Feng Shu-Fei , Zhang Fei-Yu , Wang Na , Sun Wen-Jie , Zhou Yin , Xiong Teng-Fang , Xu Xian-Lai , Yang Xiao-Ting , Zhang Xiang , Zhu Xue , Hu Xin-Tian , Xiong Lei , Liu Yun , Chen Yong-Chang

Since the epoch-making observations of circadian rhythm in Mimosaceae plants, sleep has been investigated for centuries (de  Mairan,  1729; Du  Monceau,  1758).  As  a  natural  and reversible state, sleep is marked by reduced responsiveness to  external  stimuli,  relative  inactivity,  and  loss  of consciousness.  Although  reduced  responsiveness  could potentially introduce significant danger to survival, nearly all animals in nature sleep. This strongly implies an adaptive role of sleep in increasing overall fitness of an organism. Research has  shown  that  sleep  is  responsible  for  many  vital physiological functions, including tissue repair (Oswald, 1980), skin  function  (Rechtschaffen,  1998),  thermoregulation (Parmeggiani,  1986; Rechtschaffen,  1998),  energy  saving (Siegel, 2005), insulin release and responsiveness (Spiegel et al.,  1999),  metabolic  regulation  (Sharma  &  Kavuru,  2010), immunological enhancement (Besedovsky et al., 2012; Imeri & Opp,  2009),  synaptic  plasticity  (Benington  &  Frank,  2003), neuron viability (Zhang et al., 2014a), and memory formation (Rasch  &  Born,  2013; Walker  &  Stickgold,  2006).  Sleep therefore plays an essential role in human health and is vital for physical and psychological performance (Halson & Juliff, 2017; Thun et al., 2015; Vitale & Weydahl, 2017).

449-454

Authors: Wang Xue-Hui , Song Tian-Zhang , Li Lei , Tian Ren-Rong , Zheng Yong-Tang

Intestinal biopsy is a basic experimental method for studying pathological  changes  in  the  intestinal  tract  during  human immunodeficiency virus (HIV) infection. In this study, jejunal resection and anastomosis were successfully performed in 12 Chinese  rhesus  macaques  (Macaca mulatta).  The  sampled gut  tissues  were  then  examined  by  hematoxylin  and  eosin (H&E)  staining,  electron  microscopy,  flow  cytometry, immunofluorescence  detection,  and  RNA  quality  analysis  to ensure  suitability  for  histological,  physiological,  pathological, and immunological detection, as well as mechanistic analysis at the cellular and molecular level. Importantly, the surgery did not affect the ratio or number of immune cells in peripheral blood or the concentration of lipids, proteins, and vitamins in plasma,  which  are  important  indicators  of  nutritional  status. Our  results  thus  indicated  that  jejunal  resection  and anastomosis are feasible, and that immune homeostasis and intestinal  barrier  integrity  are  not  altered  by  surgery.  All macaques  recovered  well  (except  for  one),  with  no postoperative  complications.  Therefore,  this  animal  surgery may be applicable for longitudinal intestinal research related to diseases  such  as  acquired  immunodeficiency  syndrome (AIDS).

458-464

Authors: Wang Long-Wu , Zhong Guo , He Gang-Bin , Zhang Yu-Han , Liang Wei

The egg laying behavior of brood parasites is at the heart of studies  on  host  co-evolution.  Therefore,  research  on  egg laying  behavior  can  improve  our  understanding  of  brood parasitism  and  associated  processes.  Over  a  seven  year study  period,  we  monitored  455  oriental  reed  warbler (Acrocephalus orientalis) nests during the egg laying period, 250 of which were parasitized by common cuckoos (Cuculus canorus).  We  collected  53  clear  videos  of  common  cuckoo parasitism, analyzed all recorded parasitic behavior in detail, and  summarized  the  process  of  brood  parasitism. Furthermore, based on analyses of the field video recordings, we  propose  a  new  explanation  for  egg  removal  behavior, namely the delivery hypothesis, i.e., egg pecking and biting by cuckoos  may  facilitate  fast  egg-laying  and  parasitism  by reducing host attention and attack, with egg removal a side effect of egg pecking and biting. We concluded that common cuckoos change their behavior when hosts are present at the nest, with a set of behaviors performed to deal with host attack and successfully complete parasitic egg-laying regardless of time of day.

471-475

Authors: Ma Yun-Fei , Huang Cui-Ping , Lu Fang-Ru , Li Jin-Xiu , Han Xu-Man , Adeola Adeniyi C. , Gao Yun , Deng Jia-Kun , Xie Hai-Bing , Zhang Ya-Ping

Cis-regulatory  elements  play  an  important  role  in  the development  of  traits  and  disease  in  organisms  (Ma  et  al., 2020; Woolfe et al., 2005) and their annotation could facilitate genetic  studies.  The  Encyclopedia  of  DNA  Elements (ENCODE) (Davis et al., 2018) and Functional Annotation of Animal Genomes (FAANG) (FAANG Consortium et al., 2015) offer  pioneering  data  on  regulatory  elements  in  several species.  Currently,  however,  regulatory  element  annotation data  remain  limited  for  most  organisms.  In  this  study,  we developed  a  tool  (OrthReg)  for  annotating  conserved orthologous cis-regulatory  elements  in  targeted  genomes using  an  annotated  reference  genome.  Cross-species validation  of  this  annotation  tool using  human  and  mouse ENCODE data confirmed the robustness of this strategy. To explore  the  efficiency  of  the  tool,  we  annotated  the  pig genome  and  identified  more  than  28  million  regulatory annotation records using the reference human ENCODE data. With this regulatory annotation, some putative regulatory non- coding variants were identified within domestication sweeps in European and East Asian pigs. Thus, this tool can utilize data produced by ENCODE, FAANG, and similar projects, and can be  easily  extended  to  customized  experimental  data.  The extensive application of this tool will help to identify informative single nucleotide polymorphisms (SNPs) in post-genome-wide association studies and resequencing analysis of organisms with limited regulatory annotation data.

213-219

Authors: Wong Gary , Bi Yu-Hai , Wang Qi-Hui , Chen Xin-Wen , Zhang Zhi-Gang , Yao Yong-Gang

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by infection with human coronavirus 2019 (HCoV-19 / SARS-CoV-2 / 2019-nCoV), is a global threat to the human population. Here, we briefly summarize the available data for the zoonotic origins of HCoV-19, with reference to the other two epidemics of highly virulent coronaviruses, SARS-CoV and MERS-CoV, which cause severe pneumonia in humans. We propose to intensify future efforts for tracing the origins of HCoV-19, which is a very important scientific question for the control and prevention of the pandemic.

247-257

Authors: Yu Wen-Bin , Tang Guang-Da , Zhang Li , Corlett Richard T.

The outbreak of COVID-19 started in mid-December 2019  in  Wuhan,  China.  Up  to  29  February  2020, SARS-CoV-2 (HCoV-19 / 2019-nCoV) had infected more than 85 000 people in the world. In this study, we used 93 complete genomes of SARS-CoV-2 from the  GISAID  EpiFluTM database  to  investigate  the evolution  and  human-to-human  transmissions  of SARS-CoV-2 in the first two months of the outbreak. We  constructed  haplotypes  of  the  SARS-CoV-2 genomes,  performed  phylogenomic  analyses  and estimated  the  potential  population  size  changes  of the  virus.  The  date  of  population  expansion  was calculated based on the expansion parameter tau (τ) using the formula t=τ/2u. A total of 120 substitution sites with 119 codons, including 79 non-synonymous and  40  synonymous  substitutions,  were  found  in eight coding-regions in the SARS-CoV-2 genomes. Forty  non-synonymous  substitutions  are  potentially associated  with  virus  adaptation.  No  combinations were  detected.  The  58  haplotypes  (31  found  in samples  from  China  and  31  from  outside  China) were identified in 93 viral genomes under study and could be classified into five groups. By applying the reported bat coronavirus genome (bat-RaTG13-CoV) as the outgroup, we found that haplotypes H13 and H38  might  be  considered  as  ancestral  haplotypes, and  later  H1  was  derived  from  the  intermediate haplotype H3. The population size of the SARS-CoV-2  was  estimated  to  have  undergone  a  recent expansion  on  06  January  2020,  and  an  early expansion  on  08  December  2019.  Furthermore, phyloepidemiologic  approaches  have  recovered specific directions of human-to-human transmissions and  the  potential  sources  for  international  infected cases.

273-280

Authors: Lai Xin-Lei , Zhou Wen-Liang , Gao Hua-Lei , Wang Meng , Gao Kai , Zhang Bao-Wei , Wei Fu-Wen , Nie Yong-Gang

Interspecific  killing  is  a  primary  reason  for  the  low survival  rates  of  some  animal  species.  The  giant panda  (Ailuropoda melanoleuca)  is  an  altricial eutherian mammal and thus, in comparison to other infants, panda cubs are highly vulnerable, which may significantly influence the selection of breeding sites by females. Here, we used infrared camera traps to monitor  giant  panda  dens  for  5.5  years  in  Foping National Nature Reserve (FNNR) to determine how interspecific  factors  affect  den  selection  by  wild female  pandas.  Results  indicated  that  Asian  black bears  (Ursus thibetanus),  yellow-throated  martens (Martes flavigula),  leopard  cats  (Prionailurus bengalensis),  and  masked  palm  civets  (Paguma larvata) visited the dens frequently, and the presence of these species negatively influenced den selection by  female  pandas.  Interestingly,  the  presence  of rodents and terrestrial birds appeared to indicate den safety, and female giant pandas were not averse and even preferred dens with a high abundance index of rodents and terrestrial birds. The den suitability index (DSI) was a reliable tool for evaluating whether dens were suitable for female giant pandas to give birth to and  rear  cubs,  with  preference  for  dens  with  high DSI values. This study increases our understanding of the den selection criteria of female giant pandas and  the  main  threats  to  the  survival  of  their  cubs, thus  providing  important  guidance  for  the conservation and management of this species.

292-313

Authors: Chen Jin-Min , Xu Kai , Poyarkov Nikolay A. , Wang Kai , Yuan Zhi-Yong , Hou Mian , Suwannapoom Chatmongkon , Wang Jian , Che Jing

Asian  leaf-litter  toads  of  the  genus Leptobrachella represent  a  great  anuran  diversification  in  Asia. Previous studies have suggested that the diversity of this  genus  is  still  underestimated.  During herpetological surveys from 2013 to 2018, a series of Leptobrachella specimens  were  collected  from  the international  border  areas  in  the  southern  and western  parts  of  Yunnan  Province,  China. Subsequent  analyses  based  on  morphological  and molecular data revealed three distinct and previously unknown  lineages,  which  we  formally  describe  as three new species herein. Among them, we describe a  new  species  that  occurs  at  the  highest  known elevation for Leptobrachella in China. Four species of Leptobrachella,  including  two  new  species,  are found in the same reserve. Furthermore, our results suggest that the population from Longchuan County, Yunnan, may represent an additional new species of Leptobrachella, although we tentatively assigned it to Leptobrachella cf. yingjiangensis due  to  the  small sample  size  examined.  Lastly,  we  provide  the  first description  of  females  of L. yingjiangensis.  Our results  further  highlight  that  both  micro-endemism and sympatric distributions of species are common patterns  in Leptobrachella,  that  contribute  to taxonomic  and  conservation  challenges  in  these frogs.  We  provide  an  identification  key  for Leptobrachella known to occur in Yunnan. Given the lack  of  knowledge  on  species  diversity  of Leptobrachella along international border areas, we recommend  that  future  studies  include  trans- boundary collaborative surveys.

328-340

Authors: Yang Yang , Wu Li-Na , Chen Jing-Fang , Wu Xi , Xia Jun-Hong , Meng Zi-Ning , Liu Xiao-Chun , Lin Hao-Ran

Leopard coral groupers belong to the Plectropomus genus of the Epinephelidae family and are important fish  for  coral  reef  ecosystems  and  the  marine aquaculture industry. To promote future research of this  species,  a  high-quality  chromosome-level genome  was  assembled  using  PacBio  sequencing and  Hi-C  technology.  A  787.06  Mb  genome  was assembled,  with  99.7%  (784.57  Mb)  of  bases anchored  to  24  chromosomes.  The  leopard  coral grouper genome size was smaller than that of other groupers, which may be related to its ancient status among grouper species. A total of 22 317 protein- coding  genes  were  predicted.  This  high-quality genome  of  the  leopard  coral  grouper  is  the  first genomic  resource  for Plectropomus and  should provide  a  pivotal  genetic  foundation  for  further research. Phylogenetic analysis of the leopard coral grouper and 12 other fish species showed that this fish is closely related to the brown-marbled grouper. Expanded  genes  in  the  leopard  coral  grouper genome  were  mainly  associated  with  immune response  and  movement  ability,  which  may  be related to the adaptive evolution of this species to its habitat.  In  addition,  we  also  identified  differentially expressed genes (DEGs) associated with carotenoid metabolism between red and brown-colored leopard coral groupers. These genes may play roles in skin color  decision  by  regulating  carotenoid  content  in these groupers.

345-350

Authors: Wang Jiao-Jiao , Ma Lai-Kun , Liang Wei , Yang Can-Chao

The  “call  for  help” hypothesis  proposes  that  alarm  calls produced by a bird can transmit warning information to both conspecific  and  interspecific  neighbors.  Neighbors  who  are attracted  by  social  transmission  might  benefit  from  knowing about the presence of danger or by gaining information about the presence of predators or brood parasites nearby. Brood parasite hosts can distinguish threats from different intruders and exhibit varied responses correspondingly. However, most previous studies have conducted sound playback at host nest sites and focused on conspecific individuals attracted by the alarm calls. In this study, we used random location playback to investigate  the  responses  of  different  host  species  to  alarm signals  of  oriental  reed  warblers  (Acrocephalus orientalis) toward  different  intruders  (brood  parasite,  predator,  and harmless  control)  in  order  to  reveal  how  hosts  evaluate different  threats  from  different  intruders  using  vocal information in non-nesting areas during the breeding season. We found that the alarm calls given in response to different intruders incurred similar numbers of approaching species for both conspecific and interspecific birds. However, the number of attracted individuals differed significantly among the various species,  with  conspecifics  and  vinous-throated  parrotbills (Paradoxornis webbianus)  dominating,  both  of  which  are major  hosts  of  common  cuckoos  (Cuculus canorus). Nevertheless,  interspecific  birds  did  not  present  any aggressive  behavior  according  to  the  alarm  calls,  which implied  that  visual  information  may  be  needed  for  further confirmation of threats. In addition, determining whether alarm call  structure  promoted  an  evolutionary  convergence phenomenon still needs further verification.

97-104

Authors: Zhe Zhang, Saber Khederzadeh, Yan Li

The domestic dog, as a highly successful domestication model, is well known as a favored human companion. Exploring its domestication history should provide great insight into our understanding of the prehistoric development of human culture and productivity. Furthermore, investigation on the mechanisms underpinning the morphological and behavioral traits associated with canid domestication syndrome is of significance not only for scientific study but also for human medical research. Current development of a multidisciplinary canine genome database, which includes enormous omics data, has substantially improved our understanding of the genetic makeup of dogs. Here, we reviewed recent advances associated with the original history and genetic basis underlying environmental adaptations and phenotypic diversities in domestic dogs, which should provide perspectives on improving the communicative relationship between dogs and humans.

123 - 137

Authors: Hai-Yu Shen, Yan Zhou, Qian-Jin Zhou, Ming-Yun Li, Jiong Chen

Interleukin-34 (IL-34) is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colony-stimulating factor-1 receptor (CSF-1R). However, information on the function of IL-34 in fish remains limited. In the present study, we identified an IL-34 homolog from mudskippers (Boleophthalmus pectinirostris). In silico analysis showed that the mudskipper IL-34 (BpIL-34) was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper (Epinephelus coioides) homolog. BpIL-34 transcripts were constitutively expressed in various tissues, with the highest level of expression found in the brain. Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues. The recombinant mature BpIL-34 peptide (rBpIL-34) was purified and used to produce anti-rBpIL-34 IgG. Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages (MOs/MФs) was N-glycosylated. In vitro, rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs, as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor α (BpTNF-α) and BpIL-1β in these cells. Furthermore, the knockdown of mudskipper CSF-1R1 (BpCSF-1R1), but not mudskipper BpCSF-1R2, significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФ function. In conclusion, our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

148 - 156

Authors: Ning Zheng, Zhi-Zhong Wang, Song-Wei Wang, Fang-Jia Yang, Xu-Tao Zhu, Chen Lu, Anne Manyande, Xiao- Ping Rao, Fu-Qiang Xu

The accessory olfactory bulb (AOB), located at the posterior dorsal aspect of the main olfactory bulb (MOB), is the first brain relay of the accessory olfactory system (AOS), which can parallelly detect and process volatile and nonvolatile social chemosignals and mediate different sexual and social behaviors with the main olfactory system (MOS). However, due to its anatomical location and absence of specific markers, there is a lack of research on the internal and external neural circuits of the AOB. This issue was addressed by single-color labeling and fluorescent double labeling using retrograde rAAVs injected into the bed nucleus of the stria terminalis (BST), anterior cortical amygdalar area (ACo), medial amygdaloid nucleus (MeA), and posteromedial cortical amygdaloid area (PMCo) in mice. We demonstrated the effectiveness of this AOB projection neuron labeling method and showed that the mitral cells of the AOB exhibited efferent projection dispersion characteristics similar to those of the MOB. Moreover, there were significant differences in the number of neurons projected to different brain regions, which indicated that each mitral cell in the AOB could project to a different number of neurons in different cortices. These results provide a circuitry basis to help understand the mechanism by which pheromone information is encoded and decoded in the AOS.

172 - 181

Authors: Olga Jovanović Glavaš, Paula Počanić, Vanja Lovrić, Lorena Derežanin, Zoran Tadić, Duje Lisičić

The European glass lizard, Pseudopus apodus (Pallas, 1775), is a large, legless lizard with wide distribution across south-eastern Europe and eastern and central Asia. To date, morphological diversification among populations on a geographically small scale has not yet been reported in this lizard. Thus, we investigated the morphological variations and corresponding differences in habitat utilization between two populations of P. apodus inhabiting the same biogeographical zone within a relatively close geographic area. We hypothesized that minor differences in habitat could induce a significant level of morphological differentiation, thus indicating morphological plasticity in this species on a small geographical scale. We sampled 164 individuals (92 from the Croatian mainland and 72 from the island of Cres). Results showed that P. apodus indeed exhibited morphological differences between populations in the same biogeographical zone within a relatively close geographic area, with the Cres Island individuals being generally larger than the individuals from the mainland. Some ecological characteristics were similar in both populations (e.g., soil temperature, distance to hiding place), whereas others were distinct (e.g., air temperature and humidity). In addition, vegetation cover differed between the two sites, with more vegetation present on the mainland than on the island. Furthermore, the Cres Island population showed clear sexual dimorphism, which was absent in the mainland population.

188 - 193

Authors: Yu-Lu Gan, Guo-Hua Yu, Zheng-Jun Wu

A new species of the genus Amolops, Amolops tuanjieensis sp. nov., is described from Yunnan, China. The new species can be distinguished by the following characters: dorsolateral folds present; dorsal and ventral surfaces smooth; top of head and dorsum brown-red with irregular gray and dark spots; flank green; side of head black, from tip of snout, diffusing posteriorly to axilla, continuing as black streak below edge of dorsolateral fold; SVL 39.5–40.4 mm in males, 56.8–60.7 mm in females; tympanum distinct; supratympanic fold indistinct; vomerine teeth in two oblique rows between choanae, closer to each other than choanae; vocal sacs present; nuptial pads present; outer metatarsal tubercle absent, supernumerary tubercles absent; all fingertips expanded into discs; limbs dorsally brown with dark brown bars and irregular dark brown blotches.

199-202

Authors: Huan-Zhi Chen, Hong-Yi Yang, Kai Zhong, Jia-Li Li

Changes in fine structures of the brain over a life span can have robust effects on neural activity and brain function, which both play crucial roles in neurodegenerative diseases. Clinically, however, low-resolution MRI only provides limited information about fine brain structures. Here, using high-resolution 9.4 T MRI, we established a set of structural images and explored the fine structures of the claustrum, hippocampus, amygdala complex, and subregions of the amygdala complex (BLA, including lateral, basal, and accessory basal subnuclei) in rhesus macaque (Macaca mulatta) brains. Based on these high-resolution images, we were able to discriminate the subregional boundaries accurately and, at the same time, obtain the volume of each brain nuclei. Thus, advanced high-resolution 9.4 T MRI not only provides a new strategy for early diagnosis of neurodegenerative diseases, but also provides the ability to observe fine structural changes in the brain across a life span.

208 - 212

Authors: Jing Zhang, Rong-Can Luo, Xiao-Yong Man, Long- Bao Lv, Yong-Gang Yao, Min Zheng

The Chinese tree shrew (Tupaia belangeri chinensis) is a small mammal closely related to primates. It has a small body size, low maintenance cost, and a relatively short reproductive cycle, all of which has made it the ideal model for the study of a variety of human diseases. In this study, we compared the anatomy of the skin of the Chinese tree shrew with that of the rhesus macaque, mouse and human, with the intention of providing the basic data required for the creation of skin disease models using this animal. Paraffin sections, hematoxylin-eosin (H&E) staining, masson staining and immunohistochemical techniques were used to examine the dorsal skin structure of the Chinese tree shrew. The epidermis was shown to be composed of 1–2 layers of cells. There were hair follicles, sebaceous glands and sweat glands in the dermis and the subcutaneous tissue, with apocrine glands being more common than eccrine glands. Both Keratin5 (KRT5) and Keratin10 (KRT10) were expressed in the skin of the Chinese tree shrew, with a localization in the cytoplasm. Overall, the skin morphology and histology of the Chinese tree shrew was basically the same as that of the human. We propose that the Chinese tree shrew has a strong potential to be used for creating animal models to help elucidate the molecular mechanisms underlying a variety of skin diseases.

1-2

Authors: Su-Qing Liu

Peer review plays an essential role in academic quality control of scientific journals and is central to scholarly communication. It exists in many aspects of academia, including funding applications, career building, academic performance evaluation, and publishing. In regard to publishing, different forms of publication, from traditional hardcopies and on-line journals to preprint articles (e.g., arXiv: https://arxiv.org/, BioRxiv: https://www.biorxiv.org/), continue to fine-tune the requirements of peer review, and vice versa, peer review continuously refines the quality of publications.

20-31

Authors: Hong-Zhe Li, Nan Li, Jing-Jing Wang, Heng Li, Xing Huang, Lei Guo, Hui-Wen Zheng, Zhan-Long He, Yuan Zhao, Ze-Ning Yang, Hai-Tao Fan, Man-Man Chu, Jin-Xi Yang, Qiong-Wen Wu, Long-Ding Liu

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts. Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface; however, studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models. Here, we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal hostmicrobiome niche and immune balance with steady health status. Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment. Furthermore, the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment, and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed. In addition to inducing aberrant morphology of small intestinal villi, the depletion of gut commensal bacteria led to increased proportions of CD3+ T, CD4+ T, and CD16+ NK cells in peripheral blood mononuclear cells (PBMCs), but decreased numbers of Treg and CD20+ B cells. The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes, including IL- 13, VCAM1, and LGR4.

39-50

Authors: Feng Xu, Ming-Yun Li, Jiong Chen

D-dopachrome tautomerase (DDT), a member of the macrophage migration inhibitory factor (MIF) protein superfamily, is a newly described cytokine with chemokine-like characteristics. However, research on fish DDT remains limited. In this study, we identified a DDT homolog (LjDDT) from the Japanese sea bass, Lateolabrax japonicus. Sequence analysis showed that LjDDT had typical sequence features of known DDT and MIF homologs and was most closely related to DDT of rock bream (Oplegnathus fasciatus). LjDDT transcripts were detected in all tested tissues of healthy Japanese sea bass, with the highest expression found in the liver. Upon infection with Vibrio harveyi, LjDDT transcripts were significantly down-regulated in the three tested tissues, including the liver, spleen, and head kidney. Recombinant LjDDT (rLjDDT) and the corresponding antibody (anti-rLjDDT) were subsequently prepared. The administration of 100 μg/g anti-rLjDDT had a statistically significant protective effect on the survival of V. harveyi-infected fish. Moreover, rLjDDT was able to induce the migration of monocytes/macrophages (MO/MФ) and lymphocytes both in vitro and in vivo, but without significant influence on the migration of neutrophils. rLjDDT exhibited chemotactic activity for lipopolysaccharide (LPS) -stimulated M1-type MO/ MΦ in vitro, but not for cAMP-stimulated M2-type MO/MΦ. Furthermore, the knockdown of LjCD74, but not LjCXCR4 , significantly down-regulated the rLjDDT-enhanced migration of MO/MΦ and relieved the rLjMIF-inhibited migration of MO/MΦ. These results indicate that LjCD74 may be the major chemotactic receptor of LjDDT and LjMIF in Japanese sea bass MO/MΦ. Combined rLjDDT+ rLjMIF treatment had no significant effect on the migration of MsiRNA, LjCD74si-, or LjCXCR4- sitreated MO/MΦ compared to the control group, suggesting that the roles of LjDDT and LjMIF may be antagonistic. In conclusion, our study demonstrates for the first time that DDT may play a role in the immune responses of fish against bacterial infection through chemotactic recruitment of MO/MΦ via mediation of CD74 as an antagonist of MIF.

61-69

Authors: Xing-Han Chen, Sen Yang, Wei Yang, Yuan-Yuan Si, Rui-Wen Xu, Bin Fan, Le Wang, Zi-Ning Meng

Tylorrhynchus heterochaetus is a widespread benthic polychaete worm found in coastal brackish waters of the west Pacific. It has high ecological and economic value as a biomarker of water quality and as a high-quality feed in aquaculture and fisheries and is considered a delicacy in some areas of Asia. However, it has experienced a marked reduction in recent years due to overexploitation as well as changes in the environment and climate. Here, to comprehensively understand its genetic background and thus provide insights for better conservation and utilization of this species, we assessed the genetic variability and demographic history of T. heterochaetus individuals sampled from eight locations along the coasts of southeast China and north Vietnam based on mitochondrial cytochrome c oxidase I (COI) sequences. We observed high haplotype diversity (Hd), with an average of 0.926, but relatively low nucleotide diversity (π), with a mean of 0.032 across all samples. A total of 94 polymorphic sites and 85 haplotypes were identified among 320 individuals. The pairwise genetic distances among haplotypes ranged from 0.001 to 0.067, with the high intraspecific divergence possibly reflecting geographic isolation and gene pool fragmentation. Significant genetic structures were revealed among the studied locations; specifically, the eight locations could be treated as six genetically different populations based on pairwise ΦST results (0.026–0.951, P <0.01). A significant pattern of isolation-by-distance was detected between the genetic and geographic distances (r=0.873, P=0.001). Three geographic lineages were defined based on phylogenetic tree and network analyses of COI haplotypes. AMOVA results indicated that genetic variations mainly occurred among the three lineages (89.96%). Tests of neutrality and mismatch distribution suggested that T. heterochaetus underwent recent population expansion. These results provide the first report on the genetic status of T. heterochaetus and will be valuable for the management of genetic resources and better understanding of the ecology and evolution in this species.

78-83

Authors: Wan-Cai Xia, Sheng-Nan Ji, Bao-Ping Ren, Xin-Ming He, Tai Zhong, Ali Krzton, Yun Tang, Da-Yong Li

Individual dispersal trends, unquestionably important for species ecology and evolution, are affected by multiple factors. Understanding the factors that influence female dispersal strategies offers important insight into primate dispersal mechanisms and female choice. To investigate the proximate causes of dispersal in female Yunnan snub-nosed monkeys (Rhinopithecus bieti), we observed and analyzed nine years of detailed dispersal and demographic data from a population of R. bieti in Xiangguqing, Baimaxueshan Nature Reserve, Yunnan Province, China. Results showed that females who lived long-term in a one-male unit (OMU), without giving birth and with few or no relatives, were more likely to leave that OMU. In addition, an OMU led by an outgroup male and containing more female relatives was significantly more likely to be chosen for immigration. Conversely, greater male age, longer male tenure, and more potentially fertile females discouraged immigration into an OMU. These results suggest that reproduction, male quality, and kin cooperation play the largest roles in female Yunnan snub-nosed monkey dispersal.

90-93

Authors: Ran Wang, Fu-Jia Yang, Xiao-Yan Zheng, Xian-Zheng Liao, Dong-Ying Fan, Hui Chen, Jing An

The development of an effective tetravalent vaccine against dengue viruses (DENVs) has become a world priority. We previously showed that four monovalent dengue DNA vaccines expressing premembrane (prM) and envelope (E) proteins displayed effective protection against corresponding challenges in mice. Thus, to elucidate the overall immunity and persistence of the tetravalent formulation (TetraME), we evaluated the humoral and cellular immune responses as well as the long-term protection in the current study. TetraMEimmunized mice displayed increased production of Th1/Th2- typed cytokines upon stimulation with heterologous DENV antigens. Moreover, high levels of tetravalent DENV antibodies and sterilized immunity were detected long-term (30 weeks after immunization). These findings provide feasible validation for the potential utility of this vaccine formulation.