Phylogenetic Relationships among Indigenous Sheep Populations in East Asia based on Five Informative Blood Protein and Nonprotein Polymorphisms

The phylogenetic relationships among 23 local sheep breeds and varieties in East Asia were determined based on allele frequency data for five informative and polymorphic blood protein and nonprotein loci (transferrin, arylesterase, hemoglobin-β, X-protein, and potassium transport) using electrophoresis and ion-densitometric techniques. Comparatively striking differences in the frequency distributions, especially of hemoglobin-β and X-protein alleles, were seen between the northern population of the Khalkhas, Bhyanglung, Baruwal, Jakar, Sakten, and five Chinese breeds and the southern population of the Bengal, Kagi, Lampuchhre, Myanmar, and Sipsu breeds. Clustering analyses using UPGMA and NJ methods and principal component analysis, using the data of all populations and loci tested, clearly demonstrated the difference. The two population groups are divided by the boundary of the Himalayas, and each diverged into three subgroups: Mongolian, Tibetan, and Himalayan groups in the north and Indian groups I, II, and III in the south. It was noted that the genetic differentiation of populations was more distinct in the northern group. These findings strongly suggest the existence of at least two large and phylogenetically different gene pools of sheep in East Asia.     

Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan

Background

Dog bites in humans are a public health problem worldwide. The issues of increasing stray dog populations, rabies outbreaks, and the risk of dogs biting humans have been frequently reported by the media in Bhutan. This study aimed to estimate the bite incidence and identify the risk factors for dog bites in humans, and to estimate human deaths from rabies in rabies endemic south Bhutan.

Methods

A hospital-based questionnaire survey was conducted during 2009–2010 among dog bites victims who visited three hospitals in Bhutan for anti-rabies vaccine injection. Decision tree modeling was used to estimate human deaths from rabies following dog bite injuries in two rabies endemic areas of south Bhutan.

Results

Three hundred and twenty four dog bite victims were interviewed. The annual incidence of dog bites differed between the hospital catchment areas: 869.8 (95% CI: 722.8–1022.5), 293.8 (240–358.2) and 284.8 (251.2–323) per 100,000 people in Gelephu, Phuentsholing and Thimphu, respectively. Males (62%) were more at risk than females (P<0.001). Children aged 5–9 years were bitten more than other age groups. The majority of victims (71%) were bitten by stray dogs. No direct fatal injury was reported. In two hospital areas (Gelephu and Phuentsholing) in south Bhutan the annual incidence of death from rabies was 3.14 (95% CI: 1.57–6.29) per 100,000 population. The decision tree model predicted an equivalent annual incidence of 4.67 (95% CI: 2.53–7.53) deaths/100,000 population at risk. In the absence of post exposure prophylaxis, the model predicted 19.24 (95% CI: 13.69–25.14) deaths/year in these two areas.

Conclusions

Increased educational awareness of people about the risk of dog bites and rabies is necessary, particularly for children in rabies endemic areas of Bhutan.     

The Emergence of an Endangered Species: Evolution and Phylogeny of the <Emphasis Type="Italic">Trachypithecus geei</Emphasis> of Bhutan

Golden langurs (Trachypithecus geei) are an endangered primate species in Bhutan. We discuss their evolution in terms of phylogeny, ecology, and biogeography. We test the hypothesis that rivers and mountains in Bhutan isolated a population of capped langurs (Trachypithecus pileatus) that later speciated into the morphologically distinct T. geei. Trachypithecus, the genus to which both capped and golden langurs belong, spread north from a paleorefuge in south China, and Semnopithecus (gray langurs) spread east and northward from a refuge in south India. We show that the 2 genera both arrived in Bhutan and were separated from each other by the Sunkosh River and Black Mountains. Likewise, a population of capped langurs isolated from parental populations by rivers speciated into the distinct golden langurs. We conducted field surveys covering the entire range of langurs in Bhutan. The Sunkosh River and Black Mountain range in west Bhutan isolate gray langurs and golden langurs from each other. In the east, the Manas River system (Manas-Mangde) served as a barrier between golden and capped langurs. However, it is an imperfect barrier and a contact zone between the 2 species occurred on the banks of the Mangde River. Second, we reconstructed the evolutionary history of the langurs of Bhutan via molecular phylogenetic tools. We sequenced the cytochrome b region (cyt b) of the mitochondrial DNA (mtDNA) to model a phylogeny. It revealed the distinct evolutionary paths of the golden, capped, and gray langurs. As predicted, golden and capped langurs are closely related to each other and to other species in Trachypithecus from Southeast Asia. The gray langur of Bhutan grouped into a distinct clade with conspecifics in Semnopithecus from India and Nepal. The south Indian clade of gray langurs is more ancient, with the Bhutan and Nepal gray langurs having diverged later, which fits with the glacial models of ice sheet retreats and colonization of South Asia by gray langurs from south India north toward the Himalayas. Likewise, the golden and capped langur clade are the most derived and divergent from the older groups of Trachypithecus in Southeast Asia, which also fits with paleorefuge models of recolonization by Trachypithecus into the rest of Southeast Asia and north toward the Himalayas from paleorefuges in Southeast Asia. As predicted, golden and capped langurs are closely related to each other and gray langurs are only distantly related to them. The divergence between capped and golden langurs is more recent, while the split between Trachypithecus and Semnopithecus is more ancient and took place before the Pliocene.     

In the shadows of snow leopards and the Himalayas: density and habitat selection of blue sheep in Manang,Nepal

There is a growing agreement that conservation needs to be proactive and pay increased attention to common species and to the threats they face. The blue sheep (Pseudois nayaur) plays a key ecological role in sensitive high‐altitude ecosystems of Central Asia and is among the main prey species for the globally vulnerable snow leopard (Panthera uncia). As the blue sheep has been increasingly exposed to human pressures, it is vital to estimate its population dynamics, protect the key populations, identify important habitats, and secure a balance between conservation and local livelihoods. We conducted a study in Manang, Annapurna Conservation Area (Nepal), to survey blue sheep on 60 transects in spring (127.9 km) and 61 transects in autumn (134.7 km) of 2019, estimate their minimum densities from total counts, compare these densities with previous estimates, and assess blue sheep habitat selection by the application of generalized additive models (GAMs). Total counts yielded minimum density estimates of 6.0–7.7 and 6.9–7.8 individuals/km² in spring and autumn, respectively, which are relatively high compared to other areas. Elevation and, to a lesser extent, land cover indicated by the normalized difference vegetation index (NDVI) strongly affected habitat selection by blue sheep, whereas the effects of anthropogenic variables were insignificant. Animals were found mainly in habitats associated with grasslands and shrublands at elevations between 4,200 and 4,700 m. We show that the blue sheep population size in Manang has been largely maintained over the past three decades, indicating the success of the integrated conservation and development efforts in this area. Considering a strong dependence of snow leopards on blue sheep, these findings give hope for the long‐term conservation of this big cat in Manang. We suggest that long‐term population monitoring and a better understanding of blue sheep–livestock interactions are crucial to maintain healthy populations of blue sheep and, as a consequence, of snow leopards.     

西藏布达拉宫节肢动物群落特征及其变化

为了解西藏布达拉宫古建筑内节肢动物群落特征及时空分布,为古建筑室内生物多样性资源提供基础数据,同时为布达拉宫的修缮及防虫工作提供科学依据,结合布达拉宫2015年5月至7月防虫工作的开展,在布达拉宫古建筑室内设置5个样点,对室内节肢动物群落特征和时空变化进行了初步研究。采用杀虫剂击倒法和手捡法共捕获室内节肢动物1 124只,隶属于2纲7目12科17种(类)。其中,优势种有3种,分别为拉萨索砚甲(Solskia lhasana Ren et Yu.)、棕蛛甲(Ptinus clavipes)和藏显缘短跗窃蠹(Ernobius sp.)。随时间变化,布达拉宫节肢动物物种数(S)、群落多样性指数(H)及均匀性指数(E)在5月至7月总体呈上升趋势。随空间变化,列康顶库房和扎厦三层节肢动物丰富度最大,德阳厦地垄丰富度最小。藏显缘短跗窃蠹在五个样点均有分布,是布达拉宫木材的主要害虫。     

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses

The schizothoracine fishes, also known as “mountain carps” are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a “morphologically primitive” clade and “morphologically specialized” clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the “morphologically primitive” clade in the Late Miocene and another by the “morphologically specialized” clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the “morphologically specialized” clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.     

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.     

Assembly of retinal rod or cone Na(+)/Ca(2+)-K(+) exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs

Proper control of intracellular free Ca(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca(2+) entry and Ca(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na(+)/Ca(2+)-K(+) exchanger (NCKX) is the only Ca(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments.     

万顺铅锌矿区蜈蚣草内生细菌砷氧化基因(aoxB)的多样性分析

【背景】近年来,由于金属矿的开采和冶炼、砷产品的加工与使用、煤的燃烧等各种因素,导致土壤环境中的砷污染越来越严重,导致许多人暴露于极度危险的砷毒毒害之下。【目的】研究四川的万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因的多样性,为提高土壤重金属污染生态修复效率提供理论依据。【方法】利用实时荧光定量PCR (Real-time quantitative PCR,qPCR)和限制性片段长度多态性(Restriction fragment length polymorphism,RFLP)技术,对四川省汉源县万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因表达量及多样性进行研究。【结果】qPCR结果表明,不同采样点间的蜈蚣草根组织内生细菌aoxB基因表达量存在明显差异,表现为选矿区进山口弃渣场尾矿区矿口。酶切图谱结果表明,不同采样点蜈蚣草根组织内生细菌aoxB基因多样性存在明显差异,多样性指数表现为尾矿矿口弃渣场进山口选矿区。Pearson相关分析显示,aoxB基因的表达量与重金属As之间呈显著负相关(P0.05),多样性指数则与重金属Pb和As之间呈极显著正相关(P0.01)。系统发育分析显示,aoxB基因的优势菌群为α-变形菌门(Alphaproteobacteria)。【结论】蜈蚣草根组织中存在丰富的含aoxB基因内生细菌种群,这些内生细菌表现出潜在的应用价值。