Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude

Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.     

低氧对雄性高原鼠兔性腺的影响

在人工模拟低氧环境下（低压舱模拟５０００ｍ和７０００ｍ海拔高度）,低氧暴露２４ｈ和７ｄ,观察低氧对受试动物性腺的影响。结果表明,急性低氧２４ｈ,高原鼠兔血浆雌二醇（Ｅ２）明显升高;低氧暴露７ｄ,高原鼠兔血浆Ｅ２仍维持一较高水平;５０００ｍ低氧暴露７ｄ,其睾丸指数无明显变化,７０００ｍ时却有所降低。同等条件下,大鼠睾丸指数明显增高;５０００ｍ和７０００ｍ低氧暴露７ｄ对高原鼠兔睾丸组织形态无明显影响,然而,大鼠曲细精管间隙增大,且曲细精管内各级细胞排列紊乱。低氧环境下,高原鼠兔雄体血浆Ｅ２增高,可能是其低氧适应的特征之一     

Leptin cDNA cloning and its mRNA expression in plateau pikas (Ochotona curzoniae) from different altitudes on Qinghai-Tibet Plateau

Leptin, an adipocyte-derived hormone, plays an important role in body energy homeostasis. Plateau pika (Ochotona curzoniae), an endemic and keystone species living only at 3000-5000 m above sea level on Qinghai-Tibet Plateau, is a typically high hypoxia and low temperature tolerant mammal with high resting metabolic rate (RMR), non-shivering thermogenesis (NST), and high ratio of oxygen utilization to cope with harsh plateau environment. To explore the molecular mechanism of ecological acclimation in plateau pika, we first cloned pika leptin cDNA and compared its mRNA expression in different altitudes (3200 and 3900 m) using real-time RT-PCR (Taqman probe) technology. The full-length pika leptin cDNA was 3015 with 504 bp open-reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. Pika leptin was 70-72% homologous to that of other species and was of similarly structural characteristics with other species. The pika-specific genetic diversity in leptin sequence occurred at twenty sites. With the increase in altitude, there were larger fat store and high level of ob gene expression in plateau pika. Our results indicated that leptin is sensitive to cold and hypoxia plateau environment and may play one of important roles in pika's ecological adaptation to harsh plateau environment.     

低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析

低氧性肺血管收缩反应钝化是高原鼠兔适应低氧环境的重要策略,但参与该生理代偿反应的功能基因尚不明确。间隙连接蛋白40 (Connexin40, Cx40) 在哺乳动物肺血管内皮表达。本研究对生活在海拔3 200 m的高原鼠兔进行28 d模拟海拔5 000 m低氧处理,以Sprague Dawley (SD) 大鼠为对照,采用免疫组化法分析模拟低氧处理后高原鼠兔和SD大鼠肺组织形态结构,qPCR和蛋白印记法检测Cx40基因和蛋白表达量变化,探究Cx40在高原鼠兔低氧性肺血管收缩反应钝化中的潜在作用。结果显示,低氧处理后,高原鼠兔肺泡呈空泡囊状,Cx40蛋白在支气管和肺血管中均表达,Cx40 基因mRNA水平随着低氧暴露而升高,但其蛋白质水平呈下降趋势,肺支气管Cx40蛋白无明显变化。SD大鼠肺血管和肺支气管表达的Cx40蛋白均无明显变化。暗示生活在高海拔低氧环境中的高原鼠兔,Cx40蛋白下调可抑制血管收缩信号,减弱低氧性肺血管收缩反应,使低氧性肺血管收缩反应钝化,以适应高原缺氧环境。研究结果可为高原土著动物适应高寒缺氧环境提供基础理论数据。     

高原鼠兔血清无机元素含量的分析

测定分析了高原鼠兔血清电解质、无机元素、血清铁和总铁结合力, 并与人和其它实验动物的相应指标进行了比较。得出以下主要结论: 1.高原鼠兔血清电解质浓度不受生活环境和食物条件的影响, 其机体能自身调节血清离子浓度, 以维持电解质在血液中的相对衡定; 2.高原鼠兔血清铁含量与其栖息地海拔相关, 随海拔升高血清铁含量增加; 3.高原鼠兔通过增加运铁蛋白与铁的结合力作为适应高原低氧环境的方式之一。     

青藏高原5种害鼠D型肉毒素抗性相关基因VAMP1的序列分析

突触小泡相关膜蛋白1基因(VAMP1)的变异是导致鼠类对D型肉毒梭毒素灭鼠剂产生抗性的主要原因。本研究利用转录组测序的方法,分析青藏高原地区5种主要害鼠:高原鼠兔(Ochotona curzoniae)、高原鼢鼠(Eospalax baileyi)、长尾仓鼠(Cricetulus longicaudatus)、青海松田鼠(Neodon fuscus)和喜马拉雅旱獭(Marmota himalayana)的VAMP1序列信息。同时,分别采集来自5个地理种群的58只高原鼠兔和59只高原鼢鼠,对VAMP1基因第二外显子区序列进行分析。结果显示,从转录组组装文件中成功获得5种动物的VAMP1基因全序列,长度均为357 bp,共检测到46个核苷酸变异位点和4个氨基酸变异位点,但未发现与D型肉毒素抗性相关的氨基酸位点。对高原鼠兔群体和高原鼢鼠群体的VAMP1基因第二外显子序列的分析显示,高原鼠兔所有个体的序列高度保守,而在高原鼢鼠中则存在一个同义突变位点,但两物种在D型肉毒素抗性相关位点上都未监测出位点变异。该研究结果提示,D型肉毒杀鼠剂在青藏高原地区害鼠防治方面应该可以长期发挥重要作用。     

Natural selection and adaptive evolution of leptin in the ochotona family driven by the cold environmental stress

Background

Environmental stress can accelerate the evolutionary rate of specific stress-response proteins and create new functions specialized for different environments, enhancing an organism''s fitness to stressful environments. Pikas (order Lagomorpha), endemic, non-hibernating mammals in the modern Holarctic Region, live in cold regions at either high altitudes or high latitudes and have a maximum distribution of species diversification confined to the Qinghai-Tibet Plateau. Variations in energy metabolism are remarkable for them living in cold environments. Leptin, an adipocyte-derived hormone, plays important roles in energy homeostasis.

Methodology/Principal Findings

To examine the extent of leptin variations within the Ochotona family, we cloned the entire coding sequence of pika leptin from 6 species in two regions (Qinghai-Tibet Plateau and Inner Mongolia steppe in China) and the leptin sequences of plateau pikas (O. curzonia) from different altitudes on Qinghai-Tibet Plateau. We carried out both DNA and amino acid sequence analyses in molecular evolution and compared modeled spatial structures. Our results show that positive selection (PS) acts on pika leptin, while nine PS sites located within the functionally significant segment 85-119 of leptin and one unique motif appeared only in pika lineages-the ATP synthase α and β subunit signature site. To reveal the environmental factors affecting sequence evolution of pika leptin, relative rate test was performed in pikas from different altitudes. Stepwise multiple regression shows that temperature is significantly and negatively correlated with the rates of non-synonymous substitution (Ka) and amino acid substitution (Aa), whereas altitude does not significantly affect synonymous substitution (Ks), Ka and Aa.

Conclusions/Significance

Our findings support the viewpoint that adaptive evolution may occur in pika leptin, which may play important roles in pikas'' ecological adaptation to extreme environmental stress. We speculate that cold, and probably not hypoxia, may be the primary environmental factor for driving adaptive evolution of pika leptin.     

Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and plateau pika in China

The taeniid cestode Echinococcus shiquicus n. sp. was found from the Tibetan fox Vulpes ferrilata and the plateau pika Ochotona curzoniae in the Qinghai-Tibet plateau region of China. In the adult stage, E. shiquicus from the foxes is morphologically similar to Echinococcus multilocularis. However, the new species is differentiated by its smaller rostellar hooks, fewer segments, distinct position of genital pore in the mature segment and fewer eggs in the gravid segment. Hydatid cysts of E. shiquicus found in the livers from the pikas were essentially unilocular but an oligovesicular cyst was also found. The data of mitochondrial and nuclear DNA sequences proved E. shiquicus to be a valid taxon.     

Postnatal ontogenetic size and shape changes in the craniums of plateau pika and woolly hare(Mammalia: Lagomorpha)

In the present study, postnatal ontogenetic size and shape changes in the cranium of two lagomorph species, the plateau pika(Ochotona curzoniae) and woolly hare(Lepus oiostolus), were investigated by geometric morphometrics. The ontogenetic size and shape changes of their cranium exhibited different growth patterns in response to similar environmental pressures on the Qinghai-Tibetan Plateau. The overall size change in the cranium of the plateau pika was slower than that of the woolly hare. The percentage of ontogenetic shape variance explained by size in the woolly hare was greater than that in the plateau pika. The overall shape of the cranium was narrowed in both species, and morphological components in relation to neural maturity showed negative allometry, while those responsible for muscular development showed isometric or positive allometry. The most remarkable shape variations in the plateau pika were associated with food acquisition(temporalis development), though other remarkable shape variations in the incisive and palatal foramen in the ventral view were also observed. The most important shape change in the woolly hare was demonstrated by the elongation of the nasal bones, expansion of the supra-orbital process and shape variation of the neurocranium.     

Behavioral mechanisms of male sterilization on plateau pika in the Qinghai-Tibet plateau

Fertility control is an alternative non-lethal method in the management of rodents. Previous modeling suggests that the efficacy of male sterilization depends on mating systems of animals, but behavioral mechanisms of male sterilization have not been investigated. Here we investigated the behavioral mechanism of the sterilant quinestrol in reducing the fertility of plateau pika (Ochotona curzoniae) inhabiting the Qinghai-Tibet plateau. Male pikas treated with quinestrol showed reduced aggression compared to control males, but they showed significantly higher levels of territorial behavior such as long-calls and long-chases. Levels of long-call and long-chase were negatively correlated with the number of newborn pikas in the family. Single-baiting of quinestrol effectively sterilized male pikas and reduced the pregnancy rate of female pikas; this was likely achieved by increased territorial behavior of sterilized pikas which resulted in unsuccessful invasions by fertile adult male pikas. Our study reveals a novel behavioral mechanism, increased territoriality in sterilized males, in the fertility control of plateau pikas.     

Effects of hypoxia on contents of essential elements in pika and rat heart

The effects of hypoxia on the levels of essential macroelements and trace elements (K, Na, Ca, Mg, Cu, Zn, Fe, and Mn) in the heart muscles of Wistar rats and plateau pikas (Ochotona curzoniae) were studied by atomic absorption spectrometry. Unlike the rat, the plateau pika is tolerant to hypoxia. The levels of K, Na, and the trace element Mn were not significantly changed in rat or pika hearts after exposure to hypoxia for 1, 10, or 25 d at simulated altitudes of 5000 and 7000 m. Other minerals (Ca, Mg, Cu, Zn, and Fe) were significantly affected by hypoxia and the levels followed different time-courses under different hypoxic regimes in these two animals. There were marked differences between the rat and pika in myocardial accumulation of essential elements such as Ca, which was increased to high levels in the rat but not affected in the pika. The results suggest that hypoxia affects animal physiological mechanisms by regulating the levels of essential elements.     

高寒草甸生态系统中高原鼠兔的繁殖特征

2002年4月至8月,在中国科学院海北高寒草甸生态系统定位站附近,采用标志重捕法对高原鼠免的繁殖特征进行了研究。高原鼠兔的繁殖具有明显的季节性差异,4月下旬和5月为繁殖高峰期,雄性的睾丸重而饱满,雌性的怀孕率最大。雄性成体的体重和睾丸重在繁殖期的不同时段具有显的差异,在4月11日至5月10日、6月11日至7月10日体重和睾丸重之间呈显正相关。雌性怀孕早期胚胎数与临产前胚胎数没有显差异,未发现胚胎吸收现象;同时,胚胎数在繁殖期的不同时段存在显差异。采用种群统计学中同生群的划分方法,将5～8月份出生的幼体依次记为L1、L2、L3、L4。在出生后20天内L1、L2幼体存活率明显高于L3、L4;在从出生50天至80天期间L4的存活率显高于L2。当年出生的雌雄幼体在发育上存在不同步现象,当年出生的雌性幼体性成熟早,有的可以直接参加繁殖,但当年出生的雄性幼体却无此现象。结果表明：在海北地区,经过综合治理后,高原鼠兔的生境发生改变,其繁殖策略也随之发生改变,即,减少每次繁殖活动的投入,增加繁殖次数,延长繁殖时间。     

高原鼠兔和昆明白小鼠肺组织结构比较

低氧性肺血管收缩反应钝化（blunted Hypoxic Pulmonary Vasoconstriction, bHPV）是高原鼠兔在进化过程中形成的对低氧环境的一种适应性策略。以胎儿期、幼龄期及成年期高原鼠兔和昆明白小鼠为研究对象,对两者的肺组织进行发育生物学、比较组织学和比较组织化学研究,以探讨高原鼠兔bHPV形成的组织形态学基础。结果表明：（1）高原鼠兔各时期肺组织微血管的双层弹性纤维含量均低于25%,无明显肌化现象;（2）高原鼠兔直径10~50 μm、50~100 μm和100~200 μm微血管的中膜肌层厚度与其外径比（MT/ED）随年龄增加而逐渐下降;成年高原鼠兔的肺组织微血管MT/ED10-50显著低于成年昆明白小鼠（P < 0.05）,但MT/ED50-100和MT/ED100-200均无明显的种间差异（P > 0.05）;（3）在直径10~50 μm直径范围微血管中,高原鼠兔和昆明白小鼠平滑肌细胞数与内皮细胞数的比值（SMC/EC）均随年龄增长呈上升趋势,且高原鼠兔SMC/EC的比值极显著低于同年龄组的昆明白小鼠（P < 0.01）。导致高原鼠兔bHPV发生的组织学基础可能是其肺组织中直径10~50μm微血管平滑肌细胞数量少。     

高原鼠兔低氧诱导因子- 1α的初步研究

Hypoxia-induced factor-1 plays a key role during the cell hypoxia trausduction. Hypoxia induced factor-1α (HIF-1α) is a functional subunit of hypoxia-indueed factor-1. Plateau pika ( Ochotona curron/ae), which is a Qinghai-Tibet plateau native animal lived above 3 000 m, has high ratio of oxygen utilization to adapt to plateau hypoxia environments. One fragment of the coding re-gion of eDNA sequence of plateau pika HIF-1α was obtained by RT-PCR technique using a degeneracy PCR primer based on previ-ously reported eDNA sequence in human, cattle, house mouse and Norway rat HIF-1α gene. It was directly inserted into the vector pMD18-T. DNA sequencing proved that it was highly homology with human (91%), cattle (91%), house mouse (89%) and Nor-way rat 89%) HIF-1α gene. The study provides basic important information for the HIF-1α cDNA whole sequence cloning of pla-teau pika and its functional study.     

Social and ecological influences on dispersal and philopatry in the plateau pika (Ochotona curzoniae)

Benefits and costs of dispersal and philopatry of the socialplateau pika (Ochotona curzoniae) were studied on the Tibetanplateau for 3 years. Although short-lived, plateau pikas livein cohesive family groups that occupy burrow systems in sedgemeadow habitat Most (57.8%) plateau pikas were philopatric,and dispersal movements were extremely restricted. No juvenilefemales or adult pikas moved more than two family ranges betweenyears; the greatest observed dispersal distances were by twojuvenile males that moved five family ranges from the familyof their birth. Traversing unfamiliar habitat was not a costof pika dispersal because most dispersers settled in familiesthat they could easily visit before dispersal. Dispersal movementsappeared to result in equalization of density among pika families,an expected result if competition for environmental resourcesinfluenced dispersal. Males did not disperse to gain advantagesin competition for mates, as evidenced by their moving to familieswith significantly fewer females. Females, however, moved tofamilies with significantly more males. Males provide abundantpaternal care, and significantly more offspring per female survivedto become adults from families with more adult males per adultfemale. Evidence concerning the influence of inbreeding avoidanceon natal dispersal was indirect. Some males exhibited natalphilopatry; thus some families had opportunity for dose inbreeding.Males and females that dispersed had no opposite-sex relativesin their new families. Philopatric pikas may have benefitedby remaining in families that exhibited low local densities,and philopatric females might have benefited from social cooperationwith relatives.     

The mating system and gene dynamics of plateau pikas

Evolutionary theory suggests that mating systems should have substantial effects on gene dynamics of local populations. In polygynous species, local 'breeding groups' may produce significant genetic structure, due to genetic differences among groups, and rate of loss of genetic variation from such populations may be considerably slowed. We examined possible influences of the variable mating system and family group structure on genetic properties of a population of plateau pikas (Ochotona curzoniae). Pika gene dynamics were examined via F-statistics and effective population sizes (N(e)), calculated from genetic correlations within and among individuals and families. Genetic correlations were estimated from mating patterns, population demography, and dispersal patterns. Substantial genetic structure within the population was indicated by a strongly positive F(LS). Genetic influence of natal dispersal out of pika families was indicated by a strongly negative inbreeding statistic (F(IL)=-0.34). Effective size of the population was not greatly different from the census population, whereas a traditional estimate of effective size of the population was much lower, indicating that the family structure of the pikas results in a slowed loss of genetic variation over time. Thus, even though mating patterns of plateau pikas were variable, family structure had a strong influence on pika gene dynamics.     

高原鼢鼠和高原鼠兔骨骼无机化学成分的研究Ⅱ.必需微量元素

本文报道了高原鼢鼠和高原鼠兔整体骨骼及头骨、脊柱骨和下肢骨中Ｃｕ、Ｚｎ、Ｆｅ、Ｍｎ、Ｃｏ、Ｎｉ、Ｍｏ、Ｃｒ、Ｔｉ、Ｆ、Ｓｅ和Ｇｅ１２种必需微量元素的含量,并进行了显著性差异和相关性分析。结果表明：只有Ｃｕ的含量在两种动物骨骼中没有显著性差异（Ｐ＞０．０５）,其余１１种元素均有显著性或极显著性差异（Ｐ＜０．０５或Ｐ＜０．０１）,其中Ｚｎ、Ｆｅ、Ｍｎ、Ｃｏ、Ｎｉ、Ｍｏ、Ｆ和Ｓｅ的含量,高原鼢鼠＞高原鼠兔,而Ｃｒ、Ｔｉ和Ｇｅ的含量,高原鼠兔＞高原鼢鼠。微量元素在头骨、脊柱骨和下肢骨中分布很不均衡,以头骨的微量元素最为丰富     

高原鼠兔自然寿命研究

1984至1987年2月在青海湖黑马河地区,以耳标观察法对高原鼠兔Ochotona curzoniae的
自然寿命进行了研究．其结果,高原鼠兔的生存曲线呈L型。最大寿命957天,平均寿命119.9天。1986年出生的幼体,第1胎雄鼠平均寿命108天,雌鼠106天;第2胎雄鼠为58天,雌鼠为66.3天;第3胎雄鼠为24.8天,雌鼠为15.4天．     

高原鼠兔低氧适应机制的研究概况

综述了新型实验动物——高原鼠兔对高原低氧环境的适应特点,并与移居大鼠、人类及世居高原人群的生理变化进行了比较,对于高原鼠兔的低氧适应机制,从血液学、氧的摄取、氧利用及肺循环的结构和功能,进行了较为系统的阐述,对高原适应的生理研究及发展方向也做了扼要介绍。