Telocytes in pancreas of the Chinese giant salamander (Andrias davidianus)

Telocytes (TCs), novel interstitial cells, have been identified in various organs of many mammals. However, information about TCs of lower animals remains rare. Herein, pancreatic TCs of the Chinese giant salamanders (Andrias davidianus) were identified by CD34 immunohistochemistry (IHC) and transmission electron microscopy (TEM). The IHC micrographs revealed CD34⁺ TCs with long telopodes (Tps) that were located in the interstitium of the pancreas. CD34⁺ TCs/Tps were frequently observed between exocrine acinar cells and were close to blood vessels. The TEM micrographs also showed the existence of TCs in the interstitium of the pancreas. TCs had distinctive ultrastructural features, such as one to three very long and thin Tps with podoms and podomers, caveolae, dichotomous branching, neighbouring exosomes and vesicles. The Tps and exosomes were found in close proximity to exocrine acinar cells and α cells. It is suggested that TCs may play a role in the regeneration of acinar cells and α cells. In conclusion, our results demonstrated the presence of TCs in the pancreas of the Chinese giant salamander. This finding will assist us in a better understanding of TCs functions in the amphibian pancreas.     

池养大鲵的人工催产研究

本文报道了人工培育大鲵亲本的繁殖结果。在养殖条件下,大鲵的性腺可正常发育,大鲵的性成熟年龄为5龄,性周期为一年一次,群体的繁殖高峰期出现在每年的6—8月。使用LRH-A或HCG均可诱导大鲵产卵和排精。群体的性腺发育进程存在明显的个体差异,雄性比雌性的发育时间长10—20d。在繁殖盛期进行人工催产,获产率为60%左右。人工繁育的子代,在培育为亲本时,其性腺发育的个体差异仍然存在。养殖的性成熟个体,在没有受到外源激素作用时,将不会发生自然排卵现象,雄鲵不能挤出精液。在20—23℃下,雌性大鲵的效应时间约为96—120h,雄性可在80h后挤到精液。大鲵的产卵量为400—500粒,平均为430粒。多次产卵个体的产卵量多于初次产卵个体。低剂量的药物催熟,未发现对性腺发育进程和产卵结果有明显的促进作用。     

中国大鲵研究进展

中国大鲵是我国特有濒危的两栖物种,是研究生物进化、生物多样性、性别决定分子机制等问题的好材料。近年来,人们对它的研究力度不断加大,著述颇多。本文对大鲵的生态保护、解剖发育、生理生化、遗传进化等方面的近期研究资料进行了整理和回顾,也简要探讨了今后大鲵研究的主要工作,以期为研究者们提供有价值的资料。     

大鲵的自然受精试验

众所周知,在进行家鱼人工繁殖时,关于受精的方法有两种：一种是自然交配、自然产卵、自然受精;另一种是人工采精、人工采卵、人工授精。自然受精和人工授精,各有各的优点,不能相互取代,各生产单位可根据自已的生产条件选用。在进行大鲵人工繁殖时,关于受精的方法,也应该有两种。目前全国有30余所科研院所和院校进行研究(见“大鲵成熟精、卵的形态学观察及受精卵孵化中的形态变化”《淡水渔业》1999年第3期),迄今然仍未见大鲵的自然受精方面的报道。我们做了一点工作,报道如下供参考。     

Ultrastructure of the skin mechanoreceptors of the Chinese giant salamander, Andrias davidianus

The ultrastructure of two kinds of mechanoreceptive organs, pit organs and neuromasts, in the skin of adult giant salamanders (Andrias davidianus) was studied by transmission electron microscopy. Neuromasts and pit organs differ in their types of synapses, the spatial distribution of kinocilia on sensory cells, and in the degree to which sensory cells are separated by processes of the supporting cells; the two organs probably serve complementary functions. The neuromasts in A. davidianus differ from those of other salamanders in the orientation of kinocilia, in the extent of intrusion of supporting cells into the sensory layer, in the degree of thickening of the synaptic membranes, in the distribution of synaptic spheres, and by the absence of a cupula.     

患病中国大鲵中分离到一株虹彩病毒及其特性的研究

从陕西某大鲵养殖场患病的大鲵体内分离到一株病毒。患病大鲵以体表溃疡,特别是肢体远端溃烂为主要临床特征。该病毒于10℃~30℃能在BF-2(Caudal trunk cells of blue-gillfry)、CO(Gorad cells of grass carp)、CHSE(Embryo cells of Chinook salmon)、FHM(cells of fathed minnow)等细胞中较好地增殖,最适生长温度为25℃~30℃。病毒对氯仿、热、pH3、pH10敏感,DNA抑制剂5-氟-2′-脱氧尿苷(5-fluro-2-′deoxyuridine,FUDR)能抑制病毒在细胞中的增殖,提示该病毒是有囊膜的DNA病毒。经电镜观察,在感染了病毒的细胞切片中可见到大量直径约130~150 nm有囊膜的六角形病毒颗粒成晶格排列在细胞质里,病毒呈典型的虹彩病毒形态。抽提病毒核酸后进行PCR扩增,用已知蛙病毒主要衣壳蛋白(MCP)基因的保守序列设计的引物能扩增出431bp的片段。扩增的片段测序后,和已知的几种蛙病毒属成员的主要衣壳蛋白基因中的相应片段进行比对,相似性在96%以上。血清学试验结果显示该病毒和IPNV(Infectious pancreatic necrosis virus,IPNV)、GCRV(Grass carp reovirus,GCRV)、SVCV(Spring viraemia of carp virus,SVCV)I、HNV(Infectious hematopoietic necrosis virus,IHNV)在血清学上没有相关性。以上结果提示该病毒可能是虹彩病毒科蛙病毒属的成员,暂时命名为大鲵虹彩病毒(Andrias davidianus iridovirus,ADIV)。该病毒与大鲵发病的关系有待进一步研究。     

多普勒B超鉴定中国大鲵性别

首次报道了从影像学水平上鉴定中国大鲵性别的研究结果。采用多普勒B超,常规方法直接扫描雌雄中国大鲵腹部,并记录大鲵的精巢或卵巢的形状、内部回声。实验结果表明,雌雄中国大鲵的检测结果存在明显的差畀,在雄性中国大鲵的腹部,可检测到精巢组织;在雌性中国大鲵的腹部,可检测到为卵巢组织和卵泡。初步分析了实验结果,并认为将多普勒B超鉴定法与泄殖孔法鉴定相结合可以作为中国大鲵性别鉴定的可靠方法。     

患病大鲵中弗氏柠檬酸杆菌的分离与鉴定

【目的】确定导致大鲵(Andrias davidianus)细菌性感染死亡的病原。【方法】从大鲵肝脏中分离细菌,通过Biolog微生物自动鉴定系统及分子生物学方法对纯培养的细菌进行鉴定,再用大鲵和鲫鱼分别进行人工感染试验,以确定分离菌的致病性,同时对分离到的病原菌进行药物敏感试验。【结果】从患病大鲵肝脏中分离到一株致病菌JZ01,经人工感染健康大鲵,可复制与自然发病相同的症状,且从人工感染病鲵体内再次分离到相同的病原菌。该致病菌对健康鲫鱼也有致病性。经Biolog微生物自动鉴定系统的鉴定,以及进一步的16S rDNA基因序列和系统发育分析都表明,此致病菌为弗氏柠檬酸杆菌。药物敏感性试验表明,该菌株对氨曲南、头孢三嗪、先锋噻肟等9种药物高度敏感。【结论】弗氏柠檬酸杆菌是大鲵的一种致病菌。本文在国内外首次报道了该菌对大鲵具有致病性。     

Genetic diversity of Chinese giant salamander (Andrias davidianus) based on the novel microsatellite markers

Chinese giant salamander (Andrias davidianus) is a rare amphibian species in the world. Microsatellite markers are a promising tool for accurate estimation population structure and genetic diversity. In this paper, we isolated novel microsatellite marker for Chinese giant salamander using fast isolation by AFLP of sequences containing repeats (FIASCO) method. More than 50% sequences in 132 clones had repeat number over ten times with di or trinucleeotide repeat except of (GA)₁₂. Seventy pairs of primers were designed and eleven polymorphic microsatellite loci were characterized for wild and cultivated Chinese giant salamander populations. The allele number was from 3 to 9 in different loci. Polymorphism information content was from 0.544 to 0.702 in cultivated population. The results implied more alleles and PIC were in the wild population than cultivated population. The observed heterozygosities in two populations were higher than 0.553. The data analysis suggested that the cultivated population has lower genetic diversity than wild population, which it??s perhaps owing to inbreeding in artificial breeding. To our knowledge, it??s the first time to isolated microsatellite markers for Chinese giant salamander. The result indicated that the markers were suitable for the population genetic analysis of Chinese giant salamander.     

张家界大鲵人工放流效果及其影响因素分析

大鲵(Andrias davidianus)是我国特有的两栖动物, 由于栖息地破坏和人类过度捕杀等, 种群数量急剧下降, 被列为我国II类保护野生动物。为探索大鲵人工放流的有效方法, 实现野生大鲵资源增殖, 作者对2002–2008年间在湖南张家界大鲵国家级自然保护区内进行的大鲵人工放流活动与放流效果进行了评估, 并通过对放流点的隐蔽物、水质及饵料生物等生境特点的分析, 探讨影响放流成败的因素。该保护区内共在9处大鲵原栖息地进行了11次人工放流活动, 共放流各种规格大鲵995尾, 其中4次成功。成功放流的大鲵为1–4.5 kg的性成熟个体, 雌雄比在1:1和1:1.5之间, 成功放流点均有良好的后期管理措施, 如建立保护站, 补充饵料等。虽然所选择的多数放流点生境基本能满足大鲵生长和繁殖的要求, 但由于受水质、饵料或安全某一因素的制约, 野生大鲵资源增殖效果不明显, 导致放流失败。因此我们认为依据野生大鲵繁殖的生境特征标准, 科学选择放流地点, 选择性成熟的大鲵在春季放流, 并进行完善的后期跟踪保护, 可能是大鲵人工放流成功和实现资源增殖的有效途径。     

中国大鲵生殖激素的初步研究

首次全面报道了中国大鲵6项生殖激素的研究结果.采用电化学发光技术,检测了雌雄中国大鲵在繁殖期与非繁殖期的血清中垂体泌乳素、卵泡生成激素、黄体生成激素、雌二醇、孕酮和睾酮6项生殖激素的含量.实验结果表明,雌雄性中国大鲵在繁殖期间与非繁殖期间的6项生殖激素检测结果没有明显的差异,对中国大鲵血清中的生殖激素与繁殖期的相关性进行了初步探讨,为中国大鲵生殖内分泌学的研究和人工繁殖积累了资料.     

用生理的方法促使大鲵产卵的研究

使用HCG、LRH-A、HCG LRH-A、RES LRH-A、DOM LRH-A等催产药物的五种不同组合形式,在26℃-27℃、21℃-23℃、16℃-19℃三种温度下催产大,结果表明：在21℃-23℃条件下,每千克雌注射DOM10mg LRH-A30μg催卵效果相对稳定。     

Major biological activities of the skin secretion of the Chinese giant salamander, Andrias davidianus

Amphibian skin can produce abundant secretion which contains many bioactive compounds. In this work, skin secretion of the Chinese giant salamander (Andrias davidianus) was obtained by mild electrical stimulation of the dorsal skin surface, and the main physiopathological properties of the secretion were described. Intraperitoneal administration of the skin secretion caused lethal effects in mice. Low doses of the skin secretion induced significant systemic and local effects like edema and nociception in mice. The activities of phospholipase A2 and proteolytic enzyme were likely related to the physiopathological activities observed. The work proved the complex toxic effects of the Chinese giant salamander skin secretion and provided clues to study its physiological function further.     

中国大鲵闭锁卵泡的显微和超微结构观察

用光镜和电镜观察了中国大鲵卵泡闭锁过程和闭锁小体的显微和超微结构。结果显示 ,大鲵闭锁小体是卵泡细胞侵噬卵母细胞并增殖形成细胞团 ,膜细胞未参与。在大部分卵泡处于缓慢生长期时 ,未发现卵泡闭锁现象 ;在 5、 6月份 ,卵巢内大部分卵母细胞进入卵黄形成前期 ,部分卵泡闭锁 ,但闭锁小体细胞的类固醇激素分泌结构特征不明显 ;在 7、 8月份 ,大多数卵母细胞处于卵黄形成期 ,闭锁小体细胞具有管泡状嵴线粒体、丰富的滑面内质网和脂滴、发达的高尔基体等。这些细胞学特征表明闭锁小体可分泌类固醇激素 ,以调节正常卵子的成熟。在大鲵中观察到的闭锁小体属于排卵前黄体     

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.     

中国大鲵 Dmrt 基因 DM 结构域的克隆及序列分析

Dmrt 基因家族是一个与性别决定相关的基因家族,该家族成员共有一个具有 DNA 结合能力的保守基序-DM 结构域。为了进一步探讨该家族在系统进化中的保守性,本研究通过简并 PCR 技术,扩增并克隆了中国大鲵（Andrias davidianus）基因组中的 DM 结构域。序列分析显示,中国大鲵基因组中存在 Dmrt 基因的 DM 结构域。其核酸序列与猕猴、青鳉、人、小鼠、牛、热带爪蟾相应 Dmrt 基因 DM 结构域的相似性分别为 91%、92%、92%、89%、91%、84%。其蛋白序列与上述物种的相似性均为91%,表现为4个氨基酸的变异。即第 19、34、36 和 45 位的精氨酸分别由半胱氨酸、谷胱酰胺、色氨酸和谷胱酰胺所取代。这些氨基酸的变化对其蛋白总体的三维构型没有显著影响。聚类分析结果表明,不同进化地位物种的 Dmrt 基因 DM 结构域编码序列存在高度的同源性,显示 Dmrt 基因在系统进化上的高度保守。     

大鲵肝脏组织定向cDNA文库的构建及鉴定

目前关于大鲵的胚胎发育、繁殖育种及养殖条件的优化等已有不少研究(李峰等,1 998;张育辉等,1 999) ,此外也有一些学者研究了大鲵的习性与生理生化特点(杨国华等,2 0 0 1 ;乔志刚等,2 0 0 3)。但这些研究仍停留在组织和细胞水平,加之大鲵取材来之不易,使研究具有一定的局限性。最近有学者分离了大鲵线粒体基因组全长并完成了测序工作,对于两栖类的遗传多态性研究有重要意义(Zhangetal.,2 0 0 3)。目前有关大鲵的遗传学背景资料仍然缺乏,尚未见特异的大鲵相关基因的报道,相应的遗传学工程课题更是无法开展。随着分子生物学技术的发展和人类…     

Determining threatened species distributions in the face of limited data: Spatial conservation prioritization for the Chinese giant salamander (Andrias davidianus)

The purpose of this study was to determine whether limited occurrence data for highly threatened species can provide useful spatial information to inform conservation. The study was conducted across central and southern China. We developed a habitat suitability model for the Critically Endangered Chinese giant salamander (Andrias davidianus) based on one biotic and three abiotic parameters from single‐site locality records, which represent the only relevant environmental data available for this species. We then validated model quality by testing whether increased percentage of predicted suitable habitat at the county level correlated with independent data on giant salamander presence. We randomly selected 48 counties containing historical records which were distinct from, and independent of, the single‐site records used to develop the model, and 47 additional counties containing >50% predicted suitable habitat. We interviewed 2,812 respondents near potential giant salamander habitat across these counties and tested for differences in respondent giant salamander reports between counties selected using each method. Our model predicts that suitable giant salamander habitat is found widely across central and southern China, with counties containing ≥50% predicted suitable habitat distributed in 13 provinces. Counties with historical records contain significantly more predicted suitable habitat than counties without historical records. There are no statistical differences in any patterns of respondent giant salamander reports in surveyed counties selected from our model compared with the areas of known historical giant salamander distribution. A Chinese giant salamander habitat suitability model with strong predictive power can be derived from the restricted range of environmental variables associated with limited available presence‐only occurrence records, constituting a cost‐effective strategy to guide spatial allocation of conservation planning. Few reported sightings were recent, however, with most being over 20 years old, so that identification of areas of suitable habitat does not necessarily indicate continued survival of the species at these locations.     

Reduced genetic variation in the Japanese giant salamander, Andrias japonicus (Amphibia: Caudata)

The phylogenetic relationships among 46 samples from 27 populations of the Japanese giant salamander, Andriasjaponicus and its congener, A. davidianus from China was investigated, using 3664 bp sequences of the mitochondrial genes NADH1, NADH3, cyt b and CR, partial NADH6 and intervening genes. In phylogenetic trees constructed by MP, ML, and Bayesian methods, the family Cryptobranchidae and the genus Andrias both form monophyletic groups. Japanese A. japonicus and Chinese A. davidianus are sister taxa and can be regarded as separate species despite a small degree of genetic differentiation. Andriasjaponicus is divided into central and western clades, but the phylogenetic relationships within the latter clade are unresolved. As previously reported from allozyme analyses, A. japonicus exhibits little genetic differentiation, in strong contrast to salamanders of the genus Hynobius with which their distributions overlap. This reduced genetic variability in A. japonicus is attributable to a unique mating system of polygyny, delayed sexual maturity, notable longevity, life in a stable aquatic environment, and gigantism, as well as bottleneck effects following habitat fragmentation and extinction of local populations during Quaternary glaciations. The species is thus susceptible to extinction by potential environmental fluctuations, and requires extensive conservation measures.     

Genetic variability among endangered Chinese giant salamanders, Andrias davidianus

The endangered Chinese giant salamander (Andrias davidianus) is endemic to mainland China. Genetic divergence among six populations of the species was investigated by means of isozyme electrophoresis and mitochondrial DNA (mtDNA) sequences. Forty allozyme loci were resolved for all populations; the amount of genetic divergence among populations was comparable to that in other amphibians. mtDNA sequences showed a similar level of divergence. The population from Huangshan is distinct from other populations, indicating the existence of localized divergence. Both allozyme and mtDNA data failed to associate the populations into a pattern corresponding to the three Chinese river systems, which may be the consequence of human relocation. Conservation policies should emphasize the protection of localized populations and cessation of human-facilitated introductions. Future studies should focus on investigating the divergence among localized populations from isolated mountain regions, particularly using more fine-grained techniques such as microsatellite DNA.