Protection and Utilization of Chinese Giant Salamander Germplasm Resources

Chinese giant salamander(Andrias davidianus)is salamander germplasm resource that only distributed in China.It is the material basis of the evolution principle research,genetic research and artificial breeding.Since the 1990s,overharvesting,water pollution and habitat changes have seriously destroyed the germplasm resources of A.davidianus,their population density has decreased since then.Strengthening the protection and rational utilization of A.davidianus germplasm resources are key factors in impacting the healthy,stable and sustainable development of A.davidianus industry.Under the above background,we discussed the current problems with the protection for A.davidianus germplasm resources,put forward suggestions for the conservation of A.davidianus germplasm resources.It is expected to provide a reference to effective protection for A.davidianus germplasm resources,and provide a scientific foundation for further studies,so as to promote the high-quality development of related industry.

Nutritional and medicinal characteristics of Chinese giant salamander (Andrias davidianus) for applications in healthcare industry by artificial cultivation: A review

Andrias davidianus,i.e.Chinese giant salamander(CGS),is one of the largest and oldest amphibians existing in the world and is also one of the valuable biological resources of China.Wild CGS has been threatened with extinction in the past decades due to over capturing,deterioration of natural environment,the slow breeding and growth of the wild species in nature.However,in the past twenty years,with the breakthrough and progress of artificial breeding technology by artificial insemination,the number of artificially cultivated CGS has increased rapidly.Artificially cultivated CGS can either be released to the CGS living environment to increase the population in nature or legally applied in food and medicinal industry as a feedstock due to the unique nutritional and medicinal values of CGS as recorded historically.In this review,the nutritional components,bioactive components and medicinal activities of the artificially cultivated CGS will be summarized.The mucus,skin,meat and bone of CGS contain many different bioactive substances thereby having various medicinal activities including anti-aging,anti-fatigue,anti-tumor,therapy of burn and anti-infection and other physiological functions.This paper will further discuss the potential applications of the artificially cultivated CGS in healthcare industry and prospects of future technological development.

Disordered Translocation is Hastening Local Extinction of the Chinese Giant Salamander

Biodiversity is declining globally by an unprecedented extinction rate.This is especially true for amphibians,accounting for 24.3%of all threatened vertebrates.As the largest extant amphibian species in the world,wild populations of the Chinese giant salamander(Genus Andrias)(CGS)have decreased dramatically because of overexploitation and habitat degradation.Translocation has become an important strategy for restoring threatened wild populations worldwide.However,disordered tra nsloca tion usually has negative effects on the native populations.We provide an overview of CGS translocation and show that disordered translocation can increase local population extinction.Nearly four times the estimated number of wild individuals have been released across China.There a re three types of translocation used for CGS,namely,reinforcement,reintroduction and ecological replacement,the last of which accounts for over one-third of translocations.Our genetic screening revealed that most released individuals were not from local populations,with one to four lineages detected in every release site(n=6).This disordered translocation can potentially reduce the genetic integrity of original populations.Hence,we suggest suspending current CGS translocation activities immediately,until more robust measures can be developed and implemented to improve the current translocation program,especially with respect to lineage identifica tion a nd the identifica tion of appropriate release sites.

Morganella morganii as a Causative Agent of Disease in the Chinese Giant Salamander (Andrias davidianus)

Morganella morganii strain 602-1 was isolated from a sick Chinese giant salamander （Andrias davidianus）. The strain 602-1 was identified through its physiological and biochemical properties and 16S rDNA gene amplification and analyses. Pathogenicity was proven by experimental animal infection, and histopathological examination. The results showed that the amplified 16S rDNA sequence of strain 602-1 was 1455 bp, and showed 99% identity with M. morganii. In the host infection experiment, the mortality of Chinese giant salamanders was about 70%. Pathological changes occurred in the spleen, heart, liver, kidney and intestine. This study will help the prevention, understanding and cure for M. morganii infections in amphibians.

Development and Evaluation of a Loop-mediated Isothermal Amplification(LAMP) Assay for Rapid Detection of Chinese Giant Salamander Ranavirus

Loop-mediated isothermal ampliifcation （LAMP） is a novel nucleic acid diagnostic method that can amplify rapidly a target template under isothermal conditions. In this study, a LAMP assay for rapid detection of Chinese giant salamander ranavirus（CGSRV） was developed from culture isolates and clinical samples. The LAMP assay was developed by designing one set of four speciifc primers, targeting the major capsid protein （MCP） gene of CGSRV. Reaction time and temperature were optimal for 40 min at 62°C. The developed LAMP assay is speciifc and highly sensitive for CGSRV detection, the detection limit could reach about 5 copies of cloned viral genomic fragments. The sensitivity of the LAMP assay was about 1000 and 10-fold higher than that of both conventional and nested PCR, respectively. The LAMP ampliifcation produces a typical ladder-like pattern of products on an agarose gel that can be visually inspected after addition of ethidium bromide. The LAMP assay was evaluated further with clinical samples, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for the detection of CGSRV.

Molecular Characterization and Virulence Genes of Aeromonas hydrophila Isolated from the Chinese Giant Salamander (Andrias davidianus)

The Chinese giant salamander(Andrias davidianus) is the largest living amphibian in the world. Aeromonas hydrophila strain L602 was isolated from A. davidianus. The 16S rDNA gene of this isolate was amplified using PCR,and the phylogenetic tree was constructed by the neighbor-joining method. Four virulence genes(aerA,aha1,hly and alt) of A. hydrophila were amplified by PCR and drug resistances were tested using Kirby-Bauer disk diffusion method. The results showed that the length of this 16S rDNA sequence was 1453 bp,which showed 99% homology with A. hydrophila. The GenBank accession number was JX155398. Phylogenetic analysis indicated it grouped together with A. hydrophila. Four virulence genes were all detected,indicating that strain L602 was highly virulent. This stain was resistant to four antibiotics(vibramycin,furazolidone,ampicillin and erythromycin),while it was insensitive to streptomycin. Furthermore,this strain was susceptible to six antibiotics(sulfafurazole,ciprofloxacin,penbritin,norfloxacin,florfenicol and enrofloxacin). This study will help to validate the classification and virulence of pathogenic bacteria in amphibians.

Effects of Water Quality on the Distribution of Chinese Giant Salamander(Andrias davidianus)in Guizhou Province,China

13 water quality parameters were tested from 38 reaches of 34 counties in Guizhou where Chinese giant salamander(Andrias davidianus) distributed over the past 30 years. Dissolved oxygen and p H were found to be significant determinants of the species distribution in recent years(P<0.05). There was no Chinese giant salamander distribution in the recent five years in rivers with dissolved solids of greater than 415 mg/L, conductivity of greater than 639 us/cm, salinity of greater than 0.31 ppt and total hardness of greater than 150 mg/L. Sensitive to environment, the Chinese giant salamander is an important indicator for environmental quality, so it could be used as one of the environmental indicator. Eutrophication, chemical fertilizer, pesticides and inorganic pollutants may be one of reasons driving wild CGS into extinction.

Collected Data of Local Ecological Knowledge to Predict Current Status and Potential Threats of salamander(Andrias davidianusin)in Zhengan County of Guizhou Province,China

Chinese giant salamander(CGS, Andrias davidianus) is experiencing the threat of extinction. It is disappearing gradually, because of overharvesting, environmental pollution, specially river pollution, activity of human exploitation, and so on. We conducted community interview fieldworks to collect respondent data from Xieba Country(not reserve) situated in Zhengan County of Guizhou Province, where one of the places known by historical giant salamander records in Guizhou. Through the analysis of the collected data, we created quantitative data of use for conservation management on the local status of salamander populations, and we compared our data with those available data of Y. Pan et al., who investigated the status of CGS in 3 reserves in Guizhou Province in 2015.The conclusions have a similar trend, which proved that the collected data were creditable. Through statistical analysis of the comparison data, the number of wild CGS decreased actually followed the increase of artificial framing.Because of the source of breeding to catch wild CGS, overharvesting was likely the most reason to make wild CGS decline.

Transcriptomic analysis of Andrias davidianus meat and experimental validation for exploring its bioactive components as functional foods

Andrias davidianus(Chinese giant salamander,CGS)is the largest and oldest extant amphibian species in the world and is a source of prospective functional food in China.However,the progress of functional peptides mining was slow due to lack of reference genome and protein sequence data.In this study,we illustrated full-length transcriptome sequencing to interpret the proteome of CGS meat and obtain 10703 coding DNA sequences.By functional annotation and amino acid composition analysis,we have discovered various genes related to signal transduction,and 16 genes related to longevity.We have also found vast variety of functional peptides through protein coding sequence(CDS)analysis by comparing the data obtained with the functional peptide database.Val-Pro-Ile predicted by the CDS analysis was released from the CGS meat through enzymatic hydrolysis,suggesting that our approach is reliable.This study suggested that transcriptomic analysis can be used as a reference to guide polypeptide mining in CGS meat,thereby providing a powerful mining strategy for the bioresources with unknown genomic and proteomic sequences.

大鲵皮胶原抗氧化肽的制备及分离纯化

为高效利用大鲵加工副产物大鲵皮,对大鲵皮胶原抗氧化肽的制备及分离纯化进行研究。以1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为评价指标评价其抗氧化活性,研究大鲵皮胶原抗氧化肽的制备工艺,确定最适工艺条件,即酶添加量8000 U/g、酶解pH 9.0、酶解温度55℃、酶解时间4 h。在此工艺条件下,大鲵皮胶原抗氧化肽DPPH自由基清除率为40.2%。采用超滤分离得到分子质量不同的3个组分,即P1(>6 kDa)、P2(3~6 kDa)、P3(<3 kDa),其中P3具有较强的抗氧化活性,DPPH自由基清除率为56.7%。在此基础上,进一步采用凝胶色谱技术分离,通过Sephadex G-25分离纯化P3,得到不同分子质量的3个组分(SP1、SP2、SP3),其中SP3具有较强的抗氧化活性,DPPH自由基清除率为72.3%。最后采用氨基酸自动分析仪技术进一步分析SP3的氨基酸组成,探寻大鲵皮胶原肽抗氧化活性与其氨基酸组成之间的内在联系。结果表明:大鲵皮胶原抗氧化肽氨基酸组成中,甘氨酸、脯氨酸含量较高,胱氨酸含量较低,这一特征符合胶原抗氧化肽的氨基酸组成特征;除甘氨酸、脯氨酸含量较高外,大鲵皮胶原蛋白抗氧化肽中还含有较高含量的具有明显抗氧化活性的丙氨酸、精氨酸、谷氨酸、亮氨酸等氨基酸,这可能是大鲵皮胶原抗氧化肽SP3具有较高抗氧化活性的原因。

安徽金寨西河大鲵省级自然保护区植物多样性调查

本文采用线路、样地与样方调查相结合的方法,结合查阅文献、汇总植物数据库对安徽金寨西河大鲵省级自然保护区开展植物多样性调查。结果表明,保护区植被分为6个植被型组、12个植被型、31个群系;保护区内有蕨类植物27科52属100种,裸子植物5科6属10种,被子植物142科553属1173种,苔藓植物50科95属180种;国家珍稀濒危保护野生植物15种,其中国家重点保护野生植物12种(Ⅰ级2种,Ⅱ级10种),国家珍稀濒危植物9种(2级2种,3级7种)。根据保护区的植物资源现状,从加强保护宣传、加大科学研究和强化执法巡查3个方面提出针对性的保护建议。

大鲵保护生物学及其研究进展

大鲵 (Andriasdavidianus)为我国特有的珍稀两栖动物 ,已列入国家二级保护动物名录 ,并列入CITES公约附录I中。自 2 0世纪 5 0年代起 ,由于过度收购、非法捕杀和栖息地丧失等原因 ,大鲵种群数量下降极为严重 ,湖南、安徽等地的大鲵产量在 2 0世纪 5 0年代至 70年代下降超过 80 % ,分布区也极度萎缩 ,形成了 12块岛屿状区域 ,目前分布于以我国中部山区的长江流域为主的 17个省区。部分已建立的大鲵保护区经费短缺 ,人员不足 ,管理不力 ,状况不容乐观。非法捕捉和栖息地丧失仍是威胁大鲵生存的主要因素 。

响应面法优化酶法提取大鲵皮胶原蛋白工艺

为了优化人工养殖大鲵(Andrias davidianus Blanchard)皮中胶原蛋白的提取工艺,以加酶量、液料比和酶解时间为试验因素,胶原蛋白提取率为响应值,采用Box-Behnken设计进行试验。通过响应面法考察了3个因素对胶原蛋白提取率的影响,建立了胶原蛋白提取率的二次多项式回归模型,并对提取工艺进行了优化。试验结果表明,3个因素对鲵皮中胶原蛋白提取率的影响大小次序为:液料比>酶解时间>加酶量;交互项中"液料比-酶解时间"项对提取率影响显著,其他交互项对提取率影响不显著。为满足实际需要,对模型预测得到的大鲵皮胶原蛋白最优提取工艺稍做调整,具体参数为:加酶量16.5%、液料比15 mL/g、酶解时间29 h。此条件下,大鲵皮胶原蛋白提取率可达到66.99%,与模型理论预测值(67.83%)较为一致。

中国大鲵腐皮病病原菌的分离与鉴定

目的报告中国大鲵(Andrias davidianus)腐皮病的主要病原菌的检测及其生物学特征。方法对发病濒死的大鲵病变组织进行细菌学检查及分离,对分离菌进行形态特征、理化特性16S rDNA序列的分子鉴定,并构建系统发育树;进行人工感染试验及药敏试验。结果分离到1株优势菌,根据此株分离菌的形态特征及理化特性,结合16S rDNA序列测定与系统发育分析结果,判定其为气单胞菌属的维氏气单胞菌。此菌对供试健康大鲵、斑点叉尾鮰和小白鼠有较强的致病作用,对供试27种抗菌药物中的头孢美唑等6种高度敏感,对阿奇霉素和新霉素2种中度敏感,对氟哌酸等19种药物耐药。结论所检出的维氏气单胞菌为导致中国大鲵腐皮病的病原菌。

中国大鲵四种群的遗传结构和地理分化

为了确定栖息地的破碎化和片断化引起中国大鲵的地理分化和遗传结构变异,本文测定了来自广西、河南、陕西和湖南4个地理种群的28条大鲵的mtDNAD loop基因全序列。根据这4个地理种群的地理分布,分成珠江单元(广西种群)、黄河单元(河南种群)和长江单元(湖南和陕西种群)。通过ClustalX、MEGA2.0、DnaSP4.0、Arlequin1.1分析发现,全序列长度为771bp,其中64个多态性核苷酸变异位点,占全部碱基数的8.26%。转换和颠换分别为6和2个,插入/缺失11个。27个单倍型间的序列差异平均为1.32%。3个单元的单倍型多样性指数和核苷酸多样性指数值都偏低,而且珠江单元的这两个指数值都低于长江和黄河两个单元。珠江单元和黄河、长江单元之间分化程度显著(P<0.001),而长江和黄河单元之间差异不显著(P>0.05)。地理单元内分化程度占99.31%,而单元间只有0.69%,表明遗传差异主要发生在单元内,而且各地理单元之间的基因流较频繁。构建的NJ树和MP树显示,27个单倍型呈现一种混杂的分布格局,并未分成代表3个地理单元的聚合群。

中国大鲵肌肉、尾脂营养成分分析与评价

【目的】评价大鲵的营养价值,为其营养需要的确定及配合饲料的研制提供基础资料和理论依据。【方法】采用常规分析、氨基酸分析仪、气相色谱以及原子吸收光谱法,对大鲵肌肉和尾脂中的常规营养成分、氨基酸组成、脂肪酸成分进行分析,并按照氨基酸评分(Amino acid score,AAS)、化学评分(Chemical score,CS)、必需氨基酸指数(Essential acid score index,EAAI)标准进行品质评定。【结果】新鲜大鲵肌肉中水分含量为82.04%,粗蛋白含量为17.15%,粗脂肪含量为1.73%,灰分含量为0.65%;矿质元素中,Ca含量为162.15μg/g,Zn含量为15.35μg/g,P含量为1 020μg/g,其中Zn含量远高于其他动物。大鲵肌肉中共检测出17种氨基酸,总氨基酸含量为18.64%,所检7种人体必需氨基酸含量为7.25%,占总氨基酸的比例为38.89%;鲜味氨基酸含量为6.82%,占总氨基酸的比例为36.59%。肌肉及尾脂中分别检测出11和14种脂肪酸,其中饱和脂肪酸(SFA)含量分别为27.68%,25.70%,不饱和脂肪酸(UFA)含量分别为72.32%,74.20%,大鲵尾脂中含有较高的花生四烯酸(1.69%)和花生五烯酸(1.89%)等高不饱和脂肪酸;大鲵肌肉AAS、CS分别为1.660和0.810,EAAI(65.93)较一般水生经济动物高,蛋白质结构较合理。【结论】大鲵肌肉及尾脂具有极高的营养价值和食用价值。

酶解法提取大鲵尾部油的工艺研究

为寻求我国大鲵资源利用新途径,并建立大鲵尾部油的最佳提取方法及工艺,采用酶解法从大鲵尾部脂肪组织中提取大鲵尾部油,并对所得的大鲵尾部油进行理化性质及脂肪酸组成分析。结果表明,酶解法提取大鲵尾部油的最佳工艺条件为:酶添加量0.3%,料液比1∶1.0,酶解时间1.0 h,酶解温度60℃,p H 6.5;在最佳条件下,提取率为98.53%。所提的大鲵尾部油达到我国水产行业SC/T 3502—2000粗鱼油的一级标准。酶解法所提大鲵尾部油中检测出17种脂肪酸,单不饱和脂肪酸(MUFA)含量为49.42%,多不饱和脂肪酸(PUFA)含量为29.35%。

中国大鲵养殖繁育技术的探讨

通过１９８７ ～１９９８ 年连续１２ 年对中国大鲵（ Ａｎｄｒｉａｓ ｄａｖｉｄｉａｎｕｓ） 进行养殖繁育技术的研究，取得一定的成果。养殖周年成活率达７５ ％ ～９０ ％ ，从１９８９ 年起至今已连续１０ 年产卵，从１９９１ 年起至今已连续８ 年人工孵化出大鲵幼苗。

中国大鲵资源现状及保护遗传学研究进展

中国大鲵为我国特有珍稀两栖动物,主要分布于长江、黄河、珠江中下游及其支流。近十年来虽然中国大鲵人工繁殖及规模化繁育技术取得突破性进展,种群数量也有一定的恢复,但由于对中国大鲵遗传背景缺乏了解,故不能从根本上解决其濒危问题。目前,中国大鲵的相关研究主要侧重区系分类、生态、形态、解剖、繁殖、胚胎发育、起源演化等宏观方面,而遗传学、生理生化、细胞、分子生物学等微观方面的研究较少。结合现有资料将目前大鲵保护遗传学的研究进展情况进行概述。

大鲵肝脏组织定向cDNA文库的构建及鉴定

To construct a directional cDNA library from Chinese giant salamander Andrias davidianus liver by SMART(switching mechanism at 5′ end of RNA transcript)technique, we purified the mRNA from Andrias davidianus liver and the first strand cDNA was synthesized through reverse transcription by using a modified oligo(dT)primer(contained sfi ⅠB site). We used the SMART oligonucleotide (contained sfi ⅠA site) as a template so that the first strand cDNA could be extended over the 5′ end of mRNA. The double strand cDNA was amplified by LD PCR (long distance PCR) with the above two primers and then digested by sfi Ⅰ (ⅠA and ⅠB) restriction enzyme. After cDNA fractionation through CHROMA SPIN column, the double strand cDNA was ligated into the sfi Ⅰ digested λtripIEx2 vector and then the recombinant DNA was packaged in vitro . The content of the unamplified Andrias davidianus liver cDNA library is 1 5×10 6 in which the percentage of recombinant clones is about 98 9%. The titer of the amplified cDNA library is 1 0×10 10 pfu/ml and the average exogenous inserts of the recombinants is 1 25 kb. These results show that the Andrias davidianus liver cDNA library has excellent quality.