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.

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.

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.

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.

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.

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.

Development of an eDNA metabarcoding tool for surveying the world’s largest amphibian

Due to the overexploitation of farming,as well as habitat destruction,the wild population of Chinese giant salamander(CGS)Andrias davidianus,a species with seven genetically distinct lineages,has decreased by over 80%in the past 70 years.Traditional survey methods have proven to be unsuitable for finding this rare and elusive species.We evaluated the efficacy of environmental DNA(eDNA)sampling to detect CGS indirectly from its aquatic environment.We developed several species-specific primer sets;validated their specificity and sensitivity;and assessed their utility in silico,in the laboratory,and at two field sites harboring released farm-bred CGS.We detected the presence of CGS DNA by using polymerase chain reaction and Sanger sequencing.We also sequenced an amplicon mixture of seven haplotype-represented samples using high-throughput sequencing.Our eDNA methods could detect the presence of CGS at moderate densities reported across its range,proving them as a cost-effective way to establish broad-scale patterns of occupancy for CGS.In addition,our primers enabled the detection of mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.