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

Due to the overexploitation of farming, as well as habitat loss or degradation, the wild population of Chinese giant salamander Andrias davidianus (CGS), 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 in two field sites having released farm-bred CGSs. We detected the presence of CGS DNA by using polymerase chain reaction (PCR) 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 a mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.

Bcl-xL Reduces Chinese Giant Salamander Iridovirus-Induced Mitochondrial Apoptosis by Interacting with Bak and Inhibiting the p53 Pathway

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation.

Comparative study on five kinds of cytokines in the tissues of Chinese giant salamander (Andrias davidianus) of different developmental stages

By using of the double antibody sandwich method of ELISA, the activities of five cytokines including IL-2, IL-4, IFN-α, IFN-β and TNF-α from the blood serum, liver, intestine and spleen at two developmental stages of Chinese giant salamander (Andrias davidianus) were determined to analyze the distribution of the cytokines. The result indicated that five cytokines were found in these four tissues, while their activities were different in different tissues and different ages. The highest activity of IL-2 and IL-4 was all present in blood serum of two different ages. The activity of IFN-α was the highest in blood serum of 1-year-old and in spleen of 2-year-old, respectively. The activity of IFN-β was also highest in blood serum of two different ages. The activity of TNF-α was highest in liver of two different ages. Thus, this study provides convincing reference for blood serum and liver as the most important distribution area of Chinese giant salamander.

The Immune System and the Antiviral Responses in Chinese Giant Salamander, Andrias davidianus

The Chinese giant salamander, belonging to an ancient amphibian lineage, is the largest amphibian existing in the world, and is also an important animal for artificial cultivation in China. However, some aspects of the innate and adaptive immune system of the Chinese giant salamander are still unknown. The Chinese giant salamander iridovirus (GSIV), a member of the Ranavirus genus (family Iridoviridae), is a prominent pathogen causing high mortality and severe economic losses in Chinese giant salamander aquaculture. As a serious threat to amphibians worldwide, the etiology of ranaviruses has been mainly studied in model organisms, such as the Ambystoma tigrinum and Xenopus. Nevertheless, the immunity to ranavirus in Chinese giant salamander is distinct from other amphibians and less known. We review the unique immune system and antiviral responses of the Chinese giant salamander, in order to establish effective management of virus disease in Chinese giant salamander artificial cultivation.

A high-density genetic map construction and sex-related loci identification in Chinese Giant salamander

Background The Chinese giant salamander Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci. Results In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of A. davidianus. The genetic length of LGs ranged from 17.61 cM (LG30) to 280.81 cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48 cM (LG26). The total genetic map length was 2643.10 cM with an average inter-locus distance of 0.24 cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively. Conclusion We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.

Characterization of Single Nucleotide Polymorphism Marker in the Chinese Giant Salamander Andrias davidianus

Background: Chinese giant salamander Andrias davidianus is called “baby fish” in China. Due to its palatability and nutritional value, it was widely cultured in China. The current study was to evaluate the genetic diversity of the farmed A. davidianus. Methods: 30 salamanders were collected from farm and DNA was extracted. Thirty contigs potentially containing SNPs were selected from the previously developed RAD-seq library. The primer was designed and PCR was performed, than the products was sequenced from different direction. At last the sequences were analyzed using the vector NTI 10.Result: 5824bp high-quality sequences embodied in 16 contigs were sequenced and eighty SNP loci were identified which each SNP locus exhibited bi-alleles. The effective allele number from 1.03 to 2.00 and the minor allele frequency was 0.017 to 0.500. The expected heterozygosity was from 0.0333 to 0.5091 and the observed heterozygosity was 0.0333 to 1.0000. Sixty-six of the 80 loci significantly deviated from the Hardy-Weinberg equilibrium (P less than 0.05). The results provide useful genomic resources to evaluate the genetic diversity of A. davidianus for conservation and aquaculture.