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Background: Gram-negative bacteria are the main bacterial pathogens infecting Chinese giant salamanders (Andrias davidianus; CGS) in captivity and the wild, causing substantial economic losses in the CGS industry. However, the molecular mechanisms underlying pathogenesis following infection remain unclear. Results: Spleen-derived macrophages from healthy CGS were isolated, cultured, and identified using density gradient centrifugation and immunofluorescence. A macrophage transcriptome database was established 0, 6, and 12 h post lipopolysaccharide stimulation using RNA-sequencing. In the final database 76,743 unigenes and 4,698 differentially expressed genes (DEGs) were functionally annotated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results showed that DEGs were concentrated in toll-like receptor–nuclear factor kappa B-related immune pathways. Ten DEGs were validated 12 h after lipopolysaccharide (LPS) stimulation. Although the common LPS recognition receptor toll-like receptor 4 was not activated and the key adaptor protein MyD88 showed no significant response, we observed significant up-regulation of the following adaptors: toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β, tumour necrosis factor receptor-associated factor 6, and transforming growth factor-β activated kinase 1, which are located downstream of the non-classical MyD88 pathway. Conclusions: In contrast to that in other species, macrophage activation in CGS could depend on the non-classical MyD88 pathway in response to bacterial infection. Our study provides insights into the molecular mechanisms regulating CGS antibacterial responses, with implications for disease prevention and understanding immune evolution in amphibians. [ABSTRACT FROM AUTHOR]

Abstract Background Gram-negative bacteria are the main bacterial pathogens infecting Chinese giant salamanders (Andrias davidianus; CGS) in captivity and the wild, causing substantial economic losses in the CGS industry. However, the molecular mechanisms underlying pathogenesis following infection remain unclear. Results Spleen-derived macrophages from healthy CGS were isolated, cultured, and identified using density gradient centrifugation and immunofluorescence. A macrophage transcriptome database was established 0, 6, and 12 h post lipopolysaccharide stimulation using RNA-sequencing. In the final database 76,743 unigenes and 4,698 differentially expressed genes (DEGs) were functionally annotated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results showed that DEGs were concentrated in toll-like receptor–nuclear factor kappa B-related immune pathways. Ten DEGs were validated 12 h after lipopolysaccharide (LPS) stimulation. Although the common LPS recognition receptor toll-like receptor 4 was not activated and the key adaptor protein MyD88 showed no significant response, we observed significant up-regulation of the following adaptors: toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β, tumour necrosis factor receptor-associated factor 6, and transforming growth factor-β activated kinase 1, which are located downstream of the non-classical MyD88 pathway. Conclusions In contrast to that in other species, macrophage activation in CGS could depend on the non-classical MyD88 pathway in response to bacterial infection. Our study provides insights into the molecular mechanisms regulating CGS antibacterial responses, with implications for disease prevention and understanding immune evolution in amphibians.

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.

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians. [ABSTRACT FROM AUTHOR]

B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35–54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis.

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Andrias davidianus is one of the largest amphibian species in the world. To improve our understanding of Myxovirus resistance (Mx) genes in amphibians, particularly their function in virus infection, we cloned four full-length A. davidianus (adMx) cDNA sequences and characterized these genes by bioinformatics analysis and quantitative expression techniques. The four adMx genes ranged from 2008 to 2840 bp in length, and their conserved protein domains included the signature architectural feature of the dynamin superfamily. Deduced amino acid sequences exhibited relatively high sequence identity with Mx proteins from other vertebrates and phylogenetic analysis revealed close clustering with fish. The four adMx genes were broadly expressed in healthy A. davidianus, but differentially expressed in the spleen following Chinese giant salamander iridovirus (GSIV) infection. These findings imply that the adMx genes share major sequence and protein structures and similar functions with those of other species. Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection. [ABSTRACT FROM AUTHOR]

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Abstract Determining the dynamics and sustainability of human interactions with threatened species is essential to inform evidence‐based conservation, but data can be challenging to collect across large areas and multiple user groups. Chinese giant salamanders Andrias spp. are critically depleted across China. Wild populations were exploited during the 20th century, and more recently to support a large‐scale farming industry. However, robust data remain largely unavailable on the timing of population declines in relation to changing human pressures, on primary drivers of exploitation, or on the effectiveness of conservation legislation. We conducted a series of large‐scale interview surveys across the range of giant salamanders in China, targeting potential rural and urban user groups, and stakeholders involved with giant salamander exploitation and policy management (comprising 2,932 rural households, 66 salamander farms, 115 county government officials and 835 urban consumers). Giant salamander populations were probably declining from at least the 1980s due to exploitation for food, and negative cultural values associated with these animals have not prevented rural consumption. There has been a major escalation in exploitation following the establishment of a large‐scale giant salamander farming industry in the 2000s. Our results demonstrate wide‐scale and largely unregulated illegal hunting to stock farms at a country‐wide scale in order to support demand by urban consumers for high‐prestige rare meat. We estimate there were at least 42,000 wild‐caught breeding adult giant salamanders and 164,000 wild‐caught subadults in farms across China at the time of our survey. Salamander farming probably poses unsustainable pressure on giant salamander populations. Existing legislation has clearly proved ineffective at preventing the stocking of farms with wild‐caught animals, and our findings highlight an important gap in the effectiveness of China's conservation protection for some of its highest‐priority threatened species. Tackling this problem will likely require multiple coordinated approaches, including enforcement of legislation, increased penalties for removing giant salamanders from the wild, permanent identification of captive‐bred giant salamanders, and consumer‐focused interventions to reduce urban demand. A free Plain Language Summary can be found within the Supporting Information of this article.

Abstract 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.

• OTC and CIP intervention altered the gut microbial and functional diversity of A. davidianus. • OTC and CIP increased the abundance of fluoroquinolone and multi-drug resistance genes in the gut of A. davidianus. • MGEs were implicated in the dissemination of ARGs in the gut of A. davidianus. • OTC and CIP interfered with immune-related pathways in the gut of A. davidianus.. • Potential correlations between gut gene expression and microbial composition were observed in the gut of A. davidianus. Excessive antibiotic use has led to the spread of antibiotic resistance genes (ARGs), impacting gut microbiota and host health. However, the effects of antibiotics on amphibian populations remain unclear. We investigated the impact of oxytetracycline (OTC) and ciprofloxacin (CIP) on Chinese giant salamanders (Andrias davidianus), focusing on gut microbiota, ARGs, and gene expression by performing metagenome and transcriptome sequencing. A. davidianus were given OTC (20 or 40 mg/kg) or CIP (50 or 100 mg/kg) orally for 7 days. The results revealed that oral administration of OTC and CIP led to distinct changes in microbial composition and functional potential, with CIP treatment having a greater impact than OTC. Antibiotic treatment also influenced the abundance of ARGs, with an increase in fluoroquinolone and multi-drug resistance genes observed post-treatment. The construction of metagenome-assembled genomes (MAGs) accurately validated that CIP intervention enriched fish-associated potential pathogens Aeromonas hydrophila carrying an increased number of ARGs. Additionally, mobile genetic elements (MGEs), such as phages and plasmids, were implicated in the dissemination of ARGs. Transcriptomic analysis of the gut revealed significant alterations in gene expression, particularly in immune-related pathways, with differential effects observed between OTC and CIP treatments. Integration of metagenomic and transcriptomic data highlighted potential correlations between gut gene expression and microbial composition, suggesting complex interactions between the host gut and its gut microbiota in response to antibiotic exposure. These findings underscore the importance of understanding the impact of antibiotic intervention on the gut microbiome and host health in amphibians, particularly in the context of antibiotic resistance and immune function. [ABSTRACT FROM AUTHOR]

Shewanella xiamenensis, a newly virulent zoonotic pathogen belonging to the genus Shewanella is the causative organism of emerging intra-abdominal infection, acute skin ulceration, rotten limbs and ascites in humans and animals. The global spread of S. xiamenensis entails severe economic impact. However, it was rarely reported as a cause of infection and no reports were found that S. xiamenensis isolated from clinical samples. The isolate was identified as a S. xiamenensis strain by 16S rDNA amplification and DNA sequencing identification method. Even if co-infection by other bacteria could not be ruled out, this is the first report of acute disease caused by S. xiamenensis in the Chinese giant salamander in China. By using the Kirby-Bauer disc diffusion method, the sensitivity of the isolate to clinical antibiotics was evaluated. Antibiotic susceptibility test indicated that the isolate was resistant to 32 antibacterial drugs such as kanamycin, florfenicol and ceftriaxone suggesting that the isolate was a multi-drug resistant strain. [ABSTRACT FROM AUTHOR]

Objectives: A multidrug-resistant Morganella morganii strain (CQ-M7), isolated from the kidney of a diseased Chinese giant salamander in China, was examined with whole genome sequencing to better understand drug tolerance and its pathogenicity. Methods: The draft genome of the investigated strain was assembled using HGA assembler and annotated using Rapid Annotations Subsystems Technology (RAST) server. The contigs were annotated by the appropriate bioinformatics tools available on the National Center for Biotechnology Information (NCBI) website. Antibiotic resistance genes were detected by PCR. Pathogenicity of the isolate was performed on 30 healthy Chinese giant salamanders with different infection dosages. Results: The CQ-M7 strain showed resistance to multiple antimicrobials, especially to aminoglycoside and β-lactam antibiotics. Seventeen drug-resistance genes were detected, which were related to β-lactams, aminoglycosides, fluoroquinolones, tetracyclines, peptide antibiotic, and fosfomycin resistance. Sequence analysis showed the assembled genome size to be 4 966 326 bp with 51.16% of GC content, containing 4587 protein-coding genes, 71 pseudogenes, five rRNAs, 80 tRNAs, and five noncoding RNAs. The genome sequence was deposited in GenBank under accession number RQIJ00000000. Artificial infection results indicated that the CQ-M7 strain was a low-virulence strain for the Chinese giant salamander. Conclusion: It is believed that this is the first draft genome of Chinese giant salamander original Morganella morganii strain harbouring multiple antibiotic resistance genes in China. The reported genome sequence could provide insights into antibiotic resistance mechanisms and control strategies of Morganella morganii.

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. [ABSTRACT FROM AUTHOR]

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.

Abstract Inaccurate taxonomic assessment of threatened populations can hinder conservation prioritization and management, with human‐mediated population movements obscuring biogeographic patterns and confounding reconstructions of evolutionary history. Giant salamanders were formerly distributed widely across China, and are interpreted as a single species, Andrias davidianus. Previous phylogenetic studies have identified distinct Chinese giant salamander lineages but were unable to associate these consistently with different landscapes, probably because population structure has been modified by human‐mediated translocations for recent commercial farming. We investigated the evolutionary history and relationships of allopatric Chinese giant salamander populations with Next‐Generation Sequencing methods, using historical museum specimens and late 20th‐century samples, and retrieved partial or near‐complete mitogenomes for 17 individuals. Samples from populations unlikely to have been affected by translocations form three clades from separate regions of China, spatially congruent with isolation by either major river drainages or mountain ranges. Pliocene–Pleistocene divergences for these clades are consistent with topographic modification of southern China associated with uplift of the Qinghai‐Tibet Plateau. General Mixed Yule Coalescent model analysis indicates that these clades represent separate species: Andrias davidianus (Blanchard, 1871) (northern Yangtze/Sichuan), Andrias sligoi (Boulenger, 1924) (Pearl/Nanling), and an undescribed species (Huangshan). Andrias sligoi is possibly the world's largest amphibian. Inclusion of additional reportedly wild samples from areas of known giant salamander exploitation and movement leads to increasing loss of biogeographic signal. Wild Chinese giant salamander populations are now critically depleted or extirpated, and conservation actions should be updated to recognize the existence of multiple species.

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species.

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection.

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. [ABSTRACT FROM AUTHOR]

Determining the dynamics and sustainability of human interactions with threatened species is essential to inform evidence‐based conservation, but data can be challenging to collect across large areas and multiple user groups.Chinese giant salamanders Andrias spp. are critically depleted across China. Wild populations were exploited during the 20th century, and more recently to support a large‐scale farming industry. However, robust data remain largely unavailable on the timing of population declines in relation to changing human pressures, on primary drivers of exploitation, or on the effectiveness of conservation legislation.We conducted a series of large‐scale interview surveys across the range of giant salamanders in China, targeting potential rural and urban user groups, and stakeholders involved with giant salamander exploitation and policy management (comprising 2,932 rural households, 66 salamander farms, 115 county government officials and 835 urban consumers).Giant salamander populations were probably declining from at least the 1980s due to exploitation for food, and negative cultural values associated with these animals have not prevented rural consumption. There has been a major escalation in exploitation following the establishment of a large‐scale giant salamander farming industry in the 2000s. Our results demonstrate wide‐scale and largely unregulated illegal hunting to stock farms at a country‐wide scale in order to support demand by urban consumers for high‐prestige rare meat. We estimate there were at least 42,000 wild‐caught breeding adult giant salamanders and 164,000 wild‐caught subadults in farms across China at the time of our survey.Salamander farming probably poses unsustainable pressure on giant salamander populations. Existing legislation has clearly proved ineffective at preventing the stocking of farms with wild‐caught animals, and our findings highlight an important gap in the effectiveness of China's conservation protection for some of its highest‐priority threatened species. Tackling this problem will likely require multiple coordinated approaches, including enforcement of legislation, increased penalties for removing giant salamanders from the wild, permanent identification of captive‐bred giant salamanders, and consumer‐focused interventions to reduce urban demand. A free Plain Language Summary can be found within the Supporting Information of this article. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

Chinese giant salamander Andrias davidianus has strong tolerance to starvation. Fasting triggers a complex array of adaptive metabolic responses, a process in which the liver plays a central role. Here, a high-throughput proteomic analysis was carried out on liver samples obtained from adult A. davidianus after 3, 7, and 11 months of fasting. As a result, the expression levels of 364 proteins were significantly changed in the fasted liver. Functional analysis demonstrated that the expression levels of key proteins involved in fatty acid oxidation, tricarboxylic acid cycle, gluconeogenesis, ketogenesis, amino acid oxidation, urea cycle, and antioxidant systems were increased in the fasted liver, especially at 7 and 11 months after fasting. In contrast, the expression levels of vital proteins involved in pentose phosphate pathway and protein synthesis were decreased after fasting. We also found that fasting not only activated fatty acid oxidation and ketogenesis-related transcription factors PPARA and PPARGC1A, but also activated gluconeogenesis-related transcription factors FOXO1, HNF4A, and KLF15. This study confirms the central role of lipid and acetyl-CoA metabolism in A. davidianus liver in response to fasting at the protein level and provides insights into the molecular mechanisms underlying the metabolic response of A. davidianus liver to fasting.

Abstract Background Ranaviruses (family Iridoviridae, nucleocytoplasmic large DNA viruses) have been reported as promiscuous pathogens of cold-blooded vertebrates. Rana grylio virus (RGV, a ranavirus), from diseased frog Rana grylio with a genome of 105.79 kb and Andrias davidianus ranavirus (ADRV), from diseased Chinese giant salamander (CGS) with a genome of 106.73 kb, contains 99% homologous genes. Results To uncover the differences in virus replication and host responses under interspecies infection, we analyzed transcriptomes of CGS challenged with RGV and ADRV in different time points (1d, 7d) for the first time. A total of 128,533 unigenes were obtained from 820,858,128 clean reads. Transcriptome analysis revealed stronger gene expression of RGV than ADRV at 1 d post infection (dpi), which was supported by infection in vitro. RGV replicated faster and had higher titers than ADRV in cultured CGS cell line. RT-qPCR revealed the RGV genes including the immediate early gene (RGV-89R) had higher expression level than that of ADRV at 1 dpi. It further verified the acute infection of RGV in interspecies infection. The number of differentially expressed genes and enriched pathways from RGV were lower than that from ADRV, which reflected the variant host responses at transcriptional level. No obvious changes of key components in pathway “Antigen processing and presentation” were detected for RGV at 1 dpi. Contrarily, ADRV infection down-regulated the expression levels of MHC I and CD8. The divergent host immune responses revealed the differences between interspecies and natural infection, which may resulted in different fates of the two viruses. Altogether, these results revealed the differences in transcriptome responses among ranavirus interspecies infection of amphibian and new insights in DNA virus-host interactions in interspecies infection. Conclusion The DNA virus (RGV) not only expressed self-genes and replicated quickly after entry into host under interspecies infection, but also avoided the over-activation of host responses. The strategy could gain time for the survival of interspecies pathogen, and may provide opportunity for its adaptive evolution and interspecies transmission.

The cytoplasmic helicases retinoic acid‐inducible gene I (RIG‐I) and melanoma differentiation‐associated protein 5 (MDA5) are two pattern recognition receptor genes belonging to RIG‐I‐like receptor (RLR). They play a crucial role in host sensing of pathogen invasion. Herein, we identified and characterized complete cDNA of RIG‐I and MDA5 in Chinese giant salamander (adRIG‐I and adMDA5). To evaluate their expression levels, real‐time fluorescence quantitative RT‐PCR (qPCR) was performed between normal and Chinese giant salamander iridovirus (GSIV)‐infected tissues. The full‐length adMDA5 cDNA of 3,509 bp encoded a 968‐acid amino polypeptide, and the 3567‐bp adRIG‐I cDNA encoded a 951‐acid amino protein. The amino acid sequence analysis revealed adRIG‐I and adMDA5 kept the conserved domains of RLR, and the two genes both shared relatively high similarity homologs with other species. Two genes were expressed in most tissues of Andrias davidianus, with the highest expression in spleen. After GSIV infection, both adRIG‐I and adMDA5 transcripts were initially increased then decreased in kidney and spleen. These results suggest that adRIG‐I and adMDA5 can response to GSIV infection and may play a role in antiviral infections in DNA virus. [ABSTRACT FROM AUTHOR]

Chinese giant salamander Andrias davidianus has strong tolerance to starvation. Fasting triggers a complex array of adaptive metabolic responses, a process in which the liver plays a central role. Here, a high-throughput proteomic analysis was carried out on liver samples obtained from adult A. davidianus after 3, 7, and 11 months of fasting. As a result, the expression levels of 364 proteins were significantly changed in the fasted liver. Functional analysis demonstrated that the expression levels of key proteins involved in fatty acid oxidation, tricarboxylic acid cycle, gluconeogenesis, ketogenesis, amino acid oxidation, urea cycle, and antioxidant systems were increased in the fasted liver, especially at 7 and 11 months after fasting. In contrast, the expression levels of vital proteins involved in pentose phosphate pathway and protein synthesis were decreased after fasting. We also found that fasting not only activated fatty acid oxidation and ketogenesis-related transcription factors PPARA and PPARGC1A, but also activated gluconeogenesis-related transcription factors FOXO1, HNF4A, and KLF15. This study confirms the central role of lipid and acetyl-CoA metabolism in A. davidianus liver in response to fasting at the protein level and provides insights into the molecular mechanisms underlying the metabolic response of A. davidianus liver to fasting. [ABSTRACT FROM AUTHOR]

Abstract The composition of the intestinal microbial community may vary across developmental stages. In this study, we explored how this microbial community shifted along the intestinal tract of the Chinese giant salamander (Andrias davidianus) at various ages. Next‐generation sequencing was used to sequence the bacterial 16S rRNA gene from different kind of samples, including the stomach, duodenum, ileum, and rectum. The highest mean relative abundance of the bacterial community in the gastrointestinal tract shifted in relation to age: within the first year, Bacteroidetes (47.76%) dominated the gut microbiome, whereas Proteobacteria was the most dominant at age 2 (32.88%) and age 3 (30.78%), and finally, Firmicutes was the most dominant at age 4 (34.70%). The overall richness of the gut bacterial community also generally increased from age 2 to 4. Hierarchical cluster analysis revealed that the gut microbiome at age 2 had greater variability than that at either age 3 or 4, likely representing a shift in diet from yolk or redworms as a juvenile to shrimp or crab as an adult. As these salamanders develop, their gastrointestinal tracts increase in complexity, and this compartmentalization may also facilitate an increase in microbial gut diversity.

The Chinese giant salamander Andrias davidianus is regarded as an ideal model for studying local adaptations, such as longevity, tolerance to starvation, and cutaneous respiration. Transcriptome analysis is useful for studying the large and complex genomes of amphibians. Based on the coding gene set of adult A. davidianus, dozens of A. davidianus–specific genes were identified and three signaling pathway (JAK-STAT, HIF-1, and FoxO) genes were expanded as compared with other amphibians. The results of the pathway analysis of A. davidianus–specific genes indicated that the molecular adaptation of A. davidianus may have required a more rapid evolution of the immune system. Additionally, for the first time, the gene expressions in different parts of the skin tissue were compared. The results of the comparison analysis demonstrated that lateral skin could be more focused on mucus secretion, dorsal skin on immunity and melanogenesis, and abdominal skin on water and salt metabolism. This study provides the first insight into studying longevity and starvation tolerance in A. davidianus, and offers a basis for further investigation of the molecular mechanisms of adaptations in amphibians. [ABSTRACT FROM AUTHOR]

Chinese giant salamander (CGS) has high medicinal value and long history of clinical use in ancient China. In this study, CGS skin (CGSS) collagen was extracted and purified to prepare collagen sponge by freeze-drying. TEMPO oxidized microfibrillated cellulose (TEMPO-MFC) and genipin were adopted to improve the mechanical properties of collagen sponge. The hygroscopicity, porosity, mechanical properties, hemostatic performance, morphology, and biodegradability of the resultant sponges were investigated in detail. The results indicated that CGSS collagen was type I collagen with intact triple-helical structure, and the prepared sponge had porous structure and excellent hemostatic performance with procoagulant ratio of 53.28%. However, the CGSS collagen sponge showed low tensile strength (TS) of 98.80 KPa, compression strength (CS) of 1.48 KPa, and elongation at break (E) of 4.72%. Incorporating 2.5% TEMPO-MFC into the native CGSS collagen sponge resulted in an increase of 188.26% in TS to 284.80 KPa, 166.89% in CS to 3.95 KPa, and 73.52% in E to 8.19%. The improvements were attributed to the physical filling of TEMPO-MFC in cavity and cavity wall of collagen sponge and the stable chemical linkage between carboxyl of TEMPO-MFC and amino group of collagen which effectively improved the toughening of sponge and formed good interface bonding, respectively. Subsequent 0.3% genipin treatment further improved the TS to 605.00 KPa and the CS to 8.66 KPa as a result of crosslinking reaction. Moreover, the composite reinforcing also improved the anti-degradation ability and procoagulant ratio of collagen sponge. All results suggested that the TEMPO-MFC toughened and genipin crosslinked CGSS composite collagen sponge is a promising rapid hemostatic material with high-strength and can be applicated in biomedical field. [ABSTRACT FROM AUTHOR]

• Over-expression of AdBcl-w inhibited the apoptotic process during GSIV infection while RNAi of AdBcl-w promoted this process. • Over-expression of AdBcl-w can effectively reduce the replication of GSIV. • The interaction of AdBcl-w and AdBak reduced after GSIV infection. • Over-expression of AdBcl-w restrained the mRNA expression of molecules in p53 pathway during GSIV infection. B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35–54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis. [ABSTRACT FROM AUTHOR]

Mx, Myxovirus resistance is an important interferon-stimulated protein that mediates antiviral responses. In this study, the expression and activities of Chinese giant salamander, Andrias davidianus Mx gene, AdMx, were investigated. The AdMx cDNA sequence contains an open reading frame (ORF) of 2112 nucleotides, encoding a putative protein of 703 aa. Meanwhile, AdMx possesses the conserved tripartite GTP binding motif and a dynamin family signature. qRT-PCR analysis revealed a broad expression of AdMx in vivo, with the highest expression levels in brain, kidney and spleen. The AdMx expression level in kidney, spleen and muscle significantly increased at 6 h after Chinese giant salamander iridovirus (GSIV) infection and peaked at 48 h, while that in muscle cell line (GSM) was not noticeably up-regulated until 72 h post infection. Additionally, a plasmid expressing AdMx was constructed and transfected into the Chinese giant salamander GSM cells. The virus load and gene copies in AdMx over-expressed cells were significantly reduced compared with those in the control cells. Moreover, compared to the control cells, a lower level of virus major capsid protein (MCP) synthesis in AdMx over-expressed cells was confirmed by Western blot. These results collectively suggest that Mx plays an important antiviral role in the immune responses against GSIV in Chinese giant salamander.

Chinese giant salamander (CGS) has high medicinal value and long history of clinical use in ancient China. In this study, CGS skin (CGSS) collagen was extracted and purified to prepare collagen sponge by freeze-drying. TEMPO oxidized microfibrillated cellulose (TEMPO-MFC) and genipin were adopted to improve the mechanical properties of collagen sponge. The hygroscopicity, porosity, mechanical properties, hemostatic performance, morphology, and biodegradability of the resultant sponges were investigated in detail. The results indicated that CGSS collagen was type I collagen with intact triple-helical structure, and the prepared sponge had porous structure and excellent hemostatic performance with procoagulant ratio of 53.28%. However, the CGSS collagen sponge showed low tensile strength (TS) of 98.80 KPa, compression strength (CS) of 1.48 KPa, and elongation at break (E) of 4.72%. Incorporating 2.5% TEMPO-MFC into the native CGSS collagen sponge resulted in an increase of 188.26% in TS to 284.80 KPa, 166.89% in CS to 3.95 KPa, and 73.52% in E to 8.19%. The improvements were attributed to the physical filling of TEMPO-MFC in cavity and cavity wall of collagen sponge and the stable chemical linkage between carboxyl of TEMPO-MFC and amino group of collagen which effectively improved the toughening of sponge and formed good interface bonding, respectively. Subsequent 0.3% genipin treatment further improved the TS to 605.00 KPa and the CS to 8.66 KPa as a result of crosslinking reaction. Moreover, the composite reinforcing also improved the anti-degradation ability and procoagulant ratio of collagen sponge. All results suggested that the TEMPO-MFC toughened and genipin crosslinked CGSS composite collagen sponge is a promising rapid hemostatic material with high-strength and can be applicated in biomedical field. [ABSTRACT FROM AUTHOR]

Origin and manufacturing process are key factors affecting the biological activities of chondroitin sulfate (CS), which can be utilized as a nutraceutical in dietary supplements. Herein, we extracted and purified CS from the cartilage of artificially breeding Andrias davidianus (ADCS), i.e. , Chinese giant salamander (CGS), one of the prospective functional food source materials in China. Low molecular weight CS (LMWADCS) was then prepared by free radical depolymerization of ADCS. High-performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of ADCS was 49.2 kDa, while the Mw of LMWADCS was 6.4 kDa. After the eliminative degradation of ADCS by chondroitinase ABC, strong anion-exchange high-performance liquid chromatography (SAX-HPLC) analysis showed that the disaccharide composition of ADCS was 14.6% ΔDi0S, 60.9% ΔDi6S and 24.5% ΔDi4S. Then, in vitro antioxidant assays were performed with ADCS, LMWADCS and CS from a commercial source. Our results showed that LMWADCS exerted the highest total antioxidant activity out of the total antioxidant capacity, including the capacity of scavenging DPPH radicals, hydroxyl radicals and superoxide anion radicals. From the results of this study, we can conclude that the Mw and composition of ADCS are different from those reported for bovine and shark CS, and LMWADCS can be utilized as a valuable and potential nutraceutical for the functional food industry. [ABSTRACT FROM AUTHOR]

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L) and the 58L gene (pcDNA-58L) were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS) of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%). Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA). Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN), myxovirus resistance (Mx), major histocompatibility complex class IA (MHC IA), and immunoglobulin M (IgM) were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders. [ABSTRACT FROM AUTHOR]

This study aimed to purify and identify a novel antidiabetic peptides from Chinese giant salamander (Andrias davidianus) protein hydrolysate and to evaluate its anti-diabetic properties against α-amylase and α-glucosidase. The isolation was done by gel filtration, reverse phase chromatography (RF-HLPC), and finally peptide and amino acid sequences were identified by LC/MS/MS system. Our results revealed five novel peptides that strongly inhibited α-amylase and α-glucosidase. The peptides’ amino acid sequences were Cys-Ser-Ser-Val (MW = 393.99 Da), Tyr-Ser-Phe-Arg (MW = 570.99 Da), Ser-Ala-Ala-Pro (MW = 343.89 Da), Pro-Gly-Gly-Pro (MW = 325.99 Da) and Leu-Gly-Gly-Gly-Asn (MW = 415.99 Da) possessing α-amylase inhibitory activity IC50 of 13.76 × 103, 10.82 × 103, 4.46 × 103, 4.23 × 103, and 2.86 × 103 µg/mL, respectively; and for α-glucosidase with IC50 of 206.00, 162.00, 66.90, 63.50, and 42.93 µg/mL, respectively. Intriguingly, the peptide LGGGN showed higher inhibition on both α-amylase and α-glucosidase and could be considered as a potential anti-diabetic inhibitor. [ABSTRACT FROM AUTHOR]

Thioredoxin-like protein-1 (TXNL1) is the member of thioredoxin superfamily, a family of thiol oxidoreductases. TXNL1 plays an important role in scavenging ROS and the maintenance of cellular redox balance. However, its physiological functions in Andrias davidianus have not been well understood. In the present study, the full-length cDNA encoding thioredoxin-like protein-1 (Ad TXNL1) of A. davidianus was cloned, the mRNA tissue distribution was analyzed, and the function was characterized. The Adtxnl1 cDNA contained an open reading frame (ORF) of 870 bp encoding a polypeptide of 289 amino acids with the N-terminal TRX domain, a Cys34-Ala35-Pro36-Cys37 (CAPC) motif, and the C-terminal proteasome-interacting thioredoxin domain (PITH). The mRNA of Ad TXNL1 was expressed in a wide range of tissues, with the highest level in the liver. The transcript level of Ad TXNL1 was significantly up-regulated post Aeromonas hydrophila challenge in liver tissue. Moreover, the recombinant Ad TXNL1 protein was produced and purified, and used to investigate the antioxidant activity. In the insulin disulfide reduction assay , r Ad TXNL1 exhibited strong antioxidant capability. Altogether, the thioredoxin-like protein-1 may be involved in reduction/oxidation (redox) balance and as an important immunological gene in A. davidianus. • The thioredoxin-like protein-1 gene was identified and characterized in Andrias davidianus. • Adtxnl1 was widely expressed in all tested tissues. • The Adtxnl1 transcript level after bacterial infection increased. • Recombinant Ad TXNL1 may have the antioxidant capacity. [ABSTRACT FROM AUTHOR]

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV. [ABSTRACT FROM AUTHOR]

Lysozymes as an important immune factor, play vital roles in innate immune response against pathogen infection. In the present study, one c-type and g-type lysozymes were identified from Chinese giant salamander ( Andrias davidianus ). They shared highly conserved structural features with lysozymes from other species. Spatial expression analysis revealed that AdlysC transcript was most abundant in liver and stomach, and least in muscle and brain. In contrast, the expression level of AdlysG was most abundant in liver and least in muscle and skin. The transcription level of c-type and g-type lysozymes were up-regulated after Aeromonas hydrophila infection in liver and spleen, indicating their participations in the immune response. Moreover, the recombinant AdlysC and AdlysG protein were produced and purified, and were used to investigate the lysozyme activity at different pH and temperatures. The optimal lytic activity was determined at pH 6.0 and at a temperature of 30 °C. Through the minimal inhibitory concentration test, the rAdlysC and rAdlysG exhibited apparent antibacterial activity against both Gram-positive and Gram-negative bacteria with a variable concentration. In conclusion, it is the first report of lysozymes in A. davidianus , and c-type and g-type lysozymes should be involved in the innate immune response of A. davidianus. [ABSTRACT FROM AUTHOR]

Background: Chinese giant salamander (CGS) is the largest extant amphibian species in the world. Owing to its evolutionary position and four peculiar phenomenon of life (longevity, starvation tolerance, regenerative ability, and hatch without sunshine), it is an invaluable model species for research. However, lack of genomic resources leads to fewer study progresses in these fields, due to its huge genome of ~50 GB making it extremely difficult to be assembled. Results: We reported the sequenced transcriptome of more than 20 tissues from adult CGS using Illumina Hiseq 2000 technology, and a total of 93 366 no-redundancy transcripts with a mean length of 1326 bp were obtained. We developed for the first time an efficient pipeline to construct a high-quality reference gene set of CGS and obtained 26 135 coding genes. BUSCO and homologous assessment showed that our assembly captured 70.6% of vertebrate universal single-copy orthologs, and this coding gene set had a higher proportion of completeness CDS with comparable quality of the protein sets of Tibetan frog. Conclusions: These highest quality data will provide a valuable reference gene set to the subsequent research of CGS. In addition, our strategy of de novo transcriptome assembly and protein identification is applicable to similar studies. [ABSTRACT FROM AUTHOR]