Giant Salamander Research Articles

Coordinated microRNA/mRNA Expression Profiles Reveal Unique Skin Color Regulatory Mechanisms in Chinese Giant Salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus) has been increasingly popular in the aquaculture market in China in recent years. In the breeding process of Andrias davidianus, we found that some albino individuals were extremely rare and could not be inherited stably, which severely limits their commercialization in the aquaculture market. In this study, we performed transcriptome and small RNA (sRNA) sequencing analyses in the skin samples of wild-type (WT) and albino (AL) Andrias davidianus. In total, among 5517 differentially expressed genes (DEGs), 2911 DEGs were down-regulated in AL, including almost all the key genes involved in melanin formation. A total of 25 miRNAs were differentially expressed in AL compared to WT, of which 17 were up-regulated. Through the integrated analysis, no intersection was found between the target genes of the differentially expressed miRNAs and the key genes for melanin formation. Gene Ontology (GO) and KEGG pathway analyses on DEGs showed that these genes involved multiple processes relevant to melanin synthesis and the key signal pathway MAPK. Interestingly, the transcription factors SOX10 and PAX3 and the Wnt signaling pathway that play a key role in other species were not included, while the other two transcription factors in the SOX family, SOX21 and SOX7, were included. After analyzing the key genes for melanin formation, it was interesting to note an alternative splicing form of the MITF in WT and a critical mutation of the SLC24A5 gene in AL, which might be the main reason for the skin color change of Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of individuals with specific body colors by genetic means.

Molecular cloning, tissues distribution, and function analysis of thioredoxin-like protein-1 (TXNL1) in Chinese giant salamanders Andrias davidianus

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 (AdTXNL1) 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 AdTXNL1 was expressed in a wide range of tissues, with the highest level in the liver. The transcript level of AdTXNL1 was significantly up-regulated post Aeromonas hydrophila challenge in liver tissue. Moreover, the recombinant AdTXNL1 protein was produced and purified, and used to investigate the antioxidant activity. In the insulin disulfide reduction assay, rAdTXNL1 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.

Amphibian Skin and Skin Secretion: An Exotic Source of Bioactive Peptides and Its Application

Amphibians have been consumed as an alternative protein source all around the world due to their delicacy. The skin of edible amphibians, particularly frogs and giant salamanders, always goes to waste without further utilization. However, these wastes can be utilized to extract protein and bioactive peptides (BPs). Various BPs have been extracted and reported for numerous biological activities such as antioxidant, antimicrobial, anticancer, antidiabetic, etc. The main BPs identified were brevinins, bombesins, dermaseptins, esculentins, magainin, temporins, tigerinins, and salamandrins. This review provides a comprehensive discussion on various BPs isolated and identified from different amphibian skins or skin secretion and their biological activities. The general nutritional composition and production statues of amphibians were described. Additionally, multiple constraints against the utilization of amphibian skin and secretions are reported. Finally, the prospective applications of BPs in food and biomedical industries are presented such as multifunctional food additives and/or supplements as well as drug delivery agents.

Changes of intestinal microbiota in the giant salamander (Andrias davidianus) during growth based on high-throughput sequencing.

Despite an increasing appreciation of the importance of host-microbe interaction in healthy growth, information on gut microbiota changes of the Chinese giant salamander (Andrias davidianus) during growth is still lacking. Moreover, it is interesting to identify gut microbial structure for further monitoring A. davidianus health. This study explored the composition and functional characteristics of gut bacteria in different growth periods, including tadpole stage (ADT), gills internalization stage (ADG), 1 year age (ADY), 2 year age (ADE), and 3 year age (ADS), using high-throughput sequencing. The results showed that significant differences were observed in microbial community composition and abundance among different growth groups. The diversity and abundance of intestinal flora gradually reduced from larvae to adult stages. Overall, the gut microbial communities were mainly composed of Fusobacteriota, Firmicutes, Bacteroidota, and Proteobacteria. More specifically, the Cetobacterium genus was the most dominant, followed by Lactobacillus and Candidatus Amphibiichlamydia. Interestingly, Candidatus Amphibiichlamydia, a special species related to amphibian diseases, could be a promising indicator for healthy monitoring during A. davidianus growth. These results could be an important reference for future research on the relationship between the host and microbiota and also provide basic data for the artificial feeding of A. davidianus.

Chinese Giant Salamander Iridovirus 025L Is a Viral Essential Gene.

Ranavirus is a large nucleocytoplasmic DNA virus. Chinese giant salamander iridovirus (CGSIV) belongs to the ranavirus genus, and its replication involves a series of essential viral genes. Viral PCNA is a gene closely associated with viral replication. CGSIV-025L also encodes PCNA-like genes. We have described the function of CGSIV-025L in virus replication. The promoter of CGSIV-025L is activated during viral infection, and it is an early (E) gene that can be effectively transcribed after viral infection. CGSIV-025L overexpression promoted viral replication and viral DNA replication. siRNA interfered with CGSIV-025L expression and attenuated viral replication and viral DNA replication. The Δ025L-CGSIV strain with the deletion of CGSIV-025L could not replicate normally and could be rescued by the replenishment of 025L. CGSIV-025L was proven to be an essential gene for CGSIV by overexpression, interference, and deletion mutation experiments. CGSIV-025L was found to interact with CGSIV-062L by yeast two-hybrid, CoIP, and GST pulldown. Thus, the current study demonstrated that CGSIV-025L is an essential gene of CGSIV, which may be involved in viral infection by participating in viral DNA replication and interacting with replication-related proteins.

Identification of Novel Mutations in the Tyrosinase Gene (TYR) Associated with Pigmentation in Chinese Giant Salamanders (Andrias davidianus)

The Chinese giant salamander (Andrias davidianus), an endangered amphibian species endemic to China, has been previously evaluated with regards to its phyletic evolution, zooecology, and ethology, but molecular mechanisms underlying its skin pigmentation remain unknown. Herein, a skin transcriptome database of different colored salamanders was established using RNA-seq, and a total of 47,911 unigenes were functionally annotated. Among these unigenes, a total of 1252 differentially expressed genes (DEGs) were annotated in the seven public databases, and six DEGs were validated by qPCR between five different skin colors and eight tissues. The results showed that TYR, TYRP1, and ASIP were significantly differentially expressed between different body colors, while TYR, TYRP1, and DCT were highly expressed in skin tissue. The full-length complementary DNA of TYR was cloned and analyzed between normal and yellow phenotypes. Three nucleotide sequence deletion sites were identified in the coding region of TYR, leading to premature termination of transcription and translation in yellow individuals. Our study provides useful data for the further study of the molecular mechanisms of melanin formation, and a valuable reference for the breeding of specific skin colors in other salamanders.

DNAJA4 Promotes the Replication of the Chinese Giant Salamander Iridovirus.

The DNAJ family, a class of chaperone proteins involved in protein folding, assembly, and transport, plays an essential role in viral infections. However, the role of DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) in the ranavirus infection has not been reported. This study demonstrates the function of the epithelial papilloma of carp (EPC) DNAJA4 in Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) replication. DNAJA4 consists of 1479 base pairs and encodes a 492 amino acid polypeptide. Sequence analysis has shown that EPC DNAJA4 contains a conserved J domain and shares 84% homology with Danio rerio DNAJA4 and 68% homology with Homo sapiens DNAJA4. EPC DNAJA4 was localized in the cytoplasm, and its expression was significantly upregulated after CGSIV infection. Overexpression of EPC DNAJA4 promotes CGSIV replication and CGSIV DNA replication. siRNA knockdown of DNAJA4 expression attenuates CGSIV replication and viral DNA replication. Overexpression and interference experiments have proved that EPC DNAJA4 is a pro-viral factor. Co-IP, GST-pulldown, and immunofluorescence confirmed the interaction between EPC DNAJA4 and CGSIV proliferating cell nuclear antigen (PCNA). Our results demonstrate for the first time that EPC DNAJA4 is involved in viral infection by promoting viral DNA replication and interacting with proteins associated with viral replication.

The nomenclature of Eurasian giant salamanders (Amphibia, Urodela, Andrias)

The history of the taxonomy and nomenclature of the giant Eurasian salamanders of the genus Andrias is provided, with a complete list of available nomina of all extinct and extant species, indicating their current nomenclatural status, their type-specimens and type-localities.

Predicting Sex-Related Transcripts in the Chinese Giant Salamander (Andrias davidianus): A Transcriptomics Study, Selection Gender for Preservation, Breeding and Reintroduction

The Chinese giant salamander (Andrias davidianus) is the biggest extant and endangered amphibian, which has an incongruous sex ratio during the artificial breeding process and is sex indistinguishable in appearance. The sex development mechanism of this rare species thus needs to be explored. We therefore conducted this study to explore the molecular mechanisms of sex hallmarks or gonad maintenance genes for CGS. In this study, we sequenced and analyzed the sexually mature male and female gonads of Chinese giant salamanders (CGSs) using RNA-seq technology and found a total of 15,063 differentially expressed genes (DEGs) in the testicular and ovarian tissues, of which 7973 (52.93%) were expressed in testicular and 7090 (47.07%) in ovarian tissues. Thirteen gonad DEGs were selected based on gene expression abundance and quantified in the muscle and skin tissues of both male and female CGSs. The results showed that these 13 DEGS, out of the 20 analyzed DEGs, were expressed in both the muscle and skin tissues and only two of them (Bmp15 and ZP3) were consistent in the muscle and skin compared to the gonads of CGSs of the two sexes. Overall, our study should provide an important reference for the sex-characteristics-related genes in this type of species, shedding new light on future research on this topic.

Reintroduction modifies the intraspecific variations of symbiotic microbes in captive bred Chinese giant salamander.

Microorganisms play as fundamental contributors to maintain hosts' fitness, which can be shaped by external environment. Moreover, symbiotic microbiome also varied within species (e.g., between sexes and developmental stages). However, we still need more studies to quantify whether the intraspecific variation patterns of symbiotic microbes can be modified with the change of environment. The Chinese giant salamander (CGS; Andrias davidianus) is a Critically Endangered species. Despite quantitative captive bred individuals were released to rebuild wild populations, the effectiveness is limited. More importantly, no studies have revealed the adaptation of released CGSs to the complex field conditions. In the present study, we explored whether reintroduction can reshape the intraspecific variations of symbiotic microbiota in captive bred CGSs using high-throughput amplicon sequencing of the16S rRNA gene. We found no significant difference of symbiotic microbiome in captive bred males and females, but released males and females differed significantly in skin microbiome. Juveniles had higher diversity of microbial symbiont than adults in hatchery, but lower diversity in field. Moreover, dominant bacterial taxa differed between juveniles and adults in both hatchery and field. Importantly, this symbiotic microbiome variations within species can be modified (alpha and beta diversity, and community composition) when captive bred individuals were released to the field. Overall, we observed a lower alpha diversity and higher relative abundance of Chryseobacterium, Plesiomonas, and Acinetobacter in the bacterial community of captive bred individuals. Instead, higher alpha diversity of symbiotic microbiota and higher relative abundance of S24-7 and Lactobacillus was detected in released individuals. These modifications may associate with the change of living environment, as well as the specific behavior within CGSs (e.g., movement patterns and foraging activities). Future studies can incorporate other approaches (e.g., blood physiology) to better evaluate the growth and health of reintroduced CGSs.

Wildfire in western Oregon increases stream temperatures, benthic biofilms, and juvenile coastal cutthroat trout size and densities with mixed effects on adult trout and coastal giant salamanders

Wildfire has become increasingly common and severe across forested landscapes. Shortly after wildfire, loss of riparian cover along streams and subsequent increases in light can elevate stream temperatures, a key control on metabolic rates of biota. Increased light can also increase autotrophic basal resource availability with potential bottom-up effects. We evaluated wildfire impacts on aquatic ecosystems in a replicated Before-After Control-Impact study 1 year after a severe wildfire in western Oregon, U.S. Stream temperature, chlorophyll a accrual, and age-0 coastal cutthroat trout size, density, and biomass increased in all three burned streams relative to changes in three unburned references. When streams were evaluated collectively, fire did not have an effect on larger vertebrate density or biomass. However, considering streams individually, two severely burned sites had substantial temperature increases and declines in larger vertebrate density and biomass, but the moderately burned site had modest temperature increases and adult cutthroat trout and Pacific giant salamanders increased. The loss of riparian canopies post-fire increased temperature and algae, but fish responses varied with age class and larger vertebrate responses were inconsistent.

Modelling the effect of tourism disturbance on hatching rate of the Chinese giant salamander (Andrias davidianus) by using artificial neural network

The endangered Chinese giant salamander (Andrias davidianus) is an endangered species among the conserved Chinese amphibians. Tourism-related pressures have increased for this species recently. The effect of tourism on the hatching rate of the target species was determined by experimentally observing the influences of different intensities of tourism disturbance on salamander in the Zhangjiajie Chinese Giant Salamander National Nature Reserve. Water quality factors (e.g. total nitrogen, total phosphorus, dissolved oxygen and Escherichia coli abundance) were analysed, and hatching rate was estimated. Results showed that high levels of tourism disturbance (500,000–1,200,000 visitors per year) had active effects on the hatching time and negative effects on the hatching rate. The prediction performance of artificial neural network models was validated by the low root mean square error values of 2.2539 and 3.2612 for the training and testing data and high determination coefficient values of 0.9732 and 0.9508 for the training and testing data, respectively. The potential for positive or negative feedback mechanisms in such relationships between tourists and wildlife highlights the importance of considering both sides of the complex interaction to find a balance between the development of tourism and wild animal protection.

Factors influencing the availability of artificial hellbender ( Cryptobranchus alleganiensis ) shelters in North Carolina, USA

The hellbender (Cryptobranchus alleganiensis) is a fully aquatic giant salamander experiencing population declines throughout the eastern United States. Hellbender declines are likely due to a loss of cover and breeding habitat, therefore artificial shelters have been designed for hellbenders to supplement breeding habitat and monitor reproductive success. Although several artificial shelter designs are currently in use throughout the hellbender range, no previous study has compared different designs in the field. We compared three shelter designs (modified boot design with an open bottom, modified boot design with a closed bottom, and a modified hydrodynamic design) to estimate their probability of availability (remaining in place and not obstructed by sediment) to hellbenders during variable stream conditions. We deployed 180 shelters of the three designs (n = 60 each) across six stream reaches in western North Carolina, measured habitat variables at installation points, and monitored shelters for sediment blockage, dislodgement, and hellbender occupancy. Shelter availability averaged 71% across all sampled occasions (67% modified boot closed, 69% modified boot open, 77% modified hydrodynamic). Blockage of shelters by sediment was responsible for the majority (96%) of occasions when shelters were deemed unavailable. Hellbenders rarely (range = 0 to 11% of occasions per site) used artificial shelters which precluded our ability to evaluate hellbender preference among shelter designs. Our findings suggest modified hydrodynamic shelters are slightly more resilient to the stream environment relative to modified boot designs, presumably due to their heavier weight and larger inner chamber. Additionally, our results highlight the need to consider site characteristics such as hellbender density and availability of natural cover before deploying artificial hellbender shelters.

The first identification of three AdIRAK2 genes from an evolutionarily important amphibian Andrias davidianus and their involvement in NF-κB activation and inflammatory responses

Interleukin-1 receptor associated kinases (IRAK) is the most important downstream kinases of TLRs/IL-1R signaling pathway for signal transduction and activation of inflammatory response against pathogen infections. However, the molecular identification and function characterization of IRAK2 homologs in lower vertebrate remains obscure. In this study, three IRAK2 genes (AdIRAK2a, AdIRAKb and AdIRAK2c) and their respective transcripts were identified from the Chinese giant salamander Andrias davidianus. This is the first evidence that three different IRAK2 genes exist in an ancient amphibian species, which has never been reported in other vertebrates. The complete open reading frames (ORFs) of AdIRAK2a, AdIRAK2b and AdIRAK2c were 2112 bp, 1917 bp and 816 bp encoding deduced proteins of 703 amino acids (aa), 628 aa and 271 aa, respectively. All three AdIRAK2 proteins contained two predicted conserved functional domains, including a death domain (DD) and a serine/threonine protein kinases domain (KD). Phylogenetic analysis showed that the three AdIRAK2s clustered together with other known IRAK2 of vertebrates. The three AdIRAK2s were ubiquitously expressed in all tested tissues with a similar tissues distribution pattern. After challenge of Aeromonas hydrophila (A. hydrophila), Staphylococcus aureus (S.aureus), giant salamander iridovirus (GSIV, belonging to the genus Ranavirus in the family Iridoviridae) and polyinosinic:polycytidylic acid (poly(I:C)), the expression levels of all AdIRAK2s in blood were significantly altered, however, they exhibited distinct response patterns. Moreover, the results of over-expression and RNAi of AdIRAK2s implied the involvement of AdIRAK2s in triggering NF-κB-mediated signaling pathways and inflammatory responses. This study might provide a better understanding of the presence and immune regulation function of IRAK2 in amphibians and even in vertebrates.

How will warming affect the growth and body size of the largest extant amphibian? More than the temperature–size rule

Declining body size is a universal ecological response to global warming in ectotherms. Ectotherms grow faster but mature at a smaller size at higher temperatures. This phenomenon is known as the temperature–size rule (TSR). However, we know little about the details of the relationship between temperature and size. Here, this issue was studied in the Chinese giant salamander (Andrias davidianus), one of the largest extant amphibians and a flagship species of conservation in China. Warm-acclimated A. davidianus larvae (25 °C) had accelerated development but little superiority in body growth when compared to their 15 °C counterparts when fed with red worm. This predicts a drastic decrease in adult body size with warming. However, a fish diet (more abundant lipid and protein) improved the growth performance at 25 °C. The underlying mechanism was studied. Warm-acclimated larvae had enlarged livers but shortened tails (fat depot). Their livers suffered from energy deficiencies and decreased protein levels, even when protein synthesis and energy metabolism were transcriptionally upregulated. This could be a direct explanation for their poor growth performance. Further analyses revealed a metabolic disorder resembling mammal glycogen storage disease in warm-acclimated larvae, indicating deficiency in glycogen catabolism. This speculation is consistent with their increased lipid and amino acid catabolism and explained the poor energy conditions of the warm-acclimated larvae. Additionally, a deficiency in glycogen metabolism explains the different efficiency of worm and fish diets in supporting the growth of warm-acclimated larvae, even when both diets were provided sufficiently. In conclusion, our results suggest that the relationship between temperature and body size can be flexible, which is a significant finding in terms of the TSR. The underlying metabolic and nutrient mechanisms were revealed. This knowledge can help deepen our understanding of the consequences of warming and can contribute to the conservation of A. davidianus.

Extraction Process Optimization，Purification and Characterization Analysis of Glycoprotein from the Mucus of Chinese Giant Salamander

Extraction Process Optimization，Purification and Characterization Analysis of Glycoprotein from the Mucus of Chinese Giant Salamander

On the estimation of body mass in temnospondyls: a case study using the large‐bodied Eryops and Paracyclotosaurus

AbstractTemnospondyli are a morphologically varied and ecologically diverse clade of tetrapods that survived for over 200 million years. The body mass of temnospondyls is a key variable in inferring their ecological, physiological and biomechanical attributes. However, estimating the body mass of these extinct creatures has proven difficult because the group has no extant descendants. Here we apply a wide range of body mass estimation techniques developed for tetrapods to the iconic temnospondyls Paracyclotosaurus davidi and Eryops megacephalus. These same methods are also applied to a collection of extant organisms that serve as ecological and morphological analogues. These include the giant salamanders Andrias japonicus and Andrias davidianus, the tiger salamander Ambystoma tigrinum, the California newt Taricha torosa and the saltwater crocodile, Crocodylus porosus. We find that several methods can provide accurate mass estimations across this range of living taxa, suggesting their suitability for estimating the body masses of temnospondyls. Based on this, we estimate the mass of Paracyclotosaurus to have been between 159 and 365 kg, and that of Eryops between 102 and 222 kg. These findings provide a basis for examining body size evolution in this clade across their entire temporal span.

Husbandry, Captive Breeding, and Field Survey of Chinese Giant Salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus) is the largest extant amphibian species in the world with adults capable of reaching 2m in length. Wild populations of A. davidianus have declined dramatically during the last century, making it also one of the top threatened species globally. Fortunately, aquaculture for this species developed in China during the 1970s has been extremely successful. Many relevant commercial products of A. davidianus have been produced in recent years on account of its nutritional and medicinal values. Balancing conservation and utilization will be key to the future destiny of A. davidianus. In this chapter, we describe detailed protocols for husbandry in indoor and outdoor facilities, captive breeding under natural-imitative conditions and using artificial insemination, and surveying and monitoring A. davidianus in the field. The protocols presented here aim to make the practices of A. davidianus operative and increase public awareness of this mystical and precious species.

Two new kathlaniid species (Nematoda: Cosmocercoidea) parasitic in salamanders of the genus Andrias (Amphibia: Caudata: Cryptobranchidae)

The new nematode species, Falcaustra hanzaki n. sp. and Urodelnema takanoensis n. sp. (Cosmocercoidea: Kathlaniidae), were found from the intestine of giant salamanders in Kyoto Prefecture, Japan. The first species is featured by the number and arrangement of caudal papillae (3 pairs of precloacal papillae, 8 pairs of postcloacal papillae and a single ventral precloacal papilla), the presence of a single pseudosucker, spicules equal in size (520–638 μm long), and V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The second species is characterized by the number and arrangement of caudal papillae (5 pairs of precloacal papillae, 6 pairs of postcloacal papillae and a single ventral precloacal papilla), spicules equal in size (403–593 μm long), V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The molecular phylogenetic analysis was performed using the partial 18S and 28S ribosomal DNA and the internal transcribed spacers 1 region in the nuclear DNA. This phylogenetic study raised a question about the validity of Family Kathlaniidae and related families of Cosmocercoidea.

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.