Chinese Giant Salamander Andrias Davidianus Research Articles

A time-course transcriptome analysis revealing the potential molecular mechanism of early gonadal differentiation in the Chinese giant salamander

The Chinese giant salamander (CGS) Andrias davidianus is the largest extant amphibian and has recently become an important species for aquaculture with high economic value. Meanwhile, its wild populations and diversity are in urgent need of protection. Exploring the mechanism of its early gonadal differentiation will contribute to the development of CGS aquaculture and the recovery of its wild population. In this study, transcriptomic and phenotypic research was conducted on the critical time points of early gonadal differentiation of CGS. The results indicate that around 210 days post-hatching (dph) is the critical window for female CGS's gonadal differentiation, while 270 dph is that of male CGS. Besides, the TRPM1 gene may be the crucial gene among many candidates determining the sex of CGS. More importantly, in our study, key genes involved in CGS's gonadal differentiation and development are identified and their potential pathways and regulatory models at early stage are outlined. This is an initial exploration of the molecular mechanisms of CGS's early gonadal differentiation at multiple time points, providing essential theoretical foundations for its captive breeding and offering unique insights into the conservation of genetic diversity in wild populations from the perspective of sex development.

Identification and Characterization of the Stimulator of Interferon Genes (STING) in Chinese Giant Salamander Andrias davidianus

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.

Chinese giant salamander Bcl-w: An inhibitory role in iridovirus-induced mitochondrial apoptosis and virus replication

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 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.

Effect of meal size on the postprandial metabolic response in Chinese giant salamander Andrias davidianus larvae

The digestive physiological characteristics of Chinese giant salamander Andrias davidianus have not been reported in the existing literature, and improving our knowledge in this area could help in the artificial culture of the species. The effect of meal size on the specific dynamic action (SDA) of A. davidianus larvae was assessed in this study at a water temperature of 20°C. The Chinese giant salamander was fed by test diets at meal sizes of 0.5, 1, 2, 4, 8 and 12% of its body mass (4.41 ± 0.07 g), and the postprandial oxygen consumption rate (MO2) was measured at 2 h intervals until the rate returned to its preprandial level. Peak MO2 (MO2peak) increased with meal size but levelled off at larger meal sizes within the studied range (8-12% body mass). The factorial metabolic scope increased from 1.27 to 2.11 when meal size increased from 0.5 to 12%. Time to reach MO2peak was not affected by the relative meal size. The SDA duration increased from 8.89 to 47.33 h as the relative meal size increased from 0.5 to 12%. The amount of energy expended during SDA increased linearly with increasing meal size (R2 = 0.842, p < 0.001, n = 60). There was no significant difference in the SDA coefficient among the different relative meal size groups. These data suggest that (1) A. davidianus larvae increase their energy demands during digestion by prolonging the SDA duration rather than increasing the MO2peak as meal size increases, and (2) A. davidianus showed a smaller factorial metabolic scope in the process of digesting large meal sizes, which may be related to the limited MO2peak of the species.

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

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

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

BackgroundThe 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.ResultsIn 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.ConclusionWe 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.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-β promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-β promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-β promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

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.

Identification and functional analysis of two interferon regulatory factor 3 genes and their involvement in antiviral immune responses in the Chinese giant salamander Andrias davidianus

Interferon regulatory factor 3 (IRF3), a crucial member of interferon regulatory factor (IRF) family, plays an important role in innate immunity in vertebrates. However, there are no reports on the characterization and especially their respective functional analysis of two IRF3 genes in some species. In this study, two IRF3 genes as well as their roles in the immune response were identified and investigated in Chinese giant salamander, Andrias davidianus. The complete open reading frames of AdIRF3A and AdIRF3B were 1, 113 bp and 1, 380 bp in length, encoding 370 and 459 amino acids, respectively. Both AdIRF3A and AdIRF3B protein contain an IRF and an IRF3 domain. Phylogenetic analysis indicated that AdIRF3s clustered together with other IRF3 proteins. Tissue distribution analysis showed that AdIRF3s were expressed in all tissues tested, with highest expression levels in blood. Both AdIRF3s actively responded to Chinese giant salamander iridovirus (GSIV) and poly (I:C) challenge in A. davidianus. AdIRF3A/B silencing significantly suppressed the DNA virus and viral RNA analog-induced expression of IFN-inducible genes. Luciferase reporter assay further confirmed the regulatory role of AdIRF3s in IFN signaling. These results provide new insights into the origin or evolution of IRF3 in amphibians and even in vertebrates.

Differential Proteomic Analysis of Chinese Giant Salamander Liver in Response to Fasting.

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.

Future climate change will severely reduce habitat suitability of the Critically Endangered Chinese giant salamander

Abstract Being the largest extant amphibian in the world, the IUCN Critically Endangered Chinese giant salamander Andrias davidianus is a charismatic species with great international public interest. While threats such as commercial overexploitation and habitat degradation have been extensively documented to affect natural populations of A. davidianus, still no information is available about the species sensitivity to climate change. Here, we develop an ensemble of species distribution models (SDMs) for A. davidianus and projected its habitat suitability under present‐day and future climate change scenarios. We based our SDMs on bioclimatic and topographic predictors, and recent (2012–2018) field‐collected occurrence data across the whole distribution range of the species. The ensemble SDMs exhibited good predictive capacity and suggested that slope, maximum temperature of warmest month, precipitation of driest month, and isothermality are the most influential predictors in determining distribution patterns in this species. The projections of our models point to a pronounced impact of climate changes over A. davidianus, with more than two‐thirds of its suitable range expected to be lost in all scenarios of future climates tested. In concert with the numerous other threats that are affecting this species, climate change poses a serious hindrance to the long‐term survival of A. davidianus. We emphasise the urgent need of undertaking strict measures to manage this species and safeguard the few remaining available suitable habitats. We suggest that adaptive management strategies including designation of new reserves should be considered to mitigate the impacts of climate change on A. davidianus.

RNA-seq analysis provides insight into molecular adaptations of Andrias davidianus.

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.

Spatial distribution and seasonal movement patterns of reintroduced Chinese giant salamanders

BackgroundVery little is known about the temporal or spatial movement patterns of Chinese giant salamanders (Andrias davidianus) due to their rarity, remote habitat and secretive nature. Commercial breeding farms provide a unique opportunity as a source of animals for reintroduction and spatial ecology studies, which will help inform conservation management efforts for this threatened species. We surgically implanted radio transmitters into the body cavity of 31 juvenile giant salamanders, and these salamanders were subsequently released into two small river systems (Donghe and Heihe Rivers) located in the Qinling Mountains of central China and were monitored daily from May 2013 to August 2014.ResultsOnly two salamanders survived through the end of the project at the Heihe River compared with 12 at the Donghe River, thus movement data for salamanders released at the Heihe river are described individually. The overall sedentariness (ratio of no movement to all observations) for the two salamanders at the Heihe River was 0.29 and 0.28 compared to the average sedentariness of 0.26 ± 0.01 for the 12 salamanders at the Donghe River. Mean daily movement was 15.4 m ± 0.7 at the Heihe River compared to 9.3 m ± 0.3 at the Donghe River. Overall linear home range (LHR) was 246 m and 392 m for the two salamanders at the Heihe River, compared with a mean LHR of 227.2 m ± 70.5 at the Donghe River. The Donghe salamanders exhibited different movement patterns across seasons, having higher sedentariness, shorter daily movement, and smaller LHR in winter than in summer. Up-stream dispersal and fidelity to release site were recorded at both rivers. The mean dispersal distance for the Donghe River salamanders was 145.3 m ± 61.9, while the two surviving salamanders at the Heihe River had a dispersal distance of 211 m and 205 m.ConclusionsThis project provides important insights on the movement ecology of a large aquatic salamander species, and in particular, our results may assist with reintroduction efforts by developing best management practices on when and where to release animals as a conservation strategy.

Characterization and Functional Properties of Gelatin Extracted from Chinese Giant Salamander (Andrias Davidianus) Skin

ABSTRACTThe characteristics and functional properties of gelatin obtained from Chinese giant salamander (Andrias davidianus) skin were investigated. After alkaline and acetic acid pre-treatment, yield of the gelatin by hot water extraction (60°, 8 h) was 17.63 g/100 g (wet basis), and the gelatin contained 89.46 g/100 g protein, 0.3 g/100 g fat, and 0.41 g/100 g ash (dry basis). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results of the gelatin showed clear band pattern for collagen subunits,β- and α-chains. The gelatin contained 18.85% imino acids, 28.62% hydrophobic amino acids, and 19.68% essential amino acids, which is slightly lower than porcine skin gelatin (PSG). The gelatin exhibited thermo-reversible properties, with gel strength of 192.50 ± 11.47 g, rupture distance of 10.17 ± 0.20 mm, gelling temperature of 4.98°, and melting temperature of 18.04°, Emulsifying activity index (EAI) of the gelatin diminished with increasing concentrations from 10 to 40 mg/mL (p < .05), whereas the emulsifying stability index (ESI) improved significantly (p < .05). Higher concentrations (30 and 40 mg/mL) could significantly enhance the gelatin’s foam expansion (FE) and foam stability (FS) (p < .05). Water absorption ability and oil absorption ability of the gelatin were 7.1 g water/g gelatin and 9.8 g oil/g gelatin, respectively. These results imply that the gelatin with good interfacial properties could potentially be used as a novel ingredient in food emulsion systems.Abbreviations: CGSS, Chinese giant salamander skin; CSG, Chinese giant salamander skin gelatin; PSG, porcine skin gelatin; EAI, emulsion activity index; ESI, emulsion stability index; FE, foam expansion; FS, foam stability; WAC, water absorption capacity; OAC, oil absorption capacity.

Effects of temperature and sex steroids on sex ratio, growth, and growth-related gene expression in the Chinese giant salamander Andrias davidianus

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 28:79-90 (2019) - DOI: https://doi.org/10.3354/ab00710 Effects of temperature and sex steroids on sex ratio, growth, and growth-related gene expression in the Chinese giant salamander Andrias davidianus Qiaomu Hu, Haifeng Tian, Hanbing Xiao* Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China *Corresponding author: xhb@yfi.ac.cn ABSTRACT: Andrias davidianus is one of most farmed amphibians owing to its palatability and nutritional value. In this transitional group between aquatic and terrestrial animals the effects of temperature and sex hormones on sex ratio and growth are not fully understood. Here, we quantified the sex dimorphism of A. davidianus growth with adult males approximately 30% larger than females, and for the first time determined the time of initiation of sex differentiation to be ~98 days post-hatching (dph). Mortality increased significantly with increased temperature, from 9.4% at 20°C to 40.6% at 28°C. At temperatures ≥30°C we observed 100% mortality. The proportion of males was 66.1% at 28°C, significantly higher than in the control group (p < 0.05). 17α-estradiol induced larval feminization and produced female bias of 81-100% at concentrations from 25 µg l-1 to 1000 µg l-1. However, sex bias was not observed in larvae exposed to 17α-methyltestosterone at concentrations of 50 µg l-1 and 100 µg l-1. Additionally, growth characteristics at different temperatures showed that 28°C inhibited growth and 24°C promoted growth, reflecting the expression profile of growth-related genes (GH, GHR, and IGF-1). Sex steroids including 17β-estradiol and 17α-methyltestosterone significantly inhibited growth (p < 0.05). The results suggest that water temperature and sex steroids play a vital role in gonad differentiation and growth of A. davidianus. KEY WORDS: Temperature · Sex steroids · Sex ratio · Growth · Andrias davidianus Full text in pdf format PreviousNextCite this article as: Hu Q, Tian H, Xiao H (2019) Effects of temperature and sex steroids on sex ratio, growth, and growth-related gene expression in the Chinese giant salamander Andrias davidianus. Aquat Biol 28:79-90. https://doi.org/10.3354/ab00710 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 28. Online publication date: August 08, 2019 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2019 Inter-Research.

Genome-wide RAD sequencing to identify a sex-specific marker in Chinese giant salamander Andrias davidianus

BackgroundChinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism.ResultsWe used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17β-estradiol exposed group.ConclusionThis is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.

Identification and Сharacteristics of Conserved miRNA in Testis Tissue from Chinese Giant Salamander (Andrias davidianus) by Deep Sequencing

MicroRNAs (miRNAs) are a family of ∼22-nucleotide, non-coding, single-stranded RNA molecules, which are considered as key regulators of gene expression and regulate various biological processes including gonad development, differentiation and dimorphism. Although most miRNAs studied have been identified in animals, relatively limited knowledge also exist on amphibian species. Identification of the full repertoire of miRNAs in testes of Andrias davidianus would significantly increase our knowledge on miRNA physiological functions of miRNA in gonads. In this study, a small RNA library was constructed from adult A. davidianus testis tissue and sequenced using the deep sequencing. As a result, 26 401 474 clean reads was obtained, and 1340 known conserved miRNAs were identified. Additionally, expression of 12 randomly selected miRNAs was confirmed by stem-loop qRT-PCR from 5 different tissues of A. davidianus. Of them, aca-miR-99b-5p, xtr-miR-10c, and cfa-miR-363 were highly expressed in testes. Although the physiological functions of these expressed miRNAs were still not revealed, their expression patterns indicate that they are likely to play a role in growth and development of A. davidianus testes, which is needed to be further study for confirmation. The results of this study will help in gaining understanding of the role of miRNAs in the regulation of A. davidianus reproductive system.

Identification of critical sex-biased genes in Andrias davidianus by de novo transcriptome.

The Chinese giant salamander Andrias davidianus is a protected amphibian with high nutritional and economic value. Understanding its sex determination mechanism is important for improving culture techniques and sex control in breeding. However, little information on the characterization of critical genes involved in sex is available. Herein, sequencing of ovary and test produced 40,783,222 and 46,128,902 raw reads, respectively, which were jointly assembled into 80,497 unigenes. Of these, 36,609 unigenes were annotated, of which 8907 were female-biased and 10,385 were male-biased. Several sex-related pathways were observed, including the Wnt signaling pathway. After elevated temperature and estrogen exposure, neomale and neofemale specimens were identified by a female-specific marker for the first time. RT-qPCR analysis showed the expression profile of ten selected sex-biased genes to be exhibited consistently in male and neomale and in female and neofemale, with the exception of the Amh and TfIIIa genes. Results suggested that these genes may play important roles in A. davidianus sex determination and gonad development. This provides a basis for further investigation of the molecular mechanisms of sex determination in amphibians.

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.