Cynops Orientalis Research Articles

The mRNA and microRNA Landscape of the Blastema Niche in Regenerating Newt Limbs.

Newts are excellent vertebrate models for investigating tissue regeneration due to their remarkable regenerative capabilities. To investigate the mRNA and microRNAs (miRNAs) profiles within the blastema niche of regenerating newt limbs, we amputated the limbs of Chinese fire belly newts (Cynops orientalis) and conducted comprehensive analyses of the transcriptome and microRNA profiles at five distinct time points post-amputation (0 hours, 1 day, 5 days 10 days and 20 days). We identified 24 significantly differentially expressed (DE) genes and 20 significantly DE miRNAs. Utilizing weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) enrichment analysis, we identified four genes likely to playing crucial roles in the early stages of limb regeneration: Cemip, Rhou, Gpd2 and Pcna. Moreover, mRNA-miRNA integration analysis uncovered seven human miRNAs (miR-19b-1, miR-19b-2, miR-21-5p, miR-127-5p, miR-150-5p, miR-194-5p, and miR-210-5p) may regulate the expression of these four key genes. The temporal expression patterns of these key genes and miRNAs further validated the robustness of the identified mRNA-miRNA landscape. Our study successfully identified candidate key genes and elucidated a portion of the genetic regulatory mechanisms involved in newt limb regeneration. These findings offer valuable insights for further exploration of the intricate processes of tissue regeneration.

Transcriptomic analysis of spinal cord regeneration after injury in Cynops orientalis.

Cynops orientalis (C. orientalis) has a pronounced ability to regenerate its spinal cord after injury. Thus, exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration. In this study, we established a model of spinal cord thoracic transection injury in C. orientalis, which is an endemic species in China. We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury - during the very acute stage (4 days) and the subacute stage (7 days) - and identified differentially expressed genes; additionally, we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, at each time point. Transcriptome sequencing showed that 13,059 genes were differentially expressed during C. orientalis spinal cord regeneration compared with uninjured animals, among which 4273 were continuously down-regulated and 1564 were continuously up-regulated. Down-regulated genes were most enriched in the Gene Ontology term "multicellular organismal process" and in the ribosome pathway at 10 days following spinal cord injury. We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury, indicating the importance of low metabolic activity during wound healing. Immune response-associated pathways were activated during the early acute phase (4 days), while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen, as well as tight junction proteins, was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury. However, compared with 4 days post-injury, at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated, up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle, and SHH, VIM, and Sox2 were prominently up-regulated. Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury, similar to what is observed in mammalian astrocytes after spinal cord injury, even though axolotls do not form a glial scar during regeneration. We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells, thereby inhibiting reactive gliosis, at early stages after spinal cord injury. Extracellular matrix degradation slows cellular responses, represses the expression of neurogenic genes, and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells. These ependymoglial cells act as neural stem cells: they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons. This provides the regenerative microenvironment that allows axon growth after injury.

Investigation of the activity of transposable elements and genes involved in their silencing in the newt Cynops orientalis, a species with a giant genome

Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs.

Species distribution models for predicting the habitat suitability of Chinese fire-bellied newt Cynops orientalis under climate change.

Climate change influences species geographical distribution and diversity pattern. The Chinese fire‐bellied newt (Cynops orientalis) is an endemic species distributed in East‐central China, which has been classified as near‐threatened species recently due to habitat destruction and degradation and illegal trade in the domestic and international pet markets. So far, little is known about the spatial distribution of the species. Based on bioclimatic data of the current and future climate projections, we modeled the change in suitable habitat for C. orientalis by ten algorithms, evaluated the importance of environmental factors in shaping their distribution, and identified distribution shifts under climate change scenarios. In this study, 46 records of C. orientalis from East China and 8 bioclimatic variables were used. Among the ten modeling algorithms, four (GAM, GBM, Maxent, and RF) were selected according to their predictive abilities. The current habitat suitability showed that C. orientalis had a relatively wide but fragmented distribution, and it encompassed 41,862 km2. The models suggested that precipitation of warmest quarter (bio18) and mean temperature of wettest quarter (bio6) had the highest contribution to the model. This study revealed that C. orientalis is sensitive to climate change, which will lead to a large range shift. The projected spatial and temporal pattern of range shifts for C. orientalis should provide a useful reference for implementing long‐term conservation and management strategies for amphibians in East China.

The vitellogenin genes in Cynops orientalis: New insights on the evolution of the vtg gene family in amphibians.

The vitellogenins (Vtgs) are glycolipophosphoproteins that play a key role in constituting nutritional reserves for embryo development in nonmammalian vertebrates. However, additional functional roles have been evidenced. These vtg genes are present in multiple copies, different in number and sequences in various vertebrate lineages. The comprehension of the vtg gene family evolutionary history remains a matter of intense interrogation for this field of research. In tetrapods, information about vtg genes are limited to few taxa. Up to date concerning amphibians, detailed studies have been conducted only in Anura. Therefore, in this study, to further increase knowledge about vtg genes in Amphibia class, the urodele Cynops orientalis (Amphibia: Caudata) was analyzed and four complete vtg sequences were obtained. Moreover, genomic data available for the caecilians Microcaecilia unicolor and Rhinatrema bivittatum (Amphibia: Gymnophiona) were also included. In these amphibians, our findings evidenced the presence of a vtgI sequence ortholog to that of tetrapods, absent in Anura. Moreover, microsyntenic, phylogenetic, and gene conversion analyses allowed postulating two hypotheses to explain the complex evolutionary history of this gene family.

Acquiring toxicity of a newt, Cynops orientalis

Tetrodotoxin (TTX) contents of wild-caught Chinese red-bellied newts, Cynops orientalis, and their offspring captive-reared from eggs to metamorphosed juveniles, were analysed using post-column LC-fluorescent detection (LC-FLD) and high resolution hydrophilic interaction liquid chromatography/mass spectrometry (HR-HILIC-LC/MS). TTX was detected in the parent newts and their eggs, but not in the larvae and juveniles raised under artificial condition over 20 months. However, juveniles reared in the presence of their parents, contained TTX-concentrations up to 8.05 μg/g. The origin of TTX may be implied from a close connection between the parents and their offspring.

Expression and Functional Characterization of c-Fos Gene in Chinese Fire-Bellied Newt Cynops Orientalis.

c-Fos is an immediate-early gene that modulates cellular responses to a wide variety of stimuli and also plays an important role in tissue regeneration. However, the sequence and functions of c-Fos are still poorly understood in newts. This study describes the molecular cloning and characterization of the c-Fos gene (Co-c-Fos) of the Chinese fire-bellied newt, Cynops orientalis. The full-length Co-c-Fos cDNA sequence consists of a 1290 bp coding sequence that encoded 429 amino acids. The alignment and phylogenetic analyses reveal that the amino acid sequence of Co-c-Fos shared a conserved basic leucine zipper domain, including a nuclear localization sequence and a leucine heptad repeat. The Co-c-Fos mRNA is widely expressed in various tissues and is highly and uniformly expressed along the newt limb. After limb amputation, the expression of Co-c-Fos mRNA was immediately upregulated, but rapidly declined. However, the significant upregulation of Co-c-Fos protein expression was sustained for 24 h, overlapping with the wound healing stage of C. orientalis limb regeneration. To investigate if Co-c-Fos participate in newt wound healing, a skin wound healing model is employed. The results show that the treatment of T-5224, a selective c-Fos inhibitor, could largely impair the healing process of newt’s skin wound, as well as the injury-induced matrix metalloproteinase-3 upregulation, which is fundamental to wound epithelium formation. These data suggest that Co-c-Fos might participate in wound healing by modulating the expression of its potential target gene matrix metalloproteinase-3. Our study provides important insights into mechanisms that are responsible for the initiation of newt limb regeneration.

Molecular cloning, the characterization of metallothionein and catalase, and the evaluation of testicular toxicity of Cd in the Chinese fire-bellied newt (Cynops orientalis)

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.

The transcriptome of the newt Cynops orientalis provides new insights into evolution and function of sexual gene networks in sarcopterygians

Amphibians evolved in the Devonian period about 400 Mya and represent a transition step in tetrapod evolution. Among amphibians, high-throughput sequencing data are very limited for Caudata, due to their largest genome sizes among terrestrial vertebrates. In this paper we present the transcriptome from the fire bellied newt Cynops orientalis. Data here presented display a high level of completeness, comparable to the fully sequenced genomes available from other amphibians. Moreover, this work focused on genes involved in gametogenesis and sexual development. Surprisingly, the gsdf gene was identified for the first time in a tetrapod species, so far known only from bony fish and basal sarcopterygians. Our analysis failed to isolate fgf24 and foxl3, supporting the possible loss of both genes in the common ancestor of Rhipidistians. In Cynops, the expression analysis of genes described to be sex-related in vertebrates singled out an expected functional role for some genes, while others displayed an unforeseen behavior, confirming the high variability of the sex-related pathway in vertebrates.

Characterization of glycoconjugates and sialic acid modification in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis).

Diverse glycoconjugates are expressed in the vertebrate olfactory bulb and serve as guidance cues for axons of nasal receptor neurons. Although the involvement of glycoconjugates in the segregation of the olfactory pathway has been suggested, it is poorly understood in salamanders. In this study, lectin histochemistry was used to determine glycoconjugate distribution in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis). Succinylated wheat germ agglutinin (sWGA), Ricinus communis agglutinin-I and Lens culinaris agglutinin showed different bindings in the nerve fibre layer or glomerular layer, or both, between the main and accessory olfactory bulbs. We then investigated the lectin-binding pattern after the removal of terminal sialic acids using neuraminidase. Desialylation resulted in a change in the binding reactivities with seven lectins. Wheat germ agglutinin, sWGA, soybean agglutinin (SBA) and peanut agglutinin showed different degrees of binding between the main and accessory olfactory bulbs. In addition, SBA showed a heterogeneous labelling of glomeruli in the rostral region of the main olfactory bulb. Our results suggest that terminal sialic acids mask the heterogeneity of glycoconjugates in the olfactory bulb of C.orientalis.

Comparative Morphology and Histology of the Brain in Chinese Toad ( Bufo gargarizans ) and Chinese Fire-billed Newt ( Cynops orientalis )

Comparative Morphology and Histology of the Brain in Chinese Toad ( Bufo gargarizans ) and Chinese Fire-billed Newt ( Cynops orientalis )

Integrative Analysis of MicroRNAome, Transcriptome, and Proteome during the Limb Regeneration of Cynops orientalis.

Salamanders completely regenerate their limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating regeneration in vertebrates. To investigate the influence of microRNAs (miRNAs) on newt limb regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq 2500 System during limb regeneration of Cynops orientalis at 3, 7, 14, 30 and 42 days postamputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling data sets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stage-specific regulation of miRNAs on their targets was analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration analysis will provide a powerful tool to identify the regulatory mechanisms of miRNAs and their targets. The results may have implications in understanding the complex mechanisms underlying newt limb regeneration.

Widespread occurrence of an emerging fungal pathogen in heavily traded Chinese urodelan species

AbstractUnderstanding introduction routes for wildlife pathogens is vital for the development of threat abatement plans. The chytrid fungus Batrachochytrium salamandrivorans (Bsal) has recently emerged in Europe, where it is considered to be a serious threat for urodelan conservation. If the highly diverse Chinese urodelans were to constitute a Bsal reservoir, then the significant international trade in these species may vector Bsal into naïve urodelan communities. Here, we analyzed a total of 1,143 samples, representing 36 Chinese salamander species from 51 localities across southern China for the presence of Bsal. We found Bsal was present across a wide taxonomic, geographical, and environmental range. In particular, Bsal DNA was detected in 33 samples from the genera Cynops, Pachytriton, Paramesotriton, Tylototriton, and Andrias, including the heavily traded species Paramesotriton hongkongensis and Cynops orientalis. The true Bsal prevalence across our data set was estimated between 2% and 4%, with a maximum of 50% in a population of P. hongkongensis. Even at this overall relatively low Bsal prevalence, the exportation of millions of animals renders Bsal introduction in naïve, importing countries a near certainty, which calls for the urgent implementation of proper biosecurity in the international wildlife trade.

Molecular cloning, characterization, and expression analysis of the three cysteine and glycine-rich protein genes in the Chinese fire-bellied newt Cynops orientalis

The cysteine- and glycine-rich protein (CRP) family members, including the cysteine- and glycine-rich protein 1 (CSRP1), cysteine- and glycine-rich protein 2 (CSRP2), and the cysteine- and glycine-rich protein 3 (CSRP3), have exhibited various cellular functions during cell development and differentiation. However, the sequences of the three CSRP genes and their functions are still poorly understood in newts. In this study, we cloned the complete open reading frame (ORF) sequences of the three CSRP genes from the Chinese fire-bellied newt, Cynops orientalis (C. orientalis). The complete ORF sequences of Co-CSRP1, Co-CSRP2, and Co-CSRP3 were 582, 582, and 576bp, respectively, and encoded 193, 193, and 191 amino acids, respectively. The deduced amino acid sequences of the three CRP members showed high similarities with that of other species, particularly, with amphibians. Co-CSRP1 was highly expressed in the kidney, limb, and stomach, however, the expression was low in the spleen, heart, intestine, liver, and tail (P<0.05). The mRNA expression of Co-CSRP2 was higher in the kidney and heart than that in other organs (P<0.05). It was observed that Co-CSRP3 was only expressed in the heart, limb, and tail. The mRNA expression of Co-CSRP1 and Co-CSRP3 was lower in the digits in comparison to other limb segments. However, there was no significant difference of Co-CSRP2 mRNA expression in the four limb segments. The Co-CSRP1 and Co-CSRP2 mRNA expressions were significantly increased, whereas the expression of Co-CSRP3 was remarkably decreased during the limb regeneration. This study will provide useful information for further elucidating the role of Co-CSRP genes during newt limb regeneration.

ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration

BackgroundSalamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa).ResultsOf 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding.ConclusionsThis work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.

Profiling of glycan alterations in regrowing limb tissues of Cynops orientalis.

Glycans are known to play important roles in molecular recognition, cell-cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA-E, MAL-I, SNA, UEA-I, PHA-E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL-I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL-II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.

Tetrodotoxin in Asian newts (Salamandridae)

Tetrodotoxin (TTX) and its analogues occur in a wide range of marine animals but also in terrestrial vertebrates such as frogs, toads and newts. Despite numerous studies on TTX in New World newts (Notophthalmus viridescens, Taricha spp.), few data only exist for Asian newts. Methanolic extracts of newts from China (Cynops orientalis, Pachytriton labiatus, Paramesotriton chinensis), Vietnam (Paramesotriton deloustali, P. guangxiensis), and Laos (Laotriton laoensis) were analyzed by liquid-chromatography-fluorescent detection (LC-FLD) and mass-spectrometry (LC-MS). In all species, variable amounts of TTX were detected, in most specimens also TTX-analogues like 6-epiTTX, in C. orientalis 11-oxoTTX, confirming the presence of these toxins in modern Asian newts.

The complete mitochondrial genome of Cynops orientalis (Salamandridae: Cynops)

The Oriental Fire-bellied Newt (Cynops orientalis David) has been listed as Near Threatened species (NT) recently due to habitat destruction and degradation and illegal trade in the domestic and international pet markets. We sequenced and characterized the complete mitochondrial genome (mitogenome) of C. orientalis in this paper. The complete mitogenome sequence of C. orientalis is 16,294 bp in length, containing 37 genes (13 protein coding genes, two ribosomal RNA, 22 transfer RNA genes and one control region). The new sequenced complete mitogenome of C. orientalis will provide useful information for application in conservation genetics and evolution for this near threatened species.

东方蝾螈幼体热耐受性和游泳表现的热驯化响应

东方蝾螈幼体热耐受性和游泳表现的热驯化响应

Swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures

Thermal acclimatory adjustments of locomotor performance and thermal tolerance occur commonly in ectothermic animals. However, few studies have investigated ontogenetic differences in these acclimatory responses, and thus, their causes remain unclear. In this study, juvenile and adult Chinese fire-bellied newts ( Cynops orientalis ) were acclimated to one of two temperatures (16 or 24 °C) for 4 weeks to examine ontogenetic differences in acclimation effect on burst swimming speed, and critical thermal minimum (CTMin) and maximum (CTMax). Swimming performance was thermally acclimated in both juvenile and adult C. orientalis . Adult newts had greater absolute swimming speeds than juveniles, which may simply result from their larger sizes. Cold acclimation enhanced low-temperature resistance, and warm acclimation enhanced high-temperature resistance in both juveniles and adults. Despite no ontogenetic difference in CTMin, adult newts had greater CTMax and acclimation response ratio than juveniles, indicating their greater abilities to withstand extreme high temperatures and manage rapid temperature shifts. Ontogenetic change in the thermal acclimatory responses of newts may be related to changes in the thermal environment they experience.