Androgen and glucocorticoid profiles throughout extended uniparental paternal care in the eastern hellbender salamander (Cryptobranchus a. alleganiensis).

The behavioral endocrinology associated with reproduction and uniparental male care has been studied in teleosts, but little is known about hormonal correlates of uniparental male care in other ectotherms. To address this gap, we are the first to document the seasonal steroid endocrinology of uniparental male hellbender salamanders during the transition from pre-breeding to nest initiation, and through the subsequent eight months of paternal care. In doing so, we investigated the correlates of nest fate and clutch size, exploring hellbenders' alignment with several endocrinological patterns observed in uniparental male fish. Understanding the endocrinology of hellbender paternal care is also vital from a conservation perspective because high rates of nest failure were recently identified as a factor causing population declines in this imperiled species. We corroborated previous findings demonstrating testosterone and dihydrotestosterone (DHT) to be the primary androgens in hellbender reproduction, and that cortisol circulates as the most abundant glucocorticoid. However, we were unable to identify a prolactin or a "prolactin-like" peptide in circulation prior to or during parental care. We observed âˆ¼ 80 % declines in both primary androgens during the transition from pre-breeding to nest initiation, and again as paternal care progressed past its first month. In the days immediately following nest initiation, testosterone and DHT trended higher in successful individuals, but did not differ with males' clutch size. We did not observe meaningful seasonality in baseline glucocorticoids associated with breeding or nesting. In contrast, stress-induced glucocorticoids were highest at pre-breeding and through the first two months of care, before declining during the latter-most periods of care as larvae approach emergence from the nest. Neither baseline nor stress-induced glucocorticoids varied significantly with either nest fate or clutch size. Both stress-induced cortisol and corticosterone were positively correlated with total length, a proxy for age in adult hellbenders. This is consistent with age-related patterns in some vertebrates, but the first such pattern observed in a wild amphibian population. Generally, we found that nesting hellbenders adhere to some but not all of the endocrinological patterns observed in uniparental male teleosts prior to and during parental care.

Molecular Diversity of Neuron Types in the Salamander Amygdala and Implications for Amygdalar Evolution.

The amygdala is a complex brain structure in the vertebrate telencephalon, essential for regulating social behaviors, emotions, and (social) cognition. In contrast to the vast majority of neuron types described in the many nuclei of the mammalian amygdala, little is known about the neuronal diversity in non-mammals, making reconstruction of its evolution particularly difficult. Here, we characterize glutamatergic neuron types in the amygdala of the urodele amphibian Pleurodeles waltl. Our single-cell RNA sequencing data indicate the existence of at least ten distinct types and subtypes of glutamatergic neurons in the salamander amygdala. These neuron types are molecularly distinct from neurons in the ventral pallium (VP), suggesting that the pallial amygdala and the VP are two separate areas in the telencephalon. In situ hybridization for marker genes indicates that amygdalar glutamatergic neuron types are located in three major subdivisions: the lateral amygdala, the medial amygdala, and a newly defined area demarcated by high expression of the transcription factor Sim1. The gene expression profiles of these neuron types suggest similarities with specific neurons in the sauropsid and mammalian amygdala. In particular, we identify Sim1+ and Sim1+ Otp+ expressing neuron types, potentially homologous to the mammalian nucleus of the lateral olfactory tract (NLOT) and to hypothalamic-derived neurons of the medial amygdala, respectively. Taken together, our results reveal a surprising diversity of glutamatergic neuron types in the amygdala of salamanders, despite the anatomical simplicity of their brain. These results offer new insights on the cellular and anatomical complexity of the amygdala in tetrapod ancestors.

Altered metabolic state impedes limb regeneration in salamanders.

Salamanders are unique among tetrapods in their ability to regenerate their limbs throughout life. Like other poikilothermic amphibians, salamanders also show a remarkable capacity to survive long periods of starvation. Whether the physiological reserves necessary for tissue regeneration are preserved or sacrificed in starved salamanders is unknown. In the current study, we maintained Iberian ribbed newts ( Pleurodeles waltl) under extreme physiological stress to assess the extent of regeneration and identify the molecular and cellular changes that may occur under such conditions. After 19 months of complete food deprivation, the animals exhibited extensive morphological and physiological adaptations but remained behaviorally active and vigilant. Autophagy was elevated in different tissues and the transformed gut microbiota indicated remodeling of the intestinal tract related to autophagy. Upon limb amputation in animals starved for 21 months, regeneration proceeded with progenitor cell proliferation and migration, leading to limb blastema formation. However, limb outgrowth and patterning were substantially attenuated. Blockage of autophagy inhibited cell proliferation and blastema formation in starved animals, but not in fed animals. Hence, tissue autophagy and the regenerative response were tightly coupled only when animals were under stress. Our results demonstrate that under adverse conditions, salamanders can exploit alternative strategies to secure blastema formation for limb regeneration.

Coevolution between MHC Class I and Antigen-Processing Genes in Salamanders.

Proteins encoded by antigen-processing genes (APGs) provide major histocompatibility complex (MHC) class I (MHC-I) with antigenic peptides. In mammals, polymorphic multigenic MHC-I family is served by monomorphic APGs, whereas in certain nonmammalian species both MHC-I and APGs are polymorphic and coevolve within stable haplotypes. Coevolution was suggested as an ancestral gnathostome feature, presumably enabling only a single highly expressed classical MHC-I gene. In this view coevolution, while optimizing some aspects of adaptive immunity, would also limit its flexibility by preventing the expansion of classical MHC-I into a multigene family. However, some nonmammalian taxa, such as salamanders, have multiple highly expressed MHC-I genes, suggesting either that coevolution is relaxed or that it does not prevent the establishment of multigene MHC-I. To distinguish between these two alternatives, we use salamanders (30 species from 16 genera representing six families) to test, within a comparative framework, a major prediction of the coevolution hypothesis: the positive correlation between MHC-I and APG diversity. We found that MHC-I diversity explained both within-individual and species-wide diversity of two APGs, TAP1 and TAP2, supporting their coevolution with MHC-I, whereas no consistent effect was detected for the other three APGs (PSMB8, PSMB9, and TAPBP). Our results imply that although coevolution occurs in salamanders, it does not preclude the expansion of the MHC-I gene family. Contrary to the previous suggestions, nonmammalian vertebrates thus may be able to accommodate diverse selection pressures with flexibility granted by rapid expansion or contraction of the MHC-I family, while retaining the benefits of coevolution between MHC-I and TAPs.

Full-length transcriptome assembly of andrias davidianus (amphibia: caudata) skin via hybrid sequencing.

The Chinese giant salamander, Andrias davidianus, is the largest amphibian species in the world; it is thus an economically and ecologically important species. The skin of A. davidianus exhibits complex adaptive structural and functional adaptations to facilitate survival in aquatic and terrestrial ecosystems. Here, we report the first full-length amphibian transcriptome from the dorsal skin of A. davidianus, which was assembled using hybrid sequencing and the PacBio and Illumina platforms. A total of 153,038 transcripts were hybrid assembled (mean length of 2039 bp and N50 of 2172 bp), and 133,794 were annotated in at least one database (nr, Swiss-Prot, KEGG, KOGs, GO, and nt). A total of 58,732, 68,742, and 115,876 transcripts were classified into 24 KOG categories, 1903 GO term categories, and 46 KEGG pathways (level 2), respectively. A total of 207,627 protein-coding regions, 785 transcription factors, 27,237 potential long non-coding RNAs, and 8299 simple sequence repeats were also identified. The hybrid-assembled transcriptome recovered more full-length transcripts, had a higher N50 contig length, and a higher annotation rate of unique genes compared with that assembled in previous studies using next-generation sequencing. The high-quality full-length reference gene set generated in this study will help elucidate the genetic characteristics of A. davidianus skin and aid the identification of functional skin proteins.

Energy storage in salamanders' tails: the role of sex and ecology.

In vertebrates, the main tissue devoted to energy storage is the adipose tissue. In salamanders, energy reserves can also be stored in the adipose tissues of the tail. Therefore, we evaluated if energy storage in salamanders' tails is related to individual body condition, life cycle and environmental constraints. We calculated a scaled measure of tail width for 345 salamanders belonging to six Mediterranean taxa exhibiting wide phylogenetic, behavioural and ecological variation. We related this measure to the Scaled Mass Index (SMI), a body condition index which reliably predicts body fat. We found significant relationships between the SMI and scaled tail width in the terrestrial Spectacled salamander and Alpine salamanders, independently of sex. At the same time, we found that energy storage in the tail is maximum in Alpine Salamanders, which experience reduced activity periods and restricted access to resources. Conversely, we found a significant effect of sex in Imperial cave salamanders, where females store reserves in the tail to counterbalance resource investment in parental care, and in Corsican Brook Newts, where the reproductive function of males' tails may imply a greater tail width. Finally, in the biphasic Great Crested Newt, tail width was not related to SMI in both sexes.

Salamanders: The molecular basis of tissue regeneration and its relevance to human disease.

Salamanders are recognized for their ability to regenerate a broad range of tissues. They have also have been used for hundreds of years for classical developmental biology studies because of their large accessible embryos. The range of tissues these animals can regenerate is fascinating, from full limbs to parts of the brain or heart, a potential that is missing in humans. Many promising research efforts are working to decipher the molecular blueprints shared across the organisms that naturally have the capacity to regenerate different tissues and organs. Salamanders are an excellent example of a vertebrate that can functionally regenerate a wide range of tissue types. In this review, we outline some of the significant insights that have been made that are aiding in understanding the cellular and molecular mechanisms of tissue regeneration in salamanders and discuss why salamanders are a worthy model in which to study regenerative biology and how this may benefit research fields like regenerative medicine to develop therapies for humans in the future.

Collagens made from giant salamander (Andrias davidianus) skin and their odorants.

Two collagens were made from giant salamander (Andrias davidianus) skin by using acid and pepsin extraction methods. The yields of acid-soluble and pepsin-soluble collagens were 26.9 and 58.7%, respectively. The results of spectrum, electrophoresis and amino acid analysis showed that they were type 1 collagen with two α and one ß peptides and high imino acid content. They had low solubility at a pH above 6 or salt concentration over 5%. The pepsin-soluble collagen had a better emulsion activity index. The odorants in raw skin and collagens were identified and evaluated using gas-chromatography mass-spectrometer and olfactometry methods and sensory analysis. The fishy and fatty off-odors in skin were not perceivable in the collagens. Sour, ammonia-like, and acrid off-odors were found in the collagens due to acid and enzymatic hydrolysis and protein degradation. The off-odor intensity of pepsin-soluble collagen was low. It could be considered a good and safe collagen material.

Codon usage pattern and evolutionary forces of mitochondrial ND genes among orders of class Amphibia.

In this study, we used a bioinformatics approach to analyze the nucleotide composition and pattern of synonymous codon usage in mitochondrial ND genes in three amphibian groups, that is, orders Anura, Caudata, and Gymnophiona to identify the commonality and the differences of codon usage as no research work was reported yet. The high value of the effective number of codons revealed that the codon usage bias (CUB) was low in mitochondrial ND genes among the orders. Nucleotide composition analysis suggested that for each gene, the compositional features differed among Anura, Caudata, and Gymnophiona and the GC content was lower than AT content. Furthermore, a highly significant difference (p < .05) for GC content was found in each gene among the orders. The heat map showed contrasting patterns of codon usage among different ND genes. The regression of GC12 on GC3 suggested a narrow range of GC3 distribution and some points were located in the diagonal, indicating both mutation pressure and natural selection might influence the CUB. Moreover, the slope of the regression line was less than 0.5 in all ND genes among orders, indicating natural selection might have played the dominant role whereas mutation pressure had played a minor role in shaping CUB of ND genes across orders.

Expression of Genes Involved in Offensive and Defensive Phenotype Induction in the Pituitary Gland of the Hokkaido Salamander (Hynobius retardatus).

Amphibians exhibit phenotypic plasticity, which allows flexible adaptation to fluctuating environments. Although genes involved in expression of plastic phenotypes have been identified, the endocrine bases of plastic responses are largely unknown. Larvae of the Hokkaido salamander (Hynobius retardatus) plastically display distinct phenotypes, an "offensive phenotype" characterized as larger body with broadened gape and a "defensive phenotype" characterized as enlarged gills and tail and less active behavior, in the presence of prey larval amphibians and predatory larval dragonfly, respectively. In the presence of both prey and predators, the degree of induction of both phenotypes is reduced, suggesting cross-talk between the molecular signaling pathways of these phenotypes. We conducted a transcriptomic analysis to examine how endocrine regulation affects the phenotypic expression by focusing on the pituitary gland. We found that five endocrine genes, i.e., calcitonin related polypeptide alpha (CALCA), growth hormone (GH), neuropeptide B (NPB), parathyroid hormone 2 (PTH2), and prolactin 1 (PRL1), were involved in the expression of both phenotypes. However, we conducted only RNA-seq analysis, and no confirmation of significant up-regulation or down-regulation has been conducted. These results suggest that these genes were up-regulated for induction of the offensive phenotype and down-regulated for induction of the defensive phenotype. Phylogenetic analysis indicated that possible gene duplications of PRL and CALCA have occurred during amphibian evolution. Based on these findings, it is suggested that a trade-off of molecular signaling pathways exists between the two distinct phenotypic expressions. The results also suggest that hormonal-gene duplications might have contributed to the acquisition of phenotypic plasticity in amphibians.

Species-specific and heterogeneous distribution of sialoglycoconjugates in the primary olfactory center of three species of Asian salamanders (Cynops).

Sialic acids (Sia) are terminal components of glycoconjugates that are involved in molecular and cellular interactions in the olfactory system. Diverse glycoconjugates are expressed in the salamander olfactory projection; however, their sialylation and the linkage of Sia to underlying sugars remain largely unknown. The present study aimed to determine the expression of Sia linked to galactose (Gal)-N-acetylglucosamine and N-acetylgalactosamine (GalNAc) in the olfactory bulbs of three species of salamanders using lectin binding. Abundant distribution of sialoglycoconjugates was observed in the salamander olfactory bulb by lectins, Sambucus sieboldiana (SSA) and Maackia amurensis (MAM). Moreover, SSA and MAM showed heterogeneous bindings in the primary olfactory projection of Cynops pyrrhogaster and C. orientalis. Lectin reactivities obviously decreased in all layers of the olfactory bulb after sialidase digestion, indicating selective binding to sialoglycoconjugates. Next, we examined the expression of the subterminal sugar residues, Gal and GalNAc, after terminal Sia removal. Desialylation in the olfactory bulb enhanced the reactivity of Jacalin and Vicia villosa (VVA) lectins that recognize Gal and GalNAc respectively. Together with the binding of SSA and MAM, Sia linked to Gal and GalNAc might be a major component of sialoglycoconjugates in the salamander olfactory projection.

The serotonin reuptake blocker citalopram destabilizes fictive locomotor activity in salamander axial circuits through 5-HT1A receptors.

Serotoninergic (5-HT) neurons are powerful modulators of spinal locomotor circuits. Most studies on 5-HT modulation focused on the effect of exogenous 5-HT and these studies provided key information about the cellular mechanisms involved. Less is known about the effects of increased release of endogenous 5-HT with selective serotonin reuptake inhibitors. In mammals, such molecules were shown to destabilize the fictive locomotor output of spinal limb networks through 5-HT1A receptors. However, in tetrapods little is known about the effects of increased 5-HT release on the locomotor output of axial networks, which are coordinated with limb circuits during locomotion from basal vertebrates to mammals. Here, we examined the effect of citalopram on fictive locomotion generated in axial segments of isolated spinal cords in salamanders, a tetrapod where raphe 5-HT reticulospinal neurons and intraspinal 5-HT neurons are present as in other vertebrates. Using electrophysiological recordings of ventral roots, we show that fictive locomotion generated by bath-applied glutamatergic agonists is destabilized by citalopram. Citalopram-induced destabilization was prevented by a 5-HT1A receptor antagonist, whereas a 5-HT1A receptor agonist destabilized fictive locomotion. Using immunofluorescence experiments, we found 5-HT-positive fibers and varicosities in proximity with motoneurons and glutamatergic interneurons that are likely involved in rhythmogenesis. Our results show that increasing 5-HT release has a deleterious effect on axial locomotor activity through 5-HT1A receptors. This is consistent with studies in limb networks of turtle and mouse, suggesting that this part of the complex 5-HT modulation of spinal locomotor circuits is common to limb and axial networks in limbed vertebrates.NEW & NOTEWORTHY Little is known about the modulation exerted by endogenous serotonin on axial locomotor circuits in tetrapods. Using axial ventral root recordings in salamanders, we found that a serotonin reuptake blocker destabilized fictive locomotor activity through 5-HT1A receptors. Our anatomical results suggest that serotonin is released on motoneurons and glutamatergic interneurons possibly involved in rhythmogenesis. Our study suggests that common serotoninergic mechanisms modulate axial motor circuits in amphibians and limb motor circuits in reptiles and mammals.

Translucent in air and iridescent in water: structural analysis of a salamander egg sac.

Females of some Asian salamanders of the genus Hynobius deposit in streams their eggs embedded in a translucent envelope called an 'egg sac'. The edges of the envelope exhibit a spectacular blue-to-yellow iridescent glow, which instantaneously disappears when the sac is removed from water. First, our scanning electron microscopy analyses reveal that the inner surface of the 100 µm-thick envelope displays striations (length scale of about 3 µm), which are themselves covered by much smaller (190 ± 30 nm) and quasi-periodic corrugations. The latter could constitute a surface diffraction grating generating iridescence by light interference. Second, our transmission electron microscopy and focused-ion-beam scanning electron microscopy analyses show that the bulk of the egg sac wall is composed of meandering fibres with a quasi-periodic modulation of 190 ± 60 nm along the thickness of the envelope, generating a photonic crystal. Third, Fourier power analyses of 450 electron microscopy images with varying incident angles indicate that changing the surrounding medium from water to air shifts most of the backscattered power spectrum to the ultraviolet range, hence, explaining that the egg sac loses visible iridescence when removed out of the water. Fourth, the results of our photography and optical spectroscopy experiments of submerged and emerged egg sacs rule out the possibility that the iridescence is due to a thin film or a multilayer, whereas the observed non-specular response is compatible with the backscattering expected from surface diffraction gratings and volumetric photonic crystals with spatial 1D modulation. Finally, although we mention several potential biological functions of the egg sac structural colours and iridescence, we emphasise that these optical properties might be the by-products of the envelope material internal structure selected during evolution for its mechanical properties.

Selenium Bioaccumulation Across Trophic Levels and Along a Longitudinal Gradient in Headwater Streams.

Toxic effects of selenium (Se) contamination in freshwaters have been well documented. However, study of Se contamination has focused on lentic and larger order lotic systems, whereas headwater streams have received little scrutiny. In central Appalachia, surface coal mining is a common Se source to headwater streams, thus providing a useful system to investigate Se bioaccumulation in headwater food chains and possible longitudinal patterns in Se concentrations. Toward that end, we assessed Se bioaccumulation in 2 reference and 4 mining-influenced headwater streams. At each stream, we sampled ecosystem media, including streamwater, particulate matter (sediment, biofilm, leaf detritus), benthic macroinvertebrates, salamanders, and fish, every 400 m along 1.2- and 1.6-km reaches. We compared media Se concentrations within and among streams and evaluated longitudinal trends in media Se concentrations. Selenium concentrations in sampled media were higher in mining-influenced streams compared with reference streams. We found the highest Se concentrations in benthic macroinvertebrates; however, salamanders and fish bioaccumulated Se to potentially harmful levels in mining-influenced streams. Only one stream demonstrated dilution of streamwater Se with distance downstream, and few longitudinal patterns in Se bioaccumulation occurred along our study reaches. Collectively, our results provide a field-based assessment of Se bioaccumulation in headwater food chains, from streamwater to fish, and highlight the need for future assessments of Se effects in headwater streams and receiving downstream waters. Environ Toxicol Chem 2020;39:692-704. © 2020 SETAC.

Cortisol is the predominant glucocorticoid in the giant paedomorphic hellbender salamander (Cryptobranchus alleganiensis).

Corticosterone is widely regarded to be the predominant glucocorticoid produced in amphibians. However, we recently described unusually low baseline and stress-induced corticosterone profiles in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis), a giant, fully aquatic salamander. Here, we hypothesized that hellbenders might also produce cortisol, the predominant glucocorticoid used by fishes and non-rodent mammals. To test our hypothesis, we collected plasma samples in two field experiments and analyzed them using multiple analytical techniques to determine how plasma concentrations of cortisol and corticosterone co-varied after 1) physical restraint and 2) injection with adrenocorticotropic hormone (ACTH), the pituitary hormone responsible for triggering the release of glucocorticoids from amphibian interrenal glands. Using liquid chromatography-mass spectrometry, we found that baseline and restraint-induced plasma concentrations of cortisol were more than five times those of corticosterone. We then demonstrated that plasma concentrations of both glucocorticoids increased in response to ACTH in a dose-dependent manner, but cortisol concentrations were consistently higher (up to 10-fold) than corticosterone. Cortisol and corticosterone concentrations were not correlated with one another at basal or induced conditions. The extremely low plasma concentrations of corticosterone in hellbenders suggests that corticosterone could simply be a byproduct of cortisol production, and raises questions as to whether corticosterone has any distinct physiological function in hellbenders. Our results indicate that hellbenders produce cortisol as their predominant glucocorticoid, supporting a small and inconclusive body of literature indicating that some other amphibians may produce appreciable quantities of cortisol. We hypothesize that the use of cortisol by hellbenders could be an adaptation to their fully aquatic life history due to cortisol's ability to fulfill both mineralocorticoid and glucocorticoid functions, similar to its functions in fishes. Given the large number of amphibian species that are fully aquatic or have aquatic life stages, we suggest that the broadly held assumption that corticosterone is the predominant glucocorticoid in all amphibians requires further scrutiny. Ultimately, multi-species tests of this assumption will reveal the ecological factors that influenced the evolution of endocrine adaptations among amphibian lineages, and may provide insight into convergent evolution of endocrine traits in paedomorphic species.

Proteomic analysis of eleven tissues in the Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus, CGS) is the largest extant amphibian species in the world. Global quantitative proteome analysis of multiple tissues would indicate tissue-specific physiological processes and clarify the function of each protein from a whole-organism perspective. This study performed proteome analysis of eleven tissues collected from adult CGSs using iTRAQ coupled with LC-MS/MS technology. Based on the predicted protein database from previously obtained CGS transcriptome data, 2153 proteins were identified for subsequent analysis. A weighted gene co-expression network analysis (WGCNA) clustered 2153 proteins into 17 co-expressed modules, which will be useful for predicting the functions of unannotated proteins. The protein levels of molecular complexes with housekeeping functions, such as ribosomes, spliceosomes and mitochondrial respiratory chain complexes, were tightly regulated in different tissues of the CGS, as they are in mammalian tissues. Transcription regulator, pathway and bio-functional analysis of tissue-specific proteins showed that highly expressed proteins largely reflected the physiological functions of specific tissues. Our data, as an initial atlas of protein expression of an amphibian species, will be useful for further molecular biology research on CGS.

Discovery of MicroRNAs from Batrachuperus yenyuanensis Using Deep Sequencing and Prediction of Their Targets.

MicroRNAs (miRNAs), a family of ∼22-nucleotide non-coding single-stranded RNA molecules, are considered as key post-transcriptional regulators of gene expression that regulate various biological processes in living organism. Many miRNAs have been identified in animals; however, few have been reported in Hynobiidae species. The present study is aimed to identify a full repertoire of miRNAs in Batrachuperus yenyuanensis (Yenyuan stream salamander), which would significantly increase our knowledge of miRNAs in amphibians. A small RNA library was constructed from B. yenyuanensis and sequenced using deep sequencing. As a result, 1,717,751 clean reads were obtained, representing 356 known and 80 novel miRNAs. Additionally, expression levels of eight randomly selected miRNAs in B. yenyuanensis were confirmed using the stem-loop quantitative real-time reverse transcription PCR. In addition, 13,972 targets were predicted for these identified miRNAs, although the physiological functions of many of these targets remain unknown. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that the predicted targets are involved in a variety of physiological regulatory functions in B. yenyuanensis. These results provide useful information for further research on the miRNAs involved in the growth and development of B. yenyuanensis, as well as adaptation of this species to its high-altitude habitats.

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.

Identification and Characterization of MicroRNAs in Skin of Chinese Giant Salamander (Andrias davidianus) by the Deep Sequencing Approach.

MicroRNAs (miRNA) play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. However, the miRNAs from skin of Andrias davidianus have not been reported. In this study, a small-RNA cDNA library was constructed and sequenced from skin of A. davidianus. A total of 513 conserved miRNAs belonging to 174 families were identified. The remaining 108 miRNAs we identified were novel and likely to be skin tissue-specific but were expressed at low levels. The presence of randomly selected 15 miRNAs identified and their expression in eight different tissues from A. davidianus were validated by stem-loop qRT-PCR. For better understanding the functions of miRNAs, 129,791 predicated target genes were analyzed by GO and their pathways illustrated by KEGG pathway analyses. The results show that these identified miRNAs from A. davidianus skin are involved in a broad range of physiological functions including metabolism, growth, development, and immune responses. This study exhaustively identifies miRNAs and their target genes, which will ultimately pave the way for understanding their role in skin of A. davidianus and other amphibians. Further studies are necessary to better understand miRNA-mediated gene regulation.

Fabrication and characterization of Chinese giant salamander skin composite collagen sponge as a high-strength rapid hemostatic material.

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.