Bifenthrin induces changes in clinical poisoning symptoms, oxidative stress, DNA damage, histological characteristics, and transcriptome in Chinese giant salamander (Andrias davidianus) larvae.

Bifenthrin (BF) is a broad-spectrum insecticide that has gained widespread use due to its high effectiveness. However, there is limited research on the potential toxic effects of bifenthrin pollution on amphibians. This study aimed to investigate the 50 % lethal concentration (LC50) and safety concentration of Chinese giant salamanders (CGS) exposed to BF (at 0, 6.25,12.5,25 and 50 µg/L BF) for 96 h. Subsequently, CGS were exposed to BF (at 0, 0.04, and 4 µg/L BF) for one week to investigate its toxic effects. Clinical poisoning symptoms, liver pathology, oxidative stress factors, DNA damage, and transcriptome differences were observed and analyzed. The results indicate that exposure to BF at 4 µg/L significantly decreased the adenosine-triphosphate (ATP), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) contents in the brain, liver, and kidney of CGS. Additionally, the study found that the malondialdehyde (MDA), reactive oxygen species (ROS), and 8-hydroxydeoxyguanosine (8-OHdG) contents were increased. The liver tissue exhibited significant inflammatory reactions and structural malformations. RNA-seq analysis of the liver showed that BF caused abnormal antioxidant indices of CGS. This affected molecular function genes such as catalytic activity, ATP-dependent activity, metabolic processes, signaling and immune system processes, behavior, and detoxification, which were significantly upregulated, resulting in the differential genes significantly enriched in the calcium signaling pathway, PPARα signaling pathway and NF-kB signaling pathway. The results suggest that BF induces the abnormal production of free radicals, which overwhelms the body's self-defense system, leading to varying degrees of oxidative stress. This can result in oxidative damage, DNA damage, abnormal lipid metabolism, autoimmune diseases, clinical poisoning symptoms, and tissue inflammation. This work provides a theoretical basis for the rational application of bifenthrin and environmental risk assessment, as well as scientific guidance for the conservation of amphibian populations.

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

Thioredoxin-like protein-1 (TXNL1) is the member of thioredoxin superfamily, a family of thiol oxidoreductases. TXNL1 plays an important role in scavenging ROS and the maintenance of cellular redox balance. However, its physiological functions in Andrias davidianus have not been well understood. In the present study, the full-length cDNA encoding thioredoxin-like protein-1 (AdTXNL1) of A. davidianus was cloned, the mRNA tissue distribution was analyzed, and the function was characterized. The Adtxnl1 cDNA contained an open reading frame (ORF) of 870 bp encoding a polypeptide of 289 amino acids with the N-terminal TRX domain, a Cys34-Ala35-Pro36-Cys37 (CAPC) motif, and the C-terminal proteasome-interacting thioredoxin domain (PITH). The mRNA of AdTXNL1 was expressed in a wide range of tissues, with the highest level in the liver. The transcript level of AdTXNL1 was significantly up-regulated post Aeromonas hydrophila challenge in liver tissue. Moreover, the recombinant AdTXNL1 protein was produced and purified, and used to investigate the antioxidant activity. In the insulin disulfide reduction assay, rAdTXNL1 exhibited strong antioxidant capability. Altogether, the thioredoxin-like protein-1 may be involved in reduction/oxidation (redox) balance and as an important immunological gene in A. davidianus.

A new, narrowly endemic species of swamp-dwelling dusky salamander (Plethodontidae: Desmognathus) from the Gulf Coastal Plain of Mississippi and Alabama.

We describe a new, narrowly endemic species of swamp-dwelling dusky salamander (Plethodontidae: Desmognathus pascagoula sp. nov.) from the Gulf Coastal Plain of southeastern Mississippi and southwestern Alabama based on linear morphometrics, mitochondrial DNA, and single nucleotide polymorphisms from 881 loci produced using genotype-by-sequencing. Some populations of the new species were historically referred to as D. auriculatus, a polyphyletic assemblage of at least three species in the Atlantic and Gulf Coastal Plain from Texas to North Carolina. Populations of D. auriculatus from the Gulf Coastal Plain in Louisiana and Mississippi were recently described as D. valentinei. The new species includes populations that were tentatively referred to D. valentinei, but we find it is morphologically, genetically, and geographically distinct. It is smaller, has a more defined dorsal color pattern, more irregular whitish portholes in up to three rows on the lateral surfaces of the body and tail, and a brighter orange or yellowish orange postocular stripe. At present, the new species is known from only six extant populations in the lower Pascagoula, Escatawpa, and Mobile drainages. The latter represents a distinct phylogeographic lineage. We also refer a historical collection from the northeastern side of the Mobile-Tensaw River Delta to this species, suggesting a much broader range in the past. We suspect that more populations remain to be discovered in the area, and their potential species-level distinctiveness should be tested further. This discovery increases knowledge of the biodiversity in the southeastern United States Coastal Plain, a candidate region meeting the global criteria for a biodiversity hotspot, and underscores the amount of cryptic diversity likely remaining to be discovered and described in Nearctic salamanders.

Characterization, expression pattern and antiviral activities of oligoadenylate synthetase in Chinese Giant Salamander, Andrias davidianus.

The enzyme 2'-5'-oligoadenylate synthetase (OAS) is an antiviral protein induced by interferons (IFNs), which plays an important role in IFN-mediated antiviral signaling pathway. In this study, the OAS of Chinese Giant Salamander, Andrias davidianus (AdOAS) was identified for the first time, and the expression profiles in vivo and the antiviral activities in vitro were investigated. The open reading frame (ORF) of AdOAS gene is 1185 bp in length, encoding a putative protein of 394 amino acids, in which a Nucleotidyltransferase (NTase) domain (40-143 aa) and a conserved OAS1 C superfamily domain (165-341 aa) are included. qRT-PCR analysis revealed a broad expression of AdOAS in vivo, with the highest expression level in intestine and heart. After infection with Chinese giant salamander iridovirus (GSIV), the mRNA level of AdOAS in liver increased significantly at 24 h and 48 h post infection and reached the peak at 72 h compared with the control group. The AdOAS mRNA level in kidney increased slightly at 6 h and 12 h post infection, declined to the initial level at 24 h and peaked at 48 h post infection, while in spleen it was slightly up-regulated at 6 h, inhibited at 12 h, 24 h and 48 h, and then significantly increased to the peak at 72 h post infection. In vitro, AdOAS mRNA level in Chinese giant salamander muscle (GSM) cells was not noticeably up-regulated until 24 h and then peaked at 48 h post GSIV infection. In antiviral activity test, the mRNA transcription and protein level of virus major capsid protein (MCP) in AdOAS over-expressed cells was significantly reduced compared with that in control cells by qRT-PCR and western blot analysis. In addition, ddPCR results showed that lower MCP gene copy was found in AdOAS over-expressed cells compared with the control group. These results collectively suggest that AdOAS plays a crucial role against GSIV infection in Chinese giant salamander, and provide a solid base for the further studies on the mechanism of immune defense and the control of the disease in this animal.

DNAJA4 Promotes the Replication of the Chinese Giant Salamander Iridovirus.

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

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.

Exposure to glucocorticoids alters life history strategies in a facultatively paedomorphic salamander.

Polyphenisms, where two or more alternative, environmentally-cued phenotypes are produced from the same genotype, arise through variability in the developmental rate and timing of phenotypic traits. Many of these developmental processes are controlled or influenced by endogenous hormones, such as glucocorticoids, which are known to regulate a wide array of vertebrate ontogenetic transitions. Using the mole salamander, Ambystoma talpoideum, as a model, we investigated the role of glucocorticoids in regulating facultative paedomorphosis, an ontogenetic polyphenism where individuals may delay metamorphosis into terrestrial adults. Instead, individuals reproduce as aquatic paedomorphic adults. Paedomorphosis often occurs when aquatic conditions remain favorable, while metamorphosis typically occurs in response to deteriorating or "stressful" aquatic conditions. Since glucocorticoids are central to the vertebrate stress response and are known to play a central role in regulating obligate metamorphosis in amphibians, we hypothesized that they are key regulators of paedomorphic life history strategies. To test this hypothesis, we compared development of larvae in outdoor mesocosms exposed to Low, Medium, and High exogenous doses of corticosterone (CORT). Results revealed that body size and the proportion of paedomorphs were both inversely proportional to exogenous CORT doses and whole-body CORT content. Consistent with known effects of CORT on obligate metamorphosis in amphibians, our results link glucocorticoids to ontogenetic transitions in facultatively paedomorphic salamanders. We discuss our results in the context of theoretical models and the suite of environmental cues known to influence facultative paedomorphosis.

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-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß 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.

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.

Digest: Developmental life history and adaptive radiation in salamanders.

What determines the rate at which species adapt to new climatic conditions? Weaver et al. found that the evolution of short larval periods promotes climatic niche evolution in salamanders in the genus Desmognathus.

Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.

Developmental life history is associated with variation in rates of climatic niche evolution in a salamander adaptive radiation.

Rates of climatic niche evolution vary widely across the tree of life and are strongly associated with rates of diversification among clades. However, why the climatic niche evolves more rapidly in some clades than others remains unclear. Variation in life history traits often plays a key role in determining the environmental conditions under which species can survive, and therefore, could impact the rate at which lineages can expand in available climatic niche space. Here, we explore the relationships among life-history variation, climatic niche breadth, and rates of climatic niche evolution. We reconstruct a phylogeny for the genus Desmognathus, an adaptive radiation of salamanders distributed across eastern North America, based on nuclear and mitochondrial genes. Using this phylogeny, we estimate rates of climatic niche evolution for species with long, short, and no aquatic larval stage. Rates of climatic niche evolution are unrelated to the mean climatic niche breadth of species with different life histories. Instead, we find that the evolution of a short larval period promotes greater exploration of climatic space, leading to increased rates of climatic niche evolution across species having this trait. We propose that morphological and physiological differences associated with variation in larval stage length underlie the heterogeneous ability of lineages to explore climatic niche space. Rapid rates of climatic niche evolution among species with short larval periods were an important dimension of the clade's adaptive radiation and likely contributed to the rapid rate of lineage accumulation following the evolution of an aquatic life history in this clade. Our results show how variation in a key life-history trait can constrain or promote divergence of the climatic niche, leading to variation in rates of climatic niche evolution among species.

Survival of Polyploid hybrid salamander embryos.

BACKGROUND: Animals with polyploid, hybrid nuclei offer a challenge for models of gene expression and regulation during embryogenesis. To understand how such organisms proceed through development, we examined the timing and prevalence of mortality among embryos of unisexual salamanders in the genus Ambystoma. RESULTS: Our regional field surveys suggested that heightened rates of embryo mortality among unisexual salamanders begin in the earliest stages of embryogenesis. Although we expected elevated mortality after zygotic genome activation in the blastula stage, this is not what we found among embryos which we reared in the laboratory. Once embryos entered the first cleavage stage, we found no difference in mortality rates between unisexual salamanders and their bisexual hosts. Our results are consistent with previous studies showing high rates of unisexual mortality, but counter to reports that heightened embryo mortality continues throughout embryo development. CONCLUSIONS: Possible causes of embryonic mortality in early embryogenesis suggested by our results include abnormal maternal loading of RNA during meiosis and barriers to insemination. The surprising survival rates of embryos post-cleavage invites further study of how genes are regulated during development in such polyploid hybrid organisms.

Thermal cues drive plasticity of desiccation resistance in montane salamanders with implications for climate change.

Organisms rely upon external cues to avoid detrimental conditions during environmental change. Rapid water loss, or desiccation, is a universal threat for terrestrial plants and animals, especially under climate change, but the cues that facilitate plastic responses to avoid desiccation are unclear. We integrate acclimation experiments with gene expression analyses to identify the cues that regulate resistance to water loss at the physiological and regulatory level in a montane salamander (Plethodon metcalfi). Here we show that temperature is an important cue for developing a desiccation-resistant phenotype and might act as a reliable cue for organisms across the globe. Gene expression analyses consistently identify regulation of stem cell differentiation and embryonic development of vasculature. The temperature-sensitive blood vessel development suggests that salamanders regulate water loss through the regression and regeneration of capillary beds in the skin, indicating that tissue regeneration may be used for physiological purposes beyond replacing lost limbs.

Bichirs employ similar genetic pathways for limb regeneration as are used in lungfish and salamanders.

Bichirs are a sister group to sarcopterygian and tetrapods that can fully regenerate their endochondral-skeleton-fins. Histological and transcriptomic comparison approaches have been used to investigate the morphology and genetic basis of bichir lobe-fin regeneration, with strong down-regulation of muscle-related genes and up-regulation of ECM-related genes and developmental genes being observed. Bichir limb regeneration involves similar cellular processes to those employed by lungfish and salamander, with MARCKS-like protein (MLP) that is known to be a putative regeneration-initiating molecule in salamander, also up-regulated in the early stages of bichir lobe-fin regeneration. These gene expression results suggest that limb regeneration pathways in these amphibians have a common ancestral inheritance, consistent with evolution from endochondral-skeleton-fin structures to endochondral-skeleton-limb structures of vertebrates.

Exaptation as a Mechanism for Functional Reinforcement of an Animal Pheromone System.

Animal sex pheromone systems often exist as multicomponent signals [1-11] to which chemical cues have been added over evolutionary time. Little is known on why and how additional molecules become recruited and conserved in an already functional pheromone system. Here, we investigated the evolutionary trajectory of a series of 15 kDa proteins-termed persuasins-that were co-opted more recently alongside the ancient sodefrin precursor-like factor (SPF) courtship pheromone system in salamanders [9, 12]. Expression, genomic, and molecular phylogenetic analyses show that persuasins originated from a gene that is expressed as a multi-domain protein in internal organs where it has no pheromone function but underwent gene duplication and neofunctionalization. The subsequent evolution combined domain loss and the introduction of a proteolytic cleavage site in the duplicated gene to give rise to two-domain cysteine rich proteins with structural properties similar to SPF pheromones [12]. An expression shift to the pheromone-producing glands, where expression of persuasins was immediately spatiotemporally synchronized with the already available pheromone system, completed the birth of a new pheromone. Electrostatic forces between members of both protein families likely enhance co-localization and simultaneous activation of different female olfactory neurons, explaining why persuasins immediately had a selective advantage. In line with this, behavioral assays show that persuasins increase female receptivity on their own but also exert a cumulative or synergistic effect in combination with SPF, clearly reinforcing the pheromone system as a whole. Our study reveals molecular remodeling of an existing protein architecture as an evolutionary mechanism for functional reinforcement of animal pheromone systems.

Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus) after Aeromonas hydrophila infection

Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.

Identification of the first cathelicidin gene from skin of Chinese giant salamanders Andrias davidianus with its potent antimicrobial activity.

Cathelicidins, as effector molecules, play important roles against infections and represent a crucial component of the innate immune system in vertebrates. They are widely studied in mammals, but little is known in amphibians. In the present study, we report the identification and characterization of a novel cathelicidin from Chinese giant salamander Andrias davidianus, which is the first study in Caudata amphibian. The cDNA sequence encodes a predicted 148-amino-acid polypeptide, which composed of a 20-residue signal peptide, a 94-residue conserved cathelin domain and a 34-residue mature peptide. From the multiple sequence alignments and phylogenetic analysis, AdCath shared conserved structure with other orthologs and clustered with other amphibian peptides. The tissue expression profiles revealed AdCath was highly expressed in skin. To study the function of AdCath gene, the AdCath precursor protein and mature peptide were recombinantly expressed and chemical synthesized respectively. The rAdCath protein could bind to LPS in a dose-dependent manner. When the concentrations of rAdCath protein and mature peptide were up to 22 µg/mL, they showed significantly cytotoxicity to human 293T cell lines. The rAdCath protein and synthetic peptide could exhibit antibacterial activities detected by the minimum inhibitory concentrations assay. From the SEM assay, the synthetic mature peptide could destroy the membrane of bacteria and cause loss of membrane integrity. Collectively, these findings characterized the first cathelicidin from A. davidianus, and highlighted its potential antimicrobial activities, indicating its important roles in the skin immune response against different bacteria.

High gene flow between alternative morphs and the evolutionary persistence of facultative paedomorphosis.

Paedomorphosis and metamorphosis are two major developmental processes that characterize the evolution of complex life cycles in many lineages. Whereas these processes were fixed in some taxa, they remained facultative in others, with alternative phenotypes expressed in the same populations. From a genetic perspective, it is still unknown whether such phenotypes form a single population or whether they show some patterns of isolation in syntopy. This has deep implications for understanding the evolution of the phenotypes, i.e. towards their persistence or their fixation and speciation. Newts and salamanders are excellent models to test this hypothesis because they exhibit both developmental processes in their populations: the aquatic paedomorphs retain gills, whereas the metamorphs are able to colonize land. Using microsatellite data of coexisting paedomorphic and metamorphic palmate newts (Lissotriton helveticus), we found that they formed a panmictic population, which evidences sexual compatibility between the two phenotypes. The high gene flow could be understood as an adaptation to unstable habitats in which phenotypic plasticity is favored over the fixation of developmental alternatives. This makes then possible the persistence of a polyphenism: only metamorphosis could be maintained in case of occasional drying whereas paedomorphosis could offer specific advantages in organisms remaining in water.

A novel defensin-like antimicrobial peptide from the skin secretions of the tree frog, Theloderma kwangsiensis.

Defensins are one of the major families of antimicrobial peptides (AMPs), and have been reported from prokaryotic to eukaryotic kingdoms. But defensins are seldom found in amphibian which is a major resource of novel AMPs. A novel defensin-like AMP (defensin-TK) was isolated and characterized from skin secretions of the tree frog Theloderma kwangsiensis. The cDNA encoding defensin-TK precursor was cloned from the skin cDNA library of T. kwangsiensis. The deduced precursor of defensin-TK was composed of three domains, a signal peptide of 16 residues, a spacer peptide of 1 residues and a mature peptide of 42 residues. The mature peptide of defensin-TK shared the highest identity with the salamander (Cynops fudingensis) defensin CFBD-1. The six conserved cysteines which formed intramolecular disulfide bonds of defensins also exist in defensin-TK. Phylogenetic analysis indicated that defensin-TK was closely related to fish ß-defensins. Defensin-TK showed potent and broad-spectrum antimicrobial activity. In addition, defensin-TK exerted a low hemolytic activity on human red cells. These results suggested defensin-TK might play an important role in defense the skin pathogenic microbes of tree frog T. kwangsiensis, and might be a promising candidate for development of novel antimicrobial agents.