Rana Dybowskii Research Articles

The complete mitochondrial genome information of Rana uenoi (Amphibia, Anura, Ranidae) and the phylogenetic implication

We determined the complete mitochondrial genome of Rana uenoi (Anura: Ranidae) for the first time. The whole sequences were 17,370 bp and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. The gene arrangement was completely identical to those observed from other Ranidae species. We used 11 protein-coding genes to examine the phylogenetic placement of this species in the genus Rana. Rana dybowskii was the closest sister species to R. uenoi. The clade of R. uenoi and R. dybowskii formed a cluster with Rana huarensis, which had a sister relationship with the group of Rana amurensis, Rana coreana, and Rana kunyuensis.

First detection of ranavirus in a wild population of Dybowski\u2019s brown frog (Rana dybowskii) in South Korea

BackgroundRanavirus is an emerging infectious disease which has been linked to mass mortality events in various amphibian species. In this study, we document the first mass mortality event of an adult population of Dybowski’s brown frogs (Rana dybowskii), in 2017, within a mountain valley in South Korea.ResultsWe confirmed the presence of ranavirus from all collected frogs (n = 22) via PCR and obtained the 500 bp major capsid protein (MCP) sequence from 13 individuals. The identified MCP sequence highly resembled Frog virus 3 (FV3) and was the same haplotype of a previously identified viral sequence collected from Huanren brown frog (R. huanrenensis) tadpoles in South Korea. Human habitat alteration, by recent erosion control works, may be partially responsible for this mass mortality event.ConclusionWe document the first mass mortality event in a wild Korean population of R. dybowskii. We also suggest, to determine if ranavirus infection is a threat to amphibians, government officials and researchers should develop continuous, country-wide, ranavirus monitoring programs of Korean amphibian populations.

Comparison of Gut Microbiota Diversity and Predicted Functions Between Healthy and Diseased Captive Rana dybowskii.

The gut microbiota plays a key role in host health, and disruptions to gut bacterial homeostasis can cause disease. However, the effect of disease on gut microbiota assembly remains unclear and gut microbiota-based predictions of health status is a promising yet poorly established field. Using Illumina high-throughput sequencing technology, we compared the gut microbiota between healthy (HA and HB) and diarrhoeic (DS) Rana dybowskii groups and analyzed the functional profiles through a phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. In addition, we estimated the correlation between gut microbiota structures and predicted the functional compositions. The results showed significant differences in the phylogenetic diversity (Pd), Shannon, and observed richness (Sobs) indices between the DS and HB groups, with significant differences observed in the gut microbiota composition between the DS group and the HA and HB groups. Linear discriminant analysis (LDA) effect size (LEfSe) results revealed that Proteobacteria were significantly enriched in the DS group; Bacteroidetes were significantly enriched in the HA and HB groups; and Aeromonas, Citrobacter, Enterococcus, Hafnia-Obesumbacterium, Morganella, Lactococcus, Providencia, Vagococcus, and Staphylococcus were significantly enriched in the DS group. Venn diagrams revealed that there were many more unique genera in the DS group than the HA and HB groups. Among 102 sensitive species selected using the indicator method, 33 indicated a healthy status and 69 (e.g., Acinetobacter, Aeromonas, Legionella, Morganella, Proteus, Providencia, Staphylococcus, and Vagococcus) indicated a diseased status. There was a significant and positive association between the composition and functional composition of the gut microbiota, thus indicating low functional redundancy of the frog gut bacterial community. Rana dybowskii disease was associated with changes in the gut microbiota, which subsequently disrupted bacterial-mediated functions. The results of this study can aid in revealing the effect of the R. dybowskii gut microbiota on host health and provide a basis for elucidating the mechanism of the occurrence of R. dybowskii disease.

Expression of MHCⅠ genes in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.

Environmental and host factors shaping the gut microbiota diversity of brown frog Rana dybowskii

Symbiotic microbial communities are common in amphibians, and the composition of gut microbial communities varies with factors such as host phylogeny, life stage, ecology, and diet. However, little is known regarding how amphibians acquire their microbiota or how their growth, development, and environmental factors affect the diversity of their microbiotas. We sampled the gut microbiota during different developmental stages of brown frog Rana dybowskii, including tadpoles (T), frogs in metamorphosis (M), frogs just post-metamorphosis and after eating (F), juvenile frogs in summer (Js), adult frogs in summer (As), adult frogs in autumn (Aa), and hibernating frogs (Ah). We recorded data on the environmental (ambient temperature, fasting status, habitat, and season) and host (body mass and developmental period) factors. We investigated whether the gut microbiota diversity of R. dybowskii differs according to the host developmental stage via high-throughput Illumina sequencing and whether the gut microbiota diversity is affected by environmental and host factors. We found that alpha and beta diversity varied significantly during different developmental stages. The linear discriminant analysis effect size (LEfSe) analysis identified eight phyla exhibiting significant differences: Cyanobacteria (T group), Proteobacteria (M group), Fusobacteria (F group), Firmicutes (As group), Actinobacteria (Aa group), Verrucomicrobia (Aa group), Tenericutes (Aa group), and Bacteroidetes (Ah group). The Venn diagrams showed that 49 shared OTUs were present during all stages of development, whereas 10 OTUs were present in >90% of the samples. The environmental and host factors were significantly correlated with microbial community changes. Furthermore, the AIC-based model results suggested that development was the only variable that needed inclusion in the redundancy analysis (RDA) to explain the variance in taxa. These results have broad implications for our understanding of gut microbiota development and its associations with amphibian development and environmental factors.

Diversity of Antimicrobial Peptides in Three Partially Sympatric Frog Species in Northeast Asia and Implications for Evolution.

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that play an essential role in innate immunity across taxa from invertebrates to vertebrates. The evolution system of AMP system has not been well explained in the literature. In this study, we cloned and sequenced AMP transcriptomes of three frog species, namely Rana dybowskii, Rana amurensis, and Pelophylax nigromaculatus, which are partially sympatric in northeast Asia, but show different habitat preferences. We found that each species contained 7 to 14 families of AMPs and the diversity was higher in species with a large geographic range and greater habitat variation. All AMPs are phylogenetically related but not associated with the speciation process. Most AMP genes were under negative selection. We propose that the diversification and addition of novel functions and improvement of antimicrobial efficiency are facilitated by the expansion of family members and numbers. We also documented significant negative correlation of net charges and numbers of amino acid residues between the propiece and mature peptide segments. This supports the Net Charge Balance Hypothesis. We propose the Cut Point Sliding Hypothesis as a novel diversification mechanism to explain the correlation in lengths of the two segments.

Comparative analysis of cutaneous bacterial communities of farmed Rana dybowskii after gentamycin bath.

IntroductionPathogenic bacteria limit the success of Rana dybowskii breeding. Gentamicin is used to treat R. dybowskii disease. To understand the effects of gentamicin on the composition and structure of the cutaneous bacterial community of R. dybowskii, three groups (control, gentamicin and recovery) were established in this study.Materials & MethodsThe V3–V4 hypervariable region of the 16S rRNA gene was analyzed in samples by high-throughput sequencing. Alpha diversity and beta diversity were evaluated to compare the cutaneous bacterial community diversity.ResultsA total of 1,159,668 valid sequences and 3,132 operational taxonomic units (OTUs) were obtained from these three experimental groups. The number of OTUs obtained in the control group, gentamicin group and recovery group were 2,194, 2,288, and 2,047, respectively, and the number of shared OTUs was 1,313. The alpha diversity of the cutaneous bacterial community was not significantly affected by gentamicin, while beta diversity was significantly affected.Discussion & ConclusionsThe effect of a gentamicin bath on relative species abundance was greater than the effect on the species composition. The changes in Proteobacteria, Acinetobacter, and Chryseobacterium were significant, and reductions were observed after the recovery period. Six potentially pathogenic genera were detected, including Aeromonas, Citrobacter, Chryseobacterium, Pseudomonas, Staphylococcus, and Streptococcus. Among them, Aeromonas and Chryseobacterium were significantly inhibited by the gentamicin bath. The results of this study provide a theoretical basis for the application of gentamicin in R. dybowskii breeding.

Expressions of TLR4, MyD88 and IRAK4 in the Skin of Chinese Brown Frog (Rana Dybowskii) During the Breeding and Pre-Hibernation Periods

Toll-Like Receptors (TLRs) are membrane-bound proteins that recognize invading organisms with Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Related Molecular Patterns (DAMPs)...

Effects of Seasonal Hibernation on the Similarities Between the Skin Microbiota and Gut Microbiota of an Amphibian (Rana dybowskii).

Both the gut and skin microbiotas have important functions for amphibians. The gut microbiota plays an important role in both the health and evolution of the host species, whereas the role of skin microbiota in disease resistance is particularly important for amphibians. Many studies have examined the effects of environmental factors on the skin and gut microbiotas, but no study has yet explored the similarities between the skin and gut microbiotas. In this study, the gut and skin microbiotas of Rana dybowskii in summer and winter were investigated via high-throughput Illumina sequencing. The results showed that the alpha diversity of gut and skin microbiotas decreased significantly from summer to winter. In both seasons, the microbial composition and structure differed significantly between the gut and skin, and the similarities between these microbiotas differed between seasons. The pairwise distances between the gut and skin microbiotas were greater in winter than in summer. The ratio of core OTUs and shared OTUs to the sum of the OTUs in the gut and skin microbiotas in summer was significantly higher than that in winter. The similarities between the gut and skin microbiotas are important for understanding amphibian ecology and life history.

Expressions of TLR4, MyD88, IRAK4 and NF-κΒ in the oviduct of Chinese brown frog (Rana dybowskii)

One special physiological phenomenon of the Chinese brown frog (Rana dybowskii) is that its oviduct goes through expansion prior to pre-hibernation instead of expanding during the breeding period. Our previous study discovered that some cytokines such as interleukin-1 beta (IL-1Î) and its receptor type I (IL1R1) proteins were expressed in the oviduct of Rana dybowskii. In this study, we continued to investigate the localizations and expression levels of cytokines including toll-like receptor 4 (TLR4) and its related factors, myeloid differentiation factor 88 (MyD88), interleukin- 1 receptor-associated kinase (IRAK) and nuclear factor kappa B (NF-ÎÎ) in the oviduct of Rana dybowskii during the breeding period and pre-hibernation. Morphological results showed that there were significant differences in oviductal weight and pipe diameter with significantly higher values in pre-hibernation than those of the breeding period. Strong immunostaining of TLR4, MyD88, IRAK4 and NF- ÎÎ were observed in the pre-hibernation. These proteins levels had no significant difference between these two periods except MyD88 which was significantly higher in the pre-hibernation. The mRNA expression levels of MyD88 and NF-ÎÎ were obviously higher in pre-hibernation than those of the breeding period. These findings suggested that TLR4/MyD88 signal pathway might participate in the oviductal functions of Rana dybowskii during the breeding period and pre-hibernation.

Effects of Captivity and Season on the Gut Microbiota of the Brown Frog (Rana dybowskii).

The gut microbiota of amphibians is affected by exogenous and endogenous factors. We performed a comprehensive analysis using high-throughput sequencing technology and functional predictions and observed general changes in the gut microbiota of frogs in different growth stages, seasons, and growth environments. There were no significant differences in microbial richness and diversity between juvenile and adult wild frogs, between the summer and autumn groups of captive frogs, or between wild and captive frogs. There were significant differences in the gut microbiota community structure of Rana dybowskii between the summer and autumn groups of captive frogs and between wild and captive R. dybowskii, whereas the differences between juvenile and adult wild frogs were not significant. The dominant gut bacterial phyla in frogs from both captive and wild environments included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Linear discriminant effect size (LEfSe) analysis showed that Bacteroidetes and Firmicutes were significantly enriched in captive and wild R. dybowskii, respectively linear discriminant analysis (LDA > 4). The core operational taxonomical units (OTUs) that were found in >90% of all frogs tested encompassed 15 core OTUs. The captive frogs exhibited 15 core OTUs in addition to the above overall core microbiota, whereas the wild frogs exhibited 19 core OTUs in addition to the above overall core microbiota. Predictions made using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) suggested that eleven KEGG pathways, such as infectious diseases, immune system diseases, metabolism, metabolism of other amino acids, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, neurodegenerative diseases, and transport and catabolism, were enriched in captive frogs. The relative abundance of several red-leg-syndrome-related pathogens increased significantly in captive frogs compared with that in wild frogs. To our knowledge, this is the first study on the effects of individual seasons and captivity on the gut microbiota of frogs.

An efficient and safe method for the extraction of total DNA from shed frog skin

An efficient protocol for the isolation of high-quality DNA from tissues is essential to many aspects of molecular biology. Shed skin is often overlook as a source for high-quality DNA in molecular studies involving frog. In this study, DNA from shed skin of frog was extracted using phenol–chloroform, the DNeasy extraction kit (Qiagen) and an improved salting-out protocol, in comparison with those of skeletal muscle and toe. We found that the improved salting-out procedure obtained the highest DNA yield, and avoided using toxic chemicals such as phenol, chloroform, isoamyl alcohol, and/or guanidine isothiocyanate. The quantity of DNA from shed skin was lower compared to other tissue in Rana dybowskii. Nevertheless, DNA from shed skin was useful for amplifing mtDNA and nuclear DNA using PCR, as were Rana amurensis and Bufo gargarizans. These results indicate that extrating DNA from shed frog skin by improved salting-out protocol is an efficient and safe method for the conservation genetic studies and population genetics without destroy in frogs.

Detection and comparative analysis of cutaneous bacterial communities of farmed and wild Rana dybowskii (Amphibia: Anura)

Rana dybowskii Gunther, 1876 is a dominant amphibian species in northeast China. In order to understand the composition and structure of the cutaneous bacterial communities of farmed and wild R. dybowskii, two experimental groups (farmed and wild) were investigated in this study. Following DNA extraction, the V3-V4 hypervariable region of the 16S rRNA gene was targeted and analyzed by high-throughput sequencing. The cutaneous bacterial community diversity was investigated and compared through analysis of alpha and beta diversity. A total of 524,852 valid sequences and 1,023 operational taxonomic units (OTUs) were obtained from these two experimental groups. The number of shared OTUs was 333, while there were 603 unique OTUs in the farmed group and 87 unique OTUs in the wild group. The Chao, ACE and Shannon indices of the farmed group were significantly higher than those of the wild group (p < 0.05). The composition and abundance of the dominant bacteria at the phylum and genus levels were different. The dominant phyla in the farmed group were Firmicutes, Proteobacteria, Actinobacteria, Chloroflexi, Fusobacteria, Bacteroidetes, and Cyanobacteria. The dominant phyla of the wild group were Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Furthermore, the alpha and beta diversities of the cutaneous bacterial communities of farmed and wild R. dybowskii exhibited significant differences. This study provides a theoretical basis for comprehensively understanding the composition and abundance of bacteria on the skin of farmed and wild R. dybowskii, which helps to develop its breeding industry and gain economic benefits.

Modelling the growth of the brown frog (Rana dybowskii).

Well-controlled development leads to uniform body size and a better growth rate; therefore, the ability to determine the growth rate of frogs and their period of sexual maturity is essential for producing healthy, high-quality descendant frogs. To establish a working model that can best predict the growth performance of frogs, the present study examined the growth of one-year-old and two-year-old brown frogs (Rana dybowskii) from metamorphosis to hibernation (18 weeks) and out-hibernation to hibernation (20 weeks) under the same environmental conditions. Brown frog growth was studied and mathematically modelled using various nonlinear, linear, and polynomial functions. The model input values were statistically evaluated using parameters such as the Akaike’s information criterion. The body weight/size ratio (Kwl) and Fulton’s condition factor (K) were used to compare the weight and size of groups of frogs during the growth period. The results showed that the third- and fourth-order polynomial models provided the most consistent predictions of body weight for age 1 and age 2 brown frogs, respectively. Both the Gompertz and third-order polynomial models yielded similarly adequate results for the body size of age 1 brown frogs, while the Janoschek model produced a similarly adequate result for the body size of age 2 brown frogs. The Brody and Janoschek models yielded the highest and lowest estimates of asymptotic weight, respectively, for the body weights of all frogs. The Kwl value of all frogs increased from 0.40 to 3.18. The K value of age 1 frogs decreased from 23.81 to 9.45 in the first four weeks. The K value of age 2 frogs remained close to 10. Graphically, a sigmoidal trend was observed for body weight and body size with increasing age. The results of this study will be useful not only for amphibian research but also for frog farming management strategies and decisions.

环境温度对东北林蛙不同地理种群繁殖期体温和胚胎发育的影响

环境温度对东北林蛙不同地理种群繁殖期体温和胚胎发育的影响

Quality Characteristics of Jook Prepared with Edible Frog (Rana dybowskii) Powder

Quality Characteristics of Jook Prepared with Edible Frog (Rana dybowskii) Powder

Comparison of the Bacterial and Fungal Colonies from Rana dybowskii which Collected from Inside and Outside Frog Farms and Identification of the Bacteria from the Tadpoles

Comparison of the Bacterial and Fungal Colonies from Rana dybowskii which Collected from Inside and Outside Frog Farms and Identification of the Bacteria from the Tadpoles

Expressions of IL-6, TNF-α and NF-κB in the skin of Chinese brown frog (Rana dybowskii).

The cytokine interleukin-6 (IL-6) mediates a wide range of inflammatory and immune responses. Tumor Necrosis Factor α (TNF-α) has a myriad of pro-inflammatory effects on the skin. Nuclear factor κB (NF-κB) is a transcriptional factor that regulates a battery of genes that are critical to immune system. In this study, we investigated the localizations and expression levels of IL-6, TNF-α and NF-κB in the skin of Rana dybowskii during the breeding period and pre-hibernation. Histologically, the skin of Rana dybowskii consisted of epidermis and dermis. Four kinds of cells were identified in the epidermis, while the dermis was composed of homogenous gel, mucous glands and granular glands. IL-6, TNF-α and NF-κB were immunolocalized in the epithelial and glandular cells in both periods. Western blotting showed that IL-6, TNF-α and NF-κB were significantly higher in the pre-hibernation compared to the breeding period. Real- Time PCR revealed that the relative mRNA levels of IL-6 and NB-κB in the pre-hibernation increased significantly compared with the breeding period, while the TNF-α mRNA expression levels were not significantly different between these two periods. These results suggested that IL-6, TNF-α and NF-κB might collectively be involved in the skin immune system of Rana dybowskii during the breeding period and pre-hibernation.

Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.

Diversity and phylogeography of Northeast Asian brown frogs allied to Rana dybowskii (Anura, Ranidae)

We investigated the species diversity and phylogeography of the Northeast Asian brown frogs allied to Rana dybowskii (the R. dybowskii species complex: R. dybowskii, R. pirica, and R. uenoi) using four mitochondrial and three nuclear loci. Phylogenetic analyses confirmed the existence of three distinct species in this complex; using extensive molecular data, we confirm the validity of Rana uenoi recognized as a distinct species, and infer R. dybowskii and R. pirica to be sister species. Also, we included populations from previously unsampled regions in Northeast China, and identified them to be R. dybowskii. While many species in Northeast Asia diverged due to Pleistocene glaciation, divergence-dating analyses inferred older, Miocene speciation in the R. dybowskii species complex. Ancestral area reconstruction identified the orogenic movement of the Changbai Mountain Range and the opening of the Sea of Japan/East Sea being major events influencing allopatric speciation.