Rana Dybowskii Research Articles

Differential Impact of Wildfire and Rice Straw Ash on the Skin Microbiota of Rana dybowskii Adults and Tadpoles.

Global warming has intensified severe weather conditions and increased the frequency of wildfires, posing significant threats to ecosystems. Moreover, rice straw ash, a byproduct of human agricultural activities, represents an environmental stressor that can further impact these vulnerable ecosystems. These changes particularly impact sensitive species and microorganisms, yet limited research has explored the effects of wildfire ash and agricultural byproducts, such as rice straw ash, on amphibians. This study aims to investigate the effects of both wildfire and rice straw ashes on the skin microbiota of Rana dybowskii tadpoles and adult frogs, using ash aqueous extracts (AEAs). While alpha diversity showed significant variation among tadpoles, it remained stable in adult frogs. Beta diversity analyses revealed distinct microbiota compositions, especially between control and wildfire ash-treated tadpoles. Linear discriminant analysis (LDA) Effect Size (LEfSe) analysis indicated that different ash treatments led to the enrichment of specific microbiota, reflecting the complex effects of environmental changes on amphibian skin microbiota. Specific bacterial enrichments were associated with each treatment group, and phenotypic analysis highlighted bacterial traits, including Aerobic, Anaerobic, Potentially_Pathogenic, and Stress_Tolerant, providing insights into ecological adaptations. Therefore, contrasts wildfire and rice straw ash treatments distinctly influence amphibian skin microbiota and associated bacterial traits. Our findings emphasize the impact of agricultural and wildfire ash on amphibian skin microbiota, offering key ecological insights into the challenges posed by global environmental changes.

Synergistic toxicity of cadmium and triadimefon on the microbiota and health of Rana dybowskii tadpoles

The skin and gut microbiota are crucial to amphibians. Triadimefon (TF), a widely used triazole fungicide, controls crop diseases and regulates growth, with uncertain effects on amphibian microbiota. Contamination, typically involving mixed chemicals at low concentrations, including cadmium (Cd) and TF, may detrimentally affect amphibian growth, survival, and microbiota health in both the skin and gut, but few research has examined these consequences. This research examines the impact of Cd and TF on Rana dybowskii tadpoles, focusing on survival, body mass, and microbiome changes over 28 days across four groups: control, Cd, TF, and Cd + TF groups. Results showed significant reductions in survival and body mass in Cd and TF-treated groups, with the combination group being the most affected. Microbiota analysis revealed significant dysbiosis in both gut and skin microbiomes under pollutant stress, with a marked microbiota and a shift in dominant microbial communities. Function prediction analysis based on the microbiome composition highlighted significant differences across various biological pathways, including metabolism, immune system, environmental adaptation, and disease resistance. These alterations suggest that pollutant exposure compromises the tadpoles' ability to maintain homeostasis and resist pathogens. In conclusion, this study reveals the detrimental effects of Cd and TF on the survival, growth, and microbiomes of R. dybowskii tadpoles, indicating significant environmental and health risks.

Influence of wildfire ash concentration on development, survival, and skin and gut microbiota of Rana dybowskii

Climate changes can increase wildfires and thereby endanger the habitats and survival of amphibians, but relevant research is limited. The gut and skin microbiota plays a critical role in amphibian protection. Wildfire ash may negatively impact amphibians, causing inflammation and microbiota disruption, but the impact on microbial communities is still uncertain. In this study, the impact of wildfire ash on the cutaneous and gut microbiota of Rana dybowskii was investigated over a 28-day period using five groups with aqueous extracts of ash. Polycyclic aromatic hydrocarbons in the ash were analyzed. Body mass, development, survival rates, and microbiota diversity were tested. Significant differences in body mass, development rates, and survival rates among the treatment groups were observed. The survival and development rates at lower concentrations of ash (T0 and T0_75) were more similar to those under control conditions. Analyses of alpha and beta diversity revealed significant changes in microbiota composition across ash concentrations, with specific phyla and genera affected. Linear discriminant analysis effect analysis identified distinct microbiota associated with each treatment group, demonstrating the specific influence of ash concentrations on the microbiota composition of tadpoles. BugBase analysis revealed significant differences in the same phenotypes in gut microbiota, but not in nine skin microbiota phenotypes across groups. This research underscores the sensitivity of amphibian microbiota to environmental changes and provides insights into the ecological consequences of wildfires on aquatic ecosystems.

An improved method for identifying the genotypic sex in Rana dybowskii

An improved method for identifying the genotypic sex in Rana dybowskii

Genome Survey of Male Rana dybowskii to Further Understand the Sex Determination Mechanism.

Rana dybowskii is one of the important aquaculture species in Northeast China. The fallopian tubes of female R. dybowskii are used to prepare oviductus ranae (an important traditional Chinese medicine). Therefore, R. dybowskii females have higher economical value than males. An increasing female R. dybowskii population can increase the benefits from R. dybowskii culture. However, the genome of amphibians is complex, making it difficult to investigate their sex determination mechanism. In this study, we analyzed the genome of male R. dybowskii using next-generation sequencing technology. A total of 200,046,452,400 bp of clean data were obtained, and the K-mer analysis indicated that the depth was 50×. The genome size of R. dybowskii was approximately 3585.05 M, with a heterozygosity rate, repeat sequence ratio, and genome GC content of 1.15%, 68.96%, and approximately 43.0%, respectively. In total, 270,785 contigs and 498 scaffolds were generated. The size of the contigs and scaffolds was 3,748,543,415 and 3,765,862,278 bp, respectively, with the N50 length of 31,988 and 336,385,783. The longest contig and scaffold were of the size 137,967,485 and 1,808,367,828 bp, respectively. The number of contigs and scaffolds > 10K nt was 99,620 and 451, respectively. Through annotation, 40,913 genes were obtained, including 156,609 CDS (i.e., 3.83 CDS per gene). Sequence alignment was performed with the assembled scaffolding genome in this study. Two and one fragment had high homology with two male-specific DNA molecular markers of R. dybowskii discovered previously (namely, MSM-222 and MSM-261, respectively). In addition, the Dmrt1 gene of R. dybowskii was obtained with a length of 18,893 bp by comparison and splicing. The forward primers amplifying MSM-222 and MSM-261 were located at 322-343 and 14,501-14,526 bp of Dmrt1, respectively. However, sequence alignment revealed that MSM-222 and MSM-261 were not located on Dmrt1, and only some homologous parts were observed. This indicated that in addition to Dmrt1, other important genes may play a crucial role in the sex determination mechanism of R. dybowskii. Our study provided a foundation for the subsequent high-quality genome construction and provided important genomic resources for future studies on R. dybowskii.

Determination and safety evaluation of metal element contents of Oviductus Ranae produced from <i>Rana dybowskii</i> by pond culture–forest grazing relay model in Heilongjiang Province, China

The aquatic products of grazing ecological culture exhibit a strong reliance on the production environment system, which may result in heavy metal contamination. In this study, 24 elements (Ag, Al, As, Ba, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn) were determined for Oviductus Ranae (OR) from more than 1,000 individuals of Rana dybowskii produced by pond culture–forest grazing relay mode from six major production areas in Heilongjiang Province, China. Furthermore, we assessed the associated health risks. Our findings indicate that most of these 24 metal elements were detected in OR at a detection rate of 100%, with the exceptions being As (98.0%), Ga (98.0%), Co (98.0%), V (94.0%), Cd (89.0%), Pb (76.0%), Cs (74.0%), Tl (35.0%), and U (not detectable). Variations in element concentrations were significant across the six sampling sites, with characteristic elements identified to account for spatial differences. The total target hazard quotient was determined to be 0.152 in Heilongjiang Province, thereby suggesting a relatively low health risk associated with consuming OR from this region’s production systems according to existing standards and guidelines. Nevertheless, special attention is needed for monitoring the habitat environments of R. dybowskii from two sampling locations showing heightened pollution levels.

Seasonal changes in vitamin A metabolism-related factors in the oviduct of Chinese brown frog (Rana dybowskii)

The oviduct of the Chinese brown frog (Rana dybowskii) expands during pre-brumation rather than the breeding period, exhibiting a special physiological feature. Vitamin A is essential for the proper growth and development of many organisms, including the reproductive system such as ovary and oviduct. Vitamin A is metabolized into retinoic acid, which is crucial for oviduct formation. This study examined the relationship between oviducal expansion and vitamin A metabolism. We observed a significant increase in the weight and diameter of the oviduct in Rana dybowskii during pre-brumation. Vitamin A and its active metabolite, retinoic acid, notably increased during pre-brumation. The mRNA levels of retinol binding protein 4 (rbp4) and its receptor stra6 gene, involved in vitamin A transport, were elevated during pre-brumation compared to the breeding period. In the vitamin A metabolic pathway, the mRNA expression level of retinoic acid synthase aldh1a2 decreased significantly during pre-brumation, while the mRNA levels of retinoic acid α receptor (rarα) and the retinoic acid catabolic enzyme cyp26a1 increased significantly during pre-brumation, but not during the breeding period. Immunohistochemical results showed that Rbp4, Stra6, Aldh1a2, Rarα, and Cyp26a1 were expressed in ampulla region of the oviduct. Western blot results indicated that Aldh1a2 expression was lower, while Rbp4, Stra6, RARα, and Cyp26a1 were higher during pre-brumation compared to the breeding period. Transcriptome analyses further identified differential genes in the oviduct and found enrichment of differential genes in the vitamin A metabolism pathway, providing evidences for our study. These results suggest that the vitamin A metabolic pathway is more active during pre-brumation compared to the breeding period, and retinoic acid may regulate pre-brumation oviductal expansion through Rarα-mediated autocrine/paracrine modulation.

Isolation, Identification, and Characterisation of a Novel ST2378 Aeromonas hydrophila Strain from Naturally Diseased Frogs, Rana dybowskii.

In 2023, Rana dybowskii exhibiting characteristic skin ulcers were found on a farm in northeastern China. Subsequently, two dominant bacteria, Aeromonas hydrophila Rd001 and Acinetobacter johnsonii Rd002, were isolated from naturally infected R. dybowskii. Experimental infection confirmed that Rd001 was the primary pathogen responsible for the disease in R. dybowskii, with a mean lethal dose (LD50) of 6.25 × 102 CFU/g. The virulence genotype of Rd001 was identified as ser+/aha+/lip+/nuc+/hlyA+/aer+/alt+/ast+/act+. Antimicrobial susceptibility testing indicated that Rd001 was sensitive to enrofloxacin, flumequine, and neomycin. MLST analysis showed that Rd001 belonged to a new sequence type of A. hydrophila, named ST2378. This study offered the first comprehensive investigation into the pathogenicity, virulence genotypes, antimicrobial resistance, and genetic traits of A. hydrophila isolated from R. dybowskii, providing a theoretical foundation for preventing and controlling A. hydrophila infections.

Impact of wildfire ash on skin and gut microbiomes and survival of Rana dybowskii

Climate change and human activities escalate the frequency and intensity of wildfires, threatening amphibian habitats and survival; yet, research on these impacts remains limited. Wildfire ash alters water quality, introduces contaminants, and may disrupt microbial communities, impacting gut and skin microbiota; however, the effects on gut and skin microbiota remain unclear. Rana dybowskii were exposed to five concentrations (0 g L-1, 1.25 g L-1, 2.5 g L-1, 5 g L-1, and 10 g L-1) of aqueous extracts of wildfire ashes (AEAs) for 30 days to assess AEAs' metal content, survival, and microbiota diversity via Illumina sequencing. Our results showed that the major elements in ash were Ca > K > Mg > Al > Fe > Na > Mn, while in AEA they were K > Ca > Na > Mg > As > Al > Cu. A significant decrease in amphibian survival rates with increased AEA concentration was shown. The beta diversity analysis revealed distinct shifts in microbiota composition. Notably, bacterial genera associated with potential health risks showed increased abundance in skin microbiota, emphasising the potential for ash exposure to affect amphibian health. Functional prediction analyses revealed significant shifts in metabolic pathways related to health and disease, indicating that wildfire ash exposure may influence amphibian health through changes in microbial functions. This study highlights the urgent need for strategies to mitigate wildfire ash impacts on amphibians, as it significantly alters microbiota and affects their survival and health.

Rapid molecular identification of Rana dybowskii by species-specific primers

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific.

Characterization and anti-aging activities of polysaccharide from Rana dybowskii Guenther.

Introduction: Rana dybowskii Guenther (RDG), as a traditional Chinese medicine, has been shown to have antioxidant effects. However, studies on the anti-aging effect of RDG are still limited. Methods: In this study, we prepared polysaccharides from the skin of RDG (RDGP) by hot water extraction, alcohol precipitation, ion-exchange chromatography and gel chromatography. The proteins were removed using the Sevage method in combination with an enzymatic method. The structural features were analyzed using high-performance gel permeation chromatography, β-elimination reaction and Fourier transform infrared spectra. The anti-aging effect of RDGP was investigated by using D-Gal to establish an aging model in mice, and pathological changes in the hippocampus were observed under a microscope. Results: We obtained the crude polysaccharide DGP from the skin of RDG, with a yield of 61.8%. The free protein was then removed by the Sevage method to obtain DGPI and deproteinated by enzymatic hydrolysis combined with the Sevage method to further remove the bound protein to obtain the high-purity polysaccharide DGPII. Then, DGPIa (1.03 × 105Da) and DGPIIa (8.42 × 104Da) were obtained by gel chromatography, monosaccharide composition analysis showed that they were composed of Man, GlcA, GalNAc, Glc, Gal, Fuc with molar ratios of 1: 4.22 : 1.55: 0.18 : 8.05: 0.83 and 0.74 : 1.78: 1: 0.28: 5.37 : 0.36, respectively. The results of the β-elimination reaction indicated the presence of O-glycopeptide bonds in DGPIa. The Morris water maze test indicated that mice treated with DGPIIa exhibited a significantly shorter escape latency and increased time spent in the target quadrant as well as an increase in the number of times they traversed the platform. Pathologic damage to the hippocampus was alleviated in brain tissue stained with hematoxylin-eosin. In addition, DGPIIa enhanced the activities of SOD, CAT, and GSH-Px and inhibited the level of MDA in the serum and brain tissues of aging mice. Discussion: These results suggest that RDGP has potential as a natural antioxidant and provide useful scientific information for anti-aging research.

Impact of antibiotic therapy on cutaneous and gut microbiota in Rana dybowskii amphibians: Insights and implications

Antibiotics are key in combating bacterial infections and halting disease spread, yet their effects on amphibian gut microbiomes remain poorly understood. Amphibian survival depends on their skin microbiota. Drugs used to treat amphibians may affect gut microbiota but has yet to have unexplored impacts on skin microbiota. We used Illumina sequencing to analyze the cutaneous (C) and gut (G) microbiota of healthy (H) frogs and diseased (D) frogs treated with antibiotics and evaluate their functional characteristics. The result showed that antibiotics lowered gut microbiota alpha diversity but not skin microbiota. Bray-Curtis and UniFrac measures demonstrate the significant variations in skin and gut microbiota between healthy and antibiotic-treated frogs. Bacteroidota, Firmicutes, and Proteobacteria predominated in DC and HC; Proteobacteria, Firmicutes, and Bacteroidota dominated DG and HG. Linear discriminant analysis and effect size (LEfSe) analysis identified specific bacterial taxa enriched in antibiotic-treated versus healthy frogs. Maximum likelihood (ML) analysis revealed close evolutionary relationships among certain bacterial taxa. Functional predictions indicated significant differences in metabolic pathways between groups, and BugBase analysis showed variations in bacterial phenotypes, suggesting altered microbial functions following antibiotic treatment. Antibiotic treatment in frogs significantly altered gut microbiota diversity and impacts functional pathways in both gut and skin microbiota without affecting skin microbiota alpha diversity. Antibiotics' complex effects on frog microbiota showed R. dybowskii's delicate balance between therapeutic advantages and ecological changes, which might affect conservation and health initiatives.

Rice straw ash and amphibian health: A deep dive into microbiota changes and potential ecological consequences

Rice straw is burned as a result of agricultural practices and technical limitations, generating significant volumes of ash that might have environmental and ecological consequences; however, the effects on organisms have not been researched. Amphibians depend on their gut and skin microbiomes. Ash exposure may cause inflammation and changes in microbial diversity and function in frogs' skin and gut microbiota due to its chemical composition and physical presence, but the implications remain unclear. Rana dybowskii were exposed to five aqueous extracts of ashes (AEA) concentrations for 30 days to study survival, metal concentrations, and microbial diversity, analyzing the microbiota of the cutaneous and gut microbiota using Illumina sequencing. Dominant elements in ash: K > Ca > Mg > Na > Al > Fe. In AEA, K > Na > Ca > Mg > As > Cu. Increased AEA concentrations significantly reduced frog survival. Skin microbiota alpha diversity varied significantly among all treatment groups, but not gut microbiota. Skin microbiota differed significantly across treatments via Bray-Curtis and weighted UniFrac; gut microbiota was only affected by Bray-Curtis. Skin microbiota varied significantly with AEA levels in Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes, while the gut microbiota's dominant phyla, Firmicutes, Bacteroidetes, and Proteobacteria, remained consistent across all groups. Lastly, the functional prediction showed that the skin microbiota had big differences in how it worked and looked, which were linked to different health and environmental adaptation pathways. The gut microbiota, on the other hand, had smaller differences. In conclusion, AEA exposure affects R. dybowskii survival and skin microbiota diversity, indicating potential health and ecological impacts, with less effect on gut microbiota.

The Circadian Rhythm of the Behavior and Gut Microbiota in Dybowski's Frogs (Rana dybowskii) during the Autumn Migration Period.

Many amphibian behaviors and physiological functions adapt to daily environmental changes through variations in circadian rhythms. However, these adaptations have yet to be reported in Dybowski's frog (Rana dybowskii). We aimed to elucidate the dynamic changes in the behavior and gut microbiota of R. dybowskii within a 24 h cycle during their migration to hibernation sites. Thus, we monitored their behavior at 4 h intervals and collected samples for microbiome analysis. We found that the juvenile frogs arrived at hibernation sites earlier than the adults. Among the adults, the male frogs arrived earlier. The richness and diversity of the gut microbiota in the adult R. dybowskii were lowest at 14:00. At 6:00, the differences between the males and females were most significant. At 18:00, there was an increase in the activity of Bacteroides, Coprobacillus, Ruminococcus, and Dorea in the intestinal tracts of the male frogs, whereas in the intestinal tract of the female frogs, there was an increase in the activity of Pseudoramibacter_Eubacterium, Desulfovibrio, Anaerotruncus, and PW3. This indicated diurnal rhythmic variations in the gut microbiota and significant sex-based differences in the microbial activity at different time points. Our findings contribute to the understanding of the circadian rhythm of R. dybowskii and provide crucial insights into improving breeding strategies.

Skin ulcers and microbiota in Rana dybowskii: Uncovering the role of the gut-skin axis in amphibian health

Amphibians' gut and skin microbiota are vital for host health. Ulcers may affect the skin microbiota, with unknown impacts on the gut microbiota. Skin diseases could influence both, but research on their interrelationships is limited. The cutaneous and gut microbiota of healthy and ulcerated Rana dybowskii were analyzed with Illumina sequencing. The result showed healthy frogs showed greater alpha diversity in skin microbiota than ulcerated ones, with no gut microbiota difference. Bray-Curtis and unweighted UniFrac distances revealed distinct skin microbiotas in healthy versus ulcerated groups and significant gut microbiota differences only in Bray-Curtis. Linear discriminant analysis of effect size (LEfSe) showed that ulcerated skin groups were enriched in Proteobacteria and healthy in Actinobacteria. A variety of analyses revealed that diseased frogs, unlike healthy ones, exhibit similar skin and gut microbiota, suggesting disease impacts microbiota relationships and composition. Using the indicator method, identify key bacterial species indicating healthy or diseased status in the frog's skin and gut microbiota. There were significant differences in skin and gut microbiota functions between diseased and healthy frogs, with disease impacting bacterial community structure and physiological functions. Research on R. dybowskii indicated disease aligns the skin and gut microbiota, affecting composition and function, with distinct bacterial indicators for health and disease status. This research underscores the complex interplay between skin and gut microbiota in amphibian health, highlighting implications for disease understanding and conservation strategies.

Investigation of seasonal changes in lipid synthesis and metabolism-related genes in the oviduct of Chinese brown frog (Rana dybowskii).

A peculiar physiological characteristic of the Chinese brown frog (Rana dybowskii) is that its oviduct dilates during pre-brumation rather than during the breeding season. This research aimed to examine the expression of genes connected with lipid synthesis and metabolism in the oviduct of R. dybowskii during both the breeding season and pre-brumation. We observed significant changes in the weight and size of the oviduct between the breeding season and pre-brumation. Furthermore, compared to the breeding season, pre-brumation exhibited significantly lower triglyceride content and a marked increase in free fatty acid content. Immunohistochemical results revealed the spatial distribution of triglyceride synthase (Dgat1), triglyceride hydrolase (Lpl and Hsl), fatty acid synthase (Fasn), and fatty acid oxidases (Cpt1a, Acadl, and Hadh) in oviductal glandular cells and epithelial cells during both the breeding season and pre-brumation. While the mRNA levels of triglycerides and free fatty acid synthesis genes (dgat1 and fasn) did not show a significant difference between the breeding season and pre-brumation, the mRNA levels of genes involved in triglycerides and free fatty acid metabolism (lpl, cpt1a, acadl, acox and hadh) were considerably higher during pre-brumation. Furthermore, the R. dybowskii oviduct's transcriptomic and metabolomic data confirmed differential expression of genes and metabolites enriched in lipid metabolism signaling pathways during both the breeding season and pre-brumation. Overall, these results suggest that alterations in lipid synthesis and metabolism during pre-brumation may potentially influence the expanding size of the oviduct, contributing to the successful overwintering of R. dybowskii.

Transcriptome Analysis Reveals Cross-Tissue Metabolic Pathway Changes in Female Rana dybowskii during Emergence from Hibernation

The brown frog (Rana dybowskii) exhibits a wide distribution and is extensively cultured in northeast China. This species undergoes a prolonged period of hibernation lasting from several months to half a year. The frog’s fallopian tubes are considered a valuable tonic medicine known as “Oviducts Ranae” in traditional Chinese medicine. To enhance comprehension of the molecular mechanism underlying the process of emergence from hibernation, a transcriptome-based comparative analysis was performed on differentially expressed genes (DEGs) across various organs of female frogs during hibernation and upon emergence. The organs examined encompassed the brain, liver, spleen, fallopian tube, and ovary. Subsequently, GO and KEGG enrichment analyses were performed to gain further insights into these DEGs. A total of 51,634 transcripts were identified in all five tissues. The spleen exhibited the highest number of DEGs, with 3651 members, while the liver, brain, and fallopian tube had 3182, 3115, and 3186 DEGs, respectively. The ovary had the lowest number of DEGs, with only 1661. Interestingly, only 45 genes were found to be differentially expressed in all five tissues, and GO enrichment analysis revealed distinct functional differences among the DEGs in the various tissues. Only two meaningful DEG-enriched KEGG pathways, “00310 Lysine degradation” and “05202 Transcriptional misregulation in cancer”, were present in all five tissues, and the pathway “B 09182 Protein families: genetic information processing” was significantly enriched in four tissues except the ovary, and it had the most enriched DEGs. Our findings offer insights to grasp the factors that need to be controlled in the design of overwintering sites and offer a novel perspective for the conservation and management of the overwintering population of female R. dybowskii.

Kidney cell culture of Rana dybowskii and study molecules of TRIF in Rana dybowskii cells under the stress of Aeromonas hydrophila

As a model organism, the cell culture method of Rana dybowskii (Rd) has not received widespread attention. We obtained kidney cells from Rana dybowskii by tissue block culture and enzyme digestion, and optimized the culture method by changing different culture conditions. We can more conveniently study the operation of the Rana dybowskii immune system through cell lines. One of the most important adaptor proteins in the Toll-like receptors (TLRs) signaling pathway, TIR-domain-containing adaptor inducing interferon-β (TRIF), plays a role in the apoptosis pathway and participates in the ubiquitin-mediated protein hydrolysis pathway. This rationalizes the significance of TRIF in the immune cascade signaling pathway. To obtain and analyze the full-length sequence of cDNA, sequential structures of amino acids, as well as Rana dybowskii TRIF expression profiles, the present work carried out multiple-sequence alignment, rapid amplification of cDNA ends (RACE), the establishment of a phylogenetic tree, search for the conserved domain, and qRT-PCR. Results revealed 1733-bp full-length cDNA of RdTRIF with an open reading frame (ORF) of 1281 bp, which encoded one protein containing 426 amino acid residues. The amino acid sequence of RdTRIF confirmed the presence of one Toll/interleukin-1 receptor (TIR) domain. As a kind of hydrophilic protein, the pI and molecular weight of RdTRIF were 5.67 and 24.76 kDa, respectively. We predicted 51 candidate phosphorylation sites, including 32 serine residues, 16 threonine residues along with 3 tyrosine residues. The phylogenetic tree and multiple-sequence alignment substantiated the highest homology of RdTRIF protein to Xenopus tropicalis homologs. According to the qRT-PCR results, the expression of RdTRIF was noted under the stress of Ah in the kidney.

Metabolomic Profiling Reveals Differences in Hypoxia Response between Far Eastern and Siberian Frogs.

Anoxia is a significant challenge for most animals, as it can lead to tissue damage and death. Among amphibians, the Siberian frog Rana amurensis is the only known species capable of surviving near-zero levels of oxygen in water for a prolonged period. In this study, we aimed to compare metabolomic profiles of the liver, brain, and heart of the Siberian frog exposed to long-term oxygen deprivation (approximately 0.2 mg/L water) with those of the susceptible Far Eastern frog (Rana dybowskii) subjected to short-term hypoxia to the limits of its tolerance. One of the most pronounced features was that the organs of the Far Eastern frog contained more lactate than those of the Siberian frog despite a much shorter exposure time. The amounts of succinate were similar between the two species. Interestingly, glycerol and 2,3-butanediol were found to be significantly accumulated under hypoxia in the Siberian frog, but not in the Far Eastern frog. The role and biosynthesis of these substances are still unclear, but they are most likely formed in certain side pathways of glycolysis. Based on the obtained data, we suggest a pathway for metabolic changes in the Siberian frog under anoxia.

The expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during pre-spawning and pre-hibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in pre-hibernation rather than in pre-spawning, which is one of the physiological phenomena that occur in preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during pre-spawning and pre-hibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During pre-hibernation, oviductal pipe diameter and weight were significantly larger than during pre-spawning. 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), Low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in pre-hibernation and glandular cells during pre-spawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in pre-hibernation than during pre-spawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in pre-hibernation compared to pre-spawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during pre-hibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.