Rana Dybowskii Research Articles

Decreased acetylation of HDGF improves oviduct production in Rana dybowskii, Rana amurensis, and Rana huanrenensis

The oviduct of female Rana dybowskii is a functional food and can be used as a component of Traditional Chinese medicine. The differentially expressed genes enriched was screened in cell growth of three Rana species. We quantitatively analyzed 4549 proteins using proteomic techniques, enriching the differentially expressed proteins of Rana for growth and signal transduction. The results showed that log2 expression of hepatoma-derived growth factor (HDGF) was increased. We further verified 5 specific differential genes (EIF4a, EIF4g, HDGF1, HDGF2 and SF1) and found that HDGF expression was increased in Rana dybowskii. Through acetylation modification analysis, we identified 1534 acetylation modification sites in 603 proteins, including HDGF, and found that HDGF acetylation expression was significantly reduced in Rana dybowskii. Our results suggest that HDGF is involved in the development of oviductus ranae, which is regulated by acetylation modification.

Characteristics and a comparison of the gut microbiota in two frog species at the beginning and end of hibernation.

Season has been suggested to contribute to variation in the gut microbiota of animals. The complicated relationships between amphibians and their gut microbiota and how they change throughout the year require more research. Short-term and long-term hypothermic fasting of amphibians may affect gut microbiota differently; however, these changes have not been explored. In this study, the composition and characteristics of the gut microbiota of Rana amurensis and Rana dybowskii during summer, autumn (short-term fasting) and winter (long-term fasting) were studied by high-throughput Illumina sequencing. Both frog species had higher gut microbiota alpha diversity in summer than autumn and winter, but no significant variations between autumn and spring. The summer, autumn, and spring gut microbiotas of both species differed, as did the autumn and winter microbiomes. In summer, autumn and winter, the dominant phyla in the gut microbiota of both species were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. All animals have 10 OTUs (>90% of all 52 frogs). Both species had 23 OTUs (>90% of all 28 frogs) in winter, accounting for 47.49 ± 3.84% and 63.17 ± 3.69% of their relative abundance, respectively. PICRUSt2 analysis showed that the predominant functions of the gut microbiota in these two Rana were focused on carbohydrate metabolism, Global and overview maps, Glycan biosynthesis metabolism, membrane transport, and replication and repair, translation. The BugBase analysis estimated that among the seasons in the R. amurensis group, Facultatively_Anaerobic, Forms_Biofilms, Gram_Negative, Gram_Positive, Potentially_Pathogenic were significantly different. However, there was no difference for R. dybowskii. The research will reveal how the gut microbiota of amphibians adapts to environmental changes during hibernation, aid in the conservation of endangered amphibians, particularly those that hibernate, and advance microbiota research by elucidating the role of microbiota under various physiological states and environmental conditions.

Cloning of adipor1 and adipor2 genes in Rana dybowskii and its expression pattern upon infection

Adiponectin receptor 1 (AdipoR1) and Adiponectin receptor 2 (AdipoR2) can bind to adiponectin (AdipoQ) secreted by adipose tissue to participate in various physiological functions of the body. In order to explore the role of AdipoR1 and AdipoR2 in amphibians infected by Aeromonas hydrophila (Ah), the genes adipor1 and adipor2 of Rana dybowskii were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by bioinformatics. The tissue expression difference of adipor1 and adipor2 was analyzed by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and an inflammatory model of R. dybowskii infected by Ah was constructed. The histopathological changes were observed by hematoxylin-eosin staining (HE staining); the expression profiles of adipor1 and adipor2 after infection were dynamically detected by qRT-PCR and Western blotting. The results show that AdipoR1 and AdipoR2 are cell membrane proteins with seven transmembrane domains. Phylogenetic tree also shows that AdipoR1 and AdipoR2 cluster with the amphibians in the same branch. qRT-PCR and Western blotting results show that adipor1 and adipor2 were up-regulated at different levels of transcription and translation upon Ah infection, but the response time and level were different. It is speculated that AdipoR1 and AdipoR2 participate in the process of bacterial immune response, providing a basis for further exploring the biological functions of AdipoR1 and AdipoR2 in amphibians.

Effects of fine-scale habitat quality on activity, dormancy, habitat use, and survival after reproduction in Rana dybowskii (Chordata, Amphibia)

Amphibians are facing population declines and extinctions, and protecting and supplementing refuges can help species survive. However, the microhabitat requirements of most species are unknown, and artificial shelters or burrows have not been well tested for amphibians. Some amphibians exhibit complex behaviour during the transition from post-reproductive dormancy to activity. However, little is known about the ecology, post-reproductive dormancy, and terrestrial activity of amphibians. Here, habitat site selection in experimental enclosures and the effects of shelters (stones, soil) and shade (with and without shade netting) on the activity, exposed body percentage, burrow depth, body-soil contact percentage, and survival of Rana dybowskii were investigated during post-reproductive dormancy and post-dormant activity. The results showed that R. dybowskii live individually under leaves, soil, stones or tree roots. Furthermore, although the dormant sites of frogs are significantly different, the distribution of male and female frogs in these sites is similar. Shading and shelter significantly affected the exposed body percentage, burrow depth and body-soil contact percentage of frogs compared with soil. In the stone group, soil and stone form the frog's refuge/burrow, whereas in the soil group, the refuge/burrow is composed entirely of soil. Even though the soil group has a deeper burrow and a larger area of soil contact with the body, it still has a higher exposure rate than the stone group. Frog activity frequency was affected by shelter and shade; the interaction of shelter and time and the interaction of shading and time were significant. The soil group had a higher activity frequency than the stone group, and the no-shade group had a higher activity frequency than the shade group. Shelter and shading differences do not significantly affect frog survival; however, the death rate during post-reproductive dormancy is lower than that during the active period.

Molecular Dietary Analysis of Three Sympatric Mustelidae in Northeast China

Diet analysis is essential to fully understand the biology of a species and its function within the ecosystem, as well as being key in identifying food web interactions and the population dynamics of predators and prey. The understanding of the diet of small to mid-sized carnivores remains generally lacking or uninformative due to the inability for taxonomic resolution based on morphology. The yellow-throated marten (Martes flavigula), Eurasian river otter (Lutra lutra), and Siberian weasel (Mustela sibirica) are three important Mustelidae species in ecosystems of northeast China. Based on fecal DNA and a next-generation sequencing (NGS) approach, we analyzed the vertebrate prey of these three sympatric Mustelidae. Prey included 7 mammalian taxa, 10 fishes, 2 birds, and 2 amphibians, with 85% of the taxa assigned to the species level. In total, twenty-one vertebrate prey taxa were identified from seven yellow-throated martens, eight Eurasian river otters, and two Siberian weasels. Concerning identified dietary species, 10 taxa were consumed by yellow-throated martens, 14 by Eurasian river otters, and 4 by Siberian weasels. Some prey species were identified in more than one species. Amphibians and fishes were the most dominant Eurasian river otter prey categories, whereas Eurasian badger (Meles leucurus), birds, and rodents were the main yellow-throated marten prey; amphibians and rodents were largely contained in Siberian weasel prey. Among prey items, Dybowski's frog (Rana dybowskii) and Korean field mouse (Apodemus peninsulae) were identified in all three Mustelidae species but our analyses suggest potential diet preferences among Mustelidae species. Future studies should focus on understanding the trophic relationships of these three Mustelidae species, providing valuable information for their conservation planning.

Testicular Transcriptome of Males and Pseudo-Males Provides Important New Insight into Sex Reversal of Rana dybowskii.

Simple SummaryAquaculture in Northeast China has favored Rana dybowskii (R. dybowskii) because of its regional specificity and the economic value of the female frog’s ovary, and the increasing proportion of female frogs is beneficial to improve the economic efficiency of aquaculture. Unfortunately, the sexual reversal from genotypic female to phenotypic male has led to a decrease in the proportion of females in the breeding population. In this study, differentially expressed genes between testes of sex reversal and male testes were screened by transcriptome sequencing. These findings provide a comprehensive analysis of gene expression in R. dybowskii sex reversal from genotypic female to phenotypic male and provide new ideas for sex control of R. dybowskii.Rana dybowskii (R. dybowskii) is an ecological species found in China, Japan, Korea, and Russia. Like most amphibians, R. dybowskii lacks heterotypic sex chromosomes, limiting the in-depth study of sex determination and sex reversal mechanisms. Previous studies have shown that certain environmental factors can modify R. dybowskii genotypic females into phenotypic males, but the mechanism is still unknown. Considering the difficulties in identifying and collecting sex reversal gonads at different stages of differentiation under natural conditions, testes from sexually mature wild adult R. dybowskii were taken in this study, and the genotypic sex of individuals and sex reversal were identified by two male-linked genetic markers reported in our most recent findings. Transcriptome sequencing was performed on testicular tissue from males and pseudo-males, as well as female ovary tissue. The results show that the gene expression patterns of pseudo-males’ testes were similar to those of the males but highly differed from females’ ovaries. One hundred and seventeen differentially expressed genes between testes of pseudo-males and males were found, and the up-regulation of doublesex and mab-3 related transcription factor 1 (Dmrt1) in testes of pseudo-males may play a key role in R. dybowskii sex reversal.

Cloning and tissue expression analysis of the LepROT gene of Rana dybowskii

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.

Expression of glycogenic genes in the oviduct of Chinese brown frog (Rana dybowskii) during pre-brumation

The oviduct of Chinese brown frog (Rana dybowskii) displays seasonal morphological and functional changes, which expands specifically during pre-brumation. To uncover the molecular mechanism underlying this phenomenon, we firstly confirmed the increased weight and enlarged diameter of the oviduct in pre-brumation by morphological observation. Interestingly, the glycogen content in the oviduct increased significantly during pre-brumation, indicating Rana dybowskii stores energy in the oviduct before brumation. Transcriptome analysis further identified the differentially expressed genes in the synthesis and metabolism pathways of carbohydrates in the oviduct during pre-brumation. Based on that evidence, we focused on the mRNA and protein expression of glycogenic genes in the oviduct of Rana dybowskii. qPCR confirmed that the expression of glycolysis and glycogenesis-related genes were up-regulated while gluconeogenesis-related genes were down-regulated during pre-brumation. Western blot data showed that glucose transporter GLUT1 and glycogen synthesis-regulation proteins including GYS, and p-GSK-3β were highly expressed in the oviduct during pre-brumation. Moreover, immunohistochemical data showed that GLUT1, GYS, p-GYS, GSK-3β and p-GSK-3β were expressed regionally in the oviduct of Rana dybowskii. The data suggests that glycogen synthesis may be involved in the oviductal expansion of Rana dybowskii during the pre-brumation.

Impact of photoperiods on the specific activities of immune and antioxidant enzymes in different tissues of Dybowski’s frog (Rana dybowskii)

ABSTRACT Many physiological activities in amphibians show rhythmic changes to adapt to ambient light conditions. However, the effects of photoperiod variation on the immune and antioxidant status and their possible correlation with different tissues have not been reported in Dybowski’s frog, Rana dybowskii. In this study, the specific activities of immune and antioxidant enzymes in R. dybowskii under different photoperiods were evaluated. The results demonstrated that the photoperiod significantly impacted antioxidant enzymes and immune enzymes in Dybowski’s frog. In terms of antioxidant enzyme activity, catalase (CAT) and superoxide dismutase (SOD) activities in frogs at 6 L:18D and 0 L:24D were significantly higher than those at 18 L:6D and 24 L:0D, respectively. Further, the activities of these enzymes increased during the dark phase and decreased during the light phase. The malondialdehyde (MDA) content decreased significantly with the decrease in photoperiod time. Lysozyme (LZM) activity in serum and the liver decreased with a prolonged photoperiod. LZM activity in the dark cycle was higher, but the peak activity in skin occurred at noon. Thus, long-term light exposure may cause oxidative stress in Dybowski’s frog. Accordingly, a 6 L:18D photoperiod may be suitable for optimum physiological functions of this species.

Effects of photoperiod on the melatonin cycle of Dybowski’s frog (Rana dybowskii)

ABSTRACT There are few studies on the relationship between photoperiod and the melatonin cycle in amphibians. To elucidate the role of the photoperiodically modulated endogenous melatonin cycle in different tissues of Dybowski’s frog (Rana dybowskii), its serum, liver, whole brain, skin, and eyeballs were collected under five photoperiods, and the daily variation in melatonin concentration was determined via high-performance liquid chromatography. Compared with the control group (12 L:12 D), melatonin concentration was increased under photoperiods 0 L:24 D and 6 L:18 D in serum, and the concentration peaked at 00:00 under photoperiod 0 L:24 D. This trend was synchronized with the peak melatonin concentration in the retinal, liver, and whole-brain tissues. After 24 h of light exposure, no significant daily variation was observed under photoperiod 24 L:0 D. Furthermore, we observed a significant peak at 04:00 in the serum under photoperiod 18 L:6 D. However, we observed the opposite trend in the circadian rhythm of the skin tissue. Our results underscore the high sensitivity of melatonin content to different photoperiods. These findings may have real-world implications in terms of furthering our understanding of circadian rhythms disruption induced by exposure to different photoperiods.

Structure prediction and biological activity analysis of dybowskin-1ST antimicrobial peptide in Rana dybowskii

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.

Morphology and mechanical performance between the skin surface ofRana dybowskiiandBufo gargarizans

Anuran skin is a typical natural biomaterial with multifunctional features. A specific comparison of mechanical performance and morphology related to them was performed in the skin of Rana dybowskii and Bufo gargarizans using the tensile testing technique and morphological equipment. Rana dybowskii's skin has soft smooth surface covered by polygonal epidermal cells, while the Bufo gargarizans species has tough and uneven skin surface due to numerous verrucae structures. The collagen fibre bundles in lower dermis of Bufo gargarizans have wavelike organisation while the bundles of Rana dybowskii show a parallel arrangement. The mean elastic modulus of Rana dybowskii was nine times higher than that of Bufo gargarizans. This study clarified that the arrangement of collagen fibres play an important role in the strength and elasticity of skin material.

Male heterogametic sex determination in Rana dybowskii based on sex-linked molecular markers.

Identifying the mechanism for sex determination in amphibians is challenging. Very little is known about sex determination mechanisms of Rana dybowskii, a species of importance to evolutionary and conservation biology. We screened for sex‐linked molecular markers in R. dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1. We found 2 male‐linked molecular markers in R. dybowskii, which were 222 bp and 261 bp long. The detection rates of 222 bp marker in males form Xinglong, Huadian, and Dandong were 93.79%, 69.64%, and 13.64%, respectively, while the rate in females from Huadian was 27.50%. Besides, the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%, 87.50%, and 32.73%, respectively. The inheritance patterns of sex‐linked molecular markers showed that the 2 sex‐linked molecular markers were heterozygous. Compared to the XY‐male parent, progeny from XX‐pseudo‐male parent possessed lower sex reversal ratio at the same rearing temperature, and the proportion of female froglets from an XX‐pseudo‐male parent was more than 95% at low rearing temperature (15°C). Our findings suggest that R. dybowskii displays male heterogamety, and the 2 sex‐linked molecular markers may have a guiding significance for the protection and utilization of R. dybowskii.

Daily cycle of melatonin in different tissues of dybowski’s frog (Rana dybowskii)

ABSTRACT This study is the first to analyse the daily cycle of melatonin in Dybowski’s frogs (Rana dybowskii), a species with economic importance in north-eastern China. The concentrations of melatonin in the serum, whole brain, and liver, skin, and retinal tissue samples collected from Dybowski’s frogs were measured using high-performance liquid chromatography every 4 h over a 24 h cycle. A significant melatonin peak (P< 0.05) was observed at 00:00 in the serum samples (203.25 ± 5.06 ng/L). This peak was synchronised with peak melatonin concentrations in the retinal (1513.25 ± 3.15 ng/L), liver (49.34 ± 3.67 ng/L), and whole brain (426.85 ± 6.85 ng/L) tissue samples, although melatonin concentrations in skin tissue samples peaked at 12:00 (270.02 ± 4.60 ng/L). Therefore, the time of day should be considered when examining the physiological status of this frog, given the potential effects of fluctuating daily melatonin concentrations on physiological variables.

Cooperation behavior of fore- And hindlimbs during jumping in Rana dybowskii and Xenopus laevis.

Frogs are characterized by their outstanding jumping ability, depending on the rapid extension of hindlimbs to propel their bodies into air. A typical jumping cycle could be broken into four phases: preparation, takeoff, flight, and landing. Considerable research has been performed to discuss the function of hindlimbs of frogs during takeoff phase, whereas the literature of limbs' motion in jumping between different species was limited. To profile the evolution of locomotion in anurans, it is necessary to investigate on the motion of fore‐ and hindlimbs of frogs within different taxa. In this work, we put forward a detailed description of jumping behavior of two frog species, Rana dybowskii and Xenopus laevis. High‐speed cameras were used to explore the movement of different joints in fore‐ and hindlimbs of these two animals, and kinematic analysis was operated to identify both homologous behaviors and significant differences between them. We found that the Rana dybowskii's fore‐ and hindlimbs had good cooperation during jumping, while the Xenopus laevis' uncooperative behavior in limbs may give a functional explanation for the deficiency in terrestrial jumping; besides, the R. dybowskii's landing followed the “hands‐belly‐feet slap” strategy, and Xenopus laevis had clumsy landing with “belly‐flops” sequence. The result gained here clarifies the cooperation behavior of anuran limbs and may supply a new insight into our understanding of the anuran's evolution.

Immunoreactivities of AR, ERα, ERβ and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period.

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of androgen receptors (AR), estrogen receptor α (ERα), ERβ and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERβ and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERβ protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.

Comparative research on morphology and mechanical property of integument of Rana dybowskii, Xenopus laevis and Ambystoma mexicanum

Amphibians' integument is a multifunctional organ offering protection from the exterior surroundings and facilitating the physiological change of gas, water and salts with the environment, which is a natural biomaterial with multifunctional features. Interspecies comparison of biomechanical characters and microstructure possibly related to them were performed on the integument of three species of amphibians, two anurans(Rana dybowskii and Xenopus laevis) and one urodeles(Ambystoma mexicanum) using tensile testing and morphological characterization. It was found that the integument of Rana dybowskii and Xenopus laevis was covered by polygonal epidermal cells, while the trunk surface of Ambystoma mexicanum presented irregular microstructure with the lack of keratinization. The integument of Rana dybowskii and Xenopus laevis exhibited good performance on stiffness and strength, which showed quite high mean elastic modulus, 931MPa and 1048MPa,respectively.

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.