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.

[Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection].

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

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.

Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection / 生物工程学报

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

De novo assembly and analysis of the transcriptome of the Siberian wood frog Rana amurensis.

The Siberian wood frog Rana amurensis Boulenger, 1886 is the most hypoxia-tolerant amphibian. It can survive for several months in an almost complete absence of oxygen. Little is known about the mechanisms of this remarkable resilience, in part because studies of amphibian genomes are impeded by their large size. To make the Siberian wood frog more amenable for genetic analysis, we performed transcriptome sequencing and de novo assembly for the R. amurensis brain under hypoxia and normoxia, as well as for the normoxic heart. In order to build a de novo transcriptome assembly of R. amurensis, we utilized 125-bp paired-end reads obtained from the brain under normoxia and hypoxia conditions, and from the heart under normoxia. In the transcriptome assembled from about 100,000,000 reads, 81.5 % of transcripts were annotated as complete, 5.3 % as fragmented, and 13.2 % as missing. We detected 59,078 known transcripts that clustered into 22,251 genes; 11,482 of them were assigned to specific GO categories. Among them, we found 6696 genes involved in protein binding, 3531 genes involved in catalytic activity, and 576 genes associated with transporter activity. A search for genes encoding receptors of the most important neurotransmitters, which may participate in the response to hypoxia, resulted in a set of expressed receptors of dopamine, serotonin, GABA, glutamate, acetylcholine, and norepinephrine. Unexpectedly, no transcripts for histamine receptors were found. The data obtained in this study create a valuable resource for studying the mechanisms of hypoxia tolerance in the Siberian wood frog, as well as for amphibian studies in general.

Sequencing and analysis of the complete mitochondrial genome of Rana amurensis (Anura: Ranidae).

In this study, the complete mitogenome sequence of Rana amurensis (Anura: Ranidae) is determined using long PCR. It is a circular molecule of 20,571 bp in length (GenBank accession no. MF370348). The complete mtDNA sequence of R. amurensis contained 2 rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, 13 protein-coding genes (PCGs) and 2 control regions (D-loops). The nucleotide composition was 28.6% A, 26.8% C, 14.0% G and 30.6% T. Mitochondrial genome analyses based on NJ method yield phylogenetic trees, including that 20 reported family Ranidae frogs. These molecular data presented here provide a useful tool for systematic analyses of genus Rana.