The Impact of Long-Term Hypoxia on the Antioxidant Defense System in the Siberian Frog Rana amurensis.

The Siberian frog Rana amurensis has a uniquely high tolerance to hypoxia among amphibians, as it is able to withstand several months underwater with almost no oxygen (0.2 mg/liter) vs. several days for other studied species. Since it was hypothesized that hypoxia actives the antioxidant defense system in hypoxia-tolerant animals, one would expect similar response in R. amurensis. Here, we studied the effect of hypoxia in the Siberian frog based on the transcriptomic data, activities of antioxidant enzyme, and content of low-molecular-weight antioxidants. Exposure to hypoxia upregulated expression of three relevant transcripts (catalase in the brain and two aldo-keto reductases in the liver). The activities of peroxidase in the blood and catalase in the liver were significantly increased, while the activity of glutathione S-transferase in the liver was reduced. The content of low-molecular-weight antioxidants (thiols and ascorbate) in the heart and liver was unaffected. In general, only a few components of the antioxidant defense system were affected by hypoxia, while most remained unchanged. Comparison to other hypoxia-tolerant species suggests species-specific adaptations to hypoxia-related ROS stress.

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.

Biochemical response of two earthworm taxa exposed to freezing.

Several earthworm species are known to be able to withstand freezing. At the biochemical level, this ability is based on cryoprotectant accumulation as well as several other mechanisms. In this study, we used 1H NMR to investigate metabolomic changes in two freeze-tolerant earthworm taxa, Dendrobaena octaedra and one of the genetic lineages of Eisenia sp. aff. nordenskioldi f. pallida. A total of 45 metabolites were quantified. High concentrations of glucose were present in frozen tissues of both taxa. No other putative cryoprotectants were found. We detected high levels of glycolysis end products and succinate in frozen animals, indicating the activation of glycolysis. Concentrations of many other substances also significantly increased. On the whole, metabolic change in response to freezing was much more pronounced in the specimens of Eisenia sp. aff. nordenskioldi f. pallida, including signs of nucleotide degradation.

Earthworms of Belarus (Annelida: Megadrili).

A survey of the earthworm fauna of Belarus that included most of its territory was performed in this study. One genus, three species and one subspecies were reported for the first time. The fauna of Belarus thus comprises 17 species and two subspecies. Detailed maps for all of the reported species were constructed.

Metabolomic Analysis Reveals That the Moor Frog Rana arvalis Uses Both Glucose and Glycerol as Cryoprotectants.

The moor frog Rana arvalis is one of a few amphibians that can tolerate freezing to low temperatures, up to -16 °C. In this study, we performed metabolomic analysis of the liver and hindlimb muscles of frozen and control R. arvalis. We found that the moor frog synthesizes glucose and glycerol in similar concentrations as low molecular weight cryoprotectants. This is the first such case reported for the genus Rana, which was believed to use glucose only. We found that freezing upregulates glycolysis, with the accumulation of several end products: lactate, alanine, ethanol, and, possibly, 2,3-butanediol. To our knowledge, this is also the first report of ethanol as an end product of glycolysis in terrestrial vertebrates. We observed highly increased concentrations of nucleotide degradation products, implying high level of stress. The Krebs cycle arrest resulted in high concentrations of succinate, which is common for animals. However, we found almost no signs of adaptations to reoxygenation stress, with overall low levels of antioxidants. We also performed metabolomics analysis of subcutaneous ice that was found to contain glucose, glycerol, and several other substances.

Biochemical Response to Freezing in the Siberian Salamander Salamandrella keyserlingii.

The Siberian salamander Salamandrella keyserlingii Dybowski, 1870 is a unique amphibian that is capable to survive long-term freezing at -55 °C. Nothing is known on the biochemical basis of this remarkable freezing tolerance, except for the fact that it uses glycerol as a low molecular weight cryoprotectant. We used 1H-NMR analysis to study quantitative changes of multiple metabolites in liver and hindlimb muscle of S. keyserlingii in response to freezing. For the majority of molecules we observed significant changes in concentrations. Glycerol content in frozen organs was as high as 2% w/w, which confirms its role as a cryoprotectant. No other putative cryoprotectants were detected. Freezing resulted in ischemia manifested as increased concentrations of glycolysis products: lactate and alanine. Unexpectedly, we detected no increase in concentrations of succinate, which accumulates under ischemia in various tetrapods. Freezing proved to be a dramatic stress with reduced adenosine phosphate pool and high levels of nucleotide degradation products (hypoxanthine, ß-alanine, and ß-aminoisobutyrate). There was also significant increase in the concentrations of choline and glycerophosphocholine, which may be interpreted as the degradation of biomembranes. Thus, we found that freezing results not only in macroscopical damage due to ice formation, but also to degradation of DNA and biomembranes.

Microorganisms of Two Thermal Pools on Kunashir Island, Russia.

The Kuril Archipelago is a part of the Circum-Pacific Belt (Ring of Fire). These islands have numerous thermal springs. There are very few studies on these microbial communities, and none of them have been conducted by modern molecular biological methods. Here we performed the first metagenomic study on two thermophilic microbial communities of Kunashir Island. Faust Lake is hot (48 °C) and highly acidic (pH 2.0). We constructed 28 metagenome-assembled genomes as well as 17 16S ribosomal RNA sequences. We found that bottom sediments of Faust Lake are dominated by a single species of red algae belonging to the Cyanidiaceae family. Archaeans in Faust Lake are more diverse than bacteria but less abundant. The Tretyakovsky Thermal Spring is also hot (52 °C) but only weakly acidic (pH 6.0). It has much higher microbial diversity (233 metagenome-assembled genomes; 93 16S ribosomal RNAs) and is dominated by bacteria, with only several archaeans and one fungus. Despite their geographic proximity, these two thermal springs were found to not share any species. A comparison of these two lakes with other thermal springs of the Circum-Pacific Belt revealed that only a few members of the communities are shared among different locations.

Species Delimitation of the Eisenia nordenskioldi Complex (Oligochaeta, Lumbricidae) Using Transcriptomic Data.

Eisenia nordenskioldi (Eisen, 1879) is the only autochthonous Siberian earthworm with a large distribution that ranges from tundra to steppe and broadleaved forests. This species has a very high morphological, ecological, karyological, and genetic diversity, so it was proposed that E. nordenskioldi should be split into several species. However, the phylogeny of the complex was unclear due to the low resolution of the methods used and the high diversity that should have been taken into account. We investigated this question by (1) studying the diversity of the COI gene of E. nordenskioldi throughout its range and (2) sequencing transcriptomes of different genetic lineages to infer its phylogeny. We found that E. nordenskioldi is monophyletic and is split into two clades. The first one includes the pigmented genetic lineages widespread in the northern and western parts of the distribution, and the second one originating from the southern and southeastern part of the species' range and representing both pigmented and non-pigmented forms. We propose to split the E. nordenskioldi complex into two species, E. nordenskioldi and Eisenia sp. 1 (aff. E. nordenskioldi), corresponding to these two clades. The currently recognized non-pigmented subspecies E. n. pallida will be abolished as a polyphyletic and thus a non-natural taxon, while Eisenia sp. 1 will be expanded to include several lineages earlier recognized as E. n. nordenskioldi and E. n. pallida.

Metabolic response of the Siberian wood frog Rana amurensis to extreme hypoxia.

The Siberian wood frog Rana amurensis is a recently discovered example of extreme hypoxia tolerance that is able to survive several months without oxygen. We studied metabolomic profiles of heart and liver of R. amurensis exposed to 17 days of extreme hypoxia. Without oxygen, the studied tissues experience considerable stress with a drastic decrease of ATP, phosphocreatine, and NAD+ concentrations, and concomitant increase of AMP, creatine, and NADH. Heart and liver switch to different pathways of glycolysis with differential accumulation of lactate, alanine, succinate, as well as 2,3-butanediol (previously not reported for vertebrates as an end product of glycolysis) and depletion of aspartate. We also observed statistically significant changes in concentrations of certain osmolytes and choline-related compounds. Low succinate/fumarate ratio and high glutathione levels indicate adaptations to reoxygenation stress. Our data suggest that maintenance of the ATP/ADP pool is not required for survival of R. amurensis, in contrast to anoxia-tolerant turtles.

Transcriptomic analysis confirms differences among nuclear genomes of cryptic earthworm lineages living in sympatry.

BACKGROUND: Many earthworm species demonstrate significant cryptic diversity, with several highly diverged mitochondrial lineages found within most of the taxa studied to date. The status of differences between these lineages on the nuclear level is still unclear. Because of widespread polyploidy in earthworms, most studies were limited to two nuclear loci, the ribosomal and the histone clusters. Here we attempted to elucidate the status of a set of genetic lineages within Eisenia nordenskioldi nordenskioldi, an earthworm species from Northern Asia with high intraspecific diversity. We performed RNA-seq on an IonTorrent platform for five specimens of this species belonging to five genetic lineages, as well as two outgroups from the family Lumbricidae, the congenetic E. andrei, and Lumbricus rubellus. RESULTS: We de novo assembled transcriptomes and constructed datasets of genes present in all seven specimens using broad (ProteinOrtho; 809 genes) and narrow (HaMStR; 203 genes) ortholog assignment. The majority of orthologs had identical amino acid sequences in all studied specimens, which we believe was due to strong bias towards the most conserved genes. However, for the rest of genes the differences among the lineages were lower than those between them and the congeneric E. andrei. Both datasets yielded phylogenetic trees with the same topology. E. n. nordenskioldi was found to be monophyletic. The differences on the genetic level had no concordance with geography, implying complex history of dispersal. CONCLUSIONS: We found that genetic lineages of E. n. nordenskioldi are genetically distinct on nuclear level and probably diverged long ago. Current data implies that they might even represent distinct species within the E. nordenskioldi species complex.

Mitochondrial DNA variation in Eisenia n. nordenskioldi (Lumbricidae) in Europe and Southern Urals.

There are many peregrine European earthworm species that are found in Siberia. In contrast, it is generally considered that the only Siberian species, E. n. nordenskioldi, was capable to disperse in the reverse direction, from Siberia into Europe. We studied genetic diversity of E. n. nordenskioldi in Southern Urals and Eastern Europe using the mitochondrial cox1 gene. We found that E. n. nordenskioldi from that region represents a new genetic lineage distinct from the previously known populations of this species from Siberia. Molecular clock estimates suggest that this newly found lineage separated from the rest of the species in Lower Pleistocene. Within the studied sample, we detected two geographically restricted groups, which also diverged long before the Holocene, one found in the East European Plain and the other restricted to the Urals. Those two groups were found in sympatry in only one population. Therefore, our results do not support the traditional viewpoint, suggesting that E. n. nordenskioldi is definitely not a recent invader in Europe.

Low genetic diversity in wide-spread Eurasian liver fluke Opisthorchis felineus suggests special demographic history of this trematode species.

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species.

The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda).

The complete mitochondrial genomes of the parasitic trematodes Opisthorchis felineus and Clonorchis sinensis (family Opisthorchiidae) were fully sequenced in order to develop markers for DNA diagnostics of the liver flukes infection, molecular ecology, population and phylogenetic studies. The complete sequences of mitochondrial genomes of these species comprise 14,277 and 13,875bp, respectively, and are thus the shortest trematode mitochondrial genomes sequenced to date. The gene content and arrangement are identical to that of Fasciola hepatica. ATG and GTG are used as the start-codons and TAG and TAA are used as the stop-codons. The stop-codon TAG of the C. sinensis nad1 gene overlap by 1nt with the downstream tRNA-Asn gene. Alternative structures for the Ser(UCN) tRNAs were found for both species. The noncoding control regions are separated into two parts by the tRNA-Gly gene and contain neither tandem repeats, which are characteristic for trematode control regions, nor secondary structures. In conclusion, the complete mitochondrial DNA sequences of O. felineus and C. sinensis will serve as a resource for comparative mitochondrial genomics and systematic studies of parasitic trematodes.

A novel nuclear marker, Pm-int9, for phylogenetic studies of Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis (Opisthorchiidae, Trematoda).

Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, the trematodes of the family Opisthorchiidae, are important human parasites. Two previous studies (Kang et al. Parasitol Int 57:191-197, 2008; Katokhin et al. Dokl Biochem Biophys 421:214-217, 2008) have provided evidence using ribosomal and mitochondrial sequences that O. viverrini, O. felineus, and C. sinensis are closely related. We developed a novel nuclear marker, Pm-int9, which included the ninth intron of the paramyosin gene and flanking exon sequences. Samples of O. felineus from four localities of West Siberia, C. sinensis from the Russian Far East, and O. viverrini from Thailand were genotyped by Pm-int9. Little variation was detected in exon sequences, however, intron sequences turned out to be more variable than ribosomal internal transcribed spacers. We can conclude that Pm-int9 is valuable for interspecific variation studies. Phylogenetic analysis based on Pm-int9 revealed that O. viverrini and C. sinensis were closer to each other than either of them to O. felineus, supporting the opinion that C. sinensis should be considered the sister species of Opisthorchis spp.