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The Pallas' spadefoot Pelobates vespertinus is a frog species native to eastern Europe and west Siberia. This species resists harsh winter conditions by moving up to 2 m underground. This amphibian is the first species known to withstand extreme air hypoxia. In this study, we investigated the metabolome of liver, heart, and brain of the Pallas' spadefoot after a month-long exposure of hypoxia, with oxygen levels reduced to approximately one-tenth of the air normal content. Surprisingly, our findings revealed a limited impact of hypoxia on the metabolomic profiles. Concentrations of glycolysis end products (lactate and alanine) increased only slightly compared to other amphibians under hypoxia, and no accumulation of succinate was observed. Furthermore, there were no notable changes in the content of adenosine phosphates. These results are consistent with a previous study, which indicated that the Pallas' spadefoot possesses relatively small glycogen and fat reserves before the winter compared to other frogs. It appears that this species conserves energy during winter by minimizing its metabolic activity. These findings corroborated the hypothesis that the survival of P. vespertinus under hypoxic conditions primarily relies on metabolic suppression rather than substantial energy reserves., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

The level of diversity and abundance of darkling beetles (Coleoptera, Tenebrionidae) is the main difference between the late Pleistocene and modern insect faunas of arid regions. In the Pleistocene assemblages they are extremely rare, whereas in the modern ones they predominate. It is assumed that the reason for their rarity in fossil entomological complexes is their lack of cold resistance. The supercooling points ( SCP ) and low lethal temperatures ( LLT ) of adults from five species of Altai darkling beetles that overwinter in the soil and larvae from one such species were measured in the laboratory. All beetles supercooled at negative temperatures but could not survive freezing, with the average SCP of the most cold-resistant species between -25.7 and -21.7 °C ( Bioramix picipes , Anatolica dashidorzsi , and Penthicus altaicus ). However, 50% of the individuals from different species in the experiment died after exposure during two days at temperatures ranging from -22 to -20 °C. The focal species are distributed in parts of Central Asia with an extreme continental climate, and the temperatures measured in the soil of these natural areas turned out to be lower than or close to the limit of cold resistance of the beetles. Overwintering of darkling beetles is therefore only possible in areas with deep snow: in hollows, under bushes, and under large cereals. Darkling beetles with poor cold resistance could not have existed in the colder climate of the late Pleistocene, which explains their absence from fossil fauna.

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.

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 1 H 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (Copyright © AUTHORS.)

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 1 H-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, β-alanine, and β-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.

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.

Background: Pelobates fuscus is an anuran amphibian that overwinters on land deep in soil, including regions with low sub-zero temperatures. It is not yet known if such behaviour can be explained by low cold-hardiness of the species., Objective: To measure cold-hardiness of P. fuscus and compare the results with data about its location in winter in nature., Methods: Lower lethal temperatures and supercooling points (SCP) were measured in specimens collected near the Saratov city in south-eastern European Russia., Results: SCP of P. fuscus range from -1.7 to -2.7°C. All individuals withstood exposure of 1°С for 20 days; but freezing is lethal to animals: one individual out of five withstood exposure of -1°С for 3 days, and -2°С is lethal to all animals. The condition of supercooling is unstable., Conclusion: The lower tolerable temperature is probably about 0°С. Low cold hardiness determines overwintering deep in substrates, which allowed the species to disperse over a large geographical range, which also includes cold regions with little snow in winter.

Few of the amphibian species that occur in the Subarctic and in mountains are adapted to low sub-zero temperatures; most of these species overwinter underwater. It is believed that the distribution of the species that overwinter underwater can be limited by the low oxygen levels in waterbodies covered with ice. We show that the colonisation of the coldest areas of Northern Asia (to 71°N) by the Siberian wood frog (Rana amurensis) was facilitated by a unique adaptation, the ability to survive extreme hypoxia - and probably anoxia - in waterbodies during overwintering. The oxygen content in the overwintering waterbodies that we have studied in different parts of the range of this species fell to 0.2-0.7 mg/L without causing any large-scale mortality among the frogs. In laboratory experiments the R. amurensis survived for up to 97 days in hermetically sealed containers with water that contained less than 0.2 mg/L oxygen at temperatures of 2-3 °C, retaining the ability to respond to external stimuli. An earlier study of a broad range of frog species has shown that very few of them can survive even brief (up to 5-7 days) exposure to oxygen-free water. The revealed adaptation to prolonged extreme hypoxia is the first known case of this kind among amphibians overwintering in water.

The Japanese tree frog, a representative of the Manchurian fauna, is characterized by an outstanding cold resistance among the anuran amphibian species studied so far. Almost 70% of the specimens from the population inhabiting the middle Amur River withstand the cooling down to-30°C; some animals, down to-35°C. This exceeds more than twofold the cold hardiness of the wood frog (Lithobates sylvaticus LeConte, 1825), which has been considered earlier to be the most cold-resistant species. The ability of H. japonica to survive for four months in the frozen state at low temperatures makes this species independent of the temperature overwintering conditions.

Frozen Siberian salamander safely tolerates long (45 days) stay at-35°C. Short-term (3 days) cooling down to-50°C was tolerable for 40% of adult individuals; down to-55°C, for 80% of the underyearlings. Generally, the salamanders lose about 28% of the body mass during the pre-hibernating period (before winter, at temperatures as low as 0°C) and during the process of freezing (as low as-5°C). The body weight remained constant upon further cooling (to-35°C). The frozen salamanders have no physiological mechanisms protecting from sublimation.

Background: Angarozonium amurense (Gerstfeldt, 1859) is the only one out of more than a hundred diplopod species described in Siberia and the Far East that inhabits regions with solid permafrost., Objective: To evaluate the cold hardiness of A. amurense that allows this species to inhabit permafrost regions., Methods: The survival temperature thresholds and supercooling points (SCP) were measured., Results: The temperature thresholds for adult animal survival are -8.5 C in summer and -27 C in winter. Average SCP decreases from -7.7 in summer to -16.9 in winter. Water content decreases from 55.7% in summer to 49.4% in winter., Conclusion: The cold hardiness of A. amurense sets the record in this class of animals. It allows it to overwinter in the upper 15 centimeters layer of soil in most biotopes of the coldest permafrost regions in North Asia.