Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau.

BACKGROUND: Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. RESULTS: Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. CONCLUSIONS: Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Effects of warming at embryonic and larval stages on tadpole fitness in high-altitude Rana kukunoris.

Global warming may affect the early developmental stages of high-altitude amphibians, thereby influencing their later fitness. Yet, this has been largely unexplored. To investigate whether and how the temperatures experienced by embryonic and larval stages affect their fitness at later developmental stages, we designed two experiments in which the embryos and larvae were treated with three temperatures (24, 18 and 12 °C), respectively. Then, the life history traits of the tadpoles during the metamorphotic climax in all treatments were evaluated, including growth rate, survival rate, morphology, thermal physiology, swimming performance, standard metabolic rate (SMR), oxidative and antioxidative system, and metabolic enzyme activities. The results revealed that elevated temperature accelerated metamorphosis but decreased body size at metamorphosis. Additionally, warming during the embryonic and larval stages decreased the thermal tolerance range and induced increased oxidative stress. Furthermore, high embryonic temperature significantly decreased the hatching success, but had no significant effect on swimming performance and SMR. Warming during larval periods was harmful to the survival and swimming performance of tadpoles. The effect size analysis revealed that the negative impacts of embryonic temperature on certain physiological traits, such as growth and development, survival and swimming performance, were more pronounced than those of larval temperature. Our results highlight the necessity for particular attention to be paid to the early stages of amphibians, notably the embryonic stages when evaluating the impact of global warming on their survival.

Potential Effects of High Temperature and Heat Wave on Nanorana pleskei Based on Transcriptomic Analysis.

In the context of climate change, understanding how indigenous amphibians of the Qinghai-Tibet plateau react to stresses and their coping mechanisms could be crucial for predicting their fate and successful conservation. A liver transcriptome for Nanorana pleskei was constructed using high-throughput RNA sequencing, and its gene expression was compared with frogs acclimated under either room temperature or high temperature and also heat wave exposed ones. A total of 126,465 unigenes were produced, with 66,924 (52.92%) of them being annotated. Up to 694 genes were found to be differently regulated as a result of abnormal temperature acclimatization. Notably, genes belonging to the heat shock protein (HSP) family were down-regulated in both treated groups. Long-term exposure to high-temperature stress may impair the metabolic rate of the frog and trigger the body to maintain a hypometabolic state in an effort to survive challenging times. During heat waves, unlike the high-temperature group, mitochondrial function was not impaired, and the energy supply was largely normal to support the highly energy-consuming metabolic processes. Genes were more transcriptionally suppressed when treated with high temperatures than heat waves, and the body stayed in low-energy states for combating these long-term adverse environments to survive. It might be strategic to preserve initiation to executive protein activity under heat wave stress. Under both stress conditions, compromising the protection of HSP and sluggish steroid activity occurred in frogs. Frogs were more affected by high temperatures than by heat waves.

Oxygenation properties and underlying molecular mechanisms of hemoglobins in plateau zokor (Eospalax baileyi).

The plateau zokor (Eospalax baileyi) is a species of subterranean rodent endemic to the Tibetan Plateau. It is well adapted to the cold and hypoxic and hypercapnic burrow. To study the oxygenation properties of plateau zokor hemoglobins (Hbs), we measured intrinsic Hb-O2 affinities and their sensitivities to pH (Bohr effect); CO2; Cl-, 2,3-diphosphoglycerate (DPG); and temperature using purified Hbs from zokor and mouse. The optimal deoxyHb model of plateau zokor was constructed and used to study its structural characteristics by molecular dynamics simulations. O2 binding results revealed that plateau zokor Hbs exhibit remarkably high intrinsic Hb-O2 affinity, low CO2 effects compared with human and the relatively low anion allosteric effector sensitivities (DPG and Cl-) at normal temperature, which would safeguard the pulmonary Hb-O2 loading under hypoxic and hypercapnic conditions. Furthermore, the high anion allosteric effector sensitivities at low temperature and low temperature sensitivities of plateau zokor Hbs would facilitate the releasing of O2 in cold extremities and metabolic tissues. However, the high Hb-O2 affinity of plateau zokor is not compensated by high pH sensitivity as the Bohr factors of plateau zokor Hbs were as low as those of mouse. The results of molecular dynamics simulations revealed the reduced hydrogen bonding between the α1ß1- and α2ß2-dimer interface of deoxyHb in zokor compared with mouse. It may be the primary mechanism of the high intrinsic Hb-O2 affinities in zokor. Specifically, substitution of the 131Ser→Asn in the α2-chain weakened the connection between α1- and ß2-subunit.

Convergent High O2 Affinity but Distinct ATP-Mediated Allosteric Regulation of Hemoglobins in Oviparous and Viviparous Eremias Lizards from the Qinghai-Tibet Plateau.

The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl- sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of ß2/ß1 globin and substitutions on the ß2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles.

Integrative proteome and metabolome analyses reveal molecular basis of the tail resorption during the metamorphic climax of Nanorana pleskei.

During the metamorphosis of anuran amphibians, the tail resorption process is a necessary and crucial change. One subject that has received relatively little or no attention is the expression patterns of proteins and metabolites in the different tail portions during metamorphosis, especially in highland amphibians. The mechanisms of tail resorption in three portions (the tip, middle and root) of the tail were investigated in N. pleskei G43 tadpole based on two omics (proteomic and metabolomic). Integrin αVß3 was found to be high expressed in the distal portion of the tail, which could improve the sensitiveness to thyroid hormones in the distal portion of the tail. Muscle regression displayed a spatial pattern with stronger regression in distal and weaker one in proximal portion. Probably, this stronger regression was mainly performed by the proteases of proteasome from the active translation by ribosomes. The suicide model and murder model coexisted in the tail resorption. Meanwhile, fatty acids, amino acids, pyrimidine, and purine which derived from the breakdown of tissues can be used as building blocks or energy source for successful metamorphosis. Our data improved a better comprehension of the tail resorption mechanisms underlying the metamorphism of N. pleskei tadpole through identifying important participating proteins and metabolites.

Does a Moderately Warming Climate Compensate for the Negative Effects of UV-B Radiation on Amphibians at High Altitudes? A Test of Rana kukunoris Living on the Qinghai-Tibetan Plateau.

Both the warming climate and ultraviolet-B radiation (UVBR) are considered to be notable environmental factors affecting amphibian population decline, with particular effects on tadpole development. However, the phenotypes of tadpoles living at high altitudes may be improved by moderately warming temperatures, reducing or eliminating the negative effects of oxidative damage caused by cool temperatures or strong UVBR at high altitudes. To verify this hypothesis, Rana kukunoris tadpoles, which live at high altitudes, were used to test the effect of the interaction of temperature and UVBR on their development and antioxidant systems in a fully factorial design. The tadpoles were exposed to UVBR and UVBR-free environments at 14 (cool temperature) and 22 °C (warm temperature), respectively. UVBR and a warm temperature had opposite influences on several traits of the tadpoles, including their survival, developmental rate, individual size, preferred body temperature, thermal tolerance temperature, oxidative damage, and enzymatic and nonenzymatic antioxidant systems. The moderate temperature could compensate for or override the negative effects of UVBR by increasing the tadpoles' preferred body temperature and critical tolerance temperature, thus enhancing the locomotion ability and thermal sensitivity of their antioxidant systems. Furthermore, the dark skin coloration and aggregation behavior of R. kukunoris tadpoles may also be effective strategies for allowing them to resist UVBR and helping them to better adapt to a warming environment with stronger UVBR. Thus, it is possible that a moderate degree of warming may increase the capacity of living organisms to adapt to environmental changes and thus have positive effects on the development of tadpoles living at high altitudes.

Effects of temperature on the locomotor performance and contraction properties of skeletal muscle from two Phrynocephalus lizards at high and low altitude.

Locomotor performance and skeletal muscle contraction are critical for animals and are susceptible to changes in the external thermal environment, especially for ectotherms. Phrynocephalus erythrurus, which is endemic to the Qinghai-Tibetan plateau, is known for living at the highest elevation among all reptiles in the world (4500-5300 m). In this study, which compares P. erythrurus with the lowland Phrynocephalus przewalskii, we evaluated the locomotor performance at different body temperatures, the effects of temperature and oxygen partial pressure (PO2) on the contractile properties of iliofibularis (IF) muscle in vitro, ATPase activity of IF muscle at different temperatures, and the fiber types of IF muscle. Lowland P. przewalskii runs significantly faster than highland P. erythrurus at all test body temperatures. Almost all contractile properties of the IF muscle of P. przewalskii were better than that of P. erythrurus under all test temperatures and PO2. However, P. erythrurus could achieve both optimal isometric (e.g., dPo/dt) and optimal isotonic (e.g., Vmax) contraction at a lower temperature compared with P. przewalskii. Multi-factor analysis further revealed that temperature has a significant effect on the contractile properties of IF muscle for both species. Although the proportion of fibers types and ATPase activities of IF muscle have no significant interspecies difference, the changing pattern of ATPase activities with temperature is consistent with certain contractile properties and locomotor performance. The interspecies differences in locomotor ability and contractile properties of skeletal muscle in high- and low-altitude lizards may be the results of long-term adaptation to the local environment.

Comparison of hematological traits and oxygenation properties of hemoglobins from highland and lowland Asiatic toad (Bufo gargarizans).

The Asiatic toad (Bufo gargarizans) belonging to the family of Bufonidae (Anura: Amphibia) is successfully residing on the Qinghai-Tibetan Plateau (QTP). To investigate whether the oxygen delivery undergoes adaptive adjustments to high-altitude environments in Asian toads inhabiting the QTP (Zoige County, 3446 m), choosing low-altitude populations (Chengdu City, 500 m) as control, we measured hematological traits, O2 affinities of whole blood, Hb-O2 affinities of purified Hbs, their sensitivities to temperature, and allosteric effectors (H+, Cl- and ATP). Our results showed that high-altitude Asiatic toads possessed significantly increased hemoglobin concentration, hematocrit, and red blood cell count, but significantly decreased erythrocyte volume compared with low-altitude toads. The whole blood and purified Hbs of high-altitude Asiatic toads both exhibited significantly higher O2 affinities compared with low-altitude toads. Substantially increased intrinsic Hb-O2 affinities of high-altitude Asiatic toads Hbs are likely to be the main reason for its elevated Hb-O2 affinities given the anionic cofactor sensitivities of high- and low-altitude toads were similar. The Hbs of high-altitude toads were also characterized by distinctly strong Bohr effects at the low temperature and low-temperature sensitivities. The adaptive adjustments of hematological traits could enhance the blood-O2 carrying capacity of high-altitude Asiatic toads. The increased Hb-O2 affinities could safeguard the pulmonary O2 uploading under hypoxia. The strong Bohr effects at the low temperature could help the release of O2 in metabolic tissues and cold limbs, while low-temperature sensitivity could minimize the effect of temperature fluctuation on the Hb-O2 affinity.

Suppressed LPS-mediated TLR4 signaling in the plateau zokor (Eospalax baileyi) compared to the bamboo rat (Rhizomys pruinosus) and rat (Rattus norvegicus).

Ecological immunology involves the study of the immune function of wildlife, which is seldom compared with that of model animals. Here, we evaluated and compared the level of the innate immune response in the plateau zokor (Eospalax baileyi), an indigenous underground rodent from the Tibetan Plateau, with that in the bamboo rat (Rhizomys pruinosus) and Sprague-Dawley (SD) rat (Rattus norvegicus). The spleen was observed by ordinary light and transmission electron microscopy, and the spleen index was calculated. After liposaccharide (LPS) challenge, the expression of Toll-like receptor 2 (TLR2), TLR4, and hypoxia-inducible factor 1α (HIF-1α) in the spleen was detected by Western blot analysis and immunofluorescence. The expression of nuclear factor-κB1 (NF-κB1) and mitogen-activated protein kinase 14 (MAPK14) in the spleen was detected by real-time quantitative polymerase chain reaction, and the levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-ß (IFN-ß) in the spleen were detected by enzyme-linked immunoassay. The spleen index of the plateau zokor was lower than that of the bamboo rat and SD rat. The expression of TLR4, NF-κB1, and MAPK14 and the levels of IL-6 and TNF-α in the spleen of the plateau zokor were lower than those of the bamboo rat and SD rat, while the expression of TLR2 and HIF-1α and the level of IFN-ß were higher than those of the bamboo rat and SD rat. We speculate that suppression of the TLR4 signaling pathway in the plateau zokor is an adaptation to hypoxic tunnels that decreases antigenic risk and maintains immune homeostasis. Moreover, the spleen of the plateau zokor is reduced in size, reducing the innate immunity investment in the spleen. We also noted that high levels of HIF-1α in the spleen of the plateau zokor suppressed crosstalk between HIF-1α and TLR4, promoting the innate immune response.