Brief chilling to subzero temperature increases cold hardiness in the hatchling painted turtle (Chrysemys picta)

Although many studies of ectothermic vertebrates have documented compensatory changes in cold hardiness associated with changes of season, much less attention has been paid to adjustment of physiological functions and survival limits following more acute exposure to cold. We investigated the ability of hatchling painted turtles (Chrysemys picta) to increase cold hardiness in response to brief exposure to a subzero temperature. Winter‐acclimated turtles were “cold conditioned” by chilling them in the supercooled (unfrozen) state to −7°C over a few days before returning them to 4°C. These turtles fared no better than control animals in resisting freezing when cooled in the presence or absence of ice and exogenous ice nuclei. Survival following tests of freeze tolerance (freezing for about 70 h; minimum body temperature, −3.75°C) was nominally higher in cold‐conditioned turtles than in controls (36% vs. 13%, respectively), although the difference was not statistically significant. Of the survivors, cold‐conditioned turtles apparently recovered sooner. Turtles subjected to cold shock (supercooling to −13°C for 24 h, followed by rewarming to 0°C) were strongly affected by cold conditioning: all controls died, but 50% of cold‐conditioned turtles survived. We investigated potential mechanisms underlying the response to cold conditioning by measuring changes in levels of putative cryoprotectants. Plasma levels of glucose and lactate, but not urea, were higher in cold‐conditioned turtles than in controls, although the combined increase in these solutes was only 23 mmol L−1. Cold conditioning attenuated cold‐shock injury to brain cells, as assessed using a vital‐dye assay, suggesting a link between protection of the nervous system and cold hardiness at the organismal level

Identification and expression of a putative facilitative urea transporter in three species of true frogs (Ranidae): implications for terrestrial adaptation.

Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrial R. sylvatica, intermediate in the semiaquatic R. pipiens, and quite low in the aquatic R. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments

Seasonal variation in the hepatoproteome of the dehydration- and freeze-tolerant wood frog, Rana sylvatica

Winter’s advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation

Seasonality of Freeze Tolerance in a Subarctic Population of the Wood Frog, Rana sylvatica

We compared physiological characteristics and responses to experimental freezing and thawing in winter and spring samples of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA. Whereas winter frogs can survive freezing at temperatures at least as low as −16°C, the lower limit of tolerance for spring frogs was between −2.5°C and −5°C. Spring frogs had comparatively low levels of the urea in blood plasma, liver, heart, brain, and skeletal muscle, as well as a smaller hepatic reserve of glycogen, which is converted to glucose after freezing begins. Consequently, following freezing (−2.5°C, 48 h) tissue concentrations of these cryoprotective osmolytes were 44–88% lower than those measured in winter frogs. Spring frogs formed much more ice and incurred extensive cryohemolysis and lactate accrual, indicating that they had suffered marked cell damage and hypoxic stress during freezing. Multiple, interactive stresses, in addition to diminished cryoprotectant levels, contribute to the reduced capacity for freeze tolerance in posthibernal frogs

Seasonal Variation in the Hepatoproteome of the Dehydrationand Freeze-Tolerant Wood Frog, Rana sylvatica

Winter’s advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation

Seasonality of Freeze Tolerance in a Subarctic Population of the Wood Frog, Rana sylvatica

We compared physiological characteristics and responses to experimental freezing and thawing in winter and spring samples of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA. Whereas winter frogs can survive freezing at temperatures at least as low as −16°C, the lower limit of tolerance for spring frogs was between −2.5°C and −5°C. Spring frogs had comparatively low levels of the urea in blood plasma, liver, heart, brain, and skeletal muscle, as well as a smaller hepatic reserve of glycogen, which is converted to glucose after freezing begins. Consequently, following freezing (−2.5°C, 48 h) tissue concentrations of these cryoprotective osmolytes were 44–88% lower than those measured in winter frogs. Spring frogs formed much more ice and incurred extensive cryohemolysis and lactate accrual, indicating that they had suffered marked cell damage and hypoxic stress during freezing. Multiple, interactive stresses, in addition to diminished cryoprotectant levels, contribute to the reduced capacity for freeze tolerance in posthibernal frogs

High energy Coulomb-scattered electrons for relativistic particle beam diagnostics

A new system used for monitoring energetic Coulomb-scattered electrons as the main diagnostic for accurately aligning the electron and ion beams in the new Relativistic Heavy Ion Collider (RHIC) electron lenses is described in detail. The theory of electron scattering from relativistic ions is developed and applied to the design and implementation of the system used to achieve and maintain the alignment. Commissioning with gold and 3He beams is then described as well as the successful utilization of the new system during the 2015 RHIC polarized proton run. Systematic errors of the new method are then estimated. Finally, some possible future applications of Coulomb-scattered electrons for beam diagnostics are briefly discussed.Comment: 16 pages, 23 figure

Cryoprotectants and extreme freeze tolerance in a subarctic population of the wood frog.

Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica