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. 1. Introduction Among temperate ectotherms, cold hardiness in its various forms is most strongly expressed during the winter months, coincident with the greatest need for protection from severe cold. Although seasonal variation in this trait is often pronounced, its physiological basis remains incompletely understood. Recent studies, particularly those using various “-omics” approaches [1], attest that the underpinnings are complex and involve a host of adaptations at multiple levels of biological organization. Elucidation of these mechanisms will require comprehensive study of organisms for which the fundamental adaptations of freeze tolerance are reasonably well known. The relatively robust freeze tolerance exhibited by certain woodland frogs is associated with their ability to accrue high concentrations of the cryoprotectants, glucose, and/or glycerol, which during freezing are mobilized from glycogen in the liver. These compounds limit freezing injury by colligatively lowering the equilibrium freezing/melting point ( ) of body fluids and, hence, reducing ice formation, and also by preserving the integrity of membranes and macromolecules, among other things [2, 3]. Because the hepatic glycogen store is substantially reduced following hibernation and mating, spring frogs can accrue only modest amounts of these agents, and this difference purportedly is the cause of their reduced freeze tolerance [4–8]. It was recently reported [9] that some freeze-tolerant frogs also use urea as a cryoprotectant, but
References
[1]
M. R. Michaud and D. L. Denlinger, “Genomics, proteomics and metabolomics: finding the other players in insect cold-tolerance,” in Low Temperature Biology of Insects, D. L. Denlinger and R. E. Lee, Eds., pp. 91–115, Cambridge University Press, New York, NY USA, 2010.
[2]
J. P. Costanzo and R. E. Lee, “Commentary: avoidance and tolerance of freezing in ectothermic vertebrates,” Journal of Experimental Biology, vol. 216, no. 11, pp. 1961–1967, 2013.
[3]
K. B. Storey and J. M. Storey, “Freeze tolerance in animals,” Physiological Reviews, vol. 68, no. 1, pp. 27–84, 1988.
[4]
K. B. Storey and J. M. Storey, “Persistence of freeze tolerance in terrestrially hibernating frogs after spring emergence,” Copeia, vol. 1987, no. 3, pp. 720–726, 1987.
[5]
T. A. Churchill and K. B. Storey, “Dehydration tolerance in wood frogs: a new perspective on development of amphibian freeze tolerance,” American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 265, no. 6, pp. R1324–R1332, 1993.
[6]
J. R. Layne, R. E. Lee, and M. M. Cutwa, “Post-hibernation excretion of glucose in urine of the freeze tolerant frog Rana sylvatica,” Journal of Herpetology, vol. 30, no. 1, pp. 85–87, 1996.
[7]
J. R. Layne, “Seasonal variation in the cryobiology of Rana sylvatica from Pennsylvania,” Journal of Thermal Biology, vol. 20, no. 4, pp. 349–353, 1995.
[8]
J. L. Jenkins and D. L. Swanson, “Liver glycogen, glucose mobilization and freezing survival in chorus frogs, Pseudacris triseriata,” Journal of Thermal Biology, vol. 30, no. 6, pp. 485–494, 2005.
[9]
J. P. Costanzo and R. E. Lee, “Cryoprotection by urea in a terrestrially hibernating frog,” Journal of Experimental Biology, vol. 208, no. 21, pp. 4079–4089, 2005.
[10]
J. P. Costanzo, M. C. do Amaral, A. J. Rosendale, and R. E. Lee, “Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of wood frog,” Journal of Experimental Biology, vol. 216, no. 18, pp. 3461–3473, 2013.
[11]
D. L. MacArthur and J. W. T. Dandy, “Physiological aspects of overwintering in the boreal chorus frog (Pseudacris triseriata maculata),” Comparative Biochemistry and Physiology A: Physiology, vol. 72, no. 1, pp. 137–141, 1982.
[12]
T. J. Muir, J. P. Costanzo, and R. E. Lee, “Metabolic depression induced by urea in organs of the wood frog, Rana sylvatica: effects of season and temperature,” Journal of Experimental Zoology A: Ecological Genetics and Physiology, vol. 309, no. 2, pp. 111–116, 2008.
[13]
M. P. Kirton, Fall movements and hibernation of the wood frog, Rana sylvatica, in interior Alaska [M.S. thesis], University of Alaska, Fairbanks, Alaska, USA, 1974.
[14]
B. Waldman, “Adaptive significance of communal oviposition in wood frogs (Rana sylvatica),” Behavioral Ecology and Sociobiology, vol. 10, no. 3, pp. 169–174, 1982.
[15]
R. D. Howard, “Mating behaviour and mating success in woodfrogs Rana sylvatica,” Animal Behaviour, vol. 28, no. 3, pp. 705–716, 1980.
[16]
N. J. Hudson and C. E. Franklin, “Maintaining muscle mass during extended disuse: aestivating frogs as a model species,” Journal of Experimental Biology, vol. 205, no. 15, pp. 2297–2303, 2002.
[17]
B. J. Sinclair, J. R. Stinziano, C. M. Williams, H. A. MacMillan, K. E. Marshall, and K. B. Storey, “Real-time measurement of metabolic rate during freezing and thawing of the wood frog, Rana sylvatica: implications for overwinter energy use,” Journal of Experimental Biology, vol. 216, no. 2, pp. 292–302, 2013.
[18]
E. S. Farrar and R. K. Dupre, “The role of diet in glycogen storage by juvenile bullfrogs prior to overwintering,” Comparative Biochemistry and Physiology A: Physiology, vol. 75, no. 2, pp. 255–260, 1983.
[19]
P. Koskela and S. Pasanen, “Effect of thermal acclimation on seasonal liver and muscle glycogen content in the common frog, Rana temporaria L,” Comparative Biochemistry and Physiology A, vol. 50, no. 4, pp. 723–727, 1975.
[20]
N. S. Loumbourdis and P. Kyriakopoulou-Sklavounou, “Reproductive and lipid cycles in the male frog Rana ridibunda in Northern Greece,” Comparative Biochemistry and Physiology A: Physiology, vol. 99, no. 4, pp. 577–583, 1991.
[21]
M. L. Morton, “Seasonal changes in total body lipid and liver weight in the Yosemite toad,” Copeia, vol. 1981, no. 1, pp. 234–238, 1981.
[22]
K. D. Wells and C. R. Bevier, “Contrasting patterns of energy substrate use in two species of frogs that breed in cold weather,” Herpetologica, vol. 53, no. 1, pp. 70–80, 1997.
[23]
S. C. Dinsmore II and D. L. Swanson, “Temporal patterns of tissue glycogen, glucose, and glycogen phosphorylase activity prior to hibernation in freeze-tolerant chorus frogs, Pseudacris triseriata,” Canadian Journal of Zoology, vol. 86, no. 10, pp. 1095–1100, 2008.
[24]
A. W. Pinder, K. B. Storey, and G. R. Ultsch, “Estivation and hibernation,” in Environmental Physiology of the Amphibians, M. E. Feder and W. W. Burggren, Eds., pp. 250–274, The University of Chicago Press, Chicago, Ill, USA, 1992.
[25]
K. B. Storey, “Freeze tolerance in the frog, Rana sylvatica,” Experientia, vol. 40, no. 11, pp. 1261–1262, 1984.
[26]
J. P. Costanzo, M. C. do Amaral, A. R. Rosendale, and R. E. Lee, “Seasonal dynamics and influence of hibernaculum temperature on energy reserves in the wood frog, Rana sylvatica,” meeting abstract in Society of Integrative and Comparative Biology, Charleston, SC, USA, 2012.
[27]
J. R. Layne and R. E. Lee, “Seasonal variation in freeze tolerance and ice content of the tree frog Hyla versicolor,” Journal of Experimental Zoology, vol. 249, no. 2, pp. 133–137, 1989.
[28]
W. D. Schmid, “Survival of frogs in low temperature,” Science, vol. 215, no. 4533, pp. 697–698, 1982.
[29]
J. R. Layne, “Freeze tolerance and cryoprotectant mobilization in the gray treefrog (Hyla versicolor),” Journal of Experimental Zoology, vol. 283, no. 3, pp. 221–225, 1999.
[30]
J. P. Costanzo, R. E. Lee, and P. H. Lortz, “Glucose concentration regulates freeze tolerance in the wood frog Rana sylvatica,” Journal of Experimental Biology, vol. 181, pp. 245–255, 1993.
[31]
K. B. Storey, J. G. Baust, and P. Buescher, “Determination of water “bound” by soluble subcellular components during low-temperature acclimation in the gall fly larva, Eurosta solidagensis,” Cryobiology, vol. 18, no. 3, pp. 315–321, 1981.
[32]
J. R. Layne, J. P. Costanzo, and R. E. Lee, “Freeze duration influences postfreeze survival in the frog Rana sylvatica,” Journal of Experimental Zoology, vol. 280, no. 2, pp. 197–201, 1998.
[33]
P. A. King, M. N. Rosholt, and K. B. Storey, “Seasonal changes in plasma membrane glucose transporters enhance cryoprotectant distribution in the freeze-tolerant wood frog,” Canadian Journal of Zoology, vol. 73, no. 1, pp. 1–9, 1995.
[34]
J. P. Costanzo, J. T. Irwin, and R. E. Lee, “Freezing impairment of male reproductive behaviors of the freeze-tolerant wood frog, Rana sylvatica,” Physiological Zoology, vol. 70, no. 2, pp. 158–166, 1997.
[35]
E. L. Russell and K. B. Storey, “Glycogen synthetase and the control of cryoprotectant clearance after thawing in the freeze-tolerant wood frog,” Cryo-Letters, vol. 16, no. 5, pp. 263–266, 1995.
[36]
Y. Voituron, L. Paaschburg, M. Holmstrup, H. Barré, and H. Raml？v, “Survival and metabolism of Rana arvalis during freezing,” Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, vol. 179, no. 2, pp. 223–230, 2009.
[37]
J. R. Layne, “Postfreeze O2 consumption in the wood frog (Rana sylvatica),” Copeia, vol. 2000, no. 3, pp. 879–882, 2000.
[38]
J. P. Costanzo, P. A. Callahan, R. E. Lee, and M. F. Wright, “Frogs reabsorb glucose from urinary bladder,” Nature, vol. 389, no. 6649, pp. 343–344, 1997.
[39]
W. D. Schmid, “Natural variations in nitrogen excretion of amphibians from different habitats,” Ecology, vol. 49, no. 1, pp. 180–185, 1968.
[40]
S. I. Pisarenko, E. B. Minkovskii, and I. M. Studneva, “Urea synthesis in heart muscle,” Bulletin of Experimental Biology and Medicine, vol. 89, no. 2, pp. 138–141, 1980.
[41]
J. R. Layne and S. D. Kennedy, “Cellular energetics of frozen wood frogs (Rana sylvatica) revealed via NMR spectroscopy,” Journal of Thermal Biology, vol. 27, no. 3, pp. 167–173, 2002.
[42]
A. Chakraborty, M. Sarkar, and S. Basak, “Stabilizing effect of low concentrations of urea on reverse micelles,” Journal of Colloid and Interface Science, vol. 287, no. 1, pp. 312–317, 2005.
[43]
F. O. Costa-Balogh, H. Wennerstr？m, L. Wads？, and E. Sparr, “How small polar molecules protect membrane systems against osmotic stress: the urea-water-phospholipid system,” Journal of Physical Chemistry B, vol. 110, no. 47, pp. 23845–23852, 2006.
[44]
X. Wang, W. Lingyun, M. Aouffen, M. Mateescu, R. Nadeau, and R. Wang, “Novel cardiac protective effects of urea: from shark to rat,” British Journal of Pharmacology, vol. 128, no. 7, pp. 1477–1484, 1999.
[45]
A. Pollard and R. G. W. Jones, “Enzyme activities in concentrated solutions of glycinebetaine and other solutes,” Planta, vol. 144, no. 3, pp. 291–298, 1979.
[46]
S. D. Collins, A. L. Allenspach, and R. E. Lee, “Ultrastructural effects of lethal freezing on brain, muscle and malpighian tubules from freeze-tolerant larvae of the gall fly, Eurosta solidaginis,” Journal of Insect Physiology, vol. 43, no. 1, pp. 39–45, 1997.
[47]
J. P. Costanzo, A. L. Allenspach, and R. E. Lee, “Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica,” Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, vol. 169, no. 4-5, pp. 351–359, 1999.
[48]
M. Hermes-Lima and T. Zenteno-Savín, “Animal response to drastic changes in oxygen availability and physiological oxidative stress,” Comparative Biochemistry and Physiology C: Toxicology and Pharmacology, vol. 133, no. 4, pp. 537–556, 2002.
[49]
J. Christiansen and D. Penney, “Anaerobic glycolysis and lactic acid accumulation in cold submerged Rana pipiens,” Journal of Comparative Physiology, vol. 87, no. 3, pp. 237–245, 1973.
[50]
T. V. Bagnyukova, K. B. Storey, and V. I. Lushchak, “Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation,” Journal of Thermal Biology, vol. 28, no. 1, pp. 21–28, 2003.
[51]
A. J. Clark, R. Gaddie, and C. P. Stewart, “The anaerobic activity of the frog's heart,” The Journal of Physiology, vol. 75, no. 3, pp. 321–331, 1932.
[52]
D. G. Penney, “Frogs and turtles: different ectotherm overwintering strategies,” Comparative Biochemistry and Physiology A: Physiology, vol. 86, no. 4, pp. 609–615, 1987.
[53]
P. L. Rocha and L. G. S. Branco, “Seasonal changes in the cardiovascular, respiratory and metabolic responses to temperature and hypoxia in the bullfrog Rana catesbeiana,” Journal of Experimental Biology, vol. 201, no. 5, pp. 761–768, 1998.
[54]
T. A. G. Rittenhouse, R. D. Semlitsch, and F. R. Thompson, “Survival costs associated with wood frog breeding migrations: effects of timber harvest and drought,” Ecology, vol. 90, no. 6, pp. 1620–1630, 2009.
[55]
B. Kessel, “Breeding dates of Rana sylvatica at College, Alaska,” Ecology, vol. 46, no. 1-2, pp. 206–208, 1965.
