Cryoprotection by urea in a terrestrially hibernating frog

Abstract

SUMMARY The role of urea as a balancing osmolyte in osmotic adaptation is well known, but this `waste product' also has myriad other functions in diverse taxa. We report that urea plays an important, previously undocumented role in freezing tolerance of the wood frog (Rana sylvatica), a northern woodland species that hibernates terrestrially in sites where dehydration and freezing may occur. Wood frogs inhabiting an outdoor enclosure accumulated urea to 65 mmol l-1 in autumn and early winter, when soil moisture was scarce, but subsequently urea levels fell to ∼2 mmol l-1 as the availability of environmental water increased. Laboratory experiments showed that hibernating R. sylvatica can accumulate at least 90 mmol l-1 urea under relatively dry, warm conditions. During experimental freezing, frogs synthesized glucose but did not accumulate additional urea. Nevertheless, the concentrations of urea and glucose in some tissues were similar. We tested urea's efficacy as a cryoprotectant by measuring lysis and lactate dehydrogenase (LDH) leakage in samples of R. sylvaticaerythrocytes frozen/thawed in the presence of physiological levels of urea or other osmolytes. In conferring protection against freeze/thaw damage, urea was comparable to glycerol and as good as or better than glucose, cryoprotectants found in freeze-tolerant frogs and other animals. Urea treatment also improved the viability of intact tissues frozen in vitro, as demonstrated by post-thaw measures of metabolic activity and LDH leakage. Collectively, our findings suggest that urea functions both as an osmoprotectant and a cryoprotectant in terrestrially hibernating amphibians.

Metabolic changes during hibernating period in Indian common toad, Duttaphrynus melanostictus (Schneider, 1799)

“…In those month they would probably were making them metabolically competent for the coming harsh winter. Our present findings therefore strongly indicate link between elevated urea level and cryoprotection as indicated by Costanzo and Lee (2005). It may be concluded that in the common Indian toad decline in thyroid hormone concentration has its behavioral and physiological response that leads to sustain increased cholesterol level in serum, which seems to be the most important metabolite during the hibernating phase.…”

“…Acclimatization to low temperature causes changes on their system to maintain catalytic potential or to alter the relative activities of different metabolic pathways at low temperature (Hochachka and Somero, 1984). Available reports indicate that environmental cold stress in hibernating toads causes significant changes in plasmaprotein and glucose (Churchill and Storey, 1993;Edwards et al, 2004;Costanzo and Lee, 2005), blood urea ( Jorgenensen, 1997;Costanzo and Lee, 2005 ). Effect of cold stress can also produce remarkable changes in blood urea concentration (Jorgenensen, 1997, Costanzo andLee, 2005,) which probably help the animal to cope with low temperature and act as a cryoprotectent agent.…”

“…Available reports indicate that environmental cold stress in hibernating toads causes significant changes in plasmaprotein and glucose (Churchill and Storey, 1993;Edwards et al, 2004;Costanzo and Lee, 2005), blood urea ( Jorgenensen, 1997;Costanzo and Lee, 2005 ). Effect of cold stress can also produce remarkable changes in blood urea concentration (Jorgenensen, 1997, Costanzo andLee, 2005,) which probably help the animal to cope with low temperature and act as a cryoprotectent agent. At the time of the hibernation animals such as anurans enter to a state of hypometabolism when the metabolic rate is suddenly reduced.…”

Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog

“…Among amphibians, this response is best exemplified by members of the gray treefrog complex (genus Hyla), which potentially can accumulate glycerol to concentrations of 50-80μmolml −1 blood during cold acclimation (Layne and Stapleton, 2009;Zimmerman et al, 2007). Rana sylvatica (and perhaps other species) can accrue urea to comparable levels during autumn and early winter, coincident with seasonal declines in ambient temperature and water potential (Costanzo and Lee, 2005). In the present study this species amassed urea even in the absence of osmotic stress, a potent driver of urea accumulation in amphibians (Jørgensen, 1997).…”

Regulation of theRana sylvaticabrevinin-1SY antimicrobial peptide during development and in dorsal and ventral skin in response to freezing, anoxia, and dehydration

“…In wood frogs, dehydration alone provokes a hyperglycemia response, comparable to the build-up of cryoprotectants during freezing (Churchill and Storey, 1993;Churchill and Storey, 1994a). Similarly, the build-up of urea to protect against colligative water loss is thought to be important in both dehydration and freezing (Costanzo and Lee, 2005;Muir et al, 2007). Parallel molecular responses have also been observed between wood frog dehydration and freezing, including reduced metabolism, activated liver glycogen phosphorylase and elevated PKAc and liver second messenger cAMP levels (Pinder et al, 1992;Churchill and Storey, 1994a;Holden and Storey, 1997).…”