Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.