Freezing tolerance/intolerance and cryoprotectant synthesis in terrestrially overwintering anurans in the Great...

Mechanistic bases for freezing tolerance in anurans have been well-studied only in wood frogs, Rana sylvatica, so comprehensive explanations for the mechanisms and evolution of freezing tolerance in anurans are lacking. We measured crystallization temperatures, freezing tolerance/intolerance, and tissue glucose and glycogen phosphorylase activities in frozen and unfrozen winter-acclimated Pseudacris triseriata, Bufo cognatus and B. woodhousei. Freezing occurred at higher subzero temperatures on wet substrate than on dry substrate in all species, indicating susceptibility to inoculative freezing. P. triseriata was freeze-tolerant, but survival was dependent on the level of supercooling prior to freezing. All Bufo were freezing intolerant, regardless of crystallization temperature. Glucose was significantly elevated by freezing in both liver (35-fold) and leg muscle (22-fold) in winter P. triseriata, but only liver glucose was significantly elevated in B. cognatus. However, freezing did not alter glycogen phosphorylase activity in either species. Liver phosphorylase activity was significantly higher in P. triseriata than in B. cognatus, suggesting that capacity for mobilizing glucose from liver glycogen is associated with freezing tolerance. Summer measurements of liver phosphorylase activity, however, did not differ between species. Thus, P. triseriata, but not B. cognatus, exhibited winter increment of liver phosphorylase activity that is correlated with the development of freezing tolerance.