Novel observations of "freeze resistance" and dynamic blue and green dorsal coloration in frozen and thawing Dryophytes chrysoscelis.

Abstract

Freeze tolerant animals survive the winter by tolerating the freezing and thawing of up to 70% of body water and the respective cessation and resumption of essential functions including circulation and respiration during each freeze-thaw cycle. Cope's gray treefrog Dryophytes chrysoscelis is a freeze tolerant anuran that uses a system of cryoprotectants to prevent intracellular freezing and mitigate osmotic stress during freezing and thawing episodes. Morphological features were documented in D. chrysoscelis using a repeated freeze-thaw protocol. Dorsal skin in frozen frogs was distinctly blue and green before reverting to brown during thawing. The dorsal color change in frozen frogs does not function similarly to other known color change events in amphibians. The return to brown skin color in thawing animals coincides with recovery of vital functions in freeze tolerant frogs, suggesting that dorsal color change is an indicator of postfreeze recovery in D. chrysoscelis. We also provide evidence of "freeze resistance" in D. chrysoscelis. Two individuals did not freeze following three successive bouts of ice inoculation at -2.5°C and maintained brown dorsal color despite ice crystallization on the dorsum and contact with frozen substrate. Both frogs had similar plasma osmolality, circulating cryoprotectants, and incidence of cryoinjury compared to frogs that were frozen and thawed once or three times. Freeze resistance may be explained by physical changes in the skin including lipid accumulation and dehydration. This integrative study presents novel attributes of organismal freeze tolerance in D. chrysoscelis.