Freezing temperatures pose a challenge for cold-blood animals such as frogs. Ice can damage cells from within, like soda rupturing a can left too long in the freezer. But even if it forms outside a cell, a chunk of ice leaves the remaining body fluid saltier, more concentrated and therefore less hospitable for the cells it bathes. To minimize cellular damage in the winter, some animals produce antifreeze – molecules that disrupt ice formation. For instance, the Cope's gray treefrog (Dryophytes chrysoscelis) makes glycerol, which scientists often use to preserve frozen lab samples. But whereas scientists avoid thawing their samples more than once for fear of damaging them, Cope's gray treefrogs must survive winters in the eastern United States, where they are thawed and refrozen repeatedly as temperatures regularly fluctuate above and below freezing. Elizabeth Yokum, Matthew Wascher and Carissa Krane from University of Dayton, USA, with David Goldstein from Wright State University, USA, tested these frogs by freezing and thawing them multiple times to understand how they survive the winter.

Previous research on another antifreeze-producing frog – the wood frog (Lithobates sylvaticus) – had shown that repeated cycles of freezing and thawing led that species to increase antifreeze production, consistent with studies on antifreeze-producing insects. However, the repeated freeze–thaw cycles took their toll on the insects, which accumulated more damage as they went through more cycles. Thus, Yokum, Krane and their colleagues suspected that freezing and thawing Cope's gray treefrogs multiple times would cause them to produce more antifreeze and sustain more injuries than frogs that were only frozen and thawed once.

The researchers divided Cope's gray treefrogs into three groups: one that remained unfrozen, one that was frozen and thawed once, and one that was frozen and thawed three times. During each cycle, the scientists kept the frogs frozen for a day at a time before warming the animals and monitoring them for signs of coordinated movements that would indicate successful thawing. Although all of the frozen frogs recovered, the time it took for the frogs to recover from thawing after their second or third freeze was nearly double that of frogs thawing after their first freeze. Furthermore, the scientists took blood and tissue samples from frogs in each group at the end of the experiment and analyzed them for molecules that would indicate damage to the frogs’ blood cells. They found the lowest levels of damage indicators in frogs that were never frozen and the highest levels in frogs that were frozen repeatedly. Together, these results suggest the animals do sustain more damage each time they are frozen and thawed.

However, the results were more complicated when the scientists looked for antifreeze in the blood and tissue samples they collected. All the frogs that were frozen once or frozen repeatedly had higher levels of antifreeze than frogs that were never frozen, but the levels were not always highest in the frogs that were frozen repeatedly. In fact, individual frogs showed a lot of variability in the amount of antifreeze they accumulated after being frozen, regardless of the frequency. The scientists did find a pattern though, when they compared the levels of antifreeze in the frogs with how much damage the frogs’ blood cells had sustained and how quickly the frogs recovered upon thawing. Frogs with more antifreeze in their system recovered more quickly, but also sustained more damage to their blood cells. Frogs with less antifreeze showed the opposite trends. In other words, fast recoveries were more costly, in terms of both antifreeze production and damage accumulation.

The researchers concluded that individual Cope's gray treefrogs employ different strategies to survive the winter and that variation may be a key asset for the species in this era of volatile and extreme weather.

E. E.
D. L.
C. M.
Repeated freeze–thaw in freeze-tolerant treefrogs: novel interindividual variation of integrative biochemical, cellular and organismal responses
Am. J. Physiol. Regul. Integr. Comp. Physiol.