Today we have a guest blog written by Elizabeth Evans (pictured above), a graduate student at the University of Dayton working in the laboratory of Dr. Carissa Krane. She presented her research on freeze tolerance today at the 2022 Experimental Biology conference in Philadelphia. She wrote the award-winning blog entry below which earned the 2022 Dr. Dolittle Travel Award to attend the conference. Congratulations Elizabeth!!
Stranger than science fiction: treefrogs that freeze and live to tell the story
By: Elizabeth Evans
Have you ever watched your own breath freeze in front of your face? Or been so cold that you couldn’t feel your fingers and toes? Humans and other mammals can regulate their body temperature, but what if you were a treefrog that didn’t have that ability? Cope’s gray treefrog Dryophytes chrysoscelis is one of six species of freeze tolerant frogs that can survive more than cold temperature, they can survive freezing and thawing more than half of their body fluids. Even more incredible, when frozen, they also have no heartbeat, do not breathe, and do not have functioning nerves to allow muscle movement.
These animals are not without protection from freezing conditions, however, and accumulate small molecules called cryoprotectants that act like antifreeze in a car to prevent the engine from freezing solid. When a frog comes into contact with ice, for example, cryoprotectants like glucose and glycerol prevent cells from dying and protect against physical damage that otherwise incurs in freezing conditions. To date, several studies have investigated a single round of freezing and thawing in frogs, but it is not likely that animals in their natural habitat freeze and thaw only once per year. Our recent work is one of the first to study repeated freezing and thawing in animals and improves the scientific understanding of the natural freeze tolerance in these amazing creatures.
For our work, wild-caught male treefrogs were cold acclimated in a lab setting and served as the experimental control. Two other groups were first cold acclimated and then frozen and thawed once or frozen and thawed three times. Cope’s gray treefrog uses three cryoprotectants (glycerol, glucose, and urea), which were each measured in blood plasma, liver, and muscle tissues. In both animals frozen and thawed once and three times, more of the cryoprotectants glycerol and glucose accumulated in plasma and liver tissue than in the cold control animals. Liver glycogen (a form of stored cryoprotectants) was also depleted in thawed animals. Unexpectedly, cryoprotectants did not accumulate the same way in muscle tissue. In this case, only glycerol was elevated in thawed animals, while glucose was constant. Muscle glycogen was also unchanged among groups. It is also interesting that urea, the third known cryoprotectant in Cope’s gray treefrog, did not change with freezing and thawing in any tissues analyzed. Though it was not unexpected that glycerol was the most abundant cryoprotectant in all thawed animals, it was interesting to find that glycerol explained the majority of variation in plasma osmolality, a measure of concentration. Red blood cells all contain hemoglobin, which is released into plasma when red blood cells are damaged or destroyed and can an overall measure of freezing induced damage to red blood cells. Even with increased levels of cryoprotectants in blood plasma, we still detected greater amounts of hemoglobin in repeatedly frozen and thawed animals compared to the control cold animals. Curiously, trends in plasma hemoglobin among groups did not correspond with changes in cryoprotectants.
Overall, this cryoprotectant and cryoinjury analysis indicates that cryoprotectants do not accumulate evenly among tissues with repeated freezing and thawing, and that the incidence of cryoinjury cannot be explained by trends in cryoprotectants. Animal behavior during thawing was also analyzed to determine how body movement was affected by repeated freezing and thawing. In fact, the time required for a frog to open its eyes, lift its head, move a limb, or change body position was significantly delayed with repeated freeze-thaw cycles. Incredibly, dynamic skin color changes were also documented for the first
time with freezing and thawing. Skin color changed from blue and green during freezing and gradually returned back to brown (baseline) during thawing. It was expected that repeatedly freezing and thawing would be biologically different than freezing and thawing once, but the delay in thawing and increased cryoinjury did not correlate with a simultaneous increase in cryoprotectants. By including a novel and additional experimental group with repeated freezing and thawing, this study really enhances the scientific understanding of the complexity of the natural freeze tolerance in Cope’s gray treefrog. Freeze tolerance is clearly a systemic and complex process that encompasses both behavioral and biochemical traits.