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Researchers at Miami unlock mysteries of freeze-tolerant frogs

Freeze-tolerant wood frog thawing

A freeze-tolerant frog begins to thaw in this video. Watch the process online.

Scientific research at Miami University is unlocking mysteries into how wood frogs (Rana sylvatica) can freeze in winter — with their hearts not beating while frozen — then quickly resume normal life after thawing in the spring.

The findings, reported in the Aug. 21 issue of the Journal of Experimental Biology show the freeze-tolerant frogs can survive at temperatures much lower than previously reported.

The National Science Foundation-supported research also has led to some new discoveries related to underlying physiological mechanisms that allowed frogs from the interior of Alaska to survive freezing at minus 16 degrees Celsius. They required only two days of thawing to resume normal movements, the researchers said.

Jon Costanzo, senior research scholar in the department of biology, said their work will be featured in three TV science programs: NOVA's "Making Things Colder," tentatively set to air on PBS some time in October; the David Attenborough production, "Natural Curiosities" in January; and the BBC's "Hidden Kingdoms," date unknown.

The research team has focused its attention on the differences between Ohio and Alaskan wood frogs. The study authors included Costanzo, University Distinguished Professor of Zoology Richard Lee and graduate students Clara do Amaral and Andrew Rosendale.

In August 2011, they collected dozens of frogs on the verge of hibernation near Fairbanks, Alaska, to study how they prepared for winter. What they found surprised even Costanzo, who has been studying the creatures for 25 years.

An "unexpected" finding

Back at the Miami campus, the researchers placed the frogs in programmable environmental chambers and manipulated temperature and light exposure for six weeks to simulate the frogs' normal conditions.

"We kind of faked them out as if they were being subjected to decreasing temperature and decreasing daylight like they would experience in the field," he said.

While studying how they changed physiologically, Costanzo said there was a "completely unexpected" finding: The frogs broke down muscle protein, which is surprising because they would have to breed soon after emerging from hibernation.

The frog "needs good muscle tone, good muscle structure, to be able to pull that off," Costanzo said. "Yet these frogs were using some of their muscle protein before winter."

Researchers believe that occurs so the frogs can use nitrogen in the protein to produce urea. Although humans and other creatures also produce urea, a waste byproduct, they quickly release it from their bodies. The frogs don't.

"Rather than urinating to get rid of the urea, they're hanging onto it and they really stacked it up," Costanzo said.

While the researchers have known for a while that the frogs produce urea heading into winter, they don’t yet understand how they are able to retain it the way they do.

"The concentration of urea in their blood was just huge and way more than we'd ever seen in the frogs from Ohio," he said. "We've never seen the accumulation like we've seen in these Alaskan frogs. It's really spectacular."

Some of the frogs' secrets

Urea, a cryoprotectant, can help tissues survive freezing stresses and also stabilize membranes. 

"It can help brain tissue tolerate swings in salt concentration, which you might see in freezing and thawing," he said, "so urea is probably one of their secrets."

Costanzo said urea also helps depress metabolism while the frogs hibernate for nearly eight months.

"They are not going to be feeding so depressing their metabolism during the winter is really important to survive because it's going to help them last longer on their stored energy reserves," he said.

The research also found the frogs produce glucose, which is ordinary blood sugar, as they're freezing and accumulate that to high levels, too, which appears to help the cells tolerate freezing.

Another key: dehydration. 

"We don’t know exactly how they are dehydrating their organs during freezing but we know the organs shrink," Costanzo said. "The idea is that rather than have all that water remain in the organ and freeze and become big chunks of ice, have that water freeze outside where it's not going to harm the tissue structure."

The researchers found the Alaskan wood frogs survived to temperatures of minus 16 degrees Celsius, which is 11 degrees colder than Ohio wood frogs survived in testing.

"They also survived a two-month period of freezing" at minus 4 degrees and required only two days to get back "up on their feet and looking great," Costanzo said.

The response time for the Ohio frogs was a week or longer.

"Given they came back in two days, we think they probably can go much lower than minus 16," he said.

Potential medical applications

Rosendale said that they pursue this kind of research because it's fascinating but realize that their discoveries may eventually lead to medical breakthroughs.

Scientists for years have been able to preserve simple systems such as embryos by freezing them. Regarding organ transplants, medical personnel ship and store organs on ice because they are trying to lower the temperature as much as possible to reduce damage.

"But they can't freeze organs yet," Costanzo said. 

If there is something that can be applied from the wood frog research, it is the role cryoprotectants play in improving freezing survival.

"That is something the frogs demonstrated very well," he said.

Additionally, understanding the winter biology of ectotherms such as frogs may help predict consequences of climate change for their survival, according to Lee.

Written by Margo Kissell, university news and communications, kisselm@MiamiOh.edu

Studying freeze tolerance

Jon Costanzo

Jon Costanzo

This is Jon Costanzo's 25th year at Miami doing this type of research involving frogs.  He and Richard Lee, director of the Miami's Laboratory for Ecophysiological Cryobiology, focus on how various creatures — insects, frogs, lizards, turtles and butterflies — cope with different kinds of winter stress.

Costanzo heads up the vertebrate research in the lab, with three graduate students working with him. Andrew Rosendale, who is pursuing his doctorate, brought some of the frogs to England in the spring so film crews for the Attenborough program and the BBC's "Hidden Kingdoms" could shoot time-lapse video of the freezing and thawing process.

Rosendale, who earned his master's in zoology from Miami in 2011, enjoys being part of this research project.

"I think the research we do in this lab is really unique. There aren’t a lot of labs that study freeze tolerance, especially in vertebrates, so we get to see stuff that most people have never heard of," he said. "Just the idea these frogs can survive freezing and survive that much ice in their body is just amazing to us. That’s something we (humans) can't even come close to at this point." 

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