A frog floats on top of water in what appears to be a shallow stream. The frog's legs are splayed out behind it.

Researchers unlock the mysteries of freeze-tolerant frogs

A small brown frog sits on a rough black rock.
John Costanzo’s research team studies wood frogs like the one shown here.

Jon Costanzo, senior research scholar in the Department of Biology, is helping unlock the mystery of 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.

Findings of Costanzo’s work with University Distinguished Professor of Zoology Richard Lee and graduate students Clara do Amaral and Andrew Rosendale were reported in the August 21 issue of the Journal of Experimental Biology. The researchers found that 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 research team’s work was featured on the TV science program NOVA’s “Making Things Colder” and in a David Attenborough production, “Natural Curiosities” earlier this year. A third, yet-to-be scheduled, program — the BBC’s “Hidden Kingdoms” — will also discuss the research.

Focusing on the differences between Ohio and Alaskan wood frogs, the researchers collected dozens of frogs on the verge of hibernation near Fairbanks, Alaska, to study how they prepared for winter.

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, they discovered something that surprised even Costanzo, who has been studying the creatures for 25 years.
Costanzo said the finding that the frogs broke down muscle protein at this time, was “completely unexpected” 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.”

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.

“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.

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 & Communications, Miami University. This article originally appeared August 21, 2014 on the Miami University News & Events website, and is re-used here with permission.

Photo of sitting wood frog by Meeshoo via stock.xchng. “Wood frog, floating” photo by D. Gordon E. Robertson via Wikimedia Commons.  Both used under Creative Commons license.

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