In less than 3½ minutes, an Augustana College research team can remove the pencil-eraser-sized brain of a hatchling painted turtle.
The Augie team, headed by assistant professor Tim Muir, is trying to figure out what makes the turtles tick. More precisely, Muir and his student assistants want to know how the most widespread native turtle of North America manages to survive being frozen solid.
The answer might be in the turtles’ tiny organs.
But there could be significant human consequences for Muir’s studies, too.
Doctors who perform human organ harvesting for transplant have not yet discovered a method for successfully freezing the organs. If Muir’s data uncovers the turtles’ secret of survival, it could ultimately lead to freezing methods for human organs.
For Muir, however, the goal of his research is in the data, not in the potential medical applications.
Besides, there is plenty of science to learn from the tiny turtles that make themselves available, literally, right outside Muir’s back door.
The nesting season for adult female painted turtles is from late May to July, meaning that Muir and his two student assistants have their summers cut out for them.
They use two nesting sites for trapping egg-bearing turtles: a slough on the Augustana campus and a college-owned wetland on the north shore of the Rock River in Moline. Over the past three summers, Muir and research assistant Manisha Kumar, a senior, and, more recently, James Wiebler, a junior, have become intimately acquainted with the female painted turtles that occupy the ponds.
“Manisha (Kumar) claimed she was a girly-girl in the beginning, but she’s been out there in the water, hoisting nets and handling some really stinky bait,” Muir said. “She’s skilled at dissection, too. She has a great trick with the liver.”
But first, the eggs.
“In the early summer, the females lay their eggs two to four inches under ground,” he said. “The mother then leaves, and that’s the extent of parenthood.”
However, a trapped female turtle can get her last rite of parenting decided for her.
Wiebler, of Eldridge, said he could easily detect by his second attempt whether a turtle’s eggs were mature enough to hatch. By pressing on the sides of the pregnant female, he can get a feel for the condition of the eggs inside.
If they feel like ping-pong balls, they’re ready. If they’re soft, not yet.
The egg-ready female then is placed in a bucket of warm water (so she doesn’t crush the eggs) and is administered the hormone oxytocin, which also induces labor in humans.
“We check the water every 10 minutes, because the eggs will drown if we don’t take them out of the bucket,” Muir said.
Each female turtle, which Muir’s team identifies by cutting notches into the shell, produces 10 to 12 eggs, called a clutch. They generally produce one or two clutches per season, and the adult turtles can live 50 or 60 years, meaning some could produce as many as 1,000 hatchlings in a lifetime.
This winter, 140 hatchlings spent the season in a temperature-controlled “environmental chamber” at Augustana’s Hanson Hall. In the wild, turtle eggs hatch in the fall and remain in their underground nests until spring.
Under Muir’s watch, the turtle babies are exposed to increasingly cold temperatures, just as they would be outdoors. Once they have acclimated to minus 7 degrees C, for instance, they are introduced to minus 13 degrees.
“Without first being acclimated to minus 7, none survived minus 13,” Muir said. “In the wild, their body temperature would be the outdoor temperature. The freeze line is much lower than their nests.
“All of their organs can simultaneously freeze, and they still come back. The overarching goal is figuring out how they do that.”
Brain, liver, blood
Muir didn’t have to do much talking to convince Wiebler to help with his turtle research.
“I had a class in human physiology with him, and he asked if I wanted to catch turtles in the spring, and I said, ‘Heck, yes,’” Wiebler said.
Kumar, of Davenport, was an even quicker recruit.
“I had him for a cell biology class my freshman year,” she said. “I had never heard of any of this kind of research before. Depending on where I go to medical school, I’d like to come back and check in on them.”
She already has reported some of the data from the trio’s turtle research, and Wiebler will get a chance to present more when he goes to Canada with Muir this summer for a once-every-four-years conference on ectothermic organisms.
Although interesting and exciting, the work requires considerable record-keeping and, as often is the case in laboratory research, plenty of minutia.
“We have to have permits to throw the nets when we’re trapping turtles,” Muir said. “There are long periods in the summer where we have to check those nets every single day. That’s a big commitment.”
But catching the turtles is just the start. Hatching eggs and controlling environments takes time, too. Then comes the study of tiny organs.
“When you look at the turtles frozen, it’s kind of like they’re in a comatose state,” Kumar said. “We frequently poke them to see if they’re alive.”
It’s not always easy to tell.
“You’re not going to get a lot of movement, given they are frozen, and they are turtles, after all,” Muir said. “We want to know what’s physiologically going on. We harvest the organs, using the right euthanasia methods, to measure the compounds.
“We are looking at the brain, liver and blood. From the data we’ve seen so far, it’s possible there is a correlation between freeze-tolerant survival and the level of glucose in the brain.”
Levels of lactic acid also are measured. When the temperature of turtles’ blood is below the freezing point but does not become a solid, that blood becomes a highly desirable specimen.
Whatever mysteries Muir and his evolving team of student researchers reveal, the findings will be made public and could impact the future of medicine, among other things.
“It is not my goal to find a way to successfully freeze human organs,” Muir said. “As a research scientist, I want to know how nature works. The applied sciences take it from there.”
Professor to professor
While Muir’s work at Augustana could provide useful data to other disciplines, his work also is a continuation.
When he was an undergraduate student in Michigan, a classmate shared a link to a study by Miami University professor Jon Costanzo. Muir contacted Costanzo about the possibility of doing graduate work at the Oxford, Ohio, university.
“Over the years, we’ve attracted a lot of attention,” Costanzo said of the “turtle work” that he started and Muir is continuing. “The work on painted turtles began in 1990 — in sand hills in Nebraska. Tim went there several times.
“There are very few people doing this kind of research. Most people aren’t interested in what animals are doing when they are doing nothing. How they’re passing the winter doesn’t seem like a big deal.”
But researchers such as Costanzo and Muir find the work fascinating.
“As one question is answered, more questions always come up,” Costanzo said. “What a beautiful problem that is.”
One problem the two researchers will not encounter, he said, is duplication of data, and that’s not just because Costanzo’s research now is focused on frogs.
“There would be no chance of duplication, because there are too many questions,” he said.
The relationship that started as student-professor now has graduated to a colleague-to-colleague relationship, he said, adding that he has high hopes for Muir’s research.
“Tim is really one of the best students I’ve ever had,” Costanzo said. “He’s one of the old-fashioned physiological biologists. He has this natural sense of curiosity.”
That curiosity just happened to land on a tiny turtle. Asked why he chose them for his studies, Muir gave a three-part answer:
“Hatchling painted turtles are particularly useful for studying sub-freezing biology because they are one of only a few animals that can rely on either freeze avoidance by supercooling (body-fluid temperatures drop but don’t freeze) or on freeze tolerance (body fluids freeze).
“Really, they allow us to investigate questions about what causes and protects against cold injury in the absence of any internal ice formation and to investigate how freeze-tolerant animals survive the added burden of internal freezing.
“Lastly, and importantly, it is very convenient to be able to walk 300 yards from the lab, hop in the Augie slough and catch some painted turtles!”