GLFT-funded Research Leads to First-time Discovery

Researchers have been studying Thiamine Deficiency Complex (TDC) in the Great Lakes for years, in an attempt to control the disease and its effects on the fishery population.

The disease causes fish to take in too much thiaminase, which is a protein that breaks down vitamin B. As a result, the deficiency contributes to significant losses within lake trout populations.

Those researchers, however, are now one step closer to managing TDC in lake trout. Thanks to a grant from the Great Lakes Fishery Trust, scientists at the Oregon State University Department of Fisheries and Wildlife discovered for the first time that alewives—a source of food for the lake trout—made the thiaminase enzyme de novo, or at the cellular level.

“This research will bring us one step closer to solving the TDC problem for lake trout,” said Mark Coscarelli, GLFT co-manager. “That’s why the GLFT funded it.”

Based on what the OSU researchers knew already regarding basic biology and nutrition, the leading assumption was that the thiaminase protein could be found in bacteria within the gut of alewives.

“Lake trout take in thiaminase in their diet because they eat so many alewives, which have a lot of this enzyme (thiaminase) that breaks down vitamin B,” said Allison Evans, a graduate student who was part of the research project. “Over time, that breakdown results in a vitamin B deficiency for our adult females.”

Evans worked with Scott Heppell, an associate professor of fisheries at OSU, on the research. The team worked off of previous researchers’ findings on thiamine deficiency.

“Before Scott and I came into the picture, that research group had been working on the question: ‘Why do lake trout embryos and adult lake trout not have enough vitamin B?’ What they discovered was the alewives the lake trout consume have a high amount of the enzyme thiaminase, which degrades vitamin B.”

Donald Tillitt, a senior scientist with the U.S. Geological Survey, has spent the past 15 years studying the lack of lake trout reproduction.

“The issue is very important for resource managers because we need to know what the source of thiaminase is,” Tillitt said. “We do know that thiaminase is what’s causing thiamine deficiency in some of the top predator fish. If we understand the source, then we can start to do the right kind of research to understand what controls that.”

The OSU research team ended up forming three hypotheses, with the gut bacteria theory being the leading hypothesis. The second looked at diet, speculating that alewives obtained thiaminase by eating zooplankton, which produce the enzyme. A third—and ultimately, the correct hypothesis—was that alewives have a gene that causes them to make the thiaminase protein.

“What I really like about this project is that it is really basic, hypothesis-driven, fundamental science that advances knowledge broadly,” Heppell said.

Methods used in the research included multiple biochemical techniques used to separate molecules. Evans and Heppell admit, however, that they faced challenges in separating thiaminase from all the other fish proteins.

Thiaminase made by bacteria had been characterized by other researchers, but thiaminase proteins made by fish had never been discovered previously.

“The methods that were appropriate for the bacteria were not directly translatable to the fish proteins,” Evans said. “The bacterial protein is very robust. You can add chemicals to it, you can unwind it using heat, and it’ll wind back up all on its own. Those are good things because those are the ways you can get the thiaminase, separated from other proteins. The fish proteins are different because they were a little more fragile.”

“The thiaminase in fish proved to be a lot more sensitive to the conditions they were kept in,” Heppell added.

In order to accommodate the fragile proteins, the team applied new methods in the lab to work with these unstudied fish proteins. After working out the kinks, the team was able to successfully test its third hypothesis, that alewives produce thiaminase de novo, or at a molecular level, using their own genetic machinery.

“We were sort of amazed. We didn’t know,” Evans said.” That’s why you do the work, because you don’t know what the results will be.”

The new discovery allows researchers like Tillitt to move forward with lake trout population sustainability.

“Our next steps in terms of the research is to figure out why fish produce this enzyme,” he said. “It doesn’t make sense that fish would produce an enzyme that degrades an essential nutrient that it requires. This is the first time it’s been seen in vertebrates. That’s pretty interesting. And so the next question is: Why do they produce this?”

The same question is on the minds of Evans and Heppell as well. Now that they know the cause of thiaminase in alewives, they can look at the different conditions that impact thiaminase levels and work toward managing TDC.

“We’re able to ask a lot of next-step questions,” Heppell said. “A lot of it will come down to what conditions alewives produce this enzyme under. If we find that to be the case, then we have to find out if we can either manipulate the environment or manipulate where we take action based on that information.”