Laboratory learning

Laboratory learning -- Southern Illinois University Carbondale undergraduate students (from left) Marissa Gutkowski,  Daniel Morales and Katy Banning work in the laboratory of Robin Warne, far back center, assistant professor of zoology. Warne and his students are studying ranavirus, a deadly ailment that hits several species of vertebrates, including fish, amphibians and reptiles. Here, the students are conducting solid-phase extraction to isolate glucocorticoid steroid hormones from frogs from Peru. These isolated samples will later be analyzed and hormone levels quantified by enzyme immunoassay. (Photo by Russell Bailey)

November 18, 2015

Researcher exploring spread of ranavirus

by Tim Crosby

CARBONDALE, Ill. – Most viruses are – thankfully – very specific to the animals they impact. It’s very difficult and unusual for a single virus to wreak havoc in several species. 

Not so the ranavirus. This killer hits several species of vertebrates, including fish, amphibians and reptiles. Unleashed in a local pond, the ranavirus routinely wipes out every tadpole living in it. 

A researcher at Southern Illinois University Carbondale is making this virus a priority in his work, looking at it from several angles and trying to learn more about the where it comes from and how it spreads. 

Along the way, he’s also hoping to use it to find out more in general about how disease in a given ecosystem spreads. 

Robin Warne, assistant professor of zoology, is looking at the virus’ physiological effect on animals, as well as trying to find its origins in their environment. Learning more about the virus and how it spreads is key to helping prevent mass die-offs of animals that play key roles in the food web and overall environment. 

The key to ranavirus’ success is its ability to attack several species, Warne said. 

“Most viruses are pretty species-specific. They have to have a way to invade the host and take over cells and get the cells to reproduce more virus, and there are significant barriers to each of those steps,” he said. “But unlike those, ranavirus is very general and attacks across species” 

For example, a hatchery in Missouri that specializes in growing endangered sturgeon fish recently experienced a major ranavirus outbreak that wiped out most of its livestock. There also are many instances throughout the world of the same pathogen killing all the tadpoles in ponds where it is introduced. The virus is more prevalent in the northeastern United States, but has also appeared in Southern Illinois, he said, where the common wood frog is very susceptible to the virus. 

Researchers believe the virus enters most of its victims through the gut. There are cases in which researchers have found snakes that had eaten infected frogs and were subsequently infected, for instance. Many believe the virus initially arrives in the water in which the infected animals are living in or near, but they still don’t know how. Once present in an animal, it behaves somewhat like HIV, attacking its immune system and causing death within days, depending on how much virus it ingests. 

In frogs, the virus hits hardest during the larval stage and during metamorphosis, when the water-dwelling tadpole is refashioning itself into a four-legged frog. During metamorphosis, the frog not only reshapes its body but also completely remodels its digestive tract, which will soon change from one that digests mainly plant material to one that digests insects and other organisms, too. Because researchers hypothesize the ranavirus’ main path of infection is through the frog’s gut, this time is of keen interest to Warne. 

“During this time they don’t eat for a couple weeks while remodeling their digestive tract,” Warne said. “It’s during this crazy period when a lot is going on that we’re trying to figure out how this might affect ranavirus infection and mortality. Are they more vulnerable at this time?” 

To learn more about the relationship between ranavirus infection and metamorphosis, Warne and his students have designed an experiment that pulls a switcheroo on a suspected culprit: the type of microbes lining the wood frog’s gut. 

In a lab at SIU, the team is taking eggs from common local green frogs, which are largely not susceptible to ranavirus, and are purging them of all the bacteria and fungus on and around them. Scientists theorize that such sources are where larval frogs emerging from their eggs receive their first dose of gut microbes, so the researchers are taking those away and substituting them with microbes from the ranavirus-susceptible wood frogs gut. 

“The idea is that the green frogs are getting their initial microbes when they emerge from the egg, and that they are coming from the parent who lay them,” Warne said. “We’re not sure that’s true, but it’s either that or from the environment. But that would also raise the question as to why two species from mostly the same environment, eating the same things and getting the same microbes, do not have the same reaction to ranavirus.  Do they have different microbes somehow? We think so, so we’re testing that. 

“When these green frogs hatch, if that is where they’re getting their inoculation hopefully they’ll get these wood frog microbes instead,” he said. 

Working with a research collaborator at Kansas State University, the team will then get DNA sequence data from the microbes that actually end up in the green frogs’ gut. And they’ll also test the green frogs to see if they are subsequently more susceptible to the ranavirus, much like their wood frog cousins. 

That experiment, while painstaking, won’t tell researchers how or where the ranavirus initially enters an environment, however. So Warne is also involved with research to that end. 

Working with Alessandro Catenazzi, assistant professor of zoology at SIU, Warne traveled to Peru this summer to search for answers to this question by collecting what is known as “E-DNA,” or environmental DNA. 

To do this, researchers take samples from an organism’s environment to try and isolate other organisms living there. In Warne’s case, this included taking hundreds of samples of water, leaves and other items in and around where the impacted frogs are living. 

“You take the sample and then you screen them see if you can detect virus, either in the water where they live or where they eat,” he said. “Our goal was to a lot of E-DNA surveys. We would do things like catch a frog on a leaf, and then take samples from that very leaf.” 

Back in Southern Illinois, Warne’s graduate student, Seth LaGrange, also conducted E-DNA surveys in the area looking for ranavirus’ presence. 

While that research is continuing, Warne also is using ranavirus to study how diseases behave and spread. And it’s tied up with the individual “personalities” of tadpoles. Some of them sit on the bottom near where food often is available, while others tend to swim almost nervously about. 

The ones that sit on the bottom, it turns out, are more aggressive when it comes to foraging for food. The same tadpoles have a higher metabolic rate and a lower amount of stress hormones than the ones that swim about.

“So we’re also trying to use ranavirus as a model disease system to understand more about how transmission works, and we’re trying to identify the factors that allow the disease to be transmitted, and what makes that more likely than others,” Warne said. “Tadpoles, believe it or not, actually have behaviors and personalities that are different and consistent.” 

Using a small population of wood frogs, Warne and his students infected one of them with a lethal dose of the virus and then studied how likely they were to spread the disease when they were sick, but still healthy enough to interact with their environment. So far, they’ve observed that the more aggressive tadpoles with higher metabolic rates were more likely to transmit the disease. 

Warne said understanding how the virus is impacting the invertebrates is important, as the disease is hurting many species, including endangered ones. 

“Also, amphibians are major part of the food web and ecosystem, and when they go away the system is altered,” he said. “And we rely on these ecosystems as they are. We don’t know what change will mean for us either when that happens. But it probably will be in ways we won’t know until much later.”