- The Aquatic Pathobiology Laboratory, operated by the Emerging Pathogens Institute at the University of Florida, offers investigators lab space to research infectious diseases affecting fresh- and saltwater organisms.
- A protozoan parasite has caused a mass die-off of long-spined sea urchins, Diadema antillarum, across the Caribbean and along the Florida coast. Skip ahead
- Researchers aim to understand how parasites that infect crayfish may help or hinder nonnative crayfish species when they invade new regions in the mid-West. Skip ahead.
- Tilapia lake visrus can infect tilapia by damaging tissues in the fish’s brain, spleen, liver and kidney. It is usually fatal and can devestate 90% of infected tilapia fish in aquaculture or wild environments. Skip ahead.
- Key technology used in projects stemming from the aquatic pathobiology lab include special tanks, called mesocosms, and cutting-edge CRISPR-based technology.
Infectious diseases know no boundaries, and though we often think of them as affecting animals and plants on land, they also infect organisms living in lakes, rivers and oceans.
The Aquatic Pathobiology Laboratory, operated by the Emerging Pathogens Institute at the University of Florida, offers investigators lab space to research infectious diseases affecting fresh- and saltwater organisms. The lab is directed by EPI member Andrew Kane, Ph.D., who is an associate professor at the UF College of Public Health and Health Professions.
The lab accommodates both molecular work and animal experiments. From ocean-dwelling sea urchins to landlocked crayfish and farmed tilapia, dip your toes here for highlights of the lab’s top three emerging pathogens projects.
1. Identifying a new protozoan-based pathogen that is killing off sea urchins
Scuba divers and snorkelers delight in glimpsing sea urchins move across the ocean floor in a distinctly slow, gliding gait aided by the rotation of dozens of needle-shaped spines. In addition to being fun for tourists to ogle, urchins play a key role in maintaining coral reef health.
But a mass die-off of long-spined sea urchins, Diadema antillarum, in the Caribbean that began early last year in waters around St. Thomas alarmed divers and conservationists alike. Within months the die-off spread throughout the Caribbean and to the Florida coast. Infected urchins died within days of showing symptoms.
UF researcher Donald Behringer, brought his decades of experience in identifying novel marine infectious agents to the fore of the issue. In collaborative work with colleagues at UF, Cornell University, University of South Florida and others, Behringer and the team of researchers obtained a National Science Foundation RAPID grant and National Sea Grant emergency funding to investigate. Behringer is a professor of marine and disease ecology in the School of Forest, Fisheries, and Geomatic Sciences within the UF College of Agriculture and Life Sciences and UF Institute of Food and Agricultural Sciences. He is also an EPI member and relies on space in the Aquatic Pathobiology Laboratory for his marine disease ecology work.
Behringer’s team were soon collecting sea urchin samples and data in the Caribbean. They visited sites where die-offs were reported and other sites thought to have only healthy urchin populations. Samples were brought back to the U.S. for analysis.
Because epidemics affecting urchins can burn through an area extremely fast, the RAPID and emergency grants allowed the team to work quickly and obtain samples containing the infectious agent, Behringer said.
The team then used molecular tests and challenge experiments helped identify the infective culprit as a scuticociliate, a protozoan parasite, most similar to Philaster apodigitiformis. They are also conducting a series of experiments in the Aquatic Pathology Lab using special tanks, called mesocosms, where water temperature and salinity can be manipulated to better understand the environmental factors that may have led to the die-off in the wild. The tanks, provided by the EPI, are each independent experimental units, Behringer said.
“Which is really important and difficult to come by when it comes to doing pathogens work. Because in air-breathing organisms, you can put them in a cage or isolate them when studying an airborne pathogen,” Behringer said. “But each one of our tanks is an independent unit and has its own biological filtration system, its own life support system, in addition to having the ability to manipulate environmental variables. That’s what makes it an extremely important facility to be able to do this type of work.”
Ongoing work with the mesocosms is focused on studying how the protozoan parasite is transported through the water column, to better understand how it spread so fast in the Caribbean. His team is also focused on uncovering the demographic and environmental drivers of sea urchin die-offs.
“We want to know what the consequences are for the urchin populations and the communities in which they live, while also trying to determine why it happened?” He said. “What are the drivers behind it that will help us understand or predict future outbreaks?”
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2. Invasive crayfish
The Behringer Lab for Marine and Disease Ecology uses the Aquatic Pathobiology Laboratory for other projects too. He and Lindsey Reisinger, an assistant professor also in the School of Forest, Fisheries, and Geomatic Sciences, are collaborating with Dr. Jamie Bojko from Teesside University in the UK on a project sponsored by the Wisconsin Department of Natural Resources that aims to understand how parasites that infect crayfish may help or hinder nonnative crayfish species when they invade new regions in the mid-West.
“Crayfish have been exported around the globe for different uses, sometimes by folks not realizing that crayfish have the potential to be very invasive,” Behringer said. “So invasive crayfish are now a huge problem in many different places.”
In Wisconsin, the invasive crayfish can “mow down” all the plants growing on the lake bottoms, which negatively affects native fish and invertebrate communities, he added.
Crayfish, especially those that live in lakes and ponds, lend themselves well to studying the dynamics of biological invasions. Each lake or pond can be viewed as its own experimental unit. Each lake potentially contains a slightly different mix of native and invasive species, which may have been invaded at different points in time. The virile crayfish is native to this area, and the rusty crayfish is invasive.
“The natural set up of individual lakes in this area lets us look at the relationship between parasites, parasite load, and invasion dynamics over time with these crayfish,” Behringer said.
His team plans to run a series of experiments in mesocosms back at the Aquatic Pathobiology Laboratory to characterize how temperature may play a role in these invasion dynamics.
“We have identified parasites in native crayfish that don’t infect the invasive crayfish, and vice versa, some that appeared to only infect the invasive but not the native,” he said. “So we are trying to understand the dynamics of that, the role that temperature might play, and more broadly, how these parasites affect crayfish invasion dynamics.”
3. A new CRISPR-based diagnostic test to detect tilapia lake virus
Tilapia is a popular firm, flaky-white meat for people who enjoy eating freshwater fish. It is native to lakes in Africa and is grown in aquaculture farms throughout the world. Tilapia were first found in US lakes in the mid-1970s. According to the Florida Fish and Wildlife Conservation Commission, tilapia are now widespread in Florida lakes south of Lake Okeechobee.
Tilapia can be infected by Tilapia tilapinevirus, commonly called tilapia lake virus, which damages tissues in the fish’s brain, spleen, liver and kidney. Tilapia lake virus is usually fatal and can devastate 90% of infected tilapia fish in aquaculture or wild environments. Given the $14 billion global value of farmed tilapia and the role of wild tilapia in balancing algae and mosquito larvae in wild waterways, the tilapia lake virus poses a looming threat.
“The U.S. has no surveillance network in place to monitor for tilapia lake virus,” said Kuttichantran Subramaniam, Ph.D., a research associate professor in the UF College of Veterinary Medicine. “And part of the reason for this is that we don’t have a validated diagnostic that can be done in the field [pondside] or laboratories with limited resources.”
Subramaniam, along with Ph.D. student Dorothea Megarani, plan to develop a rapid diagnostic to help fish professionals detect tilapia lake virus in the field or in laboratories with limited resources. Subramaniam directs the Wildlife and Aquatic Veterinary Disease Laboratory within the Aquatic Pathobiology Laboratory and is the principal investigator on the diagnostic development project. This project is supported by the intramural research program of the U.S. Department of Agriculture National Institute of Food and Agriculture.
The diagnostic assay uses cutting-edge CRISPR-based technology in a single tube, or “one-pot,” that eliminates the need for a central lab and specialized equipment. The one-pot test should have results ready in an hour, Subramaniam said. Work performed for this project in the Aquatic Pathobiology Laboratory helped validate the CRISPR technology for use in aquatic diseases, Subramaniam said.
Internal tissue from a fish is needed to test for the virus, and Megarani and Subramaniam are pioneering a new sampling method. Standard testing is lethal and relies on killing the fish to detect the virus, he said. But he and Megarani are testing the feasibility of using a bit of gill tissue instead. This would flip the sampling to a humane, nonlethal method.
Tissue from the gill will then be used to extract RNA, Subramaniam explained. Then it will be mixed with a reagent mixture consisting of Cas (CRISPR-associated) enzyme, primers and fluorescent tags. The reaction mixture will then go into a chamber of the testing device where a CRISPR-based reaction takes place at 62°C for an hour. The Cas enzyme and single-guide RNA (sgRNA) will probe for an amplified target region of the tilapia lake virus genome. If they find their target, the Cas enzyme will get activated, and fluorescent tags will be cleaved and light up. The fluorescence will then be read by an optic visualizer, producing a positive result. If no amplified target region of the tilapia lake virus genome is found, the device will show a negative result.
“There are other CRISPR applications developed for tilapia lake virus elsewhere in the world, but we do not yet have this in the U.S.” Subramaniam said. Subramaniam and Megarani also acknowledge Dr. Piyush Jain and his graduate student, Lilia Yang, at the UF Herbert Wertheim College of Engineering, Department of Chemical Engineering, for their technical assistance in this project.
Written by: DeLene Beeland