La Crosse Fish Health Center
Applications of Flow Cytometry for Asian Carp Management, Endangered Mussel Culture, and Fish Health Research
BY JEN BAILEY, LA CROSSE FISH HEALTH CENTER
The La Crosse Fish Health Center (LFHC) is expanding the capacity of its current flow cytometry program, which currently provides DNA ploidy determination of wild-caught invasive Asian carps to resource managers for control of grass and black carp. Biologists at LFHC are accomplishing this by increasing partnerships and advancing flow cytometry techniques that may prevent more reproductive grass and black carp from entering public waters. In addition to augmenting the LFHC’s current use of flow cytometry technology, biologists are exploring ways to use the laser technology to improve freshwater mussel culture techniques, which may aid in freshwater mussel recovery programs, and applying the technology to rapid response identification of challenges to fish health, both in culture and in the wild.
Flow cytometry is a technology that uses lasers and flourescence detection to examine multiple parameters of cells in solution. While this technology has been around for more than forty years, it has traditionally been used in human medicine for cell cycle analysis, cancer research, hematology and genetics. More recently, flow cytometry has been used to monitor phytoplankton populations in marine research, and only in recent years have conservation scientists begun developing methods to apply this technology to management of wildlife species.
Methods for flow cytometric analysis of wild caught grass and black carp were first developed by Dr. Jill Jenkins, microbiologist at the National Wetlands Research Center – U.S. Geological Survey (USGS) in 2004, when a fisheries management biologist brought samples from a free-ranging grass carp that had been captured in state waters. The reproductive status of the free-ranging fish was determined by Dr. Jenkins when she examined the nuclear DNA content of sample cells using flow cytometry. Cells of black and grass carp normally have two copies of each chromosome (diploid) in the nucleus. Diploid carp are capable of breeding when the fish reaches maturity, which can be undesirable if they are stocked into water bodies to be used as a biological control. Hatchery produced triploid fish eat and grow normally, but their reproductive cells contain three copies of each chromosome, making them unable to produce viable offspring. The difference between these two types of cells is detectable by the amount of flourescence and scattered light caused by cells as they pass through the cytometer detectors in solution. Once detected, data from thousands of cells is collected and interpreted by the analyst, and a definitive determination is provided to the resource manager. The concern for state biologists is that escaped, or free-ranging, diploid black or grass carp could have a negative impact on native fish and wildlife if successful reproduction occurs in the wild.
The analysis technique using flow cytometry was refined by Dr. Jenkins over the years through much work and collaboration of scientists at the NWRC lab at USGS, Private and University researchers as well as commercial triploid and diploid carp producers and the US Fish and Wildlife Service (FWS) Triploid Grass Carp Inspection and Certification Program. The Ploidy Analysis Program using flow cytometry was turned over to the USFWS in 2012, and was set up in the Whitney Genetics Lab just in time for the spring sampling season. Research and technical assistance continues under Dr. Jenkins leadership at the NWRC Laboratory in partnership with the program at FWS to adapt USGS-developed methods to fit the mission of the USFWS and to increase capabilities to perform fast and efficient ploidy analyses as demand and incidence of captured Asian carps continues to increase. Increasing demand is expected, as the attention of resource managers is shifting to include prevention of new introductions of reproductive grass carp in conjunction with control and eradication of previously introduced, reproductive populations.
The LFHC staff has been training on flow cytometry methods since transfer of the program from the Whitney Genetics Lab to the LFHC. The increased support will be a terrific boost to analysis capabilities during the busy spring months at LFHC. Ploidy samples are usually processed within 24 to 48 hours of receipt to ensure the highest DNA quality of sample data, so having an increased number of analysts capable of producing high quality results will be a help to colleagues and a help to the resource managers submitting samples and awaiting results. The expanded capabilities and location in the new lab has been met with enthusiasm as biologists begin to adapt flow cytometry methods to assist in problem solving in the Federal Hatchery System.
Staff at LFHC has recently teamed up with mussel biologists Jorge Buening and Nathan Eckert of Genoa National Fish Hatchery and fish biologist Mark Steingraeber of La Crosse Fish and Wildlife Conservation Office to examine juvenile freshwater mussel feeding rates in culture. Applications for detecting bacterial fish pathogens are being explored at the LFHC as well. The real time results that flow cytometry analysis provides could be a powerful tool if methods can be developed to produce test results for partners within hours of sampling sick fish. Combined with traditional methods for identifying fish pathogens in moribund fish, flow cytometry may also help provide rapid response answers to fish kills in the field. Advances in science are always important, and with the new addition of flow cytometry to the LFHC, some of those advances will help manage aquatic species right here in Region 3.