Establishing a standard ploidy assessment method using grass carp from Ohio, 2015-2016

In 2015-2016, the Ohio Division of Wildlife's undercover law enforcement purchased 1,200 grass carp (Ctenopharyngodon idella). Fish heads and eyeballs were sent overnight to U.S. Geological Survey Wetland and Aquatic Research Center for ploidy analysis. Field and laboratory standard operating procedures were established and followed. Fish lengths, fish weights, and eyeball weights were obtained from the U.S. Fish and Wildlife Service's feral carp ploidy program for grass carp and black carp (Mylopharyngodon piceus) and the Ohio grass carp. Internal 2 micrometer or 4 micrometer bead standards were used in establishing nuclear sizes from Nile tilapia (Oreochromis niloticus), known diploid (n=20) and triploid (n=20) carp blood, as well as cells derived from eyeballs from intercepted carp. Cells from the fish eyes were obtained from an ocular blood vessel as well as the vitreous humor. Nuclei were stained with propidium iodide, internal bead standards were included, and three replicate tubes per fish were analyzed by flow cytometry. The external DNA content control was fresh or cryopreserve blood from tilapia, with a diploid genome size of 2.40 pg. This standard protocol uses nuclear size as well as DNA content for reliably discriminating ploidy of interstate hauled grass carp.