28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Molecular Characterization Of The Amoeba Associated With Paramoebiasis In American Lobster (Homarus americanus) By Small Subunit Ribosomal Gene Analysis Salvatore Frasca Jr.1, Kathleen R. Nevis1, Thomas E. Mullen1, Rebecca J. Gast2 Michael T. Peglar3, Patrick M. Gillevet4 and Charles J. O’Kelly5 1Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269; 2Woods Hole Oceanographic Institution, Woods Hole, MA 02543; 3American Type Culture Collection, Manassas, VA 20110; 4Environmental Science and Public Policy Program, George Mason University, Manassas, VA 20110; 5Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME 04575 Ribosomal DNA-based studies to characterize and detect the paramoeba associated with infection of American lobsters (Homarus americanus) from western Long Island Sound (LIS) are predicated upon determination of the molecular phylogeny of members of the genus Paramoeba. Molecular phylogenetic studies based on 18S SSU rRNA gene sequences of three paramoebid and five-vexilliferid gymnamoebae derived from monoprotist cultures indicated that the Paramoebid-Vexilliferid (P-V) clade arose as a distinct lineage separate from other genera of the Gymnamoebia. Lobsters collected by trawl survey or submitted independently by fishermen or biologists in 2000 and 2001 were processed for histopathologic examination and 18S SSU rDNA amplification using order-based and genus-based primers identified by multiple sequence alignments of paramoebid and vexilliferid 18S rDNA.  An overall consensus sequence of the paramoeba from lobster was constructed by alignment of consensus sequences of three overlapping segments of SSU rDNA amplified from lobster tissues identified by histologic examination as containing paramoebae. In subsequent SSU rDNA sequence-based analyses, the lobster paramoeba demonstrated very high nucleotide sequence identity with strains of Neoparamoeba pemaquidensis, and branching of the lobster paramoeba with species of N. pemaquidensis was supported by very high distance and parsimony bootstrap values. Environmental isolates of several different strains of N. pemaquidensis have been identified from LIS, and strains of N. pemaquidensis have been implicated in other parasitic diseases of commercially relevant aquatic animal species, e.g., sea urchins and Atlantic salmon.  Primer sequences have been identified from 18S SSU rDNA sequence data that amplify rDNA of paramoeba from lobster tissues toward the development of PCR-based tests for the detection of this organism in the lobster. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page