Abstract

Mass mortalities of American lobster (Homarus americanus) occurred in western Long Island Sound (LIS) during the autumn of 1999, resulting in an estimated 90-99 percent reduction in landings compared to previous years and an estimated 11 million dead lobster. Lobster fishermen reported dead or dying "limp" lobsters, and there were concurrent reports of increased numbers of dead or dying crabs and sea urchins. Histopathologic examination of viscera and nervous tissues from "limp" lobsters revealed hemocytic polyneuritis and ganglioneuritis, multisystemic hemocytic granulomas, and intralesional amoebae. The amoebae possessed a paranuclear body that stained positively for DNA using the Feulgen technique and was subsequently demonstrated to have ultrastructural features consistent with a parasome, or "Nebenkörper," a feature associated with paramoebae of the genus ParamoebaSchaudinn, 1896. The role of this potentially pathogenic amoeba in the 1999 LIS lobster die-off has not been determined, nor have Koch's Postulates been fulfilled.

Determination of the taxonomic relationship of the lobster paramoeba to other amoebae, as well as the development of DNA-based tests for its detection in host tissues, was predicated upon determination of the molecular phylogeny of members of the genus Paramoeba, as rDNA sequence data for members of the genus were nonexistent. Monoprotist cultures of three paramoebid and five-vexilliferid gymnamoebae were obtained from major culture collections. Small subunit ribosomal DNA was amplified by PCR from extractions of genomic DNA using universal eukaryotic primers, and PCR products were isolated, ligated into TA cloning vectors, and multiple clones from each strain were sequenced. The 18S SSU rRNA gene sequences were aligned against a set of pre-aligned eukaryotic rDNA sequences available through the Michigan State Ribosomal Database Project II and Genbank using ClustalX v1.81. Phylogenetic trees were inferred by distance and parsimony optimality criteria using PAUP*.

In 2000 and 2001, lobsters collected by trawl survey or submitted independently by fishermen or biologists were processed for histopathologic examination. Frozen samples from specimens diagnosed as having microscopic evidence of the paramoeba in tissue sections were subjected to DNA extraction. Order-based and genus-based primers were identified by multiple sequence alignments of paramoebid and vexilliferid 18S rDNA and were applied in nested PCR protocols designed to amplify three overlapping segments of the 18S SSU rRNA gene. Multiple tissues from multiple lobsters were tested in triplicate, and PCR products were isolated, cloned and sequenced. An overall consensus sequence of the lobster parasite was constructed by alignment of consensus sequences of each of the three overlapping segments of SSU rDNA. Taxonomic relationships of the lobster parasite were inferred by generation of phylogenetic trees based on distance and parsimony.

Molecular phylogenetic studies resulted in a rooted tree indicating that the Paramoebid-Vexilliferid (P-V) clade arose as a distinct lineage separate from other genera belonging to the Gymnamoebia. In subsequent SSU rDNA sequence-based comparisons and molecular phylogenetic 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. Molecular phylogenetic characterization of the lobster paramoeba as a strain of N. pemaquidensis is significant to a collective understanding of the health status of the LIS lobster population. 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 netpen-reared Atlantic salmon. The 18S SSU rDNA sequence data and phylogenetic relationships from this study have been utilized in the development of DNA-based tests for the detection of this parasite in lobster tissue.

Acknowledgements

This research is supported by a grant from the University of Connecticut Research Foundation and by the Long Island Sound Research Fund, Connecticut Department of Environmental Protection, under GRANT CWF 333-R (to SF).