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Molecular Characterization of the Ichthyobodo necator Complex: An Important Fish Ectoparasite

Ichthyobodo necator is a member of the Order Kinetoplastida and an important fish ectoparasite with a broad host and ecological range. When Ichthyobodo were exposed to the anesthetic tricaine, buffered with sodium bicarbonate, parasites remained attached to the skin of fish. When parasites were exposed to unbuffered tricaine, they detached almost completely from the skin. This finding indicated that tricaine should always be buffered when fish are to be clinically evaluated to prevent a reduction or complete loss in parasite load. Unbuffered tricaine was used to collect large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female) for DNA isolation and analysis. A method for obtaining samples for DNA isolation without use of tricaine was also determined. Four preservation methods (Utermohl's solution, ethanol, formalin, freezing) and two DNA isolation methods (DNA isolation kit, crude lysate) were examined. It was concluded that ethanol-fixation was the best preservation method for DNA isolation and PCR amplification of Ichthyobodo DNA. Using these methods, genomic DNA was isolated from Ichthyobodo trophonts collected from five freshwater and one marine fish. The 18S ribosomal RNA (rRNA) gene from each isolate was PCR amplified, cloned and sequenced. The 18S rRNA gene suggested that rather than being a single species, Ichthyobodo represented a complex of at least four different species, each of which occupied a distinct geographical region. The 18S rRNA gene was also amplified and cloned for related kinetoplastid speceis. Phylogenetic relationships within the Order Kinetoplastida (suborders Bodonina and Trypanosomatina) were determined. The suborder Bodonina appeared to contain at least 3 major lineages, none of which correlated with currently recognized families. Ichthyobodo had the most divergent sequence within the Bodonina, indicating it was the most genetically distinct bodonid currently known. The study of Ichthyobodo has contributed to a new understanding of phylogenetics and systematics for the Order Kinetoplastida, as well as insight into the potential for the spread of this parasite locally, nationally and internationally.