A molecular tool for environmental monitoring of Hematodinium sp., a lethal parasite of blue crab

The blue crab (Callinectes sapidus) fishery is important to the economy and social structure near the Chesapeake and Delmarva coastal bays.  Periodically, populations of blue crab within the lower Chesapeake and coastal bays suffer mortalities associated with infections by the parasite Hematodinium sp.  These recurring outbreaks appear to be geographically localized in "hotspots," suggesting that there may be environmental or biotic reservoirs of the parasite.  To understand disease dynamics and possibly enable forecasts of disease outbreaks, methods to detect the presence of Hematodinium sp. in environmental samples are needed.  Numerous studies on the prevalence and intensity of Hematodinium sp. in blue crab have been conducted using histological techniques.  Although these techniques provide morphology and localization of parasites within a host, they are impractical for disease reservoir investigations, which may include sediment and water, and invertebrate gut contents.  Molecular (DNA)-based methodologies can be applied to any DNA sample, whether from a single organism or consortium within environmental samples.  We have developed a quantitative polymerase chain reaction (Q-PCR) assay, based on the sequence of the ITS2 region of the rRNA gene cluster, that provides the sensitivity and specificity needed to quantify Hematodinium sp. DNA in environmental or reservoir host samples.  A requirement for meaningful PCR-based quantification is a standard curve based on a dilution of the target organism that approximates the environmental samples that will be later processed.  Because the putative life stage of Hematodinium sp. that may inhabit sediment or water is not available, we created a surrogate using the cloned ITS2 target gene in the bacterium E. coli.  Q-PCR assays using DNA from "pseudo-Hematodinium" in sediment generated standard curves that parallel standard curves using purified DNA.  Using the ITS2 assay and standard curve, we are searching for evidence of Hematodinium sp. in the MD and DE coastal bays.