Detection and quantification of the free-living stage of the parasitic dinoflagellate Hematodinium sp. in laboratory and environmental samples

Species of Hematodinium are highly pathogenic parasitic dinoflagellates and can cause substantial mortalities in many decapod crustaceans. Periodic outbreaks of Hematodinium disease have significantly impacted stocks of the blue crab Callinectes sapidus throughout the Delmarva Peninsula. Using a primer set targeting the internal transcribed spacer region 1 (ITS1) of the ribosomal RNA (rRNA) gene complex of Hematodinium, we confirmed the presence of the parasite's DNA in water and suspended sediment samples collected from Virginia estuaries of the Delmarva Peninsula by polymerase chain reaction (PCR) on DNA extracted from these environmental samples. Amplicon sequences were highly conserved and 99-100% similar to the ITS1 sequence of the endemic Hematodinium sp. reported from blue crabs in the region. A sensitive real-time PCR assay was developed and validated for the detection and quantification of the parasite DNA. The assay has a limit of detection of less than one parasite cell in 100 ml of environmental water or one gram of sediment; it will provide a valuable tool for our ongoing studies on the transmission dynamics of this parasite. The real-time PCR assay was further tested on a free-living stage of Hematodinium. Sporulation resulting in the release of millions of Hematodinium dinospores from heavily diseased crabs was observed in the laboratory. Some dinospores survived up to 7 days in aquaria outside of the crab host. We speculate that the Hematodinium sp. dinospores have a short free-living stage in the water column and that transmission to new hosts must occur relatively quickly in estuarine habitats.