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Hematodinium is a genus of parasitic dinoflagellates whose species have caused significant mortalities in marine crustacean fisheries worldwide. A species of Hematodinium infects the blue crab, Callinectes sapidus on the eastern seaboard of the USA. The mode of transmission of the parasite in blue crabs is unknown. We established several continuous in vitro cultures of Hematodinium sp. isolated from the haemolymph of infected blue crabs. One isolate has been continuously maintained in our laboratory through serial subcultivation for over 12 months, and is capable of infecting new hosts when inoculated into healthy crabs. Cells of the parasite undergo characteristic developmental changes in vitro consistent with the identifiable stages of Hematodinium sp.: filamentous trophonts, amoeboid trophonts, arachnoid trophonts and sporonts, sporoblasts, prespores and dinospores (macrospores and microspores). Additionally, we describe an unusual shunt in the life cycle wherein presumptive schizonts derived from arachnoid sporonts developed into filamentous and arachnoid trophonts that can then initiate arachnoid sporonts in new cultures. This may explain the rapid proliferation of the parasite in blue crab hosts. We also found that temperature and light intensity affected the growth and development of the parasite in vitro.

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Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster.KEY WORDS: Crustacea · Viral disease · DNA probe · In situ hybridization · Florida Keys · Diagnostics Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [185][186][187][188][189][190][191][192] 2006 a labeled gene probe to a specific target nucleic acid sequence (Singer et al. 1989). ISH has been subsequently applied to diagnosis of several crustacean viruses, such as Baculovirus penaei (BP) (Bruce et al. 1993(Bruce et al. , 1994, white spot syndrome virus (WSSV) (Durand et al. 1996, Lo et al. 1997, Nunan & Lightner 1997, Chang et al. 1998, hepatopancreatic parvovirus (HPV) (Pantoja & Lightner 2001, Phromjai et al. 2002 and gill-associated virus (GAV) (Spann et al. 2003). ISH has also been applied to the diagnosis of several other pathogens of marine organisms (Stokes & Burreson 1995, Chang et al. 1996, Lo et al. 1997, Pantoja & Lightner 2001, Carnegie et al. 2003, Small et al. 2006. It is a useful tool to detect the presence of virions in infected tissues and determine tissue tropism of viral infection in host. Therefore, the objective of this study was to develop a fluorescence in situ hybridization (FISH) assay for the diagnosis of PaV1 infections in lobsters. MATERIALS AND METHODSSample collection. Juvenile spiny lobsters Panulirus argus were collected from several sites throughout the Florida Keys, USA. Tissue samples of the hepatopancreas, hindgut, foregut, gill, heart, skin, nerve, and in some cases ovary, were dissected and fixed in 10% neutral buffered formalin for approximately 48 h and then held in 70% EtOH until further process. Fixed tissues were dehydrated, embedded in paraffin and sectioned at 5 µm thickness on a rotary microtome. To verify the presence ...

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