Introduction Callinectes sapidus, blue crab, is an important fishery commodity in North America. It is economically and ecologically significant. In recent years two disease causing organisms have caused concern for hatchery and aquaculture facilities; Hematodinium sp. and a Reo-like virus. Hematodinium sp. is a parasitic dinoflagellate that is found in the hemolymph and tissue of the blue crab. It is highly lethal and eventually causes organ failure in high intensity infections. It causes seasonally re-occurring mass mortalities in wild blue crab populations. The reo-like virus, RLV is a highly lethal virus that has been found in high prevalence in stressed crabs of shedding tanks within crabbing and aquaculture facilities. This virus is suspected to play a major role in up to 50% of shedding crab mortalities. Molecular techniques are an effective way to detect both pathogens. Assays are sensitive and specific enough to detect its presence in the very small early life stages of blue crabs. Most diseases are studied in large juveniles and adult crabs due to them being more significant in the fishery. As a result, disease causing organisms in young juveniles and earlier life stages have not been well studied. Using molecular based techniques we are looking for the prevalence of two disease causing organisms in early life stages of blue crabs. Using molecular methods to monitor pathogens in early life history of blue crabs (Callinectes sapidus) Abstract Although diseases have the potential to affect blue crab populations, there have been few studies on the impact of diseases on overall mortality, especially in early life history. The protozoan parasite, Hematodinium sp., is a significant blue crab pathogen in Atlantic coastal bays of North America. It has also been recently reported that a reo-like virus (RLV) is associated with blue crab mortality in soft-shell crabs operations in from the Mid-Atlantic to Gulf of Mexico. PCR-based assays for specific diseases can permit disease prevalence assessments of large numbers of very small life stages that would otherwise be impractical to test by histological methods. With support by the NOAA-Living Marine Research Cooperative Science Center, a collaboration involving a NOAA/NCCOS lab, two undergraduate institutions, and a university research center is using specific PCR assays to detect Hematodinium sp. and RLV in megalopae and young juvenile crabs in the salt marshes near Savannah, Georgia. Collections of juvenile crabs were made throughout the summer of 2010, and megalopae were collected in the fall of Preliminary analyses indicate very low prevalence of both diseases. Figure 3. Standard curve graph of the Hematodinium sp. ITS2 q-PCR assay. This is used to calculate the number of parasite gene copies in a given sample. Results Table 1. Table shows results of the PCR assays for the juveniles measuring mm carapace width. Three juveniles from collection date showed evidence of RLV by PCR. Juveniles were collected from Country Club Creek of Savannah, GA salt marsh with n=80. Juveniles from collection date have not yet been analyzed for RLV. Acknowledgements Dr. Rosemary Jagus (UMCES-IMET) LMRCSC-UMCES-IMET, funded by NOAA-EPP References Messick & Shields 2000, Dis Aquat Org, 43: Bowers et. al. 2010, Dis Aquat Org, 93:17-19 Figure 1. Sampling area in Savannah, GA Table 2. Table shows results of the PCR assays of the collected megalopae and C1 juveniles (<5mm carapace width) from areas within Thunderbolt, Ft. Pulaski, and Savannah river. Total number of megalopae and C1 juveniles collected was 111 and 29 respectively. No evidence of Hematodinium sp. has been seen in any samples. Assays for RLV have yet to be completed. Figure % agarose gel shows the results of RLV rt-PCR assay on sample set. Samples in lanes 1-3 produced a band of the expected size. Using molecular techniques to monitor pathogens: Juveniles and megalopae can be reduced to nucleic acids which are then used in an assay to screen for either Hematodinium sp. or RLV Polymerase chain reaction (PCR) methods are rapid, specific and sensitive Issue: Blue crabs are economically and ecologically significant Seasonally re-occurring mass mortalities of blue crab due to Hematodinium sp. infections High prevalence of RLV in blue crab mortalities within soft shell and aquaculture facilities Conclusions and Future Studies Evidence of RLV seen in 3 blue crab juveniles of the mm carapace width size class, but not seen in megalopae or C1 juveniles No evidence of Hematodinium sp. in any crabs collected PCR assays indicate low prevalence of Hematodinium sp. and RLV in collected samples. Initial study shows low prevalence of both diseases however future studies with greater samples numbers may show a higher prevalence A related study planned for summer 2011 will look for Hematodinium sp. and RLV in adults and juveniles in the Northern range of blue crab Methods Collect samples Megalopae, using 500 μm plankton net suspended from dock during flood tides within Thunderbolt, Ft. Pulaski, and Savannah river, Savannah, GA Juveniles, using dip nets within Country Club Creek of Savannah, GA Extract DNA and RNA DNA for Hematodinium sp., which is eukaryotic and has its genetic information stored as DNA RNA for RLV, which is a reovirus and has its genetic information stored as dsRNA Screen for disease causing organisms Hematodinium sp. using quantitative-PCR assay RLV using reverse transcription-PCR assay Questions: Do crabs settling into nursery habitat carry Hematodinium sp. or RLV infections? How early do crabs acquire RLV and Hematodinium sp. infections? Does disease play a role in the mortality of blue crab early life stages? A. Hanif 1 *, M. Ogburn 2, S. White 3, P. Bedu 4, E. Schott 1 1 University of Maryland Center for Environmental Science-Institute of Marine and Environmental Technology 2 Savannah State University, Marine Sciences 3 NOAA Center of Excellence for Oceans and Human Health, Hollings Marine Laboratory 4 Delaware State University Collection date N life stage Hematodinium sp. prevalence (%) RLV prevalence (%) MegJuv pending pending pending pending pending pending pending pending Collection date N Hematodinium sp. prevalence (%) RLV prevalence (%) pending PCR results of blue crab megalopae and C1 juveniles measuring <5 mm carapace width PCR results of blue crab juveniles measuring mm carapace width Map of collection area Legend M Apex 100bp ladder samples 9 heavily infected crab 10cloned virus 11no template samples controls PCR product 1kb 500bp 100bp M Cycle number Copy number 1.5 % agarose gel of RLV PCR assay of sample set Chromatogram of standard curve used in Hematodinium sp. q-PCR assay Drawings, K. Forrest.VA Inst Mar Sci L. Lawrence, V. Clark, J. van Montfrans, & S. Musick Juvenile crab Mature crab Eggs Zoea Megalopa Figure 1. Out of a clutch of ~2 million eggs approximately 4-5 reach maturity. It is unknown the role disease plays in these mortalities at the early life stages. Blue Crab life cycle