Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of doublestranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in > 50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.

Hatchery production of shellfish seed is necessary to supplement natural recruitment, which is constrained by various stresses, including habitat loss, pollutant contamination, overfishing, and climate change. Bacterial diseases are considered to be a major cause of mortality in hatchery shellfish larviculture; however, overuse of antimicrobials can result in development of resistant strains of bacterial pathogens. The use of probiotics for disease prevention and improved nutrition in aquaculture is becoming increasingly popular as the demand for environmentally-friendly aquaculture grows. The objective of this study was to isolate and evaluate the efficacy of new probiotic bacteria that, incorporated into functional foods for use in shellfish hatcheries, may significantly improve larval survival. First, 26 probiotic-candidate bacteria were isolated from oysters, scallops, and a mass culture of green algae. Fifteen of these isolates (8 oyster strains and 7 bay scallop strains) inhibited known scallop-pathogen bacterial strains B183 and B122 in disk-diffusion assays. Similar to control (unchallenged) oyster larvae, survival of oyster larvae exposed to these 15 probiotic candidates for 48 h was more than 90%. The probiotic candidates were then reisolated from challenged larvae and characterized by Gram stain, colony morphology on solid agar, and the Biolog Bacterial Identification System, finding only 7 distinct strains. Using 12-well microplate assays, 5-day challenges were performed to confirm positive effects of these 7 probiotic candidates on larval survival when challenged with pathogen B183. Oyster larvae exposed to probiotic candidate OY15 had the highest survival; furthermore, survival of pathogen-challenged larvae was significantly improved by the presence of OY15 compared with pathogen alone. In addition, probiotic candidate OY15 exhibited no toxic effects on the microalgal feed strain Isochrysis sp. (T-ISO) in the range of 10 2 -10 4 cfu/mL. Future studies will confirm optimal dosage and positive effects of probiotic candidate OY15 on survival during long-term rearing of oyster larvae.

BackgroundHematodinium perezi, a parasitic dinoflagellate, infects and kills blue crabs, Callinectes sapidus, along the Atlantic and Gulf coasts of the United States. The parasite proliferates within host hemolymph and tissues, and also produces free-swimming biflagellated dinospores that emerge from infected crabs. Infections in C. sapidus recur annually, and it is not known if biotic or environmental reservoirs contribute to reinfection and outbreaks. To address this data gap, a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed to asses the temporal and spatial incidence of the parasite in Delaware and Maryland coastal bays.ResultsA previously-used PCR assay for H. perezi, based on the small subunit rRNA gene sequence, was found to lack adequate species specificity to discriminate non-Hematodinium sp. dinoflagellate species in environmental samples. A new ITS2-targeted assay was developed and validated to detect H. perezi DNA in sediment and water samples using E. coli carrying the H. perezi rDNA genes. Application of the method to environmental samples identified potential hotspots in sediment in Indian River Inlet, DE and Chincoteague Bay, MD and VA. H. perezi DNA was not detected in co-occurring shrimp or snails, even during an outbreak of the parasite in C. sapidus.ConclusionsH. perezi is present in water and sediment samples in Maryland and Delaware coastal bays from April through November with a wide spatial and temporal variability in incidence. Sampling sites with high levels of H. perezi DNA in both bays share characteristics of silty, organic sediments and low tidal currents. The environmental detection of H. perezi in spring, ahead of peak prevalence in crabs, points to gaps in our understanding of the parasite’s life history prior to infection in crabs as well as the mode of environmental transmission. To better understand the H. perezi life cycle will require further monitoring of the parasite in habitats as well as hosts. Improved understanding of potential environmental transmission to crabs will facilitate the development of disease forecasting.

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