Eric J. Schott

The Chesapeake Bay Blue Crab (Callinectes sapidus): A Multidisciplinary Approach to Responsible Stock Replenishment

The Chesapeake Bay has traditionally been one of North Americas most productive fishing grounds, supporting the worlds largest blue crab fishery. During the last several decades, fishing mortality and environmental degradation led to ∼ 70% drop in the bays blue crab abundance, 84% decline in its spawning stock, and historically low levels of juvenile recruitment as well as nursery habitats being below carrying capacity. This situation makes the Chesapeake Bay blue crab an appropriate candidate for responsible stock enhancement. A multidisciplinary, multi-institutional program was developed to study the basic biology and life cycle of the blue crab, develop hatchery and nursery technologies for mass production of blue crab juveniles, and assess the potential of using cultured juveniles to enhance blue crab breeding stocks and, in turn, bay-wide abundance and harvests. Basic biology and culture studies enabled closing the life cycle of the blue crab in captivity. Juvenile crabs have been produced year round, with excellent survival. During 2002–2006, over 290,000 cultured crabs were tagged and experimentally released into the bays nursery habitats. Cultured crabs survived as well as their wild counterparts, increased local populations at release sites by 50–250%, grew quickly to sexual maturity, mated, and migrated from the release sites to spawning grounds, contributing to the breeding stock as soon as 5 to 6 months post-release. Findings reported in this text and other articles in this volume are indicative of the feasibility of our approach of using hatchery juveniles to replenish the blue crab breeding stocks in the Chesapeake Bay.

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification, biochemical characterization, and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.

The PmSOD1 gene of the protistan parasite Perkinsus marinus complements the sod2Δ mutant of Saccharomyces cerevisiae, and directs an iron superoxide dismutase to mitochondria

The facultative intracellular oyster parasite, Perkinsus marinus, taxonomically related to both dinoflagellates and apicomplexan parasites, possesses at least two distinct genes (PmSOD1 and PmSOD2) predicted to encode iron-containing superoxide dismutases (Fe-SOD). The present study demonstrates that PmSOD1 complements a Saccharomyces cerevisiae mutant lacking the mitochondrial manganese-containing SOD (Mn-SOD), whereas PmSOD2 complements an Escherichia coli mutant lacking genes for cytosolic SOD activities. Mitochondria isolated from complemented yeast contain an SOD activity susceptible to inhibition by hydrogen peroxide, but resistant to cyanide, both characteristics of Fe-SODs. In cultured P. marinus trophozoites, indirect immunofluorescence using anti-PmSOD1 antibodies shows colocalization of PmSOD1 product with the mitochondrial marker MitoTracker Red. Further analysis of the leader sequence of the predicted PmSOD1 product revealed similarities to a mitochondrial targeting domain, an unusual observation for Fe-SODs, which are typically localized in the cytoplasm. These results suggest that PmSOD1 encodes a mitochondrial Fe-SOD, which may contribute to P. marinus resistance to exogenous oxidative damage in host phagocytes. The present study constitutes the first report of an endogenous Fe-SOD that is directed to the mitochondria, and suggests that mitochondria targeting sequences have been conserved among diverse branches of the eukaryotes, including the early protista. It also illustrates the potential of complementation-based approaches for further gene discovery and characterization in P. marinus.

ASSESSMENT OF THE NORTHERN DISTRIBUTION RANGE OF SELECTED PERKINSUS SPECIES IN EASTERN OYSTERS (CRASSOSTREA VIRGINICA) AND HARD CLAMS (MERCENARIA MERCENARIA) WITH THE USE OF PCR-BASED DETECTION ASSAYS

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.

Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus

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 double-stranded 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.

Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus

Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.

Isolation and Evaluation of New Probiotic Bacteria for use in Shellfish Hatcheries: I. Isolation and Screening for Bioactivity

ABSTRACT 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 102-104 cfu/mL. Future studies will confirm optimal dosage and positive effects of probiotic candidate OY15 on survival during long-term rearing of oyster larvae.

Structures of PmSOD1 and PmSOD2, two superoxide dismutases from the protozoan parasite Perkinsus marinus.

Perkinsus marinus, a facultative intracellular parasite of the eastern oyster Crassostrea virginica, is responsible for mass mortalities of oyster populations. P. marinus trophozoites survive and proliferate within oyster hemocytes, invading most tissues and fluids, thus causing a systemic infection that eventually kills the host. The phagocytosis of P. marinus trophozoites lacks a respiratory burst, suggesting that the parasite has mechanisms that actively abrogate the hosts oxidative defense responses. One mechanism and the first line of defense against oxidative damage is the dismutation of superoxide radical to molecular oxygen and hydrogen peroxide by superoxide dismutases (SODs). P. marinus possesses two iron-cofactored SODs, PmSOD1 and PmSOD2. Here, the crystallization and X-ray structures of both PmSOD1 and PmSOD2 are presented.

Temperature correlates with annual changes in Hematodinium perezi prevalence in blue crab Callinectes sapidus in Florida, USA

Blue crabs Callinectes sapidus were monitored biannually throughout Florida, USA, for 2 yr using a highly sensitive, quantitative polymerase chain reaction (qPCR) to determine the spatial and temporal changes in prevalence and intensity of Hematodinium perezi infections during drought years. Despite persistent drought conditions, H. perezi infections were not universally found. Overall prevalence was 25.3% (95% CI: 22.8-28.1%) in 1066 crabs sampled from 6 locations (Jacksonville, Ormond Beach, Everglades City, Tampa Bay, Steinhatchee, and Panama City) from 2011 to 2012. Presence of H. perezi was consistently highest in winter season samples, ranging from 4.2-51.1% (3 locations) in 2011, to 32-83% (5 locations) in 2012. The highest prevalence and intensities were observed in the winter samples from Everglades City. Previous studies have found that the prevalence of H. perezi in C. sapidus in temperate regions of the US East Coast shows seasonal peaks in early winter in Maryland and South Carolina and in fall and spring in Georgia. The seasonality of infections in the subtropical waters of Florida reinforces the concept that temperature is a strong factor that may override other drivers, such as drought. Seasonal H. perezi infections in Florida appear to be triggered by the parasite responding to an optimal temperature during the annual rise from the low temperature of winter when salinity is elevated. However, salinity alone is not sufficient to trigger an increase in prevalence of H. perezi in Florida.