SCECAP Overview The South Carolina Estuarine and Coastal Assessment Program ( SCECAP) was initiated in 1999 by the SC Department of Natural Resources (SCDNR) and the SC Department of Health and Environmental Control (SCDHEC) with the goal of monitoring the condition of the state’s estuarine habitats. Each year SCECAP measures water and sediment quality, and biological conditions at multiple random sites throughout the state’s coastal zone. These measurements are combined into an overall evaluation of estuarine habitat conditions at each site and throughout the entire SC coast. SCECAP provides habitat information to coastal managers as well as the public. You may visit the program's website ( for project findings and related reports. During the summer of 2005, a sub-set of the SCECAP sites that had oysters and six SCDNR long-term monitoring sites were assessed for oyster condition. We have been monitoring habitat quality, including diseases, at these six long term stations since Coen and Luckenbach (2000) and others have demonstrated that oysters are good indicators of environmental stress because of their filtering capabilities. These bivalves are useful indicators of water quality because they accumulate and concentrate metals, biotoxins and chemical pollutants (Capuzzo 1996 and Farrington 1983). Objectives Specific objectives of the SCECAP oyster assessment were to:  Determine the incidence and intensity of diseases, Perkinsus marinus (Dermo) and Haplosporidium nelsoni (MSX), at a subset of SCECAP sites and long-term monitoring stations;  Evaluate the physiological condition of oysters as a measure of organism health;  Evaluate recruitment potential, abundance and size frequency of live oysters at a sub-set of sites as a measure of population condition; and  Measure bacterial and viral densities at a sub-set of sites. Assays for these parameters were conducted by NOAA staff. See posters by Gooch et al. and Robinson et al. for results. ACKNOWLEDGEMENTS The authors wish to thank individuals in the MRRI- Shellfish Research Section (especially Michael Hodges, Steve Roth, Amanda Powers, Ben Dyar, Emma Gerald and Darin Jones), the MRRI- Environmental Research Section (George Riekerk, Marty Levisen, Pat Biondo, Leona Forbes, Steve Burns, Stacie Crowe and Virginia Irvin) for field assistance and NOAA-CCEHBR staff for their collaborative efforts with microbial and tissue contaminant work. This project was supported through funding obtained from NOAA-NMFS Grant No. NA04NMF LITERATURE CITED Anderson, M.E., Determination of glutathione and glutathione disulfide in biological samples. Methods in Enzymology 113: Bobo, M.Y., D.L. Richardson, L.D. Coen, and V.G. Burrell, A report on the protozoan pathogens Perkinsus marinus (Dermo) and Haplosporidium nelsoni (MSX) in South Carolina Shellfish populations, with an overview of these shellfish pathogens. SCDNR-MRD-MRRI Technical Report, 50 pp. Capuzzo, J.D., The bioaccumulation and biological effects of lipophilic organic contaminants. In V.S. Kennedy, R.I.E. Newell and A.F. Eble, The Eastern Oyster, Crassostrea virginica. College Park, Maryland: Maryland Sea Grant College. pp Coen, L.D. and M.W. Luckenbach, Developing success criteria and goals for evaluating oyster reef restoration: Ecological function or resource exploitation? Ecological Engineering 15: Farrington, J.W., Bivalves as sentinels of coastal chemical pollution. Oceanus 26: Gutteridge J.M.C. and B. Halliwell, The measurement and mechanism of lipid peroxidation in biological systems. TIBS 15: Howard, D. W., E. J. Lewis, B.J. Keller, and C.S. Smith, Histological techniques for marine bivalve mollusks and crustaceans. NOAA Technical Memorandum NOS NCCOS 5, 218 pp. Ray, S.A., A review of the culture method for detecting Dermocystidium marinum, with suggested modifications and precautions. Proceedings of the National Shellfisheries Association 54: Ringwood A.H., D.E. Conners, and J. Hoguet, Effects of natural and anthropogenic stressors on lysosomal destabilization in oysters Crassostrea virginica. Mar Ecol Prog Ser 166: Ringwood AH, D.E. Conners, C.J. Keppler, Cellular responses of oysters, Crassostrea virginica, to metal contaminated sediments. Mar Environ Res. 48:  P. marinus is common in SC, with prevalence levels generally >80% and intensity levels 80% and intensity levels <3.0 (scale of 0-6). Only one site had a mean intensity above 3.0.  H. nelsoni generally has low prevalence and intensity in SC and has not been a severe problem to date.  Only two of 21 SCECAP sites were positive for MSX. None of the six long-term sites had MSX infections, although when sampled in 2004, 4 of the 6 sites had infections with the highest prevalence level at 20%.  Most sites sampled had oysters with normal cellular responses. Only one site scored poorly on all three bioassays and an additional 5 sites scored poorly on two of the three bioassays.  There was a negative correlation between Dermo intensity level and glutathione concentration.  Maximum density of oysters, Crassostrea virginica, ranged from 750-5,320/m 2, which was greater than a ten-year statewide mean of 2,350/m 2.  Recruitment potential ranged from 890 to >23,500/m 2 with a mean of 7,276/m 2, which is greater compared to a statewide mean of 6,885/m 2.  For 2006 we are evaluating 26 additional SCECAP sites and the six LTD sites for oyster conditions.  An additional observation made from our recruitment data is that the crested oyster, Ostreola equestris, was present and tested positive for the parasite Bonamia sp. (testing conducted by Ryan Carnegie at VIMS). SUMMARY SCECAP MONITORING OF A CRITICAL ESTUARINE SPECIES ALONG SOUTH CAROLINA'S COAST: ASSESSMENT OF OYSTER DISEASES, CELLULAR RESPONSES AND POPULATION STATUS Donnia L. Richardson*, Loren D. Coen, M. Yvonne Bobo, Nancy Hadley, Andrew Hollis and Chuck Keppler Marine Resources Research Institute, SC Department of Natural Resources, 217 Fort Johnson Rd., Charleston, SC USA RESULTS Cellular Responses Disease Analysis and Cellular Responses Cellular Response:  Lysosomal destabilization – neutral red assay conducted with hepatopancreatic tissues following the methods described in Ringwood et al. (1998). Data were expressed as the percentage of cells with destabilized lysosomes per oyster.  Lipid peroxidation - measured using the thiobarbituric acid- malondialdehyde test (Gutteridge and Halliwell 1990). Resulting malondialdehyde (MDA) was detected and expressed as nM MDA/g weight.  Glutathione concentration- determined using the enzymatic recycling assay (see Anderson 1985, Ringwood et al. 1999). Glutathione concentrations were calculated from a standard curve. Sampling:  Twenty-one random SCECAP sites and six long-term disease (LTD) monitoring sites (Figure 1) were sampled for disease status and cellular responses.  Samples of 10 oysters (5 replicate samples of 2 oysters per site) were collected from the SCECAP stations and 25 oysters (5 replicate samples of 5 oysters per site) were collected from the six long-term sites. Diseases:  Perkinsus marinus (Dermo)- determined with gill, rectal and mantle tissues using Ray’s Fluid Thioglycollate Method (RFTM) (Ray 1966). Prevalence (% infected) and mean infection intensity were calculated for each site following Quick and Mackin’s (1971) scale of 0-6.  Haplosporidium nelsoni (MSX)- oyster cross sections were fixed and processed histologically using standard Harris hematoxylin and eosin (HHE) procedures (Howard et al. 2004). Infection and intensity levels were rated following Bobo et al Heavy Dermo Infection MSX Destabilized lysosome Stable lysosome Oyster Populations and Recruitment: Oyster Populations:  A sub-set (n=10) of the total SCECAP sites was sampled to assess the status of oyster populations.  Eight random oyster samples were collected using m 2 quadrats.  Samples were washed on a 1 mm sieve and oysters measured using digital calipers to determine the mean size and total number of live oysters per site. Recruitment:  Three replicate m 2 recruitment trays containing oyster shell were deployed at 19 sites (14 SCECAP sites) in 2005 and retrieved after months in Spring  Samples were washed and measured as above to determine the total number of spat per site. METHODS 2005 SCECAP and Long-term Monitoring Sites Figure 2A. Mean Dermo prevalence and intensity of oysters sampled at 21 SCECAP (blue bars) and six long-term stations (red bars). Error bars represent 1 standard error. Horizontal dashed lines denote Dermo grand mean intensity across all SCECAP and LTD sites. B. Mean Oyster Abundance A. Mean Oyster Shell Height C. Glutathione Concentration (GSH) Diseases Oyster Populations and Recruitment Potential Figure 3. Cellular responses of native oysters collected from 21 SCECAP and six long term monitoring sites. Error bars represent 1 standard deviation. (A). Percent Lysosomal destabilization, (B). Lipid peroxidation levels, (C). Glutathione concentrations. INTRODUCTION B. Lipid Peroxidation (LPx) Figure 4A -B. Mean shell height (A) and total number (B) of native live oysters (summed over eight m 2 samples) at 10 SCECAP stations. Error bars +1 standard error.  Half of the sites (13 out of 27) had oysters with LSD rates below 35%, which represents the normal range for healthy oysters. The remaining 14 sites had oysters with rates in the range representing exposure to toxins or serious toxicity (Figure 3A).  Half of the sites (14 out of 27) had LPx levels within the normal range for healthy oysters (<200nMol/g). The remaining 13 sites had LPx levels that represent an exposure to toxins or reflect serious stress responses (Figure 3B).  The majority of sites (24 out of 27) had oyster GSH concentrations within the range for healthy oysters (>900 nMol/g). The remaining sites had mean GSH concentrations that represent exposure to toxins. None of the sites had GSH concentrations (<600 nMol/g) associated with stress responses in oysters (Figure 3C). A. Lysosomal Destabilization (LSD) A. Perkinsus marinus (Dermo) Prevalence and Intensity Figure 2B. Prevalence of MSX in oysters sampled at 21 SCECAP and six long-term monitoring stations. B. Haplosporidium nelsoni (MSX) Prevalence LSD Rates >35% reflect serious stress responses. LPx levels >200 nMol/g indicate serious stress response. GSH concentrations <600 nMol/g, indicate stress response. Pearson Product Moment Correlations showed a negative relationship between Dermo intensity and glutathione (GSH) concentrations. No significant relationships were detected between Dermo intensity and lipid peroxidation (LPx) levels or lysosomal destabilization (LSD) rates.  Dermo was present at all stations with 20% to 100% of the animals infected at the SCECAP sites. Mean intensity levels at these sites varied from 0.30 (at Casino Creek) to 3.80 (at Calibogue Sound). The percentage of infected animals at the six long-term (LTD) sites ranged from 80% to 100% with mean intensity levels ranging from 1.48 (at North Inlet) to 2.92 (at Warsaw Flats) (Figure 2A).  Paired t-test analysis of Dermo grand means across all sites showed no statistical difference (p = 0.288) between the SCECAP sites (mean = 1.92, n = 10 oysters/ site) and long- term sites (mean = 2.30, n = 25 oysters/ site).  MSX was present in only 10% of the animals at two SCECAP sites (SC Aquarium and Ocella Creek). None of the long-term sites had MSX infections (Figure 2B). D. Mean Abundance of Oysters * * C. Mean Height of Oysters Figure 4C-D. Mean shell height (Fig. C) and total number (Fig. D) of oyster spat at 19 shellfish sites. Error bars +1 standard error (n = 3 replicate samples of m 2 trays, except sites denoted with *, n = 2). Horizontal dotted line denotes the grand mean across all sites. * * The percentage of large oysters (>60 mm) was low at all sites (2% to 9%), with maximum oyster sizes ranging from 79 mm to 122 mm. Mean shell height (SH) ranged from 12mm to 30 mm (Figure 4A). Total density of native oysters at the 10 SCECAP sites ranged from 750 to 5,320/m 2 (Figure 4B). Mean size of spat ranged from 12mm to 30 mm shell height (Figure 4C). Recruitment potential at the 19 sites ranged from 890 to >23,500/m 2 with a mean of 7,276/m 2 (Figure 4D). Cellular Responses C. Glutathione Concentration (GSH) B. Lipid Peroxidation (LPx) A. Lysosomal Destabilization (LSD) LSD Rates >35% indicate serious stress responses. LPx levels >200 nMol/g indicate serious stress responses. GSH concentrations <600 nMol/g indicate stress responses. The South Carolina Estuarine and Coastal Assessment Program (SCECAP, is a monitoring program initiated to evaluate the condition of the state's estuaries. It collects physical, chemical and biological data each year at approximately 50 random stations statewide. During the summer of 2005, we (SCDNR, NOAA) collaborated to sample native oyster populations at a subset of the above stations for diseases, recruitment, population indicators, cellular responses, microbial densities and tissue contaminants (see related posters by Gooch et al. and Robinson et al.). Here, we report on two oyster diseases, natural population assessments, and cellular bioassays. Disease status was assessed at 21 SCECAP and six long-term SCDNR sampling sites. Perkinsus marinus was present at all 27 sites, with prevalence levels ranging from 20%-100% and infection intensity levels (scale of 0-6) ranging from 0.30 (very light) to 3.80 (moderate). Only two of the 27 sites had detectable Haplosporidium nelsoni infections. Three bioassays, which may indicate chronic sublethal stress levels, were completed at the same sites. Lysozomal destabilization values ranged from 28%-40%, with 48% of the sites scoring 900 nMol/g). Recruitment potential at 19 sites ranged from 890- >23,500/m2, while the total oyster densities at 10 sites ranged from 750-5,320/m2. The percentage of large oysters (>60 mm) was low at all sites (2% to 9%), with mean sizes ranging from 12 mm-30 mm. These data will help to assess the status of SC intertidal oyster populations and identify potential restoration sites and related success criteria. ABSTRACT Mean Dermo Intensity vs. % LSD r 2 =0.136 P =0.058 Mean Dermo Intensity vs. GSH r2=0.156 P =0.041 Mean Dermo Intensity vs. LPx r2=0.038 P =0.326 (Mean = 2.30) (Mean = 1.92) (Mean=7,276) (Mean=19.18 mm) Figure 1. Map of sites sampled for oyster disease and health status. A sub-set of these sites was sampled for oyster populations and recruitment potential.