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Abstracts
Workgroup: Frontiers in Disease Research
Role of oyster lysosomal enzymes in disease resistance
Principal Investigator(s):
Fu-Lin E. Chu, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA, chu@vims.edu
Co-Investigator(s):
Peter Van Veld, Virginia Institute of Marine Science, College of William and Mary
Funding Period: May 1, 1994 to December 31, 1996.
The objectives of the project were to elucidate the role of oyster lsosomal enzymes in defense against the parasite, Perkinsus marinus and determine the effects of temperature and salinity on lysosomal enzyme activities in oysters. During the funding period, we performed experiments to: 1. determine the synergetic effects of temperature and salinity (3, 10, 20 ppt at 10 and 25ƒC) on extracellular and intracellular lysosomal enzyme activities in oysters, 2. compare the extracellular and intracellular lysosomal enzyme activities of oysters from high salinity and low salinity habitats, 3. determine if release of lysosomal enzymes occurs when oyster hemocytes are challenged with P. marinus infective particles, 4. isolate and partially purify lysozyme from oyster plasma, 5. determine the effects of partially purified lysozyme on the proliferation and function of P. marinus. The following summarizes the results from the above experiments. Both temperature and salinity affected the activities of plasma and hemocytes, but no consistent pattern was observed in the examined enzymes (lipase, acid phosphatase, L-aminopetidase) except lysozyme. While there was no interaction effect of temperature and salinity on plasma lysozyme activity, the interaction of temperature and salinity significantly affected the hemocyte lysozyme activity in oysters. Generally, no significant temperature and salinity effects were noted on lysosomal enzyme activity in oyster tissues. Oysters collected from the low salinity habitat of the Chesapeake Bay demonstrated significantly higher lysozyme activity in plasma and gill-mantle tissues than oysters collected from the high salinity habitat. Apparently the gill and mantle are sensitive to changes in temperature and salinity. When hemocytes were challenged with P. marinus meronts, no increased lysozyme activity was noted compared to the non-challenged hemocytes. A methodology was developed and established to isolate and partially purify lysozyme from oyster plasma. The partially purified plasma lysozyme was further analyzed using SDS-PAGE. Two bands were identified with molecular weights of 38.8 and 18.2 kDa. When P. marinus meronts were treated with the partially purified plasma lysozyme, however, no significant adverse effect (e.g., viability) was detected.
IMPACTS and/or BENEFITS:
Although there is not a clear cut about the role of lysosomal enzymes in defense against Perkinsus marinus, results of the project provide information for a better understanding of oyster physiology and internal defense and how the two important environmental factors, temperature and salinity affect the oyster's physiology and defense against the parasite, P. marinus. The successful development of a methodology to isolate and partially purify lysozyme from oyster plasma is critical for further study on elucidation and characterization the role of lysozyme in the internal defense of aquatic invertebrates and molluscan bivalves in particular. Currently, colleagues at Louisiana State University (Drs. La Peyre and Xue) are employing and modifying the lysozyme purification methodology established from this project to further purify the oyster plasma lysozyme and characterize its effects in against pathogenic bacteria and P. marinus. Results generated from the project have been disseminated via presentations in scientific and professional conferences and publications. Two additional manuscripts are in preparation for publication.
PROJECT PUBLICATIONS:
Volety, A. K., F-L E. Chu, and L. A. Cruz-Rodriguez. 2001. Partial purification and characterization of lysozyme-like proteins from the plasma of the eastern oyster, Crassostrea virginica. J. Shellfish Res. 20:558.
Chu, F.-L. E. 1999. Effects of temperature, salinity, and environmental pollutants on cellular and humoral responses in oysters (Crassostrea virginica). J. Shellfish Res. 18: 321.
Chu, F.-L. E., A. K. Volety, and Georgeta Constantin. 1996. Intracellular and extracellular lysosomal enzyme activities in eastern oysters (Crassostrea virginica). J. Shellfish Res. 15:514.
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