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Abstracts
Workgroup: Genetics and Oyster Populations
Combined use of a lytic peptide and protease inhibitor: a novel approach to eliminate Perkinsus marinus in eastern oysters
Principal Investigator(s):
Jerome F. La Peyre, Department of Veterinary Science, Louisiana State University. jlapeyre@agctr.lsu.edu
Co-Investigator(s):
Richard K. Cooper, Department of Veterinary Science, Louisiana State University; Terrence R. Tiersch, School of Wildlife and Fisheries, Louisiana State Univ.
Funding Period: 9/1/97-8/31/99
We identified five protease inhibitors out of ten tested (i.e., AEBSF, chymostatin, "1-antitrypsin, "2-macroglobulin, potato chymotrypsin inhibitor I and II) that decreased P. marinus protease activity and inhibited the propagation of the parasite in vitro. We found three synthetic lytic peptides out of ten tested (i.e., Phor-21, Agni-21 and Thor-21) that were lethal to P. marinus. The protease inhibitors protected the lytic activity of the peptides against degradation by the parasite protease. In addition, none of the lytic peptides and protease inhibitors (except for AEBSF at the highest concentrations) were found to be toxic to oyster cells in primary cultures.
Two protease inhibitors (chymostatin and potato chymotrypsin inhibitor I) were selected to test their effects against P. marinus in combination with the lytic peptide phor-21 because of their high inhibitory activity against the parasite, commercial availability, relatively low price, low toxicity to oyster cells, stability and solubility in saline, low molecular size and, availability of synthetic analogues and of gene sequence information. The selected lytic peptide and protease inhibitors were not found to be toxic to hemocytes or to interfere with the hemocytes' host defense related activities (i.e., hemocyte motility, phagocytosis and killing index). Our major finding was that chymostatin alone and Phor-21 with either protease inhibitor significantly reduced the number of parasites in infected hemocytes. These results suggest that entry of the protease inhibitors into infected hemocytes is critical for their activity against P. marinus since chymostatin, a low molecular weight protease inhibitor that can readily enter cells, was most effective.
The lytic peptide phor-21, the protease inhibitor chymostatin and a combination of the two were used to treat oysters and their effects on P. marinus numbers in hemolymph and whole oysters were determined. Although there was an apparent decrease in P. marinus in the hemolymph of oysters injected with chymostatin and chymostatin combined with phor-21 and an apparent increase of parasite in control oysters (saline injected) no significant difference in parasite numbers in hemolymph or whole oysters were found between treatments. The limited dose of chemicals injected and their dilution in the oyster circulatory system as well as the lack of statistical power due to the large standard deviation in parasite numbers may be responsible for the lack of a significant difference.
As part of this study, we also improved two critical techniques to determine the number of P. marinus parasites in whole oysters as well as in oyster hemolymph and developed a protocol to isolate and establish primary cultures of eastern oyster hemocytes.
IMPACTS AND/OR BENEFITS
The identification of factors lethal to the oyster parasite P. marinus in combination with factors that can inhibit the parasite protease, a putative virulence factor, is a novel approach which can lead to the development of resistant oysters by either genetic manipulation or genetic selection. We used commercially available protease inhibitors and lytic peptides synthesized at LSU to test our original hypotheses and are now in the process of identifying protease inhibitors and lytic peptides found naturally in oyster hemocytes that limit infection by P. marinus. The identification of these defenses against P. marinus will provide endogenous genes for developing resistant oysters by increasing gene expression through genetic engineering. Alternatively, the identified factors can be useful as selection markers for breeding resistance to P. marinus. This work opens the door to significant future benefits through the genetic improvement of eastern oysters.
PROJECT PUBLICATIONS
La Peyre, J. F., H. S. Kristensen, K. C. McDonough, and R. K.Cooper. 1998. Effects of protease inhibitors on the oyster pathogen Perkinsus marinus and oyster cells in vitro. #18-2. Third International Symposium on Aquatic Animal Health, August 30 - Sept. 3, 1998. Baltimore, Maryland. Book of Abstracts p. 151.
La Peyre, J. F., K. C. McDonough, and R. K. Cooper. 1998. Killing of the oyster pathogen Perkinsus marinus with synthetic antimicrobial peptides in vitro and modulation by the pathogen proteases. #S29-2. Third International Symposium on Aquatic Animal Health, August 30 - Sept. 3, 1998. Baltimore, Maryland. Book of Abstracts p. 187.
Kristensen, H. S., J. F. La Peyre, and R. K. Cooper. 1998. Effects of protease inhibitors on the oyster pathogen Perkinsus marinus in vitro. American Fisheries Society Meeting, Feb. 4-5, 1998. Bay St. Louis, Mississippi. Book of Abstracts p. 16.
La Peyre, J.F., Smith, A.K. and Cooper, R.K. 1999. Growth inhibition of isolates of the oyster pathogen Perkinsus marinus in vitro with protease inhibitors. in vitro Cellular and Developmental Biology 33(3-II):35A.
Coates, G.M., Cooper, R.K. and La Peyre, J.F. 1999. Improvement of the whole-oyster procedure for enumerating Perkinsus marinus in oyster tissues. Journal of Shellfish Research 18:328.
Nickens, A. D., and J.F. La Peyre. 2000. Optimization of quantification of Perkinsus marinus infections in oyster hemolymph. Aquaculture America 2000, U.S. Chapter, World Aquaculture Society. Feb. 2-5, 2000, New Orleans.
Nickens, A.D., T.R. Tiersch, and J.F. La Peyre. 2000. Effects of a lytic peptide and protease inhibitors on hemocyte functions of eastern oysters. Aquaculture America 2000, U.S. Chapter, World Aquaculture Society. Feb. 2-5, 2000, New Orleans.
La Peyre, J.F. and Li Y. 2000. Isolation and primary culture of eastern oyster hemocytes. Journal of Shellfish Research 19:646.
Nickens, A.D., Tiersch, T.R. and La Peyre, J.F. 2000. Effect of lytic peptide and protease inhibitors on Perkinsus marinus in infected hemocytes of eastern oysters. Journal of Shellfish Research 19:647.
Nickens, A.D., Wagner E. and La Peyre, J.F. 2000. Improved procedure to count Perkinsus marinus in eastern oyster hemolymph. Journal of Shellfish Research 19:665.
Nickens, A. D., La Peyre, J. F. and Tiersch, T. R. 2001. Treatment of Perkinsus marinus infected oyster hemocytes with protease inhibitor chymostatin in vitro. Aquaculture 2001, Orlando, FL, January 21-25. Book of Abstracts p. 476.
Nickens, A.D. 2001. Combined effects of a lytic peptide and protease inhibitors on Perkinsus marinus in the eastern oyster, Crassostrea virginica. M.S. thesis, Louisiana State University, Baton Rouge, LA.
Nickens, A. D., La Peyre, J. F., Wagner E. and Tiersch T.R. 2002. Improved procedure to count Perkinsus marinus in eastern oyster hemolymph. Journal of Shellfish Research 21:275-732.
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