Optimization Of Protease Inhibitor Assays For Eastern Oyster (Crassostrea virginica) Immune Assessments   Chris G. Earnhart and Stephen L. Kaattari   Department of Environmental Sciences, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062   The ability to quantitatively monitor specific humoral defense factors may be an effective means to monitor oyster health and disease resistance.  If successfully optimized and correlated with disease resistance, such assays may also become  valuable stock assessment tools.  For the past six years, effort has been made to elucidate the role of Crassostrea virginica protease inhibitors (PI) in the inhibition of the putative pathogenic effects of serine proteases produced by the oyster parasite Perkinsus marinus. In order to develop routine stock assessment assays for such factors, it is necessary to optimize the assay and increase its reproducibility.  The hide powder azure (HPA) assay has been used to estimate relative differences in protease inhibition in oyster stocks. The protease source for the assay is a crude, concentrated culture supernatant containing P. marinus extracellular products (ECP).  The quantity of protease is estimated by the total protein content of the ECP; therefore, media and batch differences could affect apparent inhibition by oyster plasmas.  Additionally, many low molecular weight PIs may have protease specificity, so variation in the ECP protease profiles may affect assessment of specific protease inhibition.  To optimize the HPA assay, both total protease activity and the expressed protease profile required standardization.  We investigated differences in protease expression by P. marinus P-1  when grown in three media and found significant differences in specific activity against HPA among ECPs, with one ECP having extremely low activity.  Using substrate gel electrophoresis, we investigated differences in the protease profiles elicited by the media variations.  We also investigated differences in protease profiles among geographic isolates of P. marinus.  Significant differences existed in the protease profiles expressed by P-1 grown in different media, with the highest apparent protease activity found in the ECP with the lowest specific activity in the HPA assay.  This may be due to an inhibitory molecule in that media.  There were also differences in the protease profiles of P. marinus isolates.  In standardizing the HPA assay, we elected to use the ECP from the P-1 strain grown in a chemically defined medium. Quantifying ECP by specific activity, rather than by total protein content minimizes effects of batch variations.   Future experiments will try to determine the relevance of proteases to the disease process.  Purification of proteases may allow determination of critical virulence factors and targets of C. virginica protease inhibitors, which could yield tools to test and select oyster stocks with resistance to P. marinus.  This work was supported by National Sea Grant, Oyster Disease Research Program grant NA96RG0025.