Distribution of Shell Formation Proteins in Oyster Hemolymph, Hemocytes, and Mantle Tissue
The occurrence and composition of L,3-4-dihydroxyphenylalanine-containing proteins (L-DOPA proteins) that participate in oyster shell formation has not been fully determined. It is known that the oyster mantle tissue is primarily responsible for shell formation and recent research has demonstrated the involvement of the hemolymph (blood) and hemocytes (blood cells). L-DOPA proteins are known to aid in the cross linking of shell formation proteins, in turn creating the insoluble organic matrix formed to produce the organic component of the shell. Using the biomarker amino acid L-DOPA, this research focuses on determining the localization of these shell formation proteins in hemocytes, hemolymph, and mantle tissue of Crassostrea virginica (the Eastern oyster). In order to study the localization of these proteins, rapid shell formation/repair will be induced by notching the oyster (mimicking predation) and shell protein composition and location will be determined as the oyster repairs the shell. Proteins responsible for shell formation and regeneration containing L-DOPA will be collected from the adductor muscle near the site of notching in the oysters. These proteins will be further examined after centrifugation by amino acid analysis of the cell pellet (hemocytes), supernatant (hemolymph), and mantle tissue rinsed in filtered sea water. The newly regenerated shell, like the other samples, will be extracted and analyzed for protein composition and distribution as well. All samples will be extracted at regular intervals beginning at time of induction and continuously throughout shell regeneration (t=0hrs, 48hrs, 96hrs, 168hrs, 2 weeks, 3 weeks, 4 weeks) in order to determine their amino acid composition. Amino acid analysis will be done using integrated pulse amperometryanion exchange high performance liquid chromatography.