Development of fish vaccines, which is of great importance to aquaculture, relies on a thorough knowledge of the fish immune system. Marine perciformes, such as the gilthead seabream, are of considerable economical importance to European fish farming, but knowledge of their immune functions lacks well behind that of other cultured fishes. The first objective of the proposed research is to develop B cell lines for seabream, so that some key aspects of B cell function can be studied. Using the only unequivocal surface marker of B cells in fish, membrane immunoglobulin (mIg), B cells will be isolated and cell lines will be established according to methods proven successful for catfish. B cell responsiveness to LPS or mIg cross-linking will be tested by measuring the mRNA and protein levels of secreted (s) 1g and M19. The second objective is to investigate the involvement of complement factor 3 (C3), a central component in the complement cascade, in stimulating antibody production. Complement was shown to be an important regulator of humoral immune responses in mammals. Because fish display a wide diversity in complement factors, and complement is a major contributor to host defense in fish, studying the effects of C3 on antibody production is particularly exciting and may lead to new strategies for fish vaccination. We will study the effects of various C3 isoforms isolated from seabream in combination with mIg cross-linking or antigens on Ig mRNA and protein levels.