Antibody-mediated immune response of fish during development of acquired resistance against larvae of freshwater mussels

The glochidia larvae of most freshwater mussels are obligate ectoparasites on fishes, on which they metamorphose into juveniles. We have demonstrated that bluegill sunfish (Lepomis macrochirus) develop acquired resistance to glochidial larvae of Utterbackia imbecillis after 2 infections, resulting in reduction of successful metamorphosis during the 3rd and 4th infections and significantly fewer glochidia attaching to fish during the 4th infection. Since the mechanism resulting in acquired resistance is not fully understood, one possible mechanism, antibody-mediated immune response, was examined. We collected sera from fish at 5-day intervals during 4 sequential infection periods and from naive fish. Western blots of glochidial proteins probed with the sera revealed that naive fish had natural antibodies against a ~35 kDa protein in the larval profile, a protein that also was recognized by immune sera. Immunochemical studies on whole-mount glochidia probed with serum from naive fish demonstrated that the natural antibodies recognize granular structures located between the larval mantle and shell. The antibodies in naive bluegills only recognized proteins in U. imbecillis and another anodontine mussel species. The antibodies did not recognize proteins in larvae of two other mussel species from a different unionid mussel taxon, the subfamily Ambleminae. In addition to the ~35 kDa protein, other larval proteins were recognized by antibodies in sera collected during each infection period, but not from naive fish. This study suggest that serum antibodies play a role in the development of acquired resistance in fish against glochidial larvae of freshwater mussels during multiple infections.