Comparison of the roles of IgM in systemic and mucosal immunity via tissue distribution analysis in largemouth bass ( Micropterus salmoides )

Immunoglobulin M (IgM), an important immune effector, plays a crucial role in protecting fish against various pathogens. Largemouth bass (Micropterus salmoides) is an economically important farmed fish, but the understanding of IgM characteristics and function in largemouth bass is still lacking. In the present study, the largemouth bass IgM heavy chain gene was first cloned. Sequence analysis showed that cDNA of the IgM heavy chain contains an open reading frame (ORF) of 1740 bp. The ORF of the largemouth bass IgM heavy chain encodes a polypeptide of 579 amino acids that consists of a signal peptide, a variable region (VH) and four constant regions (CH1, CH2, CH3 and CH4). Next, largemouth bass IgM was purified from serum, and polyclonal antibodies against IgM were developed using purified IgM as an antigen. Western blotting analysis showed that the polyclonal antibodies reacted with the heavy and light chains of largemouth bass IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the polyclonal antibodies could also react with membrane-bound IgM (mIgM) on the surface of B cells. Flow cytometric analysis revealed that the percentages of mIgM+ B lymphocytes in the peripheral blood, spleen and head kidney were higher than those in the gill in largemouth bass. qRT-PCR analysis showed that the gene expression of the IgM heavy chain in the head kidney and spleen was significantly higher than that in the gill, intestine and skin. Moreover, indirect ELISA showed that the IgM contents in serum were higher than those in mucus and that specific serum antibody levels were significantly increased after Aeromonas hydrophila immunization. These results demonstrated that the largemouth bass IgM gene was first cloned and characterized in this study and that IgM may play a more important role in systemic immunity than in mucosal immunity.