Discovery of two IgM subclasses in Nile tilapia (Oreochromis niloticus) provides insights into IgM evolution in teleost fish.

Immunoglobulin M (IgM) is considered the most ancient and prevalent Ig in fish, which plays important roles in systemic and mucosal immunity. In the current study, two heavy chain genes of IgM (named Ighμ1 and Ighμ2) were cloned in Nile tilapia (Oreochromis niloticus), and the amino acid identity between the constant regions of the two IgM heavy chains were 82.58 %. Cysteine and tryptophan residues that are crucial for forming disulfide bonds and the folding of the Ig domains were conserved. Genomic localization showed that the two Ighμs were adjacent within the same IgM locus. By mass spectrum detection in the native IgM purified from tilapia serum, the specific peptides in the heavy chains of each IgM could be detected. At the transcriptional level, Ighμ1 and Ighμ2 had similar expression patterns, and both were constitutively expressed in both systemic and mucosal lymphoid tissues. Detection of the expression of Ighμ1 and Ighμ2 in single IgM B cells sorted by flow cytometry revealed that Ighμ1 and Ighμ2 are mainly co-expressed by single IgM B cells. After Poly I:C, Aeromonas hydrophila or Streptococcus agalactiae treatment, Ighμ1 and Ighμ2 exhibited different expression profiles, the transcriptional level of Ighμ1 mainly involved in bacterial infection while Ighμ2 was more related to Poly I:C stimulation. These data from the genome, transcription, and protein levels have demonstrated that there exist two subclasses of IgM in tilapia, and that their heavy chain genes display different expression patterns during stimulation. Overall, our data reflect the diversity and complexity of IgM in tilapia, thus provide a better understanding of the IgM evolution in teleost fish.