Purification and sequencing of a mate-recognition protein from the copepod Tigriopus japonicus

Male copepods in the genus Tigriopus use surface proteins of female copepods to recognize appropriate mates. In a previous study of pre-copulatory mate-guarding in T. japonicus Mori, a monoclonal antibody was developed against one protein that contributed to the attractiveness of female T. japonicus as mate-guarding partners. In the present study, which was performed between December 1998 and November 2000, the antibody was used to purify two proteins from homogenates of pooled T. japonicus. A 70 kD protein was partially sequenced using mass spectrometry, revealing sequence similarity to α₂-macroglobulin. It was shown that the 70 kD protein was highly expressed in stage CV females, the most attractive stage for mate-guarding. A purified 36 kD protein was also shown to be a cognate of α₂-macroglobulin. Because the structure of both the 70 and 36 kD proteins suggested that protease activity and its inhibition might play a role in male mate choice, three protease inhibitors were tested in bioassays of male discrimination of conspecifics. The male's ability to discriminate among females was not completely blocked by the protease inhibitor, but their behavior was affected in more subtle ways. This suggests that the 70 kD protein, designated CSP70, is a variant of α₂-macroglobulin, but with more specific activity.