Cyanoprotein: Immunological properties and content changes during the development of non-diapause female bean bugs, Riptortus clavatus

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.