Hemostasis of Tiger Prawn Penaeus monodon Affected by Vibrio harveyi,Extracellular Products,and a Toxic Cysteine Protease

ABSTRACT: The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied. The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease or PBS (control) was observed for 2 h at 25 C. Of these, only the control group was clottable while all the other groups were unclottable. A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (Rα coagulogen) to neutralize the clotting ability of normal prawn hemolymph. The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels. The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using Rα coagulogen. In addition, the apparent disappearance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using Rα coagulogen or by reducing the time for pre-incubation from 2 h to 30 min. Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph. These complex events may significantly contribute to the pathogenicity of V. harveyi in the prawn.