We have previously shown that pupae of the giant silkmoth Samia cynthia have a humoral antibacterial activity, which was induced by viable, nonpathogenic gram-negative bacteria (H.G. Boman et al., 1974). We show here that this activity was formed simultaneously with a selective incorporation of amino acids into eight polypeptide chains characterized by their electrophoretic behavior. If actinomycin D or cycloheximide were given at an early time, no antibacterial activity was found. If the inhibitors were given at the time of maximum activity, there was no effect with actinomycin D but a rapid decrease of the activity in the case of cycloheximide. The results imply that the messenger ribonucleic acid was stable, but that at least one protein component was turning over. Hemolymph from immunized pupae of another giant silkmoth, Hyalophora cecropia, was fractionated by ammonium sulfate precipitation. This procedure, together with the isotope distribution after co-electrophoresis in polyarylamide gels, was used for comparing the response to injury and to different infections. Almost identical polypeptide patterns were obtained as a response to an infection with either viable Enterobacter cloacae or Bacillus subtilis. These patterns differed both qualitatively and quantitatively from the injury effect created by an injection as such. There was only a low antibacterial activity in each of the four fractions obtained by ammonium sulfate precipitation. However, a combination of three fractions restored a high killing activity. Fractionation of hemolymph from untreated pupae provided evidence for at least one preexisting factor which stimulated the killing of Escherichia coli. The osmotic pressure of the bacteria contributed to the antibacterial activity towards E. coli, but not towards B. subtitlis. The killing of E. coli was inhibited by liped A and, to a lesser extent, by an inhibitor of proteolytic enzymes. The similarities and differences with the mammalian complement system are discussed.
