Insect Immunity I. Characteristics of an Inducible Cell-Free Antibacterial Reaction in Hemolymph of Samia cynthia Pupae

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Abstract
Pupae of the silk moth, Samia cynthia , were found to contain an inducible antibacterial activity in their hemolymph. This immunity response was provoked by primary infections with either Escherichia coli K-12 or Enterobacter cloacae. In both cases the antibacterial activity was directed chiefly towards E. coli. During standard conditions, 1% of hemolymph could kill 10 3 to 10 4 viable E. coli , strain D31, within 5 min. A lower level of antibacterial activity was induced by injections of a sterile salt solution. The killing of strain D31 followed single-hit kinetics, and increasing rate constants were obtained for increasing amounts of hemolymph. The reaction was sensitive to pretreatment with trypsin and it was protected by reducing agents. The activity was inhibited by microgram quantities of lipopolysaccharide (LPS) prepared from certain LPS mutants of E. coli K-12. A comparison of the susceptibility showed that “heptose-less” LPS mutants were more sensitive to killing than other strains. During standard conditions hemolymph will lyse both E. coli and Micrococcus lysodeikticus. Lysis of E. coli followed a multi-hit kinetics and it was inhibited by LPS, whereas lysis of M. lysodeikticus was unaffected by LPS. Hemolymph was fractionated on Sephadex G-200, and the lytic activities were recovered in partly overlapping peaks. Reconstitution with pooled fractions gave synergistic effects with the killing assay.