Haemolymph immune proteins protect the insect body cavity from invading bacteria

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Injections of living, non-pathogenic Enterobacter cloacae into the body cavity of Galleria mellonella provoked the insect's non-self response system to produce antibacterial activity that eliminated the bacterial infection. Immunized larvae of Galleria contained no E. cloacae in haemolymph within 24 hr post-infection and they transmitted normally to pupae. Parallel to this protective immunity, antibacterial activity of cecropin-like polypeptides and manifold increased innate titer of haemolymph lysozyme were shown in vaccinated insects. Application of actinomycin D or cycloheximide in an induction phase of antibacterial response at a dose of 0.25 × ld 50 demonstrably reduced the lysozyme titer and entirely prevented (except for actinomycin-treated pupae) the synthesis of cecropin-like antibacterial activity. In insects with impaired antibacterial immunity, the immunizing bacteria multiplied to the titer of almost 10 9 cells/ml of haemolymph, causing death in nearly 100% of larvae due to E. cloacae bacteriaemia. No bacteriaemia of E. cloacae could be induced in larvae given actinomycin D (or cycloheximide) in an effector phase of immune response when the antibacterial proteins begin to be synthesized in invaded insects. For the expression of cell-free immunity in Galleria, there is a requirement for the de novo synthesis of antibacterial immune proteins that normally protect the insect body cavity from invading bacteria. The anti-cecropin agent from Heterorhabditis bacteriophora selectively destroyed the inducible antibacterial immunity, thereby allowing the immunizing bacteria to grow in Galleria. This implies that the cecropin antibacterial activity is primarily responsible for the elimination of E. cloacae and other bacterial invaders from the insect body cavity.