Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue
1981
The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis was bioassayed in vitro using cultured insect tissue. Larval cells of Choristoneura fumiferana (Clem.) were damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity was accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue was inversely proportional to the amount of toxin added. Tissue cells from C. fumiferana exhibited maximum susceptibility to activated delta -endotoxin after incubation for 120 h. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 mu g or less of toxin protein per 2 X 105 insect tissue cells. Activated delta -endotoxin was toxic to insects as well, as determined by mortality studies with second-instar larvae of the European corn borer [Ostrinia nubilalis (Hb.)]. Electron microscope observations of insect tissue treated with activated delta -endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.
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