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The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

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The active-toxin form of CrylAc (65 kDa) or Cry2Ab was fed to a non-susceptible insect, Lygus hesperus, in an artificial diet. Biochemical and immunocytochemical methods were used to determine the distribution of ingested toxin. The toxins did not elicit a feeding deterrent response. CrylAc and Cry2Ab were ingested; small amounts were absorbed into the hemolymph as holoproteins, but most was excreted. SDS-PAGE analysis of CrylAc and Cry2Ab incubations with salivary gland homogenate showed a small decrease in the molecular weight of the active toxins. Proteolytic processing of the toxins also occurred in vivo, within the digestive system of L. hesperus. Excreted CrylAc and Cry2Ab retained activity toward lepidopteran larvae. Immunocytochemical in vivo localization studies showed negligible association of CrylAc with L. hesperus tissues. In contrast, strong extracellular association of Cry2Ab was observed with L. hesperus midgut brush border microvilli and basement membrane, as well as with cellular outlines within the hemolymph and fat body.