Five economically important crop pests, Manduca sexta, Pieris brassicae, Mamestra brassicae, Spodoptera exigua, and Agrotis ipsilon, were tested at two stages of larval development for susceptibility to Bacillus thuringiensis toxins Cry1Ac, Cry1Ca, Cry1J, and Cry1Ba. Bioassay results for M. sexta showed that resistance to all four Cry toxins increased from the neonate stage to the third-instar stage; the increase in resistance was most dramatic for Cry1Ac, the potency of which decreased 37-fold. More subtle increases in resistance during larval development were seen in M. brassicae for Cry1Ca and in P. brassicae for Cry1Ac and Cry1J. By contrast, the sensitivity of S. exigua did not change during development. At both larval stages, A. ipsilon was resistant to all four toxins. Because aminopeptidase N (APN) is a putative Cry1 toxin binding protein, APN activity was measured in neonate and third-instar brush border membrane vesicles (BBMV). With the exception of S. exigua, APN activity was found to be significantly lower in neonates than in third-instar larvae and thus inversely correlated with increased resistance during larval development. The binding characteristics of iodinated Cry1 toxins were determined for neonate and third-instar BBMV. In M. sexta, the increased resistance to Cry1Ac and Cry1Ba during larval development was positively correlated with fewer binding sites in third-instar BBMV than in neonate BBMV. The other species-instar-toxin combinations did not reveal positive correlations between potency and binding characteristics. The correlation between binding and potency was inconsistent for the species-instar-toxin combinations used in this study, reaffirming the complex mode of action of Cry1 toxins.The ␦ endotoxins are a family of insecticidal proteins produced by Bacillus thuringiensis during sporulation. These toxins are typically found in parasporal crystals that are released into the environment with the bacterial spores. Numerous ␦ endotoxins produced by B. thuringiensis have been identified and are grouped on the basis of sequence homology and insect specificity (40). The Cry1 toxins are a group of ␦ endotoxins that principally target lepidopteran species, including several important crop pests.The mechanism of action of the Cry1 ␦ endotoxins begins with solubilization of the protoxin in the alkaline larval midgut, followed by proteolytic processing by midgut proteases (40). The stable 60-to 65-kDa toxins then bind to midgut receptors and insert into the apical membrane of brush border epithelial cells to form pores. These pores disrupt functional membrane processes and are ultimately responsible for larval death (40).Each type of Cry1 toxin has a unique spectrum of activity and targets only a small range of lepidopteran species. Within the small target ranges there are dramatic differences in potency between species that are often closely related (12,15,31). Indeed, the potency of a Cry1 toxin can significantly decrease as the larvae age (1, 38). Variations in the potencies of Cry1 toxins ...

Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 105 transformants/μg. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 102 pC194 + pUB110 transformant/μg DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and an acrystalliferous mutant of the same strain transformed at frequencies of 104–105/μg DNA with most of the plasmids tested. A cloned israelensis 27‐kDa δ‐endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.

The δ‐endotoxin crystal of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis contains four major 0‐endotoxins. Expression systems were devised to synthesize each of the four toxins at concentrations at which they formed inclusion bodies in an acrystalliferous mutant of Bacillus thuringiensis. The relative activities of these inclusions were then determined against Aedes aegypti larvae. Bioassays of mixtures of the individual toxins revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins.

The sporulation-specific penicillin-binding protein 5a was purified from Bacillus subtilis and shown to possess dd-carboxypeptidase activity in vitro.

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