Continued success of the most widely used MATERIALS AND METHODSInsects. We studied P. xylostella from 13 laboratory colonies derived from individuals collected at eight field sites in Hawaii (11,25). Larvae were fed cabbage foliage and colonies were maintained at 280C as described (25). The LAB-P colony, which was not exposed to B.t., served as the primary reference susceptible colony (26).We investigated the stability of resistance to B.t. in six laboratory colonies that were started from a field population (called NO) from Oahu, HI. The NO population had been treated repeatedly with B.t. in the field (11) and had developed moderate resistance to Dipel, a wettable powder formulation of a crystal-spore mixture of B.t. subspecies kurstaki (27,28). In the first laboratory-reared generation (F1), the LC50 (concentration required to kill 50% ofinsects tested) of NO larvae was about 25 times greater than the LC5o of larvae from the susceptible LAB-P strain (11). The NO colony was reared without exposure to B.t. for 3 generations, and then it was split into four colonies: NO-P, NO-Q, NO-R, and NO-U (23). NO-P, NO-Q, and NO-R were selected for additional resistance (see ref. 23 and description below) and then, as part of the present study, were reared without exposure to B.t. to examine the stability of extremely high resistance. To examine the stability of moderate resistance, NO-U was maintained without any additional exposure to insecticide for 35 generations. Results from the first 15 generations of rearing NO-U without exposure to B.t. were reported in detail previously (23) and are summarized here for comparison with results from NO-P, NO-Q, and NO-R. Selection and Reselection Experiments. NO-P, NO-Q, and NO-R were selected for additional resistance by feeding Abbreviations: B.t., Bacillus thuringiensis; ICP, insecticidal crystal protein; AI, active ingredient. tTo whom reprint requests should be addressed. 4120The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Selection of resistance in Spodoptera exigua (Hübner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryICresistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 g/g), whereas the 50% lethal concentration was 4.30 g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K d , and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

A soluble 106 kDa protein with aminopeptidase activity was isolated from Manduca sexta using CryIC toxin-affinity and anion-exchange chromatographies. Based on internal amino acid sequence analysis and different mobilities obtained with non-denaturing polyacrylamide gel electrophoresis, the 106 kDa aminopeptidase is distinct from a previously described 115 kDa CryIAc-binding aminopeptidase. The 106 kDa protein was preferentially precipitated by CrylC relative to CryIAc toxin. The 106 kDa form, like the 115 kDa aminopeptidase, has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol (GPI) anchor. On ligand blots, CryIAc recognized membrane bound 120 and soluble 115 kDa aminopeptidases, but not the soluble 106 kDa form. The results show that CryIC and CryIAc 6endotoxins recognize functionally related, but structurally distinct 106 kDa and 115 kDa isoforms of aminopeptidase in the M. sexta midgut.

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua andSpodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles fromS. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.