Reversal of resistance to Bacillus thuringiensis in Plutella xylostella

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 4120-4124, May 1994 Agricultural Sciences

Reversal of resistance to Bacillus thuringiensis in Plutella xylostella (Ietidal crysal protein/diam dback moth)

BRUCE E. TABASHNIK*t, NAOMI FINSON*, FRANCIS R. GROETERS*, WILLIAM J. MOARt, MARSHALL W. JOHNSON*, KE Luo§, AND MICHAEL J. ADANG§ *Department of Entomology, University of Hawaii, Honolulu, HI 96822; IDepartment of Entomology, University of Georgia, Athens, GA 30602; and tDepartment of Entomology, Auburn University, Auburn, AL 36849 Communicated by Robert L. Metcalf, February 16, 1994

helpful; their success will depend on a variety of factors, including pest movements, mating patterns, and inheritance of resistance (9, 13, 16). Provision of periods during which insects are not exposed to B.t. is a promising management option that has received limited attention. The success of such temporal refuges depends on the stability of resistance in the absence of exposure toB.t., about which relatively little is known. Initial evidence suggested that low to moderate levels of resistance to B.t. declined slowly or not at all when exposure to B.t. ceased (22-24). We report here that rapid reversal of up to 2800-fold resistance to B.t. in P. xylostella in the absence of exposure to ICPs was associated with restoration of ICP binding and increased biotic fitness. These results suggest that temporal refuges from exposure to B.t., one of the simplest resistancemanagement strategies, may be more useful for extending the effectiveness of B.t. than previously thought.

Continued success of the most widely used ABSTRACT biopesficide, Bacillus thuingiensis, is threatened by development of resistance in pests. Experiments with Plutella xylosteil (diamondback moth), the first insect with field populations resistant to B. thuringknsis, revealed factors that promote reversal of resistance. In strains of P. xylosteUa with 25- to 2800-fold resistance to B. thwingiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thningiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuningiensis toxin CryIA(c) to brush-border membrane vesides and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.

The bacterium Bacillus thuringiensis (B.t.) is becoming increasingly important for control of crop pests and insect vectors of disease (1). Insecticidal crystal proteins (ICPs) from B.t. kill insects by binding to and disrupting the integrity of midgut membranes (2). Unlike most insecticides, ICPs are not toxic to humans, most beneficial insects, and other nontarget organisms (3). Discovery of new strains of B.t. and expression of ICP genes in transgenic plants and transgenic bacteria are expected to dramatically increase the use of B.t. worldwide (1, 4). At the same time, environmental concerns, as well as resistance in >500 species of pests, are reducing the usefulness of conventional synthetic insecticides (5-9). Commercial formulations of B. t. were used for >20 years before the first cases of substantial resistance to B.t. were documented in open-field populations of any pest. Some field populations of a major vegetable pest, Plutella xylostella (diamondback moth) (10), have evolved resistance to B.t. in Hawaii, the continental United States, and Asia (11-13). Resistance to B.t. in P. xylostella is associated with reduced binding of ICP to the brush-border membranes of midgut epithelium (12) and is inherited as an autosomal recessive trait (14, 15). Laboratory selection experiments suggest that many other species of pests can also adapt to B.t. toxins (13). The potential for evolution of resistance in pests is the most serious threat to the continued success of B.t. (16-18). In some cases, resistance to one B.t. toxin did not confer crossresistance to other B. t. toxins, which suggested that one could manage resistance problems simply by switching to a new toxin or by using combinations of toxins (12, 19). Recent reports of broad-spectrum crossresistance (17) and resistance to multiple toxins (20, 21) suggest that alternations or combinations ofB.t; toxins may not delay resistance development substantially. Spatial refuges from exposure to B.t. may be

MATERIALS AND METHODS Insects. 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.

The 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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Agricultural Sciences: Tabashnik et al. cabbage disks dipped in a dilution of the active ingredient (Al) of Dipel at 25.6-512 pg/ml to -200 third-instar larvae per colony per generation (23). We reared the offspring of survivors on untreated cabbage during the first two instars. Results from the first 5 (NO-R) to 9 (NO-P and NO-Q) generations of selection are described in detail elsewhere (23). Before exposure to B.t. was stopped, we selected NO-P during 12 of 18 generations, NO-Q during 11 of 17 generations, and NO-R during 7 of 8 generations. During this initial selection experiment, the number of adults per generation ranged from 6 to 192 (mean, 70) for NO-P, from 29 to 376 (mean, 116) for NO-Q, and from 31 to 137 for NO-R (mean, 58). To compare responses to reselection for resistance with initial responses to selection, we derived two colonies (NO-QA and NO-QB) from NO-Q by reselecting with Dipel after resistance had declined. NO-QA and NO-QB were started after NO-Q had been reared without exposure to B.t. for 14 and 16 generations, respectively. Procedures for reselection were the same as for initial selection, as described above. We reselected NO-QA during 3 of 4 generations (during the second generation, 80 larvae were exposed to Dipel, but mortality was Oo; ref. 29) and NO-QB during 5 of 8 generations. The number of adults per generation during the reselection experiment ranged from 16 to 345 (mean, 128) for NO-QA and from 7 to 538 (mean, 116) for NO-QB. Realized heritability was estimated as R (the response to selection) divided by S (the selection differential) (30, 31). We estimated realized heritability of resistance based on 3 and 5 generations of reselection for resistance in NO-QA and NO-QB, respectively. These estimates were compared with previously reported estimates of realized heritability for the initial 5 generations of selection for NO-P, NO-Q, and NO-R

(31).

Toxins. We measured toxicity and binding of CryIA(c) and CryIC. CryIA(c) is one of the five ICPs in Dipel (27, 28). CryIC occurs in B.t. subspecies aizawai, but not in Dipel (27, 28). Toxins for bioassays were obtained from transformed Escherichia coli strains that each expressed a single B.t. toxin gene (21). CryIA(c) for binding assays was obtained from B.t. subspecies kurstaki strain HD-73 (32). The specific activity of 125I-labeled CryIA(c) was determined with a double-antibody sandwich ELISA (33) using a rabbit antiserum and biotinconjugated rabbit antibody against CryIA(c) toxin. The amount of labeled toxin was calculated from a standard curve for unlabeled CryIA(c) toxin. The specific activity was typically 50 mCi/mg of toxin (1 mCi = 37 MBq). CryIC for binding assays was obtained from a transformed B.t. strain (provided by Ecogen) that expressed only CryIC toxin. CryIC was labeled with Iodo-Beads (Pierce). Specific activity for 125I-CryIC was 10 mCi/mg of toxin. Bioassays. Third-instar larvae were tested for susceptibility to Dipel, CryIA(c), and CryIC by leaf residue bioassays (21, 26). Each test was replicated at least four times, with an average of 10 larvae tested per bioassay concentration. Mortality was recorded at 48 hr, which is strongly correlated with mortality at 120 hr (26). Probit analysis (34) of larval survival across a range of five concentrations and responses to single concentrations (26) were used to evaluate susceptibility. Resistance ratios were estimated as the LC50 of a particular colony divided by the LC50 of the LAB-P strain. The average LC50 of LAB-P was 2.9 ug of (Al) Dipel per ml (range, 1.8-6.0 pg/ml) (26). The average rate of change in response to Dipel per generation (R) was estimated as R = [log(final LC50) - log(initial LC50)]/n, where n is the number of generations (31). This approach enables one to make direct comparisons between decreases in resistance (as reflected by negative values of R) and increases in resistance (as reflected by positive values of R) (13, 31).

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Binding Assays. To measure binding, fourth-instar larvae from susceptible (LAB-P), revertant (NO-Q), and resistant (NO-QA) strains were stored at -700C. Larvae were thawed, midguts were removed, and brush-border membrane vesicles were prepared (12). We conducted CryIA(c) binding assays (32) in duplicate or triplicate for each strain on vesicle preparations from each of two independent samples of 1000 larvae. CryIA(c) assay mixtures (100 1.l) contained 10 pg of brush-border protein, 0.10 nM 125I-CryIA(c), and 1 of 15 concentrations ofunlabeled CryIA(c). After incubation for 30 min, the assay mixtures were centrifuged and washed. Radioactivity remaining in the tube was measured in a Beckman Gamma 4000 counter. CryIC assay mixtures (100 p1) contained 14 ,ug of brush-border protein and 0.03 nM 1251-CryIC; they were incubated for 60 min. Data from the homologous competition experiments described above were analyzed with the LIGAND computer program (35). For each strain, we also measured CryIA(c) binding by using 0.1 nM 125ICryIA(c) and duplicate samples of vesicles at concentrations ranging from 10 to 500 ,ug of protein per ml with no unlabeled

competitor.

Fitness Estimates and Model. We compared fitness parameters of revertant colony NO-Q and resistant colony NO-QA after NO-QA had been derived from NO-Q through reselection, as described above. At the time the fitness estimates were made, NO-Q and NO-QA had been separated for five generations and NO-QA had >3500-fold resistance to Dipel compared with NO-Q (29). The number of adults per generation during the five generations of this experiment ranged from 42 to 659 (mean, 305) for NO-Q and from 16 to 354 (mean, 128) for NO-QA. We used estimates of age-specific survival, age-specific fecundity, and percent egg hatch obtained from 198 females included in a previous experiment (29) to compute the finite rate of increase per individual (F.) (36) for NO-QA and NO-Q. The fitness of NO-QA relative to NO-Q was estimated as the mean Fi for NO-QA divided by the mean Fi for NO-Q. To determine whether the fitness cost associated with resistance was sufficient to account for the observed declines in resistance, we used computer simulations of a one-locus population genetics model. We assumed that resistance and the experimentally observed fitness cost were conferred by a recessive allele. Although resistance to B.t. in P. xylostella is recessive (14, 15) and appears to be controlled primarily by one or a few major loci (14), the genetic basis of the fitness cost is not known. Thus, the simulations must be considered a first approximation of reality. In three simulations (one each for NO-P, NO-Q, and NO-R) we set the initial frequency of resistant homozygotes as the proportion of survivors at 512 pg of (Al) Dipel per ml observed in each colony before exposure to B.t. was stopped. The initial proportions of susceptible homozygotes and heterozygotes were computed according to the Hardy-Weinberg equilibrium. The model was run for the number of generations during which each colony (NO-P, 7; NO-Q, 11; NO-R, 6) was reared without exposure toB.t. before survival at 512 pg of (Al) Dipel per ml was determined by bioassays. For each colony, the final proportion of resistant homozygotes in the simulation was compared with the experimental estimate of survival at 512 pg of (Al) Dipel per ml after the appropriate number of generations.

RESULTS Instability of Resistance to Dipel. In the absence of exposure to B.t., resistance declined in all four colonies (Figs. 1 and 2). The rapid reversal of resistance in the three extremely resistant colonies (NO-P, NO-Q, and NO-R) is especially noteworthy. Before selection with Dipel was stopped, the LC50 of NO-Q larvae was 2800 times greater than the LC50 of

Proc. Natl. Acad Sci. USA 91

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Table 1. Response of susceptible and revertant P. xylostella larvae to high concentrations of B.t. Dipel, n % survival jg of AI per ml Colony Susceptible 0 160 128 LAB-P 0 261 256 LAB-P 0 81 256 LAB-L 0.6 350 256 Field*

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Generation FIG. 1. Reversal of P. xylostella resistance to B.t. Resistance ratio = LC5o of NO strain/LCso of the susceptible LAB-P strain. LCso values were estimated from mortality at 48 hr of third-instar larvae fed cabbage leaf disks treated with Dipel (11). The x axis shows the number of generations without exposure to B.t. Data for NO-U were adapted from ref. 23.

the susceptible LAB-P strain, which represents one of the highest levels of resistance to B.t. ever recorded. Nonetheless, after 13 generations without selection, the LC50 of NO-Q was similar to that of the susceptible strain (Fig. 1). The average rates of decline in resistance per generation (R; see Materials and Methods) were -0.26 for NO-P, -0.30 for NO-Q, -0.28 for NO-R, and -0.06 for NO-U. For NO-P, NO-Q, and NO-R, the rate of decline in resistance (mean R = -0.28) was comparable to the rate of increase in resistance during the initial five generations of selection (mean R = 0.25; ref. 31). The decline in resistance was relatively slow in NO-U, yet the LC50 for NO-U after 15 generations without exposure to B.t. was significantly lower than the initial LC50 for NO-U (23). After 35 generations without exposure to B.t. (data not plotted), the LC50 of NO-U was 2.1 pg of (Al) Dipel per ml (95% fiducial limits = 0.7-3.5), which is not significantly different from the LC50 of the susceptible LAB-P colony [mean = 2.9 pg of (Al) Dipel per ml]. Although LC50 values declined rapidly in the extremely resistant colonies, the proportion of highly resistant individuals remained greater in all four revertant colonies than in susceptible colonies (Table 1). Survival at 128 or 256 pg of (Al) Dipel per ml was 0.2% (2/852) in susceptible colonies (Table 1). In revertant colonies, survival at 128 or 512 pg of (Al) Dipel per ml ranged from 2.5% to 7.6% after 7-39 generations without exposure to B.t. (Table 1). 100 80

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FIG. 2. Declines in survival of P. xylostella larvae at a high concentration of Dipel (512 pg of Al per ml) as a function of generations without exposure to B.t. Each point represents results from 30-193 larvae (mean, 66).

7.6 39 128 NO-U 5.0 40 512 NO-P 2.6 40 512 NO-Q 2.5 40 512 NO-R *Data were pooled for five susceptible field populations. tThe number of generations of rearing without exposure to B.t. was 20 for NO-U, 7 for NO-P, 39 for NO-Q, and 14 for NO-R.

Reselection for Resistance. After the LC50 of colony NO-Q had declined substantially, reselection with Dipel caused rapid increases in resistance. Three generations of reselection increased the LC50 by 130-fold in colony NO-QA, and five generations of reselection increased the LC50 by 3200fold in colony NO-QB. In contrast, the initial five generations of selection with Dipel increased LC50 values by 14-, 15-, and 28-fold in colonies NO-Q, NO-R, and NO-P, respectively (23). The quicker response to reselection cannot be attributed to more intense selection; the mean percent mortality per generation of selection was 59.9% during reselection (NO-QA and NO-QB) and 73.5% during initial selection (NO-P, NO-Q, and NO-R). The realized heritability of resistance to Dipel was 0.48 ± 0.08 (mean ± SE) during reselection (NO-QA, 0.56; NO-QB, 0.41) compared with 0.24 ± 0.03 during initial selection (NO-P, 0.26; NO-Q, 0.28; NO-R, 0.18; ref. 31) for resistance. These data suggest that the proportion of total variation in resistance due to additive genetic variation was about twice as high after reversal ofresistance when compared with initial levels. Toxicity and Binding of ICPs. Restoration of susceptibility to Dipel in the NO-Q colony was associated with restoration of susceptibility to CryIA(c). The LC50 to CryIA(c) was similar for susceptible LAB-P larvae and revertant NO-Q larvae after NO-Q had been reared for 28 generations without exposure to B.t. (Table 2). In contrast, after reselection for resistance to Dipel, NO-QA was extremely resistant to CryIA(c) (Table 2). Binding of CryIA(c) to brush-border membrane vesicles was restored in the NO-Q strain to a level similar to that of the susceptible LAB-P strain (Table 3; Figs. 3 and 4). Binding of CryIA(c) to vesicles from the resistant NO-QA colony was greatly reduced (Table 3; Fig. 4). At concentrations of vesicle protein ranging from 10 to 500 pg/ml, maximum binding of 1251-CryIA(c) was 1000* 3.4 12.4 6.8 Susceptible CryIC 12.5 6.0 8.8 Revertant 43.2 16.2 6.9 Resistant FL, fiducial limit. *The highest concentration tested (1000 pg/ml) killed 8%.

Slope 1.9 1.2 1.2 1.6 1.3


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Table 3. Apparent equilibrium dissociation constant (Kd), concentration of binding sites (Re), and binding (%) for B.t. proteins incubated with membrane vesicles from susceptible (LAB-P), revertant (NO-Q), and resistant (NO-QA) strains of P. xylostella

Rt, pmol/mg of Binding, Toxin Strain Kd, nM vesicle protein % CryIA(c) Susceptible 1.95 ± 0.92 1.1 ± 0.3 17.1 Revertant 0.79 ± 0.58 1.2 ± 0.1 16.3 Resistant* 1.0 CryIC Susceptible 8.8 ± 0.3 3.2 ± 0.04 25.7 Revertant 9.4 ± 1.6 3.9 ± 0.4 28.1 Resistant 8.5 ± 1.4 3.5 ± 0.3 27.0 Kd and Rt values (mean ± SE) were calculated from homologous competition experiments. Binding (%) was calculated from experiments with no unlabeled competitor. Binding (%) values are means based on seven replicates for CryIA(c) (vesicle protein at 100 Yg/ml) and two replicates for CryIC (vesicle protein at 140 Ag/ml). *Kd and Rt could not be calculated because binding was so limited in the resistant strain.

NO-Q and 29.7% for LAB-P (Fig. 4). Binding of CryIA(c) to vesicles from NO-QA was so low that the apparent equilibrium dissociation constant (Kd) and concentration of binding sites (RO) could not be calculated from homologous competition experiments. Toxicity and binding of CryIC were similar for susceptible (LAB-P), revertant (NO-Q), and resistant (NO-QA) strains (Tables 2 and 3). These results suggest that the observed differences between strains in toxicity and binding of CryIA(c) were toxin-specific and directly related to resistance.

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FIG. 4. Binding of 25I-labeled CryLA(c) as a function of the concentration of vesicle protein from P. xylostella larvae. Each point represents the mean of two replicates.

This pattern is similar to previously reported results with resistance to CryIA(b) in P. xylostella (12). Fitness. Resistance to B.t. was accompanied by decreased biotic fitness. In the absence of B.t., survival to adulthood was higher in revertant colony NO-Q (58%, n = 388) than in resistant subcolony NO-QA (44%, n = 395) (X2 = 17, df = 1, P < 0.001) (29). The mean values for finite rate of increase (F.) were 0.94 0.11 (mean SE) for the resistant colony and 1.59 0.14 for the revertant subcolony, which yields 0.59 (0.94/1.59) for the fitness of resistant insects relative to revertant insects. This difference in fitness is sufficient to cause rapid reversal of resistance in the absence of B.t. Computer simulations of a one-locus model with a recessive allele conferring resistance to B.t. and reduced fitness (0.59) in resistant homozygotes produced rates of reversal similar to the experimental results. Observed declines in survival at 512 ,ug of (AI) Dipel per ml were from 83% to 5% for NO-P in 7 generations, from 95% to 3% for NO-Q in 11 generations, and from 23% to 2% for NO-R in 6 generations. Simulations of each colony, respectively, showed declines in percentage of resistant homozygotes from 83% to 13% in 7 generations, from 95% to 10% in 11 generations, and from 23% to 4% in 6 generations. An additional fitness cost, the reduced mating success of resistant males (37), was not incorporated in the simulations because its effect on overall fitness could not be readily quantified. In competition experiments, resistant males obtained fewer total matings than did susceptible males, but resistant and susceptible males did not differ in mating duration or ability to mate with virgin females (37). We suspect that in our synchronized laboratory colonies, this type of mating disadvantage had minimal impact (37). ±

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FIG. 3. Binding of 1251-labeled CryIA(c) (A) or CryIC (B) toxin to vesicles from susceptible (LAB-P, *), revertant (NO-Q, o), and resistant (NO-QA, o) P. xylostella larvae as a function of the concentration of unlabeled homologous competitor. Each point represents the mean of two to five replicates.

Our results have important implications for understanding the biology of B.t.-toxin binding sites and for management of resistance to B.t. The decreased fitness associated with reduced binding of and resistance to CryIA(c) suggests that the reduction in binding that confers resistance to B.t. interferes with the normal function of the receptors. Various increases in concentration and affinity of binding sites have been interpreted as compensation for the loss of toxin binding associated with resistance toB.t. (19, 38). However, previous studies did not examine the fitness of insects with altered binding characteristics. The simplest explanation for our results is that alleles conferring resistance toB.t. also reduce biotic fitness, yet we cannot exclude the alternative hypotheses that the observed reduction in fitness in the resistant NO-QA strain reflects inbreeding depression or genetic drift (29). Nonetheless, if

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the mutations conferring resistance do not reduce fitness in the absence of B.t. toxins, one must find an alternative mechanism to explain the declines in resistance in replicated lines of P. xylostella. Our results show that temporal refuges of many generations during which insects are not exposed to B.t. can promote reversal of resistance. Several factors, however, may limit the effectiveness of temporal refuges for reversing development of resistance. Revertant strains responded rapidly to reselection and susceptibility was not fully restored, even after as many as 39 generations without selection. Declines in LC5o in moderately resistant strains of P. Xylostella and other insects (22-24) were much slower than those seen for the highly resistant strains of P. xylostella examined here. Three strains of P. xylostella from Japan which developed 220- to 700-fold resistance to B.t. in commercial greenhouses showed intermediate rates of decline in resistance (mean R = -0.15) when exposure to B.t. was stopped (15). We do not know the extent to which laboratory measurements of the stability ofresistance can be extrapolated to field populations. If alleles for resistance become fixed or if modifiers or novel resistance alleles ameliorate fitness costs of resistance, resistance to B.t. could become stable. When resistance is stable in the absence of exposure, temporal refuges can extend the usefulness of B.t. only by slowing the rate of increase in resistance. Because conventional applications of B.t. degrade rapidly (39), most exposure of pests toB.t. has incorporated temporal refuges, albeit inadvertently. Such refuges may be an important factor that slows development of resistance to B.t. compared with resistance to conventional synthetic insecticides, which are more persistent in the environment. With extended temporal refuges, even minor fitness costs associated with resistance can retard resistance development substantially (40). However, frequent foliar applications of B.t., such as those that produced resistance in field populations of P. xylostella (11), diminish the duration of temporal refuges. Continuous planting of transgenic varieties of crops that express B.t. constitutively could eliminate temporal refuges. When transgenic varieties are used, temporal refuges can be implemented with facultative expression of B.t. (41) or rotation of B.t.-expressing varieties with varieties or crops that do not express B.t. (8). Thus, the usefulness of one of the newest products of biotechnology-transgenic plants-may be prolonged effectively by one of the oldest strategies for pest management-crop rotation. We are grateful to C. Boake, R. Broadway, C. Chilcutt, R. Gillespie, F. Gould, D. Heckel, T. Lyttle, W. McGaughey, G. Oxford, G. Roderick, K. Spollen, and A. Taylor for helpful comments on the manuscript. T. Gonsalves, B. Helvig, and C. Yap provided technical assistance. We thank Ecogen for providing a strain of B.t. This work was supported by U.S. Department of Agriculture (USDA) Grant HAW00947H, grants from the USDA Western Region Integrated Pest Management and Pesticide Impact Assessment Programs, and the USDA/Cooperative State Research Service Special Grants in Tropical/Subtropical Agriculture Program to B.E.T. and by USDA/Cooperative State Research Service Competitive Grant 91-37302-6777 to M.J.A. This is paper 3874 of the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series. 1. Lambert, B. & Peferoen, M. (1992) BioScience 42, 112-122. 2. Gill, S. S., Cowles, E. A. & Pietrantonio, P. V. (1992) Annu. Rev. Entomol. 37, 615-636. 3. Flexner, J. L., Lighthart, B. & Croft, B. A. (1986) Agric. Ecosyst. Environ. 16, 203-254.

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