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We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella. The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C. Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D. The potency of Cry1I did not differ significantly between the resistant and susceptible strains. Cry2B did not kill resistant or susceptible larvae. By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P. NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04. Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A. Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae. In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III. High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab. Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04). The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II.

We detected binding of insecticidal crystal proteins from Bacillus thuringiensis in one susceptible strain and six resistant strains of diamondback moth, Plutella xylostella, from Hawaii. Immunohistochemical tests with tissue sections from larval midguts showed specific binding of CryIA(a), CryIA(b), and CryIA(c) to brush border membranes. CryIE, which is not toxic to P. xylostella, did not bind to midgut tissues. Larvae from one of the resistant strains ingested extremely high concentrations of a commercial formulation containing the three CryIA proteins without suffering midgut cell damage or mortality. This same resistant strain had previously been found to have greatly reduced binding of radioactively-labeled CryIA(c) to vesicles prepared from brush border membranes. The finding that binding as detected in immunohistochemical tests was not sufficient for toxicity suggests that low levels of binding can occur without harmful effects; the tests did not reflect in vivo binding, or post-binding factors contribute to resistance. Comparison with resistant strains from Florida and the Philippines, which did not bind CryIA(b) in previously reported immunohistochemical tests, suggests that more than one mechanism of resistance to B. thuringiensis may occur within a single species.

We studied effects of prolonged selection with the microbial insecticide, Bacillus thuringiensis Berliner, on diamondback moth, Plutella xylostella (L.). A laboratory colony of diamondback moth derived from a moderately resistant field population was selected with B. thuringiensis during 21 of 40 generations. Continued increases in resistance after repeated exposure to concentrations of B. thuringiensis that were high enough to kill 100% of putative heterozygotes suggest test resistance was not controlled solely by one locus with two alleles. After an apparent plateau in response to selection occurred, we evaluated the stability of resistance in the selected colony and in six isofemale lines derived from the selected colony. Resistance declined in most cases, but in one of the isofemale lines, resistance remained extremely high (resistance ratio = 5,800) after >20 generations without exposure to B. thuringiensis. These data show that at least one genotype conferring resistance to B. thurigiensis was not inherently unstable.

Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles 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.

Does adaptation to stress entail fitness costs in optimal environments? Antagonistic pleiotropy sometimes causes trade-offs in fitness across different environments in which the agent of stress is a pathogen (Lenski 1988) or a novel diet (Pashley 1988; Via 1991). It is commonly believed that evolution of resistance to insecticides also involves such trade-offs. Alleles conferring resistance are rare before exposure to insecticide and have been presumed to exert negative effects on fitness in the absence of insecticide (Crow 1957; Uyenoyama 1986; Hoffman and Parsons 1991). However, fitness costs associated with resistance to conventional synthetic insecticides appear to vary considerably among species and insecticides (Roush and McKenzie 1987; Roush and Daly 1990; Denholm and Rowland 1992). Moreover, little is known about fitness costs of resistance to insecticidal proteins from Bacillus thuringiensis, a common soil bacterium (McGaughey and Whalon 1992; Tabashnik 1994). Toxins from B. thuringiensis kill susceptible insects by binding to and disrupting the integrity of the midgut epithelium (Gill et al. 1992). Reduced binding of toxins is a primary mechanism of resistance in the diamondback moth, Plutella xylostella (Ferre et al. 1991), a major pest of cruciferous vegetables (Talekar 1992). The normal function of toxin-binding sites is not known but seems to be important because such sites are found in numerous insects (Feitelson et al. 1992). Several authors have suggested that alteration of toxin-binding sites may interfere with normal physiological functions (Van Rie et al. 1990; MacIntosh et al. 1991). Instability of resistance to B. thuringiensis in the absence of selection

The continued efficacy of environmentally safe biopesticides derived from Bacillus thuringiensis (Bt) is threatened by the potential for development of resistance in pest populations. Instability of resistance is defined here as the tendency for the frequency of resistant genotypes to decrease in a population beyond effects directly attributable to immigration or emigration. Instability can be quantified as R, the average rate of change in the logarithm of the LC50 (concentration killing 50% of individuals) per generation, which is analogous to the average rate of response to selection per generation. In seven strains of Plutella xylostella, the first insect with field populations reported to be resistant to Bt, resistance declined when exposure to insecticide ceased (mean R = − 0.19). In four other pests, resistance to Bt declined slowly or not at all (mean R = − 0.02) in the absence of exposure to Bt Reduced biotic fitness associated with resistance is the most likely cause of instability of resistance ...

In laboratory choice tests, males from a colony of diamondback moth, Plutella Xylostella (L.), resistant to Bacillus thuringiensis Berliner toxins mated fewer times than did males from a susceptible colony. Resistant and susceptible males did not differ in mating duration or ability to mate first with virgin females. Thus, the mating deficiency of resistant males was caused by reduced success in obtaining subsequent matings. Reduced mating success of resistant males is a fitness cost that may contribute to loss of resistance in the absence of exposure to toxins. Resistant females mated as frequently as susceptible females, suggesting that no change in female mating success was associated with resistance. We found no evidence for assortative mating between resistant and susceptible moths that would diminish the effectiveness of refuges in resistance management.

We addressed several key questions about duration of bioassays, diagnostic concentration, and probit regression slope for response of diamondback moth, Plutella xylostella (L.), to the microbial insecticide, Bacillus thuringiensis Berliner. Data were obtained from 54 bioassays of larvae from seven field populations and eight laboratory colonies in Hawaii. Linear regression showed that LC50s at 96 and 120 h after exposure to B. thuringiensis were associated with LC50s at 24 and 48 h. Control mortality increased as time increased, but time had little effect on slope, standard error of slope, or 95% fiducial limits for LC50 A significant portion of the variation in LC50 was accounted for by variation in mortality at a single concentration. Linear and polynomial regression showed that slope did not vary as a simple or consistent function of LC50. Variation in LC50 across all strains of diamondback moth was >5,000-fold, yet variation in LC50 for repeated bioassays of single strains was

Repeated exposure in the field followed by laboratory selection produced 1,800- to >6,800-fold resistance to formulations of Bacillus thuringiensis subsp. kurstaki in larvae of the diamondback moth, Plutella xylostella. Four toxins from B. thuringiensis subsp. kurstaki [CryIA(a), CryIA(b), CryIA(c), and CryIIA] caused significantly less mortality in resistant larvae than in susceptible larvae. Resistance to B. thuringiensis subsp. kurstaki formulations and toxins did not affect the response to CryIC toxin from B. thuringiensis subsp. aizawai. Larvae resistant to B. thuringiensis subsp. kurstaki showed threefold cross-resistance to formulations of B. thuringiensis subsp. aizawai containing CryIC and CryIA toxins. This minimal cross-resistance may be caused by resistance to CryIA toxins shared by B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai.

Two serine protease inhibitors were tested for direct effects and synergism with B. thuringiensis against larvae of diamondback moth, Plutella xylostella (L.). Concentrations of 1-1,000 mg/liter of trypsin inhibitor (Kunitz) and 1-500 mg/liter of trypsinchymotrypsin inhibitor (Bowman-Birk) caused little or no mortality to larvae in laboratory bioassays. No significant synergistic effects on mortality occurred between either protease inhibitor (100 mg/liter) and formulated B. thuringiensis in tests with larvae that were susceptible or resistant to B. thuringiensis . The lack of synergism suggests that enzymes mediating the toxicity of B. thuringiensis to diamondback moth larvae were not affected by trypsin inhibitor or trypsin–chymotrypsin inhibitor. The similar response of susceptible and resistant larvae suggests that altered processing of B. thuringiensis toxins by trypsin or trypsinlike proteases was not an important mechanism of resistance.

We tested the offspring of various crosses of Plutella xylostella (L.) to determine the mode of inheritance of resistance to a commercial formulation of the microbial insecticide Bacillus thuringiensis . The progeny of reciprocal F 1 crosses (resistant male ° susceptible female and resistant female ° susceptible male) responded alike in bioassays, indicating autosomal inheritance. The LCso of F 1 progeny was not significantly greater than the LCso of a susceptible strain, showing that resistance was recessive. Responses of progeny from a backcross (F1 ° resistant strain) corresponded more closely to predictions from a single-locus model than to predictions from models with 2, 5, or 10 loci. Deviations between observed and expected outcomes increased as the number of loci in the model increased. Estimation of the minimum number of effective factors ( sensu Lande 1981) also suggests that the number of loci influencing resistance was not large. In summary, our results suggest that resistance was controlled primarily by one or a few loci.

Laboratory selection increased resistance to B. thuringiensis in three strains established from a moderately resistant field population of diamondback moth, Plutella xylostella (L.). Five generations of laboratory selection caused 5- to 7-fold increases in LC50, resulting in 150- to 190-fold resistance compared with a susceptible laboratory colony. Nine generations of selection produced 430- to 820-fold resistance. In contrast, five foliar applications of B. thuringiensis in the field did not increase the LC50 of a moderately resistant population. Field-selected resistance to B. thuringiensis declined slowly in the absence of treatments. The rapid response to laboratory selection shows intrapopulation genetic variation in susceptibility to B. thuringiensis and suggests that intense selection may produce much higher levels of resistance to B. thuringiensis than those previously reported from the field. We hypothesize that resistance increased faster in the laboratory than in the field because selection intensity was lower in the field. Because susceptibility was not restored quickly when treatments were discontinued, rotations may not be especially effective for managing resistance to B. thuringiensis in diamondback moth. Field populations of diamondback moth developed resistance to a commercial formulation containing a mixture of B. thuringiensis toxins, an event that raises doubts about the ability of mixtures to retard resistance development. Extensive and intensive exposure of pests to B. thuringiensis toxins through transgenic crop plants or other tactics may cause widespread pest resistance. We urge judicious use of B. thuringiensis to conserve its efficacy.

Foliar applications of commercial formulations of the insecticidal spore-crystal protein complex of Bacillus thuringiensis subsp. kurstaki caused development of resistance in field populations of a major lepidopteran pest of vegetables, diamondback moth, Plutella xylostella (L.). Laboratory bioassays of larvae showed that the LC50 and LC95 for a field population of diamondback moth treated repeatedly with B. thuringiensis were 25 to 33 times greater than the respective LC50’s and LC95’s for two susceptible laboratory colonies. Mortality at the field rate of B. thuringiensis was 34-35% in two resistant populations compared with 90-100% in two susceptible laboratory colonies. The results suggest that the potential for resistance development in pest populations is an important consideration for deployment of B. thuringiensis toxin genes in genetically-engineered crop plants and use of B. thuringiensis in related tactics.

Selection with Bacillus thuringiensis subsp. kurstaki, which contains CryIA and CryII toxins, caused a >200-fold cross-resistance to CryIF toxin from B. thuringiensis subsp. aizawai in the diamondback moth, Plutella xylostella. CryIE was not toxic, but CryIB was highly toxic to both selected and unselected larvae. The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of B. thuringiensis.

Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth ( Plutella xylostella ), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.

Neither toxins fromBacillus thuringiensis Berliner nor conspecific eggs deterred oviposition by the diamondback moth,Plutella xylostella (L.) (Lepidoptera: Plutellidae), in laboratory choice tests. The finding that toxins did not deter oviposition by moths from a susceptible line shows that oviposition preference and larval survival were not associated in this line. Selection for larval resistance to toxins did not significantly alter oviposition preference, which rules out a strong genetic correlation between larval performance and oviposition preference. Failure of conspecific eggs to deter oviposition may not represent lack of association of preference and performance because other evidence suggests that larval performance may not be greatly affected by larval density. These results suggest that the ability of refuges to slow evolution of physiological resistance toB. thuringiensis toxins will not be magnified by an oviposition preference for untreated foliage.