Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+²³⁴Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+²³⁴Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Direct Determination of Copper,Lead and Cadmium in Tea Infusions by Flameless Atomic Absorption Spectrometry

Two kinds of “group A saponin,” Aa and Ab, are present as the main constituent in soybean seed. The saponin composition and content in F₁ and F₂ seeds derived from the cross parents of Aa and Ab types were analyzed. The “group A saponin” was of Aa–Ab type in all the F₁ seeds, and the ratio of Aa type : Aa–Ab type : Ab type was 1:2:1 in the F₂ seeds. From these results, it appears that Aa and Ab were controlled by codominant allelic alternatives at a single locus. An investigation of the saponin composition of the seed hypocotyls of 18 wild lines revealed some lines in which “group A saponin” was absent.     

Bt Proteins Have No Detrimental Effects on Larvae of the Green Lacewing, <Emphasis Type="Italic">Chrysopa pallens</Emphasis> (Rambur) (Neuroptera: Chrysopidae)

Biosafety of a genetically modified crop is required to be assessed prior to its commercialization. For this, a suitable artificial diet was developed and used to establish a dietary exposure test for assessing the toxicity of midgut-active Bt insecticidal proteins on Chrysopa pallens (Rambur). Subsequently, this dietary exposure test was used to evaluate the toxicity of the proteins Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa on C. pallens larvae. Temporal stability, bioactivity, and the intake of the insecticidal proteins were confirmed by enzyme-linked immunosorbent assay and a sensitive-insect bioassay. The life history characteristics, such as survival, pupation, adult emergence, 7-day larval weight, larval developmental time, and emerged male and female fresh weights remained unaffected, when C. pallens were fed the pure artificial diet (negative control) and the artificial diets containing 200 μg/g of each purified protein: Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, or Vip3Aa. On the contrary, all of the life history characteristics of C. pallens larvae were adversely affected when fed artificial diet containing boric acid (positive control). The results demonstrate that diets containing the tested concentrations of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa have null effects on C. pallens larvae. The outcome indicates that genetically modified crops expressing the tested Bt proteins are safe for the lacewing, C. pallens.     

CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins

High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (<4-fold). Resistance to Cry2Ab in both knockouts is recessive, and genetic complementary tests confirmed Cry2Ab resistance alleles are at the same locus (i.e. HaABCA2) for the two strains. Brush border membrane vesicles (BBMVs) of midguts from both knockout strains lost binding with Cry2Ab, but maintained the same binding with Cry1Ac as the SCD strain. In vivo functional evidence from this study demonstrates knockout of HaABCA2 confers high levels of resistance to both Cry2Aa and Cry2Ab, confirming that HaABCA2 plays a key role in mediating toxicity of both Cry2Aa and Cry2Ab against H. armigera.     

Toxicity and Receptor Binding Properties of Bacillus thuringiensisδ-Endotoxins to the Midgut Brush Border Membrane Vesicles of the Rice Leaf Folders, Cnaphalocrocis medinalis and Marasmia patnalis

Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated. Saturation and competition binding experiments were done with iodine (¹²⁵1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C. medinalis and M. patnalis. The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin. Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin. Iodine (¹²⁵I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C. medinalis and M. patnalis. Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C. medinalis and M. patnalis. The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C. medinalis and M. patnalis. Received: 10 April 2000 / Accepted: 23 May 2000     

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with ¹²⁵I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K_com] = 1.1 nM) for ¹²⁵I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for ¹²⁵I-Cry1Ab binding sites, though the K_com values ranged from 179 to 304 nM. Cry1Ab competed for ¹²⁵I-Cry1Ac binding sites (K_com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the ¹²⁵I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Use of an artificial diet system to study the toxicity of gut-active insecticidal compounds on larvae of the green lacewing Chrysoperla sinica

A semi-liquid artificial diet was established and found to be a suitable food source for Chrysoperla sinica larvae, comparable to aphid prey. Using the artificial diet, we established and validated a dietary exposure assay by using the insecticidal potassium arsenate (PA) as positive control. Dose-dependent responses were documented for all observed life-table parameters of C. sinica larvae such as survival rate, pupation rate, larval weight, and larval development time. Thus, the dietary assay can detect the effects of insecticidal compounds on the survival and development of C. sinica larvae. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac, and Cry2Aa proteins (which are produced by transgenic maize, cotton or rice plants) to C. sinica larvae. Artificial diets containing Galanthus nivalis agglutinin (GNA) or PA were included as positive controls. Survival and development of C. sinica larvae were not affected when the artificial diet contained purified Cry1Ab, Cry1Ac, or Cry2Aa at 200 μg/g diet. In contrast, C. sinica larvae were adversely affected when the diet contained PA and GNA. The stability and bioactivity of the Cry proteins in the diet and Cry protein uptake by the lacewing larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on green lacewing larvae. The experiments with the Cry proteins demonstrate that C. sinica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry2Aa.     

Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. ¹²⁵I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.     

A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with ¹²⁵I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Integrative Model for Binding of Bacillus thuringiensis Toxins in Susceptible and Resistant Larvae of the Diamondback Moth (Plutella xylostella)

Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.     

Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis

Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC₅₀ values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and Cry1Ac were marginally toxic to all strains, whereas the rest of the toxins tested (Cry1Ba, Cry2Aa, and Cry2Ab) were non toxic. Significant differences in susceptibility among strains were also found for Cry1Da, being the FRA strain 25-fold more susceptible than the HOL strain. Growth inhibition, as an additional susceptibility parameter, was determined in the FRA strain with the 9 toxins. The toxicity profile obtained differed from that observed in mortality assays. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa toxins produced a similar larval growth inhibition. Cry2Aa had a lower but clear effect on larval growth inhibition, whereas Cry1Ba and Cry2Ab did not have any effect.     

Interaction of Bacillus thuringiensis Toxins with Larval Midgut Binding Sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the “stacked” gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of ¹²⁵I-labeled Cry1Ab protein (¹²⁵I-Cry1Ab) and ¹²⁵I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either ¹²⁵I-Cry1Ab or ¹²⁵I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with ¹²⁵I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. ¹²⁵I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, ¹²⁵I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.