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.     

Growth and development of Bacillus thuringiensis Cry1Ab-susceptible and Cry1Ab-resistant sugarcane borer on diet and conventional maize plants

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), is a dominant maize borer pest and a major target of Bacillus thuringiensis (Bt)‐maize in Louisiana and the Gulf Coast area of Texas (USA). Growth and development of D. saccharalis on non‐toxic diet, diet treated with three low concentrations (0.01, 0.05, and 0.1 μg g^?1) of Cry1Ab toxin, and on non‐Bt maize plants were compared for five insect genotypes: a Bt‐susceptible strain (BT‐SS), a Cry1Ab‐resistant strain (BT‐RR), a back‐crossed and re‐selected resistant strain (BT‐R’R’), and two F₁ progeny of the BT‐SS and BT‐R’R’ strains. Fitness of the five genotypes was examined by infesting neonates on diet with/without Cry1Ab toxin in the laboratory and on intact non‐Bt maize plants in the greenhouse. Biological parameters measured were neonate‐to‐pupa development time and pupation rate, larval survival, larval and pupal weight, and sex ratio. Larvae of BT‐SS and BT‐R’R’ on non‐toxic diet and non‐Bt maize plants grew normally and there were no significant differences between the two strains in all measured parameters, suggesting a lack‐of‐fitness cost of the Cry1Ab resistance in D. saccharalis. Except for the development time on non‐Bt diet, all other parameters on both non‐Bt diet and non‐Bt maize plants were similar among the five genotypes. Larval development of BT‐SS was significantly affected on diet treated with Cry1Ab toxin at 0.05 and 0.1 μg g^?1, whereas the effect to BT‐RR and BT‐R’R’ was not significant. Pupal weight and sex ratio reared on Cry1Ab‐diet were similar and there were no significant differences among the five genotypes. Neonate‐to‐pupation rate decreased as Cry1Ab concentrations increased but the decrease was more significant for BT‐SS than for the other four genotypes. The lack‐of‐fitness costs of Bt resistance in D. saccharalis imply a greater challenge in managing Bt resistance for this maize borer species.     

Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.     

Presence of Cry1Ab in the Bt maize – aphid (Rhopalosiphum maidis) – ladybeetle (Propylea japonica) system has no adverse effects on insect biological parameters

Transgenic Bacillus thuringiensis Berliner (Bt) crops receive particular attention because they carry genes encoding insecticidal proteins that might negatively affect non‐target arthropods. Here, laboratory experiments were conducted to evaluate the impact of Cry1Ab‐expressing transgenic maize [5422Bt1 (event Bt11) and 5422CBCL (MON810)] on the biological parameters of two non‐target arthropods, the aphid Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae) and its predator the ladybeetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae). In a long‐term assay (three generations), no significant differences were found between R. maidis fed Bt maize and those fed a near‐isogenic line (5422) when individual parameters were compared, including nymph development time, adult longevity, aphid spawning period, and fecundity. No negative effects were detected throughout the life cycle of P. japonica in aphids’ feeding amount, development (nymphs, pupae, adults, and progeny eggs), fecundity, or egg hatching when they preyed on Bt maize‐fed aphids compared with non‐Bt maize treatments. A tritrophic assay revealed that Cry1Ab was highly diluted through the food chain (Bt maize leaves, R. maidis, and P. japonica), as detected by an enzyme‐linked immunosorbent assay (ELISA). In conclusion, although Cry1Ab concentrations in maize leaves increased as the plants developed, Cry1Ab levels were significantly reduced in the aphid R. maidis, and no traces of Cry1Ab were detected in P. japonica preying on Bt maize‐fed aphids. The two hybrids of Bt maize expressing Cry1Ab had no negative effects on the measured biological parameters of the aphid R. maidis or its predator, the ladybeetle P. japonica.     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1．0μg· g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0．065μg· g-1、Cry1Ac 0．074μg· g-1、Cry2Ab 0．133μg· g-1、Cry2Aa 11．670μg· g-1、Cry1Ah 13．010μg· g-1和Cry1Ca＞20μg· g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

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.     

Susceptibility of legume pod borer (LPB), Maruca vitrata to delta-endotoxins of Bacillus thuringiensis (Bt) in Taiwan

Baseline susceptibility of legume pod borer (LPB) to the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis, viz, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca and Cry2Aa was assessed in Taiwan. Insect bioassays were performed by incorporating the Bt delta-endotoxins into the LPB artificial diet. The efficacy of different Bt delta-endotoxins against second instar larvae of LPB showed that the toxin Cry1Ab was the most potent toxin (LC(50) 0.207ppm), followed by Cry1Ca, Cry1Aa, Cry2Aa and Cry1Ac in descending order, with LC(50)s 0.477ppm, 0.812ppm, 1.058ppm and 1.666ppm, respectively. Hence, Cry1Ab and/or Cry1Ca toxins would provide effective control of early larval stages of LPB.     

Effects of Bt maize on Frankliniella tenuicornis and exposure of thrips predators to prey-mediated Bt toxin

As a part of a risk assessment procedure, the impact of Bt maize expressing Cry1Ab toxin on the thrips Frankliniella tenuicornis (Uzel) (Thysanoptera: Thripidae) was investigated, and the potential risks for predators feeding on thrips on Bt maize were evaluated. The effects of Bt maize on F. tenuicornis were assessed by measuring life‐table parameters when reared on Bt and non‐Bt maize. The content of Cry1Ab toxin in different stages of F. tenuicornis reared on Bt maize and the persistence of the toxin in adults where determined in order to evaluate the possible exposure of predators when feeding on thrips. In addition, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) was used as a model predator to assess how the behaviour of prey and predator may influence the exposure of a natural enemy to the Bt toxin. Life‐table parameter results showed that F. tenuicornis was not affected when it was reared on Bt maize. This indicates that the potential for prey quality‐mediated effects on predators is low. Bt content was highest in thrips larvae and adults, and negligible in the non‐feeding prepupal and pupal stages. The persistence of the Cry1Ab toxin in adult F. tenuicornis was short, resulting in a decrease of 97% within the first 24 h. Predation success by young C. carnea larvae varied among the thrips stages, indicating that exposure of predators to Bt toxin can additionally depend on the prey stage. When combining the current knowledge of the susceptibility of major thrips predators with our findings showing no potential for prey quality‐mediated effects, relatively low toxin content in thrips as well as short persistence, it can be concluded that the risks for predators when feeding on thrips in or next to Bt maize fields are negligible.     

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.     

Verifying an F1 screen for identification and quantification of rare Bacillus thuringiensis resistance alleles in field populations of the sugarcane borer,Diatraea saccharalis

Using an F₁ screen, 352 feral individuals of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were examined for the presence of Bacillus thuringiensis (Bt)‐resistance alleles. These insects represented four geographical populations collected in central and northeastern Louisiana, USA, and one field population from the Gulf Coast area of Texas, USA, during 2006. The F₁ screen used various crosses between field‐collected insects and a laboratory strain of Cry1Ab‐resistant D. saccharalis, including both reciprocal crosses and group mating. F₁ neonates of the crosses were screened for Bt resistance on Bt maize leaf tissue. One field‐collected individual of D. saccharalis was shown to have a Bt‐resistance allele. Based on Bayesian analysis procedures, the Bt‐resistance allele frequency in the five populations of D. saccharalis was 0.0028 with a 95% confidence interval of 0.0003–0.0079. The successful identification of a resistance allele in a field collection of insects suggests that the F₁ screening technique could be an effective tool for detecting and monitoring rare Bt‐resistance alleles in field populations of D. saccharalis.     

Resistance of sugarcane borer to Bacillus thuringiensis Cry1Ab toxin

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), strain (F52‐3‐R) was developed from F₃ survivors of a single‐pair mating on commercial Cry1Ab Bacillus thuringiensis (Bt) corn plants in the greenhouse. The susceptibility of a Bt‐susceptible and the F52‐3‐R strain of D. saccharalis to trypsin‐activated Cry1Ab toxin was determined in a laboratory bioassay. Neonate‐stage larvae were fed a meridic diet incorporating Cry1Ab toxin at a concentration range of 0.0625 to 32 µg g^?1. Larval mortality, larval weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded on the 7th day after inoculation. The F52‐3‐R strain demonstrated a significant level of resistance to the activated Cry1Ab toxin. Larval mortality of the Bt‐susceptible strain increased in response to higher concentrations of Cry1Ab toxin, exceeding 75% at 32 µg g^?1, whereas mortality of the F52‐3‐R strain was below 8% across all Cry1Ab concentrations. Using a measure of practical mortality (larvae either died or gained no weight), the median lethal concentration (LC₅₀) of the F52‐3‐R strain was 102‐fold greater than that of the Bt‐susceptible insects. Larval growth of both Bt‐susceptible and F52‐3‐R strains was inhibited on Cry1Ab‐treated diet, but the inhibition of the F52‐3‐R strain was significantly less than that of the Bt‐susceptible insects. These results confirm that the survival of the F52‐3‐R strain on commercial Bt corn plants was related to Cry1Ab protein resistance and suggest that this strain may have considerable value in studying resistance management strategies for Bt corn.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

Susceptibility to Cry proteins of a Spanish Ostrinia nubilalis glasshouse population repeatedly sprayed with Bacillus thuringiensis formulations

Ostrinia nubilalis Hübner (Lepidoptera: Crambidae), a major pest of corn in temperate climates, can feed on other crops due to its polyphagous behaviour. In particular, this species became a serious problem in some sweet pepper commercial glasshouses in south‐eastern Spain repeatedly sprayed with Bacillus thuringiensis (Bt) products to control Spodoptera exigua Hübner (Lepidoptera: Noctuidae). The susceptibility of an O. nubilalis colony established from individuals collected in these Bt‐sprayed glasshouses was compared with a reference laboratory colony. Differences in susceptibility between the two colonies to Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa proteins were found. However, our results indicate that the O. nubilalis control failure in the glasshouse was not due to selection for resistance. Intraspecific variation probably accounts for differences between the glasshouse‐derived population and the laboratory strain. This conclusion is based on several lines of evidence: the glasshouse‐derived population retained its susceptibility to a Bt standard product and to most of its individual components (both in the form of protoxins and in the form of activated toxins), and it did not respond to laboratory selection with high doses of Cry1Ab.     

Bt Crops Producing Cry1Ac,Cry2Ab and Cry1F Do Not Harm the Green Lacewing,Chrysoperla rufilabris

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

Interaction of Bacillus thuringiensis δ-endotoxins with Midgut Brush Border Membrane Vesicles of Helicoverpa armigera

Pesticidal activity of different Bacillus thuringiensis (Bt) δ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2A, were investigated against Helicoverpa armigera infesting cotton crop worldwide. Cry1Ac toxin was found to be the most potent toxin towards H. armigera. All selected Bt toxins were found stable in vitro processing by midgut juice of H. armigera. Saturation and competition binding experiments were performed with iodine-125 labeled proteins and brush border membrane vesicles prepared from the midgut of H. armigera. The results show saturable, specific and high affinity of all toxins except for Cry2A. Both the toxins were bound with low binding affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab and Cry1Ac recognized or share the same binding site which is different from that of Cry2A. The data suggest that development of multiple toxin system in transgenic plants with toxin pyramiding, which recognize different binding sites, may be useful in the deployment strategies to decrease the rate of pest adaptation to Bt toxins in transgenic plants.     

Possibly similar genetic basis of resistance to Bacillus thuringiensis Cry1Ab protein in 3 resistant colonies of the sugarcane borer collected from Louisiana,USA

The sugarcane borer, Diatraea saccharalis (F.), is a major maize borer pest and a target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid‐southern region of the United States. Evolution of resistance in target pest populations is a great threat to the long‐term efficacy of Bt crops. In this study, we compared the genetic basis of resistance to Cry1Ab protein in 3 resistant colonies of sugarcane borer established from field populations in Louisiana, USA. Responses of larvae to the Cry1Ab protein for the parental and 10 other cross colonies were assayed in a diet‐incorporated bioassay. All 3 resistant colonies were highly resistant to the Cry1Ab protein with a resistance ratio of >555.6 fold. No maternal effect or sex linkage was evident for the resistance in the 3 colonies; and the resistance was functionally nonrecessive at the Cry1Ab concentrations of ≤ 3.16 μg/g, but it became recessive at ≥10 μg/g. In an interstrain complementation test for allelism, the F₁ progeny from crosses between any 2 of the 3 resistant colonies exhibited the similar resistance levels as their parental colonies, indicating that the 3 colonies most likely shared a locus of Cry1Ab resistance. Results generated from this study should provide useful information in developing effective strategies for managing Bt resistance in the insect.     

Inductive and synergistic interactions between plant allelochemical flavone and Bt toxin Cry1Ac in Helicoverpa armigera

Genetically engineered crops simultaneously produce defensive allelochemicals and Bacillus thuringiensis (Bt) toxin proteins to kill some of the world's most devastating insect pests. How the two types of toxins, when ingested sequentially or simultaneously, interact at both lethal and sublethal doses in these pests remains underexplored. Here, we examined the toxicological interactions between the Bt toxin Cry1Ac and the flavonoid allelochemical flavone in Helicoverpa armigera. Simultaneous exposure of H. armigera neonates to lethal doses (LC₂₅) of Cry1Ac and flavone caused a mortality significantly higher than that of either toxin alone and their expected additive mortality. Preexposure for 24 h to a sublethal dose (LC₁₀) of Cry1Ac followed by 6-d simultaneous exposure to the same dose of Cry1Ac plus a lethal dose (1.6 mg/g diets, LC₅₀) of flavone resulted in a mortality significantly higher than that of the LC₅₀ dose of flavone alone and the expected additive mortality of the LC₅₀ dose of flavone plus the LC₁₀ dose of Cry1Ac. One-day preexposure to the sublethal dose (LC₁₀) of flavone followed by 6-d simultaneous exposure to the LC₅₀ dose (6 ng/cm²) of Cry1Ac plus the LC₁₀ dose of flavone yielded a mortality significantly higher than that of the LC₅₀ dose of Cry1Ac but similar to the expected additive mortality of the LC₅₀ dose of Cry1Ac plus the LC₁₀ dose of flavone. The results suggest that Cry1Ac induces and synergizes the toxicity of flavone against H. armigera larvae.