Showing papers on "Bacillus thuringiensis published in 2015"

Optimizing pyramided transgenic Bt crops for sustainable pest management

Abstract: Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies.

MAPK Signaling Pathway Alters Expression of Midgut ALP and ABCC Genes and Causes Resistance to Bacillus thuringiensis Cry1Ac Toxin in Diamondback Moth

Abstract: Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

The food and environmental safety of Bt crops

Abstract: Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

Large-scale test of the natural refuge strategy for delaying insect resistance to transgenic Bt crops

Abstract: The 'natural refuge strategy" for delaying insect resistance to transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt) relies on refuges of host plants other than cotton that do not make Bt toxins We tested this widely adopted strategy by comparing predictions from modeling with data from a four-year field study of cotton bollworm (Helicoverpa armigera) resistance to transgenic cotton producing Bt toxin Cry1Ac in six provinces of northern China Bioassay data revealed that the percentage of resistant insects increased from 093% in 2010 to 55% in 2013 Modeling predicted that the percentage of resistant insects would exceed 98% in 2013 without natural refuges, but would increase to only 11% if natural refuges were as effective as non-Bt cotton refuges Therefore, the results imply that natural refuges delayed resistance, but were not as effective as an equivalent area of non-Bt cotton refuges The percentage of resistant insects with nonrecessive inheritance of resistance increased from 37% in 2010 to 84% in 2013 Switching to Bt cotton producing two or more toxins and integrating other control tactics could slow further increases in resistance

Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

Abstract: The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera

Abstract: Toxins from the bacterium Bacillus thuringiensis (Bt) are used widely for insect control in sprays and transgenic plants, but their efficacy is reduced when pests evolve resistance. Previous work showed that mutations in a gene encoding the transporter protein ABCC2 are linked with resistance to Bt toxins Cry1Ab, Cry1Ac or both in four species of Lepidoptera. Here we compared the ABCC2 gene of Helicoverpa armigera (HaABCC2) between susceptible strains and a laboratory-selected strain with >1,000-fold resistance to Cry1Ac relative its susceptible parent strain. We discovered a 73-base pair (bp) insertion in the cDNA of the resistant strain that generates a premature stop codon expected to yield a truncated ABCC2 protein. Sequencing of genomic DNA revealed that this insertion is an intron that is not spliced out because of a 6-bp deletion at its splicing site. Analysis of progeny from crosses revealed tight genetic linkage between HaABCC2 and resistance to Cry1Ac. These results provide the first evidence that mis-splicing of a gene encoding an ABCC2 protein confers resistance to a Bt toxin.

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

Abstract: Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.

Evidence of field-evolved resistance of Spodoptera frugiperda to Bt corn expressing Cry1F in Brazil that is still sensitive to modified Bt toxins.

Abstract: Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.

The impact of secondary pests on Bacillus thuringiensis (Bt) crops.

Abstract: Summary The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to insecticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered insect-resistant crops could mitigate many of the negative side effects of insecticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro-ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here, we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if nonsusceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible long-term ecological trophic interactions of employing this technology.

Cross-resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against the cotton bollworm

Abstract: To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the "pyramid" strategy uses plants that produce two or more toxins that kill the same pest. We conducted laboratory diet experiments with the cotton bollworm, Helicoverpa armigera, to evaluate cross-resistance and interactions between two toxins in pyramided Bt cotton (Cry1Ac and Cry2Ab). Selection with Cry1Ac for 125 generations produced 1000-fold resistance to Cry1Ac and 6.8-fold cross-resistance to Cry2Ab. Selection with Cry2Ab for 29 generations caused 5.6-fold resistance to Cry2Ab and 61-fold cross-resistance to Cry1Ac. Without exposure to Bt toxins, resistance to both toxins decreased. For each of the four resistant strains examined, 67 to 100% of the combinations of Cry1Ac and Cry2Ab tested yielded higher than expected mortality, reflecting synergism between these two toxins. Results showing minor cross-resistance to Cry2Ab caused by selection with Cry1Ac and synergism between these two toxins against resistant insects suggest that plants producing both toxins could prolong the efficacy of Bt cotton against this pest in China. Including toxins against which no cross-resistance occurs and integrating Bt cotton with other control tactics could also increase the sustainability of management strategies.

The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management.

Abstract: Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi) The RNAi-based insect control strategy depends on the selection of suitable target genes In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L) Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P xylostella strains To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management

ABCs of Insect Resistance to Bt.

Abstract: Genetically engineered crops represent one of the most controversial and rapidly adopted technologies in the history of agriculture. To improve pest control, scientists have engineered cotton, corn, and soybeans to make insecticidal proteins from the common bacterium Bacillus thuringiensis (Bt) [1]. These Bt toxins kill some devastating pests, but unlike broad-spectrum insecticides, they do little or no harm to most other organisms, including people [2,3]. The original Bt crops, first commercialized in 1996, each made a single crystalline (Cry) toxin from the Cry1 family effective against certain lepidopteran larvae. However, some of the environmental, health, and economic benefits of Bt crops have been lost because of rapid evolution of pest resistance, particularly to single-toxin Bt crops (Fig 1) [4]. Fig 1 Field-evolved resistance to Bt crops [4,20,27,29–31]. To delay resistance and broaden the spectrum of pests controlled, newer “second generation” Bt crops produce two or more Bt toxins [5]. In particular, Bt toxin Cry2Ab from the Cry2 family is used widely in combination with Cry1 toxins to kill caterpillar pests. For example, the percentage of all cotton planted that was Bt cotton producing both Cry1Ac and Cry2Ab was 69% in the US in 2012, 91% in India in 2013, and 94% in Australia in 2011 [6–8]. Despite the use of Cry2Ab in multi-toxin Bt crops since 2003 and in multi-toxin Bt sprays for decades, nearly all of what we know about Bt toxins is based on the Cry1 family. In a breakthrough that promises to accelerate progress in understanding Cry2 toxins, Tay et al., in this issue of PLOS Genetics [9], identify a gene tightly linked with resistance to Cry2Ab in Helicoverpa armigera, one of the world’s most damaging crop pests. The advance by Tay et al. is the fruit of more than a dozen years of a synergistic collaboration, integrating results from classical and molecular genetics. As part of Australia’s proactive program for monitoring resistance to Bt crops, screening of field populations for resistance to Cry2Ab began in 2002, two years before farmers there started planting Bt cotton producing this toxin in combination with Cry1Ac. Using a method called the F2 screen, the second generation progeny of single pairs of field-collected insects were tested on an artificial diet treated with Cry2Ab. In the first year of screening, the Australian team detected resistance to Cry2Ab in one of the 28 isofemale lines that were tested [10]. The 17 survivors of exposure to Cry2Ab from this isofemale line became the progenitors of a Cry2Ab-resistant strain (SP15) that was repeatedly crossed with a susceptible strain and selected with Cry2Ab [11]. The SP15 strain was so resistant that it suffered little mortality when exposed to the highest concentration of Cry2Ab tested in the artificial diet [11]. Bioassays of progeny from crosses indicated this resistance to Cry2Ab was autosomal, recessive, and probably conferred by a single genetic locus [11]. Tay et al. used genetic linkage analysis with molecular markers to narrow the source of resistance to Cry2Ab in this strain to a chromosomal region containing less than 30 genes. They found that two of these genes encode the ATP-binding cassette (ABC) transporter proteins ABCA1 and ABCA2. These genes were prime suspects because resistance to Cry1 toxins is linked with the ABC transporter protein ABCC2 in strains of seven species of Lepidoptera, including H. armigera [12–15]. Tay et al. honed in on ABCA2 because it was produced in the midgut where Bt toxin binding occurs, but ABCA1 was not. In cDNA of ABCA2 in a resistant individual from the SP15 strain, they found a 73 base pair deletion that introduces a premature stop codon. Including the SP15 strain, Tay et al. detected the same mutation in five of seven resistant lines of H. armigera, each established independently from insects collected from the field during 2002 to 2012. They found two other mutant alleles at the same locus in two other resistant lines, yielding a total of three mutant alleles that each encode a truncated ABCA2 protein. Screening of one Cry2Ab-resistant strain of the congeneric species Helicoverpa punctigera revealed a different premature stop codon in the orthologous gene. This new paper is a worthy successor to the landmark 2001 article by David Heckel’s group, which was the first to report the molecular genetic basis of resistance to Cry1Ac [16]. As that paper did for Cry1A toxins, this one will accelerate research to enhance understanding of the mode of action of Cry2A toxins. We now know that diverse mutations in cadherin and other genes can confer resistance to Cry1Ac in the field [17,18], which limits the utility of PCR-based monitoring for specific resistance mutations. Nonetheless, identification of cadherin as a key receptor for Cry1A toxins did spur genetic engineering of modified toxins that kill some insects resistant to Cry1 toxins [19,20], and this paper might inspire analogous discoveries for Cry2-resistant insects. Despite the ≥85% adoption of Bt cotton producing Cry2Ab and Cry1Ac in Australia since 2005 [7], eight years of monitoring data from the robust F1 screen method show no significant increase in the frequency of resistance to Cry2Ab (0.032 in 2007–2008 to 0.021 in 2014–2015 for H. armigera; 0.010 to 0.011 over the same eight years for H. punctigera) [21]. If the Cry2Ab resistance alleles provide protection against this toxin, why has their frequency not increased? Part of the explanation is that Cry1Ac resistance remains rare in Australia [21] and, with little or no cross-resistance, the Cry1Ac in the two-toxin cotton kills individuals resistant to Cry2Ab. This is reflected in only zero to 8.5% survival of SP15 larvae on two-toxin cotton [22]. Fitness costs associated with Cry2Ab resistance alleles could also delay the evolution of resistance in H. armigera and H. punctigera by selecting against these alleles when larvae eat non-Bt cotton or any of the other non-Bt host plants of these polyphagous species. Because insect ABC transporters often provide protection against xenobiotics, the resistance-conferring mutations disrupting these proteins may diminish their natural function, yielding higher fitness costs in the presence of toxic substances, such as plant defensive compounds and insecticides other than Bt toxins [12]. Although significant fitness costs of Cry2Ab resistance in H. armigera were not detected when larvae ate either artificial diet [23] or mature non-Bt cotton [22], survival on younger non-Bt cotton was significantly lower for resistant larvae (81%) than susceptible larvae (100%) [22], which is a substantial fitness cost. Moreover, in strains of H. armigera and H. punctigera with ABCA2 mutations, resistance to Cry2Ab is associated with significantly increased susceptibility to the organophospate insecticide chlorpyrifos and the carbamate insecticide methomyl [24]. Although Tay et al. provide compelling evidence that ABCA2 is essential for toxicity of Cry2Ab to Helicoverpa armigera, the precise role of this protein in the mode of action of Cry2Ab remains unknown. In strains of H. armigera and H. punctigera that harbor ABCA2 mutations, resistance to Cry2Ab is associated with reduced binding of this toxin [25], which implies that ABCA2 either binds Cry2Ab directly or facilitates binding of Cry2Ab to other target sites. A similar correlation with reduced toxin binding is typically seen with resistance to Cry1A toxins linked with mutations disrupting ABCC2 [12]. However, in the silkworm Bombyx mori, Cry1Ab bound equally to brush border membrane vesicles from susceptible larvae and larvae with ABCC2-linked resistance [26]. Additional work is needed to test the hypotheses of Tay et al. that ABCA2 provides both binding and pore formation functions for Cry2Ab. It will also be important to determine if resistance to Cry2Ab is associated with mutations affecting ABCA2 in other lepidopteran pests, particularly the field-evolved resistance to Cry2Ab in the US of Helicoverpa zea [4], a close relative of H. armigera. With global use of Cry2Ab increasing, more cases of field-evolved resistance are inevitable. For example, the risk is high for resistance of pink bollworm (Pectinophora gossypiella) to Cry2Ab in India, where the refuges of non-Bt host plants are scarce, resistance to Cry1Ac is widespread, and exposure to Cry2Ab is extensive [8,18,27,28]. Better understanding of the role of ABCA2 in the mode of action and mechanism of resistance to Cry2Ab may enhance our capacity to counter such resistance.

Pest control and resistance management through release of insects carrying a male-selecting transgene.

Abstract: Development and evaluation of new insect pest management tools is critical for overcoming over-reliance upon, and growing resistance to, synthetic, biological and plant-expressed insecticides For transgenic crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis (‘Bt crops’) emergence of resistance is slowed by maintaining a proportion of the crop as non-Bt varieties, which produce pest insects unselected for resistance While this strategy has been largely successful, multiple cases of Bt resistance have now been reported One new approach to pest management is the use of genetically engineered insects to suppress populations of their own species Models suggest that released insects carrying male-selecting (MS) transgenes would be effective agents of direct, species-specific pest management by preventing survival of female progeny, and simultaneously provide an alternative insecticide resistance management strategy by introgression of susceptibility alleles into target populations We developed a MS strain of the diamondback moth, Plutella xylostella, a serious global pest of crucifers MS-strain larvae are reared as normal with dietary tetracycline, but, when reared without tetracycline or on host plants, only males will survive to adulthood We used this strain in glasshouse-cages to study the effect of MS male P xylostella releases on target pest population size and spread of Bt resistance in these populations Introductions of MS-engineered P xylostella males into wild-type populations led to rapid pest population decline, and then elimination In separate experiments on broccoli plants, relatively low-level releases of MS males in combination with broccoli expressing Cry1Ac (Bt broccoli) suppressed population growth and delayed the spread of Bt resistance Higher rates of MS male releases in the absence of Bt broccoli were also able to suppress P xylostella populations, whereas either low-level MS male releases or Bt broccoli alone did not These results support theoretical modeling, indicating that MS-engineered insects can provide a powerful pest population suppressing effect, and could effectively augment current Bt resistance management strategies We conclude that, subject to field confirmation, MS insects offer an effective and versatile control option against P xylostella and potentially other pests, and may reduce reliance on and protect insecticide-based approaches, including Bt crops

A seed mixture increases dominance of resistance to Bt cotton in Helicoverpa zea

Abstract: Widely grown transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) can benefit agriculture, but adaptation by pests threatens their continued success. Refuges of host plants that do not make Bt toxins can promote survival of susceptible insects and delay evolution of resistance, particularly if resistance is inherited as a recessive trait. However, data have been lacking to compare the dominance of resistance when Bt and non-Bt seeds are planted in random mixtures versus separate blocks. Here we report results from greenhouse experiments with transgenic cotton producing Bt toxin Cry1Ac and the bollworm, Helicoverpa zea, showing that the dominance of resistance was significantly higher in a seed mixture relative to a block of Bt cotton. The proportion of larvae on non-Bt cotton plants in the seed mixture was also significantly higher than expected under the null hypothesis of random distribution. In simulations based on observed survival, resistance evolved 2- to 4.5-fold faster in the seed mixture relative to separate blocks of Bt and non-Bt cotton. These findings support previous modelling results indicating that block refuges may be more effective than seed mixtures for delaying resistance in pests with mobile larvae and inherently low susceptibility to the toxins in Bt crops.

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

Abstract: Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

Cis-mediated down-regulation of a trypsin gene associated with Bt resistance in cotton bollworm.

Abstract: Transgenic plants producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are useful for pest control, but their efficacy is reduced when pests evolve resistance. Here we examined the mechanism of resistance to Bt toxin Cry1Ac in the laboratory-selected LF5 strain of the cotton bollworm, Helicoverpa armigera. This strain had 110-fold resistance to Cry1Ac protoxin and 39-fold resistance to Cry1Ac activated toxin. Evaluation of five trypsin genes revealed 99% reduced transcription of one trypsin gene (HaTryR) was associated with resistance. Silencing of this gene with RNA interference in susceptible larvae increased their survival on diets containing Cry1Ac. Bioassays of progeny from crosses revealed that resistance to Cry1Ac was genetically linked with HaTryR. We identified mutations in the promoter region of HaTryR in the resistant strain. In transfected insect cell lines, transcription was lower when driven by the resistant promoter compared with the susceptible promoter, implicating cis-mediated down-regulation of HaTryR transcription as a mechanism of resistance. The results suggest that H. armigera can adapt to Bt toxin Cry1Ac by decreased expression of trypsin. Because trypsin activation of protoxin is a critical step in toxicity, transgenic plants with activated toxins rather than protoxins might increase the durability of Bt crops.

Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains

Abstract: Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among B. thuringiensis strains as well as B. cereus strains.

Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton

Abstract: Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.

A Transformed Bacterium Expressing Double-Stranded RNA Specific to Integrin β1 Enhances Bt Toxin Efficacy against a Polyphagous Insect Pest, Spodoptera exigua.

Abstract: Background Oral toxicity of double-stranded RNA (dsRNA) specific to integrin β1 subunit (SeINT) was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a transformed Escherichia coli expressing dsRNA specific to SeINT. Principal Findings The dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The transformed bacteria gave a significant oral toxicity to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with an ultra-sonication to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria. The larvae treated with the transformed bacteria suffered tissue damage in the midgut epithelium, which exhibited a marked loss of cell-cell contacts and underwent a remarkable cell death. Moreover, these treated larvae became significantly susceptible to a Cry toxin derived from Bacillus thuringiensis (Bt). Conclusions This study provides a novel and highly efficient application technique to use dsRNA specific to an integrin gene by mixing with a biopesticide, Bt.

Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

Abstract: More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.