Asymmetrical cross-resistance between Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests and can reduce reliance on insecticide sprays. Sustainable use of such crops requires methods for delaying evolution of resistance by pests. To thwart pest resistance, some transgenic crops produce 2 different Bt toxins targeting the same pest. This “pyramid” strategy is expected to work best when selection for resistance to 1 toxin does not cause cross-resistance to the other toxin. The most widely used pyramid is transgenic cotton producing Bt toxins Cry1Ac and Cry2Ab. Cross-resistance between these toxins was presumed unlikely because they bind to different larval midgut target sites. Previous results showed that laboratory selection with Cry1Ac caused little or no cross-resistance to Cry2A toxins in pink bollworm ( Pectinophora gossypiella ), a major cotton pest. We show here, however, that laboratory selection of pink bollworm with Cry2Ab caused up to 420-fold cross-resistance to Cry1Ac as well as 240-fold resistance to Cry2Ab. Inheritance of resistance to high concentrations of Cry2Ab was recessive. Larvae from a laboratory strain resistant to Cry1Ac and Cry2Ab in diet bioassays survived on cotton bolls producing only Cry1Ac, but not on cotton bolls producing both toxins. Thus, the asymmetrical cross-resistance seen here does not threaten the efficacy of pyramided Bt cotton against pink bollworm. Nonetheless, the results here and previous evidence indicate that cross-resistance occurs between Cry1Ac and Cry2Ab in some key cotton pests. Incorporating the potential effects of such cross-resistance in resistance management plans may help to sustain the efficacy of pyramided Bt crops

Susceptibility of Southwestern Pink Bollworm to Bt toxins Cry1Ac and Cry2Ab2 in 2005

Bt cotton is an extremely important tool for integrated pest management in the Southwest. It has been a major factor in the current historic low levels of conventional insecticide use in cotton of this region. This is due to Bt cotton’s unprecedented efficacy against the pink bollworm, Pectinophora gossypiella, and its selectivity in favor of key natural enemies of arthropod pests. Due to the pivotal importance of Bt cotton and widespread concerns about the development of pest resistance to transgenic crops, a multi-agency resistance management program was established to monitor and pro-actively manage resistance development in the pink bollworm. This report constitutes results from the ninth year of this monitoring program. Larvae were obtained from bolls collected in cotton fields located throughout the Southwest, cultured in the laboratory, and offspring tested using diet-incorporation bioassays that discriminate between susceptible and resistant pink bollworm. A total of 11 Arizona and four California collections were successfully reared and tested for susceptibility to Cry1Ac using a discriminating concentration of 10 μg Cry1Ac/ml of diet. Susceptibility to Cry2Ab2 was estimated similarly for 12 strains from Arizona and four from California using diagnostic concentrations of 1.0 and 10 μg Cry2Ab2/ml of diet. Success of pink bollworm eradication in suppressing pink bollworm populations in New Mexico and Texas precluded successful collection of samples in those states. No survivors of 10 μg Cry1Ac/ml were detected in any bioassays of 2005 strains (n=5358). The grand mean frequency of PBW survival of 10 μg Cry1Ac/ml in 2005 was 0.000%. A susceptible culture, APHIS-S, used each year as an internal control, yielded 99.3% corrected mortality in tests of 10μg/ml Cry1Ac (n=490). All twelve pink bollworm strains collected in 2005 were highly susceptible to Cry2Ab2, based on contrasts with baseline data collected from 2001-2003. There were no survivors of bioassays of either 1.0 μg Cry2Ab2/ml (n=1,000) or 10 μg Cry2Ab2/ml (n=3425). The susceptible APHIS-S culture had 82.5% corrected mortality in tests of 10 μg/ml Cry2Ab2 (n=200) and 100% mortality in tests of 10 μg/ml Cry2Ab2 (n=120). Field evaluations of efficacy of Bt cotton were conducted by the Arizona Cotton Research and Protection Council in adjacent pairs of Bt and non-Bt fields at 44 Arizona locations. Statewide, large pink bollworm larvae were found in an average of 15% of non-Bt bolls sampled from borders of refuge fields. This was on the low end of the range of infestation levels observed in refuges during the past decade. Bolls from adjacent Bt cotton (Bollgard™) fields yielded an average of 0.28% infested bolls. This value was down slightly from the previous year. Over 70% of the pink bollworm recovered from collections in Bt fields were from bolls that did not express Bt toxin. We conclude that there was no indication of problems with pink bollworm resistance to Cry1Ac or Cry2Ab2 at the locations sampled in 2005. Moreover, Bt cotton continued to exhibit exceptional field performance in Arizona

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

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.Peer reviewed: YesNRC publication: Ye

Efficacy of Bt toxins Cry1AbMod, Cry1Ac and Cry2Ab singly and in combinations against a susceptible strain of pink bollworm (APHIS-S) (see Methods for details).

a<p>All mortality values are adjusted for control mortality.</p>b<p>Observed mortality</p>c<p>Expected mortality for combinations of two or three toxins</p>d<p>Observed mortality - expected mortality; synergism causes positive values and antagonism causes negative values</p>e<p>Probability that the difference between observed and expected mortality occurred by chance based on Fisher’s exact test </p

Potency of modified Bt toxins relative to native Bt toxins against resistant and susceptible strains of pink bollworm.

a<p>LC₅₀ of a native toxin divided by the LC₅₀ of a modified toxin (based on data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080496#pone-0080496-t001" target="_blank">Table 1</a>). Potency ratios > 1 indicate the modified toxin was more potent than the native toxin; potency ratios <1 indicate the modified toxin was less potent than the native toxin.</p

Alternative Splicing and Highly Variable Cadherin Transcripts Associated with Field-Evolved Resistance of Pink Bollworm to Bt Cotton in India

<div><p>Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from <i>Bacillus thuringiensis</i> (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (<i>Pectinophora gossypiella</i>) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.</p></div

Binding and Oligomerization of Modified and Native Bt Toxins in Resistant and Susceptible Pink Bollworm

<div><p>Insecticidal proteins from <i>Bacillus thuringiensis</i> (Bt) are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV) from resistant and susceptible larvae of <i>Pectinophora gossypiella</i> (pink bollworm), a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers <i>in vitro</i> without cadherin and killed <i>P</i>. <i>gossypiella</i> larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant <i>P</i>. <i>gossypiella</i> larvae that have cadherin mutations.</p></div