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.

Figure 1. Selection for pink bollworm resistance to Cry1Ac and Cry2Ab.

We created strain Bt4R-P by selecting a subset of larvae from the Cry1Ac-resistant strain Bt4R on bolls of Bt cotton producing Cry1Ac. Next we exposed a total of 10,000 to 20,000 neonates from Bt4R and Bt4R-P to diet containing 3 μg Cry2Ab per mL diet. We started the Cry2Ab-resistant strain Bt4-R2 by pooling the adult survivors from Bt4R (n = 6) and Bt4R-P (n = 7). These 13 adults mated and produced eggs. The next two generations were reared on untreated diet. The F₃ larvae of the Bt4-R2 strain were reared on diet containing 5 μg Cry2Ab per mL diet, yielding extremely high resistance to Cry2Ab (Fig. 2 and Tables 2 and 3). We crossed AZP-R (highly resistant to Cry1Ac) with Bt4-R2 to start the AZP-R2 strain. We selected F₂ larvae of AZP-R2 on diet containing 10 μg Cry2Ab per mL diet, which yielded high, nearly homogeneous resistance to both Cry1Ac and Cry2Ab in the F₄ larvae of AZP-R2 (Table 4). AZP-R2U was a subset of AZP-R2 that was reared without additional selection on Cry2Ab.

Figure 2. Responses to Cry2Ab of pink bollworm larvae from a susceptible strain (APHIS-S), a resistant strain (Bt4-R2), and their F₁ progeny.

We scored live fourth instars, pupae, and adults as survivors after 21 d on diet. Adjusted mortality (%) is 100% minus adjusted survival (%). Adjusted survival (%) is survival on Cry2Ab-treated diet divided by survival on untreated control diet multiplied by 100%. F₁A and F₁B indicate F₁ progeny from mass crosses with male Bt4-R2 × female APHIS-S and female Bt4-R2 × male APHIS-S, respectively.

Figure 3. Responses to Cry2Ab of pink bollworm larvae from single-pair families from a susceptible strain (APHIS-S) (a), a resistant strain (Bt4-R2) (b), and their F₁ progeny (c, d).

Survival (%) on control diet (no Bt toxin, green bars) and diet treated with 10 μg Cry2Ab per mL diet (blue bars). Each of the 23 families tested (A-W) was generated by crossing a single male with a single female (n = 8–24 larvae tested per family on each diet; mean = 16 neonates per family on each diet). Asterisks indicate 0% survival on Cry2Ab-treated diet for all 17 families from APHIS-S and F₁.

Figure 4. Survival of pink bollworm larvae on field-grown cotton bolls.

Strains were susceptible (APHIS-SOM), resistant to Cry1Ac (AZP-R), moderately resistant to Cry1Ac and highly resistant to Cry2Ab (Bt4-R2), uniformly resistant to Cry1Ac and 25% of larvae resistant to Cry2Ab (AZP-R2U F₃), and highly resistant to Cry1Ac and Cry2Ab (AZP-R2 F₃ and F₄). Survival in the laboratory on bolls collected from field-grown plants of non-Bt cotton (green) and Bt cotton producing Cry1Ac and Cry2Ab (blue). Asterisks indicate 0% survival on Bt cotton bolls producing Cry1Ac and Cry2Ab for all larvae tested except AZP-R2 F₃ and F₄, which both had survival of 0.17 on two-toxin cotton relative to non-Bt cotton (see Table S1 and Methods for details).