Pink bollworm surviving Cry1Ac cotton: resistance, refuges and verification

Selection in action. Resistance alleles already exist at low frequency in the population, and most are initially recessive. On a Cry1Ac crop, susceptible and heterozygous insects die, so the only survivors are rare resistant homozygotes. Each survivor mates with another survivor, and their offspring are homozygous and fully resistant. The 92% Bt area and the 40-fold rise in LC50 show the population has responded to strong, continuous, uniform selection - an increase in the frequency of resistant alleles, which is evolution by natural selection.

Why refuge failure matters. A refuge is a block of non-Bt plants, kept free of Bt-target insecticides, that produces a large population of susceptible moths. These susceptible moths mate with any resistant survivors emerging from the Bt crop. Because resistance is recessive, the offspring are heterozygous and are killed by a high dose of toxin, so resistance stays hidden and rare. When the refuge is only a tenth of the required size and is sprayed, very few susceptible moths are produced, so resistant survivors are far more likely to mate with each other, and homozygous resistant offspring increase rapidly.

Assumptions of the strategy.

• The toxin dose is high enough to kill heterozygous resistant insects.
• Resistance is functionally recessive.
• The initial resistance allele frequency is low.
• Refuge insects and Bt-crop insects mate randomly and at the same time.
• The refuge actually produces moths, i.e. it is not sprayed with the same insecticide.
• Only one toxin is deployed, or the second toxin has no cross-resistance with the first.

If the dose is not high or resistance is dominant, a refuge alone will not stop resistance, however well it is managed.

First, separate plant failure from insect resistance. These are different problems with different fixes, so both must be tested.

Check the plants. Collect leaf or boll tissue from the very plants the larvae came from and quantify Cry1Ac by quantitative ELISA, using known standards. Compare with tissue from a known-good Bt hybrid grown in the same field and season. Low or absent toxin in the field plants means the problem is expression, not resistance. Check also for seed purity (PCR for the inserted gene), plant age (toxin titre falls late in the season), and whether surviving larvae were feeding on non-Bt plants or on refuge plants next to the sample.

Check the insects.

1. Identify the larvae to species, since other bollworms tolerate Cry1Ac.
2. Establish a susceptible laboratory colony from an unexposed area, reared on the same artificial diet.
3. Run a dose-response bioassay: neonate larvae from the field population and from the susceptible strain are placed on diet with a range of Cry1Ac concentrations, with a toxin-free control for each. Record mortality after a fixed period and calculate LC50 values by probit analysis. A resistance ratio far above 1, with non-overlapping confidence intervals, is evidence of resistance.
4. Include a diagnostic-dose test: one concentration that kills essentially all susceptible larvae; survivors from the field population confirm resistance.
5. Repeat the bioassay with a second, unrelated toxin (for example Cry2Ab) to detect cross-resistance, and repeat the whole test in a second generation to exclude maternal effects or temporary induction.
6. F1 or F2 family screening can estimate the frequency of resistance alleles in the field population, which is what monitoring programmes actually need.

Unauthorized single-gene seed: risky and unlikely to help. Such seed has no validated expression data, no dose guarantee and no resistance monitoring attached; the same selection pressure is applied over a wider area, and any cross-resistance between the new toxin and Cry1Ac will be selected immediately. Deploying another single-toxin crop across the landscape is exactly the situation that produced this problem. Stacking two toxins with different modes of action and no cross-resistance is the sounder route, but only with high dose, a refuge and regulatory oversight - not through informal seed.

Spraying Bt kurstaki: redundant and counterproductive. Bt kurstaki formulations contain Cry1 and Cry2 proteins, including toxins closely related to Cry1Ac. Spraying them on a crop already expressing Cry1Ac adds selection for the same resistance mechanism and gives only short residual activity, because the proteins are degraded by sunlight and are effective only against young feeding larvae. It wastes money and accelerates the very problem it is meant to solve.

Resistance-management plan.

• Restore the refuge: the approved non-Bt refuge, planted and managed as specified, kept free of Bt-target insecticides, with the block actually inspected.
• Use a stacked, high-dose product carrying two unrelated toxins, so a single mutation rarely confers resistance.
• Rotate modes of action for any supplementary sprays, and avoid repeated use of one insecticide class.
• Monitor: pheromone traps for adult flights, regular boll scouting, and annual bioassays of field populations against diagnostic doses.
• Cultural control: a synchronous sowing window, a firm crop-termination date, destruction of stubble and ginning waste where larvae diapause, and deep ploughing after harvest.
• Seed integrity: buy only approved seed with a label and lot number, and report unauthorized seed.

Resistance is a shared resource; an individual farmer's choice affects the whole block, so the plan only works if neighbours act together.