Pyramided genetically modified (GM) plants express two or more Bacillus thuringiensis toxin (Bt) Cry toxins that are different in structure but effective against the same insect pests. One such product, pyramided SmartStax corn Zea mays, has been recently made available to commercial growers through a joint venture between Monsanto (St. Louis) and Dow AgroSciences (Indianapolis). It expresses Cry 1A.105, Cry2Ab2 and Cry1F toxins, which are active against a variety of caterpillars (order Lepidoptera), as well as Cry34Ab1/Cry35Ab1 and Cry3Bb1 toxins that are active against corn rootworms (order Coleoptera). Although older nonstacked varieties are still grown over considerable acreages, the agbiotech industry argues that pyramided corn is highly effective in controlling its target pests and is readily adopted by farmers.

More importantly, industry argues that because pyramided plants provide such effective insecticide resistance management1, 2, 3, a substantial reduction in the size of required nontransgenic refuges is possible for such plants without jeopardizing their efficacy. Indeed, the US Environmental Protection Agency (EPA) has recently approved the reduction of the non-Bt crop refuge size to as low as 5% for pyramided Bt corn. Here, I question this decision because it is not well supported by published results from theoretical or empirical studies.

The initial registration request for reduced refuge requirements for the pyramided Bt corn was denied by the EPA4. After studying additional application materials submitted by industry scientists2, however, EPA changed its stance, agreeing with industry's push for a reduced refuge and allowing the reduction of refuge size on commercial farms from 20% to 5% of the planted area in corn-growing regions of the United States and from 50% to 20% in the regions where both corn and cotton are grown5. The refuge is supposed to be planted as a block of non-Bt plants within or adjacent to the Bt crop.

A review of registration materials I have obtained under the US Freedom of Information Act and the existing scientific literature shows that currently available evidence in support of reducing refuge for the pyramided Bt corn to 5% of the total crop area is limited to predictions of a single unpublished mathematical model, developed in-house by industry scientists. Until more scientific evidence is available, I suggest that the reduction in refuge size to 5%, currently being implemented by the EPA for pyramided Bt corn, represents a gamble.

Thus far, Bt plants have proven amazingly durable in terms of resistance compared with chemical insecticides and have outperformed predictions of their rapid failure made in the 1990s. Their sustainability is in large part due to the rigorous resistance management program mandated by the US and other governments and implemented through the joint efforts of agbiotech companies and commercial growers. Nevertheless, resistance remains a serious threat, and there are already several cases of field-evolved insect resistance to Bt plants6, 7.

The current strategy to delay the evolution of pest resistance to Bt plants relies on the so-called high dose–refuge approach. This requires planting plots of non-Bt plants in the vicinity of fields planted with Bt plants. Susceptible insects persist within these plots and mate with resistant insects that may survive on Bt plants within the main crop. The resulting offspring are heterozygous at the resistant allele(s) and, because resistance is usually inherited as at least a partially recessive trait, cannot survive the high toxin dose expressed by Bt plants6.

Pyramided Bt plants provide an additional approach to delaying resistance in pest populations6, 8. A key assumption underlying this method is that selection by one toxin does not result in cross-resistance to another toxin. Industry has taken this argument further, proposing that, when pyramided toxin genes are used, a large refuge is no longer required for successful resistance management.

On the basis of publicly available information that I obtained, only two experimental studies have ever been conducted to specifically look at pyramided plants as a resistance management technique. Results of the first study9 showed that, in the presence of the 20% refuge, pyramiding performs better than using single-toxin plants, either sequentially or concurrently as a mosaic. A follow-up study conducted using the same greenhouse microcosm8 indicated that, in the presence of the 10% refuge, the efficiency of pyramided plants declines when the plants are grown together with single-toxin plants. Neither study attempted to investigate the effects of reducing refuge size to 5%. On the contrary, because both pyramided and single-toxin corn plants are currently being grown by commercial farmers, results of the second study8 warrant extra caution in relaxing refuge requirements. In the United States, pyramided Bt cotton overlapped with single-toxin Bt cotton for several years, resulting in rapid resistance to both toxins in the corn earworm, Helicoverpa zea6. In contrast, no increase in the frequency of resistant alleles was observed in Australia, which switched completely to the pyramided Bt cotton6.

In the absence of empirical data, regulators had to rely on mathematical models predicting the insect rate of adaptation to transgenic plants. Industry submissions to the EPA1, 3 quoted peer-reviewed published results of mathematical modeling by Caprio10, Tabashnik11 and Roush12, 13, as well as unpublished results of in-house modeling2, 3 to support reducing refuge size for GM plants expressing pyramided toxins to 5%.

The choices of Caprio10 and Tabashnik11 are surprising because both articles specifically discuss experiments and simulations applied to mixtures of insecticides, but do not address pyramiding genes into Bt plants. Tabashnik's11 overall conclusion is that multiple pesticide-use tactics have many potential pitfalls and are unlikely to provide long-term solutions to pesticide resistance problems.

Mathematical modeling by Roush12 showed that when mortality of susceptible homozygotes is high, pyramiding of toxins significantly slows down evolution of resistance compared with the use of a single toxin. Subsequently, Roush13 conducted an extensive study using simulation modeling to predict insect adaptation to Bt plants under different scenarios, including varying refuge sizes and presence of cross-resistance. He found that pyramiding of two or more toxins in the same plant can delay resistance for an extended period of time, even in the presence of a refuge that would be unacceptably small for single-toxin plants. Even so, Roush13 concluded that 10% was the minimum refuge size adequate for pyramided plants, and explicitly cautioned that small refuges remain risky.

More recently, Gould et al.14 published another modeling study on insect adaptation to plants expressing multiple toxins and found that a 10% refuge size is highly effective in delaying resistance. Again, that study assumed no cross-resistance and provided no evidence that 5% of a planted area constitutes a sufficient refuge size.

Unpublished mathematical models submitted to the EPA by the industry3 specifically showed that pyramiding toxins in combination with the 5% refuge is more effective in delaying resistance evolution in the corn rootworm compared with the currently used single-toxin plants with the 20% refuge. Once again, however, the study assumes a lack of cross-resistance between the expressed toxins.

Clearly, the possibility of cross-resistance is an important issue. The industry's main argument in support of using pyramided plants as a resistance management tool is that cross-resistance to different Cry proteins is highly unlikely because they have different structure and binding patterns in insect midguts1, 3. Even so, all Cry endotoxins have essentially the same mode of action (microbial disruptors of insect midgut membranes) and might be similar enough for the development of resistance genes that can overcome toxins with different binding sites13. Moreover, resistance to multiple structurally different chemicals is also not unusual among insects. Some of the resistance mechanisms known to reduce insect susceptibility to Cry toxins, such as enhanced metabolism and regeneration of damaged midgut epithelium, are likely to affect structurally different molecules.

Indeed, several studies have produced evidence of considerable cross-resistance among structurally different Cry toxins, and even among Cry toxins and synthetic insecticides. The diamondback moth, Plutella xylostella, showed cross-resistance to Cry1Ac and a synthetic pyrethroid deltamethrin when selected by either compound in the laboratory15. Laboratory selection of the tobacco budworm, Heliothis virescens, strain with Cry1Ac caused 50-fold resistance to Cry1Ac and a 53-fold cross-resistance to structurally different Cry2Aa16. A single gene controlled &gt;1,000-fold resistance to Cry1Aa, Cry1Ab, Cry1Ac, and a structurally different Cry1F in the field population of P. xylostella17 (although sharing of binding sites could have been possible in that particular case). A more recent study18 has shown that laboratory selection of the pink bollworm, Pectinophora gossypiella, with Cry2Ab caused up to 420-fold cross-resistance to Cry1Ac, which was higher than 240-fold resistance to Cry2Ab in the same strain. Similarly, selection of the beet armyworm, Spodoptera exigua, with Cry1Ab caused more than 20-fold cross-resistance to the structurally different toxins Cry1Ca and Cry1Da19. Even the experimental data submitted by the industry to the EPA3 suggest that selection by Cry1F might have resulted in the fall armyworm, Spodoptera frugiperda, and the European corn borer, Ostrinia nubilalis, strains becoming twice as resistant to CrylA.105 as they were to the toxin with which they were selected. Positive correlations between resistance to Cry2Ab and Cry1Ac were also detected in the field populations of H. zea6. Furthermore, H. zea evolved resistance in the field to Cry1Ac and Cry2Ab in pyramided Bt cotton6.

In response to the initial rejection of its application by the EPA, the industry developed a modification to its model2 allowing various degrees of cross-resistance between the toxins. The modified model predicted that pyramided GM corn with a 5% refuge would be more durable for control of O. nubilalis, and the Southwestern corn borer, Diatraea grandiosella, than the single Bt products with a 20% refuge under all adoption, cross-resistance and efficacy scenarios.