Several lepidopteran active proteins from Bt have been commercialized in cotton. These include the crystal endotoxin proteins Cry1Ab, Cry1Ac, Cry2Ab and Cry1F, as well as vegetative proteins, such as Vip3Aa. In addition, some are expressed as fusions or hybrid proteins. One event, developed by the Chinese Academy of Sciences, expresses Cry1Ac and a cowpea trypsin inhibitor (CpT1).¹⁸ These genetic events are summarized in Table 1.¹⁹ Lepidopteran cotton pests come mainly from the taxonomic family Noctuidae. Represented in this family are the genera Helicoverpa, Heliothis, Spodoptera and Earias; most of the economic damage done to cotton globally can be traced to these genera.²⁰ Especially important are H. virescens and H. zea in the US and Helicoverpa armigera (Hübner) in sub-Saharan Africa and throughout Asia.²⁰ Another cosmopolitan lepidopteran pest is P. gossypiella, which can be found in most cotton growing regions globally and can be locally devastating to cotton production.²⁰ To some degree, all of the Bt proteins mentioned above have some activity against most lepidopteran pests, although the potency of each varies among pest species. Overall potency of a Bt cotton will depend upon the Bt proteins expressed and their levels within the plant (dose). In general terms, Cry1Ac and Cry1Ab are very effective against most genera except Spodoptera; Cry2Ab is effective against all genera; Cry1F has strong activity against Spodoptera and H. virescens; and Vip3A also has excellent activity against Spodoptera with moderate to very good activity against several other genera.^21-27 In general, first generation Bt crop products tended to express a single Bt protein, whereas subsequent generations of commercial offerings have expressed two complementary Bt proteins. Commercial trends suggest a future where most products will express multiple Bt proteins to provide robust insect control across a wider spectrum of pests species and to reduce the threat of resistance development.

As with cotton, corn hybrids expressing Bt Cry proteins for insect protection were initially developed to control lepidopteran species. Cry proteins can control the larvae of damaging US species that include the corn borer complex (O. nubilalis, southwestern corn borer Diatraea grandiosella (Dyar) and sugarcane borer [Diatraea saccharalis (Fabricus)], H. zea and fall armyworm Spodoptera frugiperda (J.E. Smith). Globally these Bt proteins are active against economically important pests such as stalk borers which include Ostrinia, Chilo, Diatraea and Sesamia, as well as earworm H. armigera and several Spodoptera species. Table 2 shows the current set of Bt proteins that have been deployed in commercial corn products to control these economic lepidopteran pests.^28,29 As with cotton, the Bt proteins in corn have varying target specificities and potencies. Cry1Ab tends to have high activity against most, but not all, stalk boring pests; Cry1F has good stalk borer and Spodoptera activity; Vip3A shows excellent activity against Spodoptera and Helicoverpa, but very little activity against some stalk borers; and Cry2Ab shows relatively broad activity across the pest lepidopteran taxa, as does Monsanto’s hybrid toxin Cry1A.105, which is composed primarily of portions of Cry1Ac and Cry1F.^22,30 As with cotton, the earliest commercial offerings expressed single Bt proteins, with the current trend moving toward the expression of multiple Bt proteins to effectively capture a broader pest spectrum and protect against resistance development.

In addition, Bt proteins have been incorporated into transgenic corn events to control corn rootworm (CRW) species. The CRW complex (western, northern and Mexican rootworms, all Diabrotica spp) is the most destructive set of insect pests of corn in the United States and is the primary target of insecticide use on corn in the US. The first commercial transgenic maize hybrid designed to control CRW larval feeding was introduced in 2003 in the US and contained Cry3Bb1.³ Additional CRW-control Bt products have been commercialized since that time: a binary protein complex that utilizes the Cry34Ab1 and Cry35Ab1 proteins, another version of Cry3Bb1 linked to a herbicide tolerance gene for improved breeding efficiency and a modified Cry3Aa protein (Table 2).

The number of Bt proteins (and independent modes of action) contained in a Bt crop, and the level and consistency of expression of each Bt protein, will strongly affect the rate of resistance evolution. Resistance should evolve more slowly to Bt crops with multiple Bt proteins than those with a single Bt protein (see more detailed discussion in the section on pyramided products). In addition, the preferred expression pattern of each Bt protein is season-long, with the level sufficiently high to be able to control target insects that are heterozygous for any resistance genes (= “high dose”).⁸ High dose expression has been achieved for some major pests of both cotton and corn in most current commercial Bt products. Examples include H. virsecens and P. gossypiella in cotton and O. nubilalis in corn.^36-40 By delivering a dose that kills all or nearly all of the susceptible heterozygous insect pests, resistance is made functionally recessive and is slow to evolve. In addition, only a few resistance mechanisms will be sufficient in these extremely susceptible insects to enable survival of target pests on these products, meaning that the frequency of resistant alleles relevant to the Bt crop will be very low. In contrast, three reports of field-evolved resistance have emerged from systems where the Bt crops contained only a single Bt protein that provided less than high dose control of the relevant pest species, presumably enabling survival of heterozygotes and subsequent increases in resistance allele frequencies.^9,10 It should be mentioned that a season-long high dose may not appear consistent with the broadly accepted principles of integrated pest management (IPM) where the use of an effective pesticide dose is targeted to specific economic action levels based upon insect pest densities.⁴¹ However, in the absence of the ability to induce the production of a high dose of Bt protein within a plant in direct response to insect damage or presence, a consistent high dose is a preferred alternative. It also should be noted that Bt crop products that control target pests at less than high dose levels still may be highly effective under certain conditions (discussed below).

The degree of dominance of a resistant allele will be determined by the nature of the resistance allele itself and the efficacy of the product, and higher dominance will tend to lead to greater survivorship of heterozygotes and more rapid resistance evolution. In most cases of Bt resistance studied so far, resistance is partially to completely recessive.⁴² Published reports of dominant or incompletely dominant resistance in Lepidoptera reveal low levels of resistance, which were not strong enough to allow survival on Bt plants.^43,44 These factors, along with the high dose nature of many Bt products, increase the likelihood that most field-relevant resistance alleles (those resulting in significant damage to a Bt crop) are functionally recessive (see above). Studies on the fitness of Bt-resistant insect colonies derived from cases of field resistance, or selected for Bt resistance in the laboratory, also suggest high fitness costs of resistance to Bt cotton, which tend to slow the rate of resistance evolution.^45,46 The separate case of Bt traits targeting Diabrotica should be mentioned because none of the current single coleopteran traits exhibit a high dose, creating a situation where resistance inheritance is likely to be functionally dominant or partially so.^14,47 IRM plans for these traits generally require larger refuges to further enhance the local populations of susceptible insects.⁴⁸

The way in which insects move between Bt and non-Bt plants determines insect exposure to the Bt toxin.⁴⁹ Both larval interplant movement and longer range adult movement have important effects on resistance evolution by affecting (1) the selection pressure for resistance, (2) the likelihood that resistant individuals will mate with susceptible insects and (3) the rate at which resistance may spread after arising. For example, studies of the biology of the major cotton pests such as heliothines indicate that they tend to disperse relatively long distances, making it likely that small pockets of resistant insects will be diluted out by susceptible immigrants.⁸ However, if areas of resistance are larger, significant migration outward may spread resistance geographically.⁵⁰ Insects whose adult dispersal and/or mating behavior occur on a more restricted spatial scale may develop pockets of resistance more readily in Bt plantings if the presence of susceptible adults is not assured through the appropriate deployment of non-Bt refuge.⁵¹ Larval interplant movement also is an important factor and is discussed below in the section on seed mix refuges.

As discussed earlier, the high dose concept represents the desirable situation in which susceptible and partially resistant target pests are effectively controlled by the Bt crop and where fully resistant survivors are rare.⁸ Bt crop products that control target pests at less than high dose levels still may be highly effective and durable but require relatively more refuge than high dose products (see discussion below).

The concept of creating a refuge for susceptible insects is an IRM strategy that is unique to Bt crops. The refuge is a source of large numbers of susceptible target insects that is located close enough to the Bt field so that resistant insects emerging from the Bt are likely to mate with susceptible insects from the refuge. The value of this approach has been demonstrated through mathematical modeling and limited field experiments.^31,52,53 Typically the refuge is an area of the crop not expressing the Bt trait that must be planted by the farmer within a certain distance of the Bt field. However, in some cases the refuge may consist of other crops or naturally occurring wild plants which are suitable hosts for the target pests.⁵⁴ In general, suitable refuge sizes and designs will vary depending on pest biology, agronomics and many other factors. For example, the placement of the refuge must anticipate the level of larval and adult movement; where adult movement is greater, the Bt crop and refuge can be separated, while still allowing interbreeding between insects emerging from the two areas. Using Bt cotton as an example, Table 3 describes the refuge requirements in place for Bt cotton products in various countries.⁵⁵ In situations where high dose critera are not met, and particularly where products contain only a single toxin, IRM plans generally call for larger refuge requirements to accommodate larger numbers of surviving heterozygotes.^31,56 Corn rootworm products containing either Cry3Bb1 or Cry34/35Ab1 illustrate this.^57,58

The importance of the role of refuge can be illustrated by a brief discussion of several instances of field resistance documented to date. In Puerto Rico in 2006, Cry1F-resistant fall armyworm, Spodoptera frugiperda (Smith), were discovered.¹⁰ Resistance to Cry1Ab corn in the maize stalk borer Busseola fusca (Full.) was reported by van Rensburg in South Africa in 2006–7.⁹ Cry1Ac-resistant populations of pink bollworm, Pectinophora gossypiella (Saunders), were discovered in Gujarat state in India in 2008–9.^11,12 Two common factors associated with these instances of field-derived resistance are the expression of a single Bt protein and the suspected reduced contribution of refuge because of non-compliance by farmers and/or specific agronomic factors. For Puerto Rico, Storer et al. suggested that a contributing influence in fall armyworm resistance to Cry1F corn may have been drought conditions which rendered irrigated Bt corn more attractive to ovipositing moths and also led to a reduction in natural grasses which normally serve as alternate hosts and, thus, a source of refuge.¹⁰ Selective irrigation of Cry1Ab corn relative to the conventional hybrids planted as refuge in South Africa likely played a role in maize stalk borer resistance due to a known preference by adult moths for high humidity.⁹ Failure by Indian farmers to plant non-Bt refuge reportedly contributed to the appearance of field-evolved resistance by pink bollworm to Cry1Ac cotton.¹¹

Resistance monitoring is another important element of the IRM programs that are in place for Bt products. Resistance monitoring involves the regular assessment of target pest populations from areas where threat of resistance evolution is considered high. This is done by testing the susceptibility of these insects to the Bt proteins present in the crop being grown and comparing the measured susceptibility to historical measures of susceptibility prior to the introduction of the Bt crop. These studies may involve laboratory feeding tests where field-collected populations are challenged with purified or semi-pure proteins in synthetic diet, or standardized lab or greenhouse testing of insects against plants expressing the Bt proteins.^14,60,61 Resistance monitoring provides early warning of resistance evolution and may help assess the effectiveness of an IRM program. Care must be taken to include relevant controls in the testing and to relate the results back to the performance of the products in question. That is, variation in susceptibility measured in the laboratory is not necessarily a demonstration of field relevant resistance; the laboratory measurements must be linked to changes in product performance in the field. When such a linkage is not evident, interpretation of monitoring results will be difficult and will lead to a variety of conclusions. For example, there have been published reports, based on laboratory assays of susceptibility, that cotton bollworm have evolved resistance to Cry1Ac in the USA; these reports have been disputed on the grounds that appropriate controls were not used in the assays and no change in product performance was observed in the field.^13,15 Nevertheless, given that monitoring programs or farmer reports may indicate that resistance is evolving to a Bt crop, appropriate remedial action plans also must be developed. These plans contain approaches for determining the nature and distribution of resistance that should dictate what sort of remedial actions are needed. In addition, they summarize alternative control measures that could be implemented to reduce the frequency of resistance alleles.

The agronomic assessment involves evaluating the local agricultural practices pertaining to, but not limited to, farm size, implementation of monocultures or intercropping, current pest control practices and expected adoption levels. As the transgenic industry matures, it will also be important to consider, if appropriate, local farmers’ current experience or history with transgenic crops. Related questions explore current farmer understanding, experience and attitudes around IPM, the concept of IRM, and the ability to influence and monitor farmer behavior as it applies to IRM stewardship activities.

An understanding of the population ecology of target pests is critical. This includes the number of generations per growing season, crop and non-crop host utilization and distribution, and adult movement and mating behavior. Also to be considered are the history of resistance in the target pest, any knowledge around the genetics of resistance, and its current exposure, if any, to existing Bt crops. As part of this assessment, it is desirable to develop two or more years of susceptibility data for key target pests to the insect-active proteins to be expressed in the introduced crop. This is often achieved by exposing geographic populations to the proteins in laboratory feeding bioassays. This can be used to establish an effective comparative “baseline” against which future populations can be assessed after product introduction.

Two or more years of adequate field data should be collected which effectively elucidate the level of control for the economically important life stages of each target pest and whether product performance meets the criteria for “high dose” (99+% control) for single mode of action products. For target pests where control is not considered complete or near complete, the probable use of insecticides to provide supplemental control should be considered as well as their use as part of an overall IPM program to control non-target pests.

In any commercial geography it is important to understand and make a plan to engage with various local stakeholders who are directly affected by, and/or have the ability to influence the discussion around, IRM plan development and implementation. These often include, but are not limited to, local academic/scientific experts, farmer organizations, crop consultants, regulators, government ministries and industry players. Local scientific experts may be engaged to leverage their knowledge of target pests in the local agronomic environment.

In light of the information gained during the risk assessment phase, several factors should be explored to establish options for the deployment of perhaps the most critical component of any IRM plan, the refuge. These factors include the relative size and placement of refuges, contribution of alternate host crops or natural vegetation, the potential for farmer compliance, and possible means of enhancing or ensuring compliance.

Discussions with stakeholders should be driven to explore the range of understanding and views on IRM. If appropriate, collaborative working relationships with local academic/scientific experts may be pursued to address any studies which may fill any knowledge gaps uncovered in the risk assessment phase.

Ultimately, an IRM plan must be proposed that considers the above-mentioned factors and may be incorporated into a local commercialization plan. In countries where IRM is a required part of regulatory submissions for a Bt crop (e.g., the US, Canada and Australia), the IRM plan must be provided to regulators for their review. The plan should consider the available science as well as practical constraints imposed by the system.

IRM stewardship activities, including education and monitoring for technology adoption and refuge compliance, should be integrated into the local commercial model and business activities. Plans to allow for the monitoring of changes in pest susceptibility, responses to product performance issues and development of remedial action plans should also occur. Funding for these activities should be ensured.

IRM and IPM messages and communication vehicles suitable for the local environment should be developed. Training materials must be provided to those charged with the job of educating farmers. If structured refuge is part of the IRM plan, resources to provide training to farmers and enable follow-up compliance monitoring must be developed and distributed. Other key stakeholders and influencers also must be educated on the basics of the IRM plan and the reasons behind it.

Very early on, systems should be put in place to assess first efforts at IRM plan implementation and education. Initial farmer responses should be evaluated and discussed, as should feedback from regulators and other key stakeholders, including internal feedback. Appropriate modifications should be considered. Post commercial monitoring needs should be determined and planned for.

This phase constitutes the ongoing stewardship phase which is required to ensure continuing compliance to the chosen plan and monitor for issues which may require action.

Monitoring adoption and use patterns. Programs should be in place to effectively monitor technology adoption levels, and farmer use patterns. Regional adoption levels will influence pest population ecology and resistance selection pressure. If the contribution of alternate hosts (crop or non-crop) were included in the IRM plan, then changes in agronomic practices which affect the availability of these hosts may influence the efficacy of the IRM plan.

Refuge compliance, obviously, will directly affect frequency levels of resistance alleles in pest populations. If a structured refuge is part of the IRM plan, then monitoring farmer compliance is an important element of post-commercial stewardship. Farmer surveys along with in-field checks are common components of a refuge compliance monitoring program. However, compliance monitoring will always be a challenge because of the resources required to visit and assess a large enough number of farmers, and the potential biases present in telephone and computer-based surveys.

Pest susceptibility should be monitored to detect changes that fall outside the normal range of variation for the species in question. These studies may involve laboratory feeding tests where field-collected populations are challenged with purified or semi-pure proteins in synthetic diet, or standardized lab or greenhouse testing of insects against plants expressing the Bt proteins.^14,60,61 Responses can be compared with historical data collected in baseline studies, or to responses made by fully susceptible laboratory colonies. Arguably the most important indicator of susceptibility changes will come from the careful assessment of a technology’s performance in the field. It is important to evaluate any evidence of changes in product efficacy. This may include collecting insects in cases of unusual survivorship followed by further studies in the laboratory or greenhouse.

From a commercial perspective, plans should be in place to respond to farmer complaints regarding product performance. Farmer complaints do not often reflect resistance events, but the possibility should be considered. As mentioned above, contingencies for evaluating truly unusual survivorship should be in place. In addition, subsequent plans of action should be developed to confirm or refute suspected resistance events, and to characterize confirmed resistance events. Finally, a collection of viable options for remediation of confirmed resistance events along with appropriate approaches for communication to stakeholders and regulators should be in place.