The design and implementation of insect resistance management programs for Bt crops

Transcription

1 review GM Crops and Food: Biotechnology in Agriculture and the Food Chain 3:3, 1-10; July/August/September 2012; 2012 Landes Bioscience REVIEW The design and implementation of insect resistance management programs for Bt crops Graham P. Head and John Greenplate Monsanto Company; St. Louis, Missouri USA This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. Keywords: compliance, dose, pyramids, refuge, seed mix, transgenic crops Cotton and corn plants with insect resistance traits introduced through biotechnological methods and derived from the bacterium Bacillus thuringiensis (Bt) have been widely adopted since they were first introduced in Because of concerns about resistance evolving to these Bt crops, they have been released with associated IRM programs that employ multiple components and reflect the input of academic, industrial and regulatory experts. This paper summarizes the current status of Bt crop technologies in cotton and corn, the principles of IRM for Bt crops and what they mean for the design of IRM programs. It describes how these IRM programs have been implemented and some of the key factors affecting successful implementation. Finally, it suggests how they may evolve to properly steward these traits in different geographies around the world. The limited number of reported cases of resistance after more than 15 y of intensive global use of Bt crops suggest that this exercise has been broadly successful. Where resistance issues have been observed, they have been associated with first generation technologies and incomplete or compromised IRM programs (i.e., inadequate structured refuge). Next generation technologies with multiple pyramided modes of action, together with the implementation of IRM strategies that are more dependent upon manufacturing and less dependent upon grower behavior, such as seed mixes, should further enhance IRM programs for Bt crops. Introduction The first cotton and corn plants with insect resistance traits introduced through biotechnological methods were commercialized in 1996 in the USA. 1,2 These products contained lepidopteran-active crystalline (Cry) insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt) and were targeted in cotton at the tobacco budworm, Heliothis virescens (Fabricus), the bollworm, Helicoverpa zea (Boddie) and the pink bollworm, Pectinophora gossypiella (Saunders). Targets in corn included the stalk borers such as the European corn borer, Ostrinia nubilalis (Hübner) and ear-feeding insects such as the corn earworm, H. zea. Since the mid 1990s, a significant number of comparable products containing an assortment of Bt proteins have been Correspondence to: Graham P. Head and John Greenplate; graham.p.head@monsanto.com and john.t.greenplate@monsanto.com Submitted: 01/11/12; Revised: 02/23/12; Accepted: 05/14/12 introduced. Of particular note was the introduction in the US in 2003 of the first Bt trait targeting below-ground coleopteran pests, specifically the corn rootworm complex (Diabrotica spp). 3 Increasingly, farmers have access to multiple choices for plantincorporated insect control traits in combination with various herbicide-tolerance traits for improved insect and weed control. 4 Bt crops have arguably revolutionized agriculture, with unprecedented levels of adoption in the USA and around the globe, 5 and have shown significant economic gains for farmers, along with substantial environmental benefits. 6,7 Prospects for future engineered crops include stacks of multiple traits built upon Bt and herbicide tolerance along with traits to intrinsically enhance yields and improve responses to environmental stress. As exciting as these prospects are, they also highlight the need to protect and properly steward Bt technologies for longevity in the marketplace. In this regard, a primary threat is the potential evolution of Bt resistance in target insect pests, practically defined in this paper as significant crop damage (product failure) due primarily to a heritable reduction in Bt susceptibility of target pest populations. Anticipating this concern, biotechnology companies have been working with academic scientists, regulators and growers to design and implement proactive insect resistance management (IRM) programs for Bt traits. 8 These IRM programs are unique in their global scope and proactive implementation, and it is a testament to these programs that only a handful of reports of field-evolved resistance have been cited globally, despite nearly two decades of high levels of adoption by farmers It is relevant to note that here, as in any field of scientific endeavor, opinions are not unanimous. Some of these reports are currently in dispute within the scientific community as being prematurely categorized as resistance This paper summarizes the current status of Bt crop technologies in cotton and corn, the principles of IRM for Bt crops and what they mean for the design of IRM programs, how these IRM programs have been implemented, some of the key factors affecting successful implementation, and how IRM programs are evolving to properly steward these traits in different geographies around the world. Current Commercial Bt Crop Products Bt cotton. 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 GM Crops and Food: Biotechnology in Agriculture and the Food Chain 1

2 Table 1. Commercially approved Bt cotton events 19 Event Bt proteins expressed Targets Provider DAS Cry1F Dow Agro Sciences DAS Cry1Ac Dow Agro Sciences COT 102 Vip3A Syngenta COT 67B Cry1Ab Syngenta MON Cry1Ac and Cry2Ab Monsanto MON 531 Cry1Ac Monsanto sgk321 Cry1A and CpT1* Chinese Acad Sci GK 12 Cry1Ac/Cry1Ab fusion Chinese Acad Sci BNLA-601 Cry1Ac CICR (Indian Govt) Event 1 Cry1Ac GFM MLS-9124 Silver Six Cry1Ac/Cry1Ab fusion unknown Bt protein unknown Bt protein *Cowpea trypsin inhibitor. Table 2. Commercially approved Bt corn events 28,29 JK Agrigenetics Ltd. (India) Nath Seeds (India) Metahelix Life Sciences (India) Myanmar Govt Event Bt proteins expressed Targets Provider MIR604 Cry3Aa Diabrotica spp Syngenta MIR162 Vip3A Syngenta MON 810 Cry1Ab Monsanto MON 863; MON MON Cry3Bb1 Diabrotica spp Monsanto Cry1A105 and Cry2Ab2 TC 1507 Cry1F DAS Cry34Ab1 and Cry35Ab1 Diabrotica spp Monsanto Dow Agro Sciences Dow Agro Sciences 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 n 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 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. Bt corn. 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). 2 GM Crops and Food: Biotechnology in Agriculture and the Food Chain Volume 3 Issue 3

3 The Principles of IRM for Bt Crops The ultimate goal of IRM programs for Bt crops as with IRM programs for any insect control technology is to slow the rate at which insect resistance evolves. IRM programs cannot be expected to prevent resistance, but they should be designed to maximize the effective life of a Bt crop. The economic benefits of this strategy are obvious and prolonged product life increases the likelihood that next-generation products can be developed and commercialized in a timely manner, creating a paradigm of continuous improvement in technologies rather than sequential replacement to keep up with resistance. Predictive mathematical models considering the pertinent factors and principles (discussed below) have become valuable tools to assist technology companies, researchers and regulators as they compare the relative merits of various IRM plans in their efforts to ultimately implement programs which compromise neither economic value nor product durability The rate at which insect resistance may evolve to Bt crops will be determined by the same factors that influence resistance evolution to conventional insecticides. These factors can be divided into: (1) the nature of the product, its performance, and how it is used (This includes the pattern of Bt expression in the crop plant and penetration of the product into the market); (2) the genetics of insect resistance (This includes the initial frequency of the resistant allele, degree of dominance of that allele and fitness costs of resistance); and (3) aspects of insect behavior that mediate how the product affects the target insects (insect movement and mating). Below we examine in more detail the factors influencing resistance, along with basic IRM program components and recommendations relative to Bt cotton and corn. The nature of the product (dose and number of modes of action). 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 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). Genetics of insect resistance to Bt. 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 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 Insect behavior. 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. Components of IRM Programs Wherever Bt crops have been commercialized, they have been released with associated IRM programs that employ multiple GM Crops and Food: Biotechnology in Agriculture and the Food Chain 3

4 Table 3. Minimum refuge size requirements for Bt cotton products in various countries, expressed as a percentage of the total cotton area except in the case of Australia 55 Country Refuge Options Bollgard II/WideStrike: no structured refuge required for heliothine pests USA a Bollgard II in PBW-only regions: 5 14% embedded non-bt cotton, 5% external unsprayed non-bt cotton, or 20% external sprayed non-bt cotton Bollgard II: 100% sprayed non-bt cotton, 10% unsprayed Australia b non-bt cotton, 5% unsprayed pigeonpea, 15% sorghum or 20% corn Brazil Brazil Bollgard: 20% sprayed non-bt cotton China China Bollgard: no structured refuge required a In the USA, no structured refuge is required for Bollgard II and WideStrike cotton in the cotton-growing area from West Texas to the east coast because heliothines are the key target pests throughout this region, and natural refuge for these pests is deemed adequate. In the southwestern USA, Pectinophora gossypiella is a key target pest and requires different refuge practices. b For Australia, refuge options are expressed as a percentage of Bollgard II area. components and reflect the input of academic, industrial and regulatory experts. Standard components of these programs are listed below. Consistently high levels of expression of Bt protein in all important tissues fed upon by the target pests. 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). Refuge for susceptible target pest insects. 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 Cry1Ac-resistant populations of pink bollworm, Pectinophora gossypiella (Saunders), were discovered in Gujarat state in India in ,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 Use of Alternative Control Measures (Placement into an IPM Context) The placement of the Bt crop within a larger framework of Integrated Pest Management (IPM) is important for several reasons. From an IRM perspective, effective integration of Bt crops into IPM programs will reduce the selection pressure on Bt crops and thereby slow the rate of resistance evolution. Alternative insect control tools also are important for the control of pests that are not targeted by Bt crops and as complementary control measures for target pests that are not fully controlled by Bt crops. 59 Monitoring and remedial action plans. 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 4 GM Crops and Food: Biotechnology in Agriculture and the Food Chain Volume 3 Issue 3

5 Figure 1. Effect of refuge size on the rate of Bt resistance evolution to a product with two Bt proteins compared with a product with one Bt protein. 31 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. Development of Additional Modes of Action and Pyramided Bt crops As Bt crops evolve, improvements are continually being made as reflected in second and third generation products which employ more than one Bt protein, often representing separate or unique modes of action. These products, where more than one unique Bt protein are expressed, are called pyramids, and are usually characterized by more robust insect protection characteristics and improved IRM value. Combining insecticidal proteins for target pest control is a strategy that can dramatically delay the evolution of resistance if the target insects are not able to develop a single mechanism of resistance that confers tolerance to both proteins simultaneously, and empirical studies with biotech crops have confirmed the value of this strategy. For example, transgenic broccoli engineered to produce Cry1C has been shown to control diamondback moth that is resistant to Cry1Ac. 62,63 In addition, mathematical models comparing alternating insecticidal products, sequential use of the same products and pyramiding proteins determined that the most effective strategy was to pyramid insecticidal proteins within a single product. 31,33,52,64 With a 5% refuge, combining Bt proteins in a single product can delay resistance approximately eight times longer than deploying the same proteins sequentially (Fig. 1). 31 Alternatively, a pyramided product with a 5% refuge can more effectively delay resistance than a single-protein product with a 20% refuge. This means that a pyramided product with multiple modes of action may potentially be released with lower refuge requirements than products with a single Bt protein (or products with multiple proteins that represent only a single mode of action). These models indicated that the strategy of pyramiding multiple Bt proteins in a single product against the same insects pests will be most effective if certain conditions apply: (1) Cross-resistance between the Bt proteins should be low. (2) The mortality of susceptible insects caused by each of the individual proteins should be high. Both individual proteins are not necessarily required to have a high dose as described above; even moderate doses in a pyramid can greatly improve IRM characteristics if susceptible insects are well controlled. 34,56 Note, however, that the effectiveness and durability of a pyramid containing two Bt proteins may be compromised if resistance to one of the Bt proteins already exists in the field. GM Crops and Food: Biotechnology in Agriculture and the Food Chain 5

6 To understand the value of any particular pyramided product for managing insect resistance, it is important to consider whether it fulfills these conditions. For example, in the case of Bollgard II cotton (expressing Cry2Ab2 and Cry 1Ac) in the US, the Cry2Ab2 protein has been shown to control Cry1Ac-resistant strains of target pests and binds differently to the insect midgut (site of action), while the Cry1Ac effectively controls Cry2Ab2-resistant target pests These data indicate that the Cry1Ac and Cry2Ab2 proteins in Bollgard II cotton have different insecticidal modes of action and therefore a very low likelihood of cross-resistance. Both of these proteins are highly active against key lepidopteran pests like H. virescens and P. gossypiella. 25 Although the high dose criterion is not met by either Bt protein for H. zea, each has good activity and the extensive presence of non-cotton refuge for this pest has been documented. 54,68,69 Therefore, Bollgard II cotton meets the desired criteria for an effective pyramided product. Similarly, several commercial Bt corn products contain the Monsanto event MON which is a single genetic insertion expressing two lepidopteran-active Bt proteins, Cry2Ab2 and Cry1A Both Cry2Ab2 and Cry1A.105 are expressed in MON corn hybrids at concentrations which provide high levels of control of target stalk-boring pests and they also show very low potential for cross resistance based upon standard evaluations of target site binding properties and experiments with resistant insect colonies. 30 As with Bollgard II, the MON pyramid can still provide significantly improved IRM characteristics over either Bt protein alone against more difficult pests like H. zea for the same reasons. As described above, pyramided insect traits have the potential to allow a reduction in refuge (Fig. 1) while still maintaining a robust IRM profile. This scenario has been realized for the products described above (Bollgard II cotton and MON corn) and other pyramided products with similar characteristics. Based upon studies which confirmed adequate refuge from noncotton crop plants and wild hosts, the requirement for a structured refuge was removed by EPA in favor of a non-structured or Natural refuge in 2007 for Bollgard II and similar pyramided Bt cotton products on all US cotton acres where heliothines (H. zea and H. virescens) are the major pests. 54,70,71 This represents the overwhelming proportion of US cotton acres and excludes only the extreme western cotton growing regions where P. gossypiella is the major pest, because it has no alternative hosts. A similar situation subsequently occurred for pyramided Bt corn products, first for MON and then for other products such as SmartStax corn (containing the Cry1A.105, Cry2Ab2 and Cry1F); the refuge requirement was reduced by the EPA from 20% to 5% for MON in the US Corn Belt in time for the 2009 growing season. Other similar pyramided Bt corn technologies also have a reduced refuge requirement. These reduced refuge requirements provide farmers the opportunity to utilize Bt technologies on a larger portion of their acres. The Next Step for Bt Crop IRM: Seed Mix Refuges (Refuge in the Bag-RIB) When considering pyramided corn traits, the effectiveness of reduced structured refuge for IRM depends on growers complying with refuge requirements. In the US, concerns have been raised by reports suggesting that approximately 25% of US corn farmers planting Bt hybrids were not fully compliant with refuge requirements. 72 In addition, as these technologies are adopted in the developing world, there is considerable uncertainty about the nature of grower compliance where practical implementation of refuge may be problematic for a number of reasons. As the probability of refuge non-compliance grows, regardless of the geography, the risk of Bt resistance evolving in insect pests will be higher. 38,73 Indeed, low compliance to established refuge standards has been implicated as a contributing factor in most cases of resistance to Bt crops reported thus far. 9-12,14 An alternative to structured refuge is the use of pyramided Bt seed mixes that already contain a proportion of non-bt seed to represent a built-in refuge, thus transferring the responsibility of refuge implementation to the company providing the technology. 74,75 Use of a seed mix ensures that an appropriate amount of a suitable refuge variety is planted in each Bt field and also distributes refuge plants relatively uniformly within Bt fields, lowering the probability of mating among Bt resistant adults compared with a separate block refuge. If presented in attractive hybrids, the convenience of planting seed mixes for growers should increase adoption of pyramided-bt toxin varieties, and thus improve the broader landscape IRM profile for target pests. 76 A primary concern relative to seed mixes is that larval movement between Bt and non-bt plants might accelerate evolution of pest resistance in seed mixes. 35 Larvae receiving a sub-lethal dose and moving from Bt plants to non-bt plants, as well as larger less susceptible larvae moving from non-bt to Bt plants, create scenarios where heterozygote fitness and, therefore, selection for resistance are both increased. In addition, movement of susceptible larvae off non-bt plants onto Bt plants reduces refuge efficiency by lowering the number of susceptible insects produced by non-bt plants. The impact of reduced refuge efficiency likely will be greatest for Bt plants with single proteins exposed to pests with highly mobile larvae, i.e., lepidopteran pests. 77,78 The larval movement concern was the primary reason that originally approved IRM programs for first-generation single-trait Bt crops in the US included external structured refuges and specifically excluded seed mix refuges. 8 However, incorporating pyramided Bt products into a seed mix with non-bt refuge makes it much less likely that larvae moving to or from Bt plants will be able to survive. Although this movement-related mortality tends to reduce the effective refuge size of a seed mix, the guaranteed compliance and improved mixing of adult insects coming from Bt and non-bt plants are expected to offset this loss and benefit IRM. 79 Seed-mix refuges may not be an appropriate strategy under all conditions but mathematical models considering various levels of trait efficacy, pest fitness, refuge size, larval movement and grower compliance, have indicated that the use of pyramided Bt proteins makes a seed mix strategy a viable IRM option in the US Corn Belt, as well as in the developing world where planting of structured refuges by growers may not be practical. 34 After evaluation of the data collected regarding the IRM properties of seed mixes, the US EPA and Canada s CFIA have recently approved (2011) a 5% seed mix refuge for certain 6 GM Crops and Food: Biotechnology in Agriculture and the Food Chain Volume 3 Issue 3

7 pyramided corn Bt traits in the US Corn Belt and Canada, including MON and SmartStax (with MON and the Cry1F protein). 80,81 Key Steps in Developing and Implementing IRM Plans for Bt Crops In the years since the first introduction of insect-protected transgenic crops in the mid 1990s, it has become clear that science, economics, the practical needs and desires of farmers, and the complex interactions of these components, all play significant roles in the development of sustainable IRM plans. Based upon nearly two decades of practical experience, we present here a basic outline which considers these elements in a reasonable sequence, or timeline, designed to result in an IRM plan which enables the sustainable use of a valuable technology. The four major phases of this effort can be described in sequence as: (1) a resistance risk assessment of the particular Bt crop and agricultural system; (2) definition and exploration of IRM options; (3) implementation of the chosen IRM option(s); and (4) maintenance of the IRM program and monitoring. These stages are discussed below in more detail along with suggestions for timing relative to the desired commercial launch of the Bt crop. Risk Assessment (Suggested Timing: 3 or More Years Prior to Projected Launch) This initial phase provides an assessment of the risk of resistance evolution to the technology in the specific geography in which it is intended to be commercialized. In doing so, we assume that the risk of resistance exists and aim to estimate the level of risk and the degree of uncertainty associated with that estimate. Agronomic assessment. 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. Target pest biology. 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. Product performance. 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. Determine key stakeholders and influencers. 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. Defining IRM Options (Suggested Timing: 2 or More Years Prior to Proposed Commercial Launch) This stage involves the utilization of the information gathered and understanding gained in the risk assessment phase to assemble reasonable IRM plan options. Further engagement of stakeholders should occur here too. Explore refuge options. 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. Engage and assess stakeholders and influencers. 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. Recommend an IRM plan. 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 Program Implementation (Suggested Timing: One or More Years Prior to Projected Launch) This stage is where the selected plan is integrated into the business activities. IRM education for all stakeholders, but especially farmers, is implemented, with a keen eye toward continuing GM Crops and Food: Biotechnology in Agriculture and the Food Chain 7

8 feedback on success. There should be flexibility and willingness to revisit decisions contingent upon the feedback received. Incorporate IRM activities into business model. 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. Implement IRM education. 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. Assess initial IRM implementation efforts. 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. Post-launch maintenance and monitoring (should begin at product launch and continue for as long as the product is on the market). 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. Monitoring refuge compliance. 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. Resistance monitoring. 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. Issues management and remediation plans. 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. Conclusions The proactive implementation of IRM programs for Bt crops in all countries where they have been commercialized has been a unique and complex effort driven by the technology developers in collaboration with public sector scientists. 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