Insect Resistance Management

The delta-endotoxins produced by Bacillus thuringiensis (Bt) act by selectively binding to specific receptors localized on the brush border midgut epithelium of susceptible insect species. Following binding, pores are formed allowing gut contents to leak into the body fluid, eventually causing death. There are no receptors for these toxins on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to their action. This activity of Bt toxins has been exploited in agriculture and forestry for over 30 years through the use of microbial sprays. More recently, a number of crop species (corn, cotton, potato and tomato) have been genetically engineered to express Bt toxin in the foliar tissue to control a range of insect pests including the European corn borer (ECB), cotton bollworm and the Colorado potato beetle. When expressed in plants, Bt toxins are made much more persistent and effective, even against insects that feed at sites difficult or impossible to reach with sprays. Approximately 11.6 million hectares of Bt crops were grown globally in 2000 (James 2000).

Despite the commercial success of Bt crops, there is widespread concern that the advantages provided by them will be short-lived because the constitutive expression of Bt toxins in plant tissues will lead to the selection and multiplication of rare Bt-resistant insects. The potential development of insect resistance to Bt, as a consequence of large-scale commercial plantings of Bt crops, is also a concern to farmers and gardeners who rely on the use of Bt microbial sprays, for which instances of pest adaptation have already been documented (Tabashnik 1994). While the observation that over 400 insect species have become resistant to at least one insecticide (Georghiou 1986) is often cited as evidence of the genetic ability of arthropods to evolve resistant strains, it is equally important to note that, to date, there have been no reports of Bt resistant insects arising as a direct consequence of the introduction of Bt plants into commercial agriculture.

Bt microbial insecticides have an enviable history of safe use and it is for this reason that regulatory authorities in the United States and Canada consider Bt toxins in microbial sprays and transgenic plants to be “in the public good” and therefore worthy of extra regulatory protection (EPA 1999; CFIA 1999). That protection is provided through the requirement for mandatory implementation of insect resistance management (IRM) plans designed to mitigate the development of Bt-resistant insect pest populations.

Insect Resistance Management Plans

In Canada and the United States, the agricultural industry has adopted what is called the “high dose/refuge strategy” as a means of delaying the onset of Bt resistance. This strategy involves exposing a portion of the pest population to Bt plants with an extremely high concentration of toxin [25 times the amount needed to kill 99% of the susceptible insects (Gould et al. 1994)], while maintaining another part of the population in a refuge where the pests do not encounter any Bt toxin (Fig. 1). This strategy has four essential assumptions:

1. Resistance genes must be nearly recessive. In other words, individuals with only a single copy of the resistance gene (i.e. RS heterozygote) have a very low survival on the Bt crop, similar to that of a fully susceptible individual (i.e. SS homozygote). Statistically, RS survival rates on Bt plants must be less than 5% of the expected survival of truly resistant individual (i.e., RR homozygote) in order for the strategy to succeed.


2. The genes conferring resistance are rare. Studies with Heliothis virescens (Gould et al. 1997) and Ostrinia nubilalis (Andow et al. 1998, 2000) support this assumption, while others question its uniform application (Tabashnik et al. 2000).


3. The refuge of non-transgenic plants will maintain a sufficient number of susceptible individuals (SS homozygotes) to outnumber the resistant individuals (RR homozygotes) during mating so that their offspring will be RS heterozygotes, thus maintaining a population that is almost exclusively RS or SS.


4. The proximity of refuges to Bt crops is sufficient to ensure nearly random mating within the typical dispersal distance of the adults.

High Dose

An EPA Scientific Advisory Panel (EPA 1998) concluded that in order to be considered high dose a Bt cultivar would have to be shown by at least two of the following methods to contain 25 times the amount of Bt toxin needed to kill 99% of the susceptible insects:

1. Serial dilution bioassay with artificial diet containing lyophilized tissues of Bt plants (tissue from non-Bt plants serving as controls


2. Bioassays using plant lines with expression levels approximately 25-fold lower than the commercial cultivar (determined by quantitative ELISA or some more reliable technique)


3. Survey large numbers of commercial plants on sentinel plots in the field (e.g., sentinel sweet corn method) to make sure that the cultivar is at the LD99.99 or higher to assure that 95% of heterozygotes would probably be killed. With this approach Bt sweet corn hybrids are used to attract high densities of ECB and cotton bollworm [Helicoverpa zea (Boddie)] (CBW/CEW) moths, sampling can be limited to sweet corn ears in the Bt plot (ca. 1/4-1/2 acre block), and a frequency of resistant phenotypes can be estimated as the ratio of density of larvae/plant in Bt sweet corn to density of larvae/plant in an adjacent planting of non-Bt sweet corn (Andow & Hutchison 1998).


4. Similar to (3) above, but would use controlled infestation with a laboratory strain of the pest that had an LD50 value similar to field strains


5. Determine if an older instar of the targeted pest could be found with an LD50 that was about 25-fold higher than that of the neonate larvae. If so, that stage could be tested on the crop plants to determine if 95% or more of the older stage larvae were killed.

It is very important that a Bt cultivar meet the requirements for high dose as moderate or low dose plants could potentially accelerate the selection of Bt resistant insect populations (Fig. 2).

Deployment of Refuges

Refuges are comprised of non-Bt crop plants that are used to maintain Bt-susceptible insects in the population. The large number of SS homozygotes that survive in the refuge will be available to mate with any rare RR insects that survive on the Bt plants. The progeny of the SS x RR mating will be RS heterozygotes and so will be susceptible to thebttoxin in the Bt plants.

Much of the current debate around IRM has focused on the size of the refuge needed, and the extent to which insect control measures can be implemented within the refuge in order to minimize the economic consequences of insect predation. These factors are variable and are dependant on the crop, regional production and rotation practices, and the phenologies of primary and secondary insect pests. For example, in the United States, Bt cotton has been deployed with either a 20% refuge of non-Bt cotton that can be sprayed with a non-Bt foliar insecticide, or a 4% refuge without insect control. Some have recommended that if farmers are allowed to spray, then refuge sizes should be as large as 50% in order to maintain a sufficient population of susceptible insects.

While various deployment strategies have been proposed to delay the onset of resistance, and many of these have been examined through computer modelling, there are few empirical data demonstrating the efficacy of the high-dose/refuge strategy.

A recent study by Shelton et al. (2000) has attempted to answer a number of questions related to refuge placement, refuge size, and the impact of insect control within refuges. Their tests involved small-scale field trials over two years using transgenic broccoli plants engineered to express the Cry1A Bt toxin, and populations of diamondback moth with resistance to this Bt toxin. The diamondback moth is the only insect that has developed resistance to the spray form of Bacillus thuringiensis in the field, and by mating resistant individuals with a laboratory susceptible strain, the authors were able to generate synthetic populations with the desired frequency of the resistance trait.

Notwithstanding the unique circumstances of each combination of crop species and insect pest and the risks inherent in over generalization based on extrapolations from a single model system, the findings of Shelton et al. (2000), as summarized below, have important consequences for pest resistance management schemes:

1. Based on the numbers of larvae on refuge plants, a 20% separate refuge was more effective at conserving susceptible larvae than a 20% mixed refuge. Mixed refuges had non-Bt plants randomly assigned within the plot.


2. Spraying plants in the 20% refuge with a related Bt formulation (Cry1C, for which there is no documented cross resistance with Cry1A), resulted in progressively higher levels of resistance (approximately 10%) over the course of the growing season than when the refuge was not sprayed.

Similar conditions, with respect to the 20% refuge size and the requirement to maintain the functionality of the refuge as a reservoir of viable susceptible insects, have been included in authorizations of Bt corn and potato cultivars in the United States and Canada. The significant difference between refuge management for Bt corn and Bt potato is that the application of insecticidal sprays has always been a necessary means of controlling Colorado potato beetle infestations (CPB), while similar control methods have not generally been used for European corn borer management.

Best Management Principles for Bt Crops

The following are best management principles for IRM of Bt crops (EPA 1999) and are reflected in joint government/industry initiatives in the United States and Canada for Bt corn:

1. A specific IRM plan is necessary to ensure long-term resistance management. Included elements of the IRM plan are: high dose, structured refuge, susceptible pest biology and ecology data, impact on secondary pests, impact on pests affecting multiple Bt crops, cross-resistance potential, resistance mechanisms, monitoring/surveillance, and remedial action.


2. A high dose/structured refuge strategy is necessary to ensure long-term resistance management.


3. Grower education, adoption, and compliance are essential to the implementation and success of a long-term resistance management strategy.


4. Bt crops are to be used as part of an integrated pest management program to enhance pest management goals.


5. Coordinated annual performance monitoring and surveillance is necessary to detect or follow resistance development.


6. Immediate and coordinated remedial action for suspected and confirmed incidents of resistance is necessary.


7. IRM strategies should be tailored to address specific regional resistance management concerns, as appropriate.


8. Deployment of IPM tactics with different modes of action, including conventional pesticides, Bt toxins expressed in crops with different modes of action, biological control methods, and other control methods, is essential for sustainable pest management goals.


9. Continued resistance management research should be conducted to evaluate the effectiveness of, and be used to modify, as necessary, IRM strategies for Bt crops.

Insect Resistance Management of Bt Corn in the United States

The following are the elements of the Industry IRM plan developed for Bt corn grown in the US (NCGA 1999). For the vast majority of corn acres (> 90%) where growers are unlikely to treat with insecticides, the Industry IRM Plan is consistent and supported by recommendations of other groups, including the USDA Regional Research Committee on the Ecology and Management of European Corn Borer (NC-205 1997, 1998), the EPA Scientific Advisory Panel Subpanel (1998), and the International Life Sciences Institute Health and Environmental Sciences Institute expert panel (ILSI 1999). In areas where growers are more likely to use insecticides on the refuge (< 10% of corn acres), the Industry IRM Plan was influenced by the need to ensure grower practicality and grower adoption of IRM Plan requirements:

1. Minimum refuge requirements will be imposed for all corn growing regions of the United States. Growers will be required to plant a minimum of 20% non-Bt corn in the corn belt states and the northern portion of the corn/cotton region. A minimum 50% refuge of non-Bt corn will be required in the southern portion of the corn/cotton growing region.


2. Although essentially the entire corn/cotton region was subject to a 50% refuge requirement during the 1999 growing season, simulation models predict that a 20% corn refuge in the northern corn/cotton region is more than sufficient to provide long-term efficacy against corn borers (these models assume three corn borer generations/year). Simulation models also predict that a 20% corn refuge maintains susceptibility of corn earworm (CEW) populations in the northern corn/cotton region due to lower overwintering survival, fewer generations/year, lower Bt cotton adoption, and a higher ratio of corn to cotton as compared to the southern portion of the corn/cotton region.


3. Plantings of Cry1Ac-producing field corn hybrids derived from Event DBT418 will continue to be restricted from cotton-growing regions of the United States as defined in the current terms and conditions of EPA Reg. No. 69575-2.


4. There are limited regions of the corn belt where conventional insecticides have historically been used to control ECB and south western corn borer (SWCB) (CEW infestations have not historically warranted treatment in field corn). Growers will have the option of applying conventional insecticide treatments to the non-Bt corn refuge, however, they will be specifically instructed to do so only if the level of pest pressure meets or exceeds economic thresholds. Additionally, growers in these limited areas who wish to retain the option to treat their refuge with conventional insecticides will be required to plant the refuge acreage within one-quarter mile of their Bt corn plantings. Growers will be specifically instructed not to use Bt-based microbial pesticides in the refuge.


5. Except as noted in (4) above, growers will be encouraged to plant their non-Bt corn acreage within one-quarter mile of their Bt corn acreage where feasible, and required to plant the refuge within one-half mile of their Bt corn acreage. Growers need this degree of flexibility in refuge placement so they can maximize their efficiency during the narrow window of time that corn is planted in most regions of the corn belt.


6. Growers who purchase Bt corn hybrids will sign a Stewardship Agreement stipulating that they will adhere to the IRM requirements. Bt corn Grower Guides that contain a uniform set of IRM recommendations and requirements will be provided to each purchaser of Bt corn.


7. The key to the success of a plan of this nature is a strong grower education program. Growers must have a clear understanding of the importance of IRM to preserve the long-term efficacy of this technology, and that their participation in this IRM stewardship program is vital to its success. Each of the seed companies participating in this plan is committed to continuing with their ongoing comprehensive education programs. The Industry IRM Plan will be communicated through seed companies, as well as reinforced through the communication and education efforts of the National Corn Growers Association, the American Crop Protection Association, the Biotechnology Industry Organization, the National Alliance of Independent Crop Consultants, state and county corn associations, land grant extension services, USDA, EPA and others. The combined efforts of these different groups communicating a uniform IRM Plan will send a strong message to the grower community regarding the importance of their implementation of this program.


8. Current Bt corn registrations require the monitoring of insect populations (e.g., ECB, CEW) to measure changes in susceptibility to the insecticidal protein (e.g., Cry1Ab, Cry1Ac) expressed in Bt corn hybrids. Many of the seed companies have been working in a cooperative fashion in this effort. Under this proposed IRM plan, populations of ECB and CEW will continue to be monitored in a similar manner on an annual basis. At such time that validated methods for monitoring SWCB become available, monitoring of SWCB populations will be initiated, as appropriate.


9. On an annual basis, surveys will be conducted to ascertain the degree of grower adoption of the IRM Plan. If the level of adoption in a region falls below expectations, the industry participants in this Plan, in concert with other stakeholders, will intensify the grower education effort in these areas. As a consequence of repeatedly ignoring the IRM Plan requirements, identified growers will be restricted from future access to this technology.


10. Customers (growers and seed distributors) will be instructed to contact the registrant or authorized distributor if incidents of unexpected levels of target insect damage occur during use of the registrant’s Bt corn products. Registrants (or their authorized distributors) will investigate and identify the cause for this damage by local field sampling of plant tissue from corn hybrids that contain the Bt corn plant-expressed protectant and sampling of local pest populations, followed by appropriate in vitro and in planta assays. Upon confirmation by immunoassay that the plants contain the appropriate Cry1A protein, bioassays will be conducted to determine whether the collected insect population exhibits a resistant phenotype.
    
    Where available and validated for a target pest species, a discriminating concentration assay will be employed to define a confirmed instance of resistance. For other target pests, until such time that a discriminating concentration assay is established and validated, registrants will utilize the following to define a confirmed instance of insect resistance:
    
    Progeny from the sampled pest population will be considered resistant if they exhibit BOTH of the following characteristics in bioassays initiated with neonates:
    
    1. An LC50 in a standard diet bioassay (incorporating the appropriate Cry1A protein) that exceeds the upper limit of the 95% confidence interval of the mean historical LC50 for susceptible pest populations, as established by the ongoing baseline monitoring program.
    
    
    2. > 30% survival and > 25% leaf area damaged in a five-day bioassay using the appropriate Cry1A-positive leaf tissue under controlled laboratory conditions.
    
    Based upon continued experience and research, this working definition of confirmed resistance may warrant further refinement. In the event that the registrants find it appropriate to alter the criteria specified in the working definition, the registrants will obtain Agency approval in establishing a more suitable definition.


11. The registrant will report all instances of confirmed pest resistance, as defined above, to the Agency within 30 days. Upon identification of a confirmed instance of resistance, registrants will take the following immediate mitigation measures:
    
    1. Notify customers and extension agents in the affected area,
    
    
    2. Recommend to customers and extension agents in the affected area the use of alternative control measures to reduce or control the local target pest population, and
    
    
    3. Where appropriate, recommend to customers and extension agents in the affected area that crop residues be incorporated into the soil following harvest, to minimize the possibility of overwintering insects.
    
    Within 90 days of a confirmed instance of pest resistance, as defined above, registrants will:
    
    1. Notify the Agency of the immediate mitigation measures that were implemented,
    
    
    2. Submit to the Agency a proposed long-term resistance management action plan for the affected area,
    
    
    3. Work closely with the Agency in assuring that an appropriate long-term resistance management action plan for the affected area is implemented, and
    
    
    4. Implement an action plan that is approved by EPA and that consists of some or all the following elements, as warranted:
       
       1. Informing customers and extension agents in the affected area of pest resistance,
       
       
       2. Increasing monitoring in the affected area, and ensuring that local target pest populations are sampled on an annual basis,
       
       
       3. Recommending alternative measures to reduce or control target pest populations in the affected area,
       
       
       4. Implementing intensified local IRM measures in the affected area based on the latest research results. The implementation of such measures will be coordinated by the Agency with other registrants; and
       
       
       5. If the above elements are not effective in mitigating resistance, registrants will voluntarily cease sale of allbtcorn hybrids subject to the Industry IRM Plan in the county experiencing loss of product efficacy and in the bordering counties until an effective local management plan approved by EPA has been implemented. During the voluntary suspension period, registrants may sell and distribute in these counties only after obtaining EPA approval to study resistance management in those counties. The implementation of such a strategy will be coordinated by the Agency with other registrants and stakeholders.
       
       If EPA agrees that an effective local resistance management plan has been implemented which mitigates resistance, the registrants can resume sales in the affected county(ies).
12. Registrants participating in the Industry IRM Plan forbtcorn will meet with the Agency on an annual basis to discuss results from the grower adoption survey, insect monitoring program, and other pertinent plan issues.

Conclusion

It is imperative that IRM plans be based on the best available science (this may include simulation modelling), reflect regional production and rotation practices, and take into account the phenologies of primary and secondary insect pests as they occur in each production zone. It is equally important the IRM plans be clear, flexible, practical and not economically punitive; if they are not, IRM plans will simply be ignored by producers and the direct and indirect benefits of the technology will be lost.

References

1. Canadian Food Inspection Agency (CFIA). (1999). Insect Resistance Management of bt Corn in Canada. Ottawa, Canada.


2. Cohen, M.B., Gould, F. & Bentur, J.S. (2000). Bt rice: practical steps to sustainable use. International Rice Research Notes 25(2), 4-10.


3. Environmental Protection Agency (EPA) (1998). FIFRA Scientific Advisory Panel, Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance Management, Feb. 9 - 10, 1998 (OPP Docket No. 00231).


4. Environmental Protection Agency (EPA) (1999). EPA and USDA Position Paper on Insect Resistance Management inbtCrops. Washington D.C.


5. Georghiou, G.P. (1986). The magnitude of the resistance problem. Pp. 14-39 in Pesticide Resistance: Strategies and Tactics for Management. National Research Council. Washington, D.C.: National Academy Press.


6. Gould, F., Follett, P., Nault, B. and Kennedy, G. (1994). Resistance management strategies for transgenic potato plants. In Advances in Potato Pest Biology and Management. Zehnder, G.W., Powelson, M.L., and Raman, R.K. (eds.). APS Press, St. Paul, MN. pp 255-277.


7. Gould, F., Anderson, A., Jones, A., Summerford, D., Heckel, D.G., Lopez, J., Micinski, S., Leonard, R. & Laster, M. (1997). Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothus virescens. Proc. Natl. Acad. Sci. USA 94, 3519-3523.


8. International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (1999). An evaluation of insect resistance management inbtfield corn: A science-based framework for risk assessment and risk management. Report of an expert panel. ILSI Press, Wash. DC.


9. James, C. (2000). Global review of commercialized transgenic crops: 2000 (Preview). International Service for the Acquisition of Agri-Biotech Applications.


10. National Corn Growers Association (NCGA) (1999). Insect Resistance Management Plan: Planting Refuges, Preserving Technology.


11. Shelton, A.M., Tang, J.D., Roush, R.T., Metz, T.D. & Earle, E.D. (2000). Field tests on managing resistance to Bt-engineered plants. Nature Biotechnology 18, 339-342.


12. Tabashnik, B.E. (1994). Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39, 47-79.