The Science of Refuges: Bt Corn and the European Corn Borer

Murray Lee Eiland

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Abstract

This paper will focus upon the science behind the

requirements to plant refuges of non-Bt corn. Considering that

many of scientific papers on this subject present rather

complicated -and at times contradictory - mathematical

models, no effort will be made to assess these data using

quantitative means. The main pest considered here is the

European corn borer (Ostrinia nubilalis, ECB). Because Bt corn is

so important in the fight against this pest, considerable

attention has been devoted to developing strategies to prolong

the use Bt corn before this pest becomes resistant. The newly

developed “refuge in a bag” technology offers the best hope of

controlling the ECB long term. Physically delimited refuges

may be a transitional phase of Bt corn technology. This is

important because the current regulatory regime for refuges is

complicated and subject to abuse. Particularly in less

developed countries with limited infrastructure, Bt crop

technology must be straightforward to utilize in order to

remain effective on a world wide basis.

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Introduction

Bt corn is now viewed as critical for the effective

control of the European corn borer (Ostrinia nubilalis, abbreviated

here as ECB). The reasons this is the case are not hard to

discern. Field spraying has a number of drawbacks, including

incomplete coverage of plants, degredation in UV radiation,

heating, and drying. For boring insects in general, sprays

are notoriously ineffective. Indeed, several commentators

suggest that genetically modified (GM) crops have allowed the

management of pests on a scale unheard of since the

introduction of synthetic organic insecticides over 50 years

ago (Kennedy 2008). The development of ever more resistant

plants to insect pests is something of an arms race. Until the

introduction of techniques of genetic manipulation, the main

constraining factor was the small number of resistant genes

that could be used. Sources of resistance were limited to

plants that could be cross pollinated with the target plant,

which were plants of the same or closely related species. In

some cases it was also possible to use “bridge species,”

manipulating ploidy levels, or use techniques such as embryo

rescue to transfer resistance from more distantly related

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plants (Smith 2005). Such manipulation could yield a

successful new cultivar after years of research. Now it is

possible to insert genes from other sources into plants in a

fraction of the time. The best known GM plants are those that

have been augmented with cry genes from Bacillus thuringiensis (Bt),

particularly cotton and maize.

Literature Review

The use of GM crops has carried with it a host of

other issues. There are ethical concerns regarding

manipulating genes. A sizable and vocal segment of consumers

fear that the toxins will damage their health. There are

lingering doubts about the long term environmental impact GM

crops will have. This is particularly the case if there are

wild relatives of crop plants that could pick up the altered

genes. The risk of outcrossing to related non-crop species

was addressed in a USEPA study, who found that the risks were

minimal for maize, cotton and potato (USEPA 2000). Use of Bt

cotton was restricted or prohibited in some parts of Florida

and Hawaii where wild relatives were common. In the case of

maize the wild plants originate from Mexico, Central, and

South America. However, the USEPA study suggested that there

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should be an isolation distance between GM and non-transgenic

maize to address the problem of cross fertilization (Sanvido et

al. 2008).

At the other end of the spectrum, there is also concern

that target pest species will, over time, develop resistance

to GM crops. Despite the promise of synthetic organic

pesticides in the middle of the last century, it was clear

that early predictions were false. Insects became resistant

to toxins, and developing new chemical agents proved to be

both costly and time consuming. While the first Bt crops to be

marketed used a single toxin, later generations of GM crops

used multiple Bt toxins to expand the number of potential pests

that could be controlled as well as to delay any potential

resistance that may develop over time. To date the target

species are lepidopteran and coleopteran (Kennedy 2008). Bt

maize and cotton as of 2007 was grown in 22 countries, and

together some 42.1 million hectares were planted (James 2007).

Considering the scale, it is certain that pest species will

develop resistance over time unless measures are taken.

Analysis/Discussion

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While toxins from other organisms besides B. thuringiensis are

under investigation or development, there is something of a

bottleneck in developing new toxins. This is because the

genetics of B. thuringiensis is well understood. In contrast it

will take time and money to develop toxins from other

organisms that can be expressed in plants. The risk involving

relying upon Bt toxins alone is expressed by Gould (1998):

“Most Bt strains produce a number of related toxins, each coded

for by a single gene. Each toxin has a very specific target

site within the insect. Both the classical most-plant

resistance and pesticide resistance literature indicate that

toxic factors that impinge on a single, specific target site

may offer less of an evolutionary barrier than toxins with

multiple effects.” However, there is some good news in that

Heckel (1994) notes that protcolysis of a toxic fragment in

the midgut of the insect involves a gain of function, and is

therefore not likely to be inherited recessively.

It is essential to delay or prevent the development of

resistance in pest populations. The two main mechanisms that

scientific papers address are high levels of toxins produced

by the plants, and the establishment of refuges. This is

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often abbreviated as the HDR (High Dose Refuge) system. In

the case of Bt toxin, this means that the plants must contain

enough toxin so that only resistant insects can survive.

Indeed, the target is that all insects will perish. It was

suggested that toxin levels must be 25 times the dose required

to kill all susceptible larvae (FIFRA, 1998). Bt corn would be

resistant to insect attack and be of direct benefit to

farmers. In contrast refuges could be described as

sacrificial plantings that maintain a supply of toxin

susceptible insects that could mate with resistant individuals

from the Bt crops. The refuge should ideally contain a rough

parity of individuals so that heterozygous offspring are

produced, leaving the next generation vulnerable to the Bt

crop. The USEPA has required plans for insect resistance

management (IRM) in order to register Bt crops for sale. These

plans change over time as more is learned about the problem.

Broadly there are three different kinds of resistance a

plant can have towards potential pests: antibiosis,

antixenosis, and tolerance. Antibiotic resistance interferes

with a pest’s metabolism. In extreme cases, the plant can

reduce target insect numbers so low that a viable population

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cannot be established. Most if not all transgenic insect

resistance is conferred by antibiosis. Antixenotic resistance

makes a plant unsuitable for feeding and/or oviposition. It

may be chemical (smell for example) or physical (hairs are one

example). This type of resistance still leaves live insects

that may attack other crops. It would also be quite difficult

to execute genetically. Tolerance refers to making the plant

more robust so that it can withstand attack. This raises the

economic injury level. While this has been a focus of much

“traditional” selective breeding, to date there are no GM

examples of this type of resistance. As is the case for Bt

crops, plants that are tolerant to insect pests exert

selection pressure for adaptation even when the pest is as

sub-economic levels.

According to Tyutyunov (et al. 2006) three main

considerations of insect resistance to Bt crops are:

1. Insects are known to become resistant to insecticides,

particularly in cases where it is applied regularly in

high doses.

2. The short life cycle of many pest species speeds up the

development of Bt susceptible insects. In the case of the

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European corn borer, four generations per year can be

achieved in optimal conditions.

3. Some pest species have been known to develop resistance

under laboratory conditions.

The theoretical basis for dealing with Bt crop resistance

was formulated decades before as the result of research with

the spread of resistance to insecticides. Comins (1977) was

the first to address, on a theoretical basis, how random

genetic exchange between two insect populations (one subject

to insecticides and one insecticide free) can retard the

spread of resistance. The critical factor of course is that

resistance must be a recessive trait. In time – well after

1977 - the terminology would become standardised. For

instance, the non-treated plots would be known as refuges.

The exact time it would take resistance to become prevalent in

a population would also be influenced by the exact dominance

level of the trait, the initial frequency of the dominance

allele, the effectiveness of the insecticide, and the extent

of insect migration. Because all these factors are difficult

to control in field experiments, mathematical models have been

the preferred method of study. All models will assume that

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the initial frequency of the resistance allele is low and that

only rare homozygous individuals survive. Those with

heterozygous resistance alleles must be at least partly

susceptible to the toxin. Finally, the refuges must be in a

location that encourages mating between individuals from the

two different populations (Tyutyunov et al. 2007).

ECB populations have been extensively studied, and from

several studies it appears that the Bt resistant alleles are

relatively scarce in populations in the US. However, genetic

studies from the upper Midwestern United States (Krumm et al.

2008) suggest that the highest gene flow exist in southern

samples, and the lowest in northern samples. This is clearly

linked with temperature and number of generations per year.

However, there is gene flow between northern and southern

groups. According to Krumm (et al. 2008) the most significant

finding was that there was very little genetic variability

between groups. This suggests that there is a relatively high

degree of gene flow. This demonstrates that for the ECB, there

is no “island phenomenon” of limited breeding pools of

individuals isolated from one another. Showers (et al. 2001)

tracked the movement of the ECB via mark and recapture

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studies. This study found 37–52% of males recaptured flew 800

m or more, 8–11% flew 3.2 km or more, and some captures of

male and female moths occurred at distances greater than 40

km. Over 99.6% of released moths were never recaptured. The

latter point may indicate that these individuals travelled

further than the geographic extent of the trapping program.

Taking the airflow across the region into account, according

to Showers (2001) and others it is likely that the moths can

disperse at a rate of at least 32 km a year. These studies

suggest that for many purposes the ECB can be considered a

single population, and that resistance could spread quickly

once established.

The EPA has devised mandatory requirements - as of the

growing season in 2000 - that commercial users of Bt corn must

follow. Agreements must be signed and growers must renew the

agreements annually. There are three broad categories of

requirement:

1. At least 20% of the area that is under Bt corn cultivation

must be matched by a corresponding refuge of non-Bt corn.

This rule still applies to most Bt crops for both corn

borers and corn rootworms, but there are now some

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exceptions. This “typical” refuge must take a specific or

structured shape, and be planted in strips in the Bt corn

field (at least 4 but 6 recommended), as a block, or as

separate fields. In counties that grow cotton as well,

the refuge must be at least 50% o the area of the Bt corn

crop. This is because there is the potential for

interaction between Bt corn and Bt cotton. The distance

the refuge must be from the Bt crop depends upon the pest.

It must be from ¼-1/2 mile if the Bt corn is designed to

control corn borers, and must be adjacent to the Bt corn

if the variety is for corn rootworm control (USEPA 2006).

2. For particular Bt crops, a structured refuge need only be

5% of the area. This is the case for the SmartStax and

Intrasect crops. The reasoning is that because these

crops use multiple independent toxins to control pests,

it is less likely that insects will develop resistance.

However, the refuges must be from ¼ to ½ mile of the Bt

corn if it is designed to control borers (Intrasect) and

adjacent to the GM corn if it is designed to control

rootworm, as in the case of SmartStax (Bessin 2010). The

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separate field system for corn borers is acceptable

because the moths are very mobile.

3. In the case of AcreMax1 and AcreMax RW, the EPA states

that Bt seed can be blended with 10% non-Bt seed. No

supplementary refuge is needed for AcreMax RW (for

rootworms). With Acremax1 a 20% structured refuge is

required if borers are the pest to be controlled, but in

this case AcreMax RW can serve as the refuge for the

AcreMax 1 fields. This technology uses multiple toxins

for each targeted pest species, and is not the same as

simply mixing the standard Bt seed with non-GM seed.

Attempts to produce mixtures oneself, with no EPA

approval may in fact increase the risk of insects

developing resistance.

Many papers that discuss mathematical models of the

development of resistance in the ECB discuss the sizes ranges

of refuges. The mandatory guidelines introduced by the EPA in

2000 were a departure from previous years, as before that date

if refuges were treated with pesticides they were required to

be bigger. It was thought that because a treated refuge

produced fewer insects they would have to be larger. After

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2000 it was recognized that farmers would not know in advance

if the ECB infestation went beyond the economic threshold. In

addition, insecticide treatments for the ECB are known not to

be particularly effective (Hurley et al. 2002). At the same

time Hurley (et al. 2002) along with most other papers stress

that the 20% refuge cannot be appreciated as an ideal, and

that if increased resistance is desired a larger refuge would

be warranted.

However, all the authors were keenly aware that the

larger the refuge, and the less economical it was, the less

likely it would be for the guidelines to be followed. As

stated in Hurley (et al. 2002): “Whether treated refuge should

be allowed in regions with historically high frequencies of

pesticide use depends on the primary objectives of the policy.

Refuge treatments should not be allowed without higher refuge

requirements if the primary goal is to limit the risk of

resistance. However, if the primary goal is to reduce

conventional pesticide use or improve agricultural production,

then allowing treatments using economic thresholds with

current refuge requirements should be sufficient.” According

to Tyutyunov (et al. 2007) a 20% refuge system with even rare

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applications of insecticide would promote insect resistance.

In contrast, a 30% (or larger) refuge system would essentially

negate the development of resistance.

Because the guidelines on planting refuges is so

complex, the National Corn Growers Association has established

on online calculator to assist planters in making decisions.

It does not replace or supplement manufacturers IRM

information. Instead it seeks to present in a more easy to

understand manner the rules (NCGA 2010). Because the site can

be updated according to the latest science, it is flexible in

ways that a printed source cannot be. However, users in

developing countries may not have access to the internet.

Hopefully the online calculator will increase compliance, but

in such a complicated regulatory landscape this is difficult.

Even seed companies can make mistakes. In 2010 Monsato agreed

to pay a $2.5 million fine for failing to notify purchasers

that their Bt cotton was not approved for planting in certain

parts of Texas with large amounts of corn under cultivation.

The notices did not appear on seed bags from 2002-2007, and

resulted in the largest civil administrative penalty imposed

under the Federal Insecticide, Fungicide, and Rodenticide Act

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(Kaskey 2010a). No information was given as to assess the

nature of the fine in relation to the profits made from

selling Bt seed to areas where it should not have been planted

for during the period in question. This example raises a

disturbing possibility that compliance may be thwarted in an

effort to maximize profits.

A particular brand of Bt seed will only gain popularity if

the product is superior to other Bt varieties on the market.

While there has been a rush to new seed that offered increased

yields, there are some indications that some new technologies

may not offer enough. An example is SmartStax produced by

Monsato. The concept is that more than one toxin is used to

suppress a pest, so that if one toxin is not successful then

another will work. Because of increased effectiveness, a

smaller refuge is required. The Monsato company stock price

fell 40% in 2010 and had the largest drop the biggest decline

on Sept 28 2010 in the Standard & Poor’s 500 Index because of

worries about SmartStax seeds. After 10% of the corn crop was

gathered, according to analysts, the new technology was

yielding 3 to 5 percent less than seeds with two or three

added traits (Kaskey 2010b). This is a serious problem, as it

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appears that the various toxic traits were expressed at the

expense of yield. This is clearly not acceptable from the

farmer’s viewpoint.

Refuges should be timed so that there are plants at the

same stage of development as the Bt corn crop. For example,

flying insects would select fields on the basis of the

developmental stage of the plants there. Within the refuge a

variety of techniques can be used to minimise pest damage.

This can include a spraying programme (according to economic

thresholds), early harvest, or weed control. The mixed refuge

technologies - so called “refuge in a bag” - such as AcreMax1

and AcreMax RW, mean that there is no difficulty in

maintaining a structured refuge. It also ensures that

compliance with requirements is straightforward. There is no

need for a structured refuge, except for corn borers and other

lepidopteran pests. The end result is that AcreMax1 is a two

“refuge in a bag” solution. The reason is that corn borers can

shift from plant to plant during their life cycle. The result

is that they could by a combination of feeding on Bt and non-Bt

plants receive a non-lethal dose of toxin and therefore

develop resistance. Press releases (from Dow in particular)

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during late 2010 state that data about new refuge in a bag

technologies that target above and below ground pests have

been submitted to EPA approval in late 2009. Pending

approval, the seeds should be made available for planting in

2012. According to statements made by Dow, a major factor in

the development of the refuge in a bag concept is the

uncertainty that surrounds compliance with current refuge laws

(Mattke 2010). It would appear from developments in

technology that the entire concept of a separate refuge will

be discarded in time.

Conclusions

From the literature it is clear that all mathematical

models are based upon assumptions regarding the origin and

spread of resistance. No matter what parameters are given, a

larger refuge with little or no pesticide use is ideal. The

recommended proportion of resistant individuals to non-

resistant ones as found by FIFRA is 500:1 (FIFRA 1998).

However, there is a considerable amount of debate about this

issue. There are also serious questions regarding the precise

arrangement of the refuges, which clearly is dictated by the

behaviour of various insect species. In the case of the ECB,

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a mobile species, there is perhaps more limited debate about

the distances refuges should be from the Bt corn.

Clearly the refuge requirements are not based primarily

upon science, and consider the crop yield of the refuge area

in economic terms as an important criterion. Indeed money it

is no doubt a critical consideration in the developed world,

and may be even more important in the developing world. With

a reduced infrastructure, without the ease of spot checking

for compliance and the imposition of penalties, it appears

that guidelines would not be uniformly followed. In high pest

areas, particularly considering the lack of effective sprays

against the ECB, there are strong economic incentives for not

planting refuges.

However, there is every reason to be optimistic. Although

it is clear that once established resistance in the ECB would

spread rapidly, in the field not a single homozygous ECB has

been found. However, given that Bt corn has only been

commercially available since 1996, there are good reasons not

to be complacent. Insect resistance to Bt maize has been

produced in the laboratory. In the end the questions of

refuges may be moot. It is in this light that the new “refuge

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in a bag” technology should be appreciated. It offers the

hope of making compliance a matter of routine.

Clearly a number of areas need further research, these

include:

1. Large scale use of Bt corn may have long term impact upon

pest populations, particularly the ECB. Studies in the

field to monitor pest levels are essential.

2. Refuge in the bag technology for the ECB is not yet for

sale, so studies that address farmer compliance with

refuge requirements are needed. While these seem to have

taken place on a limited scale it may be that certain

areas may follow particular trends. Refuge compliance in

developing countries may be particularly important. The

cosmopolitan nature of pests may make the development of

resistance a world wide issue.

3. Following from point 2, Bt corn might be designed for use

against one pest, the ECB for example, and be planted in

a country where other insect species are the target. A

pest species that might not be considered in the US may

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become a serious worldwide pest after developing

resistance in another country.

4. While there have been some studies that have identified Bt

resistance in some insect species in the laboratory (for

example Tabashnik et al. 2003) much more needs to be done

with a range of insects, particularly the ECB. The

prevalence and nature of Bt resistance in natural

populations is essential, and may take years of study to

unravel.

5. The impact of Bt corn on IPM may be significant. For

instance, a range of natural enemies that would otherwise

be sensitive to pesticides may be used in Bt corn and may

also be used in refuges. The reduction of pesticides can

also have negative effects on secondary pests - allowing

their populations to rise - that would need to be

controlled as well.

6. There have been several papers that have addressed the

impact Bt maize pollen may have non-target species, such

as butterflies and bees. These are serious issues to

address in that a disruption of the environment could

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have significant economic impact (perhaps particularly in

the case of bees).

7. The spread of Bt genes into wild maize is not an issue

that is important in the USA, as there are no wild

populations of the plant here. However, in areas where

the Bt crop are grown and wild populations exist (south of

the US border) there may be a nascent problem. The

spread of Bt genes to a wild population could affect the

development of resistance in insects.

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Comins, H. N.,1977. The development of insecticide resistance in the presence of migration. Journal of Theoretical Biology 64, pp. 177-197.

FIFRA 1998. Scientific Advisory Panel, Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticide and Resistance Management FinalReport, http://www.mindfully.org/GE/FIFRA-SAP-Bt.htm (last accessed 11/11/10).

Gould, F., 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annual Reviewof Entomology 43, pp. 701-26.

Heckel, D.G., 1994. The complex genetic basis of resistance to Bacillus thurigiensis in insects. Biocontrol Science and Technology 4, pp. 405-417.

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Kasky, J., 2010b. Monsato plunges on SmartStax corn yield. Bloomberg Sept 28 2010, http://www.businessweek.com/news/2010-09-28/monsanto-plunges-on-smartstax-corn-yield-concerns.html (last accessed 11/11/10).

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Krumm, J.T., T.E. Hunt, S.R. Skoda, G.L. Hein, D.J. Lee, P.L. Clark, and J.E. Foster, 2008. Genetic variability of the European corn borer, Ostrinia nubilalis, suggests gene flow between populations in the Midwestern United States. Journal of Insect Science 8:72 available online at: insectscience.org/8.72

Mattke, C. 2010. Dow AgroSciences showcases new refuge in a bag concept. Dow Press Release Aug 31 2010, http://www.dowagro.com/newsroom/corporatenews/2010/20100831b.htm (last accessed 11/11/10).

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Sanvido, O., F. Widmer, M. Winzeler, B. Streit, E. Szerencsits, and F. Bigler, 2008. Definition and feasibility of isolation distances for transgenic maize cultivation. Transgenic Research 17, pp. 317-335.

Showers W.B., R.L. Hellmich, M.E. Derrick-Robinson, and W.H. Hendrix III, 2001. Aggregation and dispersal behavior of marked and released European corn borer (Lepidoptera: Crambidae) adults. Environmental Entomology 30, pp. 700-710.

Smith, C.M., 2005. Plant resistance to Arthropods: Molecular and Conventional Approaches, Springer Science & Business Media, Dordrecht, The Netherlands, 423 pp.

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Tabashnik, B.E., Y. Carriere, T.J. Dennehy, S. Morin, M.S. Sisterson, R.T. Roush, A.M. Shelton and J.-Z Zhao, 2003. Insect resistance to transgenic crops: Lessons from the laboratory and field. Journal of Economic Entomology 96:4, pp. 1031-1038.

Tyutyunov, Yu V., E.A. Zhadanovskaya, R. Arditi, and A.B. Medvinsky, 2007. A spatial model of the development of pest resistance to a transgenic insecticidal crop: European Corn Borer on Bt Maize. Complex Systems Biophysics 52:1 95-113.

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