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TITLE:  Field tests on managing resistance to Bt-engineered plants
SOURCE: Nature Biotechnology, Volume 18 Number 3
        by A. M. Shelton et al.
DATE:   March 2000

-------------------- archive: http://www.gene.ch/ --------------------


Field tests on managing resistance to Bt-engineered plants

Anthony M. Shelton1, Juliet D. Tang1, 2, Richard T. Roush3, 4, 
Timothy D.
Metz5, 6 & Elizabeth D. Earle5
[see also Cornell University Press Release at
http://www.news.cornell.edu/releases/March00/BrocMoth.bpf.html]

1. Department of Entomology, Cornell University, New York State 
Agricultural Experiment Station, Geneva, NY 14456.
2. Current address: Department of Entomology and Plant Pathology, 
Mississippi State University, MS 39762.
3. Department of Entomology, Cornell University, Ithaca, NY 14853.
4. Current address: Department of Crop Protection, Waite Institute, 
PMB 1, Glen Osmond, SouthAustralia 5064 Australia.
5. Department of Plant Breeding, Cornell University, Ithaca, NY 14853.
6. Current address: Department of Biological Sciences,Campbell 
University, Buies Creek, NC 27506.
Correspondence should be addressed to A M Shelton. e-mail: 
ams5@cornell.edu.

Several important crops have been engineered to express toxins of 
Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers 
planted nearly 8 million hectares (nearly 20 million acres) of 
transgenic Bt crops approved by the EPA. Bt-transgenic plants can 
greatly reduce the use of broader spectrum insecticides, but insect 
resistance may hinder this technology. Present resistance management 
strategies rely on a "refuge"composed of non-Bt plants to conserve 
susceptible alleles. We have used Bt-transgenic broccoli plants and 
the diamondback moth as a model system to examine resistance 
management strategies. The higher number of larvae on refuge plants 
in our field tests indicate that a "separate refuge" will be more 
effective at conserving susceptible larvae than a "mixed refuge" and 
would thereby reduce the number of homozygous resistant (RR) 
offspring. Our field tests also examined the strategy of spraying the 
refuge to prevent economic loss to the crop while maintaining 
susceptible alleles in the population. Results indicate that great 
care must be taken to ensure that refuges, particularly those sprayed 
with efficacious insecticides, produce adequate numbers of 
susceptible alleles. Each insect/Bt crop system may have unique 
management requirements because of the biology of the insect, but our 
studies validate the need for a refuge. As we learn more about how to 
refine our present resistance management strategies, it is important 
to also develop the next generation of technology and implementation 
strategies.

Keywords: Insecta, Plutella xylostella, Bacillus thuringiensis, 
resistance, transgenic plants

 Expression of proteins produced by a common bacterium, Bacillus 
thuringiensis (Bt), in transgenic plants to protect them from insect 
attack is revolutionizing agriculture1. The insecticidal proteins 
produced by Bt are toxic to major pests of many of the world's most 
important crops such as cotton, rice, and corn. Of the $US 8.1 
billion spent annually on insecticides worldwide, it is estimated 
that nearly $2.7 billion could be substituted with Bt biotechnology 
applications2. At least 16 companies are presently developing 
transgenic crops with Bt genes, and at least 18 Bt-transgenic crops 
have been approved by the US Department of Agriculture (USDA) for 
field testing3.

When incorporated into plants, Bt proteins are made much more 
persistent and effective, even against insects that feed at sites 
difficult or impossible to reach with sprays4. Bt cotton was one of 
the first insecticidal plants to be approved for commercial use in 
1995, and since then the adoption of this technology has been rapid 
not only in the United States but also in Australia and China. The 
reasons for the rapid adoption of this new technology are compelling. 
For example, Bt cotton required three or fewer insecticide 
treatments, compared with historical averages of 5-12insecticide 
sprays per year for cotton in the United States5. Despite the 
considerable advantages of Bt-transgenic crops, both to the 
environment and to farm worker safety, concern is widespread that 
these gains will be short-lived because of evolution of resistance in 
the pests.

Various deployment strategies have been proposed to delay the onset 
of resistance6, and modeling studies have examined the effect of 
different deployment strategies7-10; however, few empirical data 
exist. The only commercially available strategy is use of a high dose 
of a single gene (>LC90 of heterozygous RS insects) in combination 
with a refuge. The refuge is composed of nontransgenic plants that 
will generate enough SS (homozygous susceptible) individuals to 
outnumber RR (homozygous resistant) individuals during mating, so 
that the majority of the population will remain either RS or SS. For 
cotton in the United States, "expert opinion" in 1994 and a marketing 
strategy have resulted in the nontransgenic plants being deployed as 
either a separate refuge in which 20% of the field is planted in 
nontransgenic plants that can be treated with a non-Bt foliar 
insecticide, or a 4% refuge of nontransgenic plants that are left 
untreated. The concept is that the refuge will generate enough 
susceptible insects to dilute resistant alleles while at the same 
time allowing the nontransgenic plants to generate high yields.

Recently the debate on the appropriate strategies for controlling 
insects through the use of Bt plants has focused on the size of the 
refuge needed11, or indeed whether refuges that are large enough can 
be economically acceptable to the users or sellers of Bt crops. In 
cotton, for example, some workers have called for a dramatic increase 
in refuge sizes over the Environmental Protection Agency (EPA) 
requirements, such as refuges as large as 50%, if farmers are allowed 
to spray them12.The use of current transgenic cultivars thus faces 
the following dilemma. The maximum benefits to crop production, farm 
profitability, and reduction of pesticide use may come from larger 
proportions of transgenic crops, but long-term enjoyment of these 
benefits may be feasible only by limiting the percentage of the crops 
that are transgenic. Careful modeling studies and empirical data are 
needed to address this question.

Testing a resistance management strategy is inherently difficult 
because it requires both a Bt-expressing plant and an insect that has 
developed resistance to the Bt toxin expressed in the plant. For this 
study,we have used the diamondback moth, Plutella xylostella, the 
only insect that has developed resistance to Bt toxins in the 
field13, in combination with crucifers engineered to express a 
Cry1A(c) toxin14 to study factors that influence the development of 
resistance. Resistance in this population of diamondback moth was due 
to a single autosomal recessive gene15, and the plants expressed high 
levels of the toxin14.

In green house trials16 we introduced diamondback moths that had an 
initially low Cry1A(c) resistance gene frequency into cages with 
various ratios of Bt broccoli and non-Bt broccoli plants. The insect 
populations were allowed to cycle and, after a set number of 
generations, the larvae were tested for resistance. We found that 
pure stands of Bt-expressing plants (0% refuge) resulted in rapid 
development of highly resistant diamondback moth populations, and 
increasing the size of the refuge delayed the development of 
resistance. Furthermore, the placement of the refuge plants 
significantly affected the development of resistance. When both plant 
types were mixed in a random spatial arrangement ("mixed seedling 
model"), larvae were able to move between plant types. As they moved 
from refuge plants to Bt-expressing plants, they died and caused an 
overall decline in the number of susceptible alleles. This resulted 
in a more rapid development of resistance than when plants were 
separated by a distance that limited the movement of larvae. 
Additional greenhouse and laboratory data demonstrated that resistant 
diamondback moths display similar levels of weight gain, growth, and 
survival on Bt plants as they do on non-Bt plants17.

These studies have documented that Bt-resistant insects can survive 
on Bt plants and that different management strategies will influence 
the durability of resistance. Although these studies provided some 
insight into variables that could be manipulated to delay the onset 
of resistance, the present field study was performed to provide 
further data to help identify variables that may influence resistance 
management in the field.

Results and discussion

Our 1996 field experiment examined the effect of refuge size and 
refuge placement (mixed vs. separate refuges) on the distribution of 
the larvae within the plots as well as the level of resistance in 
diamondback moths at the end of the season. Our results demonstrated 
that the cumulative number of larvae per plant on refuge plants 
through the season in the 20% mixed refuge was significantly lower 
(6.4 vs. 14.6) than the 20% separate refuge (Table 1). This finding 
indicates that, as in our previous greenhouse experiments, a separate 
refuge is more effective at conserving the number of susceptible 
alleles because larvae on these refuge plants will be more likely to 
survive to adults (either SS or RS) that can mate with RR individuals 
and thereby reduce the number of RR offspring. This finding provides 
evidence to support the use of a separate refuge for Bt-transgenic 
crops that are attacked by insects that can move between plants as 
larvae. On the Bt-expressing plants over the season, an average of 
0.3 larva was found in any of the treatments, indicating that the 
diamondback moth population was being controlled by the Bt-expressing 
plants (Table 1). This was also confirmed by the absence of any 
larvae on the Bt-expressing plants at the end of the season. In leaf-
dip assays taken through the season, no differences in susceptibility 
were detected between diamondback moths taken from any of the 
treatments (Table2). Furthermore, comparing the level of resistance 
at the beginning of the test to the level at the end, it appears that 
the insects actually became more susceptible. This was the result of 
immigration of native susceptible diamondback moths into our field 
plots, which diluted the frequency of resistant alleles of the 
released insects and prevented the establishment of resistance even 
when R allele frequencies of released larvae were as high as 0.12. 
This result was not seen in our previous greenhouse studies in which 
we had a closed system prohibiting immigration. Despite the 
differences in the number of larvae on refuge plants in the mixed and 
separate refuges in this field study (Table 1), we were not able to 
document differences in mortality (Table 2) over the relatively short 
period of thisexperiment. However, the differences in larval 
populations on the refuge plants in these treatments do lay the 
groundwork for differences in susceptibility to occur given a longer 
time period.

Our results from this field study might be taken as justification for 
not needing any refuge within a planting because of the presence of 
immigrating susceptible alleles. However, such an approach would only 
be justified if immigration patterns of susceptible insects were well 
known and had been shown to be consistent. Usually one does not know 
a priori whether such immigration of susceptible alleles will occur. 
Under conditions in which there is no such immigration, high levels 
of resistance and crop damage can occur16.

Growers may be unwilling to sacrifice large numbers of refuge plants 
to delay the onset of resistance. Thus, current recommendations allow 
the management of insects on these refuge plants through the use of 
insecticides with a different mode of action than the Bt-transgenic 
plants. The critical question in such a strategy is whether enough 
susceptible insects will survive in the refuge to provide an 
effective source of susceptible alleles.

Our 1997 field experiments examined the results of spraying the 
plants in the 20% separate refuge. Because there is no documented 
cross-resistance between Cry1C and Cry 1A Bt toxins18, 19, we 
examined how spraying the refuge with M-C (Mycogen, encapsulated 
Cry1C) affected DBM larval density and resistance on Cry1Ac broccoli. 
Our results indicate that in both 100% refuge treatments (where 
insects were released or where insects were not released), 
susceptibility increased significantly over time (Fig. 1).With a 
discriminating dose of 10 p.p.m., the population had a rate of 27% 
mortality before release into the treatments, but in both 100% refuge 
treatments the mortality at 10 p.p.m. increased to >70% by the third 
count. The similar increase in susceptibility in both treatments is 
indicative of immigration of susceptible insects into those plots, as 
was also seen in the 1996 field studies. However, despite high rates 
of immigration of susceptible insects, when resistance allele 
frequencies in the plot were high, spraying the refuge resulted in 
progressively higher levels of resistance over the course of the 
season than when the refuge was not sprayed (Fig. 1).In both the 
second and third counts, the insect population in the sprayed refuge 
had a significant and >15% lower average mortality at the diagnostic 
dose for resistance (10 p.p.m.), compared with the insects in the 
unsprayed refuge. Insects collected from the Bt plants would have a 
RR genotype for Bt var. kurstaki resistance, and we consistently 
found significantly higher numbers of Bt var. kurstaki-resistant 
larvae on the Bt plants when the refuge was sprayed than when it was 
not sprayed (Fig. 2). This is the opposite of what should occur if 
resistant alleles are to be maintained in the refuge for an effective 
resistance management strategy.

 To illustrate this further, we examined the overall diamondback moth 
population within our experimental plots of 300 broccoli plants. 
Because each 20% refuge plot had 240 Bt plants and 60 refuge plants, 
a higher number of larvae per Bt plant translated to a significantly 
higher overall populationin the plot in the second and third counts 
when the refuge was sprayed than when not sprayed (Fig. 3). The 
important point demonstrated here is that spraying the refuge reduces 
its potential to dilute resistance. By leaving the refuge unsprayed 
and giving more susceptible insects a chance to survive, short-term 
sacrifices of relatively more insects in the refuge may translate to 
seasonal reductions in resistance and reductions in the total number 
of larvae per plot. The critical question is whether such populations 
would result in unacceptable crop losses.

The high-dose/refuge strategy is the current foundation for managing 
pest resistance to Bt plants. Whereas the consensus is that the 
efficiency of this strategy depends on early implementation before 
the frequency of resistance alleles is high, evaluation under field 
conditions with this criterion is inherently difficult. We can 
approach such an evaluation by increasing the R allele frequency, as 
we did with multiple releases, and then assess changes in 
susceptibility and effectiveness of the refuge in conserving 
susceptible alleles within a field. Our results indicate that the use 
of refuges can be a sound strategy. However, this strategy will also 
depend on our ability to effectively monitor and manage susceptible 
alleles on an individual field or farm basis as well as on an 
areawide basis. Within an individual field or farm, treating the 
refuge with a highly effective insecticide may dilute the abundance 
of susceptible alleles to such an extent that the refuge is rendered 
ineffective unless there is substantial immigration of susceptible 
alleles from wild hosts or from surrounding non-Bt crops. On the 
other hand, growers may be reluctant to sacrifice a large number of 
refuge plants to insects just to maintain susceptible alleles. An 
alternative to the strategy of having a 20% refuge that can be 
sprayed (the requirement for cotton) is the EPA-approved strategy 
(also in cotton) of having a 4% refuge that remains unsprayed. 
Critical experiments need to be performed to assess which approach, 
as well as which refuge size, would be more effective in conserving 
susceptible alleles while providing acceptable crop yields, and such 
tests need to be performed in the specific insect/Bt crop system.

As we refine resistance management strategies for the currently 
available Bt crops, it is also imperative that other strategies for 
managing overall resistance to Bt be developed and implemented in the 
near future. Having Bt expressed in plants so that the insect 
population is subjected to selection pressure for particular periods 
of time (e.g., through an inducible promoter) or in particular plant 
parts (e.g., through tissue-specific promoters) may provide larger 
refuges for susceptible alleles both within the field and within a 
region while at the same time minimizing crop loss10. Although this 
appears to be technically difficult at present, it may be an approach 
that merits further development. Other options may be more feasible 
in the near future. Theoretical models suggest that pyramiding two 
dissimilar toxin genes in the same plant has the potential to delay 
the onset of resistance much more effectively than single-toxin 
plants released spatially or temporally10, 20, and may require 
smaller refuges. Other non-Bt genes may also aid in managing 
resistance to Bt crops. Currently the most promising ones being 
evaluated in transgenic plants include vegetative insecticidal 
proteins (vips), as well as various genes from other insects, 
animals, plants,and bacteria that act as inhibitors of insect 
digestive enzymes (e.g., protease inhibitors, -amylase inhibitors, 
and cholesterol oxidase21).

The development and implementation of engineered insecticidal plants 
is currently in its infancy and the only available technology is that 
of Bt-transgenic plants. Using the diamondback moth/Bt broccoli 
system as a model, we have investigated aspects important to the long-
term deployment of this novel technology. Although the diamondback 
moth/Bt broccoli system may not exactly duplicate the currently 
available insect/Btcrop systems such as cotton, corn, and potatoes, 
it can help identify areas for further work. Concurrently with more 
field studies conducted to refine the presently utilized 
recommendations, industry, public sector scientists, and farmers must 
work together to develop a second generation of technology and 
implementation strategies to ensure the even longer term durability 
of Bt-transgenic plants.

Experimental protocol

The diamondback moth. Using field-collected diamondback moth 
populations and our laboratory susceptible population (G88) we were 
able to make synthetic populations of diamondback moth with the 
desired resistance allele frequency for Cry1A toxins. Field-collected 
diamondback moth populations that showed resistance to Cry1A were 
collected in 1996 and 1997. The year the population was collected it 
was mated with G88 to create the synthetic population, then reared on 
plant material and re-released in the field. Although these synthetic 
populations were manipulated for field studies in isolated research 
plots, there was no carryover through the winter, since the 
diamondback moth does not overwinter in upstate New York where these 
tests were conducted22.

The transgenic plants. Using an Agrobacterium tumefaciens-mediated 
transformation system and a synthetic cry1A(c) gene provided by 
Monsanto, we produced diamondback moth-resistant broccoli plants23. 
The plants allowed survival of about 90% of neonate larvae from the 
resistant strain but caused 100% mortality of the F1 neonates 
heterozygous for resistance14. Mortality assays with susceptible 
larvae using leaves taken from different locations within the plant 
showed that toxin expression is fairly uniform throughout the plant 
when the plant is in its vegetative stage17. We used cytoplasmic male 
sterile transgenic plants hemizygous for the cry1A(c) gene for field 
tests.

Field tests, 1996. Four treatments with three replicates were 
arranged in a completely randomized design: 0% refuge, 20% separate 
refuge, 20% mixed refuge, and 100% refuge (plots were located at the 
New York State Agricultural Experiment Station in Geneva, NY, three 
plot replicates per treatment, 300 plants per plot, and 60 m minimum 
distance separating plots), into which we released second-instar 
diamondback moth larvae (312 larvae per plot per release) at seven 
periods over the course of the season, at an approximate R allele 
frequency of 0.12. Plants were spaced at 46 cm between plants and 90 
cm between rows. Plots with a separate refuge had two border rows on 
one side of the field, and the border rows were separated from the Bt 
plants by one blank row of bare ground. Mixed refuges had non-Bt 
plants randomly assigned within the plot. Releases were initiated on 
16 June and continued at one- to three-week intervals until 26 
August. We inoculated the plots with insects at various times to 
ensure high diamondback moth populations, with multiple and 
overlapping generations and with R alleles. We were simulating what 
would happen when insect populations immigrate into and cycle within 
a field, and are challenged by Bt plants. Additional treatments were 
included for reference to reflect native diamondback moth populations 
and were 0% refuge and 100% refuge with no insect release in either 
(one replicate each). Counts of larvae (all stages) were taken at 
five periods over the season beginning on 1 July and ending on 12 
September. Leaf-dip bioassays with JavelinWG (Bt var. kurstaki; 
Novartis) to evaluate resistance were done with progeny of the 
released larvae (time zero) and with progeny of larvae counted in the 
final collection.

Field studies, 1997. Four treatments with three replicates were 
arranged in a completely randomized design: 20% refuge, 20% sprayed 
refuge (sprays were applied five times at 8- to 13-day intervals 
throughout the growing season), and 100% refuge into which we 
released diamonback moth pupae at six periods early in the season at 
approximate R allele frequencies of 0.8 based on 10 p.p.m. Javelin. 
The fourth treatment was a 100% refuge with no release. Releases were 
initiated on 15 May and continued at one- to two-week intervals until 
25 June for the same purposes as noted in 1996. Plant and plot 
spacing were as in 1996. Larval counts (eight plants sampled 
destructively per plot) were taken at three periods over the season 
beginning on 22 July and ending on 11 August. In each sample, all 
larvae counted were taken back to the lab and reared. In each of the 
20% refuge plots, larvae were collected from equal numbers of refuge 
and Bt plants and then combined. Progeny of larval collections and 
time zero larvae (i.e., representative of what was released into the 
plots) were used in leaf-dip bioassays with Javelin to evaluate 
resistance.

Data in Table 1 on the number of larvae per plant were analyzed by 
ANOVA with the means weighted inversely by their variances. Data in 
Figures 2 and 3 were analyzed using ANOVA, and non overlap of the 
standard errors indicates significant differences. Because the plots 
were inoculated with equal numbers of insects, less than normal 
variation within a treatment probably occurred. The bioassays for 
insects were conducted using our normal protocol24, and the 
concentration or dose/mortality relationships contained in Table 1and 
Figure 1 were estimated assuming a probit model by using POLO25. 
Received 15 July 1999; Accepted 2 January 2000.

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ACKNOWLEDGMENTS
We thank Melissa Birkett, Hilda Collins, Jennifer Cooley, Bryna 
Mitchell, Sabrina Siebert, Powell Smith, and Joe Zhao for their 
assistance in this project. The research was funded by USDA NRI 
grants 91-37302-6199, 93-01977 and 95-37302-1783.



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