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Applied and Environmental Microbiology, October 2001, p. 4610-4613, Vol. 67, No. 10
Departament de Genètica, Facultad de CC
Biológicas, Universitat de València, 46100 Burjassot,
Valencia, Spain1; Department of Biology,
Imperial College of Science, Silwood Park, Ascot, Berkshire SL5 7PY,
United Kingdom2; U.S. Agricultural
Research Station, Agricultural Research Service, U.S. Department of
Agriculture, Salinas, California 939053; and
Department of Entomology, University of Arizona, Tucson,
Arizona 857214
Received 20 April 2001/Accepted 23 July 2001
So far, the only insect that has evolved resistance in the field to
Bacillus thuringiensis toxins is the
diamondback moth (Plutella xylostella). Documentation and
analysis of resistant strains rely on comparisons with laboratory
strains that have not been exposed to B. thuringiensis toxins. Previously published reports
show considerable variation among laboratories in responses of
unselected laboratory strains to B. thuringiensis toxins. Because different laboratories
have used different unselected strains, such variation could be caused
by differences in bioassay methods among laboratories, genetic
differences among unselected strains, or both. Here we tested three
unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca,
and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from
The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the
ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa
and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was
8-fold more susceptible to Cry1Ab and 13-fold more susceptible to
Cry1Da and did not differ significantly from the LAB-V strain in
response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins
were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac
being most toxic and Cry1Da being least toxic. Therefore, before
choosing a standard reference strain upon which to base comparisons, it
is highly advisable to perform an analysis of variation in
susceptibility among field and laboratory populations.
Insecticidal crystal (Cry) proteins
of Bacillus thuringiensis are contained in the
crystalline bodies produced during the sporulation phase. They are
produced as full-length proteins (protoxins) that, upon solubilization
in the insect midgut, are processed by midgut proteases to render a
protease-resistant fragment that constitutes the active toxin. The
active toxin binds to specific target sites in the insect midgut,
creating pores in the midgut membranes that eventually kill the insect
(19). Cry proteins are extremely useful because, compared
with conventional insecticides, they are more specific and thus
environmentally safer (2, 3). Transgenic crop plants that
produce Cry proteins are being used widely (10). In
addition, some insect populations resistant to chemical insecticides
have been controlled with B. thuringiensis products (2).
So far, the only insect that has evolved resistance in the field to
B. thuringiensis toxins is the diamondback moth
(Plutella xylostella) (23). Documentation and
analysis of resistant strains rely on comparisons with laboratory
strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins (Table 1). Such variation could affect not only
the absolute assessment of toxicity but also the relative resistance
levels detected for other strains. Because different laboratories have
used different unselected strains, such variation could be caused by
differences in bioassay methods among laboratories, genetic differences
among unselected strains, or both.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4610-4613.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Variation in Susceptibility to Bacillus
thuringiensis Toxins among Unselected Strains of
Plutella xylostella
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Reported LC50 and FL95
values of different toxins for four unselected laboratory strains of
P. xylostella
Each of our three laboratories has been using a different unselected P. xylostella strain as a reference strain to determine the toxicity of B. thuringiensis products and individual toxins (1, 12, 25). Here we used side-by-side comparisons to test the hypothesis that differences in susceptibility to Cry proteins between strains are genetically based. We also evaluated the effects of differences in bioassay protocols, including differences in the duration of exposure to toxins and in the source and preparation of toxins used in bioassays. Finally, we examined variations in the relative potencies of Cry proteins caused by differences in strains and bioassay procedures.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Insects. Each of the three susceptible strains had been reared for at least 10 years without exposure to Cry proteins. The LAB-V strain was collected in The Netherlands (5) and maintained in Spain; the ROTH strain was collected and maintained in the United Kingdom (18); and the LAB-PS strain was derived from the LAB-P strain, which was collected in Hawaii (13) and maintained in the United States. Larvae were reared on cabbage leaves.
Cry proteins. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da were obtained from recombinant B. thuringiensis strains EG1273, EG7077, EG11070, EG1081, and EG7300, respectively (Ecogen Inc.). Protoxin purification, trypsin activation, and protein quantification were performed as described by Sayyed et al. (18) at the University of Valencia, Valencia, Spain. Activated toxins were sent frozen to the Imperial College of Science, Ascot, Berkshire, United Kingdom. The same batch of toxins was shared and used by the above two laboratories.
Bioassays. Susceptibility to each Cry protein was tested with third-instar larvae by use of a leaf dip bioassay (21). At least five concentrations of each Cry protein were included. The replicates were performed on different days with larvae from different parents. We used three types of bioassays, each performed at a different laboratory: B1 (University of Valencia), B2 (Imperial College of Science), and B3 (University of Arizona, Tucson). The B1 bioassay used activated Cry proteins from Ecogen strains. Mortality was scored after larvae (10 per concentration) were exposed to Cry proteins for 2 days at 25°C. This bioassay was performed twice. The B2 bioassay also used activated Cry proteins from Ecogen strains, but mortality was scored after larvae (5 per concentration) were exposed to toxins for 5 days at 20°C. This bioassay was repeated eight times. The B3 bioassay used lyophilized powder containing spores and crystals from the strain that expresses Cry1Da (Ecogen strain EG7300). Two days after larvae (10 per concentration) were placed on treated leaf disks, fresh untreated leaf disks were added. Mortality was scored 5 days after the start of the bioassay. Rearing and tests for B3 were done at 28°C with 14 h of light and 10 h of dark. Four replicates of this bioassay were performed.
Before the side-by-side tests, strains were reared for at least two generations in the laboratory where the bioassays were performed at 25°C (B1, LAB-V versus LAB-PS) or 20°C (B2, LAB-V versus ROTH), at 70% relative humidity, and with a photoperiod of 16 h of light and 8 h of dark. Mortality data were evaluated by probit analysis (6) using the POLO-PC program (LeOra Software, Berkeley, Calif.) to estimate the concentrations killing 50% of the larvae tested (LC50s) and their 95% fiducial limits (FL95). LC50s were considered significantly different if their FL95s did not overlap.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Differences between strains.
Side-by-side comparisons revealed
genetic differences in susceptibility to Cry proteins between
unselected strains of the diamondback moth. Comparisons using the B1
bioassay showed that relative to LAB-V, LAB-PS was 8-fold more
susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da (Table
2). Significant differences in
LC50 between LAB-V and LAB-PS were not observed for Cry1Aa,
Cry1Ac, or Cry1Ca (Table 2). Comparisons using the B2 bioassay showed
that relative to LAB-V, ROTH was significantly more susceptible to each
of the five toxins tested (Table 3). The
differences in LC50 ranged from 17-fold for Cry1Aa to
170-fold for Cry1Ac.
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Differences between bioassays. The LC50 of Cry1Ab was significantly higher for the LAB-V strain in the B1 bioassay (exposure for and scoring at 2 days) than in the B2 bioassay (exposure for and scoring at 5 days) (Tables 2 and 3). Significant differences in LC50 did not occur for the other four toxins considered individually. However, for all five toxins, the LC50 was higher for the B1 bioassay than for the B2 bioassay (one-tailed sign test; P = 0.03). The differences in LC50 between bioassays ranged from 1.3-fold for Cry1Ac to 4.3-fold for Cry1Ab.
Relative potencies of Cry proteins.
The analysis of bioassay
data from this work showed that the relative potencies of Cry1 proteins
for the LAB-V strain followed a regular pattern. With either the B1
bioassay or the B2 bioassay, Cry1Ab and Cry1Ac were the most potent,
followed in order by Cry1Ca, Cry1Aa, and Cry1Da (Table
4). In addition, data reported for the
same strain in 1991 (5), 1994 (1), and 1996 (7) but with a diet overlay bioassay and toxins from a
different source showed the same pattern. Cry1Ab and Cry1Ac were also
the most toxic for LAB-PS and ROTH. However, Cry1Ab and Cry1Ac showed
an inverse pattern of potencies for these two strains compared with the
LAB-V strain in the same type of bioassay (Table 4). Relative potencies
for the Geneva (22) and Reunion Island strains
(17) differed greatly from those for the three strains
that we tested. For the Geneva strain, Cry1Da had the highest potency
of the five toxins tested. For the Reunion Island strain, Cry1Ab was
much more potent than the other four toxins.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results presented here show major genetic differences in susceptibility to Cry toxins between unselected laboratory strains of P. xylostella from The Netherlands (LAB-V), England (ROTH), and Hawaii (LAB-PS). In this study, genetic differences between strains in side-by-side comparisons were much larger than effects caused by differences in bioassays between laboratories.
The B1 and B2 bioassays compared here used activated toxins from the same source but differed in that the B1 bioassay was done at 25°C for 2 days whereas the B2 bioassay was done at 20°C for 5 days. As expected and in confirmation of previous results obtained with the diamondback moth (14, 20), LC50s were generally higher in shorter tests. Relative to previous studies, in the present study the extent of the difference between two time intervals might have been reduced somewhat because the temperature was higher for the shorter bioassay (B1) than for the longer bioassay (B2).
Despite differences in source of toxin and bioassay procedure and genetic differences in absolute susceptibility between strains, the patterns of relative potencies among the five toxins tested were similar for the three unselected strains tested here. For example, the pattern of relative potency for LAB-V remained similar for at least 10 years and was not affected much by the type of bioassay (leaf dip or diet overlay) or the source of toxin. Also, for LAB-PS, relative potencies were similar in bioassays with activated toxin and bioassays with crystals and spores. The use of protoxin involves additional steps over the use of activated toxins, and these have an influence on the final toxicity (8, 15). The presence of spores may also enhance the effects of toxins (12, 22). In contrast to the similar patterns seen for the three unselected strains tested here, the Geneva and Reunion Island strains showed unique relative potencies. However, in these instances, we cannot make strong inferences about the differences among strains because toxin sources and bioassay procedures varied. Side-by-side tests would be needed to determine if the differences in relative potencies were genetically based.
Side-by-side experiments performed with LAB-V and LAB-PS and with LAB-V and ROTH in different bioassay protocols revealed important variations due to genetic differences among strains. The greatest differences were obtained between LAB-V and ROTH. LAB-V and LAB-PS were rather similar with respect to their spectrum of susceptibility and also in terms of absolute LC50s.
Significant differences among conspecific populations have also been reported for other insect species. An analysis of Cry1Aa toxicity against two unselected strains of Heliothis virescens, carried out in different laboratories following similar protocols, showed about a 30-fold variation in absolute LC50s, while the toxicities of Cry1Ab and Cry1Ac showed just minor differences. These were not side-by-side studies, but they used similar protocols to test toxicity (11, 24). Moreover, two studies performed with two unselected strains of Trichoplusia ni showed about 100-fold differences in absolute LC50s for activated Cry1Ab and Cry1Ac. Although these were not side-by-side studies, they were performed in the same laboratory following essentially the same protocol and using Cry proteins from the same source (4, 9).
In conclusion, susceptibility to Cry proteins may vary among unselected populations of a given insect species. This variation affects the criteria for resistance, because a treated field population might be considered resistant or not resistant depending on the unselected reference strain used. Further, such variation could affect the standardization of potency for products based on B. thuringiensis. We strongly recommend an analysis of variation in susceptibility among unselected field and laboratory populations before a standard reference strain upon which to base comparisons is chosen.
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ACKNOWLEDGMENTS |
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We thank L. Calzada Grau for technical assistance and Ecogen Inc. for providing the recombinant strains used to prepare the toxins.
This work was supported by grants from the Spanish Agency for International Cooperation (AECI project AECI99-02-1) and the U.S.-Spain Joint Commission of Scientific and Technological Cooperation (project MAE99-0239). A.H.S. was supported by the Hundred Scholarship Scheme of the Government of Pakistan. J.G.-C. was supported by an AECI fellowship. Work in the United Kingdom was conducted under MAFF license PHL 17A/2689 (6/1998).
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FOOTNOTES |
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* Corresponding author. Mailing address: Departament de Genètica, Facultad de CC Biológicas, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain. Phone: (34) 96 386 4506. Fax: (34) 96 398 3029. E-mail: Juan.Ferre{at}uv.es.
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Applied and Environmental Microbiology, October 2001, p. 4610-4613, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4610-4613.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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