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Applied and Environmental Microbiology, September 2000, p. 3784-3789, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development and Characterization of Diamondback
Moth Resistance to Transgenic Broccoli Expressing High Levels
of Cry1C
Jian-Zhou
Zhao,1
Hilda L.
Collins,1
Juliet D.
Tang,1,
Jun
Cao,2
Elizabeth D.
Earle,2
Richard T.
Roush,3
Salvador
Herrero,4
Baltasar
Escriche,4
Juan
Ferré,4 and
Anthony M.
Shelton1,*
Department of Entomology, Cornell University,
New York State Agricultural Experiment Station, Geneva, New York
144561; Department of Plant Breeding,
Cornell University, Ithaca, New York 148532;
Department of Crop Protection, Waite Institute, South Australia
5064, Australia3; and Department of
Genetics, University of Valencia, 46100-Burjassot (Valencia),
Spain4
Received 20 March 2000/Accepted 15 June 2000
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ABSTRACT |
A field-collected colony of the diamondback moth, Plutella
xylostella, had 31-fold resistance to Cry1C protoxin of
Bacillus thuringiensis. After 24 generations of selection
with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein,
the resistance that developed was high enough that neonates of the
resistant strain could complete their entire life cycle on transgenic
broccoli expressing high levels of Cry1C. After 26 generations of
selection, the resistance ratios of this strain to Cry1C protoxin were
12,400- and 63,100-fold, respectively, for the neonates and second
instars by a leaf dip assay. The resistance remained stable until
generation 38 (G38) under continuous selection but decreased to
235-fold at G38 when selection ceased at G28. The Cry1C resistance in
this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and
recessive when evaluated using Cry1C transgenic broccoli. Saturable
binding of 125I-Cry1C was found with brush border membrane
vesicles (BBMV) from both susceptible and Cry1C-resistant strains.
Significant differences in Cry1C binding to BBMV from the two strains
were detected. BBMV from the resistant strain had about sevenfold-lower
affinity for Cry1C and threefold-higher binding site concentration than
BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it
was for BBMV from the resistant strain. These results suggest that
reduced binding is not the major mechanism of resistance to Cry1C.
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INTRODUCTION |
Microbial insecticides based on
Bacillus thuringiensis can provide a good combination of
safety and effectiveness for pest control. In 1992, over 2 million
acres of U.S. crops were treated with B. thuringiensis
sprays (16). Transgenic insecticidal crops expressing
B. thuringiensis toxins entered the U.S. market in 1996, resulting in several important economic and environmental advantages.
For example, since the commercialization of B. thuringiensis-transgenic cotton in 1996, insecticide sprays on
cotton have been reduced by approximately 3.8 million liters of
formulated product per year in the United States, and this has led to a
significant reduction in the use of more hazardous organophosphate and
pyrethroid insecticides (according to the U.S. Environmental
Protection Agency and U.S. Department of Agriculture position paper on
insect resistance management in B. thuringiensis crops
[http://www.epa .gov/oppbppd1/biopesticides/otherdocs/bt_position_paper _618.htm]).
However, there is concern that widespread use of B. thuringiensis-transgenic crops may increase the risk of resistance to both transgenic crops and B. thuringiensis microbial
spray formulations (16).
There have been no cases of insects developing resistance on B. thuringiensis-transgenic plants in the field, but the
diamondback moth, Plutella xylostella (L.), developed
resistance to B. thuringiensis toxins in foliar
sprays under field conditions (27, 31). Laboratory populations of Cry1A-resistant diamondback moth can also survive on
transgenic crucifers expressing a high level of Cry1Ac (17, 22,
38).
Although intensive research has been conducted on resistance of the
diamondback moth to B. thuringiensis, most studies have emphasized resistance to Cry1A or B. thuringiensis
formulations. Tabashnik et al. proposed a model 1 of B. thuringiensis resistance, which is characterized by more than
500-fold resistance to at least one Cry1A toxin, recessive inheritance,
little or no cross-resistance to Cry1C, and reduced binding of at least
one Cry1A toxin (33). It was suggested that the gene(s)
conferring resistance to Cry1C segregates independently of the gene
conferring resistance to Cry1Ab in the diamondback moth
(11).
To date, only a few cases of Cry1C resistance have been reported. A
high level of Cry1C resistance (>500-fold) developed in Spodoptera exigua and Spodoptera littoralis after
selection in laboratories (18, 19). Binding experiments in
S. exigua indicated that, though binding of Cry1C was
slightly reduced in the Cry1C-resistant insects compared to susceptible
insects, reduced binding was not a major mechanism of resistance to
Cry1C in the resistant strain (18). In the diamondback moth,
only low to moderate levels of resistance to Cry1C developed either in
the field (23-fold) or the laboratory (62-fold) (11, 12).
Again, Cry1C-resistant insects did not differ from susceptible insects
in terms of Cry1C binding (14).
The objective of our study was to obtain a strain of diamondback moth
with a level of resistance to Cry1C high enough to allow the insects to
complete their entire life cycle on transgenic broccoli expressing high
levels of Cry1C. Once we succeeded, we examined the pattern of
inheritance of this resistance and whether Cry1C binding site
modification was the biochemical mechanism involved.
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MATERIALS AND METHODS |
Insects.
We used two strains of diamondback moth for these
studies: our standard susceptible strain (Geneva 88) and a resistant
strain (Cry1C-Sel). Geneva 88 insects were collected in 1988 from
cabbage at the New York State Agricultural Experiment Station, Robbins Farm, Geneva, New York, and have been maintained on a wheat germ-casein artificial diet (26) for over 200 generations. While on
diet, the strain was kept in an environmental chamber at 27 ± 1°C, 50 ± 2% relative humidity, and a photoperiod of 16:8
(light/dark hours). The Cry1C-Sel strain was originally collected in
March 1997 from a collard field in Lexington, S.C., where B. thuringiensis subsp. aizawai and B. thuringiensis
kurstaki products were reported as failing. Using a leaf dip
bioassay (27), we determined it had 31-fold resistance to
Cry1C before the selection.
B. thuringiensis toxins.
The Cry1C protoxin used
for bioassays and selections during generation 2 (G2) to G14 of the
Cry1C-Sel strain was provided by W. Moar, Auburn University, Auburn,
Ala. The cry1C gene was from B. thuringiensis
subsp. entomocidus and was expressed in an acrystalliferous
strain of HD-1 (18). A liquid formulation of Cry1C protoxin
expressed in and encapsulated by transgenic Pseudomonas
fluorescens (15% active ingredient; lot no. 100021276; Mycogen,
San Diego, Calif.) was used thereafter. Binding experiments were
performed with activated Cry1C toxin from the recombinant B. thuringiensis strain EG1081 (Ecogen Inc.).
Cry1C preparation for binding experiments.
Production,
solubilization, and trypsin activation of Cry1C from the recombinant
B. thuringiensis strain EG1081 (Ecogen Inc.) have been
described elsewhere (25). For binding analyses, purification is required to remove adsorbed protoxin fragments that block iodination and affect binding parameters (15, 39). Cry1C activated
toxin (15 ml) was dialyzed against 4 liters of Tris buffer (20 mM
Tris-HCl, pH 8.6) overnight at 4°C. The dialyzed Cry1C solution
(about 2 mg/ml) was filter sterilized and loaded onto a MonoQ HR 5/5
anion-exchange column (FPLC system; Pharmacia, Uppsala, Sweden)
previously equilibrated with Tris buffer. Cry1C was eluted with a
linear NaCl gradient (0.033 mM/min). Analysis of chromatographic
fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
showed two peaks (at 10 and 18 min) with the same molecular weight as
the activated Cry1C. Preliminary experiments showed that only the protein of the peak at 18 min provided specific binding to brush border
membrane vesicles (BBMV) after 125I labeling (data not
shown). Therefore, fractions of the peak at 18 min were pooled (5-ml
total volume) and dialyzed against 200 ml of carbonate buffer (50 mM
Na2CO3, pH 8) containing 16% of polyethylene
glycol 6000 for 4 h at 4°C. Dithiotreitol was added to the
sample to a final concentration of 6.5 mM. After overnight incubation
at 4°C, the sample was centrifuged at 16000 × g for
5 min at 4°C. The supernatant was applied to a Superose 12 gel
filtration column (Pharmacia FPLC system) and eluted with carbonate
buffer containing 1 mM dithiothreitol. Cry1C was finally dialyzed for
5 h against 4 liters of phosphate-buffered saline (8 mM
Na2HPO4, 2 mM KH2PO4,
150 mM NaCl, pH 7.5).
Transgenic plants expressing Cry1C toxin.
A synthetic
truncated cry1C gene (1.9 kb in length) was introduced into
broccoli (Brassica oleracea subsp. italica, Green Comet hybrid) by Agrobacterium sp.-mediated transformation
(3). The Cry1C protein in the leaves of transgenic broccoli
plants used (lines H12 and H14) was ca. 0.4% of total soluble protein (3).
Selection on Cry1C-Sel strain.
Selection on the Cry1C-Sel
diamondback moth strain by Cry1C started in the laboratory at G2 after
collection from the field. The Cry1C protoxin in cabbage leaf dip
assays (27, 36) was used for the initial 13 generations of
selection (G2 to G14). For each selection, >1,000 second instars were
infested onto cabbage leaf disks treated with Cry1C and incubated in
plastic containers for 3 days at 27 ± 1°C. Survivors from the
leaf disks were counted and transferred onto rape plants until adult
eclosion. Leaves or plants of transgenic broccoli expressing Cry1C
toxin (3) were used for the next 23 generations (G15 to
G37). The average mortality from selection to adult eclosion caused by
the first 22 selections was 76.8% (Table
1). The mortality in G18 to G22 was
estimated based on approximate egg numbers infested on transgenic broccoli.
Leaf dip bioassays.
Cabbage leaf dip bioassays, as
previously reported (27, 37), were used for each strain of
diamondback moth using second instars for most tests. Larvae of each
strain for a bioassay were reared on oilseed rape (Brassica
napus, Dwarf Essex variety; L. L. Olds Seed, Madison, Wis.)
plants in a greenhouse. Five to six concentrations plus a control and
six disks for each concentration were included in each bioassay. Five
second instars (0.2 to 0.3 mg/larva) were placed on each of the leaf
disks. Bond spreader-sticker (Loveland Industry, Loveland, Colo.) was
added at 0.1% to all test concentrations and to the water control.
Mortality was determined after 72 h at 27 ± 1°C. Data were
analyzed with a probit model using the POLO program (23).
Where resistance ratio (RR) values were calculated (RR = LC50 / LC50 of Geneva 88, where
LC50 is the 50% lethal concentration), the resistant and
susceptible strains were tested concurrently.
Survival of resistant strain on transgenic broccoli.
Neonates of the Cry1C-Sel strain after 24 generations of selection
(G26) were infested onto Cry1C transgenic broccoli until pupation.
Nontransgenic broccoli (Green Comet hybrid) was used as a control.
There were 10 replicates for each treatment and 10 neonates for each
replicate. Each surviving pupa was weighed and placed into a 30-ml
plastic cup until eclosion. To determine the number of eggs laid per
female, 15 single male-female pairs from each treatment group were
placed in 473-ml styrofoam containers. A 10% sugar solution was
provided for the moths, and egg sheets of cabbage-treated aluminum foil
(26) were provided for laying eggs in each container.
Thirteen pairs in each treatment group laid eggs. Egg sheets were
collected at 2, 4, and 6 days, and eggs were counted. To determine the
percent hatch, 50 to 60 eggs laid on day 2 were selected from an egg
sheet with 5 replicates for each treatment group. Neonates were counted
after 2 and 3 days at 27 ± 1°C. Data were analyzed with
analysis of variance using the SAS program (24). Data were
transformed using arcsin 
P for proportion (P)
of mortality and using log(x) for other data (x)
before each analysis of variance was performed.
Stability of resistance.
A substrain of Cry1C-Sel was
established from survivors of the G26 selection (i.e., G27). Larvae of
G28 to G38 were reared on oilseed rape plants in the greenhouse without
selection. Bioassays were conducted at G28, G29, G30, G32, G34, and G38
of the reversion substrain using second instars to determine levels of
resistance to Cry1C.
Inheritance tests.
Insects of Cry1C-Sel from G28 after 26 generations of selection were used as the resistant strain for both the
crosses and backcrosses (BCs) to study the inheritance of Cry1C
resistance. Crosses and strains used for the inheritance study were as
follows: F1 = Geneva 88 (S, G240) × Cry1C-Sel
(R, G28); BC = F1 × R (G28). Following Stone's
method (28), the degree of dominance (D) for resistance was calculated using the reciprocal F1 crosses
and the pooled data. The single-concentration method (12) to
determine estimated dominance h was also used for analysis
of dominance. Both the neonates and second instars were tested in all
bioassays. There were 50 neonates (10 leaf disks and five neonates on
each disk) for each concentration, with a procedure similar to that described above. Broccoli leaves (Green Comet hybrid) grown in the
greenhouse were used for bioassays of neonates.
The
2 goodness-of-fit test was used to determine how
well the BC mortality data of the second instars observed at each
concentration
fit mortality as predicted by each model of inheritance.
For direct
testing of monogenic inheritance, calculations of expected
mortality
for the BC offspring were based on experimental data
(
21,
29).
For indirect tests of monogenic and polygenic
inheritance, we
used the methods described by Tabashnik (
29)
and Tang et al.
(
37). There were nine concentrations and 30 larvae for each
concentration in the
bioassay.
The efficacy of Cry1C transgenic broccoli on different instars of
F
1 progeny was also tested for the dominance of resistance
on
B. thuringiensis-transgenic broccoli with nontransgenic
broccoli
as a control. First, second, third, and fourth instars of
F
1 progeny
were used, with five replicates for each
treatment and 10 larvae
for each replicate. Mortality was determined
after 72 h at 27
± 1°C.
Binding assays.
Last-instar larvae were frozen at 
80°C
at Cornell University's New York State Agricultural Experiment Station
and shipped frozen to the University of Valencia. BBMV were prepared
from whole larvae by the differential magnesium precipitation method (4, 41) and then frozen in liquid nitrogen and kept at
80°C until used. Protein concentration in the BBMV was determined
with the Bio-Rad reagent (2).
Cry1C toxin was
125I labeled by the IODO-BEADS (Pierce)
method (
14), a specific activity of 0.4 mCi/mg was obtained.
BBMV were
incubated with
125I-Cry1C (2.5 nM) in 0.1 ml of
binding buffer (PBS-0.1% bovine
serum albumin) at room temperature
for 90 min. Bound toxin was
separated from free toxin by centrifugation
(
9), and the pellet
was washed twice with 500 µl of
binding buffer. Radioactivity
in the pellet was measured in a
Compugamma CS gamma irradiation
counter (LKB
Pharmacia).
Binding parameters were obtained using the LIGAND computer program
(
20). Statistical analyses of differences (
P < 0.05)
of the mean values of binding parameters were performed with
a
Student's
t test using the Prism software computer
program (GraphPad
Software, San Diego, Calif.).
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RESULTS |
Resistance development.
The initial RR of the Cry1C-Sel strain
to Cry1C protoxin based on the LC50 was 31-fold (13.7 versus 0.437 mg of the active ingredient [AI]/liter) at the second
generation (G2) reared in the laboratory after collection. After 26 generations of selection, the RR of the Cry1C-Sel at G28 increased to
63,100-fold (Fig. 1; also see Table 3).
The RR remained stable between G28 and G38 under continuous selection.
The RR to Cry1C for the neonates at G28 was 12,400-fold.
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FIG. 1.
Development of resistance to Cry1C in the Cry1C-Sel
strain ( ) of the diamondback moth and stability of resistance after
selection ceased at G28 ( ).
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When neonates of G26, after 24 generations of selection, fed on Cry1C
transgenic or on nontransgenic plants, there were no
significant
differences in mortality until pupation, weight per
pupa, eggs per
female, or percentage of eggs hatched (Table
2).
Neonates of the Cry1C-Sel strain
could complete their entire life
cycle on transgenic broccoli with a
high expression level of Cry1C,
although there were evident cumulative
disadvantages on
B. thuringiensis broccoli. The mortality of
Geneva 88 was 100% when the neonates
fed on the same Cry1C transgenic
broccoli until pupation (unpublished
data; 12% on nontransgenic
control).
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TABLE 2.
Survival and development of neonates of a Cry1C-Sel
strain on Cry1C B. thuringiensis-transgenic and a
nontransgenic (control) broccolia
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Stability of resistance.
After selection ceased at G28 of the
Cry1C-Sel strain, the Cry1C resistance decreased from 63,100-fold at
G28 to 16,900-, 2,760- and 235-fold, respectively, at G32, G34, and G38
(Fig. 1). The LC50s (95% fiducial limit [FL], milligrams
of AI/liter) for the second instars of the Cry1C-Sel strain were 3,020 (2,230 to 3,980) at G32, 663 (359 to 1,080) at G34, and 38.7 (15.5 to 69.3) at G38, respectively.
Inheritance of Cry1C resistance.
Genetic analysis of
F1 larvae indicated that the Cry1C resistance in the
Cry1C-Sel colony was inherited as an autosomal and incompletely
recessive factor or factors at the LC50 (Table
3). The degree of dominance
(D) of resistance based on pooled F1 was 0.22
for the neonate and 0.24 for the second instars. The h of
resistance using the single-concentration method (12) tended to be more recessive as the concentration increased for both the neonates and second instars. For the neonates, resistance was partially
recessive at concentrations of 10 to 100 mg of AI/liter (h = 0.49 to 0.14) and partially dominant at 0.316 to 3.16 mg of
AI/liter (h = 0.78 to 0.61). For the second instars,
resistance was recessive at concentrations of 100 mg of AI/liter or
above (h = 0), partially recessive at 31.6 (h = 0.14) or 10 (h = 0.44) mg of
AI/liter, and partially dominant at 0.316 to 3.16 mg of AI/liter
(h = 0.94 to 0.73) based on pooled F1.
The mortality of all instars of the F
1 progeny was 100%
when they were fed Cry1C transgenic broccoli for 72 h (2 to 14%
on
nontransgenic control), indicating that Cry1C resistance in the
Cry1C-Sel strain was
recessive.
In the direct test of monogenic inheritance for Cry1C resistance, five
of the nine concentrations evaluated resulted in significant
deviation
between the observed and expected mortality (Table
4).
Using the indirect method for testing
inheritance, five of the
nine concentrations also resulted in
significant deviation when
the one-locus model was used (Table
5), but deviations were significant
at
all nine concentrations tested when two- or five-locus models
were
used.
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TABLE 4.
Direct test of monogenic inheritance for Cry1C resistance
in Cry1C-Sel strain by comparing expected and observed mortality of
second instars of the BC progenya
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TABLE 5.
Indirect tests for monogenic and polygenic inheritance of
Cry1C resistance in Cry1C-Sel strain using second instars by
2 analysis (df = 1)
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Binding of 125I-Cry1C to BBMV.
Cry1C-specific
binding was tested by incubating 125I-Cry1C with various
concentrations of BBMV from the Geneva 88 and the Cry1C-Sel strains
(Fig. 2). Saturable binding was found in
both strains, with a maximum specific binding level of around 1% of
the total radioactivity added. Saturable binding in the Cry1C-Sel
strain indicated that the highly resistant insects possessed specific binding sites for Cry1C.
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FIG. 2.
Specific binding of 125I-Cry1C as a function
of BBMV concentration. Specific binding was calculated by subtracting
the nonspecific binding from the total binding. Nonspecific binding was
determined in the presence of a 120-fold excess of unlabeled toxin.
Each data point represents the mean of duplicate samples. Symbols:  ,
Geneva 88 strain;  , Cry1C-Sel strain.
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Experiments of competition of
125I-Cry1C with unlabeled
Cry1C for binding to BBMV-specific sites (Fig.
3) were performed to obtain
quantitative
estimates of the equilibrium dissociation constant
(
Kd) and binding site concentration
(
Rt). Significantly different
values for these
binding parameters were obtained for both strains
(Table
6). Cry1C showed about a sevenfold lower
affinity for
BBMV from the Cry1C-Sel strain than for that from the
Geneva 88
strain. Moreover, BBMV from Cry1C-Sel had about a threefold
higher
binding site concentration than BBMV from the Geneva 88 strain.
The overall binding affinity of the BBMV for Cry1C, estimated
as the
Rt/
Kd ratio, was also significantly
different for both
strains (Table
6), being 2.5-fold higher for the
Geneva 88 strain.
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FIG. 3.
Binding of 125I-Cry1C to diamondback moth
BBMV (0.2 mg of vesicle protein/ml) at increasing concentrations of
unlabeled Cry1C. Nonspecific binding was not subtracted. Each point
represents the mean of two independent experiments. Symbols: ,
Geneva 88 strain; , Cry1C-Sel strain.
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DISCUSSION |
Although several insect species have developed resistance to
B. thuringiensis formulations or toxins (30),
there are only three reported species for which resistant strains can
survive on transgenic insecticidal plants. Survival to maturity has
been reported for resistant strains of diamondback moth on B. thuringiensis-transgenic broccoli and B. thuringiensis-transgenic canola (17, 22, 38) and for
tobacco budworm (Heliothis virescens) and pink bollworm (Pectinophora gossypiella) on B. thuringiensis-transgenic cotton (6, 13). However, in
all these reports the resistant strains did not develop directly from
selection on B. thuringiensis-transgenic crops.
After 24 generations of selection with the Cry1C protoxin or transgenic
broccoli expressing a Cry1C protein, the Cry1C resistance in our
diamondback moth strain was so high that neonates could complete their
entire life cycle on transgenic broccoli expressing high levels of
Cry1C. This contrasts with the F1 progeny, for which the
mortality of all instars was 100% when fed the same Cry1C transgenic
broccoli. At an early stage of our laboratory selection with the Cry1C
protoxin, the RR of the Cry1C-Sel strain after six generations of
selections increased to ca. 100-fold, but the larvae could not survive
on Cry1C broccoli (3). B. thuringiensis-transgenic broccoli with a high expression level of
Cry1C rapidly accelerated the development of resistance. Considering that there may be 20 generations per year of the diamondback moth in
tropical areas (34, 35), B. thuringiensis-transgenic crucifers containing Cry1A or Cry1C
toxins could face the potential of rapid development of resistance in
the field as they enter commercial application in areas in which
B. thuringiensis products containing Cry1A or Cry1C toxins
had been used extensively. Although in our trials we gradually
increased the frequency of resistance (and perhaps the intensity of
resistance of individuals, if such does exist), it is unknown whether
populations in the field could eventually adapt to such high expression
levels as in our plants. However, our results clearly show the
potential for development of a high level of resistance to Cry1C.
The Cry1C resistance in this strain was seen to be inherited as an
incompletely recessive factor (or factors) when evaluated using a leaf
dip assay and as recessive when using Cry1C transgenic broccoli. Our
results differ from another study in which Cry1C resistance in the
diamondback moth (NO-95C) was much lower (62-fold) and inherited in an
incompletely dominant fashion (12). The h using
the single-concentration method resulted in similar trends; i.e., in
both studies the resistance tended to be more recessive as the
concentration increased. The original level of Cry1C resistance in
NO-95 collected from Hawaii was 22-fold, and there were nine generations of selection using Cry1C protoxin by a method similar to
the bioassay (11, 12). The different results on the
dominance of Cry1C resistance between the two studies may have resulted from the large difference in levels of resistance and/or from different
resistance genes in the two strains.
We could not reach a clear conclusion on monogenic or polygenic
inheritance of the Cry1C resistance based on the direct and indirect
tests. In the direct tests of monogenic inheritance, there were
significant deviations at and near the LC50 of the BC,
which suggested nonadditive polygenic inheritance or experimental error
(29). Our binding studies also suggested that there should be other important mechanisms responsible for the Cry1C resistance apart from reduced binding. But in the indirect tests, the monogenic model provided a better fit than either the two-locus or five-locus models. Alternative approaches, including repeated BC tests and molecular techniques, will be helpful for determining the mode of
inheritance for Cry1C resistance (29).
It is important that the mode of inheritance be known since the
"high dose-refuge" strategy for managing the development of insect
resistance to B. thuringiensis transgenic crops
(7; U.S. Environmental Protection Agency website)
was proposed on the basis of recessive inheritance. Our results
using Cry1C transgenic broccoli may provide the first experimental
evidence for the recessive inheritance of resistance which was selected
primarily on B. thuringiensis-transgenic plants.
A previous report indicated that Cry1C resistance in S. littoralis decreased from >500-fold to 11-fold eight generations
after selection ceased (19). A similar decline of Cry1C
resistance occurred in S. exigua eight generations after
selection ceased (18). Our results also suggest the
instability of Cry1C resistance in the diamondback moth after release
from the selection. One possible reason for the instability might be
the fitness cost of the resistance. Significant reduction was observed
in the weight per pupa and eggs per female in the Cry1C-Sel strain
compared with Geneva 88 and F1 progeny when fed on the same
nontransgenic broccoli leaves (Zhao et al., unpublished data), hinting
at some fitness costs for Cry1C resistance. However, a precise
evaluation for the fitness cost of Cry1C resistance in the Cry1C-Sel
strain is difficult, as the genetic background between each strain of the diamondback moth is different in characteristics besides the Cry1C resistance.
Binding site modification is thought to be the major mechanism of
resistance to Cry1A toxins in Pectinophora interpunctella (40), H. virescens (10), and the
diamondback moth (5, 25, 32, 36, 42), and has also been
proposed to be responsible for Cry1F resistance in the diamondback moth
(1, 8). However, this seems not to be the case for Cry1C
resistance. In S. exigua, insects highly resistant to Cry1C
(100-fold to >500-fold resistant) showed no change in Cry1C binding
site concentration and a fivefold decrease in Cry1C binding affinity
(18). Insects from a diamondback moth population
300-fold resistant to B. thuringiensis subsp. aizawai (which contains Cry1C, besides other toxins) did not
show any change in Kd or
Rt for Cry1C binding sites (42). No
significant difference in Cry1C binding was found between two
diamondback moth strains from Hawaii, one susceptible and another
selected for Cry1C resistance in the laboratory (resistance ratios at
the time of the experiments were 48-fold resistance to Cry1C protoxin and 19-fold resistance to Cry1C toxin) (14). We have found
significant differences between our resistant strain and susceptible
strain in both Kd and Rt
for Cry1C binding sites. Previous estimates of Cry1C binding parameters
in the diamondback moth gave Kd values from 4.8 to 9.4 nM (mean value of 7.9 nM) and Rt values
from 2.8 to 10.8 pmol/mg (mean value of 5.4 pmol/mg) (see Table 1 in
reference 14). The Cry1C-Sel strain has an
Rt close to the above interval, but the
Kd is considerably higher (50.5 nM), which
supports the argument for some kind of modification in the binding
site. However, taking together the decrease of affinity and the
increase in binding site concentration, the results suggest that
reduced binding is not the major mechanism of resistance to Cry1C in
the Cry1C-Sel strain. The differences in the primary sequences of the
Cry1C protein from different sources of gene (B. thuringiensis subsp. aizawai or entomocidus)
may affect the outcome of binding results, and more binding assays
using Cry1C toxin with different sources would be helpful for a better
understanding of reduced binding mechanism. More tests on other
potential mechanisms, especially on altered gut protease patterns, are
needed for a better understanding of the Cry1C resistance.
| |
ACKNOWLEDGMENTS |
This research was supported by USDA-NRI grant 990-2697.
We thank Y. X. Li for technical assistance, P. Smith for
collecting the insects, W. J. Moar for providing Cry1C protoxin
for initial selections, Mycogen for providing the M-C formulation for
tests, and Ecogen Inc. for providing the bacterial strain used to
prepare the Cry1C toxin for binding assays. We also thank B. E. Tabashnik and an anonymous reviewer for comments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456. Phone: (315) 787-2352. Fax: (315) 787-2326. E-mail: ams5{at}cornell.edu.
Present address: Department of Entomology and Plant Pathology,
Mississippi State University, Mississippi State, MS 39762.
| |
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Abstract
Introduction
