Download Mannose Phosphate Isomerase Isoenzymes Support Common in Genetic Bases of Resistance to

Mannose Phosphate Isomerase Isoenzymes
in Plutella xylostella Support Common
Genetic Bases of Resistance to Bacillus
thuringiensis Toxins in Lepidopteran
Species
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Salvador Herrero, Juan Ferré and Baltasar Escriche
Appl. Environ. Microbiol. 2001, 67(2):979. DOI:
10.1128/AEM.67.2.979-981.2001.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2001, p. 979–981
0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.2.979–981.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 67, No. 2
Mannose Phosphate Isomerase Isoenzymes in Plutella xylostella
Support Common Genetic Bases of Resistance to Bacillus thuringiensis
Toxins in Lepidopteran Species
SALVADOR HERRERO, JUAN FERRÉ,
AND
BALTASAR ESCRICHE*
Received 2 June 2000/Accepted 14 November 2000
A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A
toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A
resistance had previously been reported for a Heliothis virescens population. The fact that the two populations
share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of
homologous resistance loci in both species.
the H. virescens YHD2 population. We measured differences in
the frequencies of MPI isoenzymes before and after selection
with Cry1A toxins. A significant change in frequency for a
given MPI isoenzyme would be indicative of linkage of the
locus for this isoenzyme to the Cry1A resistance locus.
At the time of the experiments, the resistance ratio for
Cry1Ac and Cry1Ab of the PHI population had decreased
drastically and was around fourfold with respect to a susceptible control population (LAB-V) of the same species (data not
shown). Third-instar larvae from the PHI population were
selected using cabbage leaves dipped into aqueous solutions of
Cry1A toxins. Cry1Ab and Cry1Ac were obtained from B.
thuringiensis strains EG7077 and EG11070, respectively (Ecogen Inc.), and were prepared as active toxins (12). A sample of
250 larvae was selected with 4 ␮g of Cry1Ab/ml (this produced
a larva-to-adult mortality of 75%), and another sample of 700
larvae was selected with 10 ␮g of Cry1Ac/ml (this produced a
larva-to-adult mortality of 93%). Both doses produced 100%
mortality in larvae from the LAB-V population. After 2 days of
exposure to the Cry1A toxins, surviving larvae were transferred
to untreated cabbage leaves and reared until adult emergence.
Adult insects were frozen and kept at ⫺80°C. MPI isoenzyme
analysis was performed as described by Pasteur et al. (11).
Single adult insects were homogenized in 30 ␮l of 10 mM
Tris-HCl–1 mM EDTA–0.4% NADP (pH 6.8). Separation of
MPI isoenzymes was carried out using polyacrylamide gel electrophoresis (6) with 6% acrylamide in the resolving gel and 4%
in the stacking gel. The protein-denaturing agent (i.e., sodium
dodecyl sulfate) was omitted from the electrophoresis buffers.
The enzymatic activity was developed with an overlay system as
described by Pasteur et al. (11). No band was observed when
control experiments were performed by omitting either the
substrate or any of the coupling enzymes.
MPI analysis was performed for the LAB-V population, the
PHI population, the Cry1Ab-selected sample (Sel.Ab), and the
Cry1Ac-selected sample (Sel.Ac) (n ⫽ 50).
The susceptible LAB-V population and the PHI population
(Table 1) showed four MPI isoenzymes with different electrophoretic mobilities. They were designated A to D relative to
their migration patterns (A was the slowest band) (Fig. 1). The
same MPI isoenzymes plus an additional one (band E) were
The efficacy of pest control with products derived from the
bacterium Bacillus thuringiensis is limited by the capacity of the
insects to develop resistance. To date only one insect pest,
Plutella xylostella, has evolved resistance to B. thuringiensis in
open field populations (3). Laboratory selection experiments
have shown that other lepidopteran species, such as Plodia
interpunctella (9), Spodoptera exigua (10), or Heliothis virescens
(4), can also evolve resistance to B. thuringiensis formulations
or toxins. Some of these resistant populations share a similar
resistance profile: extremely high resistance to at least one
Cry1A toxin, no or minimal cross-resistance to Cry1C, recessive or partially recessive inheritance of resistance, and reduced binding of at least one Cry1A toxin to proteins of the
insect midgut. This has been called “type I” resistance (15).
Similar resistance profiles in different insect species suggest a
similar genetic basis of resistance.
A genetic analysis with isoenzyme loci was performed on the
YHD2 strain of H. virescens (5), which showed the resistance
profile described above. The authors found that the mannose6-phosphate isomerase (MPI; EC 5.3.1.8) locus mapped at 10
centimorgans from a locus (BtR-4) involved in resistance to the
Cry1Ac B. thuringiensis toxin. Considering that there is a certain degree of conserved synteny among insect species, MPI
linkage to B. thuringiensis resistance can be a common feature
in lepidopterans. Chromosomal map studies have shown the
existence of a conserved synteny of genes among different
animal or plant species (1, 18). Particularly in insects, chromosomes of mosquito species have been shown to have high levels
of synteny when mapped with restriction fragment length polymorphism (RFLP) markers (13, 14) and isoenzyme loci (8).
Obviously, some levels of chromosome rearrangement (i.e.,
chromosome translocations or inversions) would change the
linear order of the markers between species (14).
In the present work we tested the correlation of the MPI
isoenzymes to Cry1A resistance in a P. xylostella population
(PHI) (17) which showed a resistance profile similar to that of
* Corresponding author. Mailing address: Department of Genetics,
University of Valencia, Dr. Moliner 50, 46100-Burjassot (Valencia),
Spain. Phone: (34) 96 386 4506. Fax: (34) 96 398 3029. E-mail: escrichb
@uv.es.
979
Downloaded from http://aem.asm.org/ on February 19, 2014 by UNIVERSITAT DE VALENCIA
Department of Genetics, University of Valencia, 46100-Burjassot (Valencia), Spain
980
HERRERO ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 2. P values from a ␹2 test on a contingency table for
MPI isoenzyme distribution between pairs of samples
TABLE 1. Frequencies of insects showing a given isoenzyme in
samples of nonselected and toxin-selected insects
Samplea
LAB-V
PHI
Sel.Ab
Sel.Ac
Frequency of insects showing isoenzyme type:
A
0.14
0.22
0.28
0.32
B
0.38
0.54
0.38
0.36
C
0.58
0.48
0.44
0.60
D
0.18
0.12
0.28
0.32
Sample
E
b
—
—
0.14
0.16
LAB-V is a control susceptible population, PHI is a revertant population,
and Sel.Ab and Sel.Ac are toxin-selected samples from PHI.
b
—, not detected.
found in insects from the Sel.Ab and Sel.Ac samples. Isoenzyme E was detected in approximately 15% of the insects in
both selected samples but was not observed in any insect from
the unselected PHI population or in the LAB-V population.
Evidently, a low frequency of this isoenzyme form must be
present in the PHI population. We conducted single-pair mating to unravel the genetic bases of MPI isoenzymes in P. xylostella. However, no conclusions could be drawn from these
experiments, suggesting a complex genetic basis, perhaps due
to the involvement of more than one locus encoding the protein or other loci involved in the posttranslational modification
of the protein (data not shown).
No significant differences in isoenzyme frequencies (Table
2) were detected between the LAB-V and the PHI populations
or between the two selected samples (Sel.Ab and Sel.Ac).
However, MPI isoenzyme frequencies in the selected samples
were different from the frequencies found in the unselected
populations (LAB-V and PHI) (Table 2). Data from the unselected populations were pooled and compared with pooled
data from the selected samples. Analysis of the pooled data for
every single isoenzyme (using Fisher’s exact test on a contingency table for presence-absence) showed significant differences (P ⬍ 0.05) in the frequencies for the D and E forms.
Change in the frequencies of isoenzyme forms can sometimes be due to induction by exposure to a toxic agent, but then
this change is not inherited. Band E was detected only in the
selected samples; thus, if this MPI isoenzyme was due to a
physiological induction, we would not expect to observe it in
the offspring from the selected insects. A third sample of in-
FIG. 1. Enzymatically stained polyacrylamide electrophoresis gel
showing the different types of MPI isoenzymes scored. Each lane
corresponds to one single insect.
a
LAB-V
PHI
Sel.Ab
0.360
0.019
0.041
0.009
0.009
0.918
Differences were considered significant when the P value was ⬍0.05.
sects from the PHI population was selected with 50 ␮g of
Cry1Ab/ml (which produced 90% mortality); survivors were
mated, and the offspring was reared on cabbage leaves without
Cry1Ab. Analysis of a sample of the offspring (n ⫽ 35) showed
the occurrence of isoenzyme E in 8 insects (16%). This result
supports the appearance of isoenzyme E by genetic selection
and not by physiological induction.
Our results show a strong correlation between the occurrence of the D and E forms of MPI and resistance to Cry1A
toxins. Previous studies showed that a multitoxin B. thuringiensis resistance gene in P. xylostella confers resistance to at least
four Cry1 toxins: Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F (16,
17). Selection for resistance with either Cry1Ab or Cry1Ac
effected similar changes in the MPI isoenzyme frequencies, in
agreement with a common genetic basis of resistance to both
toxins.
Conserved mechanisms of resistance have been found to
insecticides such as pyrethroids or DDT. A homologous locus
for resistance to these pesticides has been found in several
insect species (2, 7). The fact that the P. xylostella PHI population and the H. virescens YHD2 population belong to the
same resistance type, along with the results with MPI isoenzymes, also suggests the occurrence of homologous resistance
loci in lepidopteran species for type I resistance to B. thuringiensis toxins.
We thank L. Calzada for help in the rearing of insect colonies and
Ecogen Inc. for providing the bacterial strains used to prepare the
Cry1A toxins.
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MPI LINKAGE TO B. THURINGIENSIS RESISTANCE