Susceptibility of Southwestern Pink Bollworm
to Bt toxins Cry1Ac and Cry2Ab2 in 2005.
Timothy J. Dennehy, Gopalan C. Unnithan, Virginia Harpold,
Yves Carrière and Bruce Tabashnik
Department of Entomology, The University of Arizona, Tucson, AZ
Larry Antilla and Mike Whitlow
Arizona Cotton Research & Protection Council, Phoenix, Arizona
Abstract
Bt cotton is an extremely important tool for integrated pest management in the Southwest. It has
been a major factor in the current historic low levels of conventional insecticide use in cotton of
this region. This is due to Bt cotton’s unprecedented efficacy against the pink bollworm,
Pectinophora gossypiella, and its selectivity in favor of key natural enemies of arthropod pests.
Due to the pivotal importance of Bt cotton and widespread concerns about the development of pest
resistance to transgenic crops, a multi-agency resistance management program was established to
monitor and pro-actively manage resistance development in the pink bollworm. This report
constitutes results from the ninth year of this monitoring program.
Larvae were obtained from bolls collected in cotton fields located throughout the Southwest,
cultured in the laboratory, and offspring tested using diet-incorporation bioassays that
discriminate between susceptible and resistant pink bollworm. A total of 11 Arizona and four
California collections were successfully reared and tested for susceptibility to Cry1Ac using a
discriminating concentration of 10 µg Cry1Ac/ml of diet. Susceptibility to Cry2Ab2 was estimated
similarly for 12 strains from Arizona and four from California using diagnostic concentrations of
1.0 and 10 µg Cry2Ab2/ml of diet. Success of pink bollworm eradication in suppressing pink
bollworm populations in New Mexico and Texas precluded successful collection of samples in
those states.
No survivors of 10 µg Cry1Ac/ml were detected in any bioassays of 2005 strains (n=5358). The
grand mean frequency of PBW survival of 10 µg Cry1Ac/ml in 2005 was 0.000%. A susceptible
culture, APHIS-S, used each year as an internal control, yielded 99.3% corrected mortality in
tests of 10µg/ml Cry1Ac (n=490). All twelve pink bollworm strains collected in 2005 were highly
susceptible to Cry2Ab2, based on contrasts with baseline data collected from 2001-2003. There
were no survivors of bioassays of either 1.0 µg Cry2Ab2/ml (n=1,000) or 10 µg Cry2Ab2/ml
(n=3425). The susceptible APHIS-S culture had 82.5% corrected mortality in tests of 10 µg/ml
Cry2Ab2 (n=200) and 100% mortality in tests of 10 µg/ml Cry2Ab2 (n=120).
Field evaluations of efficacy of Bt cotton were conducted by the Arizona Cotton Research and
Protection Council in adjacent pairs of Bt and non-Bt fields at 44 Arizona locations. Statewide,
Arizona Cotton Report (P-151) July 2007
105
large pink bollworm larvae were found in an average of 15% of non-Bt bolls sampled from
borders of refuge fields. This was on the low end of the range of infestation levels observed in
refuges during the past decade. Bolls from adjacent Bt cotton (Bollgard™) fields yielded an
average of 0.28% infested bolls. This value was down slightly from the previous year. Over 70%
of the pink bollworm recovered from collections in Bt fields were from bolls that did not express
Bt toxin. We conclude that there was no indication of problems with pink bollworm resistance to
Cry1Ac or Cry2Ab2 at the locations sampled in 2005. Moreover, Bt cotton continued to exhibit
exceptional field performance in Arizona.
Introduction
Registration of Bt cotton in the US in 1996 marked the beginning of a major change in pest management in Arizona
cotton. Pink bollworm (Pectinophora gossypiella), one of the most economically damaging pests of Arizona cotton,
is highly susceptible to the toxins produced by Bt cotton. Producer gains from use of Bt cotton in Arizona averaging
$15,000 per farm (Frisvold et al. 2000) have promoted rapid adoption of this technology. Additionally, the
environment and integrated pest management have benefited from decreased use of conventional insecticides
associated with adoption of Bt cotton. In 1995, the year preceding registration of Bt cotton, an average of over six
insecticide applications were made per acre of cotton in Arizona (Sims et al. 2001). Insecticide use in Arizona
cotton has declined to historic low levels since 1995, averaging less than two treatments per acre in 2000 and
ranging between 0-4 treatments depending on year and production region (Figure 1). These dramatic reductions are
attributable, in large measure, to the combined effects of Bt cotton used to control pink bollworm and to improved
management of whiteflies with insect growth regulators (Dennehy et al. 2002, Naranjo et al. 2003).
Loss of target pest susceptibility as a result of resistance was anticipated to be the greatest biological limitation of
transgenic insecticidal crops (Mellon and Rissler 1998). This was due foremost to the many months each year that
pests are exposed to toxins in plants producing Bt toxins. Resistance in the field seemed all the more probable
following selection in the laboratory of pink bollworm capable of surviving high concentrations of Cry1Ac (Bartlett
1995, Simmons et al. 1998, Patin et al. 1999, Liu et al. 1999, Tabashnik et al. 2000, Sims et al. 2001). Moreover,
greenhouse trials showed that the resistant pink bollworm were able to survive on Bollgard plants. Resistance to
Cry1Ac was found to be conferred by one or few major autosomal genes (Tabashnik et al. 2002). Homozygous
susceptible and F1 heterozygote individuals were killed by bioassays of 10 µg Cry1Ac/ml diet. Thus, genetic
analysis confirmed that 10 µg/ml provided a reliable discriminating concentration for monitoring resistance of pink
bollworm to Cry1Ac. Using this discriminating concentration, the frequency of pink bollworm resistance to
Cry1Ac, although unexpectedly high in 1997 collections, declined from 1998 to 2004 (Patin et al. 1999, Tabashnik
et al. 2000, Tabashnik et al. 2005).
Herein, we report the 2005 season results of Arizona’s multi-agency collaboration to monitor resistance of pink
bollworm to Bt toxins using laboratory-based bioassays, and complementary evaluations of the field performance of
Bt cotton throughout Arizona.
Materials and Methods
Susceptibility of Arizona PBW to the Bt Endotoxins, Cry1Ac and Cry2Ab2
Collections. Collections from cotton fields commenced in August and continued through December, 2005. Over
100 fields of non-Bt cotton were inspected throughout Arizona, California, New Mexico, and Texas. From these,
successful collections were obtained from 12 sites in Arizona and four sites in California (Figure 2, Table 1). An
eradication program underway in New Mexico and Texas resulted in no collections being obtained from these states.
Our objective was to establish laboratory cultures with ≥ 100 PBW from each collection site. At each location 300
to 2,000 bolls were collected from non-Bt cotton fields. Bolls were taken to the University of Arizona Extension
Arthropod Resistance Management Laboratory (EARML) in Tucson and put in boll boxes (17.6 cm x 50.4 cm x
35.2 cm).
Arizona Cotton Report (P-151) July 2007
106
Boll boxes suspended infested bolls on wire racks approximately three cm above sheets of paper toweling on the
bottom of the boxes. Fourth instar larvae cut out of infested bolls and dropped onto the paper toweling. Larvae
were transferred to pupation boxes consisting of tightly sealed, 1.7 liter rectangular Rubbermaid® containers
enclosing sheets of paper toweling. For cultures from which fewer than 100 larvae were obtained from boll boxes,
bolls were opened manually to collect additional PBW. To prevent or disrupt diapause, larvae that cut out of bolls
and webbed up were disturbed by pulling the paper toweling apart and spraying it lightly with water.
Rearing. We reared PBW using a modified version of the method of Bartlett and Wolf (1985). Offspring of fieldcollected PBW were reared singly or in pairs in one ounce cups containing approximately five g diet each.
Bioassays of susceptibility to Cry1Ac and Cry2Ab2 were conducted from November, 2005 through July, 2006.
Bioassay. Susceptibility of each collection of pink bollworm to Cry1Ac and Cry2Ab2 was determined using 21-day
diet-incorporation bioassays (Patin et al. 1999).
For Cry1Ac, MVP-II® Bioinsecticide obtained from
DowAgrosciences was diluted with sterilized, distilled water to produce a stock solution. For Cry2Ab2, the source
of toxin was freeze-dried corn leaf powder produced by Monsanto in St. Louis, MO. Our batch of leaf powder was
estimated by Monsanto to contain 9.99 mg Cry2Ab2 toxin/g of powder. Toxin was added to liquid wheat germ diet
(Adkinson et al. 1960) in amounts necessary to create final concentrations of 0 (control) and 10 µg/ml Cry1Ac/ml
diet solution, and 0 (control), 1.0 and 10 µg/ml Cry2Ab2/ml diet solution.
Diet was made in two four-liter batches, subdivided by weight into beakers, and held in water baths at 50-60°C, after
which toxin and food coloring was blended thoroughly into the liquid diet. The food coloring was added to ensure
thorough mixing of toxin in the diet. Diet was allowed to cool to room temperature and then was refrigerated at 6-8
°C for 48-72 h, after which it was cubed using a commercial cheese slicer. Cubed diet was sealed in plastic bags,
and returned to the refrigerator. Approximately five g of diet per cup was dispensed into one-ounce medicine cups
with tight fitting lids. Diet was used in bioassays within 2 weeks.
Neonate larvae were placed individually in the one ounce cups and the lids were affixed. For Cry1Ac, subjects from
each field strain were assigned to replicates consisting of 10 bioassay cups per replicate of toxin-free controls and 90
bioassay cups per replicate for 10 µg/ml Cry1Ac/ml treatments. A total of four replications per concentration were
conducted, yielding totals for each strain of 40 individuals tested in untreated controls and 360 individuals tested in
treatments of 10 µg Cry1Ac. Limitations in numbers of neonate larvae and/or robustness of cultures sometimes
altered the number of individuals tested in replicates.
Replicates of Cry2Ab2 bioassays consisted of 10 larvae isolated individually in cups containing toxin-free diet, 1.0
µg/ml, or 10 µg/ml Cry2Ab2/ml diet. A minimum of six replicates of controls and 10 replicates of 1.0 and 10 µg/ml
Cry2Ab2 were conducted, yielding totals for each strain of at least 60, 60, and 100 subjects tested in control, 1.0 and
10 µg/ml treatments, respectively.
Bioassay cups were placed in plastic trays and incubated in darkness at 29±1 °C for 21 days, after which mortality
and developmental stage of survivors (Watson and Johnson 1974) were recorded. Subjects developing to ≥ 4th instar
were scored as alive. Cups in which 4th instar larvae had exited by chewing out of the plastic were scored as alive if:
1) they contained frass of the size produced by a 4th instar; 2) the exit hole was the size produced by a 4th instar; and
3) the cups contained evidence of feeding consistent with development to 4th instar. Corrected mortality was
computed using Abbott’s formula (Abbott 1925). Bioassays were conducted on the F1-5 generations. Results
obtained from each population were pooled to obtain a single estimate of mortality for each concentration tested.
Laboratory Reference Strains. Many factors can affect the reproducibility of diet-incorporation bioassays of
arthropods. These include factors intrinsic to the pest, the toxin, the diet, the laboratory equipment utilized, and
lastly, the technician setting up and evaluating the tests. Laboratory strains of presumed stable genetic composition
should be tested concomitantly with field population to confirm reproducibility of the bioassay conditions. For this
purpose a strain susceptible to Cry1Ac and Cry2Ab2, the APHIS-S strain, was bioassayed with both Cry1Ac and
Cry2Ab2 toxins on four different dates during the period in which we evaluated the 2005 collections. This strain
has been maintained in the laboratory for at least 30 years without exposure to pesticides. Prior to 1996, field
collected pink bollworm were periodically added to the strain. Major mutations conferring resistance to Cry1Ac or
Cry2Ab2 are rare or absent in this strain. We also evaluated susceptibility of a Cry1Ac-resistant strain, AZP-R, as a
second internal standard.
Arizona Cotton Report (P-151) July 2007
107
Interpreting Cry1Ac Bioassay Results
Pink bollworm that survive 10 µg/ml discriminating concentration bioassays of Cry1Ac are homozygous for the
major Mendelian factor that confers resistant to Cry1Ac. This conclusion is based on over seven years of
investigations in Arizona. Susceptible field strains (Patin et al. 1999), susceptible laboratory strains (Tabashnik et
al. 2000), and Arizona pink bollworm heterozygous for the major resistance alleles (Tabashnik et al. 2002) had no
survivors of 10 µg/ml Cry1Ac bioassays.
Laboratory selection with Cry1Ac of pink bollworm collected in Arizona in 1997 yielded a strain (AZP-R) with high
levels of survival of 10 µg/ml Cry1Ac bioassays (Simmons et al. 1998). Tabashnik et al. (2000) subsequently
computed the frequency of resistance in Arizona field populations from statewide monitoring data based on survival
of 10 µg/ml bioassays. Greenhouse evaluations showed that the Cry1Ac-resistant AZP-R strain had 46% survival
on Bt cotton, relative to survival on non-Bt cotton (Liu et al. 2001). Morin et al. (2003) showed that resistance to
Cry1Ac in bioassays, and survival on Bt cotton in greenhouse experiments of laboratory-selected pink bollworm
from Arizona and Texas were linked with the presence of three mutant alleles of a cadherin-encoding gene. Larvae
with two of these resistance alleles in any combination were resistant, whereas those with one or none were
susceptible to Cry1Ac.
Interpreting Cry2Ab2 Bioassay Results
Baseline Data. EARML estimates of baseline susceptibility of pink bollworm to Cry2Ab2 were reported previously
(Unnithan et al. 2004, Dennehy et al. 2005). Monitoring concentrations of 1.0 and 10 µg/ml Cry2Ab2 were
identified from probit responses generated from collections made in 2001 and 2002 (Figure 3, Unnithan et al. 2004).
Nineteen pink bollworm strains from throughout the Southwest evaluated in 2003 were highly susceptible to
Cry2Ab2. Mean mortality ranged from 84.7 to 100% and from 98.3 to 100% in bioassays of 1.0 µg/ml and 10
µg/ml Cry2Ab2/ml, respectively (Dennehy et al. 2004). Only four individuals out of a total of 2040 subjects tested
survived bioassays of 10 µg/ml Cry2Ab2/ml. Thus, we have an abundance of evidence that susceptible field
populations have very high mortality in bioassays of 1.0 µg/ml Cry2Ab2 and will very rarely have survivors of 10
µg/ml Cry2Ab2/ml treatments. A composite strain established in 2001 and intensively selected with Cry2Ab2 in the
laboratory has yielded < 20% mortality in bioassays of 10 µg/ml Cry2Ab2. Studies of the inheritance and crossresistance in this Cry2Ab2-resistant strain are currently underway.
Field Efficacy of Bollgard Cotton in Arizona
These studies were conducted by the Arizona Cotton Research and Protection Council, based in Phoenix, Arizona.
Forty-four pairs of adjacent commercial fields of Bt and non-Bt cotton fields were sampled throughout Arizona from
5 August to 13 November, 2005. Each pair was sampled twice, as close as practical to the onset of harvest. On each
sampling date, 150 bolls were collected from the non-Bt (refuge) field and 500 bolls were sampled from the adjacent
Bt field of each pair, yielding total boll numbers of 300 and 1000 for the non-Bt and Bt fields, respectively. Boll
collections were made within 50 meters of the common edges of each pair of fields. No more than one boll was
sampled from any plant.
Boll samples were labeled, transported to ACRPC field offices, and placed in boll boxes (17.6 cm x 50.4 cm x 35.2
cm) in groups of 50 per box. Boll boxes suspended infested bolls on wire racks approximately three cm above
sheets of paper toweling on the bottom of the boxes. Two to three weeks after making collection, bolls were opened
to record numbers of larvae ≥3rd instar and pupae within. Additionally, counts were made of 4th instar larvae, pupae
and adults that had exited bolls in the boxes. Because non-Bt bolls often had very high infestation rates, a variable
sample size was used. When a single box of 50 bolls yielded eight or more individuals of ≥ 3rd instar, the other two
boxes from that sample of 150 bolls were not evaluated. ANOVA was used to detect differences in mean survival of
PBW between sites and years.
When possible, bolls from Bt fields in which PBW were found to have survived to ≥ 4th instar were tested for the
presence of Cry1Ac toxin. Two or three seeds of such bolls were tested individually using the ImmunoStrip test
system (Agdia, Elkhart, IN). Bolls were then designated as a) positive for Cry1Ac, b) negative for Cry1Ac, or c)
mosaic (containing seeds testing positive and negative) for Cry1Ac. Heavily damaged bolls often could not be
tested because of insufficient seed material. Archived samples of Bt and non-Bt cotton seeds served as internal
controls for these evaluations.
Arizona Cotton Report (P-151) July 2007
108
Results and Discussion
Susceptibility of Southwestern PBW to Bt Toxin Cry1Ac
Arizona Collections--2005. No survivors were observed in discriminating concentration bioassays of 10 µg/ml
Cry1Ac. Grand mean mortality of 11 Arizona strains of pink bollworm was 8.57%, and 100% at concentrations of
0, and 10 µg/ml, respectively (Table 2).
Change in AZ Collections 1997-05. Figure 4 shows the change in corrected survivorship in bioassays of 10 µg/ml
Cry1Ac from 1997 to 2005. Though undetected in 2005, low numbers of survivors of 10 µg/ml bioassays of PBW
strains were found from 2001-2004. Three points are critical regarding these rare survivors of 10 µg/ml bioassays of
Cry1Ac. First, molecular testing of survivors has been conducted since 2001 using the methodology described in
Tabashnik et al. (2006). Resistance alleles have been detected only twice: once in a strain collected in 2001 and
once in a strain collected in 2003. Both of these strains were obtained from locations outside of Arizona. Second,
selection in the laboratory of the cultures from which rare survivors of 10 µg/ml Cry1Ac were obtained has been
done since the inception of our monitoring program in 1997. Following the isolation of multiple resistant strains in
1997, selection has produced new resistant strains only in the two aforementioned cases, i.e., once in 2001 and once
in 2003. Third, despite greatly increased yearly sample sizes, the frequency of survivors of 10 µg/ml Cry1Ac has
never exceeded that observed in 1997 (Figure 4). We previously reported that Arizona pink bollworm had
significantly less mortality in 10 µg Cry1Ac bioasssays in 1997 than 1998 (P=0.031, F=5.36, df=1), 1999 (P=0.015,
F=6.95, df=1) or 2000 (P=0.007, F=8.52, df=1, Dennehy et al. 2003). Mean mortality (corrected) in bioassays of
10 µg/ml increased from 94.1% in 1997 to 98.9-100% in 1998-2005 (Figure 4). Further statistical analyses of these
results are of little consequence due to the rarity of resistant survivors of bioassays in recent years and due to the fact
that few of the rare resistant survivors turn out to possess heritable resistance. In conclusion, levels of resistance to
Cry1Ac in the Southwest were lower in 2005 than in 1997, the first year of our monitoring effort and the year that
we isolated highly resistant pink bollworm from Arizona collections.
CA Collections--2005. Two collections from the Palo Verde Valley of California and two collections from the
Imperial Valley of California yielded no survivors of 10 µg/ml Cry1Ac bioassays (Table 2).
Susceptibility of Southwestern PBW to Bt Toxin Cry2Ab2
All sixteen pink bollworm strains collected in 2005 were highly susceptible to Cry2Ab2 (Figure 5), based on
contrasts with previously published (Unnithan et al. 2004, Dennehy et al. 2005) baseline responses (see Figure 3).
Mortality was 100% in all bioassays of 1.0 µg/ml Cry2Ab2 (n=1000) and 10 µg/ml Cry2Ab2 (n=3,425) (Table 4).
Monitoring in the previous season yielded small numbers of survivors 1.0 µg/ml Cry2Ab2 bioassays. Mean
corrected mortality of individual collections made in 2004 ranged from 95.2 to 100% in bioassays of 1.0 µg
(Dennehy et al. 2005) and was 100% in all bioassays of 10 µg/ml Cry2Ab2 (Figure 5).
Laboratory Strains/Internal Standards
Of the total of 490 larvae from the APHIS strain tested with 10 µg/ml Cry1Ac there were 3 survivors, or 99.3%
mortality (Table 2). Results of bioassays of APHIS from 1998 to 2005 revealed small increases in survivors of 10
µg/ml Cry1Ac over the past two years and even greater reductions in mortality observed in bioassays of 1.0 µg/ml
Cry1Ac. These changes in 2004 and 2005 could be due to a low level of contamination of the APHIS strain, diet
constituents, toxin stability, or other aspects of our experimental procedure. The critical conclusion is that we
should now anticipate a low percentage (<1%) of the survivors of 10 µg/ml Cry1Ac may be susceptible to Cry1Ac.
Since we routinely subject all putatively resistant survivors to further genetic analysis and molecular biotyping, we
will be able to distinguish between resistant and susceptible survivors. Testing of the AZP-R strain with Cry1Ac
yielded 100% corrected survival in bioassays of 1.0 and 10 µg/ml Cry1Ac (Table 2).
The APHIS and AZP-R strains were also used as internal standards for Cry2Ab2 bioassays. Totals of 200 and 120
larvae were tested with 1.0 and 10 µg/ml Cry2Ab2, respectively, yielded corresponding corrected mortality of
82.5% and 100% (Table 4). APHIS bioassays conducted during each of the past three years have uniformly yielded
100% mortality in concentrations of 10 µg/ml Cry2Ab2 (Table 5) and mortality in 1.0 µg/ml assays has ranged from
Arizona Cotton Report (P-151) July 2007
109
72.6 to 87.9%. The AZP-R strain had 61.8% corrected mortality in bioassays of 1.0 µg/ml Cry2Ab2 (n=189) and
100% mortality in bioassays of 10 µg/ml Cry2Ab2 (n=135) (Table 4).
Field Efficacy of Bollgard Cotton in Arizona
Statewide averages for pink bollworm infestation rates in Bt and non-Bt cotton in 2005 were within the range
observed since registration of Bollgard in 1996. The grand mean of boll infestation was 0.285% for Bt fields and
15.3% for non-Bt fields (Figure 6). These values reflected a total of 6,300 non-Bt bolls and 33,000 Bt bolls
collected from 44 pairs of fields. Non-Bt bolls yielded 962 pink bollworm. Bolls from Bt fields yielded 94 PBW.
However, it is important to understand that we report any and all pink bollworm recovered from putative Bt fields,
whether the boll from which they came expressed Bt toxin in seeds or not. A high proportion of the 94 PBW
recovered from “Bt cotton samples” in 2005 developed in contaminant non-Bt bolls. Sixty of the 94 individuals
came from a single field in which immunoassay tests failed to detect Cry1Ac toxin. Through the past five years,
immunoassay tests have consistently shown that most of the bolls from Bt field in which larvae have developed do
not test positive for Cry1Ac toxin. Thus, the infestation rates we report each year are more a reflection of the
contamination levels of Bt fields by non-Bt cotton than they are an index of resistance development. We do not cull
our data because of the uncertainty that is involved in doing immunoassay test for Cry1Ac. Not infrequently, seeds
from infested bolls are fully consumed by the developing larvae and thus not suitable for testing.
As in 2004, two of Bt fields sampled in 2005 had >2% boll infestation. Immunoassay tests indicated that these
fields had either an unusually high level of non-Bt within the field or they were a non-Bt field that had been
misclassified by the producer as a Bt field. Rogue non-Bt plants within Bt fields can stem from a variety of sources.
These include: 1) volunteer plants from the previous season’s non-Bt plants; 2) on-farm mixing of Bt seed with nonBt seed in seed hoppers; 3) in-the-bag contamination of commercial seed stocks. Contrasts of 2006 results with
those from previous years revealed a significant increase in the percent infested bolls in Bt cotton from 2002 to 2004
(Figure 6). However, tests of infested bolls collected from Bt fields, conducted by Arizona Cotton Research and
Protection Council personnel, revealed that a large proportion of infested bolls did not have Cry1Ac toxin in the
seeds. Of 35 infested bolls collected from Bt fields in 2004 and tested for Cry1Ac, only two were positive for toxin.
This means that the true frequency of resistant pink bollworm surviving in Bt cotton is substantially lower than the
boll infestation rate of 0.340% we observed in Bt bolls. Non-Bt bolls in Bt fields could result from contamination of
non-Bt seed in the seed bag, contamination in the hopper of the planter at the time of planting, or volunteer non-Bt
plants originating from seed that remained in the ground from the previous season.
In conclusion, infestation levels in Bt fields remained low throughout the Southwest in 2005 and have averaged ≤
0.350% over the past ten years (Figure 6). This amounts to < four PBW per 1000 bolls. Thus, the efficacy of Bt
cotton against pink bollworm continues to be exceptional and has changed little since Bollgard was first
commercialized in 1996.
Conclusions
Extensive monitoring of field collections of pink bollworm from throughout the Southwestern US in 2005
confirmed that the frequency of resistance to Cry1Ac was so low as to be undetectable. Bollgard continued to
perform remarkably well against pink bollworm in Bt cotton fields at 44 Arizona locations evaluated. At this time
we have no indications of imminent problems with pink bollworm resistance to either Cry1Ac or Cry2Ab2 in
Arizona or elsewhere in the Southwest.
Acknowledgement
We recognize and thank the numerous contributors to this multi-agency collaboration. We thank Sarah Brink and
Brook Wood for technical assistance with the rearing and testing of pink bollworm and their student assistants,
Melanie Meyers, Josh Garcia, and Marlene Yafuso. Jerry Kerr, Penny Malone, Angie Soto, Bobby Soto, Don
Struckmeyer, Rick Webb, and Mike Woodward of the Arizona Cotton Research and Protection Council supported
the field components of the studies. Joseph Ellington and Tracy Carillo of New Mexico State University, Dan
Keaveny and Jodi Brigman of the California Department of Food and Agriculture, and Robert Staten, Joe Friesen
Arizona Cotton Report (P-151) July 2007
110
and Edward Herrere of the USDA-APHIS assisted with collections of pink bollworm. Christa Ellers-Kirk and
Robert Biggs assisted with rearing and molecular genotyping of putative resistant moths. EARML facilities are
provided and maintained by the University of Arizona. Diet for rearing of pink bollworm was provided by Thomas
Henneberry, Director of the USDA Western Cotton Research Laboratory. Funding for this project was provided by
the Arizona Cotton Growers/Cotton Incorporated, the Arizona Cotton Research & Protection Council and Monsanto
Life Sciences.
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Simmons, A. L., T. J. Dennehy, B. E. Tabashnik, L. Antilla, A. Bartlett, D. Gouge, and R. Staten. 1998. Evaluation
of B.t. cotton deployment strategies and efficacy against pink bollworm in Arizona. Proc. Beltwide Cotton
Conferences. pp. 1025-1030.
Arizona Cotton Report (P-151) July 2007
111
Sims, M. A., T. J. Dennehy, A. Patin, Y. Carrière, Y.-B. Liu, B. E. Tabashnik, L. Antilla, and M. Whitlow. 2001.
Arizona's multi-agency resistance management program for Bt cotton: sustaining the susceptibility of pink
bollworm. Proc. Beltwide Cotton Conferences. National Cotton Council, Memphis, TN.
Tabashnik, B. E., A. L. Patin, T. J. Dennehy, Y.-B. Liu, Y. Carrière, M. Sims and L. Antilla. 2000. Frequency of
resistance to Bacillus thuringiensis in field populations of pink bollworm. Proc. Natl. Acad. Sci. 97:12980-12984.
Tabashnik, B. E., Y. B. Liu, T. J. Dennehy, M. A. Sims, M. S. Sisterson, R. W. Biggs and Y. Carrière. 2002.
Inheritance of resistance to Bt toxin Cry1Ac in a field-derived strain of pink bollworm (Lepidoptera: Gelechiidae).
J. Econ. Entomol.: 95:1018-1026.
Tabashnik, B.E., T. J. Dennehy, and Yves Carrière. 2005. Delayed resistance to transgenic cotton in pink
bollworm. Proc. National Acad. Sci. 102:15389.
Tabashnik, B.E., J.A. Fabrick, S. Henderson, R.W. Biggs, C.M. Yafuso, M.E. Nyboer, N.M. Manhardt, L.A.
Coughlin, J. Sollome, Y. Carrière, T.J. Dennehy and S. Morin. 2006. DNA screening reveals pink bollworm
resistance to Bt cotton remains rare after a decade of exposure. J. Econ. Entomol. 99: 1525-1530.
Unnithan, G.C., S. Brink, B. Wood, Y. Carrière, B.E. Tabashnik, T.J. Dennehy, L. Antilla, M. Whitlow. 2004.
Susceptibility of Southwestern pink bollworm to Cry2Ab: baseline responses in 2001 and 2002. University of
Arizona Cooperative Extension. 12pp. URL:
http://epa.gov/scipoly/sap/meetings/2006/october/unnithan_et_al_04_cry2ab_baselines.pdf.
Watson, T. F. and P. H. Johnson. 1974. Larval stages of the pink bollworm, Pectinophora gossypiella. Annals
Entomol. Soc. of Amer. 67:812-814.
Arizona Cotton Report (P-151) July 2007
112
Whitefly
Pink bollworm
14
12
10
8
$217
$106
$138
$121
$111 $108
$68
$103
$85
$37
2
0
Other
$113 (ave. cost / A)
6
4
Lygus bugs
$64
$56 $62
$53
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Figure 1. Estimated number of insecticide applications made to Arizona cotton and their cost
from 1990 to 2004. The vertical line depicts the point of simultaneous introduction in 1996 of Bt
cotton effective against Pectinophora gossypiella and insect growth regulators effective against
Bemisia tabaci. From Ellsworth and Palumbo (2005).
Arizona Cotton Report (P-151) July 2007
113
>
5
5
5
5
5
5
5 5
5
5
>
5
5
5
5
5
5
Figure 2. Locations at which larvae of pink bollworm were successfully collected in 2005 and
subsequently reared in the laboratory for the purpose of monitoring resistance to Bt toxins
Cry1Ac and Cry2Ab2.
Arizona Cotton Report (P-151) July 2007
114
6.5
6
2002
(
(
)
90
)
5.5
5
(
)
(
50
)
4.5
4
10
3.5
0.01
0.1
1
10
Concentration Cry2Ab (µg/ml diet)
Figure 3. Baseline susceptibility to Cry2Ab2 of pink bollworm as determined by dietincorporation bioassays. Collections were made in 2002 in Arizona (9 strains), California (3
strains), New Mexico (1 strain), and Texas (1 strain). Probit lines with LC50 and LC90 estimates
(95% F.L.) were generated using the POLO program (Robertson and Preisler 1992). From
Unnithan et al. 2004.
Arizona Cotton Report (P-151) July 2007
115
1997
1998
1999
2000
2001
2002
2003
2004
3.90%
1997
1998
1999
2000
2001
2002
2003
2004
0.0262%
Pectinophora gossypiella
2005
0
10
20
30
No Survivors
40
Corrected Percent Survival
(+/- Standard Deviation)
Figure 4. Changes in pink bollworm susceptibility to Cry1Ac in Arizona from 1997 to 2005.
Shown are mean values (± standard deviation) of corrected survival observed in replicated
bioassays of 1.0 and 10 µg CrylAc/ml diet. The numbers of field collections evaluated each year
were: 1997 (n=9), 1998 (n=12), 1999 (n=14), 2000 (n=17), 2001 (n=17), 2002 (n=13), 2003
(n=16), 2004 (n=13) and 2005 (n=11). Testing of the diet concentration of 1.0 µg/ml was
discontinued in 2005. See Table 2 for site-specific bioassay results of all 2005 collections tested.
No larvae from any tests of 2005 strains survived treatments of 10 µg/ml Cry1Ac (n=5,358).
Arizona Cotton Report (P-151) July 2007
116
2003
2004
2005
2003
2004
2005
Pectinophora gossypiella
Figure 5. Response of Arizona pink bollworm to diagnostic concentrations of Cry2Ab2. Shown
are mean values (± standard deviation) of corrected survival observed in replicated 1.0 and 10 µg
Cry2Ab2/ml diet bioassays. The numbers of collections evaluated each year were: 2003 (n=12),
2004 (n=15) and 2005 (n=12). See Table 4 for site-specific bioassay results of all 2005
collections tested. No pink bollworm evaluated in 2005 survived either the 1.0 or 10 µg/ml
treatments.
Arizona Cotton Report (P-151) July 2007
117
0.45
0.40
Pectinophora gossypiella
50
45
Non-Bt Cotton
40
0.35
35
0.30
x1-44= 0.28%
0.25
30
25
0.20
20
0.15
x1-44= 15%
Bt Cotton
0.10
15
10
0.05
5
0
0
1995
N=5(a)
1996
N=6(a)
1997
N=7(a)
1998
N=33
1999
N=35
2000
N=36
2001
N=39
2002
2003
N=43
2004
2005
N=39
N=40
N=44
(a) Flint 1998
Figure 6. Sustained efficacy of Bt cotton in Arizona: 1995 to 2005. Data from 1995 to 1997
were reported by Flint et al. (1995) and Flint and Park (1996). All other data were collected by
the Arizona Cotton Research and Protection Council. Shown are means of percent boll
infestation (bolls with ≥ 3rd instar PBW) for pairs of Bt cotton (left axis) and non-Bt cotton fields
(right axis) sampled each year. The numbers of pairs of Bt and non-Bt fields (N) is indicated for
each year. In 44 pairs of Bt and non-Bt cotton sampled in 2005, the infestation rate in non-Bt
cotton was 15% (n=6,300 bolls), and the infestation rate in adjacent Bt cotton was 0.28%
(n=33,000 bolls).
Arizona Cotton Report (P-151) July 2007
118
Table 1. Pink bollworm strains collected in 2005 and bioassayed for susceptibility to Cry1Ac
and/or Cry2Ab2 using diet-incorporation bioassays.
State
Collection
Date
Waypoint
Cry1Ac
Cry2Ab2
Buckeye
AZ
1-Dec
O5-36
3
Eloy
AZ
30-Nov
O5-30
no
Maricopa Agricultural Center
AZ
1-Dec
O5-33
Mohave Valley
AZ
23-Oct
O5-23
North Gila Valley
AZ
12-Sep
O5-18
Parker Valley
AZ
23-Oct
O5-24
Queen Creek
AZ
1-Dec
O5-32
Sentinel/Agua Caliente
AZ
1-Dec
O5-35
Somerton
AZ
19-Sep
O5-05
Stanfield
AZ
1-Dec
O5-34
Texas Hill
AZ
23-Oct
O5-26
Tonopah
AZ
2-Dec
O5-37
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Imperial Valley #1
CA
12-Sep
O5-16
Imperial Valley #2
CA
24-Oct
O5-17
Palo Verde Valley #1
CA
12-Sep
O5-14
Palo Verde Valley #2
CA
24-Oct
O5-15
3
3
3
3
3
3
3
3
Location
Arizona Cotton Report (P-151) July 2007
119
Table 2. Susceptibility to Cry1Ac toxin of pink bollworm collected in 2005 and evaluated in
diet-incorporation bioassays.
ARIZONA
Location:
Population:
Conc Cry1Ac
µg/ml
Buckeye
05-36, F1, F2, F3.
# of Larvae
Initial
Development
1st, 2nd, Dead, Missing
3rd
4th
Pupae Adult E. Hole Survivors
Survival
Mortality
0
45
2
1
11
26
0
5
42
93.3%
6.67%
10
328
327
1
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Corrected
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
0.000%
100%
Maricopa Agricultural Center
05-33, F1.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
3
0
5
22
2
8
37
92.5%
7.50%
10
360
360
0
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Corrected
Survival
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Mohave Valley
05-23, F1, F2.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
3
0
7
19
4
7
37
92.5%
7.50%
10
360
360
0
0
0
0
0
0
0.000%
100%
Corrected
Corrected
3rd
4th
Survival
Mortality
Survival
Mortality
1
0
6
0
87.5%
0.000%
12.5%
100%
0.000%
100%
3rd
4th
Survival
Mortality
Corrected
Survival
Corrected
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
Location:
Population:
Initial
# of Larvae
0
10
40
332
Conc Cry1Ac
µg/ml
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
North Gila Valley
05-18, F2.
Conc Cry1Ac
µg/ml
Location:
Population:
Development
Initial
Development
1st, 2nd, Dead, Missing
4
332
Pupae Adult E. Hole Survivors
18
0
1
0
10
0
35
0
Parker Valley
05-24, F2.
# of Larvae
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
0
40
3
0
7
22
1
7
37
92.5%
7.50%
10
378
378
0
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Queen Creek
05-32, F2, F3.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
2
1
5
24
0
8
37
92.5%
7.50%
10
359
359
0
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Sentinel/Agua Caliente
05-35, F2.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
5
0
9
11
0
15
35
87.5%
12.5%
10
360
360
0
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
0
4
100.00%
0.00%
Location:
Population:
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Somerton
05-05, F1, F5.
Conc Cry1Ac
µg/ml
# of Larvae
0
50
Initial
Development
1st, 2nd, Dead, Missing
0
Pupae Adult E. Hole Survivors
36
0
10
50
1
30
5
16
4
2
0
3
9
30.0%
70.0%
30.0%
70.0%
10
376
360
16
0
0
0
0
0
0.000%
100%
0.000%
100%
Arizona Cotton Report (P-151) July 2007
120
Location:
Population:
Stanfield
05-34, F1, F2.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
10
344
Location:
Population:
Initial
Development
1st, 2nd, Dead, Missing
3rd
4th
Pupae Adult E. Hole Survivors
1
1
11
21
0
6
344
0
0
0
0
0
3rd
4th
Survival
Mortality
38
95.0%
5.00%
0
0.000%
100%
Survival
Mortality
Corrected
Corrected
Survival
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
Texas Hill
05-26, F3, F4.
Conc Cry1Ac
µg/ml
# of Larvae
0
40
4
0
8
19
0
9
36
90.0%
10.0%
10
366
365
1
0
0
0
0
0
0.000%
100%
0.000%
100%
3rd
4th
Survival
Mortality
Corrected
Survival
Corrected
Mortality
4
2
24
0
80.0%
0.000%
20.0%
100%
0.000%
100%
Corrected
Corrected
3rd
4th
Survival
Mortality
Survival
Mortality
0.000%
100%
Location:
Population:
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Tonopah
05-37, F1.
Conc Cry1Ac
µg/ml
# of Larvae
Initial
0
10
40
360
Development
1st, 2nd, Dead, Missing
4
358
Pupae Adult E. Hole Survivors
5
0
0
0
3
0
32
0
CALIFORNIA
Location:
Population:
Imperial Valley #1
05-16, F1.
Conc Cry1Ac
µg/ml
# of Larvae
0
30
2
1
3
13
0
11
27
90.0%
10.0%
10
360
357
3
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Initial
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Imperial Valley #2
05-17, F1, F2.
Conc Cry1Ac
µg/ml
# of Larvae
0
35
2
0
2
24
0
7
33
94.3%
5.71%
10
370
370
0
0
0
0
0
0
0.000%
100%
3rd
4th
Survival
Mortality
Location:
Population:
Initial
Development
Corrected
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Mortality
0.000%
100%
Corrected
Corrected
Survival
Mortality
Palo Verde Valley #1
05-14, F1, F2.
Conc Cry1Ac
# of Larvae
µg/ml
Initial
0
30
4
1
1
17
1
6
25
83.3%
16.7%
10
360
360
0
0
0
0
0
0
0.000%
100%
0.000%
100%
3rd
4th
Survival
Mortality
Corrected
Survival
Corrected
Mortality
0
0
2
0
83.3%
0.000%
16.7%
100%
0.000%
100%
3rd
4th
8
97
Location:
Population:
Development
1st, 2nd, Dead, Missing
Pupae Adult E. Hole Survivors
Palo Verde Valley #2
05-15, F1, F2.
Conc Cry1Ac
µg/ml
# of Larvae
Initial
0
10
30
368
Development
1st, 2nd, Dead, Missing
5
368
Pupae Adult E. Hole Survivors
14
0
0
0
9
0
25
0
2005 ARIZONA TOTALS--Cry1Ac
No. of populations: 11
Conc Cry1Ac
µg/ml
# of Larvae
0
455
Total
Development
Total
1st, 2nd, Dead or Missing
31
Pupae Adult E. Hole Survivors
223
8
88
416
Percent
Percent
Corrected
Corrected
Survival
Mortality
Survival
Mortality
91.4%
8.57%
1
30
5
16
4
2
0
3
9
30.0%
70.0%
0.000%
67.2%
10
3923
3903
20
0
0
0
0
0
0.000%
100%
0.000%
100%
3rd
4th
2005 CALIFORNIA TOTALS--Cry1Ac
No. of populations: 4
Conc Cry1Ac
µg/ml
# of Larvae
0
125
13
2
8
68
1
33
10
1458
1455
3
0
0
0
0
Total
Development
Total
1st, 2nd, Dead or Missing
Arizona Cotton Report (P-151) July 2007
Percent
Percent
Corrected
Corrected
Survival
Mortality
Survival
Mortality
110
88.0%
12.0%
0
0.000%
100%
Pupae Adult E. Hole Survivors
0.000%
121
100%
2005 TOTALS FOR ALL FIELD COLLECTIONS--Cry1Ac
No. of populations: 15
Conc Cry1Ac
µg/ml
# of Larvae
0
580
Total
Development
Total
1st, 2nd, Dead or Missing
44
3rd
4th
10
105
Pupae Adult E. Hole Survivors
291
9
121
526
Percent
Percent
Corrected
Corrected
Survival
Mortality
Survival
Mortality
90.7%
9.31%
1
30
5
16
4
2
0
3
9
30.0%
70.0%
0.000%
67.2%
10
5381
5358
23
0
0
0
0
0
0.000%
100%
0.000%
100%
Susceptible Laboratory Strain
Internal standard, APHIS strain, tested July 05 through June 06
Conc Cry1Ac
µg/ml
# of Larvae
0
140
Total
Development
Total
1st, 2nd, Dead or Missing
22
3rd
4th
Pupae Adult E. Hole Survivors
1
12
22
80
3
117
Percent
Percent
Corrected
Corrected
Survival
Mortality
Survival
Mortality
83.6%
16.4%
1
130
34
21
65
10
0
0
75
57.7%
42.3%
69.0%
31.0%
10
490
415
32
3
0
0
0
3
0.612%
99.4%
0.733%
99.3%
Cry1Ac-Resistant Laboratory Strain
Internal standard, AZP-R strain, tested July 05 through June 06
Conc Cry1Ac
µg/ml
# of Larvae
0
190
Total
Development
Total
1st, 2nd, Dead or Missing
6
3rd
4th
4
1
Pupae Adult E. Hole Survivors
47
132
0
180
Percent
Percent
Corrected
Corrected
Survival
Mortality
Survival
Mortality
94.7%
5.26%
1
84
2
2
2
31
49
0
82
97.6%
2.38%
100%
0.000%
10
214
3
2
4
109
96
0
209
97.7%
2.34%
100%
0.000%
Table 3. Corrected mortality (±SEM) of a susceptible laboratory strain, APHIS, evaluated from
1998 through 2005 in diet-incorporation bioassays of 1.0 and 10 µg Cry1Ac toxin per ml of diet.
1.0 µg/ml Cry1Ac
10 µg/ml Cry1Ac
Year
% Mortality
SEM (%)
% Mortality
SEM (%)
1998
66.2
6.0
100
0.00
1999
61.3
8.0
100
0.00
2000
73.4
23
100
0.00
2001
92.7
5.5
100
0.00
2002
98.6
2.4
100
0.00
2003
48.7
8.9
100
0.00
2004
17.0
3.0
97.7
0.68
2005
31.0
9.1
99.3
0.85
Arizona Cotton Report (P-151) July 2007
122
Table 4. Susceptibility to Cry2Ab2 toxin of pink bollworm collected in 2005 and evaluated in
diet-incorporation bioassays.
ARIZONA
Location:
Population:
Buckeye
05-36, F1-2
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
80
60
210
Location:
Population:
# of Larvae
Initial
0
1
10
70
0
215
# of Larvae
Initial
0
1
10
70
60
200
# of Larvae
Initial
0
1
10
70
60
220
# of Larvae
Initial
0
1
10
90
60
210
# of Larvae
Initial
0
1
10
60
60
230
4th
0
0
0
16
0
0
3rd
4th
0
0
0
42
0
0
3rd
4th
0
0
0
21
0
0
3rd
4th
1
0
0
32
0
0
3rd
4th
0
0
0
12
0
0
3rd
4th
1
0
0
35
0
0
3rd
4th
0
0
0
27
0
0
0
0
0
0
0
0
74
0
0
Corrected
Survival
Corrected
Mortality
92.5%
0.000%
0.000%
7.50%
100%
100%
0.000%
0.000%
100%
100%
Development
1st, 2nd, Dead or Missing
22
0
215
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
21
0
0
0
0
0
11
0
0
48
0
0
68.6%
no data
0.000%
31.4%
no data
100%
no data
0.000%
no data
100%
Development
1st, 2nd, Dead or Missing
2
60
200
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
26
0
0
0
0
0
0
0
0
68
0
0
97.1%
0.000%
0.000%
2.86%
100%
100%
0.000%
0.000%
100%
100%
Development
1st, 2nd, Dead or Missing
6
60
220
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
43
0
0
0
0
0
0
0
0
64
0
0
91.4%
0.000%
0.000%
8.57%
100%
100%
0.000%
0.000%
100.0%
100%
Development
1st, 2nd, Dead or Missing
12
60
210
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
41
0
0
0
0
0
4
0
0
77
0
0
85.6%
0.000%
0.000%
14.4%
100%
100%
0.000%
0.000%
100%
100%
Development
1st, 2nd, Dead or Missing
2
60
230
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
46
0
0
0
0
0
0
0
0
58
0
0
96.7%
0.000%
0.000%
3.33%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
90.0%
0.000%
0.000%
10.0%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
81.3%
0.000%
0.000%
18.8%
100%
100%
0.000%
0.000%
100%
100%
Queen Creek
05-32, F2
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
70
60
210
Location:
Population:
3rd
43
0
0
Mortality
Parker Valley
05-24, F1-3
Conc Cry2Ab2
µg/ml
Location:
Population:
31
0
0
Survival
North Gila Valley
05-18, F2-5
Conc Cry2Ab2
µg/ml
Location:
Population:
1
0
0
Pupae Adult E. Hole Survivors
Mohave Valley
05-23, F2-3
Conc Cry2Ab2
µg/ml
Location:
Population:
4th
Maricopa Agricultural Center
05-33, F2
Conc Cry2Ab2
µg/ml
Location:
Population:
5
60
210
3rd
Eloy
05-30, F3.
Conc Cry2Ab2
µg/ml
Location:
Population:
Development
1st, 2nd, Dead or Missing
Development
1st, 2nd, Dead or Missing
6
60
210
Pupae Adult E. Hole Survivors
28
0
0
0
0
0
0
0
0
63
0
0
Sentinel/Agua Caliente
05-35, F1-2
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
80
60
200
Development
1st, 2nd, Dead or Missing
15
60
200
Arizona Cotton Report (P-151) July 2007
Pupae Adult E. Hole Survivors
38
0
0
0
0
0
0
0
0
65
0
0
123
Location:
Population:
Somerton
05-O5, F1,5-6
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
80
60
220
Location:
Population:
# of Larvae
Initial
0
1
10
70
70
200
4th
0
0
0
14
0
0
3rd
4th
0
0
0
20
0
0
3rd
4th
0
0
0
24
0
0
3rd
4th
0
0
0
36
0
0
3rd
4th
1
0
0
27
0
0
3rd
4th
0
0
0
34
0
0
3rd
4th
1
0
0
22
0
0
3rd
4th
0
1
0
14
0
0
Pupae Adult E. Hole Survivors
56
0
0
2
0
0
0
0
0
72
0
0
Survival
Mortality
Corrected
Survival
Corrected
Mortality
90.0%
0.000%
0.000%
10.0%
100%
100%
0.000%
0.000%
100%
100%
Development
1st, 2nd, Dead or Missing
4
70
200
Pupae Adult E. Hole Survivors
Survival
Mortality
Corrected
Survival
Corrected
Mortality
46
0
0
0
0
0
0
0
0
66
0
0
94.3%
0.000%
0.000%
5.71%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
95.0%
0.000%
0.000%
5.00%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
94.3%
0.000%
0.000%
5.71%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
88.8%
0.000%
0.000%
11.3%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
98.6%
0.000%
0.000%
1.43%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
89.2%
0.000%
0.000%
10.8%
100%
100%
0.000%
0.000%
100%
100%
Survival
Mortality
Corrected
Survival
Corrected
Mortality
94.3%
0.000%
0.000%
5.71%
100%
100%
0.000%
0.000%
100%
100%
Texas Hill
05-26, F1-2
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
60
60
210
Location:
Population:
8
60
220
3rd
Stanfield
05-34, F1-2
Conc Cry2Ab2
µg/ml
Location:
Population:
Development
1st, 2nd, Dead or Missing
Development
1st, 2nd, Dead or Missing
3
60
210
Pupae Adult E. Hole Survivors
33
0
0
0
0
0
0
0
0
57
0
0
Tonopah
05-37, F1-2
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
70
60
210
Development
1st, 2nd, Dead or Missing
4
60
210
Pupae Adult E. Hole Survivors
30
0
0
0
0
0
0
0
0
66
0
0
CALIFORNIA
Location:
Population:
Imperial Valley #1
05-16, F1, 4-5
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
80
60
220
Location:
Population:
# of Larvae
Initial
0
1
10
70
60
240
44
0
0
0
0
0
0
0
0
71
0
0
Development
1st, 2nd, Dead or Missing
1
60
240
Pupae Adult E. Hole Survivors
35
0
0
0
0
0
0
0
0
69
0
0
Palo Verde Valley #1
05-14, F1, 4-5
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
83
70
220
Location:
Population:
8
60
220
Pupae Adult E. Hole Survivors
Imperial Valley #2
05-17, F1-3
Conc Cry2Ab2
µg/ml
Location:
Population:
Development
1st, 2nd, Dead or Missing
Development
1st, 2nd, Dead or Missing
8
70
220
Pupae Adult E. Hole Survivors
47
0
0
5
0
0
0
0
0
74
0
0
Palo Verde Valley #2
05-15, F1-3
Conc Cry2Ab2
µg/ml
# of Larvae
Initial
0
1
10
70
140
210
Development
1st, 2nd, Dead or Missing
4
139
210
Arizona Cotton Report (P-151) July 2007
Pupae Adult E. Hole Survivors
52
0
0
0
0
0
0
0
0
66
0
0
124
2005 ARIZONA TOTALS--Cry2Ab2
No. of populations: 12
Conc Cry2Ab2
µg/ml
# of Larvae
Total
0
1
10
870
670
2535
Development
1st, 2nd, Dead or Missing
89
670
2535
3rd
4th
3
0
0
310
0
0
3rd
4th
0
1
0
97
0
0
Total
Pupae Adult E. Hole Survivors
451
0
0
2
0
0
15
0
0
778
0
0
Percent
Survival
Percent
Mortality
Corrected
Survival
Corrected
Mortality
89.4%
0.000%
0.000%
10.6%
100%
100%
0.000%
0.000%
100%
100%
Percent
Survival
Percent
Mortality
Corrected
Survival
Corrected
Mortality
92.4%
0.000%
0.000%
7.59%
100%
100%
0.00%
0.00%
100%
100%
Percent
Survival
Percent
Mortality
Corrected
Survival
Corrected
Mortality
90.2%
0.000%
0.000%
9.80%
100%
100%
0.000%
0.000%
100%
100%
Percent
Survival
Percent
Mortality
Corrected
Survival
Corrected
Mortality
91.3%
16.0%
0.000%
8.70%
84.0%
100%
17.5%
0.000%
82.5%
100%
Percent
Survival
Percent
Mortality
Corrected
Survival
Corrected
Mortality
84.5%
32.3%
0.0%
15.5%
67.7%
100%
38%
0.000%
61.8%
100%
2005 CALIFORNIA TOTALS--Cry2Ab2
No. of populations: 4
Conc Cry2Ab2
µg/ml
# of Larvae
Total
0
1
10
303
330
890
Development
1st, 2nd, Dead or Missing
21
329
890
Total
Pupae Adult E. Hole Survivors
178
0
0
0
0
0
0
0
0
280
0
0
2005 TOTALS FOR ALL FIELD COLLECTIONS--Cry2Ab2
No. of populations: 16
Conc Cry2Ab2
µg/ml
# of Larvae
Total
0
1
10
1173
1000
3425
Development
1st, 2nd, Dead or Missing
110
999
3425
3rd
4th
3
1
0
407
0
0
Total
Pupae Adult E. Hole Survivors
629
0
0
2
0
0
15
0
0
1058
0
0
Susceptible Laboratory Strain--Cry2Ab2
Internal standard, APHIS strain, tested July 05 through June 06
Conc Cry2Ab2
µg/ml
# of Larvae
Total
0
1
10
115
200
120
Development
1st, 2nd, Dead or Missing
6
148
120
3rd
4th
0
4
0
1
6
0
Total
Pupae Adult E. Hole Survivors
8
22
0
95
4
0
1
0
0
105
32
0
Cry1Ac-Resistant Laboratory Strain--Cry2Ab2
Internal standard, AZP-R strain, tested July 05 through June 06
Conc Cry2Ab2
µg/ml
# of Larvae
Total
0
1
10
110
189
135
Development
1st, 2nd, Dead or Missing
7
124
135
Arizona Cotton Report (P-151) July 2007
3rd
4th
0
4
0
0
38
0
Total
Pupae Adult E. Hole Survivors
17
21
0
76
2
0
0
0
0
93
61
0
125
Table 5. Corrected mortality (±SEM) of a susceptible laboratory strain, APHIS, evaluated from
2003 through 2005 in diet-incorporation bioassays of 1.0 and 10 µg Cry2Ab2 toxin per ml of
diet.
1.0 µg/ml Cry2Ab2
10 µg/ml Cry2Ab2
Year
% Mortality
SEM (%)
% Mortality
SEM (%)
2003
87.9
2.8
100
0.00
2004
72.6
4.6
100
0.00
2005
82.5
3.0
100
0.00
Arizona Cotton Report (P-151) July 2007
126