Applying an Integrated Refuge to Manage Western Corn

Rootworm (Coleoptera: Chrysomelidae): Effects on Survival,

Fitness, and Selection Pressure
Author(s): Jennifer L. Petzold-Maxwell, Analiza P. Alves, Ronald E. Estes, Michael
E. Gray, Lance J. Meinke, Elson J. Shields, Stephen D. Thompson, Nicholas A.
Tinsley, and Aaron J. Gassmann
Source: Journal of Economic Entomology, 106(5):2195-2207.
Published By: Entomological Society of America
URL: http://www.bioone.org/doi/full/10.1603/EC13088

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 INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Applying an Integrated Refuge to Manage Western Corn Rootworm

(Coleoptera: Chrysomelidae): Effects on Survival, Fitness, and
Selection Pressure
JENNIFER L. PETZOLD-MAXWELL,1,2 ANALIZA P. ALVES,3 RONALD E. ESTES,4
MICHAEL E. GRAY,4 LANCE J. MEINKE,5 ELSON J. SHIELDS,6 STEPHEN D. THOMPSON,3
NICHOLAS A. TINSLEY,4 AND AARON J. GASSMANN1

J. Econ. Entomol. 106(5): 2195Ð2207 (2013); DOI: http://dx.doi.org/10.1603/EC13088

ABSTRACT The refuge strategy can delay resistance of insect pests to transgenic maize producing
toxins from Bacillus thuringiensis (Bt). This is important for the western corn rootworm, Diabrotica
virgifera virgifera LeConte (Coleoptera: Chrysomelidae), because of its history of adaptation to
several management practices. A 2-yr study across four locations was conducted to measure the effects
of integrated refuge (i.e., blended refuge) on western corn rootworm survival to adulthood, Þtness
characteristics, and susceptibility to Bt maize in the subsequent generation. The treatments tested in
this study were as follows: a pure stand of Bt maize (event DAS-59122-7, which produces Bt toxins
Cry34Ab1/Cry35Ab1), a pure stand of refuge (non-Bt maize), and two variations on an integrated
refuge consisting of 94.4% Bt maize and 5.6% non-Bt maize. Within the two integrated refuge
treatments, refuge seeds received a neonicotinoid insecticidal seed treatment of either 1.25 mg
clothianidin per kernel or 0.25 mg thiamethoxam per kernel. Insects in the pure stand refuge treatment
had greater survival to adulthood and earlier emergence than in all other treatments. Although
fecundity, longevity, and head capsule width were reduced in treatments containing Bt maize for some
site by year combinations, Bt maize did not have a signiÞcant effect on these factors when testing data
across all sites and years. We found no differences in susceptibility of larval progeny to Bt maize in
bioassays using progeny of adults collected from the four treatments.

KEY WORDS event 59122, Diabrotica virgifera virgifera, refuge strategy, resistance management

The western corn rootworm, Diabrotica virgifera vir-
gifera LeConte (Coleoptera: Chrysomelidae), is a ma-
jor pest of maize in the United States (Gray et al.
2009). Genetically modiÞed maize producing insecti-
cidal toxins derived from the bacterium Bacillus thu-
ringiensis (Bt) was commercialized for management
of western corn rootworm in 2003 (Environmental
Protection Agency [EPA] 2003). Bt maize kills several
insect pests, including western corn rootworm, and it
has been widely adopted by growers, accounting for
67% of the total area planted to maize in 2012 (U.S.
Department of AgricultureÐEconomic Research Ser-
vice [USDAÐERS] 2012). The threat of widespread
Þeld-evolved resistance to Bt maize by western corn
rootworm is signiÞcant, given its history of rapid ad-
1 Department of Entomology, Iowa State University, 13 Insectary,
Ames, IA 50011.
2 Corresponding author, e-mail: jennifer.maxwell@wartburg.edu.
3 DuPont Pioneer, 7300 NW 62nd Ave., P.O. Box 1004, Johnston, IA
50131.
4 Department of Crop Sciences, University of Illinois, 1102 S. Good-
win, Urbana, IL 61801.
5 Department of Entomology, University of Nebraska, 109B Ento-
aptation to various management practices, including
different classes of conventional insecticides and the
cultural control practice of crop rotation (Meinke et
al. 1998, Wright et al. 2000, Levine et al. 2002). In
addition, Þeld-evolved resistance to Cry3Bb1 maize
has recently been documented in Iowa (Gassmann et
al. 2011, 2012; Gassmann 2012). Thus, sustainable in-
sect resistance management (IRM) strategies are es-
sential for prolonging the effectiveness of Bt maize for
management of western corn rootworm.
Currently, the U.S. EPA mandates the refuge strat-
egy to delay development of insect resistance to Bt
maize (Gould 1998, Tabashnik et al. 2003). Under the
refuge strategy, non-Bt plants serve as a refuge for
Bt-susceptible genotypes, providing a pool of homozy-
gous susceptible individuals to mate with homozygous
resistant individuals that survive exposure to a Bt crop
(Gould 1998), thus producing heterozygous offspring.
On exposure to a high-dose event, these heterozygous
individuals are expected to die, which can delay the
onset of resistance. However, current Bt maize
products targeting the western corn rootworm (Bt

mology Hall, Lincoln, NE 68583.

maize producing Cry3Bb1, mCry3a, and Cry34Ab1/
6 Department of Entomology, Cornell University, 2130 Comstock Cry35Ab1) fail to meet the high-dose condition of
Hall, 129 Garden Ave, Ithaca NY 14853. imposing 99.99% mortality (Gassmann 2012, Devos et

0022-0493/13/2195Ð2207$04.00/0 䉷 2013 Entomological Society of America

2196 JOURNAL OF ECONOMIC ENTOMOLOGY
al. 2013). In the case of nonhigh-dose events, the rate
of resistance evolution becomes dependent on func-
tional dominance, where a greater delay in resistance
is expected as the recessiveness of the resistance trait
increases (Tabashnik et al. 2008). Recently, the EPA
approved integrated refuge, which allows Bt and ref-
uge (non-Bt) seeds to be mixed in a preestablished
percentage (EPA 2010).
The type of refuge deployed, whether integrated or
in a separate block, can affect the evolution of resis-
tance (Tabashnik 1994, Onstad 2006, Onstad et al.
2011). One beneÞt of an integrated refuge is complete
compliance by farmers in planting of a refuge. This is
important because there is evidence that some farmers
do not comply with current refuge requirements
(Jaffe 2009, Gray 2011). Simulation models show that
pests evolve resistance more quickly as the percentage
of the landscape planted to refuge decreases (Onstad
2006, Tabashnik et al. 2008, Gassmann et al. 2009, Pan
et al. 2011). Another IRM beneÞt of integrated refuge
is the potential for increased mating between adults
from refuge plants and Bt plants (Onstad et al. 2011).
In the case of western corn rootworm, adults generally
emerge from Bt plants ⬇1 wk after insects emerge
from refuge plants (Binning et al. 2010, Hibbard et al.
2010a, PetzoldÐMaxwell et al. 2012). Female western
corn rootworm adults often mate soon after they
emerge and likely before dispersal (Hill 1975, Quiring
and Timmins 1990), making males the primary dis-
persers before mating. Proximity of refuge plants to Bt
plants may promote mating between susceptible and
resistant insects, a requirement necessary for success
of the refuge strategy in delaying resistance to Bt crops
(Gould 1994, 1998).
A concern of integrated refuge is the increased
potential for larval movement between Bt and non-Bt
plants, which may increase exposure of the individuals
within the insect population to sublethal doses of the
Bt toxin and potentially increase the rate of resistance
evolution (Gould 1998, Onstad and Gould 1998). It is
generally thought that if larvae with an allele for re-
sistance move from a Bt plant to a non-Bt plant, this
could result in ingestion of sublethal amounts of Bt
toxin, leading to a Þtness advantage for partially re-
sistant insects that otherwise could not complete de-
velopment on a Bt plant (Onstad and Gould 1998).
However, because currently commercialized Bt maize
targeting western corn rootworm is not considered
high dose, survival of homozygous susceptible and
heterozygous individuals will occur in even a pure
stand of Bt maize (Siegfried et al. 2005, Meihls et al.
2008, Hibbard et al. 2010a). Thus, it is possible that
larval movement between refuge and Bt plants may
not greatly affect the rate of resistance evolution for
this species (Tabashnik 1994), although more studies
are necessary to better understand how larval move-
ment of western corn rootworm in integrated refuges
can affect evolution of resistance.
Differences in effects of block vs. integrated refuges
on Þtness of insects also may affect resistance evolu-
tion. If insects emerging from Bt plants incur sublethal
effects on Þtness, the spread of resistance genes may
Vol. 106, no. 5
be delayed compared with cases where sublethal ef-
fects are absent (Tabashnik et al. 2004). Past studies
and this study considered the life history parameters
of adult size, fecundity, and longevity to test whether
sublethal effects on Þtness are a general phenomenon
associated with Bt maize (Al-Deeb and Wilde 2005,
Murphy et al. 2011).
Few Þeld studies have examined Þtness of western
corn rootworm in a block vs. integrated refuge, or
effects on susceptibility to Bt maize in the next gen-
eration (but see Hibbard et al. 2010a; Murphy et al.
2010, 2011; Zukoff et al. 2012). In this study, survival
and Þtness of western corn rootworm adults were
measured after exposure in the Þeld to pure stands of
Bt maize (event 59122), pure stands of refuge (non-Bt
maize), and an integrated refuge. In addition, the
susceptibility of the offspring obtained from adult
corn rootworm collected from each treatment was
measured by exposing larvae to Bt and non-Bt maize
seedling mats in the laboratory.
Materials and Methods
Experimental Design. Our goals were to 1) com-
pare life history traits and survival of insects exposed
to treatments that included a pure stand of Bt maize,
a pure stand of refuge (non-Bt maize), and integrated
refuges with two rates of neonicotinoid insecticidal
seed treatments, and 2) determine susceptibility to Bt
maize for progeny of insects that emerged from each
of the treatments. Field studies were conducted in
2009 and 2010 at four locations (Ames, IA; Ithaca, NY;
Ithaca, NE; and Urbana, IL). All locations relied on
natural populations of western corn rootworm in both
years. Bt maize used in this study contained the event
DAS-59122-7, which produces the rootworm active Bt
binary toxins Cry34Ab1/Cry35Ab1 (Pioneer brand
hybrid 33W84, relative maturity: 111 d). The refuge
maize used in this research was a near isoline to the Bt
maize hybrid but lacked any rootworm-active Bt tox-
ins (Pioneer brand hybrid 33W80, relative maturity:
111 d). In the pure stand Bt and pure stand refuge
treatments, seeds were treated with Cruiser 250 (thia-
methoxam, 0.25 mg/seed, Syngenta, Basel, Switzer-
land). Both integrated refuge treatments tested in this
study contained 94.4% Bt maize and 5.6% non-Bt
maize. All of the Bt seeds in the integrated refuge
treatments were treated with Cruiser 250, while the
refuge seeds were treated with either Cruiser 250, or
Poncho 1250 (clothianidin, 1.25 mg/seed, Bayer Crop
Science, Research Triangle Park, NC). Thus, a total of
four treatments were measured in this study: 1) pure
stand refuge, 2) pure stand Bt maize, 3) an integrated
refuge in which refuge seeds were treated with
Cruiser 250 seed treatment (hereafter referred to as
integrated refuge 250), and 4) an integrated refuge in
which refuge seeds were treated with Poncho 1250
seed treatment (hereafter referred to as integrated
refuge 1250). For both years, each location consisted
of 20 plots; three were planted with pure stand refuge,
seven with pure stand Bt maize, and Þve with each
integrated refuge 250, and integrated refuge 1250. The
October 2013 PETZOLD-MAXWELL ET AL.: INTEGRATED REFUGES AND CORN ROOTWORM 2197

number of plots differed among treatments because

we anticipated greatest survival to adulthood in pure
stand refuge plots and lowest survival to adulthood in
pure stand Bt plots. Treatments were randomized
within each location. Plots consisted of four rows 3 m
long, with spacing of 0.76 m between rows, and were
separated by 3.7 m of bare ground on all sides. Plots
were kept free of weeds to ensure that rootworm
larvae fed only on maize roots. For the integrated
refuge plots, refuge (non-Bt) seeds were hand planted
into rows within plots. Plots were thinned to 18 plants
per row 2Ð3 wks after planting to achieve the desired
percentage of refuge seeds, and covered with tents
(3.7 by 3.7 m or 3.9 by 3.4 m) 6 Ð7 wks after planting
(Screenhouse 12 and Screenhouse L, Kelty, Boulder,
CO). The sides of the tents were buried to prevent
escape of rootworm adults. When tents were placed
over plots, plants were trimmed to a height of 30 Ð 45
cm, with two to three green leaves left intact. Two
plants were not trimmed so that the developing maize
ears could serve as a food source for adults.
Survival to Adulthood and Life History Measure-
ments. Adult western corn rootworm were collected
from each tent twice per week at each location (every
3Ð 4 d) with an aspirator, and within each location
adults were then pooled by treatment. Western corn
rootworm adults collected in all locations in both years
were shipped to Iowa State University where they
were separated by sex following Hammack and
French (2007), and the number of males and females
per treatment from each location at each collection
date was recorded. In 2009, tents at the Illinois location
suffered substantial damaged from a storm on 4 Au-
gust, and collection at this location was terminated Fig. 1. Number of insects that emerged per tent over
after this date (71.2% [SD ⫽ 18.2] of all beetles col- time for (A) 2009 and (B) 2010, averaged over the four
lected per tent in Illinois had emerged by this date in locations. Box plots represent the time over which insects
2010). For each combination of treatment by location were collected from the integrated refuge treatments for use
by year, mated pairs were established during the pe- in mated pairs. Within box plots, the solid line shows median
day of collection, boxes capture the 25th to 75th percentiles,
riod of peak adult emergence, and data were collected
whiskers indicate the 10th and 90th percentiles, and dots
on adult life span, fecundity, and size (measured as show outliers beyond the 5th and 95th percentile.
width of the head capsule between the outer edges of
the eyes). Collecting adults for mated pairs during
peak emergence enabled a sample to be collected pairs for each treatment within location were estab-
from the integrated refuge that coincided with emer- lished, with an average (⫾SD) per location of 20.3
gence of adults from both the pure stand Bt and pure (⫾2.9) pairs for the pure stand refuge treatment, 16.8
stand refuge treatments (Fig. 1). Thus, the adults used (⫾7.9) for the integrated refuge 250 treatment, 17.5
to generate mated pairs for the integrated refuge treat- (⫾7.9) for the integrated refuge 1250 treatment, and
ments provide a representative ratio of Bt-selected 14.1 (⫾6.1) for the pure stand Bt treatment. The total
and refuge insects, and are not biased toward only number of mated pairs established across both years
refuge insects or only Bt-selected insects. and all four locations was as follows: pure stand refuge
During each year, insects (within location and maize ⫽ 163, integrated refuge 250 ⫽ 134, integrated
treatment) were held in screened cages (18 by 18 by refuge 1250 ⫽ 140, and pure stand Bt maize ⫽ 113.
18 cm; Megaview Science, Taichung, Taiwan) in an Mated pairs were established by placing one male
environmental chamber (25⬚C and a photoperiod of and one female in a plastic 475-ml container (Del-Pak,
16:8 [L:D] h) for 1 wk after collection from the Þeld, Reynolds, Richmond, VA) covered with a modiÞed lid
after which mated pairs were established. Insects were in which the center had been removed and replaced
fed maize ear from the same hybrids they consumed with a circular piece of mesh organza fabric (diame-
as larvae (e.g., those insects emerging from pure stand ter ⫽ 6 cm) to allow for ventilation. Each container
Bt plots were fed only Bt maize ear), and 1.5% agar received maize ear (kernels, and several strands of silk
solid was provided as a source of moisture. Maize ear ⬇2 cm long) corresponding to each respective treat-
was obtained from small maize plots established close ment, 1.5% agar solid as a source of water, and an
to Ames, IA. During each year, a maximum of 26 mated oviposition substrate consisting of moistened Þnely
2198 JOURNAL OF ECONOMIC ENTOMOLOGY
sieved Þeld soil (⬍180 ␮m) in a petri dish (diameter ⫽
3.5 cm) covered with an arched piece of aluminum
foil. Mated pairs were held in incubators (25⬚C and a
photoperiod of 16:8 [L:D] h) until both individuals
died. Maize and agar were changed every 3Ð 4 d, and
oviposition dishes were moistened and replaced as
needed (every 3Ð10 d). Cups were checked every 3Ð 4
d for dead insects, which were placed in 95% ethanol,
and total life span was recorded. After the female from
a mated pair died, eggs from that female were washed
from the soil using a screen with 250-␮m openings and
enumerated using a dissecting microscope (MZ6,
Leica Microsystems, Wetzlar, Germany) and a count-
ing grid (grid size: 0.5 by 0.5 cm). Digital images of
each dead insect were captured, and head capsule
width was measured using a dissecting microscope and
accompanying image analysis software (Motic Images
Inc., British Columbia, Canada).
Susceptibility of Progeny to Bt Maize. Each year,
any insects not used to produce mated pairs for life
history measurements were placed in mass rearing
cages according to treatment within location. The eggs
collected from these adults were assayed for suscep-
tibility to Bt maize with the exception of eggs collected
from the Ames site in 2010, which experienced severe
ßooding and thus few larvae survived to adulthood and
insufÞcient eggs were collected. Insects in mass rear-
ing cages were given artiÞcial diet (F9768B-M, Bio
Serv, NJ), 1.5% agar solid, and maize leaves, as well as
an oviposition dish Þlled with moistened Þeld soil that
was changed weekly. Eggs were washed from these
dishes using a 60-␮m mesh screen and pooled by
treatment within location and year. Eggs were placed
in 45-ml plastic cups (Solo Cup Company, Lake For-
est, IL) Þlled with moistened Þeld soil sieved to a
particle size of ⬍180 ␮m, and covered with lids con-
taining four small holes. Cups were placed in sealed
plastic bags, and held at 7⬚C for at least 5 mo to break
diapause.
Eggs from each treatment within a location were
used to measure offspring susceptibility to Bt maize
event DAS-59122-7 (hereafter referred as 59122) fol-
lowing the sublethal seedling assay described in No-
watzki et al. (2008). Brießy, larvae were exposed to
either Bt (59122) or non-Bt maize seedlings by placing
eggs (⬇5Ð7 d before hatch) in plastic deli containers
(23 by 28 by 9 cm, Pactiv Corp., Lake Forest, IL)
containing 150 kernels of the appropriate seed, 200 ml
of a 1% thiophanate-methyl fungicide solution (42.25%
ai) (3336 F Turf and Ornamental Systemic Fungicide;
Cleary Chemical, Dayton, NJ), and 1,000 g Metro-Mix
200 plant growth media (ScottsÐSierra Horticultural
Products Company, Marysville, OH). A replicate con-
sisted of one container each of non-Bt maize and Bt
(59122) maize, and based on availability of eggs, one
to Þve replicates were established for each treatment
within a location and year. Containers were placed in
an incubator (25⬚C, 65% relative humidity [RH], and
a photoperiod of 14:10 [L:D] h). For all treatments
tested, a subsample of 50 Ð200 eggs (depending on egg
availability) was held in a petri dish under the same
Vol. 106, no. 5
conditions as containers to monitor initial egg hatch,
and to calculate egg viability.
Larvae were exposed to maize seedlings for 17 d
after initial egg hatch. This period of time was chosen
to maximize any developmental differences of larvae
on Bt vs. non-Bt maize (Nowatzki et al. 2008). Seedling
mats were then placed onto Berlese funnels for 4 d,
and larvae were extracted into vials containing 85%
ethanol. The total number of larvae extracted per
container was counted. For the Þrst year of the study,
because egg hatch tests were unsuccessful, proportion
survival in each replicate was calculated as the number
of larvae extracted divided by the number of eggs
placed in each container. During the second year of
the study, the number of eggs placed in a container
was Þrst multiplied by the proportion of viable eggs to
obtain number of viable eggs, and proportion survival
was calculated as the number of larvae extracted di-
vided by the number of viable eggs. A random sample
of 50 larvae per container was scored for instar using
a dissecting microscope (MZ6, Leica Microsystems)
and accompanying image analysis software (Motic
Images Inc.), according to the methods of Hammack
et al. (2003). The percentage of Þrst, second, and third
instars was calculated for each replicate.
Data Analysis. All analyses were conducted in SAS
Enterprise Guide 4.2 (SAS Institute 2009). Data from
all locations and both years were tested in each anal-
ysis, except in the analysis of susceptibility of progeny
to Bt maize, which excluded the Ames 2010 site be-
cause of lack of eggs (see above). For all data analyzed
with a mixed-model analysis of variance (ANOVA),
random effects were tested with a log-likelihood ratio
statistic (⫺2 RES log likelihood in PROC MIXED and
PROC GLIMMIX) based on a one-tailed ␹2 test as-
suming 1 df (Littell et al. 1996) and removed from the
model to increase statistical power when these factors
were not signiÞcant at a level of ␣ ⬍ 0.25 (Quinn and
Keough 2002).
Survival to Adulthood and Life History Measure-
ments. Data on the number of insects that emerged,
the Julian day that insects emerged, insect size (head
capsule width), and adult longevity (life span) were
analyzed with a mixed-model ANOVA using PROC
MIXED based on a split-plot design. Fixed effects in
the model were sex, treatment, and their interaction,
where treatment was analyzed as the whole plot factor
and sex as the split-plot factor. Random factors in-
cluded year, location, location crossed with year, the
interaction of treatment with each of these three ran-
dom factors, and sex crossed with treatment by year by
location. When signiÞcant effects were present, pair-
wise comparisons were conducted based on the TukeyÐ
Kramer method (PDIFF in PROC MIXED). Data on
number of insects that emerged were log transformed
to ensure normality of the residuals.
At each location during each year, the number of
insects expected to emerge from the integrated refuge
treatments was calculated based on the number of
insects that emerged per plant in the pure stand treat-
ments. These expected values were compared with
actual emergence from the integrated refuge treat-
October 2013 PETZOLD-MAXWELL ET AL.: INTEGRATED REFUGES AND CORN ROOTWORM 2199

ments to test the hypothesis that emergence in the Table 1. ANOVA for western corn rootworm life history mea-
integrated refuge differed from the expected emer- surements
gence. First, the number of insects that emerged per
Source dfa F P
plant from the pure stand Bt and the pure stand refuge
tents was calculated for each location within a year. Survival to adulthood
Treatment 3, 3 43.57 0.0057
Because our plots contained 72 plants, 5.6% of which Sex 1, 28 11.19 0.0024
were refuge, there were 68 Bt plants and four refuge Treatment ⫻ sex 3, 28 0.22 0.8824
plants in each integrated refuge plot. The number of Timing of emergence
insects expected to emerge per integrated refuge tent Treatment 3, 3 13.40 0.0304
Sex 1, 28 39.20 ⬍0.0001
was calculated as: (number of insects that emerged Treatment ⫻ sex 3, 28 1.03 0.3948
per Bt plant in pure stands ⫻ 68) ⫹ (number of insects Size
that emerged per refuge plant in pure stands ⫻ 4) for Treatment 3, 3 0.88 0.5369
each location within a year. Expected and actual num- Sex 1, 27 16.36 0.0004
Treatment ⫻ sex 3, 27 1.2 0.3302
ber of insects per tent for each location within a year Longevity
were compared with a paired two-tailed t-test (PROC Treatment 3, 3 1.58 0.3586
TTEST). Sex 1, 27 75.18 ⬍0.0001
Fecundity was analyzed with a mixed-model Treatment ⫻ sex 3, 27 0.12 0.9448
Fecundity
ANOVA (PROC GLIMMIX), with a log link function Treatment 3, 9 0.50 0.6930
and a Poisson distribution. Data were transformed
with the Log(n ⫹ 10) function to ensure normality of a
df: numerator, denominator.
the residuals. Treatment was a Þxed factor in the
analysis. Random factors included year, location, lo-
cation crossed with year, and the interaction of treat- ment and transformed back to the original scale using
ment with each of these random factors. To determine the EXP option. The odds ratio for each of the four
whether shipment of insects inßuenced fecundity, a treatments was as follows: ([odds of obtaining a third
mixed-model ANOVA (PROC GLIMMIX) was used, instar on non-Bt maize/odds of obtaining a Þrst or
with a log link function and a Poisson distribution. second instar on non-Bt maize]/[odds of obtaining a
Data were transformed with the Log(n ⫹ 10) function third instar on Bt maize/odds of obtaining a Þrst or
to ensure normality of the residuals. Treatment, loca- second instar on Bt maize]). A value of 1 indicates no
tion, and the interaction of treatment and location difference in development on Bt vs. non-Bt maize; a
were Þxed factors in the analysis. Random factors value ⬎1 indicates faster development on non-Bt
included year, location crossed with year, and the maize (i.e., higher odds of getting a third instar on
interaction of treatment with each of these random non-Bt maize vs. Bt maize); and a value of ⬍1 indicates
factors. The CONTRAST statement was used to com- faster development on Bt maize. Treatments were
pare fecundity of adults collected in Iowa (study lo- signiÞcantly different if they had nonoverlapping 95%
cation) with those collected from the other locations. CIs.
To determine whether insect density affected fecun-
dity, we tested the correlation between the average
Results
number of adults per tent for the pure stand refuge
treatment and the average egg production per female Survival to Adulthood. Across all locations and both
in the pure stand refuge treatment, using data from all years, there was a signiÞcant effect of treatment on
locations and both years (PROC CORR). survival to adulthood (Table 1); the number of adult
Susceptibility of Progeny to Bt Maize. A partially beetles was signiÞcantly higher in the pure stand ref-
nested mixed model ANOVA (PROC GLIMMIX) was uge than any other treatment (P ⬍ 0.01 for all com-
used to analyze survival and the proportion of third parisons) (Table 1; Figs. 1 and 2A). Relative to pure
instars. Fixed effects in the model were treatment stand refuge, survival was reduced by 88.8% (⫾10.1)
(maize treatment in the Þeld), maize hybrid in seed for integrated refuge 250, 88.5% (⫾9.0) for integrated
mats (Bt and non-Bt), and the interaction of treatment refuge 1250, and 91.2% (⫾9.0) for pure stand Bt maize.
and hybrid. Random factors included year, location, The number of insects emerging from integrated ref-
location crossed with year, the interaction of treat- uge 250, integrated refuge 1250, and pure stand Bt
ment with each of these random effects, replicate plots did not differ (P ⬎ 0.35 for all comparisons). The
nested within location by treatment by year, and the number of insects expected to emerge from the inte-
interaction of replicate by hybrid nested within loca- grated refuge tents (based on the number of insects
tion by treatment by year. Replicate was deÞned as a that emerged per Bt and refuge plant in the pure
pair of Bt and non-Bt seedling mats that were estab- stands) did not differ signiÞcantly from the actual
lished at the same time and used larvae from a single number that emerged (IR 250: t7 ⫽ 0.09, P ⫽ 0.93; IR
treatment in one location within a year. 1250: t7 ⫽ 0.61, P ⫽ 0.56) (Fig. 2B). Overall, signiÞ-
Within each replicate, data on the proportion of cantly more females survived to adulthood. The num-
third instar larvae were used to calculate an odds ratio ber of adults that emerged per tent varied consider-
and 95% conÞdence limits of obtaining a third instar ably among locations each year (Table 2). In 2009,
larva on non-Bt vs. Bt maize. Odds ratios for each survival to adulthood was greatest in Nebraska and
treatment were obtained using the ESTIMATE state- lowest in Iowa (Table 2). In 2010, survival to adult-
2200 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5

Table 2. Number of western corn rootworm adults that

emerged per tent for pure stand refuge, integrated refuge 250 (IR
250), integrated refuge 1250 (IR 1250), and pure stand Bt maize

Treatment Sex Iowa Illinois Nebraska New York

2009
Refuge F 159.0 373.0 416.0 718.0
IR 250 F 11.6 110.6 53.2 26.2
IR 1250 F 12.2 102.4 48.8 20.8
Bt F 6.6 97.3 23.9 2.9
Refuge M 125.7 339.7 883.7 554.7
IR 250 M 6.4 107.4 43.8 19.0
IR 1250 M 9.6 122.8 57.2 16.0
Bt M 4.1 105.4 13.3 4.3
2010
Refuge F 12.3 224.3 686.3 1,141.3
IR 250 F 0.2 32.6 18.2 261.0
IR 1250 F 0.6 22.0 21.3 313.4
Bt F 0.9 15.7 15.9 174.7
Refuge M 10.3 45.3 641.3 676.0
IR 250 M 0.4 5.4 14.8 149.2
IR 1250 M 0.6 3.2 15.8 140.8
Bt M 0.6 2.7 6.1 87.7

hood was greatest in New York and lowest in Iowa

Fig. 2. (A) Number of insects that emerged per tent, (B)
actual and expected number of insects that emerged per tent
for integrated refuge treatments, and (C) Julian date of
emergence for western corn rootworm. In (A) and (B), bar
heights are the sample mean and error bars the SEM. In (C),
solid and dashed lines show median and mean day of emer-
gence, respectively; boxes capture the 25th to 75th percen-
tiles, whiskers indicate the 10th and 90th percentiles, and dots
show outliers beyond the 5th and 95th percentile. Means with
same letters are not signiÞcantly different.
(because of ßooding).
Both sex of beetles and maize treatment signiÞ-
cantly affected time of adult emergence (Table 1; Fig.
2C). Average date of emergence was earlier for males
compared with females for all treatments. Average
date of emergence for insects from pure stand refuge
plots was signiÞcantly earlier than insects from pure
stand Bt plots (P ⫽ 0.03) and integrated refuge 1250
plots (P ⫽ 0.05), and marginally so for integrated
refuge 250 plots (P ⫽ 0.08). However, time of emer-
gence did not differ signiÞcantly among any treatment
containing Bt plants (pure stand or with integrated
refuge) (P ⬎ 0.38 for all comparisons) (Fig. 2C).
Adult Life History Measurements. Head capsule
width varied among locations and years (Table 3), but
across all locations and both years, treatment did not
signiÞcantly affect head capsule width (Table 1).
Overall, head capsule width was greater for females
than males (Fig. 3A), and this effect was signiÞcant
(Table 1). No other signiÞcant differences were de-
tected (Table 1). Similarly, across all locations and
both years, treatment did not signiÞcantly affect lon-
gevity (Table 1). However, females lived signiÞcantly
longer than males (Fig. 3B). At some locations, lon-
gevity was greater for pure stand refuge compared
with either integrated refuge or pure stand Bt treat-
ments (e.g., IL in 2009), and at other locations the
opposite was true (e.g., NE in 2009) (Table 3).
Over all locations and both years, females from the
refuge treatment displayed the highest average egg
production (Fig. 4), but the effect of treatment on
fecundity was not signiÞcant (Table 1). At some lo-
cations, the average number of eggs produced per
female was considerably greater for pure stand refuge
compared with the other treatments (e.g., IL in 2009);
however, fecundity was similar among treatments in
other location by year combinations (e.g., NY in 2009)
(Table 3). Fecundity did not differ between insects
collected from Iowa and insects collected from other
locations (F1,3 ⫽ 0.13; P ⫽ 0.742), indicating that ship-
 Table 3. Life history traits for western corn rootworm

N Head capsule width (mm)a Longevity (d)a Fecundity (no. eggs)a

Treatmentb Sex Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York
2009
October 2013

Refuge F 23 15 25 20 1.18 (0.01) 1.07 (0.02) 0.98 (0.01) 1.08 (0.02) 38.3 (3.6) 44.4 (3.1) 33.8 (3.0) 28.8 (2.6) 125.6 (30.5) 121.8 (45.8) 83.8 (20.1) 56.4 (14.5)
IR 250 F 13 12 25 24 1.18 (0.02) 1.11 (0.02) 1.05 (0.02) 1.12 (0.01) 39.2 (5.7) 33.5 (4.5) 36.0 (3.2) 36.4 (3.8) 45.4 (9.1) 50.5 (15.0) 61.2 (11.4) 32.7 (10.1)
IR 1250 F 18 22 26 22 1.17 (0.02) 1.09 (0.02) 1.01 (0.01) 1.12 (0.01) 43.5 (3.9) 36.6 (3.0) 34.5 (3.4) 33.2 (1.7) 38.3 (12.7) 28.4 (7.5) 36.7 (9.9) 91.1 (14.3)
Bt F 8 22 14 20 1.09 (0.03) 1.07 (0.01) 1.07 (0.02) 1.05 (0.01) 34.6 (8.6) 33.7 (3.8) 37.8 (5.9) 27.6 (2.3) 98.1 (53.2) 41.3 (13.9) 98.1 (25.6) 61.1 (16.2)
Refuge M 23 15 25 20 1.16 (0.01) 1.07 (0.02) 1.00 (0.02) 1.08 (0.01) 30.8 (3.7) 24.3 (4.6) 24.7 (4.0) 19.6 (2.3) Ñ Ñ Ñ Ñ
IR 250 M 13 12 25 24 1.11 (0.02) 1.08 (0.02) 1.03 (0.01) 1.10 (0.01) 28.2 (4.5) 18.8 (5.4) 24.5 (3.0) 23.1 (1.9) Ñ Ñ Ñ Ñ
IR 1250 M 18 22 26 22 1.12 (0.01) 1.07 (0.02) 0.97 (0.01) 1.08 (0.02) 31.7 (2.6) 24.3 (3.0) 29.0 (3.4) 26.3 (1.6) Ñ Ñ Ñ Ñ
Bt M 8 22 14 20 1.14 (0.03) 1.06 (0.02) 1.02 (0.01) 1.02 (0.01) 31.1 (6.2) 19.4 (2.9) 25.7 (5.9) 24.5 (2.3) Ñ Ñ Ñ Ñ
2010
Refuge F 20 20 20 20 1.22 (0.02) 1.25 (0.02) 1.20 (0.02) 1.28 (0.02) 42.7 (3.2) 41.1 (3.7) 41.1 (4.8) 41.6 (4.2) 38.2 (11.5) 55.1 (20.5) 74.4 (17.6) 27.6 (10.2)
IR 250 F 0 21 18 20 Ñ 1.23 (0.02) 1.31 (0.05) 1.28 (0.02) Ñ 33.4 (3.1) 43.7 (2.6) 46.5 (4.2) Ñ 32.3 (15.3) 49.1 (15.2) 20.8 (8.9)
IR 1250 F 1 11 20 20 1.17 1.20 (0.02) 1.22 (0.02) 1.32 (0.04) 39.0 38.3 (4.2) 42.9 (3.3) 43.1 (3.5) 0.0 (0.0) 26.2 (15.1) 56.7 (18.2) 20.9 (7.8)
Bt F 4 13 13 19 1.35 (0.06) 1.24 (0.04) 1.22 (0.02) 1.25 (0.01) 32.7 (9.3) 32.9 (4.1) 41.1 (4.4) 42.5 (2.8) 52.5 (30.8) 36.0 (12.1) 33.5 (12.6) 14.3 (6.7)
Refuge M 20 20 20 20 1.23 (0.02) 1.20 (0.02) 1.19 (0.01) 1.27 (0.02) 38.6 (3.6) 34.0 (3.1) 36.9 (2.5) 33.9 (4.0) Ñ Ñ Ñ Ñ
IR 250 M 0 21 18 20 Ñ 1.20 (0.02) 1.18 (0.02) 1.26 (0.01) Ñ 34.9 (2.0) 36.9 (3.2) 40.1 (2.2) Ñ Ñ Ñ Ñ
IR 1250 M 1 11 20 20 1.15 1.23 (0.04) 1.14 (0.02) 1.23 (0.01) 39.0 34.1 (5.0) 37.9 (2.6) 37.6 (4.0) Ñ Ñ Ñ Ñ
Bt M 4 13 13 19 1.13 (0.03) 1.20 (0.02) 1.23 (0.07) 1.21 (0.01) 37.5 (4.7) 32.8 (3.5) 30.2 (3.2) 34.0 (2.2) Ñ Ñ Ñ Ñ

a
Numbers in parentheses show SE.
b
Insects emerged from pure stand refuge, integrated refuge 250 (IR 250), integrated refuge 1250 (IR 1250), and pure stand Bt maize plots at four locations.

0.05).

⫺0.60; P ⫽ 0.151).

different (P ⬍ 0.05).
PETZOLD-MAXWELL ET AL.: INTEGRATED REFUGES AND CORN ROOTWORM

250 (IR 250), integrated refuge 1250 (IR 1250), and pure

refuge, integrated refuge 250 (IR 250), integrated refuge 1250

Þeld), and there was no interaction between treat-
there was no signiÞcant correlation between insect

ments (maize type from which parents emerged in the

vival did not differ signiÞcantly among the four treat-
Susceptibility of Progeny to Bt Maize. Larval sur-
density and fecundity in the refuge treatment (r7 ⫽
ping insects did not inßuence fecundity. In addition,

and both years. Bar heights are the sample mean and error
Fig. 4. Fecundity of insects emerging from pure stand
heights are the sample mean and error bars the SEM. Aster-

(IR 1250), and pure stand Bt maize plots, over all locations
isks indicate signiÞcant differences within a treatment (P ⬍
insects emerging from pure stand refuge, integrated refuge
Fig. 3. (A) Head capsule width and (B) longevity of

stand Bt maize plots, over all locations and both years. Bar

bars the SEM. Means with same letters are not signiÞcantly
2201
2202 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 5

Table 5. Odds ratio of obtaining a third instar on non-Bt maize

seedling mats over obtaining a third instar on Bt maize seedling mats

Treatment Odds ratio Lower 95% CL Upper 95% CL

Refugea 4.59 2.48 8.52
IR 250 6.43 2.97 13.90
IR 1250 4.62 2.08 10.26
Bt 4.41 1.88 10.32

a
Larvae were reared on seedling mats of non-Bt or Bt maize, and
were offspring of western corn rootworm that emerged from pure
stand refuge, integrated refuge 250, integrated refuge 1250, and pure
stand Bt maize plots.

type (Table 4). Furthermore, odds ratios did not differ

signiÞcantly among treatments (Table 5). These re-
sults indicate that survivorship and development were
not affected by the treatment from which adults
emerged during the previous generation.

Fig. 5. (A) Larval survival from seedling mats containing
either Bt (event DAS-59122-7) or non-Bt seed, and (B)
proportion of third instar larvae recovered. Larvae studied
were the progeny of adults that emerged from pure stand
refuge, integrated refuge 250 (IR 250), integrated refuge 1250
(IR 1250), and pure stand Bt maize plots. The number of
replicates for each treatment was as follows: pure stand
refuge ⫽ 16, integrated refuge 250 ⫽ 11, integrated refuge
1250 ⫽ 10, and pure stand Bt maize ⫽ 9. Bar heights are the
sample mean and error bars the SEM. Asterisks indicate
signiÞcant differences within a treatment (P ⬍ 0.05).
ment and the maize type to which larvae were exposed
in containers (Bt or non-Bt) (Fig. 5A; Table 4). For all
treatments, larval development was faster on non-Bt
seedling mats, as indicated by a signiÞcantly higher
proportion of third instar larvae recovered on non-Bt
seedling mats compared with Bt seedling mats (Fig.
5B; Table 4), and by odds ratios ⬎1 for each treatment
(Table 5). This difference in recovery of third instar
larvae on the two types of maize did not differ signif-
icantly among treatments, as indicated by the lack of
a signiÞcant interaction between treatment and maize
Discussion
In this study, Bt maize producing the Cry34/35Ab1
insecticidal protein signiÞcantly reduced survival of
western corn rootworm to adulthood and signiÞcantly
delayed the time of adult emergence. These effects
were present for both the pure stand Bt maize and the
integrated refuge treatments. In some location by year
combinations, longevity, head capsule width, and fe-
cundity were greater for pure stand refuge compared
with the integrated refuge and pure stand Bt maize
treatments. However, across all years and locations,
exposure to Bt maize did not have signiÞcant effects
on western corn rootworm adult longevity, size, and
fecundity. Furthermore, the progeny of insects col-
lected from pure stand refuge, pure stand Bt maize,
and integrated refuge did not display signiÞcant dif-
ferences in susceptibility to Bt maize in the next gen-
eration.
This study showed that the main effect of Bt maize
producing the Cry34/35Ab1 insecticidal protein on
western corn rootworm was a reduction in survival to
adulthood (Figs. 1 and 2A). The reduction in survival
observed for the integrated refuge and pure stand Bt
treatments relative to the pure stand refuge treatment
(88.5Ð91.2% relative to pure stand refuge; Fig. 2A) is
similar to what has been reported in other studies.
Storer et al. (2006) reported a 94.4 Ð96% reduction in
adult survival on maize producing Cry34/35Ab1 com-
pared with non-Bt maize (see Hibbard et al. 2010b).
Hibbard et al. (2010a) reported mortality values in the
Þeld from maize producing mCry3A (event MIR604)
that ranged from 87.54 to 99.83%, and similar decreases

Table 4. ANOVA for larvae exposed to seedling mats containing either Bt (event 59122) or non-Bt maize

Proportion recovered Proportion of third instars

Source
dfa F P dfa F P
Field treatment 3, 3 0.31 0.8172 3, 3 1.48 0.3769
Maize type in seedling mat 1, 42 3.70 0.0613 1, 42 71.32 ⬍0.0001
Field treatment ⫻ maize type 3, 42 1.18 0.3273 3, 42 0.21 0.8888

a
df: numerator, denominator.
October 2013 PETZOLD-MAXWELL ET AL.: INTEGRATED REFUGES AND CORN ROOTWORM
in survival have been reported for Bt maize producing
Cry3Bb1 (event MON863), mCry3A (event MIR604),
eCry3.1Ab (event 5307), and mCry3A pyramided with
eCry3.1Ab (Al-Deeb and Wilde 2005; Hibbard et al.
2009, 2011; Frank et al. 2011). However, because this
study relied on natural rootworm populations, we do
not know whether density-dependent mortality oc-
curred in the pure stand refuge treatment, which may
result in mortality on Bt maize to be underestimated
(Hibbard et al. 2010b).
Survival in the integrated refuge treatments was not
signiÞcantly greater than survival in the pure stand Bt
treatment, contradicting the expectation that the pres-
ence of refuge plants should result in increased sur-
vival in these treatments compared with pure stands of
Bt. However, because refuge plants only represented
4 of the 72 plants in integrated refuge treatments, it is
likely that a statistically signiÞcant difference in sur-
vival compared with pure Bt stands may be difÞcult to
obtain. The actual number of rootworm that emerged
from integrated refuge plots did not differ signiÞcantly
from the expected number (Fig. 2B). It is unclear
whether the relative proportion of insects that
emerged from Bt and refuge plants in the integrated
refuge treatments differed from those in pure stands
because we did not measure emergence from individ-
ual plants. Murphy et al. (2010) found that the pro-
portion of beetles emerging from refuge plants was
signiÞcantly less in an integrated refuge than in a pure
stand refuge; however, the proportion that emerged
from Bt plants was greater. This Þnding is similar to a
Þnding by Zukoff et al. (2012), who observed that
signiÞcantly more western corn rootworm beetles
could emerged from Bt maize plants (producing both
Cry34/35Ab1 and Cry3Bb1) when these plants were
surrounded by refuge plants on both sides as com-
pared with Bt plants surrounded by Bt plants, or refuge
plants surrounded by refuge plants. The authors pos-
tulated that movement from refuge plants to Bt plants
by larvae could produce additional susceptible insects
emerging from within the integrated refuge Þeld, be-
cause a large proportion of initial larval development
occurred on refuge plants (Zukoff et al. 2012).
To the extent that movement between refuge and
Bt plants imposes selection for resistance, thereby
decreasing the number of unselected refuge individ-
uals, resistance may evolve more quickly. By contrast,
increased survival of susceptible insects that move
from non-Bt maize to Bt maize, compared with those
that feed only on Bt maize, may delay resistance evo-
lution (Pan et al. 2011). Meihls et al. (2008) found that
progeny of larvae that were Þrst fed Cry3Bb1-produc-
ing Bt maize but Þnished their development on non-Bt
refuge plants did not have higher survival on Cry3Bb1
maize in a subsequent generation than progeny of
insects fed only non-Bt maize. However, when larvae
were fed non-Bt refuge maize for 1 wk and then fed
Cry3Bb1 maize for the rest of larval development, the
subsequent progeny did display increased survival on
Cry3Bb1 (Meihls et al. 2008). Additional studies will
be necessary to gain a more complete understanding
of how larval movement between Bt and non-Bt maize
2203
plants affects survival to adulthood and resistance evo-
lution.
In addition to imposing mortality, Cry34/35Ab1
maize also delayed adult emergence. Random mating
of insects emerging from Bt and refuge plants is im-
portant for the success of the refuge strategy. Tem-
poral asynchrony in emergence between insects from
Bt and refuge plants may reduce random mating and
diminish the extent to which refuges delay resistance
(Gould 1998). Adult emergence of western corn root-
worm on pure stands of Bt maize is generally later than
on pure stands of non-Bt maize (Storer et al. 2006,
Binning et al. 2010). Murphy et al. (2010) measured
time of emergence for western corn rootworm from Bt
maize plants (producing Cry3Bb1) and refuge plants
in Þeld plots planted as integrated refuges, block ref-
uges, and strip refuges, and found that insects from
refuge plants emerged more synchronously with in-
sects from Bt plants for integrated refuges compared
with blocks. Although we did not measure emergence
timing from individual refuge plants in our integrated
refuge treatments, our results also support more syn-
chronous emergence of males and females in inte-
grated refuges compared with pure stand refuge maize
(Fig. 2C). An integrated refuge should enhance re-
sistance management by increasing the probability of
random mating because of greater spatial and tempo-
ral proximity of insects emerging from Bt and refuge
plants (Gould 1998, Storer 2003).
Results from sublethal seedling assays indicate that
the treatment from which adults were collected in the
Þeld did not signiÞcantly affect the susceptibility of
their progeny to Bt maize (59122) in the next gener-
ation (Fig. 5; Table 4). Previous studies have shown
that populations with decreased susceptibility to Bt
maize producing Cry34/35Ab1 have faster larval de-
velopment on maize producing Cry34/35Ab1 com-
pared with susceptible populations (Nowatzki et al.
2008). The progeny of insects collected from all of our
treatments (pure stand refuge, pure stand Bt maize,
integrated refuge 250 and 1250 treatments) had odds
ratios that were ⬎1, and did not differ signiÞcantly
from each other (Table 5). In addition, there was no
interactive effect of treatment (maize type from
which parents emerged in the Þeld) and maize type
used in the assay (Bt or non-Bt) on the proportion of
third instars recovered (Table 4). This suggests that
populations collected from treatments containing Bt
maize producing Cry34/35Ab1, either in a pure stand
or in an integrated refuge, did not exhibit a decrease
in susceptibility to Bt maize (59122) after one gener-
ation of selection in the Þeld. Hibbard et al. (2010a)
tested whether western corn rootworm that experi-
enced one generation of exposure to mCry3A maize in
the Þeld exhibited increased survivorship on mCry3A
maize in the greenhouse during the next generation.
Similar to our study, Hibbard et al. (2010a) found that
there was no difference in survivorship on Bt maize for
progeny of insects that emerged from either Bt or
refuge maize in the Þeld.
There were no differences in longevity or size
among adults from any of the treatments tested (Table
2204 JOURNAL OF ECONOMIC ENTOMOLOGY
1; Fig. 3). There also was no signiÞcant difference in
fecundity among treatments over locations and years
(Table 1; Fig. 4). Pure stand refuge insects had higher
fecundity than the other treatments in several year-
by-location combinations, but in others, fecundity was
higher for pure stand Bt insects or was similar among
treatments (Table 3). The insecticidal seed treatments
thiamethoxam (0.25 mg a.i. [active ingredient] per
seed) and clothianidin (1.25 mg a.i. per seed) ap-
plied to refuge seeds in the integrated refuge did not
differ in their effects on adult fecundity, longevity,
size, emergence, or larval susceptibility to Bt maize
(Figs. 1Ð5), possibly because they were applied to a
small percentage of seeds (refuge seed equaled 5.6%
of the total seed in the integrated refuge treat-
ments).
Fecundity was lower in this study than what has
been found in other studies, which report ranges in
egg production from 125 to 441 eggs (Elliott et al.
1990), 372 to 418 eggs (Ball 1957), 385 to 516 eggs
(Fisher et al. 1991), 440 to 671 eggs (Li et al. 2009), or
an average of 353 eggs per female (Toepfer and Kuhl-
mann 2006). These studies use a variety of oviposition
substrates, and many provide artiÞcial western corn
rootworm diet, silk, and leaves as a food source. It is
possible that the small oviposition dishes used in this
study were not optimal for egg collection, or that
supplying artiÞcial diet or other forms of maize in
addition to the maize ear and small amounts of silk
would have resulted in greater egg production.
The effects of several Bt proteins on various life
history parameters of western corn rootworm, includ-
ing head capsule width, weight, longevity, and fecun-
dity, have been examined in a number of studies
(Storer et al. 2006, Oyediran et al. 2007, El Khishen et
al. 2009, Murphy et al. 2011, Zukoff et al. 2012). Studies
examining fecundity and longevity have shown neg-
ative effects of Bt maize, but these effects were often
weak and nonsigniÞcant (Wilson 2003, Al-Deeb and
Wilde 2005, Meissle et al. 2011). For example, in Þeld
studies comparing these life history traits for western
corn rootworm emerging from Bt maize (producing
Cry3Bb1) and non-Bt maize, Wilson (2003) found a
reduction in fecundity but no difference in longevity.
By contrast, Al-Deeb and Wilde (2005) found no ef-
fect on fecundity but some evidence for decreased
longevity in males on Bt maize producing Cry3Bb1. In
this study, there was a trend for smaller head capsule
widths of males (but not females) in Bt treatments
(Fig. 3A), although the interaction of sex and treat-
ment was not signiÞcant. In some cases adult head
capsule width and weight are not affected by larval
feeding on Bt maize (Storer et al. 2006, Frank et al.
2011, Meihls et al. 2011, Zukoff et al. 2012). In other
cases, larval feeding on Bt maize can reduce the size
of adults, as demonstrated by smaller head capsule
widths or a reduction in adult mass (Binning et al.
2010, Murphy et al. 2011). Head capsule width is com-
monly used as a biological Þtness parameter of root-
worm adults (Murphy et al. 2011), but it can be in-
ßuenced by density-dependent effects (Branson and
Sutter 1985). Because adult abundance was signiÞ-
Vol. 106, no. 5
cantly greater in the pure stand refuge treatment,
density-dependent effects could have confounded dif-
ferences in head capsule width, and other life history
parameters, which may have otherwise arisen be-
tween the pure stand refuge treatment and the other
treatments.
Effects of refuge conÞgurations on resistance evo-
lution can be inßuenced by the biology of the pest
species. For example, structured refuges may be more
appropriate than integrated refuges for European
corn borer (Ostrinia nubilalis Hubner) (Davis and
Onstad 2000, Siegfried and Hellmich 2012). European
corn borer larvae tend to disperse within rows rather
than between rows (Ross and Ostlie 1990), and thus
will likely encounter the same type of plant in strip or
block refuges. Larval movement of European corn
borer among Bt and refuge plants is a primary concern
in integrated refuges, especially because Bt maize is
considered a high dose for this species. If larvae with
an allele for resistance move from a Bt plant to a
non-Bt plant and ingest sublethal amounts of Bt toxin,
this could lead to a Þtness advantage for partially
resistant insects that otherwise could not complete
development on a Bt plant, thereby undermining re-
sistance management (Onstad and Gould 1998, Sieg-
fried and Hellmich 2012). By contrast, for western
corn rootworm, Bt events are not high dose and some
heterozygous individuals, as well as susceptible geno-
types, are expected to survive on Bt maize. For ex-
ample, Binning et al. (2010) found that survival of
susceptible western corn rootworm to adulthood on
Bt maize producing Cry34/35Ab1 (59122) was as high
as 5%, and for insects Þrst exposed to non-Bt corn and
then transferred to 59122 during the third instar, sur-
vival was 50%. The lack of a high dose for 59122 could
diminish the effects of larval movement on resistance
evolution (Tabashnik 1994). In addition, although in-
tegrated refuges increase random mating of adults
compared with structured refuges, this beneÞt may
depend on pest dispersal. In the case of European corn
borer, which often disperse ⬎0.8 km before mating
(Hunt et al. 2001, Showers et al. 2001), a block refuge
may be effective. By contrast, although long-distance
dispersal is possible, western corn rootworm typically
disperse ⬍40 m per day (Spencer et al. 2003, 2009),
and consequently, an integrated refuge may be most
effective at facilitating random mating for this species
(Pan et al. 2011).
To date, few studies have examined the effects of
integrated refuge on western corn rootworm survival
and life history traits in the Þeld (Murphy et al. 2010,
2011). Results from simulation models suggest that
integrated refuge can delay Bt resistance by corn root-
worm longer than block refuges (Pan et al. 2011).
Future work should examine the selection pressure of
different refuge conÞgurations on western corn root-
worm over several generations. A more thorough un-
derstanding of how natural selection on western corn
rootworm is affected by refuge deployment and insect
behavior will aid in the development of better IRM
strategies.
October 2013 PETZOLD-MAXWELL ET AL.: INTEGRATED REFUGES AND CORN ROOTWORM
Acknowledgments
We thank Jeremy Bolles, Molly Wilmes, and Patrick We-
ber for help in the laboratory and Þeld. This work was sup-
ported by DuPont-Pioneer.
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expressing the Cry3Bb1 toxin on western corn rootworm
(Coleoptera: Chrysomelidae) biology. J. Kans. Entomol.
Soc. 78: 142Ð152.
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Received 20 February 2013; accepted 23 June 2013.