10-2013
Applying an Integrated Refuge to Manage Western
Corn Rootworm (Coleoptera: Chrysomelidae):
Effects on Survival, Fitness, and Selection Pressure
Jennifer L. Petzold-Maxwell
Iowa State University, [email protected]
Analiza P. Alves
DuPont Pioneer
Ronald E. Estes
University of Illinois at Urbana-Champaign
Michael E. Gray
University of Illinois at Urbana-Champaign
Lance J. Meinke
University of Nebraska-Lincoln
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Authors
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
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
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. 2013.
Published By: Entomological Society of America
URL:
http://www.bioone.org/doi/full/10.1603/EC13088
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INSECTICIDERESISTANCE ANDRESISTANCEMANAGEMENT
Applying an Integrated Refuge to Manage Western Corn Rootworm
(Coleoptera: Chrysomelidae): Effects on Survival, Fitness, and
Selection Pressure
JENNIFER L. PETZOLD-MAXWELL,1,2ANALIZA P. ALVES,3RONALD E. ESTES,4
MICHAEL E. GRAY,4LANCE J. MEINKE,5ELSON J. SHIELDS,6STEPHEN D. THOMPSON,3
NICHOLAS A. TINSLEY,4
ANDAARON 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-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 homozyhomozy-gous 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 maize producing Cry3Bb1, mCry3a, and Cry34Ab1/ Cry35Ab1) fail to meet the high-dose condition of imposing 99.99% mortality (Gassmann 2012, Devos et
1Department of Entomology, Iowa State University, 13 Insectary, Ames, IA 50011.
2Corresponding author, e-mail: [email protected]. 3DuPont Pioneer, 7300 NW 62nd Ave., P.O. Box 1004, Johnston, IA 50131.
4Department of Crop Sciences, University of Illinois, 1102 S. Good-win, Urbana, IL 61801.
5Department of Entomology, University of Nebraska, 109B Ento-mology Hall, Lincoln, NE 68583.
6Department of Entomology, Cornell University, 2130 Comstock Hall, 129 Garden Ave, Ithaca NY 14853.
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
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
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) 6Ð7 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 30Ð45 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 3Ð4 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 after this date (71.2% [SD⫽ 18.2] of all beetles col-lected per tent in Illinois had emerged by this date in 2010). For each combination of treatment by location by year, mated pairs were established during the pe-riod of peak adult emergence, and data were collected on adult life span, fecundity, and size (measured as 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 from the integrated refuge that coincided with emer-gence of adults from both the pure stand Bt and pure stand refuge treatments (Fig. 1). Thus, the adults used to generate mated pairs for the integrated refuge treat-ments provide a representative ratio of Bt-selected and refuge insects, and are not biased toward only refuge insects or only Bt-selected insects.
During each year, insects (within location and treatment) were held in screened cages (18 by 18 by 18 cm; Megaview Science, Taichung, Taiwan) in an environmental chamber (25⬚C and a photoperiod of 16:8 [L:D] h) for 1 wk after collection from the Þeld, after which mated pairs were established. Insects were fed maize ear from the same hybrids they consumed as larvae (e.g., those insects emerging from pure stand Bt plots were fed only Bt maize ear), and 1.5% agar solid was provided as a source of moisture. Maize ear was obtained from small maize plots established close to Ames, IA. During each year, a maximum of 26 mated
pairs for each treatment within location were estab-lished, with an average (⫾SD) per location of 20.3 (⫾2.9) pairs for the pure stand refuge treatment, 16.8 (⫾7.9) for the integrated refuge 250 treatment, 17.5 (⫾7.9) for the integrated refuge 1250 treatment, and 14.1 (⫾6.1) for the pure stand Bt treatment. The total number of mated pairs established across both years and all four locations was as follows: pure stand refuge maize⫽ 163, integrated refuge 250 ⫽ 134, integrated refuge 1250⫽ 140, and pure stand Bt maize ⫽ 113.
Mated pairs were established by placing one male and one female in a plastic 475-ml container (Del-Pak, Reynolds, Richmond, VA) covered with a modiÞed lid in which the center had been removed and replaced with a circular piece of mesh organza fabric (diame-ter⫽ 6 cm) to allow for ventilation. Each container received maize ear (kernels, and several strands of silk ⬇2 cm long) corresponding to each respective treat-ment, 1.5% agar solid as a source of water, and an oviposition substrate consisting of moistened Þnely
Fig. 1. Number of insects that emerged per tent over time for (A) 2009 and (B) 2010, averaged over the four locations. Box plots represent the time over which insects were collected from the integrated refuge treatments for use in mated pairs. Within box plots, the solid line shows median day of collection, boxes capture the 25th to 75th percentiles, whiskers indicate the 10th and 90th percentiles, and dots show outliers beyond the 5th and 95th percentile.
sieved Þeld soil (⬍180m) 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 3Ð4 d, and oviposition dishes were moistened and replaced as needed (every 3Ð10 d). Cups were checked every 3Ð4 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⬍180m, 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 50Ð200 eggs (depending on egg availability) was held in a petri dish under the same
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-tailed2test
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-ments to test the hypothesis that emergence in the integrated refuge differed from the expected emer-gence. First, the number of insects that emerged per plant from the pure stand Bt and the pure stand refuge tents was calculated for each location within a year. Because our plots contained 72 plants, 5.6% of which were refuge, there were 68 Bt plants and four refuge plants in each integrated refuge plot. The number of insects expected to emerge per integrated refuge tent was calculated as: (number of insects that emerged per Bt plant in pure stands⫻ 68) ⫹ (number of insects that emerged per refuge plant in pure stands⫻ 4) for each location within a year. Expected and actual num-ber of insects per tent for each location within a year were compared with a paired two-tailed t-test (PROC TTEST).
Fecundity was analyzed with a mixed-model ANOVA (PROC GLIMMIX), with a log link function and a Poisson distribution. Data were transformed with the Log(n⫹ 10)function to ensure normality of
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 with each of these random factors. To determine whether shipment of insects inßuenced fecundity, a mixed-model ANOVA (PROC GLIMMIX) was used, with a log link function and a Poisson distribution. Data were transformed with the Log(n⫹ 10)function
to ensure normality of the residuals. Treatment, loca-tion, and the interaction of treatment and location were Þxed factors in the analysis. Random factors included year, location crossed with year, and the interaction of treatment with each of these random factors. The CONTRAST statement was used to com-pare fecundity of adults collected in Iowa (study lo-cation) with those collected from the other locations. To determine whether insect density affected fecun-dity, we tested the correlation between the average number of adults per tent for the pure stand refuge treatment and the average egg production per female in the pure stand refuge treatment, using data from all locations and both years (PROC CORR).
Susceptibility of Progeny to Bt Maize. A partially
nested mixed model ANOVA (PROC GLIMMIX) was used to analyze survival and the proportion of third instars. Fixed effects in the model were treatment (maize treatment in the Þeld), maize hybrid in seed mats (Bt and non-Bt), and the interaction of treatment and hybrid. Random factors included year, location, location crossed with year, the interaction of treat-ment with each of these random effects, replicate nested within location by treatment by year, and the interaction of replicate by hybrid nested within loca-tion by treatment by year. Replicate was deÞned as a pair of Bt and non-Bt seedling mats that were estab-lished at the same time and used larvae from a single treatment in one location within a year.
Within each replicate, data on the proportion of third instar larvae were used to calculate an odds ratio and 95% conÞdence limits of obtaining a third instar larva on non-Bt vs. Bt maize. Odds ratios for each treatment were obtained using the ESTIMATE
state-ment and transformed back to the original scale using the EXP option. The odds ratio for each of the four treatments was as follows: ([odds of obtaining a third instar on non-Bt maize/odds of obtaining a Þrst or second instar on non-Bt maize]/[odds of obtaining a third instar on Bt maize/odds of obtaining a Þrst or second instar on Bt maize]). A value of 1 indicates no difference in development on Bt vs. non-Bt maize; a value ⬎1 indicates faster development on non-Bt maize (i.e., higher odds of getting a third instar on non-Bt maize vs. Bt maize); and a value of⬍1 indicates faster development on Bt maize. Treatments were signiÞcantly different if they had nonoverlapping 95% CIs.
Results
Survival to Adulthood. Across all locations and both
years, there was a signiÞcant effect of treatment on survival to adulthood (Table 1); the number of adult beetles was signiÞcantly higher in the pure stand ref-uge than any other treatment (P⬍ 0.01 for all com-parisons) (Table 1; Figs. 1 and 2A). Relative to pure stand refuge, survival was reduced by 88.8% (⫾10.1) for integrated refuge 250, 88.5% (⫾9.0) for integrated refuge 1250, and 91.2% (⫾9.0) for pure stand Bt maize. The number of insects emerging from integrated ref-uge 250, integrated refref-uge 1250, and pure stand Bt plots did not differ (P⬎ 0.35 for all comparisons). The number of insects expected to emerge from the inte-grated refuge tents (based on the number of insects that emerged per Bt and refuge plant in the pure stands) did not differ signiÞcantly from the actual number that emerged (IR 250: t7⫽ 0.09, P ⫽ 0.93; IR
1250: t7⫽ 0.61, P ⫽ 0.56) (Fig. 2B). Overall,
signiÞ-cantly more females survived to adulthood. The num-ber of adults that emerged per tent varied consider-ably among locations each year (Table 2). In 2009, survival to adulthood was greatest in Nebraska and lowest in Iowa (Table 2). In 2010, survival to
adult-Table 1. ANOVA for western corn rootworm life history mea-surements Source dfa F P Survival to adulthood Treatment 3, 3 43.57 0.0057 Sex 1, 28 11.19 0.0024 Treatment⫻ sex 3, 28 0.22 0.8824 Timing of emergence Treatment 3, 3 13.40 0.0304 Sex 1, 28 39.20 ⬍0.0001 Treatment⫻ sex 3, 28 1.03 0.3948 Size Treatment 3, 3 0.88 0.5369 Sex 1, 27 16.36 0.0004 Treatment⫻ sex 3, 27 1.2 0.3302 Longevity Treatment 3, 3 1.58 0.3586 Sex 1, 27 75.18 ⬍0.0001 Treatment⫻ sex 3, 27 0.12 0.9448 Fecundity Treatment 3, 9 0.50 0.6930 adf: numerator, denominator.
hood was greatest in New York and lowest in Iowa (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-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 percenpercen-tiles, and dots show outliers beyond the 5th and 95th percentile. Means with same letters are not signiÞcantly different.
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
ping insects did not inßuence fecundity. In addition, there was no signiÞcant correlation between insect density and fecundity in the refuge treatment (r7⫽
⫺0.60; P ⫽ 0.151).
Susceptibility of Progeny to Bt Maize. Larval
sur-vival did not differ signiÞcantly among the four treat-ments (maize type from which parents emerged in the Þeld), and there was no interaction between
treat-Table 3. Life history traits for western corn rootworm N Head capsule width (mm) a Longevity (d) a Fecundity (no. eggs) a Treatment b Sex Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York Iowa Illinois Nebraska New York 2009 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 (1 0.2) 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 aNumbers in parentheses show SE. bInsects emerged from pure stand refuge, integrated refuge 250 (IR 250), integrated refuge 1250 (IR 1250), and pure stand Bt maize plots at four locatio ns.
Fig. 3. (A) Head capsule width and (B) longevity of insects emerging from pure stand refuge, integrated refuge 250 (IR 250), integrated refuge 1250 (IR 1250), and pure stand Bt maize plots, over all locations and both years. Bar heights are the sample mean and error bars the SEM. Aster-isks indicate signiÞcant differences within a treatment (P⬍ 0.05).
Fig. 4. Fecundity of insects emerging from pure stand refuge, integrated refuge 250 (IR 250), integrated refuge 1250 (IR 1250), and pure stand Bt maize plots, over all locations and both years. Bar heights are the sample mean and error bars the SEM. Means with same letters are not signiÞcantly different (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
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.
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 94.4Ð96% 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
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).
Table 4. ANOVA for larvae exposed to seedling mats containing either Bt (event 59122) or non-Bt maize
Source Proportion recovered Proportion of third instars
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
adf: numerator, denominator.
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
aLarvae 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.
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
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 refref-uges, 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 October 2013 PETZOLD-MAXWELL ET AL.: INTEGRATEDREFUGES ANDCORNROOTWORM 2203
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-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.
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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Received 20 February 2013; accepted 23 June 2013.
