Corn rootworm populations were sampled during the summer of 2009 in problem fields and in control fields found within Iowa, USA (). Problem fields were defined as fields with severe feeding injury to Bt maize by corn rootworm and were identified by farmers that contacted the extension service of Iowa State University. Problem fields contained plants that were goose-necked (bent at the plant-soil interface) and lodged (tilted in a pronounced manner), which are characteristic of feeding by corn rootworm larvae. Additionally, farmers noted a high abundance of rootworm adults in problem fields. Upon receiving notification of a problem field, we visited the field and sampled the western corn rootworm present in the field. In all four problem fields, the vast majority of adult Diabrotica spp. present in the field were western corn rootworm. In one case a problem field was present on an Iowa State University research farm (Northeast Research and Demonstration Farm; site P4 in and ). With the exception of this Iowa State University research farm, maize roots were dug from the problem fields to evaluate rootworm feeding injury and the presence of Bt toxin was confirmed by ELISA with a kit (Envirologix, Portland, Maine). Roots were not sampled at random but were selected to confirm the presence of rootworm feeding.
Distribution of sites sampled within Iowa during 2009.
Sampling date in 2009, corrected survival in bioassays, and history of planting in problem fields (P1–P4) and control fields (C1–C5) from 2003 to 2009.
Control fields were defined as fields not associated with unexpected feeding by corn rootworm on Bt maize. To allow for comparison with problem fields, only western corn rootworm were sampled from control fields. We sampled five control fields that were widely distributed throughout Iowa. Three of the control fields were located on Iowa State University research farms (C2, C4, C5 in ; ). One control field was identified based on a grower complaint of heavy rootworm injury to non-Bt corn (C3 in ; ). Another control field (C1) was identified as part of a survey of corn rootworm abundance in Iowa (M. Dunbar pers. obs.). This field was the only control field with a history of Bt maize but there was not apparent rootworm feeding as evidenced by an absence of lodging by maize plants (C1 in ; ). Maize roots were not examined in control fields, so the extent of rootworm feeding is unknown.
We interviewed farmers and farm managers to determine cropping history of fields from 2003 to 2009 (). Individuals were asked if Bt maize had been grown in the field, during which years, and what type of Bt maize (e.g., Cry3Bb1 or Cry34/35Ab1). No questions were asked about planting of refuge, size of refuge, or proximity of the refuge to the Bt field. For years in which Bt maize was not grown in a field, individuals were asked about the type of crop that was grown (e.g., maize or soybeans).
Adult western corn rootworm collected in the field were brought to Iowa State University where they were held in small cages (18 cm×18 cm×18 cm L×W×H) (Megaview Science, Taichung, Taiwan) and provided with food consisting of corn leaf tissue and an artificial diet (western corn rootworm diet, Bio-Serv, Frenchtown, New Jersey). The water source for the adult beetles was 1.5% agar solid, which was 98.5% water by mass, and provided water to the adult western corn rootworm when consumed. Cages were held in an incubator (25°C; 16/8 L/D) and individuals from each population were housed in separate cages. Adults were provided with an oviposition substrate that consisted of moist, finely sieved soil (<180 µm) placed in a 10 cm Petri dish. Eggs obtained from each population were placed separately in 45 mL plastic cups containing moistened sieved soil, and then sealed in a plastic bag and placed in a cold room at 8°C for at least 5 months to break diapause. Following exposure to cold, eggs were stored for one week at 25°C. Eggs were washed from the soil using a screen with 250 µm openings and then placed atop moistened sieved soil held in a 10 cm Petri dish. Neonate larvae began hatching approximately one week thereafter.
Neonate larvae from each population were evaluated in laboratory bioassays for their survival on two transgenic maize hybrids, each of which contained a unique Bt toxin targeting corn rootworm. One hybrid (DeKalb DKC 6169) produced Cry3Bb1. The other hybrid (Mycogen 2T789) produced Cry34/35Ab1. For both of these hybrids, we also evaluated rootworm survival on a near isogenic hybrid that lacked a gene for a rootworm active Bt toxin but otherwise was genetically similar to its respective Bt hybrid. In the case of Cry3Bb1 maize, the non-Bt hybrid was DKC 6172 (DeKalb) and for Cry34/35Ab1 maize the non-Bt hybrid was 2T777 (Mycogen).
Maize plants used in bioassays were grown in a greenhouse (25°C, 16/8 L/D) in 1 L containers made of clear plastic (Reynolds Food Packaging, Shepherdsville, Kentucky) with supplemental lighting provided with 400 W high-pressure sodium bulbs (Ruud Lighting Inc., Racine, Wisconsin). Containers were filled with 750 mL of a 1
1 ratio of Sunshine Sun Gro SB300 and Sunshine Sun Gro LC1 potting soils (Sun Gro Horticulture Canada Ltd., Vancouver, British Columbia). Seeds were planted one per container at a depth of ca. 4 cm. Beginning two weeks after planting, plants were fertilized weekly with 100 mL of Peters Excel 15-5-15 Cal-Mag Special (Everris International, Geldermalsen, The Netherlands) at a concentration of 4 mg per mL.
Maize seeds of 2T789 and 2T777 were coated with a seed treatment (CruiserMaxx 250, Syngenta, Basel, Switzerland), which contained the neonicotinoid insecticide Thiamethoxam. Prior to planting, this seed treatment was removed by washing ca. 50 seeds in a solution of 1 mL dish detergent (Ultra Palmolive Original, Colgate-Palmolive Company, New York, New York) and 250 mL deionized water. Seeds were placed in the detergent solution for 20 minutes and agitated gently using a stirring plate and magnetic stirring bar. This process was repeated three times with seeds rinsed four times with deionized water between each time they were washed. Seeds were then rinsed four times and allowed to dry for approximately 12 hours, followed by one hour of soaking in a 10% bleach solution, during which they were stirred every 15 minutes. After seeds were removed from the bleach solution, they were rinsed 10 times with deionized water and then allowed to dry for at least 24 hours. This process removed virtually all visible signs of the seed treatment. Insecticidal seed treatment was not applied to DKC 6169 and DKC 6172. However, to ensure that no residual insecticide was present, seeds were bleached following the methods used with 2T789 and 2T777.
Plants were grown in a greenhouse for three to four weeks, until they contained at least five fully formed leaves (V5 stage), and then moved to incubators for bioassays. For bioassays, plants were first trimmed to a height of 20 cm to allow for storage in incubators. Two to three leaves were left on each plant but were trimmed to 8 cm long. Recently hatched larvae (less than 24 hours old) were removed from the soil's surface within their Petri dish using a fine brush and placed at the base of a maize plant on a root that had been exposed by moving away a small amount of soil. Maize plants remained in their original 1 L containers throughout the bioassay. Between 10 and 20 neonates were placed on the base of each plant. Larvae were distributed equally between Bt and non-Bt maize plants. Cups containing plants and larvae were placed in an incubator for 17 days (25°C, 65% RH, 16/8 L/D), and plants were watered as needed.
After 17 days, the aboveground biomass of the plant was excised and the soil, containing roots and larvae, was removed from the 1 L plastic container and placed on a Berlese funnel to extract larvae from the soil. A length of 17 days was selected for bioassays because it allowed sufficient time for some of the fastest developing larvae to reach the third and final instar 
. Root masses were held on Berlese funnels over 4 days and rootworm larvae were collected in 15 mL glass vials containing 10 mL of 85% ethanol. The average sample sizes per population were 12.7±4.8 (mean ± standard deviation) bioassay cups for Cry3Bb1 maize and for its non-Bt counterpart, and 12.8±4.8 bioassay cups for Cry34/35Ab1 maize and for its non-Bt counterpart. We did not have sufficient western corn rootworm eggs to test one of the populations (C2 from ) on Cry34/35Ab1 maize.
Data on the number of field-years (i.e., planting of one field for single year) during which problem fields and control fields were planted to Cry3Bb1 maize were compared using a G test of independence with a Williams's correction 
For each bioassay cup, proportional survival was calculated as the quotient of the number of larvae recovered after 17 days divided by the number of neonates initially placed in a bioassay container. The mean proportional survival for each population on each type of maize was analyzed with a two-way, mixed-model analysis of variance (ANOVA) (PROC MIXED in SAS). Data for the two types of Bt maize (Cry3Bb1 maize and Cry34/35Ab1 maize) were analyzed separately. The ANOVA included the fixed factors of field type (problem field vs. control field), maize hybrid (Bt maize vs. non-Bt maize) and their interaction. Random factors in the analysis were population, which was nested within field type, and the interaction between maize hybrid and population nested within field type. Survival data were transformed by the arcsine of the square root to ensure homogeneity of variance and normality of the residuals. Pairwise contrasts were conducted using the PDIFF option in PROC MIXED.
For each population, we calculated corrected survival as the complement of corrected mortality. Corrected mortality was determined using the correction of Abbott 
, and was calculated for each population by adjusting mortality of larvae from each bioassay cup with Bt maize by the average mortality on the non-Bt near isogenic hybrid. Average corrected survival for each population was compared between control fields and problem fields for Cry3Bb1 maize and for Cry34/35Ab1 maize based on a one-way ANOVA (PROC ANOVA in SAS).
Corrected survival also was used to test the significance of three correlations among all populations sampled. We tested for the following correlations: 1) corrected survival of populations on Cry3Bb1 maize and Cry34/35Ab1 maize, 2) corrected survival for populations on Cry3Bb1 maize and the number of years populations had been exposed to Cry3Bb1 maize in the field and 3) corrected survival for populations on Cry34/35Ab1 maize and the number of years populations had been exposed to Cry34/35Ab1 maize in the field. Correlations were measured using a Pearson correlation coefficient and tested for significance against the null hypothesis of ρ
0 (PROC CORR in SAS).