General Information: Project Title: Western corn rootworm abundance, injury to corn, and resistance to Cry3Bb1 in the local landscape of previous problem fields Data Title: Local landscape data set Abtract: Data was collected between 2015 and 2018. In this study, we examined fields that had previously experienced greater-than-expected injury to Cry3 (Cry3Bb1 or mCry3A) corn roots (>1 node) (focal fields), and fields in the landscape surrounding these fields up to 2.2 km away. Fields were sampled in 2015, 2016, and 2017 to quantify rootworm abundance, root injury, Cry3Bb1 resistance, and rootworm management strategies by farmers (i.e., cultivation of corn, type of corn that was grown, and insecticide use over the previous 6 years). Additionally, a comparison of root injury to Bt and non-Bt corn within fields was made. Files are organized by analysis of interest, and each data file is paired with a text file of accompanying SAS code, which was used for analysis. Analyses are: survival data from bioassays to test for resistance to Cry3Bb1 and proportion third instar larvae in bioassays, analysis of corrected survival in bioassays, analysis of field history variables, analysis comparing root injury to Bt and non-Bt corn, analysis of root injury, adult rootworm abundance in fields, and correlations of these metrics with distance from focal fields. Authors: Coy R. St. Clair (Corresponding author) Iowa State University cstclair@iastate.edu Graham P. Head Bayer Crop Science graham.head@bayer.com Aaron J. Gassmann Iowa State University aaronjg@iastate.edu Associated publication: "Western corn rootworm abundance, injury to corn, and resistance to Cry3Bb1 in the local landscape of previous problem fields" PLOS ONE, in review Collection Information: Field data were collected summer 2015, 2016, and 2017. Bioassays were performed in spring 2016, 2017, and 2018. Fields were located in Hancock, Wright, Franklin, Grundy, Bremer, Chickasaw, Howard, Fayette, Deleware, Dubuque, and Jones counties, Iowa. FILES: NOTE: for all fields, 0=true zero (measurement). A period "." is null or missing data. File list: 1. Bioassay survival and third instar DATA.csv - bioassay survival and proportion third instar larvae in bioassays Number of variables:5 Variable name (identity): Population (population name) FieldType (Experimental field or Control) Year (year) ProportionSurve (proportion of larvae surviving the assay) PropThirdInstar (proportion of surviving larave that reached the third instar stage) Notes on variables: Population: Populations with a single number are focal fields, and populations with a number and letter are surrounding fields associated with that field number (e.g., field 1 is a focal field, and field 1a would be the closest surroudning field to field 1). FieldType: Exp=Experimental, Control=Control Year: 2015, 2016, 2017. ProportionSurv: proportion of 12 larvae that were placed on the plant that were recovered in a Berlese funnel. PropThirdInstar: proportion of the surviving larvae that reached third instar 2. Bioassay survival and third instar SAS CODE.txt - SAS code that uses File #1 above. 3. Corrected survival DATA.csv - data on corrected survival in bioassays Number of variables:5 Variable name (identity): Pop (Population name) Type (Which type of population - SURR=surrounding field, FF=Focal Field, CONTROL=susceptible control population) Cat (Category of population - EXP=Experimental, CON=Control) Year (Year the population was collected - 2015, 2016, or 2017). Corrected (Numerical measurement. Corrected survival was calculated from survival as the proportion of larvae surviving on Cry3Bb1 corn in a replicate ÷ mean proportion surviving larvae on non-Bt corn in all replicates of a population. The purpose of this was the account for variation in survival on nonBt corn). 4. Corrected survival SAS CODE.txt- SAS code for statistical analysis of corrected survival. 5. Field history DATA.csv - Raw data on field history variables Number of variables:22 Variables list: Field Year Yr_Injury Location Corn NonBt Cry3 Cry3435 Pyramid Cat FH1 FH2 FH4 FH5 FH6 FH7 FH8 FH10 FH11 FH12 Root WCR Notes on variables: Yr_Injury: Year the greater-than-expected injury to Cry3 corn occured in focal fields. Location: Identifies the farm where the field in located, as some fields were managed by the same farmer Year: 2015, 2016, 2017 Corn: Corn type planted in the field in the year of sampling, key: Non-RW Bt or Non-Bt: 0 Cry3Bb1: 1 Cry34/35Ab1: 2 mCry3A: 3 Cry34/35Ab1 + Cry3Bb1: 4 Cry34/35Ab1 + mCry3A: 5 Variables nonBt, Cry3, Cry3435, and Pyramid are binary (0=absent or 1=present) representation of information found in the "Corn" variable. "FH" is short for "Field History" over the past six years from the time the field was sampled. Key for these variables: FH1 = # years continuous corn FH2 = Proportion of years corn was planted in the field history FH4 = Was soil insecticide used in the year of sampling (1=yes, 0=no) FH5 = Proportion of years soil insecticide was used in the field history FH6 = Proportion of years non-Bt corn was planted FH7 = Proportion of years Cry3 corn was planted (either Cry3Bb1 or mCry3A) FH8 = Proportion of years Cry34/35Ab1 was planted FH10 = Proportion of years a pyramid was planted FH11 = Proportion of years Bt corn was planted with soil insecticide FH12 = Proportion of years non-Bt corn was planted with soil insecticide Root: root injury measurement for the field WCR: abundance of western corn rootworm (#/trap/day) 6. Field history SAS CODE.txt - SAS code for analysis of field history variables. 7. Injury to Bt and nonBt DATA.csv Number of variables: 5 Variable name (identity): Field (field name) FieldType (type of field, Surr=surrounding, FF=focal field) Year (year the field was sampled, 2015, 2016, or 2017) Root(Bt) (Mean root injury to Bt corn roots in the field, on a 0-3 node injury scale) Root(nonBt) (Mean root injury to nonBT corn roots int he field, on a 0-3 injury scale) 8. Injury to Bt and nonBt SAS CODE.txt - SAS code for statistical analysis of injury to bt and nonBt corn. 9. Root injury, abundance ANOVA and correlations DATA. csv Number of variables: 7 Variable name (identity): Field (Field name) Year (Year sampled) Location (Identifies the farm where the field in located, as some fields were managed by the same farmer) Category (Category of field, 0=focal field, 1=surrounding field) Distance (distance of the field from the focal field, measured from centroid to centroid) Root injury (Root injury in the field) WCR Abundance (WCR abundance in the field) 10. Root injury, abundance ANOVA and correlations SAS CODE.txt - analysis of root injury, abundance, and the correlation between distance and these metrics in SAS Methods and materials: Over three years (2015, 2016, and 2017), a total of 20 fields with a history of greater than expected rootworm injury to Bt corn expressing a Cry3 toxin (i.e., greater than one node of injury to Cry3Bb1 or mCry3A corn) were selected for study in north-central and north-eastern Iowa (referred to as “focal fields”). Sample sizes were N=5, N=6, and N=9 for 2015, 2016, and 2017, respectively. In addition to focal fields, neighboring fields in the local landscape were also selected for sampling (referred to as “surrounding fields”). We defined the “local” landscape as the area surrounding a focal field within approximately 2 km, and surrounding fields were selected based on this criterion (i.e., fields located approximately 2 km or less from the focal field). Sample sizes for surrounding fields were N=12, N=15, and N=19 for 2015, 2016, and 2017, respectively. Data collection: Abundance of western corn rootworm in all fields was measured by placing 10 unbaited yellow sticky traps (Pherocon, Trécé Inc., Adair, OK) in each field. Traps were placed on plants at ear-level, in two parallel transects of five traps each (Dunbar et al. 2016). Individual traps were 30 m apart, with approximately 15 m between transects. All traps were placed a minimum of 30 m from any field border. There were three sampling periods for each field. Periods ranged from six to 10 days (7.2 d ± 1.1 mean ± sd), and began 20 July in 2015 and 24 July in 2016 and 2017. Root injury for all fields was assessed by digging roots of corn plants and rating them on a 0-3 node injury scale (Oleson et al. 2005). Ten roots were taken from rows adjacent to where the sticky traps were placed, and were washed and rated within 48 h. A sample of leaf tissue was taken from each plant and used to determine the presence and identity of Bt proteins targeting western corn rootworm using an ELISA-based test kit (Envirologix Inc., Portland, ME). Single-plant bioassays: Bioassays consisted of two corn types: Cry3Bb1 corn (DKC 43-48) and its non-Bt genetic isoline (DKC 43-46). Plants were grown to the V4-V5 growth stage (Abendroth et al. 2011) in 0.95 L plastic cups (Placon, Madison, WI); there were 12 replicates of each corn type for each population tested. Twelve neonate larvae (>24 h old) were added to each plant by placing them gently with a paintbrush on exposed root tissue. The plants were placed in environmental chambers (24°C, 16:8h L:D, 65% R.H). Larvae were allowed to feed undisturbed for 17 d, after which time the aboveground plant tissue was removed and the contents of the cups were placed on Berlese funnels for four days. Larvae were collected in vials filled with 85% ethanol and counted using a dissecting microscope (MZ6, Leica, Microsystems, Wetzlar, Germany). Head capsule width was measured using a microscope camera and imaging software (Moticam 2500, Motic Images Plus 3.0; Motic North America, Richmond, British Columbia, Canada), and this measurement was used to determine larval instar following Hammack et al. (2003). For 2015 field populations, bioassays began (i.e., larvae were placed on the first plant) 20 April, 2016, and ended (i.e., the last vial was collected from the Berlese funnel) on 14 June, 2016. For 2016 field populations, bioassays began 24 April and ended 9 July, 2017, and for 2017 populations, bioassays began 23 April and ended 10 June, 2018. Field management history: Field management history was obtained from farmers and cooperators, which included Bt traits used (Cry3Bb1, mCry3A, eCry3.1Ab, Cry34/35Ab1, or a pyramid of multiple toxins) and soil insecticide use for a total of six years (the year of sampling and five previous years). The total number of years that corn had been grown consecutively in each field was calculated using CropScape Data Layer (National Agricultural Statistics Service, United States Department of Agriculture; available at: https://nassgeodata.gmu.edu/CropScape) from 2003, the year Bt corn was first introduced for rootworm management. Data analysis: Data provided in this archive are raw (untransformed data). All transformations occurred in SAS, provided in the accompanying code. General methods for analytical approaches found below: All data were analyzed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). For rootworm abundance, values used in analyses were calculated as [total number of adults caught/trap/sampling day] during the sampling period with the highest abundance for the field. To test the hypothesis that focal fields and surrounding fields differed in rootworm abundance and root injury, a mixed-model analysis of variance (PROC MIXED) was used. Either root injury or rootworm abundance was used as the response variable, with field type (focal field or surrounding field), year, and their interaction as fixed effects. Random effects in the model were field location (i.e., a focal field and its associated surrounding fields), location (year), field type (location), and field type (location (year)). Random terms were pooled in the model if P ≥ 0.25 for that term. Root injury and abundance were transformed using a square root transformation to improve normality of the residuals in analysis of variance. A Pearson’s correlation (PROC CORR) was used to examine the relationship between distance from focal field and either root injury or rootworm abundance (total N = 66 fields). Focal fields were included in the analysis, with a distance value of zero. For root injury, in cases where a field was planted to Bt corn, refuge plants (i.e., those lacking a Bt trait in a field planted to a mixture of Bt corn and non-Bt corn) were removed from the analysis because refuge plants are expected to have greater levels of injury than Bt plants. A secondary analysis was conducted to assess injury to non-Bt corn compared to Bt corn for fields in which both types of corn were present (N = 17). A paired t-test (PROC TTEST) was performed to compare injury to Bt and non-Bt corn, with the null hypothesis that mean injury did not differ between Bt and non-Bt plants in these fields. To analyze bioassay results, proportion survival on Cry3Bb1 corn and non-Bt corn was examined separately for each year using analysis of variance (PROC GLM). Proportion survival was the response, with population, corn type (Cry3Bb1 vs non-Bt), and their interaction as explanatory variables. Linear contrasts (CONTRAST statement) were used to compare proportion survival on Cry3Bb1 corn and non-Bt corn for each population to the susceptible controls within the year the assays were conducted. Proportion survival was transformed by the arcsine of the square root to improve normality of the residuals. To further characterize resistance, survival on Cry3Bb1 corn was compared to survival on non-Bt corn within each population using a one-tailed t-test (PROC TTEST). Likewise, the proportion of third instar larvae (i.e., the number of larvae collected at the end of the assay that reached the third instar divided by the total number of larvae collected) was calculated for each corn type within a population, and a one-tailed t-test was used to test for a lower proportion of third instar larvae on Cry3Bb1 corn compared to non-Bt corn. Proportion survival and proportion of third instar larvae were transformed by the arcsine of the square root to improve normality of the residuals. T-tests used pooled variances in cases where variances were equal between the two groups being tested. In cases where variances were unequal, the Satterthwaite method was used. Analysis of variance (PROC GLM) was used to analyze corrected survival for each year separately. Corrected survival was the response variable and population was the independent variable. Linear contrasts were used (CONTRAST statement) to compare each field population to the controls. Significantly higher corrected survival in a field population compared to controls would indicate resistance to Cry3Bb1. To test for a difference in corrected survival between focal fields and surrounding fields, analysis of variance was used (PROC GLM) with corrected survival as the response variable and year, field type, and their interaction as independent variables. To test whether field management differed for focal fields compared to surrounding fields in the year of sampling, a chi-square analysis was conducted (PROC FREQ). To test for a difference in historical management tactics, each field characteristic calculated as a proportion of management over the last six years was used in a mixed model analysis of variance (PROC MIXED), with field type (focal field or surrounding field) as a fixed effect, and field location (i.e., a focal field and its associated surrounding fields) and field type(location) as random effects. Random terms were pooled if P ≥ 0.25. Years of continuous corn was transformed by ln(y) to improve normality of the residuals. For all present and historical field management metrics, multiple regression analysis (PROC REG) was used to test the effect of management strategies on rootworm abundance and root injury. All metrics were first analyzed in a correlation matrix (PROC CORR) to determine if collinearity was present between any variables (Pearson’s correlation coefficient >0.80). Soil insecticide use in the year of sampling, proportion of years soil insecticide was used in the last 6 years, and the proportion of years soil insecticide was used on Bt corn in the last 6 years were collinear (Supp. Table 4). Soil insecticide use in the year of sampling and proportion of years soil insecticide was used on Bt corn were removed from the analysis. The remaining variable represented the variation explained by all three collinear variables, and was a measure of overall soil insecticide use in the fields. The following variables were then used as independent variables in the multiple regression, with either rootworm abundance or root injury as the response: non-Bt corn planted in the year of sampling (0=no, 1=yes), Cry3 corn planted in the year of sampling (0=no, 1=yes), Cry34/35Ab1 corn planted in the year of sampling (0=no, 1=yes), pyramided corn grown in the year of sampling (either Cry34/35Ab1 + Cry3Bb1 or Cry34/35Ab1 + mCry3A, 0=no, 1=yes), the total number of years the field was planted to corn continuously, the proportion of years the field was planted to corn in the previous six years, soil insecticide use, proportion of years non-Bt corn was planted, proportion of years single-trait Cry3 corn was planted (either Cry3Bb1 or mCry3a), proportion of years single-trait Cry34/35Ab1 corn was planted, and the proportion of years soil insecticide was used on non-Bt corn. Stepwise selection was used to identify the most appropriate model (P<0.25 for inclusion in the model, P>0.15 for retention) following Dunbar et al. [38]. Rootworm abundance and root injury were transformed using a square root transformation to improve normality of the residuals. Software: Name: SAS v9.4 Developer: SAS Institute Inc., Cary, NC, USA Licensing: This work is licensed under the Creative Commons Attribution (CC-BY) 4.0 International License. For more information visit: https://creativecommons.org/licenses/by/4.0