CHARGE QUESTIONS – CORN ROOTWORM RESISTANCE MONITORING

CHARGE QUESTION 1: (Annual Corn Rootworm Sampling to Assess Bt Susceptibility)

As a condition of registration, Bt corn registrants are required to conduct annual sampling of CRW 

populations from locations in the Corn Belt to monitor for changes in susceptibility to Bt toxins. These 

collections are obtained from random locations within three defined regions covering different western 

corn rootworm biotypes. The regions also represent areas with high Bt corn adoption and CRW pressure. 

Region 1 (rotation resistant variety) includes Indiana and eastern Illinois; Region 2 (wild type) consists of 

western Illinois, Iowa, and Missouri; Region 3 (organophosphate resistant variant) encompasses Nebraska 

and Kansas. Typically, 12-15 total populations are collected each year, mostly from Iowa, Illinois and 

Nebraska. 

In light of reports documenting field resistance to Cry3Bb1 corn since 2009 (Gassmann et al. 2011, 2012a 

and 2012b), BPPD concluded that the random sampling approach used for annual monitoring across the 

three geographic regions of the Corn Belt was not proactive enough for early resistance detection. Instead,

BPPD recommended an intensive and focused sampling approach, in which collections should be obtained 

from areas experiencing Bt crop failures or regions with high risk factors (i.e., causal factors of resistance

including continuous use of corn-on-corn with the same Bt trait, non-rotation, or poor compliance with 

refuge requirements). A purely random and less intense sampling approach could be maintained in areas of 

the Corn Belt where little or no Bt corn failures have occurred and the selection pressure is lower (i.e. 

“fringe” areas). These areas could serve as a point of comparison between the higher and lower Bt 

selection environments, which would supplement the baseline data established in the early years of Bt 

commercialization. 

a. The panel is asked to comment on sampling approaches for conducting annual CRW resistance 

monitoring that support early resistance detection. Please discuss the strengths and limitations of

BPPD’s proposal for a focused (risk based) sampling approach for the Corn Belt, supplemented with 

samples from lower risk “fringe” areas for comparison.

b. How many CRW populations should be collected from within a sampling region to adequately assess 

susceptibility?

CHARGE QUESTION 2: (Corn Rootworm Sampling in Response to Damaged Bt Fields)

When unexpected damage in Bt corn occurs, technology providers are obligated to investigate the cause of 

Bt failure. These investigations, often termed “performance inquiries,” involve a number of procedural 

steps to assess the causes and circumstances of the corn injury. If CRW are responsible and the level of 

injury exceeds a root damage trigger based on the Iowa State Nodal Injury Scale (NIS), sampling of local 

CRW populations is required to test for potential resistance. Currently, the NIS thresholds are 1.0 for single 

toxin products or 0.5 for pyramided toxins. If insufficient adults were collected during the initial 

investigation, sampling may have to occur the following season.
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For determining root damage, BPPD has recommended transect sampling through the injured field sections 

as opposed to randomly sampling plants from the entire field of concern. BPPD has reservations that 

random sampling could lead to false negatives by lowering the average root node injury score if actual Bt 

damage is localized in a portion of the field (due to clustered oviposition). In terms of adult CRW sampling, 

BPPD has recommended that collections should occur directly in the damaged Bt field(s), specifically in the 

vicinity of the damage. A concern has been raised, however, that such sampling may be biased relative to 

surrounding areas and not representative of the overall CRW population. 

a. The panel is asked to comment on methods for investigating CRW populations causing unexpected 

damage to Bt corn. Specifically, please comment on: 

i. The use of field damage ratings (NIS) as a screen for potentially resistant populations. What 

sampling triggers should be used for single toxin and pyramided Bt products? Should 

alternate techniques be considered?

ii. The use of transect sampling in damaged areas or random sampling throughout the affected 

field to assess root damage ratings.

iii. Appropriate sampling locations (i.e., in the vicinity of the damage and/or surrounding areas) 

for collections of adults if field damage triggers are exceeded. 

iv. The deployment of sentinel plots in the vicinity of damaged fields in subsequent seasons to 

1) assess the resistance allele frequency in the area, and/or 2) collect insects if no adults 

were present at time of the field investigation.

CHARGE QUESTION 3: (Diagnostic Assays)

Insect resistance monitoring for Bt corn toxins has historically been conducted with artificial diet bioassays 

using dose-response curves to obtain LC50 or EC50 measures for laboratory colonies and field populations. 

BPPD has expressed concerns that diet assays, as presently designed, are inadequate for effectively and 

proactively detecting resistant populations. Responses to toxin-incorporated diets are often highly variable 

and a functional “diagnostic” concentration (capable of detecting resistant individuals or populations) has 

not been developed for any of the registered toxins. 

Because of the inherent uncertainties (high variability, less sensitivity) with the present diet bioassay 

methodology, BPPD has recommended the use of diagnostic on-plant assays as the primary resistance 

detection tool for corn rootworm. These techniques assess CRW susceptibility directly on Bt corn plants 

and can measure lethal and sublethal variables. Two such approaches have been developed by Gassmann 

et al. (2011) and Nowatzki et al. (2008). 

a. Please comment on the strengths and limitations of diet bioassay methodologies for early 

resistance detection with CRW, considering that the currently-registered toxins are less than 

high dose. What improvements could be made to these bioassays to make them more effective 

and proactive resistance detection tools? 

b. The panel is asked to discuss the relative merits and limitations of the two on-plant assays 

(Gassmann et al. 2011 and Nowatzki et al. 2008). Please discuss the extent to which these 
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assays have different sensitivities to make early corn rootworm resistance determinations?

Should other on-plant assay approaches be considered?

CHARGE QUESTION 4: (Defining Resistance)

The nature of resistance for corn rootworm and Bt toxins is not completely understood, though it is likely to 

affect how bioassay results are interpreted for resistance determinations. A series of laboratory and 

greenhouse selection experiments with non-high dose Bt toxins have shown that CRW resistance is unlikely 

to follow a single gene, recessive model as for other target pests of Bt crops with high dose expression (e.g., 

tobacco budworm, Gould et al. 1995). Instead, research has suggested that CRW resistance may be nonrecessive (Meihls et al. 2008) or could involve multiple genes conferring varying levels of tolerance (Lefko et 

al. 2008). On-plant assays essentially function as a diagnostic screen that will identify populations with 

genetic resistance (the ability of offspring to survive on Bt plants), but may not clearly identify cases of

incomplete resistance or detect other forms of resistance such as avoidance of the toxin by selective 

feeding on roots with differential toxin expression. 

As discussed in the Agency’s white paper on CRW resistance monitoring, BPPD and industry have proposed

definitions for resistance based on the use of on-plant assays (Gassmann et al. 2011 and Nowatzki et al. 

2008). These definitions include different levels of comparisons to make a “weight-of-evidence” 

assessment -- i.e., 1) the response of a field population on Bt vs. non-Bt corn plants, or 2) the responses of a 

field population vs. a susceptible laboratory colony on Bt corn plants. To make a resistance determination, 

EPA believes that two separate measures demonstrating statistically significant reduced susceptibility to 

the Bt toxin would be needed (e.g., survival, growth). 

a. The Panel is asked to discuss the merits and shortcomings of the proposed approaches to defining 

resistance using on-plant assays. What sets of comparisons in the assays are most likely to add value 

to a weight-of-evidence approach to determining resistance?

b. What resistance allele frequency should constitute field resistance for toxins with less than high 

dose expression? Please discuss the criteria that should be used for these types of toxins (as 

opposed to high dose toxins) given that a portion of heterozygous insects will survive Bt exposure 

and drive the evolution of resistance.

c. What statistical tests, criteria, and significance levels would be best suited for early resistance 

detection with the proposed assays? Please discuss how to best avoid false negatives and/or false 

positives.

d. Please comment on the extent to which incomplete resistance can be identified with on-plant test 

systems. How should resistance definitions be adjusted to address these scenarios?

e. Please discuss the viability of resistance ratios as an option for determining resistant populations,

considering the generally low susceptibility of CRW to Bt toxins and the lack of susceptible wildtype 

populations (i.e., due to widespread adoption of Bt corn). What ratio could be considered as an 

indicator of resistance for corn rootworm using on-plant assays?
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CHARGE QUESTION 5: (Determining Remedial Action Areas)

In the event of CRW resistance, successful remediation will likely depend on the ability to geographically 

define a remedial action zone in a fashion that adequately encompasses the resistant population(s). To 

accomplish this objective, a robust scientific approach is needed to assess the extent of resistance. As 

discussed in the Agency’s white paper, this step may be complicated by the fact that corn growers use 

insecticides to suppress corn rootworm population densities (prophylactically or in response to field 

damage). These management strategies can reduce population sizes and mask the presence of resistant 

populations the following year, though resistant corn rootworm populations may be present beyond the 

visual boundary of failed Bt corn fields. One possible scientific approach to delineate a remedial action zone 

could be to use a resistance allele frequency gradient starting in the center of the area of concern and 

working outward beyond the known affected field(s). Once a resistance frequency gradient has been 

determined, a resistance threshold value could aid in demarcating the spread of resistance. 

a. Please comment on the strengths and limitations of BPPD’s proposal to use resistance allele 

gradients to define the geographic extent of a resistant population.

b. What other tools or strategies could be employed to define the remediation zone?

CHARGE QUESTION 6: (Containment/Mitigation of Resistance)

The terms and conditions for all Bt corn registrations for CRW include a generic remedial action plan, which 

is to be superseded by a specific remedial action plan (submitted to the Agency within 30-90 days) if 

resistance is confirmed. BPPD is concerned that the current generic remedial action plans may not be 

sufficiently proactive to halt resistance from spreading before a specific plan can be developed. Unless the 

resistant population is extirpated by management actions described in the generic plan, the 

implementation of targeted and specific measures (i.e., to reduce the spread of or eliminate resistant 

populations) could be hindered. 

As discussed in the Agency’s white paper, BPPD has recommended a more proactive approach of 

remediation for addressing CRW resistance. Some of the considerations include: 

• Creation of species and toxin specific remedial action plans before resistance evolves such that the

strategies can be deployed quickly if resistance is confirmed;

• Strategies to identify and address localized (i.e., “hot spots”) vs. widespread (large, continuous 

geographic areas) resistance (scenarios that could be evaluated with theoretical models);

• Additional research on CRW dispersal (i.e., proportion of long distance and pre-ovipositional 

movement) to evaluate containment strategies;

• The use of crop rotation and alternate PIP modes of action (other than the compromised toxin) as 

preferred CRW control strategies for resistance mitigation (as opposed to soil insecticide use with 

continued planting of the compromised Bt toxin);
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• Simulation modeling to help design and evaluate remediation strategies. Simulations should explore 

effects of dispersal (male dispersal and pre-ovipositional female dispersal) on the success of 

remediation and provide insight into which remedial actions are the most effective at containing or 

eliminating resistant populations.

The Panel is asked to comment on the merits and shortcomings of the current remedial action approach 

and the proposed improvements as well as alternative approaches the panel may wish to provide. 

Specifically, please discuss the following issues:

a. What remediation approaches could be taken for localized vs. area-wide resistance scenarios?

b. Which mitigation measures would be more effective in containing and/or extirpating resistant CRW 

populations? 

c. Please comment on the value of theoretical models in designing remedial action strategies for 

various resistance scenarios. 

d. The current deterministic and stochastic simulation models used for IRM purposes contain many of 

the following attributes: ecology, population biology, behavior, and genetics of pest, grower 

behavior (refuge compliance, insecticide spraying, etc), explicit spatial and probability analyses. 

What other modeling attributes would help improve the analysis of remediation strategies?