EPA’s Resistance Monitoring Program for 

Corn Rootworm: Overview of the Current 

Process, Challenges, Uncertainties, and 

Agency Recommendations

Charge to the Panel
Charge Question 1: Annual 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. 

2
Charge Question 1: (Continued)

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-oncorn with the same Bt trait, non-rotation, or poor 

compliance with refuge requirements). 

3
Charge Question 1: (Continued)

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. 

4
Charge Question 1: (Continued)

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?

5
Charge Question 2: 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. 6
Charge Question 2: (Continued)

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. 7
Charge Question 2: (Continued)

a. The panel is asked to comment on methods for 

investigating CRW populations causing unexpected 

damage to Bt corn. Specifically, please comment on: 

1. 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?

2. The use of transect sampling in damaged areas or 

random sampling throughout the affected field to 

assess root damage ratings. 

8
Charge Question 2: (Continued)

3. 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. 

4.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.

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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. 

10
Charge Question 3: (Continued)

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). 

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Charge Question 3: (Continued)

1. 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? 

2. 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 assays have different sensitivities to make 

early corn rootworm resistance determinations? Should 

other on-plant assay approaches be considered?

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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). 

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Charge Question 4: Defining Resistance

Instead, research has suggested that CRW resistance may 

be non-recessive (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.

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Charge Question 4: (Continued)

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). 15
Charge Question 4: (Continued)

1. The Panel is asked to discuss the merits and shortcomings 

of the proposed approaches to defining resistance using onplant assays. What sets of comparisons in the assays are 

most likely to add value to a weight-of-evidence approach 

to determining resistance? 

2. 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.

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Charge Question 4: (Continued)

3. 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.

4. 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?

5. 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? 17
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. 18
Charge Question 5: (Continued)

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.

1. 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. 

2. What other tools or strategies could be employed to 

define the remediation zone? 19
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.

20
Charge Question 6: (Continued)

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: 

1. Creation of species and toxin specific remedial action 

plans before resistance evolves such that the strategies 

can be deployed quickly if resistance is confirmed;

2. 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);

3. Additional research on CRW dispersal (i.e., proportion of 

long distance and pre-ovipositional movement) to 

evaluate containment strategies; 21
Charge Question 6: (Continued)

4. 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);

5. 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.

22
Charge Question 6: (Continued)

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:

1. What remediation approaches could be taken for 

localized vs. area-wide resistance scenarios?

2. Which mitigation measures would be more effective in 

containing and/or extirpating resistant CRW 

populations? 

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Charge Question 6: (Continued)

3. Please comment on the value of theoretical models in 

designing remedial action strategies for various 

resistance scenarios. 

4. 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? 

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Thank you! 

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