Scientific Uncertainties Associated With 

Resistance Monitoring for Corn 

Rootworm

Alan Reynolds

and

Jeannette Martinez

U.S. EPA, Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Washington, DC

USA
Corn Rootworm Complex

• One of the most economically important U.S. corn insects

– “Billion dollar pest”

• Western corn rootworm (Diabrotica virgifera virgifera)

– Primary target pest of Bt corn

• Northern corn rootworm (D. barberi)

• Southern corn rootworm (D. undecimpunctata howardi)

• Mexican corn rootworm (D. virgifera zea)

2
Bt Corn PIPs Registered for Corn 

Rootworm

Toxin Event Trade Name Registrant Year Initially 

Registered

Cry3Bb1 MON 863

MON 88017

Yieldgard VT 

Rootworm Monsanto 2003

Cry34/35Ab1 59122-7

Herculex 

Rootworm

Dow Agrosciences, 

Pioneer Hi-Bred 2005

mCry3A MIR 604 AgrisureRW Syngenta Seeds 2006

eCry3.1Ab 5307 Agrisure Duracade Syngenta Seeds 2012

3
Insect Resistance Management

• Required for every registered Bt corn PIP (terms and 

conditions of registration)

• Stewardship program

• Key components:

– Refuges (structured, seed blend)

– Grower Education

– Compliance Assurance Program

– Resistance Monitoring

– Remedial Action 7 Acres

10 

Acres

20 Acres

20 

Acres

20 Acres

45 

Acres

5% Refuge with Distance Requirement

75 Acres

Refuge

Refuge

30 

Acres

50 Acres

< 0.5 Mile

< 0.5 Mile

Road
Insect Resistance Monitoring - Objectives

• Monitoring for changes in susceptibility through targeted 

population sampling and testing

– “Annual” monitoring 

– Results reported to EPA

• Investigation of unexpected field damage reports

– Growers, extension agents, consultants, company agronomists

(Mel Evans, AP) (Purdue University) 5
Insect Resistance Monitoring - Main Elements

• Population Sampling

– Annual monitoring

– Unexpected damage in Bt corn fields

• Bioassays to determine susceptibility

– Diagnostic assays

– Artificial diet

– “On-plant”

• Definitions of resistance

– Bioassays

– Field measures (root damage)

• Remedial Action

– Mitigation if resistance is detected and confirmed

• Scientific uncertainties exist for each of these elements

6
Corn Rootworm – Challenges for Resistance 

Monitoring

• CRW Biology

– One generation per year

– Obligate diapause (egg)

– Larvae are subterranean root feeders

– Difficult to rear in laboratories (NCRW)

– Limits ability to conduct bioassays 

• Lack of “high dose” Bt toxins for CRW

– CRW less sensitive to Bt than other pests (Lepidoptera)

– Complicates interpretation of bioassays

• Different types of Bt corn PIP products

– Single toxin, pyramids, seed blends

– Implications for field scouting

7
• Limitations of diet bioassays 

– Response often highly variable

– Lack of a diagnostic assay to distinguish resistant 

individuals/populations

• Timing

– Assay results not available until the following year (diapause)

– Resistance determination could take 1-2 years

• Program is reactive instead of proactive

– Shifts in susceptibility (prior to field failure) not likely to be 

detected with current approaches

– More likely that resistance will be discovered in the field

– Remedial action (based on containment) may be difficult

Corn Rootworm – Challenges for Resistance 

Monitoring

8
• a) Annual monitoring

• b) Response to unexpected damage to Bt 

corn fields

• Current program

• Uncertainties

• EPA/BPPD recommendations

1) CRW Sampling

9
• Required by terms of registration for each toxin

– Cry3Bb1, Cry34/35, mCry3A, eCry3.1Ab

• Coordinated by Agricultural Biotechnology Stewardship Technical 

Committee (ABSTC)

• Three collection regions:

– Region 1 (rotation resistant variety): Indiana and eastern Illinois

– Region 2 (wild type): western Illinois, Iowa, and Missouri 

– Region 3 (organophosphate resistant variant): Nebraska and Kansas

• Focus on areas with historically large Bt corn adoption and where 

the risk of resistance evolution is presumably greatest.

CRW Sampling - Annual Monitoring

10
CRW Sampling - Annual Monitoring

• Objectives:

– 12-15 total populations per year

– ≥ 2,000 adults per population

– Populations obtained from non-Bt hosts at random locations within each 

region

• Collected populations reared in laboratory; progeny (F1) 

tested the following year

• Annual reports to EPA: Cry3Bb1, Cry34/35, mCry3A (reviews 

in docket); eCry3.1Ab (baseline data submitted)

11
CRW Sampling - Annual Monitoring: 

Uncertainties

• Random sampling

– Random approach doesn’t track populations over time

• Number of populations/genomes sampled

– Are 12-15 populations sufficient?

– Number of individuals targeted per population could be based on 

presumed resistance allele frequency

• Unknown for CRW (possibly multiple genes)

12
CRW Sampling – Annual Monitoring: 

EPA/BPPD Recommendations

• Inclusion of Wisconsin, South Dakota, Colorado, 

Minnesota in the collection regions

• Adopt a focused, non-random sampling approach for 

annual resistance monitoring in the Corn Belt

– Use of risk factors: continual corn-on-corn, use of same Bt toxin, 

low refuge compliance, insufficient refuge, previous Bt corn field 

damage

– Identify hotspots before widespread dispersal of resistance

– Limited, non-random sampling can also be conducted in “fringe” 

areas of the Corn Belt (i.e., areas with few Bt corn failures and 

lower selective pressure for resistance)

13
CRW Sampling – Response to Unexpected 

Damage

• All reports of unexpected product performance must be 

investigated (“performance inquiries”)

• Initial procedural steps:

– Confirmation that damaged corn is Bt (not non-Bt or refuge corn)

– Confirmation of appropriate expression of Bt toxin(s)

– Determination that damage is due to CRW (not other cornfeeding insects)

– Elimination of climatic, agronomic or other factors as potential 

causes of the damage

14
CRW Sampling – Response to Unexpected 

Damage

• If CRW caused damage, root sampling occurs within field 

to assess damage levels

– Iowa State Nodal Injury Scale (Oleson et al. 2005)

• Range: 0 (no injury) to 3 (heavy damage)

• Triggers for adult sampling

– Single toxin Bt corn: trigger = 1.0

– Pyramided Bt corn: trigger = 0.5

• Adults collected “from the vicinity” of the damaged field

– If too late to collect adults, attempt will be made the following 

season (if possible) 15

(Iowa State University)
CRW Sampling – Response to Unexpected 

Damage - Uncertainties

• Use of root node damage triggers (NIS) as a screening 

tool and/or potential indicator of resistance

– Non high dose toxins

– NIS can be affected by larval density and environmental factors

16
CRW Sampling – Response to Unexpected 

Damage - Uncertainties

• Where to sample

– Within the damaged field (or at the specific damage site in the 

field)

• Concern that sampling within Bt fields could bias susceptibility assessments 

due to continual exposure to Bt toxin(s)

– Neighboring or nearby fields

• Testing insects collected outside of the damaged field may not reveal much 

about the susceptibility of insects at the site of concern 

• Management tactics

– Damaged fields are typically treated (SAI, adulticides) which will 

likely suppress CRW populations and complicate sampling

– Resistance allele frequency likely to remain unless populations 

are extirpated 

17
CRW Sampling – Response to Unexpected 

Damage: EPA/BPPD Recommendations

• Focused (prescriptive) sampling scheme

– Use of transect sampling in the damaged field for collecting root node 

injury data

– Adult collections should be made within the damaged field, proximate to 

damaged plants (limited CRW dispersal)

– Take additional samples away from the damaged field (with sentinel 

plots) to determine average resistance allele frequency

– If insects can’t be collected (late in the season, CRW control measures), 

use sentinel plots the following year for sampling

18
• Current program

• Artificial diet assays

– Uncertainties

• On-plant assays

– Single plant (Gassmann) assay, Sublethal 

Seedling Assay

– Uncertainties

2) Resistance Detection Assays

19
Resistance Detection Assays

• Sampled populations (annual and unexpected damage 

collections) are tested for susceptibility to Bt toxins

• Historic approach: artificial diet bioassays

– Toxin source: microbially produced or lyophilized plant tissue

– Dose response to determine EC (growth) and LC (mortality) 

values

– Field populations compared to susceptible control colonies and 

historic baselines

– Data for Cry3Bb1, Cry34/35, mCry3A summarized in white 

paper; reviews in docket

20
Artificial Diet Assays – Concerns and 

Uncertainties

• Diet bioassays are unlikely to proactively detect 

resistance

– High variability between tested populations

• Natural variability

• Laboratory methodology

– No “diagnostic” concentrations to distinguish resistant 

individuals/populations

– LC50/EC50s of resistant colonies may not be distinguishable from 

those of the control populations

• Complications:

– Changes in toxin sources or procedures

– Dietary differences between artificial diet and corn plants

– CRW less susceptible to Bt toxins (high amounts of toxin and/or 

extrapolations needed to obtain LC50 results)

• Can diet assay methodology be improved? 21
On-Plant Assays

• Test CRW susceptibility using Bt corn plants

– Laboratory/greenhouse

– Mortality/survival or sublethal effects (growth, development)

• Two approaches developed to date

– “Gassmann on-plant assay” (Gassmann et al. 2011)

– Sublethal Seedling Assay (Notwatzki et al. 2008)

22 (Harvest Public Media)
Gassmann On-Plant Assay 

• General methodology:

– Test arenas are soil containers with one corn plant (Bt or 

non-Bt)

– Neonate larvae are distributed equally into test arenas 

(each container is a replicate)

– 17 day exposure period in an incubator (25°C, 65% RH, 

16/8 L/D)

– Larvae recovered with Berlese funnels over 4 day period

– Proportion of surviving larvae determined for each 

replicate (with Abbott’s correction)

• Initial testing conducted on Cry3Bb1 and Cry34/35 

(Gassmann et al. 2011):

– Subsequent testing with mCry3A (Gassmann et al. 

unpublished, data presented at 2012 ESA meeting)

23
Gassmann On-Plant Assay -

Uncertainties

• Standardization of the assay for all CRW toxins

– Testing conducted on Cry3Bb1, Cry34/35, mCry3A to date

• Control Colonies

– Can truly “susceptible” CRW be collected from the field?

• An issue for all CRW bioassays

– Are laboratory colonies (reared in absence of Bt) 

representative of truly susceptible wild types?

• Interpretation of results

– Statistical comparisons between Bt/non-Bt plants, 

field/control populations

– Use of resistance ratios 

24
Sublethal Seedling Assay

• Developed by Pioneer (modified from Nowatzki

et al. 2008)

• Used for Cry34/35 monitoring (since 2009, in 

conjunction with diet assays)

• General methodology:

– Seed mats (150 kernels/mat)

– Infestation: 500 larvae/mat

– 17 day exposure period in environmental chamber 

(25 °C, 65% RH, 14:10 L/D)

– Larvae collected with Berlese funnels

– Subset of 30 larvae randomly selected for analysis

25
Sublethal Seedling Assay

• Sublethal measures:

– Mean instar

– Larval size (area)

– Bt : non-Bt ratio

• If larval development on Cry34/35 equals or exceeds that on non-Bt, 

the ratio will be ≥ 1.0

• Populations suspected to be resistant if:

– Similar response when exposed to both 59122 (Cry34/35) and 

isoline at a significance level of p>0.05 for both instar and size 

data; 

– Mean instar and size ratios fall outside the 95% confidence 

interval established from data collected in previous years. 

[Additional SSA data may be needed for establish the 95% 

confidence interval for this criterion.] 26
Sublethal Seedling Assay - Uncertainties

• Assay sensitivity

– Selected colony (York-RR) didn’t meet both resistance 

criteria (2010 and 2011 testing)

– Potential for false negatives

• Interpretation of results/resistance criteria

– Assumption that resistant larvae will develop similarly on 

Bt and non-Bt

• Standardization of the assay for all CRW toxins

– Testing conducted only with Cry34/35 to date

• Control Colonies

– Truly susceptible?

27
• Current process

– General approach

– Cry3Bb1

– Uncertainties

• EPA/BPPD recommendations and new 

proposals

– Uncertainties

3) Defining Resistance 

28
Defining Resistance – Current Process

• Tiered approach

– Unexpected damage → “suspected” resistance →

“confirmed” resistance

• “Suspected” resistance

– Unexpected damage: root damage triggers (NIS)

– 1.0 single toxin, 0.5 pyramids (50% plants damaged)

– Adults collected if trigger exceeded

• “Confirmed” resistance

– Bioassays

– Process varies between registrations

29
Defining Resistance – Current Process

• Resistance confirmation – general process:

– The LC50 of the standard (diet) bioassay exceeds the 

95% confidence interval of the mean historical LC50 for susceptible pests according to baseline 

measurements

– Over 50% of Bt-expressing plants have ≥1 root nodes 

destroyed by suspected resistant populations under 

controlled lab conditions (Cry3Bb1)

– The proportion of larvae that can feed and survive on 

Bt roots from neonate to adult is significantly higher 

than the baseline proportion (mCry3A, Cry34/35)

– Trait is heritable

– Field relevance: economic thresholds, potential to 

spread

30
Defining Resistance – Current Process

• Monsanto revised approach for Cry3Bb1 (2013):

– Performance inquiry for unexpected damage

– Use of single, on-plant bioassays

– Resistance criteria:

• A statistically significant difference in measures of either 

lethality/mortality or sublethal effects (growth/development) between 

the field population and the control population on Bt corn and

• A lack of a statistically significant difference in the same measures 

of the field population raised on Bt corn and non-Bt corn plants.

– Additional testing would be needed to determine resistance if:

• The results of the single, on-plant bioassays are inconclusive (e.g., 

the results of the statistical analysis are unclear because of low 

sample sizes) or

• Another reasonable explanation for the unexpected damage exists 

(e.g., high pest pressure and/or high plant stress).

31
Defining Resistance – Uncertainties in 

the Current Process

• NIS as a predictor of potential resistance

– Appropriate thresholds

• Minimizing false negatives and positives

• Lack of high dose

– Root damage can be exacerbated by environmental factors

• Timing

– Delays inherent in the process; confirmation could take 2+ years

– Adult collections can be delayed if too late in the season

– CRW diapause

– Confirmatory testing if needed

• Sensitivity

– Natural variability

– Historic baselines complicated by changes in toxin source, 

methodology

32
Defining Resistance – EPA/BPPD 

Recommendations

• Replace artificial diet assays with on-plant assays

• SSA approach (Cry34/35)

• Single on-plant assay approach

• Statistically-relevant comparisons

– Two statistically significant comparisons needed for a positive 

resistance determination

– Sampled population’s response on Bt corn and non-Bt corn 

(statistical similarity)

– Sampled population vs. (susceptible) laboratory population on Bt 

corn (statistical difference)

– Comparisons could involve survival (Gassmann) or sublethal 

(SSA) metrics

– Resistance ratios between field and control populations(?) 33
Defining Resistance – Uncertainties with 

New Proposals

• Nature of CRW resistance not completely understood, but 

likely to affect how bioassays are interpreted

– Single, recessive gene model unlikely

• Issues with comparisons

– Comparing independent events (e.g., field population response on Bt vs. 

control colony response on non-Bt)

– Potential effects of incomplete resistance or costs of resistance

– Use of body weight – correlation with resistance? 

– Assumption that resistant larvae develop the same on Bt and non-Bt corn

– Appropriate statistical analysis/level of significance

• Resistance ratios (field population : control)

– Ratios > 10 typically indicate resistance (Tabashnik 1994), but may not be 

realistic for CRW

– CRW less susceptible; some survival of susceptible populations on Bt 34
• Current process

– “Generic” RA plans

– BMPs for fields with unexpected damage

– Uncertainties

• EPA/BPPD recommendations

4) Remedial Action

35
Remedial Action

• “Generic” plan in place for all registrations

• Superseded by specific RA plan if resistance occurs 

(within 30-90 days)

• General process for cases of confirmed resistance:

– Notification to EPA within 30 days of resistance confirmation;

– Affected customers and extension agents are to be notified 

about confirmed resistance;

– Affected customers and extension agents will be encouraged to 

employ alternative CRW control measures;

– Sale and distribution of [Bt corn] in the affected area will cease 

immediately;

– A long-term resistance management action plan will be devised 

according to the characteristics of the resistance event and local 

agronomic needs. The details of such a plan should be approved 

by EPA and all appropriate stakeholders.

36
Remedial Action

• Proactive registrant responses to unexpected field 

damage (“suspected” resistance)

• Best Management Practices are implemented during 

damage investigations (prior to resistance confirmation)

• Typical BMPs (ex. Monsanto Cry3Bb1):

– Rotation to a non-host crop (e.g., soybean);

– Use of pyramided Bt corn products (e.g. SmartStax);

– Use of Cry3Bb1 corn but with additional corn rootworm control 

tools (e.g., soil insecticides, seed-applied insecticides, 

chemigation);

– Use of an alternative (non-Cry3Bb1) rootworm-active Bt corn 

product;

– Use of non-Bt corn with soil or foliar insecticides.

– (http://www.monsanto.com/products/pages/crw-bmp.aspx)

37
Remedial Action – Challenges and 

Uncertainties

• Timing

– Resistance confirmation process

– Registrant BMPs for unexpected damage (proactive)

• Defining the geographic area for RA

– Damaged Bt fields may not necessarily demarcate the 

extent of the area affected by resistance

– Hot spot vs. area wide resistance

• Ability of RA to contain spread of resistance

– CRW dispersal

– Applied mitigation measures may suppress population 

densities and mask resistance in following years w/o 

eliminating the trait

– CRW perspective: landscape of continuous corn

– Agronomic perspective: multiple registrants and PIPs 38
Remedial Action – EPA/BPPD 

Recommendations

• Implementation of a proactive RA strategy

– Species and toxin specific plans in place before resistance evolves

– Registrant BMPs

– Separate strategies for hot spot vs. area wide resistance

• Defining the remediation zone

– Use of a resistance allele gradient

– Sentinel plots near the affected area

• RA strategies

– Rotation to a non-host crop (e.g., soybean) or alternate Bt modes of 

action are preferred;

– Insecticide use for short term population suppression;

• Research to improve RA

– Modeling to evaluate hot spot and area wide strategies

– Additional research and modeling into the effects of CRW 

movement/dispersal on RA

39
40

Thank You!!

Questions?