# 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 )

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# 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 Agrisure RW 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

(Purdue University) (Mel Evans, AP) 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

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## 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)

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## 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)

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## 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 corn feeding insects)

– Elimination of climatic, agronomic or other factors as potential causes of the damage

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## 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

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## 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

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## • Current program • Artificial diet assays

– Uncertainties 

## • On - plant assays

– Single plant (Gassmann) assay, Sublethal Seedling Assay

– Uncertainties 

# 2) Resistance Detection Assays

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# 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

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# 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

– LC 50 /EC 50 s 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 LC 50 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
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# 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?
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## • Current process

– General approach

– Cry3Bb1

– Uncertainties 

## • EPA/BPPD recommendations and new proposals

– Uncertainties 

# 3) Defining Resistance

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# 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 LC 50 

### of the standard (diet) bioassay exceeds the 95% confidence interval of the mean historical LC 50 

### 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

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# 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 3940 

# Thank You!! Questions?