CHARGE QUESTION S

– 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, an d 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 co rn 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 recommend ed an intensive and focused sampling approach , in which collections should be obtained from areas experiencing Bt crop failure s 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 failure s have occurred and the selection pr essure is lower (i.e. “fringe” areas)

. These areas could serve as a point of comparison between the higher and lowe r Bt selection environments , which would supplement the baseline data established in the early years of Bt commercialization.

a. The panel is a sked 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 Respo nse to Damage d Bt Fields)

When unexpected damage in Bt corn occurs, technology providers are obliga ted 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

. Cu rrently, the NIS threshold s 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.2 For determining root damage, BPPD has recom mended 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 sampl ing 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 adul ts if field damage triggers are exc e eded

. 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 th e 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 LC 50 or EC 50 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 method ology, BPPD has recommended the use of diagnostic on

- plant assay s 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 a pproaches 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 th at th e 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 these3 assays have different sensitivities to make early corn rootworm resistance dete rminations? 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 determin ations. 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). I nstead, 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 o ther forms of resistance such as avoidance of the toxin by selective feeding on roots wi th differential toxin expression.
As discussed in the Agency’s white paper on CRW resistance monitoring, BPPD and industry have propose d definitions for resistance based on the use of on

- plant assays (Gassmann et al. 2011 and Nowatzki et al.
20 08 ). 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 dem onstrating statistically significant reduced susceptibil ity to the Bt toxin would be needed (e.g., survival , growth)

.

a. T he Panel is asked to discuss the merits an d 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 criteri a 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

a nd 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 co mment 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?4 CHARGE QUESTION 5 : ( De termining Remedial Action Area s )

In the event of CRW resistance, success ful remediation will likely depend on the ability to geographically define a remedia l 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 su ppress corn rootworm population densities ( prophylactically or in response to field damage ). These management strategies can reduce population sizes and mask the presence of resistant populat ions 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

c ould be to use a resistance allele frequency gradi ent 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 comm ent 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 c ould be employed to define the remediation zone?

CHARGE QUESTION 6: (Contain ment / Mitigat ion of Resistance)
The terms and conditions for all Bt corn registrations for CR W 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 resist ance is confirmed. BPPD is concerned that t he 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 e liminate resistant populations) could be hindered

. As discussed in the Agency’s white paper, BPPD has recommended a more proact ive approach of remediation for addressing CRW resistance. Some of the considerations include:

• Creation of s pecies and toxin specific remedial action plans before resistance evolves such that the strategies can be deployed quickly if resistance is confir med ;

• 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 cont rol 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

.
The Panel is asked to comment on the merits and sho rtcomings 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 issue s:

a. What remediation approaches c ould 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 design ing remedial action strategies for various resista nce 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 sprayin g, etc), explicit spa tial and probability analyses. What other modeling attributes would help improve the analysis of remediation strategies?