1 

# SAP Minutes No. 201 4

- 0 1

A Set of Scientific Issues Being Considered by the Environmental Protection Agency Regarding

Scientific Uncertainties Associated with Corn Rootworm Resistance Monitoring for Bt Corn Plant Incorporated Protectants (PIPs)

De cember 4

- 5 , 201 3 FIFRA Scientific Advisory Panel Meeting

Held at the Environmental Protection Agency Conference

Center Arlington, VA2 NOTICE

These meeting minutes have been written as part of the activities of the Federal Insecticide, Fungicide, a nd Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). The meeting minutes represent the views and recommendations of the FIFRA SAP, not the United States Environmental Protection Agency (Agency). The content of the meeting minutes does not represe nt information approved or disseminated by the Agency. The meeting minutes have not been reviewed for approval by the Agency and, hence, the contents of these meeting minutes do not necessarily represent the views and policies of the Agency, nor of other agencies in the Executive Branch of the Federal Government, nor does mention of trade names or commercial products constitute a recommendation

for use.

The FIFRA SAP is a Federal advisory committee operating in accordance with the Federal Advisory Committ ee Act and established under the provisions of FIFRA as amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides advice, information, and recommendations to the Agency Administrator on pesticides and pesticide

- related issues regar ding the impact of regulatory actions on health and the

environment. The Panel serves as the primary scientific peer review mechanism of the Environmental Protection Agency, Office of Pesticide Programs (OPP), and is structured to provide balanced expert assessment of pesticide and pesticide

- related matters facing the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc basis to assist in reviews conducted by the FIFRA SAP. Further information about FIFRA SAP reports and activities c an be obtained from its website at http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305

- 5805. Interested persons are invited to contact Fred Jenkins, Jr., Ph.D., SAP Designated Federal Official,

via e

- mail at jenkins.fred@epa.gov

.

In preparing these meeting minutes, the Panel carefully considered all information provided and presented by EPA, as well as information presented by public commenters.3 TABLE OF CONTENTS

LIST OF COMMON ACRONYMS USED

................................ ................................ . 4

PARTICIPANTS

................................ ................................ ................................ .......... 7

INTRODUCTION

................................ ................................ ................................ ........ 9

PU BLIC COMMENTS

................................ ................................ ................................ . 10

SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS

.................. 1 1

DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE

.............. 2 5

REFERENCES

................................ ................................ ................................ ............. 6 64 List of Common Acronyms Used

BMPs Best Management Practices

BPPD Biopesticides and Pollution Prevention Division : CRW Corn Rootworm

EC50 E ffect C oncentration /50 percent of the test population

EPA The United States Environmental Protection Agency

GLP G ood L aboratory P ractices

IRM Integrated Resistance Management

LC50 L ethal Conc entration/ 50 percent of the test population

NCR Northern Corn Rootworm

NIS Node Injury Scale

PI Personal Injury

PIPs Plant Incorporated Protectants

USDA The United States Department of Agriculture

WCR Diabrotica v. virgifera (Western corn root worm)

## SAP Minutes No.2014-01

A Set of Scientific Issues Being Considered by the

Environmental Protection Agency Regarding Scientific

Uncertainties Associated with Corn Rootworm

Resistance Monitoring for Bt Corn Plant Incorporated Protectants (PIPs)

December 4

- 5, 2013

FIFRA Scientific Advisory Panel Meeting

Held at the

Environmental Protection Agency Conference

Center

FIFRA Scientific Advisory Pane l
Date: March 4, 2014 

## Arlington, VA

Fn

.. !Q Jenkin s,**Jr.~** Ph.D.

De s ignated Federal Otlicial

F IFRA Scien tific Advisory Panel

Date: l-1arch 4, 201467 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP)

Scie ntific Uncertainties Associated with Corn Rootworm Resistance Monitoring for Bt Corn Plant Incorporated Protectants (PIPs)

December 4

- 5 , 201 3

PARTICIPANTS

FIFRA SAP Chair

Daniel Schlenk, Ph.D. Professor of Aquatic Ecotoxicology & Environmental Toxicolo gy Department of Environmental Sciences University of California, Riverside Riverside, CA

Designated Federal Official

Fred Jenkins, Jr., Ph.D. U.S. Environmental Protection Agency Office of Science Coordination & Policy FIFRA Scientific Advisory Pan el EPA East Building, MC 7201M 1200 Pennsylvania Avenue, NW Washington, DC 20460

Phone: 202

- 564

- 3327

Fax: 202

- 564

- 8382 Email: jenkins.fred@epa.gov

FIFRA Scientific Advisory Panel Members

K. Barry Delclos, Ph.D. Pharmacologist

National Center for Toxi cological Research U.S. Food and Drug Administration Jefferson, AR8 James McManaman, Ph.D.

Professor & Chief Section of Basic Reproductive Sciences Department of Obstetrics & Gynecology, Physiology & Biophysics University of Colorado, Denver

Auror a, CO

Prakash Nagarkatti, Ph.D.

Vice President for Research Carolina Distinguished Professor University of South Carolina Columbia, SC

FQPA Science Review Board Members

Carlos Blanco, Ph.D. Biotechnologist US Department of Agriculture Animal and Pla nt Health Inspection Service Riverdale, MD

Shelby J. Fleischer, Ph.D. Professor of Entomology Pennsylvania State University University Park, PA

Susan T. Ratcliffe, Ph.D.

Director North Central Regional Integrated Pest Management Center Department of Cro p Sciences University of Illinois at Urbana

- Champaign Urbana, IL

Mark Sears, Ph.D.

Professor Emeritus Environmental Biology University of Guelph Guelph, Ontario, Canada

Stefan Toepfer, Ph.D. Integrated Crop Management Advisor / Biological Control Research Scientist CABI Europe

– Switzerland Hodmezovasarhely, Hungary9 INTRODUCTION

On December 4

- 5 the FIFRA Scientific Advisory Panel (SAP) met to address scientific

issues associated with the “ Scientific Uncertainties Associated with Corn Rootworm

Resista nce Monitoring for Bt corn Plant Incorporated Protectants (PIPs) .” As part of the Insect Resistance Management program for Bt corn Plant Incorporated Protectants (PIPs), registrants are required to conduct annual resistance monitoring of the key target

in sects. Resistance monitoring for corn rootworm (CRW) has been beset by a number of technical challenges. The PIPs registered for control of CRW are considered “non

- high dose,” meaning that a proportion of even a susceptible population can be expected to

s urvive exposure to the Bt toxin(s). Opportunities for monitoring investigations are limited because CRW have one generation per year, undergo an obligate diapause period, and can be difficult to maintain in laboratory environments. Testing with artificia l diet bioassays (as is done for lepidopteran pests of Bt corn) has yielded highly variable results which have been problematic to interpret. Taken together, these factors have complicated the establishment of a workable (regulatory) definition of “resist ance” for CRW (based on bioassay results). The Panel provided recommendations to EPA on how to improve the resistance monitoring program for CRW by addressing the scientific uncertainties

associated with this insect. Specifically, the Panel addressed the EPA’s charge to them on questions regarding the following topics: population sampling (random vs. focused), triggers (i.e., field damage to Bt corn) for investigations of potentially resistant populations, bioassay techniques, defining resistance (in the context of bioassay results), and remedial action plans in the event of resistance to contain or limit the spread of resistant populations. Opening remarks at the meeting were provided by Steven Bradbury, Ph.D., Director, Office of Pesticide Programs

, an d Robert McNally, Director, Biopesticides and Pollution Prevention Division (BPPD), OPP

.

US EPA presentations were provided by the following staff:

Alan Reynolds

Jeannette Martinez10 Public Comment er s

Oral Public comments were provided by ( provided in alphabetical order ) Analiza Alves, Ph.D. on behalf of Pioneer Hi

- Bred International Tony Burd, Ph

. D

. on behalf of the Agricultural Biotechnology Stewardship Technical

Committee Graham Head, PhD on behalf of the Monsanto Company Gregory Jaffe on behalf of the Biotech no logy Project Center for Science in the Public

Interest Clinton Pilcher, Ph.D on behalf of Pioneer Hi

- Bred International Inc. Tony Burd, PhD, Syngenta Jim Zimmerman, National Corn Growers Association

Written Public Comments were pr ovide by (provided in alphabetical order) The Agricultural Biotechnology Stewardship Technical Committee

Martin R. Barbre on behalf of the National Corn Growers Association Tony Burd, Ph.D. and Dennis P. Ward, Ph.D. on behalf of Syngenta Seeds Inc.

Christi an Krupke, Ph.D. Purdue University, West Lafeyette, IN Chris DiFonzo, PhD. of Michigan State University et al

. Center for Food Safety Aaron Gassmann, Iowa State University Michael E. Gray, Ph.D. of the University of Illinois, Urbana

- Champaign Graham Head, Ph.D., Monsanto

Monsanto Company

. Clinton D. Pilcher, Ph.D. on behalf of DuPont Pioneer

Pioneer Hi

- Bred International, Inc.

Center for Science in the Public Interest Nicholas Storer, Ph.D. on behalf of Dow Agro Sciences John F. Tooker, Ph.D of the Pennsy lvania State Extension Pennsylvania State University11 SUMMARY OF PANEL RECOMMENDATIONS

Charge Question 1 a

. Th e P a n e l is as k e d t o c o mm en t o n sam p li n g a pp r o a c h es fo r

c o n d u c t i n g a n nu al CR W r e sis t a n c e m on i t o ri n g t h at s u p

po r t e arly r e sis t a n c e d e t e c t i o n

. P l e ase d is c u ss t h e s t r en g t h s a n d li m i t a t i on s o f B PPD ’s p r o p o sal fo r a f o c u s e d ( risk

b as ed ) sa m p l i n g a p

p r o a c h f o r th e C o rn B e l t

, s up p l e m e nt e d w i t h sam p les f r o m l o w e r risk

“ f

ri n g e ” ar e as f o r c o m p a ris on

.

Panel Summary

T he Panel concurs with the Ag ency’s recommendation to expand the sampling area to include other areas with greater than expected damage including Wisconsin, South Dakota, Colorado

, and Minnesota ( BPPD 2011, 2012 ). The Panel also concurs with EPA’s primary objectives for sampling that include : 1) a ctively seeking out areas of

concern ; 2) s elect ing annual sampling locations based on risk factors ; and 3) i dentif ying

resistance issues earlier through more proactive monitoring in high risk areas. The Panel recommends that these high risk areas should entail : 1) c lose proximit ies to reported field

failures ; 2) c ontinuous corn production areas ; 3) fields that do not rotate the Bt toxin mode of action; and 4) fields that are in n on

- compliance with refuge requirements

. One panel member mentio ned that sampling according to the afore mentioned four

recommendations should ( although being focused ) still assure a certain spatial coverage (e.g. systematic grid of survey with focused sampling in each grid). The Panel disagrees

that “ fringe ” areas ar e more appropriate for the random sa m pling approach as these areas may not be lower risk depending on production practices ( John Tooker Public Comment

, Public Docket ID no: EPA

- HQ

- OPP

- 2013

- 0490

- 0038 ; Tooker and DiFonzo 2013).

Additional recommendations fro m the Panel include : 1) a focused approach to track susceptibility of individual populations or discrete geographic locations as this approach will be more likely to identify hot spots and may reduce the likelihood of area

- wide resistance ( BPPD 2012) ; 2) a proactive approach to monitoring for greater than expected damage earlier in the season by identifying high risk areas of continuous corn production or areas that lack rotation of Bt toxin modes of action based on sales records; 3) high risk (corn

- on

- corn , continuous use of the same trait) areas within 4 or 5 zones should be the only target for annual sampling, eliminating random sampling. The “fringe” zones/regions should be considered as one of the above zones and sampled in areas of highest risk; 4) in cluding n on

- compliance with refuge requirements as a factor for high risk classification since lack of refuge compliance may result in increased resistance in

areas that are not currently considered high risk ; 5) increasing the amount of acreage surveyed r egarding refuge compliance; 6) modifying the regional boundaries used in the Arent Fox surveys to better refle ct c orn rootworm population varia tions in species and resistance traits to better reflect field conditions; and 7) that the revision of “regions” boundaries for the Arent Fox survey also be applied to the sampling regions to provide consistency to allow better comparison of data obtained from surveys, sampling and greater than expected damage reports. The current sampling approach is likely to

misr epresent potential resistance since the sampling is random and populations are not

tracked over time for shifts in susceptibility. T his coupled with the associated delays in12 susceptibility studies could allow for dispersal of resistant CRW resulting in ar ea

- wide resistance prior to identification of a resistance concern.

Charge Question 1b

. H o w ma n y CR W p o pu l a t i on s s h ou l d b e c o lle c t ed f r o m w i th i n a

sam p l i n g re gi o n t o a d e q u a te ly ass e s s s u s c ept i b ili t y ?

Panel Summary

The Panel recognizes that when collecti ng samples from fields with greater than expected damage, the population samples must be adequate to generate enough larvae to

conduct all assays (diet and on

- plant assays) used to evaluate resistance

. The Panel

believes the current approach of sampling 1 2

- 15 CRW populations mostly from Illinois Iowa and Nebraska seems very low in light of the number of reported cases of suspected resistance and/or greater than expected damage ( BPPD 2011 )

. The current approach of a minimum of 2,000 adults collected for ea ch population ( EPA 2013 ) is likely not enough

.

The Panel makes the following recommend a tions : 1) collect a minimum of 4, 000 adults per population ( T his recommendation is based on the presumed resistance allele frequency for CRW ) ; 2) expand areas of ann ual sampling to include additional “high

risk” locations in W isconsin , S outh D akota , C olorado and M innesota ; and 3) annual

collections of 10

- 15 populations by each registrant from the same locations in each of the 4 or 5 zones and bioassays conducted on al l four traits presently registered.

Charge Question 2 a

. ( C o rn R o o t w o rm Sa m p li n g in R e s p on se t o D a mag e d B t F i e l d s)

Th e P a n e l is as k e d t o c o mm en t o n m e th o d s fo r i n v e s t iga t i n g CR W po p u l a t i on s c a u si n g

u n e x p e ct e d d amage t o B t c o r n

. S pe c i f

i c all y
, p lea s e c o mm e n t on :

i. Th e u se o f f ie l d d amage ra t i n gs ( N

IS) as a s c r e e n f o r p ot e nt ially r e sis t a n t

p opu l a t i o n s. W h at sam p li n g t rigg e rs s h o u ld b e u s e d fo r si n gle t o x in a n d p y rami d e d B t

p r o d u cts? S h o u ld al te r n a t e te c hn i qu es b e c on si d er e d?

The Panel agrees that the li kelihood of reports describing fields with unexpected damage will be increasing in areas where a single gene Bt product has been continuously used and where fields sown to corn following one or more years of corn has occurred. The

Panel believes that a tr igger level for further sampling of the CRW population is when a sample of corn roots produces a Node Injury Scale (NIS) rating of 1.0 for single gene expression Bt products and 0.5 for pyramided products. The Panel discussed the logistical problems of re quiring more adult sampling and larval bioassays, but concluded it is better to identify resistance in populations in “ high risk ” areas at this time so that mitigation practices can begin in those areas as soon as possible.

ii . Th e u se o f t r a n s e c t sa m p li n g in d a m ag e d ar e as o r ra ndo m sam p li n g th r ou g ho u t th e

a

f fe c t e d f ield t o ass e ss r o o t d am a ge ra t i n gs.

The Panel recommends that transect sample s be taken across the reported damaged area to help determine if resistance of the CRW population is the cause an d other factors that13 may have led to a “ hot spot ” of damage are ruled out. A transect sampling scheme is more likely to identify the severity of damage in the damaged area and, in high risk areas

which frequently lead to adult sampling as a follow up proc edure. The Panel believes it is important to reduce the frequency of false negative determinations regarding damage and subsequent resistance in CRW populations in these areas.

iii. A pp r o p ri a t e sa m p li n g l o c a t i on s ( i

. e

. , in t h e vi c i n i t y o f t h e d amage a n d /

o r s u rr o u nd i n g

a r e as) fo r c o l l e c t i o n s o f a du l ts i f

f

i e ld d amage t r igg e rs a r e e xc eed e d.

The Panel agrees that samples of adult CRW should be obtained from areas as close to the reported and identified damaged fi el d as possible. Samples should be taken as soon as feasible after damage has been determined to be greater than expected

. Clearly, because reporting of damage often occurs well after the emergence of adult beetles, samples of adults in the immediate area may not reflect the emergence of individual s responsible for the damage. In addition, beetle density may be quite low in the field and surrounding areas. Sampling for adults more than 1 mile from the damaged area simply reduced the likelihood that the population collected represents the populatio n that was responsible for the damage noted. In cases where sufficient beetles cannot be collected, a

follow

- up sample is required the following year from the same area the damage was

noted. The Panel recommends a dult sampling data should include signifi cant detail about the field history ( such as information on the field’s rotational crops, Bt expressing hybrids, use of chemical pesticides, etc.) and relation to other neighboring crops and the number of beetles collected, their sex ratio and the number o f gravid individuals.

iv

. Th e d e p l o y m e n t o f s en t i n el p l ots in t h e vi c i n i t y o f d amag e d f i e l d s in s ub s e qu e n t

s e as on s t o : 1 ) ass e ss t h e r e sis t a n c e a llele f r e q uen c y i n th e ar e a, a n d/o r 2 ) c o lle c t i n s e c ts if n o a d u l ts w e re p r e s e n t at t i m e o f t h e f ield i n v e s t i ga t i on

.

When sufficient adult CRW cannot be collected from fields with documented damage in the same season, the Panel agrees that beetles should be collected as soon as possible after their emergence the following year in the same location. This collect ion can be facilitated by the use of sentinel plots of untreated corn (or a surrogate crop like squash or pumpkin ). The reality that beetles collected from these plots may not represent those that caused the damage noted the previous year is acknowledged by the Panel. However, th is sample method is the best alternative approach for a follow

- up beetle collection.

The Panel recognizes that sentinel plots may concentrate resistant individuals that subsequently can spread considerably and increase resistan ce in the area

, but could also serve as a refuge for susceptible beetles that could subsequently dilute the resistance genes in local populations of CRW. A sentinel plot in the same area where damage was

documented the previous year planted with a non

- Bt similar maturity corn hybrid would serve as the best choice for collecting beetles the following year.

If sentinel plots were planted each year in “ high risk ” areas to serve a s sources for annual collections of adults for subsequent bioassays a rough esti mate of the change in allele frequency could be obtained by comparing bioassay results over a number of years.14 Currently, our collective knowledge of resistant allele frequency in CRW populations is limited so a more appropriate methodology is unavailable

.

Charge Question 3 a (D iag no s t ic Assa y s)

. P lease c o m m e n t o n th e s t r en g th s a n d

lim i t a t i on s o f d iet b i o assay m e t h o do l o gies fo r e arly r e sis t a n c e d e te c t i o n w i t h CR W,

c on s i d eri n g t h at th e c u rr e nt l y

- regis t e r ed to x i n s a r e less t h an h i g h do s e

. W h a t

i m p r o v e m e n t s c ou l d b e m a d e t o t he se b i o assa y s t o ma k e t he m m o re e f f

e c t ive a n d

p r o a c t i v e r e si s t a n c e dete c t i o n t oo ls?

Panel Summary

Properly conducted bioassays with artificial insect diet can provide more precise and consistent responses to screen for resistance to Bt

, than current on

- plant assays. Results

from these arti ficial diet bioassays have been established using standard statistical procedures recognized by the scientific community , including most of the parameters

used to evaluate on

- plant bioassays

. Monitori ng efforts conducted with artificial diet bioassays can occupy less space, can yield results in a few days, and be more

economical than the current on

- plant screening

. This is an advantage for most of the research facilities and for IRM mitigation attempt s.

Preliminary lethal (LC 50 ) and effective concentrations (EC 50 ) values have been already established for some of the Bt proteins used to control CRW spp. However, the Panel believes that these methods require further refinement to continue serving as hi storical

records document ing the trends of susceptibility over time. S usceptibility to Bt at a single location can be more accurately followed over time with artificial diet bioassays

than with on

- plant bioassays because of the more precise nature of the assay and the breadth of data they provide

. A single well

- maintained stock of purified Bt toxin can last a long time and perform more uniformly than plants where their Bt protein expression can be influenced by environmental and biotic factors and the cor n hybrid genetic background.

The Panel recommends keeping the bioassay approach with artificial diet to continue building some of the already observed trends, and share the results among all appropriate researchers, both public and private. Consistency i n methodology (e.g.: same type of diet, reference laboratory colony, batch of Bt toxin, proper handling of neonates, etc.), is a necessity for all types of monitoring bioassays. A solid assay can accommodate in a single replicate plate 5 or 6 conce ntratio ns of 3 different Bt proteins, providing invaluable

information. The Panel also suggests developing a standard diet where neonates can survive on assays beyond 3

- 4 days with greater than 80% survival.15 Charge Question 3b. Th e P a n e l is as k e d t o d i s c u ss t h e r e l a t i v e m e ri ts a n d l i mi t a t i on s o f

t h e tw o on

- p l a n t assa y s ( Gassma n n e t al. 20 1 1 a n d No w a t z k i e t al. 2 008 )

. P l e ase d is c u ss

t h e e x t e n t t o w h i c h the se assa y s h ave d i f f

e r e n t s e n si t i v i t ies t o ma k e e arly c o rn r oot w o rm

r e si s t a n c e d e te rm i n a t i o n s? S hou l d o t h e r o n

- p la n t a s say a pp r o a c h es b e c o n s i d er e d?

Panel Summary

The Gassmann et al. (2011) approach represents a more realistic situation of field

conditions than the Nowatzki et al. (2008) method and the artificial diet bioassay s

. The

Gassmann method may be both easier to follow by others and to confirm res ults independently, expand ing the monitoring effort by the possible inclusion of independent

scientists. However, this procedure may benefit from investigating larval behavior and mortality by inspectin g the root system over a range of 3 to 17 days after egg hatch to
determine i f there is a more optimal assay time that can yield more accurate results

. Perform ing an intense/destruc tive root sample , accounting for the total number of surviving la rvae durin g the recommended period of time and not rely ing entirely on the

larvae collected in alcohol vials after the funnel extraction, may be a great improvement

of the method

. Confirmation of the transformation event in the Bt hybrid tested and expression of th e protein in the roots is necessary to assure that the corn plants are indeed the correct hybrids. Efforts should be taken to obtain corn seed free from pesticide coating. High larv al mortality in the soil makes analysis more difficult and problematic,

i mpeding potential Bt resistance to be documented correctly. The use of 10

- 20 neonates per plant, may present a serious constrain t to the met hodology.

The Nowatzki et al. (2008) methodology, consisting of “ greenhouse plant efficacy

, ” a nd

“ sub

- lethal seed ling ” components, has greatly improved since its publication, as was

evident from the public comments during the meeting

. This method is e asier to standardize because corn seedlings are grown in containers under more consistent conditions inside a growth chamber and not in a greenhouse, also preventing possible insect escapes from the experimental unit. G rowth of seedlings may produce an expression of protein more consistent between plants, although not nec essarily similar to

field conditions. C ross

- bree ding the collected WCRs with a non

- diapausing colony enables testing over a much shorter period of time. The Panel suggests that research

efforts are continued to reduce the amount of variability in the assays, to test these methodologies with corn events containing Cry3Bb1 protein, and to develop a more Bt resistant WCRW colony

.

Gassmann et al. (2011) and Nowastzki et al. (2008) report that: “

... surviving larvae were

collected in vials with alcohol

. ” Assuming that a particular population has an unusual

hi gh survivorship, the Panel recommends collecting live samples and maintaining part of the sample in artificial diet to continue with studies to confirm resistance by other

methods. The surviving colonies would re present a great opportunity to learn more a bout the biology, behavior and genetics of the CRW

. The Panel also recommends evaluating both Gassmann and Nowatzki methodologies with pyramided events, as well as with single

- protein events separately. In both methodologies, it is necessary to obtain16 in formation about expression of the Bt toxins in the root system at different time

intervals.

Charge Question 4a

. Th e P a n e l is as k e d t o d is c u ss t h e m e r i ts a n d s ho r t c o mi n gs o f t h e

p r o p o s e d a p

p r o a c h es t o d e f

i n i ng r e sis t a n c e u si n g o n

- p l a n t assa y s. W h at s et s o f

c o m p aris on s in t h e assa y s are m o s t li k e ly t o a d d va l u e t o a w e ig h t

- o f

- e vi d en c e a pp r o a c h

t o d e t e rmi n i n g r e sis t a n c e ?

Panel Summary

Gassmann et al. (2011) and Nowatzki et al. (2008) methods measure the response of the western corn rootworm larvae to corn p lan ts that express Bt protein(s). However,

l imited/declining expression of Bt in the root system is one of the most crucial limitations to these methodologies

, a nd a better understanding of the amount of Bt protein that the larvae are exposed to through t ime, may facilitate the interpretation of

results.

Because the concentration of all the events in the currently commercialized corn hybrids

do not meet the definition of “ high dose, ” the Panel believes that the current scoring criteria of developmental i nstar proportion of surviving larvae, provides the least variable criteria to discern between putatively resistant and susceptible WCR ( Diabrotica v. virgifera ) larvae.

Charge question 4b

. W h at r e sis t a n c e all e le f r equ e n c y s h o u ld c on s t i t u t e f ield r e sis t a n c e fo r to x i n s w i t h l e ss th a n h igh do se e x p r e ssi o n ? P l e ase d is c u ss th e c ri te r ia th a t s hou l d b e

u s e d fo r t h ese t y

p es o f to x i n s ( as opp o s e d t o h igh do se t o x i n s) giv e n th a t a po r t i o n o f

h e te r o z y g ou s i n s e c ts w ill s u rvive B t e x po s u r e a n d d rive t h e e v o l ut i o n o f r e si s t a n c e

.

Panel Summary

An effective method to discern Bt resistant CRW from susceptible CRW impeded the establishment of a Bt resistance frequency prior to the release of the Bt corn events. Therefore, the resistance mechanisms in the putatively

- resis tant CRW colonies have not been thoroughly characterized, making it difficult to establish a resistant allele frequency that should trigger an action by the US E.P.A. The Panel believes that this question does not have a valid scientific answer and opens the possibility for all kinds of speculation.

Charge question 4c. W h at s t a t is t i c al te s ts, c r i te ria, a n d sig n i f

i c a n c e le v e ls w ou ld b e be s t

s u i t e d f o r e a rly r e sis t a n c e d e te c t i o n w i t h t h e p r o p o s e d assa y s? P lease d is cu ss ho w t o b e st

av o id f al s e ne g a t i v e s a n d /

o r f alse po si t i v e s.

Panel Summary

Probit analysis applied to mortality and larval development instar data obtained in bioassays with insect artificial diets, as well as assessments of the number and developmental instar of surviving larvae at different p eriods of time for on

- plant assays,17 seem to be the most adequate methods of analyzing results at the moment. The Panel suggests that bioassays with insect artificial diet should include consistency in terms of methodology, testing materials and reference colonies. Using multiple metrics ( e.g. survival, instar development, body size and weight, feeding behavior, root tissue ingestions and exploring gene expression) could be used to reduce the likelihood of false positives and false negatives. Allowing for comparisons between sampled population on

Bt and non

- Bt corn plus comparisons between sampled populations and susceptible populations (laboratory colonies) will add another indicator.

Charge question 4d. P lease c o m m e n t o n th e e x te n t t o w h i c h i n c o m p l et e r e sis t a n c e c an

b e i d e nt i f

ied w i t h o n

- p l a n t te s t s y s te ms. H o w s h o u ld r e sis t a n c e d e f

i n i t i on s b e a d j u s t e d

t o a d d r e ss the se s c en ari o s?

Panel Summary

CRW p opulations with incomplete resistance may be more fit than the control population on Bt corn, but may no t be significantly different. I f there are fitness costs for Bt resistance, the resistant population may not survive on non

- Bt cor n as well as the control colony, or delayed development may occur indicated by reduced body weight/head capsule size of resis tant larvae exposed to Bt corn

.

Resistance definitions should consider that c orn rootworm may have developed more than one response to survive on Bt corn

, complicating the use of a single diagnostic tool

.

Charge question 4e. P lease d is c u ss t h e via b il i t y o f r e sis t a n c e r a t i o s as a n o pt i o n fo r
d e te rm i n i n g r e sis t a n t p o pu l a t i o n s, c on si d er i n g th e g en e rally l o w s u s c e p t i b il i t y o f C R W

t o B t to x i n s a n d t h e l a c k o f s u s c e p t i b l e w il d t ype po p u la t i on s ( i.e., du e t o w i d es p r e a d

a do p t i o n o f B t c o r n )

. W h at ra t io c o u ld b e c on s i de r e d as an i nd i c a t o r o f r e sis t a n c e f o r

c o rn r o o tw o rm u si n g o n

- p l a n t assa y s?

Panel Summary

Different resistance ratio proposals need to be reconsidered for the current situation of

the CRW d ue to the lack of Bt corn events expressing a “ high do se. ” T he refore the

resistant ratio would need to be ~4x for the CR W for non

- high dose corn events. The re is limited historical data of the response of this insect that may serve for the calculation

of these ratios

.

CRW populations obtained from “fringe” are as may serve as “ control ” population s upon which other populations from “high risk” areas and fields with unexpected damage could be compared. Laboratory populations may be appropriate for a few years

, but concern about inbreeding and loss of behavioral tr aits reflective of field populations may be a limiting factor. The Panel believes that Bt susceptible CRW populations can be obtained in areas where the selection pressure has not been so intense such as in Europe, or from an organic corn grower with a la rge enough farm area, in the periphery of the US18 cornbelt. Also, surrogate species such as Diabrotica virgifera zea , the Mexican corn rootworm, can serve as a good indicator for susceptibility, especially if the samples are

obtained in areas where Bt corn is not registered (e.g.: Mexico).

Charge question 5a. P lease c o m m e n t o n th e s t r en g th s a n d lim i t a t i on s o f

B P P D ’s p r o po sal t o u s e r e sis t a n c e al l e le gra d i e n t s t o d e f

i n e t h e g eo gra p h ic

e x t e n t o f a r e s is t a n t p o p u la t i o n

.

Panel Summary

Mapping allele gradient s would be an attractive option if the alleles that confer resistance were well defined, and technique s existed to take m any samples from many locations in a timely fashion

. Unfortunately, resistant alleles have not been defined. Furthermore, allele pres ence does not always result in phenotypic expression of resistance. It is

feasible that the allele(s) is detected

, but not expressed in a manner that confers

resistance. A limiting factor is that resistance may be conferred via more than one allele

, and t he relative importance and interactive contributions of these alleles to resistance is

not known. Thus, it is not clear if mapping the gradient of a single allele, a small subset of alleles, or many alleles, would be necessary for inferring maps that ref lect the probability of resistant phenotypes

. Another limitation is that sample collections could be

difficult because ab undances of adults could be low or absent at the time collections are required. If w hen the constraints of defining what allele or al leles confer resistance, and

the sampling issue are resolved , the Panel recommend s that map ping the presence and frequency of those alleles should be pursued. However , other allele

- based studies using neutral genetic markers, discussed as part of the resp onse to question 5(b), could be developed to infer geographic dispersal of populations from locations where resistance is suspected.

Alternatively, it is currently feasible to measure abundance. This can be done a priori , in

areas of high rates of adopti on, and sample resolution guided by othe r data layers that suggest risk. Information could be obtained within a time frame to influence decisions about deployment of Best Management Practices (BMPs) that are currently deployed in response to suspected res istance

. Although BMPs may, or may not, influence the frequency of resistant alleles at a given location, t here is an inherent assumption that BMPs can reduce the probability of widespread Bt failure when deployed in response to a problem inquiry field. BMPs help achieve sustainability of Bt crops both at that location and at wider geographic scales, through their influence on population processes. Mapping abundances may help define the spatial scale at which these population processes are being expresse d , and test s for spatial dependence in abundance after factoring out other factors (weather, field management, etc.) that contribute to abundance may help define if problem

- inquiry fields are arising independently, or if they are more likely to arise close to nearby problem

- inquiry fields. The Panel recommends mapping population density gradients as part of Integrated Resistance Management ( IRM )

.

The Panel also recommends clarifying the goal of remedial action, and definitions that enable distinguishing a localized resistant population (a “hot

- spot”) from area

- wide19 resistance. These clarifications are necessary in defin ing the geographic extents of sampling gradients and in using gradients to define remedial action areas. Remedial

action aimed at elimi nating a resistant population would entail much longer gradients than those designed to limit and contain allelic frequencies or population abundances below predetermined thresholds. Clarifying these definitions is a necessary component of using gradients to define geographic extents of resistant populations.

Charge question 5b. W h at o t he r to o ls o r s t r a t e gi e s c ou l d b e e m p l o y e d t o d e f

i n e t h e

r e m e d ia t io n z on e?

Panel Summary

The Panel notes seven other strategies to help define remediation zones. The prim ary suggestion is to adapt from studies that delineate population structure using neutral alleles, such as those that define hybridization zones in northern Italy. Although alleles subject to selection such as those conferring resistance may not follow th e same pattern as
those detected using neutral markers

, they are the most closely related to this question of introgression of alleles among populations. If similar genotypic diversity exists in areas

of the US , then similar methods could be developed for areas of adoption. Additional strategies buil d upon field measures of abundances or bioassays, or landscape and management practices. Thus, a second strategy is to utilize dispersal kernels recently

modeled with mark

- recapture field experiments or disper sal rates based on historical patterns of abundances during early colonization. A third strategy is to utilize dispersal distances modeled from the introgression of soybean

- rotation

- resistant variants. A fourth is to spatially structure the sampling in r esponse to damage, and testing offspring to

define remedial action zones. The Panel also notes that the bioassay data provided in this

review showed temporal trends in survivorship or development rates

. I n some locations, approximately a county spatial scale, these temporal trends were positive. Thus, a fifth approach is to use spatially

- referenced temporal trends in bioassay data to help define spatial location s that could be defined as a remedial action area. A sixth approach is to develop risk maps b y categorizing relative risk for a field or land area based on expert opinion, analysis of past Personal Injury ( PI ) fields, lack

- of

- rotation, repeated use of the same trait, weather conducive to CRW development, regional corn acreage and corn density in a landscape, and other factors, and use these maps to influence remediation

zones. A seventh method is to map the probability of exceeding thresholds using presence/absence categorical data of population abundances or frequencies of specific

alleles.

T he P anel concurs with the EPA that “A primary goal of resistance monitoring is to

detect shifts ( in space and time ) in the frequency of resistant alleles (i.e., susceptibility changes) before the onset of resistance leads to widespread Bt failure.” However, the goal as stated is not feasible because we are unable to measure allele frequencies or changes in susceptibility with sufficient precision. T he Panel believes that the goal should be restated, to include metrics that we have a capacity for measuring w ith sufficient precision and in a timely fashion. Currently, the only such metric is abundance,

and some members of the P anel believe that monitoring abundance should be added,20 although not all agreed that monitoring abundance directly related to monitori ng resistance. A suggested restatement is: “A primary goal of monitoring is to detect shifts ( in abundance, susceptibility, and/or resistant allele frequency ) before the onset of resistance leads to widespread Bt failure.”

The Panel notes that the scale of remediation following the establishment of resistance, which ranged from 20 to 50 km radii based on the information available to date, may not

be feasible. Thus, achieving resistance management should focus on proactive options that avoid or delay the establishment of resistant alleles in CRW populations.

Charge question 6a. W h at r e m e d ia t i o n a p

p r o a c he s c ou l d b e t a k e n fo r l o c ali ze d vs. ar e a

w i d e re sis t a n c e s c e n ari o s?

Panel Summary

The Panel concur s with the concern stated in the EPA ( 2013 ), “...action able thresholds may not be met ( or recognized ) until resistance is widespread and effective mitigation (reducing resistant allele frequency) is impractical. In this case, managing resistance through population suppression may be the only alternative.” Th e Panel also concur s with the statement in the EPA White Paper that “BMPs ... likely need to be implemented ... irrespective of whether resistance testing has been completed (US EPA, 2013)

. ” The context for that statement dealt with responding to a problem in quiry field. The Panel

recommends widen ing the context to deal with factors that increase the risk of becoming

areas with the emergence of resistance. The Panel recommends a proactive effort of managing resistance through population suppression achieved t hrough the implementation of an integrated pest management (IPM) plan using best management practices

.

The Panel recommends developing BMPs that will address variation in production practices, species behavior and resistance to chemical and cultural pract ices such as continuous corn, rotation resistance

, and production practices. While the Panel anticipate s an overlap in BMP recommendations, the Panel believe s it is necessary to consider the population and species differences along with differences in pro duction practices and landscapes when developing the BMPs. The Panel recommends the Agricultural Biotechnology Stewardship Technical Committee (ABSTC) Insect Resistance Management Stewardship Subcommittee, the National Corn Growers’

Association and state

- based corn associations in cooperation with research and Extension

scientists and growers develop an IRM plan and BMPs using an IPM approach that would be implemented prior to suspected resistance (proactive) to reduce the likelihood of heavy selection pre ssure on corn rootworm by using the same mode of action over an extended period of time as resistance has been shown to develop within 3 to 7 years of

use in continuous corn. The Panel recommends inclusion of social scientists into the process to increase the understanding of human behavior with regard to production practices and decision

- making. The Panel note s that Extension entomologists are already developing region

- specific recommendations , for example, for the eastern corn

- growing states ( Indiana, M ichigan, New York, Pennsylvania, Ohio, Ontario) ( Tooker and DiFonzo21 2013). The Panel recommends the implementation of the proposed BMPs in advance of suspected resistance.

The Panel believe s using the IPM approach as part of BMPs may reduce the need fo r remediation although the Panel recognize s that not all producers will choose to use the BMPs as part of the production plan unless required. W ithout the required implementation of BMPs, the Panel concurs with EPA that under the current generic

remediati on approach too much time may pass before identifying resistant populations.

The Panel recommends that remediation plans be in place for all licensed products taking into consideration geographical location of the field , production practice, population

re sistance

, and species’ behaviors.

The Panel support s the most conservative remediation plans as outlined by EPA including the recommendation of the use of conventional insecticides to control the adult stage during the present field season and then selec t an alternative pest control method the following season to reduce the establishment of resistant populations , but only as a last resort to control the spread of resistant populations and not as a population abundance management tool

.

Charge question 6b

. W h i c h m i t iga t i o n m e as u r e s w ou ld b e m o r e e ffe c t ive in c o nt a i n i n g

a n d/ o r e x t ir p a t i n g r e sis t a n t CR W po p u la t i on s?

Panel Summary

To mitigate resistant populations, the Panel recommends the use of rotation with a non

host plant would be most desirable, but w ill not be effective in some geographic locations

due to rotation resistant CRW populations that will survive in first year corn by a shift in oviposition by WCR or by extended diapause in the case of N orthern C orn Rootworm (NCR)

. The Panel recommends the use of an alt ernative Bt toxin a s a viable option for rotation resistant populations

. The Panel has concern s with the recommendation to use a soil insecticide with the compromised Bt toxin as we believe this will allow resistant corn

rootworm to survive by feeding on Bt expressing roots outside of the application band of

the soil insecticide and exposing larval populations to both the compromised Bt toxin and

the soil insecticide in the band

. The Panel believes the use of a soil insecticide with a Bt hyb rid should not be done. The Panel only recommends the use of an a dult control

measure in fields with greater than expected damage during the current season in fields with resistant populations

. D ispersal to other fields, although limited, is still a conc ern as damage may not be noticed until harvest time.

To assist with earlier detection of greater than expected damage

, the Panel recommends the use of aerial monitoring using planes or drones in high risk areas especially following major weather events t hat may contribute to lodged corn. The Panel recommends development of a risk map that identifies : 1) the varying levels of greater than expected damage reports ; 2) crop production practices that contribute to resistance including the use of high dose see d treatments (may mask early resistance) ; and 3) a lack of refuge compliance.22 Charge question 6c

. P l e ase c o m m e n t o n th e val u e o f th e o r e t i c al m o d e ls in de sig n i n g

r

e m e d ial a c t i o n s t r a te gies f o r vari ou s r e si s t a n c e s c en a r i o s.

Panel Summary

CRW biology mod els are among the best developed that exist in agricultural entomology. One could reasonably argue that these models have done a good job projecting how varying management and initial biological parameters influence the emergence of

resistance. All these models show strong sensitivity to initial resistance allele frequencies, and functional dose (survivorship of heterozygotes on Bt

- corn). For some sets of parameters, models have also been predictive. W hen initial allele frequency is high, years

- to

- 50% re sistant allele frequency ranged from 5 to 8 years, which is a good approximation of the time when problem inquiry fields are being found.

We can combine what can be learned from the existing models with empirical information obtained during the years si nce commercialization to focus both future modeling and remediation strategies. First, the Panel believes that Bt resistant alleles are

not rare.

. Resistance has been selected relatively rapidly in laboratory studies. An independent line of evidence sug gesting that resistant alleles are not rare, presented by

Dr. Nicholas Miller , a faculty member at the University of Nebraska

- Lincoln, is based on the geographic distribution of problem

- inquiry fields, and contrasting that to the pattern

of rotation

- resist ant phenotypes. Therefore, the Panel must assume that presence of problems in the field is directly correlated to field

- evolved resistance. This assumption

may not always be correct, but it is a reasonable first approximation. The problem inquiry fields from Cry3Bb1 plantings show a pattern of sudden appearance in multiple locations with little to no spatial correlation. This is consistent with a process of independent selection of alleles that are not rare, and thus relatively easy to increase in frequ ency. In contrast, rotation resistance shows a pattern of being selected for in a discrete location, followed by diffusion from that location, and strong spatial dependence in the data ( Onstad et al. 1999). Often the published models start with a frequen cy of 0.001, and contrast scenarios with that as at least an initial baseline. The P anel

recommends focus ing on scenarios that show higher initial frequencies of r alleles, potentially several orders of magnitude higher.

Charge question 6d. Th e c u rr e n t d e te rm i n is t i c a n d s to c h a s t ic sim u la t i o n m o d els u s e d f o r
I R M pu r po s e s c o nt a i n m a n y o f th e fo l l o w i n g a tt r i b u te s: e c o l o g y
, p o pu l a t i o n b i o l o g y
, b e h avi o r, a n d g e n e t i c s o f p es t

, gr o w e r beh avi o r ( r e f

u ge c o m p li a n c e
, i n s e c t i c i d e s p ra y i n g,

et c)

, e x p li c i t s p a t ial a n d p r ob a b il i t y a n al y s e s. W h at o t he r m o d e li n g a t t ri b u te s w ou l d he lp

im p r o ve t h e a n al y sis o f r e m e d ia t i o n s t ra t e gies?23 Panel Summary

Although we have not exhaustively reviewed every CRW simulation model, the Panel recommends the following attributes be incl uded in simulation models :

1. Relatively high r

- allele frequencies as initial parameters.

2. Non

- random mating.

3. Multiple alleles

– polygenic (additive) resistance.

4. Pyramided traits with varying h (dominance) for each trait. Onstad and Meinke
(2010) suggest t he presence of single

- traits in the landscape reduce the benefit
that can be achieved with pyramids.

5. Varying deployments of trait packages in time: deploying each trait sequentially,

versus all

- at

- once in pyramided constructs.

6. Modeling the influence of BMP s on abundances and how that affects rates of
resistance across relevant landscapes.

In addition to simulation models, the P anel recommends statistical models describing population genetics and abun dance patterns measured in the C orn B elt

. The Panel

rec ommends that IRM models are designed to inform decisions about remedial strategies, and proactive IRM efforts . They should include the endpoints of abundance, in addition to the relative frequencies of genotypes, and couple that with efforts to measure

ab undances in the field

. This enables direct monitoring of the variable being modeled, which helps with model validation

. The Panel recommends widening the focus of both modeling, and IRM plans, to manage resistance with the use of BMPs

.

The Panel summari ze s conditions that make it unrealistic to rely upon refuges alone to achieve IRM for WRC given current Bt hybrids (adapted from Krupke

, 2012)

. :

1. Lack of a high dose: none of the traits provide a high dose. Th e survival rate reported in 2002 EPA SAP for MO N863 is 17% to 62% survival on MON868.

2. Frequency of r allele: they are not rare

3. Resistance is probably polygenic. There are multiple r alleles

4. Random mating is not occurring.

5. Cross resistance may exists between Cry3Bb1 and mCry3A

. 6. Ability to detect i ncipient locations of resistance and manage them to limit their spread is not working because :

a. LC 50 not reached until 2nd or 3rd instar (2002 SAP)

b. Bioassay problems, discussed in previous charge questions

c. Zero foot traffic following planting.

d. Lack of spa tial continuity in the occurrence of problem fields

e. Major disincentives for reporting damage

f. Measuring damage is difficult because it occurs underground

g. Method for measuring damage (NIS) is only poorly correlated to population abundance24 h. Rescue treatments d o not exist

i. Scouting is imprecise and limited

There is an implicit assumption that we can influence r

- allele frequencies and geographic distributions through implementation of BMPs

– this forms the base of utilizing BMPs following confirmation of resistan ce in problem

- inquiries. Models should focus on advancing our understanding of this assumption, with current estimates of allele frequencies and assuming polygenic resistant alleles. Questions that should be considered include: 1) What is the relationshi p between abundances and resistance

evolution? ; 2) How do BMPs (along with other management practices such as refuges and seed treatments) affect resistance evolution through their influence on abundance? Examples show that this modeling approach is feasi ble. Addressing how BMPs influence

both abundance and r

- allele frequency will require inputs defined by landscapes, which vary among regions of the corn belt. Movement among fields in relevant landscapes will

need to be considered. For example, young, re cently mated females are most likely to be moving among fields (based on captures from towers placed in fields, and sex

- ratios during early colonization). Outputs will probably need to vary across landscapes (as opposed to being expressed as a mean expect ation or distribution). This may require models to be developed in a GIS framework.25 DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE

CH A R G E Q U E ST IO N 1

A nnu a l C o rn R o o tw o rm S a m p li n g t o Ass e ss B t S u s c ept i b ili t y

As a c o n d i t i o n o f r e gis t ra t i on
, B t c o r n r e gis t r a nt s are r e qu i r e d t o c on d u c t a nn u al

sa m p li n g o f CR W po p u la t i on s f r o m l o c a t io n s in t h e C o r n B e l t t o m o n i to r f o r c h a n g e s i n s u s c ept i b ili t y t o B t to x i n s

. T h ese c o lle c t i on s are o b t ai n e d f r o m ra n do m l o c a t i o n s

w i th in t h r e e d e f i n e d r e gi on s c o v e r i n g d i f f

e r e n t w e s te rn c o rn r o ot w o rm b i o t y pe s. Th e

r e gi o n s also r e p r e s e n t a r e as w i t h h igh B t c o r n a d o p t i o n a n d CR W p r e ss u r e

. R e gi o n 1 ( r ot a t i o n r e si s t a n t v a ri et y ) i n c l u d e s I n d i a n a a n d e as t e rn Ill i no is; R e gi o n 2 (w ild t yp e)

c on s is t s o f w e s te rn Ill i no is, I o w a, a n d M iss o u ri; R e gi o n 3 ( o r g a no p ho s p h a t e r e si s t a n t
varia nt ) e n c o m p ass e s N e b ras k a a n d K a n sas. T y p i c all y
, 1 2

- 1 5 t o t al p o pu l a t i o n s a r e

c o lle c t e d e a c h y e ar, m o s t ly f r o m I o w a, Ill i no is a n d Neb ras k a.

In lig h t o f r epo r t s do c u m ent i n g f ield r e sis t a n c e t o C r y 3 B b 1 c o r n si n c e 2 00 9 ( Gassma n n

e t al. 2 011

, 2012a a n d 20 1 2b )

, B P P D c o n c l u d ed t h at t h e r a nd o m s a m p li n g a p p r o a c h u s e d

f

o r a n nu al m on i to r i n g a c r o ss the th r e e g eo g r a p h ic r e gi o n s o f t h e C o r n B e lt w as n o t

p r o a c t ive e nou g h fo r e a rly r e sis t a n c e d e t e c t i on

. I n s te a d

, B PP D r e c o mm e n d e d an i nt e n sive a n d fo c u s e d s a m p li n g a p p r o a c h

, in w h i c h c o ll e c t i on s s ho u ld b e o bt a i n ed f r o m

a

r e as e x pe ri e n c i n g B t c r o p f ai l u r e s o r r e g i o n s w i t h h igh risk f a ct o rs ( i

. e

. , c a u sal f a c to rs o f

r e si s t a n c e i n c l ud i n g c o n t i n u o u s u se o f c o r n

- o n

- c o rn w i t h t h e same B t t ra i t

, n o n

- r o t a t i on
,

o r p oo r c o m p li a n c e w i t h r efu ge r e qu i r e m e n t s )

. A pu r e ly ra n do m a n d l e ss i nt e n se

sa m p li n g a p p r o a c h c ou l d b e m a i n t ai n e d i n ar e as o f th e C o rn B e lt w h e re l i tt l e o r n o B t

c o r n f ail u r e s h ave o cc u rr e d a n d t h e se l e c t i o n p r e s s u re is l o w e r ( i.e. “ f ri n g e ” ar e as )

. Th ese a r e as c ou l d s e rve as a po i n t o f c o mp aris o n b et w ee n t h e h i g h er a n d l o w e r B t
sele c t i o n e n vi r on m e n t s, w h i c h w ou ld s u p p l e m e n t t h e b as e l i n e d a t a e s t a b lis h ed in t h e

e a r ly y e ars o f B t c o mm e r c iali z a t i on

.

a. Th e P a n e l is as k e d t o c o mm en t o n sam p li n g a pp r o a c h es fo r c o n d u c t i n g a n nu al CR W

r

e sis t a n c e m on i t o ri n g t h at s u p

po r t e arly r e sis t a n c e d e t e c t i o n

. P l e ase d is c u ss t h e

s t r en g t h s a n d li m i t a t i on s o f B PPD ’s p r o p o sal fo r a f o c u s e d ( risk b as ed ) sa m p l i n g

a

p

p r o a c h f o r th e C o rn B e l t

, s up p l e m e nt e d w i t h sam p les f r o m l o w e r risk “ f

ri n g e ” ar e as f

o r c o m p a ris on

.

The Panel is concerned that there are repeated reports of greater than expected damage in fields using Cry3Bb1 rootworm

- protected transgenic corn. Perhaps most troubling to the

Panel is that the re is a delay in identifying and reporting the failures

. This is evident

since most of the materials provided to the P anel are discussing the number of crop failures through 2010 (BPPD 2011 ). While it is possible to determine

add itional/continued failures of P lant Incorporated Protectants (P IPs ) with the Cry3Bb1

toxin from other resources , the documents provided to the P anel by the Agency do not provide an overview of the greater than expected damage for 2012

– 2013. If productio n practices that have proven to allow CRW to develop in

- field resistance have continued to26 be deployed, the number of greater than expected damage incidents are likely greater than currently documented

. Furthermore

, many fields with greater than expected damage may not have been identified/reported

. T hus

, in

- field resistance may be much greater than currently presumed in both continuous corn (Gassmann et al. 2011 ) and in first year corn in central and east central Illinois ( Gray 2013 ).

In regard to the strengths of the Agency’s sampling proposal, t he Panel concurs with the Agency’ s recommendation to expand the sampling area to include other areas with

greater than expected damage including Wisconsin, South Dakota, Colorado and Minnesota (BPPD 2011, 2012)

. Furthermore , t he Panel recommends a focused approach to track susceptibility of individual populations or discrete geographic locations as this approach will be more likely to identify hot spots and may reduce the likelihood of area

wide resistance ( BPP D 2012). The Panel also concurs with EPA’s primary objectives for sampling that include : 1) a ctively seeking out areas of concern ; 2) s elect ing annual sampling locations based on risk factors ; and 3) i dentif ying resistance issues earlier through more proa ctive monitoring in high risk areas. The Panel recommends that these high risk areas entail 1) c lose proximit ies to reported field failures ; 2) c ontinuous corn production areas ; 3) fields that do not rotate the Bt toxin mode of action; and 4) fields

that are in n on

- compliance with refuge requirements

. One panel member mentioned that sampling according to the above recommendation , although being focused, should still assure a certain spatial coverage (e.g. systematic grid of survey with focused sampling i n each grid). The Panel does not concur with the public comments specifying that “ f ringe ”

areas are more appropriate for the random sa m pling approach as these areas may not be lower risk depending on production practices ( Tooker Public Docket Id

- I D: EPA

- H Q

OPP

- 2013

- 0490

- 0038 ; Tooker and DiFonzo 2013).

The Panel recognizes that based on the timeframe of the reports of greater than expected damage, l ess than adequate samples numbers of CRW may be available due to production practices such as using soil inse cticides in conjunction with a Bt trait and conventional insecticides targeting adult populations

. The Panel recommends a proactive approach to monitoring for greater than expected damage earlier in the season by identify ing high risk

areas of continuous corn production or areas that lack rotation of Bt toxin modes of

action based on sales records. Despite proactive monitoring, o bservable damage and potentially resistant populations may be masked by use of insecticide at time of planting in conjunction wi th the continued use of a Bt hybrid with a seed treatment.

The Panel recommends that high risk (corn

- on

- corn, continuous use of the same trait) areas within 4 or 5 zones be the only target s for annual sampling . This recommendation

would eliminat e random sampling

. The “fringe” zones/regions should be considered as one of the above zones and sampled in areas of highest risk. Increasingly more “high risk” areas should be noted and examined. For example, in 2010, 27.7% of all corn planted in the U.S. foll owed corn in 2009 planted to the same acres; 11.3% of all corn acreage planted in 2010 followed corn planted in the same acreage for > 5 years previously (USDA

- NASS, 2012 ). The Panel considered and recommends the concept of sampling some zones with “high risk” areas on a continued basis in the same location for27 several ye ars wh ere unexpected damage is likely to recur year after year and thus reduce the logistical costs.

The Panel recommends including n on

- compliance with refuge requirements as a factor

for high

- risk classification since lack of refuge compliance may result in increased

resistance in areas that are not currently considered high risk

. The Panel cites the lack of potential acreage surveyed for refuge non

- compliance as described by the Arent F ox report refuge survey data. The survey criteria for participation requires that each grower plant a minimum of 20 0 acres with at least 50 acres planted to B t hybrids. The report

indicates a total of 920 individuals from the corn belt surveyed about refu ge compliance. Based on the survey criteria the amount of acreage represented could be less than 200,000 of the approximately 80 million acres of corn planted in corn in the heartland (USDA, 2013)

. The Panel recommends increasing the amount of acreage sur veyed regarding refuge compliance. The survey data provided to the Panel indicated:

77% growers surveyed adhere to refuge size requirements 62% growers surveyed adhere to refuge distance requirements 7% growers surveyed are not plan t ing refuge acreage

T he Panel recommends modifying the regional boundaries used in the Arent Fox surveys to better reflect corn rootworm population variations in species and resistance traits to

better reflect field conditions. Gray et al. (2009) shows an e xpansive area beyon d Illinois and Indiana that have populations of the western corn rootworm rotation resistant variant including eastern Iowa, south central Wisconsin, western Ohio and a few scattered fields in Michigan. This map and other resources should be used to readj ust the regional boundaries for all surveys and sampling so they are consistent between one another and better represent corn production practices and CRW populations.

The Panel recommends that the revision of “regions” boundaries also be applied to the

s ampling regions in order to provide consistency and to allow better comparison of data obtained from surveys, sampling and greater than expected damage reports. For instance

s ince the states within the Arent Fox regions surveyed do not match up with the h igh

- risk

area vs

. “ fringe areas ” for sampling

, it is difficult to determine true compliance for refuge requirement that can ultimately have an impact on the level of risk in a particular area

. The current sampling approach is likely to misrepresent potent ial resistance since the sampling is random and populations are not tracked over time for shifts in susceptibility. Consequently

, the associated delays in susceptibility studies could allow for dispersal of resistant CRW resulting in area

- wide resistance prior to identification of a resistance

concern.

b. H o w ma n y CR W p o pu l a t i on s s h ou l d b e c o lle c t ed f r o m w i th i n a sam p l i n g re gi o n t o

a d e q u a te ly ass e s s s u s c ept i b ili t y ?

The Panel recognizes that when collecting samples from fields with greater than expected dama ge, the population samples must be adequate to generate enough larvae to allow for all assays (diet and on

- plant assays) used to evaluate resistance

. The Panel28 believes the current approach of sampling 12

- 15 CRW populations mostly from Illinois

Iowa and N ebraska seems very low especially considering of the number of reported cases of suspected resistance and/or greater than expected damage ( BPPD 2011 )

. The current approach of a minimum of 2,000 adults collected for each population ( EPA

2013 ) is likely no t enough

. Considering the often limited viable offspring from these

adult collections, the Panel recommend s a minimum of 4, 000 adults per population

. This

recommendation is based on the presumed resistance allele frequency for CRW. If the phenotypic fre quency of resistance is one in 1,000 (0.001), then more than 3,000 individuals must be sampled to have a 95% probability of detecting one resistant

ind ividual (Roush & Miller 1986). However, the frequency of resistance alleles is unknown, but is expected to be higher than one in 1,000 since the Bt toxin for CRW is less than high dose and the length of product use

.

The Agricultural Biotechnology Stewardship Technical Committee ( ABSTC ) indicates that it attempts to collect multiple samples for each region, but that some collections are not successful as insufficient offspring are produced for testing ( EPA 2013 )

. In light of the number of greater than expected damage reports between 2003 and 2010, it seems necessary to increase the number of populations samp led ( BPPD 2011 ) and adopt a proactive monitoring program to detect greater than expected damage earlier in the growing season

. The Panel recommends expanding the areas of annual sampling to include additional “high risk” locations in W isconsin , S outh D ako ta , C olorado and

M innesota

.

In addition, the Panel recommends that each registrant collect annually 10

- 15 populations from the same locations in each of the 4 or 5 zones and bioassays be conducted on all

four traits presently registered. Clearly this wou ld require that registrants allow sharing of a standardized protein source and establish a common bioassay technique. Over time this would provide 50

- 75 assays of populations in each zone that would create a temporal and spatial data set that would provid e insight into the evolution of resistance.

CH A R G E Q U E ST IO N 2 :

C o rn R o o tw o rm Sa m p li n g in R e s p on se t o D a mag e d B t F i e l d s

W h en u n e x p e ct e d d am a ge in B t c o r n o cc u rs, te ch no l o gy p r o vi d e rs are o b l i ga t ed t o i n v e s t iga t e t h e c a u se o f B t f ai l u r e

. T h ese i n v e s t i g a t i on s, o f t e n te rm e d “ p e r fo rm a n c e i nqu i ri e s,” i n v o lve a nu m b er o f p r o c e d u ral s te p s t o ass e ss t h e c a u s e s a n d c ir c u ms t a n c e s o f

t h e c o r n i n j u r y

. I f CR W a r e r e s p on si b le a n d t h e l e v e l o f i n j u ry e xc eed s a r o o t d a mage

t rig g e r b as e d o n th e I o w a S t a t e N o d al I n j u ry S c ale ( N IS )

, sa m p li n g o f l o c al C R W

popu l a t i o n s is r e q u i r e d t o te s t f o r po t e n t ial r e sis t a n c e

. C u rr e nt l y
, t h e N IS th r e s ho l d s are 1

. 0 f o r si n gle to x in p r o du c t s o r 0

. 5 fo r p y rami d e d to x i n s. I f i n s u ff i c i e n t a du l t s w e re

c o l le c te d du r i n g th e i n i t ial i n v e s t ig a t i on
, sam p l i n g may h ave t o o cc u r th e f o llo w i n g

s e as o n

. F o r d ete r mi n i n g r o o t d a mag e
, the Agency h as r e c o m m end e d t ra n s e c t sa mp li n g th r o u gh t h e i n j u r e d f i e l d s e c t i ons as o p po s e d t o r a ndo m ly sam p li n g p la n t s f r o m t h e e n t ire

f ie l d o f c o n c e r n

. The Agency h as r e s e rva t i o n s t h at ra nd o m sam p li n g c o u ld l e ad t o f alse

ne g a t i v e s b y l o w e ri n g t h e av e ra g e r o o t no d e i n j u ry s c o re if a c tu al B t d amage is l o c ali z e d

i n a po r t i o n o f t h e f i e l d ( d u e t o c l u s te r e d o v i po s i t i on )

. In te rms o f a d u lt CR W sam p li n g,29 The Agency h as re c o m me n d ed th a t c o ll e ct i on s o cc u r d ir e ct ly in t h e d am a g e d B t f i e l d ( s )

,

s pe c i f i c al l y in th e vi c i n i t y o f t h e d amag e

. A c on c e rn h as b e e n rais e d

, h o w e v e r, th a t s u c h

s a m p li n g may b e b ia s e d r e l a t i v e t o s u rr o u nd i n g a r e as a n d n o t r ep r e s e n t a t i v e o f t h e o v e rall

CR W p op u la t i o n

.

a. T h e P a n e l is as k e d t o c o mm en t o n m e th o d s fo r i n v e s t iga t i n g corn rootworm ( CR W)

po p u l a t i on s c a u si n g u n e x p e ct e d d amage t o B t c o r n

. S pe c i f

i c all y
, p lea s e c o mm e n t on :

i. Th e u se o f

f

ie l d d amage ra t i n gs ( Nodal Injury Scale ( N

IS)) as a s c r e e n f o r p ot e nt ially

r e sis t a n t p opu l a t i o n s. W h at sam p li n g t rigg e rs s h o u ld b e u s e d fo r si n gle t o x in a n d

p y rami d e d B t p r o d u cts? S h o u ld al te r n a t e te c hn i qu es b e c on si d er e d?

The Panel agrees that because registrants are be ing notified of and required to

i nvestigate damaged fields, node

- injury scale ( NIS ) damage ratings will likely be high in the area of damage if CRW feeding is the cause

. Th erefore

, ratings of 1.0 for single toxin products and 0.5 for pyramided products are reasonable. The objective of the Personal Inquiry (PI) from a grower is to establish or rule out the presence of resistance

in the local CRW population so setting a relatively low trigger for further testing ( diet bioassay/ on

- plant diagnostic assay) reduces the potential of resistant populations going

un reported in fields with greater than expected damage. If mitigation of resistance is the objective of IRM for rootworms, determining that a population is less susceptible to Bt proteins when it is, in fact, not resistant (false positive), represents consi derably less risk than not establishing that a population is resistant when it actually is (false negative).

A nother potential argument is that population pressure and abundance of CRW in “high risk” areas are normally greater than that in other “ fringe ” areas of the corn belt due to geographic and edaphic factors other than population resistance so that NIS triggers of 1.5 and 1.0, respectively, would be more reasonable as greater damage is expected with heavy feeding pressure. In addition if plants fro m the entire field are included in the damage rating process

. fewer sites would require the expense and delay

of further testing

. Both scenarios would likely fail to detect pockets of incipient or resistant CRW populations. A difficult question is, what is the criteria for determining “greater than normal” population activity? It is clear to this Panel that because frequency of damaged fields will likely increase over time in the regions of greatest concern, greater than usual damage events may become mo re commonplace. If resistance to Bt toxins is the true cause, a more stringent triggering event is increasingly valuable for detecting population resistance on a local and regional scale.

An alternate view by one panelist is that in cases where systematic transect sampling is carried out across the entire field, then NIS thresholds of 1.0 for single toxin products or 0.5 for pyramided toxins might be too high to allow detection of resistance because of the usually aggregated distribution of CRW in a field. A verage

- across

- field NIS thresholds of 0.50 for single toxin products or 0.25 for pyramided toxins might be suggested as it is important to remember yield loss can occur with a root rating of 0.25

. If transect sampling is carried out only in damage

- hot

- spots of a field, then NIS thresholds of 1.0 for single toxin products or 0.5 for pyramided toxins may be too low as rootworms are often spatially aggregated (Toepfer et al. 2007), and may occur in a small30 area in such numbers that heavy root damage occurs regardless of control measures. There are many reasons to have hot spots of damage, such as a favorable egg laying

micro

- climate occurring the previous year. Then, h igh population and damage spots may not be a result of resistance. It might be even argu ed that hot

- spot sampling would not lead to any quantitatively comparable data. Therefore, a hot

- spot NIS threshold of 2 for single toxin products or 1 for pyramided toxins might be suggested. However, another panelist cautions that regardless of favorabl e egg laying micro

- climate considerations, high populations and extensive root injury could be evidence of reduced susceptibility or early resistance and disagrees with the higher NIS rating in hot spots. Conversely, it could be argued that with yield los s at a root rating of 0.25 a NIS rating of 0.5 would be appropriate for both single and pyramid toxins as suggested by a public

comment e r ( Center for Food Safety

, P ublic Comment 2013). This suggestion is supported based on recent data showing a reduced ef ficacy of Cry3Bb1 with an average root rating of 1.25 in areas with reports of greater than expected damage during the period 2012

– 2013 ( Nick Storer

, Public Docket Id

. no.

- EPA

- HQ

- OPP

- 2013

- 0490

- 0020)

The Panel suggests an alternative approach of recordin g percent plants lodging in an entire field, or the number of hot spots with lodged plants per field with 2 or 3 such spots serving as a threshold to follow up on resistance investigations. The Panel suggests a more conservative approach to increase detec tion of early resistance in the

field. By sampling plants outside of the damaged area to better represent the overall CRW population, the extent of damage is confused with the severity of damage. Because the objective of the PI visit is to mitigate the pr oblem of resistance development, if necessary, the sampling procedure should be focused on the severity

and cause of the damage. By diluting the sample outcome with surrounding area root damage ratings, the objective is blurred and the risk of making a fa lse negative decision regarding potential resistance is increased considerably.

ii. Th e u se o f t r a n s e c t sa m p li n g in d a m ag e d ar e as o r ra ndo m sam p li n g th r ou g ho u t th e

a

f fe c t e d f ield t o ass e ss r o o t d am a ge ra t i n gs.

When a PI is undertaken, the objective is to determine the cause of the observed damage. This suggests that a sample within the damaged areas of field would be more valuable to informing the question than samples of the entire field. I t would be more appropriate to sample from the damage area and w ithin the field. A transect sample of the visibly damaged plants through the damaged area of the field will lead directly to the cause of damage and provide a reference to the severity (NIS rating) of the damage that has

occurred. By randomly including d amaged and undamaged plants in the sample, an

estimate is obtained of the extent (percent of plants showing signs of damage) of the damage in that locality of the field, but dilutes the estimate of severity of attack. Both pieces of information would seem to be useful in understanding the nature of the damage reported and possibly the failure of the Bt product. The Panel recommends that a transect sample procedure through the damaged areas of the field should be required, which would also separate visibly damaged from undamaged plants and estimate the severity of damage within each of the separated groups. The combination of data from both groups would be an estimate of the extent of damage.31 The Panel recommends that a detailed description of the damaged area(s) of the field be recorded and made available in an anonymous version to the Agency’s I RM and other appropriate scientists. A suggestion from a public comment e r was that a threshold of acceptable damage would be to accept damage of 25% of plants lod ged in at least a 1 acre

area of a field ( Nick Storer; Public Docket Id no.

- EPA

- HQ

- OPP

- 2013

- 0490

- 0020 ). The Panel feels that this would be a good definition of acceptable damage in areas where cornfields are large and the geography is not diverse, but wo uld not be appropriate for areas where fields are smaller and diversity of terrain and vegetation are present ( eg . in “fringe” areas). Information included in fields with unexpected damage should include the: GPS location, area of the field and of damaged area(s), history of cropping (crop species and insecticides used) and traits used in the field, refuge compliance and disposition (structured or seed blend)

, previous PI’s for the field, and past frequency of weather events that might influence lodging.

As an example of sampling a hot spot, the Panel describes a hypothetical scenario in

which a PI is requested and the company representative determines that the area in question measures approximately 20 x 50m (65 x 165 ft. = 2 acres). The sampling of th is area would entail taking a transect through the damaged area looking for damaged plants

and select ing 12

- 15 visibly damaged plants in the transect from one corner to the other of the area and leaving at least 2 m between sampled plants. A transect coul d take the path of a V or W or be a diagonal line across the damaged area depending on the shape of the area in question. The Panel supports the idea of sequential sampling for plants with damage in the “ hot spot ” as suggested by a public comment er

. The Panel feels that this technique should be investigated further and made available to all registrants to improve their sampling efforts by shortening the decision making time when appropriate. During sampling along a transect, all plants are recorded and r ated as damaged or not. If damage exceeds a threshold after 4 or 5 samples, sampling is discontinued and the damaged area

is considered to be the result of resistant beetles. Guidelines for continued sampling would be established. Each of the damaged pla nts is tested for the presence of Bt; those that are not positive for Bt (refuge isoline plants) are discarded from further assessment. The remaining damaged plants are rated for severity of damage and the percent that exceed the established NIS rating ( f or example 1.0 for single events and 0.5 for pyramided events). If the average rating exceeds the threshold rating and greater than 50% of the plants exceed the rating, further testing for resistant populations should be initiated (sampling beetles for bi oassays).

In addition, the proportion of damaged plants in the entire transect sample could be compared with a similar transect in a surrounding area of the field to determine the extent of damage in the area or region, much like the sampling procedures discussed in Question

1. This reasoning would remove the conundrum of which type of sampling to use for determining the need for a subsequent collection of beetles and bioassay. Perhaps a combination of the two sets of transect samples would prove usefu l for both objectives covered by Questions 1 & 2.

In contrast to the above, i f a quantitative damage assessment of reaching a root damage threshold is needed, then a transect sampling over the entire field might be the only32 way. Conducting a transect sam pling only in damage

- hot

- spots of a field, would not lead to any quantitatively comparable data as there are many reasons to have hot spots of damage, such as favorable egg laying micro

- climate the previous year (Toepfer et al. 2007; Spencer et al. 2009)

. In case the hot

- spot sampling method is chosen, then the average NIS

- based damage may not be the key measure. Alternatively, any hot spot of more than 20 m diameter (plant lodging, or root damage of > 1 NIS on more than 50% of plants) should be recorded, and if two or more of such spots occur in a given field

, adult sampling is required for subsequent tests. However, o ne panelist prefers a more conservative approach that would require adult sampling if the NIS level is reached with the sampling criteria established by EPA regardless of the number of hot spots in

the field. This panelist recommends that the sampling criteria and NIS should be consistent between registrants and recognizes that single and pyramid traits may have a

different NIS

. A lthough ba sed on data suggesting reduced efficacy with Cry3Bb1 the same NIS for both single and pyramid traits in areas of greater than reported damage may be more appropriate than the varied scale.

iii. A pp r o p ri a t e sa m p li n g l o c a t i on s ( i

. e

. , in t h e vi c i n i t y o f t h e d amage a n d /o r s u rr o u nd i n g

a r e as) fo r c o l l e c t i o n s o f a du l t s i f

f

i e ld d amage t r igg e rs a r e e xc eed e d

.

The Panel feels that it would be most informative if samples of adult beetles for d iet bioassay s / plant diagnostic assay s are obtained from the damaged areas ( here damaged and adjacent fields) investigated as a result of a PI. Because of the limited medium and long distance dispersal nature of these insects, a higher proportion of t hose beetles emerging from the damaged areas will be included in the sample tha n of those beetles emerging from surrounding areas. If possible, samples of beetles should be obtained from an area no more than 1000 ft. ( 300m ) from the damage and within the same field

. Assays using the neonate offspring obtained from rearing these beet les will be the most indicative of potential resistance when data are compared with sources of beetles well away from damaged areas or from standard susceptible laboratory colonies.

It has been suggested that these beetles and their offspring have more f itness than the average beetle in the corn belt because they are surviving in high risk areas where corn

on

- corn, non

- rotation, single trait Bt, and/or minimal or no refuge fields are common. Fitness (or decreased susceptibility) relative to Bt protein in high

- risk areas is the exact issue being confronted. The objective of a resistance management strategy is to demonstrate that resistance to Cry proteins has not occurred and if that cannot be

determined then the population is resistant

. A critical issue in collection of adult beetles is the timing of collections to obtain the maximum numbers of mated, gravid females that will produce the most number of viable eggs and neonates that best represent the cause of

the previously recorded damage where resistan ce or tolerance is suspected (i.e. after the

pre

- oviposition period of 12 to 23 days ( Spencer et al. 2009) and before the population has considerably dispersed and mixed ( Spencer et al. 2007 ; Carrasco et al. 2010).

The Panel acknowledges that due to delay s in reporting damage, the logistics of examining increasing numbers of PIs

, and dispersal of CRW, sampling adult populations33 in the damaged area the during same season can be difficult. The sooner beetle samples can be taken after assessment of the damag ed area, the more likely individuals from that damaged area will be collected. Despite concerns that beetles can migrate out of as well as into the damaged areas, beetles collected close to the damaged areas are most likely to represent the local populati on that survived on plants in that area. However, i f adult sampling can be achieved in the same year to allow timely rearing of offspring for testing, then the entire cornfield could be used in order to obtain sufficient sample

numbers

. Sampling beetles from the entire field or from areas more than 2

- 3 km (1

- 2 miles) away simply (Carrasco et al. 2010) reduces the chance of fairly representing the

response in Bt protein assays of those beetles that caused the damage. This is because

WCR adult populations perform considerable inter

- field movements during their lifespan (Spencer et al. 2009), and nearly equally distribute (mix) across maize fields in an area and lay eggs accordingly.

Wher ever adult sampling must be carried out in the following year, sample s should be taken from the same field (corn

- on

- corn) or a neighboring cornfield if sufficient sample numbers are not available from the damaged field

. Samples should be taken early in the emergence of adults to maximize the collection of F1 beetles that o riginated in the previously damaged field. Use of sentinel plots of corn or squash to collect adults that may represent those that caused damage the previous year is problematic, due to supporting the spread of resistance, but would be useful under certai n conditions as

discussed below.

Sampling can be conducted anywhere in the target field, and not only in damage

- hot

spots, because the population will have moved and dispersed considerably by the time damage is accessed (Spencer et al. 2009). Moreove r, hot

- spots of adults, e.g. in search for food and/or oviposition, later in the adult season may not any more relate to hot

- spots of adult emergence due to potential Bt resistance earlier in the adult season (Toepfer et al. 2007).

iv. Th e d e p l o y m e n t o f s en t i n el p l ots in t h e vi c i n i t y o f d amag e d f i e l d s in s ub s e qu e n t

s e as on s t o: 1) ass e ss t h e r e sis t a n c e a llele f r e q uen c y i n th e ar e a, a n d/o r 2 ) c o lle c t i n s e c t s if n o a d u l t s w e re p r e s e n t at t i m e o f t h e f ield i n v e s t iga t i on

.

The Panel feels that determining the allele frequency for resistance in a population of

CRW cannot be achi eved at this time and is not easily determined until a more robust set

of data is established. These data must be based on a stable bioassay method that includes a standard control popul ation so that considerable data points for each trait can be compared in time and space. These criteria cannot be met at this time.

Because most PI follow

- ups are carried out late in the season, in many instances, beetle numbers in the damaged areas or i n the field in general may be low or nonexistent and the viability of those beetles collected may be low. Even if a late maturing field is nearby, it should not be used to collect beetles because these late fields act as attractants to beetles throughout t he region and would not likely represent the beetles from a neighboring field, let alone those that caused the34 damage. It would be imperative that a follow

- up sample of beetles be initiated the following year in the same location as that designated as the area of damage. A sentinel plot of non

- Bt corn of the same maturity as the rest of the field could be planted much like a varietal demonstration plot. These plots could be sampled for beetles at beginning

- to

- peak emergence to capture as many beetles as possible emerging from that specific area.

The Panel gives thought to the idea that by planting a sentinel crop the following season, the process would increase the spread of resistant alleles in CRW populations. In areas where local mitigation is pres cribed following a year of damage and procedures are completely followed, this may be true. However, at this time, most fields with unexpected damage are being reported from locations where

corn

- on

- corn plantings are common and not likely to diminish sign ificantly. Sentinel plots planted in the same field the season following damage and confirmed

to be resistance

- based are valuable resources of beetle populations emerging from

that localized area.

However, it should be considered that sentinel plots plan ted with non

- Bt corn and without

soil insecticide m ay allow resistant populations to build up in the area while assays are being conducted on beetles collected from fields with greater than expected damage in

the area. Conversely, the sentinel plots may p rovide a population of susceptible beetles that could mate with potentially resistant beetles emerging from Bt corn nearby. However, if female western corn rootworms mate more than once, her eggs are suggested to be mainly fertilized by the sperm of the s econd or later male s (Branson et al. 1977). Since CRW development is delayed when feeding on Bt corn, the second mating has a greater likelihood of a male that developed on Bt corn.

CH A R G E Q U E ST IO N 3 :

D iag no s t ic Assa y s

I n s e c t r e si s t a n c e m on i t o ri n g f o r B t c o rn to x i n s h a s h is t o ri c ally b e e n c o n duc te d w i t h

a r

t i f i c ial d i e t b i o assa y s u si n g do s e

- r e s p o n se c u r v e s t o o b t ain L C 50 o r E C 50 m e as u r e s f o r
l a

bo r a to ry c o l on ies a n d f i e ld p o pu l a t i on s. B PP D h as e x p r e ss e d c o nc e r n s t h at d i e t assa y s,

as p r e s e nt ly d esig n ed

, are i n a d e qu a t e fo r e f fe c t iv e l y a nd p r o a c t iv e ly d e te c t i n g r e s is t a n t

p o p u la t i o n s. R e s p on s e s t o to x i n

- i n c o r po r a te d d i e t s a r e o ft e n h ig h ly varia b le a n d a f un ct i on al “ d i ag n o s t i c ” c on c e nt r a t i o n (c a p a b le o f d e t e c t i n g r e sis t a n t i nd iv i du als o r

p o pu l a t i o n s) h as no t b e e n d ev e l o p e d f o r a n y o f t h e r e gis t e r e d to x i n s.

B e c a u se o f t h e i n h e r e n t un c e r t ai n t i e s ( h i g h vari a b ili t y
, l e ss s en s i t ivi t y ) w i t h t h e p r e s e n t
d i e t b i o as s ay m et h odo l o g y
, the Agency h as r e c o mm e n d ed t h e u se o f d ia g no s t ic o n

- p l a n t

as s a y s as t h e p rimary r e sis t a n c e dete c t i o n t oo l fo r c o rn r oot w o rm. T h e se te c hn i q u es

ass e ss CR W s u s c e pt i b ili t y d ir e c t ly o n B t c o r n p la n ts a n d c a n m e as u re le t h al a n d s ub l e th al

vari a b les. T w o s u c h a pp r o a c h es h a v e b e e n d ev e l o p e d b y Ga s sma nn e t al. ( 2 011 ) a n d

N o w a t zk i e t al. ( 2 008 )

.35 a. P lease c o m m e n t o n th e s t r en g th s a n d lim i t a t i on s o f d iet b i o assay m e t h o do l o gies fo r

e arly r e sis t a n c e d e te c t i o n w i t h CR W, c on s i d eri n g t h at th e c u rr e nt l y

- regis t e r ed to x i n s a r e less t h an h i g h do s e

. W h a t i m p r o v e m e n t s c ou l d b e m a d e t o t he se b i o assa y s t o ma k e t he m

m o re e f f

e c t ive a n d p r o a c t i v e r e si s t a n c e dete c t i o n t oo ls?

Diet bioassay strengths

The Panel points out that the approach of using bioassays with insect artificial diet and Bt

toxins have been conducted for over 40 years (Dulmage et al. 1971)

. T he scien tific community is well aware of this approach’s basic methodology . Thus

, find ing ample

information in the scientific liter ature to assess its limitations is possible

. Properly conducted bioassays, are those that include an appropriate set of dilutions, good quality insect artificial diet, kept under biologically relevant environmental conditions, and

conducted with suitable and uniform insect developmental stages and densities

. These bioassays can provide more precise and consistent responses than curre nt on

- plant assays

to screen for resistance to Bt

. R oush and Miller (1986) concluded that diet bioassays are appropriate if resistance allele frequencies are high (0.01) , but are not an effective method for detecting early resistance in a population. H ow ever, with either an on

- plant assay or a diet bioassay the insect population needed to detect a low res istance allele frequency could be substantial

.

Results of artificial diet bioassays are routinely examined with established standard statistical procedu res recognized by the scientific community. The parameters used in the evaluations of these bioassays can include those that are currently performed with on

plant bioassays (larval weight, head capsule size, etc.), but the opposite may not be true
beca use on

- plant bioassays only challenge insects on a putatively constant protein

concentration. In terms of logistics, the Panel believes that bioassays conducted with artificial diet can occupy less space and be more economical than the current on

- plant

scree ning assay . This provides an advantage for most of the research facilities.

Preliminary lethal (LC 50 ) and effective concentrations (EC 50 ) values have been established for some of the Bt proteins aiming to control CRW spp., with genetically

engineered cor n hybrids (Siegfried et al 2005; BPPD Review

, 2013 ). Although the Panel believes that these methods require refinement, particularly the inclusion of a high concentration to kill/arrest development of all homozygous and heterozygous resistant

CRW

, it also recognizes that these values already serve as historical records to document trends of susceptibility over time. Trends in susceptibility at a single location may be more precisely followed over time with artificial diet bioassays than with on

- plant

bioa ssays because of the more precise nature of the assay and the breadth of data they provide

. A single well

- maintained stock of purified Bt toxin can last a long time and perform more uniformly than plants where their Bt protein expression can be influenced by environmental, biotic and corn hybrid genetic background.36 An example of using current limited data from diet bioassay :

From reports submitted to EPA for the years 2007

- 2010, inclusive concerning results of bioassays performed on CRW populations co llected from locations of unexpected damage following PI’s, the following table is presented. The data are for EC 50 values calculated for diet assays conducted using neonate larvae over a four day period.



### Monsanto (Cry 3Bb1) Diet Bioassays 2007

- 2010

– EC 5 0 

### Comparisons

2007 2008 2009 2010 # Signif. EC 50 > Lab Strain 3/5 1/8 8/13 5/10
Repeated Bioassay Site s where EC 50 > Lab Strain Sherman Co. NE *26.17

- *41.33 *83.48

McLean Co. IL 12.68

- *37.75 48.13 Champaign, IL

- 7.73 *39.88

- Hamilton Co. IA

- - 13.90 *119.60

Howard Co. NE

- - *40.61 *73.52 Story Co. IA

- - *28.88 47.34 *significant EC50 (>95% confidence interval) from lab value for that year.
The top line indicates the number of pop ulations which had significant EC 50 (95% confidence interval) values of the total successful bioassays performed that year. The following data report the EC 50 values reported for populations sampled from the same location (County) which were repeated for two or more years. Values with an asterisk indicate significant 95% confidence intervals compared with the lab

standard.

Except for 2008, more than half of the populations sampled each year demonstrate a significant difference in growth (final weight) in the bioassays. Wh en bioassays are conducted in the same location for successive years, the trend in EC 50 values increase in

all cases. The data presented here, although limited and with certain uncertainties about the diet bioassay technique, are a good example of how diet assays can provide information on the temporal and spatial evolution of resistance in CRW populations collected from “high risk” areas.

The Panel believes that if the registrants’ on

- going efforts to produce Bt proteins in high enough concentrations are achieved, many of the current methodological hurdles stated

above could be solved. This high

- enough protein concentration may serve to establish a diagnostic concentration 1 that could transform the monitoring effort into a faster, reli able and ampler method. One panelist stressed that based on the alarming number of greater than expected damage in the field reports, time is of the essence to identify a diagnostic concentration for the Bt toxins in order for the diet bioassays to have v alue in identifying

resistance. In addition, the development of a diagnostic concentration or tool may not be practical due to selection pressure and commercialization of the Bt toxins.

The Panel agrees with the considerations in other charge questions t hat consistency in methodology (e.g.: same type of diet, reference laboratory colony, batch of Bt toxin, proper handling of neonates, etc.) is important. This is a valuable consideration that may help explain trends of susceptibility over time. There is also another advantage of this

1 A diagnostic concentration is a dose capable of killing or arrest development of most ( > 99%) of the
homozygous and heterozygous resistant insects.37 method because m any assays can be performed in a small space and entirely within a growth room that provides stable and consistent environmental conditions; a single replicate of 5 or 6 conce ntrations of 3 different Bt prote in s could be performed on a single 96

- well plate and repeated sufficiently for a substantial assay. The Panel recommends developing a standard diet where neonates can survive on assays beyond 3

- 4 days with greater than 80% survival. Composition of curren t commercial diets is a trade secret and variations of its components may not be proclaimed by the manufacturer. Such minuscule changes can greatly affect the development of insects (Blanco et al

. 2009a) , and results of bioassays containing Bt proteins (B lanco et al. 2009b). The Panel recommends keeping the bioassays with insect artificial diet to continue building some of the already observed trends, and share the results among all appropriate researchers, both public and private. However, the current cr iteria used to determine resistance using diet bioassay data is unlikely to identify early resistance that is developing in the field. To support this statement, field collected populations have

shown LC 50 s exceeding upper limit of 95% confidence, but not been deemed resistant by registrants. In addition, LC measures are highly variable ( US EPA, 2013 ( Table 2 ))

. The

EC data are less variable, but unlikely to detect early resistance due to the lack of sensitivity. In some cases, responses have varied by several orders of magnitude and much is attributed to natural variation and/or laboratory methodology

. Meihls (2010) expressed concern about the sensitivity of the LC/EC diet bioassays indicat ing that

resistant colonies can not always be distinguished from control colonies

.

In regard to the limitations of the diet assays, these artificial d iet bioassay protocol s and toxin sources, ideally, should remain consistent to allow for appropriate interpretation of results with previous assays. The Panel recognize s that science moves forward and creates opportunities for improvement s and changes. When these modifications take place, a comparator study should be performed to document the influence that methodological changes have had on the response of insect susce ptibility to Bt . A thorough documentation of each step in the protocol and material s utilized (manufacturers, batch numbers, storage conditions, etc.) in the bioassays , are necessary to understand these potential changes. The registrants and other scienti sts are familiar enough with research conducted under good laboratory practices (GLP) to know the basic

information that should be included in the protocols (US EPA.). The Panel is not recommending establishing a GLP for monitoring work

. However the Pane l is recommending that GLP is consider ed in order to ensure that these guidelines have thorough ly prepared protocols and documentation

. This is very important because the documents provided to the Panel do not contain sufficient information to judge criti cal variations between methods when changes have occurred. As described above, changes are inevitable; as such

, the search for a more concentrated source of protein, and well

documented comparator studies should be requested from registrants to thoroughly understand the impact of changes. The preliminary establishment of Bt susceptibility by Siegfried et al. (2005) and subsequent monitoring effort ( US EPA, 2010 ) performed by a different laboratory, may include substantial methodological changes. Even the most constant methodology performed simultaneously in different laboratories, can yield results with high variation (Blanco et al. 2005). The Panel recommends that these types of comparisons take into consideration the intrinsic and inevitable variation.38 In the documents provided by the Agency to the Panel, as well as within the public comments, the concept of “ effective/determination of allele frequency ” has been

mentioned. An assessment of the effective CRW diversity/number of alleles in field samples/ tested populations, although not a perfect method at the moment, can still be

measured after several years of commercialization of the Bt corn hybrids

. One approximation, and the simplest, is counting the number of the gravid females in a field sample, bu t preferably the number of those that produced subsequent generations i n the laboratory. If the field sample size recommended is at least 2,000 corn rootworm adults,

a n assessment of some (~50) of the ~1,000 field

- collected females capable of producing

pr ogeny can give an acc urate indication of the total alleles tested in a sample. If some of their progeny are Bt

- tolerant, a partial and imperfect allele frequency can still be

determined. The reproductive biology of WCR allows for accurate paternity

deter minations, because the sex ratio in the genus is ~1:1, and a great proportion of females mate only once (Spencer et al. 2009)

. Therefore, by dissecting a sub sample of

females and count ing the number of spermatophores (which is not a difficult task ) an in dication of the total number of alleles in the sample can be estimated

. Another but more elaborate and expensive method is to establish the monitoring by screening single gravid females or isofamilies. The F 1 progeny of a female, putatively the alleles o f only two individuals, are challenged on artificial diet bioassays or on

- plant assays. Each female produces sufficient larvae (Spencer et al. 2009) to perform this type of work. The response (susceptible or resistant) of the F 1 provides the necessary da ta. The basis for this work can be found on Andow and Alstad (1998), a methodological modification (Gould et al. 1997), and its applicability (Blanco et al. 2008), and cost (Blanco et al. 2009c), have been already demonstrated for Lepidoptera

.

A nother issue with the bioassays and the statistical methods employed is that it is commonly assumed that a higher proportion of resistant alleles in a bioassay do not necessarily produce a higher LC 50 /EC 50 response. In most of the cases it is true, but there

is also a pos sibility that a proportion of resistant larvae in a bioassay can produce variable

results. For example, the ~25% of resistant WCR larvae detected in some of the monitoring reports provide d to the Panel, can produce different outcomes. If these larvae happen to be tested in low enough concentrations allowing for ~25% survivorship, LC 50 values can vary up to 18% using the same data of tolerant larvae scored in different sub

lethal concentrations. With this example it is obvious that a bioassay sh ould: include : 1) at least one concentration capable of producing 100% mortality/growth inhibition, a true diagnostic concentration ; 2) a minimum of four replications to reduce variability ; and 3) a discrepancy of ~18% in the response in the current metho d which should not be attributed necessarily to a hig her resistant allele frequency

.

The Panel recognizes the seminal efforts of Dr. Blair Sieg fried on the development of the methodology with insect artificial diet (Siegfried et al. 2005), as well a s the on

- going plans by the registrants to improve it

. Understanding that Bt proteins are expensive and laborious to produce, the Panel suggests that the feeding behavior of the WCR larvae on the top of the overlaid toxin bioassay, and an assessment of the toxi n ingested

, may be necessary to understand some of the variation in the results. To partially answer this39 question, a comparative study between diet incorporated and overlaid techniques will add to our understanding on why the latter method has had many s hortcomings. It is important to note that both techniques may yield LC 50 /EC 50 results entirely different (Siegfried et al. 2007), but the intrinsic variation within a technique is what needs to be

understood. Proteins in this technique are applied to die t surface of wells on test plates that have been filled with diet to avoid denaturation of protein in diet; only the surface of the cell has the protein applied, so consumption of protein may only be at initial feeding of neonate, while subsequent feeding may be on diet below the surface, free of protein. Thus, calculation of the protein in the diet (μg/cm 2 of diet) may be inconsistent or incorrect and exposure via consumption is also variable and inconsistent. The use of dyes in diet to look for consumpti on by larvae, or the use of a diagnostic tool to detect Bt protein in the larval gut, may serve as aide

. The Panel recognizes that there is no

“ standard ” methodology to conduct this work at the moment. The report of Siegfried et al. (2005) provides more information for other s to reproduce these findings. Mortality of larvae on diet wells can become excessive or bacterial contamination may appear beyond 4 days at 24 0 C, so most tests are terminated at that time

. There might be some inconsistency of result s (3 d in the current monitoring work versus < 6 days in Siegfried et al 2005), because larvae have not all fed to the same extent or that some have died at initial contact with a high concentration of protein at the surface of the diet which does

not corre ctly represent the concentration intended.

The Panel also recommends evaluating a representative number of bioassays daily for a time lapse of 3 to 7 days. Studies have shown neonate larvae can survive 3

- 8 days without feeding ( Branson 1989 ) and Oloumi

- S adeghi and Levine (1989). It is imperative to find out if a different evaluation time, ( greater than 3 days

- after

- initiation ) may yield

better results. The Panel recommends that the laboratory currently conducting these bioassays adjust to some of the ev aluation parameters found in Siegfried et al. ( 2005) and provide comparisons.

The Panel recommends keeping the monitoring efforts utilizing bioassays with insect artificial diet as another tool to detect shifts in susceptibility. This evaluation and the on

plant efforts can complement each other under the current testing limitations. The Panel recognizes that access to the currentl y tested proteins to perform bioassays by independent r esearchers has been restricted. It has been a serious barrier for ind ependent

laboratories to contribute to the improvement of these techniques if they do not have

access to these toxins

. The Panel recommends that the toxins used in the diet bioassays be made available to academic researchers to allow comparative data from independent

sources.

Perhaps the greatest limitations of the diet bioassay are the following. Firstly, it may not accurately reflect the larva’s ability to survive on the Bt corn in the field . Secondly ;
R oush and Miller (1986) concluded diet bioassays are appropriate if resistance allele frequencies are high, but are not an effective method for detecting early resistance in a population . Thirdly, Bt toxin for corn rootworm is a non

- high dose . Lastly, the use of the diet bioassays to identify resistan ce is unlikely as a functional diagnostic40 concentration that is capable of detecting resistant populations or individuals has not been developed.

b. Th e P a n e l is as k e d t o d i s c u ss t h e r e l a t i v e m e ri t s a n d l i mi t a t i on s o f t h e t w o on

- p l a n t assa y s Gassma n n e t a l. 20 1 1 a n d No w a t z k i e t al. 2 008 )

. P l e ase d is c u ss t h e e x t e n t t o w h i c h the se

assa y s h ave d i f f

e r e n t s e n si t i v i t ies t o ma k e e arly c o rn r ootw o rm r e si s t a n c e d e te rm i n a t i o n s?

S hou l d o t h e r o n

- p la n t a s say a pp r o a c h es b e c o n s i d er e d ?

In regard to the strengths of the on

- plant (Gassmann et al. 2011), t he Panel recognizes

that the Gassmann et al. (2011) approach is more realistic of field conditions than the artificial diet bioassay s

. Although the published paper on this method has some procedural omissions, once it is understood in its entirety it may be an easy method to

follow and to confirm res ults independently from those provided by the registrants. The adoption of this method may expand the monitoring area by the inclusion of independent

scientists.

It is the understanding of the Panel that this method has been performed with different corn events, demonstrating some of its value as it may be useful in comparing durability of the registered toxins both as single toxins traits and pyramid traits

. To date, this is the only assay that has confirmed resistance in the field based on its evaluation criteria . In

addition

, it most closely replicates field conditions.

Concerning the limitations of the Gassmann o n

- p lant assay, the Panel believes that some

of these limi tations also apply to the on

- plant and sub

- lethal seedling assays described in Nowatzki et al. (2008). A paper published by Clark et al. (2006) suggests that WCR survival on Bt corn is a result of larval feeding behavior and describes that this insect

may be able to discern subtle differences in toxin levels in root tissue, thus allowing them to feed selectively on low expressing root tissues. Furthermore, Spencer et al. (2009)

also found that “ (WCR) larvae on the resistant maize moved continuously, sampli ng root hairs or root tissue but not actively feeding. These behaviors were dramatically different from those feeding on isoline maize. ” These two pieces of evidence make it imperative to address the following points in both of the current on

- plant proto cols: 1) conduct on

plant bioassays for a range of 3 to 17 days after egg hatching to find out i f there is a better (shorter) time that can yield more accurate results with this method and 2) p erform an intense/destructive representative sample of the tota l number of surviving larvae in the

root system and soil during those sample dates, and not rely entirely on the larvae

collected in alcohol vials after the funnel extraction

. The Panel believes that both methods, destructive and extraction funnels can be compared simultaneously. If the

behavior of the larvae is to avoid higher Bt concentrations in roots

, these discerning insects may hav e a delayed development result ing in smaller larvae. These discerning larvae may or may not be Bt resistant

, but due to its possible smaller size after 17 days of not actively feeding , they can be categorized as susceptible because of their smaller size

. Some of the parameters to evaluate “ resistance ” in the current methods (artificial diet and

on

- plant assays) involve th e proportion of larvae that have developed into different

instars. The Panel suggests looking carefully at the results presented by DuPont/Pioneer in the written and public comments of ( DuPont Pioneer

, Public Docket Id. no.

- EPA

- HQ

-41 OPP

- 2013

- 0490

- 0022 to us e them as an indication that shorter exposure time of larvae to roots may yield more accurate results. In addition, the Panel recommends investigating the use of the agar

- based growing medium (Clark et al. 2006 ) that would allow larval root feeding to be observed to better understand behavioral variations and consequences

.

The Panel has reviewed the publicly

- available information and finds that Bt protein expression in corn changes with time (USDA

- APHIS 2001, & 2003 ; BPPD , 2011 ), and Bt expression in cotto n plants behaves in the same manner, having a ~5x concentration difference when these plants are grown in different environments (Greenplate 1999). Plant growth conditions in greenhouses and growth chambers may also vary from

location to location and thro ughout the year, and from year to year

. It is crucial that

rese arch be initiated and published about the quantification of Bt proteins over time in different parts of the corn plant, especially in the root system. Such evaluations would be

more valuable if they are performed using the transformation events already incorporated

into commercial varieties. This may help to elucidate another source of great variation in hybrid performance and insect survivorship. The limited data available of these Bt

conce ntrations in the root system of corn plants come from the transformation event into a variety that generally, allows for effective breeding, not necessarily from a commercially

- available variety. Because this transformed event will give rise to other

vari eties by crossing it with elite lines adapted to specific geographic conditions, this may increase the possibilities that the expression of Bt may be dependent on genotype and

environmental interactions. Furthermore, some of these elite commercial varieti es may already have integrated some WCR antibiosis traits (Levine and Oloumi

- Sadeghi 1991)

, while others may not, making comparisons even more important. There is very limited data on the expression of these toxins throughout the development of the pl ants indicating

a decline of Bt expression in roots up to ~30% in a period of ~22 days (USDA

- APHIS

, 2001 & 2003 ; BPPD, 2011)

. T his lapse of time was similar to the one employed in the

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

. This may pre sen t a double challenge for these particular on

- plant bioas says, a sub

- optimal Bt expression to effectively control WCRs and its diminishing concentration during the V2

- V6 plant developmental stage, during the most actively

- feeding period by the pest. These

c hallenges would also be experienced in the field. The main problem with the Bt corn rootworm technology is that it is non

- high dose. As a result of the sub

- lethal level of toxin, the W CR are developing methods to survive exposure, thus allowing populatio ns to build and exert enough feeding pressure on the corn roots to result in greater than expected field damage.

The Panel also identified the following other areas of improvement of this methodology including:

1) The confirmation of the transformation event in the Bt hybrid and the expression of the protein in the roots is necessary to assure that the tested corn plants are indeed the correct hybrids. “ Off type ” seed is a possibility in any seed batch and the quantification of this proportion is anothe r critical issue on which the Panel recommends obtaining detailed information.42 2) This methodology would greatly improve if the corn seeds obtained are free from pesticide applications. The Panel is aware of the efforts made by Gassmann et al. (2011) rem oving the pesticide from the seeds as they come in a commercial seed bag, but no quantification of the potentially toxic residue was performed after the removal procedure. 3) High larv al mortality in the soil makes analysis more difficult and problematic

, impeding potential Bt resistance to be documented correctly. Loss of larvae that may have escaped from pots that are not sealed properly can cause greater variation in survivorship and results. The Panel is not aware if this was considered/ob served

in the Gassmann et al. (2011) and for the greenhouse plant efficacy (Nowatzki et al. 2008) research

. 4) An evaluation of the whole soil/root system at various points in time is necessary to understand this potential pitfall. 5) The use of “ between 10

- 20 n eonates ” per plant, may present a serious constrain t in

the met hodology. The influence that doubling the amount of WCR on r oot growth and protein expression may have is not known. Is it “ logical ” to assume that twice

the amount of larvae in a bioassay ma y double the quantity of roots consumed

, therefore causing a lower developmen tal time due to lack of food? Also, w hat is the influence of twice the pest population on air exchange, larval competition, etc.? These are only a few que stions that the Panel h as identified due to the inconsistent insect pressure of this method. Furthermore, t he use of larval extracti on with Berlese funnels, employed in both of the on

- plant bioassays, leaves some uncertainty. It is

not known which treatment had 10 or 20 neona tes, or an amount in between, in order

to ca lculate survivorship. Again, a thorough inspection of the soil/roots at the end of experiment

, to see if any larvae remain , could help to adjust the results. Also, how

can “ natural ” mortality be assessed w ith t his method?

Gassmann et al. (2011) and Nowastzki et al. (2008) mention that: “

... surviving larvae

were collected in vials with alcohol. ” Assuming that a particular population has an unusual high survivorship, the Panel recommends collecting live samples / maintaining part of the sample in artificial diet to continue with the studies and to confirm resistance by other method s , if that particular population demonstrates high tolerance to corn hybrids

. The surviving colonies would re present a great opportunit y to learn more about the biology, behavior

, and genetics of these interesting insects.

Curzi et al. (2012) demonstrated WCR had developed a n ability to detoxify plant compounds to allow soybean herbivory by altering gene express ion resulting in high dige stive enzyme activity

. The Panel recommends investigations to see if similar responses to Bt corn could be developed to identify resistant populations. The Panel suggests that registrants provide detailed information regarding additional testing if

suspe cted Bt resistance is identified.

Space and logistics is another consideration that the Panel believes may be of concern, especially if th e Gassmann method is compared with the sub

- lethal seedling assay proposed by Nowa tzki et al. (2008). Greenhouse spa ce to grow seedlings in pots, shelf space in growth chamber/rooms to hold the pots, space for43 extraction funnels, and maintaining consistent growing conditions, may be difficult to obtain and/or duplicate with other researchers following this methodology i n other locations, year after year. Maintaining consistency of the tested varieties with this method may be also crucial to build a historical trend of susceptibility

shifts.

Regarding the strengths of the on

- p lant assay, t he Panel recognizes the great

current improvements to the methodology of Nowatzki et al. (2008) that were submitted as public comments.

The Panel agrees that the Nowatzki et al. (2008) sub

- lethal seedling method may be

e asier to standardize because the seedlings are grown in containe rs under dark and more consistent conditions in a growth chamber. G rowth of these seedlings may produce an expression of protein more consistent between plants, although not nec essarily similar to field conditions, and because the assay is conducted in cl osed plastic boxes, it makes for a reduced opportunity for larvae to escape, while maintaining better environmental

conditions for the insects

. The methods described in Nowatzki et al. (2008) included the

verification of the correct transformation event o r its isoline, and the Bt expression in the

root was verified. The Panel recommends testing Gassmann and Nowatzki methods with pyramided events, as well as with single

- protein events separately. A great difference with Nowatzki’s method is the crossing o f the WCRs with a non

- diapausing colony, which enables the monitoring testing over a much shorter period of time. Also, as a proof of concept, the inclusion of different proportions of susceptible and “ resistant ” WCR larvae to challenge the assays, is som ething that the Panel recommends continuing with this and Gassmann’s protocols.

The Panel recognizes that some of the concerns regarding the possibility of erroneously classifying larvae as potentially resistant due to “ insensitivity ” of the assay, have been corrected either by adjusting the number of eggs or by reducing the amount of time of the assay ( DuPont Pioneer

, Public docket Id. no.

- ID: EPA

HQ

- OPP

- 2013

- 0490

- 0022). So me of the criticisms to the Nowatzki et al. (2008) method expressed by the Panel were answered during the public comment period

and in the documentation provided by DuPont Pioneer ( DuPont Pioneer

, Public

docket Id . no.

- EPA

- HQ

- OPP

- 2013

- 0490

- 0022 ).

In reference to the On

- p lant assay limitations, s creening by this method involved two

di fferent assays with live plants: a “ greenhouse plant efficacy ” assay, and a “ sub

- lethal seedling ” assay (Nowatzki et al. 2008). Be low the Panel elucidates on points that were

not previously addressed

.

The greenhouse plant efficacy assay closely resembles the Gassmann methodology. The Panel believes that some of the uncertainties expressed above (escaping larvae, insects not collected with extraction funnels, killing potentially

resistant larvae in alcohol vials, etc.), are also applicable here.

The Pane l suggests that research efforts continue to reduce the amount of variability in the assays and expand testing to include corn events containing Cry3Bb1 and Cry3A144 proteins . In addition, efforts should c ontinue to develop a WCR colony that is

considered B t resistant based on the criteria currently used when field population s from

greater than expected damage areas are tested or preferably redefine resistance to better reflect field populations that are feeding on Bt roots, inflicting extensive damage using NIS ratings and surviving. An example of lack of consistency in the current criteria for resistance is the York resistant population that had a mean instar and body area measures significantly lower on Bt than non

- Bt in 2010 and in 2011 a mean instar not significantly

different between Bt and non

- Bt, but lower body area on Bt vs. non

- Bt. During the public comment period, Pioneer indicated that this population is resistant to Cry34/35, but this population would not be considered resistant based on the cri teria Pioneer is proposing. There needs to be consistency in what is termed resistance and that criteria should be the most conservative option. One panel member recommended comparing the results from the Gassmann and Nowatzki assays using the current re gistered toxins to evaluate sensitivity and consistency of data outputs.

CH A R G E Q U E ST IO N 4 :

D ef i n i n g R e sis t a n c e

Th e n a t u re o f r e sis t a n c e fo r c o r n r oot w o rm a n d B t t o x i n s is n o t c o m p l e t e ly u n d er s to o d

, t h o u gh i t is li k e ly to a ffe c t h o w b i o assay r e s u l t s ar e i n te r p r e t e d fo r r e sis t a n c e de t e rmi n a t i o n s. A s e ri e s o f la b o ra t o ry a n d gr een h ou se s e l e c t i o n e x p erim e nt s w i t h n o n

h i g h do se B t to x i n s h ave s h o w n th a t CR W r e si s t a n c e i s un li k e ly t o fo llow a si n gle g e n e
,

r e c e ssi v e m o d e l as fo r o t h er t arg e t p e s t s o f B t c r o p s w i t h h i g h d o se e x p r e s si o n ( e

. g

. , tob a cc o b u d w o rm, G o u l d e t al. 19 9 5 )

. I n s t e a d

, r e s e ar c h h as s u gg e s t e d th at CR W

r e sis t a n c e m ay b e n o n

- r e c e ssi v e ( Me i h ls e t al. 2 008 ) o r c ou l d i n v o l v e m u l t i p le g e n e s

c o n fe rr i n g v ar y i n g lev e ls o f to l e ra n c e ( L ef k o e t al. 20 0 8 )

. O n

- p la n t assa y s e ss en t ially

f un ct i o n as a d iag n o s t ic s c r e e n th a t w ill i d e n t i f y p o pu l a t i on s w i th ge n e t ic r e sis t a n c e (t h e

a

b ili t y o f o f f

s p r i n g t o s u rvi v e o n B t p la n t s )

, b u t may n o t c learly i d e nt i f y c as e s o f

i n c o m p l e t e r e sis t a n c e o r d e te c t o t he r fo rms o f r e sis t a n c e s u c h as av o i d a nc e o f t h e to x in b y

s e l e c t i ve feed i n g o n r o o t s w i t h d i f fe r e nt ial to x i n e x p r e ssi o n.

As d is c u ss e d in t h e A g e ncy ’s w h i t e p a pe r o n CR W r e sis t a n c e m o n i t o ri n g, B PP D a n d

i n du s t ry h ave p r opo s e d d e f i n i t i on s fo r r e sis t a n c e b as e d o n th e u se o f o n

- p la n t assa y s ( Gassma n n e t al. 20 1 1 a n d No w a t z k i e t al. 2008 )

. The s e d ef i n i t i o n s i n c l ud e d i f f

e r e n t
l e v e ls o f c o m p aris on s t o ma k e a “ w e ig ht

- o f

- evi d e n c e” ass e ssm en t

- - i.e., 1 ) t h e r e s pon s e

o f a f i e l d po p u l a t i o n o n B t vs. no n

- B t c o rn p l a n t s, o r 2 ) th e r e s p o n s e s o f a f i e ld

p o pu l a t i o n vs. a s u s c ept i b l e l a bo r a to ry c o l o n y o n B t c o rn p l a nt s. T o ma k e a r e si s t a n c e d e te r mi n a t i on , E P A b elieves th a t t w o s e p ara t e m e a s u r e s de m o n s t ra t i n g s t a t is t i c ally

si g n i f i c a n t r e du c e d s u s c e p t i b i li t y to th e B t to x in w o u ld b e n e ed ed ( e .g., s u rvival,

g r o w th )

.

a. Th e P a n e l is as k e d t o d is c u ss t h e m e r i ts a n d s ho r t c o mi n gs o f t h e p r o p o s e d a p

p r o a c h es t o d e

f

i n i ng r e sis t a n c e u si n g o n

- p l a n t assa y s. W h at s et s o f c o m p aris on s in t h e assa y s are

m o s t li k e ly t o a d d va l u e t o a w e ig h t

- o f

- e vi d en c e a pp r o a c h t o d e t e rmi n i n g r e sis t a n c e ?45 The Panel has already identified several merits and shortcomings of both on

- plant assays in Charge Question 3. Both methods demonstrate the actual response of the western corn rootworm larvae to corn plants that express Bt protein(s). Limited / declining expression of Bt in the root system, addressed by the Panel in charge question 3, is one of the most crucial limitations to these methodologies. Currently, this situation of low and declining Bt expression in roots may be di fficult to overcome using the approved Bt transformation events, but a better understanding of the amount of Bt protein that the larvae are exposed to through time, and how that interacts with larval feeding behavior, may facilitate the interpretation of r esults. However, the Panel recognizes that the limited and/or declining expression of Bt in the root system is the environment that larvae are exposed to in the

field. From the public comment presentation of Dr. Analiza Alves ( DuPont Pioneer,

Public Docke t Id. no.

- EPA

- HQ

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- 2013

- 0490

- 0025 ), the Panel has learned that : 1) optimized insect density in the root system and 2) categorizing the developmental instar of surviving larvae at different periods of time, would alleviate some of the previous

variation a nd provide a more solid statistical method to differentiate between putatively resistant and susceptible larvae.

Because the concentration of all the events in the currently commercialized corn hybrids

do not meet the definition of “ high dose, ” the Panel believes that the current scoring criteria of developmental instar proportion of surviving larvae, provides the least variable criteria to discern between putatively resistant and susceptible CRW larvae. However, we must recognize that there may be some fi tness costs associated with resistant populations that may delay development and result in earlier instars than control populations. Populations surviving from on

- plant assays should be collected alive and progeny from the survivors should be tested using on

- plant assays to determine if the offspring survive thus demonstrating heritability of resistance.

b. W h at r e sis t a n c e all e le f r equ e n c y s h o u ld c on s t i t u t e f ield r e sis t a n c e fo r to x i n s w i t h l e ss th a n h igh do se e x p r e ssi o n ? P l e ase d is c u ss th e c ri te r ia th a t s hou l d b e u s e d fo r t h ese t y

p es

o f to x i n s ( as opp o s e d t o h igh do se t o x i n s) giv e n th a t a po r t i o n o f h e te r o z y g ou s i n s e c ts w ill

s u rvive B t e x po s u r e a n d d rive t h e e v o l ut i o n o f r e si s t a n c e

.

The Panel is aware that : 1) because there is not a current effective method to discern Bt resistant from susceptible CRW ; 2) this impeded the establishment of a Bt resistance frequency prior to the release of the corn events already commercialized, but more importantly ; 3) resistance mechanisms have not been thoroughly characteriz ed, allowing for speculations on the nature of its tolerance (multi

- gene, incomplete resistance) (Gassmann et al. 2011). These factors make it difficult to establish a resistant allele frequency that should trigger an action by the US EPA. These factors also do not allow for comparisons with previously suggested rare resistant allele frequencies considered for

Bt plants and Lepidoptera (e.g.: Roush and Miller 1986). The Panel believes that this question does not have a valid scientific answer and opens t he possibility of all kinds of speculation.

External laboratory selection studies have demonstrated that selection for CRW resistance to BT proteins can evolve fairly rapidly ( within 3 to 11 generations) therefore,46 the Panel assumes that BT resistance all eles are not rare in the field (Meihls et al. 2008, Oswald et al. 2011, Meihls et al. 2012, Lefko et al. 2008, Meihls et al. 2011). Modeling simula tions illustrate that if ini tial resistant allele frequency is high then resistance is predicted to evolve in 5 to 8 years even in the presence of a refuge (Pan et al. 2011). The simulations suggest that the resistanc e allele frequency in CRW populations was already high when the BT corn hybrids were introduced.

Because resistance has been detected fairly ra pidl y (3 to 11 generations), the Panel

assume s that Bt

- resistant alleles are not rare (Meihls et al. 2008, Oswald et al. 2011, Meihls et al. 2012, Lefko et al. 2008, Meihls et al. 2011). Pan et al. 2011 demonstrate s only a small influence of refuges when allele frequency is high with time to 50% resistant allele frequency ranging from 5 to 8 years , which suggests that resistant allele frequency

in the CRW populations was already high when the Bt corn hybrids were introduced.

In Charge Question 3 part 1, t he Panel discussed some of the “ imperfect ” methods that can give us an approximation to the current presence of resistant alleles in field populations (e.g.: assessing the number of females that contributed with alleles to the tested generation, establishm ent of isoline testing).

c. W h at s t a t is t i c al te s ts, c r i te ria, a n d sig n i f

i c a n c e le v e ls w ou ld b e be s t s u i t e d f o r e a rly

r e sis t a n c e d e te c t i o n w i t h t h e p r o p o s e d assa y s? P lease d is cu ss ho w t o b e st av o id f al s e

ne g a t i v e s a n d /o r f alse po si t i v e s.

Probit analysis in b ioassays with insect artificial diets (Robertson et al. 2007), and

assessments of surviving larvae at different periods of time by developmental instar ( DuPont Pioneer; Public docket Id. no.

- EPA

- HQ

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- 2013

- 0490

- 0022) for on

- plant assays, seem to be the mos t adequate method at th is time

. The Panel suggests that bioassays with insect artificial diet should include all the considerations expressed in Charge Question 3 part 1, and that the method of proportion of different developmental

instars be tested also for Cry3Bb1 events, and the assay developers continue the efforts of refining the methodology.

The Panel also suggests that to further confirm resistance consecutive additional testing should be done when: 1) results of the single, on

- pla n t bioassays are inconclusive due to unclear statistical analysis of a limited sample size ; and 2) o ther factors have the possibility of contributing to greater than expected damage, including CRW populations that might be a result of reduced susceptibility. However, the Panel suggests that it is important to recognize that higher CRW populations in certain fields could be an indication of resistance and greater survivability of the population.

Using multiple metrics (survival, instar development, body size and weight, f eeding behavior, root tissue ingestions and exploring gene expression) may be used to reduce the likelihood of false positives and false negatives could be reduced

. Allowing for comparisons between sampled population on Bt and non

- Bt corn plus comparisons between sampled populations and susceptible populations (laboratory colonies) will add

another indicator. During the public comment period (04Dec13) the Panel learned that47 some of these parameters may reduce variability and produce solid data. The Panel addressed some of the parameters in C harge Q uestion 3.

One panelist emphasizes that perhaps a more conservative approach for both on

- plant assays should be adopted initially until the importance of the possible metrics are better

understood

– using the agar medium may allow for behavior to serve as a metric.

d. P lease c o m m e n t o n th e e x te n t t o w h i c h i n c o m p l et e r e sis t a n c e c an b e i d e nt i f

ied w i t h o n

p l a n t te s t s y s te ms. H o w s h o u ld r e sis t a n c e d e f

i n i t i on s b e a d j u s t e d t o a d d r e ss the se

s c en ari o s?

Early resistan ce may be more easily determined by a diet bioassay than an on

- plant bioassay. The LC 50 value and possibly the EC 50 values by themselves could provide an estimate of resistance in WCR populations, if an acceptable value for heritable resistance

is establi shed. Over a period of time, a successive series of on

- plant assays of the same population of WCR can infer resistance by noting increasing values for both LC 50 and

EC 50 that exceed a ratio value compared with a standard population to which all registrant s agree.

Populations with incomplete resistance may be more fit than the control population on Bt corn, but may not be significantly different. In addition, if there are fitness costs for Bt resistance, the resistant population may not survive on non

- Bt corn as well as the control colony. The fitness costs for resistant populations could be reflected in the developmental stage, body weight/head capsule size, but may still be an indicator of incomplete or early resistance.

Progeny of isofamilies, a poss ibility indicated in the EPA’s white paper (US EPA. 201 3),

can be tested in a medium such as that proposed in Clark et al. (2006) that may allow

non

- destructive evaluations at different intervals. Knowledge of the proportion of larvae that die or stop dev eloping may provide an indication of the proportion of incomplete

resistance and this non

- destructive evaluation can greatly contribute to the establishment of a (late) resistance frequency.

Resistance definitions could be adjusted by considering that c orn rootworm may have developed more than one response to Bt corn allowing them to survive, such as physiological and behavioral resistance thus complicating the development of a diagnostic tool that confirms resistance on both diet bioassays and on

- plant assays. Use of field data may provide the earliest and most reliable indication of resistance or reduced susceptibility. As an example, performance of Bt corn rootworm toxins data from 2007 through 2011 from locations without greater than expected damag e shows a Cry3Bb1 average root rating using the Iowa State University node injury scale 0

- 3 (ISU NIS) of approximately 0.125 ( Nick Storer, Public Docket Id no.

- EPA

- HQ

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- 2013

- 0490

- 0020 )

Data from 2012 through 2013 from locations without reports of greate r than expected damage indicates approximately a 50% increase in average root injury (NIS 0.25) in fields planted with Cry3Bb1. Locations with greater than expected damage for the period

2012

- 2013 reflect an average root rating of 1.25, five times highe r than a root rating48 where producers can expect a yield loss (NIS of 0.25). During the public comment

session of the SAP meeting

, population resistance was defined to “ occurs when a large portion of a pest population is field

- resistant and causes the Bt corn to fail to confer economic control of the population ( Nick Storer, Public Docket Id

. no.

- EPA

- HQ

- OPP

2013

- 0490

- 0020 ”

. The data from 2012 through 2013 in locations wi th greater than expected damage meet the economic threshold of 0.25 NIS (Hodgson and G assmann 2013) and the 2012 through 2013 data from locations with greater than expected damage greatly exceed the economic threshold. These data may reflect population resistance. In the simplest terms resistance can be defined as survivability of a popul ation and the heritability of the survivability trait.

e. P lease d is c u ss t h e 1) via b il i t y o f r e sis t a n c e r a t i o s as a n o pt i o n fo r d e te rm i n i n g r e sis t a n t

p o pu l a t i o n s, c on si d er i n g th e g en e rally l o w s u s c e p t i b il i t y o f C R W t o B t to x i n s a n d 2) t h e l a c k o f s u s c e p t i b l e w il d t ype po p u la t i on s ( i.e., du e t o w i d es p r e a d a do p t i o n o f B t c o r n ) . 3)

W

h at ra t io c o u ld b e c on s i de r e d as an i nd i c a t o r o f r e sis t a n c e f o r c o rn r o o t w o rm u si n g o n

p l a n t assa y s?

1. Gassmann et al. (2011) proposed a resistance ratio (proportion of corrected su rvival of field population/proportion of corrected survival of control population) as a

determination of resistan ce Due to the nature of current Bt corn events not expressing a

“ high dose, ” the resistan ce ratio would need to be lower than the ratio of >10 x proposed by Tabashnik (1994). A suggested ratio of ~4x for the CRW for non

- high dose corn events may be reasonable at this point. The limited historical data that the BPPD IRM Team has received, may serve for the calculation of these ratios, taking int o consideration that methodological changes (different batches of Bt toxins with different concentration, discrepancy between the methods of Siegfried et al. (2005) and Custom BioProducts, etc.) may have altered the overall response of field collected and laboratory colonies.

2. A “fringe” population at this time may serve as a control population upon which other populations from “high risk” areas and fields with unexpected damage could be compared. These populations may not be sustainable over time as great er than expected damage has been reported i n Michigan and New York suggesting reduced susceptibility

in “ fringe ” areas as well, but populations from other “ fringe ” areas without reports of greater than expected damage may serve as a standard for survival, growth measures

. V iability of F 1 generations could be established for all current traits to be used in later comparisons. Laboratory populations may be appropriate for a few years but concern about inbreeding and loss of behavioral traits reflective of fi eld populations may be a limiting factor. Diet bioassays may provide realistic responses over time, but in on

- plant assays, behavioral responses may make results suspect. Bt

- susceptible Diabrotica populations can be obtained in areas where the selection pressure has not been so intense such as in Europe, or from an organic corn grower with a large enough farm area, in the periphery of the US cornbelt. Also, surrogate species such as Diabrotica virgifera zea

, the Mexican corn rootworm, can serve as a good indicator for susceptibility, especially if the samples are obtained in areas where Bt corn is not registered (e.g.:Mexico).49 3. A panelist recommends that a ratio of ~4x for the CRW for non

- high dose corn events may be reasonable at this point.

CH A R G E Q U E ST IO N 5 :

D ete rm i n i n g R e m ed ial A ct i o n A r e as

In t h e e v e n t o f CR W r e si s t a n c e
, s u cc e ss fu l r e m e d i a t i o n w ill li k e ly d ep e n d o n t h e a b il i t y

t o g eo gr a ph i c ally d e f i n e a r e m ed ial a c t i o n zon e in a f as h i o n th a t a d equ a te ly e n c o m p as s e s

t h e r e si s t a n t po p u l a t i on ( s )

. To a cc o m p li s h th is ob j e c t i v e
, a r o bu s t s c i e n t i f ic a p p r o a c h is

n e e d ed t o ass e ss th e e x t e n t o f r e sis t a n c e

. As d is c u ss e d i n t h e Ag e n cy ’s w h i t e p a p e r, th is

s t e p may b e c o m p li c a t e d b y t h e f a c t th a t c o r n gr o w e r s u se i n s e c t i c i d es t o s u pp r e ss c o rn

r o ot w o rm p o pu l a t i o n d e n s i t i e s (p r o p h y la c t i c ally o r in r e s p on se t o f ie ld d amag e )

. T h e se

m a n ag e m en t s t r a te gies c an r ed uc e p o pu l a t i o n si ze s a n d mask th e p r e s en c e o f r e s is t a nt

po p u la t i on s t h e f o ll o w i n g y e ar, t h o u gh r e sis t a n t c o rn r o ot w o rm p o pu l a t i on s may b e

p r e s e n t b e y o n d t h e vis u al b o u n d ary o f f ail e d B t c o rn f i e l d s. O n e po ss i b le s c i e n t i f ic

a p p r o a c h t o de l i ne a t e a r e m e d ial a c t i o n zo n e c ou ld b e t o u se a r e si s t a n c e alle l e f r e q uen c y

gr a d i en t s t a r t i n g in t h e c e n t e r o f t h e a r e a o f c o n c e rn a nd w o r k i n g out w a r d be y o n d t h e k no w n a f fe c t e d f iel d( s )

. O n c e a r e sis t a n c e f r e qu e n c y gra d i e n t h as b e e n det e rmi n e d

, a

r e si s t a n c e th r e s ho l d va l u e c o u ld a i d in d e mar c a t i n g t h e s p r e ad o f r e sis t a n c e

.

a. P lease c o m m e n t o n th e s t r en g th s a n d lim i t a t i on s o f B P P D ’s p r o po sal t o u s e

r e s is t a n c e al l e le gra d i e n t s t o d e f

i n e t h e g eo gra p h ic e x t e n t o f a r e s is t a n t

p o p u la t i o n

.

Unfortunately, constraints preclude measuring allele frequency from currently being a realistic option. These constraints include the following.

First, t he alleles conferring resistance have not been defined. If such a breakthrough occurred in which specific allele (s) were defined, it would be useful to map the frequency (or presence/absence) of allele (s) as gradients from problem

- inquiry fields. Mapping allele gradients would be an attracti ve option if a technique existed to take many samples from many locations, to achieve high sampling resolution. Initial locations could be defined as fields with problem inquiries, and samples taken in concentric rings surrounding that location. Maps cou ld be made if samples only were able to categorize the presence/absence of the resistant allele ( s )

. The sampling would (1) confirm the presence of resistant allele ( s ) at the location of the fields of problem inquires, and (2)

define the locations where th ese alleles are no longer present. Mapped representations of an allele frequency would have direct utility for efforts to manage gene flow and introgression. However, there are important implicit biological assumptions in the mapped realization of allele frequency for purposes of managing population abundance of resistant phenotypes. A key assumption is that the presence of the allele(s) results in expression of the resistance. It is feasible, especially for cases where multiple alleles are

involved and their expression is influenced by environmental factors, that the allele(s) is detected but not expressed in a manner that confers resistance.50 Secondly

, it is likely that resistance of CRW to the currently deployed transgenes is

conferred via more than one allele. The relative importance of each allele that contributes to resistance, and the interaction among those alleles, is not known. It is also feasible that there are gene

- by

- environment interactions, resulting in poor expression of resistance

unde r field conditions even if resistant alleles are present. T he phenotypic expression of

resistance may not be a simple function of the presence of an allele(s). Thus, it is not currently clear if mapping the gradient of a single allele, a small subset of alleles, or many alleles, would be necessary for inferring maps that reflect the probability of resistant phenotypes.

Thirdly

, sample collections could be difficult because abundances of adults could be low, or absent at the time collections are required

. Currently, collections are not always made in the year that problem

- inquiry fields are defined. It also may be difficult to obtain sufficient precision in measuring changes in susceptibility along spatial gradients in a timely fashion for proactive res istance management if many hot spots of resistance occur, or if area wide resistance occurs, due to the workload of adult sampling and running molecular assays. If resistant allele frequencies are estimated from bioassays, it is unlikely that data could b e gathered within the time frame needed to implement effective remediation due to the need to collect adults, obtain eggs, complete diapause, and run bioassays.

When the constraints of defining what allele or alleles confer resistance, and the cost of

det ermining the presence of those allele(s) in a population is reasonable, then the Panel

recommend s that efforts to map the presence and frequency of those genes be pursued

. Efforts should also include modeling the spatial patterns of those alleles, which c an be used to optimize interpolation algorithms. A spatially defined sampling approach may be applied starting from the hot spot fields (problem inquiry fields), in 0, 5, 20, km steps. This may not work if area wide resistance is detected or if multiple hot spots were detected due to the work load problem. In cases where resistant populations were found at the 20 km step, then more distant sampling is required. Then interpolated maps may be created, and remedial action zones visually defined. Assuming that the detection of resistant alleles is directly related to phenotypic expression of resistance, mapping resistance allele frequency would also help to define action zones to guide mitigation

efforts. However, mapping allele frequencies cannot proceed until the constraint of defining what gene or genes confer resistance is determined.

Alternatively, it is currently feasible to measure abundance. This can be done a priori, in areas of adoption, in the year of adoption, and sample resolution guided by o ther data layers that suggest risk (continuous corn, continuous use of similar traits, historically higher abundances, locations of problem inquiry fields, etc.). Information may be obtained within a time frame to influence decisions about deployment of B est Management Practices (BMPs). There is an inherent assumption that BMPs can reduce the probability of widespread Bt failure when BMPs are deployed in response to a problem inquiry field. Although BMPs may, or may not, influence the frequency of

resist ant alleles at a given location, BMPs help achieve sustainability of Bt

- crops both at51 that location and at wider geographic scales, through their influence on population

processes. Therefore, we suggest mapping population density gradients as part of IRM.

Although Bt resistance allele(s) have not been defined, other allele

- based studies could be used to define remedial action zones (e.g. population hybridization studies ). For the time being, the Panel advises learning from these other allele gradient st udies, such as those defining hybridization zones between different CRW populations of northern Italy. They show that hybrid populations with characteristic neutral markers of genetic diversity (microsatellites) spread about 13

- 21 km per year in intense m aize growing landscape s without geographic barriers (Bermond et al. 2012). This is due to short and medium distance dispersal, but not likely due to long distance (stratified) dispersal. The Panel recognizes that alleles subject to selection, such as all eles conferring resistance, may not follow the exact same pattern as those detected through neutral genetic diversity studies, but for the time being these studies are the most closely related to this question.

Studies using these population genetics meth ods with neutral genetic markers should be

conducted in areas at high risk of resistance (continuous corn, unmanaged populations, etc.), and areas where problem

- inquiry fields have occurred, to determine if similar spread rates exist in these landscapes an d populations. This is, however, only possible if the supposed resistant populations are genetically different (sufficient genetic diversity in the neutral markers) among surrounding populations.

For determining the size of remedial action zones, the Pan el indicates that first it is important to clarify whether the remedial action zone is aimed at elimination of resistant alleles (remedy) and/or resistance suppression and containment of resistant alleles. The latter appears more practical. The determina tion of the size of remedial action zones also requires first defining the potential control measures and the expected efficacy of each measure on population suppression.

The Panel also believe s that definitions that distinguish a “hot

- spot” versus a

“con tainment zone of area wide resistance” need to be developed. This is an issue of defining the spatial scale of resistant populations. Pa n et al. ( 2011 ) described several approaches for clarifying “hot spot” versus “containment zone of area wide resistanc e

. ”

One option is to define hot spots based on fixed distances from problem

- inquiry fields, based on dispersal kernels and/or introgression of microsatellites (see 5b, below).

The spatial scale of resistance can also be partially addressed through tests for spatial continuity of the data. Currently, resistance or problem

- inquiry fields are being discussed

as “hot

- spots”, and it is implied that they are independent. Spatial independence can be tested, and used to help distinguish independent hot

- spots v ersus larger

- scale processes. Mapping the location of problem

- inquiry fields, and the location of fields where similar traits are deployed but are not resulting in problems, results in a categorical map (locations of 0 and 1’s), from which tests of spati al autocorrelation can be applied. Moran’s I, or other regression

- based approaches to look for spatial dependence in the52 numbers as a function of intervening distance, may be used. Population genetics data may have other statistical tools for defining spa tial dependence in the data.

Mapping abundances may also help define the spatial scale at which population processes are being expressed. Although abundances are influenced by many factors other than allele frequencies, currently population abundance i s the data that has the potential to be collected in much higher spatial and temporal resolution. It may be feasible to devise a statistical approach (such as a multivariate approach) to test for spatial dependence in abundance after factoring out other s uspected factors (weather, field management, etc.)

that contribute to abundance.

It seems reasonable to look for introgression of resistant alleles among CRW populations in fields placed along a gradient as a function of distance from point

- sources that w ould be first defined as problem

- inquiry fields. However, if spatial patterns among problem

inquiry fields are best described as random with respect to distance and direction among fields, then trying to find spatial gradients among fields will be difficu lt.

b. W h at o t he r to o ls o r s t r a t e gi e s c ou l d b e e m p l o y e d t o d e f

i n e t h e r e m e d ia t io n z on e?

The Panel proposes the following seven strategies to define remedial action zones:

1. Adapt from population hybridization studies.

CRW populations with characteris tic neutral genetic markers (microsatellites) are suggested to spread about 13 to 21 km per year. This is based on a molecular genetics study modeling the spatial spread of allele frequencies of CRW hybrid populations, and

two differentiated parental popu lations in northern Italy (Bermond et al. 2012, 2013). The spatial process measured in this study is due to effects of short and medium distance dispersal but not likely due to long

- distance ( stratified ) dispersal. Using this study, an approximate 20 km radius can be suggested as a remedial action zone, but the Panel is

uncertain if this would be feasible. When assigning an approximate 20 km range as the remedial action zone, a significant reduction of the resistance alleles may be achieved,

but not with 100 % certainty. This is because (i) a small proportion of CRW disperses about 60 to 100 km per year (Baufeld and Enzian 2001) or up to 200 km/year (Gr a y et al. 2009); and (ii) efficacy of available alternative control measures far below 100%, except

for crop rotation schemes. Furthermore, the dispersal kernel of adult CRW (see 2, below), suggests that a remedial action zone using a radii of 50 km, and deployment of highly effective actions, would be needed to achieve remedy (defined as elimination of

re sistant alleles) (Carrasco et al. 2010). The Panel is uncertain if this would be feasible.

2. Adapt from dispersal kernels, used to define eradication and containment zones of newly introduced CRW in Europe

.

For remedial action zones to eradicate Bt re sistance, the experiences from European CRW eradication efforts might be useful. They divide remedial action zones (called eradications zones) into a 1 km “focus” zone around a hot spot (with highly efficient53 resistance management measures applied in that area) and additional 5 km “safety” zone (with less strict resistance management measures applied in that safety zone) ( EC Decision 2003/766/EC (Anon, 2003 )

. However, according to the dispersal kernel of adult CRW, the focus zone should be extended to 5 k m and the safety zone up to 50 km (Carrasco et al. 2010) if the intent is elimination. If this approach is to be adapted for the USA, then the suggested remedial action zone for trying to eliminate resistant populations would be 5 + 50 km per year, which is not feasible. If the detection of resistant populations requires 2 years, then the zone should be even doubled (10 + 100 km). Again, this is unlikely to be feasible because of the scale, and the lack of 100% effective

control measures.

If suppressio n of resistance rather than elimination is the aim, then a containment action zone may be created if area wide resistance is detected, and/or mitigation zones defined

around hot

- spots of resistance. For containment zones to mitigate area wide resistance, we may also learn from European experiences, where a 40 km containment zone is defined around specific CRW populations (10 km inside population, 30 km outside). Control measures are targeted at the 10 km inside populations, to suppress their populations s o much, that spread of the population becomes much less likely (integration of many different control measures). Measures targeted on the outside zones (i.e. more crop rotation) are aimed to reduce the chance of population spread . According to the

micro satellite frequency spread studies of Bermond et al. (2012, 2013), 13 to 21 km around populations would be sufficient (instead of 30 km). But according to the dispersal kernel studies of Carrasco et al. 2010, the outside population zone should be extended to 50 km (at 99% safety level, versus 5 km at 50% safety). This translates to suggested containment zones of 20 km (based on studies using microsatellites: Bermond et al. 2013) or 30 km (based on EU Legislation) to 50 km (based on dispersal kernel studi es: Carrasco et al. 2010) outside plus 10 km inside a population. The Panel questions

whether this is feasible in the USA landscape.

As for mitigation zones around hot

- spot areas of resistance, the P anel suggests also applying the 13 to 21 km suggestions by Bermond et al. 2013 (based on studies using microsatellites) or the 5 km

– focus zone suggested by Carrasco et al. (2010) considering that up to nearly 10% of a population would breach this zone (based on the dispersal kernels).

A recent review (Gray et al. 2009), and models that may be compared against measured abundances (Onstad et al. 1999, 2003a,b) suggested rates of expansion of WCR ranged from 20 to 200 km/year in North America, and was influenced by direction, prevailing winds, storm fronts, lan dscape and local management practices. However, such long distances, influenced by stratified dispersal, probably cannot be taken into account when defining remediation zones.

From these studies, we can suggest a containment zone of 20+ 10 km. But this assumes spread is emanating from a single source location, or a single front, driven by dispersal

from that source or front. In contrast, the pattern of problem

- inquiry fields appears to suggest multiple, independent initiation sites. Efforts to contain introgression of resistant54 alleles with a containment zone would be impaired by each appearance of new, independent sites harboring resistant alleles. We believe resistant alleles are not rare, making containment unlikely to be successful for long periods of time in the absence of IRM practices that significantly limit the appearance of new, independent sites harboring

resistance.

3. Adapt from rotation tolerant population spread studies

.

Lessons from the spread of rotation tolerance may be applied to de fining containment zones around area wide Bt resistance regions, thus under large scale situations. Onstad et al. (1999) modeled spread of rotation resistance at 10

- 30 km per year. As for remedial mitigation actions zones at a smaller hot spot scale, les sons from the spread of rotation tolerance may not be applied, as spatial correlations are not clear.

4. Adult CRW sampling and Bt susceptibility assessment of offspring in a series of distances from hot spots or problem

- inquiry fields

.

It is conceptuall y feasible to use the results from the sampling and bioassays in response to damage, and testing of offspring, to define remedial action zones. Spatial sampling may be applied starting from the hot spot fields (problem inquiry fields), in 0, 5, 20, km

ste ps. If resistant populations were found at the 20 km step, then more distant samplings would be required. This may not work if area wide resistance is detected or if multiple hot spots were detected due to the work load problem. Furthermore, this would require that samples be collected and analyzed in a timely fashion. These data could be used to create interpolated maps, and remedial action zones visually defined.

5. Temporal trend in bioassay information and spatial location

.

The Panel notes that bioassay data showed temporal trends in LC50s and EC50s

. I n some locations, at approximately county spatial scale, these temporal trends were positive. Thus, these temporal trends in bioassay data help define a spatial area (a county or group of counties ) that could be defined as a remedial action area.

6. Develop risk maps

.

R isk maps could be developed, and risk expressed as spatial gradients (Baufeld and Enzian 2001)

. Risk maps could be developed by categorizing relative risk for a field or

land are a based on expert opinion, analysis of past PI fields, lack

- of

- rotation, repeated use of same trait, weather conducive to WCR development, regional corn acreage and corn density in a landscape, and other factors. Such risk maps would be useful for

determi ning size of remediation zones and for guiding IRM practices and education efforts aimed at influencing human behavior in ways to minimize resistant establishment or spread. The risk could be buffered out to distances informed from the

eradication/contain ment zones in Europe, the population genetics studies from Europe, the dispersal kernel studies from Europe, or the rotation

- resistance studies from the

Midwest.55 7. Investigate mapping the probability of occurrence of exceeding thresholds, borrowing fro m statistical tools developed in the geostatistical literature.

Defining the geographic extent of a population as a gradient requires measuring the population (density, frequency of alleles, or presence/absence) with sufficient precision at discrete, know n points, so that changes over space can be detected. It is unclear if techniques currently exist to achieve that precision for alleles, but it does exist for measuring abundances.

Statis ti cally estimating density, or presence/absence, of a population at unsampled points involves interpolation. Interpolation can be based on the statistical relationship of points as a function of intervening distance (such as kriging) or assumed spatial relationships (such as inverse

- distance interpolation ). Sample pla cement can be optimized from understanding the statistical relationship of samples as a function of their distance and

direction. Any map that shows interpolated estimates should be recognized as representing statistical or graphical models.

Ideally, def ining the geographic extent of a resistant population would lend itself to interpolated mapping of resistant allele frequencies measured (with precision) at point

sources. This approach would probably require many sample sites. When positive

values are r are (which they will need to be, if used for resistance monitoring for achieving resistance management), it is important to define where the negative locations are (where resistance is absent). Without the negatives defined, interpolations will often

tend to “bleed” from positive locations over wide areas, which may be areas of false positives.

Advances in the ability to map probability of occurrences (positives) in the presence of large numbers of zeros (negatives) could be developed for this purpose. Re cta et al. (2012) provide an example for insect count data that have many zeros. Statistical approaches to mapping have also been developed that utilize other data layers (known priors) for estimating values at unsampled locations. These approaches (such as

“ indicator kriging ” ) are utilized in the geosciences for defining placement of wells, and mapping probabilities of obtaining positives (defined as economic extraction of a resource). Similar examples may exist for interpolating values for allele frequ encies, although the Panel is not currently aware of those examples.

In conclusion

, the P anel concurs with the EPA ( EPA White Paper 2013) that “ A primary goal of resistance monitoring is to detect shifts ( in space and time ) in the frequency of

resistant a lleles (i.e., susceptibility changes) before the onset of resistance leads to widespread Bt failure.” However, the goal as stated is not feasible because we are unable to measure allele frequencies or changes in susceptibility with sufficient precision

an d in sufficient time. The goal should be restated, to include metrics that we have a capacity for measuring with sufficient precision and in a timely fashion.56 Currently, the only such metric is abundance, and some members of the P anel felt that

monitor ing abundance should be added to the definition, although not all members agreed because monitoring abundance may not always be directly related to monitoring

resistance. A suggested restatement is: “A primary goal of monitoring is to detect shifts ( in a bundance, susceptibility, and/or resistant allele frequency ) before the onset of resistance leads to widespread Bt failure.”

The Panel cannot come to a final suggestion on the size and structure of remediation zones to mitigate resistance, but suggests ta king the above explained studies into account when defining such zones. The Panel did not judge the feasibility of the scale of remediation following establishment of resistance, which ranged from 20 to 50 km radii

based on the information available to da te. Thus, achieving resistance management should focus on proactive options that avoid or delay establishment of resistant alleles in CRW populations.

CH A R G E Q U E ST IO N 6

C ont a i n m e n t /M i t iga t i o n o f R e sis t a n c e

The terms and conditions for all Bt corn reg istrations 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 plan s 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 m easures (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 consideration s 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. wi despread (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 strat egies;

• 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) ;57 • 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 act ions 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

P anel may wish to provide. Specifically, please discuss the following issues:

a. What remediation approaches could be taken for localized vs. area

- wide resistance

scenarios?

The Panel concur s with the concern stated in the white paper (pg. 22), that “...actiona ble thresholds may not be met ( or recognized ) until resistance is widespread and effective mitigation (reducing resistant allele frequency) is impractical. In this case, managing resistance through population suppression may be the only alternative.” The Panel also

concur s with the statement ( US EPA, 2013 ( Page 26 )) that “BMPs ... likely need to be implemented ... irrespective of whether resistance testing has been completed

. ” The

context of this statement was addressing the situation of responding to a prob lem inquiry

field. The Panel recommends widen ing the context to deal with factors that increase higher risk of becoming areas with the emergence of resistance. The Panel recommends a proactive effort of managing resistance through population suppression through the implementation of an integrated pest management plan using BMP

.

The Agency BPPD IRM team separates population suppression from remediation ( EPA White Paper 2013 ). In this case, the separation does not hold strongly through space and

time. Th e r

- alleles are not rare. BMPs applied both prior to, and following, problem

inquiry sites and years, both only affect r

- alleles through population processes. In a high

risk setting, rotating to another crop, or different traits, will help achieve IRM wh ether they are conducted before or after a problem

- field inquiry.

Given the prioritization of BMPs to manage abundances as the primary means of influencing resistance, we now turn to consideration of proactive approaches for achieving IRM, including rap id remediation and mitigation. The Panel assume s r

- alleles are not rare, that the geographic location of problem

- inquiry fields will show low rates of spatial dependence and arise (and will continue to arise) primarily independently through

local selectio n, and secondarily through gene

- flow achieved through dispersal. The Panel

focus es on efforts to achieve proactive IRM in a situation where r

- alleles are not rare and events do not express a high dose. The Panel recognize s the value of including the

appr opriate use of transgenic cultivars in an IRM plan due to their ability to influence population densities and pesticide

- use patterns, and their labor

- saving effects.

The Panel has great concern about the viability of all Bt products for corn rootworm,

par ticularly in a landscape of continuous corn. Growers must be directly involved in the design of BMPs that will be relevant to their landscape and farming practice. Thus, there needs to be a process created to involve growers, specifically those who decid e upon58 cultivar choice. BMP practices can be encouraged through requirements placed on the

labels. These might include requirements for rotations (among traits, pyramids, sequential versus concurrent deployments of single or pyramided cultivars depending on initial r allele frequencies ( Onstad and Meinke 2010 ) , and/or crops) depending on the production p ractices. The Panel recommends developing BMPs that will address variation in production practices, species behavior and resistance to chemical and cultu ral practices such as continuous corn, rotation resistance and production practices.

While the Panel anticipate s an overlap in the BMPs recommendations, the Panel believe s it is necessary to consider the population and species differences along in conjuct ion with differences in production practices and landscapes when developing the BMPs. The

Panel recommends the Agricultural Biotechnology Stewardship Technical Committee (ABSTC)

, Insect Resistance Management Stewardship Subcommittee, the National Corn

Gro wers’ Association and state

- based corn associations in cooperation with research and

e xtension scientists and growers develop an IRM plan and BMPs using a proactive integrated pest management approach that would be implemented prior to suspected

resistance (proactive) . This proactive approach would reduce the likelihood of heavy selection pressure on corn rootworm by using the same mode of action over an extended period of time as resistance has been shown to develop within 3 to 7 years of use in

continuou s corn. The development of the IRM and IPM plans should be a process that is reviewed annually and revised as needed. Such organizations may also be able to influence the location and sequence of planting single versus pyramided traits, and use

patterns of soil insecticides, all of which influence rates of resist ance (Onstad and Meinke 2010). Involving the USDA Risk Management Agency may help facilitate options. The Panel recommends inclusion of social scientists in to the process to increase the

unders tanding of human behavior with regard to production practices and decision making. The Panel note s that Extension entomologists are already developing region

specific recommendations

, (i.e. the eastern corn

- growing states ( Indiana, Michigan, New York, Pe nnsylvania, Ohio, Ontario) ( Tooker and DiFonzo 2013).

It was suggested that the selection pressure exerted by continuous corn production, in areas that do not have rotation resistant corn rootworm populations, could be reduced by a rotation plan that allo ws producers to plant corn 4 out of 5 years by rotating corn with soybean, then a non

- Bt hybrid, non

- Bt hybrid with a soil insecticide, a pyramided Bt product and followed by a pyramided Bt product or similar rotation schemes. By implementing a rotation p lan that alternates frequency of rotation over time, management
tools and modes of action, selection pressure and abundances are greatly reduced. Reduced abundances also reduces the rate of resistance, through source

- sink dynamics interacting with movemen t rates and landscapes, as has been shown by Ca prio (2001) and Storer (2003). The combination of reduced abundances and reduced selection pressure may delay resistance to the PIPs toxins. The Panel recommends the implementation of the proposed BMPs in ad vance of suspected resistance.

The Panel believe s using the IPM approach as part of BMPs may reduce the need for

remediation although the Panel recognize s that not all producers will choose to use the BMPs as part of the production plan unless required. W ithout the required59 implementation of BMPs, the Panel concurs with EPA that under the current generic remediation approach too much time may pass before identifying resistant populations.

The Panel recommends that remediation plans for all registered p roducts take into

considera tion geographical location of the field , production practice, population

resistance and species’ behaviors. These plans may include elements outlined in the BMPs with an emphasis on crop rotation although areas with rotation res istant corn rootworm will require a multi

- year rotation plan that uses the planting of non

- Bt with rotation of soil insecticides rather than crop rotation to overcome oviposition and extended diapause populations. With the “hotspot” resistance scenarios, producers who experience higher than expected damage in their Bt field(s) in cooperation with industry representatives can control the implement at ion of a remediation plan. However, area

wide resistance will be more difficult to implement and may prove in effective as the boundaries of the resistant populations may not be known and as with refuge

, full compliance of the r emediation plan may not occur. Area

- wide remediation may require removal of the trait for which resistance exists over a wide geographic area. If the removal of a trait due to resistance occurs, the use of any pyramid traits that include the resistant trait needs to be addressed as it would be functioning as a single trait product.

IPM practices include the judicious use of tools within t he IPM toolbox as needed, not all

at once. The use of a Bt hybrid with a seed treatment and a soil insecticide does not fit in

the IPM tool box. The use of soil insecticides with a non

- Bt hybrid reduces selection pressure as band of soil insecticide prot ects roots to prevent yield loss, but provide root tissue outside of the soil insecticide band that are fed upon by CRW larvae. If larvae emerge from the soil insectic ide band, random mating with CRW adults emerging from

the non

- treated area between corn row due to the close proximity reducing the likelihood of resistant individuals from mating with one another. When soil insecticides are used with a Bt hybrid, any CRW adults emerging from the soil insecticide band have been exposed to both the soil insec ticide and the Bt toxin. CRW adults emerging from the area between cornrows have been exposed to the Bt toxin so mating between these two populations that have both survived exposure to the Bt toxin plus individuals who have been survived exposure to the soil insecticide. BMPs do not include all management options, rather BMPs are practices that provide adequate management of a pest and at the same time prevent the development of resistance thus preserving the effectiveness of the management tool. The us e of a soil insecticide with a Bt hybrid is not a BMP. The issue of using a Bt hybrid with a soil insecticide may be driven by the belief that only Bt hybrids have the elite traits that are desirable.

The Panel support s the most conservative remediation plans as outlined by EPA including the recommendation of the use of conventional insecticides to control the adult stage during the present field season and then select an alternative pest control method the following season to reduce the establishment of resistant populations , but only as a last resort to control the spread of resistant populations and not as a population abundance management tool

. Producers should not use conventional insecticides to reduce adult populations to reduce density on an annua l basis as this may lead to resistance thus eliminating the usefulness of the conventional insecticide as part of the remediation plan.60 It should be noted that the use of SmartStax with a 5% refuge will be less durable and could compromise the second toxin in areas where resistance to Cry3Bb1 is occurring

.

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

To mitigate resistant populations, the Panel recommends the use of rotation with a non

h ost plant although this is desirable it will not be effective in some geographic locations due to rotation resistant populations that will survive in first year corn by a shift in oviposition by western corn rootworm or by extended diapause in the case of northern

corn rootworm. The Panel recommends the use of an alternative Bt toxin a s a viable option for rotation resistant populations, but the Panel has concern s with the recommendation to use a soil insecticide with the compromised Bt toxin as we believe this will allow resistant corn rootworm to survive by feeding on Bt expressing roots outside of the application band of the soil insecticide and exposing larval populations to both the compromised Bt toxin and the soil insecticide

. The Panel believes the use of a soil insecticide with a Bt hybrid should not be done. The Panel recommends the use of

a dult control in fields with greater than expected damage during the current season in fields with resistant populations , but dispersal to other fields, althou gh limited, is still a concern as damage may not be noticed until harvest time. The distribution of potentially resistant populations that arise from multiple locations has a greater likelihood of being transported to new locations via extreme weather even ts (Center for Food Safety 2013).

To assist with earlier detection of greater than expected damage the Panel recommends the use of aerial monitoring using planes or drones in high risk areas especially following major weather events that may contribute to lodged corn. The Panel recommends development of a risk map that identifies the varying levels of greater than expected damage reports, crop production practices that contribute to resistance

, including the use of high dose seed treatments (may mask e arly resistance) and lack of refuge compliance.

c. P l e ase c o m m e n t o n th e val u e o f th e o r e t i c al m o d e ls in de sig n i n g r e m e d ial a c t i o n

s t r a te gies f o r vari ou s r e si s t a n c e s c en a r i o s.

WCR biology models are among the best developed that exist in agricultural en tomology. One could reasonably argue that these models have done a good job projecting how varying management and initial biological parameters influence the emergence of

resistance. All these models show strong sensitivity to initial resistance allele

fr equencies, and functional dose (survivorship of heterozygotes on Bt

- corn). Models can: 1) describe the geographic spread of rotation

- resistant phenotypes (Onstad et al. 1999), 2) show that pyramided cultivars slow resistance but that this delay is reduce d when single traits are in the landscape (Onstad and Meinke 2010), 3) show the value of fixed as opposed to random refuges sites in several scenarios (Storer 2003, Pan et al. 2011), and 4) show the affect of population dynamics and movement on rate of res istance development (Storer 2003, Caprio 2001,).61 For some sets of parameters, models are predictive. For example, the Pan et al. (2011)

model , which focuses on contrasting refuge designs, shows only a small influence of refuges when initial allele freq uency is high (0.01) (Table 4). Furthermore, when initial allele frequency is high, years

- to

- 50% resistant allele frequency ranged from 5 to 8 years, which is a good approximation of the time when problem inquiry fields are being found.

As described in detail below the Panel suggest combin ing what is learned from the existing models with empirical information obtained during the years since commercialization to focus both future modeling and remediation strategies. We

describe these next.

First, the P anel believes that Bt

- resistant alleles are not rare. Often the published models start with a frequency of 0.001, and contrast scenarios with that as an initial baseline. The Panel recommends focusing on scenarios that show higher initial frequencies of r alleles, potentially several orders of magnitude higher. Dr. N icholas . Miller , Assistant Professor/Research Entomologist at the University of Nebraska

- Lincoln summarized arguments for focusing on scenarios with a relatively high initial r frequency at t he

E ntomology Society of America (ESA) 2013 meeting in Austin, TX, and his argument is paraphrased here. The first line of evidence is that selection experiments from five studies (Meihls et al. 2008, Oswald et al. 2011, Meihls et al. 2012, Lefko et al 20 08, Meihls et al. 2011) have been able to detect resistance fairly rapidly (3 to 11 generations) from multiple popu lations across the corn belt ( including the areas of Colorado, Illinois

, Kansas, Minnesota, Missouri, Nebraska, Ohio , S outh D akota, and Wisco nsin ). The published support based on selection in the laboratory is stronger for Cr y3Bb1 than Cry34/35 and mCry3A. Onstad and Meinke (2010 ) use observed lab selection data fit to simple population genetic models to suggest an initial r allele frequency o f 0.05 > r > 0.1 (using Lefko et al 2008) or 0.2 (using Meihls et al. 2008).

A second independent line of evidence presented by Dr. Miller is based on the geographic distribution of problem

- inquiry fields, and contrasting that to the pattern of

rotation

- res istant phenotypes. In both cases, we must assume that the presence of problems in the field is directly correlated to field

- evolved resistance. This assumption is not always correct, but it is a reasonable first approximation. The problem inquiry field s from Cry3Bb1 plantings show a pattern of sudden appearance in multiple locations with

lit tle to no spatial correlation. This is consistent with a process of independent selection of alleles that are not rare, and thus relatively easy to increase in freq uency. In contrast, rotation resistance shows a pattern of being selected for a discrete location, followed by diffusion from that location, and strong spatial dependence in the data ( Onstad et al. 1999).

The ability to select for resistance rapidly, fr om many populations, and the geographic pattern of problem fields strongly support the argument that resistant alleles are not rare. This conclusion is stronger for Cry3Bb1 than Cry34/35 and mCry3A

. Models show the ability to delay resistance with refuges is poor when resistant alleles are

not rare. Examples can be seen in biologically rich models (Pan et. al

. 2011), and by simple models based on population genetics presented by Miller at the ESA in Austin,62 TX. As the initial r allele frequency is increa sed, including rates that appear realistic, the ability of refuges to influence increase in r allele frequency is dramatically dampened. With the reduction of refuge size and refuge non

- compliance the influence of refuge on resistance lessened further (Cen ter for Food Safety 2013).

One important conclusion regarding scientific uncertainty, quoted from N. Miller, is that “we ... do not know enough about WRC genetics .” We lack a sufficient understanding of which genes contribute to resistance, how they intera ct, what their current frequency is in field populations, and factors that influence the dynamics of their presence in populations.

The Panel suggest s modeling attributes to add or focus on , given our current understanding of the emer gence of resistant p opulation. However, the Panel believes that the lack of components are not the most critical element for improving the analysis of remediation strategies or for defining proactive IRM programs.. Instead, the Panel

advices that the primary requirements fo r achieving both proactive IRM and remediation

entails an identification of where to focus the initial parameters and endpoints.

d. Th e c u rr e n t d e te rm i n is t i c a n d s to c h a s t ic sim u la t i o n m o d els u s e d f o r I R M pu r po s e s

c o nt a i n m a n y o f th e fo l l o w i n g a tt r i b u te s: e c o l o g y
, p o pu l a t i o n b i o l o g y
, b e h avi o r, a n d

g e n e t i c s o f p es t

, gr o w e r beh avi o r ( r e f

u ge c o m p li a n c e
, i n s e c t i c i d e s p ra y i n g, et c)

, e x p li c i t

s p a t ial a n d p r ob a b il i t y a n al y s e s. W h at o t he r m o d e li n g a t t ri b u te s w ou l d he lp im p r o ve t h e

a n al y sis o f r e m e d ia t i o n s t ra t e gies ?

Although we have not exhaustively reviewed every WRC simulation model, the Panel recommends the following attributes be consistently included in these models :

1. Relatively high r

- allele frequencies as initial parameters. Often the models

include this amo ng the scenarios being compared. The Panel suggest focusing on those scenarios, which would improve the ability to see effects under those

conditions.

2. Non

- random mating . Storer 2003 and Pan et al. 2011 include varying development rates and mating timing t ied to when the different genotypes emerge, which results in non

- random mating. However, a caged study using

N 15 markers reported at the ESA meetings by Krupe et al. suggest higher rates

of non

- random mating, perhaps not entirely tied to development rates

. 3. Multiple alleles ( polygenic (additive) resistance )

. In regards to q uantitative genetic variation a lthough the 2002 SAP (EPA 2002) noted that “For low/moderate dose plants, any gene that confers even slight resistance is expected to be favored by natural s election...”, most models assume a single allele conferring resistance.

. 4. Pyramided traits with varying h (dominance) for each trait. Onstad and Meinke (2010) suggest the presence of single

- traits in the landscape reduce

the benefit that can be achieved wit h pyramids.

5. Varying deployments of trait packages in time : deploying each trait sequentially, versus all

- at

- once in pyramided constructs.63 6. Modeling on the influence of BMPs on abundances and how that affects rates of resistance across relevant landscapes. M odels and theory (Caprio 2001, Storer 2003) support the idea that population abundances influence the rate of resistance through source

- sink dynamics, reflecting complex interactions among population processes (mating, oviposition, and movement), and spati al patterns of Bt corn, nonBt corn, and non

- host plants in the landscape. Also, when BMPs are deployed in response to a problem inquiry field, there is an inherent assumption that BMPs can reduce the probability of widespread Bt

failure. Although BMPs ma y, or may not, influence the proportion of resistant alleles at a given location, BMPs help achieve sustainability of Bt

crops at that location and at wider geographic scales, through their influence on population abundances and processes such as the numbe r of individuals participating in dispersal. It should be noted that use of a Bt trait hybrid with a high dose seed treatment and soil insecticide should not be considered a best management practice or considered part of an integrated pest management

pro gram, but should be included in models to determine the potential for driving resistance to the Bt toxin and the insecticides. Modeling in a GIS framework using current landscapes would be useful for IRM planning and grower education.

In addition to simu lation models, the Panel recommends s tatistical model s describing population genetics and abundance patterns measured in the corn belt

. Recent examples of these modeling approaches (Miller et al

. 2005 ; Ciosi et al 2011; also see discussion for charge ques tion 5) helped elucidate origins and spread rates of populations in Europe. The review in Gray et al. (2009) notes that the Miller et al

. (2005) analysis used Bayesian approaches that integrate historical and genetic data. These approaches should be appl ied to areas associated with problem inquiry fields.

Current IRM models often focus on estimating rates of change in genotype frequencies, or time to achieve an arbitrary proportion of the population in a given genotype category,

but these measures cannot currently be compared to field data of allele frequencies for

validation. In contrast, population abundances can be used for model validation (as in Storer 2003)

, and keeping populations low contributes to resistance management by reducing the number of resistant individuals that disperse to neighboring areas

. The

Panel recommends that IRM models include modeling abundance, in addition to the relative frequencies of genotypes, and couple that with efforts to measure abundances in

the field for validation

. By validating abundances, future models may focus more on parameter values that allows them to become more predictive, at least for abundances, and hopefully also for phenotypes.

The Panel recommends widening the focus of both modeling, and IRM plans, to manage resistance by managing abundances through the use of appropriate BMPs

. This enables direct monitoring of the variable being modeled, and model validation. Although earlier reviews of IRM plans for CRW (e.g., US EPA SAP 2002 and 2009) recognized the lack of a high dose and recommended more stringent IRM plans (Center for Food Safety 2013), the current IRM plan s rel y heavily upon strategies which were64 designed to be useful for high dose. This strategy has theoretical merit and has been

efficacious for conditions of both high dose and very low frequencies of r alleles. Neither

condition is met for the Bt traits currently used for CRW management

. The Panel

summarize s conditions that make it unrealistic to rely upon refuges alone to achieve IRM

for CRW given current Bt hybrids

.

a. Lack of a high dose: none of the traits provide a high dose. Th e survival rate reported in 2002 EPA SAP for MON863 is 17% to 62% survival on MON868.

b. Frequency of r allele: T hey are not rare

c. Resistanc e is probably polygenic. There are multiple r alleles

d. Random mating is not occurring. Lack of random mating was clear in two reports at the Entomological Society of America (ESA) 2013 meeting in Austin,

TX. These include N 15 data presented by Ste ven Smith, a graduate student at Purdue University, and Sarah Hughson, a graduate student at the University of Illinois using different methods. These reports show a higher probability of phenotypes that had developed on B t plants to mate with each other.

e. Cross resistance may exists between Cry3Bb1 and mCry3A

f. The ability to detect incipient locations of resistance and manage them to limit their spread is difficult, and logistical reasons were summarized by Dr. Christian Krup k e, Associate Profes sor, Purdue University, in a symposium at the Entomological Society of America (ESA) 2013 meeting in Austin, TX, and are paraphrased here:

i. LC 50 not reached until 2nd or 3rd instar (2002 SAP)

ii. Bioassay problems, discussed in previous charge questions

iii. Zero foot traffic following planting. P lants with a NIS of 0.5 or 1.0, which are part of the current definition of resistance, may not be well detected. Under some environmental conditions, plants with this level of node

injury may not s how lodging or other signs that would trigger reporting greater than expected damage to seed dealers.

iv. Lack of spatial continuity in the occurrence of problem fields

v. D isincentives for reporting damage may be perceived by growers. These

aspec ts of human behavior are not well studied. They include reluctance to get involved with regulatory agencies, needing to switch among seed dealers, concern about being out of compliance with refuge requirements, or

other factors.

vi. Measuring damage is difficult because it occurs underground

. vii. Method for measuring damage (NIS) is only poorly correlated to population

abundance

viii. Rescue treatments do not exist

ix. Scouting is imprecise and limited

Using refuges to manage the frequency of r alleles is an indirect method. The management action (placement, size of refuges) acts on r

- allele frequency indirectly, through its influence on many population processes, such as those that affect population growth, dispersal, and mating, not throug h direct and selective action on r

- alleles. The65 Panel recommend s that proactive IRM for CRW with current PIPs must embrace

additional indirect methods appropriate for low dose traits, not

- rare r

- alleles, and polygenic r

- alleles. The Panel also recommends an approach that can be evaluated under

field conditions. This will be an approach that focus es on managing abundances (densities), to keep abundances of resistant phenotype sufficiently low.

Currently, problem

- inquiry fields trigger Best Management Pl ans (BMP). These BMPs strongly influence population dynamics

. To achieve IRM for CRW with current PIPs, the sequence of events must be reversed. IRM plans must proactively develop and deploy BMPs, and secondarily rely on detection and remediation of fie lds found to experience greater than expected damage.

The influence of BMPs upon population dynamics are modeled in several papers (Storer 2003, Crowder and Onstad 2005, Crowder et al. 2005, Onstad and Meinke 2010, Pan et al. 2011). The Panel agrees that not all management practices are BMPs, but there are

BMPs that keep WCR abundances sufficiently low (they include crop rotation, rotation among traits, deployment of pyramids perhaps prior to their deployment individually, and others)

. There is an implic it assumption that we can influence r

- allele frequencies and their spread through implementation of BMPs

– this forms the rational of utilizing BMPs following confirmation of resistance in problem

- inquiries. Models should focus on advancing our understand ing of this assumption, with current estimates of allele frequencies and assuming polygenic resistant alleles. Questions that should be

consider ed include BMP s affect s on resistance evolution through their interaction with population dynamics. Examples s how that this modeling approach is feasible. For example, Pan et al. 2013 explore how use

- patterns of seed tr eatments and soil insecticides affect population dynamics and r

- allele frequency

. Storer 2003 contrasts the addition of crop rotation to soil inse cticides, in the years prior to Bt

- corn deployment, on rates of CRW resistance (Fig. 7B and 7C ) , and suggest that “population density can have a significant effect on the rate of adaptation”.

Addressing how BMPs influence both abundance and r

- allele frequ ency will require inputs defined by landscapes, which vary among regions of the cornbelt. For example, Crowder and Onstad (2005) and Crowder et al. (2005) modeled the interaction of rotation and transgenic cultivars on CRW dynamics. Movement among fields in relevant landscapes will need to be considered. For example, young, recently mated females are most likely to be moving among fields (based on captures above from towers placed in fields, and sex

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