1 f
PRO^ Environmental Protection Agency
WHITE PAPER ON RESISTANCE IN LEPIDOPTERAN PESTS OF
BACILLUS THURINGIENSIS (Bt) PLANT-INCORPORATED
PROTECTANTS IN THE UNITED STATES
April 11, 2018
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TABLE OF CONTENTS
ABSTRACT 5
DOCUMENT ORGANIZATION 8
I. BACKGROUND 8
A. Pest of Bt corn and Bt cotton 8
B. Current IRM Strategies and Requirements for Bt Corn and Cotton 10
C. Currently Registered Bt Corn Plant-Incorporated Protectants for Lepidopteran Pests 12
D. Currently Registered Bt Cotton PIPs for Lepidopteran Pests 13
E. Current Resistance Monitoring and Mitigation Strategies for Bt Corn and Cotton 14
1. Bt Corn - Resistance Monitoring and Mitigation 14
a. Bt Corn - Annual Population Sampling and Bioassays 15
b. Bt Corn - Investigation of Reports of Unexpected Damage 17
c. Bt Corn - Resistance Mitigation 17
2. Bt Cotton - Resistance Monitoring and Mitigation 19
a. Bt Cotton - Annual Population Sampling and Bioassays 19
b. Bt Cotton - Investigation of Reports of Unexpected Damage 19
c. Bt Cotton - Resistance Determination Criteria 20
d. Bt Cotton - Mitigation 20
II. PROBLEM FORMULATION 20
A. Cases of Lepidopteran Resistance in the US 21
B. Causal Factors of Resistance 21
1. All Bt toxins are non-high dose for H. zea and expedite resistance development 21
2. Bt toxins expressed in both corn and cotton exert continuous selection pressure on some pests 21
3. Mosaic of single and pyramided traits undercuts pyramid durability 22
4. Use of seed blends in the southern US expedite resistance in ear-feeding pests of corn 22
5. Refuge non-compliance undercuts durability of pyramids 22
6. Methodology for sampling insects and diet bioassays delays resistance detection 22
7. Lack of regulatory triggers for unexpected injury delays reporting ofputative resistance 23
8. Current definition of resistance delays mitigation actions 23
9. Cross-resistance between Bt toxins reduces durability of pyramids 23
10. Non-functional pyramids lead to faster resistance development 23
11. Mitigation of resistance occurs too late 23
12. S. albicosta resistance to Bt corn 24
a. Limited availability of Cry-toxins reduce longevity of Vip3A technology 24
b. Low amount of refuge for single Bt trait reduces its durability 24
c. Seed blends and risk resistance 24
III. SCIENTIFIC UNCERTAINTIES WITH IRM FOR LEPIDOPTERA PESTS AND POTENTIAL
IMPROVEMENTS TO IRM STRATEGIES FOR BT CROPS 24
A. Lepidopteran Resistance Reports in the Continental US 24
1. H. zea resistance and field failure reports 25
2. D. grandiosella resistance reports 27
3. S. frugiperda resistance reports 28
4. S. albicosta resistance reports 29
B. Causal Factors of Resistance 31
1. Non-high dose traits and risk of faster resistance development 31
2. Toxins expressed in corn and cotton exert continuous selection pressure 32
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3. Mosaic of single and pyramided products undercuts durability ofpyramids 33
4. Seed blends in the southern US and faster resistance development in ear-feeding pests of corn 34
a. Mosaic of variable toxin expressions in kernels 34
b. Pollination pathways, survival, and development: 35
c. Maternal Bt plants: 35
i. Mosaic of kernels with variable Bt expression for a three-gene pyramid planted to pure stands 36
ii. Mosaic of kernels with variable Bt expression for single trait products planted to pure stand 37
d. Cross-pollination of refuge ears in RIBs: 37
5. Refuge non-compliance in the southern US undercuts durability of pyramids 39
6. Methodology for insect collections and diet bioassays delays resistance detection 41
a. Current Resistance Monitoring and Mitigation Strategies for Bt Corn and Cotton 41
b. Summary of H. zea Bioassay Results 41
7. Lack of thresholds for unexpected injury delays reporting ofputative resistance 47
a. Lack of regulatory UXI thresholds 47
b. Criteria for UXI thresholds 47
8. Current regulatory definition of resistance delays mitigation actions 49
9. Cross-resistance between Bt toxins reduces durability of pyramids 50
10. Non-functional pyramids lead to faster resistance development 53
11. Mitigation of resistance occurs too late 54
a. Goal of Mitigation 54
b. Uniform Regulatory Threshold Triggers for Mitigation 55
c. Use of diagnostic assay tools 55
d. Fitness cost and benefits for mitigation of resistance 56
e. Cost to resistance 57
12. Risk factors for S. albicosta resistance in Bt corn 61
a. Availability of Cry-toxins is limited 61
b. Percent refuge for single Vip3A trait reduces its durability 61
c. Seed blends and resistance risk 62
IV. EPA CONCLUSIONS AND OPTIONS TO REDUCE RESISTANCE RISKS 62
A. Options to Reduce Resistance Risks : 63
B. Additional Options to Reduce Resistance Risks : 64
C. IRM FOR S. ALBICOSTA 65
V. REFERENCES 66
VI. APPENDIX A. BT CORN RESISTANCE MONITORING STUDIES SUBMITTED TO EPA FOR H. ZEA
78
VII. APPENDIX B. BT COTTON RESISTANCE MONITORING STUDIES SUBMITTED TO EPA FOR H.
ZEA 80
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LIST OF TABLES
Table 1. Lepidopteran Pests of Bt Corn and Bt Cotton at Risk of Resistance to Bt PIPs 9
Table 2. Refuge Requirements for Bt PIPs 11
Table 3. Registered Bt Corn PIPs for Control of Lepidopteran Target Pests 13
Table 4. Registered Bt Cotton PIPs for Control of Lepidopteran Target Pests 13
Table 5. Grower Compliance with Refuge Requirements for Bt Corn in the Southern US 39
Table 6. Susceptibility of Field-Collected H. zea Populations to Bt Corn PIPs (2001 - 2015) 43
Table 7. Susceptibility of Field-Collected//, zea Populations to Bt Cotton PIPs (2003-2015) 45
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Resistance in Lepidopteran Pests to Bacillus thuringiensis (Bt) Plant Incorporated Protectants
(PIPs) in The United States: EPA's Analysis of Scientific Uncertainties Related to Resistance
Management and Options to Enhance the Current Insect Resistance Management (IRM)
Program
ABSTRACT
Reports of lepidopteran resistance to Bt (Bacillus thuringiensis) traits of corn and cotton in the
continental Unites States have been published by academic scientists in 2014 for the fall armyworm
(Spodoptera frugiperda), in 2016 for the corn earworm (Helicoverpa zed), and in 2017 for the western
bean cutworm (Striacosta albicosta). Likewise, a Bt corn registrant reported resistance for the
southwestern corn borer (Diatraea grandiosella) in resistance monitoring reports to the United States
Environmental Protection Agency (US EPA) in 2016. Based on information presented in these reports,
the Agency concluded that the following were risk factors of resistance in these cases: 1) lack of
available high dose traits; 2) use of single mode of action in Bt corn year-after-year; 3) use of corn
seed blends in the southern US; 4) poor refuge compliance for Bt corn in southern states; 5) continuous
selection with the same traits expressed in Bt corn and Bt cotton in a given year; 6) shortcomings in
current EPA recommended methodological approaches with monitoring for resistant field populations;
and 7) challenges with identifying resistance with current diet bioassay methods.
In this white paper1, the Biopesticides and Pollution Prevention Division (BPPD) within the Office of
Pesticide Programs of US EPA (hereafter referred to EPA) discusses these major risk factors believed
responsible for expediting lepidopteran adaptation to Bt toxins in the US and associated scientific
uncertainties. Furthermore, the Agency discusses shortcomings of the current insect sampling
methodologies, rearing and resistance testing of populations and the feasibility of successful mitigation
when resistance is confirmed. In addition, EPA considers whether it is warranted scientifically to
develop a resistance management plan for western bean cutworm.
EPA's overall goals as it considers the options in this document are to reduce resistance risks for
lepidopteran pests, increase the longevity of currently functional Bt traits and future technologies, and
improve the current insect resistance management program for lepidopteran pests of Bt corn and
cotton.
Although resistance could only be confirmed for D. grandiosella using EPA's regulatory process, the
agency's scientists agree with published reports by academic scientists that resistance to Bt has also
evolved in H. zea, S. frugiperda, and S. albicosta. Given the identified risk factors and resistance
occurrences, EPA is considering changes to EPA's IRM program to reduce the selection intensity on
lepidopteran pests of Bt, improve resistance monitoring approaches to confirm resistance faster and
1 Contributors from the BPPD IRM Team include J.C. Martinez, A. Reynolds, K. Welch, E. Bohnenblust, and C. Blanco.
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more reliably, and mitigate resistance where it is suspected or confirmed by academics. Management
options could address the following to lower the risk of resistance to Bt PIPs:
Risk factor 1: Single trait Bt corn in the presence of pyramided products in the landscape;
Option: Limit or otherwise manage single trait commercial products;
Option: Transition from single trait products to Bt corn pyramids.
Risk factor 2: Limited number of highly effective Bt traits;
Option: Adoption of intervening actions at "early warning resistance" in addition to actions
when field failure occurs (see "molecular assay" option);
Risk factor 3: The shift of dominance of resistance in ear-feeding pests of corn with exposure
to
Refuge-In-The-Bag (RIBs) corn products in the southern US;
Option: Potential changes to the corn breeding program to move away from hemizygous
parental corn lines, introduce pollen incompatibility between Bt and non-Bt cultivars, self-
pollinating mechanism, etc.;
Option: Manage the use of Bt corn RIB products in the southern US;
Risk factor 4: Non-compliance with refuge plantings in the southern US;
Option: The development of incentive programs to increase compliance with block refuge
requirements for Bt corn in the southern US;
Risk factor 5: Non-functional, non-high dose pyramided Bt products where individual traits
control less than 95% of susceptible insect;
Option: Implementation of best management practices (BMPs) consistent with the tenets of
Integrated Pest Management (IPM) practices for fields with Unexpected Injury (UXI);
Option: Manage the use of non-functional pyramids;
Option: Increasing the amount of refuge;
Risk factor 6: Resistance monitoring does not allow tracking the same populations year after
year;
Option: Use of sentinel plots to monitor the same populations from year to year;
Risk factor 7: Lack of standardized UXI thresholds;
Option: Development of such standards in corn and cotton would expedite insect collections,
reporting to EPA, and early mitigation;
Risk factor 8: Selection pressure on effective traits (e.g., Vip3A) in pyramids with less
effective or compromised traits;
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Option: Focus on risk in Bt corn which also drive resistance risk to Bt cotton (discussion in
section III. B. 2);
Risk factor 9: Use of diet bioassay methods for non-high dose pests;
Option: Development of molecular/DNA assays to monitor for "early warning" resistance that
allows expeditious resistance confirmation followed by early implementation of mitigation
actions;
Risk factor 10: Mitigation of resistance when field failure occurs;
Option: Establishing an early intervening threshold (e.g., "early warning resistance") would
create a greater likelihood of success for mitigation of resistance and to delay occurrences of
field failure;
Option: An additional option considered by EPA is the encouragement of IPM with IRM for
lepidopteran pests of Bt corn and cotton at the onset of a new trait deployment and throughout
its commercialization (see options in Section IV).
Risk factor 11: Protracted mitigation in response to field failure;
Options: A priori determined size for a Mitigation Action Area (MAA) based on scientifically
defensible factors relying on noctuid dispersal propensity;
Option: Implementing mitigation actions when field failure is apparent instead of after
resistance is confirmed (i.e., practical resistance);
Option: Use of best management practices (BMPs) consistent with the tenets of IPM to reduce
UXI field occurrences and delay resistance development.
In addition to the listed options above, the agency is also seeking guidance on other approaches that
would address the agency's goals and the risk factors identified in this document.
A new framework for lepidopteran pests of Bt should encourage use of IPM with IRM before
resistance is detected. Such IPM steps could include:
Increased scouting for pest densities
Use of alternate control methods when economic threshold on Bt crop is reached
Increased tilling where possible
Crop destruction methods
Multi-year management plans to control lepidopteran pests including rotation of Bt pyramided
products (different modes of action), rotation to conventional corn with insecticide use, etc.
Use of standardized regulatory thresholds defining unexpected injury levels that trigger grower
reporting to seed/trait providers
Improved and timely communication by Bt registrants through notification to growers, state
extension agents, and crop consultants of areas with early warning resistance, unexpected
injury fields, and confirmed resistance cases.
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Additionally, EPA considered whether it is warranted to design an IRM plan for S. albicosta given that
resistance has evolved to CrylF and only one other Bt trait, Vip3 A (pyramided with other Bts),
controls this pest in corn.
DOCUMENT ORGANIZATION
This white paper is organized into the following sections: 1) background; 2) problem formulation; 3)
scientific uncertainties with IRM for lepidopteran pests of Bt crops; and 4) EPA conclusions and
options to reduce risks of resistance.
The background section provides an overview of the resistance monitoring program for
lepidopteran pests of Bt corn and Bt cotton (e.g., insect collection and diet bioassay methods),
lists the current Bt toxins registered in corn and cotton, and summarizes life-history
characteristics of the lepidopteran pests of concern (Section I);
The problem formulation section presents EPA's identified risk factors of resistance to Bt corn
and Bt cotton in the continental US (Section II);
EPA's analysis in the "scientific uncertainties with IRM for lepidopteran pests and potential
improvements to IRM for Bt crops" portion of the document (Section III) discusses each
proposed risk factor with a focus on H. zea, though many also apply to other lepidopteran pests.
Subsections include agency options to address the risk of resistance and identified
uncertainties; and
Lastly, EPA identifies major aspects of the IRM program and options for improvements that, if
incorporated, could improve the durability and longevity of Bt crops in the southern US
(Section IV).
I. BACKGROUND
A. Pest of Bt com and Bt cotton
The following pests of corn and cotton are the focus of this white paper (Table 1).
Helicoverpa zea (Lepidoptera: Noctuidae, CEW (corn earworm), CBW (cotton bollworm)) is a major
economic, agricultural pest in North America, especially on sweet corn and cotton, engages in bi-
directional migrations (South to North, and back), and has between 4-6 generations per year. The
insect is highly polyphagous feeding on numerous wild hosts and cultivated crops, including corn and
cotton. Females are able to lay up to 2,500 eggs in their lifetime and deposit their eggs in clusters on
corn silks and cotton leaves. As neonates mature, they become cannibalistic, and typically one larva
(occasionally two) establishes per corn ear. Once larvae have matured, they move to the soil where
they pupate for approximately two weeks before they emerge as moths. Adults are nocturnal, live
approximately 2-3 weeks, and feed on nectar. (Capinera 2000)
Spodoptera jrugiperda (Lepidoptera: Noctuidae, fall armyworm) is a subtropical and tropical pest of
the Americas, prefers to feed on wild and cultivated grasses, and migrates northward into temperate
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North America during the growing season. In the US, it can have approximately 2 generations and in
tropical regions up to 6 generations per year. Except for subtropical regions in south Florida and Texas,
it does not overwinter in the US Although S. Jrugiperda can cause severe damage when densities are
high, it has been referred to as a sporadic pest of corn and cotton. Females are capable of laying up to
2000 eggs in their lifetime and deposit egg clutches on leaves. Once neonates hatch, they feed in
whorls of corn plants and later move up the plant into the ears of corn. Larvae are less competitive than
H. zea and may be cannibalistic occasionally. It is not atypical to find two or more larvae per corn ear.
Late-instar larvae move to the soil where they pupate and later emerge as adults (approx. 2-3 weeks).
Adults are nocturnal, capable of long-distance dispersal, and feed on plant nectar. (Purdue University
Field Crops IPM 2009a)
Diatraea grandiosella (Lepidoptera: Crambidae, southwestern corn borer) is a pest found in northern
Mexico and the southern US and prefers wild and cultivated grasses as hosts. It is a major pest of corn
with two generations per year in the US Females lay up to 400 eggs on upper and lower surfaces of
leaves and stalks; clutches consist of 2-5 eggs. Larvae feed in whirls of plants first and later bore into
the stalk. Mature larvae migrate to the base of the stalk where they pupate. Because of this behavior,
typically only one pupa is found per plant. (Chippendale & Sorenson 2017)
Striacosta albicosta (Lepidoptera: Noctuidae, western bean cutworm) is native to North America and
has recently undergone a range expansion from the west to the east of the US It has one generation per
year, feeds on reproductive parts of corn and legumes and is a sporadic pest where corn is grown on
sandy soils. Females lay eggs on the upper surface of leaves near the whorls. Neonates consume their
eggshells upon hatching. As they mature, they feed on leaves, anthers, pollen, and ultimately move on
to kernels. Scouting for larvae is challenging because of the lack of evidence for egg hatch and lack of
visible frass. Several larvae may be found on a corn ear since the pest is not cannibalistic. Kernel
damage caused by larval feeding often leads to mycotoxin contamination of ears and, therefore,
reduced quality of corn. Fully developed larvae drop off the ears and burrow into the soil where they
overwinter and undergo pupation in spring. (Purdue University Field Crops IPM 2009b)
pidopteran Pests of Bt Corn and Bt Cotton at Risk of* Resistance to Bt PIPs
Scientific name
Common name
Abbreviation
Bt host
Feeding site
Origin
H. zea
Corn earworm,
cotton bollworm
CEW, CBW
Corn,
cotton
Corn ear,
square, boll,
flower
Native to
the
Americas
S. jrugiperda
Fall army worm
FAW
Corn,
cotton
Leaf, ear
D. grandiosella
Southwestern
corn borer
SWCB
Corn
Leaf, stalk
S. albicosta
Western bean
cutworm
WBCW
Corn
Leaf, ear
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B. Current IRM Strategies and Requirements for Bt Corn and Cotton
Like other pesticides, Bt plant-incorporated protectants (PIPs) are at risk of resistance. However, Bt
PIPs are likely at greater risk than conventional pesticides targeting the same insects because they are
expressed at relatively high, though also variable levels throughout the life of the plant compared to
conventional pesticides, which typically have shorter periods of efficacy and are applied when pests
are likely to cause yield loss. Continuous exposure to Bt PIPs, often over multiple generations per
season, exerts a significant selection pressure and facilitates pest resistance development (EPA 1998,
Matten et al. 2013).
To mitigate the risk of resistance to Bt PIPs, the EPA has required insect resistance management plans
for all commercially registered Bt PIPs as part of the terms and conditions of registration. The primary
resistance mitigation strategy for Bt corn and cotton has been the use of non-Bt refuges, which serve as
sources of Bt-susceptible insects that mate with any resistant individuals emerging from Bt fields (i.e.
the high dose + refuge paradigm; US EPA FIFRA SAP 1998). The goal of the refuge strategy is to
provide sufficient susceptible insects to reduce the frequency of resistance genes in the overall pest
population Under the high dose + refuge paradigm heterozygous resistant insects die when exposed to
Bt, thus keeping the resistance allele frequency low in a population (US EPA FIFRA SAP 1998)
(further discussed below).
Three different refuge strategies have been employed for Bt PIPs. A "structured" refuge consists of a
portion of the crop that is planted to a non-Bt variety. These refuges are typically planted as discrete
fields proximate to Bt fields or as blocks or strips within or adjacent to Bt fields. A second refuge
strategy has been the use of seed blends, in which Bt and non-Bt seeds are comingled within the same
seed bags. This approach, which has been used for Bt corn, has the advantage of ensuring grower
refuge compliance, though there are concerns that durability could be reduced if larvae move readily
between plants (SAP 2011) or if the pest is an ear-feeding insect and is exposed to a mosaic of Bt
expression in kernels (Caprio et al. 2016). The third refuge type is referred to as "natural refuge" and
includes naturally occurring non-PIP plants, such as weeds and other cultivated (non-PIP) crops that
serve as sources of susceptible insects (SAP 2006a; Head et al. 2010; Matten et al. 2013). This type of
refuge strategy is used for Bt cotton.
Refuge requirements for Bt corn and cotton PIPs are summarized in Table 2 below. For Bt corn PIPs,
structured or seed blend refuges have been employed. The amount of refuge required is linked to the
resistance risk: single toxin varieties have larger refuge requirements than pyramided varieties with
multiple traits. Research has shown that pyramids of two traits with independent modes of action (i.e.,
no cross-resistance potential) and high activity (>95% of susceptible insects killed) can delay the
evolution of resistance with a reduced refuge proportion relative to single trait varieties (Roush 1998;
Zhao et al. 2003; Gould et al. 2006; summarized in Matten et al. 2013). Larger refuges have also been
required for Bt corn products grown in cotton-growing regions due to resistance concerns with H. zea,
a pest of both corn and cotton. In addition to the refuge size, structured refuges must be planted close
enough to the Bt fields to increase the likelihood of random mating between susceptible insects (from
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the refuge) and any resistant insects that might emerge from Bt fields. Refuges planted as discrete
fields must be deployed within V2 mile of Bt fields, and refuges within Bt fields must be at least four
rows wide. Insecticide use to control corn borers within the refuge is permitted provided pest pressure
reaches an economic threshold for damage. Seed blends have been approved for pyramided Bt corn
products, though a separate structured refuge must also be planted if these products are deployed in
cotton-growing regions.
For Bt cotton, a detailed analysis was conducted that demonstrated sufficient natural refuge occurred in
southeastern cotton-growing regions for resistance management (SAP 2006a; Head et al. 2010). In
western cotton-growing regions, an ongoing pink bollworm (PBW) eradication program has
supplanted the need for structured refuges (see SAP 2006b; USDA 2015).
Ideally, refuge is deployed in combination with PIPs that are highly efficacious (LC 99.99%) against
the target pests. This so called "high dose + refuge strategy" (US EPA FIFRA SAP 1998; Matten et al.
2013) ensures that only resistant homozygote (RR) genotypes will survive exposure to Bt PIPs. Under
this paradigm, resistance genes are assumed rare in the population, and thus, the likelihood for truly
resistant survivors in Bt fields is low. The few resistant individuals that survive are likely to encounter
susceptible insects from refuges which, assuming random mating, dilute the number of resistance
genes in the overall population. High dose has been defined by the 1998 SAP as "25 times the protein
concentration necessary to kill susceptible larvae", or by a lethal dose of 99.99%. For Bt corn and
cotton, the registered PIPs have generally been determined to be high dose for Ostrinia nubilalis,
Heliothis virescens, and Pectinophora gossypiella but not for H. zea. Though high dose expression is
preferable, for pests against which Bt is non-high dose, IRM can be practiced by including integrated
pest management (IPM) approaches as well as proactive resistance monitoring. The concern with non-
high dose PIPs, however, is that heterozygote genotypes (RS) survive exposure and increase the
amount of resistance genes in a population much faster than under the high dose + refuge paradigm.
Table 2. Refuge Requirements for Bt PIPs
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Crop
Bt PIP type
Refuge type
Refuge requirement
Proximity to Bt
fields
Corn
Single toxin
Structured
Corn Belt: 20%
Cotton regions: 50%
<1/2 mile
Corn
Pyramid
Structured
Corn Belt: 5%
Cotton regions: 20%
<1/2 mile
Corn
Pyramid
Seed Blend
5% (separate 20%
structured refuge required
in cotton regions)
N/A
Cotton
(Southeastern
region)
Pyramid
Natural refuge
Authorized only for
southeastern cotton-
growing regions
N/A
Cotton
(Western
region)
Pyramid
None
Under PBW eradication
program
N/A
In addition to refuge, the Bt PIPs IRM plans include the following elements:
A resistance monitoring program to sample target pests and detect potential shifts in
susceptibility to the PIP toxin(s) as well as collecting and investigating insects from grower
reported field failures;
A remedial action strategy to mitigate resistance should it occur;
A Compliance Assurance Program (CAP) for regions in which Bt corn PIP growers are required
to plant a structured (non-Bt) refuge;
Grower education activities to inform users about IRM concerns and appropriate mitigation
strategies.
C. Currently Registered Bt Cora Plant-Incorporated Protectants for Lepidopteran Pests
Table 3 below lists the currently registered Bt PIPs in corn for control of lepidopteran pests. These
PIPs target major lepidopteran stalk boring and ear feeding pests, including European corn borer, corn
earworm, and southwestern corn borer. Other lepidopteran pests, such as fall armyworm and western
bean cutworm, may also be targeted.
Bt corn varieties are predominantly field corn, though several sweet corn and popcorn uses have also
been registered. The registered PIP traits are bred into corn hybrids with genetic backgrounds that have
been optimized for different corn-growing regions. Other traits, including herbicide tolerance, may
also be stacked in the commercially available hybrids. The Bt PIPs have also been combined to create
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"pyramided" varieties (i.e., two or more toxins targeting the same pest). Commercially available
products include both single toxin and pyramided options.
The agency has drafted a "Biopesticides Registration Action Document" (BRAD) for each registered
Bt corn PIP in Table 3 below. These BRADs contain a summary of the EPA's risk assessments for
each PIP and have been updated periodically to reflect new scientific information and data. For IRM,
assessments of dose, cross resistance, and simulation modeling are contained in these documents. All
of these documents are available on-line (refer to the citations in Table 3).
,<"%»*; DII>« ¦»¦*! rf i
Bt PIP (toxin)
Event
Registrant
Year Registered
BRAD Citation
CrylAb
Btll
Syngenta
1996
U.S EPA 2010a
CrylAb
MON 810
Monsanto
1996
US EPA 2010a
CrylF
TC1507
Dow, Pioneer
2001
US EPA 2010a
CrylA.105
MON 89034
Monsanto
2008
US EPA 2010b
Cry2Ab2
MON 89034
Monsanto
2008
US EPA 2010b
Vip3Aa20
MIR 162
Syngenta
2008
US EPA 2009
D. Currently Registered Bt Cotton PIPs for Lepidopteran Pests
Registered Bt PIPs for cotton are listed in Table 4 below. As with corn, these PIPs have been combined
into pyramids. Unlike corn, commercial varieties are exclusively pyramids (no single trait products are
currently commercially available). Table 4 also includes citations for BRAD documents that contain
the agency's complete IRM assessment for each registered PIP.
e 4, Registered Bt Cotton PIPs for Control of Lepidopteran Target Pests
Bt PIP (toxin)
Event(s)
Registrant
Year Registered
BRAD
Citation
Cry 1 Ac
MON 531, MON 15985
Monsanto
1995
US EPA 2001
Cry2Ab2
MON 15947
Monsanto
2002
US EPA 2003a
CrylF
DAS-21023-5, 281-24-236
Dow
2004
US EPA 2005
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Bt PIP (toxin)
Event(s)
Registrant
Year Registered
BRAD
Citation
Cry 1 Ac
DAS-24236-5, 3006-210-23
Dow
2004
US EPA 2005
Vip3Aal9
COT102
Syngenta
2008
US EPA 2008
FLCrylAb
COT67B
Syngenta
2008
US EPA 2008
CrylAb
T3 04-40
Bayer
2012
US EPA 2012
Cry2Ae
GHB119
Bayer
2012
US EPA 2012
E. Current Resistance Monitoring and Mitigation Strategies for Bt Corn and Cotton
Through the terms of registration, Bt corn and cotton registrants are required to implement a resistance
monitoring program for the major lepidopteran target pests. A primary goal of resistance monitoring is
to detect shifts in the frequency of resistance genes (i.e., susceptibility changes) before the onset of
resistance leads to widespread Bt crop failure (see EPA 2010a).
Monitoring for resistance has consisted of two main components: 1) investigation of unexpected field
damage reports from growers, extension agents, consultants, or company agronomists, and 2)
monitoring for changes in susceptibility through targeted population sampling and testing. Unexpected
damage reports may reveal the occurrence of localized resistance (or hot spots) before the effects have
spread. Resistance monitoring through targeted field sampling can reveal changes in susceptibility of
geographically representative populations. If resistance is confirmed, a mitigation (remedial action)
plan is triggered as defined by the terms of registration (see EPA 2010a). Reports covering each
monitored pest and Bt PIP toxin must be submitted annually to the agency.
The resistance monitoring and mitigation plans for Bt corn and cotton are described below. The
strategies are conceptually similar, though the target pests and resistance standards vary between the
crops.
1. Bt Com - Resistance Monitoring and Mitigation
Resistance monitoring for Bt corn has been required for three lepidopteran pests: O. nubilalis, I),
grandiosella, and H. zea. Monitoring for a fourth pest, fall army worm, is required if an acreage
threshold for Bt sweet corn is surpassed - i.e., 5,000 or more acres per growing season in an individual
county that supports overwintering populations of FAW. To date, this acreage trigger has not been
reached and routine monitoring for FAW has not been conducted since the 2000 growing season (EPA
2010a, BPPD 2015).
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Resistance monitoring is required for each registered lepidopteran-active Bt toxin. The registered
toxins include CrylAb, CrylF, CrylA.105, Cry2Ab2, and Vip3Aa20. Monitoring efforts for the Bt
registrants have been coordinated by the Agricultural Biotechnology Stewardship Technical
Committee (ABSTC)2.
a. Bt Corn Annual Population Sampling and Bioassays
The terms of registration specify that Bt corn registrants must conduct annual sampling and
monitoring of pest populations in corn-growing areas with the highest risk of resistance
development. These regions include locations where lepidopteran active Bt traits are planted on a
high proportion of the corn acres and the target species are regarded as key pests of corn. The
number of populations to be collected is reflective of the regional importance of the pest, with
specific sampling targets identified for each pest. For O. nubilalis, a minimum of twelve (12)
populations across the sampling region must be targeted for collection at each annual sampling. For
D. grandiosella, the target is a minimum of six (6) populations. For H. zea, the target is a minimum
of ten (10) populations (EPA 201 Of).
Collections are made from multiple corn-growing states to represent different geographies and
agronomic conditions. To accomplish this goal, four sampling regions have been identified that
encompass the Corn Belt. For O. nubilalis/D. grandiosella monitoring, Region 1 ((). nubilalis
collections only) is defined as southwestern Minnesota, eastern South Dakota, southeastern North
Dakota, and northwestern Iowa. The target is to sample 6-8 O. nubilalis populations from Region 1.
Region 2 (O. nubilalis and D. grandiosella collections) covers southwestern Kansas and the
Texas/Oklahoma panhandle. From this region, 4-6 O. nubilalis populations and 3-4 I), grandiosella
(at least one from Texas) are targeted for sampling. Region 3 (O. nubilalis only) consists of
central/southeastern Iowa and north-central Illinois with a sampling target of 4-6 O. nubilalis
populations. Region 4 (D. grandiosella only) is focused on the Missouri bootheel, western
Kentucky, western Tennessee, and the southern tip of Illinois (target sampling of 3-4 D.
grandiosella populations). Individual sampling sites are determined by pest population size and are
taken at distances greater than V2 mile from Bt cornfields, to minimize the effects of elevated
resistance gene frequencies misrepresentative of the population average.
To obtain sufficient sensitivity to detect resistance alleles before they become common enough to
cause measurable field damage, the goal of each population collection is 400 insect genomes. For each
insect population to be sampled, either 200 larvae, 200 adults, 100 mated females, or 100 egg masses
can be collected to reach the objective of 400 genomes per population (egg masses are assumed to
have at least 4 genomes). However, a collection attempt can be considered successful with a minimum
of 100 genomes since it may not be possible to collect the full target number due to factors such as
2 The Agricultural Biotechnology Stewardship Technical Committee (ABSTC) is an industry consortium of Bt corn
registrants, including Monsanto, Dow, Pioneer/DuPont, and Syngenta.
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natural fluctuations in pest density, environmental conditions, and area-wide pest suppression. In such
cases, the minimum population sizes to be collected is 50 larvae, 50 adults, 25 mated females, or 25
egg masses. The specific life stage to be sampled depends on the insect and the practicality of the
collection method. Most collections occur at the peak flights of second or later generations (first
generation flights may not produce sufficient numbers for sampling).
Bioassays of the collected populations are conducted to assess susceptibility. For the lepidopteran corn
pests, these assays have been conducted with artificial diet bioassays and Bt toxin overlays (e.g.,
Siegfried et al. 1995). Two bioassay approaches have been employed: dose-response testing to
measure lethal (LCso) and sub-lethal (ECso) susceptibility, and diagnostic concentration assays with
high levels of toxin that distinguish resistant individuals from susceptible ones (e.g., Mar<;on et al.,
2000).
Any population that demonstrates unusually low sensitivity to a Bt toxin is further investigated to
determine if the population has field-relevant resistance to the trait. The following investigative
procedures and resistance determination criteria are required (taken from EPA 2010a):
i. Step 1: Re-test progeny of the collected population to determine whether the unusual
bioassay response is reproducible and heritable. If it is not reproducible and heritable, no
further action is required.
ii. Step 2: If the unusual response is reproducible and heritable, progeny of insects that survive
the diagnostic concentration will be tested using methods that are representative of
exposure to Bt corn under field conditions. If progeny do not survive to adulthood, any
suspected resistance is not field relevant and no further action is required.
If insects survive steps 1 and 2, resistance is confirmed, and further steps will be taken to
evaluate the resistance. These steps may include the following:
a. Determining the nature of the resistance (i.e., recessive or dominant, and the level of
functional dominance); estimating the resistance allele frequency in the original
population;
b. Determining whether the resistance allele frequency is increasing by analyzing
field collections in subsequent years sampled from the same site where the
resistance allele(s) was originally collected;
c. Determining the geographic distribution of the resistance allele by analyzing field
collections in subsequent years from sites surrounding the site where the
resistance allele(s) was originally collected.
iii. Step 3: Should field-relevant resistance be confirmed, and the resistance appears to be
increasing or spreading, the registrant must consult with EPA to develop and implement
a case-specific resistance management action plan.
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b. Bt Corn Investigation of Reports of Unexpected Damage
In addition to the annual population sampling described above, Bt corn registrants are required to
investigate grower reports of unexpected damage (UXD) or unexpected injury (UXI) (Andow et al.
2016) to Bt corn. EPA has established a two-step process to investigate these reports.
Once a UXD report is received, the registrant will confirm that the affected acreage was planted to a
lepidopteran-active Bt corn variety with proper toxin expression in plant tissues. Provided the insect
damage was not due to environmental (or other) reasons, the case is termed "suspected resistance."
The registrant must attempt to collect insects for bioassay testing while working with the affected
grower to implement mitigation actions to minimize spread of the insect population (see "Resistance
Mitigation" below). The terms of registration do not establish specific damage thresholds for UXD
investigations; rather it is the responsibility of the registrant to determine the level of injury that
constitutes significant field damage worthy of investigation.
As with the annual monitoring described above, dose-response and diagnostic concentration testing are
used to determine resistance with UXD cases. If the following criteria are met, the UXD population is
considered to have "confirmed resistance" (taken from EPA 2010a):
There is >30% insect survival and commensurate insect feeding in a bioassay, initiated
with neonate larvae, that uses methods that are representative of exposure to Bt corn
hybrids under field conditions (O. nubilalis and D. grandiosella only).
In standardized laboratory bioassays using diagnostic concentrations of the Bt protein suited
to the target pest in question, the pest exhibits resistance that has a genetic basis and the level
of survivorship indicates that there may be a resistance allele frequency of > 0.1 in the
sampled population.
In standardized laboratory bioassays, the LCso exceeds the upper limit of the 95%
confidence interval of the LCso for susceptible populations surveyed both in the original
baselines developed for this pest species and in previous years of field monitoring.
c. Bt Corn Resistance Mitigation
If resistance is confirmed, either in populations collected from the annual sampling or from UXD
reports, Bt corn registrants are required to implement a mitigation (remedial action) plan. The goal of
resistance mitigation is to limit or contain (extirpate if possible) the spread of the resistant population
and maintain the durability of the Bt trait in areas where it is still effective.
For resistant populations identified through the annual sampling, registrants are required to consult
with EPA to develop and implement a case-specific resistance management action plan. For UXD
cases, action levels have been established for "suspected" and "confirmed" resistance (EPA 2010a).
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In cases of suspected resistance, registrants will work with growers to:
Use alternative control measures in Bt corn fields in the affected region to control the target
pest during the immediate growing season.
Destroy Bt corn crop residues in the affected region within one (1) month after harvest with a
technique appropriate for local production practices to minimize the possibility of resistant
insects over- wintering and contributing to the next season's target pest population.
When resistance is confirmed, the following steps are required:
EPA will receive notification within 30 days of resistance confirmation;
Affected customers and extension agents will be notified about confirmed resistance within 30
days;
Monitoring will be increased in the affected area and local target pest populations will
be sampled annually to determine the extent and impact of resistance;
If appropriate (depending on the resistant pest species, the extent of resistance, the timing of
resistance, and the nature of resistance, and the availability of suitable alternative control
measures), alternative control measures will be employed to reduce or control target pest
populations in the affected area. Alternative control measures may include advising
customers and extension agents in the affected area to incorporate crop residues into the soil
following harvest to minimize the possibility of over-wintering insects, and/or applications
of chemical insecticides;
Unless otherwise agreed with EPA, stop sale and distribution of the relevant lepidopteran-
active Bt corn hybrids in the affected area immediately until an effective local mitigation
plan, approved by EPA, has been implemented;
The registrant will develop a case-specific resistance management action plan within 90
days according to the characteristics of the resistance event and local agronomic needs. The
registrant will consult with appropriate stakeholders in the development of the action plan,
and the details of such a plan shall be approved by EPA prior to implementation;
The registrant will notify affected parties (e.g., growers, consultants, extension agents,
seed distributors, university cooperators, and state/federal authorities as appropriate)
in the region of the resistance situation and the EPA approved action plan; and
In subsequent growing seasons, maintain sales suspension and alternative resistance
management strategies in the affected region(s) for the Bt corn hybrids that are affected
by the resistant population until an EPA-approved local resistance management plan is in
place to mitigate the resistance.
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2. Bt Cotton - Resistance Monitoring and Mitigation
Resistance monitoring for Bt cotton follows the same approach as Bt corn, with annual population
sampling and investigations of unexpected pest damage to Bt cotton fields. Three lepidopteran pests
are routinely monitored: tobacco budworm (H. virescens), cotton bollworm (H. zea), and pink
bollworm (Pectinophora gossypiella). H. zea is typically referred to as bollworm or cotton bollworm
as a pest on cotton and as corn earworm when infesting corn. Monitoring for this pest also occurs in
corn.
Registered Bt cotton PIP traits include Cryl Ab, Cryl Ac, CrylF, Cry2Ab2, Cry2Ae, and Vip3Aal9.
Resistance monitoring is conducted for the Cryl Ac, CrylF, Cry2Ab2, and Vip3A. Given structural
similarities between toxins, monitoring for CrylAc serves as a surrogate for Cryl Ab, while Cry2Ab2
testing also encompasses Cry2Ae. The summary of the cotton resistance monitoring strategy below
was derived from the terms of registration for Bt cotton PIPs (Table 4) (see Appendix B).
a. Bt Cotton Annual Population Sampling and Bioassays
As required by the terms of registration, Bt cotton registrants must conduct annual monitoring of the
primary lepidopteran target pests. Similar to Bt corn, registrants formed a consortium group, the
Cotton Technology Stewardship Committee (CTSC), in 2015 to coordinate the annual sampling and
bioassay activities for Bt cotton.
Registrants are required to focus collection sites for the annual monitoring in areas of high adoption of
Bt cotton in the Southeastern US, with the goal of including all states where these insects are economic
pests. The target is to collect 20 or more pest populations per growing season, with each population
consisting of at least 200-500 third instar larvae, 100-200 adults, or 200 eggs. Sampling is focused on
high risk areas, such as those with high Bt cotton adoption or low amounts of natural refuge.
Resistance detection bioassays have focused primarily on the use of diagnostic concentrations to
measure survival to set doses of toxin. Some dose-response testing to determine LCso and EC so values
has also been conducted for CrylF monitoring. Assays have been conducted by Custom Bio-Products
(H. virescens, H. zea).
b. Bt Cotton Investigation of Reports of Unexpected Damage
In addition to the annual sampling, registrants are required investigate reports of unexpected damage to
Bt cotton fields. The investigative steps are similar to those for Bt corn and include confirmation that
the affected plants are Bt-expressing varieties and that no other explanations (e.g., climatic, cultural, or
other non-targeted pests) are responsible for the damage. Registrants will attempt to sample pest
populations from the UXD fields for bioassay assessments of susceptibility (see Resistance
Determination Criteria below). As with Bt corn, EPA has not established specific damage thresholds
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for UXD investigations in the terms of registration. Each company is responsible for coordinating
scouting and damage report responses for its products and determining whether UXD cases warrant
further investigation.
Monsanto has also employed the use of "IPM-based monitoring" for its Bollgard cotton products. Crop
consultants, cotton growers, and other commercial representatives normally scout for pest damage in
cotton during the growing season. Monsanto works with these personnel to identify unusual pest
survival or damage to Bt cotton and collect insects for further investigation.
c. Bt Cotton Resistance Determination Criteria
The criteria used to make resistance determinations have varied between registrants, but generally
establish procedures for suspected ("putative") and confirmed resistance cases. Resistance is suspected
if populations collected through annual sampling or from a UXD field exhibit a statistically significant
increase in survival relative to established baseline susceptibility data. To confirm resistance,
companies conduct further study of the putative population to verify the initial bioassay results,
evaluate heritability of the observed trait, assess potential field relevance (i.e., survival on Bt cotton
plant tissue), and determine the resistance allele frequency. A population is considered resistant if the
confirmatory studies show statistically significant survival (95% confidence interval) relative to the
established LC50 baseline susceptibility for H. virescens or H. zea.
d. Bt Cotton - Mitigation
Mitigation strategies for Bt cotton essentially follow the same paradigm as those for Bt corn by
establishing procedures for "suspected" and "confirmed" resistance.
For suspected resistance, registrants conduct additional field sampling for look for further signs of
resistance, collect H. virescens or H. zea larvae for bioassays, and work with affected growers to
implement alternate control measures and crop destruct techniques for the immediate growing season.
For confirmed resistance, registrants follow the same mitigation steps developed for Bt corn (listed
above).
II. PROBLEM FORMULATION
The problem formulation identifies potential pathways of resistance among targeted insect pests from
use of Bt plant-incorporated protectants. EPA determined a list of causal factors that may accelerate
the risk of resistance development in lepidopteran target pests of Bt corn and cotton. Although the
'Causal Factors of Resistance' address mostly issues for H. zea and S. albicosta, the identified risk
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factors also apply to other pests of Bt cotton and/or corn (e.g., S. frugiperda, D. grandiosella). Based
on the identified risks and uncertainties, EPA discusses options for changes to the IRM strategies for
lepidopteran pests targeted by Bt (see Section III), which are also summarized in Section IV of this
document.
A. Cases of Lepidopteran Resistance in the US
Resistance has been reported for D. grandiosella, S. frugiperda, H. zea (non-high dose pest), and S.
albicosta in various parts of the US. With the exception of one report (D. grandiosella), resistance was
determined by academic researchers and not through the monitoring activities conducted by
registrants. It is important that the agency examine its current IRM program for lepidopteran pests of
corn and cotton to ascertain that resistance is proactively detected and effectively mitigated by
registrants. For a discussion of the resistance cases, see Section III. A.
B. Causal Factors of Resistance
1. All Bt toxins are non-high dose for 11. zea and expedite resistance development
All currently registered Bt toxins aimed at suppressing H. zea in corn and cotton express less than high
dose (see Tables 3 & 4) with Vip3A being the most efficacious trait of all (Burkness et al. 2010).
Overall, resistance can be expected to evolve faster under moderate or intermediate dose conditions
(Comins 1977; Tabashnik & Croft 1982), especially with higher population growth rates as reported
for H. zea (Caprio et al. 2009; Martinez et al. 2017). The pest has shown to have great genetic
variability with respect to susceptibility to Bt toxins (Stone and Sims 1993; Ali et al. 2006).
Populations that harbor a greater proportion of individuals with tolerance can be expected to evolve
resistance to single as well as pyramided traits more rapidly. For an in-depth discussion of this issue,
refer to Section III. B.l.
2. Bt toxins expressed in both corn and cotton exert continuous selection pressure on some
pests
Most Bt toxins expressed in corn are also deployed in cotton products. This exerts continuous selection
pressure on all traits for pests having multiple generations per year feeding on corn and cotton (e.g., H.
zea, S. frugiperda). Currently, the most efficacious trait in corn and cotton is Vip3A. Given this trait
scenario for both crops and recent resistance reports for H. zea, the risk of resistance to the most
efficacious trait(s) in the southern U.S. is of concern. For further discussion of this topic, refer to
Section III. B.2.
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3. Mosaic of single and pyramided traits undercuts pyramid durability
The southern US is a mosaic of single (corn) and pyramided Bt products that share the same traits.
Resistance to single toxin Bt crops can undercut the durability of pyramid Bt crops (Tabashnik et al.
2008). Single traits serve as stepping stones for resistance to pyramids (Zhou et al. 2003) reducing the
expected durability of the Bt technology below what could be expected if only pyramids were present
in the landscape. For an in-depth discussion of this topic, refer to Section III. B .3.
4. Use of seed blends in the southern US expedite resistance in ear-feeding pests of corn
Planting of corn seed blends (Bt and non-Bt seed in the same field) creates four different pollination
scenarios resulting in a mosaic of Bt expression in kernels. Two of these possible scenarios pose
special IRM concern in the southern US: 1) maternal Bt X paternal non-Bt and 2) maternal non-Bt X
paternal Bt. These particular pollination scenarios lead to a mosaic of Bt expressions with potentially
sub-lethal toxic exposure allowing heterozygous resistant larvae to escape control while killing
susceptible larvae only. This dynamic shifts the dominance of resistance towards higher values and can
exacerbate resistance evolution in ear-feeding pests of Bt corn. For an in-depth discussion of this issue,
refer to Section III. B.4.
5. Refuge non-compliance undercuts durability of pyramids
Publicly available data and data submitted to the agency as part of the IRM program for Bt corn
products have shown that refuge compliance has historically been low in the southern US (Reisig
2017; BPPD 2017). Full compliance has been between 12-46%, while zero compliance (no refuge
planted) ranged between 11-44% from 2011-2016. Surveys support a conclusion that knowledge of
importance of available refuges does not increase refuge compliance among southern US corn growers.
Lack of compliance increases the risk of resistance development. For an in-depth discussion of this
issue, refer to Section III. B.5.
6. Methodology for sampling insects and diet bioassays delays resistance detection
EPA receives resistance monitoring reports from biotechnology companies that are based in part on
random collections of insects from US corn and cotton growing areas. These types of samples provide
a "snap shot" in time, do not track population susceptibility over time, and are reactive in nature.
Historical diet bioassay results for H. zea have been variable and difficult to interpret in part due to the
random population sampling approach, the pest's inherent genetic variability (Stone & Sims 1993; Ali
& Luttrell 2007), lack of diagnostic concentrations for non-high dose toxins, fitness costs of resistant
insects, and other methodological challenges. Given the current challenges, it is unlikely that resistance
can be detected before it becomes visible as Bt field failure. For further discussions of this topic, refer
to Section III. B.6.
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7. Lack of regulatory triggers for unexpected injury delays reporting of putative resistance
A lack of standardized, actionable regulatory thresholds of unexpected damage in Bt corn and cotton is
likely leading to delayed reporting of problem fields and spread of resistance before mitigation can be
initiated. While each registrant has its own thresholds for unexpected injury to corn and cotton, these
are not expected to be uniform between companies and have not been shared with EPA. For an in-
depth discussion of this topic, refer to Section III. B .7.
8. Current definition of resistance delays mitigation actions
EPA's definition of pest resistance is based on heritability of the resistant trait, higher survival of
resistant individuals compared to susceptible individuals on Bt crops, and visible economic damage
caused to Bt crops in the field. This definition does not allow for proactive mitigation of resistance and
is unlikely to achieve its intended goal of slowing the spread of resistance and maintaining the
durability of Bt in surrounding areas. For an in-depth discussion of 'resistance' issue, refer to Section
III. B.8.
9. Cross-resistance between Bt toxins reduces durability of pyramids
Second generation, pyramided Bt crops produce two or more toxins active against the same pest and
may delay the evolution of resistance through redundant killing (Comins 1986). However, cross-
resistance can undermine redundant killing in pyramided crops and reduce the durability of the Bt
technology. Cross-resistance occurs when selection for resistance to one toxin causes resistance to a
second toxin. H. zea has shown documented resistance to first generation Cryl Ac and Cryl Ab Bt
toxins, which are pyramided alongside other highly homologous Bt toxins. In cases where some insects
survive on pyramids, even weak cross-resistance is expected to accelerate insect resistance evolution to
additional toxins. For an in-depth discussion of cross-resistance, refer to Section III. B.9.
10. Non-functional pyramids lead to faster resistance development
Bt resistance for multiple lepidopteran pests have been reported in the continental US (H. zea, S.
frugiperda, S. albicosta, and D. grandiosella). Pyramided products expressing these traits have been
compromised in some areas (<95% control by each toxin), yet are deployed with a relatively small
refuge of 5% (the mandated refuge for pyramids). When one of the Bt components is compromised,
then the reduced refuge will increase the risk of resistance to the second trait. For further discussion of
this topic, refer to Section III. B.10.
11. Mitigation of resistance occurs too late
Based on EPA's definition of resistance, mitigation tactics are not mandated until field resistance is
confirmed (i.e. "practical resistance" and >50% of population carrying resistance genes, Tabashnik et
al. 2014). The timing of mitigation is delayed and will complicate the success of the agency's goal to
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extend the durability of Bt in unaffected regions and contain or reduce resistance in the affected areas.
For further discussion of this topic, refer to Section III. B.ll.
12. S. albicosta resistance to Bt com
a. Limited availability of Cry-toxins reduce longevity of Yip 3A technology
S. albicosta was identified as a sporadic pest of corn, and no IRM plan is currently in place, while only
two registered Bt toxins showed activity against the pest at the time of product registration (CrylF and
Vip3Aa20). Since 2010, however, reduced susceptibility to CrylF corn has been documented in corn
growing areas. In 2017, control failure and resistance have been reported in Ontario, Canada, and
several areas in the US (Smith et al. 2017; DiFonzo et al. 2016; Ostrem et al. 2016). Vip3Aa20 remains
the only effective Bt trait available for western bean cutworm in these areas. For further information,
see Section III. B.12.a.
b. Low amount of refuge for single Bt trait reduces its durability
Vip3 A is pyramided with other toxins in Bt corn and deployed with a 5% refuge (external block or
integrated) (US EPA 2010a). This trait is, therefore, deployed with a much lower refuge (5%) than
would typically be required for single traits (20%) targeting major pests. This refuge scenario exerts a
tremendous selection pressure on the only available Bt control tool for western bean cutworm. For
further information, see Section III. B.12.b.
c. Seed blends and risk resistance
Larvae of S. albicosta feed in ears of corn, similar to H. zea. Sub-lethal expression to a mosaic of Bt
kernels might favor survival of heterozygous resistant genotypes, while killing off susceptible insects,
thereby effectively shifting dominance of resistance to higher values. Seed blends could, therefore,
present another causal factor of resistance exacerbating resistance to Vip3 A. For further information,
see Section III. B.12.C.
III. SCIENTIFIC UNCERTAINTIES WITH IRM FOR LEPIDOPTERA PESTS AND
POTENTIAL IMPROVEMENTS TO IRM STRATEGIES FOR Bt CROPS
A. Lepidopteran Resistance Reports in the Continental US
Field resistance to three out of four Lepidoptera species has been reported by academic scientists in the
continental US: H. zea (Dively et al. 2016), S. frugiperda (Huang et al. 2014), and S. albicosta (Smith
et al. 2017). Resistance to these pests of Bt did not get reported through the regulatory process in place,
likely because of methodological issues with sampling populations, confirmation of resistance, as well
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as lack of uniform regulatory triggers (see earlier discussions). Nonetheless, the available evidence
suggest resistance of these three pests has evolved to Bt in some parts of the continental US.
1. II. zea resistance and field failure reports
a. Tabashnik et al. (2008a) suggested that H. zea field resistance to Cryl Ac cotton had evolved
based on an analysis of laboratory diet bioassay data ranging from 1992 (pre-commercialization)
to 2006 (post-commercialization). The authors reported resistance ratios from 1.2 up to 578.
Moar et al. (2008) rebutted the reports of resistance by stating that the diet bioassay results from
resistance monitoring activities needed to be tied to field performance of the traits as well as
larval survival on the Bt plants. Tabashnik et al. (2008b) responded that progeny of USDA-
collected adult insects were indeed from Bt and non-Bt cotton fields, and that reduced efficacy
and increased larval survival had been measured in those Bt locations. Conversely, there was no
reported field resistance in H. zea to any of the Bt toxins by Ali et al. (2006).
b. Dively et al. (2016) conducted a 21-year and six-year field efficacy study for H. zea on CrylAb
sweet corn and CrylA.105+Cry2Ab2 sweet corn, respectively, in Maryland. The authors
collected data on population abundance and damage observed in ears (Bt and non-Bt) as well as
shifts in larval instar development across the years. The authors identified fitness cost in the
resistant population when exposed to Bt, which resulted in prolonged larval and pupal
development, lower pupal weight, and reduced survival to adulthood.
Across the years, infestation levels in the field were always high in non-Bt corn with >82% of
ears damaged and >80% of larvae reaching late instar. The damage to CrylAb ears increased
from >6% in 1996 to >85% in 2016. Mean instar size of surviving larvae in Bt also increased
over the study period with the proportion of late instars getting increasingly larger compared to
non-Bt corn. Between 1996-2006, fewer 4th - 6th instars were recorded. Between 2015-2016,
37%) of surviving larvae reached 4th - 6th instar.
Trends in Cryl A. 105+Cry2Ab2 field corn where similar to those observed for CrylAb sweet
corn. Namely, damage in Bt increased significantly compared to non-Bt controls supporting a
conclusion of reduced field efficacy (based on damage) of Bt plants. Between 2010-2012, >20%>
of Bt ears were damaged and >6%> of larvae reached 4th instar. During 2015-2016, >59%> of Bt
ears were damaged with >70%> of surviving larvae reaching 4th instar.
Assay results on the artificial diet without Bt leaf powder showed that the susceptible lab strain
developed better (avg. weight 218 g) than the 2015 field collected strain (avg. weight 159 mg).
Though the differences were not statistically significant (P-value = 0.099), this could indicate
that the susceptible strain was better adapted to consuming the artificial diet than the field strain.
When both strains were exposed to diet with incorporated Bt leaf powder at various
concentrations (10-600 mg of Bt/25 ml diet), the weight gain of both colonies was reduced, but
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the rate of decrease in weight was greater for the control colony than for the field colony (/J-value
<0.05).
The authors concluded that the rapid decrease in field efficacy and decreased susceptibility of H.
zea in recent years provide strong evidence of field-evolved resistance to multiple Cry toxins
(CrylAb, CrylA.105, and Cry2Ab2).
c. In a one-year study, Yang et al. 2017a3 investigated the susceptibility to Bt toxins of H. zea with
diet overlay assays. Populations were obtained from four areas of the southern US, such as
Mississippi (1 population from a VT2P Bt corn), Louisiana (1 population collected on Bt cotton),
Arkansas (1 population collected on non-Bt corn), and Tennessee (1 population each collected on
Bt cotton and grain sorghum).
When the resistance ratios were obtained from LCso results, the range for the susceptible lab
colonies (Benzon Research and USDA-ARS, Stonesville strain) was 1.0 - 4.9 for CrylAc and
Cry2Ab2. The resistance ratios of populations collected from non-Bt hosts ranged from 1.6 -
48.3 (CrylAc) and 1.0 - 133.3 (Cry2Ab2) and on Bt hosts from 2.1 - 7.5 (CrylAc) and 2.4 -
133.4 (Cry2Ab2). The resistance ratios could not be calculated for Vip3A. When the resistance
ratios were obtained from MIC so results, the range for the susceptible lab colonies (Benzon
Research and USDA-ARS, Stonesville strain) was 1.0-5.1 for CrylAc and Cry2Ab2. The
resistance ratios of field population resistance collected from non-Bt hosts ranged from 1.6 - 5.7
(CrylAc) and 1.0 - 133.4 (Cry2Ab2) and on Bt hosts from 9.5 - 48.3 (CrylAc) and 4.0 - 35.7
(Cry2Ab2). Any resistance ratios in excess of 10 were indicated to be significant.
d. Two locations from which Yang et al. (2017)3 collected population samples were subsequently
reported by Monsanto Company (Monsanto) as problem performance sites for 2016 Bt cotton
fields (Bollgard II, CrylAc + Cry2Ab2) in Tennessee and Louisiana (Berman et al. 2017).
Monsanto did not collect insects in TN because 6% boll damage and possibly other factors did
not meet their criteria for population collections. Likewise, in LA no collections were undertaken
by the company because no insects were present after the problem field was sprayed. Reported
boll damage was 19%. Monsanto reported that these results fell well into the range of the
historical base data for H. zea.
Monsanto also reported that two additional UXI fields occurred in North Carolina and Texas
(Berman et al. 2017). Both fields did not meet the criteria for insect collections to begin, although
those criteria were not reported to the agency.
e. Bayer CropScience (Bayer) submitted a report for four unexpected damage incidents in
TwinLink Cotton (Cry2Ae + CrylAb) in three US locations (Texas, Louisiana, and Oklahoma)
during the 2014 cotton growing season (Payne et al. 2017). The company was unable to obtain
3 EPA recognizes that these data have not been published yet and is treating them as a preliminary report of potential
resistance.
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insects in LA and OK, but collected two larval populations in Texas in 2015 and another
population in 2016 all in excess of 100 individuals. The insects were reared and testing occurred
with neonates of the F1 generation.
The LCso and MIC50 (2nd and 3rd instar) for Cry2Ae and Cryl Ab were lower than the baseline
monitoring data submitted to the agency (Payne et al. 2014). The field results were somewhat
higher than the susceptible control colonies, however. Bayer concluded that the field-collected
populations remained susceptible to the traits expressed in TwinLink Cotton.
Uncertainties
EPA did not receive information about the larval survival of the field collected populations from Texas
(UXD/UXI reports). It is unclear if potentially resistant larvae were killed during the transport to the
lab and during the rearing process. This information is needed to judge the results in their entirety and
context. The conclusion that the field populations were susceptible (yet caused calls about unexpected
damage) is somewhat non-intuitive. If the unexpected damage fields had a high insect pressure year,
this information should have also been submitted.
If heterozygous resistant individuals of H. zea behave much like their susceptible counterparts (Blanco
et al. 2008), Bayer's diet bioassay with F1 generations would not detect resistance. Both RS and SS
individuals could be expected to die from Bt exposure. An F2 screen should have been conducted to
obtain homozygous resistant survivors that then allow estimating the resistance allele frequency in the
field sample.
2. />. grandiosella resistance reports
Pioneer Hi-Bred International, Inc. (Pioneer) submitted a CrylF resistance monitoring report to EPA in
which the company reported two unexpected damage incidents from Arizona (Cochise Valley) and one
occurrence in New Mexico from Southwestern corn borer feeding in 2014 (MRIDs 497854-01 and
501749-01). The company was unable to collect population samples from the damaged fields in 2015
and obtained samples from three nearby locations in Arizona only but also obtained collections from
outside the region (e.g., OK and TN for comparison purposes). Pioneer conducted diet incorporated
bioassays on the offspring populations from Arizona field samples to estimate the EC/LC50 and
resistance allele frequency. Survivors of the diet bioassay study were reared to initiate an on-plant
feeding experiment on Herculex (CrylF) corn in the field.
27
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The LC50 could not be measured for the Arizona population. Pioneer reported that the LC50 values
were >31.0 |ig/ml of diet than the highest concentration tested and ranged from 55.3-260 |ig/ml. In
contrast, the LC50 of the susceptible lab colonies was 2.9 and 3.3 |ig/ml of diet (MRID 497854-01).
Mortality for the Cochise Valley population at the highest concentration tested was 13.9% (68.6 |ig of
CrylF/ml), while control mortality on non-Bt diet was 9.2% (0 |ig of CrylF /m). Pioneer concluded
that the Cochise population had developed resistance to CrylF based on the calculated resistance ratio
(310) and the resistance allele frequency (0.97) (MRID 501749-01).
The infestation of CrylF corn with eggs from survivors occurred over 3 days (20 eggs/day-plant).
Plants were caged, larvae were collected from non-Bt and Bt plants, and survivors (larvae + pupae)
were shipped in ethanol to the laboratory for measurements. The mean number of insects from UXD
populations did not differ on Bt and non-Bt treatments (survivorship 89.8% and 100%, respectively).
The instar distribution for the Arizona population was not significantly different on Bt and non-Bt
plants. The lab colony's survival was 0.4% on Bt and 100% on non-Bt. In this case, the distribution of
instars was significantly different on Bt compared to non-Bt (MRID 501749-01).
Pioneer concluded resistance had evolved in southwestern corn borer in the populations investigated.
Because of the Cochise valley's isolated location, mitigation of SWCB resistance to CrylF corn should
be more likely attainable. Pioneer implemented southwestern corn borer resistance mitigation plan
(planting of pyramided products only), best management practices (planting required refuge, early
planting, scouting, use insecticides when needed, destroy crops post-harvest) in Cochise valley.
Across Arizona, New Mexico and western Texas monitoring for resistance is increased and 5-7 insect
collections total will be targeted yearly. Communication with key stakeholders in the regions was
enhanced to inform them of the resistance to CrylF and recommend best management practices when
the economic thresholds were met (MRIDs 501749-01 and 501749-02).
Uncertainty:
EPA considers the mitigation approach taken by Pioneer reasonable. However, mitigation of resistance
would ideally occur with an unrelated pyramided product or a three-gene containing pyramid
expressing the compromised trait. It is unclear whether Pioneer has implemented mitigation with an
unrelated pyramided product since resistance has been confirmed.
3. S. frugiperda resistance reports
In 2006, reports of CrylF corn field failures caused by S. frugiperda were reported on the island of
Puerto Rico and resistance confirmed later (Storer et al. 2010, 2012). Haplotype similarities between
Puerto Rico and Florida insects suggested a mechanism of unidirectional migration from the island to
the continental US mainland (Nagoshi et al. 2007), though no reduced CrylF susceptibility was found
in eastern S. frugiperda population collections obtained in 2010 and 2011 (Huang et al. 2011).
28
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However, Huang et al. (2014) reported that most of the populations from Georgia, Florida, Louisiana,
North Carolina, and Texas, investigated during 2011-2013, had resistance ratios greater than 10 with
the highest ratio exceeding 270. The authors concluded that CrylF resistance had evolved in S.
jrugiperda and could be extensive in the southeastern US.
Uncertainty:
Currently, there are no IRM requirements for fall armyworm since the pest can only overwinter in a
limited portion of the southern US. If the pest becomes established and overwinters in the US, this pest
may need to be elevated to a primary pest of corn and require an insect resistance management plan.
4. S. albicosta resistance reports
a. Dyer et al. (2013) tested the survival of field collected S. albicosta from Nebraska at different
concentrations of CrylF and Cryl A toxins using toxin overlay assays. For Cryl A toxins,
survival was not significantly different than from the negative control, and no concentration
response was observed. Conversely, for CrylF, no LCso could be determined due to low
mortality at tested concentrations. An ECso (growth inhibition as evidenced by head capsule
measurements) was estimated at approximately 731 ng/cm2 after 14 days. Dose response assay
results support that larval tolerance to CrylF toxin was relatively high - even among neonates.
At 2,500 ng/cm2, it was reported that larval mortality incurred was 46.9%; 100% mortality was
never achieved across the range tested (250-25,000 ng/cm2). The authors concluded that field-
collected larvae possessed genetic variation that allowed them to feed on CrylF corn and finish
development. The concentration response results were consistent with field reports of CrylF corn
damage.
b. Ostrem et al. (2016) conducted a multi-year analysis for the purpose of developing a benchmark
susceptibility of western bean cutworm to various Cry-toxins using (diet incorporated) bioassays.
Insect samples were collected from cornfields in Nebraska (n = 5, 8) and Iowa (n = 3, 3) during
2003 and 2004 (respectively) as well as from cornfields in Nebraska (n = 5, 4), New Mexico (n =
1, 0), Texas (n = 2, 0), and Colorado (n = 0, 1) during 2013 and 2014 (respectively). Cry-toxin
concentrations ranged from 0.5-100 |ig/ml in 2003-2004 and 0.049-800 |ig/ml of diet in 2013-
2014. Most of the LCso values in 2003/4 were above the maximum concentration tested;
extrapolation was not possible because the data did not fit the Probit model. For those
populations where the LCso were measured and confidence limits could be calculated, the 2003
mean results were as low as 8.1 |ig/ml (NE) and as high as 72.7 |ig/ml (IA). In 2004, the lowest
LCso that could be measured was 5.3 |ig/ml (NE) and as high as 128.1 |ig/ml (NE). in 2013/14
the concentration of CrylF was increased to 800 |ig/ml. A meta-analysis (based on all data -
field and lab) calculated the mean LC50 at 42.2 |ig/ml (95% C.I. = 21.4-170.9) in 2003, 39.7
|ig/ml (95% C.I. = 25.5-70.7) in 2004, 514.4 |ig/ml (95% C.I. = 290.3-1156.4) in 2013, and 116.9
29
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|ig/ml (95% C.I. =70.4 -172.4) in 2014. The authors identified a 5.2-fold increase in LC50 for the
2013 and 2014 populations compared to 2003 and 2004 results.
c. Smith et al. 2017 investigated the damage caused by western bean cutworm to CrylF expressing
corn from 2011-2015 in Ontario, Canada. In addition, they reported on the susceptibility to
CrylF corn from 2015 field collections. The field trials were conducted on three farms with
sandy loam soils. In 2015 western bean cutworm moths were collected from light traps and egg
masses were obtained from naturally infested plots at their field trial locations. The moths were
reared in the lab and diet bioassays were conducted with the resulting offspring as well as with
neonates from obtained eggs using methods described by Dyer et al. (2013). The highest
concentration tested was 75 |ig/cm2 (F1 neonates) and 30 |ig/cm2 (F0 neonates from field);
mortality was assessed after 7 and 14 days, and weight of surviving larvae was also measured at
that time. LC50 (lethal concentration) and EC50 (growth inhibition) were measured or estimated
along with confidence limits.
The number of larvae found per corn ear were measured in field trials during 2013 and 2014. A
comparison between numbers revealed that there was no statistically significant difference
between densities from different treatment sites (Bt vs. non-Bt). Diet bioassays conducted at the
highest concentration did not result in 100% mortality, but Smith et al. (2017) reported that
mortality of WBCW increased with increasing concentration of CrylF. Two of the populations
had LC50 values that were higher than the highest concentration tested. All associated Chi-square
values were significant, however, indicating that the model was a poor fit. The percent mortality
at the highest concentration tested after seven days of exposure was available for two populations
only. The reported efficacy was 51.4 and 16.0% for the Fl-generation (14-day values not
reported) and 40.3 - 84.7% for FO-generation. EC50 values (day 14) were 19.8 and 12.9 |ig/cm2
for F1 and ranged from 0.1 - 4.9 |ig/cm2 for the FO-generation.
The authors concluded a visible reduction in efficacy (similar to Ostrem et al. 2016) and raised
the possibility of field-evolved resistance to CrylF corn.
EPA options to address resistance concerns
EPA concludes that CrylF resistance in S. albicosta has evolved in the western states of the US Corn
Belt (Ostrem et al. 2016) and Canada (Smith et al. 2016). In light of only one Bt toxin available to
control western bean cutworm (Vip3A) and additional CrylF field failures across northern lake states
in the US (DiFonzo 2016) and Canada, EPA considers whether it is warranted to develop an IRM plan
for S. albicosta. IPM may need to be an integral part of managing this pest in Bt due to lack of
availability of different traits (e.g., monitoring for adult densities, increased scouting for eggs in Bt
corn and use of conventional insecticides) (Smith et al. 2017). A management plan for western bean
cutworm may need to consider additional impacts on major lepidopteran pests in corn in the various
regions of concern. To protect the durability of the only efficacious trait available for control which is
30
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deployed with a 5% refuge, it may be necessary to use additional insecticide treatments for the early
instar stage of this pest. Monitoring for flight of western bean cutworm may be necessary because of
challenges associated with detecting early instar larvae.
EPA is considering whether sentinel plots in sweet corn could serve as a tool for resistance monitoring
in western bean cutworm. Such a focused sampling procedure would allow keeping track of specific
populations and could alert Bt registrants when early shifts in resistance to Vip3 A occur. Sentinel plots
would best be established in known areas of CrylF resistance and where pest pressure is expected to
be high based on environmental conditions.
Uncertainties
For H. zea and S. albicosta, the only efficacious trait available (Vip3A) is planted with a 5% refuge
because it is only available in pyramided varieties. This low refuge percentage exerts a large selection
pressure for resistance when used with pyramided varieties containing Vip3 A that are functionally
equivalent to a single trait product in areas with resistance to the other traits in the pyramid. It is
unclear whether IPM + IRM could significantly delay resistance development to Vip3 A, especially for
H. zea, since both corn and cotton express this trait in the south.
B. Causal Factors ofR.esistan.ee
1. Non-high dose traits and risk of faster resistance development
An inherent tolerance to Bt was reported for H. zea from various locations across the southern US
before the initial Bt PIP commercialization. Underlying reasons for the observed tolerance are
speculative and not well understood. One hypothesis is that H. zea may have increased types of
detoxifying enzymes because of its polyphagous nature. This might also provide greater tolerance for
Bt toxins (Stone & Sims 1993).
None of the registered Bt corn and cotton PIPs express a high-dose against H. zea (see Section I.B. for
discussion of the high dose concept). In 2008, however, the agency determined that Vip3 Aa20 (in
corn) expressed "close to a high dose" for this pest (US EPA 2009). Its analog, Vip3Aal9 (in cotton),
showed somewhat lower expression but was determined to be efficacious as well. Interestingly, even
for the more efficacious Vip toxins, H. zea susceptibility (measured as failure to molt, MIC so) varied
up to 59-fold (Ali & Luttrell 2011). The lack of available high dose PIPs in the southern US makes the
stewardship of Bt traits with refuges more challenging and resistance development in H. zea more
likely.
Single trait products pose a greater resistance risk than pyramids because pests have to overcome only
one as opposed to multiple modes of action. If resistance evolves to single trait products, pyramids that
31
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contain the compromised traits effectively become single trait products. Resistance to the second trait
can then evolve more rapidly (Zhao et al. 2005) because of the lower percent refuge requirement.
If a single trait expresses less than high-dose, resistance evolves more rapidly than for a high dose
product. The durability is lowest for traits that result in intermediate mortality and where heterozygous
resistant genotypes are likely to survive Bt exposure (Comins 1977; Tabashnik & Croft 1982). The
lowest durability occurs somewhere along the dose (susceptible mortality) range of 0.8 - 0.9 but can be
shifted towards a higher durability for the trait if the pest has a low population growth rate (Martinez et
al. 2017). Since H. zea has shown to have high population growth rates on corn and cotton (though
somewhat lower on the latter) (Caprio et al. 2009), it is even more important that growers have access
to traits that express a high dose for H. zea.
EPA options to address risks of resistance
Development of new Bts or other novel traits expressing high dose activity against H. zea would
greatly benefit the management of this economic pest in cotton. If these types of traits were
incorporated into both corn and cotton, management options should focus more on corn since the pest
funnels through that crop in the summer and spends two generations in corn in the southern US.
Other options for non-high dose single trait products are to use IPM with the deployment of single Bt
products, limit single trait commercial corn products, and transition from single trait products to Bt
corn pyramids.
Uncertainties
Stadelbacher et al. (1983) proposed the hypothesis that H. zea could tolerate Bt better than other
insects because of its polyphagous nature. Such a host adaptation could provide greater genetic
variation for detoxifying enzymes. If true, then polyphagia may not be the only mechanism to allow
pests to cope with Bt toxins. For example, corn rootworm (Diabrotica virgifera LeConte) has shown
great tolerance for Bt and is far less polyphagous and primarily feeds on maize but also cucurbits and
wheat (US EPA 2013). The causes for H. zea's greater Bt tolerance (compared to other pests) need to
be identified because this information may provide a key to developing a novel tool with which the
natural tolerance to Bt could possibly be blocked or resistance may be reduced.
2. Toxins expressed in corn and cotton exert continuous selection pressure
With the exception of Cryl Ac and Cry2Ae, all toxins expressed in Bt cotton are also expressed in Bt
corn products (see Table 3 and Table 4). But because of documented cross-resistance between some of
the toxins, it can be stated for simplicity that all toxins are expressed in both Bt crops. The presence of
the same Bt traits in corn and cotton exerts a continuous selection pressure on the multiple generations
of H. zea per year in the southern US and presents a resistance concern. Resistance management for
32
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cross-resistance conditions across multiple pest generations per year has limited options to reduce
selection pressure. The problem is further compounded with the limited availability of efficacious Bt
toxins against//, zea. If such toxins are expressed in non-functional pyramids of corn and cotton, the
durability of these traits could be reduced substantially.
EPA options to address risks of resistance
Management of efficacious Bt traits that are expressed in corn and cotton should focus on the corn
system in the south. H. zea prefers corn as its primary host in the summer, and hence, funnels through
this crop with two generations per year. During the corn growing season, the relative population
densities should be the highest on this crop compared to others in fall on Bt cotton. The subsequent
two generations have a variety of equivalent hosts to choose from; those are cotton, sorghum, soy
beans, and other cultivated crops and wild hosts. Appropriate and effective management options that
primarily focus on Bt corn could prolong the lifetime of these traits in Bt cotton where H. zea is an
economic pest of concern.
3. Mosaic of single and pyramided products undercuts durability of pyramids
Before pyramided Bt cotton (Cry 1 Ac X Cryl Ab) was introduced in Australia, the single Bt product
(Cryl Ac) was limited to 30% of cotton acres (Mahon et al. 2007). This acreage limit was implemented
to keep the Cryl Ac resistance allele frequency low in Helicoverpa. armigera (i.e., old world
bollworm) and to extend the durability of the later introduced pyramided product (Roush 1998). H.
armigera is closely related to H. zea and is a less-than high dose pest of Bt also. In the US as well as
Australia, both single Cryl Ac cotton products were subsequently removed after the introduction of the
Bt cotton pyramid. In the US, this decision of removing single traits was reached to support the
introduction of the natural refuge paradigm (EPA FIFRA SAP 2006a).
Conversely, Bt corn products are commercialized with single and pyramided traits with a block refuge,
refuge strips, or seed blends. In the southern US, the risk of resistance development and establishment
in populations of H. zea is greatest because the pest has multiple generations per year and successfully
overwinters. Single Bt corn products in the south present a resistance risk to pyramided products (Zhao
et al. 2005) in corn as well as cotton.
33
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EPA options to address risks of resistance
Single trait Bt corn planted concurrently with pyramided products reduce the lifetime of pyramids
because the pest can evolve resistance to traits sequentially and does not incur 'redundant mortality'
any longer on pyramided products. As such, reducing (or eliminating) single trait corn products will
likely increase trait durability. All currently registered Bt cotton products for commercial use are
pyramids, and the use of single trait cotton products were phased out to support the use of natural
refuge for Bt cotton (Matten et al. 2013). However, management options to mitigate this resistance risk
should focus on Bt corn because H. zea (major pest of cotton) has two generations in the southern US
that funnel through corn in the summer.
Uncertainties
EPA has concluded that H. zea developed resistance to Cryl and Cry2 toxins in some areas of the US.
The durability of Vip3A expressed in corn and cotton pyramids with such toxins will experience great
selection pressure. The effect of mitigation on the durability of Vip3A is uncertain in light of these
resistance cases.
4. Seed blends in the southern US and faster resistance development in ear-feeding pests
of com
a. Mosaic of variable toxin expressions in kernels
Seed blend refuges in southern regions are expected to increase the rate of adaptation to ear-feeding pests
of corn, such as H. zea and S. frugiperda (US EPA 2012, Caprio et al. 2016). In a single Bt RIB cornfield
consisting of 90% maternal Bt and 10% maternal non-Bt plants, corn ears could be pollinated by paternal
Bt and paternal non-Bt (pollen) at a rate of 0.9 and 0.1, respectively. However, the actual cross-
pollination rate can differ and may be affected by factors such as proximity of plant types, pollen shed
timing, and wind (Burkness et al. 2011). Cross-pollination between the two types of plants results in a
mosaic of Bt expression in kernels throughout the ears but is greatest in the ear tip area (Burkness &
Hutchison 2012). Partially resistant pests may exploit such a mosaic of expression by tasting and
rejecting toxic kernels and moving on to less toxic kernels, while truly susceptible insects die. Sub-lethal
Bt exposure can provide a pathway of selection for heterozygous resistant larvae, which have a fitness
advantage compared to susceptible individuals under this scenario. This mechanism of selection shifts
(increases) the functional dominance of the resistance alleles and increases the risk of resistance
development in ear-feeding pest. Yang et al. (2017) conducted a kernel feeding study using fall
armyworm, and their results demonstrated that RS survival in RIBs was enhanced compared to RS
survival in pure stand Bt because of cross-pollination. Conversely, early data by Burkness et al. (2011)
did not support that survival of H. zea differed on kernels obtained from maternal Bt plants that were
surrounded by either Bt or non-Bt plants. Even in a pure stand Bt field, a degree of mosaics can be
encountered that could be exploit (further discussed below). Therefore, IRM for ear-feeding pests of
34
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corn may have fundamental challenges that are not encountered for pests that exclusively feed on
maternal tissue. In seed blends, however, the problem is further compounded because the within-field
refuge is corrupted by Bt cross-pollination compromising the objective of the refuge.
b. Pollination pathways, survival, and development:
The pollination in RIBs is variable and can occur between i) Bt maternal x Bt paternal, ii) Bt maternal
X non-Bt paternal, iii) non-Bt maternal X Bt paternal, and iv) non-Bt maternal X non-Bt paternal
gametes. The first pollination scenario is the most likely to occur for Bt plants simply based on the
fraction of Bt and refuge seed planted in seed blends (90:10 or 95:5). The second scenario will add to a
reduction in toxin expression in kernels of Bt plants that are surrounded by refuge plants because one
gamete lacks Bt genes. The third scenario is the more likely pollination outcome for non-Bt plants
surrounded by a majority of Bt plants and introduces Bt exposure into refuge ears. The fourth scenario
occurs at the lowest frequency and results in true refuge kernels.
c. Maternal Bt plants:
There are four major tissues in a kernel: the pericarp, a thin layer of maternal and diploid tissue around
the embryonic tissue; the endosperm and aleurone, both triploid and embryonic tissues containing two
maternal and one paternal gamete; and the embryo, a diploid tissue with one copy of a maternal and
paternal gamete. The pericarp can be ignored for exposure purposes because it makes up a negligible
amount of thin (Bt) tissue that a larva is exposed to when feeding on a kernel (Chilcut and Tabashnik
2004). The focus of this discussion is on the diploid embryonic tissue and its genotypes and
frequencies that are possible from a cross of two Bt plants in a pure stand Bt plot for two hypothetical
products, a three gene Bt pyramid (a.) and a single gene Bt product (b.).
Punnett Squares (a. and b.) show the genotypes resulting from the cross of a Bt plant and Bt pollen under
the different trait scenarios in a pure stand Bt field where pollination occurs between Bt plants (for
discussion of unlinked traits see Caprio et al. 2016).
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i. Mosaic of kernels with variable Bt expression for a three-gene pyramid planted to pure stands
Paternal Gametes
Hypothetical, Linked Traits (A,B) X Trait C
+ +/+
++/-
/+
/
Maternal Gametes
Hypothetical, Linked Traits
(A,B) X Trait C
+ +/+
++/+,
++/+
++/-,
++/+
/+,
++/+
++/+
++/-
++/+,
++/-
++/-,
++/-
/+,
++/-
++/-
/+
++/+,
/+
++/-,
/+
/+,
/+
-/+
/
++/+,
++/-,
/,
Probabilities for embryonic genotypes for the three Bt traits:
1/16 of kernels expressing 0 copies of A_B (linked traits) and 0 copies of C;
1/16 of kernels expressing 2 copies of A_B and 2 copies of C;
4/16 of kernels expressing 1 copy of A_B and 1 copy of C;
2/16 of kernels expressing 1 copy of A_B and 0 copies of C;
2/16 of kernels expressing 0 copy of A_B and 1 copies of C;
1/16 of kernels expressing 0 copy of A_B and 2 copies of C;
1/16 of kernels expressing 2 copies of A_B and 0 copies of C;
2/16 of kernels expressing 1 copy of A_B and 2 copies of C;
2/16 of kernels expressing 2 copies of A_B and 1 copies of C.
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ii. Mosaic of kernels with variable Bt expression for single trait products planted to pure stand
Paternal Gamete
Hypothetical Single Trait (A)
+
a>
-4-i>
CD
3
O
a
a
X
Probabilities for embryonic genotypes for the three Bt traits:
1/4 of kernels expressing 0 copies of A;
1/4 of kernels expressing 2 copies of A;
1/2 of kernels expressing 1 copy of A.
d. Cross-pollination of refuge ears in RIBs:
Cross-pollination between maternal non-Bt and paternal Bt plants in RIBs can be expected to reduce the
available amount of 'true' refuge kernels. Burkness et al. (2011) found that maternal non-Bt plants
pollinated by paternal Bt (CrylAb) plants caused intermediate survival. In their preliminary study,
survivorship on non-Bt plants was determined for two ear-feeding lepidopteran pests of corn (Ostrinia
nubilalis and H. zea). Both pests experienced some mortality on refuge plants in the field that were
pollinated by surrounding paternal Bt plants. Actual field survival was reported at 43% and 63% for 0.
nubilalis and H. zea, respectively, compared to survival on pure stand non-Bt corn, though the authors
acknowledged that other factors could have contributed to observed mortality. In the lab, O. nubilalis
survival on cross-pollinated kernels was higher, for example (60%). A second, more extensive study
focused on the larval movement, development, and survival of H. zea in a seed mixture (CrylF X
CrylAb X Vip3A). The authors reported early instar inter-plant movement (2-16%), which may have
been underestimated, and delayed development and lower survival on refuge plants in RIBs compared
to structured refuges (Burkness et al. 2011, 2015).
Similarly, Yang et al. (2017b) estimated the contamination of refuge kernels to be 87% (5% RIB) and
66% (20% RIB) based on two trial locations. Survival to adulthood on cross-pollinated refuge kernels
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was 42.3 - 50% and 8.7 -10% for heterozygous resistant and susceptible genotypes, respectively (Yang
et al. 2017bc). Survival on pure stand refuge ears was similar for all genotypes and ranged from 62.5 -
13.1%. In addition, pupal development of SS and RS pupa was significantly delayed on seed blend refuge
plants compared to a pure refuge stand.
EPA options to address risks of resistance
EPA has concluded that the risk of resistance for ear-feeding pests of Bt in RIB corn needs to be
addressed in the southern US where H. zea is a major pest, has multiple generations, and overwinters.
This risk of resistance posed by RIBs for pests like H. zea, will likely compromise EPA's goals for IRM,
which are to prolong the lifetime of non-high dose traits in cotton and corn, especially in areas where the
technology is still effective, and protect the longevity of still functional and efficacious Bt trait(s). An
option would be to limit the use of Bt corn RIB products in the southern US. Though recent modeling
proposes that RIBs for H. zea may be equally if not more durable than with block refuge deployment
when incorporating grower refuge non-compliance (Onstad et al. 2018).
Seed blends could become a viable option for resistance management in the southern US if the Bt
mosaic and corruption of refuge kernels in seed blends can be eliminated or substantially reduced. A
potential solution for maternal Bt plants is to construct parental lines that are no longer hemizygous for
Bt traits and contain matching sets of Bt genes on both chromosomes. Changes to the corn breeding
program could create possible solutions for RIBs, however: 1) Move away from hemizygous parental
lines; and 2) introduce pollen incompatibilities between Bt and non-Bt cultivars, and self-pollinating
mechanisms through advances in gene editing, which create kernels with homozygous maternal traits.
Uncertainties
It is currently unknown whether kernels expressing one copy of the Bt gene express less protein than a
kernel with two copies of the gene. This information would be useful when more thoroughly
discussing and assessing the risk of resistance from hemizygous Bt plants.
If self-compatible refuge corn hybrids were developed for seed blend products, the integrity of the
refuge could be maintained for ear-feeding pests. It is unclear whether the cost of developing self-
compatible refuge plants with matching genes on both chromosomes would be economically profitable
or whether it would exceed the cost of developing an incentive program that assures grower
compliance with structured refuges.
Similarly, Bt hybrids could be developed that are only compatible with Bt pollen. Since Bt plants are
surrounded by mostly other Bt plants (95% for pyramided products), this would assure sufficient
pollen donors for Bt ears.
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5. Refuge non-compliance in the southern US undercuts durability of pyramids
Full grower compliance with refuge requirements (meaning they planted the correct percentage of
refuge corn) in the cotton region of the US has historically been lower (7-46%) than in the northern
Corn Belt (68-73%, not shown in Table 5). The percent of growers being partially compliant, meaning
they planted a refuge but less than the mandated percentage, remained variable over the course of
seven years and highest in 2017. Non-compliance more than doubled from 2015 - 2017 (BPPD 2014,
BPPD 2017, MRID 505101-01). Zero compliance (no refuge at all) was high, very similar over the
seven years reported, and lowest in 2017.
EPA concluded (BPPD 2017) that refuge compliance in the southern US had further decreased over
the past several years. This increase in non-compliance is likely caused by multiple factors, some of
which are 1) seed blend adoption in that region; 2) the mandate for a greater refuge requirement in the
south for single trait and pyramided products (50% and 20%, respectively) compared to the northern
Corn Belt; 3) lack of desired hybrids in refuge products (Reisig 2017); and 4) other factors driven by
the market and pest complexes in these areas.
Table 5. Grower Compliance with Refuge Requirements for Bt Corn in the Southern US
Southern
Growers
Surveyed who:
2011
2012
2013
2014
2015
2016
20171
(n =122)
(n =112)
(n =95)
(n =100)
(n =95)
(n =48)
(n =105)
Met refuge size
requirement
42%
42%
46%
45%
34%
7%
19%
Partially met
refuge size
26%
24%
25%
18%
25%
47%
55%
requirement
Planted no
refuge acres
32%
34%
29%
37%
41%
46%
26%
ABSTC survey data submitted to EPA before 2011 did not provide the same detail of information and, thus, was excluded
from this table.
1 Survey results were not separated for below and above-ground pests in 2017 but reported as a combined percentage of
compliance.
An independent survey conducted in North Carolina in 2014 and 2016 assessed growers' knowledge of
IRM and the willingness to plant a refuge for Bt corn (Reisig 2017). Some conclusions based on the
data were that owners of small farms were less likely to plant a refuge. The opposite was the case for
growers of large farms who planted corn and soybeans. Communicating the importance of refuges did
not increase growers' intentions to plant a refuge.
EPA options to address concerns
Growers likely favor the planting of RIB products because Refuge-In-the Bag products expedite the
planting process and reduce labor inputs. Likewise, RIB products could be favored by industry because
they guarantee that refuges for Bt corn are planted and growers remain compliant with refuge
39
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requirements. It is, therefore, not surprising that RIBs are viewed as the solution to grower refuge non-
compliance in southern states. At this point, however, there are several lines of evidence supporting the
conclusion that RIBs are less successful IRM strategies for ear-feeding pests of Bt corn (e.g., H. zea, S.
jrugiperda, etc.) than block refuges, especially in areas where pests have multiple generations per year
and overwinter. EPA's refuge requirement for single trait RIB products is lower than for the equivalent
technology deployed with a block refuge (10% vs. 50% in the south) - though an accompanying
structured refuge of 20% is also required in south. Preliminary modeling results suggest that the
addition of a structured refuge with seed blends (if planted) may not adequately delay resistance
evolution (Caprio and Martinez 2013). The agency considers options to increase grower compliance
with planting block refuges through, for example, grower incentives or reward programs, that assist in
reducing resistance occurrences in Bt crops. More research is needed into the human factors
underlying growers' behaviors that lead to non-compliance.
Uncertainties
It is uncertain whether industry will be able to successfully increase refuge compliance among
southern growers to delay resistance development sufficiently in H. zea. Delaying resistance will also
depend on the Bt trait of concern, the respective resistance levels present in the pest, whether cost to
resistance exists in H. zea, and the resistance management options implemented in Bt corn. This
demonstrates that resistance management in the southern US is complex; the ultimate resistance
outcome (measured as durability of trait) depends on multiple factors acting together in favor of
extending the Bt lifetime. However, for non-compliance with refuge requirements to decrease, a
greater number of on-farm visits may be needed. In addition to industry efforts, outreach to growers
through the National Corn Grower Association, grower consulting groups, and University extension
experts may be needed to communicate the importance of planting block refuges. Furthermore, a
greater focus on on-farm assessments could be coupled with a rewards program that motivates growers
to comply with requirements. Assessing the success (gain in years before resistance evolves) of
increased refuge compliance in the south can be simulated with complex theoretical models that
incorporate grower behavior, landscape, diverse cropping system, cost and benefits of compliance,
ecology, and other parameters. Increasing the refuge compliance should extend the life-time of Bt traits
compared to seed blends (Carriere et al. 2016 and others). Increasing the refuge compliance would lead
to a greater proportion of non-Bt corn in the landscape, which may not necessarily elicit a linear
increase in durability gains for pyramided traits (Ives et al. 2011). In addition, these models show that
pest population dynamics (e.g., density dependence, growth rate) interact with the dose of Bt and
which results in an associated increase or decrease in the expected gain in years before resistance
evolves based on the amount of available refuge (Martinez et al. 2017).
40
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6. Methodology for insect collections and diet bioassays delays resistance detection
a. Current Resistance Monitoring and Mitigation Strategies for Bt Corn and Cotton
Through the terms of registration, Bt corn and cotton registrants are required to implement a resistance
monitoring program for the major lepidopteran target pests. A primary goal of resistance monitoring is
to detect shifts in the frequency of resistance genes (i.e., susceptibility changes) before the onset of
resistance leads to widespread Bt crop failure (see EPA 2010a).
Monitoring for resistance has consisted of two main components: 1) investigation of unexpected field
damage reports from growers, extension agents, consultants, or company agronomists, and 2)
monitoring for changes in susceptibility through targeted population sampling and testing. Unexpected
damage reports may reveal the occurrence of more localized resistance before the effects might
establish regionally. Resistance monitoring through targeted field sampling can reveal changes in
susceptibility of geographically representative populations. In the event that resistance is confirmed, a
mitigation (remedial action) plan is triggered as defined by the terms of registration (see EPA 201 Of).
Reports covering each monitored pest and Bt PIP toxin must be submitted annually to the agency. A
summary of the data submitted for H. zea is detailed in the section below.
The resistance monitoring and mitigation plans for Bt corn and cotton were previously described. The
strategies are conceptually similar, though the target pests and resistance standards vary between the
crops.
b. Summary ofIL zea Bioassay Results
Historically, insect resistance monitoring for conventional pesticides has been conducted with diet
bioassays using dose/response curves to make comparisons between LD/LCsos of reference
(susceptible) strains and field-collected populations. Roush and Miller (1986) concluded that this type
of comparison was adequate if resistance allele frequencies in a population were high but that it was
not an effective method for early resistance detection. They proposed that a diagnostic assay approach,
in which susceptible individuals were killed but resistant individuals survived, would be more efficient
when monitoring for resistance. However, the authors also noted that even with diagnostic tests, the
required sample size needed to detect a resistance allele frequency of 0.01 could be very large.
Both susceptibility (LC and EC) comparisons and diagnostic assays have been incorporated into the
currently mandated IRM program for Bt PIPs to identify potentially resistant populations. For
Lepidoptera pests (e.g., ECB, SWCB), diagnostic concentrations have been established. For H. zea,
diagnostic concentrations for mortality were never achieved and a shift to measuring sub-lethal effects
was undertaken. Data for H. zea are summarized below in Tables 6 (Bt corn PIP toxins) and Table 7
(Bt cotton PIP toxins).
41
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The annual corn and cotton monitoring reports have provided no information about the sampling
locations of insect populations that served to assess susceptibility to Bt traits. Inconsistent reporting of
laboratory control susceptibility for the cotton monitoring reports makes it challenging to place the
field results in context. Additionally, insufficient description about the tested generation(s) in bioassays
does not allow calculating the number of genomes and quantifying potential non-segregation of
homozygous resistant alleles in larvae. Homozygous susceptible (SS) and heterozygous (RS) larvae
have shown to have similar responses when exposed to Cry 1 Ac (Blanco et al. 2008), and only
homozygous resistant larvae survived and developed on diagnostic concentrations. This leads to an
underestimation of resistance levels, a delay in concluding resistance has evolved, and thus, a delay in
initiating mitigation of resistance.
The bioassay methodology was changed over the years to address the need for better detection
methods. Sub-lethal endpoints were adopted because of inherent Bt tolerance by H. zea. Diagnostic
concentrations have been available for Cry 1 Ac and Cry2Ab2 since 2009 using endpoints, such as
failure to molt. The results in Table 7 support a conclusion that suppression of third instar larvae was
achieved (> 98%) from 2009-2015 using a concentration of 10 |ig/m2 for Cry 1 Ac and 10 |ig/ml of diet
for Cry2Ab2. These two toxins serve as surrogates for all registered Cryl A and Cry2A toxins because
of cross resistance and protein structure similarity. Cry IF's highest concentration tested (4,000
|ig/cm2) is not a diagnostic concentration, however; only approximately 80% of third-instar larvae
failed to molt. A more or less consistent trend of H. zea susceptibility to the three Bt proteins is
apparent.
Laboratory (negative) control colonies were not used in the early years of the IRM program (Tables 6
& 7). This makes it difficult to put the field results in context. All that can be concluded for those
results is that H. zea has variable responses to the Bt toxins tested. When negative control colonies
were used, no clear pattern was apparent for field populations. Sometimes, the sampled populations
showed lower or higher susceptibility than the lab colonies. Since the sample collections did not track
the same populations over time, this should not be too surprising. Also, it is possible that the lab
reference strains did not represent a truly susceptible field population since the insects that had been
reared in the lab for generations, may be genetically less diverse and able to deal with the lab
exposure/diet better than their natural counterparts. Typically, reference strains go through a genetic
bottleneck when established, and it is unclear whether occasional outcrossing occurs to maintain
genetic variability and relevance compared to wild populations. EPA also notes that when the field
collections turned out to be less susceptible than the lab reference strains, no follow up investigations
occurred to test for resistance in survivors (see Table 6, year 2006, ECso for CrylF).
EPA provides options to address uncertainties with insect sample collections and difficulty with
interpretation of diet bioassay results in the following sections below.
42
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Table 6. Susceptibility of Field-Collected H. zea Populations to Bt Corn PIPs (2001 - 2015) (Compiled from studies listed in
Appendix A)
CrylAb
CrylF
Vip3A
CrylA.105
Year
Population
ECso
Disc. Dose
ECso
Disc. Dose
ECso
Disc. Dose
ECso
Disc. Dose
(ng /cm2)
(% mort.)a
(ng /cm2)
(% mort.) b
(ng /cm2)
(% mort.)c
(ng/cm2)
(% mort.) d
2001
Field
0.14-0.31
>99
43.2-229.1
94.0-100
Lab
NT
NT
NT
NT
2002
Field
0.10-0.35
97.8-100
39.2- 173.5
85.3-100
Lab
NT
NT
NT
NT
2003
Field
0.20-0.46
90.8 - 100
32.1 -96.9
82.9-99.0
Lab
0.14
98.4
74.2
95.5
2004
Field
0.08-0.28
99.4 - 100
12.6-120.6
99.1-100
Lab
0.49
99.7
182.4
100
2005
Field
0.10-0.42
97.5 - 100
19.6-118.7
62.2-96.1
Lab
0.18
99.9
31.7
80.7
2006
Field
0.13-0.47
99.6 - 100
8.2-96.0
87.5-100
Lab
0.21
100
34.7
94.0
2007
Field
0.47-0.88
99.4 - 100
42.2-91.5
48.7-99.6
Lab
1.35
89.0
95.9
52.1
2008
Field
0.12-1.10
96.7 - 100
12.6-184.5
69.0-100
0.0021 -0.0447
NT
Lab
0.57
99.7
472.9
65.8
0.0034
NT
2009
Field
0.09-1.24
99.4 - 100
12.0-142.3
84.5-100
0.0007-0.0095
NT
Lab
0.47
97.2
200.8
68.2
0.0064-0.0096
NT
2010
Field
0.12-0.41
99.3 - 100
9.5-76.8
80.6-99.7
0.0016-0.0133
100
43
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CrylAb
CrylF
Vip3A
CrylA.105
Year
Population
ECso
Disc. Dose
ECso
Disc. Dose
ECso
Disc. Dose
ECso
Disc. Dose
(ng /cm2)
(% mort.)a
(ng /cm2)
(% mort.) b
(ng /cm2)
(% mort.)c
(ng/cm2)
(% mort.) d
Lab
0.74-0.81
94.9-95.7
166.5
67.4
0.0145 -0.0214
100
2011
Field
0.14-0.68
96.9 - 100
16.4-97.6
78.3 - 100
0.0012-0.0219
100
Lab
1.13-2.65
57.1 - 83.3
153.7-2764.0
77.7
0.0540-0.0741
100
2012
Field
0.14-0.65
99.3 - 100
1.3 - 115.8
42.3 -99.4
0.0018-0.0092
100
Lab
1.34
95.8
646.7
28.9-64.0
0.0200-0.0269
100
2013
Field
0.15-0.51
93.5-100
3.9-364.1
27.4-99.7
NT
100
0.0016-0.0159
100
Lab
2.79-3.27
37.6-72.6
719.5-2704.5
5.1-8.3
NT
100
0.0400
58.3
2014
Field
0.11-0.59
91.2-99.9
15.8-448.1
58.3 -99.4
NT
100
0.0014-0.0327
NR
Lab
2.45-3.33
37.1 -78.1
>2025
15.8-18.8
NT
100
0.0980-0.2600
NR
2015
Field
0.09-6.43
84.2-99.6
16.5-228.8
53.6-95.8
NT
100
0.0013 -0.0084
NR
Lab
0.10-7.83
36.0 - 100
34.4-77.3
2.1-97.6
NT
100
0.0010-0.5000
NR
a CrylAb discriminating concentration 40 ng/cm2 (2001-3); 80 ng/cm2 (2004-10)
b Cry IF discriminating concentration of 5000 ng/cm2 (2001); 7500 ng (2002-3); 54,675 ng/cm2 (2004); high concentration 4,000 ng/cm2 (2005-10)
0 Vip3 A discriminating concentration = 10 ng/cm2
d Cry 1 A. 105 discriminating concentration = 5 ng/cm2
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Table 7. Susceptibility of Field-Collected H. zea Populations to Bt Cotton PIPs (2003-2015)
(Compiled from studies listed in Appendix B)
Year
Control
Mortality
Percent Failure to Molt to 3rd Instar
% Larval
Mortality
Cryl Ac/cm2
CrylF/cm2
Cry2Ab2/ml diet
CrylAc/ml diet
10 ng
30 fig
400 (ig
4000 ng
10 Mg
30 fig
100 (ig
100 (ig
2003
75.5
2004
82
76.1
2005
67.2
89.9
2006
57.8
2007
83.2
74.2
2008
80.8
78.9
83.5
2009
6.9
0.4
0
55
16.6
83.3
0.6
98
2010
3.2
0
0
59.7
9.2
0.3
0
2011
8.7
0
0
51.5
10.9
0.3
0
2012
7.0
0
0
41.7
2
0.3
0
2013
3.9
0
0
54.4
11.4
0
0
2014
11.2
0
0
56.6
20.4
0.1
0
2015
2.3
0.4
0.3
0
0
EPA identified options to improve current methodologies
The effectiveness of the monitoring program to proactively detect resistance is largely dependent on how
population sampling is conducted. To date, insect samples have been collected in a more or less random
fashion from non-Bt hosts (e.g., refuge corn or wild hosts). This type of sampling has been used to
determine whether a change has occurred in the average susceptibility of H. zea. Because the populations
are selected randomly, however, this approach does not track susceptibility of individual populations (or
populations within discrete geographic locations) over time. Since the samples stem from non-Bt hosts, it
is also a less proactive sampling method. EPA is, therefore, considering the adoption of sentinel plots in
major cotton growing areas for resistance monitoring purposes instead of continuing with the random
sampling approach for H. zea. Such a targeted approach of collecting insects would allow, in theory, to
detect shifts in susceptibility more proactively. Such sentinel plots may need to be set up in areas or
regions with high risk factors of resistance, taking into consideration greater Bt adoption, fields with use
of same trait year-after-year, areas with low refuge compliance for Bt corn, and high H. zea pest pressure.
An analogous approach may need to be adopted for other lepidopteran pests and their IRM plans. The
primary objective of this proposed change is to actively target areas of resistance concern based on high
risk factors, gain insight into the resistance development in the same populations year after year, and
thereby identify resistance problems more proactively.
EPA is considering the use of diagnostic molecular assays for Cryl A, CrylF, Cry2A, and Vip3A toxins
targeting H. zea. Because of the speed of detection with such available approaches and cost to resistance
concerns that could lead to false negatives with diet bioassays, the agency considers molecular tools
45
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superior since they allow the use of in-field collected insects rather than their offspring generations
(Banerjee et al. 2017). This type of approach would expedite the detection of resistance, allow monitoring
for early resistance and effective implementation of mitigation, increase the accuracy of resistance
monitoring data, and improve EPA's overall resistance monitoring program for H. zea. In addition, the
development of a resistant reference colony, against which field results could be compared, should further
enhance the interpretation of monitoring results. Likely, significant research efforts will be needed to
develop such tools.
For other lepidopteran pests where diagnostic assays are available, EPA considers whether a shift to F2
screens would be useful to identify resistant populations (Andow and Alstad 1998). This option is based
on observations in the literature that RS and XY-genotypes can behave similarly in diet bioassays, which
underestimates resistance (Blanco et al. 2008). At a minimum, 400 genomes may be needed for this
approach. F2 screens have the advantage that they express homozygous resistant offspring, which survive
(unlike ^-genotypes behaving like XY-genotypes). With an appropriate sample size, the resistance allele
frequency in the population can be estimated and confidence intervals calculated.
Uncertainties
There may be additional benefit in supplementing the focused sampling approach in southern areas of the
US with sampling from northern and non-overwintering areas (H. zea) of the Corn Belt. Comparing the
Bt susceptibility of H. zea in southern populations to more northern states might provide additional
insights into how quickly resistance can establish on a continent scale.
It is unclear what the appropriate number of population samples would have to be in the southern US for
early resistance monitoring purposes. Historically, sampling targets (total populations and number of
genomes) have been based on a presumed resistance allele frequency. For example, if the phenotypic
frequency of resistance was assumed to be one in 1,000 (0.001), it was suggested that more than 3,000
individuals needed to be sampled to have a 95% likelihood of detecting one resistant individual (Roush &
Miller 1986). For H. zea and most Bt toxins, the frequency of the resistance alleles is expected to be
higher since Bt PIPs do not express high dose levels. Given these circumstances, the genome sample size
should be lower for this pest. In absence of reliable resistance allele frequency estimates, however, it will
be challenging to propose a sufficiently accurate sample size needed from each population to conduct a
meaningful F2-test.
It is uncertain whether a proper sample size can feasibly be collected for H. zea to assess 400 genomes in
an F2-test. For example, more than 50% of field-collected larvae could die before becoming adults. This
reduction of genomes can further increase if mating success and viable egg production are low during the
breeding process. This additional mortality reduces the gene pool in bioassays.
Given the knowledge about H. zea's high genetic variability and susceptibility to Bt, it is surprising not to
see any evidence of variability in the assay results for cotton monitoring (Table 7). The reasons for this
lack of variability are unclear, but EPA hypothesizes that contributing factor may be the elimination of
resistant genotypes during the rearing process. Typically, companies do not report mortality of field
46
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collected individuals prior to starting diet bioassays; this information would be needed to further
understand the lack of variability seen in assay results. Also, it is unclear what the methodological
differences were between assays leading to results listed in Tables 6 and 7.
7. Lack of thresholds for unexpected injury delays reporting of putative resistance
An unexpected injury (UXI) threshold serves as an alert that a pest may have evolved resistance to Bt.
Such injury (typically known as Bt failure) warrants further investigation by the company whose product
was affected. The UXI threshold is not to be mistaken with "economic injury level" (EIL) (i.e., lowest
population density that will cause economic damage) or "economic threshold" (ET) (i.e., amount of injury
that justifies the cost of artificial control) (Stern et al. 1959), which are terms and tools of integrated pest
management (IPM) options. The threshold for UXI should be higher than EIL and ET.
a. Lack of regulatory UXI thresholds
As described in the Background section, the registration terms for Bt PIPs do not specify lepidopteran
UXI levels that would trigger investigative actions. Without established regulatory threshold triggers for
unexpected Lepidoptera injury in Bt corn and Bt cotton, it is unlikely that timely collections of relevant
insect samples and follow-up bioassays can occur. Resistance is, therefore, provided an opportunity to
spread unchecked and before mitigation actions can be initiated in a timely manner.
Likely all companies have their own thresholds for unexpected injury in corn and cotton, though these
have not been shared with EPA and may not be uniform across industry. The terms of Bt registration
simply state that each company needs to conduct follow up investigations when unexpected damage
occurs. The terms do not prescribe what the particular thresholds should be.
b. Criteria for UXI thresholds
UXI thresholds may be toxin, pest, and/or crop specific. Damage caused in Bt cotton and corn by the
same pest likely has distinct thresholds simply based on the different plant tissues that are affected. A
non-high dose trait may have a higher threshold than a high dose toxin because some damage should
always be expected for the former.
Cotton
A uniform regulatory threshold needs to be established for unexpected crop injury in Bt cotton; such a
threshold has to be 'stand-alone' since no refuge cotton is planted and can serve as an infestation and
damage comparator (natural refuge paradigm for pyramided Bt cotton). Currently, 6 - 7% damage to bolls
is considered unexpected Bt injury in some southern US areas (Porter and Reisig, 2017 personal
communication).
47
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Corn
Likewise, there is a need for a regulatory and uniform threshold for UXI in Bt corn. Field entomologists
compare the damage in Bt cornfields to that observed in the refuge fields, since high pest pressure could
lead to false positives based on UXI in Bt alone (resistance suspected, yet low resistance allele frequency
in high density population). Conversely, low pest pressure could result in missing resistance development
(false negative, yet high resistance allele frequency in population). The comparison to a check (i.e.,
refuge) should in theory account for pest pressure.
Growers may not scout for H. zea damage in field corn, however, since they do not consider it a major
pest (Storer, 2017 pers. comm.). Given the lack of concern about//, zea damage in field corn of the
southern US, a resistance problem to Bt trait may not become apparent until the resistant population
moves into Bt cotton and UXI becomes apparent there.
EPA options to address concerns
EPA is considering the adoption of uniform unexpected injury thresholds in Bt corn and Bt cotton based
on percent damage in ears and stalks (corn) and squares and/or bolls (cotton) for the various major
lepidopteran pests. When those thresholds are triggered, insects will need to be collected for resistance
testing. For Bt corn, additional information about refuge damage should be collected to estimate pest
pressure representative of a resistant problem. If it is impractical to establish thresholds for H. zea in Bt
corn because growers do not scout for this pest, monitoring for pest abundance may need to occur with
adult traps. Such trap samples could be utilized to screen for resistance frequencies in populations when
adult number triggers are exceeded.
Uncertainties
Depending on the trait and its effectiveness against a target pest (intermediate vs. high dose), thresholds
may differ between traits and require flexibility and adjustment. In addition, the same trait may have
different expression in cotton than in corn (e.g., Vip3 A). In such a case, thresholds may need to be crop
specific.
For cotton, damage to bolls incurred may be of economic significance but may not be the best measure for
unexpected damage. Bolls that have incurred early damage are aborted by the cotton plant (Reisig, 2017
pers. comm.). Hence, an UXI threshold based on boll damage alone would underestimate actual damage
and may not be proactive enough. It may, therefore, be more relevant to scout for square damage when
looking for Bt performance in cotton.
If growers do not typically scout for H. zea damage in field corn, the UXI approach may not be practical,
and an alternate approach would be needed. Not scouting for zea damage in field corn will delay
resistance detection and puts additional risk on the durability of Bt cotton.
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8. Current regulatory definition of resistance delays mitigation actions
The Food and Agricultural Organization of the United Nations (FAO 1979) defined resistance as "the
inherited ability in a strain of a pest to tolerate doses of toxicant that would prove lethal to a majority of
individuals in a normal population" and further stated that lab resistance did not necessarily equal loss of
efficacy in the field. The National Research Council of the National Academy of Sciences (1986) defined
resistance broadly as "any heritable decrease in sensitivity to a chemical within a pest population" but
specified that it could occur on a spectrum of slight to severe as well as locally rare to widespread.
Tabashnik et al. (2009) elaborated on field-evolved resistance with respect to Bt crops and explained that
it did not always imply economic loss. Tabashnik et al. (2014) presented five categories of Bt resistance
with each being successively more severe and having an increasing proportion of resistant individuals in
the pest population. These categories are:
1) Incipient resistance: a statistically significant increase in resistance allele frequency (Downes et al.
2010), and <1% of individuals in a population are resistant;
2) Early warning resistance: a statistically significant increase in resistance allele frequency (Zhang
et al. 2011), and 1-6% of individuals in a population are resistant (warrants consideration to
manage resistance);
3) >6-50% of the population resistant;
4) >50% of the population resistant, reduced efficacy is expected but not reported (Tabashnik et al.
2012); and
5) Practical resistance '. >50%) of the population resistant, and reduced efficacy is reported with
practical implications for pest control (Tabashnik et al. 2012).
The US EPA's definition for Bt resistance includes three criteria that have to be met before resistance is
confirmed and mitigation initiated. The following needs to be demonstrated scientifically:
1. Higher survival of resistant individuals on Bt compared to susceptible individuals;
2. Heritability of the resistant trait; and
3. Field relevance (i.e., economic damage to Bt crops).
The agency's definition of Bt resistance describes the most severe state of field-evolved resistance
described by Tabashnik et al. (2014), namely "practical resistance".
EPA notes that if "confirmed field-resistance" is used as the trigger for initial mitigation, then the
likelihood of success is greatly diminished. Once >50%> of individuals in the population are resistant, it
will be exceedingly difficult to mitigate effects of resistance, especially for lepidopteran species with
great dispersal abilities (e.g., Noctuidae, Crambidae). For EPA's IRM program to achieve its intended
goal (to contain, reduce, or delay the spread of resistance and extend durability of Bt in non-affected
areas), a new mitigation trigger (other than confirmed resistance) should be considered that allows for
timely and effective implementation of mitigatory strategies (also see discussion in Section III. A.l 1).
Monitoring for resistance can be an effective stewardship tool if mitigation of putative resistance is
initiated at lower resistance allele frequencies (e.g. category 2 "early warning resistance").
49
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EPA options to address concern
EPA is considering the adoption of two triggers for mitigation of resistance. The first trigger ("early
warning resistance") would result in proactive mitigation actions that delay the development and spread of
resistance and maintain the Bt trait longevity in areas where the insects are still susceptible. The second
trigger (UXI, field failure, etc.) would initialize immediate actions based on best IPM practices before
resistance was confirmed in the lab. The goals of this immediate intervention when field failure is visible
is to reduce the spread of resistant individuals in the landscape by managing the population densities.
Once resistance is confirmed, a lepidopteran-specific mitigation plan would be implemented and a
mitigation action area established.
An additional option considered by EPA is the encouragement of IPM with IRM for lepidopteran pests of
Bt corn and cotton at the onset of a new trait deployment and throughout its commercialization (see
options in Section IV).
Uncertainties
It may not be expeditious to employ diet bioassays on insects for investigating field failures and making
early resistance determinations. Insect rearing takes time, especially with EPA's proposed option to adopt
the more resource intensive F2 screens (Section III. B. 6). By the time, EPA receives a resistance report
from industry, at least one growing season will have passed and official mitigation cannot begin until year
two. While developing the molecular tools to conduct quick assays for resistance screening of field
collected insects may be laborious, it would ultimately speed up the resistance determination and
subsequent implementation of mitigation. Thus, it may be of benefit to abandon the diet bioassay methods
in the long run and consider a shift to more rapid and more advanced analytical tools.
Mitigation of lepidopteran resistance is unlikely to succeed with a trigger for mitigation relying on
"practical resistance". Noctuid moths, specifically H. zea, engage in migratory flight behavior on a
continental scale (Beerwinkle et al. 1995) as well as substantive inter-field dispersal. Computer
simulations for a pest with more limited dispersal propensities than H. zea have shown that once
resistance is widespread, regional mitigation of resistance may have minimal impact on slowing resistance
from increasing and extending the lifetime of the compromised Bt trait (Martinez and Caprio 2016). By
extrapolation, resistance should spread even faster through a landscape for a more mobile pest like H. zea.
If practical resistance from UXI cases is confirmed and resistance is widespread, it may useful to rename
the regulatory action from "mitigation of resistance" to "management of resistance".
9. Cross-resistance between Bt toxins reduces durability of pyramids
Cross-resistance is more likely to occur between Bt crystalline (Cry) toxins with high amino acid
sequence similarity, based on the predominant Bt mode of action that presumes such toxins share binding
sites in the insect midgut (Tabashnik et al. 1996; Hernandez-Rodriguez et al. 2013; Adang et al. 2014; and
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Carriere et al. 2015). For CrylAc, Welch et al. (2015) confirmed that sequence homology within domain
II, the domain linked with receptor binding, was more closely associated with cross-resistance than
similarity of domains I and III in a resistant colony of H. zea. A comprehensive review corroborated this
trend for cross-resistance among Cry toxins homologous at domain II in 10 major pests of Bt crops
(Tabashnik et al. 1996; Carriere et al. 2015).
Some studies have observed shared binding sites among Cry toxins expressed in Bt crops for H. zea and
H. armigera. The six toxins currently expressed by Bt crops with activity against H. zea include CrylAc,
CrylA.105, CrylAb, Cry IF, Cry2Ab, and Vip3Aa. Starting with CrylA.105 these toxins respectively
share 99, 99, 51, 15, and 0 percent sequence similarity to CrylAc at domain II (Carriere et al. 2015).
Competitive binding data show that CrylAc and Cry IF share a receptor in H. zea and H. armigera
(Hernandez and Ferre 2005) and CrylAb and CrylAc share a receptor in H. armigera (Estela et al. 2004).
Additional binding studies found CrylA.105 shares a receptor with CrylAb, CrylAc, and CrylF in O.
nubilalis and S. frugiperda (Hernandez-Rodriguez et al. 2013). These results support that cross-resistance
among CrylAc, CrylA.105, CrylAb, and CrylF may be explained by reduced binding to a shared
receptor.
Cross-resistance is likely an important factor predicting the rate of resistance evolution in H. zea given
observed CrylAc and CrylAb "practical resistance" (Tabashnik et al. 2008, but also see Moar et al. 2008;
Reisig and Reay-Jones 2015; Dively et al. 2016) and non-functional pyramid crops (<95% mortality of
susceptible insect, Roush 1998). Since no Bt toxin expresses a high dose for H. zea, even weak cross-
resistance is expected to greatly accelerate resistance evolution.
Five studies report varying degrees of cross-resistance in Cry 1 Ac-selected H. zea strains (Burd et al.
2003; Anilkumar et al. 2008; Caccia et al. 2012; Brevault et al. 2013; Welch et al. 2015). The literature
indicates consensus regarding cross-resistance between CrylAc and CrylAb (Anilkumar et al. 2008;
Caccia et al. 2012; Welch et al. 2015). Cross-resistance between these two toxins is expected based on the
mechanism outlined above as these proteins share more than 99% amino acid sequence similarity at
domain II and may share a midgut receptor. However, results vary concerning other toxins.
Limited evidence of strong cross-resistance has been found between CrylAc and CrylA.105, the latter
being used in many Bt corn pyramids. Cryl A. 105 is a chimeric protein with domain II and most of the C-
terminus taken from CrylAc (Biosafety Clearing-House 2008). One study has assayed a MVP II (a
formulation 98.5% identical to CrylAc) selected strain of H. zea against CrylA.105 and found high levels
of cross-resistance (Welch et al. 2015). This is not surprising because of the high sequence homology
between CrylAc and CrylA.105.
Cry2Ab is found in both corn and cotton pyramids. CrylAc and Cry2Ab exhibit relatively low sequence
homology at domain II (15%). Cryl Ac-selected strains of H. zea have been found to have weak cross-
resistance to Cry2A group toxins (Burd et al. 2003; Welch et al. 2015) but not in all cases (Anilkumar et
al. 2008; Brevault et al. 2013). Responses to CrylAc and Cry2Ab were positively correlated in two
studies of field-collected populations of H. zea (Jackson et al. 2006; Ali and Luttrell 2007). Higher
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survivorship of Cryl Ac resistant individuals has been found in plant bioassays with Cryl Ac + Cry2Ab
cotton as well (Brevault et al. 2013). Laboratory assays in conjunction with consistent field results
indicate that selection of H. zea with Cryl Ac causes weak cross-resistance to Cry2A toxins.
CrylF is expressed in single toxin corn as well as pyramid corn and cotton. Selection to CrylF in single
toxin corn favors resistance development to other similar Cry toxins in pyramided crops. Cryl Ac shares
approximately 50 percent sequence homology at domain II with CrylF. One study reports weak, non-
significant cross-resistance between Cryl Ac and CrylF in H. zea based on conservative criterion of non-
overlapping 95% fiducial limits with associated LCsos between selected and susceptible reference strains
(Welch et al. 2015). This report likely underestimates cross-resistance between the toxin pairing due to
low susceptibility to the non-high dose trait CrylF in the unselected control strain. Cross-resistance
between CrylF and either Cryl Ac or Crylb was significant in 16 documented cases of a total of six
different pest species (Carriere et al. 2015).
Cross-resistance is not expected between Vip3Aa and Cryl Ac because there is no sequence overlap
between the two families of toxins. As expected, Anilkumar et al. (2008) reported no cross-resistance
between these two Bt toxins. Non-significant cross-resistance was, however, detected by Welch et al.
(2015) based on the conservative criterion of non-overlapping 95% fiducial limits for the LCso measures
of the resistant and susceptible reference strains. However, there was higher survivorship in the Cryl Ac
resistant strain when assayed against Vip3Aa than anticipated (Welch et al. 2015). Studies with H.
armigera, Trichoplusia ni (cabbage looper), and H. virescens have also reported weak cross-resistance to
this toxin pairing (Carriere et al. 2015). Based on the sum of these observations, weak cross-resistance
cannot be ruled out between Cryl Ac and Vip3Aa (Welch et al. 2015; Carriere et al. 2015). Any cross-
resistance between these two unrelated toxins implies that other factors could be responsible that are not
typically considered under the prevailing view of Bt mechanism of action.
EPA options to address concern
For pests with low susceptibility to Bt toxins, even weak cross-resistance can accelerate the evolution of
resistance. To protect the remaining most efficacious Bt trait(s), managing non-high dose single toxin
products in Bt corn should be the focus, which should also reduce the risk of resistance to pyramided Bt
cotton in the southern US.
Under ideal conditions, resistance genes for all toxins in a pyramid should be rare at the time of
commercialization. While this is not always achievable, resistance management is more effective with a
high dose profile and low resistance allele frequencies. PIP registrants as well as growers should benefit
from the development of high dose traits against lepidopteran pests of corn and cotton and maximize the
durability of such products.
Companies that are developing new traits should avoid pyramiding Bt proteins that share high sequence
homology or chimeric toxins composed of first generation proteins like Cryl A. 105. Registrants should
also consider including full-length protoxins in lieu of activated toxins in Bt crops (Tabashnik et al. 2015;
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Soberon et al. 2016). For best outcomes, developing transgenic plants with novel traits would improve the
options and success of IRM in a challenging and diverse cropping area such as the southern US.
Uncertainties
Cross-resistance is expected to accelerate the rate of resistance to Bt toxins in H. zea, however, there are
several uncertainties that may undermine this claim. It is generally agreed that cross-resistance is less
likely between toxins that differ markedly in structure and target sites (Ferre and van Rie 2002). However,
the molecular mechanisms resulting in cross-resistance are still somewhat uncertain and may be irrelevant
depending on the course of resistance evolution in field scenarios. Resistance to Bt crops can develop
through numerous pathways, not all of which may be linked to receptor binding affinity. Not all Bt-
resistant H. zea with known mechanisms of resistance showed reduced binding of Cry 1 Ac as an important
factor contributing to resistance (Karim et al. 2000; Anilkumar et al. 2008). Since weak cross-resistance
has been documented for Vip3Aa and Cryl Ac (Welch et al. 2015) despite the fact that they do not share a
receptor, cross-resistance between these toxins is caused by other mechanisms not currently explored in
the literature. Bt toxins may share low-affinity receptors (Hernandez-Rodriguez et al. 2008), which could
affect the development of weak cross-resistance between Bt toxins even in strains where reduced binding
affinity does not fully explain the mechanism of resistance.
The source of toxins used in laboratory selection and bioassays may skew research results. Bt crops
express activated Bt proteins, yet studies published in the scientific literature used either full length
protoxins or activated toxins for selection and comparison. Many laboratories select H. zea with MVP II
because it is readily available in large quantities (Brevault et al. 2013; Welch et al. 2015). MVP II is a
formulation containing a hybrid protoxin that is identical to Cryl Ac in the active portion of the toxin and
98.5% identical overall. Protoxins, however, are more effective against lepidopteran pests than their
activated counterparts (Tabashnik et al. 2015; Soberon et al. 2016), which implies that studies using
protoxin selected H. zea strains may have overestimated the magnitude of cross-resistance potential.
There are conflicting reports of fitness costs among Bt selected H. zea strains (Storer et al. 2003; Orpet et
al. 2015). This is discussed in more detail in Section III. B. 12. d. However, any mechanism that
undermines the fitness of resistant individuals compared to susceptible individuals will delay resistance
evolution (Gassmann et al. 2009), whether on Bt or non-Bt, and may negate the consequences of cross-
resistance.
10. Non-functional pyramids lead to faster resistance development
If individual Bt traits in pyramids each achieve >95% mortality of susceptible (SS) target genotypes,
Roush (1998) proposed that a non-Bt refuge could be as low as 10% and offer "superior delays in
resistance" compared to sequential release of the single toxins with larger refuges. However, the modeling
results also showed that reducing the refuge could still carry a significant risk for resistance. Hence,
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Roush concluded that refuges should be kept as large as could be justified economically to avoid reducing
a pyramid's lifetime. EPA scientists refer to pyramids as "functional" when they meet the mortality
standard set by Roush (1998) and "non-functional" if they do not.
Historically, Bt corn traits were first commercialized as single Bt products. Subsequently, these traits
were pyramided with each other in various combinations. Thus, target pests were offered an initial
opportunity to adapt to Bt traits before being exposed to their respective pyramids. Such a
commercialization strategy effectively lowers a pyramid's efficacy or lifetime and is especially
problematic for non-high dose pests such as H. zea. In the southern US, the corn refuge for pyramids was
set to 20% (compared to 5% in the Corn Belt), however, to mitigate resistance concerns for H. zea.
EPA options to address concern
Theoretical analyses may need to explore new management options for non-functional Bt corn pyramids
(less than 95% mortality provided by individual traits), including cost to resistance, community block
refuges (in light of grower non-compliance with requirements), bigger refuges, grower incentive programs
to plant refuges, etc.
Uncertainties
It is unclear by how much new management strategies in Bt corn and greater compliance with block
refuges would delay product failure for compromised pyramids in cotton. Density dependent effects in a
structured refuge coupled with high pest growth rates (as documented for H. zea) may result in lower than
expected durability gains (Martinez et al. 2017).
11. Mitigation of resistance occurs too late
Lepidopteran resistant cases to Bt have increased in the US over the past decade (see Section III. A.). This
phenomenon could generally be addressed by implementing 1) effective and proactive strategies that
delay the onset of product failure (see Section III. B. 8 & Section IV) and 2) reactive strategies that allow
farmers to manage pest densities in wide-spread product failure scenarios.
a. Goal of Mitigation
From a scientific perspective, the goal of mitigation is to implement a series of effective management
actions that substantially reduce insect densities in the sites of concern and limit the spread of resistance
(Andow et al. 2016). If subsequent recolonization into the previously identified resistant sites occurs with
primarily susceptible individuals, then overall resistance levels are likely mitigated (Comins 1977).
Depending on the population dynamics, ecology, and biology of the organism, however, extirpation of
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localized resistance may or may not be an option and likely depends heavily on the timing of resistance
detection and mitigation.
EPA's regulatory goal for mitigating Bt resistance is to reduce resistance in the site of concern and
maintain the durability of those traits in areas where they are still effective (US EPA 2013). For
mitigation actions to make a significant impact (e.g. extend overall trait durability in area), several life-
history parameters should be known and certain methodological tools need to be in place. Some of these
biological parameters are the spatial scale occupied by the resistant population (e.g. random mating unit)
and the typical fraction of individuals in a population engaging in non-trivial dispersal (i.e., leaving natal
habitat despite availability of food) (Southwood 1962). The first factor aids in delineating a potential
Mitigation Action Area (MAA) for emerging resistance in a particular location (i.e., hot spot). The second
factor allows for estimating the rate with which resistance spreads from a resistant site. In addition,
reliable, high resolution, and rapid resistance confirmation tools and uniform regulatory threshold triggers
for unexpected damage have to be in place to allow timely implementation of mitigation.
b. Uniform. Regulatory Threshold Triggers for Mitigation
The risk of having a late mitigation trigger (e.g., at field failure) is that resistance can spread and develop
to high levels in the landscape before it is detected. This is especially of concern for Noctuid moths,
which have a great dispersal propensity (Beerwinkle et al. 1995; Westbrook 2008). Theoretical work
suggests that once resistance is widespread (r-frequency = 0.1), it would be difficult to extend the
durability of Bt sufficiently even with 100% of fields mitigated in an affected areas and limited pest
dispersal (Martinez & Caprio 2016). Thus, early mitigation of resistance should be the primary focus of a
resistance management program to delay the onset of Bt field failure or practical resistance. Pest
management rather than mitigation of resistance will likely be the strategy when practical resistance is
widespread.
Proactive mitigation needs to be initiated when an early warning threshold for resistance is met; such a
threshold would be independent of field performance or plant damage and solely rely on resistance levels
in populations. Furthermore, this threshold would preferably be combined with high resolution, molecular
tools and set at a low level of resistance (1-6% in a population).
Since trait failure is unlikely to be avoided and resistance to Bt will eventually evolve, a second and
uniform regulatory threshold is needed. Such a trigger would initiate sample collections and assay
investigations to confirm resistance, while at the same time triggering best management practices (IPM)
to control pest densities. This particular threshold should rely on plant damage or Bt plant performance.
c. Use of diagnostic assay tools
i) Where diagnostic concentration assays are available for high dose lepidopteran pests (e.g. Heliothis
virescens, D. grandiosella), and ^-genotypes survive like /^-genotypes, an F1 screen may suffice. If
/tt'-genotype survival is similar to that of SS-genotypes, an F2 screen is recommended. Two independent
tests should be used to measure and determine resistance levels of field-collected insects (e.g., percent
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mortality at the highest Bt concentration tested (positive and negative control and field collections); sub-
lethal measure comparisons on Bt and non-Bt leave tissue for the same population (positive and negative
control and field collection) (see BPPD 2013). When between 1-6% of individuals in any population
tested exhibit resistance (early warning threshold), then an agreed upon, early mitigation plan should be
implemented. The agency considers resistant (positive control) colonies to be an imperative tool to
improve interpretation of these assays in addition to susceptible (negative control) colonies.
ii) Where diagnostic concentration assays are not available and lepidopteran species are non-high dose
pests of Bt (e.g., H. zea), the goal to detect early warning resistance is unattainable with the current
bioassay methods (and resistance is likely much higher already). In addition, cost to resistance (Dively et
al. 2016) will hamper efforts to detect resistance proactively with diet bioassays. For this reason, EPA
proposes that molecular (e.g., microsatellites, SNPs, etc.) assays be developed to identify resistance genes
in such non-high dose pests, possibly analogous to work done for fall armyworm (Baneijee et al. 2017).
Such tools could be used on the collected field insects rather than having to go through the mating and
rearing process.
Until such tools are available for H. zea, focusing on the following should aid in reducing resistance
reports: 1. use of IPM with IRM (e.g. increased scouting, alternate control tools, etc.; see Section IV.A).
2. use of alternate pesticides when economic thresholds on Bt have been reached, and 3. immediate
mitigation of field incidents where growers report to have had high pest pressure and crop injury by
managing population density and switching to other Bt products the following year. In the absence of
confirmed resistance, best management practices relying on the tenets of IPM should be used around the
field and encompassing area (e.g. preferably the size of random mating population) to proactively manage
such incidents (see EPA Options Section below).
If molecular tools and methodologies can be developed to reliably estimate resistance allele frequencies in
H. zea populations, there is sufficient evidence to support that EPA's IRM program could benefit from
adopting monitoring for "early warning resistance" (likely applicable for new technologies). Such a
change will initiate mitigation actions when a shift in susceptibility becomes apparent and much earlier
than field failure will occur. Early mitigation actions do not presume that Bt products are compromised
but rather alert stakeholders and growers in such areas that these stewardship steps are aimed at further
extending the durability of these traits.
d. Fitness cost and benefits for mitigation of resistance
Evolutionary trade-offs arising from the development of pesticide resistance may be key to slowing or
possibly reversing resistance in pest populations (Gould et al. 2006; Gassmann et al. 2009). Fitness costs
occur when resistant individuals have reduced fitness on Bt (incomplete resistance) or in the absence of
selection (cost-to-resistance). The focus hereafter is on fitness costs that occur for resistant insects in
absence of selection.
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e. Cost to resistance
If cost to resistance is present in resistant populations, theoretical simulations have shown that resistance
levels can be reduced when single traits were removed from the landscape and only pyramids remained
(Gould et al. 2006). When all toxins were high dose, initial resistance allele frequencies were low,
susceptible insects from refuges mated at random, and fitness costs were associated with resistance, then
resistance did not evolve. Even with moderate Bt dose, the presence of fitness costs in resistant
populations reduced the resistance allele frequency in these modeling scenarios. When just a small
proportion of single toxin plants were in close proximity to pyramid crops, however, cost to resistance
became ineffective and resistance evolved (Gould et al. 2006).
The presence and magnitude of fitness costs in field collected and/or laboratory-selected strains is further
discussed for key lepidopteran pests of Bt and how these pest attributes may influence the development of
and monitoring for resistance.
Examples of fitness cost
H. zea
Fitness costs associated with resistance to Cryl Ac have been found in most cases for H. zea (Orpet et al.
2015; Moar et al. 2010; Anilkumar et al 2008; Jackson et al. 2006; Jackson et al. 2004; Burd et al. 2003;
but see Brevault et al. 2013). Studies comparing other Cryl Ac laboratory selected strains to the
unselected strains demonstrated reduced larval weight, larval duration, pupal weight, and pupal duration,
along with an increase in percentage of malformed adults and percentage reversion to susceptibility
(Anilkumar et al. 2008; Moar et al. 2010; Orpet et al. 2015). Field collected family lines resistant to both
Cryl Ac and Cry2A toxins demonstrated fitness costs as well as slower growth rates than expected (Burd
et al. 2003; Jackson et al. 2004; Jackson et al. 2006). In conclusion, fitness costs appear to be prevalent
amongst//, zea resistant strains.
The magnitude of fitness costs differed between artificial diet and on-plant bioassays (Bird and Akhurst
2004) for H. armigera (a close relative of H. zea). In on-plant assays, there was a measurable
developmental delay between resistant and susceptible individuals on non-Bt cotton plants but none was
measured on artificial diet (Bird and Akhurst 2004) whereas with other colonies, fitness costs were found
with both on plant assays and artificial diet with higher costs associated with higher levels of resistance
(Liang et a. 2008). Fitness costs were higher for some host plants than others, though these observations
were not necessarily linked to nutritional suitability. For example, susceptible H. armigera had equivalent
fitness among cotton, pigeon pea, and sorghum; however, Bt resistant individuals had more dominant
fitness costs on cotton and sorghum (Bird and Akhurst 2007). These results indicate that laboratory
bioassays may sometimes underestimate the costs of resistance, while the magnitude of fitness costs
measured via on-plant assays may change depending on the host plant selected.
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S. frugiperda
Numerous studies have found fitness costs in S. frugiperda associated with Cry IF (Horikoshi et al. 2016;
Dangal et al. 2015; Jakka et al. 2014), Vip3A (Bernardi et al. 2016), and pyramid corn containing CrylF,
CrylA.105, and Cry2Ab (Bernardi et al. 2017). Fitness costs in S. frugiperda included longer
development time, reduced survival, lower reproductive capacity, reduced larval weight, and skewed sex
ratio (Bernardi et al. 2017; Bernardi et al. 2016; Horikoshi et al. 2016; Dangal et al. 2015; Jakka et al.
2014). Some field collected strains had no significant fitness costs associated with CrylF resistance in
laboratory bioassays (Santos-Amaya et al. 2017) or on non-Bt corn leaves (Leite et al. 2016). One CrylF
resistant strain demonstrated heterosis (Velez et al. 2014), which may accelerate resistance evolution or be
a result of a laboratory artifact of increased diversity when crossing strains. S. frugiperda has more
variable responses to developing fitness costs on Bt diets than Helicoverpa spp.
EPA options to address concerns with delayed mitigation of resistance
Early Warning Resistance and Mitigation Strategies in Corn
As discussed in an earlier section of this document, resistance development for H. zea is driven by corn
since the pest funnels through this crop in summer (two generations). Hence, any mitigation targets
discussed here should focus on this crop. Not all proposed options would be equally effective at
mitigating resistance, and it is unclear what is currently incorporated already. In addition, some may be
preferred financially, while others may have more scientific support and environmental benefits. An early
resistance mitigation program could incorporate the following:
Addressing the risk of single traits and seed blends in the southern US to extend the durability of
pyramids: such actions would reduce the number of field failures requiring mitigation and help
delay the onset of resistance to still functional traits. For areas where pyramids remain partially
efficacious, addressing these risks should also benefit growers and decrease the number of
problem fields, though it may be necessary to incorporate additional control tools. In areas with
widespread resistance to numerous toxins, rotating to other traits may not be a viable strategy and
management of pest densities may be the primary focus of 'mitigation' efforts.
Alternate control measures. These are:
o Mixture strategy for chemical pesticides on conventional corn: this is analogous to
pyramiding Bt traits. If no high dose, chemical pesticides are available, this option would
provide another tool with more than one chemistry to control a pest;
o Rotation of pyramids: this strategy works if rotated products have no cross resistance and
if at a minimum two functional traits are expressed in plants;
o Rotation of traits with rotation of mixture strategies: a combination of pyramided Bt and
pyramided chemistries could be an option for pest outbreaks or other unusual density
phenomena;
o Rotation of traits with conventional (non-Bt) corn, where growers would use chemical
control tools (etc.) when the economic threshold is reached;
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o Pheromone strategies: this option is costly but could be a used with other density
management approaches; and
o Planting of trap crops.
Addressing mechanisms to increase refuge compliance for Bt corn in the southern US. To further
quantify the potential durability gains from higher refuge compliance, EPA recommends that more
complex probabilistic modeling be conducted for the southern corn growing areas incorporating
pest density dependence, delayed adult emergence from Bt compared to refuge fields, etc.
Improving communication between industry, seed dealers, growers, extension entomologist, and
crop consultants will assure that the latest field observations and performance issues are shared.
Growers will then be able to make informed decisions about what to anticipate and how to prepare
for next season.
Field Failure and Additional Mitigation
When the threshold for unexpected injury is met, insects should be collected and tested for resistance,
preferably from the generation causing the injury. Since UXI could be an early indication of field
resistance, EPA is considering mitigation options, which would be implemented on the affected farm(s)
even before resistance investigations are concluded (e.g., adulticide spraying to avoid spread of resistance,
rotation of control strategies the following year, and other mitigatory measures designed to limit the
spread of the population and reduce selection pressure for further resistance).
Once resistance has been confirmed, strategies that could be employed are listed above in the "Early
Resistance and Mitigation" section. In addition, planting an increased amount of non-Bt expressing plants
may assist by reducing resistance allele frequencies in areas if cost to resistance is present. Likewise, if a
pyramid is chosen to mitigate resistance, and it contains the compromised trait, then the associated refuge
size may need to be reconsidered. Possible new mitigation options in addition to the ones in place could
be:
Limit the use of the compromised trait in the affected area
Planting a greater block refuge: this could be challenging in the southern US given the historical
grower non-compliance;
Adjusting planting time of Bt crop to reduce egg laying by pest of concern;
Use of trap crop;
If compromised traits are part of the pyramids, or each trait is less than 95% effective, or there is
cross resistance between pyramided traits, the refuge may need to be increased to protect the still
functional trait(s).
If a non-related pyramid is planted, yet one or more traits do not result in >95% mortality, then the refuge
size may need to be reconsidered.
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Fitness Costs
Fitness costs have the potential to delay field-evolved resistance to Bt crops in lepidopteran pests but not
in the presence of single toxin Bt (Gould et al. 2006). In the case of H. zea, due to low inherent
susceptibility to Bt toxins, documented field-evolved resistance (Tabashnik et al. 2013), and often lack of
structured refuges in southern corn growing areas, the presence of fitness costs may not be enough to
extend durability of Bt crops significantly.
Developing Bt or other new technology crops with high toxicity to pests like H. zea that have shown to
have low inherent susceptibility to Bt, would provide options to exploit fitness costs and delay resistance
(Gould et al. 2006; Carriere et al. 2010). EPA is considering addressing the risk posed by single toxin Bt
corn to pyramided Bt and lack of available non-Bt refuges. However, differences in density dependent
survival in refuge and Bt fields have shown to reduce the expected durability gain of pyramids (Ives et al.
2012), especially when the pest growth rate is high, as for H. zea (Caprio et al. 2009; Martinez et al.
2017).
Fitness costs causing mortality during the rearing process affect the ability to detect shifts in susceptibility
using diet bioassays as monitoring tools and, thus, produce results that are not necessarily reflective of
field conditions. Prior to rearing, the collections of insects exposed to non-Bt corn diets during the
transport to the lab could also remove resistant individuals from the samples. These are examples of how
the resistance allele frequency and resistance levels can be underestimated in the presence of fitness costs.
As described in Section III.B. 6. EPA considers other options than diet bioassays that would allow
resistance assessment on field-collected insects.
The mechanism by which resistance evolves may differ from selection under field conditions versus
laboratory condition (US EPA FIFRA SAP 2009). If fitness costs are linked to the mechanism of
resistance, the results of such studies may not be applicable to field scenarios. For example, poor sperm
competition affecting mating success in Cry 1 Ac-resistant H. armigera fed on non-Bt diet was related
specifically to cadherin mutations leading to Bt resistance (Zhang et al. 2014). Bt resistance related to
cadherin mutations, and thus gut permeability, may be more likely to incur fitness costs in non-Bt arenas
due to increased susceptibility to host plant defenses (Carriere et al. 2010). If resistance evolves by
another mechanism in field scenarios, such results may not be relevant.
Threshold Triggers
Field failure is a function of pest pressure and resistance levels. When using thresholds triggers for
determining UXI in corn, a subsequent comparison between Bt and non-Bt damage should always be
undertaken after a threshold has been met. High pest densities can lead to field failure with lower
resistance allele frequencies. Conversely, no field failure may be observed at higher resistance levels if
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the pest densities are low. This uncertainty supports the option to shift mitigation of resistance to an
earlier point in the pest's evolutionary time, namely when 'early warning resistance' is identified.
Life-History
Prolonged development of resistant field populations on artificial diet with Bt (Dively et al. 2016) may
reflect a possible delay in emergence for H. zea coming out of Bt fields compared to non-Bt fields. It is
unclear if and how much assortative mating takes place in the field; no such data are available. This
information is needed to explore theoretically whether a greater refuge size could be an effective
mitigatory tool.
Moths in the family Noctuidae show great propensity for movement and are able to engage in long
distance migration on the scale of the North American continent (Westbrook 2008). Flight mill studies
support that male Noctuid moths are capable of uninterrupted flight lasting three hours. This duration is
estimated to be an equivalent of 18.8 km (n = 132) (Beerwinkle et al. 1995). Given this long-distance
dispersal potential, it needs to be determined what fraction of a typical H. zea population engages in this
behavior. These data are currently not available but would be needed to estimate the spread of resistance
genes out of a resistant site and the establishment of resistance in the landscape.
12. Risk factors for S. albicosta resistance in Bt com
Hutchison et al. (2011) identified several underpinning factors that seem to have contributed to the rapid
range expansion of western bean cutworm across the northern Corn Belt. Some of those are reduced
tillage leading to greater overwintering survival, reduced chemical pesticide use with expanding Bt corn
adoption, the pest's propensity for long distance dispersal, and possibly climate change facilitating the
expansion. Increased Bt corn adoption contributes to greater risk of resistance development in S.
albicosta. Several factors already discussed for H. zea are further contributors to greater resistance risk.
Those are lack of high dose Bt traits, 5% refuge for single trait acting on S. albicosta, and possibly seed
blends.
a. Availability of Cry-toxins is limited
Cry IF has never fully controlled S. albicosta but was added to the label in 2003. EPA concluded at the
time of review that CrylF at best suppressed the pest (US EPA 2003b). Since S. albicosta has historically
not been considered a major pest of Bt but a sporadic pest, registrants may not have developed Bt toxins
with this pest in mind. This would explain why there are so few Bt tools available today.
Vip3 A was developed by Syngenta to control Lepidoptera pest of corn and cotton and registered by the
EPA in 2009. This particular toxin has shown to have good activity against S. albicosta. It is currently the
only Bt PIP tool available to reduce larval densities in corn.
b. Percent refuge for single YipJA trait reduces its durability
Pyramided Bt corn products are deployed with a 5% refuge in the northern Corn Belt (external block and
seed blends) (US EPA 2010) and where S. albicosta has developed a tolerance towards CrylF (Smith et
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al. 2017), one of only two Bt toxins that has historically shown some efficacy against this pest. Vip3A is
pyramided with other lepidopteran Bt traits in corn, none of which suppress or control S. albicosta.
Therefore, this toxin is essentially deployed as a single trait product with a much lower refuge (5%) than
is typically required (20%). This exerts a tremendous selection pressure on the only available Bt PIP tool
for western bean cutworm.
c. Seed blends and resistance risk
S. albicosta feeds in ears of corn with weak intra-specific density dependence (Ostrem et al. 2016). The
resistance risk to Vip3 A may be higher in S. albicosta because only one viable trait is expressed in the
maternal tissue. In a seed blend environment, kernels have a 25% probability of expressing both genes or
no genes and a 50% probability of expressing one Bt gene. Thus, sub-lethal exposure to a Bt mosaic may
present a resistance risk for this pest, as was described for H. zea.
EPA options to address risks of resistance
EPA is considering whether western bean cutworm should be viewed as a primary pest of corn and if a Bt
corn IRM plan is warranted for the insect. Such a plan should consist of the following post-registration
requirements: monitoring for and mitigating resistance (insect collections, assay development, and
mitigation strategies) and grower education. EPA notes that IPM with IRM is another option that would
delay resistance development in S. albicosta.
Uncertainties
Since western bean cutworm feeds on maternal corn tissue first as a young instar larva, it should,
therefore, always be exposed to Bt expressed in maternal tissue after hatching. Therefore, it could be
argued that selection occurs early and on the leaf tissue rather than the kernels and that the potential for
sub-lethal expression in a mosaic environment may not be relevant. Seed blends would then not pose an
additional resistance risk to western bean cutworm.
If western bean cutworm is elevated to a primary pest of corn, then refuge proportion may need to be
reconsidered for pyramided Bt products that express only one functional toxin for the control of this
insect. Modeling analyses may be necessary to assist with such an option.
IV. EPA CONCLUSIONS AND OPTIONS TO REDUCE RESISTANCE RISKS
It has been reported that four lepidopteran species developed resistance to Bt in the continental United
States over the last decade. Four academic publications found resistance in corn earworm, fall armyworm,
and western bean cutworm (Dively et al. 2016; Huang et al. 2014; Ostrem et al. 2016; Smith et al. 2017).
A PIP registrant reported to the US EPA that resistance was found in southwestern corn borer (MRIDs
497854-01 and 501749-01). EPA agrees with all four resistance claims, although only one was confirmed
using the regulatory process in place. Based on scientific concerns for risks of resistance development in
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non-high dose pests of Bt corn and Bt cotton, EPA is proposing that changes be adopted into EPA's IRM
program for lepidopteran pests of Bt to reduce the selection intensity and improve the resistance
monitoring approach and success of mitigation of resistance.
A. Options to Reduce Resistance Risks:
Potential options for developing an improved IRM program would have to address the following
identified risk factors (RF):
Risk factor 1: Single trait Bt corn in the presence of pyramided products in the landscape;
Option: Limit or otherwise manage single trait commercial products;
Option: Transition from single trait products to Bt corn pyramids.
Risk factor 2: Limited number of highly effective Bt traits;
Option: Adoption of intervening actions at "early warning resistance" in addition to actions when
field failure occurs (see "molecular assay" option);
Risk factor 3: The shift of dominance of resistance in ear-feeding pests of corn with exposure to
Refuge-In-The-Bag (RIBs) corn products in the southern US;
Option: Potential changes to the corn breeding program to move away from hemizygous parental
corn lines, introduce pollen incompatibility between Bt and non-Bt cultivars, self-pollinating
mechanism, etc.;
Option: Manage the use of Bt corn RIB products in the southern US;
Risk factor 4: Non-compliance with refuge plantings in the southern US;
Option: The development of incentive programs to increase compliance with block refuge
requirements for Bt corn in the southern US;
Risk factor 5: Non-functional, non-high dose pyramided Bt products where individual traits
control less than 95% of susceptible insect;
Option: Implementation of best management practices (BMPs) consistent with the tenets of
Integrated Pest Management (IPM) practices for fields with Unexpected Injury (UXI);
Option: Manage the use of non-functional pyramids;
Option: Increase the amount of refuge;
Risk factor 6: Resistance monitoring does not allow tracking the same populations year after
year;
Option: Use of sentinel plots to monitor the same populations from year to year;
Risk factor 7: Lack of standardized UXI thresholds;
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Option: Development of such standards in corn and cotton would expedite insect collections,
reporting to EPA, and early mitigation;
Risk factor 8: Selection pressure on effective traits (e.g., Vip3A) in pyramids with less effective
or compromised traits;
Option: Focus on risk in Bt corn which also drive resistance risk to Bt cotton;
Risk factor 9: Use of diet bioassay methods for non-high dose pests;
Option: Development of molecular/DNA assays to monitor for "early warning" resistance that
allows expeditious resistance confirmation followed by early implementation of mitigation
actions;
Risk factor 10: Mitigation of resistance when field failure occurs;
Option: Adoption of an early intervening threshold when resistance allele frequencies are still low
(e.g., "early warning resistance");
Risk factor 11: Protracted mitigation in response to field failure;
Options: A priori determined size for a Mitigation Action Area (MAA) based on scientifically
defensible factors relying on noctuid dispersal propensity;
Option: Implementing mitigation actions when field failure is apparent instead of after resistance
is confirmed (i.e., practical resistance);
Option: Use of best management practices (BMPs) consistent with the tenets of IPM to reduce
UXI field occurrences and delay resistance development.
B. Additional Options to Reduce Resistance Risks:
A new framework for lepidopteran IRM could further benefit from the use of IPM with IRM and
increased communication between stakeholders and other proactive actions such as:
Increased scouting of pest densities
Use of alternate control methods in addition to Bt when economic threshold is reached
Increased tilling where possible and crop destruct
Multi-year management plan to control major pests including rotation of Bt pyramided products
(different modes of action), Bt corn with conventional corn and insecticide use, etc.
Use of standardized regulatory thresholds defining unexpected injury levels that trigger insect
population investigation (collections, BMPs, and assay follow up to test for resistance)
Implementation of best management practices (consistent with tenets of IPM) when unexpected
injury levels are triggered
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Improved and timely communication through notification to inform growers, state extension
agents, and crop consultants of areas with early warning resistance, unexpected injury fields, and
confirmed resistance cases.
C. IR.M for 5*. albicosta
The agency is considering whether an insect resistance management plan should be developed for S.
albicosta and Bt corn incorporating the options in the previous two sections including the use of IPM with
IRM (see IV. 1 & 2 ).
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V. REFERENCES
Adang, M. J., N. Crickmore, and J. L. Jurat-Fuentes. 2014. Diversity of Bacillus thuringiensis crystal
toxins and mechanisms of action. Adv. Insect Physiol. Vol. 47: 39-87.
Ali, M. I., R. G. Luttrell, and S. Y. Young. 2006. Susceptibilities of Helicoverpa zea and Heliothis
virescens (Lepidoptera: Noctuidae) populations to CrylAc insecticidal proteins. J. Econ. Entomol. Vol.
99 (1): 164-175.
Ali, M. I. and R. J. Luttrell. 2007. Susceptibility of bollworm and tobacco budworm (Lepidoptera:
Noctuidae) to Cry2Ab2 insecticidal protein. J. Econ. Entomol. Vol. 100: 921-931.
Ali, M. I. and R.G. Luttrell, 2011. Susceptibility of Helicoverpa zea and Heliothis virescens
(Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot cotton. J. Invert. Path. Vol.
108: 76-84.
Andow, D. A., S. G. Pueppke, A. W. Schaafsma, A. J. Gassmann, T. W. Sappington, L. J. Meinke,
P. D. Mitchell, T. M. Hurley, R. L. Hellmich and R. P. Porter. 2016. Early detection and mitigation of
resistance to Bt maize by western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. Vol.
109(1): 1-12.
Andow, D. A. and D. N. Alstad. 1998. F2 screen for rare resistance alleles. J. Econ. Entomol. Vol. 91(3):
572-578.
Anilkumar, K.J., A. Rodrigo-Simon, J. Ferre, M. Pusztai-Care, S. Sivasupramaniam, and W.J.
Moar. 2008. Production and characterization of Bacillus thuringiensis Cryl Ac-resistant cotton bollworm
Helicoverpa zea (Boddie). Appl. Environ. Microbiol. Vol. 74: 462-469.
Banerjee, R., J. Hasler, R. Meagher, R. Nagoshi, L. Hietala, F. Huang, K. Narva, and J. L. Jurat-
Fuentes. 2017. Mechanism and DNA-based detection of field-evolved resistance to transgenic Bt corn in
fall armyworm {Spodoptera frugiperda). Scientific Reports, Vol.7(10877), doi:10/1038/s41598-017-
09866-y
Beerwinkle, K. R., J. D. Lopez Jr., D. Cheng, P. D. Lingren, and R. W. Meola. 1995. Flight potential
of feral Helicoverpa zea (Lepidoptera: Noctuidae) males measured with a 32-channel, computer-
monitored, flight-mill system. Env. Entomol. Vol. 24(5): 1122-1130.
Bernardi, D., O. Bernardi, R. Horikoshi, E. Salmeron, D. Okuma, J. Farias, A. Nascimento, and C.
Omoto. 2017. Selection and characterization of Spodoptera frugiperda (Lepidoptera: Noctuidae)
resistance toMON 89034x TC1507x NK603 maize technology. Crop Protect. Vol. 94: 64-68.
Bernardi, O., Bernardi, D., Horikoshi, R., Okuma, D., Miraldo, L., Fatoretto, J., Medeiros, F.,
Burd, T., and C. Omoto. 2016. Selection and characterization of resistance to the Vip3 Aa20 protein
from Bacillus thuringiensis in Spodoptera frugiperda. PestMgmt. Sci. Vol. 72: 1794-1802.
66
-------
Berman, K., D. Sumerford, and P. Price. 2017. Monitoring the susceptibility of field populations of
Heliothis virescens and Helicoverpa zea to Cryl Ac and Cry2Ab2 proteins: 2016 monitoring results.
Unpublished report submitted by Monsanto to the EPA, dated August 28, 2017.
Biosafety Clearing-House, 2008. Gene and DNA Sequence. Record ID 43771
(4.3.2015).
Blanco, C. A., O. P. Perera, F. Gould, D. V. Sumerford, G. Hernandez, C. A. Abel, and D. A.
Andow. 2008. An empirical test of the F2 screen for detection of Bacillus thuringiensis-resistant alleles in
tobacco budworm (Lepidoptera: Noctuidae). J. Econ. Entomol. Vol. 101(4): 1406-1414.
BPPD. 2017. BPPD review of ABSTC's 2014-2016 compliance assurance program reports for Bt corn
products targeting corn borer and corn rootworm. Memo from J.C. Martinez to M. Weiner on December
6, 2017. MRIDs 495595-01, 498470-01, and 501716-01.
BPPD. 2015. Review of 2011 - 2013 lepidopteran monitoring data submitted by ABSTC for Bt corn
registrations. Memorandum from A. Reynolds to M. Mendelsohn, September 30, 2015.
BPPD. 2014. EPA review of ABSTC's 2011-2013 corn insect resistance management compliance
assurance program. Memo from J.C. Martinez to A. Sibold on April 9, 2014. MRIDs 487328-01, 490484-
01, and 493069-01.
Bird, L. J., and R. J. Akhurst. 2004. Relative fitness of Cryl A-resistant and-susceptible Helicoverpa
armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton. J. Econ. Entomol. 97: 1699-
1709.
Brevault, T., S. Heuberger, M. Zhang, C. Ellers-Kirk, X. Ni, L. Masson, X. Li, B. E. Tabashnik, and
Y. Carriere. 2013. Potential shortfall of pyramided Bt cotton for resistance management. Proc. Natl.
Acad. Sci. U.S.A. Vol. 110: 5806-5811.
Burd, A. D., F. Gould, J. R. Bradley, J. W. Van Duyn, and W. J. Moar. 2003. Estimated frequency of
non-recessive Bt resistance genes in bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in
eastern North Carolina. J. Econ. Entomol. Vol. 96: 137-142.
Burkness, E. C. and W. D. Hutchison. 2012. Bt pollen dispersal and Bt kernel mosaics: integrity of non-
Bt refugia for Lepidopteran resistance management in maize. J. Econ. Entomol. Vol 105(5): 1773-1780.
Burkness, E. C., T. M. Cira, S. E. Moser, and W. D. Hutchison. 2015. Bt maize seed mixtures for
Helicoverpa zea (Lepidoptera: Noctuidae): larval movement, development, and survival on non-
transgenic maize. J. Econ. Entomol. Vol. 108(6): 2761-2769.
Burkness, E. C., P. K. O'Rourke, and W. D. Hutchison. 2011. Cross-pollination of non-transgenic corn
ears with transgenic Bt corn: efficacy against Lepidopteran pests and implications for resistance
management. J. Econ. Entomol. Vol. 104(5): 1476-1479.
67
-------
Burkness, E. C., G. Dively, T. Patton, A. C. Morey, and W. D. Hutchison. 2010. Novel Vip3 A
Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera:
Noctuidae) under field conditions: implications for resistance management. GMCrops, Vol. 1(5): 337-
343.
Caccia, S., W. J. Moar, J. Chandrashekhar, C. Oppert, K.J. Anilkumar, J. L. Jurat-Fuentes, and J.
Ferre. 2012. Association of Cryl Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline
phosphatase levels in the midgut lumen. Appl. Environ. Microbiol. Vol. 78: 5690-5698.
Capinera, J. L. 2000. University of Florida, http://entnemdept.ufl.edu/creatures/veg/corn earworm.htm.
Last updated June 2017.
Caprio, M. A., J. C. Martinez, P. A. Porter, and E. Bynum. 2016. The impact of inter-kernel
movement in the evolution of resistance to dual-toxin Bt-corn varieties in Helicoverpa zea (Lepidoptera:
Noctuidae). J. Econ. Entomol. Vol. 109(1): 307-319.
Caprio, M. A., C. D. Parker, and J. C. Schneider. 2009. Future fitness of female insect pests in
temporally stable and unstable habitats and its impacts on habitat utility as refuges for insect resistance
management. J. Insect Sci. Vol. 9: 1-10.
Caprio M. A. and J. C. Martinez. 2013. Using seed mixes in conjunction with structured refuges. MSE
Vol. 6: 1-11.
Carriere, Y., J. A. Fabrick, and B. E. Tabashnik. 2016. Can pyramids and seed mixtures delay
resistance to Bt crops? Trends in Biotech. Vol. 34(4): 291-302.
Carriere, Y., N. Crickmore, and B. E. Tabashnik. 2015. Optimizing pyramided transgenic Bt crops for
sustainable pest management. Nat. Biotech. Vol. 33: 161-168.
Carriere, Y., D. Crowder, and B. Tabashnik. 2010. Evolutionary ecology of insect adaptation to Bt
crops. Evol. Appl. Vol. 3(5- 6), 561-573.
Chilcutt, C. F. and B. E. Tabashnik. 2004. Contamination of refuges by Bacillus thuringiensis toxin
genes from transgenic maize. PNAS Vol. 101(20): 7526-7529.
Chippendale, G. M. and C. E. Sorenson. 2017. https://ipmworld.umn.edu/chippendale
Comins, H. N. 1986. Tactics for resistance management using multiple pesticides. Ag. Ecosyst. Environ.
Vol. 16: 129-148.
Comins, H. N. 1977. The development of insecticide resistance in the presence of migration. J. Theor.
Biol. Vol. 64:177-197.
Crespo, A. E., Z. Pan, J. Staley, C. Pilcher, and R. Binning. 2015. Monitoring the susceptibility of
southwestern corn borer, Diatraea grandiosella, collected from unexpected damage fields containing
68
-------
event DAS-01507-1. Unpublished study submitted by Pioneer Hi-Bred International, Inc. to the US EPA,
dated November 30, 2015. MRID 497854-01.
Crespo, A., E. Huang, and C. Pilcher. 2017. Monitoring the susceptibility of southwestern corn borer
(Diatraea grandiosella) to the CrylF protein in southeastern Arizona: 2015 collection. Unpublished study
submitted by Pioneer Hi-Bred International, Inc. to the US EPA, dated January 30, 2017. MRID 501749-
01.
DiFonzo, C, C. Krupke, A. Michel, E. Shields, K. Tillmon, and J. Tooker. 2016. Open letter regarding
efficacy of CrylF trait on western bean cutworm. Last accessed Nov. 21, 2017.
http://msue.anr.msu.edu/news/efficacv of crylf trait on western bean cutworm
Dangal, V. and F. Huang. 2015. Fitness costs of CrylF resistance in two populations of fall armyworm,
Spodopterafrugiperda (JE Smith), collected from Puerto Rico and Florida. J. Invert. Path. Vol. 127: 81-
86.
Dively, G., P. Venugopal, and C. Finkenbinder. 2016. Field-evolved resistance in corn earworm to Cry
proteins expressed by transgenic sweet corn. PLoS One doi: 10.1371/journal.pone.0169115
Dyer, J. M., T. W. Sappington, and B. Coates. 2013. Evaluation of tolerance to Bacillus thuringiensis
toxins among laboratory-reared western bean cutworm (Lepidoptera: Noctuidae). J. Econ. Entomol. Vol.
106(6): 2467-2472.
Downes, S., T. Parker, and R. Mahon. 2010. Incipient resistance of Helicoverpapunctigera to the
Cry2Ab Bt toxin in Bollgard II cotton. PLoS One, Vol. 5(9): el2567.
Estela, A., B. Esriche, and J., Ferre. 2004. Interaction of Bacillus thuringiensis toxins with larval
midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. Vol.
70: 1378-1384.
Ferre, J. and J. van Rie. 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis.
2002. Annu. Rev. Entomol. Vol. 47: 501-533.
FAO [Food and Agriculture Organization]. 1979. Pest resistance to pesticides and crop loss
assessment. PAO Plant Production and Protection Paper, No. 6 (2). FAO, Rome, Italy.
Gassmann, A., Y. Carriere, and B. E. Tabashnik. 2009. Fitness costs of insect resistance to Bacillus
thuringiensis. Ann. Rev. Entomol. doi: 10.1146/annurev.ento.54.110807.090518
Gould, F., M. B. Cohen, J. S. Bentur, G. G. Kennedy, and J. VanDuyn. 2006. Impact of small fitness
costs on pest adaptation to crop varieties with multiple toxins: a heuristic model. J. Econ. Entomol. Vol.
99(6): 2091-2099.
69
-------
Head G., R. E. Jackson, and J. Adamczyk. 2010. Spatial and temporal variability in host use by
Helicoverpa zea as measured by analyses of stable carbon isotope ratios and gossypol residues. J. Appl.
Ecol. Vol. 47(3):583-592.
Heckel D. G., L. C. Gahan, F. Gould, and A, Anderson. 1997. Identification of a linkage group with a
major effect on resistance to Bacillus thuringiensis Cryl Ac endotoxin in the tobacco budworm
(Lepidoptera: Noctuidae). J. Econ. Entomol. Vol. 90:75-86.
Hernandez, C. S. and J. Ferre. 2005. Common receptors for Bacillus thuringiensis toxins Cryl Ac,
CrylFa, and Cryl Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua. Appl. Environ.
Microbiol. Vol. 71: 5627-5629.
Hernandez-Rodriguez, C. S., P. Hernandez-Martinez, J. Van Rie, B. Esriche, and J. Ferre. 2013.
Shared midgut binding sites for Cryl A. 105, CrylAa, CrylAb, Cryl Ac and CrylFa proteins from Bacillus
thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda. PLoS One,
e68164. http://dx.doi.org/10.1371/journal.pone.0068164.
Horikoshi, R.., O. Bernardi, D. Bernardi, D. Okuma, J. Farias, L. Miraldo, F. Amaral, and C.
Omoto. 2015. Near-isogenic CrylF-resistant strain of Spodoptera frugiperda (Lepidoptera: Noctuidae) to
investigate fitness cost associated with resistance in Brazil. J. Econ. Entomol. Vol.109: 854-859.
Huang, F., D. A. Andow, and L. L. Buschman. 2011. Success of the high-dose/refuge resistance
management strategy after 15 years of Bt crop use in North America. Entomol. Exp. Appl. Vol. 140: 1-16.
Huang, F., J. A. Qureshi, R. L. Meagher Jr., D. D. Reisig, G. P. Head, D. A. Andow, X. Ni, D.
Kerns, G. D. Buntin, Y. Niu, F. Yang, and V. Dangal. 2014. CrylF resistance in Fall armyworm
Spodoptera frugiperda: single gene versus pyramided Bt maize. PLoS One Vol. 9(11): el 12958.
https://doi.org/10.1371/iournal.pone.0112958
Hutchison, W. D., T. E. Hunt, G. L. Hein, K. L. Steffey, C. D. Pilcher and M. E. Rice. 2011.
Genetically Engineered Bt corn and range expansion of the western bean cutworm (Lepidoptera:
Noctuidae) in the United States: a response to Greenpeace Germany. JIPM Vol. 2(3): 1-8.
Ives, A. R., P. R. Glaum, N. L. Ziebarth, and D. A. Andow. 2011. The evolution of resistance to two-
toxin pyramid transgenic crops. Ecol. Appl. Vol. 21(2): 503-515.
Jackson, R.E., F. Gould, L. Bradley, and J. Van Duyn. 2006. Genetic variation for resistance to
Bacillus thuringiensis toxins in Helicoverpa zea (Lepidoptera: Noctuidae) in eastern North Carolina. J.
Econ. Entomol. Vol. 99:1790-1797.
Jackson, R., J. Bradley, and J. van Duyn. 2004. Performance of feral and Cryl Ac-selected Helicoverpa
zea (Lepidoptera: Noctuidae) strains on transgenic cottons expressing one or two Bacillus thuringiensis
spp. kurstaki proteins under greenhouse conditions. J. Entomol. Sci. 39:46-55
70
-------
Jakka, S., V. Knight, and J. Jurat-Fuentes. 2014. Fitness costs associated with field-evolved resistance
to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae). J. Econ. Entomol. Vol. 107: 342-351.
Karim, S., S. Riazuddin, F. Gould, and D. H. Dean. 2000. Determination of receptor binding properties
of Bacillus thuringiensis delta-endotoxins to cotton bollworm {Helicoverpa zea) and pink bollworm
(Pectinophora gossypiella) midgut brush border membrane vesicles. Pest. Biochem. Physiol. Vol. 67:
198-216.
Kim, M., A. J. Smith, and A. Vulcan. 2018. 2017 Insect resistance management (IRM) compliance
assurance program report for corn borer-protected Bt corn, corn rootworm-protected Bt corn, corn
borer/corn rootworm-protected stacked and pyramided Bt corn. Unpublished report submitted by ABSTC
to EPA on 1-26-2018. MRID 505101-01
Leite, N., S. Mendes, O. Santos- Amaya, C. Santos, T. Teixeira, R. Guedes, and E. Pereira. 2016.
Rapid selection and characterization of CrylF resistance in a Brazilian strain of fall armyworm. Entomol.
Exp. Appl. Vol. 158: 236-247.
Liang, G., K. Wu, H. Yu, K. Li, X. Feng, and Y. Guo. 2008. Changes of inheritance mode and fitness
in Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae) along with its resistance evolution to Cryl Ac
toxin. J. Invert. Path. Vol. 97: 142-149.
Mahon, R. J., K. M. Olson, S. Downes, and S. Addison. 2007. Frequency of alleles conferring
resistance to the Bt toxins Cryl Ac and Cry2Ab2 in Australian populations of Helicoverpa armigera
(Lepidoptera: Noctuidae). J. Econ. Entomol. Vol. 100(6): 1844-1853.
Marion, P.C.R.G., B D. Siegfried, T. Spencer, and W.D. Hutchison, 2000. Development of diagnostic
concentrations for monitoring Bacillus thuringiensis resistance in European corn borer (Lepidoptera:
Crambidae). J. Econ. Entomol. Vol. 93: 925-930.
Martinez, J. C., M. A. Caprio, and N. A. Friedenberg. 2017. Density dependence and growth rate:
evolutionary effects on the resistance development to Bt (Bacillus thuringiensis). J. Econ. Entomol. doi:
10.1093/jee/tox323, https://academic.oup.com/iee/advance-
article/doi/10.1093/iee/tox323/4769330?guestAccessKev=12f5898b-b857-4a26-8c2e-6843641afcdc
Martinez, J. C. and M. A. Caprio. 2016. IPM use with the deployment of a non-high dose Bt pyramid
and mitigation of resistance for western corn rootworm (Diabrotica virgifera virgifera). Environ.
Entomol. Vol. 45(3): 747-761.
Matten, S. R., R. J. Frederick, and A. H. Reynolds, 2013. United States Environmental Protection
Agency Insect Resistance Management Programs for Plant-Incorporated Protectants and Use of
Simulation Modeling. In: Regulation of Agricultural Biotechnology: The United States and Canada. C.
Wozniak and A. McHughen, eds. (Springer) p. 175-267.
71
-------
Moar, W., R. Roush, A. Shelton, J. Ferre, S. Macintosh, B. Rogers Leonard, and C. Abel. 2008.
Field evolved resistance to Bt toxins. Nat. Biotech. Vol. 26(10): 1072-1074.
Nagoshi, R. N., P. Silvie, R. L. Meagher, J. Lopez, and V. Machado. 2007. Identification and
comparison of fall armyworm (Lepidoptera: Noctuidae) host strains in Brazil, Texas, and Florida. Ann.
Entomol. Soc. Am. Vol. 100(3): 394-402.
NRC [National Research Council]. 1986. Pesticide resistance strategies and tactics for management.
National Academy Press, Washington DC.
Onstad, D. W., A. L. B. Crespo, Z. Pan, P. R. Crain, S. D. Thompson, C. D. Pilcher, and A. Sethi.
2018. Blended Refuge and insect resistance management for insecticidal corn. Env. Entomol., Vol.47(1):
210-219.
Orpet, R., B. Degain, G. Unnithan, K. Welch, B. E. Tabashnik, and Y. Carriere. 2015. Effects of
dietary protein to carbohydrate ratio on Bt toxicity and fitness costs of resistance in Helicoverpa zea.
Entomol. Exper. Appl. Vol. 156 (1): 28-36.
Ostrem, J. S., Z. Pan, J. L. Flexner, E. Owens, R. Binning, and L. S. Higgins. 2016. Monitoring
susceptibility of western bean cutworm (Lepidoptera: Noctuidae) field populations to Bacillus
thuringiensis CrylF protein. J. Econ. Entomol. Vol. 109(2): 847-853.
Payne, G., T. Dennehy, and C. Sansone. 2017. Unusual survival report: susceptibility of bollworm
(Helicoverpa zea) to Cry2Ae and Cryl Ab Bacillus thuringiensis (Bt) proteins - 2016. Unpublished report
submitted to the EPA.
Payne, G., S. Piper, T. Dennehy, and C. Sansone. 2014. Baseline susceptibility of bollworm
(Helicoverpa zea) and tobacco budworm (Heliothis virescens) larvae to Cryl Ab and Cry2Ae Bt proteins -
2013. Unpublished report submitted to the EPA in 2014.
Purdue University Field Crops IPM. 2009a.
https://extension.entm.purdue.edu/fieldcropsipm/insects/fall-armyworm.php
Purdue University Field Crops IPM. 2009b.
https://extension.entm.purdue.edu/fieldcropsipm/insects/western-bean-cutworm.php
Reisig, D. and F. Reay-Jones. 2015. Inhibition of Helicoverpa zea (Lepidoptera: Noctuidae) growth by
transgenic corn expressing Bt toxins and development of resistance to Cryl Ab. Environ. Entomol. Vol.
44(4): 1275-1285.
Reisig, D. D. 2017. Factors associated with willingness to plant non-Bt maize refuge and suggestions for
increasing refuge compliance. J. Integ. PestManagm. Vol. 8(1): 1-10, doi. 10.1093/jipm/pmx002
Roush, R. T. and G. L. Miller. 1986. Considerations for design of insecticides resistance monitoring
programs. J. Econ. Entomol. Vol. 79: 293-298.
72
-------
Roush, R. T. 1998. Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding
succeed where pesticide mixtures have not? Philos. Trans. Roy. Soc. B Biol. Sci. Vol. 353: 1777-1786.
Santos-Amaya, O. F., C. S. Tavares, J. V. C. Rodrigues, T. C. Souza, N. Rodriguez-Silva, R. N. C.
Guedes, A. P. Alves, and E. J. G. Pereira. 2017. Magnitude and allele frequency of CrylF resistance in
populations of the fall armyworm (Lepidoptera: frugiperda) in Brazil. J. Econ. Entomol. Vol. 110(4):
1770-1778.
Siegfried, B. D., P. C. R. G. Marion, J. F. Witkowski, and G.W. Warren, 1995. Susceptibility of field
populations of the European corn borer, Ostrinia nubilalis (Hiibner) (Lepidoptera: Pyralidae), to the
microbial insecticide Bacillus thuringiensis Berliner. J. Agric. Entomol. Vol. 12: 257-263.
Smith, J., M. Lepping, D. Rule, Y. Farhan, and A. Schaafsma. 2017. Evidence for field-evolved
resistance of Striacosta albicosta (Lepidoptera: Noctuidae) to CrylF Bacillus thuringiensis protein and
transgenic corn hybrids in Ontario, Canada. J. Econ. Entomol. Vol. 110(5), doi:10.1093/jee/tox228
Soberon, M., R. Monnerat, and A. Bravo. 2016. Mode of action of Cry Toxins from Bacillus
thuringiensis and resistance mechanisms. In: P. Gopalakrishnakone et al. (eds.), Microbial Toxins,
Toxicology, doi 10.1007/978-94-007-6725-6_28-l
Southwood, T. R. E. 1962. Migration of terrestrial arthropods in relation to habitat. Biol. Rev. Vol. 37:
171-214.
Stadelbacher, E. A., H. M. Graham, V. E. Harris, J. D. Lopez, J. R. Phillips, and S. H. Roach. 1983.
Heliothis populations and wild host plants in the southern US Southern Cooperative Series Bulletin, Vol.
316: 54-74.
Staley. 2017. Herculex I resistance management plan for southwestern corn borer in southeastern
Arizona. Unpublished study submitted by Pioneer Hi-Bred International, Inc. to the US EPA, dated
January 31, 2017. MRID 501749-02.
Stern, V. M., R. F. Smith, R. van den Bosch, and K. S. Hagen. 1959. The integrated control concept.
Hilgardia, Vol. 29: 81-101.
Stone, T. B. and S. R. Sims. 1993. Geographic susceptibility of Heliothis virescens and Helicoverpa zea
(Lepidoptera: Noctuidae) to Bacillus thuringiensis. J. Econ. Entomol. Vol. 86: 986-994.
Storer, N. P., M. E. Kubiszak, J. E. King, G. D. Thompson, and A. C. Santos. 2012. Status of
resistance to Bt maize in Spodopterafrugiperda: Lessons from Puerto Rico. J. Invert. Path. Vol. 110:
294-300.
Storer, N. P., J. M. Babcock, M. Schlenz, T. Meade, G. D. Thompson, J. W. Bing, and R. M.
Huckaba. 2010. Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda
(Lepidoptera: Noctuidae) in Puerto Rico. J. Econ. Entomol. Vol. 103: 1031-1038.
73
-------
Storer, N., Peck, S., Gould, F., Van Duyn, J. W., and G. Kennedy. 2003. Spatial processes in the
evolution of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton in a
mixed agroecosystem: a biology-rich stochastic simulation model. J. Econ. Entomol. Vol. 96(1); 156-172.
Tabashnik B. E. and Y. Carriere. 2017. Search in resistance to transgenic crops and prospects for
sustainability. Nat. Biotech. Vol. 35(10): 926-935. doi: 10.1038/nbt.3974
Tabashnik B. E., M. Zhang, J.A. Fabrick, Y. Wu, M. Gao, F. Huang, J. Wei, J. Zhang, A. Yelich, G.
C. Unnithan, A. Bravo, M. Soberon, Y. Carriere, and X. Li. 2015. Dual mode of action of Bt proteins:
protoxin efficacy against resistant insects. Sci. Rep. Vol. 5:15107.
Tabashnik B. E., D. Mota-Sanchez, M. E. Whalon, R. M. Hollingworth, and Y. Carriere. 2014.
Defining Terms for proactive management of resistance to Bt crops and pesticides. J. Econ. Entomol. Vol.
107(2): 496-507.
Tabashnik, B. E., S. Morin, G. C. Unnithan, A. J. Yelich, C. Ellers-Kirk, V. S. Harpold, M. S.
Sisterson, P. C. Ellsworth, T. J. Dennehy, L. Antilla, L. Liesner, M. Whitlow, R. T. Staten, J. A.
Fabrick, X. Li, and Y. Carriere. 2012. Sustained susceptibility of pink bollworm to Bt cotton in the
United States. GMCrops, Vol. 3(3): 1-7.
Tabashnik B. E., J. B. J. Van Rensburg, and Y. Carriere. 2009. Field-evolved insect resistance to Bt
crops: definition, theory, and data. J. Econ. Entomol. Vol. 102: 2011-2025.
Tabashnik, B. E., A. J. Gassmann, D. W. Crowder, and Y. Carriere. 2008. Insect resistance to Bt
crops: evidence versus theory. Nat. Biotech. Vol. 26(2): 199-202.
Tabashnik, B. E. and B. A. Croft. 1982. Managing pesticide resistance in crop-arthropod complexes:
interactions between biological and operational factors. Environ. Entomol. Vol. 11: 1137-1144.
USDA [United States Department of Agriculture]. 2015. Cotton pests. Last accessed 11-17-2017.
https://www.aphis.usda.gov/aphis/ourfocus/planthealth/plant-pest-and-disease-programs/pests-and-
diseases/cotton-pests/ct cotton pests home
(overview of pink bollworm eradication program)
US EPA [United States Environmental Protection Agency]. 2013. White paper on corn rootworm
resistance monitoring for Bt Plant-Incorporated Protectants. Last accessed 11-17-2017.
http://www.regulations.gov/document?D=EPA-HQ-OPP-2013-0490-0030
US EPA [United States Environmental Protection Agency]. 2012. Biopesticide Registration Action
Document: PLANT-INCORPORATED PROTECTANTS: Bacillus thuringiensis CrylAb protein and
the Genetic Material Necessary for its Production in Event T304-40 Cotton [PC Code 006525, OECD
Unique Identifier: BCS-GH004-7]; Bacillus thuringiensis Cry2Ae protein and the Genetic Material
Necessary for its Production in Event GHB119 Cotton [PC Code 006600, OECD Unique Identifier: BCS-
GH005-8]; and TwinLink Cotton (T304-40 and GHB119 Combination PIP product) [OECD Unique
74
-------
Identifier: BCS-GH004-7 x BCS-GH005-8], Last accessed 11-17-2017.
https://www.regulations.gov/document?D=EPA-HO-OPP-2Ql 1-0988-0008.
US EPA [United States Environmental Protection Agency]. 2010. Terms and Conditions for Bt Corn
Registrations. Last accessed 11-17-2017.
https://www3.epa.gov/pesticides/chem search/reg actions/pip/bt-corn-terms-conditions.pdf
US EPA [United States Environmental Protection Agency]. 2010a. Biopesticide Registration Action
Document: Cryl Ab and CrylF Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants. Last
accessed 11-17-2017. https://www3.epa.gov/pesticides/chem search/reg actions/pip/crylf-crylab-
brad.pdf
US EPA [United States Environmental Protection Agency]. 2010b. Biopesticide Registration Action
Document: Cryl A. 105 and Cry2Ab2 Insecticidal Proteins and the Genetic Material Necessary for Their
Production in Corn [PC Codes 006515 (Cry2Ab2), 006514 (Cryl A. 105)]. Last accessed on 11-17-17.
https://www3.epa.gov/pesticides/chem search/reg actions/pip/mon-89034-brad.pdf
US EPA [United States Environmental Protection Agency]. 2009. Biopesticides Registration Action
Document: Bacillus thuringiensis Vip3 Aa20 Insecticidal Protein and the Genetic Material Necessary for
Its Production (via Elements of Vector pNOV1300) in Event MTR162 Maize (OECD Unique Identifier:
SYN-IR162-4). Last accessed on 11-17-17.
https://www3.epa.gov/pesticides/chem search/reg actions/registration/decision PC-006599 3-Apr-
09.pdf
US EPA [United States Environmental Protection Agency]. 2008. Biopesticide Registration Action
Document: Bacillus thuringiensis modified CrylAb (SYN-IR67B-1) and Vip3Aal9 (SYN-IR102-7)
insecticidal proteins and the genetic material necessary for their production in COT 102 X COT67B
cotton. Last accessed on 11-17-17.
https://www3.epa.gov/pesticides/chem search/reg actions/registration/decision PC-006529 12-Aug-
08.pdf
US EPA [United States Environmental Protection Agency]. 2005. Biopesticide Registration Action
Document: CrylAb and CrylF Bacillus thuringiensis (Bt) corn plant-incorporated protectants (updated in
2010). Last accessed November 11, 2017.
https://www3.epa.gov/pesticides/chem search/reg actions/registration/decision PC-006491 1-Sep-
10.pdf
US EPA [United States Environmental Protection Agency]. 2003a. Biopesticide Registration Action
Document: Bacillus thuringiensis Cry2Ab2 protein and its genetic material necessary for its production
in cotton. Last accessed on November 17, 2017.
https://www3.epa.gov/pesticides/chem search/reg actions/registration/decision PC-006487 4-Mar-
03.pdf
75
-------
US EPA [United States Environmental Protection Agency]. 2003b. Review of Pioneer Hi-Bred and
Mycogen/Dow AgroSciences' request to add Western bean cutworm to the label of Herculex I corn.
Memo from A. Reynolds to M. Mendelsohn, dated May 28, 2003.
US EPA [United States Environmental Protection Agency]. 2001. Biopesticides Registration Action
Document: Bacillus thuringiensis (Bt) Plant-Incorporated Protectants. Last accessed on November 17,
2017. https://www3.epa.gov/pesticides/chem search/reg actions/pip/bt brad.htm
US EPA FIFRA SAP [United States Environmental Protection Agency Scientific Advisory Panel].
2009. Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held February 23-24, 2009 on
the Evaluation of the resistance risks from using a seed mix refuge with Pioneer's Optimum AcreMax 1
corn rootworm protected corn. SAP Minutes May 6, 2009. Last accessed on November 17, 2017.
https://www.regulations.gov/document?D=EPA-HQ-OPP-2008-0836-0085
US EPA FIFRA SAP [United States Environmental Protection Agency Scientific Advisory Panel].
2006a. Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held June 13 - 15, 2006 on the
Analysis of a Natural Refuge of Non-Cotton Hosts for Monsanto's Bollgard II Cotton. Last accessed on
November 17, 2017. https://www.regulations.gov/document?D=EPA-HO-OPP-2006-Q217-0042
US EPA FIFRA SAP [United States Environmental Protection Agency Scientific Advisory Panel].
2006b. Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held October 24, 2006 on the
Evaluation of the Resistance Risks from Using 100% Bollgard and Bollgard II Cotton as Part of a Pink
Bollworm Eradication Program in the State of Arizona. Last accessed on November 17, 2017.
https://archive.epa.gov/scipolv/sap/meetings/web/pdf/october2006finalmeetingminutes.pdf
US EPA FIFRA SAP [United States Environmental Protection Agency Federal Insecticide,
Fungicide and Rodenticide Act Scientific Advisory Panel]. 1998. Transmittal of Final Report of the
FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and resistance
management. Meeting held on February 9 and 10, 1998. Memo from Dr. E. Milewski (DFO) to L.
Goldman (AA Office of Prevention, Pesticides and Pollution Prevention). Last accessed on November 17,
2017. https://archive.epa.gov/scipolv/sap/meetings/web/pdf/finalfeb.pdf
Velez, A., T. Spencer, A. Alves, A. Crespo, and B. Siegfried. 2014. Fitness costs of CrylF resistance in
fall armyworm, Spodopterafrugiperda. J. Appl. Entomol. Vol. 138: 315-325.
Welch, K., G. Unnithan, B. Degain, J. Wei, J. Zhang, X. Li, B. E. Tabashnik, and Y. Carriere. 2015.
Cross-resistance to toxins used in pyramided Bt crops and resistance to Bt sprays in Helicoverpa zea. J.
Invert. Pathol. Vol. 132: 149-156.
Westbrook, J. K. 2008. Noctuid migration in Texas within the nocturnal aeroecological boundary layer.
Integr. Comp. Bio. Vol. 48(1): 99-106.
76
-------
Yang, F., D. Kerns, J. Gore, A. Catchot, G. Lorenz, and S. Stewart. 2017a. Susceptibility of field
populations of the cotton bollworm in the southern US to four individual Bt proteins. BWCC Proceedings.
Yang, F., D. L. Kerns, S. Brown, G. P. Head, and F. Huang. 2017b. Pollen contamination in seed
mixture increases the dominance of resistance to Bt maize in Spodoptera frugiperda (Lepidoptera:
Noctuidae). PestManagm. Sci. Vol. 73(11): 2379-2385.
Zhang, H. W. Yin, J. Shao, L. Jin, Y. Yang, S. Wu, B. E. Tabashnik, and Y. Wu. 2011. Early warning
of cotton bollworm resistance associated with intensive planting of Bt cotton in China. PLoS ONE, Vol. 6:
e22874
Zhao, J. Z., Y. X. Li, H. L. Collins, and A. M. Shelton. 2002. Examination of the F2 screen for rare
resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae). J.
Econ. Entomol. Vol. 95:14-21.
Zhao, J. Z., J. Cao, Y.X. Li, H. L. Collins, R. T. Roush, E. D. Earle, and A. M. Shelton. 2003.
Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat.
Biotech. Vol. 21: 1493-1497.
Zhao, J. Z., J. Cao, Y.X. Li, H. L. Collins, R. T. Roush, E. D. Earle, and A. M. Shelton. 2005.
Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect
adaptation to pyramided plants. Proc. Natl. Acad. Sci., Vol. 102: 8426-8430.
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VI. APPENDIX A. Bt CORN RESISTANCE MONITORING STUDIES SUBMITTED
TO EPA FOR H. ZEA
Year
Toxin(s)
MRU)# or Citation
1997
CrylAb, Cry 1 Ac
MRID#s 444754-01, 444756-01
1998
CrylAb
MRID# 447753-01
1999
CrylAb
MRID# 450568-01
2000
CrylAb
No report submitted
2001
CrylAb
Custom Bio-products, 2002a. Monitoring Bt susceptibility of
H. zea to CrylAb: 2002 Final Report. (Report for 2001
season.) Unpublished studies submitted to EPA - No MRID #.
2001
CrylF
Custom Bio-products, 2002b. Monitoring Bt susceptibility of
H. zea to CrylFa: 2002 Final Report. (Report for 2001 season.)
Unpublished studies submitted to EPA - No MRID #.
2002
CrylAb
Custom Bio-products, 2002c. Monitoring Bt susceptibility of
H. zea to CrylAb: 2002 Collections and Assays. (Report for
2002 season.) Unpublished studies submitted to EPA - No
MRID#.
2002
CrylF
Custom Bio-products, 2002d. Monitoring Bt susceptibility of
H. zea to CrylFa: 2002 Collections and Assays. (Report for
2002 season.) Unpublished studies submitted to EPA - No
MRID#.
2003
CrylAb
Custom Bio-products, 2003a. Monitoring Bt susceptibility of
H. zea to CrylAb: 2003 collections and assays. Unpublished
study submitted to EPA - No MRID #.
2003
CrylF
Custom Bio-products, 2003b. Monitoring Bt susceptibility of
H. zea to CrylFa: 2003 collections and assays. Unpublished
studies submitted to EPA - No MRID #.
2004
CrylAb
Custom Bio-products, 2004a. Monitoring Bt susceptibility of
H. zea to CrylAb: 2004 collections and assays. Unpublished
study submitted to EPA - No MRID #.
2004
CrylF
Custom Bio-products, 2004b. Monitoring Bt susceptibility of
H. zea to CrylFa: 2004 collections and assays. Unpublished
studies submitted to EPA - No MRID #.
2005
CrylAb, CrylF
MRID# 468749-01
2006
CrylAb, CrylF
MRID# 471184-01
2007
CrylAb, CrylF
MRID# 474139-01
2008
CrylAb, CrylF
MRID# 478418-01
2009
CrylAb, CrylF
MRID# 482082-01
2009
CrylA.105
MRID# 482074-01
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Year
2010
2010
2011
2011
2011
2012
2012
2012
2013
2013
2014
2014
2015
Toxin(s)
CrylAb, CrylF
CrylA.105
CrylAb, CrylF
CrylA.105
Vip3A
CrylAb, CrylF
CrylA.105
Vip3A
CrylAb, CrylF,
Vip3A
CrylA.105
CrylAb, CrylF,
Vip3A
CrylA.105
CrylAb, CrylF,
Vip3A
MRID# 485938-01
MRID# 485888-01
MRID# 489261-01
MRID# 489232-01
MRID# 485912-01
MRID# 492070-01
MRID# 492046-01
MRID# 492064-01
MRID# 494600-01
MRID# 495010-01
MRID# 497848-01
MRID# 497463-01
MRID# 503241-01
MRID# or Citation
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VII. APPENDIX B. Bt COTTON RESISTANCE MONITORING STUDIES SUBMITTED TO
EPA FOR H. ZEA
Year
Toxin(s)
MRU)# or Citation
1996-1998
Cry 1 Ac
MRID# 448633-01
1999-2000
Cry 1 Ac
Public literature reviewed in: EPA, 2001. Bt Plant-Incorporated
Protectants Biopesticides Registration Action Document.
October 15, 2001. Available at:
httD://www3.eDa.sov/Desticides/chem search/reg actions/DiD/1-
overview.odf
2001
Cry 1 Ac
MRID# 456863-01
2002
Cry 1 Ac
Hardee, D.R., et al., 2003. Monitoring for Cry 1 Ac susceptibility
among field populations of cotton bollworm and tobacco
budworm during the 2002 growing season. Unpublished study
submitted to EPA. No MRID# assigned.
2002-2003
Cry2Ab2
MRID# 462720-02
2003
Cry 1 Ac,
Cry2Ab2
MRID# 462720-03
2004
Cry 1 Ac,
Cry2Ab2
MRID# 465476-01
2005
Cry 1 Ac,
Cry2Ab2
MRID# 468266-01
2005
Cry2Ab2
MRID# 468266-02
2005
CrylF
MRID# 469387-01
2006
Cry2Ab2
MRID# 471664-01
2006
Cry 1 Ac,
Cry2Ab2
MRID# 471664-03
2006
CrylF
MRID# 472235-01
2007
Cry 1 Ac,
Cry2Ab2
MRID# 474608-01
2007
Cry 1 Ac,
CrylF
MRID# 474622-01
2008
Cry 1 Ac,
Cry2Ab2
MRID# 477913-02
2008
Cry2Ab2
MRID# 477913-03
2008
Cry 1 Ac,
Cry2Ab2,
CrylF
MRID# 477913-04
2008
Cry 1 Ac,
CrylF
MRID# 477911-01
80
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Y
2009
2009
2009
2010
2010
2010
2010
2011
2011
2011
2011
2012
2012
2012
2012
2013
2013
2013
2013
2014
2014
2014
2014
2015
2015
Toxin(s)
MRU)# or Citation
Cry 1 Ac
MRID# 481673-03
Cry2Ab2
MRID# 481673-04
Cry 1 Ac,
CrylF
MRID# 481714-01
Cry 1 Ac
MRID# 485241-03
Cry2Ab2
MRID# 485241-04
Cry 1 Ac,
Cry2Ab2
MRID# 485241-05
Cry 1 Ac,
CrylF
MRID# 486241-01
Cry 1 Ac
MRID# 488789-03
Cry2Ab2
MRID# 488789-04
Cry 1 Ac,
CrylF
MRID# 489571-01
Vip3A
MRID# 485912-01
Cry 1 Ac
MRID# 491632-03
Cry2Ab2
MRID# 491632-04
Cry 1 Ac,
CrylF
MRID# 492250-01
Vip3A
MRID# 492064-01
Cry 1 Ac
MRID# 494195-03
Cry2Ab2
MRID# 494195-04
Cry 1 Ac,
CrylF
MRID# 494778-01
CrylAb,
CrylF, Vip3A
MRID# 494600-01
Cry 1 Ac
MRID#s 496603-03, 497102-01
Cry2Ab2
MRID#s 496603-04, 497102-01
Cry 1 Ac,
CrylF
MRID# 497242-01
Vip3A
MRID# 497848-01
Cry 1 Ac,
Cry2Ab2
MRID# 499587-01
Cry 1 Ac,
CrylF
MRID# 500345-01
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