2

EXECUTIVE SUMMARY AND CONCLUSIONS

The BPPD IRM team has analyzed annual corn rootworm (CRW) resistance monitoring data submitted 

by Monsanto for the Cry3Bb1 toxin. These data include populations sampled from the Corn Belt during 

the 2011 growing season and investigations of unexpected damage in Cry3Bb1 fields in part of the Corn 

Belt. 

BPPD concluded that the 2011 field populations (collected at random within the Corn Belt) had mean 

EC50 values similar to those exhibited by the lab reference strain. No comparison could be made to the 

historical EC50 range (2007-20010) because Monsanto switched its Cry3Bb1 protein source. BPPD 

concluded that four samples collected from Cry3Bb1 corn instead of non-Bt host plants had significantly 

lower mortality rates and higher average body mass than the reference strain at the highest (diet bioassay) 

concentration tested. Although Monsanto implemented Best Management Practices during 2012 for these 

locations (Floyd Co., IA; Hamilton Co., IA; Buchanon Co., IA; and Stanton Co., NE), BPPD recommends 

continued monitoring of these sites in subsequent growing seasons.

In 2011, Monsanto investigated CRW populations from fields with unexpected damage using a new onplant assay methodology (similar to Gassmann et al. 2011). After reviewing those data, BPPD identified 

one population (Trent, SD) that was likely Cry3Bb1-resistant, given that there was no difference in 

percent mortality, body weight, and head capsule width between CRW exposed to non-Bt and Cry3Bb1 

plants. The remaining three populations (Shell Rock, Onslow, and Mankato) had also no significant 

difference in survival on Bt and isoline corn, while average head width and body weight differed 

significantly. These results together are inconclusive, and it cannot be concluded that the other three 

populations are resistant. BPPD recommends that Monsanto monitor these four locations for further field 

failures in the coming future.

Monsanto has made significant progress towards developing an on-plant assay as a diagnostic tool that 

could be used for future corn rootworm resistance monitoring. BPPD agrees with Monsanto that a 

comparison between the distributions of instar development should provide greater power than a 

comparison between survival on Bt and non-Bt seedling mats. BPPD notes, however, that the 

distributions of the lab and field populations on Bt seedling mats do not need to be completely nonoverlapping before a field population is called resistant. Rather, a statistically significant difference in the 

two distributions should suffice to make that determination. Furthermore, BPPD proposes that Monsanto 

use a second measure (i.e., body mass) to confirm resistance. Two independent measures taken together to 

identify resistant corn rootworm populations should reduce false positives (i.e., concluding a population is 

resistant when it is not).

In July, 2013, Monsanto’s Cry3Bb1 registrations were amended to include a new resistance detection 

strategy. This revised approach incorporates field damage triggers for unexpected crop damage as well as 

the use of on-plant bioassays to determine resistance. These monitoring procedures were not in place 

when Monsanto conducted testing of the 2011 CRW populations (tests were performed in 2012); 

however, Monsanto did conduct on-plant assays of six populations collected from performance inquiry 

fields during 2011 (as described above). Though four of the populations appeared to partially meet the 

criteria for resistance, there was insufficient information to make a conclusive determination.

Preliminary (not yet published) reports from three research teams suggest potential cross resistance 

between Cry3Bb1 and mCry3A. A number of cases of field-evolved Cry3Bb1 resistance have been 

recently documented (e.g., Gassmann et al. 2011) and in some cases, Cry3Bb1-tolerant populations have 
3

also shown decreased susceptibility to mCry3A (Gassmann et al. 2012, Meinke et al. 2012, Zukoff and 

Hibbard 2012). The BPPD IRM Team recognizes that additional data will be needed to fully assess 

potential cross resistance and implications for Insect Resistance Management.

Monsanto provided an update on the resistance monitoring program for northern corn rootworm (NCRW) 

which demonstrated that there are significant logistical challenges including difficulties with sampling, 

rearing, egg production, and testing with bioassays. For this reason, the BPPD IRM team concurs with 

industry that, at the present time, it is not possible to effectively monitor NCRW for changes in toxin 

susceptibility. Despite the technical hurdles presented by this pest, BPPD continues to recommend that Bt 

corn registrants work to develop a functional resistance monitoring program for this insect and that 

ABSTC continue their efforts to collect NCRW from representative regions of the Corn Belt. 

RECOMMENDATIONS

Monsanto noted that some methodological changes for diet bioassays were made in addition to switching 

the protein source and testing of a new highest concentration. It was unclear to BPPD what those 

additional changes were. BPPD recommends that Monsanto specifically communicate all modifications 

of resistance monitoring methodologies.

When body weight results are obtained with Bt diet bioassay for the purpose of determining whether a 

corn rootworm population is resistant, BPPD recommends that the comparisons be made between the 

field population and a resistant reference colony rather than the susceptible control colony. More 

information can be extracted from the first comparison.

BPPD recommends that the registrant use a second measure for sublethal seedling assays to confirm 

resistance (i.e., body mass).

Monsanto investigated the feasibility of a single on-plant assay (using MON 88017 seedlings) for 2011 

problem fields and fields in high-pressure areas. BPPD recommends that Monsanto also consider using 

MON 862 plants for on-plant assays because of the greater toxin expression, which could allow further 

separating resistant from susceptible individuals. 

The registrant chose to measure decreased Cry3Bb1 susceptibility with “percent (larval) recovery” for onplant assays. BPPD recommends that Monsanto use percent mortality for on-plant assays because 

“percent larval recovery” has not been a reliable meassure for identifying putative resistant from 

susceptible individuals. Head capsule width and average dry weight were two additional measures that 

showed greater promise in separating susceptible from resistant populations. Some aspects of the 

methodology were not described in Monsanto’s submission, and it was, therefore, unclear how similar 

Monsanto’s assay was to Dr. Gassmann’s. If applicable, BPPD recommends that Monsanto consider 

using a 17-day assay period since this duration allowed for insects to reach third instar development

Gassmann et al. (2011). 

BPPD concludes that while Monsanto’s BMPs are a reasonable mitigatory approach to dealing with 

unexpected damage in Bt corn fields, most of the BMPs are not a long-term solution for managing 

Cry3Bb1resistance and/or putative resistance in corn rootworm (except crop rotation and switching to 

another Bt trait). Furthermore, if Cry3Bb1 resistance is known to have evolved in a particular population

or region, continued planting of the compromised toxin or pyramids contain Cry3Bb1 deployed with a 5% 
4

refuge is not recommended. To protect the longevity of a single toxin, theoretical modeling supports 

increasing the seed blend to a minimum of 10% (BPPD 2010a).

I. BACKGROUND

Corn rootworm (CRW, Diabrotica spp.) is among the most serious economic insect pests of corn in the 

United States (Levine & Oloumi-Sadeghi, 1991). Western corn rootworm (Diabrotica virgifera virgifera, 

WCRW) is found from Mexico to the U.S. Corn Belt and Canada and is widely distributed; Northern corn 

rootworm (D. barberi, NCRW) is found in the Midwest of the U.S and has a localized distribution. 

Together, they are the most prevalent Diabrotica pests in the U.S. The Mexican corn rootworm (D. 

virgifera zea, MCRW) has a distribution from Central America to the southern U.S. and is a sporadic 

problem in central Texas and southern Oklahoma. 

In February 2003, the Agency approved the first Bacillus thuringiensis (Bt) toxin targeting corn 

rootworm: Monsanto’s Cry3Bb1 protein (event MON 863 (YieldGard Rootworm, EPA Reg. No. 525-

528)) and the genetic material (Vector ZMIR13L) necessary for its production. Cry3Bb1 was the first Bt

PIP product registered to protect crops against corn rootworm (CRW) species. 

In December 2005, MON 88017 was approved for registration. The Cry3Bb1 toxin expressed in MON 

88017 is equivalent (99.8% similar) to that in MON 863. The Cry3Bb1 protein produced in MON 88017 

and MON 863 is a variant of the wild-type Cry3Bb1 protein from Bt subspecies kumamotoensis. The 

primary difference between the hybrids is that MON 88017 also expresses a gene for resistance to 

glyphosate (Roundup) based herbicides. 

One “pyramided” Bt corn product has been registered that includes Cry3Bb1. SmartStax is a stacked PIP 

product consisting of a three-gene pyramid (Cry1A.105xCry2Ab2xCry1F) targeting lepidoptera and a 

two-gene pyramid (Cry3Bb1xCry34Ab1/35Ab1) aimed at controlling corn rootworm. It was 

conditionally registered by the Agency in July 2009 with a 5% structured refuge for lepidoptera and corn 

rootworm. Subsequently, the Agency approved registration of a SmartStax product with a 5% seed blend 

(“refuge-in-the-bag”). At present, there are no other pyramided products that contain the Cry3Bb1 toxin.

At the time of this review, there are three other registered Bt toxins active against CRW (mCry3A, 

Cry34Ab1/35Ab1, and eCry3.1Ab). Similar to Cry3Bb1, none of these toxins express a high dose against 

corn rootworm. Simulation modeling predicts and empirical studies confirm that resistance evolves

fastest when the dominance of the resistance allele is increased by lowering the dose of toxin. The greater 

risk of resistance evolution to Bt crops can be expected when toxins are less than high dose (Roush 1998, 

Tabashnik et al., 2004). The first academic reports of corn rootworm resistance to Cry3Bb1 emerged in 

2009, six years after the commercialization of the toxin.

In the 2012 resistance monitoring review for Cry3Bb1, BPPD concluded that as in previous years, 

Monsanto’s diet bioassays showed that field-collected populations (collected from Illinois, Iowa, 

Minnesota, and Nebraska) had generally higher EC50 values than the susceptible laboratory colony 

although the values (with one exception) fell within the range of previous years’ collections (BPPD 

2012). The BPPD IRM team’s analysis of monitoring data from 2007 to 2010 indicated a general trend of 

reduced susceptibility to Cry3Bb1. There were concerns, however, that the diet bioassay approach was

likely insufficient to reliably detect resistant populations in a timely manner. BPPD also summarized 
5

reports of Cry3Bb1 corn rootworm resistance reported by academia in Iowa in 2011 - 2012 and in two 

counties in western Illinois during the 2011 growing season. The BPPD IRM team concurred with the 

conclusions of the researchers that the tested CRW collections from Iowa and Illinois represented

Cry3Bb1-resistant populations; these populations did, however, not meet the current regulatory definition 

of resistance (see discussion in BPPD 2012). To improve the current Cry3Bb1 monitoring program the 

BPPD IRM team recommended the following enhancements be adopted: 

• Expansion of the annual sampling plan to include the states of Wisconsin, South Dakota, and 

Colorado. Corn rootworm damage to Cry3Bb1 fields has been reported in parts of these three states.

• The use of high risk factors (causal factors of resistance) to determine annual sampling locations. 

These considerations should include areas in close proximity to reported field failure/unexpected 

rootworm damage, fields with a history of corn-on-corn planting (i.e., lack of crop rotation with a 

non-CRW host plant), repeated (e.g., prophylactic) use of Cry3Bb1, and/or high non-compliance 

with refuge requirements.

• Standard use of an on-plant assay (such as those developed by Gassmann et al. 2011 and/or

Nowatzki et al. 2008) in lieu of diet bioassays as the primary resistance detection tool. 

In 2010, the terms and conditions of all Bt corn registrations were amended to require that registrants with 

corn rootworm PIP registrations investigate the feasibility of using a sublethal seedling assay as a 

diagnostic assay. A progress report towards this requirement needed to be submitted within six months 

from the date of the amended registration (BPPD 2010b). Monsanto has subsequently submitted several

reports (Letters dated 8-31-2012 and 9-28-2012, MRIDs 484368-01 and 486367-01), in which the 

company further communicated their progress towards an on-plant diagnostic assay. Additionally 

Monsanto submitted their 2011 rootworm resistance monitoring report for Cry3Bb1. Monsanto’s latest 

submissions are summarized and discussed in this review.

II. BPPD SUMMARY OF MONSANTO’S SUBLETHAL SEEDLING ASSAY PROGRESS

REPORTS AND UPDATE ON THE CONDITIONS OF REGISTRATION FOR MON 

88017

a. Monsanto 3/3/2011 submission (MRID 484368-01)

Monsanto’s report was submitted in response to BPPD’s request that the registrant detail their progress on 

developing a sublethal seeding assay methodology (BPPD 2010a). In this first submission Monsanto 

asserted that MON 88017 seedlings were not practical for use in SSA because western corn rootworm 

larval development (mortality and development) was less affected than what was observed in the field. 

The experimental design employed in method development followed other currently published and 

unpublished procedures (Nowatzki et al. 2008 and Head & Storer 2008). Seeds of MON 88017 and nonBt corn were planted in separate plastic containers and grown under appropriate growth chamber 

conditions. Larvae (or eggs) from susceptible colonies and one resistant western corn rootworm colony

(Hibbard, USDA-ARS lab) used to infest the root mats in the plastic containers. Developing larvae were 

allowed to feed for 5-10 days before they were extracted from the root mats with Berlese funnels. Larvae 

were collected into vessels containing an ethanol solution, counted, and their head width and instar 

determined. Afterwards larvae were freeze dried and their weights were determined. The initial results 

using this methodology were, however, inconclusive. Differences in mortality and development 
6

parameters measured on rootworm larvae from Cry3Bb1 and non-Bt plants were not great enough to 

clearly discriminate susceptible and resistant larvae on Bt treatments. Method development was on-going

at the time of the report. 

In response to BPPD’s sampling concerns (BPPD 2010a), Monsanto reported that ABSTC’s 2010 adult

collections were expanded to include Minnesota and further areas in Iowa. Additionally, the registrant 

also made collection in northeastern Iowa and two locations in South Dakota during the 2010 season.

As indicated in the terms and conditions of Monsanto’s Cry3Bb1 registration, BPPD requested that 

research be conducted to determine if IRM strategies developed for NCRW and WCRW were appropriate 

for the Mexican rootworm (MCR) (BPPD 2010a). Monsanto reported that all available data continue to 

indicate that WCRW and MCR are genetically very similar (e.g., Szalanski et al., 1999), though WCRW is 

more abundant and widespread making WCRW a natural surrogate for MCR. Where possible, Monsanto has 

conducted efficacy testing in areas expected to have MCR to confirm that the efficacy of Monsanto's corn 

rootworm products against MCR is comparable to that against WCRW (e.g., Moore, 2004), and therefore that 

WCRW is a reasonable surrogate for MCR. The efficacy of these products has been uniformly good in these 

areas, indicating that efficacy against MCR is indeed comparable to efficacy against western corn rootworm. 

However, rootworm pressure is consistently light in these areas making field testing and collecting MCR, 

problematic. Furthermore, rearing methods have not been worked out for MCR, as is the case for NCRW and,

therefore, field efficacy work remains the logical focus for MCR assessments.

b. Monsanto 9/28/2011 submission (MRID 486367-01)

Monsanto indicated that MON 88017 seeds did not allow for successful discrimination between Cry3Bb1 

resistant and susceptible insects and determined that seeds from a different event, such as MON 862, had 

much higher toxin expression levels than MON 88017 seeds and, therefore, showed greater promise to 

better discriminate between susceptible and putative resistant insects. Hence, appropriate changes were 

made to the experimental protocol described in MRID 484368-01 to include the different seed source 

from MON 862. Root expression data from MON 862 and MON 88017 are shown at different growth 

stages in Table 1 below. MON 862 seeds were limited in the next trial, and therefore only 15 Bt and nonBt seeds were planted per treatment container. Infestation and duration of allowed feeding was initiated as 

described in the earlier report as well.

The experimental design of the SSA was modeled after Nowatzki et al. (2008) and Head & Storer (2008). 

SSA results with MON 862 seedlings showed great promise (see Table 2). The percent larval recovery 10 

days after infestation was very low in Bt treatment containers for larvae from susceptible colonies but was 

36 to 52-fold higher in non-Bt treatment containers. In comparison, resistant larvae (USDA lab selected 

colony) were recovered in similar numbers from Bt and non-Bt seedling mats as expected. In both cases, 

the percent recovery of resistant larvae was similar to the percent recovery of susceptible larvae from nonBt seedling mats. The response of resistant and susceptible larvae on the Bt seedling mats was very 

different (see Figure 1). Larval recovery was low for the susceptible larvae indicating that larval recovery 

does not appear to be a suitable metric for this assay system if the discrimination between resistant and 

susceptible larvae is to be effective.

The observed age structure percentages of surviving resistant and susceptible larvae on MON 862

seedling mats were non-overlapping (see Table 3). Susceptible larvae (both colonies) did not develop to 

third instar, while 65% of the USDA resistant colony reached third instar by the end of the assay (see 

Figure 2). Monsanto concluded that with MON 862 the difference in development between resistant and 
7

susceptible populations might be large enough to consistently discriminate between susceptible and 

putative resistant larvae. 

Since the MON 862 seed source was limited, another comparable Cry3Bb1 expressing line was used to 

test whether it could successfully replace MON 862. The registrant continued to improve and optimize the 

SSA methodology. Monsanto had further concerns that the greenhouse selected USDA resistant colony 

might not reasonably represent the resistance encountered in the field. Hence, the registrant had

collaborated with Dr. Aaron Gassmann from Iowa State University to obtain additional resistant western 

corn rootworm colonies.

Monsanto also updated BPPD that the rearing and laboratory testing of MCR and NCR remained difficult 

and problematic. Available data continued to indicate that all three corn rootworm species were

genetically and biologically very similar, and control of MCR on Cry3Bb1 expressing corn was similar to 

the control observed for Western corn rootworm. WCRW, however, remained the most abundant and 

widespread species of the corn rootworm complex. 

An expert meeting was organized by member companies of ABSTC at the 2012 Entomological Society 

Meeting in Reno, NV, to discuss rearing and bioassay methods for NCRW and whether they could be 

improved successfully to allow monitoring for resistance in NCR. Monsanto’s submission indicated that a

summary report would be provided to the EPA (summarized in next section).

Table 1 Expression Levels of Cry3Bb1 in MON 862 and MON 88017 Root Tissues

Seed Type Growth Stage Avg. Expression Level

(ppm of Cry3Bb1)

MON 88017

V2 25

V3 30

V4 29

MON 862

V2 55

V3 45

V4 38

Taken from MRID 486367-01

Table 2 Western Corn Rootworm Larval Recovery 10 Days after Infestation onto Seed Mats Generated with

MON 862 and Non-Bt seed

Population % Recovered from MON 862 % Recovered from Non-Bt

Crop Characteristics (CC) susceptible colony 0.33 ±0.33 12 ±9.39

Brookings USDA susceptible colony 0.50 ±0.50 26 ±10.9

Columbia USDA resistant colony 11 ± 2.86 13.66 ±3.63

CC and Brookings are susceptible colonies; USDA resistant colony supplied by Hibbard Lab.

Taken from MRID 486367-01
8

Table 3 Age Structure of Western Corn Rootworm on MON 862 and Non-Bt Seedling Mats

Population % Mean First Instar % Mean Second Instar % Mean Third Instar

CC Susceptible on non-Bt 0 0 100

CC Susceptible on MON 862 0 100 0

Brookings on non-Bt 0 4.2 95.8

Brookings on MON 862 0 100 0

Resistant USDA on non-Bt 0 8.9 91.1

Resistant USDA on MON 862 0 35 65

Modified from MRID 486367-01

Figure 1 Western Corn Rootworm Recovery 10 Days after Infestation with 2 Susceptible Colonies and 1 

Resistant Colony on MON863 Seedling Mats (MRID 486367-01)
9

Figure 2 Age Structure of Western Corn Rootworm larvae Recovered on MON 862 and Non-Bt Seedling 

Mats (MRID 486367-01)

c. Monsanto 8/31/2012 submission:

EPA received updates on the progress of developing a proactive resistance monitoring program for 

northern corn rootworm (Diabrotica barberi). ABSTC held a workshop with expert entomologists in 

November 2012 at the Entomological Society of America meeting to discuss obstacles associated with 

northern corn rootworm field collections, rearing, and diet bioassays. The group concluded that current 

technical and logistic constraints make it impossible to reliably monitor for northern corn rootworm. 

There are no currently available established techniques for collecting and rearing of this rootworm 

species. An action plan was proposed to address the present constraints and should be available by 2013. 

d. Monsanto 9/28/2012 submission:

Monsanto summarized the conclusions of the prior two SSA updates and their efforts to establish a single 

on-plant assay. In their SSA-update, the registrant reported that the first seed increase for MON 862 was 

successful but would take an additional 1-2 generations to generate sufficient amounts of seed for further 

experiments. Monsanto expected that by October 2012 sufficient seed should be available to continue 

their work with SSAs and Cry3Bb1 expressing seedlings. 

Monsanto also investigated the feasibility of single on-plant assays as a diagnostic tool for Cry3Bb1 

resistance detection. The methodology was tested on six populations collected from either high-pressure 

fields or from performance inquiry sites in 2011; the Bt seeds utilized in the on-plant assay were from 

MON 88017 stock. On-plant results were compared to diet bioassay results. Both assays were designed to 

evaluate western corn rootworm survival and growth inhibition. Survival in the diet bioassay was assessed 
10

as percent mortality and in the on-plant assay as percent recovery. Growth inhibition was assessed as 

average weight of survivors in diet bioassays and as head capsule width and dry weight for larvae that had 

recovered after the on-plant assay exposure time. 

Monsanto’s Results and Conclusions:

Diet bioassays: the registrant stated that average percent mortality and/or average weight of survivors 

indicated that all populations were less susceptible than expected based on the historical baseline data 

(except Niebur, CO) (see Table 4). 

On-plant assay: Monsanto reported that on-plant results indicated that Niebur (CO) and Humphrey (NE) 

populations remained susceptible to Cry3Bb1. The remaining four populations, however, had lower 

susceptibilities based on their limited response to MON 88017 expressing plants (see Table 4). 

Monsanto concluded diet bioassay and on plant-assay results were generally comparable with one 

exception (Humphrey population, NE). Monsanto continued to develop, reconcile, and optimize their 

assay systems for use in corn rootworm resistance monitoring. Additional field populations would be 

tested side-by-side using both assays. The results would be compared with results of mechanistic assays 

(assays of resistance mechanisms) to determine which assay best represents the susceptible status of a 

rootworm population.

Table 4 Diet Bioassay and Sublethal Seedling Assay Results1 for CRW from High-Pressure and/or 

Performance Inquiry Fields (2011 Season)

Location

Diet Bioassay Results Plant Bioassay

(Control compared to MON 88017)

EC502

(95% CL)

Diagnostic 

Concentration

(170.8 μg/cm2) % Larval Recovery Ave Head Caps. Width Ave Dry Weight

Avg.%

mort.

Avg.Wt

per

Surviv. Control MON 

88017

PValue control MON 

88017

PValue control MON 

88017

PValue

Niebur Field 

(CO)

24.99 

(14.4-35.2) 46 0.16 23 6 .012 0.48 0.38 .0002 0.77 0.24 0.0002

Onslow (IA) * 43 0.32 9 12 .710 0.48 0.42 .004 0.73 0.39 0.004

Shell Rock 

(IA) * 29 0.36 17 11 .352 0.51 0.47 .009 0.98 0.59 0.002

Mankato 

(MN) * 51 0.37 33 23 .135 0.51 0.39 .001 0.81 0.60 0.002

Humphrey 

(NE) * 22 0.30 29 8 .002 0.52 0.35 <0.05 0.86 0.19 <0.05

Trent (SD) * 19 0.27 26 24 .836 0.47 0.43 0.274 0.67 0.46 0.314

Historical

Range3

7.3-119.6

(2.1-153.7) 24-89 0.01-0.13 - - - - - - - - -

Modified from Monsanto submission dated 9/28/2012 (p. 3)

*Data failed to converge: EC50 could not be determined 1 Diet bioassay and plant-based bioassay results from Monsanto report: 2011 western corn rootworm resistance monitoring for Cry3Bb1 2 EC= Effective Concentration; CL Confidence Limit 3 Historical = Ranges from routine monitoring results from 2007-2010
11

III. MONSANTO’S 2011 WESTERN CORN ROOTWORM RESISTANCE MONITORING 

DATA FOR CRY3Bb1 (DIET BIOASSAYS) (MRID 489283-01)

Monsanto submitted the 2011 Cry3Bb1 resistance monitoring report containing diet bioassay results for 

the routine annual monitoring program as well as follow up investigations for fields with unexpected 

Cry3Bb1 damage in 2010 or 2011. In addition to conducting diet bioassays, the registrant also conducted 

on-plant assays on a subset of the populations to provide a more in-depth assessment of populations in 

question. Monsanto also submitted a summary of the 2011 Problem Investigations (PI) and their Best 

Management Practices (BMPs) implemented in those fields during the 2012 corn growing season.

a. Summary of the 2011 random population sampling results

DM Crop Research Group and Monsanto Company made corn rootworm collections throughout the US 

Corn Belt in 2011 (on behalf of ABSTC). Thirteen geographically distinct populations of WCRW were 

collected from Nebraska, Iowa, and Illinois where Cry3Bb1 adoption and western corn rootworm 

infestations have been historically high. All insects were sent to Custom BioProducts and maintained in 

the lab for egg collections; eggs were maintained in a diapausing condition until initiation of bioassays in 

March 2012. Bioassay tests were conducted on the progeny of the 12 populations with typically four to 10 

replicates to test the susceptibility of corn rootworm to the Cry3Bbl protein (Table 5). A non-diapausing 

laboratory population maintained by Monsanto produced enough neonates for four replications. In an 

effort to improve bioassays a new lot of protein was developed with a concentration of 9.9 mg/ml. This 

new lot of protein allowed for the diagnostic (highest) concentration to increase from 170 μg/cm2 to 341.6 

μg/cm2

. Both concentrations were used to determine percent mortality and average weight per population

(see Table 6). Further methodology descriptions were provided in Appendix 1 of MRID 489283-01 and 

will not be discussed here.

Monsanto concluded the 2011 diet bioassay results suggested that four of the 13 populations obtained 

from high Bt adoption areas had reduced Cry3Bbl susceptibility relative to the historical range and other 

2011 populations tested. Those populations with reduced Cry3Bb1 susceptibility were from Floyd (IA), 

Hamilton (IA), Buchanon (IA), and Stanton (NE) counties (see Table 6). These routine monitoring 

collections were intended to come from non-Bt expressing corn or other non-corn host plants in areas of 

high CRW-trait adoption in Illinois, Iowa, and Nebraska. Since the corn plants from which the beetles 

were collected in 2011 were not specifically identified (gene-checked), Monsanto further investigated. 

They determined that the populations with reduced susceptibility for Cry3Bb1 were obtained from 

Cry3Bb1 expressing corn fields. It was, therefore, concluded that the reduced susceptibility to Cry3Bbl

observed in these populations was not unexpected and not representative of populations collected from 

non-Bt fields. Going forward these four fields will be treated as Performance Inquiry (PI) fields. 

Monsanto collected western corn rootworm from these locations in 2012. For 2013, one of the BMPs 

identified by Monsanto for PI fields will be implemented for each of these fields. In addition, since the 

routine monitoring collections for 2012 were already complete Monsanto was also gathering similar 

product information for fields where beetles were collected from in 2012. The process for making these 

collections will be amended through ABSTC to include identification of the corn product in the relevant 

field for collections in 2013.

Monsanto reported that there were six populations (McLean, Champaign and Piatt in IL, Scott and Cerro 

Gordo in IA, and Sherman, NE) that had EC50 results that fell within historical ranges and were 

comparable to the laboratory control colony. Two populations (Story, IA and York, NE) were below the 

historical range, while one population had an elevated EC50 (Stanton, NE). EC50s could not be estimated 
12

for four populations (Floyd, Hamilton and Buchanon, IA, and Clay, NE) because only one replicate was 

available for these sites. Considering survival and larval mass at the previously used diagnostic 

concentration (170.8 ug/cm2

), there were seven populations (Champaign and Piatt (IL), Scott and Story 

(IA), and Clay, Sherman and York (NE)) for which the mortality and average larval were within the

historical range (Table 5). There were four populations (McLean, IL and Cerro Gordo, Floyd and 

Hamilton (IA)) for which the mortality was within the historical range; the weight was, however, higher 

than the historical range at 170.8 ug/cm2

. Two populations (Stanton, NE and Buchanon, LA) had average

mortality and larval weights outside the historical range at this concentration. 

Table 5. Population Mean EC50s, Percentage Mortality, and Mass of Surviving Larvae at 170 

μg/cm2 from Routine Monitoring in 2011

Population

EC50

1 Response at 170 μg/cm2 Field History

(95% CI) 

μg

Range 

status

%

mortality

Range 

status

Ave 

weight/ 

survivor

Range 

status 2011 2012

Mc Lean Co, IL 19.92

(5.1-35.3) Within 64 Within 0.17 Above VT32 Rotate

Scott Co, IA 14.45

(5.6-21.7) Within 69 Within 0.08 Within Non-Bt Non-Bt

Champaign Co, 

IL

9.72

(2.7-16.8) Within 54 Within 0.07 Within Competitor product SSX RIB2

Piatt Co, IL 15.71

(4.0-26.8) Within 61 Within 0.07 Within Competitor product VT3+SAI2

Floyd Co, IA * N/A 32 Within 0.27 Above VT3 SSX RIB

Hamilton Co, IA * N/A 29 Within 0.32 Above DeKalb2 VT3

Story Co, IA 1.08

(0.1-3.5) Below 49 Within 0.12 Within DeKalb Competitor product

Clay Co, NE * N/A 67 Within 0.07 Within Corn2 Corn2

Sherman Co, NE 25.97

(17.9-33.5) Within 60 Within 0.13 Within VT3 VT3

York Co, NE 5.66

(1.4-11.0) Below 87 Within 0.03 Within Unknown Competitor product

Stanton Co, NE 151.99

(4.0-26.8) Above 22 Below 0.24 Above VT3 VT3

Cerro Gordo Co, 

IA

12.93

(9.5-16.4) Within 60 Within 0.19 Above Corn2 Rotate

Buchanon Co, IA * N/A 20 Below 0.34 Above VT3P2 VT3

Monsanto Lab 

colony

10.23

(3.1-16.7) N/A 89 N/A 0.11 N/A N/A N/A

Historical Range3 

2007-2010 at 170 

μg/cm2

7.30-119.60

(2.1-153.7) N/A 24-89 N/A 0.01-0.13 N/A N/A N/A

Modified from MRID 489283-01 (Table 1, p. 9) *Data failed to converge: EC50 could not be determined

Green shaded populations were collected from within Cry3Bb1 expressing maize fields.

Pink shaded fields indicate populations that have EC50s, average weights above and/or % mortality below the historical range. 1 Effective concentration of 341.6 μg/ml; 2 VT3=VT Triple Pro (MON 88017 x MON810); SSX RIB=SmartStax Refuge in the Bag 

(MON89034 x TC1507 x MON 88017 x DAS-59122-7); SAI=Soil applied insecticide; DeKalb product, trait unknown; Corn=Research field 

used to trap CRW beetles, no trait planted, or trait (if any) unknown; VT3P=VT Triple Pro (MON89034 x MON 88017); 3 Ranges from 

routine monitoring results from 2007-2010
13

Table 6. Comparison of 2011 Percent Mortality at Two Different Highest Concentrations Tested

Location New Diagnostic Concentration

(341.6 μg/cm2)

Previous Diagnostic Concentration 

(170.8 μg/cm2)

% mortality Average weight % mortality Average weight

Mc Lean Co, IL 65 0.07 64 0.17

Scott Co, IA 79 0.01 69 0.08

Champaign Co, IL 68 0.02 54 0.07

Piatt Co, IL 74 0.02 61 0.07

Floyd Co, IA 39 0.14 32 0.27

Hamilton Co, IA 32 0.22 29 0.32

Story Co, IA 67 0.04 49 0.12

Clay Co, NE 100 -- 67 0.07

Sherman Co, NE 58 0.05 60 0.13

York Co, NE 77 0.02 87 0.03

Stanton Co, NE 35 0.15 22 0.24

Cerro Gordo Co, IA 75 0.05 60 0.19

Buchanon Co, IA 22 0.28 20 0.34

Monsanto Lab colony 2011 90 0.122 89 0.112

BPPD modified Table from MRID 489283-01, Appendix 1

Green shaded populations were collected from within Cry3Bb1 expressing maize fields.

Pink shaded fields indicate populations that have EC50s, average weights above and/or % mortality below the historical range. 1 Susceptible lab control colony is a non-diapausing strain maintained by Monsanto 2 Weight of susceptible colony on isoline corn

Shaded populations identified by BPPD are likely Cry3Bb1 resistant

b. Diet bioassays of collections from performance inquiry fields

Monsanto’s objective was to characterize western corn rootworm populations in areas of the Corn Belt 

where elevated levels of corn root feeding had occurred. In an effort to improve diet bioassays, a new lot 

of protein was developed with a concentration of 9.9 mg/ml. This new lot of protein allowed for the 

highest (diagnostic) bioassay concentration to increase from 170 μg/cm2 to 341.6 μg/cm2. All 2011 diet 

bioassay results were obtained with this new protein source. In addition, a new diet plate recipe was 

developed (no additional information was provided regarding this change in MRID 489283-01). 

Three populations were collected from problem fields in 2011 and eleven fields where high corn 

rootworm pressure was previously observed in 2010 but where no insects had been collected at the time. 

Insects were sent to Custom BioProducts and maintained in the lab for egg collections; eggs were 

maintained in a diapausing condition until initiation of bioassays in March 2012. Of the total eggs 

collected from the 14 populations, only 12 populations produced enough eggs to conduct bioassays. A 

non-diapausing laboratory population maintained by Monsanto produced enough neonates for four 

replications. Further methodology descriptions were provided in Appendix 2 of MRID 489283-01 and are 

not discussed here.

Three populations had mean EC50s that fell within the historical range for diet bioassays and were 

comparable to the results obtained for the laboratory control treatments (Niebur, CO; Prophetstown, IL;

and Gayville, SD). One population (Livengood, CO) had a mean EC50 that fell below and another 

population’s EC50 (Schutte, CO) fell above the historical range. For seven populations, the mean EC50

could not be estimated (Maynard, Shell Rock, and Onslow (IA); Chatfield and Mankato (MN); 

Humphrey, (NE); and Trent (SD)) (see Table 7).
14

Table 7. Population Mean EC50s, Percent Mortality, and Mass of Surviving Larvae at 170 μg/cm2 from 2011 

Problem Fields

BPPD modified Monsanto’s Table 1 (p. 9 of MRID 489283-01). 1 Effective concentration of 341.6 μg/ml

2 VT3=VT Triple (MON 88017 x MON810); SSX RIB=SmartStax Refuge in the Bag (MON89034 x TC1507 x MON 88017 x DAS-59122-

7); SAI=Soil applied insecticide; DeKalb product, trait unknown; VT3P=VT Triple Pro (MON89034 x MON 88017); 3 Ranges from routine monitoring results from 2007-2010

Using survival and body mass at the previously highest concentration tested (170.8 μg/cm2), one 

population was within the historical range (Livengood, CO) (see Table 8). For eight populations, the 

average mortality was within the historical range but average weight/survivor was above the historical 

Populations

EC50

1 Response at 170 μg/cm2 Field History

(95% CI) 

μg

Range 

status

Percent 

mortality

Range 

status

Ave weight/ 

survivor

Range 

status 2010 2011 2012

Maynard, IA * N/A 33 Within 0.27 Above High

pressure

Rotate 

& trials 

Rotate & 

trials 

Shell Rock, 

IA * N/A 29 Within 0.36 Above Highpressure

Rotate 

& trials SSX RIB

Humphrey, 

NE * N/A 22 Below 0.30 Above Highpressure

Rotate 

& trials Competitor

Onslow, IA * N/A 43 Within 0.32 Above High

pressure

Rotate 

& trials 

VT32/

VT3P8+SAI4

Livengood,

CO

3.30

(1.1-6.5) Below 46 Within 0.13 Within Unknown

CRW 

claim 

field in 

2011

Rotate, 

SSX RIB3, 

competitor

Niebur, CO 24.99

(14.5-35.2)

Within

46

Within

0.16 Above Unknown

CRW 

claim 

field in 

2011

VT32/ 

VT3P+SAI4

competitor

Schutte, CO 140.67

(95.1-246.2) Above 29 Within 0.30 Above Unknown Rotate & trials 

Rotate, 

SSX RIB

Gayville, SD 28.87

(19.7-37.8) Within 43 Within 0.17 Above Highpressure

Rotate 

& trials Rotate

Trent, SD * N/A 19 Below 0.27 Above High

pressure

Rotate 

& trials SSX RIB

Chatfield, 

MN * N/A 19 Below 0.20 Above Highpressure

Rotate 

& trials SSX RIB

Mankato, MN * N/A 51 Within 0.37 Above High

pressure

Rotate 

& trials Rotate

Prophetstown, 

IL

50.0

(25.1-78.5) Within 58 Within 0.18 Above Highpressure

High 

press.

Rotate & 

trials

Amber, IA No results – not enough insects to run bioassays High

pressure

Rotate 

& trials 

VT3/VT3P

+ SAI

Oelwein, IA No results – not enough insects to run bioassays High

pressure

High 

press.

Rotate & 

trials

Monsanto 

Lab colony

10.23

(3.1-16.7) N/A 89 N/A 0.11 N/A N/A N/A N/A

Historical 

Range3

2007-2010 at 

170 μg/cm2

7.30-119.60

(2.1-153.7) N/A 24-89 N/A 0.01-0.13 N/A N/A N/A N/A
15

control. Three populations (Humphrey (NE), Trent (SD), and Chatfield (MN)) were above the historical 

range for percent mortality and average weight/survivor. Monsanto concluded that collectively these 

results indicated that eight populations (Maynard, Shell Rock, and Onslow (IA), Schutte Fields (CO), 

Chatfield and Mankato (MN), Humphrey (NE) and Trent (SD)) of the 12 populations obtained from fields 

with PIs had potentially reduced Cry3Bb1 susceptibility compared to the historical range and other 2011 

population samples obtained from routine monitoring efforts. 

Table 8. Comparison of 2011 Percent Mortality of PIs at Two Different Effective Concentrations 

Location Effective Concentration 

(341.6 μg/cm2)

Effective Concentration 

(170.8 μg/cm2)

% mortality Average weight % mortality Average weight

Maynard, IA 50 0.24 33 0.27

Shell Rock, IA 42 0.34 29 0.36

Humphrey, NE 25 0.36 22 0.30

Onslow, IA 39 0.37 43 0.32

Livengood, CO 40 0.12 46 0.13

Niebur, CO 58 0.17 46 0.16

Schutte, CO 44 0.27 29 0.30

Gayville, SD 53 0.17 43 0.17

Trent, SD 15 0.29 19 0.27

Chatfield, MN 19 0.28 19 0.20

Mankato, MN 54 0.31 51 0.37

Prophetstown, IL 64 0.23 58 0.18

Monsanto Lab Colony1 2011 90 0.122 89 0.112

Modified from MRID 489283-01 (Appendix1)

Pink shaded populations identified by Monsanto as populations with reduced Cry3Bb1 susceptibility. 1

Susceptible lab control colony is a non-diapausing strain maintained by Monsanto 2 Weight of susceptible colony on isoline corn

c. On-Plant assay results from high pressure areas or problem fields identified in 2010

Six of the populations collected in 2011 came from 2010 high pressure or performance inquiry fields. 

These populations were also tested with on-plant assays at a Monsanto facility (Chesterfield, MO). A 

single seed of MON 88017 or non-Bt expressing corn variety was planted into a three liter pot (between 

3-10 pots (or replicates) per population) and maintained under conditions favorable for growing corn. At 

the V4-V5 stage of the plant, infestations were made with 30 neonates/plant.

Two of the populations remained highly susceptible to Cry3Bb1 (see Table 9) as judged by the relative 

survivorship between MON 88017 and non-Bt expressing plants (Humphrey, NE; Niebur, CO). The 

remaining four populations had reduced susceptibility on MON 88017 plants (Shell Rock and Onslow, 

IA; Trent, SD; and Mankato, MN) but lower larval survival on Bt than on non-isoline plants (except for 

Onslow population). In addition to lower survival, all populations had smaller head capsule width and 

lower larval weights (except for Trent population) than larvae from isoline plants. If the four populations 

with reduced susceptibility exhibited resistance, then it appeared to be incomplete resistance in all cases. 

Monsanto concluded that these results were generally in agreement with the results obtained from diet 

bioassays (except for the Humphrey, NE population).
16

Table 9. On-Plant Assays Conducted in 2011 with Populations from High Pressure or PI Fields (2010 Data)

Population % Larval Survival Average Head Width (mm) Average Dry Weight (mg)

Isoline Cry3Bb1 P-value Isoline Cry3Bb1 P-value Isoline Cry3Bb1 P-value

Shell Rock, IA 17 11 0.352 0.51 0.47 0.009 0.98 0.59 0.002

Humphrey, NE 29 8 0.002 0.52 0.35 <0.05 0.86 0.19 <0.05

Onslow, IA 9 12 0.710 0.48 0.42 0.004 0.73 0.39 0.004

Niebur field, CO 23 6 0.012 0.48 0.38 0.0002 0.77 0.24 0.0002

Trent, SD 26 24 0.836 0.47 0.43 0.274 0.67 0.46 0.314

Mankato, MN 33 23 0.135 0.51 0.39 0.001 0.81 0.60 0.002

Waterman lab colony 14* 0 0.027 0.39 -- N/A 0.42 -- N/A

Table has been modified from Monsanto submission MRID 489283-01(Tables 2-4, p. 41)

Red shading indicates suspected resistant populations; green shading indicates resistant population (BBPD determination).

* BPPD notes low survival of reference strain on isoline corn. No explanation was provided in the submission.

d. Implementation and performance of Monsanto’s CRW Best Management Practices (BMPs) in 

2011

The majority of PIs in 2011 occurred in Colorado, Illinois, Iowa, Kansas, Nebraska, and Minnesota. 

Monsanto reported that as a conservative response to the increased number of PIs reported, a program of 

BMPs was implemented in these PI fields. Some PIs were also reported in Michigan, Montana, 

Oklahoma, and South Dakota. The BMPs were recommended to growers in the following order:

1. Rotation of the affected field to a non-host crop such as soybeans, which breaks the CRW cycle;

2. Use of Genuity SmartStax® corn hybrids on PI field because of dual CRW modes-of-action 

pyramid and high level of CRW control;

3. If rotation or switching to Genuity SmartStax corn are not an option for growers, use of

soil- or foliar-applied insecticides with MON810 x MON 88017 (VT3) and MON89034 x MON 

88017 VT3P) to manage larvae and adult populations of CRW; or

4. Growers also have a choice of planting Monsanto’s MON89034 Lepidopteran protected PIP 

product, a competitor’s CRW protected PIP products or non-Bt corn with soil and foliar applied 

insecticide.

Monsanto followed up three times with every grower who experienced CRW PIs in 2011 to ensure that 

they understood, chose, and implemented the right BMPs. Monsanto surveyed all PI fields in 2012 to 

verify that the BMPs were implemented but also to assess the CRW damage during the following growing 

season. Figure 3 below identifies the percentage of growers who chose BMPs 1-4 in the five states 

identified with the greatest amount of PIs.

Corn rootworm collections from 2010/2011 PI fields were obtained during the 2011 season, and best 

management practices were implemented (as described above) during the 2011 and/or 2012 season (see 

Table 7 for field history). Of the 2010 PI fields that were rotated to soybean in 2011, a small set of fields 

were used for a replicated test of the BMPs, which provided the insect source for 2011 collections. In 

2012, these PI fields were once more subjected to BMPs or test plots. The three PI fields from 2011 had 

beetles collected, were planted to corn again with appropriate BMPs during 2012, and surveyed the 

following year for root damage. All sites had effectively implemented some BMP strategy during 2012 
17

and had therefore reduced CRW densities and controlled any putative Cry3Bb1 resistance at those 

locations. 

Figure 3. Distribution of BMPs by State: CO = Colorado, IA = Iowa, IL= Illinois, KS = Kansas, MN = Minnesota, NE = Nebraska, 

and SD = South Dakota (Figure taken from MRID 489283-01)

IV. BPPD IRM TEAM REVIEW OF MONSANTO’S SUBMISSIONS AND OVERALL 

CONCLUSIONS

a. 2011 Sublethal Seedling Assay

The 2011 Sublethal Seedling Assays update (see Section II) demonstrates that Monsanto has made 

substantial progress towards developing a Cry3Bb1 seedling assay as a functional bioassay for resistance 

detection (MRIDs 484368-01 and 486367-01). Based on the protein expression data of MON 88017 and 

MON 862 seedlings, BPPD concurs that MON 862 is a better choice for sublethal seedling assays and 

should provide greater separation between susceptible and potentially resistant larvae. Based on the data 

submitted, BPPD agrees with the registrant that a comparison between the distributions of instar 

development should also provide greater power than a comparison between survival on Bt and non-Bt 

seedling mats. BPPD questions, however, whether the distributions of the lab and field populations on Bt 

seedling mats need to be completely non-overlapping before a field population is called resistant. In 

particular, BPPD is concerned with possible cases of incomplete resistance (visible on Bt corn only) and 

cost to resistance (visible on no-Bt corn only) that may delay development and cause partial overlap of the 

two distributions (control and field colony). Cost to resistance in western corn rootworm has so far been

identified with respect to fecundity for laboratory selections of field-derived populations (Oswald et al. 
18

2012 and Meihls et al. 2012). If statistical criteria for resistance determinations are too stringent, BPPD is 

concerned that this would limit the effectiveness of the resistance monitoring program to detect resistance.

Resistance evolution in the field is likely to select for different resistance genes than artificial selection 

experiments carried out in the laboratory (SAP 2009). BPPD agrees with Monsanto that it will be 

important to test field resistant populations from Dr. Gassmann’s lab with the registrant’s SSA method to 

see whether the results of the USDA resistant colony can be replicated. Since there are still uncertainties 

regarding mechanisms and type of resistance for corn rootworm on Bt toxins (though it is suspected that 

resistance may be incomplete (Gassmann et al. 2011)), BPPD proposes to test for “a significant difference 

in age distributions” when concluding whether a population is resistant rather than requiring that 

“distributions are non-overlapping”. BPPD also proposes that the registrant use a second measure (i.e., 

body mass) to confirm resistance. In other words, two independent measures taken together would be 

needed to identify resistant corn rootworm populations. Once the SSA methodology has been fully 

worked out by Monsanto, it will be important to develop and implement a statistical approach that 

proactively identifies resistant populations.

b. 2011 On-Plant Greenhouse Assay 

In 2012, Monsanto investigated the feasibility of an on-plant assay using MON 88017 seedlings for 2011 

problem field insect collections and insect populations from high pressure areas, while also using diet 

bioassays as a comparison. BPPD reviewed the preliminary results submitted to the Agency (Letter dated 

9-28-2012) and was unclear as to the distinction between a Cry3Bb1 problem field and a Cry3Bb1 field 

that experienced unusually high insect pressure. Typically one would expect both observations to occur in 

fields with heavy damage. The registrant chose to measure decreased Cry3Bb1 susceptibility with 

“percent recovery” for on-plant assays and “percent mortality” for diet bioassays. BPPD recommends that 

Monsanto use percent mortality for on-plant assays because data have shown that percent recovery was

not reliable at identifying putative resistant individuals. Cry3Bb1, as are all currently registered corn 

rootworm toxins, is non-high dose, which may explain why even susceptible neonates could recover from 

toxic exposure to the toxin. Aspects of the on-plant methodology were unclear, such as the assay duration.

BPPD recommends that Monsanto consider using a 17-day period, as was done by Gassmann’s lab, since 

this time period allows for insects to reach third instar development. Head capsule width was another 

measure used in the on-plant assays. BPPD notes that four out of six field populations were identified to 

have (statistically significant) smaller average head capsule widths compared to the susceptible lab 

colonies. Hence “head capsule width” as well as “dry weight” appear to show greater promise in 

separating susceptible from resistant populations. BPPD recommends that Monsanto also use MON 862

plants for on-plant assays since the greater toxin expression in this Cry3Bb1 seed may provide greater

sensitive to separate resistant from susceptible individuals. Percent mortality, average dry weight, and/or

head capsule measurements are recommended for the on-plant assays.

c. 2011 Diet and On-Plant Assays

In their 2011 annual monitoring report (MRID 489283-01), Monsanto tested a new highest concentration 

(341.6 μg/cm2

) with a new protein source in addition to 170.8 μg/cm2, the maximum concentration used 

in previous monitoring assays. Because of the switch in protein source, a direct comparison should, 

therefore, not be made between the 2011 diet bioassay results and the 2007-2010 results (listed in the 

same table). Changes in toxin sources have been a periodic challenge for diet bioassays and resistance 

monitoring. In such situations, the comparisons could only be made between results of control and field 
19

populations within a specific year. Figure 4 shows that the percent mortality and average weight of 

surviving insects of a population were more strongly correlated when 341.6 μg/cm2was used instead of 

the previously established 170.8 μg/cm2. The variance was greater at the lower concentration; the R2 at 

the (new) highest concentration was 0.9089 compared to the R2 of 0.7685 at 170 μg/cm2. This suggests 

that the higher concentration likely provided a better estimate of susceptibility for two measures 

(mortality and weight of survivors). BPPD included all field populations from the annual routine 

monitoring report (Table 6) in the correlations shown in Figure 4 (excluding Clay Co, NE because all

individuals tested died). BPPD excluded the results of the non-diapausing laboratory colony because it 

was unclear how representative these were of truly susceptible western corn rootworm field populations. 

BPPD notes, however, that even with the new higher concentration, diet bioassay results were still 

variable. For example, two populations had lower percent mortality at 341.6 μg/cm2 than at 170.8 μg/cm2

(York and Sherman, NE). 

Although Monsanto implemented BMPs in 2012 on four fields identified with potentially resistant corn 

rootworm populations (Floyd Co (IA), Hamilton Co (IA), Buchanon Co (IA), and Stanton Co (NE)), 

BPPD recommends that the registrant monitor these sites in 2013. These four corn rootworm populations 

where collected from Bt rather than non-Bt crops (or alternate hosts) during the annual monitoring 

activities and had a lower percent mortality (range 22%-39%) and higher average weight (range 0.14 mg -

0.28 mg) than the laboratory control populations (see Table 5 and Table 6). Also, Stanton (NE) had a 

much higher mean EC50 (151.99 μg/cm2

) compared to the lab colony (10.23 μg/cm2). These results 

suggest that these four populations may be resistant or in the process of developing resistance. 

BPPD notes that the registrant was unable to obtain EC50s with the new protein source for seven out of 14 

populations. This is further evidence that the current diet bioassay methodology does not provide a 

functional resistance monitoring tool for corn rootworm. 

Furthermore, when body weight results are obtained with Bt diet bioassay for the purpose of determining 

whether a corn rootworm population is resistant, BPPD recommends that the comparisons be made 

between the tested field population and a resistant reference colony rather than the susceptible control 

colony. More information can be extracted from the first comparison.
20

Figure 4. Correlation between percent mortality and average weight of survivors in a particular population

at two highest concentrations tested (BPPD generated graph using Monsanto’s data from Table 6 of this 

review)

Monsanto used their improved diet bioassay methodology for 2011 populations that originated from 

unexpected damage sites as well as high-pressure areas identified in 2010. Monsanto further followed up 

on six of the PIs with their single on-plant assay and measured percent mortality, head capsule width, and 

average dry weight for each population raised on Bt and non-Bt expressing corn plants. The statistical 

analysis was done for the field populations on the two treatments. The registrant reported that four 

populations were of concern (greater survival on Bt but smaller head capsule width and average weight) 

and could exhibit incomplete resistance. Those populations were: Shell Rock (Butler Co, IA), Onslow 

(Jones Co, IA), Mankato (Blue Earth Co, MN), and Trent (Moody Co, SD). BPPD identified one 

population where at least two measures from the on-plant assay indicated that there was no significant 

difference between treatments for determining potentially resistant populations and concludes that the 

Trent, Moody Co (SD) population is likely resistant to Cry3Bb1. This population had no significant 

difference in % survival, average head capsule width, and average dry weight on Bt and non-Bt plants. 

The remaining three populations (Shell Rock, Onslow, and Mankato) had no significant difference in 

survival on Bt and isoline corn, while average head width and body weight differed significantly. These 

results together are inconclusive, and it cannot be concluded that the other three populations are resistant. 

BPPD recommends, however, that Monsanto monitoring these four locations for further field failures in 

the coming future.

R2 = 0.9089

R2 = 0.7685

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 20 40 60 80 100

Ave weight of survivors

Percent mortality

341.6 ug/cm2 170.8 ug/cm2
21

Figure 5. 2013 Updated map showing the general areas in the US Corn Belt that have had Cry3Bb1 fields with heavy damage and/or resistant 

western corn rootworm populations (2009-2012)

Kansas

Oklahoma

Colorado

Nebraska

South Dakota

Minnesota

Wisconsin

Iowa

Illinois
22

d. Modifications to the Cry3Bb1 Resistance Assessment Procedures

In July, 2013, Monsanto’s Cry3Bb1 registrations were amended to include a new resistance detection 

strategy. This revised approach incorporated field damage triggers for unexpected crop damage as well as 

the use of on-plant bioassays to determine resistance. Major aspects of this approach include the following 

(paraphrased from the terms and conditions of registrations 524-552, 524-576, and 524-606):

• Monsanto is required to investigate “performance inquiries” (i.e., reports of unexpected corn 

rootworm damage to Cry3Bb1 corn) from growers. Fields with unexpected damage that are 

confirmed to be Cry3Bb1-expressing and experienced root damage with a Node Injury Score (NIS) > 

1.0 on at least 50% of plants sampled are subjected to further investigation.

• CRW are collected from the field in question (at least 250, ideally 500 or more). Collections may be 

extended to any bordering cornfields, if necessary to obtain sufficient CRW adult individuals. If 

sampling is unsuccessful, Monsanto will visit the farm or field the following year to reattempt 

collections (provided the grower is still a Monsanto customer). Collected populations are 

investigated for potential resistance using an on-plant bioassay.

• Monsanto works with the affected grower to implement CRW management practices. These options 

include (but are not limited to): rotation to non-host crop (e.g., soybean), use of pyramided products, 

use of additional CRW control tools (e.g., soil insecticides, seed-applied insecticides, chemigation), 

and use of alternate CRW PIPs.

• A population is considered resistant if the following criteria are met:

1. An initial performance inquiry investigation exceeds the root damage threshold and Cry3Bb1 

protein levels are found to be within the documented range for that hybrid; and

2. Single, on-plant bioassays of insect collections from the affected fields show the following:

i. A statistically significant difference in measures of either lethality/mortality or 

sublethal effects (growth/development) between the field population and the control 

population on Cry3Bb1 corn and

ii. A lack of a statistically significant difference in the same measures of the field 

population raised on Bt corn and non-Bt corn plants.

• In the event the on-plant bioassays are inconclusive (e.g., the results of the statistical analysis are 

unclear because of low sample sizes) or another reasonable explanation for the unexpected damage 

exists (e.g., high pest pressure and/or high plant stress) another year of CRW adult collections and 

additional testing will be conducted to determine resistance.

These monitoring procedures were not in place when Monsanto conducted testing of the 2011 CRW 

populations (tests were performed in 2012). Monsanto, however, did conduct on-plant assays of six 

populations collected from performance inquiry fields during 2011 (see Table 9). Four of these populations 

appeared to meet the second criterion for resistance in that the response to one measure (% larval survival) 

was statistically similar on Bt and non-Bt corn plants. One population (Trent, SD) had no significant 

different responses on Bt and non-Bt for all three measures (% larval survival, average head width, and 

average dry weight). On the other hand, it could not be determined from the report whether the % larval 

survival values of the field populations were statistically different from the control population (the first 

resistance criterion).
23

e. Monsanto’s Best Management Practices 

Monsanto reported that they implemented BMPs in all Cry3Bb1 cornfields in 2011 that had experienced 

unusually high root damage and that were reported to the company as PIs. BPPD agrees with Monsanto’s 

approach to take immediate action before a resistance situation is confirmed. BPPD further agrees that crop 

rotation in problem fields is the best solution to unexpected Bt failure but understands that not every farmer 

can resort to this type of management practice for logistical reasons. Crop rotation with a non-corn rootworm 

host plant should extirpate the offspring of a putative resistant corn rootworm population. BPPD, however,

cautions that these problem fields do not occur in a vacuum but rather are planted in a landscape of 

continuous corn. Any management practice will only alleviate the corn rootworm problem for a short period 

of time if resistance has spread outside the managed field. Because of this, it would be more effective to 

implement BMPs on a wider geographic scale (if possible). Re-colonization of adult corn rootworm from the 

continuous corn landscape will spread putative resistance genes back into the managed fields once corn is 

grown there again. BPPD understands the limitations faced when implementing BMPs and that it is not 

possible for Monsanto to require that non-Monsanto customers (i.e., neighboring growers of a managed 

field) follow their best management practices.

Using SmartStax as the second management practice to Cry3Bb1 unexpected damage, as proposed by 

Monsanto, could be a good option but will increase selection pressure on the second toxin (Cry34/35) with 

the current 5% refuge requirement. In areas with resistance to Cry3Bb1, SmartStax is essentially a single 

trait PIP with a 5% refuge that was designed for an effective two-toxin pyramid. The better scientific strategy 

would be to deploy the pyramid containing the compromised toxin with a 10% refuge.

Monsanto’s third option to apply SAI and adulticides will reduce the susceptible and resistant individuals 

proportionally. This BMP will suppress the population density but is not expected to mitigate resistance 

unless the field is the ‘remedial action area’ and the population is extirpated. If corn rootworm populations 

rebound, managed problem fields will likely fail again if they are planted to Cry3Bb1. 

BPPD concludes that while Monsanto’s BMPs are a reasonable mitigatory approach to dealing with 

unexpected damage in Bt corn fields, most of the BMPs are not a long-term solution for managing Cry3Bb1

resistance and/or putative resistance in corn rootworm (except crop rotation or switching to another Bt trait). 

Furthermore, if Cry3Bb1 resistance is known to have evolved in a particular population, continued planting 

of the compromised toxin in that area is not recommended.

f. Northern Corn Rootworm

Monsanto submitted an update on the resistance monitoring program for northern corn rootworm (NCRW). 

The BPPD IRM team recognizes that NCRW presents significant logistical challenges for resistance 

monitoring including difficulties with sampling, rearing, egg production, and testing with bioassays. For this 

reason, the BPPD IRM team concurs with ABSTC’s conclusions that at present time, it is not possible to 

effectively monitor NCRW for changes in toxin susceptibility. Despite the technical hurdles presented by 

NCRW, BPPD continues to recommend that Bt corn registrants work to develop a functional resistance 

monitoring program for this insect. BPPD notes that one NCRW population was tested with the Sublethal 

Seedling Assay approach by another registrant with a different corn rootworm toxin (BPPD 2013) and that 

the assay may hold promise as a practical resistance detection tool. The greater challenge with NCRW,

however, appears to be in sampling and laboratory rearing. ABSTC attempted to make multiple NCRW 
24

collections during 2009-2011, but of the total 24 identified locations only 10 resulted in successful 

collections. Of those 10 populations, only one produced enough eggs for testing. The BPPD IRM team 

recommends that ABSTC continue the efforts to collect NCRW from the representative regions of the Corn 

Belt. Any successful collections should be tested by a suitable bioassay (on-plant) but can also be used to 

refine and improve NCRW rearing techniques in the lab. BPPD also recommends that any progress towards 

developing a resistance monitoring program be reported to the EPA on an annual basis.

V. ACADEMIC REPORTS OF CROSS-RESISTANCE BETWEEN CRY3Bb1 AND mCRY3A 

At the 2012 Entomological Society of America meeting in Tennessee, three independent research teams 

presented data (currently unpublished) indicating potential cross resistance between mCry3A and Cry3Bb1. 

For example, Dr. Aaron Gassmann (Iowa State University) exposed 17 different Cry3Bb1 field resistant 

colonies to mCry3A and Cry3Bb1 expressing plants and subsequently compared the observed (corrected) 

survival of each population to both toxins. When plotting the survival on mCry3A against the survival on 

Cry3Bb1 plants for all 17 populations, a positive and strong correlation emerged (P-value < 0.0001) and lead 

Gassmann to conclude that Cry3Bb1 field resistant western corn rootworm populations exhibited cross 

resistance to mCry3A (Gassmann et al. 2012). Dr. Lance Meinke (University of Nebraska-Lincoln) reported 

that the Wayne County western corn rootworm population inflicted comparable root damage and beetle 

emergence to both Cry3Bb1 and mCry3A field plots (Meinke et al. 2012). Sarah Zukoff (University of 

Missouri) conducted a laboratory selection experiment in which CRW selected for resistance on Cry3Bb1 

corn were exposed to mCry3A in greenhouse seedling assays. These larvae were found to also be tolerant of 

mCry3A, though not in all populations (Zukoff and Hibbard 2012). While these reports are preliminary and 

have not been subjected to peer review or publication, they are strongly suggestive that cross-resistance 

between Cry3Bb1 and mCry3A may occur in resistant CRW populations. The mechanism of resistance in 

CRW to Bt toxins is not well understood, so it is unclear whether cross-resistance is due to a common 

physiological trait or other means. Additional data will be needed to fully assess potential cross resistance 

and implications for insect resistance management. 

BPPD is concerned with these preliminary reports of cross-resistance between Cry3Bb1 and mCry3A and 

the planting of CRW pyramided corn expressing Cry3-Bt toxins in areas with Cry3Bb1 resistant corn 

rootworm populations. Such pyramids are effectively reduced to a single toxin product deployed with a 

much lower refuge (5%). There are currently four registered corn rootworm toxins, two of which (mCry3A 

and Cry3Bb1) would be ineffective in corn rootworm resistant areas. The selection pressure in these regions 

on the remaining toxin, Cry34/35, will be tremendous if deployed in a pyramid with Cry3Bb1 and a reduced 

refuge of 5%.
25

VI. CITATIONS

BPPD. 2013. Review of corn rootworm resistance monitoring submissions and data (2009-2011) for Bt corn 

products containing Cry34/35Ab1. Memo from A. Reynolds to A. Sibold, dated March 20, 2013

BPPD. 2012. BPPD IRM Team review of Monsanto’s 2010 corn rootworm monitoring data, unexpected 

damage reports for Cry3Bb1 expressing Bt Corn and academic reports of Cry3Bb1 field failures as well as 

corn rootworm resistance. Memo from JC Martinez to A. Sibold, dated October 11, 2012.

BPPD. 2011. Updated BPPD IRM review of reports of unexpected Cry3Bb1 damage, Monsanto’s 2009 corn 

rootworm monitoring report, and revised corn rootworm resistance monitoring plan for MON 88017, MON 

88017xMON 810, MON 863, MON 863xMON 810, MON 89034xTC1507xMON 88017xDAS-59122-7, 

and MON89034xMON 88017. Memo from JC Martinez to M Mendelsohn, dated Nov 22, 2011.

BPPD. 2010a. Science assessment of Pioneer’s modeling addendum in support of their application of 

registration for Optimum AcreMax1 Insect Protection. Memo from JC Martinez to M. Mendelsohn, dated 

February 4, 2010.

BPPD. 2010b. Review of Monsanto’s 2008 corn rootworm monitoring report and revised corn rootworm 

resistance monitoring plan for MON 88017, MON 88017xMON 810, MON 863xMON 810, MON 

89034xTC1507xMON 88017, and MON89034xMON 88017. Memo from JC Martinez to M Mendelsohn, 

dated Jun 30, 2010.

BPPD. 2010c. Terms and conditions for Bt corn registrations. September 30, 2010. 

http://www.epa.gov/oppbppd1/biopesticides/pips/bt-corn-terms-conditions.pdf

BPPD. 2009. Review of Monsanto’s corn rootworm monitoring reports from 2005-2007 for susceptibility to 

Cry3Bb1 and revised corn rootworm resistance monitoring plan for MON 88017, MON 88017xMON810, 

and MON 89034xMON 88017. Memo from JC Martinez to M Mendelsohn, dated August 24, 2009.

EPA Scientific Advisory Panel meeting 2009. Transmittal of 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 AcreMax1 corn rootworm-protected corn. Memorandum from J.E. 

Bailey (DFO) to Dr. D. Edwards (Director of OPP) on May 6, 2009.

Gassmann A. 2012. Western corn rootworm and Bt maize: a case study illustrating the need for IPM and 

IRM. Presentation at the ESA in Knoxville, Tennesse.

Head G and PA Price. 2011 Western corn rootworm resistance monitoring for Cry3Bb1. Unpublished report 

submitted to EPA, MRID 489283-01

Head G and N Storer. 2008. Insect resistance management plan for MON 89034 x TC1507 x MON 88017 x 

DAS-59122-7. Unpublished registrant report submitted to EPA, MRID 47444911

Jenkins D. 2012b. September 2012 update on the conditions of registration for MON 88017, MON 88017 x 

MON 810, MON 89034 x MON 88017, MON 89034 x TC1507 x MON 88017 x DAS-59122-7, MON 

89034 x TC1507 x MON 88017 x DAS-59122-7 RIB Complete, and MON 89034 x MON 88017 RIB 

Complete, Monsanto Company is submitting a progress report on the development of a sub-lethal seedling 
26

assay (SSA). Letter from Monsanto to Kimberly Nesci (Acting Chief of MPB/BPPD), dated September 28, 

2012b.

Jenkins D. 2012. Extension of the condition of registration for developing a northern corn rootworm 

resistance monitoring program: conditions of registration for MON 88017, MON 88017 x MON 810, MON 

89034 x MON 88017, MON 89034 x TC1507 x MON 88017 x DAS-59122-7, MON 89034 x TC1507 x 

MON 88017 x DAS-59122-7 RIB Complete, and MON 89034 x MON 88017 RIB Complete, Monsanto 

Company is submitting a progress report on the development of a sub-lethal seedling assay (SSA). Letter 

from Monsanto to Kimberly Nesci (Acting Chief of MPB/BPPD), dated August 31, 2012a.

Meinke, L, T Hunt, G Kruger, R Wright, D Wangila, and K Miwa, 2012. Evaluation of western corn 

rootworm susceptibility to rootworm Bt traits in Nebraska. Presentation at the Entomological Society of 

America Annual Meeting. November 13, 2012.

Meihls LN, ML Higdon, MR Ellersieck, BE Tabashnik, and B Hibbard. 2013. Greenhouse selected 

resistance to Cry3Bb1-producing corn in three western corn rootworm populations. PLoS One. Vol. 7(12):1-

11

Moore GC. 2004. Efficacy of MON 88017 and YieldGard (MON 863) Corn Rootworm Events, Force 3G and 

Poncho 1250 on Mexican Corn Rootworm, Ellis County, Texas. Texas A&M Cooperative Extension Report.

Nowatzki T, SA Lefko, RR Binning, SD Thompson, TA Spencer, and BD Siegfried. 2008. Validation of a

novel resistance monitoring technique for corn rootworm (Coleoptera: Chrysomelidae) and event DAS59122-7 maize. J. Appl. Entomol. Vol. 132:177–188.

Oswald KJ, BW French, C Nielson, and M Bagley. 2012. Assessment of fitness cost in Cry3Bb-resistant and 

susceptible western corn rootworm (Coleoptera: Chrysomelidae) laboratory colonies. J. Appl. Ent., Vol. 

136(10): 730-740

Roush R. 1998 Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding 

succeed where pesticide mixtures have not? Phil. Trans. R. Soc. London, B, Vol. 353: 1777-1786

Szalanski A.L., RL Roehrdanz, DB Taylor, and L Chandler. 1999. Genetic variation in geographical populations 

of western and Mexican corn rootworm. Insect Molecular Biology, Vol. 8: 519-525

Tabashnik BE, F Gould, and Y Carriere. 2004. Delaying evolution of insect resistance to transgenic crops by 

decreasing dominance and heritability. J. Evol. Biol., Vol. (17): 904-912

Zukoff, S.N. and B.E. Hibbard, 2012. Evaluation of the potential development of cross resistance between 

eight transgenic corn types in western corn rootworm (Diabrotica virgifera virgifera). Presentation at the 

Entomological Society of America Annual Meeting. November 13, 2012.