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Impact of the Bt Corn Proteins Cry34/35Ab1 and Cry3Bb1,

Alone or Pyramided, on Western Corn Rootworm (Coleoptera:

Chrysomelidae) Beetle Emergence in the Field
Author(s): A. J. Hitchon, J. L. Smith, B. W. French, and A. W. Schaafsma
Source: Journal of Economic Entomology, 108(4):1986-1993.
Published By: Entomological Society of America
URL: http://www.bioone.org/doi/full/10.1093/jee/tov125

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Impact of the Bt Corn Proteins Cry34/35Ab1 and Cry3Bb1,

Alone or Pyramided, on Western Corn Rootworm (Coleoptera:
Chrysomelidae) Beetle Emergence in the Field

J. Econ. Entomol. 108(4): 1986–1993 (2015); DOI: 10.1093/jee/tov125

ABSTRACT Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a major pest of corn,
Zea mays L. The effect of the Bt proteins Cry34/35Ab1 and Cry3Bb1, alone or pyramided in corn hybrids
on D. v. virgifera adult emergence was evaluated in field experiments for 3 yr. Experiments were infested
artificially with 2,500 viable D. v. virgifera eggs per row meter of corn. The reduction in beetle emer-
gence compared with non-Bt controls, from Cry34/35Ab1, Cry3Bb1, and the pyramided hybrids ranged
from 64.3 to 97.4%, 91.1 to 95.2%, and 98.1 to 99.6%, respectively. The sex ratio of emerged beetles was
usually female-biased from the Cry3Bb1 and pyramided treatments, but not from Cry34/35Ab1
treatment alone. Emergence from all Bt hybrids was delayed compared with the control, with the delay
longest from the pyramided hybrid. In 2013, three egg infestation levels were tested, with density-
dependent mortality observed at 1,250 viable eggs per row meter. The effect of Bt proteins on the emer-
gence timing and sex ratio of D. v. virgifera may impact the suitability of resistance management plans,
specifically the effectiveness of the refuge strategy. Susceptible males emerging from refuge might not
be synchronized to mate with potentially resistant females emerging later from Bt corn hybrids.

KEY WORDS Diabrotica, maize, resistance management, refuge, mating

Transgenic corn, Zea mays L., that produces insecti- implemented to delay resistance development
cidal proteins derived from the soil bacterium, Bacillus (Macdonald and Yarrow 2003). The current IRM is
thuringiensis Berliner (Bt), is planted widely by Cana- based on the “refuge strategy” that is also widely
dian corn producers for insect control. Three Bt pro- adopted in the United States for both D. v. virgifera
teins Cry3Bb1, Cry34/35Ab1, and mCry3A are and O. nubilalis (Gould 1998, Huang et al. 2011).
available commercially and control larvae of western Despite IRM, as prescribed by the U.S. Environ-
corn rootworm, Diabrotica virgifera virgifera LeConte mental Protection Agency, field-evolved resistance of
(Coleoptera: Chrysomelidae) and northern corn root- D. v. virgifera to the Cry3Bb1 and mCry3a proteins de-
worm, Diabrotica barberi Smith and Lawrence, major veloped after six generations of selection in some areas
pests of corn (Gray et al. 2009). The use of rootworm- of the U.S. Corn Belt (Gassmann et al. 2011, 2014).
active Bt corn increased in Canada from 45.1 to 60.2% Resistance evolved faster in areas with high D. v. virgi-
of total corn acreage from 2009 to 2013, mainly in the fera populations and continuous corn production, espe-
provinces of Ontario and Quebec (Dunlop 2013). cially when the same Bt proteins were planted
Bt corn use has desirable environmental, economic, consecutively in a field for >3 yr (Gassmann et al.
and agronomic benefits, and reduces pesticide use and 2011, 2014; Cullen et al. 2013). Resistance has not
exposure (Rice 2004, Hutchison et al. 2010). Develop- been confirmed in Canada, and smaller farm size and
ment of resistance by target pests is the primary con- greater diversity in crop rotation and landscape may
cern associated with Bt crops. Historically, foliar delay the evolution of resistance to Bt in the eastern
applications of formulated Bt, while not targeting corn Corn Belt compared with the western United States
rootworm, heightened concern that insects developing (DiFonzo et al. 2013). In Ontario, three or more crops
resistance to Bt crops might compromise the use of fo- are often included in the rotation, whereas in much of
liar Bt insecticides, especially in organic agriculture the U.S. Midwest, corn and soybean are the two pri-
(Roush 1994, Belzile 2002). As a condition of Bt crop mary crops (Knight et al. 2005).
registration, the Canadian Food Inspection Agency re- IRM prescriptions are based on models that predict
quired insect resistance management (IRM) plans be resistance evolution using knowledge and assumptions
about the biology and behavior of the pest (Storer
2003, Onstad and Meinke 2010, Devos et al. 2012).
Recently, for D. v. virgifera, key assumptions made in
Department of Plant Agriculture, Ridgetown Campus, University these models, including that of random mating, are
of Guelph, 120 Main St. E., Ridgetown, ON N0P 2C0, Canada. proving to be incorrect (Spencer et al. 2013). Random
North Central Agricultural Research Laboratory, 2923 Medary
Ave., Brookings, SD, 57006. mating of susceptible males with resistant females
Corresponding author, e-mail: aschaafs@uoguelph.ca. relies on a synchrony in emergence between the sexes
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
All rights reserved. For Permissions, please email: journals.permissions@oup.com

from both refuge and Bt hybrids. A delay in emergence on each side of the row, while a “U”-shaped drag chain
of adults from Bt hybrids compared with those emerg- covered eggs with a small layer of soil.
ing from non-Bt refuge has been reported (Storer et al. Experiments were configured in a randomized com-
2006; Hibbard et al. 2010b, 2011; Clark et al. 2012) in- plete block design with four replications and repeated
creasing the probability of assortative mating. These twice in each of 2010 and 2012 at Ridgetown (42.44908
data were generated in the U.S. Corn Belt, and their N, 81.879821 W) and Centralia, ON (43.316060 N,
relevance to Ontario, in the north-eastern range of dis- 81.500023 W). In 2010 and 2012, plots were infested
tribution for D. v. virgifera, is uncertain. with a rate of 2,500 viable eggs per row meter to limit
To address this question, experiments were con- density-dependent mortality (Hibbard et al. 2010a). In
ducted to compare the number of beetles emerging, 2013, one experiment was conducted at Ridgetown,
timing of emergence, and sex ratio, from hybrids ex- ON, as a split-plot design, with three replications. Bt
pressing single Bt proteins (Cry3Bb1 and Cry34/ hybrid was the main plot and three levels of egg infes-
35Ab1), a pyramid of these two proteins (“SmartStax” tation (625, 1,250, and 2,500 viable eggs per row meter)
corn) and a non-Bt control. Our hypothesis was that Bt were assigned randomly to subplots to test for density-
proteins expressed in corn would alter these variables dependent mortality effects.
with the overarching goal to provide better emergence Experiments were planted into clay loam soil follow-
data as input for insect resistance modeling for D. v. ing a previous crop of winter wheat, Triticum aestivum
virgifera in their north-eastern distribution. L., where it was assumed no feral rootworm eggs were
present. Experiments were planted at Ridgetown, ON,
on 31 May 2010, 12 June 2012, and 16 July 2013 and
at Centralia, ON, on 1 June 2010 and 15 June 2012.
Materials and Methods
Planting was delayed in 2013 due to a wet spring and
The timing and number of beetles emerging from corn in other years due to delays in obtaining import permits
hybrids with a similar genetic background, but differing in and seed. However, because laboratory sourced eggs
expression of the Bt proteins Cry34/35Ab1 and Cry3Bb1, were used and applied on the day of planting, delayed
were compared with emergence from a non-Bt hybrid in planting was assumed not to influence results. Plots
field trials at two locations in 2010 and 2012, and one loca- were fertilized by broadcast application of 300 kg/ha of
tion in 2013 in Ontario, Canada. Hybrids tested had the fol- 6-24-24 NPK, incorporated by cultivation before
lowing insect protection: non-Bt with no D. v. virgifera planting. Weeds were controlled using a post-emergent
protection, Herculex Xtra (Event DAS-59122-7  TC1507, application of 4 liter/ha of Primextra II Magnum (s-
expressing Cry34/35Ab1), YieldGard VT Triple (Event metolachlor; 400 g/liter and atrazine and related
MON 88017  MON 89034, expressing Cry3Bb1), and triazines; 320 g/liter, Syngenta Canada Inc., Guelph,
SmartStax (MON 89034  TC1507  MON 88017  DAS- ON, Canada) plus 0.3 liter/ha of Callisto 480 SC (meso-
59122-7, expressing Cry34/35Ab1 and Cry3Bb1 as trione; 480 g/liter, Syngenta Canada Inc., Guelph, ON,
pyramided traits). Seed was provided by Dow AgroSciences Canada) up to the three-leaf stage. Corn seeds were
(Indianapolis, IN), commercially pretreated with Cruiser planted in egg-infested rows using a two-row cone-
Extreme 250 [thiamethoxam (0.250 mg a.i./seed), azoxystro- seeder at a rate of eight seeds per row meter (105,260
bin (0.005 mg a.i./seed), fludioxonil (0.013 mg a.i./seed), and plants per ha). Plots were eight rows wide, spaced
mefenoxam (0.010 mg a.i./seed)] provided by Syngenta 0.76 m apart, and 4 m in length, consisting of a pure
Crop Protection (Greensboro, NC). stand of each hybrid. All seed was provided as pure hy-
Immediately before planting, experiments were in- brids in separate packaging free from blended refuge
fested artificially with eggs from a Bt-susceptible labo- seed by the technology provider. Plots were thinned at
ratory population of diapausing D. v. virgifera, from the two-leaf stage to 25 plants per row and side-dressed
USDA-ARS North Central Laboratory (Brookings, with 300 kg/ha of 46-0-0 NPK. Bt protein expression
SD). Each year, six laboratory hatch tests of 50–100 was not verified; it was assumed seed was genotypically
eggs were used to estimate egg viability in the month pure.
before egg infestation. Percent hatch was 68.9% Eureka external screen houses (4.08 by 2.88 m)
(SEM 6 4.06) and 72.3% (SEM 6 5.19) in 2012 and (Johnson Outdoors Canada, Burlington, ON, Canada)
2013, respectively; results from 2010 hatch tests were were placed in the center four rows of each plot cover-
discarded because of a high incidence of nonviable ing 16.32 row meter (80 plants) at the five-leaf stage
eggs due to fungal infection. at Ridgetown, ON, on 5 July 2010, 5 July 2012, and
Before infestation, eggs were suspended uniformly 13 August 2013 and at Centralia, ON, on 6 July 2010
in a 0.15% agar solution, quantified, and adjusted to and 12 July 2012. Tent sides were buried in a 15 cm
the desired concentration by dilution. A two-row culti- deep trench and covered with soil to limit beetle es-
vator was modified and calibrated to apply 125 ml of cape or entry, and secured with rope tethers attached
agar solution per row meter to the center four rows to metal stakes. Plots were hand weeded as needed to
of each plot metered by a ground-driven pump (Model prevent larval feeding on alternate hosts. Tents were
MP-466, Demco Manufacturing Co., Boyden IA). checked daily for first beetle emergence, upon which
These pumps dispense regular aliquots of slurries by all but 12 central corn plants in each tent were cut to
positive displacement with little to no pressure. “S”- 30 cm in height to increase beetle collection effi-
tine cultivator teeth fitted with 1.9-cm-diameter deliv- ciency. Beetles were collected twice weekly from plants
ery tubes, placed eggs 2.5 cm below the soil surface and tent walls using a modified cordless wet/dry

vacuum (Model DC515, DeWalt Industrial Tool Co., were calculated for the survival function (Hall and
Baltimore, MD). Diabrotica barberi were counted and Wellner 1980). Pairwise comparisons made with the
discarded. Beetles were transferred to labeled vials in- Sidak correction identified differences among hybrids
side the tent from which collection occurred and frozen (Šidák 1967). Probability curves were compared using
until counted and sexed under a stereomicroscope the log-rank test. Emergence at Centralia 2010 was
based on abdominal characteristics (White 1977). Col- inexplicably delayed and not included in the survival
lection ceased on 24 September 2010, 18 September analysis. A Type I error of 0.05 was set for all statistical
2012, and 24 October 2013 at Ridgetown, ON, and on tests.
20 September 2010 and 24 September 2012 at Cen-
tralia, ON, respectively, when beetle emergence was
close to zero and the corn growing season was ending.
Adult weights were not measured.
Given late planting dates of our experiments, ther- Number and Sex Ratio of Beetles Emerged. The
mal time provided a more meaningful measure than number of beetles emerged was significantly affected by
calendar date to compare data among environments hybrid (males: F ¼ 244.67; df ¼ 3,42; P  0.0001;
(Schaafsma et al. 1993). Degree–day (DD) accumula- females: F ¼ 168.86; df ¼ 3,42; P  0.0001), location
tion was calculated from the date of egg infestation by (males: F ¼ 4.96; df ¼ 4,14; P ¼ 0.0106; females:
subtracting a base temperature of 11.1 C (Levine et al. F ¼ 6.29; df ¼ 4,14; P ¼ 0.0041), and their interaction
1992) from the 24 h average ambient air temperatures (males: F ¼ 3.20; df ¼ 12,42; P ¼ 0.0025; females:
recorded at screen height at each location (data pro- F ¼ 2.87; df ¼ 12,42; P ¼ 0.0057; Table 1). Bt hybrids
vided by Weather INnovations Consulting LP, Chat- consistently reduced emergence when compared with a
ham, ON, Canada). Using soil temperature would have non-Bt hybrid. The pyramid occasionally further
been more informative, but these data were not avail- reduced emergence, compared with single trait hybrids
able. However, Schaafsma et al. (1993) showed that air (Table 1). Emergence from Cry34/35Ab1 or Cry3Bb1
temperature correlated well enough with soil tempera- was similar. The mean number of beetles emerging per
ture to predict eclosion. row meter ranged from 0.2 to 1.5, 1.2 to 4.2, 3.5 to 7.4
Data Analysis. The number of beetles emerged, (with an outlier of 38.2), and 26.2 to 112.4 for hybrids
reduction in emergence compared with the non-Bt expressing the pyramid, Cry3Bb1 alone, Cry34/35Ab1
control, and 2013 egg density data were analyzed sepa- alone, and the non-Bt expressing hybrid, respective-
rately using PROC MIXED of SAS (Windows version ly.The percent reduction in beetle emergence was
9.4, SAS Institute, Cary, NC). Reduction in emergence affected by hybrid (male: F ¼ 842.36; df ¼ 3,42;
from each Bt hybrid compared with the control was P  0.0001; female F ¼ 512.97; df ¼ 3,42; P  0.0001),
calculated by dividing the number of beetles that and the interaction between hybrid and location (male:
emerged from each hybrid by the number that F ¼ 4.40; df ¼ 12,42; P ¼ 0.0002; females: F ¼ 3.85;
emerged from the non-Bt control and subtracting from df ¼ 12,42; P ¼ 0.0006). Percentage reduction of beetle
1 for each replicate within each location. Beetle counts emergence compared with the non-Bt expressing hybrid
were log (x þ 1) transformed, and percentage reduc- ranged from 64.3 to 97.4%, 91.1 to 95.2%, and 98.1 to
tions were arcsine-transformed to meet the assump- 99.6% for hybrids expressing the pyramid, Cry3Bb1
tions of normality and homogeneity of variances; alone, and Cry34/35Ab1 alone, respectively, and was
nontransformed values are reported. The standard similar for both males and females . The pyramid
infestation rate (2,500 viable eggs per row meter) was resulted in the greatest reduction of overall adult emer-
used for analysis of total beetle emergence and the gence, exceeding 98% in all location–years.
reduction in emergence compared with the non-Bt Hybrid (P  0.0001) and the interaction between loca-
control. The linear statistical model contained location, tion and hybrid (P ¼ 0.0336) affected the sex ratio of
hybrid (non-Bt, Cry34/35Ab1, Cry3Bb1, and the pyra- emerging beetles. A female-biased sex ratio was observed
mid), and the interaction between location and hybrid in Cry3Bb1 and pyramid plots in the order of two or
as fixed effects and replication within location as a ran- three females to one male. The female-bias was signifi-
dom effect. The 2013 density study model contained cant in 6 of 10 comparisons for these hybrids. A female-
egg density, hybrid, and the interaction between egg biased sex ratio was not apparent for beetles emerging
density and hybrid as fixed effects with replication from non-Bt or Cry34/34Ab1 hybrids (Table 2).
within location as a random effect. For all analyses, The total number of D. barberi emerging from the
least squares means (LSMEANS) of fixed effects non-Bt hybrid was 0, 0, 9, 1, and 203 for Centralia
were calculated for each location using the 2010, Centralia 2012, Ridgetown 2010, Ridgetown
SLICE option; comparisons were performed using the 2012, and Ridgetown 2013, respectively. The high
Tukey–Kramer method (PDIFF option in PROC number of D. barberi emerging in 2013 was unex-
MIXED). A paired t-test was used to test differences in pected, ranging from 0.9 to 2.3 beetles per row meter
male and female emergence from each location. Sur- across all plots. Mean emergence with blocks increased
vival analysis (PROC LIFETEST of SAS) was used to from 1 to 1.8 beetles per row meter moving east to
compare the emergence probability over time from west, with the western block adjacent to a continuous
each of the hybrids and to estimate the time to 50% corn plot. There was no relationship between numbers
emergence based on the survival function computed by of D. barberi emerging and the number of eggs of
the product-limit procedure. Hall-Wellner bands (95%) D. v. virgifera applied.

Table 1. Mean number of D. v. virgifera male and female beetles emerging per row meter
from artificially infested corn plots and the reduction in beetles emerging from Bt hybrids com-
pared with a non-Bt hybrid at Ridgetown and Centralia, Ontario

Hybrida Beetles emerging per row Reduction in emergence

meter (mean 6 SEM) (mean % 6 SEM)
Malesb Females Males Females
Centralia 2010
Non-Bt 20.1 6 4.62a 23.6 6 4.87a
Cry34/35Ab1 1.6 6 0.56b 2.4 6 0.56bc 91.9 6 2.79 89.9 6 2.37
Cry3Bb1 1.1 6 0.33b 2.3 6 0.28b 94.7 6 1.64 90.1 6 1.20
Pyramid 0.2 6 0.07c 0.5 6 0.14c 99.0 6 0.36 97.8 6 0.60
Centralia 2012
Non-Bt 57.6 6 13.98a 54.7 6 13.47a
Cry34/35Ab1 16.5 6 4.01a 21.3 6 5.23a 71.3 6 6.69a 61.2 6 9.56a
Cry3Bb1 1.2 6 0.33b 3.0 6 0.61b 97.9 6 0.57b 94.5 6 1.11b
Pyramid 0.4 6 0.21b 1.0 6 0.42b 99.3 6 0.36b 98.1 6 0.77b
Ridgetown 2010
Non-Bt 14.8 6 3.72a 21.2 6 7.11a
Cry34/35Ab1 3.6 6 1.00a 3.8 6 1.15b 75.9 6 6.73a 82.0 6 5.40a
Cry3Bb1 0.7 6 0.23b 1.4 6 0.56bc 95.3 6 1.53b 93.2 6 2.65ab
Pyramid 0.2 6 0.04b 0.3 6 0.03c 98.8 6 0.24b 98.6 6 0.16b
Ridgetown 2012
Non-Bt 12.3 6 3.57a 13.8 6 5.02a
Cry34/35Ab1 1.4 6 0.20b 2.1 6 0.59b 88.8 6 1.66a 84.5 6 4.29a
Cry3Bb1 0.3 6 0.16c 0.8 6 0.31b 97.6 6 1.31b 93.9 6 2.23ab
Pyramid 0.0 6 0.00c 0.2 6 0.09c 100.0 6 0.0b 98.9 6 0.69b
Ridgetown 2013
Non-Bt 29.9 6 7.08a 27.5 6 4.41a
Cry34/35Ab1 0.8 6 0.49b 1.0 6 0.69bc 97.3 6 1.63 96.5 6 2.49
Cry3Bb1 0.7 6 0.43b 2.3 6 1.17b 97.7 6 1.43 91.5 6 4.23
Pyramid 0.1 6 0.04b 0.2 6 0.04c 99.8 6 0.12 99.4 6 0.15
Hybrids express no-Bt (non-Bt), single rootworm-active Bt proteins (Cry34/35, Cry3Bb) or both
proteins (pyramid).
Means followed by the same or no letter are not different within each location–year. (P < 0.05
Tukey’s honestly significant difference [HSD] test).

Table 2. Proportion of female D. v. virgifera beetles emerging from artificially infested corn
plots at Ridgetown and Centralia, Ontario, in 2010, 2012 and 2013

Location Proportion of femalesa ( 6 SEM) t P

Centralia 2010 54.7 6 1.97 2.05 0.1327
Centralia 2012 48.6 6 1.37 1.08 0.3603
Ridgetown 2010 55.2 6 6.24 1.11 0.3483
Ridgetown 2012 51.8 6 2.79 0.64 0.5669
Ridgetown 2013 48.7 6 2.74 0.74 0.5354
Centralia 2010 63.5 6 6.63 2.57 0.0825
Centralia 2012 56.1 6 0.62* 3.53 0.0386
Ridgetown 2010 48.8 6 6.34 0.49 0.6566
Ridgetown 2012 57.9 6 6.70 1.24 0.3027
Ridgetown 2013 49.9 6 4.00 0.69 0.5616
Centralia 2010 70.4 6 4.12* 7.56 0.005
Centralia 2012 71.3 6 2.58* 5.01 0.0153
Ridgetown 2010 67.2 6 6.25 1.71 0.1866
Ridgetown 2012 79.5 6 7.13* 3.36 0.0437
Ridgetown 2013 84.5 6 7.77 2.27 0.1511
Centralia 2010 78.5 6 7.38* 4.20 0.0246
Centralia 2012 75.8 6 8.59 2.29 0.1061
Ridgetown 2010 63.4 6 3.51* 4.90 0.0163
Ridgetown 2012 100.0 6 0 1.61 0.2062
Ridgetown 2013 77.8 6 11.1* 5.00 0.0377
Compared with the proportion of males with a t-test, df ¼ 3.
Hybrids express no-Bt (non-Bt), single rootworm active Bt proteins (Cry34/35, Cry3Bb) or both
proteins (pyramid).
*Significant at P < 0.05.

Timing of Beetle Emergence. The pattern of Discussion

emergence differed for males from the non-Bt com-
In the single experiment at Ridgetown in 2013,
pared with females from each Bt hybrid (v: 3,172.98;
where egg density was varied, density-dependent mor-
df ¼ 3; P  0.0001), with non-Bt males emerging signifi-
tality in the non-Bt hybrid was observed at a rate of
cantly earlier (Fig. 1). Among the Bt hybrids, female
1,250 viable eggs per row meter. Hibbard et al. (2010a)
emergence was similar from Cry3Bb1 and Cry34/
oberved no density-dependent mortality at infestation
35Ab1, but delayed even further on the pyramid. The
rates of 82 to 1,967 viable eggs per row meter. We can-
time to 50% emergence was 544.8, 654.0, 657.7, and
not rule out density-dependent mortality at 625 eggs
724.6 DD11.1 C for males from the non-Bt hybrid, and
per row meter as no lower densities were tested.
for females from the Cry34/35Ab1, Cry3Bb1 or pyra-
Although egg viability was assessed in the laboratory, it
mided hybrids, respectively (Fig. 1).
could not be confirmed how many eggs of those placed
Density-Dependent Mortality. The percentage of
actually hatched in the field or may have been dam-
beetles emerging from plots infested 625, 1,250, or
aged during infestation. Assuming that this one experi-
2,500 viable eggs per row meter was impacted by
ment supports density-dependent mortality at lower
hybrid (males: F ¼ 97.06; df ¼ 3,22; P  0.0001;
egg densities, density-dependent mortality cannot be
females: F ¼ 54.91; df ¼ 3,22; P ¼ < 0.0001), infestation
ruled out at the infestation rate of 2,500 viable eggs per
rate (males: F ¼ 12.74; df ¼ 2,22; P ¼ 0.0002; females:
row meter used in our other experiments.
F ¼ 9.36; df ¼ 2,22; P ¼ 0.0011), and their interaction
Petzold-Maxwell (2013a) reported a reduction in
(males F ¼ 10.14; df ¼ 6,22; P  0.0001: females
survival to adulthood of 71% for Cry3Bb1 maize in
F ¼ 6.27; df ¼ 6,22; P ¼ 0.0006).
the absence of density-dependent mortality assum-
From the non-Bt hybrid, the percentage of viable
ing, according to Hibbard et al. (2010a), that
eggs emerging as beetles was highest (8.7%) at the density-dependent mortality is unlikely to occur
infestation rate of 625 eggs per row meter followed when root injury exceeds 1 node on the 0–3 node
by 5.7 and 2.3% for 1,250 and 2,500 eggs per row injury scale (Oleson et al 2005). For Cry34/35Ab,
meter rates, respectively (Table 3). As egg infesta- the reduction in survival was close to 10% (Petzold-
tion rate increased in the Cry3Bb1 and pyramided Maxwell 2013b), but in this case, density-dependent
hybrids, the total beetle emergence decreased mortality was not accounted for. Head et al. (2014)
(Table 3). examined emergence under low natural infestation

Probability of Emergence (%)



Non-Bt males
25 Cry34/35Ab1 females
Cry3Bb1 females
. . . . Pyramid females

400 600 800 1000
Degree Days (°C)
Fig. 1. Probability of emergence for individual D. v. virgifera beetles across two years and two locations plotted against
DD11.1 C from time of infestation for each experiment with 95% Hall–Wellner bands. Time to 50% emergence was 544.8,
654.0, 657.7, and 724.6 DD11.1 C for males from non-Bt, and females from Cry34/35Ab1, Cry3Bb1, and the pyramid hybrids,
respectively. Total emergence across the four experiments trials was 6,995, 1,823, 460, and 106 beetles for males from non-Bt
corn and females from Cry34/35Ab1, Cry3Bb1 and pyramid hybrids, respectively.

Table 3. Mean percentage of beetle emergence from artificial infestation at three egg densities at Ridge-
town, ON, in 2013

Egg density (viable Emergence percentage ( 6 SEM)

eggs per row meter)
Malesa Females Total
625 4.23 6 1.016a 4.41 6 0.697a 8.73 6 1.710a
1,250 2.99 6 0.478b 2.64 6 0.276b 5.70 6 0.679b
2,500 1.20 6 0.447c 1.10 6 0.176c 2.33 6 0.447c
625 0.26 6 0.108 0.34 6 0.057 0.61 6 0.108
1,250 0.12 6 0.048 0.18 6 0.078 0.30 6 0.124
2,500 0.03 6 0.020 0.04 6 0.027 0.07 6 0.047
625 0.082 6 0.023 0.25 6 0.059 0.33 6 0.803a
1,250 0.07 6 0.043 0.12 6 0.058 0.19 6 0.101b
2,500 0.03 6 0.017 0.09 6 0.047 0.12 6 0.064b
625 0.01 6 0.006 0.05 6 0.015 0.06 6 0.020a
1,250 0.01 6 0.004 0.01 6 0.006 0.02 6 0.009b
2,500 0.01 6 0.002 0.00 6 0.001 0.01 6 0.003b
Means within a column and protein followed by the same or no letter are not different (P < 0.05 Tukey’s HSD
test) within Bt protein.
Hybrids express no-Bt (non-Bt), single rootworm-active Bt proteins (Cry34/35, Cry3Bb) or both proteins

levels and at a high artificial infestation rate in sepa- Wet conditions increase larval mortality, decrease move-
rate experiments. Comparing percent reduction in ment, and reduce establishment, which may explain some
emergence from the non-Bt hybrid in the naturally differences in adult emergence across sites (Macdonald
infested experiments, values ranged from 59.1 to and Ellis 1990). Precipitation at the sites with the lowest
95.9%, 40.1 to 92.2%, and 80.8 to 100%, for the emergence was 42% greater than the 30-yr average in
Cry34/35Ab1, Cry3Bb1, and pyramid hybrids, July near Ridgetown and Centralia, whereas the sites
respectively. These values are considerably lower with high emergence (Ridgetown 2013 and Centralia
than those obtained by Head et al. (2014) or us 2012) had 32 and 11% lower than average precipitation,
under artificial infestation. respectively (Environment Canada 2014a,b).
Density-dependent mortality might occur at lower egg Differences in the time (DD11.1 C) to 50% emer-
densities than first believed, perhaps before larvae are gence between males from the non-Bt control and
exposed to the Bt proteins, immediately following eclo- females from the pyramid were particularly striking.
sion, as they competed for feeding sites. If this mortality The pyramid reduced beetle emergence, delayed emer-
is not accompanied by a fitness cost to survivors, it would gence, and often increased the proportion of female
mainly be independent of mortality because of exposure beetles. These findings cast doubt on one assumption
to Bt protein. Kang et al. (2014), in their predictions, of the “refuge strategy” used in IRM – that males
applied density-dependent mortality after exposure to Bt emerging from non-Bt refuge will disperse through the
proteins, which may need to be reconsidered in predict- Bt corn and outcompete less abundant, potentially
ing resistance evolution. resistant males, emerging from the Bt corn resulting in
We advise caution in the interpretation of the random mating with potentially resistant females
density response we observed. D. barberi were present at emerging from Bt plants. (Spencer et al. 2009). From
20-fold higher numbers at Ridgetown in 2013 compared an IRM perspective, delays in males emerging from
with levels of 0–9 beetles emerging per location in other non-Bt refuge may contribute to assortative mating and
years. Nevertheless, we assume they had limited impact increase the evolution of resistance.
because they are weak competitors when they co-occur Kang et al. (2014) suggest that the effects of a
with D. v. virgifera (Woodson 1994). Extended diapause female-biased sex ratio and delayed emergence of bee-
in D. barberi is not common in Ontario. We suspect that tles from Bt corn, both observed in this study, may be
D. barberi beetles moved from adjacent continuous corn insignificant on the evolution of resistance under a
into the winter wheat stubble where soil moisture was blended refuge. Others predicted an impact on the
more attractive for oviposition than in corn under drought evolution of resistance in a structured refuge (Onstad
conditions, a phenomenon we have observed previously et al. 2001, Pan et al. 2011). Typically, males are protan-
in our region. For the same reason, feral D. v. virgifera drous, becoming sexually mature within 7 d of emer-
could also not be ruled out. The effect of rootworm-active gence and completing mating by day 17, resulting in
Bt proteins on beetle emergence and sex ratio were con- overlap with sexually mature females (Guss 1976,
sistent over three years and two locations. However, total Quiring and Timmins 1990, Kang and Krupke 2009).
emergence varied across years and locations. Because Females emerging late from the Bt hybrids will have
gene expression was not verified, some plants may not less opportunity to mate with susceptible males emerg-
have been expressing Bt protein; however, the corn seed ing from the refuge. This gap was most apparent in the
and egg source were the same for both sites each year. pyramid where a delay of 15.8 d to reach 50%

emergence was observed (calculated based on an aver- pyramided traits, may support greater durability for this
age daily temperature of 22.5 C). This delay falls within new product than might be expected for Smartstax, espe-
the sensitivity range tested by Kang et al. (2014). Nota- cially under structured refuge. These contradicting results
bly, our study did not determine whether late emerging suggest adult survival patterns may vary between traits,
females were susceptible or resistant genotypes. when used singly or when pyramided, adding another
A similar study using a higher rate of egg infestation layer of complexity to insect resistance modeling.
found emergence was reduced in Cry34/35Ab1,
Cry3Bb1, and pyramided corn ranging from 94.2 to
99.2%, 96.2 to 99.96%, and 98.2 to 99.97%, respectively
(Head et al. 2014). Their percent reduction from the
pyramid was similar to that observed in this study; Acknowledgments
however, we observed greater emergence from both We thank Todd Phibbs for technical assistance in planting and
Cry34/35Ab1 and Cry3Bb1 corn at most sites. Clark infesting experiments and research assistants for tent set-up and
et al. (2012) reported that larval mortality in Cry3Bb1 beetle collection. We are grateful to Chad Nielson (USDA-ARS
corn averaged 98.5% (4.4 points higher than the 94.6% Brookings, USA) for supplying D. v. virgifera eggs. This project
we report) and 50% emergence for both sexes combined was funded jointly by Monsanto Canada Inc. and Dow Agro-
was delayed by18.3 d when compared with non-Bt corn Sciences Inc., and access to their seed technology is gratefully
acknowledged. The experimental design was developed following
(6 d longer than we report also for both sexes com-
consultation with the sponsors and consideration of similar refer-
bined). Storer et al. (2006) reported 99.1–99.98% mor- eed studies. Neither sponsor was involved in the decision to pub-
tality of western corn rootworm, and a short delay (3 to lish. We thank the anonymous reviewers for helpful comments
7 d) in time to 50% adult emergence in two of three that greatly improved this manuscript. Mention of trade names or
studies where corn expressing Cry34/35Ab1 was tested. commercial products in this publication is solely for the purpose
While the reported delays in emergence are consistent of providing specific information and does not imply recommen-
with our results, the reported mortalities are higher than dation or endorsement by the U.S. Department of Agriculture
we observed (64.3–97.4 or 83.3% when averaged across (USDA). USDA is an equal opportunity provider and employer.
locations) for corn expressing Cry34/35Ab1. Differences
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