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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 INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

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

Alone or Pyramided, on Western Corn Rootworm (Coleoptera:
Chrysomelidae) Beetle Emergence in the Field
A. J. HITCHON,1 J. L. SMITH,1 B. W. FRENCH,2 AND A. W. SCHAAFSMA1,3

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-
cidal proteins derived from the soil bacterium, Bacillus
thuringiensis Berliner (Bt), is planted widely by Cana-
dian corn producers for insect control. Three Bt pro-
teins Cry3Bb1, Cry34/35Ab1, and mCry3A are
available commercially and control larvae of western
corn rootworm, Diabrotica virgifera virgifera LeConte
(Coleoptera: Chrysomelidae) and northern corn root-
worm, Diabrotica barberi Smith and Lawrence, major
pests of corn (Gray et al. 2009). The use of rootworm-
active Bt corn increased in Canada from 45.1 to 60.2%
of total corn acreage from 2009 to 2013, mainly in the
provinces of Ontario and Quebec (Dunlop 2013).
Bt corn use has desirable environmental, economic,
and agronomic benefits, and reduces pesticide use and
exposure (Rice 2004, Hutchison et al. 2010). Develop-
ment of resistance by target pests is the primary con-
cern associated with Bt crops. Historically, foliar
applications of formulated Bt, while not targeting corn
rootworm, heightened concern that insects developing
resistance to Bt crops might compromise the use of fo-
liar Bt insecticides, especially in organic agriculture
(Roush 1994, Belzile 2002). As a condition of Bt crop
registration, the Canadian Food Inspection Agency re-
quired insect resistance management (IRM) plans be
1
Department of Plant Agriculture, Ridgetown Campus, University
of Guelph, 120 Main St. E., Ridgetown, ON N0P 2C0, Canada.
2
North Central Agricultural Research Laboratory, 2923 Medary
Ave., Brookings, SD, 57006.
3
Corresponding author, e-mail: aschaafs@uoguelph.ca.
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
implemented to delay resistance development
(Macdonald and Yarrow 2003). The current IRM is
based on the “refuge strategy” that is also widely
adopted in the United States for both D. v. virgifera
and O. nubilalis (Gould 1998, Huang et al. 2011).
Despite IRM, as prescribed by the U.S. Environ-
mental Protection Agency, field-evolved resistance of
D. v. virgifera to the Cry3Bb1 and mCry3a proteins de-
veloped after six generations of selection in some areas
of the U.S. Corn Belt (Gassmann et al. 2011, 2014).
Resistance evolved faster in areas with high D. v. virgi-
fera populations and continuous corn production, espe-
cially when the same Bt proteins were planted
consecutively in a field for >3 yr (Gassmann et al.
2011, 2014; Cullen et al. 2013). Resistance has not
been confirmed in Canada, and smaller farm size and
greater diversity in crop rotation and landscape may
delay the evolution of resistance to Bt in the eastern
Corn Belt compared with the western United States
(DiFonzo et al. 2013). In Ontario, three or more crops
are often included in the rotation, whereas in much of
the U.S. Midwest, corn and soybean are the two pri-
mary crops (Knight et al. 2005).
IRM prescriptions are based on models that predict
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
these models, including that of random mating, are
proving to be incorrect (Spencer et al. 2013). Random
mating of susceptible males with resistant females
relies on a synchrony in emergence between the sexes
August 2015 HITCHON ET AL.: CORN ROOTWORM EMERGENCE IN BT CORN
from both refuge and Bt hybrids. A delay in emergence
of adults from Bt hybrids compared with those emerg-
ing from non-Bt refuge has been reported (Storer et al.
2006; Hibbard et al. 2010b, 2011; Clark et al. 2012) in-
creasing the probability of assortative mating. These
data were generated in the U.S. Corn Belt, and their
relevance to Ontario, in the north-eastern range of dis-
tribution for D. v. virgifera, is uncertain.
To address this question, experiments were con-
ducted to compare the number of beetles emerging,
timing of emergence, and sex ratio, from hybrids ex-
pressing single Bt proteins (Cry3Bb1 and Cry34/
35Ab1), a pyramid of these two proteins (“SmartStax”
corn) and a non-Bt control. Our hypothesis was that Bt
proteins expressed in corn would alter these variables
with the overarching goal to provide better emergence
data as input for insect resistance modeling for D. v.
virgifera in their north-eastern distribution.
Materials and Methods
The timing and number of beetles emerging from corn
hybrids with a similar genetic background, but differing in
expression of the Bt proteins Cry34/35Ab1 and Cry3Bb1,
were compared with emergence from a non-Bt hybrid in
field trials at two locations in 2010 and 2012, and one loca-
tion in 2013 in Ontario, Canada. Hybrids tested had the fol-
lowing insect protection: non-Bt with no D. v. virgifera
protection, Herculex Xtra (Event DAS-59122-7  TC1507,
expressing Cry34/35Ab1), YieldGard VT Triple (Event
MON 88017  MON 89034, expressing Cry3Bb1), and
SmartStax (MON 89034  TC1507  MON 88017  DAS-
59122-7, expressing Cry34/35Ab1 and Cry3Bb1 as
pyramided traits). Seed was provided by Dow AgroSciences
(Indianapolis, IN), commercially pretreated with Cruiser
Extreme 250 [thiamethoxam (0.250 mg a.i./seed), azoxystro-
bin (0.005 mg a.i./seed), fludioxonil (0.013 mg a.i./seed), and
mefenoxam (0.010 mg a.i./seed)] provided by Syngenta
Crop Protection (Greensboro, NC).
Immediately before planting, experiments were in-
fested artificially with eggs from a Bt-susceptible labo-
ratory population of diapausing D. v. virgifera, from
USDA-ARS North Central Laboratory (Brookings,
SD). Each year, six laboratory hatch tests of 50–100
eggs were used to estimate egg viability in the month
before egg infestation. Percent hatch was 68.9%
(SEM 6 4.06) and 72.3% (SEM 6 5.19) in 2012 and
2013, respectively; results from 2010 hatch tests were
discarded because of a high incidence of nonviable
eggs due to fungal infection.
Before infestation, eggs were suspended uniformly
in a 0.15% agar solution, quantified, and adjusted to
the desired concentration by dilution. A two-row culti-
vator was modified and calibrated to apply 125 ml of
agar solution per row meter to the center four rows
of each plot metered by a ground-driven pump (Model
MP-466, Demco Manufacturing Co., Boyden IA).
These pumps dispense regular aliquots of slurries by
positive displacement with little to no pressure. “S”-
tine cultivator teeth fitted with 1.9-cm-diameter deliv-
ery tubes, placed eggs 2.5 cm below the soil surface
1987
on each side of the row, while a “U”-shaped drag chain
covered eggs with a small layer of soil.
Experiments were configured in a randomized com-
plete block design with four replications and repeated
twice in each of 2010 and 2012 at Ridgetown (42.44908
N, 81.879821 W) and Centralia, ON (43.316060 N,
81.500023 W). In 2010 and 2012, plots were infested
with a rate of 2,500 viable eggs per row meter to limit
density-dependent mortality (Hibbard et al. 2010a). In
2013, one experiment was conducted at Ridgetown,
ON, as a split-plot design, with three replications. Bt
hybrid was the main plot and three levels of egg infes-
tation (625, 1,250, and 2,500 viable eggs per row meter)
were assigned randomly to subplots to test for density-
dependent mortality effects.
Experiments were planted into clay loam soil follow-
ing a previous crop of winter wheat, Triticum aestivum
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.
Planting was delayed in 2013 due to a wet spring and
in other years due to delays in obtaining import permits
and seed. However, because laboratory sourced eggs
were used and applied on the day of planting, delayed
planting was assumed not to influence results. Plots
were fertilized by broadcast application of 300 kg/ha of
6-24-24 NPK, incorporated by cultivation before
planting. Weeds were controlled using a post-emergent
application of 4 liter/ha of Primextra II Magnum (s-
metolachlor; 400 g/liter and atrazine and related
triazines; 320 g/liter, Syngenta Canada Inc., Guelph,
ON, Canada) plus 0.3 liter/ha of Callisto 480 SC (meso-
trione; 480 g/liter, Syngenta Canada Inc., Guelph, ON,
Canada) up to the three-leaf stage. Corn seeds were
planted in egg-infested rows using a two-row cone-
seeder at a rate of eight seeds per row meter (105,260
plants per ha). Plots were eight rows wide, spaced
0.76 m apart, and 4 m in length, consisting of a pure
stand of each hybrid. All seed was provided as pure hy-
brids in separate packaging free from blended refuge
seed by the technology provider. Plots were thinned at
the two-leaf stage to 25 plants per row and side-dressed
with 300 kg/ha of 46-0-0 NPK. Bt protein expression
was not verified; it was assumed seed was genotypically
pure.
Eureka external screen houses (4.08 by 2.88 m)
(Johnson Outdoors Canada, Burlington, ON, Canada)
were placed in the center four rows of each plot cover-
ing 16.32 row meter (80 plants) at the five-leaf stage
at Ridgetown, ON, on 5 July 2010, 5 July 2012, and
13 August 2013 and at Centralia, ON, on 6 July 2010
and 12 July 2012. Tent sides were buried in a 15 cm
deep trench and covered with soil to limit beetle es-
cape or entry, and secured with rope tethers attached
to metal stakes. Plots were hand weeded as needed to
prevent larval feeding on alternate hosts. Tents were
checked daily for first beetle emergence, upon which
all but 12 central corn plants in each tent were cut to
30 cm in height to increase beetle collection effi-
ciency. Beetles were collected twice weekly from plants
and tent walls using a modified cordless wet/dry
1988 JOURNAL OF ECONOMIC ENTOMOLOGY
vacuum (Model DC515, DeWalt Industrial Tool Co.,
Baltimore, MD). Diabrotica barberi were counted and
discarded. Beetles were transferred to labeled vials in-
side the tent from which collection occurred and frozen
until counted and sexed under a stereomicroscope
based on abdominal characteristics (White 1977). Col-
lection ceased on 24 September 2010, 18 September
2012, and 24 October 2013 at Ridgetown, ON, and on
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-
mal time provided a more meaningful measure than
calendar date to compare data among environments
(Schaafsma et al. 1993). Degree–day (DD) accumula-
tion was calculated from the date of egg infestation by
subtracting a base temperature of 11.1 C (Levine et al.
1992) from the 24 h average ambient air temperatures
recorded at screen height at each location (data pro-
vided by Weather INnovations Consulting LP, Chat-
ham, ON, Canada). Using soil temperature would have
been more informative, but these data were not avail-
able. However, Schaafsma et al. (1993) showed that air
temperature correlated well enough with soil tempera-
ture to predict eclosion.
Data Analysis. The number of beetles emerged,
reduction in emergence compared with the non-Bt
control, and 2013 egg density data were analyzed sepa-
rately using PROC MIXED of SAS (Windows version
9.4, SAS Institute, Cary, NC). Reduction in emergence
from each Bt hybrid compared with the control was
calculated by dividing the number of beetles that
emerged from each hybrid by the number that
emerged from the non-Bt control and subtracting from
1 for each replicate within each location. Beetle counts
were log (x þ 1) transformed, and percentage reduc-
tions were arcsine-transformed to meet the assump-
tions of normality and homogeneity of variances;
nontransformed values are reported. The standard
infestation rate (2,500 viable eggs per row meter) was
used for analysis of total beetle emergence and the
reduction in emergence compared with the non-Bt
control. The linear statistical model contained location,
hybrid (non-Bt, Cry34/35Ab1, Cry3Bb1, and the pyra-
mid), and the interaction between location and hybrid
as fixed effects and replication within location as a ran-
dom effect. The 2013 density study model contained
egg density, hybrid, and the interaction between egg
density and hybrid as fixed effects with replication
within location as a random effect. For all analyses,
least squares means (LSMEANS) of fixed effects
were calculated for each location using the
SLICE option; comparisons were performed using the
Tukey–Kramer method (PDIFF option in PROC
MIXED). A paired t-test was used to test differences in
male and female emergence from each location. Sur-
vival analysis (PROC LIFETEST of SAS) was used to
compare the emergence probability over time from
each of the hybrids and to estimate the time to 50%
emergence based on the survival function computed by
the product-limit procedure. Hall-Wellner bands (95%)
Vol. 108, no. 4
were calculated for the survival function (Hall and
Wellner 1980). Pairwise comparisons made with the
Sidak correction identified differences among hybrids
(Šidák 1967). Probability curves were compared using
the log-rank test. Emergence at Centralia 2010 was
inexplicably delayed and not included in the survival
analysis. A Type I error of 0.05 was set for all statistical
tests.
Results
Number and Sex Ratio of Beetles Emerged. The
number of beetles emerged was significantly affected by
hybrid (males: F ¼ 244.67; df ¼ 3,42; P  0.0001;
females: F ¼ 168.86; df ¼ 3,42; P  0.0001), location
(males: F ¼ 4.96; df ¼ 4,14; P ¼ 0.0106; females:
F ¼ 6.29; df ¼ 4,14; P ¼ 0.0041), and their interaction
(males: F ¼ 3.20; df ¼ 12,42; P ¼ 0.0025; females:
F ¼ 2.87; df ¼ 12,42; P ¼ 0.0057; Table 1). Bt hybrids
consistently reduced emergence when compared with a
non-Bt hybrid. The pyramid occasionally further
reduced emergence, compared with single trait hybrids
(Table 1). Emergence from Cry34/35Ab1 or Cry3Bb1
was similar. The mean number of beetles emerging per
row meter ranged from 0.2 to 1.5, 1.2 to 4.2, 3.5 to 7.4
(with an outlier of 38.2), and 26.2 to 112.4 for hybrids
expressing the pyramid, Cry3Bb1 alone, Cry34/35Ab1
alone, and the non-Bt expressing hybrid, respective-
ly.The percent reduction in beetle emergence was
affected by hybrid (male: F ¼ 842.36; df ¼ 3,42;
P  0.0001; female F ¼ 512.97; df ¼ 3,42; P  0.0001),
and the interaction between hybrid and location (male:
F ¼ 4.40; df ¼ 12,42; P ¼ 0.0002; females: F ¼ 3.85;
df ¼ 12,42; P ¼ 0.0006). Percentage reduction of beetle
emergence compared with the non-Bt expressing hybrid
ranged from 64.3 to 97.4%, 91.1 to 95.2%, and 98.1 to
99.6% for hybrids expressing the pyramid, Cry3Bb1
alone, and Cry34/35Ab1 alone, respectively, and was
similar for both males and females . The pyramid
resulted in the greatest reduction of overall adult emer-
gence, exceeding 98% in all location–years.
Hybrid (P  0.0001) and the interaction between loca-
tion and hybrid (P ¼ 0.0336) affected the sex ratio of
emerging beetles. A female-biased sex ratio was observed
in Cry3Bb1 and pyramid plots in the order of two or
three females to one male. The female-bias was signifi-
cant in 6 of 10 comparisons for these hybrids. A female-
biased sex ratio was not apparent for beetles emerging
from non-Bt or Cry34/34Ab1 hybrids (Table 2).
The total number of D. barberi emerging from the
non-Bt hybrid was 0, 0, 9, 1, and 203 for Centralia
2010, Centralia 2012, Ridgetown 2010, Ridgetown
2012, and Ridgetown 2013, respectively. The high
number of D. barberi emerging in 2013 was unex-
pected, ranging from 0.9 to 2.3 beetles per row meter
across all plots. Mean emergence with blocks increased
from 1 to 1.8 beetles per row meter moving east to
west, with the western block adjacent to a continuous
corn plot. There was no relationship between numbers
of D. barberi emerging and the number of eggs of
D. v. virgifera applied.
August 2015 HITCHON ET AL.: CORN ROOTWORM EMERGENCE IN BT CORN 1989

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
a
Hybrids express no-Bt (non-Bt), single rootworm-active Bt proteins (Cry34/35, Cry3Bb) or both
proteins (pyramid).
b
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

b
Non-Bt
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
Cry34/34Ab1
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
Cry3Bb1
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
Pyramid
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
a
Compared with the proportion of males with a t-test, df ¼ 3.
b
Hybrids express no-Bt (non-Bt), single rootworm active Bt proteins (Cry34/35, Cry3Bb) or both
proteins (pyramid).
*Significant at P < 0.05.
1990 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 4

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

100
Probability of Emergence (%)

75

50

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

0
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.
August 2015 HITCHON ET AL.: CORN ROOTWORM EMERGENCE IN BT CORN 1991

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
Non-Btb
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
Cry34/35Ab1
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
Cry3Bb1
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
Pyramid
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
a
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.
b
Hybrids express no-Bt (non-Bt), single rootworm-active Bt proteins (Cry34/35, Cry3Bb) or both proteins
(pyramid).

levels and at a high artificial infestation rate in sepa-
rate experiments. Comparing percent reduction in
emergence from the non-Bt hybrid in the naturally
infested experiments, values ranged from 59.1 to
95.9%, 40.1 to 92.2%, and 80.8 to 100%, for the
Cry34/35Ab1, Cry3Bb1, and pyramid hybrids,
respectively. These values are considerably lower
than those obtained by Head et al. (2014) or us
under artificial infestation.
Density-dependent mortality might occur at lower egg
densities than first believed, perhaps before larvae are
exposed to the Bt proteins, immediately following eclo-
sion, as they competed for feeding sites. If this mortality
is not accompanied by a fitness cost to survivors, it would
mainly be independent of mortality because of exposure
to Bt protein. Kang et al. (2014), in their predictions,
applied density-dependent mortality after exposure to Bt
proteins, which may need to be reconsidered in predict-
ing resistance evolution.
We advise caution in the interpretation of the
density response we observed. D. barberi were present at
20-fold higher numbers at Ridgetown in 2013 compared
with levels of 0–9 beetles emerging per location in other
years. Nevertheless, we assume they had limited impact
because they are weak competitors when they co-occur
with D. v. virgifera (Woodson 1994). Extended diapause
in D. barberi is not common in Ontario. We suspect that
D. barberi beetles moved from adjacent continuous corn
into the winter wheat stubble where soil moisture was
more attractive for oviposition than in corn under drought
conditions, a phenomenon we have observed previously
in our region. For the same reason, feral D. v. virgifera
could also not be ruled out. The effect of rootworm-active
Bt proteins on beetle emergence and sex ratio were con-
sistent over three years and two locations. However, total
emergence varied across years and locations. Because
gene expression was not verified, some plants may not
have been expressing Bt protein; however, the corn seed
and egg source were the same for both sites each year.
Wet conditions increase larval mortality, decrease move-
ment, and reduce establishment, which may explain some
differences in adult emergence across sites (Macdonald
and Ellis 1990). Precipitation at the sites with the lowest
emergence was 42% greater than the 30-yr average in
July near Ridgetown and Centralia, whereas the sites
with high emergence (Ridgetown 2013 and Centralia
2012) had 32 and 11% lower than average precipitation,
respectively (Environment Canada 2014a,b).
Differences in the time (DD11.1 C) to 50% emer-
gence between males from the non-Bt control and
females from the pyramid were particularly striking.
The pyramid reduced beetle emergence, delayed emer-
gence, and often increased the proportion of female
beetles. These findings cast doubt on one assumption
of the “refuge strategy” used in IRM – that males
emerging from non-Bt refuge will disperse through the
Bt corn and outcompete less abundant, potentially
resistant males, emerging from the Bt corn resulting in
random mating with potentially resistant females
emerging from Bt plants. (Spencer et al. 2009). From
an IRM perspective, delays in males emerging from
non-Bt refuge may contribute to assortative mating and
increase the evolution of resistance.
Kang et al. (2014) suggest that the effects of a
female-biased sex ratio and delayed emergence of bee-
tles from Bt corn, both observed in this study, may be
insignificant on the evolution of resistance under a
blended refuge. Others predicted an impact on the
evolution of resistance in a structured refuge (Onstad
et al. 2001, Pan et al. 2011). Typically, males are protan-
drous, becoming sexually mature within 7 d of emer-
gence and completing mating by day 17, resulting in
overlap with sexually mature females (Guss 1976,
Quiring and Timmins 1990, Kang and Krupke 2009).
Females emerging late from the Bt hybrids will have
less opportunity to mate with susceptible males emerg-
ing from the refuge. This gap was most apparent in the
pyramid where a delay of 15.8 d to reach 50%
1992 JOURNAL OF ECONOMIC ENTOMOLOGY
emergence was observed (calculated based on an aver-
age daily temperature of 22.5 C). This delay falls within
the sensitivity range tested by Kang et al. (2014). Nota-
bly, our study did not determine whether late emerging
females were susceptible or resistant genotypes.
A similar study using a higher rate of egg infestation
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;
however, we observed greater emergence from both
Cry34/35Ab1 and Cry3Bb1 corn at most sites. Clark
et al. (2012) reported that larval mortality in Cry3Bb1
corn averaged 98.5% (4.4 points higher than the 94.6%
we report) and 50% emergence for both sexes combined
was delayed by18.3 d when compared with non-Bt corn
(6 d longer than we report also for both sexes com-
bined). Storer et al. (2006) reported 99.1–99.98% mor-
tality of western corn rootworm, and a short delay (3 to
7 d) in time to 50% adult emergence in two of three
studies where corn expressing Cry34/35Ab1 was tested.
While the reported delays in emergence are consistent
with our results, the reported mortalities are higher than
we observed (64.3–97.4 or 83.3% when averaged across
locations) for corn expressing Cry34/35Ab1. Differences
in results may be owing to selection intensity of Bt pro-
teins, varying with environment (Hibbard et al. 2011).
An additive relationship of Cry3Bb1 and Cry34/
35Ab1 was not observed by Hibbard et al. (2011); they
reported survivorship on pyramid of 1.8%, 12.4-fold
greater then would be expected if Cry34/35Ab1and
Cry3Bb1 were independently influencing survivorship
Similarly, Head et al (2014) show a 19-fold increase in
survivorship in a pyramid of the same two traits. How-
ever, multiplying Cry34/35Ab1 and Cry3Bb1 survivor-
ship in this study (0.167  0.054 ¼ 0.009), a 0.9%
survivorship would be expected on the pyramid; this is
close to the 1.1% we observed. These results suggest the
effects of both proteins (Cry34/35Ab1 and Cry3Bb1) in
the pyramid were additive when compared with each
expressed singly under the conditions of our study.
We accept our hypothesis that Bt proteins expressed in
corn would alter beetle emergence, sex ration, and time
of emergence: beetle emergence 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,
compared with non-Bt controls; the sex ratio of emerged
beetles was usually female-biased from the Cry3Bb1 and
pyramided treatments, but not from Cry34/35Ab1 treat-
ment alone; and emergence from all Bt hybrids was
delayed compared with the control, with the delay longest
from the pyramided hybrid. However, a new pyramid
combining two other proteins, mCry3A and eCry3.1Ab,
is expected to be released in Canada in the future.
Hibbard et al. (2011) found a 99.9% reduction in emer-
gence and delayed emergence by 4.6 d in plots of this
pyramid compared with a non-Bt. When eCry3.1Ab and
mCry3a were evaluated individually, the delay of the sin-
gle traits was 8 and 4.1 d, respectively. Higher mortality
and more synchronous beetle emergence with non-Bt
corn, in the absence of cross-resistance between
Vol. 108, no. 4
pyramided traits, may support greater durability for this
new product than might be expected for Smartstax, espe-
cially under structured refuge. These contradicting results
suggest adult survival patterns may vary between traits,
when used singly or when pyramided, adding another
layer of complexity to insect resistance modeling.
Acknowledgments
We thank Todd Phibbs for technical assistance in planting and
infesting experiments and research assistants for tent set-up and
beetle collection. We are grateful to Chad Nielson (USDA-ARS
Brookings, USA) for supplying D. v. virgifera eggs. This project
was funded jointly by Monsanto Canada Inc. and Dow Agro-
Sciences Inc., and access to their seed technology is gratefully
acknowledged. The experimental design was developed following
consultation with the sponsors and consideration of similar refer-
eed studies. Neither sponsor was involved in the decision to pub-
lish. We thank the anonymous reviewers for helpful comments
that greatly improved this manuscript. Mention of trade names or
commercial products in this publication is solely for the purpose
of providing specific information and does not imply recommen-
dation or endorsement by the U.S. Department of Agriculture
(USDA). USDA is an equal opportunity provider and employer.
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Received 26 November 2014; accepted 25 April 2015.