Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize
et al. (2014) Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus
Pyramided Bt Maize. PLoS ONE 9(11): e112958. doi:10.1371/journal.pone.0112958
Cry1F Resistance in Fall Armyworm Spodoptera frugiperda : Single Gene versus Pyramided Bt Maize
Fangneng Huang 0
Jawwad A. Qureshi 0
Robert L. Meagher Jr. 0
Dominic D. Reisig 0
Graham P. Head 0
David A. Andow 0
Xinzi Ni 0
David Kerns 0
G. David Buntin 0
Ying Niu 0
Fei Yang 0
Vikash Dangal 0
Kun Yan Zhu, Kansas State University, United States of America
0 1 Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, United States of America, 2 Department of Entomology and Nematology, University of Florida, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, United States of America, 3 Insect Behavior and Biocontrol Unit, USDA-ARS CMAVE, Gainesville, Florida, United States of America, 4 Department of Entomology, North Carolina State University, Vernon G. James Research and Extension Center, Plymouth, North Carolina, United States of America, 5 Monsanto Company , St. Louis , Missouri, United States of America, 6 Department of Entomology, University of Minnesota , St. Paul , Minnesota, United States of America , 7 USDA , Agricultural Research Service, Crop Genetics and Breeding Research Unit , Tifton , Georgia , United States of America , 8 UGA-Griffin Campus , Department of Entomology, the University of Georgia , Griffin, Georgia , United States of America
Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012-2013 exhibited 18.8-fold to .85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had .85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of crossresistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Competing Interests: The authors have read the journals policy and have the following competing interests. F.H. received research funding related to this
project from the Louisiana Soybean and Feed Grain Promotion Board, Monsanto, and the hatch funds from the USDA National Institute of Food and Agriculture.
G.P.H. has an affiliation to one of the financial supporters of this study. This does not alter the authors adherence to PLOS ONE policies on sharing data and
Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a
wellknown long-distance migratory insect that is distributed from
Argentina to Canada . In the U.S., populations from
overwintering areas in south Texas (TX) and south Florida (FL)
migrate annually into various regions across the country . S.
frugiperda is a major target of both Bt maize and Bt cotton in
North and South America [3,4]. In 2013 alone, approximately 50
MHa of Bt crops were planted in the Americas for insect pest
management [5,6]. Effective insect resistance management (IRM)
is crucial to ensure the long-term durability of these Bt crops .
Resistance monitoring must be addressed in IRM plans for Bt
crops . Although disagreements over the definition of field
resistance still exist [9,1113], the possibility of field resistance
should be considered when there is a field control failure or
significantly reduced efficacy . Based on this criterion, field
resistance to Bt crops has been clearly documented in at least four
cases around the world [9,11], including resistance of S.
frugiperda to Cry1F maize in Puerto Rico [14,15].
In recent years, unexpected survival of S. frugiperda on Cry1F
maize has been reported on several occasions in the southeastern
U.S. and in Brazil (F.H., R.L.M., J.A.Q., and D.D.R.,
unpublished data). However, scientific documentation of field resistance
to Bt maize in S. frugiperda has not been reported anywhere
except Puerto Rico . During 20112013, an F2 screen,
dietincorporated bioassays, greenhouse tests, and field studies with
various maize products (Table S1) were conducted in four
southeastern U.S. states and the results documented that the
unexpected survival of S. frugiperda on Cry1F maize in the region
was due to resistance. The occurrence of field resistance of S.
frugiperda in the U.S. mainland indicates a great challenge in
resistance management for migratory targets of Bt crops.
Results and Discussion
In 2011, a total of 142 F2 two-parent families of S. frugiperda
were established using single-pair mating of field-collected
individuals, which included 70 families from Rapides and Franklin
parishes in central and northeast Louisiana (LA) and 72 families
from Collier County in south FL. F2 neonates of these families
were screened on leaf tissue of Herculex I (HX1) maize expressing
the Cry1F protein. The F2 screen showed that Cry1F resistance
alleles were not rare in the LA and FL populations. The parents of
47.2% families in the two populations were found to carry $1
resistance allele (Table 1). For the LA population, parents of 49
families were negative for the presence of resistance alleles
(genotype SSSS), 14 families carried 1 resistance allele (RSSS),
and 7 families carried 2 resistance alleles (RSRS or RRSS). Among
the 72 FL families, only 26 were negative, while 15, 25, 5, and 1
families were identified to carry 1, 2, 3 (RRRS), and 4 (RRRR)
resistance alleles, respectively. The Cry1F resistance allele
frequency estimated using a multinomial Bayesian statistical
model (Methods S1) was 0.103 with a 95% credibility interval
(CI) of 0.070 to 0.141 for the LA population and 0.293 with a 95%
CI of 0.242 to 0.347 for the FL population. The resistant families
initiated with five neonates per plant in the greenhouse grew well
and survived on 4080% of the Cry1F maize plants after 13 d
(Table S2). A significant level of resistance (.270-fold) was also
observed when these families were tested against purified Cry1F
protein in diet-incorporated bioassays (Table 2).
We interpreted the high Cry1F resistance allele frequency
estimated by the F2 screen in the FL population as an indication of
field resistance as defined above. To confirm this hypothesis, 13
additional populations of S. frugiperda were collected from LA,
Georgia (GA), FL, and North Carolina (NC) during 20122013
(Table 2), which included 9 populations (2 LA, 2 GA, 5 FL) from
non-Bt maize and 4 populations from Cry1F maize. Two of the
four populations from Cry1F maize were collected from fields that
showed unexpected damage by feral populations of S. frugiperda,
which included one from FL and one from NC. The other two of
the four populations from Cry1F maize were collected from two
field trials in FL in 2012 and 2013. Diet-incorporated bioassays
showed that, relative to the Cry1F-susceptible (SS) strains, larvae
of S. frugiperda collected from non-Bt maize were 3.6- to
.85.4fold less susceptible to purified Cry1F protein (Table 2). All four
populations collected from Cry1F plants in FL and NC were
highly resistant (.85.4-fold) to Cry1F protein. No significant
mortality was observed at the Cry1F concentration of 31.6 mg/g,
the highest concentration tested, for any of the four populations.
The results confirmed that the unexpected damage by S.
frugiperda observed in the fields in FL and NC was due to
resistance to the Cry1F protein in the plants.
There also was clear evidence of Cry1F resistance in the field
when trials were conducted in 2012 and 2013 at the location in FL
where S. frugiperda were collected for the F2 screen. In 2012, an
average leaf injury rating of 4.3 (Davis 19 scale)  due to the
damage by S. frugiperda was recorded on Cry1F maize plants
during the V2V10 plant stages  (Table S3). Additional
Table 2. Susceptibility of Spodoptera frugiperda collected from multiple locations to purified Cry1F protein using diet-incorporated
% growth inhibition at
Resistance ratio 10 mg/g (Mean SEM)
0.37 (0.27, 0.49)
23.1 (17.3, 34.2)
10.9 (8.2, 15.3)
1.33 (1.00, 1.76)
4.94 (1.50, 75.6)
6.97 (4.12, 14.4)
7.35 (5.35, 10.8)
29.5 (18.9, 55.8)
20.7 (13.4, 41.0)
Larval mortality was calculated as the number of dead larvae divided by the total number of larvae in the test. During this study, three Cry1F-susceptibile (SS) strains
(SSFL, SS-LA, and SS-TX) were used as references. SS-FL was initiated from larvae collected from Hendry Co., FL in 2011; SS-LA was established from insects collected from
Franklin Parish, LA in 2008; and SS-TX was developed from insects collected from Hidalgo Co., TX in 2013. All three SS strains were highly susceptible to both Cry1F
maize plants and Cry1F protein in diet. Because the overall performance on maize plants and diet were similar among the three strains, SS was used to denote all three
strains unless mentioned specifically. FL-HD-nBt-12 was collected from a heavily infested non-Bt sweet corn field that was close to an early-planted Bt maize field. The Bt
maize field was heavily damaged by S. frugiperda and the population infesting the non-Bt sweet corn was believed to be the progeny of moths that came out of the Bt
maize field. LA-RD-24 and FL-39 were two resistant families isolated from populations from Rapides Parish, LA and Collier Co., FL, respectively, using the F2 screen.
FL-CLnBt-12, FL-CL-nBt-13, FL-CL-Bt-12, and FL-CL-Bt-13 were collected from non-Bt and Bt plants in two field trials in Collier Co., FL in 2012 and 2013. LA: Louisiana, FL:
Florida, GA: Georgia, NC: North Carolina. NBt: non-Bt maize, Bt: Bt maize. The LC50 value of a population was considered to be greater than the highest Cry1F
concentration tested if its larval mortality was ,50% at the highest concentration in the bioassays. Limited by the cost of Cry1F protein, the highest concentrations used
in the bioassays varied depending on the sources of the populations. The highest concentration assayed for LA-RD-24 and FL-39 was 100 mg/g, while it was 31.6 mg/g
for other populations. Mortality at 100 mg/g was 20.663.9% for LA-RD-24 and 0.060.0% for FL-39. Mortality at 31.6 mg/g was 7.165.1% for FL-SC-Bt-13 and zero for
FLHD-nBt-12 and all other populations collected from Bt maize plants. Resistance ratio was calculated as the LC50 of the field populations divided by that of the SS strain.
Analysis of variance for growth inhibition: F14,46 = 59.75, P,0.0001. Mean values for growth inhibition followed by a common letter were not significantly different at
a = 0.05 (Tukeys HSD test). n/a: Data are not available.
greenhouse tests showed that five out of 20 Cry1F plants each
infested with 10 F1 neonates of S. frugiperda collected from
nonBt plants in the FL field trial were heavily injured, with a leaf
injury rating of 69, and five live 4th5th instars were recovered
from the five plants (1 larva/plant) after 12 d (Table S4). In
contrast, the Cry1F plants killed all of the SS larvae placed on
them and had virtually no leaf injury. More importantly, the field
trial in 2013 showed that Cry1F plants were essentially ineffective
against the feral populations of S. frugiperda (Fig. 1). There were
no significant differences in the leaf injury ratings and the
percentage of plants containing live larvae of S. frugiperda
between the non-Bt and Cry1F maize (HX1) plants. Both non-Bt
and Cry1F plants were heavily injured by S. frugiperda, with a leaf
injury rating of 8.24 on the non-Bt maize and 8.09 on HX1 at V9
V12 and .80% plants at the R1 plant stage contained large, live
larvae (most of which were 5th instars) (Fig. 1). Diet-incorporated
bioassays showed that the larvae collected from the non-Bt maize
plants had 18.8-fold (for FL-CL-NBt-2012) and 55.9-fold
(FL-CLNBt-2013) resistance to Cry1F protein (Table 2). As described
above, for the two populations (FL-CL-Bt-2012 and
FL-CL-Bt2013) collected from the Cry1F plants, no mortality was observed
at 31.6 mg/g of diet. Thus, the performance of the Cry1F maize in
the 2012 trial showed reduced efficacy of Cry1F because the
nonBt maize plants had significantly greater leaf injury, while the 2013
trial demonstrated failure of Cry1F against S. frugiperda. The
results of the field trials confirmed that field resistance to Cry1F
maize in S. frugiperda had occurred in FL and NC.
The geographical range and distribution of Cry1F resistance in
S. frugiperda in the mainland U.S. remains unknown. A recent
independent study found an resistance allele frequency of 0.132 to
Cry1F in three samples of S. frugiperda collected from Palm
Beach and Hendry counties in FL in 2011 and 2012 . They
found no unexpected field survival, but one population collected
from Palm Beach in 2012 showed a resistance allele frequency of
0.247. Although unexpected field survival of S. frugiperda has not
been reported in LA, the resistance allele frequency (0.103) of the
LA populations detected in this study was also relatively high. The
results of our study, together with other published data, indicate
that the range of Cry1F resistance in S. frugiperda may be
geographically extensive in the southeast coastal region of the U.S.
The factors that led to the field resistance of S. frugiperda to
Cry1F maize in FL and NC are unknown. Local selection pressure
due to the planting of Bt maize appears not to be a major factor
driving the development of field resistance. In most years, S.
frugiperda in the U.S. mainland overwinters only in south FL and
south TX [2,19]. Maize is not a major crop in FL, which had a
total planting area of ,40,000 ha/year . A high proportion of
maize in the state is sweet corn, and most sweet corn does not
contain Bt genes. In addition, S. frugiperda is a polyphagous insect
with a wide host range . For these reasons, local selection
pressure by Bt maize in FL should be limited. Although it is
unclear if local selection caused by the use of Bt microbial
insecticides is a contributing factor, the more plausible reason for
the field resistance appears to be the migration of resistant
populations from Puerto Rico through other Caribbean islands to
FL. Northerly movement of FL populations along the U.S. East
Coast has been documented for years . This hypothesis is
supported by a recent comparative study of mitochondrial
haplotype ratios in S. frugiperda . The study showed that
the Puerto Rico populations of S. frugiperda had only very limited
interactions with TX populations, but could have substantial
exchanges with FL populations. In addition, the areas with
unexpected damage by S. frugiperda on Bt maize also match the
expected migration patterns of S. frugiperda from the Caribbean
islands to the mainland U.S. that were generated based on weather
While further studies are warranted to reveal the geographical
ranges and factors leading to field resistance in the U.S. mainland,
effective management of Cry1F- resistant populations of S.
frugiperda is needed to ensure the continued success of Bt crop
technologies. To generate essential information for IRM,
additional F2 screen, and laboratory, greenhouse, and field studies
were performed to analyze the cross-resistance to other commonly
used Bt proteins and Bt maize products containing single and
We analyzed the cross-resistance of S. frugiperda between HX1
and five other Bt maize products based on the survival of the 142
families in an F2 screen that was performed simultaneously with
the F2 screen against HX1 mentioned above. The five Bt maize
products included two experimental Bt maize lines, Cry1A.105Ln
(Cry1A-P) and Cry2Ab2Ln (Cry2A-P), as well as three commercial
products: Genuity VT Double (VT2P), Genuity SmartStax (SMT),
and Agrisure Viptera 3111 (VIP3). Cry1A-P and Cry2A-P
produce a single Bt protein, Cry1A.105 and Cry2Ab2,
respectively, whereas VT2P expresses both Cry1A.105 and Cry2Ab2 (Table
S1) . SMT produces six Bt proteins including the two in VT2P
and Cry1F for controlling Lepidoptera plus Cry3Bb1 and Cry34/
35Ab1 for Coleoptera. VIP3 produces three Bt proteins including
Vip3A and Cry1Ab for Lepidoptera and mCry3A for Coleoptera.
Correlation analysis showed that there was a significantly (P,
0.05) positive relationship in larval survival of the 142 families
between Cry1F maize and three other maize products, namely,
Cry1A-P, VT2P, and SMT, but not with Cry2A-P or VIP3
(Fig. 2). The correlation coefficients calculated based on larval
survivorship between HX1 and other products were 0.534 (P,
0.05) for Cry1A-P, 0.461 (P,0.05) for VT2P, and 0.491 (P,0.05)
for SMT, but only 0.021 (not significant) for Cry2A-P (Table S5).
No correlation coefficients could be calculated with VIP3 which
killed all of the F2 larvae in the 142 families and has previously
been reported to be extremely toxic towards S. frugiperda [23
25]. The results suggest that some level of cross-resistance exists
between HX1 and Cry1A-P, VT2P, and SMT, but not VIP3 and
To understand the cross-resistance patterns observed in the F2
screen, diet-incorporated bioassays were conducted to determine
the susceptibility of a known Bt-susceptible strain collected from
TX in 2013 (SS-TX) and a resistant strain (FL-39) to five
individual purified Cry proteins: Cry1Aa, Cry1Ab, Cry1Ac,
Cry1A.105, and Cry2Ab2. FL-39 was isolated from a two-parent
family of the FL population using the F2 screen mentioned above.
Relative to SS-TX, FL-39 exhibited 4.8-fold less susceptibility to
Cry1A.105, while susceptibility to Cry2Ab2 was similar between
SS-TX and FL-39 (Table 3). Individual Cry1Aa, Cry1Ab, and
Cry1Ac proteins were not very effective against either strain.
LC50s of the three proteins were $23.8 mg/g against the two
strains, and larvae of both strains showed a considerable weight
gain at 31.6 mg/g. Survival of SS-TX and FL-39 was also
evaluated in the greenhouse on whole plants of YieldGard corn
borer (YG), VIP3, and three Bt maize lines: Cry1A-P, Cry2A-P,
and Cry2Ab2Hn (Cry2A-HP, an experimental line expressing a
high level of Cry2Ab2 protein). In these tests, YG was virtually
ineffective against S. frugiperda, with an average of 58.3% plants
containing live larvae at 12 d after infestation with five neonates of
SS-TX or FL-39 per plant (Table 4). In contrast, no larvae of
either strain survived on VIP3. Cry1A-P also killed all of the
SSTX larvae, while 37.5% of the Cry1A-P plants infested with FL-39
contained live larvae. SS-TX and FL-39 survived on 18.8% and
31.3% of the Cry2A-P plants, respectively, but no survivors of
either strain were observed on Cry2A-HP. The results of the
greenhouse tests further confirmed that some level of
crossresistance exists in S. frugiperda between Cry1F and Cry1A.105,
but not between Cry1F and Cry2Ab2 or Vip3A.
The observed cross-resistance in the F2 screen between HX1
and the two pyramided products VT2P and SMT is likely due to
the similar (Cry1A.105) and/or shared (Cry1F in SMT) protein
domains in the products. VT2P and SMT contain the same
Cry1A.105 gene . Cry1A.105 is a chimeric protein
incorporating domains I and II from Cry1Ab or Cry1Ac, domain III from
Cry1F, and the C-terminal domain from Cry1Ac . Limited by
the technology available, the expression levels of Cry1F or
Cry1A.105 in the pyramided Bt maize plants were not
determined. Based on the gene structures, the overall amino acid
sequence identity of Cry1A.105 to Cry1Ac, Cry1Ab, and Cry1F is
93.6%, 90.0%, and 76.7%, respectively . As shown in both the
diet-incorporated bioassays and the whole-plant tests, both
Cry1Ab and Cry1Ac were ineffective against S. frugiperda. Thus,
if S. frugiperda develops resistance to Cry1F protein, Cry2Ab2 is
the only protein in VT2P and SMT still fully active against S.
frugiperda (with incomplete resistance to Cry1A.105). Because
Cry2Ab2 has a mode of action distinct from that of Cry1F or
28.3 (12.3, 141.8)
23.8 (14.5, 56.7)
29.2 (12.7, 347.8)
43.5 (27.6, 84.1)
17.7 (7.4, 260.4)
12.5 (5.8, 88.6)
% growth inhibition at
31.6 mg/g (mean SEM)
SS-TX was developed from insects collected from Hidalgo Co., TX in 2013 and documented to be susceptible to Cry1F maize and Cry1F protein. FL-39 was a resistant
family isolated from an FL population collected in 2011 using an F2 screen. n = total number of neonates assayed. Limited by the amount of Cry proteins available, the
highest concentrations used in some bioassays didnt cause a 50% or greater larval mortality. LC50 value of an insect strain was considered to be greater than the
highest Cry concentration assayed if its larval mortality was ,50% at the highest concentration. Mortality at 31.6 mg/g was 6.761.1% for SS-TX and 26.169.5% for FL-39
for Cry1Aa, and 24.1610.2% for SS-TX for Cry1Ac. Resistance ratios for a Cry protein were calculated by dividing the greater LC50 value by the smaller one. A negative
sign was given if the LC50 of FL-39 was smaller than that of SS-TX. Analysis of variance for growth inhibition: F4,29 = 22.19, P,0.0001 for protein; F1,29 = 12.18, P = 0.0016
for insect strain; and F4,29 = 4.79, P = 0.0043 for the interaction. Mean values followed by a common letter in a column were not significantly different at a = 0.05 (Tukeys
Leaf injury rating after 7 d
% plants containing live larvae after 12 d
Analysis of variance
F1,75 = 89.73, P,0.0001
F8,75 = 196.09, P,0.0001
F8,75 = 31.6, P,0.0001
F1,75 = 14.78, P = 0.0003
F8,75 = 66.72, P,0.0001
F8,75 = 6.39, P,0.0001
Data were pooled for three non-Bt maize hybrids/lines which included DKC 61-22 (NBt-2), N78N-GT (NBt-5), and ExpH (NBt-6). n/a: Information is not available. Mean
values followed by a common letter within a parameter measured were not significantly different at a = 0.05 (Tukeys HSD test).
Cry1A , cross-resistance between Cry2Ab2 and Cry1F or
Cry1A is unlikely [16,2831]. The results of our study are
consistent with those of a recent protein binding study  which
showed that S. frugiperda shares binding sites for Cry1A.105 and
Cry1F. The high effectiveness of VIP3 against S. frugiperda is
most likely due to the Vip3A protein. As mentioned above, neither
YG plants nor purified Cry1Ab protein are very effective against
S. frugiperda, indicating a limited activity of Cry1Ab protein in
VIP3 for the insect. Cross-resistance between Cry1F and Vip3A is
unlikely because the two proteins do not share binding sites 
and was not seen in the case of the Puerto Rico Cry1F-resistant
population of S. frugiperda .
In North and South America, pyramided Bt maize products are
becoming prevalent and thus it is necessary to know the
performance of these products in order to effectively manage
Cry1F-resistant populations of S. frugiperda. In a greenhouse trial,
we observed no larval survival of either SS-TX or FL-39 on three
pyramided Bt products (VT2P, SMT, and VIP3) (Table 4). Niu et
al.  also showed that these pyramided Bt maize products were
effective in controlling a Puerto Rico Cry1F-resistant population of
S. frugiperda in the greenhouse. To validate the performance of
these products in the field, the HX1 field trial in Collier Co., FL in
2013 was extended to include VT2P, SMT, and VIP3 along with
closely related non-Bt maize hybrids. As described above, the feral
population of S. frugiperda at the trial site (FL-CL-nBt-13 and
FLCL-Bt-13) was highly resistant to both HX1 maize (Fig. 1) and
purified Cry1F protein (Table 2). The field trial showed that the
natural population of S. frugiperda caused very limited leaf injury
on the pyramided (VT2P, SMT, and VIP3) Bt-plants (1.72.8 on
the Davis scale), with 2.520.0% of the plants containing live
larvae (Fig. 1). Some larvae could have moved between plots, but
sampling was avoided at the plot ends where this risk was high. A
positive correlation was observed between the survival in the F2
screen and the open field trial for the three pyramided products,
suggesting that the low level of cross-resistance to Cry1A.105 could
allow limited survival of Cry1F-resistant S. frugiperda on maize
plants with pyramided traits related to Cry1A.105.
Our documentation of field resistance of S. frugiperda to Cry1F
maize in the continental U.S. indicates that the Cry1F-based crop
technologies may face a great challenge due to the migration of the
Cry1F-resistant populations of S. frugiperda. It appears that
geographic isolation and withdrawal of Cry1F maize (TC1507)
from Puerto Rico  were not enough to stop the spread of
resistance. Cry1F maize was first registered in 2001 in the U.S.
and later commercially planted in Puerto Rico in 2003 for
controlling lepidopteran pests including S. frugiperda, which is the
most important maize pest in the territory [14,34]. With the
extensive use of TC1507 maize products along with several other
factors [11,25], field resistance to Cry1F maize occurred in Puerto
Rico in 2006 [14,15]. Upon an initial confirmation of field
resistance in 2006 and as a part of IRM, the commercial sale of
Cry1F maize seeds was stopped in Puerto Rico [14,35]. However,
resistance is still persistent after several years of not planting
TC1507 products [15,25,36]. In addition, unlike Bt resistance in
most other insects, the Cry1F resistance in S. frugiperda is likely
complete resistance  and not associated with any fitness costs
[16,37]. Thus, the Cry1F based Bt maize and Bt cotton products
currently planted in North and South America could be at risk.
For example, Cry1F-resistant S. frugiperda could migrate north
and damage Bt maize fields. The resistance observed in NC in this
study may be a good example of such a situation. In the southern
US, resistant populations of S. frugiperda could impact
WideStrike cotton that contains the Cry1F protein. In Brazil and
Argentina, .18 MHa of Bt crops were planted in 2013, much of it
targeted against S. frugiperda . Therefore, effective IRM for S.
frugiperda and other similar migratory polyphagous pests will
require careful consideration of their movement patterns and of
possible Bt crop deployment strategies.
Materials and Methods
Third to fifth instars of S. frugiperda were collected during
20112013 from multiple locations in four southeastern U.S.
states: LA, GA, FL, and NC. Insects collected in 2011 were used to
establish two-parent families for an F2 screen . A total of .
1,200 larvae of S. frugiperda were collected from sorghum fields in
Franklin and Rapides parishes in LA and from non-Bt sweet corn
fields in Collier Co. in south FL. Field-collected larvae were reared
individually on a meridic diet as described in Yang et al .
Newly emerged virgin male and female adults derived from the
field collections were paired. Progeny (F1) produced by each pair
were separately reared on diet and the F1 adults were sib-mated
within each two-parent family to produce F2 offspring. The
number of viable F1 pupae in each family ranged from 55 to 80
with an average of 76.561.0 (mean 6 SE) for the LA populations
and 50 to 80 with an average of 67.961.7 (mean 6 SE) for the FL
population. The F2 neonates were used in an F2 screen on Bt
maize leaf tissue as described below.
In addition, 13 field populations of S. frugiperda were collected
during 20122013 from Bt and non-Bt maize fields in 10 locations
in LA, GA, FL, and NC (Table 2). Sample size was 35 larvae for
one population (FL-CL-Bt-13) and 92300 for other populations.
Field-collected larvae were reared on a meridic diet  and F1
progeny of the field-collected populations, except NC-13, were
used to determine the susceptibility to purified Cry1F protein. For
NC-13, F3 progeny were used in the bioassay. Purified (99.9%)
Cry 1F protein was obtained from Case Western Reserve
University, Cleveland, Ohio, USA .
A total of 142 two-parent families of S. frugiperda were
established from the field collections in 2011, which included 70
families from LA and 72 from FL (Table 1). Among the 70 LA
families, 47 families were collected from Rapides Parish and 23
were from Franklin Parish. F2 neonates of the families were
screened on leaf tissues of HX1, Cry1A-P, Cry2A-P, VT2P, SMT,
and VIP3 maize as described in Yang et al . Limited by the
technology available, expression levels of Bt proteins in plants were
not measured, but Cry protein expression for a maize hybrid/line
was confirmed using the ELISA-based assays (EnviroLogix,
Quantiplate kits, Portland, ME. In each family, 96 neonates were
placed in 24 wells (4 neonates/well) (Bio-Smart-32, CD
International, Pitman, NJ) containing leaf tissue excised from
greenhouse-grown maize plants at V4V9. The decision to use
four neonates per well was based on a previous study to minimize
larval cannibalism . All bioassay trays containing maize leaf
tissue and larvae of S. frugiperda were incubated in environmental
chambers maintained at 28uC, ,50% RH and a 16-h: 8-h (L:D)
photoperiod. Fresh leaf tissue was added every 23 d. Larval
survival and development were recorded after 7 d. Live larvae
were separated into two groups based on their growth: small (1st or
2nd instars) and large ($3rd instars).
Definition of potential positive families possessing
resistance alleles to Cry1F maize
During the study, three SS strains (SS-FL, SS-LA, and SS-TX)
were used as references for laboratory bioassays and greenhouse
tests. SS-FL was initiated from larvae collected from non-Bt maize
fields in Hendry Co., FL in 2011 ; SS-LA was established from
cotton and maize fields in 2008 in LA ; and SS-TX was
developed from insects collected from non-Bt maize in TX in
2013. All three SS strains were highly susceptible to both Cry1F
maize plants and Cry1F protein in diet. Because the overall
performance on maize plants and diet were similar among the
three strains, SS was used to denote all three strains unless
mentioned specifically. Baseline survival assays showed that all
three Cry1F-susceptible strains (SS) of S. frugiperda survived well
on non-Bt maize leaf tissue after 7 d with an average survivorship
of 63.4% and a larval mass of 44.2 mg/larva (Table S6). In
contrast, on HX1 leaf tissue, only a small percentage (2.3%) of
larvae survived and all survivors were 1st or 2nd instars. The results
suggested that survivorship of large larvae ($3rd instars) in the F2
screen on HX1 leaf tissue could be used to identify potential
positive families carrying resistance alleles to Cry1F.
Correspondingly, #2nd instars that survived the F2 screen were treated as dead
larvae in determining resistance alleles.
Theoretically, if one of the two parents of a family contains a
recessive resistance allele, 6.25% of the F2 larvae are expected to
be homozygous (RR) for Bt resistance and should survive in the F2
screen . Based on the baseline survival data of SS, an average
of 3.59 [ = 96 (neonates screened)66.25% (RR frequency)659.9%
(baseline survivorship on HX1)] live larvae were expected in a
family in the F2 screen if one parent of the family possessed a
resistance allele. A x2-test showed that a survival of one larva in a
family was not significantly (P.0.05) different from the expected
survivorship (3.59 larvae/family), and thus a family with one or
more survivors was considered as a potential positive family for
resistance alleles to Cry1F maize.
Cry1F resistance confirmation
Based on the larval survival in the F2 screen, 21 of the 70 LA
families of S. frugiperda and 46 of the 72 FL families were
identified to be potential positive families (Table S7). To confirm if
a potential positive family actually possessed resistance alleles, six
strains were established from the survivors of six potential positive
families including three families (LA-RD-24, LA-RD-34, and
LARD-37) from Rapides Parish, LA and three families (FL-13,
FL37, and FL-39) from FL (Table S8). To increase the chance of
success in the strain establishments, all F2 survivors (both large and
small larvae) of a family were transferred to the diet  and
reared in varied temperatures to synchronize their development.
Progeny of the strains established were then selected on Cry1F
maize leaf tissue for 12 times using the similar methods as
described in the F2 screen. Initial confirmation for the six potential
positive families was performed by measuring larval survival of the
potential positive families and SS on HX1 leaf tissue using the
same method as described in the F2 screen. Then, resistance of
three potential positive families (LA-RD-24, LA-RD-34, and
FL39) was reconfirmed on whole plants of greenhouse-grown HX1
plants (Table S2). In the reconfirmation tests, five neonates of a
potential positive family were placed into the whorl of a plant at
the V6VT stages. Leaf injury ratings, larval survival, and larval
mass were recorded 1214 d after the initial insect infestation. In
addition, non-Bt maize and SS-FL were also included in the tests
as the controls of the experiment. A potential positive family was
considered to actually possess resistance alleles if it showed a
significant survivorship with live $3rd instars on the leaf tissue and
on whole plants in the confirmation tests.
In addition, susceptibility to purified Cry1F protein of two
families (LA-RD-34 and FL-39) that were already confirmed to be
resistant to Cry1F maize was examined, along with SS, using a
diet-incorporated bioassay  (Table 2). In the bioassay, larval
survival (both small and large larvae) and masses of live larvae
were recorded 7 d after neonate infestations. Corrected dose/
mortality data  of SS were subjected to probit analysis [38,44]
to determine LC50 and 95% CL. For the two resistant families, the
LC50 value was considered to be greater than the highest Cry
concentration (100 mg/g) tested because the larval mortalities were
,50% at 100 mg/g. Resistance ratios were calculated using the
LC50 value of a HX1-resistant strain divided by the LC50 of SS. In
addition, the percentage of larval growth inhibition at 10 mg/g was
calculated as described in Huang et al . Growth inhibition
data were analyzed using a one-way analysis of variance
(ANOVA) . Comparison among insect strains was made
using the Tukeys HSD test at a = 0.05.
Estimate of Cry1F resistance allele frequency
Results of the resistance confirmation studies showed that all six
potential positive families examined possessed resistance alleles
against HX1 maize plants. Diet-incorporated bioassays further
confirmed that the survival of S. frugiperda on HX1 maize plants
was due to resistance to the Cry1F protein in the plants.
Therefore, all of the 21 LA and 46 FL potential positive families
identified in the F2 screen were considered to carry resistance
alleles. Revisiting the F2 screen data (Table S7), we found that the
survivorship of F2 progeny of some families in the F2 screen was
much greater than the expected survival of 3.59 larvae/family,
suggesting that there was .1 resistance allele in the two parents of
some families. To accurately estimate the resistance allele
frequency, a x2-test with the assumption of single-gene Mendelian
inheritance was used to determine the number of resistance alleles
in the two parents of each family (Tables S7, S9, S10, S11). We
then framed a Bayesian statistical model [39,46,47] as a
multinomial problem to calculate the expected resistance allele
frequency and the corresponding 95% CI (Methods S1).
Susceptibility of field populations of S. frugiperda to
The surprisingly high Cry1F resistance allele frequency in the
populations of S. frugiperda detected in the F2 screen, especially
for the FL population, suggests that it should be possible to detect
resistance using a convenient dose-response bioassay method .
During 20122013, a total of 13 field populations of S. frugiperda
were collected from LA, GA, FL, and NC (Table 2). Susceptibility
of these field populations, along with SS, to purified Cry1F protein
was determined using the diet-incorporated bioassay method as
described above. Limited by the cost of Cry1F protein, these
populations were assayed with Cry1F concentrations up to only
31.6 mg/g. LC50s and larval growth inhibition (%) were analyzed
using the methods mentioned above.
Survival and leaf injury of natural populations of S.
frugiperda on non-Bt and HX1 under field and
Larval survival and plant injury of natural populations of S.
frugiperda were evaluated in 2012 and 2013 in the same field (26u
289N, 81u 269W) at the Southwest Florida Research and
Education Center, University of Florida in Collier Co., FL where
insects were collected for the F2 screen. The field trials were
permitted by the Southwest Florida Research and Education
Center, University of Florida. The field work did not involve any
endangered or protected species. No human participants,
specimens or tissue samples, or vertebrate animals, embryos or tissues
were involved in the study. A randomized completely block (RCB)
design with four replications was used in both years. There were
200 plants/replication in 2012 and 504 plants/replication in 2013.
Only an HX1 hybrid and a closely related non-Bt maize hybrid
were included in the trial in 2012, while the test in 2013 also
contained three pyramided Bt maize traits (VT2P, SMT, and
VIP3) along with closely related non-Bt maize hybrids (Fig. 1).
Leaf injury by S. frugiperda was rated using Davis 19 scale 
in V2V10 plant stage for the trial in 2012 and V9V12 plant
stages for the trial in 2013. In addition, in the 2013 trial, larval
occurrence of S. frugiperda was recorded at the R1 plant stage,
when the plants showed maximum leaf injury. Occurrence of S.
frugiperda was not recorded for the trial in 2012. Transformed
data  on leaf injury ratings and percentage plants containing
live larvae were analyzed using a one-way ANOVA .
Treatment means for each trial were separated using Tukeys
HSD test at a = 0.05.
In addition, susceptibility to Cry1F protein of field-collected
populations (F1) from non-Bt and Bt maize plants of the two trials
was determined using the diet-incorporated bioassay as described
above. Larval survival and plant injury of the field population (F1
of FL-CL-nBt-12) collected from non-Bt maize plants in the trial in
2012 along with SS were also tested on whole plants of
greenhouse-grown HX1 and non-Bt maize plants to demonstrate
the biological activity of HX1 against susceptible S. frugiperda
and resistance in the field-collected population. In the greenhouse
tests, 10 neonates of FL-CL-nBt-12 and SS were placed into the
whorl of a plant at V5V7. Larval survival and leaf injury ratings
were recorded at 12 d after insect infestation. A RCB was used in
the test with four replications and 5 plants/replication.
Transformed data  were analyzed using a two-way ANOVA (38).
Treatment means were separated using Tukeys HSD test at
a = 0.05.
Determination of cross-resistance
Cross-resistance of Cry1F-resistant S. frugiperda was examined
using two methods. First, correlation and regression analyses 
were performed to examine if there were significant relationships
in the survivorship of the 142 families of S. frugiperda in the F2
screen on leaf tissue of HX1 and the five other Bt maize products.
A significant positive correlation would suggest the existence of
cross-resistance among Bt maize products. Second, susceptibility
of FL-39 and SS-TX to five common individual Cry proteins
(Cry1Aa, Cry1Ab, Cry1Ac, Cry1A.105, and Cry2Ab2) was
determined using the diet-incorporated bioassay method as
described above. All five proteins were provided by Monsanto
Company (St. Louis, MO, USA).
Larval survival and plant injury of SS and Cry1F-resistant
S. frugiperda on Bt maize plants containing single and
Both greenhouse and field studies were utilized to evaluate if
pyramided Bt maize and other related traits were effective against
the Cry1F- resistant S. frugiperda. In the greenhouse study, larval
survival and plant injury of FL-39 and SS-TX were investigated
on four non-Bt and eight Bt maize products using a method similar
to that described in Niu et al . The eight Bt maize products
included five commercial products (HX1, YG, VT2P, SMT, and
VIP3) and three experimental lines (Cry1A-P, Cry2A-P, and
Cry2A-HP). Expression/non-expression of Bt proteins for a maize
hybrid/line was also confirmed using the ELISA-based assays
mentioned above. The four non-Bt maize products were
genetically closely related to 12 of the Bt products. At the V5
V7 plant stages, five neonates of a S. frugiperda strain were placed
into the whorl of a plant. Leaf injury was rated with Davis 19
scale at 7 d after larval release, and larval survival was recorded
after 12 d. A RCB was used in the tests containing four
replications with four plants/replication. Transformed data 
were analyzed using a two-way ANOVA . Treatment means
were separated using Tukeys HSD test at a = 0.05.
Non-Bt and Bt maize products evaluated in
Table S2 Plant injury and larval survival of a
Cry1Fsusceptible (SS-FL) strain and three Cry1F-resistant
families (LA-RD-24, LA-RD-34, and FL-39) of Spodoptera
frugiperda on whole plants of non-Bt and Cry1F maize
hybrids in the greenhouse.
Table S3 Leaf injury ratings (mean SEM) of non-Bt
and HX1 plants caused by feral populations of
Spodoptera frugiperda in a field trial in Collier Co., FL in 2012.
Table S4 Leaf injury rating and survival (mean SEM)
of Cry1F-susceptible strain (SS-FL) and a field
population (FL-CL-NBt-2012) of Spodoptera frugiperda collected
from non-Bt maize from a field in Collier Co., FL in 2012
and tested in the greenhouse.
Table S5 Correlation coefficients of larval survivorship
of 142 two-parent families of Spodoptera frugiperda in
an F2 screen with maize leaf tissue containing single or
pyramided Bt genes.
Table S6 Baseline survivorship (mean SEM) of a
susceptible strain (SS-FL) of Spodoptera frugiperda on
leaf tissue of Bt and non-Bt maize plants.
Table S7 Potential positive families (PPF) possessing
resistance alleles (RAs) to Cry1F maize that were
identified in the F2 screen in three populations of
Spodoptera frugiperda collected from Louisiana (LA)
and Florida (FL).
Table S8 Larval survivorship (%) of potential positive
families of Spodoptera frugiperda on leaf tissue of Cry1F
Table S9 Baseline survival (mean SEM) of
Cry1Fsusceptible (SS-FL), -resistant (RR), and -heterozygous
(RS) genotypes of Spodoptera frugiperda on leaf tissue of
HX1 and non-Bt (NBt) maize plants.
Table S10 Expected frequency of genotypes of in the F2
progeny of a two-parent family of Spodoptera
Table S11 Six different parental (P1) crosses of
twoparent families and the related frequencies and genetic
This article is published with the approval of the Director of the Louisiana
Agricultural Experiment Station as manuscript No. 2014-234-15414.
Conceived and designed the experiments: FH JAQ GPH. Performed the
experiments: FH JAQ RLM DDR XN DK GDB YN FY VD. Analyzed
the data: FH DAA JAQ. Contributed reagents/materials/analysis tools:
FH JAQ RLM DDR GPH DAA XN DK GDB. Contributed to the writing
of the manuscript: FH GPH JAQ DAA. Discussed the results and
commented on the manuscript: FH JAQ RLM DDR GPH DAA XN DK
GDB YN FY VD.
1. Sparks AN ( 1979 ) A review of the biology of the fall armyworm . Fla Entomol 62 : 82287 .
2. Pashley DP ( 1988 ) The current status of fall armyworm host strains . Fla Entomol 71 : 2272234 .
3. Buntin D , Flanders K ( 2012 ) Bt corn products for the southeastern United States . http://www.caes.uga.edu/commodities/fieldcrops/gagrains/ documents/2012BtcornSEBtcorntraitstableNov21.pdf. Accessed 17 July 2014 .
4. Frizzas MR , Neto SS , de Oliveira CM , Omoto C ( 2014 ) Genetically modified corn on fall armyworm and earwig populations under field conditions . Ciencia Rural 44 : 2032209 .
5. NASS (National Agricultural Statistics Service) ( 2013 ). Acreage . USDA, Washington DC. USA. http://usda01.library.cornell.edu/usda/current/Acre/ Acre- 06 - 28 - 2013 .pdf. Accessed 17 July 2014 .
6. James C ( 2013 ) Global status of commercialized biotech/GM crops: 2013 . ISAAA Brief No . 45. ISAAA: Ithaca, NY, USA.
7. Gould F ( 1998 ) Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology . Ann Rev Entomol 43 : 7012726 .
8. Matten SR , Frederick RJ , Reynolds AH ( 2012 ) United States Environmental Protection Agency insect resistance management programs for plant-incorporated protectants and use of simulation modeling . In: Wozniak CA, McHughen A , editors. Regulation of agricultural biotechnology: The United States and Canada . Springer. 1752267.
9. Tabashnik BE ., Brevault T , Carrie`re Y ( 2013 ) Insect resistance to Bt crops: lessons from the first billion acres . Nat Biotechnol 3 : 5102521 .
10. US-EPA (U.S. Environmental Protection Agency) ( 2001 ) Biopesticide registration action document: Bacillus thuringiensis plant-incorporated protectants . http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad2/1-overview.pdf. Accessed 17 July 2014 .
11. Huang F , Andow DA , Buschman LL ( 2011 ) Success of the high dose/refuge resistance management strategy after 15 years of Bt crop use in North America . Entomol Exp App 140 : 1216 .
12. Tabashnik BE , van Rensburg JBJ , Carriere Y ( 2009 ) Field-evolved insect resistance to Bt crops: definition, theory, and data . J Econ Entomol 102 : 2011 - 2025 .
13. Sumerford DV , Head GP , Shelton A , Greenplate J , Moar W ( 2013 ) Fieldevolved resistance: assessing the problem and ways to move forward . J Econ Entomol 106 : 152521534 .
14. Storer NP , Babcock JM , Schlenz M , Meade T , Thompson GD , et al. ( 2010 ) Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico . J Econ Entomol 103 : 103121038 .
15. Storer NP , Thompson GD , Head GP ( 2012 ) Application of pyramided traits against lepidoptera in insect resistance management for Bt crops . GM Crop 3 : 1542162 .
16. Velez AM , Spencer TA , Alves AP , Moellenbeck D , Meagher RL , et al. ( 2013 ) Inheritance of Cry1F resistance, cross-resistance and frequency of resistant alleles in Spodoptera frugiperda (Lepidoptera: Noctuidae) . Bull Entomol Res 103 : 7002713 .
17. Davis FM , Ng SS , Williams WP ( 1992 ) Visual rating scales for screening whorlstage corn for resistance to fall armyworm . Miss Agric Forestry Exp Stn Tech Bull 186.
18. Ritchie S W , Hanway JJ , Benson GO , Herman JC ( 1993 ) How a corn plant develops . Iowa State Uni Coop Ext Ser Sp Rep No . 48, Ames, Iowa, USA.
19. Capinera JL ( 1999 ) Fall armyworm . Uni. Fla. Publi. no. EENY-98 . http:// entnemdept.ufl.edu/creatures/field/fall_armyworm.htm. Accessed 17 July 2014 .
20. Mitchell ER , McNeil JN , Westbrook JK , Silvant JF , Lalanne-Cassou B , et al. ( 1991 ) Seasonal periodicity of fall armyworm (Lepidoptera: Noctuidae) in the Caribbean basin and northward to Canada . J Entomol Sci 26 : 39251 .
21. Nagoshi RN , Meagher RL , Jenkins DJ ( 2010 ) Puerto Rico fall armyworm has only limited interactions with those from Brazil or Texas but could have substantial exchanges with Florida populations . J Econ Entomol 103 : 3602367 .
22. DiFonzo C , Cullen E ( 2012 ) Handy Bt trait table . http://www3.ag.purdue.edu/ agry/PCPP/Documents/Entry%20forms/Handy_Bt_Trait_Table.pdf. Accessed 17 July 2014 .
23. Burkness EC , Dively G , Patton T , Morey AC , Hutchison WD ( 2010 ) Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions . GM Crop 1 : 1 - 7 .
24. Yang F , Huang F , Qureshi JA , Leonard BR , Niu Y , et al. ( 2013 ) Susceptibility of Louisiana and Florida populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) to transgenic Agrisure Viptera 3111 maize . Crop Protect 50 : 37239 .
25. Niu Y , Yang F , Dangal V , Huang F ( 2014 ) Larval survival and plant injury of Cry1F-susceptible, -resistant, and -heterozygous fall armyworm (Lepidoptera: Noctuidae) on non-Bt and Bt corn containing single or pyramided genes . Crop Protect 59 : 22228 .
26. Biosafety Cleaning-House. Gene and DNA sequence, Cry1A .105. https://bch. cbd.int/database/record.shtml?documentid=43771. Accessed 10 October 2014 .
27. Storer NP , Kubiszak ME , King JE , Thompson GD , Santos AC ( 2012 ) Status of resistance to Bt maize in Spodoptera frugiperda: Lessons from Puerto Rico . J Invert Pathol 110 : 2942300 .
28. Sivasupramaniam S , Moar WJ , Ruschke LG , Osborn JA , Jiang C , et al. ( 2008 ) Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests . J Econ Entomol 101 : 546 - 554 .
29. Brevault T , Prudent P , Vaissayre M , Carrie`re Y ( 2009 ) Susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry1Ac and Cry2Ab2 insecticidal proteins in four countries of the West African cotton belt . J Econ Entomol 102 : 2301 - 2309 .
30. Wu X , Leonard BR , Zhu Y-C , Abel CA , Head GP , et al. ( 2009 ) Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins . J Invert Pathol 100 : 29234 .
31. Sumerford DV , Head GP ( 2014 ) Patterns of cross resistance among insecticidal Bacillus thuringiensis proteins and their implications for the durability of pyramided Bt crops. Crop Prot . In press.
32. Hernandez-Rodrguez CS, Hernandez-Martnez P. Van Rie J , Escriche B , Ferre J ( 2013 ) Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda . PLoS ONE 8 ( 7 ), e68164. doi:10.1371/journal.pone.0068164.
33. Sena JDA , Hernandez-Rodriguez CS , Ferre J ( 2009 ) Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites . App Environ Microbiol 75 : 223622237 .
34. US-EPA (U.S. Environmental Protection Agency) ( 2007 ) TC1507 maize and fall armyworm in Puerto Rico , MRID 47176001. USEPA, Washington, DC, USA.
35. Matten SR , Head GP , Quemada HD ( 2008 ) How governmental regulation can help or hinder the integration of Bt crops within IPM programs . In: Romeis J, Shelton AM , Kennedy GG editors. Integration of insect resistant genetically modified crops with IPM programs . Springer USA. 27239.
36. Niu Y , Meagher Jr RL , Yang F , Huang F ( 2013 ) Susceptibility of field populations of the fall armyworm (Lepidopteran: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and corn leaf tissue containing single and pyramided Bt genes . Fla Entomol 96 : 7012713 .
37. Jakka SRK , Knight VR , Jurat-Fuentes JL ( 2014 ) Fitness costs associated with field-evolved resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae) . J Econ Entomol 107 : 3422351 .
38. SAS Institute Inc ( 2010 ) SAS/STAT: 9.3 User's Third Edition . Cary, NC, USA.
39. Andow DA , Alstad DN ( 1998 ) F2 screen for rare resistance alleles . J Econ Entomol 91 : 5722578 .
40. Yang F , Qureshi JA , Leonard BR , Head GP , Niu Y , et al. ( 2013 ) Susceptibility of Louisiana and Florida populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) to pyramided Bt corn containing Genuity VT Double Pro and SmartStax traits . Fla Entomol 96 : 714 - 723 .
41. Zhang L , Huang F , Leonard BR , Chen M , Clark T , et al. ( 2013 ) Susceptibility of Cry1Ab maize.-resistant and -susceptible strains of sugarcane borer (Lepidoptera: Crambidae) to four individual Cry proteins . J Invertebr Pathol 112 : 2672 272.
42. Hardke JT , Leonard BR , Huang F , Jackson RE ( 2011 ) Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis Cry proteins . Crop Protect 30 : 168 - 172 .
43. Abbott WS ( 1925 ) A method of computing the effectiveness of an insecticide . J Econ Entomol 18 : 2652267 .
44. Finney DJ ( 1971 ) Probit analysis (Cambridge University Press, England).
45. Huang F , Ghimire MN , Leonard BR , Zhu Y-C , Head G ( 2012 ) Susceptibility of field populations of sugarcane borer from non-Bt and Bt maize plants to five individual Cry toxins . Ins Sci 19 : 5702578 .
46. Andow DA , Alstad DN ( 1999 ) Credibility interval for rare resistance allele frequencies . J Econ Entomol 92 : 7552758 .
47. Stodola TJ , Andow DA ( 2004 ) F2 screen variations and associated statistics . J Econ Entomol 97 : 175621764 .
48. Huang F Detection and monitoring of insect resistance to transgenic Bt crops (2006) Ins Sci 13 : 73284 .