Baseline Susceptibility and Monitoring of Brazilian Populations

of Spodoptera frugiperda (Lepidoptera: Noctuidae) and Diatraea

saccharalis (Lepidoptera: Crambidae) to Vip3Aa20 Insecticidal
Protein
Author(s): Oderlei Bernardi, Douglas Amado, Renan S. Sousa, Fabiana Segatti, Julio
Fatoretto, Anthony D. Burd and Celso Omoto
Source: Journal of Economic Entomology, 107(2):781-790.
Published By: Entomological Society of America
URL: http://www.bioone.org/doi/full/10.1603/EC13374

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 INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Baseline Susceptibility and Monitoring of Brazilian Populations of

Spodoptera frugiperda (Lepidoptera: Noctuidae) and Diatraea
saccharalis (Lepidoptera: Crambidae) to Vip3Aa20 Insecticidal Protein
ODERLEI BERNARDI,1 DOUGLAS AMADO,1 RENAN S. SOUSA,1 FABIANA SEGATTI,2
JULIO FATORETTO,3 ANTHONY D. BURD,4 AND CELSO OMOTO1,5

J. Econ. Entomol. 107(2): 781Ð790 (2014); DOI: http://dx.doi.org/10.1603/EC13374

ABSTRACT The genetically modiÞed maize expressing Vip3Aa20 insecticidal protein from Bacillus
thuringiensis Berliner is a biotechnological option for the control of Spodoptera frugiperda (J.E. Smith)
and Diatraea saccharalis (F.) in Brazil. To support an Insect Resistance Management program, we
conducted studies of baseline susceptibility and monitoring of Brazilian populations of S. frugiperda
and D. saccharalis to the Vip3Aa20 insecticidal protein. Neonates were exposed to Vip3Aa20 applied
on artiÞcial diet surface. Mortality and growth inhibition were assessed after 7 d. All populations were
susceptible to Vip3Aa20. The LC50 ranged from 92.38 to 611.65 ng Vip3Aa20/cm2 for 16 populations
of S. frugiperda (6.6-fold variation), and between 61.18 and 367.86 ng Vip3Aa20/cm2 for 6 populations
of D. saccharalis (sixfold variation). The EC50 ranged from 21.76 to 70.09 and 48.65 to 163.60 ng
Vip3Aa20/cm2 for S. frugiperda and D. saccharalis, respectively. There was a low interpopulation
variation in susceptibility to Vip3Aa20, which represents the natural geographic variation in the
response, and not the variation caused by previous exposure to selection pressure. For these two pests,
the diagnostic concentrations of 2,000 and 3,600 ng of Vip3Aa20/cm2 caused high mortality. These
diagnostic concentrations will be used in resistance monitoring programs in Brazil.

KEY WORDS fall armyworm, sugarcane borer, Vip3A, Bt corn, insect resistance management

In Brazil, corn (Zea mays L.) is planted on ⬎14 million
hectare per year distributed in two planting times
called the Þrst and second season, and in areas with
center pivot irrigation such as that of the Brazilian
Midwestern region, the corn is also cultivated during
the winter season (Barros et al. 2010). Among the pests
attacking this crop, the fall armyworm, Spodo-
ptera frugiperda (J.E. Smith) (Lepidoptera: Noctu-
idae), and the sugarcane borer, Diatraea saccharalis
(F.) (Lepidoptera: Crambidae), stand out as the main
lepidopteran pests. S. frugiperda is the most destruc-
tive species of corn, mainly in the Central and South
American countries, causing severe losses in yield
(Pogue 2002). However, D. saccharalis is the main
species of corn stalk borer in South America (Capin-
era 2001). This insect pest has a great potential to
damage corn (Cruz 2007), but its occurrence has been
limited because of drastic reduction in populations
caused by intense adoption of Bt corn by farmers.
1 Departamento de Entomologia e Acarologia, Escola Superior de
Agricultura “Luiz de Queiroz” (ESALQ/USP), Av. Pádua Dias 11,
Piracicaba, São Paulo 13418 Ð900, Brazil.
2 Syngenta Seeds Ltda, Rodovia BR 452, Km 142, Uberlândia, Minas
Gerais 38400-974, Brazil.
3 Syngenta Crop Protection, Av. Nações Unidas 18001, Santo
Amaro, São Paulo 04795-900, Brazil.
4 Syngenta Crop Protection, Greenboro P.O. Box 18300, Greens-
boro, NC 27419.
5 Corresponding author, e-mail: celso.omoto@usp.br.
The main Integrated Pest Management (IPM) strat-
egy for control of S. frugiperda and D. saccharalis has
been the use of corn hybrids that express Bacillus
thuringiensis (Berliner) (Bt) proteins, which cur-
rently is grown on ⬎85% of the corn-growing area in
Brazil (Céleres 2012). Most corn events for commer-
cial use express Cry proteins (␣-endotoxins), and only
two events express Vip proteins (Vegetative Insecti-
cidal Protein). The Bt corn events that express Vip
proteins are MIR 162 (Vip3Aa20) and Bt11 ⫻ MIR
162 ⫻ GA21 (Cry1Ab ⫹ Vip3Aa20 ⫹ mEPSPS; Comis-
são Técnica Nacional de Biossegurança [CTNBio]
2009).
The Vip insecticidal proteins present a different
mode of action compared with the ␣-endotoxins,
which offer a promising alternative for Insect Resis-
tance Management (IRM) (Lee et al. 2003). These
proteins are exotoxins produced and secreted during
the vegetative growing stage of B. thuringiensis, and
present a mode of action distinct from the Cry toxins,
which are produced in the sporulation stage of B.
thuringiensis (Estruch et al. 1996, Selvapandiyan et al.
2001, Kurtz 2010). Moreover, the Vip insecticidal pro-
teins have distinct binding properties and lack se-
quence homology with any Cry proteins (Estruch et
al. 1996, Lee et al. 2003). These differences cause pore
formation with unique properties, indicating a low
potential for cross-resistance (Jackson et al. 2007, Sena

0022-0493/14/0781Ð0790$04.00/0 䉷 2014 Entomological Society of America

782 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2

Table 1. Identification, location, and date of collection of S. frugiperda and D. saccharalis populations from major Brazilian cornfields
used to establish the baseline susceptibility data to Vip3Aa20 insecticidal protein

Population
City, state Farm Latitude Longitude Date
code
S. frugiperda
Lab Sete Lagoas, MG Embrapa Milho e Sorgo 19⬚28⬘05⬙ S 44⬚14⬘51⬙ W Ð
RS Santo Ângelo, RS Tabuleiro 28⬚18⬘01⬙ S 54⬚15⬘48⬙ W Dec. 2011
SC Chapecó, SC Irineu Igatto 27⬚05⬘48⬙ S 52⬚37⬘07⬙ W Feb. 2013
PR-1 Castro, PR Chacará Azaléia 24⬚47⬘32⬙ S 50⬚00⬘42⬙ W Jan. 2012
PR-2 Arapoti, PR Van Noort 24⬚08⬘43⬙ S 49⬚49⬘07⬙ W Jan. 2012
SP Palmital, SP Santa Lúcia 22⬚47⬘30⬙ S 50⬚12⬘18⬙ W May 2012
MS Dourados, MS Boa Vista 22⬚13⬘18⬙ S 54⬚48⬘23⬙ W April 2012
MG Uberlândia, MG Cossissa 18⬚54⬘41⬙ S 48⬚15⬘44⬙ W Dec. 2011
GO-1 Rio Verde, GO Oliveiras 17⬚47⬘22⬙ S 50⬚53⬘13⬙ W Dec. 2011
GO-2 Planaltina, GO Agro-Garça 15⬚29⬘02⬙ S 47⬚38⬘21⬙ W Dec. 2011
BA-1 Luṍs Eduardo Magalhães, BA Campanholi 12⬚05⬘58⬙ S 45⬚47⬘54⬙ W Dec. 2011
BA-2 Barreiras, BA Sete Belo 12⬚08⬘51⬙ S 44⬚59⬘42⬙ W April 2012
MT-1 Campo Verde, MT Pirassununga 15⬚32⬘44⬙ S 55⬚09⬘58⬙ W April 2012
MT-2 Sinop, MT Aeroporto 11⬚52⬘23⬙ S 55⬚29⬘53⬙ W April 2012
TO Palmas, TO Rodeio 10⬚10⬘08⬙ S 48⬚19⬘54⬙ W June 2012
PI Uruçuṍ, PI Boa Esperança 7⬚13⬘46⬙ S 44⬚33⬘22⬙ W Jan. 2013
D. saccharalis
Lab Ð Ð Ð Ð Ð
RS Santo Antônio do Planalto, RS Capão da Moeda 28⬚24⬘04⬙ S 52⬚41⬘37⬙ W April 2011
PR Sabáudia, PR Santa Maria 23⬚19⬘28⬙ S 51⬚33⬘53⬙ W Mar. 2012
SP Jaboticabal, SP Santo Antônio 21⬚15⬘19⬙ S 48⬚19⬘21⬙ W May 2012
MS Douradina, MS Boa Vista 22⬚02⬘59⬙ S 54⬚36⬘42⬙ W May 2012
MT Campo Verde, MT Agropecuária Otelhar 15⬚32⬘44⬙ S 55⬚09⬘58⬙ W April 2011

et al. 2009, Kurtz 2010, Gouffon et al. 2011). In this
context, the Vip insecticidal proteins may be efÞcient
in the control of insects resistant to Cry proteins, and
could be introduced in crops that express Cry proteins
to produce an exceptionally robust stacking.
The presence of alleles conferring resistance to
Vip3A in different insect populations has already been
documented. A high intensity of resistance (⬎1,000-
fold) to a Vip insecticidal protein was documented in
Heliothis virescens (F.) population selected in labora-
tory; but this resistance was unstable in the absence of
exposure to the insecticidal protein, suggesting the
existence of a Þtness cost associated to resistance
(Gulzar et al. 2012). Another study reported a high
frequency of resistance alleles in Þeld populations of
Helicoverpa armigera (Hübner) and Helicoverpa punc-
tigera (Wallengren) before commercial release of Bt
cotton, which expresses Vip3A in Australia (Mahon et
al. 2012). Therefore, the risk of resistance evolution to
other target pests should be considered mainly in
situations with intensive Brazilian corn production
systems.
In Brazil, because of tropical climatic conditions,
crops can be cultivated all year round. Therefore, the
selection pressure is very intense and evolution of
resistance to any pest control agent is much higher
than in temperate climate regions. Resistance of S.
frugiperda to conventional insecticides, such as pyre-
throids and organophosphates, has already been doc-
umented in Brazil (Diez-Rodrṍguez and Omoto 2001,
Carvalho et al. 2013). For Bt crops, the adoption of
refuge strategy by the growers has been very low in
Brazil (C. O., personal communication), which can
speed up the resistance evolution. Therefore, the re-
sistance monitoring of target pests to Vip3Aa20 will be
an important step for implementing IRM programs. It
is crucial to understand the natural response of distinct
geographic populations of the target pests, by char-
acterizing the baseline susceptibility (Fischhoff 1996).
With baseline susceptibility data, it is possible to estimate
the diagnostic concentrations for the resistance moni-
toring, which allows identifying potential changes in the
susceptibility of the populations or small changes in the
resistance gene frequency in response to the selection
pressure exerted by the Bt plant (Sims et al. 1996). Based
on this, the objective of the current study was to establish
a baseline susceptibility to Vip3Aa20 insecticidal protein
in geographically distinct populations of S. frugiperda
and D. saccharalis from major corn-growing regions in
Brazil and to validate the diagnostic concentrations for
resistance monitoring programs.
Materials and Methods
Populations. For establishing the baseline suscep-
tibility data to the Vip3Aa20 insecticidal protein, pop-
ulations of S. frugiperda and D. saccharalis were col-
lected in distinct geographic regions of greater
representativity of corn growing in Brazil (Table 1 and
Fig. 1). In regions where corn is cultived in two season
per year and in large areas, two populations of S.
frugiperda were evaluated. All populations were col-
lected in commercial plantings of non-Bt corn (500 Ð
700 larvae per species), with the exception of the S.
frugiperda population collected in Piauṍ State (popu-
lation PI), which was collected in soybean. After the
collections, S. frugiperda and D. saccharalis larvae
were taken to the laboratory and kept on artiÞcial diets
proposed by Kasten et al. (1978) and King and Hartley
(1985). In addition to the Þeld populations, a suscep-
tible reference population (Lab) of each pest also was
tested. The Lab populations were being kept in the
April 2014 BERNARDI ET AL.: S. frugiperda AND D. saccharalis SUSCEPTIBILITY TO Vip3Aa20 783

RR
AP

AM PA
MA CE RN

PB
PI
AC PE

AL
RO TO
SE
BA
MT

Pop. code State GO

DF
RS Rio Grande do S ul

SC Santa Catarina
MG
PR Paraná MS
ES
SP São Paulo
SP
MS Mato Grosso do Sul
RJ
MG Minas Gerais PR
N
GO Goiás
BA Bahia SC Spodoptera frugiperda
MT Mato Grosso
RS Diatraea saccharalis
TO Tocantins

PI Piauí

Fig. 1. Locations of collection of S. frugiperda and D. saccharalis populations from major cornÞelds (gray area) in Brazil
used to establish the baseline susceptibility data to Vip3Aa20 insecticidal protein.

laboratory for ⬎10 yr, free of selection pressure by
insecticides and Bt proteins.
Baseline Susceptibility in Diet Overlay Bioassays.
The Vip3Aa20 insecticidal protein was provided by
Syngenta Seeds Ltd. (Uberlandia, Brazil) with 86.5%
of purity and stored in a freezer at ⫺20⬚C. For the
bioassays, we used the artiÞcial diet proposed by Kas-
ten et al. (1978), commonly used for rearing S. fru-
giperda. This same diet was used in the bioassays with
D. saccharalis because it is easier to prepare and the
larval growth is comparable with the standard diet.
After preparation, the diet was poured on the bioassay
trays (BIO-BA-128, CD International Inc., Pitman,
NJ), containing 128-wells (1 ml per well). Afterwards,
the Vip3Aa20 protein was diluted in distilled water to
prepare the different concentrations to be tested. Tri-
ton X-100 at 0.1% was added to obtain a uniform spread
of the solution over the diet surface. The control
treatment was composed of distilled water ⫹ surfac-
tant. For each population of S. frugiperda and D. sac-
charalis, Þve to eight concentrations of Vip3Aa20 were
tested, which were applied on the diet surface with a
replication pipette (30 ␮l per well). The diet surface
area in each well was 1.5 cm2. After a drying period,
one S. frugiperda or D. saccharalis neonate larvae
(0 Ð24 h old) was added to each well using a Þne brush.
The trays were sealed with self-adhesive plastic sheets
(BIO-CV-16, CD International Inc.) that allow for gas
exchange with the external environment and then
placed in a climatic chamber (temperature 27 ⫾ 1⬚C,
60 ⫾ 10% relative humidity, and a photoperiod of 14:10
[L:D] h). The bioassays were repeated twice for each
population, with each concentration being repeated
twice per bioassay (a total of four replications of 16
neonates per concentration). The biological activity
of the Vip3Aa20 insecticidal protein was assessed after
7 d. Mortality (with larvae that did not go beyond the
Þrst instar also being considered dead) and the weight
of the surviving larvae were used as response criteria.
Validation of Candidate Diagnostic Concentra-
tions. The bioassay procedure for the validation of the
diagnostic concentrations of Vip3Aa20 insecticidal
protein for resistance monitoring programs was iden-
tical to the previously described. The bioassays used a
reference susceptible population (Lab) of each target
pest, and Þeld populations of S. frugiperda and D.
saccharalis. The Þeld populations were from regions
with the greater representativity of the corn growing
area in Brazil, during the second season of 2011Ð2012
(nine and Þve populations of S. frugiperda and D.
784 JOURNAL OF ECONOMIC ENTOMOLOGY
saccharalis, respectively) and the Þrst season of 2012Ð
2013 (10 populations of S. frugiperda). In the Þrst
season of 2012Ð2013, D. saccharalis were not collected
because there was a low incidence of the pest, even in
areas of cultivation of non-Bt corn, making sampling
of a representative number of insects difÞcult. For
each pest species two diagnostic concentrations of
Vip3Aa20 insecticidal protein were designated, which
were deÞned from the joint analysis of the baseline
susceptibility data. For each diagnostic concentration
1,024 neonates per population were tested (64 repli-
cations of 16 neonates per diagnostic concentration
and 4 replications of 16 neonates in the control treat-
ment). The mortality was assessed after 7 d, using the
same criteria described above.
Statistical Analysis. To estimate the lethal concen-
tration (LC50 and LC90) and the respective CIs, the
concentrationÐmortality data of each population were
submitted to Probit analysis (PROC PROBIT, SAS
Institute 2000, Cary, NC). A likelihood ratio test was
conducted to test the hypothesis that the LCp values
(lethal concentration at which a percentage mortality
P is attained) were equal. If the hypothesis was re-
jected, pairwise comparisons were performed and the
signiÞcance was declared if CIs did not overlap (Savin
et al. 1977, Robertson et al. 2007). The signiÞcance of
differences among slopes was determined by likeli-
hood ratio test for parallelism and equality (Savin et al.
1977). For the estimate of the diagnostic concentra-
tions, the concentrationÐmortality data of all popula-
tions were analyzed jointly, according to the method
proposed by Sims et al. (1996). In the joint analysis,
mortality data were Þtted with a binomial model using
the logÐlog complement connection function (gom-
pit; PROC PROBIT, SAS Institute 2000). Through this
analysis, LC99 and the respective CIs were estimated.
The candidate diagnostic concentrations for the re-
sistance monitoring of S. frugiperda and D. saccharalis
to Vip3Aa20 insecticidal protein were designated
based on the upper CIs of ⬇2 times the estimate of the
LC99. For the validation of the candidate diagnostic
concentrations, the mortality percentage (x) of S. fru-
giperda and D. saccharalis in each diagnostic concen-
tration and population was transformed in arcsine
冑x/100, submitted to the variance analysis and the
means were compared by the Tukey test (P ⱕ 0.05;
PROC ANOVA, SAS Institute 2000).
For the estimate of effective concentration (EC50
and EC90) and respective CIs, the weight of the sur-
viving larvae in each concentration was submitted to
a nonlinear regression analysis with the software JMP
(SAS Institute 2010). The nonlinear logistic models
used for the computation of EC50 (Sims et al. 1996)
and EC90 (adapted from Sims et al. 1996) were, re-
spectively:
Weight ⫽ W o/关1 ⫹ 共dose/EC 50兲 b]
Weight ⫽ W o/关1 ⫹ 共dose/EC 50兲兴 关log 9/(log共EC50/EC90兲兲]
Vol. 107, no. 2
Results
Baseline Susceptibility in Diet Overlay Bioassays.
The Vip3Aa20 insecticidal protein showed high bio-
logical activity against neonates of all populations of S.
frugiperda and D. saccharalis. For both species, the
concentration of 20 Ð2,000 ng Vip3Aa20/cm2 caused
mortality between 10 and 100%, respectively. For 16
populations of S. frugiperda, the LC50 ranged from
92.38 (population SC) to 611.56 (population TO) ng
Vip3Aa20/cm2. The LC90 of S. frugiperda ranged from
370.35 (population MS) to 1,490.17 (population TO)
ng Vip3Aa20/cm2. The variation in susceptibility
among the S. frugiperda populations to Vip3Aa20 dem-
onstrates the existence of a signiÞcant geographic vari-
ation in the susceptibility (P ⬍ 0.05), as evidenced by
the 6.6- and 4.0-fold differences for the LC50 and LC90,
respectively (Table 2). For six populations of D. sac-
charalis, the susceptibility in terms of LC50 ranged
from 61.18 (population Lab) to 367.86 (population
MT) ng Vip3Aa20/cm2 (Table 3). For the LC90, there
was a variation of 291.17 (population MS) to 1,414.64
(population TO) ng Vip3Aa20/cm2. This difference in
the susceptibility to Vip3Aa20 among populations of
D. saccharalis represents a signiÞcant geographic vari-
ation in the susceptibility (P ⬍ 0.05), as evidenced by
the 6.0- and 4.8-fold differences for the LC50 and LC90,
respectively (Table 3). The variation in the suscepti-
bility among populations of D. saccharalis was very
near to that for the populations of S. frugiperda.
For the larval growth inhibition, there was similar
susceptibility to Vip3Aa20 between S. frugiperda and
D. saccharalis populations (Tables 4 and 5). For both
pest species, the concentrations that inhibited the
larval growth ranged from 20 to 1,120 ng Vip3Aa20/
cm2, with an inhibition between 15 and 95%, respec-
tively. For S. frugiperda, the EC50 ranged from 21.76
(population SC) to 70.09 (population GO-1) ng
Vip3Aa20/cm2. For the EC90, there was a variation of
94.16 (population PR-2) to 861.35 (population BA-2)
ng Vip3Aa20/cm2 (Table 4). The variation in the sus-
ceptibility indicated by the larval growth inhibition
was different from that seen for the lethal concentra-
tions, 3.2 and 9.0-fold differences for the EC50 and
EC90, respectively. For D. saccharalis, the EC50 ranged
from 48.65 (population Lab) to 163.60 (population
MT) ng Vip3Aa20/cm2. For the EC90, there was a
variation between 172.55 (population Lab) to 412.52
(population PR) ng Vip3Aa20/cm2. Similarly to S. fru-
giperda populations, the variation in susceptibility of
D. saccharalis populations indicated by the larval
growth inhibition was also different from that of the
lethal concentrations, 3.3 and 2.4-fold differences for
the EC50 and EC90, respectively (Table 5).
For the estimate of the LC99, the data of concen-
trationÐmortality of all populations were pooled and
analyzed jointly. By the joint analysis, the LC99 was
estimated to be 1,711.34 (FL 95% [1,436.33Ð2,100.51])
ng Vip3Aa20/cm2 (n ⫽ 12,259; Slope [⫾SE] ⫽ 1.90
[⫾0.10]; ␹2 ⫽ 11.33; df ⫽ 8) for the S. frugiperda
populations. For the populations of D. saccharalis the
LC99 was 1,652.91 (FL 95% [1,219.07Ð2,178.11]) ng
April 2014 BERNARDI ET AL.: S. frugiperda AND D. saccharalis SUSCEPTIBILITY TO Vip3Aa20 785

Table 2. Concentration–mortality response (LC; ng/cm2) of S. frugiperda neonates exposed to the Vip3Aa20 insecticidal protein
overlaid on artificial diet

Population
Generation n Slope ⫾ SEa LC50 (95% FL)a,b LC90 (95% FL)a,b ␹2c dfd
code
Lab Ð 512 2.50 ⫾ 0.33cde 221.41 (171.79Ð267.71)bcd 718.80 (577.44Ð989.86)ab 6.00 6
RS F4 384 2.22 ⫾ 0.19cd 101.01 (68.37Ð129.78)ab 380.52 (284.66Ð628.86)a 5.94 4
SC F1 512 1.76 ⫾ 0.16b 92.38 (74.85Ð112.18)a 492.27 (369.79Ð723.53)a 8.35 5
PR-1 F4 512 2.68 ⫾ 0.25def 153.86 (129.04Ð179.70)b 461.66 (381.99Ð579.62)a 8.16 6
PR-2 F4 512 1.84 ⫾ 0.17bc 124.46 (97.23Ð153.86)ab 618.28 (476.56Ð869.09)ab 4.06 6
SP F2 511 2.16 ⫾ 0.18c 254.06 (216.81Ð297.97)cd 997.27 (783.00Ð1368.08)b 6.74 6
MS F2 447 1.99 ⫾ 0.27bc 94.23 (61.33Ð121.57)a 370.35 (239.46Ð605.42)a 8.85 5
MG F4 448 1.72 ⫾ 0.16b 94.83 (74.33Ð116.76)a 527.03 (404.75Ð745.83)a 5.93 5
GO-1 F4 445 3.07 ⫾ 0.62def 116.42 (80.38Ð143.35)ab 504.35 (443.89Ð660.25)a 4.42 5
GO-2 F4 384 1.62 ⫾ 0.20ab 104.54 (76.34Ð134.92)ab 643.09 (452.90Ð969.45)ab 3.17 4
BA-1 F4 510 1.34 ⫾ 0.15a 169.11 (122.70Ð223.63)bc 1,031.22 (898.03Ð1345.72)b 4.63 6
BA-2 F2 447 2.27 ⫾ 0.27cde 318.08 (244.74Ð390.36)cd 1,116.33 (918.99Ð1433.38)b 4.75 5
MT-1 F2 448 2.02 ⫾ 0.25bc 189.19 (150.69Ð229.48)bc 783.13 (575.20Ð951.16)ab 7.06 5
MT-2 F2 511 1.83 ⫾ 0.19bc 226.59 (170.01Ð285.82)bcd 1,118.74 (861.91Ð1367.03)b 9.32 6
TO F2 384 3.26 ⫾ 0.41def 611.65 (533.55Ð710.92)e 1,490.17 (1248.30Ð2917.18)b 5.21 4
PI F2 448 1.49 ⫾ 0.16ab 109.03 (88.64Ð137.62)ab 387.16 (272.45Ð593.77)a 5.53 5

a
LC50 and LC90 values designated by different letters within a column are signiÞcantly different from each other through nonoverlap of
95% Þducial limits. SigniÞcance of differences among slopes determined by likelihood ratio test of equality followed by pairwise comparisons
using nonoverlapping Þducial limits.
b
LC50: concentration of Vip3Aa20 (ng/cm2) required to kill 50% of larvae in the observation period of 7 d. Similarly, LC90 is the concentration
of Vip3Aa20 required to kill 90% of larvae tested.
c
P ⬎ 0.05 in the goodness-of-Þt test.
d
Degrees of freedom.

Vip3Aa20/cm2 (n ⫽ 4,328; Slope [⫾SE] ⫽ 1.71
[⫾0.12]; ␹2 ⫽ 12.56; df ⫽ 7). From the LC99, the
candidate diagnostic concentrations of 2,000 ng
Vip3Aa20/cm2 (within the FL [95%] of LC99) and
3,600 ng Vip3Aa20/cm2 (⬇2 times the LC99) were
designated for the resistance monitoring of S. fru-
giperda and D. saccharalis to the Vip3Aa20 insecticidal
protein in Brazil.
Diagnostic Concentrations for Resistance Monitor-
ing. There was low variation in the susceptibility of
Þeld populations of S. frugiperda to the diagnostic
concentrations of Vip3Aa20 (Table 6). In the second
season of 2011Ð2012, signiÞcant differences in mortal-
ity were found among populations of S. frugiperda in
the diagnostic concentrations of 2,000 (F ⫽ 2.85; df ⫽
9, 310; P ⫽ 0.0031) and 3,600 ng Vip3Aa20/cm2 (F ⫽
4.67; df ⫽ 9, 310; P ⬍ 0.0001; Table 6). At the smallest
diagnostic concentration, the population of S. fru-
giperda from Uberlândia-MG was signiÞcantly less sus-
ceptible than the Lab, Caiapônia-GO and Santa Bár-
bara do Sul-RS populations. Likewise, at the highest
diagnostic concentration, only the population from
Uberlândia-MG was less susceptible, differing from
the remaining populations.
In the Þrst season of 2012Ð2013, signiÞcant differ-
ences were also veriÞed in the population mortality of
S. frugiperda at the two diagnostic concentrations,
2,000 (F ⫽ 19.30; df ⫽ 10, 341; P ⬍ 0.0001) and 3,600
ng Vip3Aa20/cm2 (F ⫽ 16.80; df ⫽ 10, 341 P ⬍ 0.0001;
Table 6). At the diagnostic concentration of 2,000 ng
Vip3Aa20/cm2, the populations of S. frugiperda from
Chapadão do Sul-MS and Lucas do Rio Verde-MT
were signiÞcantly less susceptible than the remaining
populations. The population from Não-Me-Toque-RS
presented intermediate susceptibility, being less sus-
ceptible than the Lab, Casa Branca-SP, and Ara-
guari-MG populations, but was more susceptible than
the population from Chapadão do Sul-MS. However,

Table 3. Concentration–mortality response (LC; ng/cm2) of D. saccharalis neonates exposed to the Vip3Aa20 insecticidal protein
overlaid on artificial diet

Population
Generation n Slope ⫾ SEa LC50 (95% FL)a,b LC90 (95% FL)a,b ␹2c dfd
code
Lab Ð 607 1.42 (⫾0.11)a 61.18 (40.34Ð80.49)a 565.39 (360.86Ð729.88)ab 9.74 7
RS F4 527 1.39 (⫾0.18)ab 173.50 (116.09Ð236.28)b 940.32 (809.09Ð1143.16)c 6.87 6
PR F2 448 1.83 (⫾0.22)bc 106.24 (76.96Ð136.66)ab 529.10 (393.25Ð807.15)b 2.27 5
SP F2 447 2.17 (⫾0.20)c 74.77 (60.40Ð90.06)a 291.17 (232.00Ð390.88)a 3.39 5
MS F1 448 3.15 (⫾0.49)d 132.14 (100.46Ð158.92)b 367.08 (278.60Ð470.60)ab 5.16 5
MT F8 511 1.90 (⫾0.39)abc 367.86 (160.91Ð555.88)c 1,414.64 (1074.03Ð1850.67)c 9.73 6

a
LC50 and LC90 values designated by different letters within a column are signiÞcantly different from each other through nonoverlap of
95% Þducial limits. SigniÞcance of differences among slopes determined by likelihood ratio test of equality followed by pairwise comparisons
using nonoverlapping Þducial limits.
b
LC50: concentration of Vip3Aa20 (ng/cm2) required to kill 50% of larvae in the observation period of 7 d. Similarly, LC90 is the concentration
of Vip3Aa20 required to kill 90% of larvae tested.
c
P ⬎ 0.05 in the goodness-of-Þt test.
d
Degrees of freedom.
786 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 107, no. 2

Table 4. Concentration–stunting response (EC; ng/cm2) of S. frugiperda neonates exposed to the Vip3Aa20 insecticidal protein
overlaid on artificial diet

Population
Generation n EC50 (95% FL)a,b EC90 (95% FL)a,b
code
Lab Ð 340 52.50 (35.71Ð78.84)bc 413.74 (209.06Ð965.93)bcd
RS F4 223 40.32 (36.53Ð44.58)b 117.63 (97.88Ð144.65)a
SC F1 260 21.76 (15.09Ð27.86)a 116.65 (66.43Ð267.24)ab
PR-1 F4 323 35.89 (29.30Ð42.89)ab 143.84 (102.43Ð219.98)ab
PR-2 F4 296 28.93 (24.99Ð33.39)a 94.16 (70.31Ð135.32)a
SP F2 365 69.08 (51.56Ð93.32)c 735.98 (504.29Ð915.91)d
MS F2 273 57.65 (39.08Ð85.58)bc 299.79 (162.10Ð421.11)b
MG F4 272 40.94 (35.28Ð47.54)b 165.35 (127.29Ð225.27)ab
GO-1 F4 237 70.09 (57.72Ð86.65)c 465.12 (296.71Ð668.70)cd
GO-2 F4 278 48.27 (36.30Ð65.38)bc 443.48 (236.81Ð688.13)cd
BA-1 F4 306 57.09 (52.23Ð62.48)c 506.38 (374.87Ð747.60)cd
BA-2 F2 350 62.59 (48.71Ð80.63)c 861.35 (549.43Ð1052.11)d
MT-1 F2 407 24.59 (15.61Ð34.59)a 311.47 (176.64Ð496.77)bc
MT-2 F2 389 48.73 (37.87Ð63.23)bc 299.29 (188.08Ð427.40)bc
TO F2 447 51.41 (36.12Ð72.62)bc 384.51 (209.95Ð533.22)bcd
PI F2 241 46.67 (33.54Ð51.53)bc 316.67 (183.33Ð476.75)bc

a
EC50: effective concentration of Vip3Aa20 (ng/cm2) required to cause 50% growth inhibition in the observation period of 7 d. Similarly,
EC90 is the effective concentration of Vip3Aa20 required for 90% inhibition of growth.
b
EC50 and EC90 values designated by different letters within a column are signiÞcantly different from each other through nonoverlap of
95% Þducial limits.

at the concentration of 3,600 ng Vip3Aa20/cm2, only
the population from Chapadão do Sul-MS presented a
signiÞcant difference in susceptibility (Table 6). How-
ever, similar values of susceptibility were found among
the populations of S. frugiperda from the second sea-
son of 2011Ð2012 and Þrst season of 2012Ð2013, indi-
cating that this small variation represents the natural
difference of the response to Vip3Aa20.
Similarly to S. frugiperda populations, there was also
signiÞcant differences in the mortality of neonates
when the populations of D. saccharalis were exposed
to the diagnostic concentration of 2,000 ng Vip3Aa20/
cm2 (F ⫽ 2.88; df ⫽ 5, 186; P ⫽ 0.0156; Table 7). At this
diagnostic concentration, the populations from
Campo Verde-MT and Santo Antônio do Planalto-RS
were signiÞcantly less susceptible. However, at the
diagnostic concentration of 3,600 ng Vip3Aa20/cm2,
all populations presented similar susceptibility (F ⫽
1.41; df ⫽ 5, 186; P ⫽ 0.2210; Table 7).
Discussion
The neonates of all populations of S. frugiperda and
D. saccharalis were susceptible to Vip3Aa20 applied on
the surface of the artiÞcial diet. For S. frugiperda
populations, the variation in susceptibility to Vip3Aa20,
in terms of LC50 was of 92.38 Ð 611.56 ng Vip3Aa20/
cm2. A larger variation in the susceptibility of S. fru-
giperda was reported for the Vip3Aa protein obtained
through different protocols of extraction and activa-
tion, with the LC50 ranging between 41 and 340 ng
Vip3Aa20/cm2 (Chakroun et al. 2012). Lee et al.
(2003) reported a LC50 of 55.9 ng Vip3A/cm2 for S.
frugiperda. In another study, S. frugiperda presented
greater susceptibility to Vip3Af and Vip3Aa than that
found in this study with LC50 of 21 and 49.3 ng/cm2,
respectively (Sena et al. 2009). In the same study,
Vip3Af and Vip3Aa were more toxic for S. frugiperda
than Cry1Ab and Cry1Fa. In addition, Vip3A ex-
pressed in cotton was efÞcient in the control of S.
frugiperda (Adamczyk and Mahaffey 2008). Vip3Aa1
and Vip3Ac1 were also toxic to S. frugiperda (Fang et
al. 2007).
In general, the Vip3 insecticidal proteins have
shown high toxicity against species of Spodoptera and
other lepidopteran pests. Vip3Aa presented high in-
secticidal activity against Spodoptera albula (Walker),
Spodoptera cosmioides (Walker), Spodoptera eridania

Table 5. Concentration–stunting response (EC; ng/cm2) of D. saccharalis neonates exposed to the Vip3Aa20 insecticidal protein
overlaid on artificial diet

Population code Generation n EC50 (95% FL)a,b EC90 (95% FL)a,b

Lab Ð 516 48.65 (37.66Ð62.10)a 172.55 (74.17Ð293.18)a
RS F4 310 84.76 (60.59Ð120.32)ab 280.62 (187.86Ð425.11)a
PR F2 252 70.74 (56.28Ð89.81)ab 412.52 (266.31Ð743.88)a
SP F2 240 86.08 (59.46Ð145.11)a 406.76 (288.78Ð567.32)a
MS F1 263 98.00 (42.34Ð132.66)ab 299.73 (211.21Ð376.32)a
MT F8 385 163.60 (75.22Ð252.02)b 378.75 (269.76Ð497.82)a

a
EC50: effective concentration of Vip3Aa20 (ng/cm2) required to cause 50% growth inhibition in the observation period of 7 d. Similarly,
EC90 is the effective concentration of Vip3Aa20 required for 90% inhibition of growth.
b
EC50 and EC90 values designated by different letters within a column are signiÞcantly different from each other through nonoverlap of
95% Þducial limits.
April 2014 BERNARDI ET AL.: S. frugiperda AND D. saccharalis SUSCEPTIBILITY TO Vip3Aa20 787

Table 6. Mortality of S. frugiperda neonate larvae at two candidate diagnostic concentrations of Vip3Aa20 insecticidal protein overlaid
on artificial diet

City, state Generation % mortalitya,b

2
Second season 2011/2012 2,000 ng/cm 3,600 ng/cm2
Susceptible reference (Lab) Ð 99.4 ⫾ 0.4a 100.0 ⫾ 0.0a
Santa Bárbara do Sul, RS F2 99.4 ⫾ 0.3a 99.5 ⫾ 0.3a
Palotina, PR F2 98.8 ⫾ 0.4ab 99.7 ⫾ 0.2a
Palmital, SP F2 98.7 ⫾ 0.4ab 100.0 ⫾ 0.0a
Chapadão do Sul, MS F2 98.2 ⫾ 0.6ab 99.1 ⫾ 0.3ab
Uberlândia, MG F2 97.4 ⫾ 0.5b 97.9 ⫾ 0.5b
Sinop, MT F2 98.9 ⫾ 0.3ab 99.5 ⫾ 0.3a
Caiapônia, GO F2 99.5 ⫾ 0.2a 100.0 ⫾ 0.0a
Barreiras, BA F2 98.6 ⫾ 0.4ab 99.4 ⫾ 0.3a
Palmas, TO F2 97.6 ⫾ 0.7ab 99.1 ⫾ 0.3ab
First season 2012/13
Susceptible reference (Lab) Ð 99.8 ⫾ 0.1a 100.0 ⫾ 0.0a
Não-Me-Toque, RS F2 97.3 ⫾ 0.7bc 99.4 ⫾ 0.2a
Chapecó, SC F1 99.8 ⫾ 0.1ab 100.0 ⫾ 0.0a
Tibagi, PR F2 99.7 ⫾ 0.2ab 99.8 ⫾ 0.2a
Casa Branca, SP F2 99.9 ⫾ 0.1a 100.0 ⫾ 0.0a
Chapadão do Sul, MS F1 92.1 ⫾ 1.2d 95.7 ⫾ 0.9b
Araguari, MG F2 100.0 ⫾ 0.0a 100.0 ⫾ 0.0a
Lucas do Rio Verde, MT F2 96.9 ⫾ 0.6c 98.7 ⫾ 0.4a
Flores de Goiás, GO F2 99.7 ⫾ 0.2ab 99.9 ⫾ 0.1a
São Desidério, BA F2 98.3 ⫾ 0.6abc 99.7 ⫾ 0.2a
Uruçuṍ, PI F2 99.2 ⫾ 0.4abc 100.0 ⫾ 0.0a

a
1,024 neonates were tested per candidate diagnostic concentration per Þeld pop.
b
Values represent means ⫾ SE. Means followed by the same letter in each season and each candidate diagnostic concentration do not differ
statistically (Tukey test, P ⱕ 0.05).

(Cramer), and S. frugiperda with LC50 of 3.90, 2.78,
3.44, and 24.66 ng/cm2, respectively (Bergamasco et
al. 2013). In the same study, Vip3Aa and Cry1Ia10
presented synergistic effects for S. albula and S. cos-
mioides, but demonstrated an antagonist effect for S.
eridania by competing for the same receptor in the
action site. Vip3Aa was also toxic for Spodoptera litura
(F.), Spodoptera exigua (Hübner), and H. armigera
(Chen et al. 2003). The Vip3Aa16 protein, another
Vip3 insecticidal protein, was active against Spodo-
ptera littoralis (Boisduval) with LC50 of 305 ng/cm2
(AbdelkeÞ-Mesrati et al. 2011). The Vip3Aa1 protein
was more efÞcient against S. litura (45 ng/cm2) than
for Agrotis ipsilon (Hufnagel) (80 ng/cm2) and Plu-
tella xylostella (L.) (220 ng/cm2; Doss et al. 2002). The
similar toxicity of the Vip3 proteins against species of
Spodoptera may be explained by the small difference
in the amino acid sequence between these proteins; an
example of that is the similarity between the native
protein Vip3Aa1 and Vip3Aa20, which differ only by
two amino acids, in the positions 129 and 284 (simi-
larity ⬎99%; Raybould and Vlachos 2011).
The susceptibility of the sugarcane borer to Vip
proteins had not yet been reported. In this study, the
Vip3Aa20 protein demonstrated high toxicity against
D. saccharalis, with LC50 ranged from 61.18 to 367.86
ng/cm2. However, for neonates of Ostrinia nubilalis
(Hübner), another species of Crambidae that attacks
corn stalks, the Vip3Aa1 and Vip3Ac1 proteins did not
demonstrate any detectable toxicity (Fang et al. 2007).
In the same study, the chimerical protein Vip3AcAa
has the N-terminal region of Vip3Ac1 and the C-ter-
minal region of Vip3Aa1, demonstrated a small toxicity
to O. nubilalis. In another study, Vip3Ba1 was not toxic
to O. nubilalis (Rang et al. 2005).
In this current study, there was a low interpopula-
tion variation in the susceptibility of S. frugiperda and
D. saccharalis to Vip3Aa20. Unlike this study, there
was a large variation in the susceptibility of Helicov-
erpa zea (Boddie) and H. virescens populations to

Table 7. Mortality of D. saccharalis neonate larvae at two candidate diagnostic concentrations of Vip3Aa20 insecticidal protein
overlaid on artificial diet

City, state Generation % mortalitya,b

2
Second season 2011/12 2,000 ng/cm 3,600 ng/cm2
Susceptible reference (Lab) Ð 99.0 ⫾ 0.3ab 99.7 ⫾ 0.3a
Santo Antônio do Planalto, RS F4 98.2 ⫾ 0.6b 99.8 ⫾ 0.1a
Sabáudia, PR F4 98.8 ⫾ 0.4ab 99.7 ⫾ 0.2a
Jaboticabal, SP F4 100.0 ⫾ 0.0a 100.0 ⫾ 0.0a
Douradina, MS F4 99.1 ⫾ 0.3ab 100.0 ⫾ 0.0a
Campo Verde, MT F9 98.0 ⫾ 0.5b 100.0 ⫾ 0.0a

a
1,024 neonates were tested per candidate diagnostic concentration per Þeld pop.
b
Values represent means ⫾ SE. Means followed by the same letter in each candidate diagnostic concentration do not differ statistically
(Tukey test, P ⱕ 0.05).
788 JOURNAL OF ECONOMIC ENTOMOLOGY
Vip3A in the United States, 75- and 132-fold differ-
ences for the LC50, respectively (Ali and Luttrell
2011). In Brazil, the fall armyworm presented a larger
variation in susceptibility to Cry1A.105 (15-fold for six
populations; Salmeron et al. 2008a). However, the
populations of D. saccharalis exposed to Vip3Aa20
presented less interpopulation variation, compared
with that found for seven populations of this species
when exposed to Cry1A.105 (40-fold), and similar
variation in the response to Cry1Ab (Þvefold; Salm-
eron et al. 2008b). For another species of Crambidae
of the United States, Diatraea grandiosella (Dyar),
there was also similar variation in the susceptibility to
Cry1Ab (Þvefold for eight populations; Trisyono and
Chippendalle 2002). A smaller difference in the sus-
ceptibility was found for Brazilian populations of H.
virescens, only fourfold when exposed to Cry1Ac pro-
tein (Albernaz et al. 2013), and for populations of O.
nubilalis in the United States, approximately fourfold
for Cry1Ab and Cry1Ac proteins (Marçon et al. 1999).
However, there was a large variation in the suscepti-
bility of H. zea to Cry1Ac in the United States, 441-fold
for 11 populations (Luttrell et al. 1999). In China, the
variation in susceptibility of H. armigera to Cry1Ac
was also high, 100-fold difference in 23 populations
(Wu et al. 1999).
The interpopulation variation in the susceptibility
to chemical or microbial insecticides is a common
phenomenon when bioassays are repeated (Robert-
son et al. 1995). This variation may be associated to
several factors, among which include the source of the
insecticidal protein (Ali et al. 2006), bioassay method
and pest population (Luttrell et al. 1999), time of
exposure (Dulmage et al. 1978), temperature (Rob-
ertson et al. 1996), and artiÞcial diet (Blanco et al.
2009). However, because of the small variation in the
susceptibility to Vip3Aa20 in this initial comparison, it
seems probable that the differences in response may
be caused by previous exposure to selection pressure,
being more likely to represent the natural variation of
the susceptibility of S. frugiperda and D. saccharalis
populations. However, from an IRM perspective, the
variation in susceptibility is also an indication of the
ability of the insects to adapt to the toxin, especially in
those regions with the highest LC values. In this con-
text, Carrière et al. (2010) asserts that gene ßow be-
tween populations with repeated colonization of Bt
Þelds provides opportunities for rapid local adapta-
tion. In this current study, the populations SC, MS, and
MG were more susceptible than the Lab population;
the reason for this result is unknown. Understanding
this reason should be a research topic for future ex-
periments.
To subsidize an IRM program, the monitoring of the
susceptibility of S. frugiperda and D. saccharalis to
Vip3Aa20 is of critical importance to maintain the
efÞciency and control of this insecticidal protein. For
susceptibility monitoring, the diagnostic concentra-
tions of 2,000 and 3,600 ng Vip3Aa20/cm2 caused a
high mortality of the neonates for these pest insects.
The use of these diagnostic concentrations may pro-
vide consistent data about susceptibility of Þeld
Vol. 107, no. 2
populations of S. frugiperda and D. saccharalis to
Vip3Aa20. For a more effective monitoring, the
growth inhibition could also be used as a response
criterion because it would increase the possibility to
detect the evolution of the Þeld resistance (Sims et al.
1996). However, in this study there was no evident
advantage in the use of the growth inhibition as a
response criterion. In other words, the diagnostic con-
centrations caused high mortality, making this bioas-
say procedure fast, practical, and compatible to the
large scale bioassays in the IRM programs, demon-
strating that this bioassay technique was adequate to
evaluate the susceptibility of S. frugiperda and D. sac-
charalis populations to the Vip3Aa20 protein. The ef-
Þciency of this bioassay technique was also reported
for other Bt proteins and insect pests (Marçon et al.
1999, Liao et al. 2002, Siegfried et al. 2007, Ali and
Luttrell, 2011).
The compilation of this susceptibility database for
populations of S. frugiperda and D. saccharalis and the
validation of the diagnostic concentration to Vip3Aa20
will be useful in the follow up of possible changes in
the susceptibility of these pests from repeated expo-
sure to Vip3Aa20 in the Þeld. Future efforts should be
concentrated on representative samples of popula-
tions of these target pests, especially in those regions
where corn is cultivated intensively, and posterior
exposure of neonates to the diagnostic concentrations
deÞned here, for the monitoring of the frequency of
resistant insects in the Þeld.
Acknowledgments
We thank Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) (Project number 2011/22743-0) for
granting postdoctoral scholarship to the Þrst author and Con-
selho Nacional de Desenvolvimento CientṍÞco e Tec-
nológico (CNPq) (Project number 475748/2011-5) for the
partial Þnancial support.
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Received 27 August 2013; accepted 22 January 2014.