Low susceptibility of Spodoptera cosmioides, Spodoptera eridania and Spodoptera frugiperda (Lepidoptera: Noctuidae) to genetically-modified soybean expressing Cry1Ac protein

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Crop Protection 58 (2014) 33e40

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Crop Protection

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Low susceptibility of Spodoptera cosmioides, Spodoptera eridaniaand Spodoptera frugiperda (Lepidoptera: Noctuidae) togenetically-modified soybean expressing Cry1Ac protein

Oderlei Bernardi a,*, Rodrigo J. Sorgatto a, Alexandre D. Barbosa a, Felipe A. Domingues a,Patrick M. Dourado b,1, Renato A. Carvalho b,1, Samuel Martinelli c,2, Graham P. Head c,2,Celso Omoto a

aDepartamento de Entomologia e Acarologia, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ/USP), Av. Pádua Dias 11, Piracicaba,São Paulo 13418-900, BrazilbMonsanto do Brasil Ltda, São Paulo, BrazilcMonsanto LLC, Saint Louis, MO, USA

a r t i c l e i n f o

Article history:Received 10 May 2013Received in revised form18 December 2013Accepted 2 January 2014

Keywords:Transgenic soybeanNon-target effectLife history traitsIntegrated Pest Management

* Corresponding author. Tel.: þ55 19 8190 3275.E-mail address: [email protected] (O

1 Current address: Monsanto do Brasil Ltda, Av. NaçSP 04578-910, Brazil.

2 Current address: Monsanto LLC, 800 North Lind63167, USA.

0261-2194/$ e see front matter � 2014 Published byhttp://dx.doi.org/10.1016/j.cropro.2014.01.001

a b s t r a c t

Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) and Spodoptera frugiperda (J. E. Smith) havecaused significant damage on soybean Glycine max (L.) Merrill in Brazil. Genetically-modified MON87701 � MON 89788 soybean that expresses the Cry1Ac protein is potentially an alternative tool for themanagement of these species. Purified protein bioassays were done to evaluate the susceptibility ofS. cosmioides, S. eridania and S. frugiperda to Cry1Ac protein. The level of efficacy of the Bt soybean plantsin controlling these species was measured through laboratory and greenhouse trials under high artificialinsect infestations. The biology of these insects was evaluated over their development cycles to under-stand their life history when fed on Bt soybean. Purified Cry1Ac protein at the maximum concentrationtested (100 mg Cry1Ac mL�1 diet) resulted in low mortality of S. cosmioides and S. eridania (<13%) andintermediate mortality of S. frugiperda (50%). No significant effects of the Bt soybean plants wereobserved in the life table parameters of S. cosmioides and S. eridania. However, S. frugiperda fed on Btsoybean plants had a prolonged larval stage (by 5 days), reduced larvae viability, increased mean gen-eration time (by 8 days) and reduced intrinsic rate of increase. In general, the Bt soybean plants showedpoor control of Spodoptera species when evaluated by leaf-disc bioassay and greenhouse trials. Conse-quently, other control tactics must be used in combination with MON 87701 � MON 89788 soybean inthe field for the efficient management of S. cosmioides, S. eridania and S. frugiperda.

� 2014 Published by Elsevier Ltd.

1. Introduction

Caterpillars from the Spodoptera genus have caused damage tosoybean fields Glycine max (L.) Merrill (Fabaceae: Phaseoleae) inBrazil during recent years (Bueno et al., 2011). Within the Spo-doptera complex, Spodoptera eridania (Stoll), Spodoptera cosmioides(Walker) and Spodoptera frugiperda (J. E. Smith) are prominent incausing damage. They have attacked soybeans in the Cerrado

. Bernardi).ões Unidas, 12.901, São Paulo,

bergh Blvd, Saint Louis, MO

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region, Central and Southern Brazil (Hoffmann-Campo et al., 2000;Santos et al., 2005). The great potential for defoliation of soybeanplants (Bueno et al., 2011) and damage to flowers and pods(Hoffmann-Campo et al., 2000) by Spodoptera species requireadoption of control tactics to prevent loss of grain yield. Control isachieved with insecticides, often indirectly as result of sprays forvelvetbean caterpillar Anticarsia gemmatalis Hübner, soybeanlooper Chrysodeixis includens (Walker) and tobacco budwormHeliothis virescens (F.). The use of organophosphates, carbamatesand pyrethroids has been the main control strategy for Spodopteraspecies in soybean in Brazil, but these chemicals have low efficacyand control failures are common due to high natural tolerance ofthese pests to insecticides (Diez-Rodríguez and Omoto, 2001;Carvalho et al., 2013). The genetically modified insect-resistantand glyphosate-tolerant soybean (event MON 87701 � MON

O. Bernardi et al. / Crop Protection 58 (2014) 33e4034

89788) (CTNBio, 2010) is potentially an alternative tool to managethe population of Spodoptera species because of its expression ofthe Cry1Ac protein, but there are no data available in the literaturedemonstrating the impact of this protein on the life-history pa-rameters of Spodoptera species. This information is important dueto the threat of resistance evolution to Bt crops. The evolution ofresistance to Bt crops by natural selection is dependent upon atleast three conditions: (i) variation among individuals in survivalon Bt crops, (ii) inheritance of survival on Bt crops, and (iii) fitnessdifferences consistently associated with the variation in survival onBt crops (Carrière et al., 2010). The potential risk for Bt-resistanceevolution is high for Spodoptera species because the Braziliancrop-production system has temporal and spatial overlap of Bt-transformed host plants for Spodoptera species such as corn (Zeamays L.) (S. frugiperda), cotton (Gossypium hirsutum L.) and soybean(S. eridania, S. cosmioides and S. frugiperda). In the field these cropspotentially expose population of Spodoptera species to intense se-lection pressure in each insect generation, increasing the risk ofselecting for Bt-resistant individuals. Gene flow between sourceand sink habitats allows for repeated colonization of Bt fields andprovides opportunities for local adaptation (Carrière et al., 2010), orit could function as a source of susceptibility and delay resistanceevolution (Head et al., 2010). Additional important aspects that canfavor the evolution of resistance are the natural tolerance of Spo-doptera species to Cry1Ac protein (Luttrell et al., 1999;Sivasupramaniam et al., 2008) and the rapid ability to evolveresistance to Cry1F in the field (S. frugiperda) (Storer et al., 2010).This inherent tolerance of insect pest species to certain Bt proteinscould impact the selection advantage of resistance by eitherreducing the selection coefficient (Gould, 1998) or allowing selec-tion of incomplete resistance (Gassmann et al., 2011).

These considerations highlight the need for studies to under-stand the effects of MON 87701 � MON 89788 soybean on Spo-doptera species and on the potential risk for Cry1Ac resistanceevolution. This information is particularly important to supportInsect Resistance Management (IRM) programs and best agricul-tural practices in a crop-production system where transgenic soy-bean and cotton events both express the Cry1Ac protein. Toevaluate the susceptibility of S. eridania, S. cosmioides andS. frugiperda to Cry1Ac and the biological effects of MON87701�MON 89788 soybean against these species, Cry1Ac proteinbioassays, leaf-disc and greenhouse trials were conducted and lifetable parameters were evaluated when those insects were fed on Btsoybean.

2. Materials and methods

2.1. Susceptibility to Cry1Ac diet-incorporation bioassay

Susceptible reference populations of S. cosmioides, S. frugiperdaand S. eridaniawere collected in soybean (200 larvae per species) inPelotas-RS, Rondonópolis-MS and Ibiporã-PR, respectively, inDecember 2008. In the laboratory, these populations were kept freeof selection pressure by insecticides or Bt proteins for at least 3years (>15 generations) and then they were used to evaluate thespecies’ susceptibility to Cry1Ac protein. All populations weremaintained on artificial diet based on white bean, wheat germ andyeast (adapted from Greene et al., 1976), at 25 � 1 �C with a 14:10 hlight:dark photoperiod. Purified Cry1Ac protein was provided byMonsanto of Brazil Ltda at a concentration of 1.4 mg of activeCry1Ac mL�1 and stored in a freezer at �80 � 5 �C. After thawing,Cry1Ac protein was diluted in buffer consisting of 50 mM 3-(cyclohexylamine)-1-propanosulphonic acid at pH 10.25, 1 mMbenzomidine-HCl, 1 mM tetraacetic diamine ethylene acid and2.5 mM dithiothreitol. For bioassays, the insecticidal protein and

buffer solution were added to artificial diet when the diet tem-perature reached 45e50 �C. Cry1Ac was incorporated into artificialdiet using a Vortex-type tube mixer for 2 min. The diet containingthe incorporated protein was kept in a water bath at z55 �C forlater distribution into 128-well bioassay trays (BIO-BA-128, CD In-ternational, Inc., Pitman, NJ) (1 mL diet/well). A total of five con-centrations, 1.8, 10, 18, 32 and 100 mg Cry1Ac mL�1 diet, were used.After diet solidification and cooling, one neonate larvae (<24 h old)was placed into each well using a fine brush. Bioassay trays weresealed with self-adhesive plastic sheets (BIO-CV-16, CD Interna-tional Inc.) allowing gas exchange with the external environmentand placed in a climatic chamber (temperature: 27 � 1 �C; relativehumidity: 60 � 10%; photoperiod: 14:10 h light:dark). The experi-mental design was completely randomized with 8 replicates perconcentration (16 larvae/replicate). Mortality caused by Cry1Acwasassessed by counting the number of dead larvae after seven days,including as ‘dead’ all larvae still in the first instar. The weight ofsurviving larvae was also recorded. The percentage mortality wascorrected on the basis of the mortality in the control treatment,which consisted of artificial diet þ buffer (Abbott, 1925). The per-centage of growth inhibition was calculated as larval weightreduction relative to the control treatment. Corrected percentmortality and growth inhibition were transformed using

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

xþ 0:5p

because of non-normal distributions of residuals. After trans-formation, mortality and growth inhibition data was subjected totwo-way ANOVA with species, diet concentration, and the inter-action as fixed effects. Means were compared using the Tukey test(P � 0.05) (PROC ANOVA, SAS Institute, 2000).

2.2. Leaf-disc bioassays

MON 87701 � MON 89788 soybean and near-isogenic negativechecks of maturity groups 5.5 (recommended for planting insouthern Brazil) and 8.3 (recommended for planting in midwesternBrazil) were sown in 12 L plastic pots (4 seeds/pot) in the green-house. Completely expanded leaves were removed from the upperthird of the plants when they reached the phenological stages V3eV4, V5eV6 and R1eR2 (Farias et al., 2007). Leaf discs 2.4 cm indiameter were cut using a metallic cutter and placed on a non-gelled mixture of watereagar 2.5% (1 mL/well) in acrylic plates(Costar�) with 12 wells (Corning, Tewksbury, MA, USA). Leaf discswere separated from the water-agar layer by a filter paper disc.Separate bioassays were performed for S. cosmioides, S. frugiperdaand S. eridania. In each bioassay one neonate larvae (<24 h old) wasplaced on each soybean leaf-disc using a fine brush. Plates weresealed with plastic film (Magipack�) and placed in a climaticchamber (temperature: 27 � 1 �C; relative humidity: 60 � 10%;photoperiod: 14:10 h light:dark). The experimental design wascompletely randomized with 8 replicates per treatment; eachreplicate consisted of 12 neonate larvae for a total of 96 neonatelarvae tested for each species per phenological stage. Larval mor-tality was assessed after five days. Mortality on MON 87701�MON89788 soybean was corrected on the basis of the mortality in thenear-isogenic negative check (Abbott, 1925). Corrected percentagemortality of each replicatewas transformed using

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

xþ 0:5p

becauseof non-normal distribution of residuals. The mortality data onMON87701 � MON 89788 soybean and the respective near-isogenicnegative check for each species and phenological stage werecompared by t-test (P � 0.05) (PROC TTEST, SAS Institute, 2000).

2.3. Biological parameters of Spodoptera species when fed on MON87701 � MON 89788 soybean

MON 87701 � MON 89788 soybean and near-isogenic negativechecks of maturity groups 5.5 and 8.3 were sown in the greenhouse

Table 1Mortality and growth inhibition of Spodoptera species neonates exposed to Cry1Acprotein incorporated in artificial diet.

Species n Response criteriaa

Corrected mortality (%) Growth inhibition (%)b

1.8 mg Cry1Ac mLL1 dietSpodoptera cosmioides 124 2.9 � 0.8 b 13.9 � 8.6 bSpodoptera eridania 128 3.2 � 0.8 b 12.7 � 8.5 bSpodoptera frugiperda 128 5.3 � 0.8 b 14.1 � 9.9 b10 mg Cry1Ac mLL1 dietSpodoptera cosmioides 127 3.6 � 1.3 b 16.1 � 7.7 bSpodoptera eridania 121 5.2 � 1.9 b 15.5 � 9.9 bSpodoptera frugiperda 127 8.2 � 3.7 b 18.9 � 10.5 b18 mg Cry1Ac mLL1 dietSpodoptera cosmioides 128 4.1 � 2.0 b 18.7 � 10.8 bSpodoptera eridania 127 6.9 � 2.4 b 17.9 � 9.3 bSpodoptera frugiperda 128 9.7 � 4.0 b 19.8 � 9.6 b32 mg Cry1Ac mLL1 dietSpodoptera cosmioides 125 4.9 � 0.1 b 38.6 � 10.6 bSpodoptera eridania 129 7.4 � 1.2 b 29.6 � 11.3 bSpodoptera frugiperda 126 30.9 � 7.5 a 44.2 � 13.3 b100 mg Cry1Ac mLL1 dietSpodoptera cosmioides 129 8.6 � 3.2 b 44.5 � 8.3 bSpodoptera eridania 129 12.3 � 4.8 b 30.1 � 9.3 bSpodoptera frugiperda 128 50.0 � 5.0 a 80.2 � 8.2 a

a Values represent means� SE. Means followed by the same letter in each columnare not significantly different.

b The percentage of growth inhibition was calculated as larval weight reductionrelative to the control.

O. Bernardi et al. / Crop Protection 58 (2014) 33e40 35

as described previously. Beginning at R1, completely expandedleaves were removed from the middle third and top of the plantsand placed into glass tubes (8.5 cm length � 2.5 cm diameter)previously sterilized and buffered with hydrophobic cotton. Sepa-rate bioassays were performed for S. cosmioides, S. frugiperda andS. eridania. In each bioassay one neonate larvae (<24 h old) wasplaced into the glass tube using a fine brush. Glass tubes weresealed with hydrophobic cotton and placed in a climatic chamber(temperature: 27� 1 �C; relative humidity: 60� 10%; photoperiod:14:10 h light:dark). Leaves were changed every 48 h over the larvaldevelopment period. The experimental design was completelyrandomized with 10 replicates, each one consisting of 10 larvae. Foreach treatment, the following biological parameters were evalu-ated: duration and survival rates of egg, larval and pupal periods;total cycle duration (egg to adult); larval weight 14 days afterinfestation; pupae weight 24 h after pupation; sex ratio; femalelongevity; duration of pre-oviposition, oviposition and post-oviposition periods; daily fecundity (eggs/female/day) and totalfecundity (total eggs/female). Egg viability and duration of egg,larval and pupal periods and total cycle were determined in dailyobservations. Female longevity and fecundity were evaluated fromthe formation of 10 pairs/species/treatment that were kept in PVCcages (23 cm height � 10 cm diameter) internally coated by papertowel (oviposition substrate) and closed at the topwith a voile-typefabric. Adults were fedwith a 10% honey/aqueous solution providedon cotton. Number of eggs and mortality of adults were assesseddaily. To determine the embryonic period and viability, 100 eggswere obtained from the second oviposition of each pair. Eggs wereplaced into glass tubes with flat bottoms (8.5 � 2.5 cm). A piece ofpaper (2 � 1 cm) moistened with distilled water daily was placedinside the tube, which was closed at the top with plastic film. Eggsand the number of larvae hatched were counted daily. Egg viabilityand duration of egg, larval and pupal stages and the egg-adultperiod (total cycle) of the three Spodoptera species were trans-formed using

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

xþ 0:5p

scale because of non-normal distributions ofresiduals. The data on MON 87701 � MON 89788 soybean and thenear-isogenic negative check for each species were compared by t-test (P � 0.05) (PROC TTEST, SAS Institute, 2000). The putativedeviation in the sex ratio was compared using the Chi-square test(c2) (P � 0.05) (PROC FREQ, SAS Institute, 2000). A life table wascalculated by estimating the mean generation time (T), the netreproductive rate (Ro), the intrinsic rate of increase (rm) and thefinite rate of increase (l). The life table parameters were estimatedby the “Jackknife” method using “Lifetable.sas” (Maia et al., 2000)and compared using a bilateral t-test (P � 0.05) (SAS Institute,2000).

2.4. Greenhouse trials

MON 87701 � MON 89788 soybean plants were infested withS. eridania or S. frugiperda in separate greenhouse trials. Treatmentsconsisted of MON 87701 � MON 89788 soybean and near-isogenicnegative checks of maturity groups 5.5 and 8.3 sown at a density of13 seeds linear/meter, in a randomized block design. The experi-ment consisted of four blocks of the four treatments, with four rowsof soybean/block. Within each block, the lines of soybean (3.0 mlength � 0.5 m between rows) represented the experimental rep-licates (one replicate per treatment per block). From the V5eV6phenological stage, plants were kept in a containment system ofnylon net cages (16� 18mesh) 13.0 m length� 3.5 mwide� 2.9 mheight which remained until the end of the study. For S. frugiperda,when the plants reached the reproductive stage R1, all plants ineach plot were infested with a piece of paper (oviposition sub-strate) containing 30 eggs which were fixed (clamped) on the apexof the plants. For S. eridania, 1300 pupae (subdivided into four

acrylic boxes) were placed inside the cage at four points on top ofwooden stands 1.0 m in height. Larval incidence and defoliationwere assessed at 14 days (S. frugiperda) or 43 days (S. eridania) afteregg infestation and pupae introduction, respectively. Larval inci-dence (number of larvae per plant) and defoliation were estimatedfor every plant located in the central 2 m of each plot. To assessplant defoliation, leaves of each plant were compared with a soy-bean defoliation scale (Willson, 2009). Larval incidence data (con-verted into larvae per meter) and the percentage of defoliationwere transformed using

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

xþ 0:5p

because of non-normal distri-butions of residuals. After transformation, the data on MON87701 � MON 89788 soybean and respective near-isogenic nega-tive check were compared by t-test (P � 0.05) (PROC TTEST, SASInstitute, 2000).

3. Results

3.1. Susceptibility to purified Cry1Ac protein

Statistically significant interaction between concentrations ofCry1Ac and Spodoptera species was detected for mortality (F¼ 5.00,df ¼ 14, 105, P < 0.0001) (Table 1). Minimal mortality (<10%) forSpodoptera species was observed at concentrations of 1.8, 10 and18 mg Cry1Ac mL�1 diet. Nevertheless, higher mortality wasobserved at concentration of 32 and 100 mg Cry1Ac mL�1 diet forS. frugiperda (31 and 50%, respectively), whereas it was less than13% for S. cosmioides and S. eridania (Table 1). Additionally, due tothe low susceptibility to Cry1Ac protein, it was not possible to es-timate the LC50 (LC e Lethal Concentration) for any of the speciesbecause of low mortality observed at the maximum concentrationof 100 mg Cry1Ac mL�1 diet.

Similar to mortality response, a statistically significant interac-tion between concentrations of Cry1Ac and Spodoptera specieswere also detected for growth inhibition (F ¼ 2.95, df ¼ 14, 105,P ¼ 0.0051) (Table 1). Indistinguishable growth inhibition rateswere observed at concentrations of 1.8, 10, 18 and32 mg Cry1Ac mL�1 diet. In contrast, at a concentration of

O. Bernardi et al. / Crop Protection 58 (2014) 33e4036

100 mg Cry1Ac mL�1 diet a higher growth inhibition was detectedfor S. frugiperda (80%), than for S. cosmioides (44%) and S. eridania(30%) (Table 1). In conclusion, S. frugiperdawas more susceptible toCry1Ac protein than the other two species.

3.2. Leaf-disc bioassays

After five days infestation, mortality of S. cosmioides andS. eridania on MON 87701 � MON 89788 soybean was lower than10% for all phenological stages and both soybean maturity groupsand not different from on the near-isogenic negative check(Table 2). However, MON 87701 � MON 89788 soybean leavescaused higher mortality of S. frugiperda larvae than on the near-isogenic negative check for all phenological stages and bothmaturity groups, with mortality of 41e47% and 32e35% for matu-rity groups 5.5 and 8.3, respectively (Table 2).

3.3. Biological parameters of Spodoptera species when fed on MON87701 � MON 89788 soybean

There was no significant difference in the duration and survivalof egg and pupal stages for S. cosmioides, S. eridania and S. frugiperdawhen fed on MON 87701 �MON 89788 soybean and near-isogenicnegative check (Fig. 1). There also was no significant difference inlarval stage duration, total cycle (eggeadult), and larval survival forS. cosmioides and S. eridania. However, the larval stage was signif-icantly longer (5 days) for S. frugiperda (Fig. 1E). This caused a sig-nificant increase of 5.5 days in the time required to complete thelife cycle on MON 87701 � MON 89788 soybean. Larval survival ofS. frugiperda also was significantly lower on MON 87701 � MON89788 soybean (37%) than the near-isogenic negative check (82%)(Fig. 1F). This affected the number of insects that completed the lifecycle, with less than 27% of the insects reaching the adult stage.

Feeding on MON 87701 � MON 89788 soybean caused a sig-nificant reduction in larval weight for all three species, but thedifferences were much larger for S. frugiperda than for the othertwo species (Table 3). Pupae of S. frugiperda originating from larvaefed on MON 87701 �MON 89788 soybean leaves were lighter thanthose from the near-isogenic negative check. However, differencesin 14-day larval weight did not affect pupal weight for S. cosmioidesand S. eridania. In other words, there was some developmentaldelay after 14 days in Bt soybean for S. cosmioides and S. eridania,but larvae reached full size by pupation.

The sex ratio was not affected for any of Spodoptera species(Table 3), nor was the longevity of females, timing of pre-oviposition, oviposition and post-oviposition for the three Spo-doptera species (Table 4). Furthermore, feeding on MON87701 � MON 89788 soybean leaves did not affect the oviposition

Table 2Percentage mortality of neonate larvae of three Spodoptera species after five-days feedincompared with near-isogenic negative checks in laboratory trials.

Treatment Maturity group 5.5a

V3eV4 V5eV6

Spodoptera cosmioidesMON 87701 � MON 89788 soybean 3.9 � 1.1 a 6.9 � 3.1 aNegative check 2.0 � 1.2 a 1.2 � 0.9 aSpodoptera eridaniaMON 87701 � MON 89788 soybean 4.3 � 2.9 a 4.3 � 4.1 aNegative check 3.1 � 1.5 a 2.1 � 1.4 aSpodoptera frugiperdaMON 87701 � MON 89788 soybean 41.5 � 5.1 a 47.5 � 5.5 aNegative check 2.1 � 1.4 b 6.2 � 2.6 b

a Values represent means � SE. A separate t-test (P� 0.05) was conducted between MOeach phenological stage and Spodoptera species (For each pair of means, those followeddifferent).

capacity of S. cosmioides and S. eridania. In contrast, S. frugiperdafemales had oviposition capacity reduced 65% when fed on MON87701 � MON 89788 soybean, reflecting the reduced pupal size.

MON 87701 � MON 89788 soybean did not affect life tableparameters of S. cosmioides and S. eridania species when fed onMON 87701�MON 89788 soybean leaves comparedwith the near-isogenic negative check (Table 5). In contrast, for S. frugiperda therewas an increase in the mean generation time (T) (by 8 days) andreduction in net reproductive rate (Ro) indicating that the devel-opment of the caterpillars on Bt soybean reduced populationgrowth by 81%. Based on these results, it is estimated that after 44.5days (T) of S. frugiperda development on Bt soybean approximately59 females are expected to result from each female, whereas on thenegative check 341 females are expected after 36.3 days (T).Furthermore, the development of S. frugiperda on this Bt soybeanreduced the intrinsic rate of increase (rm) by 41%. The finite rate ofincrease (l) of S. frugiperda also was 43% lower on Bt soybean.

3.4. Greenhouse trials

Moderate larval incidence of S. eridania and S. frugiperda wasfound on MON 87701 � MON 89788 soybean and the respectivenear-isogenic negative check (Table 6). No significant differences inlarval incidence and defoliation by S. eridaniawere found on the Btsoybean and the near-isogenic negative checks for both maturitygroups. Similarly, larval incidence of S. frugiperda on the Bt soybeandid not differ significantly from the respective near-isogenicnegative checks for both maturity groups. However, defoliationby S. frugiperda on the Bt soybean was significantly lower than onthe near-isogenic negative checks for both maturity groups(Table 6).

4. Discussion

S. cosmioides, S. eridania and S. frugiperda exhibited low to nosusceptibility to MON 87701 �MON 89788 soybean containing theprotein Cry1Ac. Of the three species, S. frugiperda showed somesusceptibility to purified Cry1Ac, but even the maximum concen-tration tested did not cause high mortality. Therefore, these Spo-doptera species show higher tolerance to the Cry1Ac protein thansome other Lepidoptera species, such as C. includens, H. virescensand Helicoverpa zea (Boddie) (Luttrell et al., 1999), although thistolerance varies within and among populations of Spodoptera spp.(Luttrell et al., 1999; Adamczyk et al., 2008; Santos et al., 2009;Greenberg et al., 2010). This has been shown for populations ofS. frugiperda from Mexico, Brazil and Colombia, which differ insusceptibility to Bt strains, with some populations apparentlylacking receptors for certain insecticidal proteins (Monnerat et al.,

g on leaf-discs of MON 87701 � MON 89788 soybean of maturity groups 5.5 and 8.3

Maturity group 8.3a

R1eR2 V3eV4 V5eV6 R1eR2

5.5 � 4.1 a 4.0 � 2.1 a 6.9 � 3.1 a 5.4 � 2.5 a5.2 � 1.5 a 1.2 � 0.9 a 1.3 � 0.9 a 4.2 � 2.2 a

4.0 � 2.1 a 4.3 � 2.1 a 4.3 � 2.5 a 6.5 � 4.1 a1.9 � 1.1 a 4.2 � 2.8 a 3.1 � 2.2 a 4.2 � 2.2 a

41.3 � 4.4 a 35.2 � 7.6 a 34.4 � 4.1 a 32.9 � 3.8 a4.2 � 2.2 b 8.3 � 2.2 b 3.1 � 2.2 b 8.3 � 2.7 b

N 87701�MON 89788 soybean and the respective near-isogenic negative check forby the same letter in each column for each Spodoptera species are not significantly

MON 87701 × MON 89788 soybean Negative check

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Fig. 1. Duration and survival rates of life stages of Spodoptera cosmioides (A, B), Spodoptera eridania (C, D) and Spodoptera frugiperda (E, F) fed on MON 87701 � MON 89788 soybeanand a near-isogenic negative check. Pairs of columns with the same letters are not significantly different by t-test (P � 0.05).

O. Bernardi et al. / Crop Protection 58 (2014) 33e40 37

2006) or at least having a low number of binding sites in themidgut, as was observed for S. frugiperda to Cry1Ab (Aranda et al.,1996; Flannagan et al., 2005). Low susceptibility of Spodopteraspecies to Cry1Ac also may be associated with inactivation ofinsecticidal protein by proteases in the midgut (Rahman et al.,2012).

Overall, S. cosmioides and S. eridaniawere not susceptible to theCry1Ac protein in MON 87701 � MON 89788 soybean when eval-uated by leaf-disc bioassay and greenhouse infestation. No effect onbiological parameters was observed for these two species. How-ever, S. frugiperda had intermediate mortality when exposed toMON 87701 � MON 89788 soybean and showed reduced larvalbiomass, extended larval development and longer total life cycles.Similar results were observed when S. frugiperdawas fed on cottonleaves expressing Cry1Ac, resulting in lower larval biomass due tochanges in nutritional intake, digestion and food absorption(Ramalho et al., 2011). In another study, S. frugiperda had similar lifetable parameters on soybean leaves compared to corn and millet

(Pennisetum glaucum (L.) R. Brown), but lower performance oncotton, due the mortality in the early larval stages on cotton plants(Barros et al., 2010).

Within the IPM context, alternative strategies will be necessaryto control Spodoptera species when significant infestation occurs onMON 87701�MON 89788 soybean in the field. Considering that allthree Spodoptera species had reduced larval size at 14 days whenfed on MON 87701 � MON 89788 soybean (Table 3), larvae may bemore exposed to, and partially managed by, biotic and abiotic fac-tors, including insecticides sprayed for other insect pests. Moreover,the use of selective insecticides in conjunction with natural bio-logical control may increase the mortality of Spodoptera larvae onMON 87701 � MON 89788 soybean, because Cry1Ac has no nega-tive impacts on key natural enemies (Head et al., 2005; Romeiset al., 2006).

The high survivorship of Spodoptera species on MON87701 � MON 89788 soybean enables a large number of insects tocomplete their life cycle. Therefore, coefficients of selection for

Table 3Larval weight at 14 days after infestation (DAI), pupal weight and sex ratio of Spodoptera species fed onMON 87701�MON 89788 soybean and a near-isogenic negative check.

Biological parametera MON 87701 � MON 89788 soybean Negative check P value

Spodoptera cosmioidesLarval weight 14 DAI (mg) 305.04 � 18.73 378.53 � 13.06 0.0060Pupae weight (mg) 290.27 � 10.76 284.52 � 8.54 0.2539Sex ratio (\/\þ_) 0.48ns 0.49ns 0.8922Spodoptera eridaniaLarval weight 14 DAI (mg) 178.72 � 8.83 228.62 � 13.18 0.0056Pupae weight (mg) 218.89 � 9.46 204.52 � 2.80 0.1626Sex ratio (\/\þ_) 0.51ns 0.54ns 0.1983Spodoptera frugiperdaLarval weight 14 DAI (mg) 87.05 � 6.20 232.89 � 7.37 <0.0001Pupae weight (mg) 114.19 � 7.97 170.15 � 3.17 <0.0001Sex ratio (\/\þ_) 0.48ns 0.47ns 0.2928

nsThere were no statistically significant differences based on a Chi-square test (c2) (P > 0.05).a Values represent means � SE. A separate t-test (P � 0.05) was conducted between MON 87701 � MON 89788 soybean and the near-isogenic negative check for each

biological parameter.

Table 4Biological parameters of adult females of Spodoptera species fed on MON 87701 � MON 89788 soybean and a near-isogenic negative check.

Biological parametera MON 87701 � MON 89788 soybean Negative check P value

Spodoptera cosmioidesAdult female longevity (days) 15.00 � 1.00 15.25 � 1.10 0.9000Pre-oviposition (days) 4.50 � 0.42 3.00 � 0.58 0.0872Oviposition (days) 7.67 � 0.98 5.75 � 1.44 0.4583Post-oviposition (days) 3.33 � 1.03 4.00 � 0.89 0.7297Eggs/female/day 296.86 � 85.19 234.38 � 35.18 0.0937Total eggs/female 1744.47 � 373.04 1336.38 � 294.33 0.5670Spodoptera eridaniaAdult female longevity (days) 17.10 � 1.68 15.70 � 1.39 0.4465Pre-oviposition (days) 3.20 � 0.49 3.10 � 0.38 0.8735Oviposition (days) 8.90 � 1.04 8.30 � 0.97 0.6301Post-oviposition (days) 5.00 � 1.26 4.00 � 1.09 0.5560Eggs/female/day 167.72 � 13.56 186.66 � 21.84 0.4700Total eggs/female 1575.20 � 210.19 1433.80 � 105.95 0.5555Spodoptera frugiperdaAdult female longevity (days) 17.29 � 1.21 17.50 � 1.82 0.8463Pre-oviposition (days) 4.67 � 1.20 3.60 � 1.60 0.6030Oviposition (days) 9.67 � 1.07 8.11 � 1.65 0.4109Post-oviposition (days) 5.67 � 2.88 7.67 � 2.55 0.6501Eggs/female/day 31.59 � 10.83 129.45 � 41.30 0.0041Total eggs/female 377.70 � 133.75 1061.00 � 326.93 0.0001

a Values represent means � SE. A separate t-test (P � 0.05) was conducted between MON 87701 � MON 89788 soybean and the near-isogenic negative check for eachbiological parameter.

Table 5Fertility life table parameters of Spodoptera species fed onMON 87701�MON89788soybean and a near-isogenic negative check.

Biologicalparametera

MON 87701 � MON 89788soybean

Negative check P value

Spodoptera cosmioidesT (days) 46.35 � 0.35 46.51 � 0.33 0.6494Ro (\/\) 358.72 � 59.96 320.86 � 70.75 0.3355rm (\/\*day) 0.126 � 0.004 0.125 � 0.003 0.2800l 1.138 � 0.003 1.133 � 0.005 0.1833Spodoptera eridaniaT (days) 41.68 � 0.30 42.02 � 0.41 0.0530Ro (\/\) 475.30 � 34.98 457.58 � 59.60 0.8035rm (\/\*day) 0.149 � 0.002 0.144 � 0.004 0.3046l 1.160 � 0.003 1.155 � 0.005 0.3044Spodoptera frugiperdaT (days) 44.54 � 1.28 36.33 � 3.90 0.0250Ro (\/\) 53.78 � 21.27 292.99 � 93.81 0.0323rm (\/\*day) 0.091 � 0.010 0.154 � 0.023 0.0276l 1.095 � 0.012 1.166 � 0.027 0.0324

a Values represent means � SE. T ¼ mean generation time; Ro ¼ net reproductiverate; rm ¼ intrinsic rate of increase and l ¼ finite rate of increase.

O. Bernardi et al. / Crop Protection 58 (2014) 33e4038

Cry1Ac resistance will be low, and selection pressure minimal,particularly for S. eridania and S. cosmioides. However, the length-ened life cycle of S. frugiperdawill affect population growth. The useof other Bt crops such as corn and cotton also may affect the pop-ulation dynamics of this pest and reduce the flow of moths amongdifferent host crops. These aspects may reduce the populationdensity of Spodoptera species, facilitating the use of other effectivepest control tactics, but also have implications for resistance-management strategies.

The current challenge is to preserve the current level of sus-ceptibility of Spodoptera species to the Bt proteins currentlydeployed in Brazil, because S. frugiperda is being exposed to Btproteins expressed in corn (Cry1Ab, Cry1A.105, Cry2Ab2, Cry1F andVip3A), and S. frugiperda, S. eridania and S. cosmioides will beexposed to proteins in soybean (Cry1Ac) and cotton (Cry1Ac,Cry1Ab, Cry1F, Cry2Ab2 and Cry2Ae). Consequently, susceptibilitymonitoring to Bt proteins will be an important part of IRM pro-grams to ensure the sustainability of this control strategy.

Pyramiding of Bt proteins is a promising alternative for moreeffective control of Spodoptera species in soybean in the future.Among the potential insecticidal proteins are Cry1A.105, Cry1F,Cry2Ab and Vip3A, which all have greater toxicity to Spodoptera

Table 6Larval incidence and defoliation on MON 87701 � MON 89788 soybean of different maturity groups compared with near-isogenic negative checks after infestation withSpodoptera species in greenhouse trials.

Treatment Larval incidence (larvae/meter)a % Defoliationa

Spodoptera eridaniaMON 87701 � MON 89788 soybean (Maturity group 5.5) 18.75 � 5.19 a 24.75 � 8.13 aNegative check (Maturity group 5.5) 19.00 � 12.25 a 38.50 � 6.89 aMON 87701 � MON 89788 soybean (Maturity group 8.3) 13.50 � 12.84 a 22.75 � 8.15 aNegative check (Maturity group 8.3) 20.75 � 12.03 a 32.75 � 8.10 aSpodoptera frugiperdaMON 87701 � MON 89788 soybean (Maturity group 5.5) 3.88 � 2.20 a 0.63 � 0.32 bNegative check (Maturity group 5.5) 6.75 � 0.92 a 13.99 � 2.18 aMON 87701 � MON 89788 soybean (Maturity group 8.3) 2.00 � 1.19 a 0.05 � 0.05 bNegative check (Maturity group 8.3) 6.50 � 1.76 a 13.28 � 4.31 a

a Values represent means� SE. A separate t-test (P� 0.05) was conducted between MON 87701 �MON 89788 soybean and the respective near-isogenic negative check foreach variable evaluated (means followed by the same letter in each column for each Spodoptera species and maturity group are not significantly different).

O. Bernardi et al. / Crop Protection 58 (2014) 33e40 39

species than Cry1Ac (Siebert et al., 2008; Sena et al., 2009; Storeret al., 2010). In particular, the Vip-like proteins can producerobust Bt pyramids for the next generation of Bt soybean, becausethese proteins do not present any sequence homology with Cryproteins and causes pore formation with unique properties,evidencing a low potential for cross-resistance (Estruch et al., 1996;Lee et al., 2003; Gouffon et al., 2011). However, polyphagous insectspecies such as these Spodoptera species may exploit several cropplants in the landscape resulting in cross-habitat fluxes of insects inthe field. Therefore, the challenge is to deploy strategies to manageinsect resistance in the agricultural landscape by deploying Btproducts with highly efficacious and novel/unique modes of action.

Acknowledgments

We thank Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq) for granting a doctoral scholarship to the seniorauthor.

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