Does Bt maize cultivation affect the non-target insect community in the agro ecosystem?

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ww w . r b e n t o m o l o g i a . c o m

REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

Biological

Control

and

Crop

Protection

Does

Bt

maize

cultivation

affect

the

non-target

insect

community

in

the

agro

ecosystem?

Daniela

Chaves

Resende

a

_,

_Simone

_Martins

_Mendes

a,∗

_,

_Rosangela

_C.

_Marucci

b

_,

Alessandra

de

Carvalho

Silva

c

_,

_Mônica

_Matoso

_Campanha

a

_,

_José

_Magid

_Waquil

d

a_Embrapa_Milho_e_Sorgo,_Sete_Lagoas,_MG,_Brazil

b_Universidade_Federal_de_Lavras,_Lavras,_MG,_Brazil

c_Embrapa_{Agrobiologia,}_Seropédica,_RJ,_Brazil

d_Consulting_and_Training_in_Integrated_Pest_Management,_Sete_Lagoas,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16December2014

Accepted20November2015

Availableonline19December2015

AssociateEditor:MárioA.NavarroSilva

Keywords: Biosafety

GMmaize

Tri-trophicinteractions

Integratedpestmanagement

a

b

s

t

r

a

c

t

ThecultivationofgeneticallymodifiedcropsinBrazilhasledtotheneedtoassesstheimpactsofthis technologyonnon-targetspecies.Underfieldconditions,thepotentialeffectoninsectbiodiversitywas evaluatedbycomparingahomogeneouscornfieldwithconventionalandtransgenicmaize,expressing differentBtproteinsinsevencountiesofMinasGerais,Brazil.Therichnesspatternofnon-targetinsect species,secondarypestsandnaturalenemieswereobserved.Theresultsdonotsupportthehypothesis thatBtproteinaffectsinsectbiodiversity.Therichnessanddiversitydataofinsectsstudiedwere depend-entonthelocationandotherfactors,suchastheuseofinsecticides,whichmaybeamajorfactorwhere theyareused.

Introduction

Transgenicstrategiesforprotectingcropsagainstpestsdepend onthetransferandexpressionofdefensegenestothecropspecies ofinterest.Amongthemostwidelyknownandstudiedexamples of inducedresistance are those based ontheuse of the delta-endotoxinof thebacteriumBacillusthuringiensisBerliner,1915, alsoknownasBtcrops.Thisbacteriumoccursnaturallyinsoiland hastheabilitytoformcrystalproteinsduringthestationaryand/or sporulationphase(Vasconcelosetal.,2011).Afteringestionand solubilizationofthecrystalsinthemidgutoftheinsect,its degrada-tionoccursfromtheactionofproteases,releasingdelta-endotoxins orCryproteins,whichadheretospeciﬁcreceptors(Carneiroetal.,

2009).

Bttoxins have highspeciﬁcity,both forspeciﬁcreceptors in thegutand forthedegradationofprotein crystalsby the alka-linepHinsusceptiblespecies.Fordecades,Btbio-pesticideshave beenusedformosquitoandinsectpestcontrolinagriculturaland reforestationareas, and there have beennoreports of adverse effectsrelatedtotheiruse.However,thereisatleastone impor-tantdifferencebetweentheBtbio-insecticideand Bttransgenic

∗ Correspondingauthor.

E-mail:[email protected](S.M.Mendes).

plants.Thefirstcasedealswithamixtureofsporesandcrystals, sprayedonplants,and theymustbeactivatedinthegutofthe insects,whereasingeneticallymodified(GM)plants,theprotein isproducedalreadyactivatedinitstoxicform.Thus,thequestion concernsthoseherbivorousinsects thatdo notprovidesuitable conditionsintheirdigestivetracttoactivatetheproteinspresent inthebio-insecticides:maytheystillbeaffectedbythetoxinof theBttransgenicplant,iftheyhavespecificreceptors(Fontesetal.,

2003)?

ThecommercialintroductionofGMcropshasledtotheneed toassessthepossibleimpactsofthistechnologyonthe environ-ment,andamongthelikelyundesirableimpactsaretheeffectson non-targetorganisms.Somestudieshaveindicatedpossibletoxic effects ofBtinsecticidal proteinsonnon-target species, includ-ingotherherbivores,scavengers,predators,parasitoids andsoil fauna(Hilbecketal.,1998;Loseyetal.,1999;Schuleretal.,1999;

O’Callaghanetal.,2005;Romeisetal.,2014).However,mostof

thesestudies tested theeffectof theseproteinson thespecies inunnaturalconditions,notconsidering,forexample,ecological interactionsand theactuallevel ofexposureofsensitivestages undernaturalconditions(Daleetal.,2002).Morestudies, consider-ingmultivariatesystemsandexposureconditions,similartothose presentintheﬁeld,mayprovidemorerealisticinformationabout theharmfuleffectsofBtcropsonnon-targetspecies(forexample,

seeSearsetal.,2001).

http://dx.doi.org/10.1016/j.rbe.2015.12.001

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In addition, a number of studies have shown theimpact in somespeciﬁccases.Hilbecketal.(1998)reportedthatCry1 Ab-producingBtmaizeand pureCry 1Abproteinharmedlarvaeof Chrysoperlacarnea(Neuroptera:Chrysopidae),butinthisreview,

Romeisetal.(2014)showthatthereissufﬁcientinformation

avail-abletodaytoconcludethatBtmaizecontainingCry1Abdoesnot harmC.carnea.Theseauthorsdiscussthenecessitytodevelop con-ceptualﬁeldmodels,whichshouldbebasedonproperlydesigned studies that can be reproduced with a minimal probability of falsepositivesor negatives.Thus, studiesintheﬁeld shouldbe focused.

With regard to natural enemies – key species within agro ecosystemsthatprovidethebiological pestcontrolservice– Bt plantscouldaffectthemdirectly,bytheinsectfeedingonplant partsthatexpresstheprotein(asinthecaseofpredatorywasps andparasitoidsthatfeedonpollen)orindirectly,bytheuseofprey thathavefedonthetransgenicplants(Piresetal.,2003;Frizzasand

Oliveira,2006).Thesearchforpreyinparasitoidspeciesmayoccur

associatedwiththeperceptionofvolatilesproducedbyplantsasa functionofherbivory,whichalsorepresentsasourceofimpact,if GMplantshavetheirattractivenessmodiﬁed(Schuleretal.,1999). Foranyevent,theactualreductionofthepredatedpopulations,due tothepresenceoftheinsecticidalproteinintheGMplant,perse, mayrepresentanimpactonthepopulationstructureofthespecies ofparasitoidsandpredators(WhiteandAndow,2005).Froman environmentalpoint ofview,onepossibleadvantageoftheuse ofGMmaize wouldbeareduction ininsecticideapplications– especiallythebroadspectrumactiveingredient–sincetheeffect ofthesecanbemoreimpactfulonthepersistenceof theinsect community(Dively,2005;Naranjo,2005).

StudiesofBt-transgeniccropshaverevealedthatexposureto Cryproteinsvarieswidelyamongdifferentherbivorefeedingguilds andspecies(Raybouldetal.,2007;Romeisetal.,2009).Arthropods suchaspredatorsorparasitoidsaremainlyexposedtothe plant-producedtoxinswhenpreyingonorparasitizingherbivoresthat havefedonGM crops.Thereisevidencethattheconcentration ofthearthropod-activecompoundisusuallydilutedasitmoves upthefoodchainanddoesnotaccumulate(Romeisetal.,2009;

MeissleandRomeis,2009,2012).Despiteanypossibleadvantage

associatedwiththeuseofGMOs,thecommercialreleaseofthese organismsisprecededbysafetyassessmentstudiescarriedoutin eachcase.InBrazil,theNationalBiosafetyTechnicalCommission (CTNBio)isinchargeofthesafetyassessmentofGMO cropsRegard-ingenvironmentalriskassessment,fewdataunderﬁeldconditions areavailable,somoreresearchisneededtosupporteffective mod-elstoanticipatepotentialchangesintheagroecosystem.Capalbo

etal.(2009)emphasizethattheBraziliansystemdoesnotrequire

aspecificevaluationprocess,whichallowstheuseofanyorganism model,aslongasthechoiceisdescribedandjustified.For scien-tificdevelopment,thecontinuousprocessofanalysisandselection

highlightstheneedforexpost-releasemonitoringofBtriskand impactsonthenon-targetcommunity.

However,sinceGMcropsrepresentarecenttechnological inno-vationandanovelevolutionarystrategy,itisessentialtomaintain aprocessofcontinuousmonitoringandevaluationofitsefﬁcacy andeffectsontheenvironment,especially independent posteri-oririsk assessments(Bauer-Panskusand Then, 2014).Thus, the aimofthisstudywastoassesspossibleimpactsofBtmaizeon theinsectbiodiversitypresentintheagroecosystemindifferent regionsofMinasGerais,comparingcorn-ﬁeldsgrowing conven-tionalandtransgenicmaize,expressingdifferentBtproteins.The workinghypothesiswasthatthepresenceoftheBtproteinsdoes notaffecttherichnessanddiversityofinsectspresentincrops.

Materialandmethods

Collectionofbiologicalmaterial

ThisworkinvolvedmonitoringtheincidenceofS.frugiperda– theprimarytargetpestofmaize–infestingthewhorlsandears, andtheinsectcommunityonconventionalandBtmaize express-ingdifferentproteins,insevendifferentcountiesofMinasGerais

(Table1).

Inordertobalancethetechnologicallevel usedineach corn fieldfromdifferentsamplingareas,sampleswerecollectedfrom cropareasofmorethan350haofmaizewithexpectedproductivity around200bags/ha.Toenablecomparisonoftheinsect commu-nity,collectionsfromcornfieldscultivatedwithconventionaland Btmaize,expressingdifferentproteins,wereconducted.The col-lectionsweremadeinNovemberandDecemberof2010.Thecrop fieldwithconventionalmaizereceivedthreeinsecticide applica-tionsandtheBtmaizereceivednone.

Thecollectionofbiologicalmaterialwasperformedina sys-tematic wayinorder toenable comparisonof therichness and diversityobservedonconventionalandBtmaize.Ineachsample cornﬁeld,threesamplingpointswereselectedandusedas replica-tion.Ateachsamplingpoint,usingthemethodproposedbyWaquil

(1997),whorls,earsandtasselsof10randomlychosenplantswere

collected.ThecollectedmaterialwastakentotheEmbrapaMaize andSorghumlaboratory,inSeteLagoas,MG.Insectsfoundinthe collectedmaterialwerestoredin70%ethanol,separatedand iden-tiﬁedusingbibliographicmaterialavailableandwiththeassistance ofspecialistsindifferentgroups.Thematerialwasidentiﬁed,when possible,atspecieslevel.

Statisticalanalyses

ToevaluatethecultivationeffectofBtmaize(differentproteins) onthe abundanceof S.frugiperda, variance analyses were per-formedontwofactors,consideringtheeffectoftreatment(maize

Table1

LocationsofconventionalandBtmaizeexpressingproteinsstudiedindifferentcountiesoftheStateofMinasGerais.

County Stateregion Coordinates Btprotein

TrêsCorac¸ões South/SouthwesternMinas 21◦₄₁₄₁_S,₄₅◦₁₅₁₉ _Cry1F,_Cry1A105₊_Cry2Ab2,_Cry1Ab

Nazareno CampodasVertentes 21◦₁₂₅₇_S,₄₄◦₃₆₃₉ _Cry1F,_Cry1A105₊_Cry2Ab2,_Cry1Ab

Iguatama WesternMinas 20◦1026S,45◦4239 Cry1Ab,

Cry1A105+Cry2Ab2,Cry1F

Inhaúma MetropolitanregionofBeloHorizonte 19◦₂₉₂₇_S,₄₄◦₂₃₂₄ _Cry1F,

Cry1Ab

Matozinhos MetropolitanregionofBeloHorizonte 19◦₃₃₂₈_S,₄₄◦₄₅₁ _Cry1F,

Cry1Ab

VarjãodeMinas NorthwesternMinas 18◦2240S,46◦155 Cry1F,

Cry1Ab

IraídeMinas MinasTriangle/AltoParanaíba 18◦₅₉₂_S,₄₇◦₂₇₃₉ _Cry1F,

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Três Corações ≤ 1cm 1 to 2 cm >2 cm -2 0 2 4 6 8 10 12 Larvae abundance Iguatama ≤ 1cm 1 to 2 cm >2 cm Inhaúma ≤ 1cm 1 to 2 cm >2 cm Iraí de Minas ≤ 1cm 1 to 2 cm >2 cm Matozinhos ≤ 1cm 1 to 2 cm >2 cm Nazareno ≤ 1cm 1 to 2 cm >2 cm Varjão de Minas ≤ 1cm 1 to 2 cm >2 cm

Conv. crops Cry1Ab Cry1F Cry(1A105+2Ab2)

Fig.1. AbundanceofdifferentsizesoflarvaeofSpodopterafrugiperdainwhorls

ofconventionalandtransgenicmaize(Cry1Ab,Cry1FandcombinedCry2Ab2and

Cry1A105proteins)fromcornﬁeldsofdifferentcountiesinMinasGerais.Bars

rep-resenta95%conﬁdenceinterval.

hybrid)andthesizeofthelarvae,accordingtoFernandesetal.

(2003).Theseanalyseswereperformed separatelyfor data

col-lectedinthewhorlandearandgroupedatthelocation(county), sincetheexperimentaldesignwasincomplete,becausenotallcrop ﬁeldsusedthesamehybridseeds.

SpeciesrichnesswasestimatedusingtheJackknifeprocedure, sincethismethodaddressestheintrinsicdefectof underestima-tionthatoccursinthistypeofdata(HeltsheandForrester,1983). TheJackknifeprocedure,beingare-samplingtechnique,provides inadditiontospeciesrichnessestimation,anestimateofthe conﬁ-denceinterval,allowingastatisticalcomparisonbetweendifferent locations.To estimatethediversity,theShannon–Wienerindex

(Krebs,1999)wasused.TheEstimateS8.0software(Colwell,2000)

wasusedfortheanalyses.

ToevaluatetheeffectofBtmaizecultivationontheestimated richness and diversity of secondary pestsand natural enemies, varianceanalyseswereperformedontwofactors,consideringthe effectof treatment(type ofprotein)and the type oforganism. Theseanalyseswereperformedseparatelyfordatacollectedinthe whorl,earandtasselandgroupedatthelocation(county),since theexperimentaldesignwasincomplete,becausenotallthecrops usedthesameseedtype.Tofacilitateinterpretationoftheresults, datafrom all cornﬁeldswith differentBt maize crops are pre-sentedtogetherincharts,thusconsideringtheconventionalandBt treatments.

Regressionanalyseswerealsoperformedtoevaluateifthe rich-ness of natural enemies present in the cropﬁelds was related totherichnessofsecondary pestsand ifthetotal areasownin eachcornﬁeldaffectedtheinsectrichnessbylocation.Toevaluate theinsecticidesprayingaffectedontheestimatedinsectrichness, Pairedt-testanalyseswereperformed.

Results

AbundanceofS.frugiperda

TheabundanceofS.frugiperdalarvaeofdifferentsizescollected inwhorlsampleswasdependentonthelocation(N=252,F=1.56, p<0.05,Fig.1).TheabundanceofS.frugiperdainthecornfieldwith Btmaizewaslowerinthethreestudiedcounties–Inhaúma,Três CoraçõesandNazareno.InInhaúma,thedifferenceoccurredonly forlarvaewitha bodysizesmallerthan1cm, inTrêsCorações,

Três Corações ≤ 1cm 1 to 2 cm >2 cm –3 –2 –1 0 1 2 3 4 5 6 7 8 Larvae abundance Iguatama ≤ 1cm 1 to 2 cm >2 cm Iraí de Minas ≤ 1cm 1 to 2 cm >2 cm Nazareno ≤ 1cm 1 to 2 cm >2 cm Varjão de Minas ≤ 1cm 1 to 2 cm >2 cm

Conv. crops Cry1Ab Cry1F Cry(1A105+2Ab2)

Fig.2.AbundanceofdifferentsizelarvaeofSpodopterafrugiperdainearsof

conven-tionalandtransgenicmaize(Cry1Ab,Cry1FandcombinedCry2Ab2andCry1A105

proteins)fromcornﬁeldsofdifferentcountiesinMinasGerais.Barsrepresenta95%

conﬁdenceinterval.

wherethedifferencewasforlarvaesmallerthan2cm,andﬁnallyin Nazareno,wheretheconventionaltreatmentshowedhigher abun-danceofS.frugiperdaindependentoflarvalsize.

Thetreatmenteffectsonfrequencyofthedifferentlarvalsizes ofS.frugiperdaintheearsweresignificantlydifferentamongthe samplingsites(N=162,F=1.96,p<0.01,Fig.2).InTrêsCorações,for larvaesmallerthan1cm,theabundanceofS.frugiperdawashigher inthecornfieldwithconventional maize.For larvaelargerthan 2cm,theabundanceofS.frugiperdawashigherintheconventional cornfieldthaninthefieldwithBtmaize,exceptintheBtcornfield withCry1Abprotein.InIraídeMinas,forlarvalbodysizesmaller than1cminthecornfieldwithconventionalmaize,theabundance ofS.frugiperdawaslowerthan thein cornfieldwithtransgenic maize.Moreover,inthecornfieldwithCry1Fprotein,the abun-danceofS.frugiperdawashigherthaninthecornfieldwithCry1Ab protein.Forlarvalbodysizebetween1and2cm,theabundance ofS.frugiperdawashigherinthecornfieldwithCry1Abprotein, whereasforlarvalbodysizegreaterthan2cm,theconventional cornfieldhadquiteahighabundanceofS.frugiperda.Andfinally, inVarjãodeMinasforlarvaesmallerthan1cm,thecornfield culti-vatedwiththeBthybridexpressingCry1Abproteinshowedhigher abundanceofS.frugiperdathantheothertreatments,whereasfor larvaelargerthan2cm, theconventional cornfieldhad ahigher abundanceofS.frugiperdathanthecornfieldwithCry1Ftransgenic maize.

Overallinsectrichness

Theoverallpatternofestimatedrichnessofinsectsinthe differ-entplantpartsofmaizewasdistinctamongthesevencropﬁelds studied,andtheresultsdidnotsupportthehypothesisofBt pro-teinshavinganegativeeffectoninsectrichness.

Consideringtheinsectcommunityinthewhorl,areductionin richnesswasobservedintheBtcornfields,withhybridsexpressing Cry1AbproteinfromthecountiesofNazareno(Fig.3),Varjãode Minas(Fig.4)andIraídeMinas(Fig.5).InTrêsCorações(Fig.6) andInhaúma(Fig.7),theestimatedinsectrichnessinthecornfield withBtmaizewashigherthaninconventionalmaize.Comparing onlytheBtcornfieldsinTrêsCoraçõesandIguatama,the trans-genicmaizewithCry2Ab2andCry1A105proteins, theobserved richnesswaslowerin thefirstcrop,only whencompared with acornfieldexpressingCry1Fprotein.In InhaúmaandNazareno,

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Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F 0 2 4 6 8 10 12 14 16 18 20 22 24 Estimated richness Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105

Whorl Ear Tassel

Fig.3.Estimatedrichnessofinsectsinthewhorl,earandtasselofconventional(Conv.)andtransgenicmaize(Cry1Ab,Cry1FandcombinedCry2Ab2andCry1A105proteins)

fromNazarenocounty,MG.Barsrepresent95%conﬁdenceinterval.

theestimatedinsectrichnessinacornﬁeldwithCry1Ftransgenic maizewaslowerthanintheotherlocationswithtransgenicmaize expressingthecombinedproteinsCry2Ab2andCry1A105.

Thedatafromtheearsshowedareductioninestimatedrichness ofinsectsintheBtcornfieldinNazareno,exceptfortheBt(Cry1Ab) cornfield(Fig.3)andinVarjãodeMinas,onlyinthecornfieldwith Cry1Abprotein(Fig.4).InTrêsCorações(Fig.6),thecornfieldwith conventional maize had a lower estimated richness compared to transgenic maize with Cry2Ab2 and Cry1A105 proteins. In

Iguatama(Fig.8),onlyintheBt(Cry1F)cornﬁeldwastheinsect richness lower than in other treatments, whereas in Nazareno (Fig.3),theBt(Cry1Ab)cornﬁeldpresentedhigherrichnessthan inothertransgenictreatments.

Finally,consideringthedatafromtassels,theestimatedinsect richnesswaslowerintheconventionalcornfieldinTrêsCorações (Fig.6),IraídeMinas(Fig.5),Nazareno(Fig.3)andVarjãodeMinas (Fig.4)when comparedto therichnessobserved in allBtcorn fields.Inothercropfields,therewasnodifferenceinestimated

Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F

2 4 6 8 10 12 14 16 18 20 Estimated richness

Whorl Ear Tassel

Fig.4.Estimatedrichnessofinsectsinthewhorl,earandtasselofconventional(Conv.)andtransgenicmaize(Cry1AbandCry1Fproteins)fromVarjãodeMinascounty.

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Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F Conv. Cry1Ab Cry1F 0 2 4 6 8 10 12 14 16 Estimated richness

Whorl Ear Tassel

Fig.5.Estimatedrichnessofinsectsinthewhorl,earandtasselofconventional(Conv.)andtransgenicmaize(Cry1AbandCry1Fproteins)fromIraídeMinasGeraiscounty.

Barsrepresent95%conﬁdenceinterval.

insectrichnessbetweenconventionalandBtcornfields.Comparing onlythetreatmentswithBt(Cry1Ab)maizeinTrêsCoraçõesand Iguatama,therichnesswashigherthanintheothertreatments, whereasinVarjãodeMinastherichnesswashigherthantheother treatmentsinaBtcornfieldwithCry1Fprotein(seeFig.9). Richnessofnon-targetinsects:secondarypestsandnatural enemies

Consideringthedatafrommaizewhorls,therewasno inter-actionbetweenthetreatments’effectontheestimatedrichness

ofsecondarypestsandnaturalenemies(N=155,F=1.63,p<0.05,

Fig.10).Itcouldbeobservedthatinthecornfieldfromthethree counties(TrêsCorações,InhaúmaandMatozinhos),therichnessof secondarypestswashigheronBtthanonconventionalmaize.In Iguatama,therewasareductioninthesecondarypests’richness bygrowingBtmaize,whileinNazareno,boththesecondarypests’ andnaturalenemies’richnesswerehigherintheconventionalcorn field(Fig.10B).

Similarly,therewasaninteractionbetweentreatmenteffectson diversity–estimatedbytheShannonindex–ofsecondarypestsand naturalenemies(N=155,F=1.85,p<0.01,Fig.11).Itwaspossible

0 2 4 6 8 10 12 14 16 Estimated richness Whorl Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105 Tassel Ear

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 Estimated richness Conv. Whorl Tassel Cry1F

Cry1Ab Conv. Cry1Ab

Fig.7.Estimatedrichnessofinsectsinthewhorlandtasselofconventional(Conv.)

andtransgenicmaize(Cry1AbandCry1Fproteins)fromInhaúmacounty,MG.Bars

represent95%conﬁdenceinterval.

toobserveareductioninthesecondarypests’andnaturalenemies’ diversityincropfieldswithBtmaize,inIguatamaandNazareno counties.InVarjãodeMinas,therewasareductiononlyin sec-ondarypests’diversity.InTrêsCoraçõesandMatozinhos,inturn, wefoundanincreaseinthediversityofsecondarypestsintheBt cornfield.

Regarding ears, there was also no interaction between the treatmenteffectontheestimatedrichnessof differenttypesof organismsandlocation(N=108,F=2.35,p<0.01,Fig.12).Infourof thefivecropfieldsstudied,thepatternobservedwassimilar,with nodifferencebetweentherichnessestimatedinconventionaland Btcornfields,despitehigherestimatedrichnessofsecondarypest speciesthanthenaturalenemy.However,inVarjãodeMinas,the estimatedsecondarypestrichnesswashigherintheconventional

Conv. Cry1Ab Cry1F Conv. Cry1F

4 6 8 10 12 14 16 Estimated richness Whorl Tassel

Fig.9. Estimatedrichnessofinsectsinthewhorlandtasselofconventional(Conv)

andtransgenicmaize(Cry1AbandCry1Fproteins)fromMatozinhoscounty,MG.

Barsrepresent95%conﬁdenceinterval.

cornfield than in the Bt cornfield, while the natural enemies’ estimatedrichnesswashigherintheBtcornfield.

Thetreatments’effectsoninsectdiversity–estimatedbythe Shannon index – wasdifferent between studied sites (N=108, F=3.75, p<0.001, Fig. 13). In the cornfields in Três Corações, IguatamaandIraídeMinastherewerenodifferencesinestimated diversitybetweentheconventionalandBtcornfields.Inthecrop fieldsstudiedinNazarenoandVarjãodeMinas,theestimated sec-ondarypests’diversitywashigherintheconventionalcornfield and,moreover,inVarjãodeMinas,thenaturalenemydiversitywas higherintheBtcornfield.

Finally,considering therichness onplanttassels,once again thetreatmenteffectsonthetypeoforganismswassigniﬁcantly dependentonthestudiedsites(N=132,F=5.22,p<0.001,Fig.14).

2 4 6 8 10 12 14 16 18 Estimated richness Tassel Whorl Ear Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105 Cry2Ab2/ Cry1A105

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Três Corações

N.E. S.P.

N.E.

S.P. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

N.E.

S.P. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

–2 0 2 4 6 8

A

B

Estimated richness Iguatama Inhaúma Iraí de

Minas Matozinhos Nazareno

Varjão de Minas Três Corações –2 –1 0 1 2 3 4 5 6 7 Estimated richness Iguatama Inhaúma Iraí de

Varjão de Minas Conv. Cry1F Cry1Ab Cry(1A105+2Ab2)

Conventional Bt

Fig.10.Estimatedrichnessofsecondarypests(S.P.)andnaturalenemies(N.E.)inconventionalandtransgenicmaizewhorlsforCry1Ab,Cry1FandcombinedCry2Ab2and

Cry1A105proteins(A)andestimatedrichnessinconventionalmaizeandBtmaize(B)indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdenceinterval.

InthecornfieldsfromTrêsCorações,Iguatama,IraídeMinasand VarjãodeMinastherewerenosignificantdifferencesbetween con-ventionalandBtcornfieldseitherforsecondarypests’orfornatural enemies’estimatedrichness.InInhaúmaandNazareno,the esti-matedrichness ofsecondarypestswashigherintheBtthanin theconventionalcornfield,whileinMatozinhosboththerichness ofsecondarypestsaswellasnaturalenemieswashigherinthe conventionalcornfield.

Again,therewasaninteractionbetweentreatmenteffectson theestimateddiversity oforganismsonplanttasselsandstudy location(N=132,F=5.44,p<0.001,Fig.15).Insixcropﬁelds,there wasnodifferenceinestimateddiversity,eitherforsecondarypests orfornaturalenemies,betweenconventionalandBtcornﬁelds. However,itisworthnotingthatinInhaúma,therewasa consider-ableincreaseinthediversityofnaturalenemies.InMatozinhos,in turn,thesamepatternwasnotobservedsincethediversity,both

ofsecondarypestsaswellasnaturalenemies,washigherinthe conventionalthanintheBtcornﬁeld.

Among the insect species observed in different studied parts of maize plants, Carpophilus sp. (Coleoptera: Nitiduli-dae) and Euxesta sp. (Diptera:Ulidiidae) were the species that showedanapparentreductioninabundanceintheBtcornfield. Amongthe predators,the earwigDoru luteipes (Scudder, 1876) (Dermaptera:Forficulidae)andminutepiratebugOriusinsidiosus (Say,1832)(Hemiptera:Anthocoridae)showedconsiderable fluc-tuationin abundanceamong thedifferenttreatmentsevaluated

(Table2).

Localcharacteristics

TheeffectsofBtmaizeontheoverallrichnessanddiversityof insectsappeartobedependentonthegeographicallocationand

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Três Corações

N.E. S.P.

N.E.

S.P. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

N.E.

S.P. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

–0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

A

B

Shannon diversity Iguatama Inhaúma Iraí de

Varjão de Minas Três Corações –0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Shannon diversity Iguatama Inhaúma Iraí de

Varjão de Minas Cry1Ab

Conv. Cry1F Cry(1A105+2Ab2)

Conventional Bt

Fig.11.Estimateddiversitysecondarypests(S.P.)andnaturalenemies(N.E.)inconventionalandtransgenicmaizewhorlsforCry1Ab,Cry1FandcombinedCry2Ab2and

Cry1A105proteins(A)andestimateddiversityinconventionalmaizeandBtmaize(B)indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdenceinterval.

cropmanagementintheagroecosystem.Sothereisanindication thatotherfactors,suchassprayinginsecticidesonthecornﬁeldor not,mayexertastrongerinﬂuenceonthisprocess.

Theestimatedrichnessofnaturalenemies,however,appearsto beinﬂuencedbytherichnessofsecondarypests,inspiteofthe possibilityof a limitednumber of samplingstoshowthis rela-tionship (R2₌_0.51, _b₌_0.71, _t₌_2.28, _p₌_0.07, _Fig.₁₆_)._Among_all

thesitesstudied,onlyInhaúmaisfurtherremovedfromthe pro-posedmodel,presentingahigherrichnessofnaturalenemiesthan wouldbeexpectedbasedonthenumberofsecondarypests,sothat, excludingthisanalysislocation,thisrelationshipwasconﬁrmed (R2₌_0.75,_b₌_0.86,_t₌_3.46,_p_<_0.05).

Thetotalareasownineachcornﬁelddidnotinﬂuencethe esti-matedrichness of insects (R2₌_0.00;_b₌_0.00;_t₌_0.009,_p₌_0.99).

Similarly, there wasnoeffect of theuse of insecticidesonthe estimatedinsect richness(N=7,t=0.48, p=0.64),for secondary

pests (N=7,t=0.31, p=0.76) or natural enemies (N=7, t=1.63, p=0.15).

Discussion

TheeffectoftheuseofBtmaizecontrollingS.frugiperdawas dependentontheBtevent consideredandthelocation studied during all samplingtimes. These data suggest that the species community mayrespondtothepresenceof Btmaize linkedto interactions among different factors like local adaptations that generatethecombinationsofdifferentpopulations(Busatoetal.,

2004,2005).Forsamplesfromthewhorl,onlythreeoftheseven

cornfieldsstudiedshowedenoughinfestationtodiscriminatethe treatments that reduced theabundance of larvae onBt maize. Furthermore,inthesecornfieldstheabundanceoflarvaeofsmall size(upto2cm)inconventionalmaizewassignificantlygreater

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Três Corações S.P. N.E. –1 0 1 2 3 4 5 6 7

A

B

Estimated richness

Iguatama Iraí de_Minas Nazareno Varjão de_Minas

S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

Três Corações –1 0 1 2 3 4 5 6 7 Estimated richness Iguatama Iraí de Minas Nazareno Varjão de Minas

S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

Conv. Cry1F Cry1Ab Cry(1A105+2Ab2)

Conventional Bt

Fig.12. Estimatedrichnessofsecondarypests(S.P.)andnaturalenemies(N.E.)in

earsofconventionalandtransgenicmaizeforCry1Ab,Cry1FandcombinedCry2Ab2

andCry1A105proteins(A)andestimatedrichnessinconventionalmaizeandBt

maize(B)indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdence

inter-val.

thantheabundanceoflargerlarvae.AccordingtoWaquilet al.

(2004)andMendesetal.(2011),theuseofBtmaizeoftendoes

notleadtotheimmediatedeathofcaterpillars,butreducestheir development,making theinsect more vulnerable tobiotic and abiotic mortality factors. Another hypothesis for this situation maybeassociated withtheresultsencounteredby Paulaet al.

(2014),indicatingthatthelepdopteranChlosynelacinia,in

exper-imentalconditions,transferredtodescendantstheBtinsecticidal protein,increasing the mortalityand developmental time. It is noteworthythat all conventional cornﬁelds weresprayed with insecticides,whichshowsonceagainthatdifferentlocationsmay present different difﬁculty levels regarding the control of this pest.

Theoverallpattern ofestimated richnessof secondarypests andnaturalenemieswasverydifferentamongthesevencornfields studied,andthevariation observedwasnotconsistentwiththe hypothesisoftheBtproteins’effectonthestructureoftheinsect community. In many cases, the insect richness estimated for conventionalandBtcornfieldswasnotsignificantlydifferentor waslowerthanonBtmaize.Again,consideringthatconventional cornfieldsunderwentinsecticidespraying,itislikelythatthelow insectrichnessinthesefieldsistheresultofthisimpact.However, although other studieshave already shown that the impact of

Três Corações

S.P. N.E.

S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

S.P. N.E. S.P. N.E. S.P. N.E. S.P. N.E.

–0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

A

B

Shannon diversity Iguatama Iguatama Iraí de Minas Iraí de Minas Nazareno Nazareno Varjão de Minas Varjão de Minas –0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Shannon diversity

Conventional Bt

Fig.13.Estimateddiversityofsecondarypests(S.P.)andnaturalenemies(N.E.)in

earsofconventionalandtransgenicmaizeforCry1Ab,Cry1FandcombinedCry2Ab2

andCry1A105proteins(A)andestimateddiversityinconventionalmaizeandBt

maize(B),indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdence

interval.

insecticideusemaybestrongeronthestructure ofinsect com-munitiesthantheimpactsoftransgenicBtcrops,inthepresent study,theestimatedinsectrichnesswasnotsignificantlyaffected byinsecticideuseonthestudiedcornfields(Dively,2005).Since allthesecroplandsarealreadyoccupiedbyBtmaizeformuchof theircommercialproduction,itcanbeseenthattherehasbeena reductionintheuseofthesepesticides.Inonlythreeoftheseven cropfieldsstudiedhavebroad-spectrum insecticidesbeenused and,insuchcases,onlyonconventionalmaizecultivars.Moreover, inonlyfourofthesecornfieldswasinsecticidesprayedmorethan once,withamaximumofthreeapplications(inInhaúmaandTrês Corações).Thisrepresentsalowuseofinsecticideifcomparedwith otherregions,whereitiscommontohaveuptoeightsprayings duringacropseason.

The abundance of Carpophilus sp. was reduced in Bt crops, regardlessoftheplantpart.Thisresultisconsistentwithother studiesshowingthatdamagereductioninBtcultivation–fromthe targetpest–resultsinalowercolonizationbysaprophagicspecies

(Dively,2005).SpeciesofDiptera,genusEuxesta,mayalsoundergo

thiseffect,becauseovipositionofthisspeciesappearstobe stimu-latedbythepreviousplantdamageoccurrence,whichhappensless inBtmaizeﬁelds(Cruzetal.,2011).Theapparentﬂuctuationsin theabundanceofpredatorsamongthedifferenttreatmentsstudied

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Table2

Totalabundanceofinsectbyspecies(herbivoresandnaturalenemies)sampledinplants(whorl,earandtassel)ofconventionalandtransgenicmaizeexpressingCry1Ab,

Cry1FandcombinedCry2Ab2andCry1A105proteinsfromdifferentMinasGeraiscounties.

Taxon Conventional Cry1Ab Cry1F Cry(1A105+2Ab2)

Whorl

Herbivores

DalbulusmaidisDeLong,1923 4 3 2 1

Peregrinusmaidis(Ashmead,1890) 0 1 0 0

Deoisﬂavopicta(Stal,1854)andMahanarvasp. 8 11 1 2

DiabroticaspeciosaGermar,1824 0 5 6 1

DiabroticaviridulaFabricius,1801 1 0 1 0

Thrips(Thripidae) 1 1 0 0

Lagriavillosa(Fabricius,1781) 0 11 3 0

Rhopalosiphummaidis(Fitch,1856) 688 1101 329 109

Colapsissp. 20 33 9 0 Elateridae 2 0 0 0 Euxestasp. 8 4 6 0 Carpophilussp. 25 13 3 5 Naturalenemies Reduviidae1 1 1 2 1 Reduviidae2 0 1 0 1

Oriusinsidiosus(Say,1832) 2 0 1 0

Miridae 0 2 0 0

Lebiasp. 1 1 2 0

Anthicidae 1 0 0 0

Callidascutellaris 1 1 0 0

DoruluteipesScudder,1876 34 30 4 2

Chrysopidae 0 16 1 0

Geocorispunctipes(Say,1832) 10 4 1 2

Ear

Herbivores

DalbulusmaidisDeLong,1923 0 0 2 0

Thrips(Thripidae) 0 1 0 2

Lagriavillosa(Fabricius,1781) 0 1 0 1

Colaspsissp. 1 0 9 7 Euxestasp. 105 49 54 73 Carpophilussp. 229 183 59 49 Naturalenemies Reduviidae1 17 8 3 0 Reduviidae2 0 1 1 0 Nabissp. 1 1 0 0

Lebiasp. 0 0 0 1

Cyclonedasanguinea(Linnaeus,1763) 1 2 1 0

Chrysopidae 1 0 0 1

Euboreliaannulipes(Lucas,1847) 0 1 0 0

Tassel

Herbivores

DalbulusmaidisDeLong1923 0 2 3 1

Peregrinusmaidis(Ashmead,1890) 0 4 0 0

DiabroticaviridulaFabricius,1801 7 11 0 0

Thrips(Thripidae)sp1 0 0 1 0

Thrips(Thripidae)sp2 0 1 0 0

Colaspsissp. 2 4 1 0 Euxestasp. 1 0 0 0 Carpophilussp. 77 53 22 0 Naturalenemies Reduviidaesp.1 7 7 0 0 Reduviidaesp.2 2 0 0 0 Nabissp. 1 0 0 0

Lebiasp. 0 1 0 0

Anthicidae 0 1 0 0

Callidascutellaris 0 4 0 0

Cyclonedasanguinea (Linnaeus,1763) 7 1 1 0

Chrysopidae 1 0 0 0

Cantharidae 2 0 0 0

Euboreliaannulipes(Lucas,1847) 1 4 0 0

Syrphidae 2 0 0 0

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Três Corações

S.P.N.E.

S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E.

S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E.

–2 –1 0 1 2 3 4 5 6 7 8 9

A

B

Estimated richness

Iguatama Inhaúma Iraí de_{Minas Matozinhos Nazareno}Varjão de_Minas

Três

Corações Iguatama Inhaúma Iraí de

Minas Matozinhos Nazareno Varjão de Minas –2 –1 0 1 2 3 4 5 6 7 8 9 Estimated richness

Conventional Bt

Fig.14.Estimatedrichnessofsecondarypests(S.P.)andnaturalenemies(N.E.)

intasselsofconventionalandtransgenicmaizeforCry1Ab,Cry1Fandcombined

Cry2Ab2andCry1A105proteins(A)andestimatedrichnessinconventionalmaize

andBtmaize(B),indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdence

interval.

appearsconsistentwiththevariationobservedintheabundanceof othernon-targetherbivores,whichisalsoconsistentwithresults alreadyreportedintheliterature,sincepredators–generalist nat-uralenemies–seemtobelessaffectedbythepresenceofBtmaize thanparasitoids(Pilcheretal.,2005;Romeisetal.,2014).

Thediscussionontheimpactorenvironmentalriskassociated withtheuseof genetically modiﬁed*cropsin agriculturemust startwiththedeﬁnitionofthereferencestartingpoint(Frizzasand

Oliveira,2006)andbefollowedbymonitoringasstatedbyCTNBio.

Thisisbecausetheuseoftransgenicplantsisjustonemoremethod amongvariousIntegratedPestManagement(IPM)strategies avail-able.Regardingcommunity diversity,therichness of secondary pestswasdirectlyrelatedwiththerichness ofnaturalenemies. Also,thedataandrecentliteratureindicatenosignificanteffectof Btproteinsonnaturalenemies.Variouspracticesusedinmodern agricultureareassociatedwiththedeclineofbiodiversityinagro ecosystems,especiallythoserelatedtointensiveagriculture,such asmonoculture,useoffertilizersandpesticides.Thesimplification ofthelandscaperelatedtoagriculturereducesthediversityoffauna andthestructureofcommunitiesassociatedwithagroecosystems andthefindingsofthisstudyreinforcethisidea.

Três Corações

N.E. S.P.

N.E.

S.P. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E.

N.E.

S.P. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E. S.P.N.E.

–0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

A

B

Shannon diversity Iguatama Inhaúma Iraí de

Minas Matozinhos Nazareno Varjão de

Minas

Três

Corações Iguatama Inhaúma Iraí de

Minas Matozinhos Nazareno Varjão de Minas –0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Shannon diversity Conventional Bt

Fig.15.Estimateddiversityofsecondarypests(S.P.)andnaturalenemies(N.E.)

intasselsofconventionalandtransgenicmaizeforCry1Ab,Cry1Fandcombined

Cry2Ab2andCry1A105proteins(A)andestimatedrichnessinconventionalmaize

andBtmaize(B),indifferentcountiesinMinasGerais.Barsrepresent95%conﬁdence

interval. Iguatama Inhaúma Iraí de Minas Matozinhos Nazareno Três Corações Varjão de Minas 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5

Estimated richness of secondary pests

7 8 9 10 11 12 13 14 15 16

Estimated richness of natural enemies

Fig.16.Relationshipbetweenestimatedrichnessofsecondarypestsandestimated

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Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ToEmbrapaMilhoeSorgoandespeciallytotheﬁeldand labo-ratorysupportteam:EustáquioFranciscoSouzadeOliveira,Ismael M.MacielandAdemilsonS.daRochafortheircollaborationinthis work.

References

Bauer-Panskus,A.,Then,C.,2014.Casestudy:industryinﬂuenceintherisk

assess-mentofgeneticallyengineeredmaize1507.TestbiotechBackground10,1–32.

Busato,G.R.,Grützmacher,A.D.,Garcia,M.S.,Giolo,F.P.,Zotti,M.J.,StefanelloJr.,G.J.,

2005.Biologiacomparadadepopulac¸õesdeSpodopterafrugiperda(J.E.Smith)

(Lepidoptera:Noctuidae)emfolhasdemilhoearroz.Neotrop.Entomol.34, 743–750.

Busato,G.R.,Grützmacher,A.D.,Oliveira,A.C.,Vieira,E.A.,Zimmer,P.D.,Kopp,M.M.,

Bandeira,J.M.,Magalhães,T.R.,2004.Análisedaestruturaediversidade

molecu-lardepopulac¸õesdeSpodopterafrugiperda(J.E.Smith)(Lepidoptera:Noctuidae) associadasàsculturasdemilhoearroznoRioGrandedoSul.Neotrop.Entomol. 33,709–716.

Capalbo,D.M.F., Dusi, A.N.,Pires,C.S., Paula, D.P., Arantes,O.M.N., Melo, I.S.,

2009.OGMeBiosseguranc¸aAmbiental.In:Costa,M.A.F.,Costa,M.F.B.(Eds.),

Biosseguranc¸a de OGM: Uma visão integrada. Publit, Rio de Janeiro, pp.

190–219,Availableat:http://www.ﬁocruz.br/ioc/media/101027Biosseguranca

%20de%20OGMV1.pdf.

Carneiro,A.A.,Guimarães,C.T.,Valicente,F.H.,Waquil,J.M.,Vasconcelos,M.J.V.,

Carneiro,N.P.,Mendes,S.M.,2009.MilhoBt:Teoriaepráticadaproduc¸ãode

plantastransgênicasresistentesainsetos-praga.EmbrapaMilhoeSorgo,Sete Lagoas(CircularTécnica135).

Colwell,R.K.,2000.Statisticalestimationofspeciesrichnessandsharedspecies

fromsamples(EstimateS)[6.0b1],Availableat:http://viceroy.eeb.uconn.edu/

estimates(accessed15.04.13).

Cruz,I.,Silva,R.B.,Figueiredo,M.L.C.,Pentedo-Dias,A.M.,DelSarto,M.C.L.,Nuessly,

G.S.,2011.Surveyofearﬂies(DipteraUlidiidae)inmaize(ZeamaysL.)andanew

recordofEuxestamazorcaSteyskalinBrazil.Rev.Bras.Entomol.55,102–108.

Dale,P.H.,Clarke,B.,Fontes,E.M.G.,2002.Potentialfortheenvironmentalimpactof

transgeniccrops.Nat.Biotechnol.20,567–574.

Dively,G.P.,2005.ImpactoftransgenicVIP3ACry1AbLepidopteran-resistantﬁeld

cornonthenontargetarthropodcommunity.Environ.Entomol.34,1267–1291.

Fernandes,O.D.,Parra,J.R.P.,Neto,A.F.,Pícoli,R.,Borgatto,A.F.,Demétrio,C.G.B.,

2003.Efeitodomilhogeneticamentemodiﬁcadomon810sobrea

lagarta-do-cartuchoSpodopterafrugiperda(JESmith,1797)(Lepidoptera:Noctuidae).Rev. Bras.MilhoSorgo2(2),25–35.

Fontes,E.M.G.,Pires,C.S.S.,Sujii,E.R.,2003.Oimpactodeplantasgeneticamente

modiﬁcadasresistentesainsetossobreabiodiversidade.In:Pires,C.S.S.,Fontes, E.M.G.,Sujii,E.R.(Eds.),Impactoecológicodeplantasgeneticamente modiﬁ-cadas:oalgodãoresistenteainsetoscomoestudodecaso.EmbrapaRecursos GenéticoseBiotecnologia,CNPq,Brasília,pp.65–83.

Frizzas,M.R.,Oliveira,C.M.,2006.Plantastransgênicasresistentesainsetose

organ-ismosnão-alvo:Predadores,parasitóidesepolinizadores.Univ.Cienc.Saude4, 63–82.

Heltshe,J.F.,Forrester,N.E.,1983.Estimatingspeciesrichnessusingthejackknife

procedure.Biometrics39,1–11.

Hilbeck,A.,Baumgartner,M.,Fried,P.M.,Bigler,F.,1998.Effects oftransgenic

Bacillusthuringiensiscorn-fedpreyonmortalityanddevelopmenttimeof immatureChrysoperlacarnea(Neuroptera:Chrysopidae).Environ.Entomol.27, 480–487.

Krebs,C.J.,1999.EcologicalMethodology,2nded.Addison-WesleyEducational

Pub-lishers,Inc.,Vancouver.

Losey,J.E.,Rayor,L.S.,Carter,M.E.,1999.Transgenicpollenharmsmonarchlarvae.

Nature399,214.

Mendes,S.M.,Boregas,K.G.B.,Lopes,M.E.,Waquil,M.S.,Waquil,J.M.,2011.Respostas

dalagarta-do-cartuchoamilhogeneticamentemodiﬁcadoexpressandoatoxina Cry1A(b).Pesq.Agropec.Bras.46,239–244.

Meissle,M.,Romeis,J.,2009.Theweb-buildingspiderTheridionimpressum(Araneae:

Theridiidae)isnotadverselyaffectedbyBtmaizeresistanttocornrootworms. PlantBiotechnol.7,645–656.

Meissle,M.,Romeis,J.,2012.NoaccumulationofBtproteininPhyllonetaimpressa

(Araneae:Theridiidae)andotherarthropodsinBtmaize.Environ.Entomol.41, 1037–1042.

Naranjo,S.E.,2005.Long-termassessmentoftheeffectsoftransgenicBtcottonon

thefunctionofthenaturalenemycommunity.Environ.Entomol.34,1211–1223.

O’Callaghan,M.,Glare,T.R.,Burgess,E.P.J.,Malone,L.A.,2005.Effectsofplants

geneticallymodiﬁedforinsectresistanceonnon-targetorganisms.Annu.Rev. Entomol.50,271–292.

Paula,D.P.,Andow,D.A.,Timbó,R.V.,Suiii,E.R.,Pires,C.S.S.,Fontes,E.M.G.,2014.

UptakeandtransferofaBttoxinbyaLepidopteratoitseggsandeffectsonits offspring.PLoSOne9,1–7.

Pilcher,C.D.,Rice,M.E.,Obrycki,J.J.,2005.ImpactoftransgenicBacillusthuringiensis

cornandcropphenologyonﬁvenontargetarthropods.Environ.Entomol.34, 1302–1316.

Pires,C.S.S.,Sujii,E.R.,Fontes,E.M.G.,2003.Avaliac¸ãoecológicaderiscode

plan-tasgeneticamentemodiﬁcadasresistentesainsetossobreinimigosnaturais. In:Pires,C.S.S.,Fontes,E.M.G.,Sugii,E.R.(Eds.),Impactoecológicodeplantas geneticamentemodiﬁcadas:oalgodãoresistenteainsetoscomoestudodecaso. EmbrapaRecursosGenéticoseBiotecnologia,Brasília,pp.85–115.

Raybould,A.,Stacey,D.,Vlachos,D.,Graser,G.,Li,X.,Joseph,R.,2007.Non-target

organismsriskassessmentofMIR604maizeexpressingmCry3Aforcontrolof cornrootworms.J.Appl.Entomol.131,391–399.

Romeis,J.,Meissle,M.,Raybould,A.,Hellmich,R.L.,2009.Impactofinsect-resistant

transgeniccropsonabove-groundnon-targetarthropods.In:Ferry,N., Gate-house,A.M.R.(Eds.),EnvironmentalImpactofGeneticallyModiﬁedCrops.CAB International,Wallingford,pp.165–198.

Romeis,J.,Meissle,M.,Naranjo,S.E.,Li,Y.,Bigler,F.,2014.Theendofamyth–Bt

(Cry1Ab)maizedoesnotharmgreenlacewings.FrontPlantSci5,1–10.

Schuler,T.H.,Potting,R.P.J.,Denholm,I.,Poppy,G.M.,1999.Parasitoidbehaviorand

Btplants.Nature400,825–826.

Sears,M.K.,Hellmich,R.L.,Stanley-Horn,D.E.,Oberhauser,K.S.,Pleasants,J.M.,

Mattila,H.R.,Siegfried,B.D.,Dively,G.P.,2001.ImpactofBtcornpollenon

monarchbutterﬂypopulations:ariskassessment.Proc.Natl.Acad.Sci.U.S. A.98,11937–11942.

Vasconcelos,M.J.V.,Carneiro,A.A.,Valicente,F.H.,2011.EstudodecasoemmilhoBt.

In:Borém,A.,Almeida,G.D.(Eds.),Plantasgeneticamentemodificadas:desafios eoportunidadespararegiõestropicais.UniversidadeFederaldeViçosa,Viçosa, pp.311–332.

Waquil,J.M.,1997.AmostragemeabundânciadecigarrinhasedanosdeDalbulus

maidis(DeLong&Wolcott)(Homoptera:Cicadellidae)emplântulasdemilho. An.Soc.Entomol.Bras.26,27–33.

Waquil,J.M.,Vitousek,P.M.,Siegfried,B.D.,Foster,G.N.,2004.Atividadebiológicadas

toxinasdoBt,Cry1A(b)eCry1FemSpodopterafrugiperda(SMITH)(Lepidoptera: Noctuidae).Rev.Bras.MilhoSorgo3,153–163.

White, J.A., Andow, D.A., 2005. Host–parasitoid interactions in a transgenic

landscape:spatial proximityeffects ofhostdensity.Environ.Entomol. 34, 1493–1500.