Preference of Neoseiulus californicus (Acari: Phytoseiidae) for volatiles of Bt maize induced by multiple herbivory

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

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

Biological

Control

and

Crop

Protection

Preference

of

Neoseiulus

californicus

(Acari:

Phytoseiidae)

for

volatiles

of

Bt

maize

induced

by

multiple

herbivory

Jéssica

Mayara

Cofﬂer

Botti

,

Mayara

Loss

Franzin

,

Marcos

Antonio

Matiello

Fadini

∗

,

Júlio

Onésio

Ferreira

Melo

UniversidadeFederaldeSãoJoãoDelRei,CampusSeteLagoas,SeteLagoas,MG,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9April2019 Accepted23September2019 Availableonline17October2019 AssociateEditor:MarianeA.Nickele Keywords:

Indirectinduceddefense Tetranychusurticae Secondaryplantcompounds Plantresistance

Fallarmyworm

a

b

s

t

r

a

c

t

Plantindirectinduceddefensesagainstherbivoresarecharacterizedbytheproductionofplantvolatiles thattoattractnaturalenemies.Theobjectiveofthisstudywastoevaluatewhethertheattackofthe two-spottedspidermiteTetranychusurticaeorthemultipleherbivoryofT.urticaetogetherwiththe fallarmywormSpodopterafrugiperdaareabletoelicitindirectinduceddefenseinconventionalandBt maizeplants.TheexperimentwascarriedoutinthelaboratoryusingY-tubeolfactometer,evaluatingthe predatorymiteNeoseiuluscalifornicusolfactorypreferenceforplantvolatiles.Thetreatmentsinvolved: CleanConventionalPlant;ConventionalPlantInfestedwithT.urticae;CleanBtPlant;BtPlantInfested withT.urticae;ConventionalPlantInfestedwithT.urticae+fallarmyworm;BtPlantInfestedwithT. urticae+fallarmyworm.ForthechemicalanalyzestheTraceGCUltragaschromatographwasused, pairedwiththePolarisQmassspectrometer,GC MSsystem.Neoseiuluscalifornicuswasalsounableto distinguishbetweenvolatilesfrombothconventionalandBtinfestedmaizeplants.Moreover,therewas nodiscriminationofthepredatormitebetweenplantsundersingleandmultipleinfestations,bothin conventionalandBtmaize.WhencomparingconventionalandBtplants,bothwithmultipleinfestation, thepredatormiteN.californicushadnopreferenceamongthesesourcesofodors.However,therewas observedchemicalchangesofthevolatilesamongthegroupsofplantsstudied.Thus,itissuggested thatthegroupsofplantsunderstudyhavechemicalmodiﬁcations,buttheyarenotabletoattractN. californicus.Inaddition,BtplantsresponsewassimilartoconventionalplantsonattractingN.californicus. ©2019PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeEntomologia.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Introduction

Theplantsdevelopedadiversityofdefensemechanismsagainst

differentconditions,suchasherbivorousattack(Bergelsonetal.,

2001).A mechanism against herbivoresis the indirectinduced

defense,characterizedbytheproductionofplantvolatilesafter

herbivorythatareabletoattractnaturalenemiesofherbivores

(Arimuraetal.,2005;Schaller,2008).

In addition to induced defenses, plants have constitutive

defensesthatareexpressedcontinuouslyandindependentof

her-bivorousattack(Priceetal.,1980).OneexampleisBtplants,since

theycontinuouslyexpressthedefenseprotein.Forexample,30F35

HxmaizeexpressestheCry1Fproteinthatconferslepidopteran

resistance.However,notonlythetargetherbivoresareexposed

to toxins, non-target herbivores and their natural enemies are

∗ Correspondingauthorat:UniversidadeFederaldeSãoJoãoDelRei.MG424,km47.SeteLagoas,MG,35701-970,Brazil.

E-mails:jessicabotti@hotmail.com(J.M.Botti),mayarafranzin@gmail.com(M.L.Franzin),fadini@ufsj.edu.br(M.A.Fadini),onesiomelo@gmail.com(J.O.Melo).

consequentlyexposed(TorresandRuberson,2008).Althoughthe

use of genetically modiﬁed plants hasadvantages (Kouser and

Qaim,2011),theimpactoftheseplantsontheforagingbehavior

ofnaturalenemieshasbeenquestioned(Poppyand Sutherland,

2004).Hence,itisimportanttounderstandtheinﬂuenceofBtplant

proteinsonnon-targetorganisms.

Further,thecultivationofBtplantspromotesadecreaseinthe

useofinsecticides(Cattaneoetal.,2006).Inthisway,speciesof

non-targetpestsoflessereconomicimportance,suchasTetranychus

urticae(Koch)(Acari:Tetranychidae)inmaize,maybecomemore

important,becausetheloweruseofinsecticidesappliedtotarget

pestsmayincreasetheirpopulation.

Therefore,itisimportanttounderstandifBt-basedcornplants

arecapableableofreleasingvolatilestoattractthenatural

ene-miesofT.urticae.Thisquestioningisperformedbecauseindirect

https://doi.org/10.1016/j.rbe.2019.09.003

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induceddefensehascostsfortheplants(SteppuhnandBaldwin,

2008).Totriggerthisdefense,theplantneedstoallocate

physiolog-icalandecologicalresources,whichcanaffectrelevantprocessesof

theplant,suchasgrowthandreproduction(SteppuhnandBaldwin,

2008).

Asecondconcernsistheinteractionamongdifferenttypesof

plantdefensesmechanisms(Kantetal.,2015;Priceetal.,1980).

Studieshavedemonstratedsigniﬁcantdifferencesintheamount

ofvolatilesemittedbetweenBtandnon-Btmaizeplants(Deanand

DeMoraes,2006;Turlingsetal.,2005).Thisshowsthatplantswith

constitutivedefenses(e.g.Bt)canaltertheproductionofvolatiles

and,consequently,theattractionofnaturalenemies.Thissuggests

anegativeinteractionbetweenthesedefenses(Shimoda,2010).

Furthermore,anotherfactorthatcanalterinduceddefenseis

themultipleattacksofherbivorespecies,affectingpreferenceand

performance of herbivoresand of their natural enemies(Dicke

etal.,2009).Innature,plantsarenotexposedtoasingle

herbi-vore.Theattackofmultipleherbivorescanaltertheemissionof

compounds,bothpositivelyandnegativelyintheattractivenessof

naturalenemies(Kaplanetal.,2009;Rodriguez-Saonaetal.,2005;

Viswanathanetal.,2005).Thisworkevaluatedwhetherboth

con-ventionalandBtmaize plantsinducetheproductionofvolatile

compoundsattractivetoNeoseiuluscalifornicus(McGregor)(Acari:

Phytoseiidae)afterherbivoryofT.urticae.Andifconventionaland

Btmaizeplantsinducedbysimple (T.urticae)andmultiple

her-bivory(T.urticae+fallarmyworm),producevolatilecompounds

attractivetothepredatorymiteN.californicus.

Materialsandmethods

Rearingoftwo-spottedspidermite,fallarmywormandpredatory

mite

Infested leavesof Sorghum plants with T. urticae were

col-lectedinthegreenhouseatEmbrapaMilhoeSorgofacilities,and

takentothe EntomologyLaboratories ofthe Universidade

Fed-eral de São João Del-Rei (UFSJ), in Sete Lagoas, Minas Gerais,

Brazil.Toestablishthecolony,adultmiteswerecollected,

individ-uallyusingastereomicroscopeandahairsbrush,thentransferred

tojack bean plants (Canavalia ensiformis)with two completely

expandedcotyledonleavesandwithoutanyphytosanitary

treat-ment.

Thebeanplantsusedwereplantedin1.0Lplasticpotsusing

Terral Solo® substrate. Infested plants were kept isolated in

screenedcagesinordertopreventexternalinfestationwithother

herbivores.Irrigationwasdonewhenever necessary.Cleanjack

bean plants were added to the rearing colony when needed.

TheSpodopterafrugiperda(Smith) (Lepidoptera:Noctuidae)was

acquired from rearing colony at Embrapa Milho e Sorgo. The

predatorymite, N.californicus, was acquired from the Koppert

company.

Plantingofmaizeseeds

Themaizeseedsusedforplantingwerethecommercialhybrids

Pioneer®30F35(conventionalmaize)andPioneer®30F35Hx(Bt

maize).Twoseedsweresownin plastic potswithavolume of

1.0L,usingTerralSolo®substrateandkeptinagreenhouse.After

15daysofsowing,thinningandfertilizationwereprovidedwith

0.2gofammoniumsulphateperpot.Themaizeplantsweresplit

into3 groups: clean plants, plants infested withT. urticae and

plantsinfestedsimultaneouslybyT.urticaeand fallarmyworm.

ForT.urticaeinfestation,20daysaftersowing,10femaleswere

onone leaf,usingentomological glue. Theglue was appliedat

theleafbase,notallowingthefemalestomovetotherestofthe

plant.

ThegroupofplantsinfestedwithT.urticae+fallarmyworm,

besidestheinfestationofthemitethatwasidenticaltotheprevious

group,athirdinstarlarvaewasisolatedwitha“clipcage”.Thiswas

carriedoutonadifferentleafofT.urticaeinfestation,also20days

aftersowing.

Olfactometertest

Plantsafter48hofinfestationwereusedtocarryoutthetestsin

Y-tubeolfactometer,withthefollowingtreatments:Clean

Conven-tionalPlantXConventionalPlantInfestedwithT.urticae;CleanBt

PlantXBtPlantInfestedwithT.urticae;ConventionalPlantInfested

withT.urticaeXBtPlantInfestedwithT.urticae;ConventionalPlant

InfestedwithT.urticaeXConventionalPlantInfestedwithT.urticae

+fallarmyworm;PlantBtInfestedwithT.urticaeXBtPlantInfested

withT.urticae+fallarmyworm;ConventionalPlantInfestedwith

T.urticae+fallarmywormXPlantBtInfestedwithT.urticae+fall

armyworm.Eachtreatmentconsistedofthreetruereplicates(i.e.

odorsources)then15responsesofpredatorymiteswereobtained

fromeachreplicate, totaling45 releasesofpredatorymitesper

treatment.

AY-tubeolfactometerwasusedtostudytheresponseofN.

cal-ifornicus.TheglassY-Tube(withdimensionsof21.0cmoflength

ofeacharmand3.5cmofdiameter)hadaY-shapedmetalwire

insidetofacilitatethemovementofmites.Containersconsisting

ofa plasticbox wereattachedtotheY-tubeboth arms.The

Y-tubebasewasconnectedtoavacuumpumpthatdirectedtheﬂow

fromthecontainerstotheY-tubearms.Aﬂowmeterwasconnected

tothecontainersairoutletandwasusedtocalibratetheairinlet

speedineacholfactometerarm.Thewindspeedwas0.50m/son

eachY-tubearm.Thepredatorswereindividuallyintroducedatthe

startingpointonthesteelwireandthenobserveduntilapredator

reached1/3ofanarm,wherethemiteresponsewasconsidered.

Predatorymiteswhodidnotpass1/3ofeitherarmwithin5min

were_{accounted ¨unresponsive¨.}Aftereveryﬁveresponses,theodor

sourcespositionswerereversed.

Chemicalanalysisofplantvolatiles

For chemicalanalysis,a leafsample of0.250gof eachplant

wastaken.Thechemicalanalysiswasperformedusingsolidphase

microextraction(SPME) in headspacemodeandTrace GCUltra

gaschromatograph(ThermoScientiﬁc,SanJose,CA)pairedwith

thePolarisQmassspectrometer(ThermoScientiﬁc,SanJose,CA),

GC–MSsystem,withanion-traptypeanalyzer(Garciaetal.,2016).

Forthisanalysisatruereplicatewasdrawnonwhichaleafsample

(0.250g)waswithdrawnfromeachplant,whichweretransferred

intoaheadspaceﬂask(25mL)andmaceratedwithaglassstick.The

ﬂasksweresealedwithaluminumsealandrubberseptumandleft

inthefreezerat−18◦_C_until_analysis._Each_sample_was_heated_for

5minat65◦Cbeforeexposureofthesolidphasemicroextraction

ﬁber(SPME).Then,abovethemaceratedmaizeleaf,thesemi-polar

polydimethylsiloxane/divinylbenzene(PDMS/DVB)65␮mﬁber

wasexposedtothegasphaseintheheadspaceﬂasksat65◦Cfor

20min.AfterexposureofthePDMS/DVBﬁberwiththesample,

thesamewasleftinsertedintheinjectorofthechromatographat

200◦C,for5min,fordesorptionofthevolatilescollected.

Theconditionsforthegaschromatographareinjector

temper-ature,200◦C;injectioninsplitlessmode;splitlesstime,5min;ion

sourcetemperature, 200◦C; interfacetemperature,275◦C;

pro-grammedtemperatureheating (40◦C for2min,thena gradient

of8◦C /minto100◦C, atwhichtemperaturetheisothermwas

maintainedfor 2min and then 15◦C / min to180◦C, at which

temperature it maintained the isotherm for 2min, and ﬁnally

a gradient of 15◦C / min to245◦C, at which temperature the

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scanmode(fullscan,from30to300),usingtheelectron-impact

ionization(EI) technique, with energy of 70eV. The

chromato-graphiccolumnusedwasHP-5MScapillarycolumn(5%phenyland

95%methylpolysiloxane),withthefollowingdimensions:length

(30m),internaldiameter(0.25mm)andﬁlmthickness(0.25␮m)

(AgilentTechonolgiesINC,Germany),speciﬁcformass

spectrom-etry analysis.The componentsidentiﬁcationwas performedby

comparingthemassspectrapresentintheNIST/EPA/NIH(2005)

libraries.

Statisticalanalyzes

Fortheolfactometertests,thestatisticaldesignwascompletely

randomized,with6treatmentsand3replicatespertreatment.

Forthechemicaldata,theretentiontimeandpeakintensity

val-uesweretakenfromtheThermoElectronCorporationXcalibur1.4

software.Thepeakswerethenselectedbycuttingninecompounds

oflargerareaineachchromatogram.Acompoundwasrelatedto

eachretentiontimebasedonmassspectraandretentionindexes.A

tablewascreatedwithsamplesintherowsandcompoundsinthe

columnsandinsidethematrixwereplacedtherelativeintensities

ofthepeaks,obtainedinthechromatographicanalysis.Thedata

wereassessedbyanalysisofthemainPCAcomponents(Crawley,

2013).

Predatorymitebehaviordataonolfactometerweresubmittedto

chi-squaretestsforcategoricaldata(Crawley,2013).Predatormites

thatdidnotchooseoneoftheolfactometerarmsafterﬁveminutes

werenotconsideredforanalysis.TheR(RDevelopmentCoreTeam

2014)softwarewasusedtoperformthestatisticalanalyzesand

plotthegraphs.

Results

OlfactorypreferenceofN.Californicus

Predatorymitesdidnotdiscriminatebetweenodorsfrom

non-infestedandinfested maizeplants,both inconventional andBt

plants.TheN.californicuswasalsounabletodistinguishbetween

theodorsfrominfestedconventionalandBtmaizeplants(Fig.1).

There was no preference of N. californicus predatory mites

amongthevolatilesof T.urticaeinfestedmaize plantsversusT.

urticae+S. frugiperda multipleinfestation, both in conventional

maizeplantsand inBtplants.Thesameoccurredinthe

prefer-enceofvolatilesofplantsofconventionalandBtmaizeonmultiple

infestation(mite+fallarmyworm)(Fig.2).

Chemicalparameters

Fortheanalysisofmaizeleavesvolatilechemicalproﬁle,nine

compoundswiththehighestamountineachsamplewereselected.

IncleanplantsversusinfestationwithonlyT.urticae15volatile

compoundswerefound,11ofwhichwerepre-identiﬁed

belong-ingtotheterpenoidgroup(3monoterpenesand1sesquiterpene),

3aldehydes,1ether,1ketone,1alcohol and1ester(Fig.3).In

thesimpleversusmultipleinfestations,13compoundswerefound

inthefourgroupsofplants,ofwhich10werepre-identiﬁedas3

monoterpenes,3aldehydes,1ether,1ketone,1alcoholand1ester

(Fig.4).

The principal componentanalysis of volatilecomposition in

conventional andBtleaves, cleanedandinfestedwithT.urticae

explained65.0%ofthevariation(Fig.5).Principalcomponent1

sep-aratedthecleanBtplantsfromtheothers.Theprincipalcomponent

2separatedtheinfestedBtplantsfromtherest.Theprincipal

com-ponentanalysis(PCA)ofvolatilecompositionofconventionaland

Btmaizeleaves,withsingleandmultipleinfestations,explained

49.8%of thevariation (Fig.6).Principalcomponent1separated

conventional plants withsingle infestationfrom Btwith single

infestation.However,themaincomponent2didnotseparatethe

groupsofplants.

Discussion

Themite didnotdistinguishamongtheodorsfrominfested

anduninfestedplants byT.urticae,bothconventional andBt.It

29 (C) 24 21 1 0.5 0.5 1 Proportion of choice 16 21 24 X2_{=3.76, df=1, p=0.05} X2_{=0.20, df=1, p=0.65} X2_{=0.20, df=1, p=0.65} (Bt) (C+Tu) (Bt+Tu) (Bt+Tu) (C+Tu)

Fig.1. PredatorymiteNeoseiuluscalifornicuspreferenceforvolatilecompoundsintwo-choiceolfactometertests,comparing:conventionalmaizeplantsuninfested(C); conventionalmaizeplantsinfestedbyTetranychusurticae(C+Tu);Btmaizeplantsuninfested(Bt);andBtmaizeplantsinfestedbyT.urticae(Bt+Tu);Numbersinbars representindividualpredatorymitesthatchoosetheindicatedodor.Nosigniﬁcantp≥0.05.

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X2_{=0.02, df=1, p=0.88} X2_{=0.55, df=1, p=0.46} X2_{=0.02, df=1, p=0.88} (C+Tu+Sf) (C+Tu) (Bt+Tu) (C+Tu+Sf) (Bt+Tu+Sf) (Bt+Tu+Sf) 23 23 25 23 22 20 1 0.5 0.5 1 Proportion of choice

Fig.2.PredatorymiteNeoseiuluscalifornicuspreferenceforvolatilecompoundsintwo-choiceolfactometertests,comparing:conventionalmaizeplantsinfestedby Tetrany-chusurticae(C+Tu);conventionalmaizeplantsinfestedbyTetranychusurticaeandSpodopterafrugiperda(C+Tu+Sf);BtmaizeplantsinfestedbyT.urticae(Bt+Tu);Btmaize plantsinfestedbyT.urticaeandS.frugiperda(Bt+Tu+Sf).Numbersinbarsrepresentindividualpredatorymitesthatchoosetheindicatedodor.Nosigniﬁcantp≥0.05.

RT: 0,00 - 26,19 100 50 1 2 5 6 7 7 10 12 12 12 12 10 10 10 11 89 2 2 2 5 6 1 1 1 3 4 5 6 1315 13 14 14 15 15 50 50 50 0 0 0 10u 10u Relative abundance 10u 0 0 2 4 6 8 10 Time (min) 12 14 16 18 20 22 24 26 (C+Tu) (C) (Bt) (Bt+Tu)

Fig.3.Chromatogramsofvolatilecompoundsextractedfromleavesofconventionalmaizeplantsuninfested(C);conventionalmaizeplantsinfestedbyTetranychusurticae (C+Tu);Btmaizeplantsuninfested(Bt);andBtmaizeplantsinfestedbyT.urticae(Bt+Tu).Thecompoundsobservedinanalysiswere1=(E)-Hex-2-enal;2=heptanal; 3=(E)-Oct-2-enal;4=␣-pineno;5=␤-cisocimeno;6=␤-Ciclocitral;7=1-metil-6-(3-metilbuta-1,3-dienil)-7-oxabiciclo[4,1,0]heptano;8=noidentificated=C8;9=no identificated=C9;10=␤-ionona;11=Ciclosativena;12=(E)-7-tetradecen-1-ol;13=noidentificated=C13;14=Linolenicacidethylester;15=noidentificated=C15.

didnotdistinguishbetweenodorsfromconventionalinfestedand

uninfestedplantswithT.urticae.

StudieshaveshownthatplantsinfestedwithT.urticaeproduce

volatilescapableofattractingnaturalenemies.Oneexamplewas

theattractionofthepredatorymiteN.californicustobeanplants

infestedwithT.urticae(Shimodaetal.,2005).Thisshowsthatthe

sameherbivoremayormaynottriggerdefensesondifferentplants.

Inaddition,Gardineretal.(2005)studiedHumuluslupulus(L.)

(Ros-ales:Cannabaceae)hopsplantsinfestedbyT.urticaeversusclean

plants,veriﬁedthatinthesameplantdifferentiationinthe

pref-erenceofdistinctpredatorscanoccur.Wherethepredatorymite

Neoseiulusfallacis(Garman)(Acari:Phytoseiidae)wasattractedby

infestedplants,butthepredatorymiteGalendromusoccidentalis

(Nesbitt)(Acari:Phytoseiidae)wasnot.Thisallowsfurtherresearch

ontheforagingofothernaturalenemiesbyT.urticaeinfestedmaize

plants.

Thecultivarisanotherfactorthatis correlatedwithindirect

defense induction (Dicke et al., 1990).Tahmasebi et al. (2014)

studiedfourbeancultivarsinfestedwithT.urticaeandobserved

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RT: 0,00 - 26,19 100 50 0 10u 50 0 10u 50 0 10u 50 0 10u₀ ₂ ₄ ₆ ₈ ₁₀ ₁₂ ₁₄ ₁₆ ₁₈ ₂₀ ₂₂ ₂₄ ₂₆ Time (min) Relative abundance 1 2 5 6 7 8 10 1113 8 10 13 13 12 1113 12 8 8 10 10 7 1 2 5 6 1 1 2 2 4 5 34 5 6 6 (C + Tu) (C + Tu+ Sf) (Bt + Tu) (Bt + Tu + Sf)

Fig.4.ChromatogramsofvolatilecompoundsextractedfromleavesofconventionalmaizeplantsinfestedbyTetranychusurticae(C+Tu);conventionalmaizeplantsinfested byTetranychusurticaeandSpodopterafrugiperda(C+Tu+Sf=+);BtmaizeplantsinfestedbyT.urticae(Bt+Tu);BtmaizeplantsinfestedbyT.urticaeandS.frugiperda (Bt+Tu+Sf).Thecompoundsobservedinanalysiswere1=(E)-Hex-2-enal;2=heptanal;3=(E)-Oct-2-enal;4=˛-pineno;5=ˇ-cisocimeno;6=ˇ-Ciclocitral;7= 1-metil-6-(3-metilbuta-1,3-dienil)-7-oxabiciclo[4,1,0]heptano;8=ˇ-ionona;9=noidentificated=C9;10=(E)-7-tetradecen-1-ol;11=noidentificated=C11;12=Linolenicacidethyl ester;13=noidentificated=C13. DIm2 (24.1%) 2 0 -2 0.0 Dim1 (40.9%) 2.5 5.0

Fig.5. Projectiontoprincipalcomponentanalysis(PCA)basedontheheadspace compositionofvolatilesofconventionalmaizeplantsuninfested(C=o); conven-tionalmaizeplantsinfestedbyTetranychusurticae(C+Tu=•);Btmaizeplants uninfested(Bt=);andBtmaizeplantsinfestedbyT.urticae(Bt+Tu=N),using theﬁrsttwoprincipalcomponents(Dim)withexplainedvarianceinbrackets.

predatorymitePhytoseiuluspersimilis(Athias-Henriot)(Acari:

Phy-toseiidae). This demonstrates a variation in the production of

volatileswithinthesameplantspecies.So,thisresultsindicatethe

needforfurtherstudieswithvariousotherscultivars.

For the analysis of conventional and Bt plants with single

andmultipleinfestation,thepredatormiteN.californicusdidnot

presentapreferencebetweenvolatiles.Thissuggeststhatmultiple

herbivorydoesnotalterthepredatorresponse,orthepredatory

mitedidnotdistinguishbetweenvolatilesfromconventionaland

Btplantswithmultipleinfestation.Studieshaveshownthatplants

mayresponddifferentlywhenattackedbyoneormoreherbivores

(Dickeetal.,2009).Theattractionofthenaturalenemycanincrease

whentheplantisundermultipleherbivory.Forexample,pepper

2 1 0 -1 -2 -3 -2 -1 0 1 Dim1 (28.1%) 2 3 DIm2 (21.7%)

Fig.6. Projectiontoprincipalcomponentanalysis(PCA)basedontheheadspace compositionofvolatilesofconventionalmaizeplantsinfestedby Tetranychus urticae(C+Tu=•);conventionalmaizeplantsinfestedbyTetranychusurticaeand Spodopterafrugiperda(C+Tu+Sf=+);BtmaizeplantsinfestedbyT.urticae(Bt+Tu =N);BtmaizeplantsinfestedbyT.urticaeandS.frugiperda(Bt+Tu+Sf=*),using theﬁrsttwoprincipalcomponents(Dim)withexplainedvarianceinbrackets.

plantsinfestedwithonlyoneherbivore,T.urticaeorgreenpeach

aphidMyzus persicae(Sulzer)(Hemiptera:Aphididae),wereless

attractivetothepredatorMacrolophuscaliginosus(Wagner)

(Het-eroptera:Miridae)thanplantsinfestedwiththetwoherbivores

simultaneously.Thisshowsthatthevolatilesproducedby

multi-pleherbivoryaremoreattractivetothepredatorthanthevolatiles

inducedbyonlyoneherbivore(Moayerietal.,2007).

Besides that, multipleinfestations may not alter the

attrac-tiveness of natural enemies, as found in the present study. A

studycomparingneutralityin theattraction ofnaturalenemies

showsthatthepredatorymiteNeoseiulusbaraki(Athias-Henriot)

(Acari:Phytoseiidae)doesnotdistinguishfruitodorsfromcoconut

treesinfestedbyAceriaguerreronis(Keifer)(Acari:Eriophyidae)or

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Tarsonemidae)fromodorsofthefruitinfestedsimultaneouslyby

theseherbivores(Limaetal.,2016).

Thevolatilesreleasedinresponse toherbivoryaregenerally

detectableandindicatorsofthepresenceofherbivoreswhichmay

potentiallyattractnaturalenemiesneartheirprey(DeMoraesetal.,

2000).However,asT.urticaeisnotapestthatcausessevere

dam-agetothemaizecrop,theseplantsmaynotcoevolvetoproduce

efﬁcientcompoundstoattractpredatorymites.Moreover,when

theplantswereundermultipleherbivory,achangeoccurredinthe

productionofcompounds,whichwerenotattractivetothe

preda-tormiteN.californicus,butthischangemaybespeciﬁctosome

naturalenemyofthefallarmyworm,whichthemaizeplanthas

agreaterinteractionandconsequentlythecoevolutionprocessis

morelikely.

Inthepresentstudy,theBttechnologydidnotaltertheforaging

ofthepredatorymiteN.californicus,butstudiesshowthatBttoxins

mayhaveindirecteffectsonnaturalenemies,anexampleishaving

thenutritionallyinadequatebeingprey(Divelyetal.,2004).

ConventionalandBtmaize,cleanedandinfestedbyT.urticae,

showedachemical differentiationof thegroupsof plantunder

study. However, this difference was not able to attract the

predatory mite. Compounds detected in conventional infested

plantswere:(E)-Hex-2-enal;heptanal;␤-cisocyme;␤-Cyclocitral;

1-methyl-6-(3-methylbut-1,3-dienyl)-7-oxabicyclo [4.1.0]

hep-tane; ␤-ionone; (E) -7-tetradecen-1-ol. While, in Bt were: (E)

-Hex-2-enal;heptanal;(E)-Oct-2-enal;␣-pinene;␤-cisocyme;

␤-Cyclocitral;␤-ionone;(E)-7-tetradecen-1-ol;Linolenicacidethyl

ester.These chemicalresultsarenot compatiblewithT. urticae

inducedvolatilesinbeanplantswhereitwasdetectedthe

follow-ingcompounds(E)-Hex-2-enal,(Z)-3-hexen-1-ol,(Z)-3-hexenil

acetate,methylene salicylate, linalool, (Z)-␤-ocimene, (Z)

-4,8-dimethyl-1,3,7-nonatriene,(E)-4,8-dimethyl-1,3,7-nonatriene,

␤-caryophyllene, (E, E) -4,8,12-trimethyl-1,3,7,11-tridecatetraene.

ThissetofcompoundswasabletoattractthepredatorymiteN.

californicus(Shimodaetal.,2005).Thisindicatesthat,eventhough

maizeplantshavechangedchemicallyafterT.urticaeinfestation,

thevolatilesproduced areunable toattractN.californicus.This

chemicalchangeallowsnewstudieswithdifferentpredators,since

thevolatilesproducedcanbeattractivetoothernaturalenemies.

ConventionalandBtmaizeleaveswithsingleandmultiple

infes-tationsshowedachemicaldifferentiationoftheplantgroupswith

singleinfestation. ThisindicatesthatBt plantsdifferchemically

whentheyareonT.urticaeherbivory.However,thepredatormite

N.californicushadnopreferenceamongthesegroupsofplants.The

BtplantsrequireenergyresourcestoproducetheCry1Ftoxinthat

mayaltertheinvestmentofsecondarydefensecompoundssuch

asvolatiles,possiblythechemicalproﬁledifferenceoftheseplants

wereduetotheinteractionofconstitutiveandinduceddefenses

(Kantetal.,2015;Priceetal.,1980).However,thepredatordoes

notdistinguishbetweentheodorsreleasedbyplantsinsingleand

multipleinfestations,whichindicatesthattheconstitutivedefense

(BtMaize)didnotaltertheforagingofthepredatorunderstudy.

Nonetheless,this chemical changefoundallows furtherstudies

withothernaturalenemies.

Besidesthemaizevolatilesalreadymentionedintheliterature,

inthe presentstudy 1-methyl-6- (3-methylbuta-1,3-dienyl)

-7-oxabicyclo[4.1.0]heptane,(E)-7-tetradecen-1-ol andlinolenic

acidethylesterwereﬁrstidentiﬁedinmaize.Thiswaspossibly

duetothefactthattherewerenoreportsinthevolatileliterature

ofPioneer30F35and30F35Hxcorn.Degenetal.(2004)showed

thatamong31varietiesofmaizethereisadifferentiationintotal

amountofvolatilesandvolatileproﬁlesarehighlyvariableineach

genotype.

Theevidencefromthisstudyindicatesachemical

differentia-tioninthegroupsofplantsstudied.However,theN.californicus

arenotabletodistinguishthem.Thisfactpotentiatesthechance

ofT.urticaetoreachpeststatusinmaizebyreducingthechanceof

encounterbetweenpredatorandprey.N.californicusdoesnot

dif-ferbetweensingleandmultipleinfestationsinmaizeplants.This

factindicatesthatmultipleinfestationsdoesnotalterforagingof

thepredatormite.

Acknowledgments

TheauthorsthanktheGraduateProgramin Agricultural

Sci-ences,ofFederalUniversityofSãoJoãodel-Rei(UFSJ/PPGCA),for

thescientiﬁc support.WethankEmbrapaMilhoeSorgoforthe

structureprovided fortheexperiment, andFapemig

(CAG-APQ-00556-16)andCapesfortheﬁnancialsupport.Also,theauthors

thankDr.J.M.Waquil,fortheEnglishreviewandsuggestionsin

originalmanuscript.

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