Copaene is an attractant, synergistic with quercivorol, for improved detection of Euwallacea nr

20 Pages·2017·5.74 MB·English

RESEARCHARTICLE
α-Copaene is an attractant, synergistic with
quercivorol, for improved detection of
Euwallacea nr. fornicatus (Coleoptera:
Curculionidae: Scolytinae)
PaulE.Kendra1*,DavidOwens1,WayneS.Montgomery1,TeresaI.Narvaez1,Gary
R.Bauchan2,ElenaQ.Schnell1,NurhayatTabanca1,DanielCarrillo3
a1111111111 1 UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,SubtropicalHorticultureResearch
Station,Miami,FL,UnitedStatesofAmerica,2 UnitedStatesDepartmentofAgriculture,Agricultural
a1111111111
ResearchService,BeltsvilleAreaResearchCenter,ElectronandConfocalMicroscopyUnit,Beltsville,MD,
a1111111111
UnitedStatesofAmerica,3 UniversityofFlorida,TropicalResearchandEducationCenter,Homestead,FL,
a1111111111
UnitedStatesofAmerica
a1111111111
*[email protected]
Abstract
OPENACCESS
Theteashot-holeborer,EuwallaceafornicatusEichhoff,isanambrosiabeetleendemicto
Citation:KendraPE,OwensD,MontgomeryWS,
NarvaezTI,BauchanGR,SchnellEQ,etal.(2017) Asiaandapestofcommercialtea,Camelliasinensis(L.)Kuntze.Recently,acomplexof
α-Copaeneisanattractant,synergisticwith speciesmorphologicallysimilartoE.fornicatushasbeenrecognized,whichincludesnew
quercivorol,forimproveddetectionofEuwallacea
pestsestablishedinIsraelandtheUSA,bothinCaliforniaandFlorida.Collectivelytermed
nr.fornicatus(Coleoptera:Curculionidae:
Scolytinae).PLoSONE12(6):e0179416.https:// E.nr.fornicatus,thesecrypticspeciescarrysymbioticFusariumspp.fungi,someofwhich
doi.org/10.1371/journal.pone.0179416 causediebackdiseaseinsusceptiblehosts,whichincludeavocado,Perseaamericana
Editor:RaulNarcisoCarvalhoGuedes, Miller.Duetothethreattothiseconomicallyimportantcrop,researchwasinitiatedtoevalu-
UniversidadeFederaldeVicosa,BRAZIL ateefficacyofkairomone-basedluresfordetectionofthebeetleinFlorida(termedthe
Received:April24,2017 Floridateashotholeborer,FL-TSHB).Aseriesoffieldtestswereconductedin2016in
commercialavocadogrovesknowntohaveFL-TSHBatvariouspopulationlevels.Alltests
Accepted:May30,2017
evaluatedlurescontainingquercivorol(p-menth-2-en-1-ol)andα-copaene,presentedsepa-
Published:June13,2017
ratelyandincombination;andonetestevaluatedeffectoftraptypeonbeetlecaptures.In
Copyright:Thisisanopenaccessarticle,freeofall addition,electroantennography(EAG)wasusedtoquantifyfemaleolfactoryresponsesto
copyright,andmaybefreelyreproduced,
lureemissions.Thisstudyidentified(-)-α-copaeneasanewattractantforFL-TSHB,equiva-
distributed,transmitted,modified,builtupon,or
otherwiseusedbyanyoneforanylawfulpurpose. lentinefficacytoquercivorol(thestandardlureforEuwallaceadetectionintheUSA);how-
TheworkismadeavailableundertheCreative ever,thecombinationoflurescapturedsignificantlymoreFL-TSHBthaneitherlurealone.
CommonsCC0publicdomaindedication.
Thiscombinationresultedinsynergisticattractionattwofieldsitesandadditiveattractionat
DataAvailabilityStatement:Allrelevantdataare athirdsite.StickypaneltrapscapturedmoreFL-TSHBthancomparably-baitedLindgren
withinthepaperanditsSupportingInformation
funneltraps.Femalesengagedinhost-seekingflightfrom11:00to16:00hr(EST),with
files.
peaknumbersobservedbetween12:00and13:00hr.EAGanalysesconfirmedolfactory
Funding:Thisresearchwassupportedbythe
chemoreceptionofbothkairomones,withahigherresponseelicitedwiththecombinationof
UnitedStatesDepartmentofAgriculture,
volatiles.ResultsindicatethatdetectionofpestE.nr.fornicatusinFloridacanbeimproved
AgriculturalResearchService(USDA-ARS)
AppropriatedFunds(MitigationoftheInvasivePest byusingatwo-componentlureconsistingofp-menth-2-en-1-oland(-)-α-copaene.
ThreatfromtheAmericanTropicsandSubtropics)
andbyanappointmenttotheARSResearch
ParticipationagreementbetweentheU.S.
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 1/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
DepartmentofEnergy(DOE)andtheU.S. Introduction
DepartmentofAgriculture(USDA).ORISEis
AmbrosiabeetlesintheweevilsubfamilyScolytinae(Coleoptera:Curculionidae)typicallyhave
managedbyORAUunderDOEcontractnumber
DE-SC0014664.Thefundershadnoroleinstudy abroadrangeofhosts,buttargetonlystressedanddyingtrees.Theyarewoodborersthat
design,datacollectionandanalysis,decisionto excavatebroodgalleriesinthehostxylemandinoculatethosegallerieswithsporesofsymbi-
publish,orpreparationofthemanuscript. oticfungi[1].Theresultantfungalgardensprovidethesolenutritionalsourceforbothadult
Competinginterests:Theauthorshavedeclared andlarvalbeetles,butthiscolonizationprocessalsoplaysavitalecologicalrolebyinitiating
thatnocompetinginterestsexist. andacceleratingdecompositionofdeadwood.Ingeneral,ambrosiabeetlesseldominjure
healthytreeswithintheirendemicrange;however,withanincreaseinglobalcommerce,
coupledwiththechallengeofdetectingwood-boringinsectsatportsofentry[2],exotic
ambrosiabeetlesposeaneverincreasingthreattoforestandagriculturalecosystemsworld-
wide.Althoughbenignintheirnativehabitats,somespeciesareacquiringpeststatusinnewly
establishedlands,astheirfungalsymbiontsarenowpathogenicinnew(naïve)hosttrees[3].
EuwallaceafornicatusEichhoff,theteashot-holeborer(TSHB),isanambrosiabeetlenative
toSoutheastAsiaandawidespreadpestofcommercialtea,Camelliasinensis(L.)Kuntze[4].
Inrecentyears,acomplexofbeetlesmorphologicallyindistinguishablefromE.fornicatushave
becomeestablishedaspestsofavocado,PerseaamericanaMiller,andnumerousothertreesin
Israel[5],intworegionsofsouthernCalifornia[6–7],andinsouthernFlorida[8–9].Members
ofthiscrypticgrouparereferredtocollectivelyasE.nearfornicatus;allappeartobehostgen-
eralistsandcarrycombinationsofsymbioticfungiinthegeneraFusarium,Graphium,and
Acremonium[9–11].Someofthosesymbiontsarephytopathogenic,causinglocalizedlesions
andbranchdiebackdiseaseininfestedtrees[5–6,9–10].Aninitialphylogeneticanalysisof
thiscomplexsuggeststhatthereareatleastfivegeneticallydistinctbeetlespeciesandninefun-
galsymbionts,withE.nr.fornicatussp.#1(giventhecommonnamepolyphagousshothole
borer,PSHB)foundinIsraelandCalifornia(originallyestablishednearLosAngeles),sp.#2in
Florida,sp.#3inAustralia,sp.#4inSriLanka(presumablytheoriginalTSHB),andsp.#5(the
Kuroshioshotholeborer,KSHB)alsoinCalifornia,butsouthofthefocalestablishmentofsp.
#1,inthevicinityofSanDiego[12].Morerecently,aphylogeographicanalysissuggeststhat
populationsofE.nr.fornicatusinFlorida,Hawaii,Thailand,Australia,andPapuaNewGuinea
allhaveacommonsource,andcorrespondtotheTSHB[13].
Duetoalackofconsensusregardingspeciesstatus,andpotentialbehavioraldifferences
amongE.nr.fornicatuspopulationsthataregeographicallyisolated,thespecieswestudiedwill
bereferredtohereastheFloridateashotholeborer(FL-TSHB).Thisbeetlewasfirstdetected
inSouthFloridaavocadogrovesin2010,butthespecieswasfoundinlownumbersandnot
observedtocauseseriousdamageatthattime[8].However,intheearlyspringof2016,one
grovewasobservedtohavenumerousinfestationsofFL-TSHB(~1500trees)andconsiderable
branchdieback[9].Inresponsetothischangeinpeststatus,researchwasinitiatedtogaina
betterunderstandingofthechemicalecologyoftheFloridaspecies,includingevaluationsof
kairomone-basedlures.Atpresent,lurescontainingquercivorol(p-menth-2-en-1-ol)arethe
standardfordetectionandmonitoringofE.nr.fornicatuspopulationsinbothCaliforniaand
Florida[14].However,inafieldtesttargetingredbayambrosiabeetle(Xyleborusglabratus
Eichhoff,theprimaryvectoroflaurelwilt)inFloridaavocadogroves,itwasdiscoveredthat
FL-TSHBwascapturedintrapsbaitedwithlurescontainingα-copaene[15],asesquiterpene
thatfunctionsastheprincipalhost-locationcuefordispersingfemaleX.glabratus[16–17].
Inthisreport,wesummarizeresearchconductedinseveralFloridaavocadogrovesin2016
toevaluatetheefficacyofcommerciallyavailablequercivorolandα-copaenelures,presented
aloneandintandem,forattractionofin-flightfemaleFL-TSHB.Fieldsitesincludedawell-
maintainedgrovethatwasfreeoflaurelwilt,agroveaffectedbylaurelwiltinwhichsymptom-
atictreeswereactivelyremoved,andanothergrovewithlaurelwiltwhichcontainednumerous
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 2/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
standingtreeskilledbylaurelwilt.Thus,studysitesvariedconsiderablyintheirpopulation
levelsofFL-TSHB.Datacollectionalsodocumentedthedailyflightpatternsofthispestbeetle
insouthernFlorida.Inaddition,quantitativeelectroantennographicanalyseswereperformed
toconfirmchemoreceptionofthesetwosemiochemicals,andtocomparefemaleolfactory
responsestovolatileemissionsfromtheluretreatmentsevaluatedinthefield.
Materialsandmethods
Ethicsstatement
FieldtrialswereconductedatthreeprivatelyownedavocadogrovesinMiami-DadeCounty,
FL,withpermissionofthelandowners.Fieldstudiestargetedinsectpests,anddidnotinvolve
anyprotectedorendangeredspecies.
Trapsandlures
Commerciallurescontainingquercivorolandα-copaenewereusedinallfieldandlaboratory
experiments.Bothluresconsistedofclearplasticbubbledispensers,29mmindiameter,
loadedwitheither1-mlsamplesofquercivorolor2-mlsamplesofα-copaene(SynergySemio-
chemicalsCorp.,Burnaby,BC,Canada).Fieldtestswereconductedusingstickypaneltraps,
comparabletothoseusedpreviouslyforevaluationofluresforX.glabratus[18].Twowhite
stickycards(23x28cm,Sentrywingtrapbottoms;GreatLakesIPM,Vestaburg,MI,USA)
werewiredback-to-backalongtheupperedge,securedwithabinderclipontheloweredge,
andhungfromtreelimbsusings-shapedwirehooks.Lureswerefastenedtothewirehookjust
abovethestickycards,andthefinalassemblywastoppedwithaninvertedclearplasticplate
(24cmdiam.)toprovideaprotectivecovering.
Onefieldtestalsoincorporatedfour-unitLindgrenfunneltraps(BioQuip,RanchoDomin-
guez,CA,USA).Withthistrapdesign,lureswerefastenedtooneofthesidesupportarms
midwaybetweenthesecondandthirdfunnel.Thecollectioncupswerefilledwith300mlof
anaqueoussolutionof10%propyleneglycol(Low-Toxantifreeze;Prestone,Danbury,CT,
USA)toretainandpreservecapturedbeetles.
Fieldtests
Fivefieldtestswereconductedwithinthreeavocadogrovesduring2016,andeachtest
includedfourtreatments:aquercivorollure,anα-copaenelure,acombinationofquercivorol
andα-copaene(2separateluressecuredsidebyside),andanunbaitedcontroltraptoassess
randomcaptures.Testsfollowedarandomizedcompleteblockdesign,withfivereplicate
blocks(inallbutonetest;seebelow).Eachblockconsistedofarowoftrapshung~1.5m
aboveground[19],withaminimumspacingof10mbetweenadjacenttraps.Wellshaded
locationswerechosenforplacementofeachtrap.
InitialfieldtrialswereconductedinthegrovewhereahighpopulationofFL-TSHBwas
firstdetectedin2016[9](25o30’106”N,80o29’372”W).Thisgrovewaswellmaintained,
andthreeshorttests(amountingto8weekstotaltrappingeffort)werecoordinatedaroundthe
maintenancescheduleofthegrovemanager.Fieldtests1and2were2-weektestsconducted
from24Februaryto9March,andfrom4to18May,respectively.Trapswerecheckedthree
timesaweekinbothtests.Fieldtest3wasa4-weektest,conductedfrom30Juneto28July,
withtrapscheckedonceaweek.Ateachsamplingdate,thestickypanelswerecollected,trap
positionswererotatedsequentiallywithineachrow(tominimizepositionaleffectsonbeetle
captures),andnewstickypanelswerehung.
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 3/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
Fieldtest4wasconductedinanavocadogroveaffectedbylaurelwilt,wherediseasedtrees
werecutdownanddestroyedbythegrovemanager(25o29’418”N,80o28’593”W).Thiswas
an8-weektest,servicedweeklyfrom21Julyto15September,followingthegeneralmethods
describedabove.However,sinceinitialsurveysindicatedthatFL-TSHBwaspresentatvery
lowlevelsinthisgrove,tenreplicateblocksweredeployedtoimprovesensitivityofbeetle
detection.
Fieldtest5wasconductedinanothergrovewithlaurelwilt(25o35’894”N,80o27’847”
W),butthissitehadmanydeadandsymptomatictrees,withmoderatelevelsofbeetleinfesta-
tion.Thiswasa6-weektestcheckedweeklyfrom10Novemberto22December.Treatments
werethesameasthoseusedinprevioustests,butthistestincludedanadditionaltreatment
consistingofafunneltrapbaitedwiththecombinationlure.Forbothtestsconductedinlaurel
wiltgroves,blocksweresetupinportionsofthegrovecontaininghealthy(asymptomatic)
treeswhichprovidedshadecomparabletothatfoundinthegrovelackinglaurelwilt.
StickypanelscollectedfromfieldtestsweretakentotheUSDA-ARSlaboratory(Miami,
FL)andinspectedunderadissectingmicroscope.Forfieldtests1through4,allspeciesofSco-
lytinaewereremovedfromthepanels,soakedbrieflyinhistologicalclearagent(Histo-clearII;
NationalDiagnostics,Atlanta,GA,USA),counted,andidentifiedbasedonmorphological
characters[20–21].Forfieldtest5,onlyspecimensofFL-TSHBwerecounted.
Determinationofflightpatterns
Duringthecourseoffieldtest1,datawerecollectedondailyflightpatternsoffemaleFL-
TSHB.Onthreesamplingdates(4,8,9March2016),everytrap(alltreatmentscombined,
n=20)wasinspectedat30-minuteintervalstorecordthenumberofin-flightbeetlescaptured
throughouttheday.Observationswereinitiatedat10:00hoursEST(whennewstickypanels
werehung)andcontinueduntil16:30hours(whencapturesceased).Foreachcollectionday,
thenumberscaughtweresummedforeachinterval,andthenconvertedtopercentagesofthe
totalcapturesforthatday.
Electroantennography
BeetlesusedforEAGanalyseswereFL-TSHBfemaleslaboratoryrearedfrominfestedavocado
wood.Brancheswerecutfrommaturetreesandheldinplasticemergencechambers[44gal
(167L)Brute1containers;RubbermaidCommercialProducts,Winchester,VA,USA].To
preventcondensation,ventilationholeswerecutinthesideandlidofeachcontainerandcov-
eredwithfinemeshscreening.Tofacilitatecollectionofnewlyemergedbeetles,eachcontainer
wasfittedwithawidemouth1quart(0.95L)masonjar(BallCorp.,Broomfield,CO,USA).
Thiswasdonebycuttingacircularhole(8.5cmdiameter)inthesideofthecontainer,and
thengluingthemetalringlidinplace,permittingeasyremovalofthejar.Moisttissuepaper
(Kimwipes,Kimberly-Clark,Roswell,GA,USA)wasplacedinsideeachjar,andbeetles(cid:20)24
hrpost-emergencewerecollectedeachmorning.
Testsubstratesconsistedofcommercialluresofquercivorolandα-copaene(asdescribed
above).Thestandardreferencecompoundwasethanol(5ml95%;Pharmo-Aaper,Brookfield,
CT,USA),whichhasbeenshownpreviouslytoelicitstrongEAGresponsesinfemaleambrosia
beetles[22].Threesamplebottleswereprepared:quercivorol,α-copaene,andquercivorol+α-
copaene.Substrateswereplacedintoseparate250mlglassbottlesequippedwithalidcontain-
ingashortthru-hullport(Swagelok,Solon,OH,USA)andsiliconeseptum(Alltech,Deerfield,
IL,USA).Samplebottleswerethensealedandequilibratedfor2hrat24˚Ctoallowforhead-
spacesaturationwithvolatiles.
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 4/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
PeripheralolfactoryresponseswererecordedwithaSyntechEAGsystemandEAG2000soft-
ware(SyntechOriginalResearchInstruments,Hilversum,theNetherlands)usingthegeneral
methodspreviouslydescribedforanalyseswithXyleborusspp.[18,22].Theantennalholdercon-
sistedofagold-plated2-prongedprobe(SyntechCombi-Probe)whichwasmodifiedbysoldering
athingoldwireontothedifferentelectrode[22].Eachpreparationconsistedofasingle,excised
antenna(meanlengthof0.468±0.008mm,n=20)andasmallportionoftheheadcapsulesur-
roundingthesocket.Thebasalportionwasmountedontotheindifferentelectrodeusingsalt-free
conductivegel(Spectra360;ParkerLaboratories,Fairfield,NJ,USA),andtheflexiblewire,coated
withathinlayerofgel,wasmanipulatedtomakeminimalcontactwiththeapicalantennalclub.
TofacilitateappropriateEAGpreparations,low-temperaturescanningelectronmicroscopy(LT-
SEM)wasusedtovisualizetheantennalmorphologyoffemaleFL-TSHB,usingpublishedmeth-
ods[23–24].LT-SEMimages(Fig1A–1D)indicatedthattheolfactorysensillaarenotdistributed
evenlyovertheclub,butprimarilyconcentratedontheventralsurfaceinconcentricrings.There-
fore,antennaewerepositionedwiththeventralsurfacefacingupwards,andthegoldwireelec-
trodeincontactwiththedorsumoftheclubtopreventcoatingthesensillawithconductivegel.
Antennalpreparationswereplacedunderastream(400ml/min)ofhumidifiedair,purified
withactivatedcharcoalgranules(grainsize1–2mm).Usinggas-tightsyringes(SGEAnalytical
Science,Victoria,Australia),samplesofsaturatedvaporwerewithdrawnfromtestbottles,
injectedintotheairstream,anddeliveredtotheantennae.Ineachrecordingsession,thean-
tennawaspresentedwithsamplesinthefollowingorder:theethanolstandard(2mldose),
samplesofvolatilesemittedbytestlures,anegativecontrol(cleanairinjectionequalinvolume
tothetestsamples),andafinalethanolstandard.Injectionsweredeliveredat2minintervals,
withacleanairflushinbetween,tominimizeantennaladaptation.
Testresponseswererecordedinitiallyinmillivolts(peakheightofdepolarization)andthen
normalizedtopercentagesrelativetotheresponseobtainedwiththeethanolstandard.Nor-
malizationcorrectsfortime-dependentdeclineinantennalperformance,andalsoallowsfor
comparisonofrelativeEAGresponsesobtainedwithdifferentsubstrates[25–27]andwithdif-
ferentcohortsofinsects[28–29].Anyresponseobtainedwiththenegativecontrolwassub-
tractedfromthenormalizedresponse,andthefinalcorrectedvalueswereusedtomake
comparisonsamongtestsamples.
TwoEAGexperimentswereconductedwithlurevolatiles.Thefirstexperimentquantified
dose-dependentresponsestovolatilesemittedfromthequercivorollure,usingfivevolumetric
dosesrangingfrom0.5to4.0ml.Basedontheresultsofthisinitialtest,thesecondEAGexper-
imentusedfixeddosestocompareolfactoryresponsestovolatilesfromthethreeluretreat-
ments.Responseswererecordedfrom10femalestoconstructthequercivoroldose-response
curve,andfrom15femalesinthecomparativeEAGexperiment.
Aftertheexperimentswereconcluded,gaschromatographic(GC)analysiswasperformed
toquantifyheadspacevolatilesfromtheEAGsamplebottles.Aninternalstandard,600μlhex-
adecane(99%,SigmaAldrich,St.Louis,MO,USA),wasintroducedintoeachbottleusinga1
mlsyringe(SGEAnalyticalScience),andbottleswereleftundisturbedfor>48hrtoallowvol-
atilestoequilibratepriortosampling.Headspacesamples(10μleach,20replicatespersample
bottle)werecollectedwitha25μlsyringe(PressureLok;VICIPrecisionSampling,Baton
Rouge,LA,USA)andanalyzedusingaTRACEGCModel2000(ThermoElectronNorth
AmericaLLC,WestPalmBeach,FL,USA)fittedwithaJ&WDB-5MScolumn,25mx0.25mm
x0.25μm(AgilentTechnologies,SantaClara,CA,USA).Thesplit/splitlessinjectorwassetat
225˚C;heliumwasthecarriergassetataconstantflowof0.8ml/min.Theflameionizationde-
tector(FID)wassetat250˚C.Theovenwassetupwithatemperaturerampprogram,starting
at50˚Candincreasingat15˚C/minto130˚C,thenat10˚C/minto220˚Candholdingatthat
temperaturefor2min.
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 5/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
Fig1.ScanningelectronmicrographsofantennaefromfemaleEuwallaceanr.fornicatus.(A)Ventralviewofantenna,showing
arrangementofsensillainconcentricringsontheapicalendofantennalclub(450xmagnification).(B)Lateralviewofantenna,showingthe
diagonalelongationofthedorsalsurfaceoftheclubandconcentrationofsensillaontheventralsurface(500xmagnification).(C)Portionofthe
lowerconcentricring,showingtwotypesofsensilla:longserratedsensillatrichoideaalongtheouteredge,andmorenumerousbluntly-ended
sensillabasiconicathroughouttheinterior(2000xmagnification).(D)Portionoftheupperconcentricring,showingthreetypesofsensilla:
serratedsensillatrichoidea,bluntsensillabasiconica(withfinesculpturing),andshorterbulboussensillabasiconica(withdeepgrooves)(5000x
magnification).
https://doi.org/10.1371/journal.pone.0179416.g001
Statisticalanalysis
Regressionanalysiswasusedtodescribetherelationshipbetweensubstratedoseandresponse
ofolfactoryreceptorstovolatilesemittedfromthequercivorollure(firstEAGexperiment),
andalsotodocumenttherelationshipbetweentimeofdayandthenumberoffemaleFL-
TSHBobservedinflight.One-wayanalysisofvariance(ANOVA)wasusedtotesttheeffectof
treatmentonattraction(fieldtests)andolfactoryresponse(comparativeEAGexperiment);
significantANOVAswerethenfollowedbymeanseparationwiththeTukeyHSDtest.To
evaluatepotentialsynergismbetweenlures,analysisbyt-testwasusedtocomparethefield
capturesobtainedwiththecombinationluretothesummedcapturesobtainedwithindividual
α-copaeneandquercivorollures.Inaddition,analysisbyt-testwasusedtocompareEAG
responsesobtainedwithadjacentdosesofquercivorol.Whennecessary,dataweresquareroot
(x+0.5)-transformedtostabilizevariancepriortoanalysis.Allanalyseswereperformedusing
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 6/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
SystatSoftware[30].Resultsarepresentedasmean±SEM;probabilitywasconsideredsignifi-
cantatacriticallevelofα=0.05.
Results
Fieldtests
Infieldtest1(Fig2A,S1Table),fieldtest2(Fig2B,S2Table),andfieldtest3(Fig2C,S3
Table)–thetrialsconductedinanavocadogrovewithoutlaurelwilt–thereweredifferencesin
meancaptureofFL-TSHBamongthefourtreatmentsevaluated(F=17.018,df=3,16,P<
0.001;F=12.970,df=3,16,P<0.001;F=9.398,df=3,16,P<0.001,respectively).Inallthree
tests,highestcaptureswereobtainedwiththecombinationofquercivorolandα-copaene
lures,intermediatecaptureswithsingleluresofeitherquercivorolorα-copaene,andfewest
captureswiththeunbaitedcontroltrap.Thecombinationofα-copaeneandquercivorol
resultedinanadditiveeffectoncapturesofFL-TSHB.Capturesofnon-targetScolytinaewere
few.InadditiontoFL-TSHB,tenotherspecieswerecaptured,withAmbrosiodmus devexulus
(Wood)beingthespeciesmostcommonlyobserved(Table1).Withthecollectivecaptures
fromthisfieldsite,FL-TSHBcomprised89.0,95.8,and96.0%ofthescolytinebeetlesinter-
ceptedintrapsbaitedwithα-copaene,quercivorol,andthecombinationlure,respectively.
Infieldtest4(Fig3,S4Table),conductedinthemanagedgrovewithlaurelwilt,therewere
alsodifferencesinmeancapturesofFL-TSHBamongluretreatments(F=24.494,df=3,36,
P<0.001).Aswithtests1–3,captureswerehighestwiththecombinationlure.However,at
thissitewithverylowlevelsofFL-TSHB,captureswiththesinglelureswerenotstatistically
greaterthanthoseobtainedwiththeunbaitedcontrol.Consequently,meancaptureswiththe
combinationlureweresignificantlygreaterthanthesummedcapturesfromthetwotraps
baitedwithsinglelures(t=3.736,df=18,P=0.002),suggestingsynergisticattraction.Incon-
trasttothepreviousfieldsite,capturesofnon-targetScolytinaewerenumerous;thirteenother
specieswereencountered,withthemostcommonincludingXyleborusbispinatusEichhoff,
Ambrosiodmus devexulus,andXyleborusvolvulusEichhoff(Table2).Trapsbaitedwithα-
copaeneandthecombinationlurealsocaughtseveralspecimensofX.glabratus.FL-TSHBwas
thesecondmostabundantspecies,butcomprisedonly7.7,11.3,and35.4%ofthescolytine
beetlescaughtwithα-copaene,quercivorol,andthecombinationlure,respectively.
Consistentwithpreviousresults,meancapturesofFL-TSHBinfieldtest5(Fig4,S5Table)
werealsosignificantlydifferentamongtreatments(F=28.222,df=4,20,P<0.001).Highest
captureswereobtainedwithstickytrapsbaitedwiththecombinationlure,intermediatecap-
tureswithsinglequercivorolorα-copaenelures,andlowestcaptureswiththeunbaitedcon-
trol.Lindgrenfunneltrapsbaitedwiththecombinationlurecapturedsignificantlyfewer
beetlesthanequallybaitedstickytraps,andcaptureswithbaitedfunneltrapswerenodifferent
thanthoseobtainedwithunbaitedstickypanelcontrols.Asintheothergrovewithlaurelwilt,
synergisticattractionofFL-TSHBwasobservedwiththecombinationlure;meancaptures
withthecombinationluredeployedinstickytrapsweresignificantlyhigherthanthesummed
capturesfromthetwosingle-baitedtraps(t=3.318,df=8,P=0.011).Atthissitewithlaurel
wilt,asingleX.glabratuswascapturedinatrapbaitedwiththecombinationlure,andseveral
X.glabratuswerecapturedwithα-copaeneluresinaninitialtestconductedwithinthisavo-
cadogrovein2015[15].
Temporalpatternsinhost-seekingflight
FemalesofFL-TSHBwereobservedinflightasearlyas11:00hoursEST,withpeakflightfrom
12:00to13:00hours(Fig5,S6Table).Numbersgraduallydeclinedthroughouttheafternoon,
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 7/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
Fig2.Mean(±SEM)capturesoffemaleEuwallaceanr.fornicatusinfieldtestsconductedinawell
maintainedavocadogroveinMiami-DadeCounty,FL.(A)2-weektestinFeb-Mar2016.(B)2-weektestin
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 8/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
May2016.(C)4-weektestinJun-Jul2016.Luretreatmentsconsistedofaquercivorolbubblelureandanα-
copaenebubblelure,deployedseparatelyandincombinationinstickypaneltraps.Thecontroltreatment
consistedofanunbaitedstickytrap.Barstoppedwiththesameletterarenotsignificantlydifferent(Tukey
HSDmeansseparation,P<0.05).
https://doi.org/10.1371/journal.pone.0179416.g002
withnocapturesobservedafter16:00hours.Thispatternwasbestfitbyregressionwitha
Gaussianpeakmodel(y=16.95e-0.5[(x-12.77)/1.19]2,R2=0.882).
Electroantennography
TheEAGtechniquesdevelopedforXyleborusspp.[22]workedwellforquantificationofolfac-
toryresponsesfromEuwallaceaantennae,withrepresentativeelectroantennogramsshown
inFig6A.Therelationshipbetweendoseofvolatilesfromthequercivorollureandtheampli-
tudeofEAGresponsewasbestfitbyregressionwithahyperbolicmodel[y=68.96x/(0.98+
x),R2=0.976;Fig6A,S7Table].Amplitudeincreasedwithdosageupthroughthe1-mldose
(meanEAGresponsetothe1-mldosewasgreaterthanresponsetothe0.5-mldose;t=-4.62,
df=14,P<0.001),andthenbegantoleveloff.EAGresponsesdidnotincreasesignificantlyas
dosedoubledfrom1to2ml(t=-1.35,df=14,P=0.198)orfrom2to4ml(t=-1.63,df=14,
P=0.126).ThisobservedplateauinEAGresponsesuggestedthatthedoserangeevaluated
wassufficienttoelicitmaximalantennalresponse(saturationofolfactoryreceptors),anda
fixed2-mldosewaschosenforthecomparativeEAGexperiment.
Thereweresignificantdifferencesinantennalresponsestovolatileemissionsfromthe
threeluretreatments(F=5.997;df=2,33;P=0.006;Fig6B,S8Table).MeanEAGresponse
elicitedwiththecombinationofquercivorolandα-copaenewassignificantlygreaterthanthe
responsesobservedwitheitherlurealone.Meanantennalresponseswerecomparablewhen
volatilesfromthetwolureswerepresentedseparately.
QuantificationofheadspacevolatilesfromtheEAGsamplebottles(S9Table)indicatedthat
saturatedvaporfromtheα-copaenelurecontained7.92±0.44mg/mlofα-copaene.Saturated
vaporfromthequercivorollurecontainedtwocomponents:adominantisomer(cis-p-menth-
2-en-1-ol,86.6%)andasecondaryisomer(trans-p-menth-2-en-1-ol,13.4%),quantifiedat
4.06±0.42and0.63±0.06mg/ml,respectively.Analysisofheadspacefromthebottlecontain-
ingbothluresindicatedacompositionof47.4%α-copaene,44.3%cis-p-menth-2-en-1-ol,and
Table1. SummedcapturesofScolytinae(Coleoptera:Curculionidae)fromthreefieldtestsconductedinawell-managedavocadogroveinMiami-
DadeCounty,FL(8wktotaltrappingeffort,Feb-Jul2016).
Species Quercivorol+α-Copaene Quercivorol α-Copaene UnbaitedControl
TribeXyleborini
Ambrosiodmusdevexulus(Wood) 34 8 31 4
AmbrosiodmusleconteiHopkins 1 1
Euwallaceanr.fornicatus 1514 620 573 52
PremnobiuscavipennisEichhoff 1
Theoborusricini(Eggers) 7 4 13 8
Xyleborinusandrewesi(Blandford) 1 1
XyleborinusgracilisEichhoff 8 1 5
Xyleborinussaxesenii(Ratzeburg) 1 1 2 1
XyleborusbispinatusEichhoff 1 1
Xylosandruscrassiusculus(Motschulsky) 6 2
TribeCryphalini
Hypothenemusspp. 6 9 17 3
https://doi.org/10.1371/journal.pone.0179416.t001
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 9/20
NewattractantandimprovedlureforEuwallaceanr.fornicatus
Fig3.Mean(±SEM)capturesoffemaleEuwallaceanr.fornicatusinan8-weekfieldtest(Jul-Sep2016)
conductedinamanagedavocadogrovewithlaurelwiltinMiami-DadeCounty,FL.Luretreatmentsconsisted
ofaquercivorolbubblelureandanα-copaenebubblelure,deployedseparatelyandincombinationinstickypanel
traps.Thecontroltreatmentconsistedofanunbaitedstickytrap.Barstoppedwiththesameletterarenotsignificantly
different(TukeyHSDmeansseparation,P<0.05).
https://doi.org/10.1371/journal.pone.0179416.g003
Table2. SummedcapturesofScolytinae(Coleoptera:Curculionidae)froman8-wkfieldtestconductedinanavocadogroveaffectedbylaurelwilt
inMiami-DadeCounty,FL(Jul-Sep2016).
Species Quercivorol+α-Copaene Quercivorol α-Copaene UnbaitedControl
TribeXyleborini
Ambrosiodmusdevexulus(Wood) 45 4 29 5
AmbrosiodmusleconteiHopkins 1 1
Euwallaceanr.fornicatus 101 16 13 2
Theoborusricini(Eggers) 1
Xyleborinusandrewesi(Blandford) 3 1 2
Xyleborinussaxesenii(Ratzeburg) 1 3 3 2
XyleborusaffinisEichhoff 2 8 7 6
XyleborusbispinatusEichhoff 89 79 78 49
Xyleborusferrugineus(F.) 1 3 3 4
XyleborusglabratusEichhoff 2 2
XyleborusvolvulusEichhoff 21 15 14 5
Xylosandruscompactus(Eichhoff) 2 2 2 1
Xylosandruscrassiusculus(Motschulsky) 9 3 1
TribeCryphalini
Hypothenemusspp. 9 7 13 2
https://doi.org/10.1371/journal.pone.0179416.t002
PLOSONE|https://doi.org/10.1371/journal.pone.0179416 June13,2017 10/20