Non-Native Ambrosia Beetles as Opportunistic Exploiters of Living but Weakened Trees

21 Pages·2015·0.99 MB·English

RESEARCHARTICLE
Non-Native Ambrosia Beetles as
Opportunistic Exploiters of Living but
Weakened Trees
ChristopherM.Ranger1*,PeterB.Schultz2,StevenD.Frank3,JuangH.Chong4,Michael
E.Reding1
1 USDAAgriculturalResearchService,HorticulturalInsectsResearchLab,andDepartmentofEntomology,
OhioAgriculturalResearchandDevelopmentCenter,TheOhioStateUniversity,Wooster,Ohio,United
StatesofAmerica,2 HamptonRoadsAgriculturalResearchandExtensionCenter,VirginiaPolytechnic
InstituteandStateUniversity,VirginiaBeach,Virginia,UnitedStatesofAmerica,3 Departmentof
Entomology,NorthCarolinaStateUniversity,Raleigh,NorthCarolina,UnitedStatesofAmerica,4 PeeDee
ResearchandEducationCenter,ClemsonUniversity,Florence,SouthCarolina,UnitedStatesofAmerica
* [email protected]
Abstract
OPENACCESS
Citation:RangerCM,SchultzPB,FrankSD,Chong ExoticXylosandrusspp.ambrosiabeetlesestablishedinnon-nativehabitatshavebeen
JH,RedingME(2015)Non-NativeAmbrosiaBeetles associatedwithsuddenandextensiveattacksonadiverserangeoflivingtrees,butfactors
asOpportunisticExploitersofLivingbutWeakened
drivingtheirshiftfromdying/deadhoststolivingandhealthyonesarenotwellunderstood.
Trees.PLoSONE10(7):e0131496.doi:10.1371/
journal.pone.0131496 Wesoughttocharacterizetheroleofhostphysiologicalconditiononpreferenceandcoloni-
zationbytwoinvaders,XylosandrusgermanusandXylosandruscrassiusculus.Whengiven
Editor:GarretSuen,UniversityofWisconsin-
Madison,UNITEDSTATES free-choiceunderfieldconditionsamongfloodedandnon-floodeddeciduoustreespecies
ofvaryingintolerancetoflooding,beetlesattackedflood-intoleranttreespeciesovermore
Received:March22,2015
tolerantspecieswithin3daysofinitiatingfloodstress.Inparticular,flood-intolerantflower-
Accepted:June1,2015
ingdogwood(Cornusflorida)sustainedmoreattacksthanflood-tolerantspecies,including
Published:July2,2015 silvermaple(Acersaccharinum)andswampwhiteoak(Quercusbicolor).Ethanol,akey
Copyright:Thisisanopenaccessarticle,freeofall host-derivedattractant,wasdetectedathigherconcentrations3daysafterinitiatingflooding
copyright,andmaybefreelyreproduced,distributed, withinstemsoffloodintolerantspeciescomparedtotolerantandnon-floodedspecies.A
transmitted,modified,builtupon,orotherwiseused
positivecorrelationwasalsodetectedbetweenethanolconcentrationsinstemtissueand
byanyoneforanylawfulpurpose.Theworkismade
availableundertheCreativeCommonsCC0public cumulativeambrosiabeetleattacks.WhenadultX.germanusandX.crassiusculuswere
domaindedication. confinedwithno-choicetostemsofflood-stressedandnon-floodedC.florida,moreejected
DataAvailabilityStatement:Allrelevantdataare sawdustresultingfromtunnelingactivitywasassociatedwiththeflood-stressedtrees.Fur-
withinthepaperanditsSupportingInformationfiles. thermore,livingfoundresses,eggs,larvae,andpupaewereonlydetectedwithingalleries
Funding:ThisresearchwasfundedbytheUSDA createdinstemsofflood-stressedtrees.Despiteacapabilitytoattackdiversetreegenera,
FloricultureandNurseryResearchInitiativeandbase X.germanusandX.crassiusculusefficientlydistinguishedamongvaryinghostqualitiesand
fundsassociatedwithUSDAARSResearchProject
preferentiallytargetedtreesbasedontheirintoleranceoffloodstress.Non-floodedtrees
3607-22000-012-00D(NationalProgram304-Crop
werenotpreferredorsuccessfullycolonized.Thisstudydemonstratesthehost-selection
ProtectionandQuarantine),andtheHorticultural
ResearchInstitute.Thefundershadnoroleinstudy strategyexhibitedbyX.germanusandX.crassiusculusinnon-nativehabitatsinvolves
design,datacollectionandanalysis,decisionto detectionofstress-inducedethanolemissionandearlycolonizationoflivingbutweakened
publish,orpreparationofthemanuscript.
trees.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0131496 July2,2015 1/21
AmbrosiaBeetle-HostTreeInteractions
Introduction
Non-nativeambrosiabeetles(Coleoptera:Curculionidae)inthetribeXyleborinihavebeenreg-
ularlyinterceptedatportsofentryandseveralspeciesarenowestablishedandabundantin
NorthAmerica[1–9].Acrypticnature,dietofsymbioticfungi,sib-mating,haplodiploidy,
broadhostrange,andinteractionswithnaïvehostslikelyaidintheirintroductionandestab-
lishmentinnon-nativehabitats[1–9].Inparticular,twospeciesnativetoSoutheastAsia,Xylo-
sandrusgermanus(Blandford)andXylosandruscrassiusculus(Motschulsky),havebecome
widelyestablishedacrossregionsofNorthAmerica[2,10].Asof2010,X.germanusandX.cras-
siusculuswerefoundin32and29of50states,respectively[10].Xylosandrusgermanushasalso
becomeestablishedthroughoutmuchofEurope[11–16].Xylosandruscrassiusculuscurrently
hasanarrowerdistributioninEurope,butisestablishedinCentralAmericaandtheCaribbean,
EastandWestAfrica,andOceania[17,18].
XylosandrusgermanusandX.crassiusculusexhibitacapabilitytoattackadiverserangeof
>200and>120species,respectively[19,20],andhaveextensivelyattackedlivingandappar-
ently-healthytreesgrowingwithinnon-nativeornamental,horticultural,andforestedsettings
[9,11,12,21–26].However,thebasisfortheseattacksisnotwellunderstood,particularlysince
bothspeciescolonizedyinganddeadtreesintheirnativehabitats[4].Overadozenadditional
speciesofambrosiabeetlesthatnormallyattackdyingordeadtreeshavealsoshiftedtoattack-
inglivingtreesinnon-nativehabitats;therebyleadingtospeculationaboutthemechanisms
underlyingtheirselectionoflivingtreesashosts[4,9,27].
AswithotherScolytinae,host-derivedolfactorycues,particularlyethanol,playanimpor-
tantroleduringhostselectionbyX.germanusandX.crassiusculus[9,28–30].Ethanolisakey
host-derivedattractantandbaiting,irrigating,orinjectingtreeswithethanolinducedattacks
onspecifictrees[9,28,31–33].Xylosandrusgermanusalsoefficientlylocatedandattackedetha-
nol-injectedtrees,butrarelylandedonanddidnotattackadjacenttreesnotemittingethanol
[9].Apositivecorrelationwasalsodemonstratedbetweenconcentrationofethanolinjected
intoatreeandcumulativeambrosiabeetleattacks[31],alongwithapositivecorrelation
betweenemissionratesfromluresandcorrespondingtrapcaptures[29].Inadditiontousing
host-derivedvolatiles,someScolytinaeandPlatypodinaeambrosiabeetlesproduceaggregation
pheromonestoattractconspecificsandoverwhelmtreedefenses[34,35].However,thisstrat-
egyisuncommonamongtheXyleboriniambrosiabeetlesandX.germanusandX.crassiusculus
arenotanticipatedtoproduceaggregationpheromones[9].
Ethanolisrapidlyinducedbytreesinresponsetoavarietyofabioticandbioticstressors,
includingfloodanddroughtstress,girdling,freezing,pathogens,rootandcrowndisturbance,
andpollutants[36–43].Inthecaseofflooding,rootssubjectedtolittleornooxygenwillswitch
fromaerobictoanaerobiccellularrespiration[41,42].Pyruvateformedduringglycolysisis
thenconvertedintoethanolandeithermetabolizedbyalcoholdehydrogenaseoremittedfrom
theepidermis.Fieldandexperimentalobservationshavedocumentedthepotentialforflood
stresstopredisposetreestoattackbynon-nativeambrosiabeetles.Forinstance,extensive
ambrosiabeetleattacksweredetectedinMay2011onfieldgrowndogwood(Cornusflorida×
Cornuskousa)treesthatweresubjectedtowaterloggingandpoorsoildrainageattwoorna-
mentalnurseriesfollowingrecordsettingprecipitation[43].Subsequentexperimentsdemon-
stratedflood-stressedC.florida,whichisconsideredintolerantofflooding,weremore
attractiveandpreferentiallyattackedbyX.germanusandX.crassiusculus[43].Ethanolwas
alsodetectedwithinthevasculartissueandemittedfromtheepidermisofC.florida,butnot
fromnon-floodedtrees.
Comparativelyhighconcentrationsofethanolcanbeassociatedwithfloodintoleranttrees,
butsometolerantspeciesdelayoravoidaccumulatingethanol[44,45].Thus,allowingX.
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germanusandX.crassiusculustochooseamongtreespeciesvaryingintheirtoleranceofflood
stresscouldprovideinsightintotheirpreferencefortreesinaspecificphysiologicalcondition.
PreviousstudiesindirectlysuggestedX.germanuscoulddistinguishamongevenslightdiffer-
encesintreequality[9,21,22].Furthermore,theextenttowhichX.germanusandX.crassiuscu-
lusutilizehealthynaïvetreesinnon-nativehabitats,especiallyintheabsenceofweakened
ones,isalsopoorlyunderstoodandunsubstantiated.Attackshavebeensuspectedtooccuron
apparently-healthytrees[10,21,46–48],butsuchtreesmayactuallyhavebeeninapparently-
stressedatthetimeofattack[9].Comparingcolonizationsuccessonflood-stressedandnon-
floodedtreescouldtherebyprovidecrucialinsightintohostutilizationwithinnon-nativehabi-
tatsandaidincharacterizingfactorscontributingtotheinvasivenessofX.germanusandX.
crassiusculus.
TheoverallgoalofthisstudywastoassessfactorsinvolvedwithX.germanusandX.cras-
siusculustargetinglivingtreesinnon-nativehabitats,particularlytheroleofhostphysiological
conditiononpreferencebehaviorandcolonizationsuccess.Tocharacterizehostpreference
specificity,wetestediftreespeciesintolerantoffloodingwouldbepreferentiallyattacked
underfreechoiceconditionsoverspeciesmoretolerantofflooding.Ethanolinstemtissuesof
non-floodedandflood-stressedtreeswasalsoanalyzedtodetermineifelevatedconcentrations
wereassociatedwiththemoreintolerantspecies.Toelucidatelimitationsimposedbytreecon-
ditiononcolonizationsuccess,wealsocomparedthecapabilityofX.germanusandX.crassius-
culustocolonizeflood-stressedandnon-floodedtrees.WehypothesizedthatX.germanusand
X.crassiusculuscandistinguishamongtreesofvaryingphysiologicalconditionsandpreferen-
tiallyattackspeciesintolerantoffloodstress,butexhibitnon-preferenceandpoorcolonization
ofnon-floodedtrees.Wealsohypothesizedinterspecificvariabilityinethanolstemconcentra-
tionswouldberelatedtointoleranceoffloodstress.
MaterialsandMethods
PreferenceamongTreesVaryingintheirToleranceofFloodStress
Free-choiceexperimentswereconductedinOhioandVirginiatocharacterizetheroleofhost
physiologicalconditiononpreferenceofX.germanusandX.crassiusculusandtodetermineif
treespeciesintolerantoffloodingwouldbepreferentiallyattackedovermoderatelytolerantor
tolerantspecies.FloweringdogwoodC.florida((cid:1)20mmstemdiam.at15.24cmabovesoil),
JapanesesnowbellStyraxjaponicusS.etZ.((cid:1)23mm),andeasternredbudCerciscanadensisL.
((cid:1)25mm)wereselectedasrepresentativesofflood-stressintolerantspecies[49–51];flowering
cherryPrunusserrulataLindl.((cid:1)28mm),AmericanelmUlmusamericanaL.((cid:1)19mm),and
riverbirchBetulanigraL.((cid:1)21mm),wereselectedasmoderatelytolerantspecies[49,52,53];
andswampwhiteoakQuercusbicolorWilld.((cid:1)19mm)andsilvermapleAcersaccharinumL.
((cid:1)19mm)wereselectedastolerantspeciesfordeploymentinOhio[49,51].Cornusfloridaand
C.canadensiswereselectedasintolerantspecies;P.serrulataandM.virginianarepresented
moderatelytolerantspecies;andQ.bicolorandA.saccharinumrepresentedtolerantspeciesfor
deploymentinVirginia.Treeswere2−3yearsoldandmaintainedin19-Lpots.
Floodingconditionswereimposedusingapot-in-potsystemaccordingtoRangeretal.
[43],wherebya26-Lpotwasfirstlinedwithaplasticwastebagof3mil(0.076mm)thickness.
A19-Lpotcontainingasingletreewasthenplacedwithintheplasticlinedpot.Floodstress
wasinitiatedbyirrigatingthemediawithintheinternalpotuntilstandingwaterpooledaround
thebaseofthetrees.Treeswerecheckeddailytoensurestandingwaterwasmaintainedinthe
selectedpotsthroughoutthedurationofexperiments.Excessplasticlinersurroundingtheedge
ofthefloodedpotwasdrapedaroundtheinternalcircumferenceofthepotandwrapped
aroundthebaseofthestemtopreventbeetlesfromlandinginthestandingwater.
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Non-floodedandfloodedspecimensofA.saccharinum,B.nigra,C.canadensis,C.florida,P.
serrulata,Q.bicolor,S.japonicus,andU.americanaweredeployedineightrandomizedcom-
pleteblockswithinawoodlotinOhio(40°45’41.97”N81°51’20.89”W).Permissionwasprovided
byTheOhioStateUniversitytousethedescribedfieldsites.Therewasapproximately3m
betweentreeswithineachblockand10mbetweenadjacentblocks.Floodstresswasinitiatedon
29April2013andmaintaineduntil14May2013.Similarly,non-floodedandfloodedspecimens
ofA.saccharinum,C.canadensis,C.florida,M.virginiana,P.serrulata,andQ.bicolorandwere
deployedinsixrandomizedcompleteblocksalongtheedgeofawoodlotinVirginia(37°
17’17.64”N76°39’0.07”W).PermissionwasprovidedbytheDepartmentofParksandRecrea-
tion,YorkCounty,Virginia.Floodstresswasinitiatedon8April2013andmaintaineduntil23
April2013inVirginia.Treeswereinspectedforambrosiabeetleattacksonedayafterinitiating
floodstressandtheneverytwotothreedaysuntiltheexperimentwasterminated.
TreesdeployedinOhiothatsustainedatleastoneambrosiabeetleattackwerecutatthe
rootsunderfieldconditionsonthelastdayoftheexperimentandsubsequentlytransferredtoa
walk-inrefrigeratorheldat5°C.Treeswerethendissectedtorecoverfoundressambrosiabee-
tlesfromthetunnelsandgalleries,andspecimenswerepreservedin95%ethanolforidentifica-
tion.AttackedtreesdeployedinVirginiawerealsocutatthebaseonthelastdayofthe
experimentandstemsectionswereheldinplasticcontainersatroomtemperatureuntilbeetles
wereexcavatedforidentification.Noprotectedspeciesweresampledduringthecourseofthis
study.
AssociationofEthanolwithTreesVaryingintheirToleranceofFlood
Stress
Ethanolwasquantifiedwithinthevasculartissueoftheflood-stressedandnon-floodedtrees
deployedinOhioandVirginiaaspartoftheexperimentevaluatingambrosiabeetlepreference
fortreesvaryingintheirtoleranceofflooding.BasedonRangeretal.[43],threedaysafteriniti-
atingfloodinganOsbornearchpunch(C.S.Osborne&Co.,Harrison,NewJersey)wasusedto
takefourtissuecoresamples(1mmdepth,5mmdiam.)at10cmabovethebasefromflooded
andnon-floodedspecimensofA.saccharinum,B.nigra,C.canadensis,C.florida,P.serrulata,
Q.bicolor,S.japonicus,andU.americanadeployedinOhio.Similarly,tissuesampleswerealso
takenthreedaysafterinitiatingfloodingfromflood-stressedandnon-floodedspecimensofA.
saccharinum,C.canadensis,C.florida,P.serrulata,Q.bicolor,andM.virginianadeployedin
Virginia.Thesuperficialtissuecoresamplesincludedtheouterbark,phloem,andvascular
cambium,butnotthesapwoodandheartwood.Tissuecoresampleswereplacedin2mL
Eppendorftubesimmediatelyaftersampling,whichwerethencoveredamongdryiceuntil
transportedbacktothelaboratory.Tissuesampleswerestoredat-80°Cuntilanalysis.Tissues
samplestakeninVirginiawerepackedwithdryiceandshippedovernighttotheUSDA-AR-
S-HIRLforanalysis.
Solidphasemicroextraction-gaschromatography-massspectrometry(SPME-GC-MS)was
usedtoanalyzeethanolcontent[9,43].Inshort,phloemandvascularcambiumtissuesfrom
thefourcoresampleswereplacedin2mLglassvialsandsuspendedinawaterbathat100°C
for30min.VialswereremovedfromthewaterbathandaSPMEfiberwasexposedtothehead-
spacewithinthevialfor5min.Thefiberwasretractedaftersamplingandthesyringewas
immediatelycappedwithasealedsectionofPTFEmicroboretubing(0.568mminnerdiam.×
1.07mmouterdiam.,Cole-Parmer,VernonHills,Illinois)topreventcontaminationofthe
fiber.Acoatingofcarboxen-polydimethylsiloxane(CAR/PDMS;75μmcoating;Sigma-
Aldrich,St.Louis,Missouri)wasused,whichisidealforgasesandlowmolecularweightcom-
pounds(MW30−225)[54].
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Immediatelyaftersampling,syringeswereinsertedintotheinjectionportofaGC(Varian
CP-3800;VarianInc.,PaloAlto,California)equippedwithaMerlinMicrosealseptumsystem
(Sigma-Aldrich)andthermallydesorbedfor2minat250°C.Theinjectionportwasoperated
insplitlessmodefrom0–2minandthensplitataratioof1:20fortheremainderoftherun.A
capillarynonpolarDB-5column(0.25μm×30m×0.25μm;cross-linked/surfacebonded5%
phenyl,95%methylpolysiloxane;AgilentJ&W,SantaClara,California)wasusedforanalysis
accordingtothefollowingprogram:40–60°Cat3°C/minand60–230°Cat20°C/min.AVarian
2200MSdetectorwasoperatedinelectronimpactmodewithascanrangeof14−415m/z.Sys-
temcontrolwasaccomplishedwithStarChromatographyWorkstationsoftware(VarianStar
Toolbar,version6.8).Fiberswereconditionedbeforeeachanalysisbyexposurewithinthe
injectionportfor20minat250oC.
Theexternalstandardmethod[9,43]wasusedfordeterminingconcentrationsofethanol
associatedwiththetissuecoresamples.Serialdilutionsofethanolrangingfrom100g/Lto
0.0001g/Lweremadeinwaterandaliquotscontainingknownamountsofethanolwere
appliedtofilterpaperdiscs(5mmdiam.)sealedinglass2mLautosamplervialsforsampling
purposes.PeakareasassociatedwiththeethanolstandardsweremeasuredusingtheStarChro-
matographyWorkstationsoftware.Astandardconcentrationcurvewasthendevelopedand
usedtodetermineconcentrationsofethanolassociatedwiththetissuesamples.
PreferenceforAttackingTreeParts
Todocumentspeciespreferencesforattackingcertaintreeparts,wecomparedthepropensity
ofambrosiabeetlestoattackabove-groundaerialpartsofflood-stressedandnon-floodedC.
florida.Treeswereflood-stressedaspreviouslydescribedandpermissionwasprovidedby
OSUtodeploytreesinawoodlotinOhio(40°45’41.97”N81°51’20.89”W).Floodedandnon-
floodedtreeswerearranged1mapartinpairswith5mbetweenadjacentpairs.Sixpairsof
replicatedtreesweredeployedandtreeswereheldunderfieldconditionsfor15daysfrom7
July2014to22July2014.Attackedtreeswerethenexcisedatthebaseandtransferredtoa
walk-inrefrigeratorheldat5°C.Tunnelsandgallerieswithintreeswerethendissectedto
recoverfoundressambrosiabeetles,whichwerequantifiedandidentifiedtospecies.Presence
ofthewhiteambrosialform(i.e.conidiaandsproutcells)ofthesymbioticfungus[55]and
ambrosiabeetleeggswithinthegallerieswerealsorecorded.
ColonizationSuccessonFlood-StressedandNon-FloodedTrees
No-choiceexperimentswereconductedinOhiousingX.germanusandinVirginiausingX.
crassiusculustocomparecolonizationsuccessonflood-stressedandnon-floodedC.florida.
BottletrapsdescribedbyRangeretal.[28]wereusedforobtainingfield-collectedadults,but
insteadofpropyleneglycol,amoistenedpapertowelrolledintoatubewasplacedinthebot-
tomcollectionvesselofthetraptomaintainambrosiabeetlespecimensduring24hrsunder
fieldconditions.Ambrosiabeetleswerethenreturnedtothelaboratoryandtransferredtopar-
afilm-sealedpetridishescontainingmoistenedfilterpaperandstoredfor24to48hrsat3.3°C.
Floodingofcontainergrowntreeswasestablishedusingthepreviouslydescribedpot-in-pot
systemon19May2014inagreenhouseonthecampusoftheOARDCandon12May2014on
thecampusoftheHRAREC.Threedaysafterinitiatingflooding,anindividualadultfemaleX.
germanusfield-collectedinOhioorX.crassiusculusfield-collectedinVirginiawereplaced
insideofachambermadeofpolytetrafluoroethylene(PTFE)tubingthatwascutlongitudinally
(2.5cm×1cm×0.9cm;l×w×h)andsealedatbothendswithMoldedThermogreenLB-2
Septa(Supelco,Bellefonte,PA)cutintoasemi-circle(Fig1).Cablestieswereusedtosnugly
securethechambersagainstthesteminparallel,therebyconfininganindividualbeetleto
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Fig1.ChamberdesignedforconfiningadultfemaleX.germanusandX.crassiusculustostemsof
flood-stressedandnon-floodedCornusflorida.
doi:10.1371/journal.pone.0131496.g001
2.5cm2ofbarktissue.SmoothbarkoftheC.floridatreesallowedforclosecontactbetweenthe
tissueandchambers,therebyeffectivelyconfiningthebeetlespecimens.Tenchamberspertree
wereplacedonsixflood-stressedandsixnon-floodedC.floridatreesstartingfromthebase
andextendinglinearlyabout60cmupthemainstemwithabout1.5−2cmbetweencages.Gen-
erally,onetotwounresponsiveorinjuredbeetlespertreewereremovedandreplacedduring
thefirstday.Chambersconfiningfoundressambrosiabeetleswereheldinplacefor25days,
afterwhichthestemswerecutatthebaseandtemporarilystoredat5°Cuntilfurtheranalysis.
Asanindicationoftunnelingactivity,chamberswerecarefullyremovedfromthestems1‒2d
laterandejectedsawdustwithineachcagewasweighed.Stemsectionsassociatedwitheach
chamberaspartoftheexperimentconductedinOhiowerealsodissectedtodetermineifthe
foundresswasstillaliveandassessthepresence/absenceofeggs,larvae,pupae,andfungal
growthwithineachtunnel/gallery.Floodedtreesweredrained15daysintotheexperimentto
avoidtreedeathandthenwateredaccordinglyfortheremaining10days.
StatisticalAnalyses
Cumulativeambrosiabeetleattacksontreesvaryingintheirtoleranceoffloodingwerefirst
examinedusingarepeatedmeasuresANOVAtotestforbetween-subjecteffects[56].Whena
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significantbetween-subjecttreatment×timeeffectwasdetected(P<0.05),thenumberof
attackspertreatmentatagiventimepointwereanalyzedbytwo-wayANOVAusingtreespe-
ciesandfloodlevelasmaineffectsandtreespecies×floodlevelasaninteraction[56].Differ-
encesamongtreatmentsweredeterminedusingleast-squaresmeans(α=0.05).Two-way
ANOVAandleast-squaresmeanswerealsousedtocompareethanolconcentrationsassociated
withtheflood-stressedandnon-floodedtreespecies(maineffects=treespeciesandflood
level;interaction=treespecies×floodlevel).Regressionanalysiswasusedtotestforacorrela-
tionbetweenday3ethanolconcentrationsandcumulativeattackspertreeassociatedwith
flood-stressedandnon-floodedtrees.Two-wayANOVAandleast-squaresmeanswereusedto
comparecumulativeattacksandambrosiabeetlespecimensexcavatedpertreefromstemsand
branchesofflood-stressedandnon-floodedtrees(maineffects=treepartandfloodlevel;
interaction=treepart×floodlevel).Anunpairedt-test(α=0.05)wasusedtocompareejected
sawdust,andthepercentageofgalleriespertreewithlivingfoundresses,eggs,larvae,pupae,
andfungalgrowthbetweenflood-stressedandnon-floodedtrees.Alldataweresquareroot
transformedpriortoanalysis,butuntransformeddataarepresented.
Results
PreferenceamongTreesVaryingintheirToleranceofFloodStress
Within3daysofimposingfloodstress,ambrosiabeetlesbeganrapidlyattackingtreespecies
intolerantofflooding,C.floridaandS.japonicus,whengivenfreechoiceamongflooded
andnon-floodedtreesofvaryingtolerance(Fig2A).Asignificantbetween-subject
treatment×timeeffectwasdetectedincumulativeambrosiabeetleattacksfortreesdeployed
inOhio(F=22.23;df=15,112;P<0.0001)(Fig2A).By5days,asignificantinteractioneffect
wasdetectedbetweentreespecies×floodinglevel(F=12.27;df=7;P<0.0001),alongwithsig-
nificantmaineffectsassociatedwithtreespecies(F=11.54;df=7;P<0.0001)andflooding
level(F=42.18;df=1;P<0.0001).Specifically,S.japonicusandC.floridasustainedsignifi-
cantlymoreattacks5daysafterinitiatingfloodingthanallotherfloodedandnon-floodedspe-
cies,whileC.canadensisreceivedmoreattacksthanalltheremainingtreatments.By15days,
C.floridaandS.japonicussustainedthehighestnumberofcumulativeattacksfollowedbyC.
canadensis(species×floodinginteraction:F=15.07;df=7;P<0.0001;treespecieseffect:
F=14.59;df=7;P<0.0001;floodingeffect:F=71.12;df=1;P<0.0001).Noattacksoccurred
onflood-stressedB.nigraandQ.bicolororanyofthenon-floodedtreespeciesthroughoutthe
durationoftheexperiment.
Non-nativeX.germanuswasthepredominantspeciesexcavatedfromgalleriescreatedinC.
floridaandS.japonicusdeployedinOhio,butspecimensofthenon-nativespeciesAmbrosiod-
musrubricollis(Eichhoff)andnativeMonarthrummali(Fitch)werealsorecovered(Table1).
Xylosandrusgermanus(n=102specimens)represented98.0%ofthetotalambrosiabeetles
recoveredfromC.florida,followedby1.0%forA.rubricollis(n=1)and1.0%forM.mali
(n=1).OnlyX.germanuswasrecoveredfromflood-stressedS.japonicus(n=112),C.cana-
densis(n=35),P.serrulata(n=8),A.saccharinum(n=2),andU.americana(n=1)
(Table1).
Asignificantbetween-subjecttreatment×timeeffectwasalsodetectedforflood-stressed
andnon-floodedtreesdeployedinVirginia(F=158.34;df=11,60;P<0.0001)(Fig2B).
Attacksbegantooccuronflood-stressedC.floridathreedaysafterinitiatingflooding.By7
days,asignificantinteractioneffectwasdetectedbetweentreespecies×floodinglevel
(F=43.8;df=5;P<0.0001),alongwithsignificanteffectsassociatedwiththemainfactorsof
treespecies(F=42.8;df=5;P<0.0001)andfloodinglevel(F=48.6;df=1;P<0.0001).
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Fig2.Cumulativemean(±SE)attacksbyambrosiabeetlesonflood-stressedtreeswithvaryingintoleranceoffloodingdeployedin(A)Ohioand
(B)Virginia.Datafornon-floodedtreesarenotpresentedinthefigure.Differentlettersindicatesignificantdifferencesamongtreatmentsatday15bytwo-
wayANOVAandleast-squaresmeans(Pvalueprovidedfortreespecies×floodinginteractioneffect;seeResultssectionforremainingoutput;n=8and
n=6treespertreatmentforOhioandVirginia,respectively).
doi:10.1371/journal.pone.0131496.g002
Cornusfloridasustainedmoreattacks7daysafterinitiatingfloodingthanallotherfloodedand
non-floodedspecies.
By15daysafterinitiatingflooding,morecumulativeattacksoccurredonflood-stressedC.
floridathanallotherfloodedandnon-floodedtrees(species×flooding:F=107.1;df=5;
P<0.0001;treespecieseffect:F=106.3;df=5;P<0.0001;floodingeffect:F=162.2;df=1;
P<0.0001).Considerablyfewerattacksoccurredonflood-stressedC.canadensisandP.serru-
lata,whichwerehigherthanalltheremainingnon-floodedandfloodedspeciestreatments
exceptflood-stressedM.virginiana.Nodifferenceinattacksweredetectedamongnon-flooded
A.saccharinum,C.canadensis,C.florida,M.virginiana,P.serrulata,andQ.bicolor,alongwith
flood-stressedA.saccharinum,M.virginiana,andQ.bicolor.Noattacksoccurredonflood-
stressedA.saccharinumandQ.bicolor,ornon-floodedC.florida,M.virginiana,andP.serru-
lata.However,onenon-floodedC.canadensissustainedoneattack.
Onlynon-nativeambrosiabeetlesattackedexperimentaltreesdeployedinVirginia.Xylo-
sandrusgermanuswasthepredominantspeciesexcavatedfromgalleriescreatedinC.florida
deployedinVirginia,butspecimensofnon-nativeX.crassiusculusandnon-nativeXylosandrus
compactus(Eichhoff)werealsorecovered(Table2).Xylosandrusgermanus(n=241speci-
mens)represented70.3%ofthetotalambrosiabeetlesrecoveredfromC.florida,followedby
28.6%forX.crassiusculus(n=98)and1.2%forX.compactus(n=4).
AssociationofEthanolwithTreesVaryingintheirToleranceofFlood
Stress
At3daysafterinitiatingflooding,differencesweredetectedinethanolconcentrationsassoci-
atedwithtreesvaryingintheirtoleranceofflooding.Asignificantinteractioneffectonethanol
concentrationswasdetectedbetweentreespeciesandfloodingfortreesdeployedinOhio
(F=10.29;df=7;P<0.0001)(Fig3A).Significantmaineffectswerealsoassociatedwithtree
species(F=9.85;df=7;P<0.0001)andfloodstress(F=59.41;df=1;P<0.0001).Higherlev-
elsofethanolweredetectedintissuescollectedfromflood-stressedC.florida,S.japonicus,and
Table1. Mean(±SE)cumulativeattacksandambrosiabeetlespecimensrecoveredperflood-stressedtreedeployedinOhio.
Scolytinaerecoveredpertree
Flood-stressedtreespeciesa CumulativeAttacks Xylosandrusgermanus Ambrosiodmusrubricollis Monarthrummali
Styraxjaponicus 19.6±5.9 14.0±5.9 0.0±0.0 0.0±0.0
Cornusflorida 18.9±6.3 12.8±4.6 0.1±0.1 0.1±0.1
Cerciscanadensis 6.6±1.6 4.4±1.5 0.0±0.0 0.0±0.0
Prunusserrulata 1.3±0.7 1.0±0.6 0.0±0.0 0.0±0.0
Acersaccharinum 0.6±0.4 0.3±0.2 0.0±0.0 0.0±0.0
Ulmusamericana 0.1±0.1 0.1±0.1 0.0±0.0 0.0±0.0
Betulanigra 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Quercusbicolor 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
aNoattacksoccurredonnon-floodedtrees(n=6treespertreatment).
doi:10.1371/journal.pone.0131496.t001
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Table2. Mean(±SE)cumulativeattacksandambrosiabeetlespecimensrecoveredperflood-stressedtreedeployedinVirginia.
Scolytinaerecoveredpertree
Flood-stressedtreespeciesa CumulativeAttacks Xylosandrusgermanus Xylosandruscrassiusculus Xylosandruscompactus
Cornusflorida 187.8±23.3 40.2±7.0 16.3±7.3 0.7±0.7
Cerciscanadensis 3.7±3.1 1.0±0.8 0.0±0.0 0.0±0.0
Prunusserrulata 2.8±1.9 0.0±0.0 0.0±0.0 0.0±0.0
Magnoliavirginiana 0.8±0.7 0.0±0.0 0.0±0.0 0.0±0.0
Acersaccharinum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
Quercusbicolor 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
aNoattacksoccurredonthenon-floodedtrees,exceptfor0.2±0.2attacksonnon-floodedC.canadensis(seeResultssection;n=6treespertreatment).
doi:10.1371/journal.pone.0131496.t002
C.canadensiscomparedtoalltheremainingtreatments(Fig3A).Ethanollevelsassociated
withA.saccharinumwerehigherthantheremainingtreatments,whilenodifferenceswere
detectedamongfloodedandnon-floodedQ.bicolor,B.nigra,U.americana,andP.serrulata,
andnon-floodedA.saccharinum,C.canadensis,C.florida,andS.japonicus.Asignificantposi-
tivecorrelation(r2=0.52)wasdetectedbetweenday3ethanolconcentrationsandcumulative
attacksontheflood-stressedandnon-floodedtrees(F=129.03;df=1,126;P<0.0001)(Fig
4A).
Asignificantinteractionwasalsodetectedbetweentreespeciesandfloodinglevelonetha-
nolconcentrationsassociatedwithtreesdeployedinVirginia(F=12.30;df=5;P<0.0001),
alongwithsignificantmaineffectsfortreespecies(F=9.94;df=5;P<0.0001)andflooding
level(F=32.92;df=1;P<0.0001)(Fig3B).Higherlevelsofethanolweredetectedinflood-
stressedC.floridatissuescomparedtoalltheremainingfloodedandnon-floodedtreatments
(Fig3B).Ethanollevelsassociatedwithflood-stressedP.serrulatawereconsiderablylower
thanC.florida,buthigherthanalltheremainingtreatmentsexceptforC.canadensis.Asignifi-
cantpositivecorrelation(r2=0.76)wasdetectedbetweenday3ethanolconcentrationsand
cumulativeattacksontheflood-stressedandnon-floodedtrees(F=222.61;df=1,70;P
<0.0001)(Fig4B).
PreferenceforAttackingTreeParts
Stemsofflood-stressedC.floridadeployedunderfieldconditionsinOhiowerepreferentially
attackedoverbranchesofflood-stressedtrees,andstemsandbranchesofnon-floodedtrees
(Fig5A–5D).Ameanof199.8and35.7attacksoccurredonstemsandbranchesofflood-
stressedtrees,while1.5and0.0attacksoccurredonstemsandbranchesofnon-floodedtrees,
respectively(Fig5A).Asignificantinteractionwasdetectedbetweentheinfluenceoftreepart
andincidenceoffloodstressoncumulativeattacks(F=25.18;df=1;P<0.0001),alongwith
significantmaineffectsassociatedwithtreepart(F=34.08;df=1;P<0.0001)andfloodstress
(F=145.70;df=1;P<0.0001)(Fig5A).Attackswerenotobservedtooccuronanytreeparts
otherthanstemsandbranches.
Stemsontwoofthesixnon-floodedC.floridatreessustainedoneandeightcumulative
attackspertree;noattacksoccurredontheremainingnon-floodedtrees.Thetwonon-flooded
treesattackedbyambrosiabeetleswerepairedwiththetwomostheavilyattackedflood-
stressedtreesthatsustained338and337attackspertree,respectively,comparedtotheremain-
ingflood-stressedtreeswith173,260,120,and185attackspertree.Onlysuperficialtunneling
occurredonthetwonon-floodedtreesandnoambrosiabeetleswereexcavatedfromthe
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