Feeding on Host Plants with Different Concentrations and Structures of Pyrrolizidine Alkaloids

27 Pages·2015·1.09 MB·English

RESEARCHARTICLE
Feeding on Host Plants with Different
Concentrations and Structures of Pyrrolizidine
Alkaloids Impacts the Chemical-Defense
Effectiveness of a Specialist Herbivore
CarlosH.Z.Martins1,2☯,BeatrizP.Cunha3‡,VeraN.Solferini3‡,JoséR.Trigo1☯*
1 LaboratóriodeEcologiaQuímica,DepartamentodeBiologiaAnimal,InstitutodeBiologia,UNICAMP,
CaixaPostal6109,13083–970,Campinas,SãoPaulo,Brazil,2 ProgramadePós-GraduaçãoemBiologia
FuncionaleMolecular,InstitutodeBiologia,UNICAMP,CaixaPostal6109,13084–970,Campinas,São
Paulo,Brazil,3 DepartamentodeGenéticaeEvolução,InstitutodeBiologia,Unicamp,CaixaPostal6109,
13083–970,Campinas,SãoPaulo,Brazil
☯Theseauthorscontributedequallytothiswork.
‡Theseauthorsalsocontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Citation:MartinsCHZ,CunhaBP,SolferiniVN,Trigo Sequestrationofchemicaldefensesfromhostplantsisastrategywidelyusedbyherbivo-
JR(2015)FeedingonHostPlantswithDifferent
rousinsectstoavoidpredation.LarvaeofthearctiinemothUtetheisaornatrixfeedingon
ConcentrationsandStructuresofPyrrolizidine
unripeseedsandleavesofmanyspeciesofCrotalaria(Leguminosae)sequesterN-oxides
AlkaloidsImpactstheChemical-Defense
EffectivenessofaSpecialistHerbivore.PLoSONE ofpyrrolizidinealkaloids(PAs)fromthesehostplants,andtransferthemtoadultsthrough
10(10):e0141480.doi:10.1371/journal.pone.0141480 thepupalstage.PAsconferprotectionagainstpredationonalllifestagesofU.ornatrix.
Editor:PeterSchausberger,UniversityofNatural AsU.ornatrixalsousesotherCrotalariaspeciesashostplants,weevaluatedwhetherthe
ResourcesandLifeSciences,AUSTRIA PAchemicaldefenseagainstpredationisindependentofhostplantuse.Wefedlarvae
Received:March26,2015 fromhatchingtopupationwitheitherleavesorseedsofoneofeightCrotalariaspecies
(C.incana,C.juncea,C.micans,C.ochroleuca,C.pallida,C.paulina,C.spectabilis,and
Accepted:October8,2015
C.vitellina),andtestedifadultswerepreyeduponorreleasedbytheorb-weavingspider
Published:October30,2015
Nephilaclavipes.Wefoundthattheprotectionagainstthespiderwasmoreeffectivein
Copyright:©2015Martinsetal.Thisisanopen
adultswhoselarvaefedonseeds,whichhadahigherPAconcentrationthanleaves.The
accessarticledistributedunderthetermsofthe
exceptionswereadultsfromlarvaefedonC.paulina,C.spectabilisandC.vitellinaleaves,
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany whichshowedhighPAconcentrations.WithrespecttothePAprofile,wedescribeforthe
medium,providedtheoriginalauthorandsourceare firsttimeinsect-PAsinU.ornatrix.ThesePAs,biosynthesizedfromthenecinebaseretro-
credited.
necineofplantorigin,ormonocrotaline-andsenecionine-typePAssequesteredfromhost
DataAvailabilityStatement:Allrelevantdataare plants,wereequallyactiveinmothchemicaldefense,inadose-dependentmanner.These
withinthepaper.
resultsarealsopartiallyexplainedbyhostplantphylogeny,sincePAsofthehostplantsdo
Funding:Financialsupportwasprovidedby haveaphylogeneticsignal(cladeswithhighandlowPAconcentrationsinleaves)whichis
FAPESPtoJRT(11/17708-0)andVNS(12/02526-7),
reflectedintheadultdefense.
andCNPqtoJRT(306103/2013-3).Thefundershad
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0141480 October30,2015 1/27
ChemicalDefenseinaSpecialistHerbivore
Introduction
Defensesevolvedbyanimalstoavoidpredationareubiquitousinnature,anddifferentdefen-
sivestrategieshaveevolvedinresponsetodifferentlifestyles.Themyriaddefensivestrategies
includeavoidingdetection,preventingattack,anddeceivingpredators[1].Someherbivorous
insectsfeedingonchemicallyprotectedhostplantsareabletoovercometheseplantdefenses
bysequesteringplantsecondarycompounds,andusingthemfortheirowndefenseagainst
predatorattack[2–4].Sequestrationmayhaveevolvedindependentlyindifferenttaxaofher-
bivorousinsects.ItpredominatesinColeopteraandLepidoptera,butalsooccursfrequentlyin
theHeteroptera,Hymenoptera,OrthopteraandSternorrhyncha[4].Thesequestereddefensive
compoundscompriseavastarrayofnaturalproductsofdifferentbiosyntheticpathways,such
ascardenolides,cyanogenicandiridoidglycosides,aristolochicacids,glucosinolatesandpyrro-
lizidinealkaloids.Thesesubstancesareeffectiveagainstavarietyofpredators,rangingfrom
invertebratesincludingspidersandants,tovertebratesincludingbirdsandmammals[2–5].
Generally,theeffectivenessofchemicaldefensesagainstpredationisdose-and/orstruc-
ture-dependent[3].Highconcentrationsofsequesteredcompoundsintheherbivorousprey
aremoreeffectiveagainstpredators,andtheireffectivenessisusuallyrelatedtotheconcentra-
tionofthesecompoundsinthehostplants.Differentstructuresofthesameclassofcom-
poundsmayalsoshowdifferentactivitiesagainstpredation.Forinstance,apalatability
spectrumofthemonarchandqueenbutterflies,DanausplexippusandD.gilippus,wasfound
tobeassociatedwiththeamountofcardenolidessequesteredfromdifferenthostplants[6,7].
ThispatternhasbeenalsoobservedintheadultsofthenymphalidbutterflyEuphydryaspha-
eton,whichacquireddefensiveiridoidglycosidesaslarvaefromtwodifferenthostplants[8,9];
andintheadultsoftheheliconiinebutterflyHeliconiuserato,whoselarvaefeedonfourspecies
ofPassiflora[10].Additionally,SilvaandTrigo[11]demonstratedthatpyrrolizidinealkaloids
haveapositivedose-dependentactivityintheprotectionofinsectsagainsttheorb-weavingspi-
derNephilaclavipes(Nephilidae).Inthesamestudy,theyshowedthatdifferentPAstructures
haddifferentantipredationactivities.
Amongthedefensivecompoundssequesteredbyherbivorousinsects,theroleofpyrrolizi-
dinealkaloids(PAs)hasbeenwelldocumented[12].Thesecompoundsareproducedbyplants
inseveralfamilies(e.g.Asteraceae,BoraginaceaeandLeguminosae),andaresequesteredby
specialistgrasshoppers,hemipterans,beetles,mothsandbutterflies[5,13,14],wheretheyshow
defensiveandsexualcommunicationfunctions[14].PAsinspecializedinsectsarealwayspres-
entinN-oxideform[15].ManyarctiinemothsconvertPAssequesteredfromtheirlarvalhost
plantsinto“insect-PAs”inwhichtheacidcomponentsofthealkaloidsarereplacedbysmall,
branchedaliphatic2-hydroxyacidsofinsectorigin[16].ThesePAsofinsectoriginareprecur-
sorsofthemalesexpheromoneinthesemoths[16].
ThearctiinerattleboxmothUtetheisaornatrixisoneofthemost-studiedspecieswith
respecttoitsecologicaldependenceonPAs[5,14,17–19].U.ornatrixoccursintheNeotropics
andwarmNearcticregions[20],whereitfeedsonmanyspeciesofCrotalarialegumes[21].
ThegenusCrotalariahasaPantropicaldistribution,andsomemembershavecolonizedthe
warmerpartsoftheNearcticregion[22].ThelarvaeofU.ornatrixsequesterthesealkaloids,
andpassthemtopupaeandadults.Duringmating,malestransferPAstofemales,whichtrans-
ferthemtotheeggs.Therefore,alllifestagesofU.ornatrixareprotectedbyPAsagainstpreda-
tors[14].Eisner[23]firstdemonstratedthattheunpalatabilityofU.ornatrixadultsisdueto
PAsintheirtissues,andthatthealkaloidoriginatesfromthelarvalhostplant,Crotalariapal-
lida(formerlyC.mucronata).EisnerfoundthatadultsofU.ornatrixwereprotectedagainst
theorb-weavingspiderNephilaclavipes,aswellasfromotherspiders,andbirds.Additional
evidenceofthedefensiveroleofPAsinU.ornatrixcamefrombioassays,withPAstopically
PLOSONE|DOI:10.1371/journal.pone.0141480 October30,2015 2/27
ChemicalDefenseinaSpecialistHerbivore
appliedonpalatableorganismsorbyofferingdietswithorwithoutPAstoPA-specialist
insects,andtestingtheseorganismsagainstvariouspredators[5].
AlthoughitissuggestedthattheunpalatabilityofU.ornatrixmaybecloselyrelatedtothe
amountofPAsintheirhost-planttissues,fewstudieshavedemonstratedthisrelationship.Eis-
neretal.[14,23–25]testedtheadults,withandwithoutPAs,againstspidersandbirds,butno
dose-activitybioassaywascarriedout.Ferroetal.[26]foundthatthedifferencesinadultpalat-
abilityincreasedwhentheirlarvaefedonunripeseedsandleavesofC.pallida.Theleaveshad
alowerPAcontentthanunripeseeds,andconsequentlythelarvaefedonleavesweremore
consumedbyN.clavipesthanthosefedonunripeseeds.Likewise,HristovandConner[27]
alsoshowedthatU.ornatrixfedonleavesofC.spectabilisweremorepredatedbythebatEpte-
sicusfuscuscomparedtomothsfedonseeds;mothsraisedinadietfreeofPAswerepalatable
tothebats.
AnimportantissuethatremainsunclearistheroleofPAstructureinthechemicaldefense
ofU.ornatrix.Dodifferentstructuresconferdifferentlevelsofdefense?Eisneretal.[23–25]
raisedU.ornatrixonitsusaraminePA-containinghostplant(C.pallida)andobservedthatthe
mothwasrejectedbythespidersN.clavipesandLycosaceratiola,whilemothsraisedonadiet
freeofPAswerepreyedupon.SimilarresultswerefoundforL.ceratiola,whichreleasedU.
ornatrixraisedonadietsuppliedwithPAmonocrotaline,themainPAofC.spectabilis[24,25].
IntwootherstudiesonchemicaldefenseofU.ornatrixadults,larvaewerealsoraisedonC.pal-
lidaorC.spectabilis,andthereforethePAsinvolvedinthedefensewererespectively,usara-
mineandmonocrotaline[26,27].However,larvaeofU.ornatrixcanfeedonmanyCrotalaria
species[21,28],withdifferentPAconcentrationsandprofiles[29].ThefindingthatC.pallida
andC.spectabilisconferredsimilarlevelsofdefenseinspiteofcontainingdifferentPAsledus
tohypothesizethatthestructureisunimportantindeterminingpalatability.Ourpredictions
were:(1)adultsfromlarvaethatfedonplantsorplantpartswithhighPAcontentswouldbe
betterdefendedthanthosethatfedonplantsorplantpartswithlowPAcontents,and(2)PA
structurewouldplaynoroleinthemothchemicaldefenses.
Anotherpointthathasnotbeenexaminedinstudiesofchemicaldefensesofherbivores
sequesteredfromtheirhostplantsistheroleofhostplantphylogeny.Itwouldbeexpectedthat
phylogeneticallyrelatedhostplantsproducesimilarcompoundsavailableforsequestrationby
insects.Thereforewecannotconsidereachhostplantspeciesasindependentforcomparative
analysis(e.g.[30]).Ifphylogeneticinertiaisstrong,thepotentialadaptationsthatrelatedspe-
ciesmayevolvewillbesimilarlyconstrained,withtheeffectthatspeciescannotberegardedas
independentofeachother[31].Consequently,itwasnecessarytotakeintoaccountthehost
plantphylogeny,inordertocomparethesequesteredchemicaldefensesinanherbivorous
insect.Toourknowledge,thisapproachhasneverbeentakeninpublishedstudies.Forexam-
ple,evenforthemonarchbutterfly,awell-studiedmodelofsequesteredchemicalcompounds,
theconnectionbetweeninsectdefensesandhostplantphylogeneticrelationshipshasnever
beenexplored,althoughthephylogenetictrendsrelatedtochemicaldefensesarewellknownin
Asclepias[32,33].Therefore,weexploredthequestionofwhetherCrotalariaphylogenydirects
anytrendforthechemicaldefensesofU.ornatrix.Wehypothesizedthatmothsthatfedon
phylogeneticallyrelatedhostplantswouldshowsimilardefensivepatterns.
Toaddressourhypothesisandpredictions,wefedlarvaeonleavesorseedsofeightdifferent
CrotalariaspeciesfoundintheNeotropics,whichwerenative,non-nativeorofuncertainori-
gin,withdifferentPAconcentrationsandprofiles[29].Theadultsthatemergedfromlarvae
fedthedifferentdietswereofferedtothespiderN.clavipesinapredationbioassay.Weana-
lyzedthePAconcentrationsandprofilesforU.ornatrixadultsandCrotalariaspecies,andcor-
relatedthePAconcentrationsofadultswiththePAcontentsoftheplantpartstheyfedonas
larvae.WealsocorrelatedthePAconcentrationandprofilebetweenthemothsandthespider
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ChemicalDefenseinaSpecialistHerbivore
responseinthepredatorbioassay.Thiscorrelationwillorwillnotsupportourfirstprediction.
SincewefounddifferentclassesofPAsinU.ornatrix,wealsobioassayedthethreemostcom-
monclassesagainstthespidertotestoursecondprediction.Finally,wemappedthePAprofiles
andconcentrationsinCrotalariaspecies,thePAconcentrationsinthemoth,andthedefensive
responseofN.clavipesagainstanindependentphylogenetichypothesisfortheeightCrotalaria
species,inordertotestourlasthypothesis.
MaterialsandMethods
StudySystem
TherattleboxmothUtetheisaornatrix(Erebidae:Arctiinae)isprimarilyNeotropicaland
extendstowarmerareasoftheNearcticregion[20].U.ornatrix,togetherwithfivespeciesthat
occuronlyintheGalapagosIslands,aretheextantUtetheisaspeciesintheNeotropics[34,35].
TheadultsaregenerallyfoundflyingnearpatchesofCrotalariainpastures,theedgeofwoods
androadsides,wherethelarvaecanbefoundfeedingonbothleavesorseedsinsidetheunripe
podsofCrotalaria[21,26,28].
Crotalaria(Leguminosae:Papilionoideae:Crotalarieae)isaPantropicalgenusofweeds,
comprisingapproximately702species[22,36].IntheNeotropics,particularlyinBrazil,31
nativeand11non-nativespecieshavebeenrecorded[37].CrotalariaspeciesarerichinPAs
[34],whicharefoundinhigherconcentrationsinseedsthaninleaves[26].InadditiontoPAs,
Crotalariaspecieshaveotherdefensivetraitsagainstherbivoresincludingantifeedingdeter-
rentssuchaslectins[38],non-proteinaminoacids[39]andproteaseinhibitors[40].Theyalso
haveextrafloralnectaries(EFNs)thatattractpredatoryantsandwasps[26,41–43].Weused
eightspeciesofCrotalaria,includingthreenatives,threenon-natives,andtwowithuncertain
origins(Table1),thathavebeenplantedinanopenareaneartheAnimalBiologyDepartment,
InstituteofBiologyattheStateUniversityofCampinas,Campinas,SãoPaulo,Brazil(22°
49'15.38"S,047°04'8.87"W).Innaturalenvironments,wehaveobservedU.ornatrixusingC.
incana,C.micans,C.spectabilis,C.pallidaandC.vitellineashostplants.Fortheotherthree
species,wehavenoinformationabouttheirnaturalusebyU.ornatrix.
Theneotropicalorb-weavingspiderNephilaclavipes(Nephilidae)isapredatorthatbuilds
itswebinforestclearingsandcorridors,whichareflightpathsforinsects[44].Thisspider
preysongrasshoppers,bees,wasps,moths,andbutterflies,butisabletodiscriminatePA-
containinginsects,releasingthemunharmed[5](Fig1).Intheedgesofwoods,thisspiderco-
occurswithU.ornatrix.
ThepermitforresearchwithwildanimalswasprovidedbyIBAMA-ICMBio(Ministériodo
MeioAmbiente,Brazil).
RearingUtetheisaornatrixLarvae
WeobtainedlarvaeofU.ornatrixfromadultscollectedontheFazendaSantaMariana,Campi-
nas,SãoPaulo,Brazil(22°47’02.91”S,47°00’36.03”W),wherethehostplantC.pallidaisabun-
dant.Webroughttheadultstothelaboratory,sexedthemfollowingTravassos[45],and
allowedthemtomate(20malesand10femalespercage)inapapercage(ca.3.2L)following
Cogni[28].Wesuppliedfoodinavialcontaining5.0%aqueoushoney.After3–4days,the
adultsstartedtoovipositonthepaper-cagesurfaces.Theeggsweremixedtorandomizethe
parentalorigin.Aftereclosion,werearedthefirstinstarsonleavesorunripeseedsofoneofthe
eightspeciesofCrotalaria.WealsorearedagroupoffirstinstarsonaPA-freediet,following
Cognietal.[46].Foreachtreatment(dietsofleavesorunripeseedsofeachspeciesofCrota-
laria),werearedca.20individualsinaplasticcontainer(6cmhigh,20cmdiameter)until
pupation.Forsometreatments,dependingontheavailabilityofseedsandleaves,wehad2or
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ChemicalDefenseinaSpecialistHerbivore
Table1. SpeciesofCrotalariausedtofeedlarvaeofUtetheisaornatrixforNephilaclavipesbioassayandPAanalysis.
Species Section Nativerange
C.incanaL. Chrysocalycinae Pantropical(uncertainorigin)2
C.junceaL. Calycinae India,Asia2
C.micansLink Chrysocalycinae Neotropics1,2
C.ochroleucaG.Don Hedriocarpae TropicalAfrica2
C.pallidaAiton Hedriocarpae Pantropical(uncertainorigin)2
C.paulinaSchrank Calycinae Neotropics1,2
C.spectabilisRoth Crotalaria Asia2
C.vitellinaKerGawler Chrysocalycinae Neotropics1
1Flores[38]
2Polhill[22]
doi:10.1371/journal.pone.0141480.t001
3containers,giving40–60individualspertreatment.Thelarvaewerefedadlibitum.We
inspectedthecontainersdaily,removingfecesanddeadindividuals,andreplacingtheleavesor
seedswithnewones.Wemovedthepupaetoanothercontainerwiththesamedimensions
untiladultemergence,sincethelarvaecancannibalizepupae[47].Wecarriedoutthesame
procedureforpupaefromthePA-freediet.Wemovednewlyemergedadultstopapercages,
separatingthembytreatmentandsex,untilthespider-predationbioassays.Wekeptboththe
papercageswithadultsandtheplasticcontainerswithlarvaeandhostplants,orpupae,at
roomtemperature.
PredationBioassaywithLivingMoths
WecarriedoutthebioassayswithNephilaclavipesinasmallpatchofwoodsinCampinas(22°
48'21.26"S,47°4'43.12"W)fromMarchtoMayin2012and2013,whenthespiderswereabun-
dant.Weconducedallbioassaysbetween09:00and16:00hs.Weusedonlyadultfemalespi-
dersthatrespondedimmediatelywhenanypreywastossedintotheirweb.Wedidnotuse
spidersthatwereinthecourseoffeedingonapreyinsect,butwedidnotcontrolforsatiation
beforeeachbioassay.Weusedaround80spidersforthebioassays.Sincethenumberofspiders
wasalimitingfactor,sometimeswerepeatedthebioassayswiththesamespider,buteachindi-
vidualwasusedonlyonceperweek.Webioassayed481adultmoths,testingaround20individ-
ualsforeachsex,hostplantandplantpart.Wemadeasmallcutinthemoth'shindwings
beforeplacingitontheweb,topreventthemothfromflyingawayifreleasedbythespider.We
placedthemothontheweb,andrecordedifitwaseatenorreleased.Weconsideredthespider
responsetobepredationwhenitbit,wrappedandkilledthemoth.Whenthespidercutthe
webaroundthemothaftertouchingitandfreeditunharmed,werecordedtheresponseasa
release.WedidnotrecordtherejectionbehaviordescribedbyVasconcellos-NetoandLewin-
sohn[48](thepreywasinitiallysuckedandthenfreed),sincethepreywasremovedfromthe
webbeforethisstage.Ifthespiderreleasedthemoth,weofferedapalatablefreeze-killedmeal-
wormTenebriomolitor(Coleoptera:Tenebrionidae)asacontrol.Werecordedthetrialasa
rejectiononlyifthespiderfedonthemealworm.Ifthespiderkilledtheexperimentalmoth,we
didnotusethecontrol.Whenpredationwasrecorded,weimmediatelyremovedthemoth
fromthewebandplaceditina1.5-mLEppendorftubefilledwithMeOHforfurtherPAanaly-
sis.Allmothswererecoveredintact;eventhepartsbittenbythespiderhadnovisiblesignsof
damage.WealsopreservedthereleasedmothsinMeOHasabove.Weanalyzed310individu-
als;171werenotusedforPAanalysis,sincetheywerelostafterthespiderbioassay.
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ChemicalDefenseinaSpecialistHerbivore
Fig1.FemaleofNephilaclavipeshandlinganadultmaleofUtetheisaornatrix.Notethatthespidertouchesthemothwithherpedipalps,probably
evaluatingthecontentofdefensivepyrrolizidinealkaloids.
doi:10.1371/journal.pone.0141480.g001
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PyrrolizidineAlkaloids:Extraction,Quantification,Characterizationand
Isolation
ToquantifythePAs,weextractedthefreeze-driedsamplesofunripeseeds,leavesofhostplants
oradultmothsthreetimeswithEtOH(10xvolume:weight).WecentrifugedtheEtOHextract
at10,416rcffor10min,andrecoveredtheEtOHlayer.WecompletedtheEtOHlayerto20
mLandtookanaliquot(0.1or1.0mL)tocarryoutthecolorimetricanalysisaccordingto
Trigoetal.[49,50].Weusedmonocrotalineforthereferencecurve.ThetotalPAconcentration
wasgiveninμgofPAs/mgofdryweight.
ForPAcharacterizationbygaschromatography-massspectrometry(GC-MS),weextracted
theplantorinsectsamplesusingtheacid-baseproceduresdescribedbyTrigoetal.[49,50].
TheGC-MSanalysiswascarriedoutinelectronimpactmodeaccordingtoFloresetal.[29].
Theretentionindicesandmassfragmentationpatternswerecomparedwithpublisheddescrip-
tions(seeTable2,andreferences[29,51,52]).
ForPAcharacterizationbyliquidchromatography-massspectrometrythesampleswere
extractedinEtOHasabove.TheEtOHlayerwasevaporatedinarotaevaporatorat40°C,recov-
eredwith1.5mL2%aqueousaceticacidandcleaned3timeswiththesamevolumeofhexane.
Theacidsolutionwasaddedtoaclean2-mLvial,capped,andstoredat-20°CuntilLC-MS
analysis.WeusedanAgilent1260InfinityQuaternaryLC,equippedwithanEclipsePlusC-18
column,4.6x250mm,5μm,andaguardcolumnwiththesamephase,coupledwithanAgi-
lent6130singlequadrupoleinelectrosprayionizationmode.Thecolumnwaskeptat40°C,
andtheinjectionvolumewas5to50μl.ThePAwasseparatedusingalineargradientcontain-
ingaqueous20mMammoniumacetateandMeOHatflowrate0.5mL/min.Thegradient
startedat95%ammoniumacetate:5%MeOH(3min),MeOHwasraisedto100%in25min,
andkeptat100%for3min.Themassspectrometerwasruninpositivemode,scanningthe
productionsfrom100–500amu(seeTable2).
ForthepredationbioassayswithpurePAsinN-oxideform,weisolatedmonocrotaline
fromC.spectabilis,integerriminefromSeneciobrasiliensis(Asteraceae:Senecioneae)andthe
putativemixtureinsectPAscreatonotineB:iso-creatonotineB(Fig2)fromadultsofU.orna-
trixthatfedaslarvaeonleavesandunripeseedsofC.vitellina.WeextractedPAsusingthe
acid-baseprocedureasdescribedabove,andisolatedthemusinganadsorptioncolumnchro-
matograph(40cmlength,2.5cmdiameter),usingSilicaGel70–325meshasthestationary
phase,andagradientfromCHCl toCHCl :MeOH:Et N85:14:1asthemobilephase.Wefol-
3 3 3
lowedthePAsin10-mLfractionsbysilica-gelthin-layerchromatographywithCHCl :MeOH:
3
NH OH85:14:1aseluentandDragendorff'sreagentfordetection.WeN-oxidizedthePAsand
4
purifiedthemusingtheproceduredescribedbyCraigandPuroshothaman[53].
PredationBioassaywithPurePyrrolizidineAlkaloids
WefoundthatadultsofU.ornatrixgenerallycontainedPAN-oxidesofmonocrotalineand
integerriminetypes,andthespecificmixtureofinsectPAscreatonotineB:iso-creatonotineB
(seeResults).WecarriedoutpredationbioassayswithN.clavipestodetermineifPAN-oxides
withdifferentstructureshadsimilaractivityagainstthespider.Thebioassayswerecarriedout
inthesameareawherewecarriedoutthepredationbioassaywithlivingmoths.
Wetreatedpalatablefreeze-killedlarvaeofthemealwormT.molitor(hereafterbaits)with
3.0μg/mgdryweightofeachalkaloid,whichwereobtainedasdescribedabove.Thisconcen-
trationwasdeterminedbycalculatingthelogisticregressionoftheN.clavipesresponseinrela-
tiontoPAconcentrationinadultsofU.ornatrixfedaslarvaeononeoftheeightdifferenthost
plants(seeStatisticalanalyses),whereat3.0μg/mgtheprobabilityofreleasebythespiderwas
87%.ToevaluatewhetherthePAconcentrationalsoplayedanyroleinthechemicalprotection
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ChemicalDefenseinaSpecialistHerbivore
Table2. MassfragmentationpatternofpyrrolizidinealkaloidsfoundinCrotalariaspeciesandinadultsofUtetheisaornatrixfedonthesehost
plants. Theanalyseswerecarriedoutbygaschromatography-massspectrometry(GC-MS)inelectronimpactmodeandliquidchromatography-massspec-
trometry(LC-MS)inelectrosprayionizationmode.
Pyrrolizidinealkaloids RIa Rtb DiagnosticionsforGC-MSc,m/z(%) Diagnosticionsfor Referencefor
LC-MSd,m/z(%) GC-MSe
Retronecine 1487 6.085 [M+]155(23),111(60),94(17),80(100) [2M+H]+343(9),[M [29]1
+H]+172(100)
9-(2’-Hydroxy)-ethanoylretronecine- 1795 nd [M+]227(2),183(6),138(62),120(6),93 nd [29]1
like (100),80(40)
9-(2-Hydroxy-3-methylpentanoyl)- 1831 nd [M]+255(2),142(6),125(10),124(100),96 nd [52]1
trachelanthamidine (4),83(20),82(12)
CreatonotineB-like 1840 nd [M]+255(3),211(7),138(99),124(13),120 nd [51]2
(11),94(40),93(100),80(25)
7-Senecioylretronecine-like 1861 nd [M]+237(5),137(29),136(18),94(25),93 nd [29]1
(10),80(100)
9-Senecioylretronecine-like 1890 nd [M]+237(3),193(9),154(15),138(27),137 nd [29]1
(28),136(20),94(24),93(100),80(20)
Iso-creatonotineB 2024 22.075 [M]+269(3),251(10),138(40),124(17), [M+Na]+308(11),[M [51]1
120(26),111(61),106(51),94(26),80 +H]+286(100)
(100)
7-Octanoylretronecine-like 2031 nd [M]+281(50),220(19),154(8),136(22), nd [29]1
124(22),111(69),106(47),94(24),80
(100)
CreatonotineB 2048 22.969 [M]+269(<1),251(1),225(5),138(100),93 [M+Na+]308(6),[M [51]1
(88),80(18) +H]+286(100)
9-Octanoylretronecine-like 2052 nd [M]+281(5),236(6),138(62),120(26),106 nd [29]2
(13),94(47),93(100),80(44)
1,2-DihydrocreatonotineB 2064 nd [M]+271(4),210(12),171(20),140(25), nd [52]1
139(11),114(10),96(34),95(70),82(100)
9-(5’-Hydroxy)- 2082 24.162 [M]+283(1),224(4),155(24),138(65),93 [M+Na]+322(14),[M [29]1
heptanoylretronecine-like (100),80(19) +H]+300(100)
Crispatine-like 2175 22.598 [M]+309(2),222(7),136(88),120(83),119 [M+Na]+348(11),[M [29]3
(100),93(62),80(29) +H]+326(100)
Unknownmonocrotaline-type 2243 24.936 [M]+323(3),236(4),208(10),136(97),120 [M+Na]+362(9),[M [29]3
(87),119(100),93(54),80(27) +H]+340(100)
Incanine-like 2315 27.280 [M]+337(6),264(11),250(5),222(8),136 [M+Na+]376(15),[M [29]1
(100),120(76),119(79),93(49),80(27) +H+]354(100)
Monocrotaline 2336 18.610 [M+]325(1),254(3),236(46),136(52),120 [M+Na]+364(6),[M [29]1
(100),93(38),80(19) +H]+342(100)
Senecionine/Integerrimine 2339/ 26.789/ [M]+335(6),291(12),248(12),220(21), [M+Na)+]374(13),[M [29]1
2410 25.111 136(97),120(100),93(83),80(34) +H]+352(100)
Trichodesmine-like 2341/ 25.326 [M]+353(6),264(100),136(31),120(35), [M+Na]+392(17),[M [29]1
2348 93(44),80(14) +H]+370(100)
Unknownmonocrotaline-type 2346 26.050 [M]+337(2),222(5),136(58),120(89),119 [M+Na]+376(20),[M [29]3
(100),93(40),80(18) +H]+354(100)
Unknownmonocrotaline-type 2346 nd [M]+353(2),264(45),136(75),120(100), nd [29]2
93(70),80(25)
Senecionine-like 2376 nd [M]+337(6),222(18),136(75),120(100), nd [29]2
93(67),80(32),55(47)
Methylmonocrotaline-like 2384 nd [M]+339(1),250(58),136(53),120(100), nd [29]1
93(39),80(20)
Senecionine-like 2386 nd [M]+321(13),247(7),136(98),120(100), nd [29]1
93(76),80(37)
Trichodesmine-like 2418 23.415 [M]+353(2),264(60),136(47),120(100), [M+Na]+392(13),370 [29]1
93(35),80(18) [M+H]+370(100)
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0141480 October30,2015 8/27
ChemicalDefenseinaSpecialistHerbivore
Table2. (Continued)
Pyrrolizidinealkaloids RIa Rtb DiagnosticionsforGC-MSc,m/z(%) Diagnosticionsfor Referencefor
LC-MSd,m/z(%) GC-MSe
Incanine-like 2430 nd [M]+337(5),264(27),222(20),136(62), nd [29]1
120(100),93(65),80(27)
Trichodesmine-like 2437 nd [M]+353(2),264(83),222(8),136(58),120 nd [29]1
(100),93(34),80(18)
Senecionine-like 2540 nd [M]+337(1),155(12),138(71),136(28),93 nd [29]2
(100),80(19),55(27)
Senecionine-like 2551 nd [M]+351(31),220(10),136(43),120(100), nd [29]2
119(90),93(75),80(33)
PlatyphorineC-like 2556 25.260 [M]+383(2),281(13),267(11),252(100), [M+Na]+422(19),[M [52]1
138(24),136(27),120(51),93(51),80(18) +H]+400(100)
Unknownseco-PA 2615 nd [M]+365(20),321(15),276(22),238(100), nd [29]1
168(70),122(38),110(32),94(31),83(46)
Retrorsine/Usaramine 2621/ 22.989/ [M]+351(7),246(5),136(100),120(99), [M+Na]+390(7),[M [29]1
2647 22.731 119(84),93(80),80(35) +H]+368(100)
Senecionine-like 2675 nd [M]+351(3),224(8),143(100),136(65), nd [29]2
120(86),119(67),93(51),80(20)
Senecionine-like 2684 nd [M]+351(2),143(24),136(51),120(100), nd [29]2
119(91),93(79),80(23)
Unknownseco-PA 2728 nd [M]+381(25),338(55),320(63),250(58), nd [29]1
238(77),168(100),150(32),122(52),110
(44)
Unknownseco-PA 2815 nd [M]+379(43),334(26),238(22),168(74), nd [29]1
151(29),139(57),122(100),110(62),94
(57)
Unknownseco-PA 2866 nd [M]+421(31),376(22),168(77),150(79), nd [29]1
139(67),122(100),110(64),94(45),43
(96)
Unknownseco-PA 2907 nd [M]+437(34),250(79),226(41),197(78), nd [29]1
183(78),168(44),122(100),110(52),94
(52)
a.RetentionindexinGC-MSanalyses.
b.Retentiontime(min)inLC-MSanalyses.SomecompoundsdetectedbyGC-MSwerenotdetected(nd)byLC-MS.
c.infreebaseform.
d.inN-oxideform.
e.CharacterizationofPAsbymassspectrausing:1Massspectramatchingwithliterature,2.Interpretationofthemassspectrumfromliterature,
3Erroneouslydescribedin29asunknownsenecionine-type.
doi:10.1371/journal.pone.0141480.t002
againstthespider,wealsobioassayedthesePAN-oxidesinaone-tenthconcentration.Addi-
tionally,weconductedbioassayswith5.0μg/mgN-oxidefortheinsectPA.Allbioassayswere
conductedusing10baitsforeachPAconcentration.ThePAsweredilutedinMeOHand
appliedtopicallywitha10-μLsyringeonthesurfaceofthebait,whichwaskilledbyfreezing.
WeusedaMeOH-treatedbaitasacontroltodeterminewhethertoacceptthetrial,as
describedabovefortheN.clavipespredationbioassaywithlivingmoths.
HostPlantPhylogeneticHypothesisandCharacterMapping
WemappedthecharactersofPAsinvolvedinthechemicaldefensesofU.ornatrixinaninde-
pendentphylogenetichypothesisfortheeightCrotalariaspecies.Thisproceduremayreveal
theevolutionarytrendsofmappedcharacters[54].Wedidthisonlyforthosethatwerefedon
leaves,asallindividualsfedonseedswerewellprotectedagainstthespider,regardlessofthe
PLOSONE|DOI:10.1371/journal.pone.0141480 October30,2015 9/27
ChemicalDefenseinaSpecialistHerbivore
Fig2.Somepyrrolizidinealkaloidsfoundinthisstudy.ThealkaloidsaredrawnintheirN-oxideform.Formorestructures,see,e.g.,Floresetal.[29].
doi:10.1371/journal.pone.0141480.g002
hostplant.AsPAcharactersweusedPAconcentration(high>3.0μg/mgandlow<1.0μg/
mg)andPAtype(monocrotaline,senecionine,senkerkineorinsectPA)inalleightCrotalaria
speciesandU.ornatrix,andtheresponseofthepredatorN.clavipesinrelationtoU.ornatrix
(percentageofrelease).
WeconstructedaphylogenetichypothesisbasedonITSsequencesretrievedfromGenBank
forsevenCrotalariaspeciesandfortheout-groupBolusiaamboensis[36].TotalgenomicDNA
ofC.vitellina(unavailableinGenBank)wasextractedfromfreshleaves,collectedinthegarden
ofUNICAMP,usingthetwo-foldhexadecyltrimethylammoniumbromide(CTAB)method
[55].ThenuclearribosomalITSregion(ca.700bp)wasamplifiedasdescribedbyleRouxetal.
[36],usingtheprimersITS17SEandITS26SE[56,57].PCRproductswerepurifiedwitha
GFXPCRDNAandGelBandPurificationKit(GEHealthcare)andsequencedattheCenterof
PLOSONE|DOI:10.1371/journal.pone.0141480 October30,2015 10/27