Host plant forensics and olfactory-based detection in Afro-tropical mosquito disease vectors

21 Pages·2017·3.44 MB·English

RESEARCHARTICLE
Host plant forensics and olfactory-based
detection in Afro-tropical mosquito disease
vectors
VincentO.Nyasembe1,2,DavidP.Tchouassi1,ChristianW.W.Pirk2,CatherineL.Sole2,
BaldwynTorto1,2*
1 InternationalCentreofInsectPhysiologyandEcology,Nairobi,Kenya,2 DepartmentofZoologyand
Entomology,UniversityofPretoria,Hatfield,SouthAfrica
*[email protected]
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a1111111111 Abstract
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Theglobalspreadofvector-bornediseasesremainsaworryingpublichealththreat,raising
theneedfordevelopmentofnewcombatstrategiesforvectorcontrol.Knowledgeofvector
ecologycanbeexploitedinthisregard,includingplantfeeding;acriticalresourcethatmos-
quitoesofbothsexesrelyonforsurvivalandothermetabolicprocesses.However,theiden-
OPENACCESS tityofplantspeciesmosquitoesfeedoninnatureremainslargelyunknown.Bytestingthe
Citation:NyasembeVO,TchouassiDP,PirkCWW, hypothesisaboutselectivityinplantfeeding,weemployedaDNA-basedapproachtargeting
SoleCL,TortoB(2018)Hostplantforensicsand trnH-psbAandmatKgenesandidentifiedhostplantsoffield-collectedAfro-tropicalmos-
olfactory-baseddetectioninAfro-tropicalmosquito
quitovectorsofdengue,RiftValleyfeverandmalariabeingamongthemostimportantmos-
diseasevectors.PLoSNeglTropDis12(2):
quito-bornediseasesinEastAfrica.TheseincludedthreeplantspeciesforAedesaegypti
e0006185.https://doi.org/10.1371/journal.
pntd.0006185 (dengue),twoforbothAedesmcintoshiandAedesochraceus(RiftValleyfever)andfivefor
Anophelesgambiae(malaria).Sinceplantfeedingismediatedbyolfactorycues,wefurther
Editor:BrianL.Weiss,YaleSchoolofPublic
Health,UNITEDSTATES soughttoidentifyspecificodorsignaturesthatmaymodulatehostplantlocation.Usingcou-
pledgaschromatography(GC)-electroantennographicdetection,GC/massspectrometry
Received:October2,2017
andelectroantennogramanalyses,weidentifiedatotalof21antennally-activecomponents
Accepted:December20,2017
variablydetectedbyAe.aegypti,Ae.mcintoshiandAn.gambiaefromtheirrespectivehost
Published:February20,2018
plants.WhereasAe.aegyptipredominantlydetectedbenzenoids,Ae.mcintoshidetected
Copyright:©2018Nyasembeetal.Thisisanopen mainlyaldehydeswhileAn.gambiaedetectedsesquiterpenesandalkenes.Interestingly,
accessarticledistributedunderthetermsofthe
themonoterpenesβ-myrceneand(E)-β-ocimenewereconsistentlydetectedbyallthemos-
CreativeCommonsAttributionLicense,which
quitospeciesandpresentinalltheidentifiedhostplants,suggestingthattheymayserveas
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal signaturecuesinplantlocation.Thisstudyhighlightstheutilityofmolecularapproachesin
authorandsourcearecredited. identifyingspecificvector-plantassociations,whichcanbeexploitedinmaximizingcontrol
DataAvailabilityStatement:Thehaplotypes strategiessuchassuchasattractivetoxicsugarbaitandodor-baittechnology.
generatedfromthisstudyhavebeendepositedin
GenBankunderaccessionnumbersMG573108,
MG573126-MG573131(RVFvectorshostplant
trnH-psbAgenesequences),MG573132-
MG573139(denguevectorhostplanttrnH-psbA Authorsummary
genesequences),MG573109-MG573125
Plantsplayanimportantroleinthefitnessofmosquitodiseasevectors,yettheidentityof
(malariavectorhostplanttrnH-psbAgene
plantspeciesthattheyfeedonintheirnaturalhabitatsremainslargelyunknown.Inthis
sequences),andKY308115-KY398121(malaria
vectorhostplantmatKgenesequences).
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
Funding:ThisstudywasfundedthroughSwedish
InternationalDevelopmentCooperationAgency study,weemployedDNAbarcodingtoidentifytheplantspeciesfeduponbyAedes
(Sida)studentshiptoVON.Partialfundingwasalso aegypti,Aedesmcintoshi,AedesochraceusandAnophelesgambiaeintheirnaturalhabitats.
receivedfromtheUK’sDepartmentfor
Sinceplantfeedingismediatedbyolfactorycues,withpotentialapplicationasattractant-
InternationalDevelopment(DFID),theSwiss
basedtoolsforvectorsurveillance,weidentifiedspecificodorsignaturesthatmaymodu-
AgencyforDevelopmentandCooperation(SDC),
latehostplantlocation.Ourfindingsshowedpreferenceinodordetectionamongthe
andtheKenyanGovernment.Theviewsexpressed
hereindonotnecessarilyreflecttheofficialopinion vectorsfordifferentcompoundclasses;benzenoidsforAe.aegypti,aldehydesforAe.mcin-
ofthedonors.Thefundershadnoroleinstudy toshiandsesquiterpenesandalkenesforAn.gambiae.Thisstudyhighlightstheutilityof
design,datacollectionandanalysis,decisionto molecularapproachesinidentifyingspecificvector-plantassociations,aknowledgewhich
publish,orpreparationofthemanuscript.
canbeexploitedinmaximizingvectorcontrolstrategiessuchasattractivetoxicsugarbait.
Competinginterests:Theauthorsdeclarethatno Furthermore,theelucidationofpotentialodorsignaturelaysfoundationfordevelopment
competinginterestsexist. ofplantodor-baittechnologywhichiscriticalforsurveillanceofdifferentmosquitodis-
easevectorsofvaryingphysiologicalstatesandthepathogenstheytransmit.
Introduction
Therehasbeenanincreaseintheincidenceofvector-bornediseases,keyamongthemarbo-
viraldiseasessuchasdengue,chikungunya,RiftValleyfever(RVF)andzika.Whiledengue
predominantlyaffectsAsiancountries,increasingoutbreaksinEastAfricancoastalregions
havebecomeevidentinrecenttimes[1–3].RiftValleyfeverwhichmainlyoccursinAfrica,
withfociinEastAfrica,israpidlyspreadingeastwardsintoAsiaandtheArabianPeninsula
[4–6].Therecentupsurgeindengueincidencehasbeenattributedtorapidandunplanned
urbanizationwhichcreatesconducivebreedinghabitatsforthekeymosquitovectorofthedis-
ease,Aedesaegypti[3,7].Ontheotherhand,RVFisanepizooticdiseasemainlyassociated
withdevastatingoutbreaksfollowingwidespreadelevatedrainfall,leadingtofloodingthatcre-
atesfavorablebreedingsitesfortheprimarymosquitovectorsAedesmcintoshiandAedes
ochraceus[5,8].InEastAfrica,inparticularKenya,thepublichealthburdenduetothesearbo-
viraldiseasesisfurthercompoundedbytheendemicityoftheparasiticmalariadiseasetrans-
mittedbycertainspeciesoftheAnophelesmosquito[9].Globally,vector-bornediseasespose
riskofinfectiontomorethanhalfoftheworldpopulationwithmorethanamilliondeaths
annually[10].Consequently,thereisrenewedefforttocomeupwithnewdiseasecontrolmea-
suresandvectorcontrolformsakeypillarintheseefforts.Detailedunderstandingofthevec-
torecologyisneededinsearchfornovelcontrolstrategies.
Plantfeedingplaysacriticalroleinthebio-ecologyofmosquitodiseasevectors.Several
studieshavedemonstratedthatbothsexesofdifferentmosquitospeciesforageonplantsto
obtaincarbohydratesrequiredformetabolicprocessesvitalfortheirsurvival[11–13].Besides
providingareadysourceofenergyforflight,fecundityandcellmetabolism[12,14,15],plant
carbohydratesarealsoutilizedduringdiapausebymosquitoessuchasCulexpipienstosynthe-
siselipidreserves[16–18].Theavailabilityofhostplantshasalsobeenshowntoextendsur-
vivalofAnophelesgambiaeandAn.sergentii,likelyallowingforthecompletionofsporogonic
cycleofmalariaparasitesandtherebyincreasingdiseasetransmissionpotentialofthesevectors
[19–22].Ontheotherhand,abundanceoffloweringplantshasbeenlinkedtoreducedhuman
bitingbehaviorbymosquitodiseasevectors,whichcanimpacteitherpositivelyornegatively
ondiseasetransmissionpotentialdependingontheinfectionstatusofthemosquitoes[13,20,
23,24].Inaddition,studieshaveshownthatbothmosquitoesandsandfliesimbibeplantsec-
ondarymetabolitesduringplantfeeding,someofwhichreduceparasiteloadinthevector
[21,25,26].Thishasledtothehypothesisofpossibleselfmedicationbythesediseasevectors
[15,26]openingupnewavenuesforexploitingphytochemicalsindevelopmentofnovel
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
chemotherapeuticsagainstthepathogenstheytransmit.Thus,beyondtheprovisionofnutri-
ents,understandingplantfeedingindiseasevectorsofferspromisingopportunitiesfordevel-
opmentofnewcontrolstrategiesagainstthemyriadofvectorbornediseases.Despitethis,
littleisknownaboutplantfeedingbehaviorofmosquitovectorsofdengueandRVF.
Previousstudieshavedemonstratedthatmosquitoesarehighlyselectiveintheirchoiceof
plants[27–30].Theseinferencesweredrawnfromsemi-fieldandfieldexperimentswhich
eitherinvolvedfeedingmosquitoesonrandomlyselectedperi-domesticplantstodetermine
theiracceptability[27,28]ordeterminingtheattractionofmosquitoestorandomlyselected
fruits/seedpodsandfloweringplants[29–31].Determinationofplantfeedingamongvarious
mosquitospecieshasmainlybeenbasedonanalyticaltechniquessuchascoldanthroneteststo
detectfructoseinthecropoffieldcollectedmosquitoes,chromatographicmethodstodetect
plantsugarsandcellulosestainingtodetectplanttissuefeeding[12,16,19,23,24,28,32],with
littletonodirectfieldobservationsofmosquitoesfeedingonplants[33,34].Whilecontribut-
ingimmenselytowardsourunderstandingoftheroleofplantfeedinginthevectorialcapacity
ofmosquitoes,thesemethodsare,however,limitedbytheirinadequacyindeterminingthe
precisehostplantsinthenaturalmosquitohabitats.Asdiverseplantsoftenoccurineachhabi-
tat,thecriticalquestionofwhichplants,ifany,areforageduponbymosquitodiseasevectors,
remainsunanswered.
RecentadvanceshaveseentheapplicationofDNAbarcodingtargetingspecificgenesto
profileplantspeciesfeduponbydiseasevectors[35,36].However,thishasnotbeenapplied
foranyAfro-tropicaldiseasevector,thusfar.ByemployingDNAbarcodingtargetingmultiple
geneloci,wetestedthehypothesisthatAfro-tropicaldiseasevectorsfeedoncertainplantsin
theirrespectiveecologies.Wefocusedonfourmosquitospecieswhichtransmitdengue(Aedes
aegypti),RiftValleyfever(AedesmcintoshiandAedesochraceus)andmalaria(An.gambiae)[3,
37–39].Thesediseasesrankamongthemostimportantvector-bornediseasesinKenyawith
somehavingbeenassociatedwithlargeoutbreaksaffectinghumansintherecentpast[2,5,6,
9].Giventhecentralroleofolfactorycuesinlocatingthiskeyplantresource[40–42],wefur-
therusedcoupledgaschromatography/massspectrometryandelectrophysiologicalassaysto
testthehypothesisthatthesediseasevectorsuseuniqueodorbouquettolocatetheirsuitable
naturalhostplant.OurresultsshowthatthefourAfro-tropicalmosquitospeciesfeedoncer-
tainplantspecieswithintheirecologicalrangeanddetectcommonandspecificchemicalcues
tolocatetheirsuitablehostplants.Thisstudyprovidesusefulinsightthatcaninformvector
controlstrategiestargetingplantfeedingbehaviorsuchasattractivetoxicsugarbaitandodor
baittechnology.
Matherialsandmethods
Mosquitosamplecollection
MosquitosampleswereobtainedfromthreesitesinKenya:Ae.aegyptifromKilifi(3.6333˚S
and39.8500˚E)inthecoastalregionwithhighdengueendemicity[2,3],Ae.mcintoshiand
Ae.ochraceusfromIjara(1.5988˚Sand40.5135˚E)innortheasternKenyawhichisaRiftVal-
leyfeverendemicregion[5,6]andAn.gambiaes.l.fromAhero(0˚10’S,34˚55’E)whichisa
malariaendemicareainwesternKenya[9].Thetrappingmethodsusedtocollectmosquito
samplesaredescribedindetailinNyasembeetal.[42].Briefly,unlitCDCtrapsseparately
baitedwithlinalooloxide(LO),BioGent(BG)lureandHONAD(amixtureofheptanal,octa-
nal,nonanalanddecanal)formulatedfrommammalianodorbyTchouassietal.[37]were
usedtotrapAe.mcintoshi,Ae.ochraceusandAn.gambiae;whileBGsentineltrapsseparately
baitedwithLOandBGlure,wereusedtotrapAe.aegypti.Thetrapswerealsoeitherbaited
withorwithoutcarbondioxideintheformofdryiceatallthethreesites.Alltrappingswere
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
carriedoutoutdoors.InAheroandKilifi,trapswereplacedinthreedistinctsettingsi)closeto
thehomestead,ii)closetobreedingsitesaprioriidentifiedaspositiveforthespecificmosquito
larvae,andiii)invegetationawayfromhumandwelling[3,39,43,44].InIjara,thetrapswere
setattwodistinctsettingsi)nextto‘dambos’whichserveasbreedingsitesforAe.mcintoshi
andAe.ochraceusandii)inbushygrasslandswherepastoralistsgrazetheirlivestock[6,37,
38].InKilifi,trappingswerecarriedoutbothduringthedayandnight(informedbythediur-
nalnatureofAe.aegypti)whileinAheroandIjaratrappingswerecarriedoutduringthenight
only(duetothenocturnalnatureofmosquitovectorsintheselocalities).Trapswereemptied
after12handthecollectedmosquitoesimmobilizedbyplacingondryice,immediatelyfrozen
inliquidnitrogenandtransportedtoicipelaboratoriesinNairobiforfurtherprocessing.
Samplepreparation
Topreparethesamplesforbiochemicalandmolecularanalyses,individualmosquitoeswere
submergedinasolutionof0.5%hypochlorite,agitatedgentlyfor1minwithforceps,andthen
rinsedindoubledistilledwater(ddH 0)for1min.Thiswastoremoveanyplantdebristhat
2
mayhavebeenontheoutsideoftheinsect,whichcouldotherwisecontaminatethesample.
Themosquitoeswerethenplacedindividuallyina1.5mlsterileEppendorftubeandmacer-
atedusinground-tippedglassrodssterilizedthroughtheflameofaBunsenburner.Onehun-
dredmicrolitresofabsoluteethanolwasaddedandthesolutionhomogenized.Twosetsof
controlswereusedasfollows:a)laboratory-rearedAn.gambiaes.s.fedonPartheniumhystero-
phorus(Asteraceae)overnight,andb)An.gambiaeaspirateddirectlyfromP.hysterophorus
fieldinAherousingabackpackaspirator(3”IN-LINEBLOWER,JohnW.HockCompany,
Gainesville,FL,USA).Allmosquitoesfromthecontrolswerepreparedasdescribedabove.
Determinationofevidenceofrecentplantfeedinginfieldcollected
mosquitoes
ThiswasdoneusingthecoldanthronetestasdescribedbyvanHandeletal.,[45]asaquick
initialtesttodetectfructose.Aliquots(50μl)ofthepreparedmosquitohomogenatewereindi-
viduallyplacedinthewellsofaflatbottomed96-wellmicrotiterplatefollowedby300μlofthe
reactionsolutioncomprising0.15%anthrone(Sigma)(wt/vol)in71.7%sulphuricacid.This
wasincubatedat25oCfor60minbeforebeingexaminedforcolorchanges.Inthepresenceof
fructose,thereactionmixturechangeditscolorfromyellowtoblue.Theremainingaliquotof
fructose-positivesampleswassubjectedtoplantDNAextractionasdescribedbelow.
ExtractionofplantDNAfrommosquitoes
PlantDNAwasextractedfromthehomogenizedsamplesoffructose-positivemosquitoesonly
usingthemanufacturer’sprotocoldescribedbyDNeasyPlantMinikit-(QIAGEN,USA)with
aminormodification.TheincubationperiodwithlysisbufferAP1andRNasewasextended
by30minwhilethatwiththeelutionBufferAEwasextendedfor3hr.TheextractedDNAwas
storedat-20˚CuntiluseinPCRamplification.
PCRamplificationandsequencing
PlantDNAextractedfromthefructose-positivemosquitospecimenswasamplifiedtargeting
thetrnH-psbAintergenicspacerregionandmaturaseK(matK)gene(Table1)usingestab-
lishedprimers.Theuseofmorethanonetargetwastomaximizeonthedetectionpossibility
asindividualgenesselectivelyamplifycertainplantfamilies[46].EachPCRreaction(carried
outinavolumeof20μl)consistedof7μltemplateDNA,10μl2xHotStarTaqMasterMix
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
Table1. Forwardandreverseprimersequencesforthreegenetargetsusedtoidentifynaturalhostplantsofdengue,RiftValleyfeverandmalariamosquitodisease
vectors.
Primer Direction Sequence(5’-3’) Reference
trnH-psbA trnH CGCGCATGGTGGATTCACAATCC Shawetal.,2005
psbA GTTATGCATGAACGTAATGCT Shawetal.,2005
matK 2.1forward CCTATCCATCTGGAAATCTTAG Kressetal.,2005
5reverse GTTCTAGCACAAGAAAGTCG Kressetal.,2005
https://doi.org/10.1371/journal.pntd.0006185.t001
(HotStarTaqPlusMasterMixKit,Qiagen),0.5μMofeachprimer,and2μlofRNasefree
water.APCRnegativecontrol(RNase-freewater)wasroutinelyused.Sampleswereamplified
usingVeriti96-wellThermalCycler(Singapore).FortrnH-psbA,thecyclingparameterswere
94oCfor1min,followedby45cyclesof94oCfor1min,55oCfor40secand72oCfor1min,
andfinalextensionat72oCfor10min.SimilarcyclingconditionswereusedformatKamplifi-
cationwiththeannealingtemperaturesetat48oC.
SuccessfulamplificationswereconfirmedbyvisualizingPCRampliconsin1%agarosegel
electrophoresis.TheywerepurifiedusingtheExo/SAP-ITKitforPCRproduct(Affymetrix
Inc.,USA)asperthemanufacturer’sinstructionsandoutsourcedforbidirectionalsequencing
toInqabaBiotechnologicalIndustries(Pty)Ltd(Pretoria,SouthAfrica).
TheobtainedplantDNAsequencesforeachgenewerecleaned,editedandcomparedto
referencesequencesintheGenBankdatabase[47].InGenBank,the‘megablast’searchoption
ofnucleotideBasicLocalAlignmentSearchTool(BLASTn)[48]algorithmwasusedwiththe
defaultsearchparameters.Thehitswithsequenceidentityabove96%wereretrievedand
addedtotheoriginalsamplequerysequences.ThesequenceswerealignedusingClustalWin
MEGA6[49].Alignedmatriceswereusedtoconstructp-distancephylogenetictreeusingthe
NeighborJoiningmethodforindividualgeneswith1000bootstraps.Nodalsupportwasevalu-
atedbybootstrappingwithvaluesof95%ormoreconsideredsignificant.
Confirmationofidentifiedmosquitohostplants
Furtherstepstoconfirmtheplantidentityincludedon-siteidentificationwithinthespecific
ecologiesfromwherethemosquitoesweresampledbyaplanttaxonomist(SimonMathenge,
retiredfromtheHerbarium,DepartmentofBotany,UniversityofNairobi)andcomparison
toestablishedbotanicaldatabasebytheNationalMuseumofKenya(http://www.museums.
or.ke).
Leavesandflowers(whereapplicable)ofidentifiedplantsweresampledfromtherespective
fieldsitesforDNAextractionandsequencing.Thesampleswerecleanedusingdoubledistilled
waterbeforeobtainingapproximately100mgwetweightofthesamplewhichwereplacedin
sterile1.5mlEppendorftubes.TheplantsampleswerehomogenizedandDNAextracted
usingtheDNeasyPlantMiniKitasdescribedabove.TheobtainedplantDNAwassimilarly
amplifiedfortrnH-psbAandmatKgenes,processedandsentforsequencingasdescribed
above.Thesequenceswerethenalignedwiththosefromthemosquitoesandphylogenetic
treesobtained.Nodalsupportwasevaluatedbybootstrappingwithvaluesof95%ormorecon-
sideredsignificant.
ThehaplotypesgeneratedfromthisstudyhavebeendepositedinGenBankunderaccession
numbersMG573108,MG573126–MG573131(RVFvectorshostplanttrnH-psbAgene
sequences),MG573132–MG573139(denguevectorhostplanttrnH-psbAgenesequences),
MG573109–MG573125(malariavectorhostplanttrnH-psbAgenesequences),and
KY308115—KY398121(malariavectorhostplantmatKgenesequences).
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
Collectionofheadspacevolatilesfromtheidentifiednaturalmosquitohost
plants
HeadspaceVOCswerecollectedfromfiveoftheconfirmednaturalhostplantsforAe.aegypti,
Ae.mcintoshi,Ae.ochraceusandAn.gambiae.ThefiveplantsincludedPithecellobiumdulce
(Fabaceae),Opuntiaficus-indica(Cactaceae),Leonotisnepetifolia(Lamiaceae),Sennaalata
(Fabaceae)andRicinuscommunis(Euphorbiaceae).Thiswasdonebycollectingtheheadspace
volatilesfromtheseplantsinsituattheirnaturalhabitatsusingaportablefieldpump(Analyti-
calResearchSystems,Gainesville,Florida,USA).Theaerialpartsofanintactplantweregently
enclosedinanair-tightovenbag(Reynolds,Richmond,VA,USA)andcharcoalfilteredair
passedovertheplantataflowrateof350ml/minintoaSuper-Qadsorbenttrap(30mg,Ana-
lyticalResearchSystems,Gainesville,Florida,USA).Theaerialplantpartsenclosedinthe
ovenbagsincludedleaves,flowersandpodsofP.dulceandS.alata,leavesandflowersofL.
nepetifolia,leavesandleafstalksofR.communis,andleaves,flowersandfruitsofO.ficus-
indica.Forallplantspecies,volatileswerecollectedfor12hrduringthedayand12hratnight
andreplicatedthreetimesusingdifferentplantsineachreplicate.TheSuper-Qtrapswere
elutedwith200μlGC/GC-MS-gradedichloromethane(DCM)(BurdickandJackson,Muske-
gon,Michigan,USA)andtheeluentsstoredat-80˚Cuntilanalysis.
Analysisofvolatiles
Forquantificationandidentificationoftheconstituentcompoundsoftheplantvolatiles,an
aliquot(1μl)ofeachsamplewasinjectedintoagaschromatograph(Agilenttechnologies-
7890)coupledtoinertXLEI/CImassspectrophotometer(5975C,EI,70eV,Agilent,PaloAlto,
Califonia,USA)(GC/MS)inasplitlessinjectionmode.TheGCwasequippedwithanHP-5
column(30mx0.25mmIDx0.25μmfilmthickness,Agilent,PaloAlto,California,USA),
withheliumasthecarriergasataflowrateof1.2ml/min.Theoventemperaturewasheldat
35˚Cfor5min,thenprogrammedtoincreaseat10˚C/minto280˚Candmaintainedatthis
temperaturefor10min.Thevolatileorganiccompoundswereidentifiedbycomparingtheir
massspectrawithlibrarydata(Adams2.L,Chemecol.LandNIST05a.L)andwiththoseof
authenticstandardswherepossible(seesourcesandpurityunderchemicalsectionbelow).
TheabsoluteareasofeachconstituentascalculatedbytheNIST05a.Lsoftwarewasusedto
estimatetheiramountsusinganexternalcalibrationequationgeneratedfromknownamounts
ofauthenticcompounds.
Electrophysiologicalassays
ToisolatethespecificVOCsthataredetectedbythedifferentmosquitodiseasevectorsand
theirpreferrednaturalhostplants,wildcaughtadultAe.aegypti,Ae.mcintoshiandAn.gam-
biaes.l.,werecollectedfromtheirrespectivehabitatsusingmethodsdescribedaboveand
transportedalivetotheicipelaboratoriesinNairobiunderhighcontainmentlevelanddirectly
usedinelectrophysiologicalassays.Thetrappedmosquitoeswereaspiratedinto30x30x30
cmcagesandprovidedwith10%glucosesolutionsoakedincottonwoolduringtransporta-
tion.Thetopsofthecageswerecoveredwithamoisttoweltomaintainhighhumidity.Once
aticipe,themosquitoeswerekeptinahighcontainmentanimalrearingunitatatemperature
of27–31˚Candaveragehumidityof80%.Onlyfemalemosquitoeswereusedforelectrophysi-
ologicalassaysandtheywerestarvedfor2hrbeforeexperimentation.Anophelesgambiaes.s.
antennalresponsestoR.communisheadspacevolatileshadbeentestedinourpreviousstudy
[40],hencewasnotrepeatedinthisstudy.Inaddition,Ae.ochraceuswasnotusedinthese
studiesasnonewerecollectedduringthisfieldsampling.
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
Coupledgaschromatography/electro-antennographicdetection(GC/EAD)analyseswere
performedasdescribedbyNyasembeetal.[40].Briefly,5μlofvolatilesampleswereanalyzed
usingaHewlett-Packard(HP)5890SeriesIIgaschromatographequippedwithanHP-5col-
umn(30mx0.25mmIDx.0.25μmfilmthickness,Agilent,PaloAlto,California,USA)with
nitrogenasthecarriergasat1ml/min.Volatileswereanalyzedinthesplitlessmodeatan
injectortemperatureof280˚Candasplitvalvedelayof5min.Theoventemperaturewasheld
at35˚Cfor3min,thenprogrammedat10˚C/minto280˚Candmaintainedatthistempera-
turefor10min.Thecolumneffluentwassplit1:1afteradditionofmake-upnitrogengasfor
simultaneousdetectionbyflameionizationdetector(FID)andEAD.ForEADdetection,sil-
ver-coatedwiresindrawn-outglasscapillaries(1.5mmI.D.)filledwithRingersalinesolution
servedasreferenceandrecordingelectrodes.Livemountinginwhichthemosquitowas
restrainedwithanadhesivetapewiththereferenceelectrodeconnectedtothebaseofthehead
andtherecordingelectrodeconnectedtothetipoftheantennae.Theanaloguesignalwas
detectedthroughaprobe(INR-II,Syntech,Hilversum,theNetherlands),capturedandpro-
cessedwithanintelligentdataacquisitioncontroller(IDAC-4,Syntech,theNetherlands),and
lateranalyzedwithEAG2000,software(Syntech).FIDsignalsfromtherespectivehostplant
volatilesthatelicitedrepeatedantennalresponsesinatleastthreereplicatesusingfreshanten-
naeweredesignatedasEAD-activecompoundsandidentifiedbymatchingthemwithcorre-
spondingGC/MSdataandthoseofauthenticstandards.
EAGpuffswereusedtoconfirmthedetectionofsevenEAD-activecomponentswhichelic-
itedantennalresponsesusingsyntheticstandards.Thesevencompoundswereselectedbased
oneitherbeingdetectedbymorethanonemosquitospeciesfromthevolatilesfromtheir
respectivehostplants,orbythesamemosquitospeciesfromvolatilesofdifferenthostplants.
Thesyntheticstandardswerepreparedataconcentrationof1ng/μl,2ng/μland4ng/μlin
dichloromethane(SigmaAldrich,99.9%)andseparatelydeliveredaspuffson1cmX1cmfil-
terpaperplacedinPasteurpipettes.Thepuffsweredeliveredat1mininterval,allowingthe
antennaetoequilibratepost-exposure.Tocorrectforvariabilityinresponse,responsesto
blanks(filterpaperlacedwithsolventonly)weresubtractedfromeachsampleandantennal
responsevalueswerenormalizedtoastandardstimulussetat100%(2ng/μl1-octen-3-ol,cho-
senbasedonitsknownattractivenesstohematophagousinsects[41].EAGpuffswererepli-
catedninetimesforeachdoseofeverystimulus.
Chemicalsused
ThesyntheticstandardsofthefollowingEAD-activecompoundswereused:hexanal(Sigma
Aldrich,99%),(E)-2-hexenol(Aldrich,96%),benzaldehyde(SigmaAldrich,99.5),β-myrcene
(SigmaAldrich,99%),ocimene(InternationalFlavorsandFragrance,NewYork,USA,(Z)-β-
ocimene=27%,(E)-β-ocimene=67%andallo-ocimene=6%),linalooloxide(SigmaAldrich,
mixtureofstereoisomerswithfuranoidform,99.5%and0.5%pyranoidform),indole(Sigma
Aldrich,99%)and1-octen-3-ol(FlukaChemica,racemicmixtureofRandS98%).
Statisticalanalysis
Todetermineiftherewasanysignificantdifferenceinthevolatileprofilesofthefiveplantspe-
cies,tenmostabundantvolatileconstituentsineachplantspecieswereselected.Attempts
werethenmadetoretrieveeachofthesecompoundsfromtheVOCsanalyzedfortherestof
theplantspecies,yieldingatotalof26differentcompounds.Theabsoluteareasofthesecom-
poundswerethenmeasuredandconvertedintoapercentageofthetotal.Thesepercentages
werethensubjectedtoPrincipalComponentAnalysis(PCA)todeterminewhichones,ifany,
areimportantinexplainingthevariationintheodorprofilesofthefivedifferentplantspecies.
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
QuantitativedifferencesinVOCsofthefivedifferentplantspeciesweredetectedusingUni-
variateanalysisofvarianceandTukeyposthoctest.Differencesbetweentheantennaldose
responsesandbetweenthethreedifferentmosquitospecies,weredetectedusingANOVAand
Tukeyposthoctest.Allstatisticalanalyseswerecarriedoutat95%confidenceintervalusingR
2.15.1software[50].
Results
Evidenceofplantfeedingamongwild-caughtAfro-tropicalmosquito
species
Byapplyingthecoldanthronetesttodetectfructoseasevidenceofrecentplantfeeding,we
establishedthedegreeofplantfeedingamongthefemalesoffourAfro-tropicalmosquitospe-
ciesAedesaegypti(denguevector),Aedesmcintoshi,Ae.ochraceus(RVFvectors)andAnopheles
gambiaes.l.(malariavector)trappedfromdifferenthabitatsinKenyaduringthelongrainy
season(April-June,2014).SincenomalemosquitoeswerecollectedforRVFandmalariavec-
tors,thisanalysiswaslimitedtoonlyfemalemosquitoesforallthefourspecies.Wefoundevi-
denceofrecentplantfeedinginAe.aegypti(17%,n=245),Ae.mcintoshi(56%,n=68),Ae.
ochraceus(65%,n=50)andAn.gambiae(24%,n=146).
Afro-tropicalmosquitospeciesfeedondiverseplantspeciesintheir
naturalhabitats
Todeterminetheidentitiesoftheplantspeciesfeduponbythesemosquitospeciesintheir
naturalhabitats,wesubjectedaliquotsofsamplesthattestedpositivefortheanthronetestto
DNAextractionfollowedbyamplificationtargetingtwoplantgenes;trnH-psbAandmatK;
andthensequencing.WeobservedthatthesuccessratesinamplificationofplantDNAfrom
themosquitocropdifferedsignificantlybetweenthetwogenetargets;trnH-psbA(24.5%)and
matK(8.8%)(P<0.05;Table2).Similarly,sequencingsuccessratesdifferedsignificantly
betweentrnH-psbA(16.4%)andmatK(1.9%)(P<0.05;Table2).Thesequencedfragment
sizesrangedfrom276–617bpfortrnH-psbAand133–846bpformatKgenes.
BlastsearchesofthesequencesforeachtargetinGenBankandfurtherphylogenyshowed
strongsupport(bootstrapvalues95%andabove)andidentifiedhostplantsasPithecellobium
dulce(Fabaceae),Sennauniflora(Fabaceae)andHibiscusheterophyllus(Malvaceae)forAe.
aegypti(Fig1A);Opuntiaficus-indica(Cactaceae)forAe.mcintoshi;andO.ficus-indicaandan
unidentifiedplantspeciesforAe.ochraceus(Fig1A);andSennaalata(Fabaceae),Sennatora
(Fabaceae),Ricinuscommunis(Euphorbiaceae),Partheniumhysterophorus(Asteraceae)and
Leonotisnepetifolia(Lamiaceae)forAn.gambiae(Fig1Aand1B).Theplantidentitieswere
Table2. VariablesuccessratesoftwogenetargetsinamplifyingandsequencingplantDNAinthecropofdiffer-
entmosquitospecies.
Mosquitospecies N Amplified(Sequenced)
trnH-psbA(total(success)) matK(total(success))
Aedesaegypti 42 8(6) 0(0)
Aedesmcintoshi 38 2(2) 2(0)
Aedesochraceus 32 11(3) 9(0)
Anophelesgambiaes.l. 35 18(15) 3(3)
Overall 147 39(26) 14(3)
N=numberofmosquitoesfromwhichplantDNAwereextracted.
https://doi.org/10.1371/journal.pntd.0006185.t002
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
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Hostplantforensicsandolfactory-baseddetectioninAfrotropicalmosquitodiseasevectors
Fig1.NJphylogenetictreesfromtwogenetargetsshowingplantspeciesidentifiedasnaturalhostplantsoftheAfro-
tropicalmosquitospecies.A)PlantspeciesidentifiedusingtrnH-psbAgenetargetsashostplantsforAedesaegypti,Aedes
mcintoshi,AedesochraceusandAnophelesgambiae.B)PlantspeciesidentifiedusingmatKgenetargetsashostplantsfor
Anophelesgambiae.PlantspeciesnameswithprefixAafromAedesaegypti,AmfromAedesmcintoshi,AofromAedesochraceus
andAgrepresentthosethatwereidentifiedfromAnophelesgambiae,thenumbersbeingsampleID.Plantspecieswithprefix
P1-4representtheplantsamplessequencestoconfirmtheidentityofthemosquitohostplantswhilethosewithsuffixesare
outgroupsfromGenBankwithextensionbeingaccessionnumbers.
https://doi.org/10.1371/journal.pntd.0006185.g001
furthercorroboratedbyon-sitebotanicalidentificationtoconfirmtheirpresenceandinclu-
sionofmatchedsequencesofextractedDNAintheanalyses.Theseandputativesequences
fromthemosquitogutclusteredtogetherwithstrongbootstrapsupportinthephylogenetic
analysis(Fig1Aand1B).
Diversevolatileorganiccompoundscharacterizethenaturalhostplantsof
Afro-tropicalmosquitospecies
WeanalyzedheadspacevolatilesfromfiveoftheidentifiednaturalhostplantsvizP.dulce,O.
ficus-indica,L.nepetifolia,S.alataandR.communis.TheVOCsofthefivedifferentplantspe-
ciesweredifferentiatedbyuniquechemicalconstituentsofvaryingabundance(Fig2A).Prin-
cipalComponentAnalysis(PCA)resolvedthesechemicalconstituentsintothreeclusters
whichaccountedformorethan90%ofthetotalvariation(Fig2B).PC1explained38%ofthe
variation;PC2explained32%whilePC3explained22%ofthevariation.PC1wasweighedpos-
itivelybymonoterpenoidsandbenzenoids,predominantlyuniquetoP.dulce,whilePC2was
positivelycontributedtobysesquiterpeneswhichwerecharacteristicallyabundantinL.nepoti-
folia(S1Table).PC3waspositivelycharacterizedbymonoterpeneswhichwerethekeyconstit-
uentsdetectedintheVOCsofR.communis(S1Table).Theheadspacevolatileconstituentsof
S.alataandO.ficus-indicacontainedbenzenoids(S1Table).(E)-β-Ocimenewaspresentin
theVOCsofallthefivehostplantspecies,whilehexanal,(E)-2-hexen-1-ol,β-myrcene,benzal-
dehyde,α-pinene,nonanal,linalooloxide,decanal,methylsalicylate,(E)-β-caryophylleneand
germacreneDwerevariablypresentinthevolatilesoftwoormoreplantspecies(S1Table).
Multivariateanalysisofvariancerevealedsignificantquantitativedifferencesinthevolatile
profilesofthefiveplants(F =142.907,P<0.001;Fig2C).
(4,1095)
Afro-tropicalmosquitospeciesdetectuniquevolatileorganiccompounds
fromtheirnaturalhostplants
TotestifAfro-tropicalmosquitospeciesdetectodorsoftheirnaturalhostplants,weemployed
coupledgaschromatography/electroantennographicdetection(GC/EAD)andGC/massspec-
trometrytoisolateandidentifyVOCsthataredetectedbyantennaeofAe.aegypti,Ae.mcin-
toshiandAn.gambiae.Ourassaysrevealedthattheantennaeofthethreedifferentmosquito
speciesdetectedatotalof21differentVOCs,someofwhichwereuniquetotheirpreferred
hostplantswhileotherswerecommonacrosstwoormoreoftheplantspecies.Antennaeof
Ae.aegyptidetected8componentsinP.dulceheadspacevolatiles(Fig3A),with12compo-
nentsdetectedbyAe.mcintoshifromO.ficus-indica(Fig3B)whilethoseofAn.gambiaes.l.
detected13and7componentsinthevolatilesofL.nepetifoliaandS.alata,respectively(Fig3C
and3D).β-Myrceneandocimeneweredetectedbyantennaeofallthethreedifferentmos-
quitospeciesfromtheirrespectivehostplantswhilehexanal,(E)-2-hexenol,andlinalooloxide
isomers,andbenzaldehydewerevariablydetectedbythethreedifferentmosquitospecies.On
theotherhand,antennaeofthethreedifferentmosquitospeciesalsodetecteduniquecom-
poundsfromtheirrespectivehostplantswhichincludedbenzenoids(benzylalcoholand
indole)byAe.aegypti,aldehydes(octanal,nonanalanddecanal)byAe.mcintoshi,and
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