treatments55556.com

RESEARCHOpenAccessFresh,driedorsmokedRepellentpropertiesofvolatilesemittedfromethnomedicinalplantleavesagainstmalariaandyellowfevervectorsinEthiopiaFitsumFikruDube2,3,KassahunTadesse3,GranBirgersson1,EmiruSeyoum3,HabteTekie3,RickardIgnell1andSharonRHill1*AbstractBackground:Inthesearchforplant-basedmosquitorepellents,volatileemanationswereinvestigatedfromfiveplantspecies,Corymbiacitriodora,Ocimumsuave,Ocimumlamiifolium,OleaeuropaeaandOstostegiaintegrifolia,traditionallyusedinEthiopiaasprotectionagainstmosquitoes.
Methods:Thebehaviouroftwomosquitoes,themalariavectorAnophelesarabiensisandthearbovirusvectorAedesaegypti,wasassessedtowardsvolatilescollectedfromtheheadspaceoffreshanddriedleaves,andthesmokefromburningthedriedleavesinatwo-choicelandingbioassayandinthebackgroundofhumanodour.
Results:Volatileextractsfromthesmokeofburningdriedleaveswerefoundtobemorerepellentthanthosefromfreshleaves,whichinturnweremorerepellenttomosquitoesthanvolatilesfromdriedleaves.
Ofallsmokeandfreshvolatileextracts,thosefromCo.
citriodora(52-76%)andOc.
suave(58-68%)werefoundtobethemostrepellent,Os.
integrifolia(29-56%)tobeintermediatewhileOl.
europaea(23-40%)andOs.
integrifolia(19-37%)weretheleastrepellent.
OnevolatilepresentineachofthefreshleafextractsofCo.
citriodora,Oc.
suaveandOs.
integrifoliawas-ocimene.
Thelevelsof-ocimenereflectedthemosquitorepellentactivityofthesethreefreshleafextracts.
Femalehost-seekingmosquitoesrespondeddose-dependentlyto-ocimene,bothphysiologicallyandbehaviourally,withamaximalbehaviouralrepulsionat14%-ocimene.
-ocimene(14%)repelsmosquitoesinour6-minutelandingassayscomparabletothesyntheticinsectrepellentN,N-diethyl-m-toluamide(10%DEET).
Conclusions:VolatilesinthesmokeofburningaswellasfreshleavesofCo.
citriodoraandOc.
suavehavesignificantrepellentpropertiesagainsthostseekingAn.
arabiensisandAe.
aegyptimosquitoes.
-ocimene,presentinthefreshleafheadspaceofCo.
citriodora,Oc.
suaveandOs.
integrifolia,isasignificantlyeffectivevolatilemosquitorepellentinthelaboratory.
Inadditiontoitsrepellentproperties,-ocimenehaslongapprovedsafeforuseinfoodandcosmetics,makingthisvolatileanintriguingcompoundtopursueinfurthertestsinthelaboratoryandfieldtovalidateitsmosquitorepellentactivityandpotentialforuseinacommercialproduct.
Also,thelandingbioassaywithhumanisedmembranesisapotentiallyusefulrepellentscreeningtechniquethatdoesnotrequiretheexposureofhumanstothevectors,howeverfurthertestsinparallelwithconventionaltechniquesareadvised.
*Correspondence:sharon.
hill@slu.
se1DivisionofChemicalEcology,DepartmentofPlantProtectionBiology,SwedishUniversityofAgriculturalSciences,23053Alnarp,SwedenFulllistofauthorinformationisavailableattheendofthearticleDubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/3752011Dubeetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
BackgroundThehealthrisksassociatedwitharthropoddiseasevec-torshavelongencouragedresearchintomethodsforprotectioninendemicareas,inboththegrassroots[1]andscientificcommunities.
Diligentinvestigationsintosuchgrassrootsprotectionmethodsbythescientificcommunityisleadingtothedevelopmentofnewbio-rational,effectiveandaffordableproductsaswellasincreasingknowledgeandconfidenceintraditionalpro-tectionmethodsandreducingvector-bornedisease.
Oneofthemosteffectivestrategiestominimizevector-bornediseaseispersonalprotection,whichfocusesonthebehaviourofbothpeopleandmosquitoestominimizehumanexposuretovectors[2].
Theuseofinsecticide-treatednets(ITNs)isthemostpowerfulmethodforper-sonalprotectioncurrentlyavailableforeffectiveinfectionreduction[3].
Evenso,ITNshavetheirlimitations;pri-marily,thattheydonotprotectagainstexophagicvectors,orthosevectorsthatbiteattimeswhenpeoplearenotsleepingundertheirbednets.
[4].
Diseasevectors,suchasAnophelesarabiensis,thepri-marymalariavectorinsemi-arideasternsub-SaharanAfrica[5,6],Aedesaegypti,themainvectorofdengueandyellowfever,aswellasthemalariavectorsAnophelesfar-autisensulatoandAnophelesdarlingi[7,8],haveadaptedtheirpeakbitingactivitiestotheearlyeveningandearlymorning,whentheirpotentialhostsarelessprotected.
Infactinsomeregions,An.
darlingihasbecomeexclusivelyexophagic,arguablyinresponsetoindoorresidualspray-ing(IRS)[8]whichhasreducedthenumberofendophilicspecies[9].
Suchbehaviouraladaptationstothesecurrentprotectionmethods,emphasizetheneedforanotherlineofdefenceagainstdiseasetransmission.
Mosquitorepel-lentshaveauniqueroleundertheseconditions.
Easilyaccessible,safeandeffectivemosquitorepellentsprovideavaluablesupplementtoIRSandITNuse,andinareaswithday-biting,exophagicvectors,thismaybetheonlyoptionforreducingthelevelofdiseasetransmission[10].
Plant-basedmosquitorepellentsareaviablesourceofmaterialforuseinprotectionagainstmosquitoesandmosquito-transmitteddiseases[11]andhavesomeadvan-tagesoverthecurrentgold-standardsyntheticrepellent,N,N-diethyl-m-toluamide(DEET)[10].
Avarietyofplantshavebeenidentifiedfortheirmosquitorepellentproper-tiesthroughbothgrassrootsandscientificinvestigations[10,11].
VolatilesfromessentialoilsofLamiaceae(culinaryherbs),Poaceae(aromaticgrasses)andPinaceae(pineandcedartrees),areeffectiveagainstvarioushaematophagousarthropodsandsomeessentialoils,ortheircomponents,formthebasisofcommercialrepellentformulations[11,12].
Themostnotableoftheseisp-menthane-3,8-diol(PMD),ahydro-distilledcompoundfromthelemoneuca-lyptusplant,Corymbiacitriodora[12].
Theburningand/orhangingoffreshanddriedleavesfromLamiaceae,PoaceaeandPinaceaearoundandwithinthehometoprovideprotectionagainstmosquitobitesiswidelyusedthroughoutruralEthiopia[13,14]aswellasothertropicalregions[15-19].
Smokefromsomeoftheseplantsiseffectiveinrepellinganophelinemosquitoes:e.
g.
Ostostegiaintegrifolia(90.
1%)[20],Oleaeuropaea(79.
8%)[20],Co.
citriodora(78.
7%)[16,21]andOcimumsuave(44.
5%)[16].
TheleavesofOc.
canumprovided63.
6%pro-tectionfrommosquitobiteswhenhungfreshinthehomesinGuineaBissau,WestAfrica[17].
InwesternKenya,Seyoumetal[15,16,19]foundlivepottedplantsofOc.
americanum,Oc.
kilimandscharicumandOc.
suavetoberepellentprovidingonaverageof39.
7%,44.
45%and44.
45%[15,16,19]protectionfrombites,respectively.
Inlightofthesestudies,thisinvestigationwascarriedouttoevaluatethepotentialofvolatilesfromtheleavesofCo.
citriodora,Oc.
suave,Oc.
lamiifolium,Os.
integrifo-liaandOl.
europaeatorepeltheday-bitingvectorsAn.
arabiensisandAe.
aegypti,importantvectorsofmalariaanddengue/yellowfeverinEthiopia,respectively.
MethodsExperimentalinsectsAcolonyofAe.
aegypti[Rockefellerstrain]wasmaintainedattheSwedishUniversityofAgriculturalSciences(SLU),Sweden.
Larvae(200-300)ofAe.
aegyptiwererearedintrays15cmwide*30cmlongwith2-3cmwaterindepth,andfedonceadayonadietofflakes(0.
2-0.
5g/tray)fromfishfoodBestFriend(BestFriendGroup,Finland).
InEthiopia,Ae.
aegypti(colonyfromAkliluLemmaInstituteofPathobiology)andAn.
arabiensis(col-onyfromWHOMalariaControlCentre)weremaintainedattheWHOMalariaControlCentreinNazareth,Ethio-pia;larvaewererearedintrays15cmwide*30cmlonginwater2-3cmdeep,andfedonceadayonfedonFaffapowder(0.
2-0.
5g/tray;FaffaFoods,Ethiopia).
Allcolonieswererearedunderstandardinsectaryconditionsof27±2°C,75±5%R.
H.
,L:D12:12h.
Adultsofbothspeciesweremaintainedincagesconstructedfromplasticbucketswithmeshlids(20cmdiameter*30cmheight)andweregivenadlibitumaccessto10%sucrosesolution.
Adultnon-blood-fedfemalemosquitoesusedforexperimenta-tionwerebetween4and6dayspost-emergenceandstarvedfor12hpriortotesting.
OdourcollectionInthisstudy,leavesofmatureCo.
citriodora,Oc.
suaveandOc.
lamiifoliumwerecollectedfromWondoGenetEssentialOilsResearchCentreinSouth-CentralEthiopia(latitude7.
0862,longitude38.
6190)andgrownintepidhumidhighlandconditions(agro-ecologyH3)wherethemajorsoiltypesareluvisols(sandyloamwithPHof7.
2).
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page2of14WondoGenetEssentialOilsResearchCentreisatanaltitudeof1780mabovesealevelwithatemperaturebetween10°Cand30°Candamaximumrainfallof2000mmandaminimumof700mm.
Leavesfromthetwootherspecies,Os.
integrifoliaandOl.
europaea,werecol-lectedfromAddisZemen,Ethiopia(latitude12.
143,long-itude37.
779)grownat1975mabovesealevelunderthesameconditionsasWondoGenetEssentialOilsResearchCentrestatedabove.
Leavesfromthesespecieswerecho-sentobeusedinthevolatilecollections,aspreviousstu-dieshavedemonstratedtheirpotentialasrepellents[15,16,19-21].
Volatileswerecollectedfromfresh,driedandsmokingdriedleavesusingstandardheadspacesam-plingmethods[22].
Volatileswerecollectedfromleavesthatwerefreshlycutandthosethatweredried,aswellasfromthesmokeofburningdriedleaves,representingthedifferentwaystheseleavesarecurrentlyusedinhomesasprotectionagainstmosquitoes.
Freshanddriedleafheadspacevolatileswerecollectedfor3hfromtheleaves(10g)placedinclosedglassbottleswithanactivatedcharcoalfilteredairinletandanoutletleadingtotheTefloncolumn(55mm*3mminnerdia-meter)filledwithSuperQabsorbent(35mg,mesh80/100,Alltech,Deerfield,IL,USA).
Volatilesfromtheburn-ingdriedleaves(10g)werecollectedbyplacingdriedleavesontotheburningcharcoalunderaninvertedglassfunnelfor10minallowingallleafmaterialtobecon-sumed(Figure1).
Inordertofilteroutthenon-volatileparticulatesemittedinthesmoke,arollofglasswool(5cm)wasplacedinsidethetubeofthefunnelupstreamofthevolatile-collectingcolumnfilledwithSuperQabsor-bentasabove(Figure1).
Volatileheadspacewasalsocol-lectedfromcharcoalsmokealoneasabioassaycontrolfortheburnedleaves,asabove.
Aftertheodourcollection,thevolatileswereelutedbyadding300μlhexanetothecol-umnstoobtainanextractofthevolatiles.
Thesamplesweresealedin1.
5mlglassvials(SkandinaviskaGenTecAB,Sweden)andstoredat-18°Cuntilusedinthebeha-viouralstudies.
LandingbioassayswithhumanisedmembranesMembranefeeders,commonlyusedtoprovideabloodmealtomosquitoesininsectaries[23],wereusedtomea-suretherepellenceofplantvolatileextractstomosqui-toes[24].
Humanodourwasusedasanattractantandaddedtobothcontrolandtreatmentmembranes(to'humanise'them)toensurethatmosquitoeswouldbeattractedtothemembraneintheabsenceofanyrepel-lentcompound(Figure2)[25].
InSweden,thebeha-viouralresponseofAe.
aegyptiwastestedbyusingtheHemotek("store-bought")feedingmembranes(DiscoveryWorkshops,Accrington,UK),whichwererubbedfor1minontheexperimenter'spalms,washedbynon-per-fumedsoap(Lactacyd,GlaxoSmithKline,UK)24hbeforetheexperiment,shiftingbetweenthehandseveryhalfminute.
Priorcontrolexperiments,bothnochoice(Figure2b-c)andchoiceassays(Figure2d),indicatedthattherewasminimalvariationinmosquitoattractiontotheeightpeopleassayed.
Thepersonchosentohuma-nisethemembranesintheexperimentsreportedherewastestsubjectAinordertokeepthebackgroundlevelofattractiontothemembranesconsistent.
Eachtreat-mentwasappliedatarateof10μlextractper19.
625cm2area,whichWakaetal.
[24]identifiedastheoptimaldoseperunitarea.
Odoursfromleafheadspaceweredilutedto5%byusinghexanegiving0.
3minequivalents(i.
e.
20sequivalents)appliedforfreshanddriedleaves,and0.
017minequivalents(i.
e.
1sequivalents)forthesmoke.
Everytreatmentwastestedinatwo-choiceland-ingassayinthepresenceofanegativecontrol(ahuma-nisedmembranetowhich10μlofthesamesolventisadded).
Forthepositivecontrolexperiments,DEET(5%and10%)wastestedinthesametwo-choicebioassay,alsointhepresenceofthenegativecontrol.
ThesolventFigure1Apparatusforcollectingvolatilesfromthesmokeofburningleaves(a)inacharcoalbrazier(b).
VolatileswerecollectedonaTefloncolumn(55mm*3mminnerdiameter)filledwithSuperQabsorbent(35mg,mesh80/100)(c)whichisprotectedfromnon-volatilesmokeparticulatesbyaglasswoolplug(50mm)(d)intheneckofthefunnel.
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page3of14Figure2Controlexperimentsforthetwochoicelandingassaywiththehumanisedmembrane.
(a)Rateofattractionoftwentyfemalemosquitoes(Aedesaegypti)becomesconstantafter4minofexposuretountreatedhumanisedmembranes.
ThenumberofAe.
aegypti(b)andAnophelesarabiensis(c)femaleslandinginthetwo-choiceassayonmembranesbothhumanisedbydifferenttestsubjectsvariesminimally.
ThenumberofAe.
aegyptifemaleslandinginthetwo-choiceassayonmembranes,onehumanisedbytestsubjectAandtheotherbyeithertestsubjectBorC,doesnotsignificantlydifferbetweentreatments(d).
ThenumberofAe.
aegyptiandAn.
arabiensisfemaleslandinginthetwo-choiceassayonmembranesbothhumanisedbytestsubjectA(e).
TherateoflandingonmembraneshumanisedbytestsubjectAremainsconstantfrom4minto14minafterbothspeciesofmosquitoeshavebeenexposedtothemembranes(f).
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page4of14usedintheDEETexperimentswasdichloromethane,aneffectivesolventforthelong-termstorageofDEET.
Duetoitslowboilingpoint(40°C),dichloromethaneevapo-rateswithinsecondsofapplicationtothemembrane,andthus,likehexane,willnotinterferewiththelandingbioassayresultsasmorethan4minhavepassedafterapplicationtothemembranebeforedatacollectionbegins.
TwodosesofDEETwereusedsincetheyarecommonincommercialpreparationsusedonbothchil-drenandadults.
Thelandingbioassay,carriedoutinSweden,usedtwoHemotekchambers(6cmdiameter;Figure3a)withhumanisedmembranes,onewithsolventaddedasacontrolandtheotherwiththetestextract,whichwereplacedagainstthetopnettingofthecage(Figure3b).
Twentynon-blood-fedfemalemosquitoeswerereleasedintoa30cmcubicgauzecagefor3htoacclimatizeandthenplacedina30cmcubictestcage.
Experimentswerecarriedoutunderstandardinsectaryandlabora-torytestingconditions[11]duringthemosquitoes'mostFigure3Behaviouralbioassayequipmentusedinthisstudy.
Membranefeedingchambers(a)fromthestore-boughtapparatus(Hemotek)aredisplayedhere.
Landingbioassaychambersfromastore-bought(Hemotek)usedforAedesaegyptiinSweden(b)andalab-constructedmembranefeedingapparatususedforbothAe.
aegyptiandAnophelesarabiensisinEthiopia(c).
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page5of14activeperiods:atdawnanddusk(06:00-08:00and17:00-19:00)forAe.
aegyptiandduskforAn.
arabiensis(17:00-19:00).
After4minofexposuretothetreatments,thetimeittakestoreachaconstantrateofmosquitoattractiontothehumanisedmembranes(Figure2a),thenumbersofmosquitoeslandingonboththeextracttreatedandthesolventtreatedhumanisedmembraneswerecountedat1-minintervalsfor6min.
Choiceindices(CITandCIC),aswellasarepellenceindex(R),weredeterminedforeachtreatmentasfollows:CIT=T/(T+C);CIC=-C/(T+C);and%R=(C-T)/C*100%[26-28];whereTisthetotalnumberofmosqui-toeslandingontheextracttreatedhumanisedmem-braneeachminutefor6minandCisthetotalnumberofmosquitoeslandingonthesolventtreatedhumanisedmembraneeachminutefor6min.
Theexperimentswerereplicated3-5times.
Thetreatmentandcontrolchamberlocationswerealternatedbetweeneachtesttocontrolforanypotentialpositioneffect.
Abioassaychamber,similarinconstructiontothestore-boughtHemotekapparatus(Figure3b),wasbuilttoconductthelandingassaysinEthiopiausingametalwaterbath,regulatoryheater,pumpsandTeflontubes(Figure3c).
Thewaterbathwasfittedwithtwocham-bers(6cmindiameter)protrudingdownwardsfromitsbaseandmadefrommetalpipes(10cminlength).
Insidethebath,theheaterwasadjustedto37±3°C,tosimulatehumanbodytemperature.
Inordertomaintainthetemperatureatauniformlevelthroughoutthebathandtwochambers,thetwopumpsinsidewerecon-nectedwithTeflontubestocirculateheatedwater.
Thislab-constructedchamberalsomadeuseoftheHemotekbrandmembraneandbothAe.
aegyptiandAn.
arabien-siswereevaluatedfortherepellencepotentialofvola-tilesfollowingsimilarproceduresasfortheHemoteklandingbioassaydescribedabove.
ChemicalanalysisVolatileextractsfromtheleavesofallfiveplantswereassessedusinggaschromatography(GC)and,subse-quently,freshleafextractswereevaluatedbycombinedGCandmassspectrometry(GC-MS).
ExtractswereinjectedontoaHP6890gaschromatograph(AgilentTechnologies,PaloAlto,CA,USA)fittedwithasplit-lessinjector(220°C)andflameionizationdetector(FID)(220°C).
Volatileswereseparatedonafusedsilicacapillarycolumn(30m*0.
25mminnerdiameter)coatedwithDB-WAX(df=0.
25μm).
Hydrogenwasusedasthemobilephase(speed45cms-1).
Theoventemperaturewasheldat40°Cfor2minandthenincreasedat10°Cmin.
-1toafinaltempera-tureof230°C,whichwasheldfor10min.
TheidentificationofactivecompoundsintheextractswasperformedbyGC-MS.
Eachextract(2μl)wasinjectedontoa6890Ngaschromatograph(AgilentTechnologies)coupledtoa5975massspectrometer(AgilentTechnologies).
CompoundswereseparatedonasimilarcapillarycolumnasintheGC-analysisabove.
Themobilephasewashelium(speed35cms-1).
Theoventemperaturewasheldat40°Cfor2minandthenincreasedat10°Cmin-1toafinaltemperatureof230°C,whichwasheldfor10min.
Theidentityofactivecom-poundswasdeterminedbycomparisonwithreferencesfrommassspectrallibraries(e.
g.
NIST05,AgilentTech-nologies)andKovatsindices.
PhysiologicalanalysisTheGCwasfittedwithasplitattheendofthecolumn,deliveringhalftheeffluenttotheFIDandtheotherhalfthroughaheatedtransferline(230°C)intotheairstreampassingoverthemosquitoantennamountedforelectroan-tenno-detection(GC-EAD).
AglasscapillaryreferenceelectrodefilledwithBeadle-EphrussiRingerandgroundedthroughasilverwirethatwasinsertedintothebaseoftheheadofamosquito.
Asimilarrecordingelectrode,con-nectedtoahighimpedanceDCamplifierwithautomaticbaselinedriftcompensation,wasplacedoverthedistalcutendofanantenna.
TheantennalsignalwasstoredandanalysedonaPCequippedwithanIDAC-cardandtheprogramEADversion2.
3(Syntech,Kirchzarten,Germany).
Arepeatableresponse,indicatinganactivecompound,wasdefinedasadepolarizationoftheantennalsignalatthesameretentiontimeinatleastthreetrials.
FollowingtheputativeidentificationofantennalactivecompoundsinfreshleafextractsusingGC-EADandGC-MS,onecompoundsharedbythoseextractswasfoundtobebehaviourallyrepellent,-ocimene,andwasusedforfurtheranalysis.
Variousamountsofsynthetic-ocimenewereusedintheelectroantennographicassaytoconfirmitsphysiologicalactivityandtodeterminewhetheritinducedadoseresponse.
-Ocimenewasseriallydilutedinredistilledhexaneindecadicsteps(0.
001-10%).
Tenmicro-litresofeachdosewasaddedtoa0.
5cm2pieceoffilterpaperthenplacedintotheendofaglassPasteurpip-etteandallowedtoequilibrateforatleast20minprioruse.
Thetipofthesestimuluscartridgeswasthenplacedintotheairflowovertheantennaandtheairdivertedthroughthecartridgefor0.
5s.
Eachstimulusresponsehastheaverageoftwosolventblankresponses,onepriorandonefollowingthestimuluspulse,subtractedtodeterminetheantennalresponsetothetestvolatile.
Antennalresponsesarepresentedasaratioofmaximalresponse.
SyntheticchemicalsSyntheticvolatilesarecommonlyusedtoconfirmphysio-logicalandbehaviouralactivityofcompoundsidentifiedfromnaturalextracts[22].
Intheseexperiments,syn-thetic-ocimenewasusedtoconfirmtheactivityofthiscompoundputativelyidentifiedfromtheodourextractsDubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page6of14offreshleaves.
N,N-diethyl-m-toluamidewasusedasacontrolinthelandingassaystoindicatethemaximalrepellentbehaviourofthemosquitoes.
-ocimene(3,7-dimethyl-1,3,6-octatriene)andN,N-diethyl-m-toluamide(DEET),werepurchasedfromInternationalFlavorsandFragrances,R&D(No.
00151353;>90%)andSigma-Aldrich(LaborchemikalienGmbH,Seelze,Germany)respectively.
Dilutionsof-ocimeneandDEETforbioas-saysweremadeinre-distilledhexane.
StatisticalanalysisTheeffectivenessofvolatilecollections(treatment)wasevaluatedagainstsolventalone(control).
Therepellenceindex(R)wasestimatedas%R=(C-T)/C*100%,whereCandTarethemeannumberofmosquitoeslandingonthecontrolandthetreatmentmembranes,respectively[26-28].
Comparisonsofrepellenceindicesamongmosquitospecies,landingassaytype,plantspe-ciesandleaftreatmentswereanalysedbytheunba-lancedgenerallinearmodeloftheanalysisofvariance(ANOVA)statedasfollows:ABCC*AC*BDD*AD*BD*CD*A*CD*B*C;whereAisthemosquitospe-cies(Ae.
aegypti,An.
arabiensis),Bisthebioassay(store-bought,lab-constructed),Cistheplantspecies(Co.
citriodora,Oc.
suave,Oc.
lamiifolium,Os.
integrifo-lia,Ol.
europaeaandhandrubcontrol)andDisthetreatment(smoke,fresh,driedandcharcoalcontrol).
Dunnett'ssimultaneousposthoctestswereconductedtocompareresponsestotheextractswithasolventcon-trolinthelandingassays,andTukey'sposthoctestswereusedtocompareamongalloftheresponsestovariousdosesof-ocimeneinthephysiologicalandbehaviouralassaysasrequiredusingMINITABstatisti-calprogramversion14.
12.
0(Minitab2004).
ResultsBehaviouralresponsetothehumanisedmembraneUnderno-choiceconditionswithasinglehumanisedmembrane,therateofattractionofAe.
aegyptifemalesstabilisedafter4min(5±1perminute;Figure2a).
Mosquitoesweretestedforattractiontomembraneshumanisedbyeightdifferenttestsubjects.
Inano-choiceassaywithtwohumanisedmembranes,attractiontomembraneshumanisedbydifferenttestsubjectswasnotsignificantlydifferentineitherspecies(Ae.
aegyptic20.
1360,df2;An.
arabiensisc20.
3625,df6;Figure2band2c).
Inatwo-choiceassay,testsubjectAwasnotsignificantlymoreattractivethaneithertestsubjectB(pairedt-testt=0.
3492,df5)orC(pairedt-testt=0.
5058,df11)totheAe.
aegyptifemales(Figure2d).
TestsubjectAwasthereforechosentohumaniseallmembranesinthesubsequentexperiments.
Inno-choiceassayswithtwomembraneshumanisedbytestsubjectA,thelandingrateperminuteover10mindidnotdifferbetweenthespecies(unpairedt-testt=1.
247,df18;Figure2e).
TherateofattractiontothehumanisedmembraneswasdeterminedtobeconstantinAe.
aegypti(R2=0.
99987)andAn.
arabiensis(R2=0.
99981)over10min(Figure2f)followingthe4-minacclimatisa-tionperiod(Figure2a).
NeitherAe.
aegyptinorAn.
ara-biensisdifferedinthenumberoflandingsmadeonhumanisedmembraneswitheithertheextractfromthecharcoalsmokeheadspacecollectionorsolventcontrol(datanotshown).
BehaviouralresponsetoplantextractsThetwo-choicelandingbioassaysusingmembranefee-ders(Figure3)wereconductedunderlaboratorycondi-tionsonAe.
aegyptiandAn.
arabiensisfemales.
Ofthemosquitoesthatwereactivatedtofly(≥80%),thenum-berofmosquitoeslandingoneitheroftheprofferedmembraneseachminutefor6minallowedforthecal-culationofchoiceindicesassociatedwiththecontrol(CIC)andtreated(CIT)membranes(Figure4)aswellastherepellenceindex(R).
Agenerallinearmodel(GLM)fortheanalysisofvar-iance(ANOVA)oftherepellenceindiceswasdevelopedincludingfourfactors(bioassay,mosquitospecies,plantspeciesandleaftreatment).
PriortothegenerationoftheGLM,therepellenceindicesweredeterminedtofol-lowanormaldistribution(D'Agostino-Pearsonnormal-itytest,P>0.
05).
ThismodeldeterminedthatthelandingbehaviourofAe.
aegyptididnotdifferbetweenthestore-boughtandlab-constructedbioassays(F=0.
06;DFn=1;DFd=144;P=0.
81;Figure4).
Thepat-ternoflandingbehaviourofthefemalemosquitoes,whetherAe.
aegyptiorAn.
arabiensis,inresponsetoalltheextractsinthetwo-choiceassaydidnotsignificantlydiffer(F=2.
98;DFn=1;P=0.
087;Figure4).
TheGLMindicatedthattherewasnosignificantinteractionamonganyofthefactors(mosquito*plant,F=0.
15,DFn=5,P=0.
98;bioassay*treatment,F=0.
61,DFn=2,P=0.
55;plant*bioassay,F=0.
05,DFn=5,P=1.
00;bioassay*treatment,F=0.
95,DFn=15,P=0.
50;mos-quito*plant*treatment,F=0.
17,DFn=15,P=1.
00;bioassay*plant*treatment,F=0.
12,DFn=15,P=1.
00).
Therefore,furthercomparativeanalysesoffemalemos-quitoresponsesweremadeconsideringplantspecies(F=5.
93,DFn=5,P0.
001;Figure4).
GaschromatographyandmassspectroscopyThesmokedleafextractsresultedinhighlycomplexchromatogramsthatweredifficulttointerpret,whiledriedleafextractsappearedtohaveverylowlevelsofvolatilecompoundsonthechromatogram.
Forthesereasons,theinvestigationsfocusedonthefreshleafextracts(Figure5).
TheGC-EADwasperformedusingthefreshleafextractonAe.
aegyptiantennae(datanotshown).
TherepeatedphysiologicalresponseoftheantennaetooneGCpeak(10.
94min.
)wassharedamongstthemostrepellentheadspaceextracts,Co.
citriodora,Oc.
suaveandOs.
integrifolia,andthusOcimumsuaveCorymbiacitriodoraOstostegiaintegrifolia0'"1#('0"*'"1Figure5Identificationof-ocimene.
Totalionchromatograms(TIC)oftheheadspacecollectedfromfreshleavesofplantswhoseextractswereshowntoberepellentintwo-choiceassays.
ThearrowsindicatethepeakthathassincebeenidentifiedasZ--ocimeneusingmassspectrometry.
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page9of14promptedfurtherinvestigation.
Theputativeidentifica-tionofthiscompoundthroughGC-MSdetermineditwasZ--ocimene.
TherelativeamountsofZ--ocimenepresentintheheadspaceofthesethreespecies,andnotfoundintheothertwo,reflectedtheheadspacerepel-lencedeterminedinthebioassays(Figures4and5).
AlthoughotherGCpeaksweresharedamongthesethreespecies(e.
g.
at17.
5min.
),noneoftheseshowedconsistent,repeatablephysiologicalresponsesduringGC-EADanalyses,thereforethesepeakswerenotpur-suedfurtherinthisstudy.
Synthetic-ocimeneAmixtureofbothgeometricisomers(E-andZ-)of-ocimeneisdetectedbytheantennaeoffemalesofbothmosquitospeciesunderinvestigation,An.
arabiensisandAe.
aegypti.
Usingelectroantennogram(EAG)record-ings,theantennalresponseofAn.
arabiensisandAe.
aegyptitosyntheticocimenewasfoundtobedosedependent(r2=0.
9325andr2=0.
998,respectively;F=26.
8,DFn=6,P0.
05).
Thebeha-viouralsensitivityofbothmosquitospeciestoDEETisapproximately10timesthatof-ocimene,asestimatedbyED50(0.
1%DEETand1-2%-ocimene).
Whilethereisnosignificantinteractionbetweenspeciesanddose,thetwomosquitospeciesappeartobehavesignificantlydifferentlyto-ocimene(2-wayANOVA;F=7.
279;DFn=6,P=0.
010)withAe.
aegyptibeingmarginallymoresensitiveto0.
7%-ocimene(TukeyposthocP=0.
05;Figure7).
DiscussionThepresentstudydemonstratesthatodoursfromtheleavesoffiveplantstraditionallyusedinEthiopiaaspro-tectionfrommosquitobiteshaverepellentproperties.
Theheadspaceextractsofsmokefromburningthese/7557595;5=56555-555565-55565-5565-565-6665655+0A)'1!
/0A1Figure6Antennalresponsetosynthetic-ocimene.
FemaleAnophelesarabiensis(diamonds;n=6)andB.
Aedesaegypti(circles;n=4)antennalresponseto-ocimeneusinganelectroantennogram(EAG).
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page10of14leavesweremorerepellentthanthosefromfreshordriedleavesofthesameplantspeciestohost-seeking(i.
e.
non-blood-fed)femalemosquitoesofbothAn.
ara-biensisandAe.
aegypti,theprimarymalariaanddengue/yellowfevervectorsinEthiopia,respectively.
Previousstudies,inwhichOs.
integrifolia[20],Ol.
europaea[20],Co.
citriodora[16,21]andOc.
suave[16]wereburnedtorepelmosquitoes,havealsodemonstratedalargereduc-tioninthenumberofmosquitoeslanding.
Notonlythese,buttherearealsomanyotherexamplesofburn-ingleavestodecreasethenumberofmosquitoesinthehouse,someofwhichhavealsoresultedinthereduc-tionofotherarthropodvectordensitiesindoors,suchasthesandflyandblackfly[29,30].
AccordingtoHoeketal[31]thereisasignificantlylowerriskofmalariainhouseholdsusingtraditionalsmokesandfumigants,suchastheburningleavesinthisstudy,comparedwiththosethatdidnot.
Thesepreviousstudiesontheeffectsofplant-derivedsmokeonarthropodvectorssupportourpresentfindings,whichclearlydemonstratethepotentialfortheuseofcombustion-releasedvolatilesagainstmosquitoes.
Somepossiblemechanismsfortheactionofplant-derivedsmokehavebeenproposed[1].
Thesmokemaydisguisehumankairomonecuesusedbythevectorstotargettheirhosts,itmaydisrupttheconvectioncurrentsessentialformosquitohostlocation,and/ortheburningoftheseleavesmayreleasevolatilecompoundsthatactasrepellentsorirritantsagainstthemosquito.
Smokefromcommonfirewoodhasbeenreportedtobeamos-quitorepellentbydeterringmosquitoesfromroostinginhouses[30,32]suggestingthatmaskingandcurrentdis-ruptionplayaroleintheefficacyofleafsmoke/fumi-gants.
Thecurrentstudy,however,investigatedthethirdpremiseandhasdemonstratedthattheprotectionagainstthemosquitoesresultingfromtheburningofdriedleavesappearstobeduetothereleaseofvolatilecompoundsduringcombustion.
Moreover,theback-groundodoursfromthecharcoalfuelusedforsmokeodourcollectiondemonstratednorepellentpropertieswhentestedinthelandingbioassay.
Thus,atleastaportionoftherepellentpotentialofsmokeheadspaceoriginatesfromtheplantvolatilesthemselves.
Heat,convectioncurrentsandparticulatesinthesmokemayalsoplayaroleintheprotectionprovidedbyplant-derivedsmokeasdiscussedabove,butthiswasnottestedinthisstudy.
Mosquitomembranefeedershavepreviouslybeenusedtotestarthropodrepellenceinbioassaysusingani-malbloodorskinasthebackgroundattractantforthemosquitoes[24,33].
Inthisstudy,analternativemethodforscreeningrepellentswasfoundtobe'humanisingthemembranes'byrubbingthefeedingmembranewithhumanhandstotransfertheappropriatehostodours.
565758595:5;556556655-55565-5565-565-6665655)0A1!
/0A1Figure7Behaviouralresponsetosynthetic-ocimene.
FemaleAedesaegypti(circles,n=10)andAnophelesarabiensis(diamonds,n=5)landingbehaviourinresponseto-ocimene(circlesanddiamonds)andDEET(triangles,n=3)addedtoahand-rubbed,heatedmembranecomparedtoahand-rubbed,heatedmembranealoneinatwo-choiceassay.
Dubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page11of14Oneadvantagewiththehumanisedmembranesisthattheresultsobtainedfromthesebioassaysrelatedirectlytotheefficacyofrepellentforhumanratherthananimalprotection.
Emanationsfoundassociatedwithhumanhands,suchassweat,aredetectedby[34,35],andshowntobebehaviourallyattractiveto,mosquitoes[36].
Also,humanisingthemembranesisfast,ethicallysafeandcanbeeasilydoneunderlaboratoryorfieldcondi-tions.
Thetwolandingbioassayarenasusedinthisstudy,onestorebought(Hemotekmembranefeeder,DiscoveryWorkshops,UK)andtheotherselfassembled(F.
F.
Dube,AddisAbabaUniversity,ET),werefoundtobeequallyefficient,demonstratingagainthesimplicityandeaseofuseofthistechnique.
Amongtheplantspeciestested,theheadspaceextractsfromburningthedriedleavesofCo.
citriodoraandOc.
suavewerethestrongesthost-seekingfemalemosquitorepellent(>65%)bothforAn.
arabiensisandAe.
aegyptiinthelaboratory.
TheheadspaceextractsofCo.
citriodoraandOc.
suavefreshleaveswerealsoshowntobesignificantlyrepellent(>50%)whilethedriedleavesofnoneofthespecieswerestronglyrepel-lent(<30%).
ThesmokefromleavesofCo.
citriodorawaspreviouslysuggestedasastrongmosquitorepellentincontrolledsemi-fieldstudiesusingvolatilesexpelledthroughheatingtheleavesonmetalplate[15,16,21].
Theincreasedrepellentpotencyoftheheadspaceofburningleavesmaybeduetotheincreasedreleaserateofrepellentvolatilecompounds,eitheralreadypresentinthefresh/driedleafand/orcreatedduringthecom-bustionprocess.
Thatrepellentcompoundsarepresentintheleavesofmanyplantspecieshasbeenwelldocumented[12,37,38].
Boththejuicesandtheessentialoilextractsfromleaveshavearthropodrepellentproperties.
Forexample,theessentialoilextractedfromCo.
citriodoracanactdirectlyasanaturalinsectrepellenttoprovideprotectionagainstmosquitoesandotherharmfularthropods[37].
Infact,PMD,acompoundfoundintheessentialoilofCo.
citrio-dora[12],istheonlynaturallyderivedactiveingredienttobecertifiedasaninsectrepellentinEuropeandtheUSAthatiscommerciallyavailable.
ThejuicefromtheleavesofOc.
suaveandOc.
canumspreadonthelegsofhumanvolunteershasapproximately50%reductionofmosqui-toeslanding[38].
Z--ocimenewasidentifiedfromthefreshleafhead-spaceextractsofCo.
citriodora,Oc.
suaveandOs.
integri-foliaasaneffectivemosquitorepellentcompoundinthehumanisedmembranelaboratoryrepellenceassay.
Amixtureofbothgeometricisomersof-ocimene(14%)wasnotsignificantlydifferentinrepellentactivityfromDEET(10%)over6mininthelandingassay.
-ocimenehasbeenidentifiedpreviouslyinvolatileemissionsofCo.
citriodora(ca.
10%-ocimene)[39].
Inthesearchforrepellents,butmoreoftenforinsecticides,-ocimenehasbeenidentifiedasamajorcomponentofleafessentialoils,e.
g.
Oc.
suave(13.
5%Z--ocimene,[40])aswellasotherplantspecies[41-44].
Whilecommonpracticeassessestheentireessentialoilforitsbioactivity,-oci-meneitselfhasbeenshowntobeaneffectiveinsecticideagainstsomecroppests[45]andhoneybeemites[46].
Essentialoilscontainingahighproportionof-ocimenehavebeenassessedforlarvicidalactivityagainstmosqui-toes[43,44,47-49],howeveruntilnow,-ocimenealonehasnotbeenevaluatedforitsefficacyasarepellentofhost-seekingmosquitoes.
Theresultsindicatethat-oci-meneisaninterestingbiologicallyactivevolatilethatshouldbeincludedinfurtherexaminationsofplant-derivedmosquitorepellents,includingconfirmationofmosquito-repellentactivityusingconventionalrepellenceassays[10,11]aswellasdurationofactionandefficacyinblendswithothernaturallyderivedcompounds.
As-ocimeneisahighlyvolatilecompound,itsformulationforuseasaskin-appliedrepellentiscriticalwhenitcomestoitsdurationofaction.
Someformulationmeth-odstoreducethevolatilityofsuchcompoundshavebeendescribed[50].
Forexample,theadditionofalargemole-culesuchasvanillincansubstantiallyextendthedurationofactionofothernatural,buthighlyvolatile,repellents[50].
Thecommonuseof-ocimeneasapleasingscentincommercialproducts,e.
g.
alcohol-andcream-basedperfumes,whichneedtocontinuetoreleasetheirscentoveranumberofhoursafterapplication,bodeswellforthepossibilityoffindinglong-lasting,nottomentionpleasantsmelling,repellentformulationscontaining-ocimene.
-ocimenemaybeacheapandlocallyavailablemosquitorepellentwithaninoffensiveodourthatgov-ernmentandregulatorybodies,suchastheEUEFSA[51]andUSFDA[52],havealreadycertifiedassafeforuseinproductsappliedtohumanskinatconcentrationsupto20%(withtherecommendeddoseof5%topreventthepossibilityofskinirritationorsensitisation[51]).
Theseconcentrations,currentlyusedinmanycommer-ciallyavailableproducts(e.
g.
perfume,soapanddeodor-ant)appeartobewithintherepellentactivityagainstbothmosquitospeciestestedinthelaboratory.
ConclusionsTherefore,furtherstudiesareproposedtocharacterizetherepellentpotentialof-ocimeneandtoidentifyotherpotentialvolatilesfromheadspaceleafextractsofCo.
citriodoraandOc.
suave.
Theuseofatwo-choicelandingbioassaywithhumanisedmembranesprovidingtheback-groundemanationpromisestobeanefficientscreeningtechniquefortheassessmentofecologicallyrelevantpotentialmosquitorepellents,betheyheadspaceextracts,essentialoilsorsyntheticcompounds.
Whilethistechni-querepresentsapotentiallyusefulrepellentscreeningDubeetal.
MalariaJournal2011,10:375http://www.
malariajournal.
com/content/10/1/375Page12of14techniquethatdoesnotrequiretheexposureofhumanstothevectors,furthertestsinparallelwithconventionaltechniquesareadvised.
AcknowledgementsTheauthorswouldliketothankLinnaeus-PalmeStudentExchangeProgrammeandtheInsectChemicalEcology,EthologyandEvolution(IC-E3)togetherwithfundsfromFormasandSLUfortheirfinancialsupport,withoutwhichthecollaborationbetweenAddisAbabaUniversityandtheSwedishUniversityofAgriculturalSciences,necessaryforthecompletionofthiswork,wouldnothavebeenpossible.
MeridNegashisacknowledgedforhistechnicalcontributioninplantandvolatilecollection.
ThanksisofferedtotheWHOMalariaControlCenterinNazarethEthiopia,WondoGenetEssentialOilsResearchCentre,AkliluLemmaInstituteofPathobiology,andInstituteofBiodiversityConservationforaccesstotheirfacilitiesandmosquitocoloniesaswellasforinformativediscussionsthroughoutthisstudy.
Authordetails1DivisionofChemicalEcology,DepartmentofPlantProtectionBiology,SwedishUniversityofAgriculturalSciences,23053Alnarp,Sweden.
2DepartmentofPlantScience,McGillUniversity,LakeshoreRoad,Ste-Anne-de-Bellevue,QuébecH9X3V9,Canada.
3DepartmentofBiology,AddisAbabaUniversity,POBox1176,AddisAbaba,Ethiopia.
Authors'contributionsFFDcarriedoutthebehavioural,GC,GC-EADandGC-MSexperiments,andconstructedthemembrane-landingassayinAddisAbaba.
GBcarriedouttheGCandGC-MSanalysestogetherwithFFD.
KTcarriedoutthedoseresponseEAGexperimentswith-ocimene.
ES,togetherwithRIandSRHconceivedthestudyandparticipatedinthestudydesign.
ESandHTcoordinatedtheEthiopianstudies,whileRIandSRHdidthesameinSweden.
SRHperformedthestatisticalanalyses.
FFDandSRHdraftedthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
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