a pyrethroid-treated bed net increases host attractiveness for

1

Apyrethroïd-treatedbednetincreaseshostattractiveness1

forAnophelesgambiaes.s.carryingthekdralleleinadual-2

choiceolfactometer3

Angélique PORCIANI1,2,*,¶, Malal DIOP1,2,¶, Nicolas MOIROUX3, Tatiana KADOKE-LAMBI2, Anna4

COHUET3,FabriceCHANDRE3,LaurentDORMONT4,CédricPENNETIER1,25

1: MiVEGEC (UM1-UM2-CNRS 5290-IRD 224), Institut de Recherche pour le Développement (IRD)6

Cotonou,Bénin.7

2:CentredeRechercheEntomologiquedeCotonou(CREC),Bénin8

3:MiVEGEC (UM1-UM2-CNRS 5290-IRD 224), Institut de Recherche pour le Développement (IRD),9

Montpellier,France.10

4:CNRSUMR5175,Centred’EcologieFonctionnelleetEvolutive(CEFE),Montpellier,France.11

*Correspondingauthors12

Email:[email protected]

¶Theseauthorscontributedequallytothiswork14

15

.CC-BY-NC-ND 4.0 International licensenot peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was. http://dx.doi.org/10.1101/077552doi: bioRxiv preprint first posted online Sep. 26, 2016;

http://dx.doi.org/10.1101/077552

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

Abstract16

Theuseoflonglastinginsecticidenets(LLINs)treatedwithpyrethroïdisknownforitsmajor17

contributioninmalariacontrol.However,LLINsaresuspectedtoinducebehavioralchangesin18

malariavectors,whichmayinturndrasticallyaffecttheirefficacyagainstPlasmodiumsp.19

transmission.InsubSaharanAfrica,wheremalariaimposestheheaviestburden,themainmalaria20

vectorsarewidelyresistanttopyrethroïds,theinsecticidefamilyusedonLLINs,whichalso21

threatensLLINefficiency.Thereisthereforeacrucialneedfordecipheringhowinsecticide-22

impregnatedmaterialsmightaffectthehost-seekingbehaviorofmalariavectorsinregardsto23

insecticideresistance.Inthisstudy,weexploredtheimpactofpermethrin-impregnatednetonthe24

hostattractivenessforAnophelesgambiaemosquitoes,eithersusceptibletoinsecticides,orcarrying25

theinsecticideresistanceconferringallelekdr.Groupsoffemalemosquitoeswerereleasedinadual-26

choiceolfactometerandtheirmovementstowardsanattractiveodorsource(arabbit)protectedby27

insecticide-treated(ITN)oruntreatednets(UTN)weremonitored.Kdrhomozygousmosquitoes,28

resistanttoinsecticides,weremoreattractedbyahostbehindanITNthananUTN,whilethe29

presenceofinsecticideonthenetdidnotaffectthechoiceofsusceptiblemosquitoes.Theseresults30

suggestthatpermethrin-impregnatednetisdetectablebymalariavectorsandthatthekdrmutation31

impactstheirresponsetoaLLINprotectedhost.Wediscusstheimplicationoftheseresultsfor32

malariavectorcontrol.33

34


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Introduction35

Anophelesgambiaeisoneofthemajormosquitovectorsofhumanmalariaparasitesinsub-36

SaharanAfrica.Itsremarkablevectorialcapacity[1]mainlyreliesonitshighdegreeofanthropophily.37

Moreover,An.gambiaepreferstobitehumansindoorsandoftenrestsinsidehousesafterblood38

feeding[2–4].Thesebehavioralpreferencesledtothedevelopmentofinsecticide-basedindoor39

vectorcontrolmeasures,suchasinsecticide-treatedbednets(ITNs)andindoorresidualspraying40

(IRS),tolimitthehuman-vectorcontactsandreducemosquitosurvival.Todate,fourinsecticide41

familiesareavailableforIRS(organochlorides,organophosphates,carbamatesandpyrethroïds),42

whereasonlypyrethroïdsarerecommendedformosquitonetsbecauseoftheirlowmammalian43

toxicityandhighinsecticidalpotency[5].44

Tokill,insecticidemoleculesmustcontactandpenetratethroughthemosquitocuticle/gut45

tothenreachandinteractwiththeirtargetbeforebeingdegraded.Anyphysiologicalorbehavioral46

mechanismthatmayinterferewithoneofthesestepscanleadtoinsecticideresistance.The47

widespreaduseofpyrethroïd(PYR)insecticidesinmalariavectorcontrolandagriculturehasfavored48

thedevelopmentofresistanceinmalariavectorspecies[6].Oneofthemoststudiedphysiological49

mechanismsinvolvedinPYRresistanceisthereducedsensitivityofthevoltage-gatedsodium50

channelstoPYRbindingcausedbynon-silentmutations,knownasknockdownresistance(kdr)51

mutations[7].BehavioralresistanceisanothermechanisminvolvedinPYRresistance.Thiscanbe52

definedasamodificationofthemosquitobehaviortoavoidcontactwithalethaldoseofinsecticide53

[8].Todate,behavioralresistancetoinsecticidesremainspoorlydocumented,despiteofitshuge54

potentialimpactonmalariatransmission.55

Behavioraladaptationstopesticidescanbeclassifiedasstimulus-dependentor-independent56

[9].Stimulus-independentadaptationsarenotassociatedwiththeperceptionofchemicals,butmore57

probablywithmodificationsofthevectorintrinsicbehavior,suchaschangesinhost-seeking58


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behaviorpreferences(levelofanthropophily,endophagy,endophilyorhourlybitingactivities).Such59

behavioralmodificationshaverecentlybeenobservedinthecontextofITNwidespreaduse:60

mosquitovectorsmaypostponetheirbloodfeedinguntilthemorning,whenhumanhostsare61

protectedbyITNsanymore[10–12].Thesechangesmaylimitthecontactbetweenaggressive62

malariavectorsandtreatedsurfaces,thusthreateningtheefficiencyofindoorvectorcontroltools.63

Conversely,stimulus-dependentbehavioraladaptationsarespecificallylinkedtothedetectionof64

chemicals.Stimulus-dependentinsecticideavoidancecanbedefinedasa“flyaway”behaviorto65

leavetheimmediatetoxicenvironmentaftercontact(irritancy)ornot(repellence)withthetreated66

surface[13–15].AvoidancebehaviorfollowingcontactwithPYRhasbeenreportedinsomecases67

[16–20],butsimilarbehaviorintheabsenceofdirectcontactwiththeinsecticidehasbeenpoorly68

documented.OnlyindirectobservationssuggestadetectionandavoidanceofITNsbymalaria69

vectors:mosquitoentryrateswerefoundreducedinexperimentalhutscontaininginsecticide-70

treatednetscomparedtoentryratesincontrolhuts,moreovertheobservedratesweredependent71

onthekdrallelepresenceinthemosquitoes[21–23].Althoughtheeffectsofpyrethroïdson72

differentpartofhostseekingbehaviorhasbeenalreadystudied[20,24–26],theirinfluenceonthe73

relativehostattractivenesshasbeenneglecteddespiteitsimportanceinhostchoiceandonmalaria74

transmission.Therefore,inordertoadequatelyevaluateanduseITNs,ithasbecomeurgentto75

investigatethepossiblemodulationofthehost-seekingbehaviorinpresenceofindoorvectorcontrol76

toolsinregardstootherinsecticideresistancemechanisms77

Inthisstudy,weexaminedthelong-rangehost-seekingbehaviorofAn.gambiaemosquitoes78

todeterminewhethertheattractivenessofavertebratehost(arabbit)inadual-choiceolfactometer79

wasinfluencedbyphysicaland/orchemicalbarriers(insecticide-treatedanduntreatednets)andby80

themosquitokdr(L1014F)genotype.81

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Methods83

Ethicsstatement84

RabbitswerehandledandblooddrawninaccordancetotheprotocolapprovedbyNationalComity85

forEthicandResearch(CNERS)andHealthministryofBenin(N°023).Thisstudywascarriedoutin86

strictaccordancewiththerecommendationsofAnimalCareandUseCommitteenamed“Comité87

d’éthiquepourl’expérimentationanimale;LanguedocRoussillon”andtheprotocolwasapprovedby88

theCommitteeontheEthicsofAnimalExperiments(CEEA-LR-13002fortherabbits).Rabbitswere89

notsubjectedtoanesthesia,analgesiaorsacrifice.90

Mosquitoes91

TwolaboratoryreferencestrainsofAnophelesgambiaesensustricto(formerlycalledSmolecular92

form)(20)wereusedinthisstudy.TheKisumureferencestrain,isolatedinKenyain197593

(VectorBase,http://www.vectorbase.org,KISUMU1),isfreeofanydetectableinsecticideresistance94

mechanism.Thekdr-kisstrainwasobtainedbyintrogressionintotheKisumugenomeofthekdr-west95

allele(L1014F)[27]thatoriginatedfromaPYR-resistantpopulationcollectedinKouValley,Burkina96

Faso,whichwasusedtoestablishastrainnamedVKPer.Introgressionwasobtainedthrough1997

successiveback-crossesbetweenKisumuandVKPer[28].VKPerstraindisplayedthesameexpression98

levelofmetabolicresistanceenzymeasKisumu[29].Kisumuandkdr-kismosquitoesaretherefore99

homozygoussusceptible(SS)andhomozygousresistant(RR)atthekdrlocus,respectively. The100

heterozygousgenotypeRSwasobtainedbycrossingKisumuSSfemaleswithkdr-kisRRmales.101

Mosquitoeswererearedininsectaryconditions(27±3°C,60-80%relativehumidityanda12:12light102

anddarkcycle).Groundcatfoodwasusedtofeedlarvaeand10%sucrosesolution(withrabbit103

bloodtwiceperweek)tofeedadultfemales.Forbehavioralexperiments,5-12dayoldfemales,104

withoutprioraccesstoabloodmeal,werestarvedfor4hbeforetheassay.105


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Experimentalset-up106

Thedual-choiceolfactometerwasadaptedfromGeierandBoeckh(1999)[30].Itwasmadeof107

Plexiglasandwasdividedinfourparts:releasezone(RZ),flightchamber(FC)andonecollectingzone108

ineachofthetwoarms(A1orA2)(Fig1).RotatingdoorsmadefrommeshgauzeintheRZandin109

botharmsallowedmosquitoreleaseorcapture.Theupwindpartoftheexperimentalset-upwas110

composedofawidechamberwhereanattractivehost(arabbit)canbeplaced,andthatwas111

connectedtotwotreatmentboxesthatcontainedornotthenets.Eachtreatmentboxwas112

connectedtoonearmoftheolfactometer.Inordertoavoidanyperturbationontheairflowbythe113

treatment,fanswereplacedonthedownwindfacesoftheexperimentboxesandextractedtheair114

fromthetreatmentboxestotheolfactometer,providingtheodor-ladenaircurrent.Atthebeginning115

ofeachexperiment,theairflowwasmeasuredinarm1and2andinthereleasezoneusingaTesto©116

435-1multifunctionalmeter(Testo,Forbach,France)andthermo-anemometricprobe(m.s-1)and117

adjustedat0.20±0.03m.s-1.Duringtheexperiment,athickblacktarpaulincoveredtheolfactometer118

tokeepallthesystemindarknessandavoidvisualdisturbance.119

Fig1.Experimentalset-up.120Dual-choiceolfactometer(rightside)connectedtothetreatmentboxes(middle)andthewide121chamber(leftside).122123

Experimentaldesign124

Fourexperiments,summarizedinTable1,wereperformedusingSS,RRandRSmosquitoes.The125

treatmentboxesandthewidechamberwereemptyduringthefirstexperiment.Fortheother126

experiments,thewidechambercontainedarabbitasodorsource.Thetreatmentboxescontained,127

dependingontheexperiment,nothingor2m2ofuntreated(UTN)orinsecticide-treatednet(ITN,128

Olyset®Netimpregnatedwith1000mg/m2ofpermethrin).Netsweredividedin50piecesof129

20x20cmandhungonametallicstructureperpendicularlytotheairflow.Thesamenetswereused130

duringallexperiment,theOlyset®wasconservedat4°Cbetweeneachdayofexperiment.Thenets131


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wereplacedinboxesthatcouldnotbevisibleformosquitoes,sothatnovisualclueswereavailable132

tomosquitoesduringtheexperiments.133

Table1:Descriptionoftheexperimentaldesign134

135

136

137

138

(UTN:untreatednet,ITN:insecticide-treatednet)139

Assaysforthefourexperimentswereperformedeverydayfor20daysbetween10:00amand140

14:00pm(correspondingtomosquitostrainfeedingtimeinlaboratory).Wealwaysstartedwith141

assaysofexperiment1,tocheckpossibleodororinsecticidecontaminations.Whenpossible(i.e.,142

whentheinsectaryproductionwassufficient),femalesofthethreegenotypesweretestedthesame143

dayforthefourexperiments,otherwiseatleasttwogenotypesweretestedthesameday(a144

summaryoftheassaysispresentedinsupplementarydata).Eachday,inassaysforexperiments3145

and4,treatmentswererotatedonetimebetweenboxestopreventanyarmeffect.Between146

rotations,theboxeswerecarefullycleanedwithethanoltoavoidanyresidualinsecticideeffect.147

Moreover,theolfactometerwascleanedwithethanoleveryday.Theexperimenterworelatex148

glovestoavoidcontamination.Thesamerabbitwasusedasodorsourceduringallthestudy.Itwasa149

1-yearoldfemalerearedinthesameconditionsasthoseusedininsectariestofeedmosquitoes.CO2150

concentrationandrelativehumidity(RH)weremonitoredineacharmswithaTesto©435-1151

multifunctionalmeter(Testo,Forbach,France)equippedwithanIndoorAirQuality(IAQ)probe152

[%RH;range:0to+100%RH;accuracy:±2%RH(+2to+98%RH)],[CO2;range:0to+10000ppm;153

accuracy:(±75ppmCO2±3%ofmv)(0to+5000ppmCO2)].Theroomwaskeptataconstant154

temperatureof25°Cduringthestudy.155

Experimentno.

Experimentname

Odorsource Treatment box1

Treatment box2

1 Empty None Empty Empty2 Rabbitalone Rabbit Empty Empty3 Rabbit+UTN Rabbit Empty UTN UTN Empty4 Rabbit+ITN Rabbit ITN UTN UTN ITN


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Foreachassay,abatchof20-23femaleswasreleasedintheRZfor5minforacclimation.The156

rotatingdoorswerethenopenedandfemaleswerefreetoflyintheolfactometer.After5minutes,157

therotatingdoorswereclosedandthenumbersofmosquitoesinRZ(NRZ),FC(NFC),A1andA2(NA1158

andNA2)wererecorded(Figure1).159

Behavioralindicators160

Theindicatorsusedinthisstudydescribethemosquitoprogressinsidetheolfactometerand161

therelativeattractiveness(RA)oftreatmentsorarms.162

Twoindicatorsoftheprogressioninsidetheolfactometerwerecalculated.First,upwind163

flight(UF)thatistheproportionoffemalethatleftthereleasezone(i.e.collectedinFC,A1andA2)164

relativetothetotalnumberofreleasedmosquitoes(N).Secondisthelocalization(L)ofodorsource165

thatistheproportionoffemalethatreachedA1andA2(NA1andNA2),relativetothenumberof166

mosquitoesthatlefttheRZ(N-NRZ).Theseindicatorswerecalculatedforeachreleaseandforeach167

odorsource(none,rabbitwithoutITNandrabbitwithITN).168

Theupwindflightandlocalizationvaluesmeasuredinexperiment1(emptyset-up,cleanair)are169

baselineindicatorsoftheanemotacticresponseofthethreemosquitogenotypestoairflow.The170

influenceofrabbitodoronmosquito’sprogressionwasdeterminedbycomparingthevaluesof171

upwindflightandlocalizationrecordedintheemptysystem(experiment1)withthoserecordedin172

thesystemwithoutITN(mergedUFandLvaluesofexperiments2and3).Themergedupwindflight173

andlocalizationvaluesrecordedinexperiments2and3(rabbitodor,noITN)werecomparedto174

thoserecordedinexperiment4(rabbitodorandITN)todetermineITNodorinfluenceonmosquito175

behavior.176

Therelativeattractiveness(RA)ofonearmversustheotherwascalculatedastheproportionof177

mosquitoesinA1orA2(NA1orNA2)relativetothesumofthemosquitoescollectedinbotharms.In178


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ordertoverifythesymmetryoftheexperimentalset-up,wemeasuredRAexp2inexperiment2(rabbit179

asanodorsource,emptyboxes)asfollowandexpectedittonotbedifferentthan0.5:180

!"#$%& =()*

(()* + ()&)181

RelativeattractivenessofUTNversusemptybox(RAexp3)andITNversusUTN(RAexp4)werealso182

calculatedfromexperiments3and4,respectively,usingthefollowingequations:183

!"#$%. =(/01

(23%45 + (/01184

!"#$%6 =(701

(/01 + (701185

whereNUTNisthenumberofmosquitoescollectedinthearmwiththeboxcontainingtheUTN186

(experiment3or4),NEmptyisthenumberofmosquitoesinthearmwiththeemptybox(experiment187

3)andNITNisthenumberofmosquitoescollectedinthearmwiththeboxcontainingtheITN188

(experiment4).ThemeasureofRAexp3allowedustoassessthepossibleeffectoftheUTNasa189

physicalbarrierforthediffusionofodorcomingfromtherabbittotheolfactometer.190

Statisticalanalysis191

AllanalyseswereperformedusingtheRsoftware,version3.0.2[31],withthelme4package[32].We192

analyzedupwindflightandlocalizationusingbinomiallogisticmixed-effectmodels.Thedayof193

releasewassetasrandominterceptbecausereleasesperformedonasamedaymightnotbe194

independentandbecauseallthreegenotypeshavenotbeentestedeachday.Thekdrgenotypes(SS,195

RSorRR),thedifferentodorsources(none,RabbitwithoutITN,andRabbit+ITN)andinteractions196

betweenthemwereincludedinthemodelsasexplanatoryvariables.Upwindflight(UF)and197

localization(L)modelswerewrittenasfollow:198

logit(=>@ABCDE) = FG +FCH#IJ45%# + FD

KLJM + FCH#IJ45%#×FD

KLJOM + PE 199


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,whereUForLijkistheproportionUForLrecordedforgenotypeiwithodorsourcejonday200

k,FCH#IJ45%#denotestheeffectonthelogitoftheclassificationincategoryi(SS,RSorRR)of201

Genotype;FDKLJMdenotestheeffectoftheclassificationincategoryjofOdorsource:Empty202

(experiment1),RabbitwithoutITN(experiment2and3),orRabbit+ITN(experiment4);anddk203

representstherandominterceptfordayk.Eachcombinationofcategoriesiandjoftheexplanatory204

variableswassuccessivelyusedasreferenceclassformultiplecomparisonsamonggenotypesand205

odorsources.Oddsratiosandtheir95%confidenceintervals(CI)werecomputed.206

Weverifiedthesymmetryoftheexperimentalset-upbymodellingtherelativeattractiveness207

measuredinexperiment2(RAexp2)usingabinomialmixed-effectmodelwiththereleasedayas208

randomeffect:209

logit(!"QRS&,CE) = FG +FCH#IJ45%# + PE 210

,whereRAexp2,ikistheproportionRAinA1forgenotypeiinexperiment2ondayk,FCH#IJ45%# isthe211

effectonthelogitoftheclassificationincategoryi(SS,RSorRR)ofGenotype;anddk,therandom212

interceptfordayk.213

RelativeattractivenessofUTNvs.emptyboxandITNvs.UTNwereanalyzedusingasimilarmodel214

that,inaddition,allowedforrandomeffectsoftheboxthatreceivedthetreatment:215

logit(!"CEU) = FG +FCH#IJ45%# + VU + PE 216

,whereRAiklistheproportionRAexp3orRAexp4forgenotypeiondaykwiththetreatmentplacedin217

boxl,FCH#IJ45%# indicatestheeffectonthelogitoftheclassificationincategoryi(SS,RSorRR)of218

Genotype;bl,theeffectonthelogitofthetheboxlthatreceivedthetreatment(UTNorITNfor219

RAexp3andRAexp4,respectively)anddk,therandominterceptfordayk.Eachgenotypewas220


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successivelyusedasreferenceclassformultiplecomparisons.Oddsratiosandtheir95%CIwere221

computed.222

CO2concentrationswerecomparedbetweenarmsusingtheWilcoxonsigned-ranktestforpaired223

data.RHvalueswerecomparedbetweenarmsusingthepairedTtest.224

Results225

Overall,6286mosquitoeswereincludedintheassays(2621SS,1268RSand2397RR)during226

47,49,84and98releasesforexperiments1to4respectively(Table2).227

Table2:Numberofreleasesperformedpergenotypeandexperiment228

Experiment GenotypesSS RS RR Total

1-Empty 19 9 19 472-Rabbitalone 20 10 19 493-Rabbit+UTN 34 18 32 844-Rabbit+ITN 40 20 38 98Total 113 57 108 278

(UTN:Untreatednet,ITN:Insecticide-treatednet)229

DoAn.gambiaefemalesrespondtotheairflow?230

Wefirstinvestigatedtheresponsetotheairflow(anemotacticresponse)bycalculatingthe231

proportionofupwindflight(UF)femalesandthoselocated(L)inarmsintheemptyset-up232

(Experiment1).Overall,theprobabilitytoleaveRZ(UF)was0.43(95%CI[0.38–0.48];Fig2A).233

Amongtheactivatedmosquitoes,10%(95%CI[6–17])reachedA1orA2(Fig2B).Inspiteofsimilar234

upwindflightproportionamonggenotypes,theprobabilityoflocalization(L)forRSanopheleswas235

higherthanthoseofRRmosquitoes(Figure2B;ORL=2.15,95%CI[1.04,4.41]).236

Fig2:Upwindflightandlocalizationindicatorsforthethreegenotypesinrelationtotreatment237(Mean±95%ConfidenceInterval).***p<0.001,**p≤0.01,*p≤0.05,ns=notsignificant.238


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DoAn.gambiaefemalesrespondtoanattractiveodorsource?239

Thepresenceofarabbitasanattractiveodorsource(experiments2and3)didnotchangethe240

proportionofupwindflightmosquitoescomparedtotheexperimentswithoutattractantodor241

(experiment1),independentlyoftheirgenotype(Table3).However,thecomparisonoftheupwind242

flightprobabilitybetweengenotypesshowthatforRSmosquitoes,UFprobabilitiesbecame243

significantlyhigherthanforSSandRRindividuals(Fig2A;ORRSvsSS=1.2495%CI[1.01,1.54];ORRSvsRR=244

1.2995%CI[1.04,1.59]).Moreover,thelocalizationprobabilitysignificantlyincreasedforall245

genotypesinthepresenceofanodorstimuluscomparedtonoodor(Table3),independentlyof246

genotypes(Fig2B).Therabbitodorhadaneffectonmosquitobehavioronlywhentheywereclose247

toarmslikelybecauseoftheodorconcentrationthatwasmoreimportantinarmsthaninthe248

releasezone.249

250

Behavioralindicator OdorsourcescomparisonsGenotypefor

thekdrmutation

OddsRatios[95%ConfidenceInterval] p-value

Upwindflight(UF)

Rabbitavsnoodorb

SS 1.09[0.87,1.37] ns

RS 1.27[0.91,1.77] ns

RR 1.08[0.85,1.37] ns

Rabbit+ITNcvsRabbita

SS 1.02[0.85,1.21] ns

RS 1.05[0.82,1.34] ns

RR 1.12[0.94,1.35] ns

Localization(L)

Rabbitvsnoodorb

SS 2.63[1.67,4.15] ***RS 1.96[1.14,3.36] *RR 4.63[2.67,8.02] ***

Rabbit+ITNcvsRabbita

SS 1.3[0.99,1.69] ns

RS 1.01[0.72,1.42] ns

RR 1.01[0.76,1.33] ns

Table3:ResultsoftheUpwindflight(UF)andlocalization(L)generalizedlinearmodels.Comparison251ofmosquitoes’progressfirstwhentherabbitwasaddedasanodorsource(vs.noodor)andthenwhenITN252waspresent(vs.rabbitalone).aexperiments2and3,bexperiment1,cexperiment4(seeTable1);***p<0.001,253**p≤0.01,*p≤0.05,ns:notsignificant.ITN:insecticide-treatednet.SS:homozygousforthesusceptibleallele,254RS:heterozygous,RR:homozygousfortheresistantallele.255


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Ismosquitoresponseinfluencedbyinsecticide-treatednets?256

Totestwhethertheinsecticideonnetfibersaffectedmosquitoprogression,wecompared257

upwindflightandlocalizationprobabilitiesinthepresence(experiment4)orabsence(experiments2258

and3)oftheITN.TheprobabilitiesweresimilarinpresenceorabsenceoftheITN,regardlessofthe259

genotype(Table3;Fig2A,2B).Nevertheless,thecomparisonbetweengenotypesshowedthat260

upwindflightprobabilitiesforheterozygousRSmosquitoesremainedhigherthanthoseofthetwo261

othergenotypes,bothinthepresenceorabsenceofinsecticide(Fig2A;ORRSvsSS=1.2895%CI[1.01,262

1.62],ORRSvsRR=1.2095%CI[0.94,1.53]).263

Istheexperimentalsetupsymmetric?264

Analysisofthearms’relativeattractivenessdatafromexperiment2(Rabbitodor;twoempty265

boxes)showednosignificantdifferencesbetweenthenumberofmosquitoescollectedinA1vs.A2,266

regardlessofthegenotypes(Fig3A;RAexp2,SS=0.58,95%CI[0.34,0.79];RAexp2,RS=0.62,95%CI[0.34,267

0.83];RAexp2,RR=0.54,95%CI[0.30,0.76]).NodifferenceinRAexp2wasobservedamonggenotypes268

(ORSSvsRS=1.1695%CI[0.46,2.94];ORSSvsRR=0.8595%CI[0.38,1.94],ORRSvsRR=0.7395%CI[0.28,1.90]).269

Moreover,CO2concentrationandRHwerenotdifferentbetweenarms(p>0.05;S1).Theseresults270

demonstratedthattheolfactometerwassymmetrical.Moreover,analysesofRAexp3andRAexp4,271

(resultsdescribedbelow),showednoeffectrelativetotheboxreceivingthetreatment(i.e.variable272

nosignificantinthemodel),indicatingthatsymmetrywasmaintainedduringexperiments3and4.273

Figure3:Relativeattractivenessrates274RA:numberofmosquitoesfoundinonearmrelativetothetotalnumberofmosquitoesfoundin275botharms.(A)Experiment2(rabbitonly).(B)Experiment3(rabbit+UTNoremptybox).(C)276Experiment4(Rabbit+UTNorITN).Asterisksshowdifferenceto0.5,traducingachoiceforone277treatmentratherthantheother.Errorbarsshowthe95%CI;**p≤0.01,*p≤0.05.UTN:Untreated278net,ITN:Insecticide-treatednet.SS:homozygotefortheL1014Sallele(insecticide-susceptible),RS:279heterozygousfortheL1014Fallele,RR:homozygousforL1014Fallele(insecticide-resistant).280


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IstheattractivenessoftheodorsourceinfluencedbytheUTN?281

Inexperiment3(oneemptyboxandoneboxwith2m²ofUTN,bothinpresenceofrabbitodor),the282

emptyboxwasmoreattractiveforSSandRRmosquitoesbutnotforRS(RAexp3,SS=0,3195%CI283

[0.20,0.46],p-value=0.013;RAexp3,RR=0.27,95%CI[0.17,0.42],p-value=0.002)(Fig3B).Nosignificant284

differenceofmosquito’sproportioninarmswasevidencedbetweengenotypes(ORSSvsRS=1.24,285

95%CI[0.61,2.50];ORSSvsRR=0.83,95%CI[0.41,1.66],ORRSvsRR=0.67,95%CI[0.31,1.47]).CO2286

concentrationwasnotdifferentbetweenarms,whileasignificant1%differenceinRHwasobserved287

(63.9%intheUTNarmand64.9%intheemptyarm,pairedTtestp-value=0.007).288

IstheattractivenessoftheodorsourceinfluencedbytheITN?289

AnalysisofRAexp4fromexperiment4(Rabbitodor;oneboxwith2m²ofUTNandonewith2m²of290

ITN)showedthatRRmosquitoespreferablychosetheITNarmwithprobability0.63(95%CI[0.53-291

0.73],p-value=0.01;Fig3C).ThisprobabilitywassignificantlyhigherthanthoseobservedbothforRS292

(RAexp4,RS=0.4795%CI[0.34-0.60];ORRRvsRS=1.95,95%CI[1.06,3.57],p-value=0.03)andSSgenotypes293

(RAexp4,SS=0.595%CI[0.40-0.61];ORRRvsSS=1.71,95%CI[1.03,2.83],p-value=0.04).CO2concentration294

andRHwerenotdifferentbetweenarms.295

Discussion296

Thehost-seekingbehaviorofmosquitoestowardshumanssleepingunderabednetispoorly297

understood.Particularly,itisnotknownwhetherspecificvolatilechemicalsemanatingfromtreated298

netsmightmodulatethisbehavior.Here,weusedadual-choiceolfactometertoassesswhetherthe299

presenceofpermethrin-treatednetsmayinfluencethehostattractivenessformosquitoesof300

differentkdrgenotypes.Wefoundthatnetsrepresentbothaphysicalandachemicalsignalthat301

modulatemosquitoactivationand/orchoice.Moreover,thethreekdrgenotypesbehaveddifferently302

inresponsetohostodorsinthepresenceofITNsorUTNs.303


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Physicalbarrier&environmentalcues 304

Inexperiment3,mosquitoespreferablychosethearmconnectedtotheemptyboxrather305

thantheboxwithUTNs.NodifferenceinCO2quantitywasnotedbetweenarms.However,the306

humiditylevelwasslightlyhigherinthearmconnectedwiththeemptybox.Ashumidityisknownto307

attractmosquitoes[33],theobservedpreferencefortheemptybox(higherhumidity)wasnot308

surprising.ThisdifferencecouldhavebeencausedbythephysicalbarrierformedbyUTNsthatmay309

absorbhumiditycomingfromtherabbitbox.Inaddition,thenetstructurecouldalsohaveretained310

volatilechemicalsemanatingfromrabbitwhichareimportantinmosquitoorientationandchoice311

[34,35].312

Chemicalecology&ITNs313

Ourresultsraisedthequestionofthevolatilechemicalsemanatingfromnetsthatmaydrive314

aspecificbehaviorinresistantmosquitoes.Permethrinisnotknownasaclassicalvolatilecompound315

becauseofitslowvaporpressure(5.18x10-8mmHgat25°c).Nevertheless,Bouvieretal[41]recently316

detectedpermethrininindoorairsamples(11and18.8ng/m3fortrans-permethrinandcis-317

permethrinrespectively)indicatedthatsuchpyrethroidmightbepresentintheaireventheyare318

semi-volatileorganiccompounds.Moreaccurately,astudybyBomannetal.[42]fromtheBayer319

companymeasuredameanconcentrationofcyfluthrin(apyrethroidwithamolecularstructureclose320

tothepermethrin)of0.000021mg/m3at1mawayfromatreatednet.Suchconcentration321

correspondsto3.46x109molecules/cm3.Angioyetal[36]foundthatonly6moleculesofa322

pheromoneenteredincontactwiththeolfactorysensillumofmothspeciesmayinducea323

physiologicalresponse.Wethereforehypothesizethatmosquitomaydetectverylowconcentration324

ofpyrethroidintheair.325

Moreover,somenasaltrouble(i.erunnynose)havebeenrecordedwhenLNswereusedfor326

thefirsttime[37].SuchobservationsreinforcethehypothesisthatLNsemitchemicalsintheair.327

Regardlessthesechemicalsareinsecticideitself,additivechemicals(i.e.fragrances),degradation328


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products,thatcomposedthenet,theymaybedetectedbymosquitoesandelicitbehavioral329

modulation.330

Thebehaviorofinsects,suchasmosquitoes,isdrivenbytheperceptionandintegrationof331

odorantsignalsinantennaeandhigherbraincenter.Inourstudy,weobservedthatkdrresistant332

mosquitoesweremoreattractedbyhostodorsemanatingbehindapermethrin-treatednetthan333

hostodorsbehindanuntreatednet(Fig3C),itindicatesthattheyperceivedatdistanceadifference334

betweenITNandUTNandbehaveddifferentlyinresponse.Wethenhypothesizethatmosquitoes335

areabletodetectchemicalsreleasedbynetwitholfactoryreceptors(Ors)tunedtorespondtothese336

chemicals.Asanexample,authorsrecentlyidentifiedoneolfactoryreceptoractivatedandanother337

inhibitedbysyntheticpyrethroïdinAedesaegypti[38],suggestingthatsuchORmayalsoexistin338

Anophelesmosquitoes.Themajorresearchperspectiveraisedbyourresultsisthereforetostudythe339

chemicalandbehavioralecologyrelativetovectorcontroltoolsalreadywidespreadinendemic340

country.341

Insecticideresistance&hostseekingbehavior342

Ourresultsalsoclearlyindicatedthatthekdrmutation,orcloselylinkedpolymorphisms[39],343

modulatedthehostchoiceofAn.gambiaemosquitoesinthepresenceofaITN.Thestrainsusedin344

ourstudysharethesamegeneticbackground.ExceptifgenescodingforORssensitivetoLN-related345

odorantsarelocatedinflankingregionofKdrmutation,,onlyapleiotropiceffectofthekdrmutation346

affectingthetransmissionorintegrationoftheneuronalsignalinhomozygousmosquitoescould347

explainthedifferentbehaviorsbetweengenotypes.Thekdrmutationmayinfluencethetransmission348

ofanodorantsignaltowardshigherbrainregionsbyenhancingtheclosed-stateinactivationofthe349

voltage-gatedsodiumchannel,whichplaysacentralroleinmessagepropagationinthenervous350

system.Asaconsequence,areductionofneuronalexcitabilitycouldbeobservedinkdrmutantsin351

comparisontosusceptibleindividuals[40].Allchemicalsignalsaretransducedbyspikefrequencies352


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intheolfactorysensoryneurons[41]andtheinformationsentbystimulatedorinhibitedneuronsis353

treatedintheantennallobe[42].Therefore,iftheneuronalexcitabilitydiffersinhomozygouskdr354

genotypes,theresponsepatternoftheolfactoryneuronsandsubsequentlythesignalintegration355

andprocessinginthecentralnervoussystemcouldbealtered,leadingtoamodifiedmotorresponse,356

inthiscase,adifferenceinhostchoice.357

Thepresentstudysuggeststheexistenceofinteractionsbetweenthephysiological358

mechanismsthatallowmosquitoestosurviveacontactwithinsecticideandthebehavioralresponse359

toolfactorycues.Theseinteractionsmayhavemajorimplicationsinmalariacontrol.Asanexample,360

chemicalsemanatingfromtheITNsarestronglyrelatedtothepresenceofhumanbeings.Shouldit361

beintegratedasanattractivecueforresistantmosquitoes?Thismaydramaticallyaffectsthe362

personalandcommunityprotectiongivenbythemassiveuseofITNs.Ourstudyonlyfocusedonthe363

Kdrmutation,buttheresistancepatterninwildAnophelespopulationsisfarmorecomplex[43].It364

wouldbeinterestingtoinvestigatetheinteractionbetweeneachresistancemechanismsisolatedin365

specificstrainsbeforegoingtostudythisinteractionbetweenresistance,behaviorandITNsinsemi-366

fieldandnaturalconditions.Recentpapersweremodelingandquestioningtheriskconferredby367

resistance,basedonsurvivaltoinsecticideexposure[44],buttheimpactofsuchresistanceon368

behaviorisalsotobeinvestigatedurgently[45].369

Weusedrabbitasanodorsourcebecausemosquitoeswerefedonrabbitsatthelaboratory,370

andwerelikely“selected”torespondtorabbit’sodor.Butinthefield,Anophelesgambiaeprefersto371

bitehumanwhenavailable[46].Whetherthesameexperimentconductedwithhumansasanodor372

sourcewillprovidesimilarresultsremaintobeexperimentallyevaluated.Ifweusedahumaninstead373

oftherabbitwechangethecompositionofodorplume(quantityandqualityofsemiochemicals).374

ThereforetheinteractionbetweenchemicalsreleasedbyLLINandhumanodorshouldinducea375

differentbehavior.Nevertheless,ourexperimenthighlightedtheinvolvementofLLINinhostseeking376


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behaviorandemphasizedtheneedtostudyingtherelationbetweenLLIN,hostodorsandmosquito377

hostseekingbehavior.378

Kdrgenotypes&behavior379

HeterozygousRSmosquitoesweremoreactivethanSSandRRmosquitoes.Theadditionof380

anattractantdidnotchangetheproportionofRSleavingtheRZ,suggestingthatthisbehaviormight381

berelatedtoabetteranemotacticresponse(i.eresponsetoairflow)orspontaneousflightactivity382

thanabetterperceptionofodorantsinRSmosquitoes.Thishypothesisisstrengthenedbythe383

absenceofdifferenceintheprogressiontowardstheolfactometerarmsamonggenotypes.Inother384

words,heterozygousmosquitoesflymore,butmightnotsmellbetter.Ontheonehand,byflying385

moretheymightincreasetheprobabilityofencounteringahostodorantplumewhichmightbe386

advantageous.SuchheterozygousadvantageforthekdrlocusinAn.gambiaes.s.hasbeenrecently387

documentedalsoforanotherbehavioraltrait:theabilitytofindaholeinapieceofbednet[24]and388

formalemating[47].Inotherhand,itcouldrepresentacostformosquitoesifenergyspentduring389

flightisnomoreavailableforothertraitscloselyrelatedtofitnessasfertility,fecundityand390

longevity.Thistradeoffmustbedeeplyinvestigatedasthismighthavegreatinfluenceon391

Plasmodiumtransmission.392

Thebehaviorofkdrheterozygousindividualsinourstudymustbeinterpretedwithcaution393

becauseotherloci,distinctfromthekdrlocus,couldalsoinfluencethisbehavioraltrait.Introgression394

andselectionthekdralleletoproducethehomozygousresistantstrainwasindeedlikelytoalsohave395

selectedlinkedpolymorphisms[45].396

Conclusion397

Inconclusion,ourstudyshowedthattheAnophelesmosquitoesdetectedthepresenceof398

bothphysicalandchemicalbarriersofITNS.Facetothisresults,iturgestodecipherwiththe399


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interactionbetweenhost-seekingbehavior,insecticideresistanceandvectorcontroltools.Themost400

overlookedpartofthepuzzleisthechemicalecologyinacontextoflargevectorcontrolmeasure401

deployment.Thisresearchavenuewillbechallengingforthevectorcontrolcommunitybutiscrucial402

nottowasteforcesinwrongdirections.403

Acknowledgement404

We thank TeunDekker for helpful discussion.Wewould like to thank the IEMTV staff in Abomey405

Calavi(Benin)fortechnicalassistance.406

407


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Supportinginformation535

S1:Effectoftreatmentonenvironmentvariables536537538539


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61#cm#

Rota*ng#doors#

8#cm#

32#cm#45#cm#

30.5#cm#

33.5#cm#

34#cm#

30#cm#

23.5#cm#

Fan#Box#with#treatment#

Release#zone#(RZ)#

Flight#chamber#(FC)#Arm#1#and#2#


Figure 1

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No-odor (Experiment 1)

Rabbit (Experiment 2+3)

Rabbit+ITN (Experiment 4)

0.0

0.2

0.4

0.6

0.8

1.0P

ropo

rtion

of f

emal

es

SSRS

RR

* * *ns

ns nsns ns

ns

Upwind flight

No-odor (Experiment 1)

Rabbit (Experiment 2+3)

Rabbit+ITN (Experiment 4)

0.0

0.2

0.4

0.6

0.8

1.0

Pro

porti

on o

f fem

ales

nsns *

ns nsnsns ns ns

Localisation

A

B


Figure 2

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0.00.20.40.60.81.0

0.00.20.40.60.81.0

0.00.20.40.60.81.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.0

Proportion of female found in arm

A

B

C

Empty box 1 Empty box 2

UTN Empty box

ITN UTN

*

**

**

RS

SS

RR


Figure 3

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a pyrethroid-treated bed net increases host attractiveness for

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