REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Medical
and
Veterinary
Entomology
Oviposition
of
Aedes
aegypti
Linnaeus,
1762
and
Aedes
albopictus
Skuse,
1894
(Diptera:
Culicidae)
under
laboratory
and
field
conditions
using
ovitraps
associated
to
different
control
agents,
Manaus,
Amazonas,
Brazil
William
Ribeiro
da
Silva
a,
Joelma
Soares-da-Silva
b,
Francisco
Augusto
da
Silva
Ferreira
a,
Iléa
Brandão
Rodrigues
a,
Wanderli
Pedro
Tadei
a,
João
Antonio
Cyrino
Zequi
c,∗aInstitutoNacionaldePesquisasdaAmazônia,ProgramadePós-Graduac¸ãoemEntomologia,LaboratóriodeControleBiológicoeBiotecnologiadaMaláriaeDengue,Manaus,AM,
Brazil
bUniversidadeFederaldoMaranhão,CursodeCiênciasNaturais,CampusVII,Maranhão,MA,Brazil
cUniversidadeEstadualdeLondrina,DepartamentodeBiologiaAnimaleVegetal,LaboratóriodeEntomologiaMédica,Londrina,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received26April2018 Accepted10August2018 Availableonline25August2018 AssociateEditor:AnaCampos Keywords: Chikungunya Control Dengue Insecticides Zika
a
b
s
t
r
a
c
t
TheaimofthisstudywastoanalyzetheeffectivenessofdifferentcontrolagentsofAedesaegyptiand
Aedesalbopictusassociatedwithovitrapsunderlaboratoryandfieldconditions.Fivetreatmentswere
used:grassinfusion+Bacillusthuringiensisisraelensis(gI+Bti),grassinfusion+Saccharopolysporaspinosa
(gI+Ss),grassinfusion+Pyriproxyfen(gI+P),distilledwater+Toxorhynchiteshaemorrhoidalis(dW+Th),
andgrassinfusion(gI)(control).Thehighestmeannumberofeggsofbothspecieswereobtainedwith
grassinfusioninthelaboratory.Amongcontrolagents,thelowestmeanofA.aegyptieggsoccurredwith
gI+SsandthelowestmeanofA.albopictuseggsoccurredwithdW+Th.Therewasnodifferencebetween
treatmentsinA.aegypti(P=0.4320)andA.albopictus(P=0.7179).Inthefield,thehighestmeannumber
ofeggsforbothspecieswereobtainedwithgI+Ss,andthelowestvalueswereobtainedwithgI+P
(P=0.0124).Thetreatmentscanbeappliedtoboththesurveillanceandthecontrol,butovitrapswith
biologicallarvicideBtiweremoreeffectiveandsaferconsideringthenumberofeggslaidandselectivity
ofpathogensformosquitoes.
©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Aedes(Stegomyia)aegyptiLinnaeus,1762andAedes(Stegomyia) albopictusSkuse, 1894(Diptera: Culicidae)are particularly sus-ceptibletoseveralarbovirusesthat arethreats topublichealth inseveralregionsoftheworld(Alahmed,2012;Forattini,2002; Rossatietal.,2015).Bothspecieshavewideepidemiological impor-tance in the Americas due to implications in the transmission ofmanyarbovirusresponsibleforhighhospitalizationratesand deaths. They are distributed throughout several regions in the continent(PAHO,2018).Thesemosquito-bornepathogensaffect economiesduetoanimpairedworkforceandtheneedtotreatsick people(Calvoetal.,2016;Gubler,2005;Vega-Rúaetal.,2014).
∗ Correspondingauthor.
E-mail:joaozequi@gmail.com(J.A.Zequi).
DerivedfromAfricawheretherearewildandurbanpopulations, A.aegyptiiscurrentlywidespreadandisfoundinallstatesandthe FederalDistrictofBrazil(CarvalhoandMoreira,2017).Thisspecies isthemainvectorofDengue,Chikungunya,Zika,andUrbanYellow FevervirusesintheAmericas.Thisanthropophileisadaptedfor urbanenvironments,whereitinhabitslaundrytanks,containers, barrels,bottles,potsofplantsandothercontainers(Pinheiroand Tadei,2002;Soares-da-Silvaetal.,2012).
These oviposition sites are different from those used by A. albopictus,whichinhabitssiteswithnativeorsecondary vegeta-tionnear humanpopulations (Martinset al., 2013; Silvaet al., 2006).Consideringitsepidemiologicalrole,thisspecieshas poten-tialfortransmittingUrbanYellowFeverandVenezuelanEquine Encephalitisviruses;laboratoryassayshavecheckedthisspecies susceptibilitytomorethan20arbovirusesincludingtheDengue virus (the primary vector in Asia) and the Chikungunya virus (Gubleretal.,2001;Moore andMitchell,1997;Vega-Rúaetal., 2014).ThefirstrecordofthisspeciesinBrazilwasin1986inthe
https://doi.org/10.1016/j.rbe.2018.08.001
0085-5626/©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
stateofRiodeJaneiro;afewyearslaterithadalreadyspreadtoall statesoftheSoutheasternregion.Itiscurrentlydistributed nation-wide(Martinsetal.,2013;Santos,2003).
Vectorcontrolpracticesthatarelessdamagingtothe environ-mentandareeffectiveformosquitolarvaehaveincreasedoverthe lastyears.Theseincludenaturalpredators,bacteria,growth regu-lators,andmorerecently,fungiandplantextracts(Darbroetal., 2011; Ferreiraet al., 2015;Lopes, 1999;Medeiros etal., 2013; Soares-da-Silva et al., 2015; Soares-da-Silva et al., 2017; Zequi etal., 2015).New attempts atmosquito controlhave advanced tosuppresspopulationsofmedicalimportanceincludingtheuse of transgenic mosquitoes (Beech et al., 2009; Carvalho et al., 2015)andthesymbioticbacteriumWolbachia,whichinhibitsthe developmentofDenguevirusinsidetheinsectthusblockingits transmission(Dutraetal.,2016).
Vectorspeciesmonitoringisessential.Studyingmosquito dis-tributionisessentialtoidentifycriticallarvalreproductionandegg layingsites.Theuseofovitrapsbeganin1965.Ovipositiontrapsfor A.aegyptipopulationswereshowntobeeffectiveforlarvalresearch andstudiesonvectorfrequency(FayandEliason,1966;Regisetal., 2008).
Ovitrapsprovideusefuldataonthespatio-temporal distribu-tionofmosquitoesbecausethismonitoringallowsonetocheckthe presenceanddensityofthevectoratalocalscale(Fisheretal.,2017; Nunesetal.,2011).Inaddition,ovitrapsmightbeemployedfor vec-torcontrolbecausetheyremoveeggsfromtheenvironment;they areasensitiveandlow-costtooltoaidinepidemiologicalstudies (Depolietal.,2016;Gomes,2002).
Dataobtainedwiththistoolcanmonitortheimpactofcontrol measuressuchastheuseofinsecticides.Theycanalsohelp moni-tortheeffectivenessofvectorreductionprogramsinurbanareas. Although ovitrapsare effective, they are a short-term method, replacementisrequiredwithinapproximately5–7dayssothat lar-valhatchingdoesnotoccurandtrapsdonotbecomeoviposition sites(Alarcónetal.,2014;Gomes,1998;Gomes,2002;Regisetal., 2008).
The association of ovitraps and larval control agents could improve field traps and preventpotential larval hatching. This wouldensurethesafetyofthesetraps(Depolietal.,2016;Regis etal.,2013).However,itisnecessarytoanalyzethebestcontrol agentassociatedwithovitrapsbecausetheselectionofapotential ovipositionsiteinvolvesvisual,olfactory,andtactileresponses.In thisrespect,grassinfusionisanimportantattractantforlayingeggs (Reiteretal.,1991)asisthepresenceofaconspecificlarvaehas beenreported(Nunesetal.,2011;Santanaetal.,2006).
ManausisanAmazoncitywithclimaticconditionsthatfavorA. aegyptiandA.albopictusyear-round.Ithasabundantrainandhigh temperaturesaswellasadiversityofnaturalandartificiallarval habitat.Therefore,integratingamonitoringmodelthatcombines ovitrapswithcontrolagentsmightbeaneffectivealternativefor surveillanceandcontrolofmosquitoesthatarevectorsofhuman pathogens.Thus,theaimofthisstudywastoanalyzethe effec-tivenessofdifferentcontrolagentsassociatedwithovitrapsforA. aegyptiandA.albopictusunderlaboratoryandfieldconditions.
Materialsandmethods
ObtainingAedesaegyptiandAedesalbopictusmosquitoes
Toobtainfemales,A.aegyptiandA.albopictuspopulationswere keptandstabilizedinaninsectaryoftheLaboratoryofBiological ControlandBiotechnologyof Malaria andDengue(LCBBMD) of the National Institute of Amazonian Research (INPA), Manaus, Amazonas,Brazil.Theinsectarystartedobtainingeggsusing ovipo-sitiontraps,alsoknownasovitraps.Ovitrapsareblackandround
plasticpotsmeasuring9x11cmwithacapacityof500mL.Inside theovitraps,thereisafive-mmthickDuratreepaddle,Eucatex® brand,15cmlongbythreecmwide.Thisisplacedverticallywith therough surfaceof thematerial facingoutwardsto providea substrateforovipositionandeggadherence.Themainattractant offemalesinthetrapswasagrassinfusionwithtotalavolumeof 300mLat1.2%ofColoniãograss(MegathyrsusmaximusJacq) fer-mentedforsevendaysatameanroomtemperatureof32◦C(range of38◦Cand20◦C)accordingtoKoeppen’sclassification(1948).
Twenty ovitrapswere mounted and distributed in theINPA Campus.Afteroneweek,ovitrapswerecollectedand the Dura-treepaddlescontainingtheeggsweresubmergedindistilledwater forlarvalhatching.Thelarvaewerefedwithcatfood(Whiskas®) mincedinfineparticles.Thewaterinthecontainerswaschanged everytwodaystopreventfeedfermentation—thiscandamagegas exchangeduringtheimmaturestages.Afteremergence,theadults werecapturedwithaCastrograbber,andthespeciesidentitywas confirmedusingexternalmorphologicalcharacteristicsofadults, especiallyinthethoraxusingstereoscopicmicroscope,wherethey wereidentifiedwiththehelpofamosquitoidentificationguide (ConsoliandLourenc¸o-de-Oliveira,1994;Forattini,2002;WRBU, 2018).
Theadultsweresubsequentlymaintainedinacageformating andmaintenanceofadultindividuals.Cottonwrappedingauzeand soakedinsugaredwaterat12%wasaddedasasourceof carbohy-drates,andfemaleswerefedabloodmealtwiceaweekusingan adequatelyanesthetizedhamster(Mesocricetusauratus)following theprocedureapprovedbytheINPAEthicalCommitteefortheUse ofAnimals(CEUA:02/2014–“Breedingofvectorsunderlaboratory conditions”).
ObtaininglarvaeofToxorhynchiteshaemorrhoidalisFabricius, 1787
Toxorhynchiteshaemorrhoidalisisanaturalpredatorofculicids andotherinsects.ToobtainimmatureT.haemorrhoidalis,eightcut tires(25×15×11cm),containingwellwaterandasmallamountof litterweredistributedthroughouttheINPACampusandmonitored daily.Wealsoactivelysearchedforimmaturestagesinthenatural larvalhabitat.ThelarvaewerekeptintheinsectaryoftheLCBBMD andfedonthethirdandfourthinstarlarvaeofAedesspp.;56third instarlarvaeofT.haemorrhoidaliswereused.
Substratesusedintheexperiment
Table1liststheassociationsusedintheovipositionprocessof A.aegyptiandA.albopictusunderlaboratoryandfieldconditions.
Controlagentsweredilutedusingfalcontubesanddissolved with a Vortex agitatorand a BRANSONIC® ultrasonic until the desiredconcentrationswerereachedbasedonthemanufacturers’ recommendations(Table1).
Laboratoryexperiments
FromastabilizedA.aegyptiandA.albopictusinsectary,25 fertil-izedfemalesfromgenerationF1werepreviouslyselectedfromeach
speciesforeachexperiment.Fivedaysafterthebloodmeal,females wereplacedinanaluminum-screenedcage(55×47×47cm).Five 160mLwhiteplasticcupswereplacedinside—oneforeach treat-mentontaining50mLofeachtreatmentandapaperfilterasthe oviposition substrate (Table 1). Subsequently, a container with 12%sugar inwater wasadded asasourceofcarbohydratesfor females.
Containerswereplacedinthefourcornersofthecageandone inthecenter.Thecontainerpositionswerechangedclockwisedaily forfourdaystodiscardanyexternalinfluenceonoviposition;three
Table1
TreatmentsusedinovipositionexperimentswithAedesaegyptiandAedesalbopictusunderlaboratoryandfieldconditionsintheperiodrangingfromMarchtoJuly,2015, Manaus–Amazonas–Brazil.
Treatments Controlagents Concentration Batch/validity
Grassinfusion+Vectobac®WG Bacillusthuringiensisisraelensis(gI+Bti)* 0.004g/L 237-445-PG–Jan/2016
Grassinfusion+NatularTMDT Saccharopolysporaspinosa(gI+Ss)* 0.007g/L 1309190010–Oct/2015
Grassinfusion+Sumilar® Pyriproxyfen(gI+P)* 0.01g/L 4303F425–Jan/2016
Distilledwater+T.haemorrhoidalis T.haemorrhoidalis (dW+Th)*
Grassinfusion Control(gI)* 1.2%
Legend:*Entomopathogenicbacterium:Bacillusthuringiensisisraelensis;*Entomopathogenicbacterium:Saccharopolysporaspinosa;*InsectGrowthRegulator(IGR):
Pyriprox-yfen;Predatoryinvertebrate:Toxorhynchiteshaemorrhoidalis;Control:GrassInfusion.
replicateswereperformed.Thefemaleswerecapturedand sac-rificedattheendofeachreplicate.Experimentswereconducted undercontrolledtemperature,humidityandphotoperiod condi-tions(27±2◦C;80–90%;12L:12D).
The eggs obtained at the end of each replicate were dried onabsorbent paperfor60minutesat27±2◦C and humidityof 80–90%.Theywerethenstoredunderthesametemperatureand humidityconditionsin750mLcupsforsubsequenteggcounting usingaZEISSStemi200050Xstereoscopicmicroscope.
Fieldexperiments
Forthefieldovipositiontests,50ovitrapsweredistributed(10 foreachtreatment)(Table1).TrapswereinstalledintheINPA cam-pus.Theywereplacednearbuildingsatgroundlevelwithshelter fromthesunandrain.Wefocusedonareasfreeofconstant move-mentofpeopleandanimals;theywerelocated30metersapart fromeach other.Theexperimentlastedfiveconsecutiveweeks, andtheDuratreepaddles werereplacedeverysevendays.Eggs werecountedusingastereoscopemicroscope(ZEISSStemi2000 50X).
Aftereggcounting,paddlesweresubmergedseparatelyin dis-posable600mLflasks containing 400mL ofdistilled water and 0.0055gcatfood(Whiskas®)groundintomincedinfineparticles tofeedthelarvaeandobtainadults.Aftertheadultsemerged,the specieswereidentifiedusingexternalmorphologicalanalysis.The specimensthatwerenotidentifiedbecauseofthedifficultyin visu-alizingthepatternsofornamentationofthescaleswereforwarded liveoneaftertheothertoastereoscopicmicroscope,wherethey wereidentifiedwiththehelpofamosquitoidentificationguide (ConsoliandLourenc¸o-de-Oliveira,1994;Forattini,2002;WRBU, 2018).
Statisticalanalysis
Thefollowingindiceswerecalculated:OPI–ovitraps positiv-ityindex,EDI–eggdensityindex,andVDI(meaneggs)–vector densityindex.TheOPIistheratiobetweenthenumberofpositive trapsandthenumberoftrapsexaminedmultipliedbyone hun-dred(Gomes,1998).TheEDIistheratiobetweenthenumberof eggsandthenumberofpositivetraps(Gomes,2002).TheVDIis themeannumberofeggsineachtypeoftreatmentobtainedby theratiobetweenthenumberofeggsandthenumber oftraps examined(Avendanha,2007).In relationtothedataof number ofeggsobtainedineachtreatment,ananalysiswasfirstmadeto knowifthedatahadanormaldistributionornot,tolaterverify whichstatisticwouldbethemostadequate,parametricor non-parametric.Aftertheseinitialtests,ANOVAwasusedforthedata that presented normal distribution, followed by Tukey’s multi-plecomparisontest(p<0.05),whilethedatathatdidnotpresent normalitywerechosenbynon-parametricKruskaltestsWallis, fol-lowedbytheDunntest(p<0.05).ThestatisticalsoftwareSPSS® 14.0forWindows®(SPSSInc.2005Headquarters,Chicago,IL,USA) wasusedforanalysisassistance.
Results
Ovipositionatthelaboratory
ThenumberofA.aegyptieggscollectedinalltreatmentswas higher than the number of A. albopictus eggs (4215 and 2074 eggs,respectively).ThishadastatisticaldifferenceusingTukey’s test(F=21.836;P=0.0019).No differenceswerefoundbetween themeannumberofA.aegypti(F=1.046;P=0.4320)eggsandA. albopictuseggs(F=0.5312;P=0.7179)eitherinthegrassinfusion (control)orwithcontrolagentsusingTukey’stestat5%significance level(Table2).
Bothspecieshadthehighestmeannumberofeggswithgrass infusion(control);however,A.aegyptihadthehighestmeanin ovi-trapswithdistilledwater+T.haemorrhoidalisandthelowestvalue withgrassinfusion+S.spinosa.ResultsforA.albopictuswereexactly theopposite,but thisdifferencewasnotstatisticallysignificant (Tukey’s,p>0.05).
Ovipositioninthefield
Thehighestmeannumberofeggsforbothspecieswasobtained withgrassinfusion+S.spinosa,andthelowestvaluewasobtained withgrassinfusion+Pyriproxyfen.Thisresultedinamean num-berofeggsthatwastwiceaslowasgrassinfusion+S.spinosa;this corroboratesthesignificantdifferencebetweentreatments(Tukey, P=0.0124) (Table 3).Except for the treatmentwithgrass infu-sion+Pyriproxyfen,alltreatmentshadahighermeannumberof eggsthanthegrassinfusion(control)(Table3);however,therewas nostatisticaldifferencebetweentreatments(Tukey,p>0.05).
Nearly all treatments had an OPI of 100% except distilled water+T.haemorrhoidalis,whichobtained97%ofpositivity(Fig.1). The EDI values of both species showed that biotic agents received a higher amount of eggs than the chemical agent Pyriproxyfen (Fig. 1).Grass infusion+S. spinosa and grass infu-sion+B.thuringiensisisraelensisshowedhigherEDIs(150and130, respectively; Fig. 1). On the other hand, treatment with grass infusion+Pyriproxyfen hadthelowest density(70eggs; Fig.1). The same result wasobserved for theVDI, which was highest withgrass infusion+S. spinosaand the lowest withgrass infu-sion+Pyriproxyfen.
Themosquitoemergencerateobtainedfromeggscapturedin thefieldwashigherforA.albopictus:4734specimenscomprisingof 2270malesand2464females(1:1ratio).Incontrast,494specimens wereobtainedforA.aegypti:272malesand222females(1:1ratio). Therefore,A.albopictuspredominatedthroughoutthestudyarea.
Therateofmosquitoemergencewasanalyzedinthetreatments. The highest number of adults occurredwith grass infusion+S. spinosaandgrassinfusion(control).Inthesetreatments,the num-berofadultA.albopictuswasapproximatelytentimesashighas thatobtainedforA.aegypti(Fig.2).Thecontrolagentshadlittle interferenceintheeggphaseoftheseinsects.
Thelowestvaluesforadultemergencewereseenwithgrass infusion+B.thuringiensisisraelensis(Fig.2)despitethistreatment
Table2
MeannumberofAedesaegyptieggsandAedesalbopictuseggsobtainedwithovitrapscontaininggrassinfusionanddifferentcontrolagentsunderlaboratoryconditions,in theperiodrangingfromMarchtoApril,2015,Manaus–Amazonas–Brazil.
Treatments Aedesaegypti Aedesalbopictus
Mean Maximum Minimum Mean Maximum Minimum
*Grassinfusion+B.thuringiensisisraelensis 270.a* 345 171 124a 254 56
*Grassinfusion+S.spinosa 222.a 421 92 164.7a 208 135
*Grassinfusion+Pyriproxyfen 249.3a 279 193 117.3a 177 50
*Distilledwater+T.haemorrhoidalis 289.7a 306 269 98a 220 32
*Grassinfusion(Control) 373.7a 440 287 187.3a 278 94
Legend:*SamelettersinthecolumndonotdifferaccordingtotheTukey’stestat5%significancelevel.
Table3
Totalmeanofeggs(AedesaegyptiandAedesalbopictus)obtainedbytheassociationofgrassinfusionanddifferentcontrolagentsusingovitrapsunderfieldconditionsinthe periodrangingfromMarchtoApril,2015,Manaus–Amazonas–Brazil.
Treatments Mean Maximum Minimum
*Grassinfusion+B.thuringiensisisraelensis 1322.8ab* 2025 831
*Grassinfusion+S.spinosa 1528.2a 1932 1121
*Grassinfusion+Pyriproxyfen 712.6b 903 380
*Distilledwater+T.haemorrhoidalis 1158.ab 1525 765
*Grassinfusion(Control) 988.2ab 1299 533
Legend:*SamelettersinthecolumndonotdifferaccordingtotheTukey’stestat5%significancelevel.
100 160 140 120 100 80 60 40 20 0 90 80 70 60 50 40 30 20 10 0 gl + Bti gl + Ss dW + Th gl + P gl (control) OPI EDI Ovitr
aps positivity inde
x OPI (%)
Egg density inde
x -EDI
Fig.1. OvitrapsPositivityIndex(OPI)andEggDensityIndex(EDI)withassociation ofgrassinfusionwithdifferentcontrolagentsusingovitrapsintheperiodranging fromJunetoJuly2015,Manaus–Amazonas–Brazil.Legend:*(gI+Bti)–grass
infu-sion+Bacillusthuringiensisisraelensis;*(gI+Ss)–grassinfusion+Saccharopolyspora
spinosa;*(dW+Th)–distilledwater+Toxorhynchites haemorrhoidalis;*(gI+P)–
grassinfusion+Pyriproxyfen;*(gI)–grassinfusion(control).
1200 1000 800 600 400 200 0 gl + Bti gl + Ss gl + P gl (control) Treatments Aedes albopictus Aedes aegypti dW + Th Number of adults
Fig.2. TotalAedesaegyptiandAedesalbopictusadultsobtainedthroughegg collec-tioninovitrapscontaininggrassinfusion(control)andassociatedwithdifferent controlagents.Legend:*(gI+Bti)–grassinfusion+Bacillusthuringiensis
israelen-sis;*(gI+Ss)–grass infusion+Saccharopolysporaspinosa; *(dW+Th)–distilled
water+Toxorhynchiteshaemorrhoidalis;*(gI+P)–grassinfusion+Pyriproxyfen;*(gI)
–grassinfusion(control).
having the second highest mean number of eggs in the field (Table3).Nodifferenceswereseeninlarvalhatchingratebetween grass infusion(control) and control agents eitherfor A. aegypti (P=0.1481;H=6.78)norforA.albopictus(P=0.093;H=7.96)using theKurskallWallistestwithasignificancelevelof5%(Fig.2).
Discussion
Biologicalcharacteristicsaredeterminingfactorsfor oviposi-tionandmightdirectlyinfluencethehighernumberofA.aegypti eggsobservedinthelaboratory(Phasomkusolsiletal.,2014) espe-ciallybecausemosquitosareanintradomiciliarandanthropophilic insect.Femalesofthisspecies completetheirgonotrophiccycle withtwobloodmeals.Themeannumberofeggsateachoviposition is120;A.albopictusneedsmultiplemealstogenerateanaverage of60to65eggs(Clements,1999;Forattini,2002).
Thehighnumberofeggsseeninbothspeciesinovitrapswith grassinfusion(control)mightbeexplainedbytheproven effective-nessofthissubstrateasanattractanttofemaleAedesspp.(Nunes etal.,2011;Reiteretal.,1991;Santanaetal.,2006).
Thedifferencefoundinthetreatmentwithdistilledwater+T. haemorrhoidaliswithhigheraveragenumberofA.aegyptieggscan beexplainedbythebehaviorofthisspecies.Itovipositsin pre-existingimmature-containingbreedingsites,sinceinmosturban environments,specimensdonotestablishstrongecological rela-tionsofcompetitionwithotherinsects,suchascoexistencewith predatoryinvertebrates,andthusbecomecompletelygeneralistin relationtoovipositionsites(Forattini,2002).Ontheotherhand,A. albopictuscoexistswithT.haemorrhoidalisinthesuburban environ-mentofManaus.T.haemorrhoidalisisanaturalpredatorofculicids invegetatedareas—siteswhereA.albopictusoccurs.Itconsequently colonizesdifferenttypesofnaturalbreedingsitesestablishing eco-logical relationshipswithotherinsects, e.g.habitatcompetition (Baileyetal.,1983;Forattini,2002;WHO,1984).Thismightexplain thelownumberofA.albopictuseggsinovitrapscontaininglarvae ofthispredatoryspecies.
Thechoiceofpotentialoviposition sitesisrelated toseveral environmentalfactorsincludingwatersprayintensity,sizeofthe watersurfacerelativetolight,presenceofimmatureculicidstages, andcertainchemicalcomponentsthatmightactasrepellentsor attractantsdependingontheconcentration(AllanandKline,1995; Chadeeetal.,1990).
Thelaboratoryexperimentshadcontrolledconditions.It iso-latedthedifferentfactorsrelatedtoovipositionandmaximizedthe treatmentdifferences.Thisallowedustorelatethefactorsfound tobeeffectiveinovitrapsforeachcontrolagent.Theseagentscan attractmorefertilizedfemalesleadingtobetterstudyandcontrol ofthesespecies.
Underfield conditions,moreeggswerecollectedinovitraps withgrassinfusion+S.spinosa.OnestudycarriedoutinMexico alsoshowedtheeffectivenessofthisbacteriumasanattractant formosquito females inthefield where theoviposition rateof A.aegyptiwashigherthanintreatmentswithtemephosor dis-tilledwater (Solís-Valdez et al., 2015). However, in this study, grassinfusion+S.spinosaonlydifferedstatisticallyfromthe chem-icalPyriproxyfen(Tukey,P=0.0124;Table3).Ovitrapswithgrass infusion+B.thuringiensisisraelensis,werepreferredforoviposition corroboratingpriorfieldresults(Carrierietal.,2009;Depolietal., 2016;JahanandSajjadSarwar,2013;Stoops,2005).
Spinosad® isanewgenerationlarvicidederivedfromthe bac-teriumS.spinosa.Itistoxictoseveralinsectspeciesoftheorders Lepidoptera, Thysanoptera, and Diptera including mosquitoes (Bond et al., 2004;Diaset al., 2017;Huseyin et al.,2005).The effectivenessofS.spinosawasverifiedinthefieldforA. aegypti over13 weeksduring thedryseasonand 10 weeksduringthe rainyseason(Marinaetal.,2011).Thisisimportantinformation froman ecological standpoint becauseit shows feasibility at a largescale.Persistenceinthefield—combinedwiththe effective-nessofthebacteriumasanattractanttofemalesandcontrolof immaturestages—makesthistoolanimportantalternative.This information isimportant consideringthe climaticconditions in Manaus.Ithasahighsolarincidenceandabundantrainsevery year,butthesedonotlimittheuseofovitrapsassociatedwithS. spinosa.
However,themajorconcerninusingthisbacteriumfor biologi-calpestorvectorcontrolistheselectionofresistantinsects.Thishas alreadybeenobservedinagriculturalpests(Moultonetal.,2000; RehanandFreed,2014;Rinkevich andScott,2009)andinCulex quinquefasciatusSay,1823(SuandCheng,2012,2014a,2014b)and A.albopictus(Khanetal.,2011).
Theeffectiveness of B.thuringiensis israelensis is proven and selectiveforthecontrolofdifferentdipteralgroupssuchas Culici-dae,Simuliidae,andChironomidae(Bravoetal.,2011).Ithasbeen widelyemployedsuccessfully in differentregionsof theworld. Therewasalimitedselectionofresistantinsectsduetothespecific mechanismofactionandsafety(theseeffectsarenot accumula-tive;Bravoetal.,2011;Gill,1995).ByintegratingB.thuringiensis israelensis,ourresultspointtowardanimportantalternativeasa surveillancetool.ThistoolcouldaidinA.aegyptiandA.albopictus controlinurbanManaus.
Thelowest mean ovipositionwas observedin ovitrapswith grassinfusion+Pyriproxyfen.Thiscorroboratesotherstudies find-ingapreferenceforovipositioninsubstratescontainingbiological products(Quiroz-Martínezetal.,2012;Solís-Valdezetal.,2015). According to Forattini (2002), the microbial activity attracts female mosquitoes to lay their eggs in containers with this substrate.
Thelow numberofeggsobtainedinovitrapscontaining dis-tilledwater+T.haemorrhoidalis,isinaccordancewiththeresults obtainedforA.albopictusunderlaboratoryconditions.Itis impor-tanttoemphasizethatManausisacitywithstronganthropogenic influence,withurbanoccupationamongnativeandsecondary veg-etationmosaics,acharacteristicthatispresentinthestudyarea, withnaturaloccurrenceofT.haemorrhoidalis,whichcoexistswith themosquito speciestested. Thiscoexistencemighthave influ-enced thelow effectiveness of these traps in thefield, due to thewell-knownpredatorypotentialofthisspecies,mainlyofA. albopictus.
The number of eggs obtainedhere using ovitrapsand their respectiveOPI, EDI,and VDI indices highlight theeffectiveness of this tool in monitoring these vectors. The removal of eggs fromtheenvironment is analogous tothecontrol ofimmature stageswithacontrolagent.Thismakesthismethodmore effec-tiveandsaferformosquito-controlprograms.Therewereahigh numberofeggsfoundinthedifferentcontrolagentswithno repel-lents,andthisstrategycouldmonitorvectorsandassistinvector control.
StudiesusingovitrapstoestimatedensityofA.aegyptiandA. albopictusshowthatorganicmatterinfusions,mostlythosemade ofgrass,havebeenusedtomaximizetheeffectofthetrap, serv-ingasanattractanttofemales(Reiteretal.,1991;Santanaetal., 2006;Villasecaetal.,2001).AccordingtoNunesetal.(2011),grass infusionassociatedtoovitrapismoreeffectivethanovitrapswith water.Thiswasalsoobservedinourstudywithovitraps contain-ingonlygrassinfusionandgrassinfusionassociatedwithdifferent controlagents.
Studiesdemonstratethefeasibilityofovitrapsformonitoring andcontrolofA.aegyptiinthelong-term(ChismandApperson, 2003;Perichetal.,2003;Rapleyetal.,2009).Thesetraps, asso-ciatedto enthomopathogens, causereduction in thedensity of mosquitoesofmedicalimportance,preventinglarvaefrom devel-oping,thusensuringmoretimeintheenvironmentandpreventing trapsfrombecomingapotentialbreedingsite(Depolietal.,2016; Regisetal.,2008).
Althoughsomemosquitoes’populationsresistanttoNatularTM
DT– S.spinosahave beenreported,this productshouldnotbe excludedfromcontrolprograms,asithasbeenshowntobe effec-tivewhenassociatedtoovitraps.However,thislarvicideismore indicatedinspecificsituations,i.e.,whereithasnotbeen previ-ouslyusedandwhensurveillanceisrequiredbecauseofitsexcess use.
OvitrapsassociatedtoVectobac® WG–B.thuringiensis israe-lensisalsoplayanimportantroleintheovipositionofA.aegypti andA. albopictus.Thefactthat itis highlyselectiveforthe tar-getorganisms,thatnoselectionofresistantmosquitoeshasbeen detected,andthatitdoesnotrepelfemalesintheoviposition pro-cessmakestheintegrationofthisbiolarvicidewithovitrapsthe bestsurveillance/controltoolforAedes.
Conflictofinterest
Theauthorsdeclarenoconflictofinterest.
Acknowledgements
The authors thank the team of the Laboratory of Biological ControlandBiotechnologyofMalariaandDengueoftheNational InstituteofResearchoftheAmazon,FoundationforResearch Sup-portoftheStateofAmazonas,andNationalCouncilfortheScientific andTechnologicalDevelopmentfortheirfinancialsupport.
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