ContentslistsavailableatScienceDirect
Acta
Tropica
j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a
Deltamethrin-mediated
survival,
behavior,
and
oenocyte
morphology
of
insecticide-susceptible
and
resistant
yellow
fever
mosquitos
(Aedes
aegypti)
Nadja
Biondine
Marriel
a,
Hudson
Vaner
Ventura
Tomé
b,
Raul
Carvalho
Narciso
Guedes
b,
Gustavo
Ferreira
Martins
a,∗aDepartamentodeBiologiaGeral,UniversidadeFederaldeVic¸osa,CampusUniversitário,Vic¸osa,MinasGeraisCEP36570-900,Brazil bDepartamentodeEntomologia,UniversidadeFederaldeVic¸osa,CampusUniversitário,Vic¸osa,MinasGeraisCEP36570-900,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received24December2015
Receivedinrevisedform26February2016 Accepted27February2016
Availableonline2March2016 Keywords: Aedesaegypti Behavior Deltamethrin Insecticideresistance Oenocytes
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InsecticideuseistheprevailingcontroltacticforthemosquitoAedesaegypti,avectorofseveralhuman viruses,whichleadstoever-increasingproblemsofinsecticideresistanceinpopulationsofthisinsect pestspecies.Theunderlyingmechanismsofinsecticideresistancemaybelinkedtothemetabolismof insecticidesbyvariouscells,includingoenocytes.Oenocytesareectodermalcellsresponsibleforlipid metabolismanddetoxification.Thegoalofthisstudywastoevaluatethesublethaleffectsofdeltamethrin onsurvival,behavior,andoenocytestructureintheimmaturemosquitoesofinsecticide-susceptible andresistantstrainsofA.aegypti.Fourthinstarlarvae(L4)ofbothstrainswereexposedtodifferent concentrationsofdeltamethrin(i.e.,0.001,0.003,0.005,and0.007ppm).Afterexposure,L4were sub-jectedtobehavioralbioassays.Insecticideeffectsoncellintegrityafterdeltamethrinexposure(at0.003 or0.005ppm)wereassessedbyprocessingpupaloenocytesfortransmissionelectronmicroscopyor TUNELreaction.TheinsecticideresistantL4survivedallthetestedconcentrations,whereasthe 0.007-ppmdeltamethrinconcentrationhadlethaleffectsonsusceptibleL4.SusceptibleL4werelethargicand exhibitedlessswimmingactivitythanunexposedlarvae,whereastheresistantL4werehyperexcited followingexposureto0.005ppmdeltamethrin.Nosublethaleffectsandnosignificantcelldeathwere observedintheoenocytesofeithersusceptibleorresistantinsectsexposedtodeltamethrin.Thepresent studyillustratedthedifferentresponsesofsusceptibleandresistantstrainsofA.aegyptifollowing expo-suretosublethalconcentrationofdeltamethrin,anddemonstratedhowthebehavioroftheimmature stageofthetwostrainsvaried,aswellasoenocytestructurefollowinginsecticideexposure.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
Themosquito Aedes aegypti isof great importancetopublic healthasoneofthemainvectorsofnumerousarboviruses, includ-ingthosecausingdengueandtheurbanyellowfeverviruses(Braga andValle,2007).Todate,vectorcontrolistheprincipalmethod ofpreventingdengueandotherdiseases.Amongthemethodsfor thecontrolofmosquitovectors,includingthoseemployedforA. aegypticontrol, theuseof insecticides,suchasdeltamethrin, is widespread(Rose,2001).However,thedevelopmentofresistance todeltamethrininvectorpopulationshasbeenreported(Gayathri andMurthy,2006;Pereraetal.,2008).
∗ Correspondingauthor.
E-mailaddress:gmartins@ufv.br(G.F.Martins).
Insecticideresistanceininsectvectors,includingmosquitoes, occursbecauseofastrictlygeneticmechanism.Themainmutations affectthetargetproteinsofinsecticides,whichmayalsoberelated toinsecticidemetabolism,andthismayresultinthereductionin insecticideexposureoruptakeand sequestrationofthe insecti-cideawayfromthetargetedproteins.Themainpyrethroidsitesof actionaretheneuronvoltage-gatedsodiumchannels(Narahashi, 1996, 2002; Soderlund,2012), which affect theperipheral and central nervous system,stimulating cells to produce repetitive dischargesandeventuallycausingparalysisandtheknockdown effect(BragaandValle,2007;Santosetal.,2007).However,there aremechanismsofresistancetopyrethroidsthatreduceneuronal sensitivitytotheinsecticide;onesuchmechanism isknownas knockdownresistance(KDR)(Soderlundand Bloomquist,1990). TheKDRleadstoapproximatelya10-to20-folddecreaseinthe sensitivityofthesodiumchannelinsecticide(Daviesetal.,2007a).
http://dx.doi.org/10.1016/j.actatropica.2016.02.021 0001-706X/©2016ElsevierB.V.Allrightsreserved.
InthehouseflyMuscadomestica,forexample,achangeinasingle nucleotideinthesodiumchannelsresultsinaKDRmutation(Ingles etal.,1996;Knippleetal.,1994;Miyazakietal.,1996;Williamson etal.,1993,1996).Insomecases,mutationsrepresenta100-fold increase in pyrethroid resistancewhat is knownas super-KDR resistance(Daviesetal.,2007a).Mutationsinthesodiumchannel areresponsibleforKDRandhavebeendocumentedinseveral agri-culturalpestanddiseasevectorinsects(Daviesetal.,2007b;Dong, 2007).Theselectivepressureofinsecticides,theirfrequencyofuse, andtheinheritabilityofresistancetothemcontributetothespread ofinsecticideresistanceamonginsectpopulations,compromising controlefforts(HemingwayandRanson,2000;Lietal.,2007).
Thebehaviorofinsects,aswellasallanimals,isgovernedby interactionsbetweenneuronsandbetweenthenervoussystem, andtheenvironment.Thereareneurotoxicinsecticides(suchas deltamethrin,atypeIIpyrethroid)thatactatspecificsitesinthe insectnervoussystemwhileinteractingwiththesodium chan-nelsinneurons(Narahashi,1996;RayandFry,2006).Therefore, neurotoxicinsecticidescanaffectthebehaviorofinsectsevenat lowconcentrations(Guedesetal.,2016;Haynes,1988).Sublethal exposuretoinsecticidescanalsoalterpredator-preyinteractions (Clements and Newman, 2002; Dell’omo, 2002; Guedes et al., 2016;Teplitskyetal.,2005;VanGossumetal.,2009),and inter-ferewithdevelopment,survival,andmobility,asobservedinA. aegypti(Toméetal.,2014).Inaddition,variousactivities,suchas seekingrefuge,evasion,breathing,foraging,andlocomotion,can beaffectedwhen insectsareexposed toneurotoxicinsecticides (Brackenbury,2001;Guedesetal.,2016;JanssensandStoks,2012). Insectbehavioralresponsestoinsecticidesarediverseandmay berelatedtoinsectmobility(Davidson,1953;Guedesetal.,2016; HaubrugeandAmichot,1998;Lockwoodetal.,1984),andthese behavioralresponsescanbeevaluatedbyexcito-repellencytests (Robertsetal.,1997).Irritabilitybycontactexposuretakesplace when adults of A. aegypti are directly exposed to insecticides, suchas deltamethrin, and themosquitoes quicklyescape from insecticide-contaminatedareas,whichcanhavesignificanteffects ontheeffectivenessofmosquitocontrolanddiseasetransmission (Kongmee etal.,2004).Therefore, othereffectsof deltamethrin shouldbeevaluatedtounderstandmosquitobiology,includingthe behavioraleffectsofinsecticideexposure.
Themetabolismofxenobioticsisoneofthemaindetoxification mechanismsofpesticidesininsects.Severalinsecttissuesandcells areinvolvedinthedetoxificationprocesses,includingthe integu-ment,midgut, fat bodies,and oenocytes.Oenocytesarecells of ectodermaloriginthatareallegedlyinvolvedininsectprotection againstinsecticides(ClarkandDahm, 1973;Lycettetal.,2006). Therefore, as detoxification enzymes present on oenocytes are potentiallyinducedbychemicalstress,thesecellsareapromising targettoelucidateinsecticideactionandinsectresponseto insec-ticideexposure.Hereinwereportonacomparativestudyoftwo populations(onesusceptibleandtheotherresistanttopyrethroids) oftheyellowfevermosquitoA.aegypti.Thegoalsofourstudywere todeterminetheirsurvival,larvalbehavior,and(pupal)oenocyte morphologyfollowing exposuretovarioussublethal concentra-tionsofdeltamethrin.
2. Materialandmethods
2.1. Mosquitoes
Fourthinstarlarvae(L4)andpupaeofA.aegyptiwereobtained fromtwodifferentstrains:thepyrethroid-susceptiblePPCampos strain(CamposdosGoytacazes),andthepyrethroid-resistantF2 Oiapoque strain (Rio de Janeiro). Mosquitoes were maintained in theinsectary of theDepartment of General Biology,Federal
Fig.1.SurvivalcurvesforL4ofpyrethroid-susceptibleA.aegypti(A)andresistant (B)strainssubjectedtoincreasingsublethalconcentrationsofthepyrethroid insec-ticidedeltamethrin.SurvivalcurvesofdifferentcolorsdifferentbyHolm-Sidak´ıs test(P>0.05).
University of Vic¸osa(Vic¸osa,MG, Brazil) undercontrolled tem-perature(25±2◦C),relativehumidity(60±2%),andphotoperiod (12:12L:D).
2.2. Survival
OnehundredL4ofbothpopulationsweremaintainedin 500-mL glass containers with 200mL of water and 5mg of turtle food(ReptoLife)and weresubjectedtodifferentconcentrations of deltamethrin (0.001, 0.003, 0.005,and 0.007ppm). The con-trolgroupunderwentthesametreatment,exceptforexposureto theinsecticide.Deltamethrin(25giaL−1emulsifiableconcentrate, BayerCropScience,SãoPaulo,SP,Brazil)wasdilutedwithdistilled watertoobtainthedesiredconcentrations.Afterexposure,the lar-vaewerekeptinthesameenvironment,monitored,andevaluated untilemergenceofalladults12daysfollowingexposure.The sur-vivalcurveswereestimatedbytheKaplan-Meyerdesignusingthe softwareSigmaPlotv.12.0(Systat,JanJose,CA,US).
2.3. Swimmingbehavior
For the behavioral assays, six independent replicates were performed as follows: six glass vials containing 25 L4 of both strains–pyrethroidsusceptibleandresistant–wereexposedto 0.003and0.005-ppmofthepyrethroidinsecticidedeltamethrin.
Fig.2.Linearregressionsillustratingthevariationofpixelsovertimeinsusceptible (A)andresistant(B)L4Aedesaegyptiat1,3,5,7,9,and24haftertheexposureto 0.003or0.005ppmdeltamethrin.
Unexposedindividualsofthetwostrainswereusedasseparate controls.Exposedandunexposedindividualsweremonitoredat differenttimeintervals(1h,3h,5h,7h,9h,and24hafter insec-ticideexposure).L4weretransferredbrieflytoarenasconsisting ofPetridishesplacedonawhitesurfaceandcontaining20mLof distilledwater.Aftereachrecording,larvaewerereturnedtotheir respectivevials(Toméetal.,2014).
Thelocomotionactivityofthelarvaeinthearenawasrecorded for15minby anautomatedvideo monitoringsystemequipped withaCCDcamera(ViewPointLifeSciencesInc.,Montreal,Canada) connectedtoacomputer.Therecordedparameterineacharena wasthelevelofactivity,measuredinpixels.For eachreplicate, thecontrolandtreatmentgroupswererandomlyplacedand inter-spersedinfourdifferentpositionstopreventanypossibleposition effects,forexample,subtledifferencesinlightintensityor temper-aturegradient(Toméetal.,2014).
2.4. Oenocytes
Oenocytesweredissectedfrompupaeofthetwostrains (treat-mentandcontrol)whoseL4werepreviouslytreatedwith0.003and 0.005ppmdeltamethrin.Toobtainlargeamountsofcells, oeno-cytesweredissectedfrompupae48hafterinsecticideexposure. Atthisstage,theoenocytesarelooselyclusteredcellsthathave notyetspreadwithinthefatbody,makingthemeasiertoisolate (Martinsetal.,2011c).Withtheaidofastereoscopemicroscope, pupaeweredissectedonglassslidesinphosphatebuffer(PBS0.1M, pH7.2).Theclustersofoenocyteswerecollectedwiththeaidofa micropipette(1–10L)andfixed(Martinsetal.,2011c).
2.5. Transmissionelectronmicroscopy(TEM)
Dissectedoenocytes(treatmentandcontrol)weretransferred tomicrocentrifugetubescontaining1mLof2.5%glutaraldehydein 2%sucroseand0.1McacodylatebufferpH7.2foratleast2h.After fixation,cellswerewashedthreetimesinPBS,andpost-fixedin1% osmiumtetroxidefor2hinthedark.Thecellswerethenplaced in LRWhite resin (London Resin Company Ltd.) in gelatin cap-sules.Beforeeachsolutionexchange,themicrotubescontainingthe oenocytesweresubjectedtoashortspin(upto5000rpm)toobtain pellets(Martinsetal.,2011c).Ultrathinsectionswerestainedwith uranylacetateandleadcitratefor15and8min,respectively.The materialwasanalyzedundertheTEMZeissEM109attheNúcleo deMicroscopiaeMicroanáliseatUniversidadeFederaldeVic¸osa (NMM/UFV),oratthePlataformadeMicroscopiaEletrônicaRudolf BarthdoInstitutoOswaldoCruz(FIOCRUZ,RJ)undertheTEM JEOL-JEM-1011.
2.6. TUNELreaction
Oenocytesweredissectedandtransferredtopreviouslycleaned glassslidesandallowedtorestfor30minforcelladhesionatroom temperature. Adherent cells were fixedwith 4% paraformalde-hyde (pH 7.4) for 30min, washed three times with PBS, and stored in distilled waterat 4◦C (Martinset al., 2011c).For the detectionofcleavageofthenuclearDNA,fixedoenocyteswere sub-jectedtoTUNEL(theterminaldeoxynucleotidyltransferasedUTP nickend labeling) reactionfor 1hat37◦C usingthekit InSitu CellDeathFluorescein(RocheMolecularBiochemicals,Mannheim, Germany)accordingtothemanufacturer’sinstructions.Theslides withadheredcellsweremountedinMowiolantifadingmedium (Fluka,Steinheim, Germany),analyzed,andphotographedusing thefluorescencemicroscopeZeissplus AxiostarwithCCD cam-eraAxioCamMRMatNMM/UFV.Forthenegativecontrolforthe TUNELreaction, oneslidewithoenocytesof each experimental group(totalof4)wasincubatedwithoutthetransferaseenzyme. 2.7. Statisticalanalyses
Larval activity data of the two strains were subjected to repeatedmeasureanalysesofvariance todeterminetheeffects of deltamethrin concentrations. Differences were evaluated by Fisher’s exact test(PROC ANOVA;SAS Institute, 2008).Survival curveswerecomparedusingtheHolm-Sidak’stestusingSigmaPlot.
3. Results
3.1. Survival
ThesurvivalofA.aegyptiwasrecordedforsusceptibleand resis-tantstrainssubjectedtodifferentconcentrationsofdeltamethrin (0.001,0.003,0.005,and0.007ppm).AccordingtoKaplan-Meyer estimators, the survival of the susceptible strain differed sig-nificantly among the tested concentrations (2=301.97, df=4;
P<0.001)(Fig.1).Forthesusceptiblestrain,100%ofexposed lar-vaediedat0.007-ppmdeltamethrinbysevendaysafterexposure. Thus,0.007ppmisalethalconcentrationandpreventsthe insecti-cidesusceptiblelarvaefromreachingtheadultstage.Fortheother concentrations, theinsecticide-susceptibleindividuals exhibited differentsurvivalrates(Fig.1A).
Theresistantstrainrespondeddifferentlytothedeltamethrin thandidthesusceptiblestrain.Bothdeltamethrin-exposedand -unexposedindividualsoftheresistantstrainhadaconstantsurvival rateof100%,beingunaffectedbythefourconcentrations(2=3.78,
df=4;P=0.43)(Fig.1B).Deltamethrinconcentrationsof0.003ppm and0.005ppmwerechosenforsubsequentsublethalassessments
ofbehaviorandpupaloenocytesofA.aegyptibecausetheyledto theaverageandhighestmortalityvaluesobtained,respectively. 3.2. Swimmingbehavior
Behavior of insecticide-susceptible and resistant L4 was recorded at1, 3, 5,7, 9, and 24hafter deltamethrin exposure. RepeatedmeasuresperformedforlocomotoractivityofL4 indi-cated significant differences between treatments and controls (controlvs.deltamethrinconcentrations:F2,15=125.54,P<0.0001),
timeafterexposure(Wilk’s␥>0.11;F>16.69;dfnum/den=5/11; P<0.0001),andinteractionsbetweensourcesofvariation(Wilk’s ␥>0.05;F>7.36;dfnum/den=10/22;P<0.0001)forthe suscepti-blestrain.Repeatedmeasuresanalysesalsoindicatedsignificant differences between treatments (control vs. deltamethrin con-centrations:F2,15>17.28,P<0.0001),timeafterexposure(Wilk’s
␥>0.33; F>4.32; df num/den=5/11; P=0.02), and interaction between the sources of variation (Wilk’s ␥>0.08; F>5.33; df num/den=10/22;P=0.0005)fortheresistantstrain.
For both insecticide-susceptible and resistant strains, linear regressionswerenotsignificant(P>0.005);therefore,therewas notime-dependentvariationinlocomotoractivityforexposedand unexposedindividuals.Althoughtherewasnovariationovertime, theactivity(measuredinpixels) ofsusceptibleL4waslowerin thedeltamethrin-exposedinsectsthanintheunexposedinsects (Fig.2A).InresistantL4,hyperexcitationwasobserved24hafter exposurewith0.005ppmdeltamethrinincomparisontothe con-trol.Ontheotherhand,resistantL4treatedwith0.003ppmshowed adecreasein activity(Fig.2B).Apparently,theunexposed indi-viduals of the resistant strain exhibit higher activity than do thesusceptibleindividuals.However,whentheexperimentwas repeatedforthesameparameterstocomparethesusceptibleand resistantcontrols,therewerenosignificantdifferencesbetween theiractivitylevels.
3.3. Celldamage
Therewereveryfewoenocytespositive(∼1.5%ofcells)forthe TUNELreactionineithersusceptible(Fig.3A–F)orresistant indi-viduals(Fig.3G–L).Approximately600cellswereanalyzedforeach strain,andtherewerenodifferencesregardingpositivecells(with agreenfluorescentnucleus)ofL4treatedwiththeconcentrations of0.003ppmor0.005ppm.
3.4. Transmissionelectronmicroscopy
Pupaloenocytesofsusceptibleorresistantindividuals exhib-itednochangesintheirultrastructurecomparedtotheirrespective unexposedcontrols(Figs.4and5).Thesecellshadextensive mem-braneinvaginations(Figs.4A,5B)andelectron-densecytoplasm, withlipiddropletsinthecytoplasmandmitochondria(Figs.4A–F and5A–F).Oenocyteshavethecharacteristicsofametabolically activecell,withacentralnucleusandawell-developednucleolus, andpredominantnon-condensedchromatin(Figs.4B,C,F,and5
A,C,F).Sometreatedcells(at0.005ppmdeltamethrin)ofthetwo strainshaddamagedmitochondria(Figs.4Hand5H)andnuclei (Fig.4G).Inthesecases,thecompletedisorganizationofthenuclear chromatinandmembranewasobserved,andtherewerechanges inthemorphologyofmitochondrialcristae(Figs.4G–H;5H).
4. Discussion
ThehighmortalityofsusceptibleA.aegyptiL4afterdeltamethrin exposureat differentconcentrations is in accordance withthe observationsofToméetal.(2014),whoshowedthelethaleffects of deltamethrin in the same population and stage, suggesting
that this insecticide can be very toxic to the mosquito larvae. In general, despite the potentially low pyrethroid penetration through thelarvalintegument,the pyrethroidcouldbe quickly absorbedviathedigestivetractafteringestionorviathe respira-torytract(Soderlundetal.,2002).Afterabsorption,theinsecticide causedimmediateparalysisandmortality(knockdown)(Santos et al.,2007).Unlike susceptibleindividuals, allof the resistant-individualsexposedreachedtheadultstage.Thedeltamethrinis effectiveandbroadlyrecommendedasadulticideinthecontrolofA. aegypti(WHO,2005;Kumaretal.,2011).However,thispyrethroid insecticideisalsoeffectiveasalarvicidewithreportedeffectson mosquitodevelopmentandbehavior(e.g.,GayathriandMurthy, 2006; Pereraet al., 2008; Tomé et al.,2014).Although we did not identifythemechanism involved in mosquito resistance, it hasbeenbroadlyconfirmedthatresistanceinA.aegypti popula-tionsmaybeassociatedwithmutationsinsodiumchannelsorby enhanceddetoxificationofxenobiotics(Kumaretal.,2002;Martins etal.,2009;Montellaetal.,2007;Rodríguezetal.,2005; Saavedra-Rodriguezetal.,2007).
TheexposureofsusceptibleA.aegyptiL4to0.003or0.005ppm deltamethrinledtoreducedlocomotoractivitieswhencompared withthose ofthe unexposedcontrols.This lethargiceffect was probablyassociatedwiththeneurotoxicmechanismofactionof pyrethroids(Narahashi,1996;RayandFry,2006).ThelethargicL4 haddifficultyinswimming,whichinturncouldleadtothe impair-mentoftheirforagingactivity.Inaddition,becauseofthedifficulty inswimmingandmoving,L4maybemorefrequentlyattackedby predatorsandwouldhavedifficultyinsearchingforrefuge,thereby decreasingtheirchancesofsurvival(Brackenbury,2001;Janssens andStoks,2012).Forexample,itwasobservedthatthepyrethroid treatmentofCulexpipiensmolestuslarvaecompromisedtheiralarm responsetopredators,increasingthelikelihoodoflarvalmortality causedbypredation(Reynaldietal.,2011).
The behavior responses related to the pyrethroid resistant strainofA.aegyptidifferedfromthoseobservedinthesusceptible strain.TheresistantL4exposedto0.003ppmdeltamethrin exhib-itedreducedlocomotoractivitywhencomparedtotherespective control; however,theydidnot demonstratethelethargiceffect observedinsusceptibleL4.Moreover,resistantL4exhibited hyper-excitabilitywhencomparedwiththecontrol24hafterexposureto 0.005ppmdeltamethrin.Inadditiontoactingonthesodium chan-nelsofnervecells,thetypeIIpyrethroids,suchasdeltamethrin, may also interactwith the␥-aminobutyric acid (GABA) recep-tors, an important inhibitory neurotransmitter in the central nervoussystem,asobservedinAnophelesgambiaeadultstreated with deltamethrin (Bradberry et al., 2005; Santos et al., 2007; Taylor-Wellsetal.,2015;Veliseketal.,2006).GABAleadsto hyper-polarizationofthepost-synapticterminal,andhyperexcitationin resistantL4probablymaybeaconsequenceoftheblockageofGABA receptorsbydeltamethrin(MacdonaldandOlsen,1994).
Not surprisingly, the negative effects of exposure to deltamethrinweremoreevidentforthesusceptibleindividualsof A. aegypti,oncetheinsecticide impairedswimmingmovements of theL4. On the other hand, in the resistant individuals, the treatmentledtohyperexcitability,whichapparentlydidnothave significant negativeimplications because all of the exposed L4 weremovingnormally,withnodifficultyswimmingorforaging.
TheA.aegyptioenocytesallegedlyparticipateinthe detoxifi-cationofexogenousmoleculesbyexpressingproteintranscripts of thecytochrome P450 superfamily (over 8%) (Martins et al., 2011a)orbythesuperexpressionofcytochromeP450reductase enzymeinA.gambiaeand Drosophilamelanogaster(Lycettetal., 2006).Ourworkfocusedontheanalysisofpossiblesideeffects ofdeltamethrinintheoenocytescausedbytheirallegedrolein xenobioticdetoxification(Martinsetal.,2011a;Lycettetal.,2006). However,nosignificantcelldamagewasdetectedusingtheTUNEL
Fig.3.PupaloenocytesofA.aegyptideltamethrinsusceptible(A–F)andresistant(G–L)strainssubjectedtoTUNELreaction:(A,B,G,andH):clustersofoenocytes(oe) ofcontrol(unexposedindividuals)withnonuclearfluorescence(*).(C,D,J,andI):clustersofoenocytesafter0.003ppmdeltamethrintreatment.(E,F,K,andL):clusters ofoenocytesfromtreatedindividualswith0.005ppmdeltamethrin.ThenucleusofoenocyteswithDNAdamage(arrows)aregreen-fluorescent.Arrowsindicatecells. Bars=20m.
assayandTEManalysis.TherewerefewTUNEL-positivecells in thepupaeofsusceptibleandintheresistantindividualsin compari-sontotheirrespectiveunexposedcontrols.Oenocyteultrastructure remained unaltered in exposed individuals of the two strains comparedwithunexposedcontrols.Eveninexposedindividuals, thecells maintainedtheiroriginalcharacteristicsandpreserved metabolicallyactivecellswiththecytoplasmfilledwithtranslucent vesicles,whichcorrespondtotheaccumulationoflipids(Gutierrez etal.,2007)andinvaginations oftheplasmamembrane, which areresponsiblefortheincreaseofthecellsurface(Martinsetal., 2011a,b).
Toobtainalargeramountofoenocytes,thecellsweredissected frompupae, where theyweregrouped, moreconspicuous, and eitherlooselyintegratedornotintegratedintofatbody(Martins etal.,2011c).The deltamethrinexposurestartedatL4, andthe individualswerecontinuouslyexposedtoinsecticideuntil pupa-tionand dissection of oenocytes.It was not possibleto search for any deltamethrin effect on larval oenocytes because they
disappearduringpupation.Therefore,anyeventualindirecteffect onoenocytesformationshouldappearlater,onceoenocyte differ-entiationoccursdenovoduringpupation(Wigglesworth,1933). Deltamethrinexposureappearstoaffectcells differently.Inthis case, different targets(oenocytes or thenervous system) were notaffectedin thesame way.Contrarytotheexpectedresults, deltamethrinexposuredidnotleadtooenocytedeathinthe sus-ceptiblestrain,orevenalteredoenocyteultrastructure,indicating thatcellintegrityisnotrelatedtoinsecticideresistance. Further-more,seriousimpairmentwasobservedofmotoractivitiesofthe susceptiblestrainandhyperexcitationinresistantL4treatedwith 0.005ppmdeltamethrin.
5. Conclusion
DeltamethrindamagetoDNAintegrityandoenocyte ultrastruc-turewasverylimitedintheimmaturelarvaofbothsusceptibleand resistantstrainsofA.aegypti.Thisconfirmedthattherewereno
Fig.4. TransmissionelectronmicrographsofpupaloenocytesofA.aegyptioftheinsecticide-susceptiblestrain.(AandB):Oenocytes(e)ofthecontrol(unexposedindividuals). (C,D,andE):oenocytesofindividualsexposedto0.003ppmdeltamethrin.(F,G,andH):oenocytesofindividualsexposedto0.005ppmdeltamethrin.(A):Fourgrouped oenocyteswithmembraneinvaginations(in).(I):intercellularspace.(B,C,andF):Generalviewofoenocyteswithnosignofcelldamagewithalargenucleus(n)with non-condensedchromatin(nc),atranslucentcytoplasmfilledwithvesiclesresemblinglipiddroplets(L),andmitochondria(M).(D):Detailsofintactmitochondriainacell ofanindividualexposedto0.003ppmdeltamethrin.Arrowsindicatetheroughendoplasmicreticulum.(E):cellcortexwithmembraneinvaginations(in).(G–H):Abnormal nucleus(n)andmitochondrion(m)incellsoftreatedindividualswith0.005ppmdeltamethrin.Notethechangesinthepatternsofchromatinandnuclearmembraneand mitochondrialcrests.
Fig.5. Transmissionelectronmicrographsofpupaloenocytesoftheinsecticide-resistantstrainsofA.aegypti.(AandB):oenocytes(e)ofcontrol(unexposedgroup).(C,D, andE):oenocytesofanindividualexposedto0.003ppmdeltamethrin.(F,G,andH):oenocytesofanindividualexposedto0.005ppmdeltamethrin.(A,C,andF):General viewofoenocyteswithnosignofcelldamagewithalargenucleus(n)withnon-condensedchromatin(nc),atranslucentcytoplasmfilledwithvesiclesresemblinglipid droplets(L),andmitochondria(M).(B):Invaginations(in)ofthecellmembrane.(D):mitochondriaofintactcellsofanindividualexposedto0.003ppmdeltamethrin.(E): Lipiddroplets(L).(G):mitochondriainintactcellsofanindividualexposedto0.005ppm.(H):Abnormalmitochondria(m)withabnormalcrestsinacellofanindividual exposedto0.005ppmdeltamethrin.
significantsecondaryeffectsofdeltamethrinonoenocytesunder the conditions tested in this study that would contribute to pyretroidresistance.Therefore,oenocytespreservetheirfunctions eveninsusceptibleindividuals,confirmingthatthedifferencein thesusceptible and resistantstrainsis probablycaused by tar-getsite differencesinthenervoussysteminsteadofdamageon oenocytes.Incontrast,survivalandswimmingunderdeltamethrin exposuredifferbetweenthemosquitostrains,indicatingthatthe resistance insects are able tobetter cope with the insecticide. Finally,ourworkcontributedtotheelucidationoftheresponses related todeltamethrin exposureand theunderstandingofthe varioussublethaleffectsoftheinsecticideinsusceptibleand resis-tantstrainsofA.aegyptiandtheirrelationshiptoneurologicaland physiologicalprocessesthatdirectlyaffectmosquitobehaviorand ecology.
Acknowledgments
WethankDr.Ademir deJesusMartinsJúniorandJoséBento PereiraLima(InstitutoOswaldoCruz,IOC-Fiocruz,RiodeJaneiro) forkindlyprovidingthemosquitoeggsoftheF2Oiapoquestrain, NMM/UFVandIOC/FiocruzfortechnicalassistancewithTEM,and FAPEMIGandCAPES(PVE39088881.030429/2013-01)forfinancial support.
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