Fitness cost in field Anopheles labranchiae populations associated with resistance to the insecticide deltamethrin

REVISTA

BRASILEIRA

DE

Entomologia

Medical

and

Veterinary

Entomology

Fitness

cost

in

field

Anopheles

labranchiae

populations

associated

with

resistance

to

the

insecticide

deltamethrin

Ahmed

Tabbabi

,

Jabeur

Daaboub

DepartmentofHygieneandEnvironmentalProtection,MinistryofPublicHealth,Tunis,Tunisia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25October2017 Accepted18December2017 Availableonline29December2017 AssociateEditor:MariaAniceSallum Keywords: deltamethrin Insecticideresistance Fitnesscost Anopheleslabranchiae Malariavector Tunisia

a

b

s

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t

Weevaluatedinthepresentstudytheeffectofdeltamethrinresistanceonthefitnesscostofthefiled populationsofAnopheleslabranchiae.Asusceptiblepopulationwasusedasreferencetododifferent com-parisons.WeselectedthemostresistantlarvaepopulationcollectedfromnorthernTunisia.Eggswere usedforstudyoflifehistorytraitsincludingdevelopmentaltime,larvaemortality,fertility,hatchability andadultsex-ratio.Ourresultsshowedthatdeltamethrinresistanceaffectednegatively(p<0.05)the developmentaltimewiththemedianrangeof70h,mortalitywiththerateof7foldsinresistant popula-tionandhatchabilitywhicharelowerthaninsusceptiblepopulation.Whereas,nosignificantdifferences weredetectedinadultsex-ratioandfertilityofthetwostudiedpopulations.Ourresultscouldhelpto determinetheevolutionofpopulationdynamicsoftheresistantstudiedpopulationintheareaswhere insecticideresistanceisreportedandresistancemanagementisneeded.

©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The Plasmodium, which is transmitted by mosquito, is the biggestkillermosquitoesborndiseases(WHO,2014).Itremains aserioushealthproblemformanycountriesclassifiedasmalaria freethroughcasesimportedfromendemicregions.Inthecaseof Tunisia,severalstudiesshowedtheincreaseoftheannualincidence ofimportedcasesofmalariarelatedtotheexistenceofAnopheles mosquitoesinnumbershighenoughtoensuretheriskofa resump-tionofthediseasetransmissioninTunisia(Chadlietal.,1985;Ben Rachidetal.,1984;Gmara,2006;Bouratbineetal.,1998).

Anopheles(An.) labranchiae (Falleroni, 1926) is knownas an important vector of malaria throughout its global distribution (Beckeretal.,2010).Itsroleasvectorhasbeensuggestedin north-ernTunisiawheremalariawastransmitteduntilitseliminationin 1980(Tabbabietal.,2015).Historically,theirabilitiestotransmit strainsofPlasmodiumfalciparuminnaturaandunderlaboratory conditionshavebeensuggested(Totyetal.,2010).Thereis evi-dencealsooftheircapacitiestotransmitPlasmodiummalariae(Toty etal.,2010).Theirresponsibilitiesinthetransmissionof Plasmo-diumvivaxhavebeenrecentlyreported(Baldarietal.,1998).

∗ Correspondingauthor.

E-mail:tabbabiahmed@gmail.com(A.Tabbabi).

The control of An. labranchiae can be difficult. This species remainsestablishedinTunisiadespitetheeradicationofmalaria in1980(Tabbabietal.,2015).Chemicalandbiologicalpesticides includingDDT,and laterdeltamethrin, fentrothion, pyrethroids, and larvivorous fish such as Gambusiahave beenused against mosquitoes’ larvaebut An.labranchiaeis stillpersistingin high densitiesinnorthernpartsofthecountry(Tabbabietal.,2015).

Itisknownthatthemassiveapplicationofinsecticideduring malaria eradication program actually tendsto favor insecticide resistancewhichremainsaseriousthreatinmosquitoescontrol programs(BenCheikhetal.,1998;Nauen,2007;Daaboubetal., 2008).Thetoxiceffectofinsecticidescouldbeexpressedininsects by differentways suchasbehavioral, physiological and genetic expressions(KliotandGhanim,2012).Fitnesscostsresultingfrom resistancetoinsecticideshasbeenreportedinmanyinsectsfrom differentordersincludingmosquitoes (TabbabiandBenCheikh, 2017;SanilandShetty,2012;Brownetal.,2013;Jaramilloetal., 2014).However,otherstudiesdidnot observeanybiotic disad-vantageinresistantstudiedpopulations(Okoyeetal.,2007;Bielza etal.,2008;Lyonsetal.,2016).

Accordingtoourknowledge,theeffectofinsecticideresistance ontheAn.labranchiae lifehistory traitshasnot beenexplored. Differentparametersincludingdevelopmentaltime,larvae mor-tality,fertility,hatchabilityandadultsex-ratiowerestudiedusing twofieldcollectedpopulationsthatpreviouslyshowedhighand lowlevelofresistancetodeltamethrin,respectively(Tabbabiand Daaboub,2017).

https://doi.org/10.1016/j.rbe.2017.12.003

0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Tunisia Algeria Libya Ben arous Le kef 2: 1:

2

1

Fig.1.GeographicoriginofTunisianpopulationsofAnopheleslabranchiae.

Materialsandmethods

Mosquitoes

TwopopulationsofAn.labranchiaewerecollectedin2016in northwestern(LeKef)andnortheastern(BenArous)Tunisia(Fig.1) and identified assusceptible and resistantpopulations, respec-tively.Wechoosethemostresistant(frequentcontrolmosquitoes usingchemicalinsecticides)andthemostsusceptiblepopulation (absenceofmosquitoescontrol)todocomparisonsofstudied bio-logicalparameters.

Table1

ResistancetodeltamethrininAn.labranchiaefromTunisia.

Population LC50(␮g/L) RR50 95%CI Slope±SE Susceptible population 0.12(0.05–0.17) 2.10±0.32 – Resistant population 1.50(0.50–2.20) 1.22±0.17 12.50 (10.20–14.10) RR50,resistanceratioatLC50(RR50=LC50ofthepopulationconsidered/LC50of

sen-sitivestrain).

Mosquitorearing

Eggsweretakeninbreedingsitesofresistantandsusceptible populationsandtransferredtoplasticbasinscontainingwaterand rabbitcropwhichservedasfoodforhatchedlarvae.Rearing pro-ceededuntiltheadultstage.

Larvalbioassays

Resistancetodeltamethrininsecticide(99.5%[AI])wasevaluate in late third and early fourth instar larvae from both popula-tionsaccordingto standard methodsof Raymond et al.(1986). Lethalconcentrations(LCs)andresistanceratios(RR50andRR95) (Table 1)were calculatedvia probitanalysis ofRaymond et al. (1993)basedonFinney(1971).Bioassaysincluded5 concentra-tions(100,10,1,0.1,and0.01ppm)providingbetween0and100% mortalityand3replicatesperconcentrationonsetsof20larvaein atotalvolumeof100mLofwatercontaining1mLofethanol solu-tionofeachtestedinsecticide.Werepeatedtheassayiftherateof mortalityinthecontrolgroupexceeded10%.Itshouldnotethatthe studywascarriedoutunderlaboratoryconditionsandnotunder fieldconditions.

Fertility

Fertilitywasmeasuredasnumberoflarvaehatchedfromeach egg.Eggswerequalifiedasbigwhenthenumberoflarvaeexceeded 150,averagewhenthenumberoflarvaewaslocatedbetween150 and100andsmallwhenitnotexceeded100larvae.

Hatchability

Thislifehistorytraitwasmeasuredinpercentageasthenumber ofhatchedeggsbythetotalnumberofcollectedeggs.

Larvaldevelopmentaltimeandmortalityrate

Developmental time and larvae mortality were assessed by followinglarvaldevelopmentofresistantand susceptible popu-lationsfromfirstinstarslarvaetoemergenceofadults.Weused three ranges to neutralize the effect of density: low density (50larvae/500mL),averagedensity(100larvae/500mL)andhigh density(200larvae/500mL).Mortalitywasrecordeddaily. Adultsex-ratio

Thepupae weretransferred daily toa small cupcontaining 200mLtobeabletoidentifytheminmaleandfemales.

Statisticalanalysis

Data obtainedforeach parameterevaluated werecompared usingttestsforquantitativeparametersand2_analysisfor quali-tativevariables,asindicatedintheresults.

Table2

Hatchabilityandeggfertilityofsensitiveandresistantstrains.

Numberoffertileeggs Eggsizea _{Numberoffertileeggs/numbertotal(%) Bigeggs(%) Averageeggs(%) Smalleggs(%)}

Susceptiblepopulation 78 122.17±(38.15) 98.45 8.55 28.45 64.00 Resistantpopulation 24 89.42±(52.22) 35.27 12.18 33.79 54.03

a_{Standarderrorsinparentheses.}

Table3

Averagenumberoflarvae,percentageofemergedadults,mortalityrateanddevelopmenttimeofresistantstrain.

Density Larvae Adults Males Females Mortalityrate Developmenttime(h)

Male Females ALDR 50±(0.00) 45±(7.89) 21.55±(1.27) 23.45±(5.25) 5.00±(7.88) 301.87±(18.26) 298.15±(14.24) 304.00±(13.07) AMDR 100±(0.00) 50.47±(11.32) 20±(3.79) 30.47±(9.04) 50.53±(14.97) 412.75±(6.27) 390.55±(17.45) 422.99±(16.50) AHDR 200±(0.00) 35.33±(11.07) 15.75±(6.37) 19.58±(8.74) 164.66±(12.45) 450.33±(17.08) 390.00±(14.78) 420.33±(20.87) AA 116.66±(76.37) 43.6±(14.35) 19.1±(2.59) 24.5±(12.07) 73.33±(13.28) 388.31±(54.77) 359.56±(44.58) 382.44±(55.22) h,hours;standarderrorsinparentheses;ALDR,AverageofthethreeLow-DensityRepetitions;AMDR,AverageofthethreeMediumDensityRepetitions;AHDR,Averageof thethreeHigh-DensityRepetitions;AA,AverageofAverages.

Table4

Averagenumberoflarvae,percentageofemergedadults,mortalityrateanddevelopmenttimeofsensitivestrain.

Density Larvae Adults Males Females Mortalityrate Developmenttime(h)

Male Female ALDR 50±(0.00) 49.33±(4.53) 20±(5.69) 29.33±(5.48) 1.66±(4.72) 150.66±(24.45) 120.00±(9.65) 180.87±(22.19) AMDR 100±(0.00) 96.66±(3.48) 29.66±(5.87) 57.33±(10.42) 3.66±(8.40) 185.50±(17.43) 145.50±(28.77) 220.08±(17.38) AHDR 200±(0.00) 175±(4.79) 75±(3.78) 100±(6.45) 25±(8.08) 320.05±(22.37) 290±(20.41) 350±(12.27) AA 116.66±(76.37) 106.99±(32.27) 41.55±(21.07) 62.22±(8.77) 10.10±(5.27) 218.73±(75.88) 185.16±(42.78) 250.31±(75.85) h,hours;standarderrorsinparentheses;ALDR,AverageofthethreeLow-DensityRepetitions;AMDR,AverageofthethreeMediumDensityRepetitions;AHDR,Averageof thethreeHigh-DensityRepetitions;AA,AverageofAverages.

Results

Insecticideresistancestatusofstudiedpopulations

As shown in Table 1, the lethalconcentration (LC50)of the resistantpopulationisveryhighcomparedwiththesusceptible populationcalculatedbylogprobitanalysis(1.50(0.50–2.20)and 0.12(0.05–0.17) ug/L,respectively).

Hatchability

Themeanhatchingrateofsusceptiblepopulationwas98.45%, whereas, resistant population scored a mean range of 35.27% (Table 2). Statistical analysis showed a significant difference betweenthetwopopulations(p<0.05).

Fertility

Nosignificantdifferenceinfertilitywasobservedbetweenthe twostudiedsamples(p>0.05,Table2).

Developmentaltime

Itshouldbenotethatthestudyofthedevelopmenttimeofthe twostudiedpopulationswasinvestigated untiltheadult emer-gence.Thecomparisonofthis traithistoryparametershoweda significant differencebetween the two populations (2_=298.7, df=1, p<0.05, Tables 3 and 4). The larval developmental time

for resistantpopulationwaslongest(median=388h) compared tosusceptiblepopulation(median=218h).Ontheotherhand,the densityseemedtoaffectthedevelopmenttime.Indeed,thislife his-torytraitwasshorterinlowdensitiesthaninhighones.Itshouldbe notethatanysignificantdifference(p>0.05)wasdetectedbetween developmenttimeofmalesandfemales.

Mortalityrate

Mean larval mortalities were 73.33 and 10.10 for resistant andsensitivepopulations,respectively(Tables3and4).Statistical analysisrevealedsignificantdifferenceinmortalityratebetween strains(p<0.05).Ontheotherhand,mortalityratewashigherin highdensitiesthaninlowandmediumonesofthetwostudied populations.Thesignificantdifferenceinlarvaldevelopmenttime ofthetwostudiedpopulationscouldbeexplainedbytheimportant mortalityrateofresistantpopulation.

Adultsex-ratio

Basedonourresults,unbalancedsex-ratiowasobservedinall studiedpopulations(Tables3and4).Theeggstendtogivemore femalesthanmales.Thisdifferencewassignificantlyobservedin resistantstrain(p<0.05).

Discussion

In the present study, we tried to estimate the effects of deltamethrin resistance on several life histories of two An. labranchiaepopulations(resistantandsusceptible)collectedfrom

NorthernTunisia.Itshouldbenotethatthefitnesscostofseveral mosquitovectorsduetoinsecticideresistancehasbeenreported. However,studiesonfitnesscostofAn.labranchiaehavenot,toour knowledge,beenexploreddespitetheirpublichealthimportance (Tabbabietal.,2015).Our findingshowedthenegativeeffectof deltamethrinresistanceonsomelifehistoryparametersofresistant population.Similarresultswerefoundonothermosquitospecies usingdifferentinsecticides(Martinsetal.,2012;Jaramilloetal., 2014).

Amongtheaffectedlifehistoryparameters,anincreaseofthe developmenttimewasrecordedintheresistantpopulation.These findingareinagreementwithpreviousstudieswhichreportedan increaseofthelarvaldevelopmenttimeinresistantAedesaegypti (Diniz et al., 2015).In contrast, Plernsub et al. (2013) showed thatresistance didnotaffect larval developmenttime ofAedes mosquitoesinThailand.Itisimportanttomentionthatthis param-eteraffectthedisseminationofmosquitopopulationinthefield (Martinsetal.,2012).Innatural environment,itmayaffectthe adaptiveadvantagesofanindividualbycauseofseveral extrin-sicfactors,suchasphysicaldestructionofbreedingsites(Berticat etal.,2004),andthepresenceofpredatorsorparasitescanreduce thelarvalsurvivalrate(AgnewandKoella,1999)whichhelpinthe reductionofgenerations.Weshouldnotethatdevelopmentaltime variedaccordingtothesex.Indeed,slowingofthedevelopmentof femalecouldbeexplainedprobablybytheirneedtoaccumulate resourcesforreproduction(Clements,1992).

Ourresultsshowedthatresistancetendstoincreasethe mor-talityrateofAn.labranchiae.Similarresultshavebeenobserved inmanypreviousstudiesonmosquito’sspecies(Guillemaudetal., 1998;Lenormandetal.,1998).Thefactthatmortalitywashigherin thehigh-densityletussuggesttheroleplayedbydensityinhigher mortality.However,previousstudiesshowedthatlarvaldensity affectslarvalgrowthdramatically,butlarvalmortalitywas inde-pendentoflarvaldensity(DuffyandEpifanio,1994).Itshouldnote thatfoodplayedalsosomeroleinthelarvaldeathbypollutedwater quicklyenoughtokill.Ontheotherhand,afemale-biasedsexratio hasalsobeenreportedinAnophelesgambiaefromKenyia(Mutuku etal.,2006).Skewedsexratiosoffemalesathighlarvaldensities havebeenreportedinmanyinsects.Cipollini(1991)statedthat male-biasedmortalityathigherlarvaldensitiesproduceda female-biasedsex-ratioinAcanthoscelidesobtectus.Inthepresentstudy, themagnitudeofdensity-dependentdevelopmentandmortality thuscausedthedistortedsexratio.

Inourstudy,changesinbehavioralaspectswerealsoobserved. Indeed,areductioninhatchabilityandfertilityofresistant pop-ulation was recorded. Similar results were observed in Culex pipiensand Aedes aegypti (Li et al.,2002; Martinset al., 2012). In contrast, some reproductiveadvantages have been reported inpyrethroidresistantmalariavector,An.funestus(Okoyeetal., 2007).

ThedetectionofhighdeltamethrinresistanceinAn.labranchiae mosquitoesfromTunisia canbeexplainedbydeltamethrin/DDT cross-resistance.Infact,theDDTwasusedasthemainthemain insecticideintheframeworkoftheNationalProgramforthe Erad-icationofMalariaduringthe60sand70sagainstmalariavectors. Previousstudy(TabbabiandDaaboub,2017)showedthe involve-ment of target site alteration (Kdr mutation) in the recorded resistance.However,detoxificationenzymeswerenotinvolved.In supportoftheseresults,theresistancemechanisminvolvedexhibit a reduction infitness. Authorsnotedthat theincrease of mor-talityrateand thedecreaseof fecundityfemaleswereprobably duetothemodifiedacetylcholinesterasethatappearsassociated withahighercostthanthatassociatedwithoverproducedesterase (Lenormandetal.,1998;LenormandandRaymond,2000).Inthis context,weshouldnotethatitisnotcleariftheinsecticide resis-tancecanaffectthevectorialcapacityofmalariavectors.However,

Aloutetal.(2013)havereportedthenegativeimpacton compe-tencyofAn.gambiaetotransmitP.falciparum.

Theevaluationoffitnesscostparameterswascarriedoutinthe laboratoryconditionswhichdifferwithfieldconditions.The arti-ficialandoptimumconditionscansur-and/orunder-estimatethe results.Indeed,severalenvironmentalfactorsincludingthequality offood,larvaldensity,temperatureandhumiditycouldbe delete-riousforresistantpopulationsinthenature(BelinatoandMartins, 2016).

Conclusion

Thenegativeimpactofinsecticideresistanceondifferentfitness costparameters isveryimportantagainst themaintenance and dispersionoftheresistantindividualsinthefield.Further inves-tigationsareneededtoevaluatethisimpactonvectorialcapacity ofAn.labranchiaewhichisanimportantmalariavector.Itshould benotedthatourresultscouldhelptodeterminetheevolution ofpopulationdynamicsoftheresistantstudiedpopulationinthe areaswhereinsecticideresistanceisreportedandresistance man-agementis needed.However,furtherinvestigations areneeded to elucidate the mechanism of resistance using molecular and biochemicalmethodsandthereforeknowingwhetherinsecticide resistancemutationsinvolvefitnesscosts.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WearegratefultotheRegionalDirectoriesofPublicHealth.A specialthankstoDr.S. Fessi,Dr.A.Houerbi,Dr.L.Sakhri,Dr.T. Barhoumi,Mrs.S.Kilani,H.Hajlaoui,A.Mribai,H.Dellaii,H.Aloui, A.Rezeigui,N.Mejri,S.BenBdirafortheirprevioushelpandeffort toidentifybreedingsitesofAnophelesmosquitoesinNorthernand CentralTunisia.

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