Behavioral patterns, parity rate and natural infection analysis in anopheline species involved in the transmission of malaria in the northeastern Brazilian Amazon region

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

Behavioral

patterns,

parity

rate

and

natural

infection

analysis

in

anopheline

species

involved

in

the

transmission

of

malaria

in

the

northeastern

Brazilian

Amazon

region

Ledayane

Mayana

Costa

Barbosa

_,

_Raimundo

_Nonato

_Picanc¸

_o

_Souto

_,

Ricardo

Marcelo

dos

Anjos

Ferreira

_,

_Vera

_Margarete

_Scarpassa

a_{LaboratóriodeArthropoda,DepartamentodeCiênciasBiológicasedaSaúde,UniversidadeFederaldoAmapá,RodoviaJuscelinoKubitschek,km02,Jardim} MarcoZero,CEP68.903-419Macapá,Amapá,Brazil

b_{LaboratóriodeGenéticadePopulac¸õeseEvoluc¸ãodeMosquitosVetoresdeMaláriaeDengue,Coordenac¸ãodeBiodiversidade,InstitutoNacionalde} PesquisasdaAmazônia,AvenidaAndréAraujo,n◦_{2936,BairroAleixo,CEP69.067-375Manaus,Amazonas,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received7June2016

Receivedinrevisedform6September2016 Accepted17September2016

Availableonline19September2016 Keywords:

Transmissiondynamic Anophelesspecies Malaria

BrazilianAmazonregion

a

b

s

t

r

a

c

t

Thecharacterizationofbehavioralpatternsallowsabetterunderstandingofthetransmissiondynamics andthedesignofmoreeffectivemalariavectorcontrolstrategies.Thisstudyanalyzedthebehavioral patternsoftheAnophelesspeciesoftheCorac¸ãodistrictsituatedinthenortheastoftheBrazilianAmazon region.Thebehavioralpatternsoftheanophelinespeciesweremeasuredbasedonthe36collectionsites ofthisdistrictfromDecember2010toNovember2011.Collectionsoffourhoursforthreeconsecutive nightseachmonthandfour12-hcollections,comprisingtwointherainyseasonandtwointhedryseason, wereperformed.Furthermore,toinfertheanthropophilyandzoophilyindexes,fouradditional four-hourcollectionswereperformed.Thesampleswerealsoevaluatedforparityrateandnaturalinfectivity forPlasmodiumspp.Atotalof1689anophelineswerecaptured,comprisingofninespeciesandtwo subgenera(Nyssorhynchus–sixspecies,andAnopheles–threespecies).Anophelesdarlingiwasthemost abundantandwidelydistributedspeciesinthearea,followedbyA.braziliensisandA.marajoara.Anopheles darlingiandA.marajoaraweretheonlyspeciespresentinthefourcollectionsof12-h,butonlyA.darlingi showedactivitythroughoutnight.Anophelesdarlingiwasthemostanthropophilicspecies(AI=0.40),but thezoophilyindexwashigher(ZI=0.60),revealinganeclecticandopportunisticbehavior.Ofthesix mostfrequentspecies,A.nuneztovaris.l.wasthemostzoophilicspecies(ZI=1.00).Allcapturedspecies showedpredominancetowardsbitinginoutdoorenvironments.AnophelesdarlingiandA.braziliensis showedmultimodalbitingpeaks,whereasA.marajoararevealedastablepattern,withthebitingpeak aftersunset.UsingthePCRtechnique,noanophelinewasfoundinfectedwiththemalariaparasite.SinceA. darlingiandA.marajoaraarerecognizedasimportantvectorsinthisregion,thedistrictofCorac¸ãomaybe consideredasahighlypotentareafortransmissionofmalaria,therefore,thepreventionandsurveillance measuresshouldbetakenconstantlytopreventthesame.TheroleofA.braziliensisasmalariavector needstobeurgentlyinvestigated.

PublishedbyElsevierB.V.

1. Introduction

Nowadays, malaria is a major publichealth problemin the

worldand is widely distributed in thetropical and subtropical

regions.Althoughintherecentyearsthenumberofmalariacases

∗ Correspondingauthorat:Coordenac¸ãodeBiodiversidade,InstitutoNacional dePesquisasdaAmazônia(INPA),AvenidaAndréAraujo,2.936,BairroAleixo,CEP 69.067-375Manaus,Amazonas,Brazil.

E-mailaddress:vera@inpa.gov.br(V.M.Scarpassa).

worldwidehasdecreasedbyapproximately47%,thedataarestill

alarming.Around3.2billionpeoplein97countriesand

territo-riesareattheriskofbeinginfectedwiththemalariaparasite.In

2015,214millioncasesofthediseasewererecorded,88%ofthem

inAfrica.Itcausedthedeathof438,000peopleworldwide,among

which306,000werechildrenunderfiveyears(MS,2013;WHO,

2015).InBrazil,therewasa75%decreaseinthenumberofcases

from2002to2013,andin2014(intheBrazilianAmazonregion)the

lowestnumberofmalariacases(143,552)inthelast35yearswere

recorded(MS,2015).However,despitethereductioninthenumber

ofcases,thereisstillevidencesofhighriskofoutbreaks,especially

http://dx.doi.org/10.1016/j.actatropica.2016.09.018 0001-706X/PublishedbyElsevierB.V.

2006;Tadeietal.,1998;TadeiandDutary-Thatcher,2000).These

parameters togetherwiththeecologicalconditions of areaand

the social and cultural factors of the local human populations

provide a base for management and a more effective vector

control.

Asalreadymentionedabove,overthelastyearsmalaria

trans-missionhasdecreasedintheBrazilianAmazonregion,especially

asaresultofmoreinvestmentscomingfromthefederal,stateand

municipalgovernments, which improvedthecontrolprograms.

Thestrategiesandtargetsoftheseprogramsincludedthetraining

ofhealth technicians,earlydiagnosisfollowed bydisease

treat-ment,besidesthepreventionbymosquitocontrol(MS,2013,2015;

WHO,2015).Infact,vectorcontrolisstilloneofthemostsuccessful

waysofcontrollingmalaria.

InthestateofAmapá,althoughtherewasanoverallreductionin

malariacases(MS,2015),themunicipalitiesofMazagão,Calc¸oene,

PedraBrancadoAmapari,SerradoNavioandTartarugalzinhowere

classifiedashigh-riskareasforoutbreaks(MS,2015).Thissituation

isworrying,becausethemainmalariavector,A.darlingi,iswidely

distributedinthestateofAmapáandhasbeencapturedathigh

den-sitiesinmanylocalitiesofthisState(Tadeietal.,1998;Voorham,

2002;Galardoetal.,2007,2009;Zimmermanetal.,2013).Recently,

highdensity of A. darlingi wasalso observed in thedistrict of

Corac¸ão(Barbosaetal.,2014).Furthermore,studiesconductedin

thisStatehavereportedhighdiversity(ahotspot)andabundance

ofotheranophelinespecies(Tadeietal.,1998;Póvoaetal.,2001),

suchasAnophelesmarajoara,Anophelesnuneztovaris.l.,Anopheles

braziliensis,Anophelesoswaldois.l.andAnophelestriannulatuss.l.,

havingbeenappointedasimportantlocal,secondaryandpotential

malariavectors(Voorham,2002;Zimmermanetal.,2006,2013;

Galardoetal.,2007,2009;Póvoaetal.,2001;Barbosaetal.,2014).

ThisscenariojustifiesfurtherstudiesinthestateofAmapá,inorder

tosupportanefficientvectormonitoringandtargetcontrol.The

presentstudyanalyzedthebehavioralpatternsoftheanopheline

speciesfromthedistrictofCorac¸ão,aperi-urbanareawithrecent

urbandevelopment,locatedinthestateofAmapá,Brazil,

determin-ingtheirhourlyhematophagicactivity,endophagyandexophagy

levels,anthropophilyandzoophilyindexes,aswellastheparity

rateandnaturalinfectionforPlasmodiumspp.Thedataofthisstudy

arethepartofawiderproject,thefirstpartofwhichisrelated

tocomposition, abundanceandaspectsoftemporal variationof

theAnophelesspecies,includingtheinfluenceofclimaticfactors,

waspreviouslypublishedbyBarbosaetal.(2014).Thus,the

behav-ioralpatterns,parityrateandnaturalinfectionanalysespresented

hereweregeneratedfromthedatasetpublishedbyBarbosaetal.

(2014).

climaticclassification,theclimateistypeAf(super-humid

equato-rial).Theregionischaracterizedbyahumidtropicalclimate,with

ahighannualrainfallratecombinedwithasmallannual

tempera-turerange,asitislocatedontheequatorialbelt(SUDAM,1984).The

naturalenvironmentsurroundingthedistrictofCorac¸ãoconsistsof

savannah,fieldsandislandsofnon-floodedforestandgallery

for-est.Themajorityofthelandscapeis coveredwith“buriti”palm

(Mauritiaflexuosa)trees.Thesurroundingwetlandsareunderthe

influenceofprecipitationandoftheMatapiRiver.Drainagesinthe

galleryforestconnectwiththeriver,whichmaintainsaminimum

waterlevelduringthedryperiods.Theclimateconditionsofthe

regionaretypicallyhotandhumid(meanhumidity∼85%),with

temperaturesrangingfrom22◦Cto32◦C.Therainyanddry

sea-sonsextend,respectively,fromJanuarytoJulyandfromAugust

toDecember.Climaticdataof themonthlyaccumulated

precip-itation(mm), meanrelative humidity (%)and temperature(◦C)

wereobtainedfromtheInstitutoNacional dePesquisas

Espaci-ais/SistemaNacionaldeDadosAmbientais(INPE/SINDA).

The mosquitoes were captured throughout the district of

Corac¸ão, totaling36 collectionsites(Fig.1),betweenDecember

2010andNovember2011,coveringtherainyanddryseasonsin

theBrazilianAmazonregion.The36collectionsiteswererandomly

chosen,withanaveragedistanceof∼1500mbetweeneachother.

Thecollectionswereperformedmonthlyduringthreeconsecutive

nights,eachcollectionperiodlastingforfourhours,from6:00to

10:00p.m.Undertheseconditions,themosquitoeswerecaptured

concomitantlyin theintra (indoor) and peridomestic(outdoor)

environments. Additionally,four uninterrupted12-h collections

werealsoperformedfrom6:00p.m.to6:00a.m.Twoofthemwere

performedduringtherainyseason(FebruaryandMay,2011)and

twointhedryseason(AugustandNovember,2011).Inthe12-h

col-lections,theanophelineswerecapturedonlyintheperidomestic

environment.Toestimatetheanthropophily(AI)andzoophily(ZI)

indexes,fourextracollectionsoffourhoursweremade,between

6:00p.m.and10:00p.m.Twoofthemwereperformedduringthe

rainy season (March and June, 2011) and two in the dry

sea-son(SeptemberandOctober,2011).Fortheanthropophilyindex

(AI),thecollectionsweremadeonlyintheperidomestic

environ-ment,andthemosquitoeswerecollectedwhentheywerenearor

attemptinglandonthecollector’sskin(Tadeietal.,1993;Forattini

etal.,1999).Thezoophilyindex(ZI)wasmeasuredinthe

extra-domiciliaryenvironment,andthemosquitoeswerecapturedwhen

theywererestingnearthecattlecorralsorpigpensandchicken

coops (Tadei etal., 1993).In this study,theperidomestic

envi-ronmentwasdefined asupto∼30maway fromthedwellings

(Barrosetal.,2010),whereastheextra-domiciliaryenvironment

was_∼30–100mawayfromthedwellings(Barrosetal.,2010).Inthe

Fig.1. CollectionsitesinthedistrictofCorac¸ãoarea,stateofAmapá,Brazil.A:InleftcorneristhesmallermapofBrazilshowingthelocalizationoftheStateofAmapá(in red);MapoftheStateofAmapáshowingthestudyarea(redcircle);B:SatelliteimageofthedistrictofCorac¸ãoarea.The36collectionsitesareindicatedasredcircles(For interpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.).

Sources:GoogleEarth,http://www.portalpolitico.com.br/ap/index.htmandhttp://mapasblog.blogspot.com.br/2012/01/mapas-do-amapa.html.

Table1

AbsoluteandrelativefrequenciesofAnophelesspeciescapturedinthemonthlycollections,four12-hcollections,andfouradditionalfour-hourcollections.

Species Monthlycollections(%) 12-hcollections(%) Anthropophilyand

zoophilyindexes(%)

Total(%)

Anopheles(Nyssorhynchus)marajoara 132(67.35) 18(9.18) 46(23.47) 196(11.6)

Anopheles(Nyssorhynchus)braziliensis 307(85.04) 27(7.48) 27(7.48) 361(21.37)

Anopheles(Nyssorhynchus)darlingi 377(52.00) 202(27.86) 146(20.14) 725(42.92)

Anopheles(Anopheles)intermedius – 1(20.00) 4(80.00) 5(0.30)

Anopheles(Anopheles)mediopunctatus/Anopheles(Anopheles)forattinii 2(100.00) – – 2(0.12)

Anopheles(Nyssorhynchus)nuneztovaris.l. 10(5.62) 1(0.56) 167(93.82) 178(10.54)

Anopheles(Nyssorhynchus)oswaldois.l. – – 5(100.00) 5(0.30)

Anopheles(Anopheles)peryassui 41(43.16) 34(35.79) 20(21.05) 95(5.63)

Anopheles(Nyssorhynchus)triannulatuss.l. 69(56.56) 9(7.38) 44(36.06) 122(7.22)

Total(%) 938(55.53) 292(17.29) 459(27.18) 1689(100.00)

‘–’nomosquitoescollected.

Inallcollections,themosquitoeswerespottedwiththeaidofa flashlightandcapturedwithamanual-typecapturer(Castro). Ento-mologicalnetsandmini-puc¸átype(networkofconicalshapefixed onarim)werealsoused.Themosquitoescollectedweretransferred intoplasticcups,properlylabeledwithdate,hourandcollection method.Attheendofthecaptures,thespecimensweretransported aliveinsidetightlyclosedisothermalboxestotheArthropoda Lab-oratoryoftheUniversidadeFederaldoAmapá,Macapá,Brazil.On thenextday,thespecimenswerekilledinthefreezerat−20◦_C,

andthenwereidentifiedusingmorphologicalkeysfor Neotrop-icalAnophelesspecies (Faranand Linthicum, 1981; Consoli and Lourenc¸o-de-Oliveira,1994;Forattini,2002).Currently,A.

nunezto-vari,A.oswaldoiandA.triannulatusarerecognizedascrypticspecies

complexesandinthepresentstudytheseparationoftheir

mem-berswerenot possible;thus, thesespeciesare herementioned

assensulato.Incaseofthespecimensmorphologicallyidentified

asA.albitarsiscomplex,thesequencingfortheBarcoderegionof

theCOI geneindicated theexistenceofA. marajoarainthe

dis-trictofCorac¸ão.Thus,inthepresentstudythesespecimenswere

namedasA.marajoara.AnophelesmediopunctatusandAnopheles

forattiniiarealmostisomorphicspeciesintheadultstage(females)

andinthisstudytheirseparationalsowasnotpossible;therefore,

bothspecieswerecitedseparatedbyslash.Allcollectionsiteswere

geo-referencedusingGlobalPositionSystems(GPS),GARMINMap

brand,model60CSX.

Themethodologyusedinthepresentstudywassubmittedto

theResearchEthicsCommitteeofInstitutoNacionaldePesquisas

daAmazônia(INPA),Manaus,Brazil,whichitwasreviewedand

approvedunderprocessnumber036/2010.

2.2. Parityrate

Theovarydissectionswereperformedinallcollectedspecies,

sampledbycollectionhourandmethod.Afterremovingthehead

andthoraxfromthefemalesforinfectivityanalysisforPlasmodium

spp.,asdescribedbelow,theabdomensweredissectedto

deter-minetheparityratebasedonthepresenceorabsenceofovary

tracheafilaments(Detinova,1962).Theovariesweredissectedin

adropofPBSbuffersolutiononaslide,andthenexaminedunder

anopticalmicroscope(BelPhotonics)ata40×/0.65magnification.

Basedontheovarytracheafilaments,theoniparousandnulliparous

ratesweredetermined.

2.3. Naturalinfectionanalyses

NaturalinfectivitybyPlasmodiumfalciparum,Plasmodiumvivax

andPlasmodiummalariaewasevaluatedusingthePCR(Polymerase

ofChainReaction)technique onthepooledheads andthoraces

of up to ten anophelines of the same species. After the

mor-phologicalidentification,theheads andthoraceswereremoved

frommosquitoesandstoredintoeppendorftubes.Thepoolswere

separated by species, date and hour of capture and collection

method.Genomic DNAwasextractedfromeachpoolaccording

reac-Table 2 Absolute frequency of Anopheles species captured in the indoor and outdoor environments distributed monthly. Species Dec/10 a Jan/11 Feb/11 Mar/11 Apr/11 May/11 Jun/11 Jul/11 I P I P I P I P I P I P I P I P Anopheles marajoara – 1 1 20 22 12 – 8 1 17 – 3 – 2 2 7 Anopheles braziliensis – 2 – 12 3 5 – – – – – 3 – 5 11 98 Anopheles darlingi – – – 2 13 9 – 4 – 9 – 4 – 9 – 26 Anopheles mediopunctatus /Anopheles forattinii – – – – – – – – – 2 – – – – – – Anopheles nuneztovari s.l. – – 1 – 2 – – 6 – – – – – 1 – – Anopheles peryassui – – – – – – – – – 2 – 2 – 1 – 2 Anopheles triannulatus s.l. – 1 – – 1 1 – – – 3 – 2 – 1 – 1 Total – 4 2 34 41 27 – 18 1 33 – 14 – 19 14 134 a Months of collections; I: Indoor; P: Outdoor; –: none mosquito collected.

calculationweredividedbythetotal numberofcollectiondays

(HBR=N/NC/TC;Forattini,2002).Thiscalculationwasemployed

forthe12hcollections.

Theanthropophily(AI)andzoophily(ZI)indexeswere

calcu-latedasfollow;theformer(AI)wascalculatedastheproportionof

allcollectedindividualsofagivenspeciesinthismethoddivided

bythetotalnumberofspecimens ofthesamespeciescaptured

inbothmethods(AI+ZI).Thezoophilyindex(ZI)wascalculated

astheproportionofallindividualsofagivenspeciescollectedin

thismethoddividedbythetotalnumberofspecimensofthesame

speciescollectedinbothmethods(AI+ZI).

Theparityrate(PR)wascalculatedasfollow:thenumberof

parousfemalesmultipliedby100anddividedbythetotalnumber

offemalesdissected.Thiscalculationwasperformedseparatelyfor

eachcollectionmethod(monthlycollectionsand12hcollections).

3. Results

Atotalof1689specimenscorrespondingtoninespeciesand

twosubgenera,Nyssorhynchus(sixspecies)andAnopheles(three

species),werecapturedinthetwelvemonthlycollectionsoffour

hourseach(144h),four12-hcollections(48h),andfouradditional

four-hourcollections (16h)forassessingtheanthropophilyand

zoophilyindexes,totaling208hofcollections.Takingintoaccount

allcollectionmethods,A.darlingiwasthemostabundantspecies

(n=725;42.92%),followedbyA.braziliensis(n=361;21.37%),A.

marajoara(n=196;11.6%),andA.nuneztovaris.l.(n=178;10.54%)

(Table1).Theotherspecies,A.intermedius,A. mediopunctatus/A.

forattinii,A.oswaldois.l.,A.peryassuiand A.triannulatuss.l.,had

frequenciesbelow10%.Anophelesmediopunctatus/A.forattiniiwas

capturedonlyinthemonthlycollections(inApril)andhadavery

lowfrequency(twospecimens),whereasA.oswaldois.l.was

cap-turedfeedingontheanimals(inSeptember)andalsohadalow

frequency(fivespecimens).

3.1. BehavioralPatternsoftheSpecies

3.1.1. Hourlybitingactivityandindoorandoutdoor

environments

Table1andFig.2presentthebehavioralpatternsofthespecies

capturedduringthefour12-hcollectionsintheperidomestic

envi-ronment.Inthismethod,atotalof292anophelineswerecollected,

which were distributed in seven species. Anopheles darlingi, A.

peryassui,A.braziliensisandA.marajoarawerethemostfrequent

species.Ofthese,A.darlingiandA.marajoarawereonlyspecies

capturedinthefour12-hcollections,whereasA.peryassuiwas

col-lectedinonlyonecollection(August2011).Anophelesdarlingiwas

capturedinallhourlycollectionsandshowedamultimodal

Fig.2. Hourlyhematophagicactivityduringthe12-hcollections;(A)forallAnophelesspeciescaptured;(B)forthethreemostfrequentAnophelesspecies.

and11:00p.m.,3:00a.m.and4:00a.m.,and5:00a.m.and6:00a.m.

Similarly,A.braziliensisalsoshowedamultimodalpattern,with

biting peaks between 7:00p.m. and 8:00p.m., 11:00p.m. and

00:00a.m.,4:00a.m.and5:00a.m.;however,itwasnotcapturedin

someofthehourlycollections(8:00p.m.and10:00p.m.,00:00a.m.

and2:00a.m.).Anophelesmarajoaraexhibitedawell-defined

pat-tern,withabimodalpeakbetween10:00p.m.and11:00p.m.,and

between3:00a.m.and 04:00a.m.It was howevernot captured

in thehourlycollections between 00:00a.m.and 3:00a.m. and

between4:00a.m.and6:00a.m.Anophelesperyassuiexhibited a

unimodalpeakbetween00:00a.m.and1:00a.m.Theotherspecies

werecollectedatlowfrequenciesanddidnotshowadefined

pat-tern.

Table2showstheabsolutefrequenciesofthespeciescaptured

intheintra(indoor)andperidomestic(outdoor)environments

dur-ingthemonthlycollections.Theresultsindicatehigherfrequencies

forallspeciesintheoutdoorenvironment,withtheexceptionof

February2011,whichshoweda higherfrequency ofA.darlingi,

A.marajoaraandA.nuneztovaris.l.intheindoorenvironment.In

December2010andMarch/May/June/October2011,however,no

specimenwascapturedindoors.Noanophelinewascapturedin

November2011ineitheroftheenvironments.Meanstudent’sttest

revealedsignificantdifferencesbetweentheindoorandoutdoor

environments(t=−2.103;p=0.040).

Table3showsthefrequencyofanophelinescapturedintheintra

(indoor)andperidomestic(outdoor)environments,distributedby

fourhourscollections(6:00p.m.to10:00p.m.).Overall,therewas

ahigherfrequencyofthespeciesintheoutdoorenvironment.In

thefirsthourlycollections(6:00p.m.to7:00p.m.),allA.darlingi,A.

peryassuiandA.triannulatusspecimenswerecapturedoutdoors;a

similarresultwasfoundforA.braziliensisinthe4thhourly

col-lection.However,A.marajoara wasactivein allthefourhourly

collectionsandinbothenvironments.Anophelesnuneztovaris.l.was

collectedbothindoorsandoutdoors;however,itsfrequencywas

verylow.TheresultofStudent’sttestshowedastatistically

sig-nificantdifferencebetweentheindoorandoutdoorenvironments

(t=−2.225;p=0.033).

3.1.2. Humanbitingrate(HBR)

Table4showstheHBRforthesevenspeciesinthe12-h

collec-tions.Similarlyforthemonthlycollections,A.darlingiexhibitedthe

highestHBRcomparedtotheotherspeciescapturedinthismethod.

Anopheles darlingi was the only species captured in all hourly

collectionsand had anHBR varyingfrom 2.67to0.08 between

7:00p.m. and 8:00p.m. and between 00:00a.m. and 1:00a.m.,

respectively.AnophelesbraziliensisshowedthehighestHBR(0.42)

inthehourlycollectionsbetween11:00p.m.and00:00a.m.and

between4:00a.m.and6:00a.m.,whereasinA.marajoaraitwas

0.33inthehourlycollectionsbetween10:00p.m.and11:00p.m.

AnophelesperyassuihadthehighestHBR(0.92)between00:00a.m.

and1:00a.m.

3.1.3. Anthropophilyandzoophilyindexes

Table5showstheanthropophily(AI)andzoophily(ZI)indexes.

Atotalof459anophelinescorrespondingtoeightspecieswere

cap-tured.Ofthese,77(16.78%)werecapturednearhumandwellings

(peridomestic) and 382 (83.22%) in cattle corrals, pens and/or

chicken coops (extra-domiciliary). In theextra-domiciliary

col-lections,therewasahighfrequencyofanophelinesinallhourly

collections.Themostanthropophilicspecieswere:A.darlingi,A.

marajoara,A.braziliensis,A.perysassuiandA.triannulatuss.l.The

highestAIwasforA.darlingi(0.40),followedbyA.marajoara(0.23),

whereasthehighestZIwasforA.nuneztovaris.l.(1.00).AlthoughA.

darlingihadthehighestAI,italsoshowedahighZI(0.60),

indicat-ingitseclecticismandopportunism.TheresultofStudent’sttest

(mean)wasstatisticallysignificant(t=−2.642;p=0.033),

reflect-ingsignificantdifferencesbetweenthetwoindexes.Table6shows

thesedatacoveringtherainyanddryseasons.ThehighestZIwas

observedin September2011(dryseason)with294mosquitoes

captured in outdoor condition nearcattle corrals, whereas the

lowest AI and ZI were obtained in November 2011, with one

mosquitocollected(A.triannulatuss.l.)intheanthropophilic

con-dition.Anophelesintermedius,A.nuneztovaris.l.andA.oswaldois.l.

werecollectedonlyinzoophilycondition(ZI=1.00)inSeptember

2011.AnophelesperyassuiwascollectedonlyinJune2011,where

twospecimens(AI=0.10)werecapturedintheanthropophilic

con-ditionand18(ZI=0.90)inthezoophilycondition(chickencoop).

3.1.4. TemporaldistributionoftheAnophelesspecies

FromAugust2011on,therewasanincreaseinthedensityof

anophelines,reachingthemaximumpeakinSeptember2011.

Dur-ingthistime,thenumberofmalariacasesincreasedinthedistrict

of Corac¸ão(MS, 2011).The correlationtests betweenthethree

mostfrequentspeciesandthenumberofmalariacaseswere

per-formed.ForA.darlingiandA.braziliensis,thecorrelationbetween

thedensityandthenumberofmalariacaseswaspositive,butnot

significant(rs=0.251;p=0.429;rs=0.5188;p=0.084,respectively).

Fig.3.FrequencyofAnophelesspeciesandthe∗_{numberofmalariacasesbetweenDecember2010andNovember2011.}

∗_{Source:MinistériodaSaúde(MS):SecretariadeVigilânciaemSaúde.2011.SIVEP-Malária–SistemadeInformac¸ãodeVigilânciaEpidemiológica–Notificac¸ãodeCasos.} 2010a2011.Site:http://www.portalsaude.saude.gov.br.AccessedatAugust17,2016.

thenumberofmalariacaseswasnegativeandnotsignificant(rs=−

0.288;p=0.363)(Fig.3).

3.1.5. Parityrate

Allspecimenscollectedinthisstudyweredissected,makingit

possibletoanalyzethetrachealfilamentsof1077(87.56%)females.

Intheremainingspecimens(12.44%),thisanalysiswasnotpossible

becausethefemaleswereinadvancedeggdevelopmentstage.

Ofthe938anophelinescapturedinthemonthlycollections,it

waspossibletoanalyzethetrachealfilaments(nulliparousor

oni-parous)of827(88.17%)females(Table7).Ofthese,310(37.48%)

belongedtospeciesA.darlingi,289(34.95%)toA.braziliensis,118

(14.27%)toA.marajoara,65(7.86%)toA.triannulatuss.l.,36(4.35%)

toA. peryassui,7(0.85%) toA. nuneztovaris.l.,and 2 (0.24%)to

A.mediopunctatus/A.forattinii.InA.darlingi,theparityratevaried

from47.36to76.42inthefourhoursofcollections, thehighest

valuesbeingobservedinthelaterhours(between8:00p.m.and

10:00p.m.).ThespecieswiththesecondhighestparityratewasA.

marajoarathatvariedfrom65.00to76.00,withhighvaluesinall

hourlycollections.Similarly,A.braziliensispresentedratherhigh

parityratevaluesinallhourlycollections,rangingfrom55.81to

65.97.

Ofthe292anophelinessampledinthe12-hcollections,itwas

possibletodissectthetrachealfilamentsof250(85.62%)females,

whicharedistributed asfollows:171(68.40%)ofA.darlingi, 33

(13.20%)ofA.peryassui,22(8.80%)ofA.braziliensis,14(5.60%)ofA.

marajoara,9(3.60%)ofA.triannulatuss.l.,and1(0.40%)ofA.

inter-medius(Table8).Anophelesdarlingidisplayedoniparousfemales

inallhourlycollections,exceptbetween00:00a.m.and1:00a.m.,

withaparityratebetween15.38and50.00.InA.braziliensis,the

parityraterangedfrom33.33to100.00,withthehighestvalues

observedbetween2:00a.m.and4:00a.m.InA.marajoara,the

find-ingswereirregular,buttheparityratewashigh(100.00).Anopheles

peryassuiexhibitedahighparityrate,rangingfrom33.33to100.00

andpresentingthehighestvaluesbetween1:00a.m.and2:00a.m.

andbetween4:00a.m.and6:00a.m.Thehighparityratevalues

(100.00)observedforA.marajoara andA.peryassuiwere

proba-blyinfluencedbythelownumberofspecimenscapturedinthese

hourlycollections.

3.1.6. InfectionrateoftheanophelinespeciesforPlasmodium

spp.

Inthisstudy,397poolscontainingupto10headsandthoraces

wereanalyzedusingthePCRtechnique,distributedasfollows:136

ofA.darlingi,86ofA.braziliensis,72ofA.marajoara,41ofA.

tri-annulatuss.l.,31of A.peryassui,26ofA. nuneztovaris.l.,2ofA.

intermedius,2ofA.oswaldois.l.,and1ofA.mediopunctatus/A.

forat-tinii.Allpoolsshowednegativeresults,indicatingthatnoneofthese

specieswereinfectedwithPlasmodiumspp.,however,abandfor

thepositivecontrolwasobservedinallanalyses.The

entomologi-calinoculationrates(EIRisdefinedasthenumberofinfectivebites

perpersonpertimeperiod)werenotcalculated,becausenoneof

themosquitoeswerepositiveforPlasmodiumspp.

4. Discussion

Theresultsofthisstudyrevealedthat,ofthethreemost

abun-dantspecies,two(A.darlingiandA.marajoara)havebeenappointed

asthemostimportantmalariavectorsinthestateofAmapá(Póvoa

etal.,2001;Connetal.,2002;Galardoetal.,2007).Thelocalities

(LagoadosIndios/GranjaAlvesandthreeruralvillagesalongthe

MatapiRiver)investigatedbyConnetal.(2002)andGalardoetal.

(2007)arelocatednearthedistrictofCorac¸ão.Inthepresentstudy,

A.darlingiwasthemostabundantandwidelydistributedspecies

inthedistrictofCorac¸ão,followedbyA.braziliensisandA.

mara-joara.AnophelesdarlingiandA.marajoara weretheonlyspecies

presentinthefour12-hcollections,butonlyA.darlingishowed

activitythroughoutthenightandithadthehighestAIandHBR.

Thesefindingsraiseconcern,becauseA.darlingiisthemost

anthro-pophilicandefficientmalariavectorintheBrazilianAmazonregion

anditiswidelydistributedinthestateofAmapá(Deane,1986,

1988;Lourenc¸o-de-Oliveiraetal.,1989;Tadeietal.,1998;Tadei andDutary-Thatcher,2000;Galardoetal.,2007;Moutinhoetal., 2011;Martins-Camposetal.,2012).

Allspeciescapturedinthisstudyshowedatendencyofbiting

intheoutdoorenvironment(outdoorfeeding=exophagic)as

com-paredtotheindoor environment(indoor feeding=endophagic).

Both environments were statisticallydifferent (considering the

months,t=−2.103,p=0.040;consideringthehourlycollections,t

Table 3 Absolute and relative frequencies of the Anopheles species captured indoors and outdoors in the monthly collections, during the four hours collections. Species 6:00–7:00 p.m. 7:00–8:00 p.m. 8:00–9:00 p.m. 9:00–10:00 p.m. I (%) P (%) T I (%) P (%) T I (%) P (%) T I (%) P (%) T Anopheles marajoara 2 (9.52) 19 (90.48) 21 16 (25.81) 46 (74.19) 62 7 (20.59) 27 (79.41) 34 1 (6.67) 14 (93.33) 15 Anopheles braziliensis 1 (1.70) 58 (98.30) 59 9 (8.82) 93 (91.18) 102 5 (5.32) 89 (94.68) 94 – 52 (100.00) 52 Anopheles darlingi – 42 (100.00) 42 3(6.25) 45 (93.75) 48 7 (4.40) 152 (95.60) 159 4 (3.13) 124 (96.87) 128 Anopheles mediopunctatus /Anopheles forattinii – – – – 2 (100.00) 2 – – – – – – Anopheles nuneztovari s.l. – – – 1 (25.00) 3 (75.00) 4 1 (20.00) 4 (80.00) 5 1 (100.00) – 1 Anopheles peryassui – 8 (100.00) 8 1 (4.55) 21 (95.45) 22 – 7 (100.00) 7 – 4 (100.00) 4 Anopheles triannulatus s.l. – 20 (100.00) 20 1 (4.00) 24 (96.00) 25 – 16 (100.00) 16 – 8 (100.00) 8 Total (%) 3 (2.00) 147 (98.00) 150 31(11.70) 234 (88.30) 265 20 (6.35) 295 (93.65) 315 6 (2.88) 202 (97.12) 208 I: Indoor; P: Outdoor; T: Total; –: no mosquito collected. Within parentheses are the percentages.

bitingoutdoors is significantly higherthan that of those biting

indoors.Thepredominanceofthemalariavectorsintheoutdoor

environmenthasalsobeenobservedinotherregionsofthe

Brazil-ianAmazon(Tadeietal.,1998;McGreevyetal.,1989;Giletal.,

2003;Moutinhoetal.,2011),Colombia(Naranjo-Diasetal.,2013)

andVenezuela(Morenoetal.,2007).TadeiandDutary-Thatcher

(2000) argued that the endophilic/endophagic behavior of the

anophelinevectorstendstodecreaseasaresultofchangesinthe

humanhabitsandalsolikelyduetothebehavioraladaptationsof

theanophelines,inducedbythemassiveuseofinsecticidesinthe

past.Inthepresentstudy,thepredominanceofanophelines

cap-turedintheoutdoorenvironmentcanbeexplained,atleastinpart,

bythefactthattheactivitypeakofmosquitovectorscoincideswith

thetimesofdaywhenpeopleareoutsidetheirhomes,involvedin

socialandculturalactivities,andalsobythekindofdwellingsin

thedistrictofCorac¸ão,sincemostofthemhavewalls,andwith

doorsandwindowsprotectedbyscreens,blockingtheentryofthe

mosquitoes;thus,thisscenariocontributestotheextra-domiciliary

transmission,withdirectconsequencesonthemalariaincidence

andcontrol.

Inthisstudy,A.darlingiwasthemostanthropophilicspecies

(AI=0.40), but it also showed accentuated zoophily (ZI=0.60),

revealingitseclecticand opportunisticbehavior. Anopheles

dar-lingialsoshowedamultimodalpatterninitsbitingpeak,andhad

thehighestHBR(perhour),whereasA.marajoararevealedamore

stablepattern.WhilestudyingthebitingactivitypatternsofA.

dar-lingiandA. marajoarapopulationsfromthemunicipalityofSão

RaimundodoPirativa,anareanearthedistrictofCorac¸ão,Voorham

(2002) described a complex multimodal pattern for A. darlingi,

whereasA.marajoarashowedawell-pronouncedpeakduringthe

firstthreehoursofthenight(crepuscularpeak).Theresultsofthis

studyarecompletelycongruentwithVoorham’sfindings(2002),

whichindicatethatecologicaldeterminants(environmental

vari-ables)playanimportantrole;asaconsequence,thismultimodal

behaviorcanincreasethepotentialofthisspeciesasmalariavector

andredoubletransmissionintheseareas.

Inthisstudy,thehighestdensityofthecapturedanophelines,

especiallyA.darlingi,occurredduringthedryseason(September)

soonaftertherainfall,asituationthathasbeenobservedinmany

localities of the Brazilian Amazon (Tadei and Dutary-Thatcher,

2000;Póvoa etal.,2009;Moutinhoet al.,2011)where abouta

monthlateranincreaseinthemalariacasesoccurs(Tadeietal.,

1998).Thisscenariosuggeststhatinterventionsforvectorcontrol

shouldbeimplementedattheendoftherainyseason.

Curiously,A.braziliensiswasoneofthethreemostabundantand

anthropophilicspeciesinthedistrictofCorac¸ão.Itwascaptured

inbothindoorandoutdoorenvironments,althoughit

predomi-natedinoutdoors.Thisresultcompletelydiffersfromthestudyof

Galardoetal.(2007, 2009),whichwasconductedinthreerural

villagesalongtheMatapiRiver,localitiesneartheareaofpresent

study.Intheselocalities,theauthorsdidnotreportpredominance

and/oranthropophilyforA.braziliensis.Otherpreviousstudieshave

reportedA.braziliensisasazoophilicspecieswithnoorlittle

impor-tanceinmalariatransmission(ConsoliandLourenc¸o-de-Oliveira,

1994;Forattini,2002)orasecondaryvector(Deaneetal.,1948).It

has,however,beenfoundinfectedwiththehumanmalaria

para-siteinthestatesofAmazonas(TadeiandDutary-Thatcher,2000;

Martins-Camposetal.,2012),Amapá(Póvoaetal.,2001),Rondônia (Lourenc¸o-de-Oliveiraetal.,1989)andRoraima(Silva-Vasconcelos

etal.,2002).Takentogether,A.braziliensismayplaysomerolein

malariatransmissioninthedistrictofCorac¸ão,whenathigh

den-sityandco-occurringwithA.darlingi.Nonetheless,furtherstudies

areurgentlyneededtoclarifytheroleofA.braziliensisinmalaria

transmissioninthisdistrictandinotherareasofAmapá.

Inthisstudy,A.nuneztovaris.l.wasthemostzoophilicspecies,

Ama-Anthro. Zoo. Anthro. Zoo. Anthro. Zoo. Anthro. Zoo. Anthro. Zoo. Anophelesmarajoara 3 9 7 19 1 7 – 1 11 36 0.23 0.77 Anophelesbraziliensis 1 5 4 12 – 4 – – 5 21 0.19 0.81 Anophelesdarlingi 25 11 17 40 16 30 – 7 58 88 0.40 0.60 Anophelesintermedius – – – 4 – – – – – 4 – 1.00 Anophelesnuneztovaris.l. – 25 – 32 – 79 – 31 – 167 – 1.00 Anophelesoswaldois.l. – 1 – 3 – 1 – – – 5 – 1.00 Anophelesperyassui 1 2 1 11 – 5 – – 2 18 0.10 0.90 Anophelestriannulatuss.l. – 2 1 11 – 16 – 14 1 43 0.02 0.98 Total 30 55 30 132 17 142 – 53 77 382

Anthro.:anthropophily;Zoo.:zoophily;AI:AnthropophilyIndex;ZI:ZoophilyIndex;–:nomosquitocollected.

Table6

AnthropophilyandzoophilyindexescalculatedforthespeciesofAnophelesinthefourcollectionscoveringtherainyanddryseasons.

Species March2011 June2011 September2011 November2011

AI ZI AI ZI AI ZI AI ZI Anophelesmarajoara 0.16 0.83 0.31 0.69 – 1.00 – – Anophelesbraziliensis 1.00 – 0.23 0.77 – 1.00 – – Anophelesdarlingi – 1.00 0.77 0.23 – 1.00 – – Anophelesintermedius – – – – – 1.00 – – Anophelesnuneztovaris.l. – – – – – 1.00 – – Anophelesoswaldois.l. – – – – – 1.00 – – Anophelesperyassui – – 0.10 0.90 – – – – Anophelestriannulatuss.l. – – – – – 1.00 1.00 – Total(%) 0.11 0.89 0.51 0.49 – 1.00 1.00 –

AI:AnthropophilyIndex;ZI:ZoophilyIndex;–:nonemosquitocollected.

Table7

ParityratecalculatedfortheAnophelesspeciesinthemonthlyfour-hourcollections.

Species ParityRate

6:00–7:00p.m. 7:00–8:00p.m. 8:00–9:00p.m. 9:00–10:00p.m.

Anophelesmarajoara 65.00 73.33 76.00 69.23

Anophelesbraziliensis 64.28 65.97 55.81 64.00

Anophelesdarlingi 47.36 64.28 76.42 74.76

Anophelesmediopunctatus/Anophelesforattinii – 50.00 – –

Anophelesnuneztovaris.l. – 33.33 – 100.00

Anophelesperyassui 50.00 42.85 – 100.00

Anophelestriannulatuss.l. 61.90 56.52 84.61 37.50

‘–’Noanophelinewascollectedornofemalewasoniparous.

Table8

ParityratecalculatedfortheAnophelesspeciescapturedinthefour12-hcollections.

Species ParityRate

6:00 7:00 7:00 8:00 8:00 9:00 9:00 10:00 10:00 11:00 11:00 00:00 00:00 1:00 1:00 2:00 2:00 3:00 3:00 4:00 4:00 5:00 5:00 6:00 Anophelesmarajoara 100.00 – – 100.00 – 100.00 – – – – – – Anophelesbraziliensis 50.00 50.00 – – 33.33 50.00 – – 100 100 75.00 66.66 Anophelesdarlingi 50.00 15.38 27.27 20.00 29.16 33.33 – 20.00 40.00 29.41 16.66 44.44 Anophelesintermedius – – – – – – – – – – – – Anophelesperyassui 57.14 42.85 – – – – 40.00 100 – 33.33 100 100 Anophelestriannulatuss.l. – 66.66 – 50.00 – – – – – 100 – –

zonregion(Deaneetal.,1948;TadeiandDutary-Thatcher,2000; Silva-Vasconcelosetal.,2002).However,thisspecieswasreported

tocarrythemalariaparasiteatseveralsitesoftheBrazilianAmazon

andwasappointedasanimportantlocalvectorinAmapá,whenat

highdensity(Galardoetal.,2007).Itispossiblethat,inthedistrictof

Corac¸ão,A.nuneztovaris.l.alsocontributestomalariatransmission,

especiallywhenathighdensity.Inthepresentstudy,A.triannulatus

s.l.,A.oswaldois.l.,A.peryassui,A.intermediusandA.

mediopuncta-tus/A.forattiniiweresampledatlowdensities.Thisresultmayhave

beeninfluencedbythecollectionmethodsused(which

predomi-natedintheintraandperidomesticenvironments)andthetypeof

studyarea(urbanized),sincethesespeciesarereportedas

predom-inantlyzoophilicandareoftenfoundintheforestoftheBrazilian

Amazon(Tadeietal.,1998),anenvironmenttowhichtheyarewell

adapted.

Inadditionthebehavioroftheanophelines,theparityrateand

agecomposition offemalesareparamountparameters,because

they reflect the epidemiological importance of vector

popula-tions.Inmalariatransmissionareas,forexample,eachbloodmeal

increases the chance of a female becoming infected with the

malariaparasite,soitsepidemiologicalimportanceincreases

pro-portionally toher physiological age.However, due to thehigh

mortalityrateoffemalesineachgonotrophiccycle,onlyasmall

proportionoffemales surviveuptoa moreadvanced age.Asa

consequence,thereisadirectrelationbetweentheageofa

popu-lationofmosquitoesanditsepidemiologicalimportanceinmalaria

transmission.Forexample,intheoldermosquitopopulationsthere

aregreaterchancesofcontaininginfectedfemalesreadyto

trans-mittheparasite,buttheyareinverylowfrequency(Tadei,1993).

Thus,theepidemiologicalimportanceofthefemalescanbe

esti-matedbasedontwomainpoints:1)thePlasmodiumcyclewithinan

anophelinefemale(vector)takesaround10–15days;2)theperiod

betweenthepreviousandthenextbloodmeal(eachgonotrophic

cycle)canbeestimatedtobefrom3to4days.Therefore,afemale

(vector)canbeconsideredabletotransmitthemalariaparasite

afterthethirdbloodmeal,ifthisfemaleholdsthefirstmealina

personwithgametocytes.

Inthepresentstudy,theparityratedatadidnotshowadefined

patternforthreeofthemostfrequentspecies,possiblybecauseit

shouldneedofalongerperiodofobservation.ForA.darlingi,

how-ever,itwaspossibletoverifyinthemonthlycollections(fourhours)

thattherewasahigherfrequencyofoniparousfemalesinthelater

hours,between8:00p.m.and10:00p.m.,indicatingthattheolder

femalesweremoreprevalentinthelaterhourlycollections.In

con-trast,thenulliparousfemalesweremorecommoninthefirsthourly

collections,suggestingthatthenewlyemergedfemalesare

feed-ingbloodbeforetheolderfemales.Asimilarresultwaspreviously

reportedforthisspeciesfromanotherareaoftheBrazilianAmazon

(Barrosetal.,2007)andthesefindingsarecrucialforthemalaria

epidemiologyandinterventionmeasures.

Inthisstudy,noneoftheanalyzedmosquitoeswereinfected

withPlasmodiumspp.Thisresultcanbeaconsequenceofvarious

factors.First,thedensityofanophelineswasslightlylowduring

thestudy.Second,it iswellknownthatthenumberofinfected

mosquitoes(females)islowinthefield(Tadeietal.,1998;

Silva-Vasconceloset al., 2002; Barroset al., 2010; Vezenegho et al.,

2014).Third, thenumber of malaria casesrecorded in the

dis-trictofCorac¸ãoduringthisstudy(Dec.2010–Nov.2011)waslow,

with71 cases (MS, 2011), withthe highest number of malaria

casesbeingrecordedinAugust(17),September(24)andOctober

(18).Four,therewerewildfiresaroundofthedistrictofCorac¸ão

betweenSeptemberandOctober,2011thatresultedinadrastic

decreaseofanophelines,whichcanbeobservedinNovember,2011

(Tables2and6).Takentogether,thissetoffactorscanexplainthe

absenceofanophelinesinfectedwithPlasmodiumspp.inthe

dis-trictofCorac¸ão,aswellasthenon-significantcorrelationbetween

densityof A.darlingiandthenumber ofmalaria cases.Aresult

similartoourstudywasdetectedinthemunicipalityofJuruti,in

thestateofPará,Brazil(Póvoaetal.,2009).Negativeresultswere

alsofoundinamalariatransmissionareainFrenchGuiana,where

noinfectedA.darlingispecimenwasobserved(Vezeneghoetal.,

2014).Itisnotunusualtofindnegativeresultsintheinfectivity

testsduringmalariaoutbreaks,duetotheoccurrenceofsmall

num-bersofinfectedmosquitoesinthefield(Barrosetal.,2010).On

theotherhand,differentresultswerereportedinthe

municipal-ityofAnajá,stateofPará,Brazil(Santosetal.,2005).Theauthors

foundthat,althoughA.darlingiwascapturedatalowdensityin

thatarea, theinfectivity ratewashigh(5.9%).Thesedifferences

inthemalariatransmissiondynamicsamongthelocationscanbe

relatedtothehumanhabits,suchassocialandculturalactivities

(densityandexposureofhost),whichprovidegreatercontactwith

vectorsthatmayresultinthehighinfectionrateoftheanophelines.

Otherimportantfactorsinvolvedarealsothedensityandbehavior

oftheanophelinevectors,climaticconditions,typesofdwellings,

andthepresenceofotheranimalsintheperidomesticareaandthe

efficiencyofthecontrolmeasures.

In conclusion,of thenine species capturedin thedistrictof

Corac¸ão, A. darlingi, A. braziliensis and A. marajoara were the

mostabundantandanthropophilicspecies.Anophelesdarlingiwas

widelydistributedin thestudyareaand showedbitingactivity

throughoutthe night,aswell asit alsohad thehighestAI and

HBR.ThesefindingssuggestthatA.darlingiislikelytobethemain

malariavectorinthestudiedarea.Anophelesmarajoaramayalsobe

animportantvectorinthisareaandA.braziliensismayequallyplay

someroleinmalariatransmission.Nonetheless,furtherstudiesare

neededtoclarifytheroleofA.braziliensisasmalariavectorinthis

district.Allcapturedanophelinespeciesshowedthepredominance

forfeedingintheoutdoorenvironment,indicatingthatmeasuresof

vectorcontrolshouldbeimplementedsimultaneouslyinboth

out-doorandindoorenvironments,especiallyattheendoftherainy

season.

Thus,thedistrictofCorac¸ãohasimportantconditionsthatallow

ustoclassifyitasahighlypotentareaofmalariatransmission;

therefore,thepreventionandsurveillancemeasuresshouldbe

con-stantlytakentopreventit.

Authors’contributions

LMCB,RNPS, VMS:designed thestudy. LMCB, RNPS, RMAF:

collected the anophelines in the field and identified them.

LMCB:generatedand analyzedthedata.LMCB,VMS:wrotethe

manuscript.

Acknowledgments

Thisresearch was supportedby INPA/MCTI-Projeto Grandes

Vultos,byUniversidadeFederaldoAmapá;byFAPEAM-Universal

Amazonas (grant number: 3111/2012) to VMS, by

FAPEAM-PAPAC (grant number: 062.01832/2015) to VMS and by

PRO-EQUIPAMENTO-CAPES,Brazil. LMCBreceiveda fellowshipfrom

CAPES.

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