12/19/2016 IsolationandidentificationofculturablebacteriafromwildAnophelesculicifacies,afirststepinaparatransgenesisapproach

Isolation and identification of culturable bacteria from wild Anopheles culicifacies, a first step in a
paratransgenesis approach
Ali Reza Chavshin, Mohammad Ali Oshaghi, [...], and Olle Terenius

Abstract

Background
Duetotheeffectofmidgutbacteriaonproliferationofparasitesandtheirpotentialasparatransgenesistools,theiridentificationinmalariavector
mosquitoesisimportant.Anophelesculicifaciess.l.isoneofthemainmalariavectorsinAsiahowever,itsmidgutmicrobiotaremainsunstudied.
Thisworkwasprimarilydesignedtoisolatepotentialcandidatesforuseinaparatransgenesisapproach,butalsotogiveapictureofthemidgut
microbiotaofwildcaughtAn.culicifacieslarvaeandadultsfromthesoutheastcornerofIran,whichhasthehighestmalariaendemicityinthe
country.

Methods
Atotalof68larvaeand34adultfemales(newlyeclosedandolder)fromthreedifferentbiotopesinIranwereanalyzedfortheirmidgutmicroflora.
Themosquitoeshadtheirmidgutbacterialcontentsplatedonthreedifferentculturemedia(brainheartagar,nutrientagarandbloodagar)yielding
57bacterialisolates.The16SrRNAgenesoftheisolatesweresequenceanalyzedforspeciesdesignation,whichthenwasconfirmedby
biochemicalanalysis.

Results
Atotaloftwelvebacterialgenerawereidentified:Acinetobacter,Aeromonas,Bacillus,Chryseobacterium,Delftia,Exiguobacterium,Kurthia,
Microbacterium,Pseudomonas,Staphylococcus,ThorselliaandVariovorax.Inolderfemales,onlyGramnegativebacteriawerefound,whereas
larvaeandnewlyeclosedadultsalsoharboredGrampositivebacteria.Thediversityofisolatesalsovariedbetweensamplingsitesandmosquito
stages,withthelargestnumberofgenerafoundintheAnguridistrictandinlarvae,respectively.Pseudomonaswasthemostcommongenus
retrievedfromallsamplingsites,andinbothlarvaeandadults,suggestingapotentialtransstadialpassageofthesebacteria.Interestingly,identical
16SsequencesofPseudomonaswerefoundinmosquitoesoriginatingfromdifferenthabitatsatleast45kmapart,whichcouldsuggestthatthese
bacteriahavebeenadaptedtothemosquitoes.

Conclusions
Thestudyofvectormosquitomicrobiotahasrecentlygatheredincreasedinterestbecauseofthepotentialinfluenceonvectorcompetence.Byadding
datafromahithertouncharacterizedmalariamosquito,abetterpictureofgutflorainvectormosquitoeswasobtained.Furthermore,somespeciesof
thepredominantgenusPseudomonaswillbeevaluatedfortheselectionofaparatransgenesiscandidate.

Keywords:Midgutmicrobiota,16SrRNA,Anophelesculicifacies,Malaria,Paratransgenesis

Background
Vectorbornediseasescausehealthproblemsinseveralpartsoftheworldandamongthemmalariaisthemostimportantwithalmost700,000deaths
annually[1].Malariacontrolprogramsfocusedonmosquitovectorshavereducedmortalityandmorbidity,butemerginginsecticideresistantvectors
andenvironmentalissuesrelatedtoapplicationofpesticideshavenecessitateddevelopmentofnewcontrolstrategieswithlessenvironmental
impacts/damageandhigherefficacy[2,3].

Oneofthecontrolapproachesunderdevelopmentisparatransgenesis,whichhasbeendefinedasusingsymbioticorganisms(suchasbacteria)to
deliverantiparasiticeffectormoleculestowildvectorpopulations[4].Initialstepsofdevelopingparatransgenesisagainstmalariahavebeentaken
inlaboratoryexperimentsonAn.gambiaemosquitoes.Inanearlystudy,asinglechainantibodytargetingPlasmodiumbergheiookinetePbs21was
linkedtothelyticpeptideShiva1andexpressedinEscherichiacoli,whichresultedin95.6%transmissionblockage[5].Morerecently,itwas
shownthatengineeredPantoeaagglomeransbacteriaisolatedfrommosquitoesinhibiteddevelopmentofthehumanmalariaparasitePlasmodium
falciparumandtherodentmalariaparasitePlasmodiumbergheibyupto98%[6].

Inmosquitoes,obligatesymbiontsareyettobefoundtherefore,afirststepinparatransgenesisistoidentifythenormalmidgutmicrobiotaof
mosquitoesandtoisolatecandidatesforfurthermodification[7,8].Sofar,alimitednumberofstudieshavebeencarriedoutonthemicrobiotaof

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Anophelesmosquitoes[9].Thestudiedspeciesinclude:laboratoryrearedAnophelesstephensi,An.gambiae,andAn.albimanus[10],fieldcollected
An.albimanusfromsouthernMexico[11],fieldcollectedAn.gambiaes.s.,An.arabiensisandAn.funestusfromwesternKenya[12],field
collectedAn.darlingifromBrazil[13],laboratoryrearedandwildcaughtAn.stephensifromIndia[14],semifieldcollectedAn.gambiaefrom
Kenya[15],fieldcollectedAn.maculipennisandAn.stephensifromIran[16],andfieldcollectedAn.stephensifromIran[17].

An.culicifaciesGilesisoneofthemainmalariavectorsinthetropicalpartsofSouthandSouthwestAsia[1823],whereittransmitsboth
PlasmodiumfalciparumandP.vivax[24].Inthisstudy,culturablemidgutbacteriaisolatedfromwildcaughtAn.culicifaciesinsoutheasternIranare
identifiedusing16SrRNAsequenceanalysiswiththeaimofselectingpotentialcandidatesforparatransgenesis.

Methods

Ethics statement
PriortotheapprovalofallprojectsbytheTehranUniversityofMedicalSciences(TUMS),theyarereviewedandendorsedbytheethical
committeeoftheTUMS.Mosquitocollectionwascarriedoutfromprivatedwellings.Atleastoneweekpriortoanymosquitocollection,theowners
wereinformedbytheLocalHealthSystemofficers.TheresearchanditsobjectiveswereexplainedbyAliRezaChavshin(ARC),toresidents.
Ownersoftheland(forlarvalcollection)anddwellings(foradultmosquitocollection)gavepermissiontoconductthestudyonthesesites.After
theirpermission,thesampleswerecollectedatanagreeddateandtime.Thewholeprocesswascoordinated,managedanddocumentedbythe
LocalmanagerofmalariacontrolprogramandLocalHealthSystemofficerinthestudyareasandsignedbyARC.Alsoitisdeclaredthatthe
collectedspeciesisnotendangeredorprotectedintheareaofinvestigation.

Field collection of An. culicifaciesand isolation of midgut bacteria

WildcaughtmosquitosampleswerecollectedduringJulySeptember2010,from1)theIranshahrdistrict,anurbanregion2)theAnguridistrict,a
mountainousorhillyruralregionwithtemporalriversand3)theSaraydandistrict,aruralplainregion(Table1).Thedistancesbetweenthethree
sitesare4590km(IranshahrtoSaraydan45km,SaraydantoAnguri50km,andIranshahrtoAnguri90km).AllthreeareasarepartsoftheSistan
andBalouchistanProvince,whichborderstoPakistanandcomprisesthemostimportanturbanandruralmalariafociinsoutheasternIran(Figure1).

Table1
Thegeographicalandecologicalpropertiesofthesamplingsites

Figure1
MapofIranindicatingthelocationsoftheSistanvaBaluchistanProvince ,andthedistricts/villageswherethespecimenswerecollected.1)
Iranshahr,2)Anguri,and3)Saraydan.

Astandarddippingtechniquewasusedforlarvalsamplecollection(350mldipper).Adultmosquitoeswerecollectedbyaspirationfromthewalls
andtheroofsofdwellingsandpitshelterswhereadultsnormallyrestafterfeeding[25].Thelivespecimensweretransferredtothelaboratoryof
Iranshahr,NationalInstituteofHealthResearch(NIHR).Allcollectedsampleswereidentifiedtospecieslevelusingstandardmorphologicalkeys
[26].InthispartofIran,An.culicifaciessubspeciesAisthemostabundant[27].Amongtheidentifiedspecies,theAn.culicifaciess.l.were
separatedandanalyzedformidgutmicrobiota.Specimensanalyzedformidgutmicrobiotawerefromthreesources:1)wildcaughtfemales,2)wild
caughtfourthinstarlarvae,and3)newlyemergedfemaleadults(immediatelydissectedafteremergencefrompuparearedfromasubsetofthe
larvaein2).

Preparation,sterilizationanddissectionofspecimensweredoneaccordingtoapreviouslydescribedmethod[28].Obtainedmidgutsweremashed
andsuspendedin500LofBrainHeartInfusion(BHI).A100Laliquotofthecontentswasseriallydilutedupto106andplatedontodifferent
media:1)BrainHeartagar(BHA),2)Nutrientagar(NA),and3)Bloodagar(BA)(Merck,Germany)andincubatedat282Cfor2448hours.
Thesterilityofallreagentswascheckedandcontrolsfortheefficiencyofsterilizationweretreatedliketheothersamples.Continuoussubculture
ofeverygrownbacterialcolonywasperformedinordertoisolatesinglepurifiedcoloniesofthebacteria.Thesinglecoloniesofthebacteriawere
laterusedforDNAextractionandPCR,andbiochemicalinvestigations.

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DNA extraction, 16S rRNA gene amplification and sequencing

EachpurifiedbacterialcolonywassubjectedtogenomicDNAextractionusingtheQIAGENDNeasyKit(Qiagen,Germany)accordingtothe
manufacturersinstructions.The16SrRNAuniversalprimers16suF5GAGTTTGATCCTGGCTCAG3and16suR5
GTTACCTTGTTACGACTT3[29]wereusedtoamplifyabout1500bplongsequences.ThePCRprogramhadaninitialdenaturationat94Cfor
10min,followedby35cyclesofdenaturationat95Cfor30s,annealingat56.5Cfor40s,andextensionat72Cfor30s,followedbyafinal
extensionat72Cfor8min.AllampliconsweresentforsequencingtoSeqLab(Germany).TheMallardprogram(http://www.bioinformatics
toolkit.org)wasusedforallacquiredsequencestocheckthepresenceofprobablechimericsequencesandthespecimenswithsuspicioussequences
wereremovedfromthedataset.TheresultantsequenceswerecomparedtothedatabasesoftheRibosomalDatabaseProject(RDPIIMichigan
StateUniversity,http://rdp.cme.msu.edu)andtheGenBank(http//:http://www.ncbi.nlm.nih.gov/BLAST)forconfidentsequenceanalysisandtheir
seqmatchandsequencessimilaritychecktoolswereused.

BasedonsequencecomparisonwiththeGenBankandRDPIIentries,identificationofisolatesandtheirclassificationatgenusandspecieslevelwas
done99percentorhighersequenceidentitieswiththeGenBankentrieswereassumedforspeciesdelineation[30].Allisolateswerealsoidentified
usingclassicalphenotypingandbiochemicalmethodssuchasGramstaining,oxi/fermtestsandusingselectivecultivationmedia[31].Theresultsof
biochemicalandphenotypingmethodswerecomparedtothesequencingresultsandonlythosebacteriathatconfirmedthesequencingresultsare
presented.AllconfirmedsequencesweresubmittedtoGenBank.AllGenBankreferencenumbersaredisplayedinFigure2.

Figure2
Phylogeneticanalysisof16SrRNAsequences(~1500bp)ofallbacteriaisolatedfromAn.culicifaciesusingMaximumLikelihood(ML)basedon
theTamura3parametermodel[33].ThefirstpartofthesequenceIDindicatesfromwhichlifestagethe...

TheMEGA5.05[32]softwarewasusedforphylogeneticanalysisandtreeconstruction.PhylogenetictreeswerebuiltusingMaximumLikelihood
(ML)basedontheTamura3parametermodel[33](1000bootstrapreplicates)analyses.

Results
Inthisstudy,midgutsofatotalof102specimensofAn.culicifacies(68larvaeand34adults)fromthreedifferentsamplingsiteswereanalyzed
(Table1).Thespecimensweredissectedandscreenedforcellfreecultivablebacteriaondifferentmediaresultinginatotalof57bacterialisolates
(Table2).FortyoneisolatesweremembersofsevengeneraofGramnegativebacteriaandtheremaining16isolatesbelongedtoGrampositive
bacteria.Gramnegativebacteriawereisolatedfromlarvaeandbothadultstages,butGrampositivebacteriawereonlyisolatedfromlarvaeand
newlyemergedadults.ThedominationofGramnegativebacteriawasstatisticallysignificantinlarvaeandinmosquitoesfromtheIranshahrarea
(Table3).Sequencedataofthebacteriathatwerefoundinthemidgutoflarvalandadultstageswereusedforphylogeneticanalysis.

Table2
Thenumberofpurifiedcoloniesfrommidgutsof An.culicifaciesinrelationtotheculturemediaandlifestageofmosquitoes

Table3
NumberofGramnegativeandGrampositiveisolatesindifferentmosquitolifestagesandareas

Atotalof19bacterialspecieswereisolatedandidentifiedfromthedifferentstagesofthecollectedAn.culicifaciesspecimens.InFigure2,allnon
identical16Ssequencesarerepresentedwithinformationonstageandsamplinglocality.Accordingtothenumberofisolatedandidentifiedspecies,
itwasshownthatthelarvalstageofthewildcaughtsampleshadahigherdiversitywith17speciesdividedin12generaofbacteria:Acinetobacter,
Aeromonas,Bacillus,Chryseobacterium,Delftia,Exiguobacterium,Kurthia,Microbacterium,Pseudomonas,Staphylococcus,Thorselliaand
Variovorax.Incontrast,inwildcaughtadultsonlythreespeciesfromthreegenerawereidentified:Brachybacterium,PseudomonasandVariovorax.

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Inthenewlyemergedadultsfromwildcaughtlarvaefourspeciesfromthreegeneraofbacteriawereidentified:Acinetobacter,Bacillus,and
Pseudomonas(Table4).

Table4
Theisolatedgeneraofbacteriafromthemidgutof An.culicifaciesinrelationtomosquitolifestageandsamplinglocations

ThegenusPseudomonaswasthemostfrequentlyisolatedbacteriainthisstudy(20/57,57%)andmembersofPseudomonashavebeenalsoidentified
inothermalariavectors[10,13,14,16,17].Toshowtherelationshipbetweensequencedataandoriginofsamples,allthesequencesbelongingto
thegenusPseudomonasfromAn.darlingi,An.gambiae,An.maculipennisandAn.stephensiavailableinGenBankwereretrievedandanalyzed
togetherwiththeonesfoundforAn.culicifaciesinthisstudy(Figure3).

Figure3
Phylogeneticanalysisof16SrRNAsequences(~1500bp)of PseudomonasstrainsusingMaximumLikelihood(ML)basedontheTamura3
parametermodel[33]fromdifferent Anophelesmosquitoes: An.culicifaciesfromIran(Ir_Cuthisstudy),An.darlingi...

Discussion
Identificationofgutbacteriainvectormosquitoeshasreceivednotableattentionsincerecentstudiessuggestthatthecompositionofthevectorgut
microbiotaaffectstheoutcomeofmosquitoinfectionwithPlasmodiumparasites[34,35].Thiscorroboratesearlierresults,forexamplethatsome
bacteriahaveanaturalantiPlasmodiumeffect[10,11,36].Althoughanumberofstudieshavebeencarriedouttoidentifythebacterialmicrobiota
ofAnophelesmosquitoes,severalimportantvectorsremainunstudied.AmongthosewasAn.culicifacies,whichhasbeeninvestigatedinthecurrent
study,andfromwhichaerobicmidgutbacteriahavebeenisolatedandidentified.Strikingly,asseenbythelackofbranchlengthdifferencesbetween
theisolatesinFigure2,manyareverysimilardespitecomingfromdifferentstagesanddifferentareas.Thissuggeststhatthebacteriaacquiredby
An.culicifaciesisnotarandomselection,butinsteadpointstopossiblecoadaptationbetweenbacteriaandmosquitoesand/ortheirbreedingwaters.

Albeitthenumberofisolatesinthisstudyismoderate,thedatasuggestthatGramnegativebacteriadominatethefloraofAn.culicifacies(Table3).
ThisagreeswithearlierresultsfromculturebasedstudiesonAn.stephensiandAn.maculipennisinIran[16]andAn.gambiaeandAn.funestusin
KenyaandMali[37],andalsodatafromsequencebasedstudiesonAn.darlingiinBrazil[13]andAn.gambiaeinKenya[15]allshowing
predominanceforGramnegativebacteria.InWangetal.[15],theproportionofGramnegativebacteriaincreasesfromlarvaetoadults,similarto
whatweseeforAn.culicifacies.

Comparingtheresultsofthisstudywithothersimilarstudiesshowedthatsomebacteriaarecommonamongseveralimportantvectors.Forinstance
thegenusThorsellia,whichwasisolatedfromAn.arabiensis[12]andAn.stephensi[14]andalsofoundinAn.gambiaes.l.[15,38],hasnowbeen
identifiedinAn.culicifacies(thisstudy).IntwodifferentKenyanpopulationsofAn.gambiaemosquitoes,Thorselliawasthedominantgenus[15,
38].Brionesetal.[38]consistentlyisolatedT.anophelisfromthewatersurfacemicrolayer(SML),i.e.,whereAnopheleslarvaefeed,aswellas
in40%oftheadults,whileWangetal.[15]foundthatalmost70%ofthebacteriainyoungadultsbelongedtotheThorselliagenus.Thorselliais
adaptedtoalifeinmosquitoeswithhightoleranceforthealkalineconditionsfoundinthelarvaeandwithincreasedgrowthratesinbloodmedium
[38].Thorselliahasonlybeenfoundinvectormosquitoesandtheirbreedingwaters,andappearstobeauniquegenusofbacteriaonlydistantly
relatedtoothermembersofgammaproteobacteria[39].

AnothercommonbacterialgenusisPseudomonas,whichhasbeenfoundamongseveralmosquitovectorsinAsiaandtheAmericas[11,13,14,17,
36]however,arecentstudyfoundPseudomonasonlyatalowlevelinKenyanmosquitoes[40].Inthisstudy,weidentifiedPseudomonasasthe
mostfrequentlyisolatedbacteriafromAn.culicifacies.ThesamefindingwasmadepreviouslyinAn.stephensimidguts[17].Itshouldbenotedthat
althoughPseudomonasisthemostfrequentlyisolatedspecies,othernonculturablespeciesmaybeimportantconstituentsofthemidgutmicrobiotain
An.culicifacies.However,eveninstudiesonmosquitogutfloramainlyusingPCRbasedmethodsforidentification,thosebacteriapossibletogrow
underlaboratoryconditionsdominatethegutflora[13,15,40].ThehighfrequencyofPseudomonasisolatesispromisingforaparatransgenesis
approachalsobecauseofitspossibilitytogrowincellfreeandordinaryculturemediaandsuitabilityforgenetictransformation[41].However,
characteristicssuchastransstadialtransmission,nonpathogenicity,immunologicalandphysiologicaladaptationtomosquitomidgutconditions,
colonizationinthemosquitomidgutincludingeffectivecompetitionwithresidentbacteriaandpersistencyinthegutforareasonabletime,shouldbe
studiedbeforeselectionoftheisolatesforparatransgenesis.Anoteofcautionisthatamongthemorethan100Pseudomonasspeciessomeare

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pathogenicforexample,Pseudomonasaeruginosaisanopportunistichumanpathogeninseverelyimmunocompromisedpatients[42].Therefore,
caremustbetakensothatforaparatransgenesisapproachonlyPseudomonasspeciesthatarenonpathogenicshouldbeselected.

Inaparatransgenesisstrategyagainstmosquitobornediseases,itwouldbeanadvantageifthetransformedbacteriaexpressingeffectormolecules
couldremaininthevectorpopulation.Somekindoftransstadialtransmissionwouldthereforebeanessentialcharacteristicforanideal
paratransgenesiscandidate.However,itwassuggestedthatmostbacteriaarelostduringmetamorphosisinthepupalstage[43].Inthisstudy,the
sequenceandphylogeneticanalysisshowedthatsomeisolatesofthegenusPseudomonaswerecommonandpresentinfieldcollectedlarvae,adults,
andnewlyemergedadultsfromfieldcollectedlarvae.SomeofthePseudomonasisolatesfoundindifferentstagesofAn.culicifaciesfromthesame
areahave16Ssequenceswith100%sequenceidentitysuggestingtransstadialtransmission.Thisfindingissimilartoapossibletransstadial
transmissionofsomePseudomonasisolatesinAn.stephensi[17],butneedstobefurtherinvestigated.

Conclusions
ThisisthefirststudyonmicrobiotainAn.culicifacies,animportantvectorofmalariainAsia.Furtherstudiesareneededregardingthebiological
characteristicsofthebacteriaandinteractionsbetweenthegutmicrobiotaandthehost.ThefactthatPseudomonasbacteriawithidentical16S
sequencesarepresentbothindifferentlocationsanddifferentstagescouldsuggestthatthisspecieshasadaptedtoalifeinAn.culicifacies.The
potentialsymbioticrelationshipbetweenAn.culicifaciesandPseudomonasmakesitagoodcandidateforparatransgenesis.

Acknowledgment
TheauthorswouldliketothankthestaffoftheIranshahrresearchcenterandNationalInstituteofHealthResearch(NIHRTUMS)fortheir
valuablehelpandsupport.ThisworkwassupportedbytheTehranUniversityofMedicalSciences(TUMS)andtheSwedishUniversityof
AgriculturalSciences(SLU).

Footnotes
Competing interests

The authors declare that they have no competing interests.

Authors contributions

ARC, AR, HV and MAO designed the study. ARC collected the samples, carried out the laboratory process, analyzed the results and wrote the draft of the manuscript. MRP
participated in the species designation of bacteria and related biochemical tests. AR facilitated field work. ARC and OT carried out the phylogenetic analysis. HV, OT and MAO
supervised the project and finalized the manuscript. All authors read the manuscript and approved its contents.

Contributor Information
AliRezaChavshin,Email:chavshin@gmail.com.

MohammadAliOshaghi,Email:moshaghi@sina.tums.ac.ir.

HasanVatandoost,Email:hvatandoost1@yahoo.com.

MohammadRezaPourmand,Email:mpourmand@tums.ac.ir.

AhmadRaeisi,Email:ahmadraeisi@yahoo.com.

OlleTerenius,Email:Olle.Terenius@slu.se.

Article information
Parasit Vectors. 2014; 7: 419.
Published online 2014 Sep 4. doi: 10.1186/1756-3305-7-419
PMCID: PMC4261757

Ali Reza Chavshin, Mohammad Ali Oshaghi, Hasan Vatandoost, Mohammad Reza Pourmand, Ahmad Raeisi, and Olle Terenius

Social Determinants of Health, Research Center, Urmia University of Medical Sciences, Urmia, Iran
Department of Medical Entomology and Vector Control, School of Public Health, Urmia University of Medical Sciences, Urmia, Iran
Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261757/ 5/8
12/19/2016 IsolationandidentificationofculturablebacteriafromwildAnophelesculicifacies,afirststepinaparatransgenesisapproach

Department of Medical Biotechnology, School of Advanced Medical Technology, Tehran University of Medical Sciences, Tehran, Iran
Department of Ecology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
Ali Reza Chavshin, Email: chavshin@gmail.com.
Contributor Information.
Corresponding author.

Received 2014 Aug 13; Accepted 2014 Aug 17.

Copyright Chavshin et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
stated.

This article has been cited by other articles in PMC.

Articles from Parasites & Vectors are provided here courtesy of BioMed Central

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