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A comparative test of ixodid tick identification by a network of European researchers.

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ContentslistsavailableatScienceDirect

Ticks

and

Tick-borne

Diseases

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / t t b d i s

Original

article

A

comparative

test

of

ixodid

tick

identification

by

a

network

of

European

researchers

A.

Estrada-Pe ˜

na

a,∗

,

G.

D’Amico

b

,

A.M.

Palomar

c

,

M.

Dupraz

d

,

M.

Fonville

e

,

D.

Heylen

f

,

Habela

M.A.

g

,

S.

Hornok

h

,

L.

Lempereur

i

,

M.

Madder

j

,

M.S.

Núncio

k

,

D.

Otranto

l

,

M.

Pfaffle

m

,

O.

Plantard

n

,

M.M.

Santos-Silva

k

,

H.

Sprong

e

,

Z.

Vatansever

o

,

L.

Vial

p

,

A.D.

Mihalca

b

aDepartmentofAnimalHealth,FacultyofVeterinaryMedicine,MiguelServet177,50013,Zaragoza,Spain

bUniversityofAgriculturalSciencesandVeterinaryMedicineCluj-Napoca,DepartmentofParasitologyandParasiticDiseases,Cluj-Napoca,Romania

cCenterofRickettsiosisandArthropod-BorneDiseases,HospitalSanPedro-CIBIR,Logro˜no,LaRioja,Spain

dMIVEGECUMR5290IRD-CNRS-UM1-UM2CentreIRD,911AvenueAgropolis,BP64501,34394Montpellier,France

eLaboratoryforZoonosesandEnvironmentalMicrobiology,NationalInstituteforPublicHealthandEnvironment(RIVM),Bilthoven,TheNetherlands

fUniversityofAntwerp,DepartmentofBiology,EvolutionaryEcologyGroup,Antwerpen,Belgium

gParasitology&ParasiticDiseases,DepartmentofAnimalHealth,FacultyofVeterinaryMedicine,UniversityofExtremadura,10071,Cáceres,Spain

hUniversityofVeterinaryMedicine,DepartmentofParasitologyandZoology,Istvanu.2.,1078Budapest,Hungary

iLaboratoryofParasitologyandParasiticDiseases,FacultyofVeterinaryMedicine,UniversityofLiège,Liège,Belgium

jDepartmentofVeterinaryTropicalDiseases,FacultyVeterinaryScience,UniversityofPretoria,PrivateBagX04,Onderstepoort,0110,SouthAfrica

kInstitutoNacionaldeSaúdeDr.RicardoJorge,CentrodeEstudosdeVectoreseDoenc¸asInfecciosasDr.FranciscoCambournac,Av.daLiberdade,5,

2965-575,ÁguasdeMoura,Portugal

lDepartmentofVeterinaryMedicine,UniversityofBari,Str.prov.perCasamassimakm3,70010Valenzano,Bari,Italy

mKarlsruheInstituteofTechnology,ZoologicalInstitute,DepartmentofEcologyandParasitology,76131Karlsruhe,Germany

nBIOEPAR,INRA,Oniris,LaChantrerie,44307,Nantes,France

oKafkasUniversity,FacultyofVeterinaryMedicine,DepartmentofParasitology,Kars,Turkey

pCIRAD,UMRCMAEE,F-34398Montpellier,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29August2016

Receivedinrevisedform4March2017

Accepted6March2017

Availableonline8March2017

Keywords: Comparativetest Identification Morphology Molecular Ixodidticks WesternPalearctic

a

b

s

t

r

a

c

t

ThisstudyreportstheresultsofacomparativetestofidentificationofticksoccurringinWesternEurope andNorthernAfrica.Atotalof14laboratorieswerevoluntarilyenrolledinthetest.Eachparticipant receivedbetween22and25specimensofadultandnymphalticksof11species:Dermacentormarginatus, D.reticulatus,Haemaphysalispunctata,Hyalommalusitanicum,Hy.marginatum,Ixodesricinus,I.hexagonus, Rhipicephalusannulatus,R.bursa,R.rossicus,and/orR.sanguineuss.l.Ticksweremorphologically identi-fiedbythreeoftheco-authorsandtheidentificationconfirmedbyafourthco-authorwhousedmolecular methodsbasedonseveralgenes.Thentickswererandomlyselectedandblindlydistributedamong par-ticipants,togetherwithaquestionnaire.Onlyspecimenscollectedwhilequestingand,ifpossible,inthe samesurvey,werecirculated.Becauseoftherandomnatureofthetest,aparticipantcouldreceive sev-eralspecimensofthesamespecies.Speciesinthedifferentgenerahadvariablemisidentificationrates (MR)of7%(Dermacentor),14%(Ixodes),19%(Haemaphysalis),36%(Hyalomma),and54%(Rhipicephalus). Withingenera,theMRwasalsovariablerangingfrom5.4%forI.ricinusor7.4%forD.marginatusor D.reticulatusto100%forR.rossicus.Thetestprovidedatotalmisidentificationrateof29.6%ofthe speciesofticks.TherearenosignificantdifferencesinMRaccordingtothesexofthetick.Participants

∗ Correspondingauthor.

E-mailaddresses:aestrada@unizar.es(A.Estrada-Pe ˜na),gianluca.damico@usamvcluj.ro(G.D’Amico),ampalomar@riojasalud.es(A.M.Palomar),marlene.dupraz@ird.fr

(M.Dupraz),manoj.fonville@rivm.nl(M.Fonville),Dieter.Heylen@uantwerpen.be(D.Heylen),mahabela@unex.es(M.A.Habela),

hornok.sandor@univet.hu(S.Hornok),llempereur@hotmail.com(L.Lempereur),maximemadder@hotmail.com(M.Madder),sofia.nuncio@insa.min-saude.pt(M.S.Núncio),

domenico.otranto@uniba.it(D.Otranto),miripfaeffle@web.de(M.Pfaffle),olivier.plantard@oniris-nantes.fr(O.Plantard),m.santos.silva@insa.min-saude.pt

(M.M.Santos-Silva),hein.sprong@rivm.nl(H.Sprong),zativet@gmail.com(Z.Vatansever),laurence.vial@cirad.fr(L.Vial),amihalca@usamvcluj.ro(A.D.Mihalca).

http://dx.doi.org/10.1016/j.ttbdis.2017.03.001

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wererequestedtoperformasecondroundofidentificationsonthesamesetofticks,usingonlypurposely preparedkeys(withoutillustrations),circulatedtotheenrolledparticipants,including2speciesofthe genusDermacentor,8ofHaemaphysalis,10ofHyalomma,23ofIxodes,and6ofRhipicephalus.Theaverage MRinthesecondroundwas28%:0%(Dermacentor),33%(Haemaphysalis),30%(Hyalomma)18%(Ixodes), and50%(Rhipicephalus).Specieswhicharenotreportedinthecountriesofaparticipatinglaboratory hadalwayshighestMR,i.e.purelyMediterraneanspecieshadhighestMRbylaboratoriesinCentraland NorthernEurope.Participantsexpressedtheirconcernsaboutacorrectidentificationforalmost50%of theticksofthegeneraHyalommaandRhipicephalus.Theresultsrevealedlessthantotalconfidencein identifyingthemostprominentspeciesofticksintheWesternPalearctic,andunderpintheneedfor referencelibrariesforspecialistsinvolvedinthistask.Resultsalsoshowedthatacombinationofcertain genesmayadequatelyidentifythetargetspeciesofticks.

©2017ElsevierGmbH.Allrightsreserved.

1. Background

Ticksareknowntotransmitalargevarietyofpathogensof med-icaland veterinaryconcernandare amongthemostimportant disease-transmittingarthropods(Estrada-Pe ˜naanddelaFuente, 2014).Fieldstudiesonticksshouldbebasedonacorrect identi-ficationofthespecimenscollected,asacrucialstepinachainof microbiologicalorepidemiologicalstudies.Theidentificationhas beencommonlydoneonlybymorphologicalexamination,andthe useof“molecularonly”protocolsarestilluncommoninEurope.For example,inarecentliteraturereviewontheoccurrenceofticksand tick-bornepathogensinEurope(Maiolietal.,2012),onlystudies thatusedmorphologicalkeysfortickidentificationwere consid-ered.Morethan60speciesofthefamilyIxodidaearepresentin Europe.Themorphologicalidentificationofticksisnottrivial,as somespeciesformcomplexeswithcrypticorsiblingspecies,such astheRhipicephalussanguineusgroup(Dantas-Torresetal.,2013) orshowalargerangeofmorphologicalvariability,whichisnot cap-turedbyuntrainedresearchers,forexamplethegenusHyalomma. Overthis backgroundof unstablecriteria fortick identification, newspecies are beingrecognised or interspecific hybridization betweentaxaisreported,abiologicaleventthatmayposeadditinal difficultiesforspecificidentification,evenwhenusingmolecular markers(Kovalevetal.,2015).Moreover,morphologicalkeysfor tickscommonlycoveronlythespeciesofmedicalinterest,anddon’t alwaysincludeallthestages(Arthur,1963;NosekandSixl,1972; Cordasetal.,1993;Hillyard,1996;Filippova,1997;Manilla,1998; Estrada-Pe ˜naetal.,2004;Cringolietal.,2005;Pérez-Eid,2007). Additionally,someofthemmaybeunreliablebecausetheydonot includethemostrecentconceptsaboutthespeciesidentity. There-fore,recentstudiesoncomparativemorphologyofticksinEurope arescarce(Heylenetal.,2014).

Theavailabilityofdifferentmethodshasprovidedinsightsin theuseofcuticularhydrocarboncomposition(Estrada-Pe ˜naetal., 1996)orMALDI-TOF(matrixassistedlaserdesorption ionization-time of flight mass spectrometry) (Yssouf et al., 2013) for the identificationof ticks,whilemostreportsfocusedontheuseof adequategeneticmarkers,like16SrRNA,12SrRNA,orcytochrome c oxidase I(coxI). Although these technologies are useful, they willalways relyonreference specimens for which morpholog-icalidentification needstobecorrectly conducted(Navaet al., 2009;Araya-Anchettaetal.,2015).“Garbagesequences”obtained fromunreliablyidentifiedspecimensthataccumulateindatabanks areasourceofmolecularmisidentification.Theymayintroducea backgroundnoisewhenincludedinthecontextofaphylogenetic reconstructiontickspecies(ZhangandZhang,2014)orproducean incorrectidentificationofindividualspecimens.

Severalpointsjustifytheassessmentofthecomparative capac-ityofresearchersworkingontheidentificationofticks,namely: i) the specific associations between certain tick species and

medically significant pathogens;ii) theconcern for the spread of ticks beyondtheirhistorical ranges; and iii) theimportance of observing harmonised criteria for the identification of ticks. Blindtestsofqualityassessmentareoftenappliedforthe unbi-aseddeterminationofeventsindifferentfacetsofthescience.The protocol involvesthedistributionof specimens toparticipating laboratoriesbyavalidatingteam,whoestablishtherequired stan-dardandcollateandcirculatetheresults(EllisandCross,1981). Biologicalscienceshavebenefitedfromblindtestsfor the iden-tificationoforganisms,theprocedurebeinggenerallyappliedto comparethedegreeofsimilaritybetweentheopinionsofseveral specialistsabouttheclassificationoforganisms.Mosttestsof non-morphologicalmethodsusedfortheidentificationofsomeparasitic arthropodsalwaysusedtheblindapproach,comparingtheresults ofacandidatemethodagainstthebackgroundofmorphological identificationbyspecialists(i.e.Diemeetal.,2014;Yssoufetal., 2013).Examplesofthisapproachtotickssofarcovertheuseof DNA-barcodingforthedetectionofthebloodmealsource(Gariepy etal.,2012).Nevertheless,themorphologicalidentificationofticks isroutinelyusedinlaboratoriesaroundtheworld,morecommonly thanmolecularmethods,mainlyinlargesamplesets,which sim-plifytheflowofworkandreducesthecosts.Insummary,thereis noobjectivemeasureofthecomparativereliabilityofresearchers inthefieldinrecognisingcommonspeciesofticksinalargeregion. We aimed to candidly test thecomparative performance of 14teamsofresearchersinvolvedin thestudyofticksand tick-bornepathogensinEuropeonthemorphologicalidentificationof 11speciesofticksreportedasestablishedinEuropeandNorthern Africa.OurstudyaimstoidentifythechallengesinEuropewhen dealingwiththeidentificationofticks,thecausesfor misidentifica-tions,andthebestproceduresforharmonisedresults.Theselection ofparticularspeciesoftickswasinitiallydonebymorphological identificationbythreeco-authorspossessingmarkedtaxonomic expertise,followedbyconfirmationusingmolecularmethodsby afourthco-author(thevalidatingteam)andthendistributionfor performanceassessmentto14laboratorieswithrelevantresearch interestsinticks.Special attentionwaspaidtoreliability ofthe identificationaccordingtothedistributionoftheticks(e.g.species existinginthecountryofresidenceoftheparticipant,andtherefore familiartotheresearcher)andtheapproachusedinthe identifica-tionofthespecimens(e.g.usingdedicatedbooks,reprints,voucher specimens,etc.).Asecondaryaimwastoestimatetheconfidence oftheparticipantswiththeiridentificationsascomparedwiththe rateofmismatches,aratiothatexpresseshowaccuratelytheycan identifyspeciesnotencounteredbefore.Wefurtherevaluatedthe reliabilityandusabilityofacomprehensivekeyforallthespecies ofticksreportedintheWesternPalearcticasameansofincreasing accuracyofidentification.

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Table1

PCRprimerpairsandconditionsusedforthegeneticidentificationofticks.

Tagetgene Primersequence(5->3) Meltingtemperature (◦C)

Fragmentsize(basepairs) Reference 16SrRNA 16S+1:

CTGCTCAATGATTTTTTAAATTGCTGTGG 16S−1:CCGGTCTGAACTCAGATCAAGT

48 54

456 BlackandPiesman(1994)

12SrRNA T1B:AAACTAGGATTAGATACCCT

T2A:AATGAGAGCGACGGGCGATGT

51 53

approx.360 BeatiandKeirans(2001)

ITS2 RIB-4F: CCATCGATGTGAAYTGCAGGACA RIB-R: GTGAATTCTATGCTTAAATTCAGGGGGT 55 variable, 800–1100

Zahleretal.(1995);Mclainetal. (1995)(fromLabrunaetal.,2002)

2. Materialandmethods

2.1. Speciesofticks

Foranadequaterepresentation ofthemostcommonspecies reportedin the Western Palearctic(including Northern Africa), the following species have been included in the test: Derma-centor marginatus (Panzer) (14♀, 13♂), Dermacentor reticulatus (Fabricius)(14♀,14♂),HaemaphysalispunctataCanestrini& Fan-zago(14♀,12♂),HyalommalusitanicumKoch(7♀,11♂),Hyalomma marginatumKoch(14♀,12♂),Ixodes hexagonusLeach(14♀,13♂, and 15 nymphs), Ixodes ricinus (Linnaeus) (14♀, 11♂, and 12 nymphs),Rhipicephalusannulatus(Say)(14♀,11♂),Rhipicephalus bursaCanestrini&Fanzago(12♀,12♂),Rhipicephalusrossicus Yaki-movandKohl-Yakimova(12♀,15♂),andRhipicephalussanguineus s.l.(9♀,17♂).Thetestwasexplicitlyfocusedonthetickfaunafrom thetwolargebiogeographicalregionsofthetargetterritory:the countriesborderingtheMediterraneanbasin,whichalsoinclude speciesfromNorthernAfrica,andcountriesinCentraland North-ernEurope.Wedidnotincludetickspeciesthatarerestrictedtoa limitedregion(i.e.Ha.hispanicaGil-Collado,R.pusillusGil-Collado, I. ventalloi Gil-Collado, I. lividus Koch) or species inadequately describedorrarelyreported(i.e.I.eldaricusDzhaparidze,I.festai Tonelli-Rondelli,I.kaiseriArthur).Thespecieschosenforthestudy havespecialsignificanceinboth humanandanimalhealth, and theyarewell-knownvectorsofpathogenstohumansoranimals (JongejanandUilenberg,2004).

Thestandardizationof thebatches ofticks circulatedtothe participantswasa point ofspecial concern.Thisprevented the inclusionofsomespeciesofpotentialimportanceinthetest,for exampleIxodespersulcatusSchulze,becausetheavailable speci-menswereinavariabledegreeofrepletion,orobtainedfroma widevarietyofsources,farfromthestandardsrequiredforthe pro-tocol.Onlyunengorgedtickswereused.Specimenswerealways collectedwhile questing toavoid thedistortionof morphologi-calproportions.In thecase ofR.annulatus,whichis aonehost species,specimenswerecollectedasengorgednymphsoncattle, andallowedtomoultinthelaboratorytoflatadults.Althoughthis isnotalwaysaroutineprocedureduringsampletickidentification, wehaveconsideredthisapproachtoprovideparticipantswitha morehomogeneoussamplebatch,inwhichallthetickspecimens areunengorged.Allthespecimensofthesamespecieswere col-lectedin thesamelocality andin thesame samplingevent,to obtainthemosthomogeneoussamplesetpossible.Every speci-menwithmorphologicalabnormalitieswasremovedfromthetest. SpecimensofR. sanguineuss.l. werecollectedonthewallsof a kennelintheMediterraneancoastofSpain,toensureonly spec-imensconformingtotheclassicdescriptionbyFilippova(1997). Thisdescriptionoverlapswiththatforthemorphologyofthe“type II”specimensreportedbyDantas-Torresetal.(2013).

2.2. Initialidentificationandfurthervalidationoftheticks

Allspecimens (n=306)weredeterminedbyoneofthree co-authors(noneoftheseparticipatedintheblindtest)andconfirmed bythetwoothers.Afourthco-authorenrolledintheblindtest, performedamoleculardeterminationofeveryspecies,andresults were100%in agreementwiththemorphologicaldetermination ofthethree co-authorsmentioned above.Themolecular identi-ficationwasdonebyaPCRtargetingthetickmitochondrial16S ribosomalRNAgene (16SrRNA)(BlackandPiesman,1994).For somespecimens,e.g.tickspeciesinwhichthisgenefragmenthad notbeencharacterized,orwasnotspecificenough,PCRassaysfor themitochondrial12SrRNAgeneandthenuclear5.8S-28SrRNA intergenictranscribedspacer2(ITS2)werealsoperformed(Beati and Keirans,2001; Labruna et al.,2002).In the case ofR. san-guineuss.l.themolecularidentityofthespecimensconfirmedthat theybelongedtotheso-calledtemperateclade.Thegenestargeted andtheprimersusedformolecularidentificationareincludedin

Table1. Table2includes themaximumidentities ofsequences obtainedinthisstudyforthemolecularidentificationoftheticks. 2.3. Samplerandomization

Thedistributionofsamplestoeachparticipantwasblindand random.Onceeachindividualtickhadbeenidentifiedbythe val-idatingteam,itwasplacedseparatelyinasmallvialcontaining 70%ethanol.Auniquecodewasrandomlyallocatedtoeach spec-imen(vial).Thespecimensweresenttooneoftheparticipating laboratorieswhichwereidentifiedbyarandomnumber.Each par-ticipantreceived22–25specimens.Becauseoftherandomnature ofthetest,aparticipant couldreceiveseveralspecimensofthe samespecies,withoutregardtotickgenderorstage.Participants wereinformedofthischaracteristicofthetest,andinstructedthat evenifaspecieshadalreadybeenidentified,thesamespeciescould appearagain,ornot,inthereceivedmaterial.

Sixteenresearchers,from14laboratories(oneoftheminvolved only in the molecular identification) enrolled for the test. All ofthem havea longstandingexperiencein tickresearch,either becausethey workprimarily onthe ecologyof ticks, in deter-minationoftick-transmittedpathogens,orareinvolvedinissues ofanimaland/orpublichealth,andhavea backgroundof peer-reviewedpublicationsonthetopic.Onlythreeoftheauthors(those that conducted the primary/initial identificationof ticks) were awareoftheidentityoftheotherparticipants,toavoidexchangeof informationduringtheblindtest.Onlyoneoftheauthorsknewthe completecorrespondencebetweenspeciesofticks,identification numbersofvials,anddetailsoftheenrolledlaboratories.

2.4. Identificationofticks

The identificationperformance test comprised two steps. In thefirststep,identificationoftickswasperformedusingalready

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Table2

Maximumidentitiesofsequencesobtainedforthegeneticcharacterizationoftickspecies.

Tickspecies Genesequences;%identity&GenBankno(noofsequences)

16SrRNA 12SrRNA ITS2

Dermacentormarginatus 99.5JX051098(1) – –

99.5JX051097(1) 100AF031848(1) 99.6FN296278(1)

Dermacentorreticulatus 99.8JF928493(1) – –

Haemaphysalispunctata 99.5KR870978(3) 100AF483218a(3)

Hyalommalusitanicum 99.7–100Z97881(3) – –

Hyalommamarginatum 99.5L34307(1) – –

Ixodeshexagonus 100JF928502(1) 99.6AF081828(1) 100GQ924083(1)

Ixodesricinus 100GU074616(1) 100AF150029(1) – 100GU074606(1) 100KF197118(1) – 99.8GU074590(1) 100JN248424(1) – 100GU074589(1) – – Rhipicephalusannulatus 99.8L34311(2) – –

Rhipicephalusbursa 100KU664351(4) 100KC243833(2) –

Rhipicephalusrossicus 100KP866202b;99.6KU848178(1) 100AF150021(1) 99.3AF271282c(1)

Rhipicephalussanguineuss.l. 100KT382469(2) – –

aIdentifiedasHaemaphysalissp.

bR.sanguineussequence,onlyashortsequencefromR.rossicusavailableinGenBank(KU848178).

c SequenceidentifiedasRhipicephaluspumilio.NoITS2sequencefromR.rossicusavailableinGenBank.

publishedreferences,accordingtothedecisionofeachparticipant. Afterthefirststep, theparticipantswererequestedtoperform asecond roundof identificationonthesamesetofticks,using onlythekeysspecificallypreparedforthestudy,andtosubmitthe resultsagain.Thesekeysweretailoredforeverystageandspeciesof IxodidaefoundinthePalearcticregion,withoutillustrations.The keys(preparedbyoneoftheco-authors) includedtheadultsof 2speciesofgenusDermacentor,8speciesofHaemaphysalis,10of Hyalomma,23ofIxodes,and6ofRhipicephalus,aswellaskeysfor nymphsof23speciesofIxodes.

We receivedresponses from the14 laboratories in thefirst roundbutonly 11responsesinthesecondround. Thisresulted in306ticksidentifiedbymorphologicalmethodsinthefirstround and259inthesecond.

2.5. Questionnaire

The samples were circulated together with a printed ques-tionnairetobefilled-inindividuallyforeachidentifiedspecimen, in both steps.These questionnaireswere pre-labelled withthe number of the vial,and included questions about the identity of the specimen, its gender and stage. We specifically aimed to collect details about the process of identification, involving theproceduresfollowed bytheparticipantregardingtheuseof keys/reprints/monographs,andhowconfidenttheyfeltaboutthe identification.Thecompletequestionnaireisincludedinthe sup-plementarymaterial.

2.6. Calculationofratesofincorrectidentificationsandderived statistics

Wecalculatedtotalratesofincorrectidentifications (misiden-tificationrate=MR)bytickspecies,inbothrounds.Additionally, wecalculatedthespecificMRforeachgenus(toevaluatewhether somegenerahadpooreridentificationratesthanothers)andbysex (tocheckwhethermalesorfemaleshaddifferentMR).Wefurther comparedwhetherMRarehigherforspeciesthatdonotexistin thegeographicalareaofeachparticipatinglaboratory,definingas “endemic”theticksthatwerereportedfromthecountryofthe par-ticipant,and“non-endemic”theticksthatdonothavepermanent populationsinthatterritory.Inotherwords,wetestedwhether participatinglaboratoriesareabletoidentifypotentiallyinvasive ticks.Theconfidenceoftheparticipantswiththeiridentifications

wascomparedwiththerateofindividualMR.Thisratioexpresses thesatisfactionofparticipantsevenwithinaccurateidentifications. Everyparticipantwasconfidentiallyinformedofhis/her identi-ficationsuccessrate,inbothrounds.Wedidnotconsiderthatsome speciescouldbemoredifficulttoidentifythanothers,andtherefore themisclassificationrate(MR)isacrude,unweightedpercentage. Therelativeperformanceoftheparticipantsisnotincludedinthis study.

3. Results

3.1. Misidentificationrate(MR)

Theidentitiesofallticksclassifiedonmorphologicalgrounds beforedistributiontotheparticipantswereconfirmedbymolecular methods(Table2).Regardingidentificationbyparticipantsmade onmorphologicalgrounds,everyspecimen(exceptone)was cor-rectlyidentifiedtogenuslevel.Misidentificationswerefoundonly atthelevelofspeciesorstage.ThetotalspecificMRinthefirst roundwasof29.6%,whichdecreasedto28.5%inthesecondround. TheMRofstageswas1.6%(5outof306)and0.8%(2outof259)in thefirstandsecondrounds,respectively.TwomalesofD. margina-tuswereinitiallyconsideredasfemalesduringthefirstroundof identification.

Fig. 1 shows thespecific MR aggregated by genera, in both thefirstandsecondrounds.Itmustbenotedthatthenumberof responsesbyparticipantswaslowerinthesecondroundthaninthe first(306vs.259ticks,respectively).Inthefirstround,thespeciesin thegeneraDermacentorandIxodesobtainedthelowestMR:7.27% and13.92%,respectively.However,everyspecimenofDermacentor wascorrectlyidentifiedinthesecondroundbuttheMRforgenus Ixodesincreasedto18.03%.Forthesetwogenera,atspecieslevel, theMRvariedbetween5.4%forI.ricinusand7.4%forD.marginatus orD.reticulatus.

Species of the genus Haemaphysalis had MR of 19.23% and 33.33%inthefirstandsecondrounds,respectively.Thespeciesof HyalommaandRhipicephalushadthehighestMRinbothrounds, withsimilarfigures,around36%inHyalommaand54%in Rhipi-cephalus.TheMRbyspeciesareincludedinFig.2.Thespecieswas adequatelyidentifiedifthespecimenwasamalein71%and72%of cases(firstandsecondround,respectively),orin68%ofthecasesin females(bothrounds).TheMRwas100%forR.rossicusinthefirst

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Fig.1.Themisidentificationrates(MR)ofticks,bygenera(in%)inboththefirstand

secondroundofmorphologicalidentification.

round,aneglectedspecieswhichisrarelyconsideredinstudiesin Europe.

3.2. Correlationofthemisidentificationrates(MR)withthe questionnaireresponses

WecomparedtheMRwiththepresence/absenceofthetickin thenationalterritoryofeachparticipatinglaboratory.Only8.49%of “endemic”ticksweremisidentifiedinthefirstround,avaluethat increasedto10.45%inthesecondroundofidentifications. How-ever,theMRof“non-endemic”tickswere21.1%and13.6%,inthe firstandsecondrounds,respectively.

Regardingtheuseofbibliographicalresources,21%ofspecimens wereidentifiedwithoutthehelpoffurtherreferences,because par-ticipantswerefamiliarwiththe tick,49% usedreprintsfor the identification(listedseparately in thesupplementarymaterial), 5%usedgeneralistbook(s)thatcompile(s)dataonspeciesfrom particularregions,and24%usedbothreprintsandbooks.

Theself-perceptionoftheparticipantsaboutthereliabilityof identificationswasvariable.Theparticipantsjudgedthat27%of specimenshadbeenreliablyidentifiedafterafirstlookbecause theywerefamiliarwiththetick,andthat49%ofspecimenshad beenidentifiedcorrectlyaftercheckingthebibliographical refer-ences.Theparticipantshad“seriousconcernsaboutthereliability

Fig.3.Thedegreeofself-perceptionbyparticipantsaboutthereliabilityofthe

identificationofticks,bygenusofthetick(in%).

Fig.4.Thepercentoferroneouslyidentifiedticks(groupedbygenera)accordingto

theself-perceptionbyparticipantsaboutthereliabilityoftheidentificationofticks.

ofthemorphologicalidentification”of23%ofthespecimens(see

Fig.2).Comparing these figuresabout self-perceptionwiththe MR,2%ofspecimenswereerroneouslyidentifiedinthefirst cat-egory, 7% in thesecond, and 15% in the third. However,these roughfigureswerehighlyvariablewhenconsideredbytickgenus (Figs.3and4).Mostparticipantsfeltthatidentificationsofspecies ingenera Dermacentorand Ixodes werereliable.Self-perception

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ofidentificationreliabilitydecreasedforHaemaphysalisspp.Most participantsexpressed“seriousconcerns”aboutthereliabilityof identificationtospeciesforbothHyalommaandRhipicephalus.The comparisonofthesecrudefigureswiththeaccuracyofthe identifi-cationsprovidessomesignificantfindings:participantswereaware thatahighpercentageoftheticksinthegeneraRhipicephalusand Hyalommawereprobablywronglydetermined.

4. Discussion

Thisstudyreports theresultsofa comparativeblindtest of identificationoftickscarriedoutby14self-enrolledlaboratories inEurope.Thestudywasintendedtoevaluatethecapacityto iden-tifyboththewell-establishedspeciesofticksthatarecommonin thecountryoftheparticipants,andtoquantifythecompetences of theEuropean researchteams tocope with potentially inva-sivespecies.ThetestincludedspeciesofthegeneraDermacentor, Haemaphysalis,Hyalomma,IxodesandRhipicephalus,distributedin EuropeandNorthernAfrica.Thestudywasintendedtofocuson themostprominentspeciesofixodidticksofthetargetregionto clearlydelineatethepotentialoftheresearchersinthe manage-mentoftickspecieswiththehighestimpactonhumanoranimal health.

ResultsshowedthatspeciesinthegeneraIxodesand Dermacen-torhadthelowestMR,whilespeciesofHyalommaandRhipicephalus hadthehighestratesof unreliableidentifications.Cruderesults showedabout30%ofmisidentifications,whichdecreasedonlyto 28%whenaspecifically-preparedkeywithoutillustrationswas cir-culatedamongparticipantsforasecondround.Claimsaboutthe possibilityof spreadof tickscurrentlyreportedin the Mediter-raneantonorthernlatitudes(Jaensonetal.,2012)arethusaconcern aftertheseresults,since21%ofthese“non-endemic”specieswere unreliablyidentified.

Thereare severalfactorsthat couldtheoretically biasresults insuchastudyasthis.Themostobviousisthe“goldstandard” establishedfor theidentityofeachspecimen.Thiswasdoneby threecoauthors,whousedmorphologicalmethods.Theyinitiated thestudy,distributedthespecimensanddidnotcontributedata totheidentificationanalysis.Theywereawareofthe geograph-icalorigin of theticks, which otherparticipantswere not, and theiridentificationswereconfirmed100%byafourthparticipant, usingmolecularmethods.Foreveryotherstep,weadheredtothe randomnessof bothbatchesof ticksandparticipants,therefore eliminatingpotentialbiases.Evenatthestageofmanuscriptediting andfinalagreementofthesubmittedpaper,theco-authorswere unawareoftheratesobtainedbytheotherparticipants.

Itisinterestingtonotice thatthegeneraDermacentor,Ixodes andHaemaphysaliswerewell-knownbyalmosteveryparticipant, withlowratesofmisidentifications.However,thegenera Rhipi-cephalusandHyalommaaccumulatedthehighestratesofmistakes. In other words, the purely Mediterranean species were inade-quatelyidentifiedbymostoftheparticipatinglaboratories. Itis howeverimportanttomentionthanonly6laboratoriesin Mediter-raneancountrieswereinvolvedinthetest,ofatotalof14.Inthe caseofRhipicephalus,theerrorsinclassificationweremainlydue tothelackofaharmoniseddefinitionoftheR.sanguineuss.l.group, whichneedsare-descriptionandthedesignationofaneotypeofR. sanguineuss.s.(Navaetal.,2015).Asafurtherproofoftheinherent identificationdifficultieswithinthegenusRhipicephalus,no par-ticipantwasabletocorrectlyidentifytheadultsoftheneglected R.rossicus(rarelyincludedinmostbooksusedbyEuropeantick researchers)inthefirstround,andonlyonelaboratorymanageda correctidentificationafterakeycontainingthespecieswas circu-latedamongtheparticipants.Therefore,weshouldconsiderthat thehighMR ofboth Hyalommaand Rhipicephaluswerederived

from:i)alackoffamiliarityofmorethan50%ofparticipantswith theseticks,ii)thedeficientcoverage ofthesespeciesin papers commonly usedforidentificationof ticks,iii)theunavailability ofcoherentcriteriaforidentificationofthespeciescolonizingthe targetterritory.

Of particular interest is the fact that the circulation of the keyswithoutillustrationsdidnotsignificantlyimprovetherate of reliable identifications, and, in some cases, introduced even higher rates of misidentifications. The second round produced pooreridentificationratesbysomeparticipantswhohadalready accuratelyidentified aspecimeninthefirstround. Theobvious interpretationisthat:i)theinclusionofmorespeciesofticksin thekeyproducedabackgroundnoisethatconfusedthe partici-pants(i.e.IxodesorHaemaphysalis),ii)thelackofillustrationsisa seriousissuewhenonlythecrudetextisusedforidentification.

Beingunfamiliarwiththese“new”species,researcherstended toidentifyticksbyclosesimilarityratherthanbycompleteidentity, intheabsenceofillustrationsguidingtheprocess.Thisexplanation isfurtherconfirmedbytheMRof“endemic”versus“non-endemic” species.Thekeyscouldprobablyhelptheparticipantstoidentify thetickswithwhichtheywerenotfamiliar,butintroduceda fur-thercomplicationwhendealingwithspecieswhichtheyalready knew, becauseseveral such species were included in the keys addinga“backgroundnoise”intheidentification.

Itisnecessarytostressthatparticipantswereawareinmost of the casesof their unreliableidentifications. Highest rates of confidencewereobtainedforthegeneraDermacentorandIxodes, meaningthatparticipantsweresatisfiedwiththeiridentifications. Highestratesofconcernaboutthevalidityofidentificationwere obtainedforspeciesofHyalommaandRhipicephalus.Again,thereis ahighagreementbetweentheratesofconcernaboutthevalidityof classificationsandtheactualunreliableidentificationsforspecies ofthesegenera.

Resultsfromthiscomparativetestshowtheimportanceofan adequatesourceofinformationfortheresearchersinvolvedinthe identificationof ticks.Studiesinmultiple fieldsrelated toticks wouldbenefitfromadequateidentificationsofticks,whichshould beideallybasedoni)acuratedlibraryofspecimensforreference, includingeveryspeciesandstagespresentatcontinentalscale,and ii)a setof trustworthymolecularsequenceseitherproducedin houseorobtainedfromGenBank.The5regionofthemitochondrial genecytochromecoxidasesubunitI(coxI)isthestandardmarkerfor DNAbarcoding(Hebertetal.,2004).Nevertheless,the16SrRNA geneisareliablemarkerforthetickidentificationatthespecies level(Lvetal.,2014a,b),andsequencesoffragmentofthisgeneare themostcommoninGenBank.Moreover,othermarkerssuchas the12SrRNAgeneorinternaltranscribedspacers(ITS)canbe com-plementaryfortickclassification(Lvetal.,2014a,b).Undoubtedly, thesuccessofDNAbarcodingforanyparasitespeciesidentification reliesheavilyonaccuratemorphologicalidentificationofreference specimens.Indeed,barcodingofticksusingthemolecularapproach alonecouldleadtoinconsistentresults(Lvetal.,2014a)anda com-binationofthreeDNAmarkers(coxI,16SrRNA,and18SrRNA)could efficientlyseparateseveralspeciesofticks(Lvetal.,2014a,b).The specificidentificationofticksbymolecularmethodsshouldnotbe consideredasdefinitivesinceitrequirespersonalexperienceand adequatelibraries,leadingtotheneedofdepositionofvoucher specimens(i.e.Beatietal.,2013;Navaetal.,2014).Theprocedures usedinthepresentstudyshowthatanadequatecombinationof severalgenesandoftheportionsthatproducethehighest phylo-geneticinformationissuitableforidentificationofticks.

Whileawarenessofticksandtick-bornepathogensincreases worldwide,thereisalackofadequateknowledgeaboutthe iden-tityofthemostprominentspeciescolonizingextensiveregionsin theWesternPalearctic.Asfarasweknow,asimilarcomparative testhasneverbeenperformedinotherpartsoftheworld.Itthus

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remainsofinteresthowresearchersofotherregionsaddressthe issue,andhowlesserknownspeciesareidentifiedbyspecialists. Althoughotherspeciesofticksmaylackmedicalinterest,theymust beconsideredpotentiallyconfusingentitieswhencomparedwith thefocalspecies,introducing‘noise’inreporting.Wewantedto candidlypresenttheseresults,whileurgingtheneedforadequate trainingofexpertsinvolvedintheidentificationofticks,astep nec-essarytobothaddressepidemiologicalstudiesandtocopewiththe riskposedbyinvasivespecies.

Acknowledgments

Thisstudyhasbeencarriedoutundertheumbrellaofthe Euro-peanCOSTActionTD1303,“EurNegVec”.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.ttbdis.2017.03. 001.

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Figure

Fig. 2. The misidentification rates (MR) of species of ticks (in%) in both the first and second round.

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