First Record of Black Band Disease in the Hawaiian Archipelago PDF
Preview First Record of Black Band Disease in the Hawaiian Archipelago
RESEARCHARTICLE First Record of Black Band Disease in the Hawaiian Archipelago: Response, Outbreak Status, Virulence, and a Method of Treatment GretaS.Aeby1,3*,ThierryM.Work2,ChristinaM.Runyon1,3,AmandaShore-Maggio1,4, BlakeUshijima1,4,PatrickVideau4,SilviaBeurmann1,4,SeanM.Callahan1,3,4 1 Hawai‘iInstituteofMarineBiology,Kāne‘ohe,Hawaii,UnitedStatesofAmerica,2 U.S.GeologicalSurvey, NationalWildlifeHealthCenter,HonoluluFieldStation,Honolulu,Hawaii,UnitedStatesofAmerica,3 Marine BiologyGraduateProgram,UniversityofHawai‘i,Honolulu,Hawaii,UnitedStatesofAmerica,4 Microbiology Department,UniversityofHawai‘i,Honolulu,Hawaii,UnitedStatesofAmerica * [email protected] OPENACCESS Abstract Citation:AebyGS,WorkTM,RunyonCM,Shore- MaggioA,UshijimaB,VideauP,etal.(2015)First Ahighnumberofcoralcolonies,Montiporaspp.,withprogressivetissuelosswerereported RecordofBlackBandDiseaseintheHawaiian fromthenorthshoreofKaua‘ibyamemberoftheEyesoftheReefvolunteerreportingnet- Archipelago:Response,OutbreakStatus,Virulence, work.Thediseasehasadistinctlesion(semi-circularpatternoftissuelosswithanadjacent andaMethodofTreatment.PLoSONE10(3): darkband)thatwasfirstobservedinHanaleiBay,Kaua‘iin2004.Thedisease,initially e0120853.doi:10.1371/journal.pone.0120853 termedMontiporabandedtissueloss,appearedgrosslysimilartoblackbanddisease AcademicEditor:ChristianRVoolstra,King (BBD),whichaffectscoralsworldwide.Followingtheinitialreport,arapidresponsewasini- AbdullahUniversityofScienceandTechnology, SAUDIARABIA tiatedasoutlinedinHawai‘i’srapidresponsecontingencyplantodetermineoutbreakstatus andinvestigatethedisease.Ourstudyidentifiedthethreedominantbacterialconstituents Received:October30,2014 indicativeofBBD(filamentouscyanobacteria,sulfate-reducingbacteria,sulfide-oxidizing Accepted:January27,2015 bacteria)incoraldiseaselesionsfromKaua‘i,whichprovidedthefirstevidenceofBBDin Published:March16,2015 theHawaiianarchipelago.Arapidsurveyattheallegedoutbreaksitefounddiseasetoaf- Copyright:Thisisanopenaccessarticle,freeofall fect6-7%ofthemontiporids,whichishigherthanapriorprevalenceoflessthan1%mea- copyright,andmaybefreelyreproduced,distributed, suredonKaua‘iin2004,indicativeofanepizootic.TaggedcolonieswithBBDhadan transmitted,modified,builtupon,orotherwiseused averagerateoftissuelossof5.7cm2/dayoveratwo-monthperiod.Treatmentofdiseased byanyoneforanylawfulpurpose.Theworkismade availableundertheCreativeCommonsCC0public colonieswithadoublebandofmarineepoxy,mixedwithchlorinepowder,effectivelyre- domaindedication. ducedcolonymortality.Withintwomonths,treatedcolonieslostanaverageof30%lesstis- DataAvailabilityStatement:All16SrRNAgene suecomparedtountreatedcontrols. sequencesanddsrAsequencesidentifiedinthis studyweredepositedintheGenBankdatabase underaccessionnumbersKM924158-160, KJ914890-91,KM258122-127,KM924161-163,165. Allotherrelevantdataarewithinthepaperandits SupportingInformationfiles. Introduction Funding:Fundingwasprovided,inpart,bythe IntheCaribbean,declinesinlivecoralbeganinthelate1970swhendiseaseoutbreaksthataf- Hawai‘iCoralReefInitiativeandtheHawai‘iDivision fectedthemajorreef-formingcorals,AcroporapalmataandA.cervicornis,werefirstreported ofAquaticResourcesawardedtoGSA.Further supportwasprovidedbytheUniversityofHawaii. [1].Sincethattime,coralcoverhasdeclinedprecipitouslyfromanaverageof50%inthe1970s PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 1/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Thefundershadnoroleinstudydesign,data toanaverageof10%in2002[2]andcoraldiseaseshavecontributedsignificantlytothatde- collectionandanalysis,decisiontopublish,or cline[3].CoraldiseasesarenowdegradingreefsoftheIndo-Pacific[4–6];numerousdisease preparationofthemanuscript. outbreakshavebeenreportedfromAustralia[7,8],Philippines[9],MarshallIslandsandPalau CompetingInterests:Theauthorshavedeclared [10],andeventheremotereefsofPalmyraAtoll[11].ThereefsoftheIndo-Pacificappearto thatnocompetinginterestsexist. bestartingdownthesamepathwayofdisease-induceddestructionexperiencedbythereefsin theCaribbeanandFloridaKeys. WithinHawai‘i,baselineinformationoncoraldiseaseshasbeenestablished;diseaseiswide- spreadonreefsbutoccursatalowprevalence[5,12,13].However,diseaseoutbreaksarestarting tooccurwithincreasingfrequency.In2003,anoutbreakofAcroporawhitesyndromecausedmas- sivemortalityofthetablecorals(Acroporacytherea)atFrenchFrigateShoalsinthenorthwestern HawaiianIslands(PapahānaumokuākeaMarineNationalMonument)[14,15].Additionally, outbreaksofMontiporawhitesyndromeoccurredinKāne‘oheBay,O‘ahuin2006[16]and ‘Āhihi-Kīna‘u,Mauiin2008[17].SincecoraldiseasesareanemergingissueinHawai‘i,theState developedarapidresponsecontingencyplan(RRCP)togivemanagersthecapacitytorespondto bleachingordiseaseeventsinatimelyandefficientmanner[18].Protocolswereestablishedtoin- vestigatebleachingordiseaseeventsandaidindeterminingthesignificance,epizootiology,and potentialcausallinkagesofoutbreaks.Acitizenscienceprogram,theEyesoftheReefNetwork (http://eorhawaii.org/),wasalsodevelopedtotraincommunitymembersinidentifyingandalert- ingmanagerstotheoccurrenceofunusualdiseaseeventsonHawai‘i’scoralreefs. Inthesummerof2012,amemberoftheEyesoftheReef(EOR)networkreportedahigh numberofcoralcolonies,Montiporaspp.(M.capitata,M.patula,M.flabellata),onthenorth shoreofKaua‘iwithprogressivetissueloss.Thecoralshaddistinctdiseaselesions(semi- circularpatternoftissuelosswithanadjacentdarkband)(Fig.1)thatwerefirstobservedin HanaleiBay,Kaua‘iin2004[19].Thelesion,initiallytermedMontiporabandedtissueloss,ap- pearedgrosslysimilartoblackbanddisease(BBD),whichaffectsnumerouscoralgeneraworld- wide[3].BBDiscausedbyamicrobialconsortium,visuallydominatedbyfilamentous cyanobacteria,thatcreatesthecharacteristicblackband[20].OtherconstituentsoftheBBDle- sionincludesulfide-oxidizingbacteria(Beggiatoaspp.),sulfate-reducingbacteriathatinclude membersoftheDesulfovibriogenus,andnumerousheterotrophicbacteria[21–23].Thecyano- bacteriaandsulfide-oxidizingbacteriaexhibitverticalmigrationwithinthemicrobialmat underchangingdiellightconditions,whichresultsindynamicverticalmicrogradientsinoxy- genandsulfide[24,25].Thesulfate-reducingbacteriaareresponsibleforthehighlyconcentrat- edsulfideandanoxicconditionsunderneaththeBBDmatthatislethaltocoraltissue[25,26]. FollowingtheinitialreportbyanEORmember,arapidresponsewasinitiated,asoutlinedin Hawai‘i’sRRCP,todetermineoutbreakstatusandcollectdataforfollow-upstudies.Theobjec- tivesofthisstudywereto1)conductrapidsurveystodetermineoutbreakstatusofthedisease, 2)determinediseasevirulencebymeasuringrateoftissuelossonmarkedcolonies,3)testlesion occlusionasamethodofdiseasetreatment,and4)determinewhetherthedominantmicrobial constituentsofthediseaselesionwereconsistentwithBBDreportedfromotherregionsofthe world.OurresultsindicatetheoutbreakstatusofthefirstreportofBBDintheHawaiianarchi- pelagoandshowasuccessfulmethodarrestingprogressionoflesions.Theabilitytoidentify andrespondquicklytothisdiseaseoutbreakwasfacilitatedbythepriordevelopmentofpro- gramssuchastheEyesoftheReefnetworkandHawai‘i’sRapidResponseContingencyPlan. Methods Ethicsstatement ThisresearchwasconductedunderSpecialActivityPermitNo.2013–16issuedbytheHawai‘i DivisionofAquaticResources. PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 2/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig1.Montiporacapitatacolonywithsignsofblackbanddisease. doi:10.1371/journal.pone.0120853.g001 Rapidsurveys Todeterminediseaseprevalence(numberofinfectedcolonies/totalcoloniesexamined)at Anini,whichisthereportedoutbreaksite(22.227°N,159.456°W),adiverswamfromshore outtothereefcrestandallMontiporaspp.coloniesalonganapproximately6meterwide swathwereexaminedandenumerated.Alldiseasedcolonieswerephotographedandenumer- ated.SurveyswereconductedinOctober2012. Diseasevirulenceandlesionocclusionfordiseasetreatment Therateoftissuelossondiseasedcolonies(n=8)andcoloniestreatedusingthelesionocclu- sionmethod(n=8)wasmeasuredbytaggingandphotographingcolonies(M.capitata) thoughtime.Tomeasurediseaseprogression,abandofmarineepoxy(ZSPARSplashZone) wasappliedtotheeightcontrolcoloniesonbareskeletonroughlytwocentimetersbehindthe leadingedgeofthelesion.Fortheeighttreatmentcolonies,thebacterialmatassociatedwith diseaselesionswasloosenedfromthecoralsurfaceusingtheflatedgeofadiveknifeandre- movedbysuctionwithasterile50ccsyringeandusedforidentificationofselectbacteriaas PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 3/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig2.Thelesionocclusionmethodofdiseasetreatment.A)InfectedM.capitatabeforetreatment.B)Colonywithmarineepoxyoverlesionandwitha bandofepoxyplacedasa“firebreak”approximately5cmaway.C)Samecolonytwomonthspost-treatment.Notethatthemarineepoxyisovergrownwith algaeandtheedgeofthelesionisstartingtogrowovertheepoxy.D)ControlinfectedM.capitatabeforemarkingwithmarineepoxy.E)Colonywithmarine epoxyapproximately5cmbehindlesion.F)Controlcolonyaftertwomonths. doi:10.1371/journal.pone.0120853.g002 describedbelow.Toincreasetheprobabilityofkillinganypotentialbacterialpathogens,the marineepoxywasmixedwithchlorinepowder(calciumhypochlorite)(~15mL/50mLepoxy), andthenspreadovertheborderoflivetissueandbareskeleton(primaryband).Anotherband ofmarineepoxywasappliedtoanareaofhealthycoralca.twotofivecentimetersbeyondthe edgeoftheprimarybandasa“firebreak”orasecondattempttoblockdiseaseprogressionif theprimarybandfailedtohaltdiseaseprogression(secondaryband)(Fig.2). Weconductedapreliminarytrialusingthisdouble—bandmethodforatissuelossdisease elsewhereandfoundthattreatmentwasmoreeffectiveiftheepoxywasmixedwithchlorine powder.Thereisthepossibilitythatthechlorinecandiffuseoutofthemarineepoxyandaffect adjacentcoraltissue.However,therewaslittlemortalityofcoraltissuebeyondthe2ndtreat- mentbandssuggesting,chlorine-impregnatedepoxycausedminimaldamagetothecolony. Thiswasconfirmedafterseveralmonths,whereuponcoralswereobservedre-growingback overtheepoxy,thatitself,wascolonizedbyfilamentousandcrustosecorallinealgae(Fig.2) suggestingminimallonger-termtoxicity. Allcolonies(controlandexperimental)werephotographedbeforeandaftermarkingor treatmentandre-photographedtwomonthslater.Allofthetaggedcolonieswereflat,encrust- ingorplatingcolonies,therebyallowingrateoftissuelossperdaytobemeasureddigitally (ImageJsoftware,v1.46NIH).Theperimeterofthehealthytissuewasdigitallyoutlinedand theencircledareacalculatedincm2.Digitalmeasurementswereconductedintriplicatefor eachphotographandtheaverageswereusedforanalysis. PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 4/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment IdentificationofbacteriaindicativeofBBD TodetermineifthedominantmicrobialconstituentsinBBDwerepresentinthelesionschar- acteristicofMontiporabandedtissueloss,microscopy,culture,andPCRwereusedtoidentify representativefilamentouscyanobacteria,sulfide-oxidizingbacteria,andsulfate-reducingbac- teriafromlesionmaterial. Isolationandgrowthofcyanobacteria.. Filamentouscyanobacteriaidentifiedunder10X magnificationwereremovedfromdiseasesamplesusingsterileforceps,rinsedthriceinauto- clavedfilteredseawater(FSW),platedontothecenterofaplateofsolidASW:BG-11medium [27],andincubatedat28°Cunder2.0%CO and30to40μE/m2/seccoolwhitefluorescent 2 lighting.Asubpopulationoffilamentsthathadmigratedtotheperipheryoftheplatewas transferredtofreshmediumweekly.Bacteriawerepassagedfivetimesbeforeidentification. Standardmicroscopyoffilamentousbacteriawasconducted[28]. Isolationandgrowthofsulfide-oxidizingbacteria.. Whitefilamentousbacteriacontain- inglargeinclusionbodies,grosslysimilartoBeggiatoaspp.,wereremovedfromdiseasesam- plesusingamicropipettorandplacedatthesurfaceofJ3mediadeeps[29]withinsterile polyethyleneconicals,andbufferedtopH8.4withTrisbase,amediumdesignedtoenrich sulfide-oxidizingbacteria.InoculatedJ3deepswereincubatedat28°C.Bacteriathatwereable tomigrateintotheJ3deepwouldindicateamotilebacteriumcapableoflivinginamicro-oxic, sulfide-richenvironment,suchasBeggiatoaspp..Bacteriathatmigratedintothedeepwerere- isolatedwithasyringepenetratingthroughthesideoftheconicalandre-inoculatedontothe surfaceoffreshmedium.Bacteriawerepassagedseventimesbeforeidentification. Molecularidentificationofbacteria.. Toidentifymarinecyanobacteria,filamentseither removeddirectlyfromsampledlesionsorstrainsculturedonASW:BG-11,wereboiledinsterile Milli-Qwaterforfiveminutes,centrifugedat5000xgfortwominutes,andthesupernatantwas usedastemplateDNAforPCRamplificationofthe16SrRNAgenesusingthecyanobacteria- specificprimersCYA106FandCYA781R(a)[30].PCRproductsfromcyanobacterialfilaments weresequenceddirectlyusingprimerCYA106F. Toidentifyfilamentoussulfide-oxidizingbacteriathatwereculturedasdescribedaboveor takendirectlyfromlesions,filamentswereboiledinMilli-Qwaterforfiveminutes,centrifuged at5000xgfortwominutes,andthesupernatantwasusedastemplateDNAforPCRamplifica- tionofthe16SrRNAgeneusinguniversalbacterialprimers8Fand1513R[31]. ThedsrAgeneencodessubunitAofdissimilatorysulfitereductaseandhasbeenusedtoin- dicatethepresenceofsulfate-reducingbacteriainenvironmentalsamples,includingmaterial fromBBD[23,32–34].TotalDNAwasextractedfromcorallesionmaterialusingthePower- SoilDNAExtractionkit(MoBio)asperthemanufacturer’sinstructions.DNAfromlesions wasusedasatemplateforPCRamplificationofanapproximately200bpregionofthedsrA geneusingprimersDSR1-FandDSR-R[32],whichtargetasubsetofdsrAgenesequencespre- viouslyidentifiedfromsulfate-reducingbacteria[35]andfromDesulfovibrionacaeofBBDmi- crobialmats[23,32–34]. PCRproductsderivedfromthe16SrRNAgeneoffilamentoussulfide-oxidizingbacteriaor dsrAgenesofsulfur-reducingbacteriawereclonedintotheSmaIsiteofpBlueScriptSK+as previouslydescribedandsequencedusingtheprimersM13FandM13R[36]. Dataanalyses Therateoftissuelossofinfectedcolonieswascomparedbetweentreatedandcontrolcolonies ofM.capitata.Thedatawerenotnormallydistributedsoanon-parametricMann-Whitney two-grouptestwasusedtoassesstreatmenteffectiveness.Theproportionofthecolonythat PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 5/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment washealthytissueattimezero(T0)andaftertwomonths(T1)wascomparedfortreatedand controlcorals. PhylogeneticanalysisofDNAsequenceswasconductedaspreviouslydescribed[37].Briefly, sequenceswerealignedusingBioEdit[38]andamaximumlikelihoodtreewasconstructed usingthegeneralizedtime-reversible(GTR)algorithm[39]and1000bootstrapreplicateswere performedusingMEGA5[40].Allpositionscontaininggapsandmissingdatawereeliminated usingtheMaximumLikelihoodmethod[41].AlldsrAsequenceswerecheckedmanuallyfor chimerasusingalignmentinMEGA5andBLASTbeforeanalysis.All16SrRNAgenesequences anddsrAsequencesidentifiedinthisstudyweredepositedintheGenBankdatabaseunderac- cessionnumbersKM924158–60,KJ914890–91,KM258122–127,KM924161–163,165. Results Rapidsurveys AtAninisiteone,133montiporidcolonieswereexaminedand8showedsignsofthedisease (prevalence=6.0%).AtAninisitetwo,170montiporidswereexaminedand13werefoundin- fected(prevalence=7.6%). Rateoftissuelossincontrolandtreatedcolonies M.capitatacoloniestreatedwithadoublebandofmarineepoxylostsignificantlylesstissueas comparedtotheuntreatedcontrolcolonies.(Mann-Whitneytest,W=97.0,p=0.003).Aver- agetissuelosswas1.1(SE±0.4)cm2/dayfortreatedcoloniescomparedtountreatedcolonies averaging5.7(SE±1.1)cm2oftissuelossperday. Colonysizesweresimilarbetweengroupswithcontrolcolonieshavinganaveragetotalsur- facearea(healthyanddead)of692.7cm2(range148.6–2881.1cm2)andtreatedcolonieshadan averageof703.4cm2(range72–1556.2cm2).Theaverageproportionofdeadareaoncolonies atthebeginningofthestudywasalsosimilarinbothcontrolandtreatmentgroups(9.7±2.5% and10.4±1.2%respectively,Mann-Whitney,W=60.0,p=0.431).Aftertwomonths,however, untreatedcontrolcoloniesexhibitedsignificantlymoremortality(avg.reductioninareaof healthytissue=69.7±10.6%)comparedtotreatedcolonies(avg.=23.4±7.1%)(Mann- Whitneytest,W=94.0,p=0.007)(Fig.2)andtwocontrolcoloniesexperienced100%mortality (casefatalityrate=25%). Fortreatedcolonies,onecolonyhadnotissuelossbeyondtheprimarybandandfouraddi- tionalcoloniesshowednotissuelossbeyondthesecondarybandduringtheobservationperi- od.Theremainingthreeoutofeighttreatmentcoloniesexperiencedminimaltissuelosspast thesecondaryband(avg.0.52cm2/day). IdentificationofthepathogensindicativeofBBD Cyanobacteriaassociatedwithdiseaselesions.. Thevisuallydominantfilamentousmi- croorganismfromthediseaselesionsexhibitedagrossmorphologyandtypicalautoflorescence characteristicofcyanobacteria(Fig.3).16SrRNAgenesequencesfromfilamentstakendirectly fromdiseaselesionsandtwoculturedstrains,Kaua‘iisolatesOCN073andOCN074,were identicaltoeachother(KJ914890).ThesequencesfromKaua‘icyanobacterialisolateswere 99%identicaltothatofPseudoscillatoriacoraliistrainBgP10_4S,acyanobacteriumassociated withBBDfromtheRedSea[42],andOscillatoriasp.RMS2,thecyanobacteriumassociated withBBDfromPalau[43].TheKaua‘iisolatesclusteredwithcyanobacteriaassociatedwith BBDfromregionsintheIndo-PacificbutdidnotclusterwiththeGeitlerinemasp.isolate PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 6/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig3.Microscopyimagesofcyanobacteriaisolatedfromdiseaselesions.A)Cyanobacteriaunderlight microscope.B)Cyanobacteriaunderfluorescencemicroscope.Redautofluorescenceisindicativeof photosyntheticpigments.Imagesweretakenat60xmagnification.Blackbarrepresents20μm. doi:10.1371/journal.pone.0120853.g003 (previouslydescribedasPhormidiumcorallyticum),theoriginalBBDcyanobacteriumisolated fromtheFloridaKeys[44](Fig.4). Beggiatoaassociatedwithdiseaselesions.. Whitefilamentousmicroorganismsthatcon- tainedapparentsulfurgranulesandexhibitedglidingmotility,consistentwithBeggiatoa,were PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 7/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig4.PhylogeneticrelationshipofcyanobacteriumOCN074,isolatedfromdiseaselesionsfromKaua‘i,toothercyanobacteria.All16SrRNAgene sequencesfromthethreeKaua‘iBBDcyanobacterialisolateswereidentical.AphylogenetictreewasgeneratedusingtheMaximumLikelihoodmethod.The ‘*’indicatescyanobacteriaassociatedwithBBDofPalau(*[42])theRedSea(**[41]),andtheFloridaKeys(***[43]).Thetreewiththehighestloglikelihood isshown,and1000bootstrapreplicateswereused.NCBIaccessionnumbersareinbracketsandbootstrapvaluesareindicatedatbranchnodes.Scalebar representstwosubstitutionsper100nucleotidepositions. doi:10.1371/journal.pone.0120853.g004 observedfromsamplesofcorallesions(Fig.5).InoculationofJ3medium,whichisrichinhy- drogensulfide,resultedinthemigrationoffilamentstwotothreecentimetersdownintothe deep,consistentwiththemotilebehaviorofsulfide-oxidizingBeggiatoa[28].Sequencesof16S rRNAgenesfromfilamentsculturedonJ3deeps(KM924160,KM924158,KM924159)or takendirectlyfromlesionmaterial(KJ914891)clusteredtogether(99to100%identical)and withotherBeggiatoaspecies(Fig.6).Survivalandbehaviorinhydrogensulfiderichmedium, thepresenceoflargegranules,glidingmotility,and16SrRNAgenesequenceareallconsistent withtheseisolatesbelongingtothegenusBeggiatoa. Sulfate-reducingbacteriaassociatedwithdiseaselesions.. Adiversegroupofsulfate- reducingbacteriawerepresentintheKaua‘icorallesions.ThedsrAgenewasusedasaproxy forthepresenceofsulfate-reducingbacteria[35,45]andpartialdsrAsequenceswererecovered byPCRusingdsrA-specificprimersfromlesionmaterial.Thirteensequencesweredetermined PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 8/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig5.BrightfieldimageofBeggiatoasp.filamentsremovedfromdiseaselesions.Imagetakenat60xmagnification.Blackbarrepresents20μm. doi:10.1371/journal.pone.0120853.g005 andcomparedtothosefromotherBBDsamplesandknownsulfate-reducingbacteria(Fig.7). Twogeneralgroupsofsulfate-reducingbacteriawereidentifiedafterphylogeneticanalysis. TwelvesequencesfromtheKaua‘iBBDlesion(KM258124,KM258125,KM258127, KM258122(2clones),KM258123,KM924162,KM924163,KM924161(2clones),KM924165 (2clones))clusteredwithsequencesretrievedfromtheRedSeaBBDlesionmaterial[23]and sequencesfromotherDesulfovibriostrains.OneKaua‘idsrAsequence(KM258126)clustered withdsrAsequencesretrievedfromtheGreatBarrierReefBBDlesionmaterial[34]. Discussion Thisstudyestablishesthepresenceofblackbanddisease(BBD)onmontiporidsintheHawai- ianarchipelago.ThethreemajormicrobialcomponentsindicativeofBBD(filamentouscyano- bacteria,sulfide-oxidizingbacteriaandsulfate-reducingbacteria)wereidentifiedindisease lesionsfromcoralsonKaua‘i.Aphototrophic,filamentous,motilecyanobacteriumcultured fromBBDlesionsinKaua‘iwasgeneticallysimilartobothPseudoscillatoriaandOscillatoria, PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 9/17 BlackBandDiseaseOutbreakinHawaii:ResponseandTreatment Fig6.PhylogeneticrelationshipofBeggiatoaspp.isolatedfromdiseaselesionsfromKaua‘itootherBeggiatoa.Aphylogenetictreewasgenerated usingtheMaximumLikelihoodmethodwithsequencesfrom14otherBeggiatoaandcloselyrelatedsulfur-oxidizingstrainsincludingrepresentativetype strainsfromtheThiotrichaceaefamily.No16SrRNAgenesequencesfromBeggiatoafoundinBBDfromotherregionswereavailableforcomparison.The treewiththehighestloglikelihoodisshown,and1000bootstrapreplicateswereused.NCBIaccessionnumbersareinbrackets,andbootstrapvaluesare indicatedatbranchnodes.Scalebarrepresentsfivesubstitutionsper100nucleotidepositions. doi:10.1371/journal.pone.0120853.g006 cyanobacteriafoundinBBDreportedfromotherareasoftheIndo-Pacific[43].Motile,white filamentousbacteriafromKaua‘iBBDlesionsweremorphologicallysimilartootherstrainsof Beggiatoa[46],andcorresponding16SrRNAgenesequenceswereidentifiedasBeggiatoa. PriorstudiesonBBDhaveexclusivelyusedthedistinctmorphologyofBeggiatoatoidentify thisbacterium[24,25].Ourstudyisthefirsttousemorphology,behaviorandmolecularanaly- sisforidentification.Lastly,arangeofdsrAgenesequenceswereamplifiedfromtheKaua‘i BBDlesions,indicativeofadiversegroupofbacteriathatcanreducesulfate.NoDNAse- quenceswereavailableforBeggiatoafoundinBBDfromotherregionsforourcomparison. However,consistentwithotherstudies,wefoundthatalthoughBBDisfoundonreefsworld- wide,thespecificbacterialspeciesassociatedwiththispolymicrobialdiseasethathavebeenge- neticallyidentified(cyanobacteriaandsulfate-reducingbacteria)variesacrossregions[23,47, 48].IthasbeensuggestedthatthespecificmicrobialcommunitiesfoundinBBDmaybe PLOSONE|DOI:10.1371/journal.pone.0120853 March16,2015 10/17
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