First Record of Black Band Disease in the Hawaiian Archipelago

17 Pages·2015·1.75 MB·English

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.
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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
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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.
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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
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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
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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
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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
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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,
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Fig6.PhylogeneticrelationshipofBeggiatoaspp.isolatedfromdiseaselesionsfromKaua‘itootherBeggiatoa.Aphylogenetictreewasgenerated
usingtheMaximumLikelihoodmethodwithsequencesfrom14otherBeggiatoaandcloselyrelatedsulfur-oxidizingstrainsincludingrepresentativetype
strainsfromtheThiotrichaceaefamily.No16SrRNAgenesequencesfromBeggiatoafoundinBBDfromotherregionswereavailableforcomparison.The
treewiththehighestloglikelihoodisshown,and1000bootstrapreplicateswereused.NCBIaccessionnumbersareinbrackets,andbootstrapvaluesare
indicatedatbranchnodes.Scalebarrepresentsfivesubstitutionsper100nucleotidepositions.
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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
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