Does Dark-Spot Syndrome Experimentally Transmit among Caribbean Corals?

16 Pages·2016·2.67 MB·English

RESEARCHARTICLE
Does Dark-Spot Syndrome Experimentally
Transmit among Caribbean Corals?
CarlyJ.Randall1*,AdánG.Jordán-Garza1¤,ErinnM.Muller2,RobertvanWoesik1
1 DepartmentofBiologicalSciences,FloridaInstituteofTechnology,Melbourne,Florida,UnitedStatesof
America,2 MoteMarineLaboratory,Sarasota,Florida,UnitedStatesofAmerica
¤ Currentaddress:UniversidadVeracruzana,Tuxpan,Veracruz,México
*[email protected]
Abstract
Overthelasthalf-century,coraldiseaseshavecontributedtotherapiddeclineofcoralpop-
ulationsthroughouttheCaribbeanregion.Somecoraldiseasesappeartobepotentially
infectious,yetlittleisknownabouttheirmodesoftransmission.Thisstudyexperimentally
testedwhetherdark-spotsyndromeonSiderastreasidereawasdirectlyorindirectlytrans-
missibletoneighboringcoralcolonies.Wealsotestedwhetheropenwoundswereneces-
sarytofacilitatediseasetransmission.Atthecompletionoftheexperiments,wesampled
bacterialcommunitiesondiseased,exposed,andhealthycoralcoloniestodetermine
OPENACCESS
whetherbacterialpathogenshadtransmittedtothesusceptiblecolonies.Wesawnoevi-
Citation:RandallCJ,Jordán-GarzaAG,MullerEM, denceofeitherdirectorwaterbornetransmissionofdark-spotsyndrome,andcoralsthat
vanWoesikR(2016)DoesDark-SpotSyndrome
receivedlesionsbydirectcontactwithdiseasedtissue,healedandshowednosignsof
ExperimentallyTransmitamongCaribbeanCorals?
infection.Therewerenosignificantdifferencesamongbacterialcommunitiesonhealthy,
PLoSONE11(1):e0147493.doi:10.1371/journal.
pone.0147493 exposed,anddiseasedcolonies,althoughnineindividualribotypesweresignificantly
higherindiseasedcoralscomparedwithhealthyandexposedcorals,indicatingalackof
Editor:ChristinaA.Kellogg,U.S.GeologicalSurvey,
UNITEDSTATES transmission.Althoughourexperimentsdonotfullyrefutethepossibilitythatdark-spotsyn-
dromeisinfectiousandtransmissible,ourresultssuggestthatinsitumacroscopicsignsof
Received:October29,2015
dark-spotsyndromearenotalwayscontagious.
Accepted:January5,2016
Published:January20,2016
Copyright:©2016Randalletal.Thisisanopen
accessarticledistributedunderthetermsofthe Introduction
CreativeCommonsAttributionLicense,whichpermits
Overthelasthalf-century,marinediseaseshavecontributedtothewide-spreaddeclineof
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare Caribbeancorals[1–4].Despitedecadesofresearch,theetiologiesofmostcoraldiseasesare
credited. poorlyunderstood.Severalcoraldiseasesareknowntobeinfectious,andareassociatedwith
pathogenssuchasbacteria,protists,fungi,orwithaconsortiumofmicrobes[5–16].Yetthe
DataAvailabilityStatement:Alldataarecontained
withinthepaperorthesupplementaryfiguresand degreetowhichinfectiousdiseasesarecontagiousishighlyvariable[17],andfewstudieshave
tables. experimentallytestedthepotentialtransmissionofseveralcommoncoraldiseasesintheCarib-
bean.Understandingthedynamicsofcoral-diseasetransmissionisnecessarytoaccuratelypre-
Funding:Fundingforthisstudywasprovidedbythe
NationalScienceFoundationaward#OCE-1219804 dictfutureoutbreaksofcoraldiseases,andtopotentiallymitigatethespreadofthosediseases.
awardedtoRvW. Infectiousdiseasesarecausedbytheinvasionofahostbyapathogenicagent[17–18].In
veterinaryepidemiology,therearetwotypesofinfectiousagents:exogenouspathogensand
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. endogenouspathogens[17].Exogenouspathogensarepresentintheexternalenvironment,
PLOSONE|DOI:10.1371/journal.pone.0147493 January20,2016 1/16
TestingTransmissionofCaribbeanDark-SpotSyndrome
andareacquiredbyexposuretoacarrierorganism,toanon-biologicalvector,ortoaninfected
host.Thesepathogenscausediseasesthathaveclearclinicalsignsandhavedistinctpathologi-
callesions[17].Bycontrast,endogenouspathogensoftenarepresentinhealthyhosts,but
causediseaseonlywhenthehostbecomesstressed.Formostcoraldiseases,itisunknown
whetherthediseasesarecausedbyexogenousorendogenouspathogens,orifinstead,theyare
theresultofnon-infectiousdiseases[19].
Addressingthequestionofcoral-diseasetransmissionhasproveddifficultformanyreasons,
includingthefollowing:firstly,pathogensformanydiseasesareunknownorareinconsistently
diagnosed[20–22].Secondly,thediagnosisofinfectionisbasedonthesubjectiveidentification
ofmacroscopicsigns,potentiallyofdifferentorigins[21,23].Thirdly,complexinteractions
amongthepathogens,thehostcorals,andtheenvironmentmayexist,whichcouldresultin
successfultransmissionofadiseaseunderonesetofenvironmentalconditions,andinunsuc-
cessfultransmissionofadiseaseunderanothersetofenvironmentalconditions[24].Fourthly,
unknownandpotentiallycomplexstagesofadiseasecanexistwherebytransmissionsucceeds
whenthediseaseisinonestage,andfailswhenthediseaseisinanotherstage[25].Fifthly,
thereisthepotentialexistenceofvectors,orcarriers,inwhichdiseasesignsdonotmanifest
[26].Despitethesedifficulties,examiningcoral-diseasetransmissionisnecessaryforelucidat-
ingdiseaseetiology.
Inthisstudywedefinecontagiousdiseasesasthosewhicharetransmissiblethroughdirect
physicalcontactbetweencorals,orwhicharetransmissibleindirectlythroughavector[27].
GiventhatcoralsaresessileandthatCaribbeanreefscurrentlysupportlowcoralcover[28],
physicalcontactbetweencoralsislimited,anditislikely,therefore,thatdirecttransmissionof
contemporarycoraldiseasesisuncommon[29].Itismorelikelythatifcoraldiseasesarebeing
transmittedthroughouttheCaribbeanregion,thetransmissionwouldbeindirect,eitherviaa
waterbornepathogenorthroughareef-associatedvector.
Dark-spotsyndromeisoneofthemostprevalentandubiquitouscoraldiseasesintheCarib-
bean.Althoughthisdiseaseisthoughttobepotentiallyinfectiousandtransmissible,dark-spot
transmissionhasyettobeexperimentallydemonstrated[30–37].Dark-spotsyndromeaffects
primarilySiderastreasiderea,O.faveolata,andStephanocoeniaintersepta,althoughithasbeen
observedonatleastseventeenspeciesofscleractiniancoralsintheCaribbean[30,34,38,39],
andsomeresearchershavearguedthattheextentofdark-spotsyndromeisanimportantindica-
torofoverallreefhealth[33,40].Acausativeagentofdark-spotsyndromehasnotbeenidenti-
fied,althoughseveralinfectiousagentshavebeenproposed,includingbacteriafromthegenera
Vibrio[32],Corynebacterium,Photobacterium,andAcinetobacter,[36],bacteriafromthefamily
Parvularculaceae[36],fungiresemblingAspergillusspp.[35]andRhytismaspp.[36],andcyano-
bacteriaresemblingOscillatoriaspp.[36].AstudyrecentlypublishedbyKelloggetal.[37]found
nosignificantdistinctionsbetweenbacterialcommunitiesinhealthyanddark-spotaffectedS.
sidereafromSt.John,USVIandtheDryTortugas,suggestingthatbacterialpathogenswerenot
thecausativeagentsofthedisease.Someassessmentssuggestthatdark-spotsyndromeaffects
primarilythesymbioticzooxanthellaewithinthecoraltissue,causingimpairmentofmitosis,and
areductioninthedensityofsymbionts[31].Yet,fieldstudieshavefoundthatdark-spotsyn-
dromeoncoralscanresultfromthephysicalabrasionofthecoraltissue,andthatdiseasesigns
donotalwayscausetissuemortality[33,41].Thishasledsomeresearcherstosuggestthatdark-
spotsyndromeisastressresponse,ratherthananinfectiousdisease[19,33].
Theobjectivesofthepresentstudywereto:(1)testdirect-contacttransmissionandwater-
bornetransmissionofdark-spotsyndromeonSiderastreasiderea;and(2)comparethebacte-
rialcommunitiesondiseased,disease-exposed,andhealthySiderastreasidereacoloniesatthe
completionoftheexperimenttoexaminewhetherpotentialbacterialpathogenstransmittedto
theexposedcolonies.
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TestingTransmissionofCaribbeanDark-SpotSyndrome
MaterialsandMethods
Ethicsstatement
CoralcollectionstookplaceunderapermitissuedbytheFloridaKeysNationalMarineSanctu-
arynumberedFKNMS-2013-086.Noethicalapprovalwasrequiredforthelaboratoryresearch
describedinthisstudy.
Coralcollectionandexperimentalsystem
AseriesoflaboratoryexperimentswereconductedonSiderastreasidereatodeterminewhether
dark-spotsyndromeistransmissible.S.sidereawasselectedforthetransmissionexperiments
becauseitisoneofthemostcommonhostsofdark-spotsyndrome[30,31,38,39].Diseased
coralswereselectedbasedontheirmacroscopicclinicalsignsandonthepresenceofpathologi-
callesions.AsperWorkandAeby’s[42]guidelines,thecasedefinitionofdark-spotsyndrome
was:multifocal,circularorirregularlyshapedpurpletobrowncoloredlesionsthatarecentrally
orperipherallylocated,andrangefrommild(1–20%)tomoderate(21–50%)extentonindivid-
ualcolonies.Somelesionswereassociatedwithcentrallylocatedtissueloss,andthelesions
mostcloselyresembledTypeIandTypeIVlesionsasdefinedbyBorger[33](Fig1).
Usingahammerandchisel,fragmentsofS.sidereacolonieswerecollectedfromWonder-
landReefinthelowerFloridaKeys(N24.56028°,W81.50127°)inJuly2013.Eightdiseased
andfourapparentlyhealthyS.sidereafragmentswerecollectedatadepthof6–7m.Thenum-
berofdiseasedcoloniesavailableforsamplingwaslimitedbythenumberofdiseasedcolonies
atthesiteofcollection(whichwasapproximately200mby100m).Inaddition,tenhealthy
colonies(20–90cm2)werecollectedfromtheinshorecoralnurseryinKeyWest,Florida,to
limitdamagetocoralsonWonderlandReef.Allwoundsproducedbysamplingwerecovered
byanepoxyresintominimizefurtherdamagetothecorals.Allfragmentswerecollectedby
Fig1.Donorcoralcolonies.Representativedonorcolonies(aandb)sampledforuseinexperimentstestingthetransmissionofdark-spotsyndromeon
Siderastreasiderea.Insetimagesshowdiseaselesionsindetail.
doi:10.1371/journal.pone.0147493.g001
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TestingTransmissionofCaribbeanDark-SpotSyndrome
diverswearingsurgicalgloves,andhealthyfragmentswerecollectedpriortodiseasedfrag-
ments,topreventthepotentialexposureofhealthyfragmentstodiseaseduringthecollection
process.AllcoralswereindividuallypackagedinZiploc1bagswithseawaterandtransported
inseparatecoolers,forhealthyanddiseasedcorals,toMoteMarineLaboratoryinSummerland
Key.Immediatelyfollowingcollectionandtransportation,coralswerefragmented,witha
bleach-rinsed(25%bleachsolution)wet-tilesaw,into~16cm2colonies,mountedon2.5cm
wideby2.0cmtallpolyvinylchloride(PVC)baseswithPlastilina1modelingclay,andplaced
intorandomlyassignedtreatmenttanks.Allcoralswerehandledwithsurgicalgloves,andall
equipmentwasrinsedwitha25%bleachsolutionin-betweenthehandlingofeachcoral
fragment.
Theoutdoorexperimentalsystemwassuppliedwithflow-throughseawaterfromMote
MarineLaboratory’swell-watersystem[43].Seawaterwasoff-gassedinahead-tanktomain-
tainpHat7.9–8.1,andwasmechanicallyfilteredwithasand-particlefilter.Seawaterentering
theflow-throughsystemwasultraviolet-light(UV)sterilizedwithan18WCoralife1Tur-
botwistUVSterilizertoensurethatthesupplyofwaterwasfreeofpathogens.Thesystemwas
coveredwithshadeclothtomatchtheirradiancelevelsintheaquariaascloselyaspossible
withtheirradiancelevelsthatweremeasuredatthesiteofcoralcollection(58μmolquantam-2
s-1atapproximately10:50am).Allexperimentswereconductedbetween10Julyand14August
2013atMoteMarineLaboratoryinSummerlandKey,Florida.
Experiment1:Waterbornetransmission
Thesystemforthewaterborne-transmissionexperimentsconsistedofsixty(previously
unused)2.7Lacrylicaquaria,set-upinacascadingdesign(Fig2).Thisdesignallowedforuni-
directionalflowtotestwaterborne-diseasetransmissionbyexposingcoralsinthe‘lowercham-
bers’towaterthathadbeenpreviouslycirculatedinthe‘upperchambers’housingdiseased
corals.Flowratesinallaquariaweremeasuredandcalibrateddaily.Theflowratesaveraged
28±4Lhr-1(anaverageof2cmsec-1with11volumeturnoversperhour,andaresidencetime
of6minutes).Watertemperaturesweremeasureddailywithastandardlaboratorythermome-
terandaveraged27.4°C(±0.6°CSD;Fig3).
Fourtreatmentswereincludedintheexperimentaldesign:(i)transmissionfromadiseased-
coralcolonytoahealthy-coralcolony(experimental);(ii)transmissionfromahealthy-coral
colonytoahealthy-coralcolony(control1);(iii)transmissionfromarubblefragmenttoa
healthy-coralcolony(control2);and(iv)transmissionfromanemptychambertoahealthy-
coralcolony(control3).Sevenreplicatesofeachtreatmentpairweretested.Thewaterborne-
transmissionexperimentwasrunfor11days,afterwhichtheexperimentalcoralswereplaced
inaholdingtankandmonitoredforanadditional17days(foratotalof28days).Allcoralcol-
oniesweremonitoredforsignsofdiseaseandphotographeddaily.
Tocomparethephotochemicalyieldsofcolonieswithineachtreatment,pulseamplitude
modulated(PAM)fluorometrymeasurementsweretakenthreehoursaftersunset(i.e.,three-
hourdark-acclimated)onDays6and9oftheexperiment.Twotechnical-replicatemeasurements
weretakenoneachcolonyondifferentlocationswithinthesamecolony,andwereaveragedto
obtainonemeasurementofphotochemicalyieldpercolony.PAMmeasurementsofthediseased
colonyweretakendirectlyontissuewithvisiblelesions.ThePAMprobewasrinsedwitha10%
bleachsolution,andthenrinsedwithsterilizedseawaterin-betweeneachmeasurement.
Arepeatedmeasuresanalysisofvariance(RM-ANOVA)wasusedtoassessdifferencesin
photochemicalyieldsamongtreatmentsonthesamecoralsthroughtime.Photochemical-yield
datamettheassumptionsofRM-ANOVA,andstatisticalanalyseswereconductedusingbase
R,andthepackages‘car’[45]and‘userfriendlyscience’[46]inthestatisticalprogramR[47].
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TestingTransmissionofCaribbeanDark-SpotSyndrome
Fig2.Cascadingexperimentaldesign.Thedesignhadunidirectionalwaterflow(arrows)fromupperchambers(2.7Leach)tolowerchambers(2.7L
each),whichallowedthehealthycoralsinthelowerchamberstobeexposedtowaterfromtheupper-chambershousingdiseasedcorals.Allupperchambers
weresuppliedwithUVsterilizedandmechanicallyfilteredseawater,andalleffluentwaterwastreatedwithUVsterilizationandbleach,beforeallowingthe
watertodrainintoanearbycanal.
doi:10.1371/journal.pone.0147493.g002
Experiment2:Direct-contacttransmission
Thesystemforthedirect-contacttransmissionexperimentconsistedofsix(previouslyunused)
25Lplasticaquaria.Allaquariareceivedflow-through,filtered,andUV-sterilizedseawater,as
describedabove.Flowratestoallaquariaweremeasuredandcalibrateddaily,andaveraged
48±6Lhr-1(anaverageof2cmsec-1,witha2-volumeturnoverperhour,andaresidence
timeof32minutes).Watertemperaturewasmeasureddailyusingastandardlaboratoryther-
mometerandaveraged27.5°C(±0.8°CSD)(Fig3).
Twotreatmentswereincludedintheexperimentaldesign:(1)direct-contactfromadis-
eased-coralfragment(~1cm2)toahealthy-coralcolony(~16cm2)(experimental);and(2)
direct-contactfromahealthy-coralfragment(~1cm2)toahealthy-coralcolony(~16cm2)
(control).Threereplicatesweretestedforeachtreatment.OnDay1,smallfragmentsofS.side-
rea,(~1cm2,eitherdiseasedorhealthydependingonthetreatment)wereplaceddirectlyon
thetopofhealthyS.sidereacolonies(~16cm2),withdirecttissue-to-tissuecontact(Fig4).The
coralfragmentswereleftindirectcontactwiththehealthy-coralcoloniesforthreedays,and
werethenremoved.Afteratotaloffivedays(i.e.,twodaysfollowingfragmentremoval),no
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TestingTransmissionofCaribbeanDark-SpotSyndrome
Fig3.Thermalhistoryofthecoralstestedinaseriesoflaboratory-basedtransmissionexperiments.a.Meanmonthlysea-surfacetemperature
(dottedline)andtheminimumandmaximumofthe10-yearmonthlymean(grayrange)obtainedfromHadIsea-surfacetemperaturerecordsforthesiteof
collection[44].b.Meandailysea-surfacetemperaturefromaHobo1dataloggerdeployednearthesiteofcollection(dataloggedevery15minutes)andthe
standarddeviationaroundthemean(grayrange).c.Meandailyexperimentaltemperaturesmeasuredwithastandardlaboratorythermometerforthe
durationofthetransmissionexperiments.Grayrange=standarddeviationaroundthemean;W=waterbornetransmission,D=direct-contacttransmission.
doi:10.1371/journal.pone.0147493.g003
dark-spottransmissionwasobserved.Therefore,onDay5,lesionswerecreatedonthehealthy
S.sidereacoloniestotestthehypothesisthatdirect-contacttransmissiononlyoccurswhena
woundispresent,allowingforpathogenentry.Lesionswerecreatedbyforciblygrindingthe
coralfragmentsintothehealthy-coralcolonies,therebybreakingthehealthytissueanddamag-
ingthecoralskeleton,sothatpotentialpathogens,wherevertheymaybelocatedinthecolony,
wouldbeexposedtothehealthycoralcolonies(Fig4,Day5).Coralfragmentswereleftincon-
tactwiththeinjuredtissueofthehealthy-coralcoloniesfor13hourstofacilitatepotentialpath-
ogentransmission,andthenremoved.TheS.sidereacoloniesweremonitoredforsixmore
days(foratotalof11days)andwerephotographeddailyforthedurationoftheexperiment,
afterwhichallS.sidereacolonieswereplacedinaholdingtankandmonitoredforanaddi-
tional17days(foratotalof28days).
Examinationofthetransmissionofpotentialbacterialpathogens
Immediatelyfollowingcompletionofthewaterborne-transmissionexperiments(Experiment
1),threeeach,ofdiseased,exposed,andhealthycoloniesofS.sidereawererandomlyselected
forbacterial-communityanalyses,todeterminewhetherpotentialbacterialpathogenshad
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Fig4.Time-seriesimagesoftwohealthy-coralcolonies(~16cm2)ofSiderastreasidereainadirect-contacttransmissionexperiment.Thefirst
columnofimagesisaclose-upofthefragments(~1cm2)thatwereplacedindirectcontactwiththecoralcolonies.Thefirstrowofimagesshowahealthy-
coralcolony(~16cm2)indirectcontactwithahealthyfragment(~1cm2)(control).Thesecondrowofimagesshowahealthy-coralcolonyindirectcontact
withafragmentwithdarkspotlesions(~1cm2)(experimental).NotethatDay1imagesweretakenpriortotheadditionofthedirect-contactcoralfragment.
Notealsothedirect-contactlesionscarsonS.sidereaonDay5.
doi:10.1371/journal.pone.0147493.g004
transmittedtotheexposedcolonies.Theninecoralcolonieswereplacedinindividual,sterile
whirl-paksat-80°CandthenweretransportedondryicetoMoteMarineLaboratoryinSara-
sota,Florida.
Tissuewasremovedfromtheskeletonofthepreserved-coralcoloniesusingaPaasche1air-
brushwith10mLofsterileseawater.Thetissueslurrywascollectedinasterile50mLFalcon1
tubeandhomogenizedusingavortex.Thetissuehomogenatewasthenspundownintoapellet
usingacentrifugesetat10,000rpm.Thepelletwasre-suspendedin2mLofsolutionC1and
DNAwasextractedusingaPowersoilDNAextractionkit(MoBIOLaboratoriesInc.Lot
#PS14F19).ExtractedDNAwasthensenttoMRDNALaboratory(www.mrdnalab.com,Shal-
lowater,TX,USA)forIllumina1sequencing(20,000readsperassay)usingtheuniversalbac-
terialprimers27F/519Rwithabarcodeontheforwardprimer.The16SrRNAgeneontheV1–
V3hypervariableregionwasamplifiedbyapplyinga30cyclepolymerasechainreaction(PCR)
withtheHotStarTaqPlusMasterMixKit(Qiagen,USA).PCRwasappliedusingthefollowing
protocol:(1)94°Cfor3minutes,(2)28cyclesof:94°Cfor30seconds,53°Cfor40seconds,and
72°Cfor1minute,and(3)afinalelongationstepat72°Cfor5minutes.Afteramplification,
PCRproductswereconfirmedin2%agarosegelstodeterminethesuccessofamplificationand
therelativeintensityofthebands.Multiplesampleswerepooledtogetherinequalproportions
basedontheirmolecularweightandDNAconcentrations.Pooledsampleswerepurifiedusing
calibratedAmpure1XPbeads.ThenthepooledandpurifiedPCRproductwasusedtopre-
pareDNAlibrariesbyfollowingtheIllumina1TruSeqDNAlibrarypreparationprotocol.
SequencingwasperformedusingtheIllumina1sequencingplatformatMRDNA(www.
mrdnalab.com,Shallowater,TX,USA)followingthemanufacturer’sguidelines.Sequencedata
wereprocessedusingastandardizedanalysispipeline.Briefly,sequenceswereinitiallydepleted
ofbarcodes.Thensequenceslessthan150bporwithambiguousbasecallswereremoved.
Operationaltaxonomicunits(OTUs)weregenerated,andchimeraswereremovedusing
UCHIME[48].OTUsweredefinedbyclusteringat3%divergence(i.e.,showing97%similar-
ity)usingadenovomethod.FinalOTUsweretaxonomicallyclassifiedusingBLASTnagainst
thecuratedNationalCenterforBiotechnologyInformation(NCBI)databaseandtheRibo-
somalDatabaseProject(RDP).
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Fig5.Time-seriesimagesofSiderastreasidereafromwaterborne-transmissionexperiments.Thetoprowrepresentsdiseasedorhealthy-coral
colonies(~16cm2),dependingonthetreatment,fromtheupperchambers.Thebottomrowrepresentshealthy-coralcolonies(~16cm2)fromthelower
chambers.
doi:10.1371/journal.pone.0147493.g005
Therelative-percentagecontributionsofeachgenus(andhighertaxonomiclevels)tothe
bacterialassemblageswerecalculatedforeachoftheninecoralcolonies.Differencesincoral-
bacterialcommunitiesamongtreatmentsweretestedusingapermutationalmultivariateanaly-
sisofvariance(PERMANOVA).Theresultswerevisualizedusingnon-metricmultidimen-
sionalscalingplots.Differencesinmeanbacterialrichnessamongtreatmentsateach
taxonomiclevelweretestedusingananalysisofvariance(ANOVA),orusingaKruskal-Wallis
testincaseswheretheassumptionsofANOVAcouldnotbemetthroughdatatransforma-
tions.Shannon-Weinerdiversityindiceswerecalculatedforallsamples,ateachtaxonomic
level,anddifferencesindiversityamongtreatmentsweretestedusingANOVA.Differencesin
therelativeabundanceofeachbacterialtaxonamongtreatmentswerecalculatedusingaKrus-
kal-Wallisrank-sumtestwithachi-squaredistribution.Analyseswereconductedusingthe
packages‘vegan’[49]and‘ggplot2’[50]inR[47].
Results
Dark-spotsyndromedidnotvisuallytransmittohealthycoralsinanyexperimentinthepres-
entstudy(Figs4and5).Therewasnoapparenttransmissiontothelesionsthatweregenerated
bydirectcontactwithdiseasedfragments,andlesionswerecompletelyhealedbyDay28(Fig
4).AnalysisoftheIllumina1sequencingdatarevealednosignificantdifferencesamongthe
bacterialcommunitiesonhealthy,exposed,anddiseasedcoralsatthecompletionofthewater-
borne-transmissionexperiment(Table1,S1andS2Figs).Furthermore,nosignificantdiffer-
encesinbacterialrichnessweredetectedamongtreatments(Table2,S3Fig),andno
significantdifferencesintherelativeabundanceofsixpurportedpathogenswerefoundamong
treatments(S1Table).However,therelativeabundanceofninebacterialtaxaincreasedsignifi-
cantlyindiseasedcoralscomparedwithhealthyandexposedcorals,pointingtopotentialpath-
ogens,butindicatingalackoftransmission(S4Fig).
Experiment1:Waterbornetransmission
Nowaterbornetransmissionofdark-spotsyndromeonS.sidereawasobservedduringthefirst
11days(Fig5),andallexposedcoralsremainedhealthyintheholdingtanksfollowingthe
experiments.Throughouttheexperiment,thesize,color,andshapeofthedark-spotlesions
remainedunchanged,indicatingthattherewasnoprogressionorregressionofthelesions
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Table1. PERMANOVAresultsofbacterial-communityanalysesondiseasedandhealthycorals.
Dark-spotsyndrome df SS MS Pseudo(F) R2 P(perm)
Genus 2 0.35 0.18 1.73 0.37 0.057
Family 2 0.26 0.13 1.83 0.38 0.082
Order 2 0.25 0.13 1.96 0.39 0.069
Class 2 0.13 0.07 1.47 0.33 0.246
Permutationalmultivariateanalysesofvariance(PERMANOVA)comparingbacterialcommunitiesamonghealthy,exposed,anddiseased-coralcolonies
(~16cm2)testedinlaboratory-baseddarkspot-transmissionexperimentsonSiderastreasiderea.Differencesinbacterialcommunitieswereexaminedat
eachoffourtaxonomiclevels.df=degreesoffreedom;SS=sumofsquares,MS=meansumofsquares;Pseudo(F)=permutedF-value;P(perm)=
permutedp-value.
doi:10.1371/journal.pone.0147493.t001
throughtime(Fig5).Furthermore,therewerenosignificantdifferencesinthephotochemical
yieldamongtreatmentsorthroughtime(F=2.17,df=2,p=0.125fortreatment,andF=1.46,
df=1,p=0.233fortime;Fig6).
Experiment2:Direct-contacttransmission
Nodirecttransmissionofdark-spotsyndromeonS.siderea,wasobservedduringthefirst11
days(Fig4),andallexposedcoralsremainedhealthyintheholdingtanksfollowingtheexperi-
ments(Fig4).AllhealthyS.sidereacoloniesshowedpartialrecoveryfromtheirartificially-
inducedlesionsbyDay11,anddidnotdevelopdarkspots.Followingtherecoveryperiod,all
healthy-coralcolonieshadcompletelyhealedfromtheirartificially-inducedlesions,andno
dark-spotsyndromehaddevelopedafter28days(Fig4).
Examinationofthetransmissionofpotentialbacterialpathogens
Anaverageof98%oftheDNAsequencedfromeachoftheninecoralcolonieswasofProkary-
oticorigin,and4,103operationaltaxonomicunits(OTUs)thatrepresented71differentclasses
weredetectedacrosstheninesamplesofS.siderea(S2andS3Tables).Thedifferencesamong
thebacterialcommunitiesondiseased,exposed,andhealthy-coralcolonies,afterallowingfor
waterbornetransmission,wereexaminedatfourtaxonomiclevels(genustoclass)usingPER-
MANOVA.Nosignificantdifferencesinthebacterialcommunitiesweredetectedatanytaxo-
nomiclevel(Table1,Fig7,S1andS2Figs).Furthermore,nosignificantdifferencesinbacterial
richnessorspeciesdiversityweredetectedatanytaxonomiclevel(Table2,S3Fig),andnosig-
nificantdifferencesintherelativeabundanceofsixpurportedpathogentaxawerefound
amongtreatments(S1Table).ThesamefivebacterialclassesAlphaproteobacteria,Clostridia,
Table2. Meanbacterialrichnessondiseasedandhealthycorals.
Healthy Exposed Diseased ANOVA/Kruskal-WallisResults
Taxa Average LowerCI UpperCI Average LowerCI UpperCI Average LowerCI UpperCI df F-value Chi-sq p-value
Genus 314 262 367 312 247 378 297 236 358 2 0.096 n/a 0.910
Family 145 120 170 144 124 165 139 119 159 2 n/a 1.681 0.432
Order 77 63 91 76 65 86 74 68 79 2 n/a 0.090 0.956
Class 39 31 48 39 33 46 36 32 40 2 0.270 n/a 0.722
Averagebacterialrichnessatfourtaxonomiclevelsinhealthy,exposed,anddiseased-coralcolonies(~16cm2)testedinlaboratory-baseddark-spot
syndrometransmissionexperimentsonSiderastreasiderea.CI=95%confidenceinterval.Resultsofanalysisofvariance(ANOVA)(orthenon-parametric
Kruskal-WallistestwhenANOVAassumptionswerenotmet)comparingbacterialrichnessamongtreatments,ateachtaxonomiclevelarepresented.
doi:10.1371/journal.pone.0147493.t002
PLOSONE|DOI:10.1371/journal.pone.0147493 January20,2016 9/16
TestingTransmissionofCaribbeanDark-SpotSyndrome
Fig6.Photochemicalyieldoncoralstestedinawaterbornedisease-transmissionexperiment.Mean
photochemicalyieldof3-hourdark-adaptedcoloniesofSiderastreasidereatestedinadark-spottransmission
experimentontwodays.Measurementsofdiseased-coralcoloniesweretakendirectlyontissuewithdark
spots.Wheren=7,anderrorbarsdenotestandarddeviation.
doi:10.1371/journal.pone.0147493.g006
Flavobacteriia,GammaproteobacteriaandPlanctomycetiaweremostabundantinhealthyand
diseasedtissuesofS.siderea.Ofthe100mostabundantbacterialgeneraindark-spotS.sidereatis-
sue,whichtogetherconstituted94%oftherelativeabundance,nonewereabsentinhealthyS.side-
reatissue.However,taxon-specificKruskal-Wallisrank-sumtestsidentified14outof934taxa,
withabundancesthatdifferedsignificantlyamongtreatments.Nineofthosetaxaweresignifi-
cantlymoreabundantindiseasedcoralscomparedwithhealthyandexposedcorals,andincluded
Alteromonas(96%homology),Aquabacterium(95%homology),Arthrobacter(84%homology),
Bermanella(95%homology),Haliscomenobacter(87%homology),Litoreibacter(97%homology),
Oscillatoriales(92%homology),Pseudomonas(96%homology),andSorangiineae(93%homol-
ogy).Twotaxa,Azospirillum(90%homology)andPsychroflexus(89%homology)weresignifi-
cantlylessabundantindiseasedcoralscomparedwithhealthyandexposedcorals(S4Fig).
Discussion
Dark-spotsyndromewasfirstdescribedinthe1990s[51,52],yetitremainsunknownif,and
how,thisdiseaseistransmittedthroughcoralpopulations.Thepresentstudytesteddirectand
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