Denitrification potential of the eastern oyster microbiome using a 16S rRNA gene based metabolic

21 Pages·2017·3.05 MB·English

RESEARCHARTICLE
Denitrification potential of the eastern oyster
microbiome using a 16S rRNA gene based
metabolic inference approach
AnnArfken1☯*,BongkeunSong1☯,JeffS.Bowman2,MichaelPiehler3
1 DepartmentofBiologicalSciences,VirginiaInstituteofMarineScience,GloucesterPoint,Virginia,United
StatesofAmerica,2 IntegrativeOceanographyDivision,ScrippsInstitutionofOceanography,Universityof
California,SanDiego,California,UnitedStatesofAmerica,3 InstituteofMarineSciences,UniversityofNorth
CarolinaatChapelHill,MoreheadCity,NorthCarolina,UnitedStatesofAmerica
a1111111111
a1111111111 ☯Theseauthorscontributedequallytothiswork.
a1111111111 *[email protected]
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a1111111111
Abstract
Theeasternoyster(Crassostreavirginica)isafoundationspeciesprovidingsignificanteco-
systemservices.However,therolesofoystermicrobiomeshavenotbeenintegratedinto
OPENACCESS
anyoftheservices,particularlynitrogenremovalthroughdenitrification.Weinvestigatedthe
Citation:ArfkenA,SongB,BowmanJS,PiehlerM
compositionanddenitrificationpotentialofoystermicrobiomeswithanapproachthatcom-
(2017)Denitrificationpotentialoftheeastern
oystermicrobiomeusinga16SrRNAgenebased bined16SrRNAgeneanalysis,metabolicinference,qPCRofthenitrousoxidereductase
metabolicinferenceapproach.PLoSONE12(9): gene(nosZ),andN fluxmeasurements.Microbiomesoftheoysterdigestivegland,theoys-
2
e0185071.https://doi.org/10.1371/journal.
tershell,andsedimentsadjacenttotheoysterreefwereexaminedbasedonnextgenera-
pone.0185071
tionsequencing(NGS)of16SrRNAgeneamplicons.Denitrificationpotentialsofthe
Editor:XiangzhenLi,ChengduInstituteofBiology,
microbiomesweredeterminedbymetabolicinferencesusingacustomizeddenitrification
CHINA
geneandgenomedatabasewiththepaprica(PAthwayPRedictionbyphylogenetICplAce-
Received:May15,2017
ment)bioinformaticspipeline.Denitrificationgenesexaminedincludednitritereductase
Accepted:September6,2017 (nirSandnirK)andnitrousoxidereductase(nosZ),whichwasfurthersubdividedbygeno-
Published:September21,2017 typeintocladeI(nosZI)orcladeII(nosZII).Continuousflowthroughexperimentsmeasuring
N fluxeswereconductedwiththeoysters,shells,andsedimentstocomparedenitrification
Copyright:©2017Arfkenetal.Thisisanopen 2
accessarticledistributedunderthetermsofthe activities.Papricaproperlyclassifiedthecompositionofmicrobiomes,showingsimilarclas-
CreativeCommonsAttributionLicense,which sificationresultsfromSilva,GreengenesandRDPdatabases.Microbiomesoftheoyster
permitsunrestricteduse,distribution,and
digestiveglandsandshellswerequitedifferentfromeachotherandfromthesediments.
reproductioninanymedium,providedtheoriginal
Therelativeabundanceofdenitrifyingbacteriainferredbypapricawashigherinoystersand
authorandsourcearecredited.
shellsthaninsedimentssuggestingthatoystersactashotspotsfordenitrificationinthe
DataAvailabilityStatement:Sequencesgenerated
marineenvironment.Similarly,theinferrednosZIgeneabundanceswerealsohigherinthe
inthisstudymaybedownloadedfromtheNCBI
SequenceReadArchive,accessionnumber oysterandshellmicrobiomesthaninthesedimentmicrobiome.GeneabundancesfornosZI
SRP106715. wereverifiedwithqPCRofnosZIgenes,whichshowedasignificantpositivecorrelation
Funding:FundingwasprovidedbytheNational (F1,7=14.7,p=6.0x10-3,R2=0.68).N2fluxratesweresignificantlyhigherintheoyster
ScienceFoundationDivisionofBiological (364.4±23.5μmolN-N m-2h-1)andoystershell(355.3±6.4μmolN-N m-2h-1)compared
2 2
Oceanography,https://www.nsf.gov/funding/pgm_ tothesediment(270.5±20.1μmolN-N m-2h-1).Thus,bacteriacarryingnosZIgeneswere
summ.jsp?pims_id=11696,grantnumber 2
foundtobeanimportantdenitrifier,facilitatingnitrogenremovalinoysterreefs.Inaddition,
1321373toBS;NationalScienceFoundation
DivisionofBiologicalOceanography,https://www. thisisthefirststudytovalidatetheuseof16Sgenebasedmetabolicinferenceasamethod
nsf.gov/funding/pgm_summ.jsp?pims_id=11696,
PLOSONE|https://doi.org/10.1371/journal.pone.0185071 September21,2017 1/21
Denitrificationpotentialoftheoystermicrobiome
grantnumber1233327toMP;NationalScience fordeterminingmicrobiomefunction,suchasdenitrification,bycomparinginferenceresults
FoundationDivisionofPolarPrograms,https:// withqPCRgenequantificationandratemeasurements.
www.nsf.gov/div/index.jsp?div=PLR,grantnumber
1641019toJSB;aswellascontributionsfromthe
NationalScienceFoundation,theCommonwealth
ofVirginiaEquipmentTrustFundandtheOfficeof
NavalResearch.Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. Introduction
Competinginterests:Theauthorshavedeclared ChesapeakeBay,thelargestestuaryintheUnitedStates,isoneofmanysystemsthathasexpe-
thatnocompetinginterestsexist. riencedthedetrimentaleffectsofexcessnitrogen(N)andculturaleutrophication,including
bottomwaterhypoxia,reducedfisheriesharvests,andlossofsubmergedaquaticvegetation
[1,2].Overthelastseveralyears,restorationoftheeasternoyster(Crassostreavirginica)tothe
BayhasgainedmomentumasapotentialmeanstoenhanceNremovalandmitigateeutrophi-
cationbyincreasingratesofdenitrification[3,4].Denitrificationisthemicrobially-mediated
stepwisereductionofnitrate(NO -)andnitrite(NO -)togaseousnitricoxide(NO),nitrous
3 2
oxide(N O)anddinitrogen(N )[5].
2 2
Themajorityofstudiesaddressingdenitrificationassociatedwithoystershaveprimarily
focusedonwhetheroystersenhancedenitrificationinsedimentswithinandadjacenttooys-
terreefs[6–9].Oystersmaystimulatedenitrificationbysupplyingorganiccarbon(C)andN
intheformofbiodepositstodenitrifyingcommunitiesinsediments[4,10,11].Ammonium
(NH +)remineralizedfromoysterbiodepositsandexcretionscanbenitrifiedtoNO -,which
4 3
supportsdenitrification[3,12].Inaddition,theoysteritselfcanprovideamicrobialhabitat
fordenitrification(oysterdenitrification).Liveoystershavebeenshowntohavesignificantly
higherratesofdenitrificationthansediments[13].Oystergutorgansinparticular,maybe
hotspotsfordenitrification,asgutorgansofseveralinvertebratesincludinginsects,earth-
worms,andmusselshaveshowntoexhibitdenitrificationactivity[14–17].Denitrificationin
theinvertebrategutisthoughttobearesultoftheanoxicconditionsandavailabilityoflabile
organiccarbonprovidedwithinthegutenvironment[15,18].Oystershellswerealsofound
tohavedenitrificationactivityeventhoughtheratesweremuchlowerthanthosemeasured
inliveoysters[19].Shelldenitrificationmaybeinfluencedbyfactorssimilartothoseimpact-
ingsedimentarydenitrification.Likeoysterreefsediments,theshellmicrobiomeisexposed
toincreasedCandNfrombiodepositsandexcretions,whichmayenhancedenitrification.
Boththegutandshellmicrobiomesarelikelyimportantcontributorstooysterdenitrifica-
tion,however,nopreviousstudieshaveidentifieddenitrifyingtaxaorgenesintheoyster
microbiome.
Studiesinvestigatingthecompositionofoystermicrobiomesarealsolimitedcomparedto
thoseregardingsedimentmicrobiomes.Previousexaminationsofoystermicrobiomesby
cloningandsequencingof16SrRNAgenes,DNAfingerprintingandfluorescentinsitu
hybridization(FISH)revealedProteobacteriaandFirmicutesasdominanttaxaindifferentoys-
terspecies,butwererestrictiveinscaleorresolution[20–25].Kingetal.[26]wasoneofthe
firststudiesusinghigh-throughputnext-generationsequencing(NGS)of16SrRNAgene
ampliconstocharacterizetheintestineandstomachmicrobiomeoftheeasternoyster.This
studyshowedadominanceofMollicutesorPlanctomycetesintheoysterstomach,whileintes-
tineswerefoundtobemorespeciesrichandlargelycomposedofthephylaChloroflexi,Proteo-
bacteria,Verrucomicrobia andPlanctomycetes[26].Follow-upmicrobiomestudiesusing16S
NGSincludedfurtherexaminationoftheoystergutmicrobiome,aswellasmicrobiomesof
oystergills,mantleandhemolymph[27–30].Forexample,inLokmeretal.[28]higherabun-
dancesofGammaproteobacteriawerereportedinthegut,gill,mantle,andhemolymphmicro-
biomescomparedtothesurroundingseawater.However,noneofthestudiestodatehave
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Denitrificationpotentialoftheoystermicrobiome
attemptedtoconnecttheoystermicrobiomestructuretoitsfunctionusingNGSof16SrRNA
geneamplicons.
Exploringthelinkagebetweenthestructureandfunctionofmicrobiomespresentsafinan-
cialandlogisticalchallenge.Whole-genomeshotgunmetagenomicsoffertheabilitytoidentify
communitystructureandfunctionalgenesrelatedtometabolicprocessesinanenvironment,
suchasthoseofmicrobiomes.Wide-scale,whole-genomemetagenomicstudieshowever,are
oftenprohibitivelycostlyandmaynotbesufficientforlargesamplesetsorforsampleswhere
prokaryoticgeneticcontributiontothemetagenomeislow[31].Asaresult,manymicrobiome
studiesrelyonmuchlessexpensiveandaccessible16SrRNAgenebasedampliconsequencing,
whichtraditionallyhasofferedlittleinsightintofunctionality.Toaddressthisshortcoming
with16SrRNAgenesequencing,bioinformaticprograms,PhylogeneticInvestigationofCom-
munitiesbyReconstructionofUnobservedStates(PICRUSt)[32],andmorerecentlyPAthway
PRedictionbyphylogenetICplAcement(paprica)[33],havebeendevelopedtoinfermetabolic
pathwaysfrom16SrRNAgenesequences.Severalrecentstudieshaveusedmetabolicinference
programstoinfermicrobialmetabolismsinmarinemicrobiomessuchasthoseofmacrobiota
biofilms[34],sponges[35],andcorals[36].Somekeydifferencesintheprogramsareinthe
assignmentofpathwaysanduserflexibility.PICRUStusesancestralstatereconstructionto
infertheprobablemetabolism(accordingtotheKEGGontology[37])ofextantGreengenes
operationaltaxonomicunits(OTUs)[38].Incomparison,papricadescribescommunitystruc-
turethroughphylogeneticplacementwithpplacer[39]ontoareferencetreecreatedfromall
completedgenomeinGenbank[40].Papricathenusesapre-computeddatabasetoassign
genomicfeatures(includinggenesandmetabolicpathwaysviatheMetaCycontology[41]).
Papricaisdesignedtomaximizeuserflexibilityandhasoptionsforaddingreferencedraft
genomesandcustomizingtheenzymecommission(EC)numbersassociatedwithreference
genomes.
Wecombinedacustomizeddatabaseofgenomesanddenitrificationgeneswiththepaprica
programtolinktheoysterdigestivegland(gut),shell,andreefsedimentmicrobiomestruc-
turestodenitrificationbycharacterizingthecompositionofmicrobiomesandidentifying
potentialdenitrifiersfrom16SrRNAampliconsequences.Ourmainobjectiveswereto(1)
comparetheoystermicrobiomes’taxonomicclassificationsdeterminedbypapricaandother
taxonomicdatabases,(2)examinethestructureanddiversityoftheoystermicrobiomesusing
ataxonomicallyindependentOTUanalysis,and(3)connecttheoystermicrobiometoratesof
denitrificationbycomparingtherelativeabundancesandcompositionofdenitrificationgenes
ineachmicrobiometomeasuredN fluxes.Acustomizedpapricadatabasewasconstructed
2
withdissimilatorynitritereductasegenes(nirSandnirK),andnitrousoxidereductasegene
(nosZ)identifiedfromcompletedordraftgenomes.NirSandnirKencodeenzymesresponsi-
bleforthereductionofnitrite(NO -)tonitricoxide(NO),whilenosZencodesforenzymein
2
thereductionofnitrousoxide(N O)tonitrogengas(N )inthedenitrificationpathway.The
2 2
nosZgeneclassificationwasfurtherdividedintotwoseparateclades;cladesI(nosZI)andII
(nosZII).GenecladesnosZIandnosZIIdifferbasedonvariationsinsignalingpeptides,phy-
logeny[42],andresponsestoenvironmentalconditions[43,44].Continuousflowexperiments
wereperformedwithliveoysters,emptyshells,andreefsedimentstomeasuretheassociated
denitrificationactivity.
Materialsandmethods
Samplecollectionandflow-throughexperiment
Triplicatesamplesofliveoysters,pairsofemptyoystershells,andintertidalsurficialsediment
corestakenwithinoysterreefs[45]werecollectedon7July2013atlowtidefromHoopHole
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Denitrificationpotentialoftheoystermicrobiome
Creek(Latitude34.706483,Longitude76.751931),atidalcreeklocatedinAtlanticBeach,NC,
andimmediatelytransportedtotheUniversityofNorthCarolinaInstituteofMarineSciences
(UNCIMS).OysterssampleswereacquiredaccordingtoconditionsdetailedinUNCIMS’s
researchcollectionpermitfromNCDivisionofMarineFisheries.Temperature,salinity,dis-
solvedoxygen(DO)weremeasuredusingaYSIwaterqualitysonde(YSI,Inc.).Waterwasfil-
teredthroughWhatmanGF/Ffilters(25mmdiameter,0.7lmnominalporesize)andthe
filtratewasanalyzedwithaLachatQuick-Chem8000automatedionanalyzerforNO -.
3
Sedimentcoreswereleftinawaterbathovernightwithcontinuousaerationwithairstones.
Oystersandshellswerestoredovernightinracewayflumesandthenaddedtoindividualcores
andcappedthefollowingmorning.Continuous,flow-throughcoreincubationexperimentsto
measureN fluxeswereconductedunderdarkconditionsinanenvironmentalchamberheld
2
atconstantsitewatertemperaturesusingeachofthecollectedsamples.Thetreatmentscon-
sistedof:(1)liveoyster,(2)oystershellsonly,and(3)sediment.Samplesfromthebypassline
(floweddirectlyfromreservoirto5mlgroundglassvial)andeachcore’soutflowwerecollected
followingtheacclimationperiod.Inflowwaterandoutflowwaterleavingthecoreswereana-
lyzedfordissolvedN ,O andArusingaBalzersPrismaQME200quadruplemassspectrome-
2 2
ter[46].ConcentrationsofO andN weredeterminedusingtheratiowithAr[46,47].
2 2
Followingtheexperiment,oysters,oystershells,and50mLofsedimentfromthecoreswere
frozenandshippedtotheVirginiaInstituteofMarineScience,wheretheywerestoredat
-80˚C.
Wholeoysterswerepartiallythawedatroomtemperatureforapproximately30minutes
beforedissection.Dissectionswerecarriedoutusingsterilescalpelblades.Digestiveglands
werecarefullyexcised,transferredto2.0mLmicrocentrifugetubes,andfrozenat-80˚C.Fol-
lowingdissection,theremainingoystertissuewasremovedfromitsshell,andtheinteriorof
theshellwasscrubbedwith75%ethanol.Oystershellsfromliveoysters(shell(live))and
pairedoystershellscollectedfromthereef(shell(only))werecrushedintoroughly0.5–5.0
mmsizedpiecesusingsterilizedhammerstohomogenizetheexteriorshellbiofilm.Shellfrag-
mentswerethentransferredto50mLfalcontubes,andfrozenat-80˚C.
DNAextractionandamplification
DNAwasextractedfrom0.25–0.30gramsofdigestiveglandusingtheQiagenDNAstoolmini
kit(Qiagen,Hilden,Germany)followingthepathogendetectionprotocol.Shell(0.40–0.60
grams)andsediment(0.50–0.75grams)extractionswereconductedusingMoBIOPowersoil
extractionkits(Mo-BioLaboratories,Inc.,Carlsbad,CA)followingthemanufacture’sproto-
col.Asaresultofvariationinthesourcematerial,differentkitswereusedtoextractDNA
fromtheoysterdigestiveglandandtheoystershellorreefsedimentinordertooptimizeDNA
qualityandDNAyieldforPCRandsequencingefficiency.Whilethismayintroducesome
bias,thesebiasestendtohaveaminimalimpacton16SNGSmicrobiomestudies[48].Overall,
12DNAsampleswereextracted:triplicateDNAsamplesfrom(1)oysterdigestivegland,
(2)shellfromliveoysters,(3)collected(empty)pairedshells,and(4)oystersediment.
InitialamplificationofthetargetedhypervariableV4regionofthe16SrRNAgenewasper-
formedonextractedDNAusingforwardprimer515Fandmodified,barcodedreverseprimer
806R[49],adaptedforusewiththeIonTorrentPersonalGenomeMachine(PGM).Thebasic
manufacturer’sPCRprotocolwasusedwithTaqDNAPolymerase(Invitrogen,Carlsbad,CA)
tocreateaPCRmastermixwiththefollowingmodification:1mMdNTPmixturewasusedin
placeof10mMforafinalconcentrationof0.02mMdNTP.Thermalcyclingconditionscon-
sistedofaninitialdenaturationstepat94˚Cfor3min,followedby30cyclesof94˚Cfor1min,
54˚Cfor1min,68˚Cfor2min.Afinalelongationstepof68˚Cfor10minwasaddedtoensure
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Denitrificationpotentialoftheoystermicrobiome
completeamplification.TheamplifiedproductsweregenecleanedusingtheUltraCleanGel-
SpinDNAPurificationKit(Mo-BioBioLaboratories,Inc.,Carlsbad,CA).Theresulting
ampliconlibrarieswerethenusedastemplatesforsequencingwiththeIonS5platformfollow-
ingthemanufacture’sinstruction(ThermoFisherScientific,Waltham,MA).Sequencesgener-
atedinthisstudymaybedownloadedfromtheNCBISequenceReadArchive,accession
numberSRP106715.
Bioinformaticanalyses
Anoverviewofthebioinformaticpipelineusedforthe16SrRNAbasedmicrobiomeanalyses
isshowninsupplementarymaterials(S1Fig).Removalofbarcodesandprimersfromraw
sequencesandtrimmingofsequencelengthwereconductedusingtheRibosomalDatabase
Project(RDP)pipelineinitialprocess[50](http://rdp.cme.msu.edu)withaminimumquality
scoreof20,minimumlengthof200bases,andamaximumlengthof500.Mothurv1.35.1[51]
wasusedtofurthertrimsequencesagainsttheSILVAv123[52]alignmenttemplate,preclus-
ter,andscreenforchimericsequencesusingtheuchimedenovoprogram[53].Unknowntaxa,
mitochondria,chloroplast,archaea,andeukaryoticsequenceswereremovedfromanalysis
usingSILVAv123referencetaxonomyandtheWangclassificationmethod[54]withan80%
minimumidentity.Archaeawereexcludedfromthisanalysisduetotheirlowabundances;
archaeacomprised<1.0%ofthetotaloverallsequencingreadsandmadeup<3.8%ofthe
readsinanyonesample.Furtheranalysesfocusedonhighqualitybacterialsequencesonly.
PhylotypeanalysesusingMothurwereconductedonhighquality,trimmedbacterial
sequencestodeterminethetaxonomicalcompositionofoysterdigestivegland,oystershell,
andoysterreefsedimentmicrobiomes.SequenceswereclassifiedwithSILVAv123,Green-
genesv13_5,orRDPv14referencetaxonomydatabasesusingtheWangclassificationmethod
describedpreviously.Forallphylotypeanalyses,resultingtaxonomicrelativeabundancesfrom
triplicatemicrobiomesampleswereaveragedtogether,withoystershellsfromliveoysters
(shell(live))andcollectedpairedshells(shell(only))combinedtogethertofromtheoyster
shellmicrobiome.Inaddition,anoperationaltaxonomic(OTU)analysiswasconductedon
themicrobiomesequencestoassessmicrobiomediversity.Sequenceswereclusteredinto
OTUsbasedona97%identityusingtheaverageneighborclusteringalgorithm.Toremove
samplingintensityerrorandnormalizesamples,individualsamplereadswererandomlysub-
sampledtothelowestnumberofreadsfoundinthesampledataset(n=66,687).Alldiversity
metricsarebasedonmicrobiomeaverages.Fordiversitymetrics,bothshell(live)andshell
(only)treatmentsdescribedpreviouslywerecombinedtoformtheshellmicrobiome;forprin-
ciplecoordinateanalysis(PCoA),shell(live)andshell(only)microbiomeswereanalyzedsepa-
ratelytodetermineshellmicrobiomestructuresimilarity.
Toconductphylotypeanddenitrificationgeneinferenceanalysesusingpaprica,acustom-
izedpapricadatabasewasconstructedwith5,445completeand222draftbacterialgenomes
(S1andS2Tables).Highqualitydraftgenomes,whereavailable,wereselectedforinclusionin
thedatabasebasedontheirrelevancetooystermicrobiometaxonomicalstructuresdeter-
minedbytheSilva,Greengenes,andRDPphylotypeanalyses.Alldraftgenomesweredown-
loadedfromGenBank(https://www.ncbi.nlm.nih.gov/genbank/).Eachindividualgenomewas
curatedforthepresenceofnirS,nirK,andnosZgenesusingeithertheKEGGdatabasefor
completedgenomesorgeneannotationsfordraftgenomes.Constructionofthepapricarefer-
encedatabaseandinclusionofthegene-specificinferenceswereconductedfollowingthe
instructionsfoundonthedeveloper’swebsite(http://www.polarmicrobes.org/building-the-
paprica-database/).Phylotypeandgeneinferenceanalyseswereperformedbyfirstaligningthe
qualitycontrolledqueryreadstothereferencealignmentwithInfernal,thenplacingthemon
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thephylogeneticreferencetreewithpplacer[39].Taxonomicalclassificationandgeneinfer-
enceswerebasedonedgeplacementandconsensusidentitywitheitherinternalorterminal
nodesasdescribedinBowmanandDucklow[33].Resultingabundancesfrompapricawere
givenaseithervaluesnormalizedto16SrRNAgenecopynumberorasuncorrectedvalues.
Normalizedvalueswerecalculatedasthemeasuredabundancedividedbythenumberof16S
rRNAgenecopiespredictedforeachtaxon.Uncorrectedvalueswereusedforthephylotype
analysistoperformanequivalentcomparisonwiththeMothurphylotypeanalyses,whilenor-
malizedvalueswereusedwithgeneabundancestobettercapturepotentialdenitrifiers.Dis-
tinctionsbetweennosZIandnosZIIgeneabundancesandtaxonomicclassificationwerebased
onedgetaxonomiesonly.
QuantitativePCR
QuantitativePCR(qPCR)assayswereperformedonoysterandsedimentsamplestodeter-
minetherelativeabundanceofnosZIgenes.RelativeabundancesofnosZIgenesineachsam-
plewerecalculatedusingtheratioofnosZIabundancetotheabundanceof16SrRNAgenes.
GeneabundancesfornosZIand16SrRNAweredeterminedusingthe6FlexReal-TimePCR
system(ThermoFisherScientific,Waltham,MA).16SrRNAgeneqPCRassayswerecarried
outinavolumeof20μLconsistingof10μLof2XSYBRgreenbasedGoTaqqPCRMaster
Mix,0.05μLCXRreferencedye(PromegaCorporation,Madison,WI),0.01mg/mLBSA(Pro-
megaCorporation,Madison,WI),0.5μMeachof16SrRNAspecificprimersEU341Fand
685RtargetinghypervariableregionsV3,and1uLoftemplateDNA.Thermalcyclingcondi-
tionsconsistedofaninitialdenaturingstepat95˚Cfor10min,followedby30cyclesof95˚C
for15s,55˚Cfor30s,and72˚Cfor30swithfluorescencedetection.QuantificationofnosZI
wasperformedusingthesamereactionvolumesandcomponentsdescribedfor16S,with
nosZIspecificprimersnosZ1FandnosZ1R[55].ThermalcyclingconditionsfornosZIqPCR
werethesameas16Swiththeexceptionthattotalcyclenumberwasincreasedto50cycles,
elongationstepat55˚Cwasincreasedto45s,andadditionalstepat80˚Cwithfluorescence
detectionwasadded.Allreactionswereperformedon96-wellplateswithduplicatenegative
controlsandstandards.Standardswerepreparedbyseriallydilutingplasmidscarryingeither
the16SornosZIgeneandquantifiedwiththeAgilent220TapeStationSystem(AgilentTech-
nologies,SantaClara,CA).Standardcurvesandgelelectrophoresiswereusedtoconfirmreac-
tionspecificity.
Statisticalanalyses
Forallresults,variationwithineachmicrobiomeisreportedasthestandarddeviation.Diver-
sitystatisticsincludingcoverage,ChaoI,andShannonwereconductedusingthesummary.
singlecommandinMothur.Aprinciplecoordinateanalysis(PCoA)andtheAdonisfunction
forPermanova(non-parametricpermutationalmultivariateanalysisofvariance;Anderson
[56])usingBray-CurtisdissimilaritywereperformedonOTUdistributionswiththePhyloseq
package[57]inR(version3.1,https://wwww.R-project.org).Fluxdatawasassessedfornor-
malityusingtheqqplotfunctionandShapiro-Wilknormalitytest(p<0.05).One-wayanalysis
ofvariance(ANOVA)andapost-hocTukeyhonestsignificantdifference(HSD)testswere
performedonfluxmeasurementstotestforsignificantdifferences.Aone-tailed,pairedt-test
wasusedtodeterminedifferencesbetweennosZIandnosZIIwithintheshellandsediment
microbiomes,andaone-tailed,Welch’st-testwasusedtocomparegeneabundancesbetween
theshellandsedimentmicrobiomes.Forcomparisonsbetweentheoyster,shell,andsediment
microbiomes,relativeabundancesofthedigestiveglandmicrobiomeandshell(live)micro-
biomewerecombinedtoformtheoystermicrobiome.Asimplelinearregressionanalysiswas
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Denitrificationpotentialoftheoystermicrobiome
conductedtocomparetherelativeabundancesofnosZImeasuredbyqPCRandtheuncor-
rectednosZIgeneabundancespredictedbypaprica;uncorrectedpapricavalueswereusedso
thatequivalentcomparisonsbetweengeneabundancesandqPCRrelativeabundancescould
bemade.UnlessotherwisestatedallstatisticswereconductedinRandsignificancewasbased
onp<0.05.
Results
Phylotypecomparisonofmicrobiomes
Atotalof982,504trimmed,highquality16SrRNAgenesequenceswereobtainedfrom
theoysterandsedimentmicrobiomesamples.Sequencingdepthaveragesforeachmicro-
biomewere85,640±1.5x104foroysterdigestivegland,86,745±1.6x104forshell,and
68,378±1.5x103forsediment.Amongthe4databases,papricaclassifiedthegreatestnumber
ofsequencesatthefamilylevel(85.4±9.8%),followedbySilva(76.5±18.9%),Greengenes
(75.8±18.5%),andRDP(57.2±18.7%).Allfourdatabasesshowedanoverallsimilarpattern
atthefamilyclassificationlevelfortheaveragerelativeabundanceofsequences(cid:21)1%(Fig1).
Withtheexceptionofoneshellintheshell(only)treatmenthavingaslightlydifferentprofile
Fig1.Averagerelativeabundancesofbacterialfamiliesintheoyster-relatedmicrobiomes,classifiedbydifferentreference
databases.Familieswith(cid:21)1%relativeabundanceinsamplesareshown.Shellmicrobiomeconsistsofshell(live)andshell(only)
treatments.
https://doi.org/10.1371/journal.pone.0185071.g001
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Denitrificationpotentialoftheoystermicrobiome
(S2Fig),phylotypecomparisonsbetweentheshell(live)andshell(only)microbiomeswere
similarintaxonomyandrelativeabundance,andwerethuscombinedtogethertoformthe
shellmicrobiome.Oftheoyster-relatedmicrobiomes,thesedimentmicrobiomeshowedthe
greatestnumberoffamilies(n=12.5±1.7)andthelowestpercentofsequencesidentified
(47.7±6.7%),theoysterdigestiveglandmicrobiomeshowedthelowestnumberoffamilies
(n=1.3±0.5)andthehighestnumberofsequencesidentified(73.1±24.5%),andtheoyster
shellmicrobiomefellsomewhereinthemiddle(n=8.8±2.5;59.7±7.7%)(Fig1).Eachofthe
fourdatabasesconsistentlyidentifiedfamilyMycoplasmataceae fromphylumTenericutes as
thedominantfamilyinthedigestiveglandmicrobiome.Papricawastheonlymethodtoalso
includetheclassificationofOdoribacteraceaeasanotherdominantfamilymemberinthe
digestiveglandmicrobiome.Withintheoystershellmicrobiome,allfourdatabasesshoweda
dominanceoffamiliesSphingomonadaceae, Erythrobacteraceae,andRhodobacteraceaefrom
phylumProteobacteria,andFlammeovirgaceae,Flavobacteriaceae,andSaprospiraceaefrom
phylumBacteroidetes.DesulfobacteraceaeandRhodobacteraceaefromphylumProteobacteria,
andFlavobacteriaceaeandSaprospiraceaefromphylumBacteroidetes,werethedominantfami-
liesconsistentlyidentifiedinthesedimentmicrobiomesacrossallfourdatabases.Thegreatest
variationamongthedatabasesintheclassificationoffamiliesoccurredinpaprica’sidentifica-
tionofsequencesfromphylumBacteroidetesandGreengenes’sidentificationofsequences
fromphylumProteobacteria.However,atthephylumlevel,identificationofsequencesfor
eachphylumwasrelativelyconsistentamongthefourdatabases.
DiversitycomparisonofmicrobiomesusingOTUanalysis
All12microbiomesamplesweresubsampledto66,687sequencestoconductanOTUdiversity
analysis(Table1andFig2).Averagecoverageofsequencesrangedfrom89.1±0.9%inthesedi-
mentmicrobiometo99.6±0.0%intheoysterdigestivegland.Significantdifferencesamongthe
microbiomesweredetectedwithPermanova(F =8.19,p=0.001)anddemonstratedusing
2,11
PCoA(Fig2),whichexplained65.8%ofthevariationfound.Theoysterdigestivegland,shell,
andsedimentsamples,formeddistinctmicrobiomes,clusteringseparatelybasedonsample
type.Thegreatestdissimilarityoccurredbetweentheoysterdigestiveglandandthesediment
microbiome.Therewerenodifferencesbetweentheshellmicrobiomes,whethertheshellcame
Table1. Summarystatisticsof16SrRNAgeneampliconsequencingforoyster-relatedmicrobiomes.
Sample No.ofOTUsa Coverage(%) ChaoIndex ShannonDiversity
DigestiveGland1 477 1.00 1038.17 1.06
DigestiveGland2 545 1.00 1292.67 1.10
DigestiveGland2 552 1.00 1372.33 1.33
Shell(Live)1 6,508 0.93 18777.19 5.46
Shell(Live)2 7,491 0.93 22004.59 6.31
Shell(Live)3 6,387 0.94 19435.59 5.90
Shell(Only)1 7,027 0.93 18966.56 6.24
Shell(Only)2 5,616 0.94 16288.26 5.37
Shell(Only)3 4,555 0.96 12681.37 5.19
Sediment1 10,946 0.89 33237.59 6.88
Sediment2 9,417 0.90 29882.63 6.57
Sediment3 11,106 0.89 32986.13 7.00
Allmetricsarebasedonsubsamplesofn=66,687.
aOTUsarebasedon97%sequenceidentityusingMothur’saverageneighborclusteringalgorithm
https://doi.org/10.1371/journal.pone.0185071.t001
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Denitrificationpotentialoftheoystermicrobiome
Fig2.Principalcoordinateanalysis(PCoA)ofoyster-relatedmicrobiomes.PCoAbasedon16SrRNAgenesequencesusingBray-
Curtissimilaritymatrix.
https://doi.org/10.1371/journal.pone.0185071.g002
fromaliveoysteroradiscarded,emptyshell.Similartrendswerefoundamongthemicro-
biomesregardingChaoIrichness,Shannondiversity,andOTUabundances(Table1).Sediment
microbiomeshadthehighestlevelofdiversityandrichnessthanallothermicrobiomes(Chao
1=32,035±1.8x103,Shannon=6.8±0.2),andanaverageOTUabundanceof10,489±9.3x102.
Shellmicrobiomehadmoderatediversityandrichness(Chao1=18,025±3.2x103,Shan-
non=5.7±0.5)withanaverageOTUabundanceof6,264±1.5x103,andtheoysterdigestive
glandshadthelowestlevelsofdiversityandrichness(Chao1=1,234±1.7x102,Shan-
non=1.2±0.1)withanaverageOTUabundanceof525±4.1x101.
Microbiomedenitrificationgeneinferenceswiththepapricadatabase
Thesedimentandshellmicrobiomeshadaninferredaveragerelativeabundanceof
23.8±2.8%and26.1±3.0%,respectively,ofdenitrificationgenes(Fig3).Thedigestivegland
microbiomewascomprisedofa(cid:20)0.1%relativeabundanceofdenitrificationgenes.Thegreat-
estdifferencesamongthemicrobiomeswerefoundintherelativeabundancesofthenirK,
nirS,ornosZgenesonly.Combined,organismscarryingoneofthesegenesweremore
PLOSONE|https://doi.org/10.1371/journal.pone.0185071 September21,2017 9/21
Denitrificationpotentialoftheoystermicrobiome
Fig3.Predictedaveragerelativeabundancesofdenitrificationgenesbypapricaforoyster-relatedmicrobiomes.
Shellmicrobiomeincludesshell(live)andshell(only)treatments.Eachfullcirclerepresentsarelativeabundanceof
26.1%.
https://doi.org/10.1371/journal.pone.0185071.g003
dominantthanorganismscarryingbothnirSandnosZornirKandnosZgenes.Betweenthe
shellandsedimentmicrobiomes,theshellmicrobiomehadasignificantlyhigherrelative
abundanceofbacteriacarryingthenirKonlygene(unpairedt-testt =6.48,p=2.6x10-5),
5
whilethesedimenthadasignificantlyhigherabundanceofthenirSonly(unpairedt-testt =
7
8.75,p=2.6x10-5)andahigher,butnotsignificant,abundanceofnosZonly(unpairedt-test
t =2.74,p>0.05)genes.Amongthemicrobiomes,theaveragerelativeabundanceoforgan-
7
ismscarryingnosZIIgenewasoverallhigherthanthosecarryingthenosZIgene(Fig4).Inthe
sedimentmicrobiome,thisdifferencewassignificant(pairedt-testt=7.14,p=9.5x10-3),but
itwasnotsignificantintheshellordigestiveglandmicrobiomes.Taxonomically,nosZIbacte-
riawereprimarilyfromclassAlphaproteobacteria,whilenosZIIbacteriawerefromclasses
CytophygiaandFlavobacteriiaintheshell,andGammaproteobacteria,Cytophygia,andFlavo-
bacteriiainthesediments(S3Fig).
N fluxexperiments
2
Sitewaterphysicalandchemicalparametersusedinthefluxexperimentswereasfollows:
30˚Ctemperature,30pptsalinity,6.8mg/Ldissolvedoxygen(DO),and0.51μmolN/LNO -.
3
LiveoystercoreshadthehighestaveragefluxofN at364.4±23.5μmolN-N m-2h-1,followed
2 2
bytheshellonlycoresat355.3±6.4μmolN-N m-2h-1,andsedimentcoreswiththelowestat
2
270.5±20.1μmolN-N m-2h-1(Fig5).TherewerenosignificantdifferencesintheN fluxes
2 2
betweentheliveoysterandshell,butbothweresignificantlyhigherthanthesedimentcores
(ANOVA,F =23.7,p=1.4x10-3;TukeyHSD,p<0.05).
2,6
MicrobiomenosZIgeneinferencecomparisontofluxmeasurementsand
qPCR
TheratesofN fluxesfollowedasimilartrendtotheaveragerelativeabundanceofnosZI
2
genesinferredinoyster,shell,andsedimentmicrobiomes(Figs5and6B).Oystersandshells
hadsimilarlyhighN fluxratesandnosZIgenes,whilesedimentsampleshadlowerratesofN
2 2
fluxandlowerabundancesofnosZIgenes.Thistrendwasnotfoundintheaveragerelative
abundanceofthenosZIIgenesorinoverallnosZgeneabundance(Figs5,6Aand6C).Asignif-
icant,positivelinearcorrelationwasdeterminedbetweenthecopynumberofnosZIgenes
quantifiedintheshellandsedimentmicrobiomesbyqPCRandtherelativeabundanceof
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