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Bacterial-invertebrate symbioses [Elektronische Ressource] : from an asphalt cold seep to shallow water / vorgelegt von Luciana Raggi Hoyos

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Bacterial-invertebratesymbioses: from an asphaltcold seep to shallow watersDissertationzur Erlangung des Grades einesDoktors der Naturwissenschaften- Dr. rer. nat. -dem Fachbereich Biologie/Chemie derUniversit¨at Bremenvorgelegt vonLuciana Raggi HoyosBremenSeptember 2010Die vorliegende Arbeit wurde in der Zeit von April 2007 bis August 2010 inder Symbiose Gruppe am Max-Planck Institut fur¨ marine Mikrobiologie inBremen angefertigt.1. Gutachterin: Dr. Nicole Dubilier2. Gutachter: Prof. Dr. Ulrich FischerTag des Promotionskolloquiums: 18. Oktober 2010To Pabloto my family‘La simbiosis, la uni´on de distintos organismos para formar nuevoscolectivos, ha resultado ser la m´as importante fuerza de cambio sobre laTierra’L. Margulis & D. Sagan, 1995‘La uni´on hace la fuerza’- Frase popularAbstractSymbiotic associations are complex partnerships that can lead to new metaboliccapabilities and the establishment of novel organisms. The diversity of these as-sociations is very broad and there are still many mysteries about the origin andthe exact relationship between the organisms that are involved in a symbiosis(host and symbiont). Some of these associations are essential to the hosts, suchas the chemosynthetic symbioses occurring in invertebrates of the deep-sea. Inothers the host probably would rather not be the host, as in the case of parasiticmicrobes.

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Publié par
Publié le 01 janvier 2010
Nombre de lectures 74
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Abstract

Symbioticassociationsarecomplexpartnershipsthatcanleadtonewmetabolic
capabilitiesandtheestablishmentofnovelorganisms.Thediversityoftheseas-
sociationsisverybroadandtherearestillmanymysteriesabouttheoriginand
theexactrelationshipbetweentheorganismsthatareinvolvedinasymbiosis
(hostandsymbiont).Someoftheseassociationsareessentialtothehosts,such
asthechemosyntheticsymbiosesoccurringininvertebratesofthedeep-sea.In
othersthehostprobablywouldrathernotbethehost,asinthecaseofparasitic
microbes.MyPhDresearchfocusesonsymbioticandparasiticassociationsin
chemosyntheticandnon-chemosyntheticinvertebrates.Thisthesisdescribesand
discussesthreedifferentaspectsofassociationsbetweenbacteriaandmarinein-
vertebrates.Thefirstaspectfocusesonchemosyntheticassociationsfromaunique
asphaltseepcalledChapopoteintheGulfofMexico(GoM).Phylogeneticanalyses
ofhostgenes(cytochrome-c-oxidasesubunitI)andbacterialgenes(16SrRNA)in
twoBathymodiolusmusselspeciesandanEscarpiatubewormshowedthatboth
thehostsandtheirchemosyntheticsymbiontsareverysimilartotheircongeners
fromthenorthernGoM.Unexpectedly,anovelsymbiontmostcloselyrelatedto
hydrocarbondegradingbacteriaofthegenusCycloclasticuswasdiscoveredinB.
heckerae.StablecarbonisotopevaluesinB.heckeraetissuesoflipidstypicalfor
Cycloclasticusspp.wereconsistentlyheavierby2.5thanotherlipidsindicating
thatthenovelsymbiontmightuseisotopicallyheavyhydrocarbonsfromtheas-
phaltseepasanenergyandcarbonsource.Thediscoveryofanovelsymbiontthat
maybeabletometabolizehydrocarbonsisparticularlyintriguingbecauseuntil
nowonlymethaneandreducedsulfurcompoundshavebeenidentifiedasenergy
sourcesinchemosyntheticsymbioses.Thelargeamountsofhydrocarbonsavailable
atChapopotewouldprovidethesemusselsymbioseswitharichsourceofnutri-
tion.Thesecondaspectofthisthesisdealswithbacteriathatinfectthenucleiof

vi

marineinvertebratesandwererecentlyfoundtobewidespreadindeep-seaBathy-
modiolusmussels.Becauseoftheirpotentiallylethaleffectonbivalvepopulations,
Ilookedforthepresenceofintranuclearbacteriaineconomicallyimportantand
commerciallyavailablebivalvespecies,i.e.oysters(Crassostreagigas),razorclams
(SiliquapatulaandEnsisdirectus),bluemussels(Mytilusedulis),Manilaclams
(Venerupisphilippinarum),andcommoncockles(Cerastodermaedule).Fluores-
cenceinsituhybridization(FISH)revealedthepresenceofintranuclearbacteriain
allinvestigatedbivalvesexceptoystersandbluemussels.Preliminarytestswith
real-timePCRshowedmassiveamountsofintranuclearbacteriainsomeofthe
bivalvespecies,raisingthequestionifthesemightaffectnotonlythehealthof
thebivalvesbutpossiblyalsoofthehumansthateatthem.Inthethirdand
finalaspectofmythesis,Iexaminedthegeneraldiversityofbacteriainthegill
tissuesofdeep-seaandshallow-watermusselsandclams.Comparative16SrRNA
sequenceanalysisandcultivationexperimentsrevealedamuchhigherdiversity
thanpreviouslyrecognized.Thisthesisshowsthatbivalvesareidealmodelsfor
studyingthemicrobiotaofmarineinvertebratesbecauseofthehighdiversityof
bothhighlyspecificandmoregeneralizedsymbioticandparasiticbacteriaintheir
tissues.gill

Zusammenfassung

SymbiotischeAssoziationensindkomplexePartnerschaften,diezuneuenmetabo-
lischenF¨ahigkeitenundderEtablierungneuartigerOrganismenf¨uhrenk¨onnen.
DieVielfaltdieserAssoziationenistsehrhoch,undinvielenF¨allenbleibenihr
UrsprungunddiegenaueBeziehungzwischendenindieSymbioseeingebunde-
nenOrganismen(WirtundSymbiont)ungekl¨art.EinigedieserVerbindungensind
unverzichtbarf¨urdenWirt,wieetwadiechemosynthetischeSymbionten,diebei
InvertebrateninderTiefseevorkommen.Ineinigenanderenw¨arederWirtwohl
liebernichtderWirt,wieimFallvonparasitischenMikroorganismen.DieFor-
schungmeinerDissertationkonzentriertsichaufsymbiotischeundparasitische
Assoziationeninchemosynthetischenundnicht-chemosynthetischenWirbellosen.
DievorliegendeArbeitbeschreibtunddiskutiertdreiverschiedeneAspek-
tederAssoziationenzwischenBakterienundmarinenInvertebraten.Dererste
AspektkonzentriertsichaufchemosynthetischeAssoziationenaneinemeinzigarti-
genAsphaltvulkan,demChapopoteimGolfvonMexico(GoM).Phylogenetische
AnalysenvonWirtsgenen(Cytochrom-c-OxidaseUntereinheitI)undbakteriel-
lenGenen(16SrRNA)inzweiBathymodiolus-MuschelartenundeinemEscarpia-
R¨ohrenwurmhabengezeigt,dasssowohldieWirtealsauchihrechemosynthe-
tischenSymbiontenihrenArtverwandtenausdemn¨ordlichenGoMsehr¨ahnlich
sind.UnerwarteterweisewurdeinB.heckeraeeinneuerSymbiontentdeckt,der
amn¨achstenmitdenKohlenwasserstoffeabbauendenBakteriendesGenusCy-
cloclasticusverwandtist.DiestabilenKohlenstoffisotopederf¨urCycloclasticus
typischenLipideindenGewebenvonB.heckeraewarendurchg¨angigum2.5
schwereralsbeianderenLipiden.Diesdeutetdaraufhin,dassderneuartigeSym-
biontisotopenschwereKohlenwasserstoffeausdemAsphaltvulkanalsEnergie-und
Kohlenstoffquellenutzenk¨onnte.DieEntdeckungeinesneuartigenSymbionten,
derinderLageseink¨onnte,Kohlenwasserstoffezumetabolisieren,istbesonders

viii

faszinierend,dabishernurMethanundreduzierteSchwefelverbindungenalsEner-
giequelleinchemosynthetischenSymbiosenidentifiziertwordensind.Diegroßen
MengenvonKohlenwasserstoffen,diebeiChapopoteverf¨ugbarsind,w¨urdendieser
MuschelsymbioseeinereichhaltigeN¨ahrstoffquellezurVerf¨ugungstellen.
DerzweiteAspektdieserArbeitbesch¨aftigtsichmitBakterien,diedieZellker-
nevonmarinenInvertebrateninfizierenundvorKurzemweitverbreitetinBathy-
modiolus-MuschelnderTiefseegefundenwurden.Wegenihrerpotentiellt¨odlichen
AuswirkungenaufBivalven-PopulationenhabeichbesondersnachderPr¨asenzvon
intranuklearenBakterienin¨okonomischbedeutsamenundkommerziellerh¨altlichen
Muschelspeziesgesucht,d.h.inAustern(Crassostreagigas),Schwertmuscheln(Si-
liquapatulaundEnsisdirectus),Miesmuscheln(Mytilusedulis),Venusmuscheln
(Venerupisphilippinarum)undHerzmuscheln(Cerastodermaedule).DieFluores-
zenz-in-situ-Hybridisierung(FISH)brachteintranukleareBakterieninallenunter-
suchtenMuschelnzumVorschein,außerinAusternundMiesmuscheln.Vorl¨aufige
TestsmitHilfederReal-timePCRzeigtenhoheMengenvonintranuklearenBak-
terienineinigenderBivalvenspezies,wasdieFrageaufwirft,obdiesenichtnurdie
GesundheitderMuscheln,sondernm¨oglicherweiseauchdiedersieverzehrenden
Menschenbeeintr¨achtigenk¨onnten.
ImdrittenundletztenAspektmeinerDoktorarbeithabeichdieallgemeine
Diversit¨atvonBakterienindenKiemengewebenvonTiefsee-undFlachwassermu-
schelnuntersucht.Vergleichende16SrRNA-SequenzanalyseundKultivierungsex-
perimentehabeneinedeutlichh¨ohereDiversit¨atenth¨ullt,alsvorherbekanntwar.
DieseDissertationzeigt,dassBivalviaaufgrundderhohenDiversit¨atvonsowohl
hochspezifischenalsauchgeneralisiertensymbiotischenundparasitischenBakteri-
eninihrenKiemengewebenidealeModellorganismensind,umdieMikrobiotavon
marinenInvertebratenzustudieren.

sttenCon

Aboutthestructureofthisthesis...................

nIIductiontro

1Invertebrate-bacteriaassociations
1.1Thedifferentmodels.......................
...........................nsectsI1.1.11.1.2Squid...........................
1.1.3Gutlessoligochaetes...................
1.1.4Vesicomyidclams.....................
1.2Summary:Theroleofsymbioses................
Concept-Box1:Symbiosisandsymbiology.............

abitatsH22.1Deep-seacoldseeps........................
2.1.1GulfofMexico......................
2.1.2Chapopote........................
2.2Shallow-watercoastalzone....................

ostsH33.1Deep-seaBathymodiolusmussels.................
3.2Deep-seaEscarpiatubeworms..................
3.3Shallow-waterbivalves......................
Concept-Box2:Immunologyofbivalves...............

x

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345901013141

1551618181

2002227282

TABLEOFCONTENTS

4BacterialSymbionts
4.1Chemosyntheticsymbionts....................
4.1.1Thiotrophicsymbionts..................
4.1.2Methanotrophicsymbionts................
4.2Hydrocarbondegraders......................
4.3Intranuclearparasites.......................
5Methodsofstudy
5.1Cultivation............................
5.2Molecularmarkers:16SrRNA,aprA,pmoA..........

Aims

299213314363399393

41

IIResultsandDiscussion44
6Studiesfromanasphaltcoldseep45
6.1PhylogenyoftubewormsandmusselsfromChapopote.....46
6.2PhylogenyofchemosyntheticBathymodiolusandEscarpiasym-
bionts...............................46
6.3NovelsymbiontsinBathymodiolusmussels...........50
6.4Host-bacteriaspecificity.....................53
6.5Metabolismofthesymbioses...................54
65.............................Summary6.67Bacteriaassociatedwithbivalves58
7.1Intranuclearbacteria.......................58
7.2Diversityofbacteriaassociatedwithbivalves..........60
7.3Bacterialcultivation.......................66
7.4SummaryandOutlook......................68

IIIManuscripts72
Resultingmanuscriptsfromthisthesisworkandcontributions:74

xi

TABLEOFCONTENTS

ManuscriptI:BacterialsymbiontsofBathymodiolusmusselsand
EscarpiatubewormsfromChapopote,anasphaltseepinthe
southernGulfofMexico.....................76
ManuscriptII:Anintranuclearbacterialparasiteinshallowwater
bivalves..............................117
ManuscriptIII:Minireview:Bacterialdiversityofshallow-water
bivalves..............................134
ManuscriptIV:Widespreadoccurrenceofanintranuclearparasite
inbathymodiolinmussels.....................148

remarksConcludingIV

8GeneralSummary,ConclusionsandOutlook
8.1SymbiontdiversityinChapopote................
8.2TheSandPconcept.......................
8.3Conclusions............................

yBibliograph

Glossary

Actswledgemenkno

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168

169961071271

173

195

197

FiguresofList

1.1Aphid-Buchnerasymbiosis....................
1.2Squid-Vibriosymbiosis......................
1.3Gutlessoligochaetesymbiosis..................
1.4Calyptogena-thiotrophssymbiosis................

2.1GulfofMexico-Chapopote...................
2.2Diapirism-saltdomes......................
2.3Bivalvesintheirhabitattiers..................

3.1PhylogenyofBathymodiolusmussels..............
3.2Phylogenyofvestimentiferantubeworms............

4.1Symbiosisinbathymodiolinmussels...............
4.2Sulfuroxidation..........................
4.3Methaneoxidation........................
4.4Thiotrophicandmethanotrophicphylogeny...........
4.5IntranuclearbacteriainBathymodiolusspp...........

6.1Escarpiaandsymbiontsphylogeny...............
6.2Bathymodiolusandsymbiontsphylogeny............
6.3Escarpiatubewormsandbathymodiolinmusselssymbioses..
6.4Isotopicvaluesofthemusselsandtubeworms.........
6.5Metabolicmarkergenes.....................

7.1Bacteria16SrRNAtree.....................
7.2Gammaproteobacterialdiversity.................

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0332335373

7484514575

9526

FIGURESOFLIST

xiv

7.3

4.7

7.5

6.7

Alpha-andEpsilonproteobacterialdiversity

Bacteroidetesdiversity......................

..........

SpirochaeteandFusobacterialdiversity.............

NIX-clade

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64

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96

List

3.1

3.2

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acterialB

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tubewormsandtheirsymbionts............

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52

56

Preface

Aboutthestructureofthisthesis

Thisthesisiscomposedoffourgeneralparts.PartIistheIntroduction,
wherealltheconceptsonwhichthisthesisisbasedaresummarized.Within
thispartChapter1describesthemainmodelsofsymbiosisinageneral
context.Chapters2-5describethehabitats,hosts,symbiontsandmethods
relevanttothisthesis.TheaimsofthisthesisareexplainedinChapter6.
PartIIisthesummaryanddiscussionoftheresultsobtainedduringthePhD
period.Threemanuscriptsareanticipatedasaresultofthisthesisworkand
theyareincludedinPartIII.PartIVistheconclusionofthethesis.Herein
Isummarizeandbringuptheoutlookofmyareaofinvestigation.Themain
objectiveofthisthesisisaccomplishedinthemomentyoureaderhavefun
learningaboutsymbiosisthroughthesepages.

xvi

Abbreviations

APR-Dissimilatoryadenosine-5’-phosphosulfatereductase
aprA-Genecodingforthealpha-subunitoftheAPR
denosine-5’-phosphosulfateA-APSBLAST-BasicLocalAlignmentSearchTool
CARD-Catalyzedreporterdeposition
Methane-CH4CO2-Carbondioxide
COI-MitochondrialcytochromecoxidasesubunitI
CTAB-Hexadecyl-trimethyl-ammoniumbromide
DAPI-4’6-diamidino-2-phenylindole
DNA-Deoxyribonucleicacid
EDTA-Ethylendiamintetraaceticacid
ormamideF-FAFISH-Fluorescenceinsituhybridization
GoM-GulfofMexico
H2S-Hydrogensulfide
RNAMessanger-mRNAMTPH-Methyl-toluene-phenolhydroxylase
NIX-NuclearinclusionX
PCR-Polymerasechainreaction
pmoA-GenecodingforthepMMOactivesubunit
pMMO-Particulatemethanemonooxygenase
RNA-Ribonucleicacid
ROV-RemotelyOperatedVehicle
rRNA-RibosomalRNA

xviii

SOTa24

4

q−-

-

Sulfate

Thermus

aquaticus

TIONSABBREVIA

xix

Patr

troIn

I

duction

2

1Chapter

Invertebrate-bacteriaassociations

Symbioticbacteriaarewidespreadwithinalmostallinvertebrateanimals.
Insectsarethemoststudiedgroupandtheyoverwhelmthepoolofdescribed
invertebratespecies,1millionspeciesareformallydescribedbutitisesti-
matedthatthereareabout3to30millionspecies(Gaston1994).Farless
isknownaboutthebiodiversityofmarinespeciesthanterrestrialonesbut
itisestimatedthatthereare1-10millionspeciesofonlydeep-seainverte-
brates(May1992),andthatmarineinvertebrateshavethegreatestphylo-
geneticdiversityamonganimals(BruscaandBrusca1990,McFall-Ngaiand
Ruby2000).Thus,itislikelythatthegreatestvarietyofanimal-bacterial
symbiosesoccurswithinthisgroup.Marinebacteria-invertebrateassoci-
ationshavebeengreatlystudiedinmarineannelidslikeRiftiapachyptila
(Cavanaughetal.1981,DiMeoetal.2000,BrightandSorgo2003,Bright
andBulgheresi2010),Olaviusalgarvensis(Dubilieretal.2001,Ruehland
etal.2008)orEscarpiaandLamellibrachiavestimentiferans(reviewedby
McMullinetal.2003,BrightandBulgheresi2010),insponges(Vaceletand
Donadey1977,Friedrichetal.1999,RadjasaandSabdono2009),andamong
themollusks,thesquidEuprymnascolopes(McFall-NgaiandKimbell2001,
McFall-Ngaietal.2010),clams(Southward2009,Fisher1990,Newtonetal.
2007),mytilids(Distel1994,Nelsonetal.1995,VanDoverandTrask2000,
Duperronetal.2009)andothermusselssuchasLyroduspedicellatus(Distel
etal.2002).Threetypesofmetabolicinteractionshavebeenrecognizedin
symbiosesingeneral,andalsoinbacteria-marineinvertebratessymbiosesin
particular:‘phototrophic’-wherebacterialikecyanobacterialiveassociated

3

ODUCTIONINTR

tosponges,ascidians,orechiuroidwormsandgainenergyfromlight(Usher
2008);‘heterotrophic’-wherebacteriauseorganiccompoundsascarbon
source.Examplesaresponges(e.g.Friedrichetal.1999)andOsedaxspp.
symbioses(e.g.Rouseetal.2004);and‘chemosynthetic’-wherebacteria
convertoneormorecarbonmolecules(usuallycarbondioxideormethane)
andnutrientsintoorganicmatterusingmethane(methanotrophs)orinor-
ganiccompoundssuchashydrogensulphide(thiotrophs)aselectrondonors
(forareviewonchemosynthesisseeDubilieretal.2008).Chemoautotrophic
bacteria(asthiotrophs)woulduseCO2ascarbonsource.Ifwetrackback
andobservethesymbioticassociationsinthewholeinvertebrategroupwe
findthattheinsectsymbiosisresearchistheoldestwithinthesymbiology
studies(HertigandWolbach1924,Buchner1965).Thisisthecuttingedge
areaandIthinkweshouldlearnaboutitanddiscussgeneralresultscom-
paredwithinsectmodels.Then,wewillbeabletostandardizenamesand
conceptsandexpandthesymbiologystudieswithabetterfoundation.

1.1Thedifferentmodels

Thissectionsummarizessomeofthemostimportantmodelsofinvertebrate
symbiosis.Theyarethemoststudiedmodelsandthemostadvancedinthe
senseofinformationandunderstanding;thereforetheyarethemostcom-
plete.Ihavechosenexamplestoincludeoneofeachcaseofsymbiosis:het-
erotrophic,chemoautotrophic,mixed,intracellular,extracellular,obligatory,
andfacultative(seeGlossaryforexplanationofconcepts).Thefocusofthis
thesisisonmarinesymbioses,howeverIstartbyintroducingaterrestrial
modelbecauseofitsgreatimportancetosymbiologystudies:theinsect-
bacteriasymbioses.IdonotchooseonlyoneinsectmodelbecauseIwould
liketoshowthesymbiosisdiversityininsectstudies.Thesediverseassocia-
tionsarenotstrictandareevendynamic,makingtheirstudymoredifficult
andchallenging,howeverconceptsarebroadandwellaccepted.Forexample
the‘S’concept,aboutfacultativesymbionts,(moredetailsinSection1.1.1)
isaverydynamicconceptthatleavesthedooropentoincludemanydiffer-
entassociationsandgivesimportancetothenon-obligatoryassociations.We

4

ODUCTIONINTR

findininsect-bacteriaassociationsallthemaindifferentsymbiosesdescribed
sofar:intracellularorextracellular,obligateorfacultative,mutualistic,com-
mensalisticorparasitic.Someofthesebacteriahavebeencultivated,which
permitsabetterunderstandingaboutthetransmissionprocess,theecological
importance,andthephysiologicalintricaciesofthedifferentsymbioses.We
knownowthatbacterialsymbiontsinfluencemanyphysiologicalfunctions
ofinsects.Inconclusion,insectstudiesteachusmanybiochemicalpath-
waysusedbytheinsect-bacteriaassociation,theexperimentaldesignused
fortheirstudy,andtheecologicalimportancethattheymighthave.Perhaps
wewouldfindallthesefunctionsinbacterialsymbiontsfrommarineorgan-
ismsbutthestudiesarefartoofewincomparison.Comparisonsofmarine
andterrestrialsymbiosesshouldimproveourunderstandingofboth.Innext
section(Section1.1.2)Igodirectlytothemarinesystemsandintroducethe
heterotrophicsymbiosisofsquid-Vibriobacteria.Asthisbacteriumhasalso
beencultivated,thestudyatthemolecularandphysiologicallevelisremark-
able,beingperhapsthemostunderstoodmarinesymbiosesatthemolecular
level.InSection1.1.3Idoasynthesisofthegutlesswormsymbiosis,as
thismightbeoneofthemoststudiedmodelswherethereisthepresenceof
bothchemoautotrophicandnon-chemoautotrophicbacteria.Thisisavery
particularsymbiosisbecauseitisawellstudiedextracellularbutendogenous
marinesymbiosis.Finallyinsection1.1.4Iintroducevesicomyidsclamsas
theymaintainaverywellstudiedchemoautotrophicsymbiosis.Itisaone-
to-one(bynarian)host-symbiontobligatoryassociationandtheyareagroup
close-relatedtothemaingroupofinterestinthisthesis,theBathymodiolus
mussels,whichIwillbeintroducinginSection3.1.Also,theybelongtothe
bivalves,whicharethefocusofthethirdmanuscript.

Insects1.1.1

Insectsarethelargestdescribedgroupofeukaryoticorganismswheresymbi-
oticmicroorganismsareuniversallypresent.Itisbelievedthattheyhavethe
mostdiversifiedsymbioticassociations,bothinsideandoutsidetheirbodies
(BourtzisandMiller2003).Symbiontsinfluenceinsectnutrition,develop-

5

ODUCTIONINTR

ment,reproductionandspeciation,immunologicalresponses,andhabitat
selection(BourtzisandMiller2003,Sioziosetal.2008,Bourtzis2008,Buch-
ner1965),makinginsectsthemostversatileorganismsonEarth.Insect
symbiontsareclassifiedundertwocategories:‘primary’(P)and‘secondary’
(S)symbionts,basedoncharacteristictraitsthatforS-symbiontsarecom-
plexandthereforedifficulttodefine.P-symbiontsarelargebacteriahosted
inspecializedhostcells(bacteriocytes),transmittedinaverticalmode(from
parentstooffspring),andhaveacoevolutionaryhistorywiththeirhosts.
InsectswithP-endosymbiontshaveanutrient-poordiet,thereforetheirsym-
biontsarenutritionallyimportanttogainessentialaminoacids,vitamins,
andothercofactors.S-symbiontsareaveryheterogeneousgroupbecause
theyareusuallyincidentalinfectionswithahighlyvariablefunction(Bau-
mann2005,Bourtzis2008).Bothpositiveandnegativeeffectsonthehost
havebeenobservedinsymbioticassociationsinvolvingsecondarysymbionts.
Someofthepositiveeffectsarethecapacityofinfectedhoststosurviveheat
stress,developresistancetoparasiticwasps,orexhibitalteredhostplant
preference(e.g.Montlloretal.2002,Oliveretal.2005,Oliveretal.2003,
Scarboroughetal.2005,Tsuchidaetal.2004).Inothercases,thefacultative
symbiontsaffectgrowth,reproduction,andlongevityofthehost(Chenetal.
2000;MinandBenzer1997,Stouthameretal.1999).Theimportanceofthe
S-symbiontisundeterminedinpartbecauseofthedynamismthatasym-
biosiscanhave,e.g.‘replacement’canoccurinaphids:anS-symbiontcan
takeoverthenutritionalroleofthedisappearedP-endosymbiont(Kogaetal.
2003).Furthermore,bacterialikeWolbachiathataremembersoftheobligate
intracellularrickettsialesforgenotonlyparasiticrelationshipswitharthro-
pods,butalsomutualisticrelationships,primarilywithnematodes(Mer¸cot
andPoinsot2009).TodateonlyS-symbiontshavebeencultivated(e.g.
Burkhordeliaofthebroad-headedbugRiptortusclavatus(Heteroptera:Aly-
didae)).Nevertheless,non-cultivatingmethodslikewholegenomesequencing
letusnowgaininsightintoseveralspecies,andevenmorecompletely,the
genomesequencingofboth,thehostandsymbioticbacteria.Asexamplewe
havenowtheaphid-Buchneraassociationthathavebeensequencedtwice
(InternationalAphidGenomicsConsortium2010,Shigenobuetal.2000).

6

ODUCTIONINTR

Figure1.1:Aphid-Buchnerasymbiosis.Intheinset,theaphidschemeshowing
aphidBuchnerabadinomengreen.reveLaledeftibymageFISHshowspingecificsymprobbiones.t-contBlueainingisagbacterioeneralcytesDNAwsithintain,
highlightingaphidnuclei,ingreenBuchnerabacteria(P-symbionts)andinred
Rcellsegiellawithin(S-symabbionacteriots).cyteIn(thepinkrigharrotws)image,andanerbymicrographbactetioshocyteswingcontelongateainingRegielBuch-la
nerbarsaare(greeninmarroicrons.ws).(FrBlacomkaIArroCws2010)indicatethebacteriomecellmembrane.Scale

Buchneragenomeanalysisuncoveredalargenumberofgenesthatlikely
codeforaminoacidbiosynthesisgenesandalmostnonefornon-essential
aminoacids.Italsorevealedthatobligatebacterialendosymbiontsofin-
sectshavelostmanygenesandareamongthesmallestofknownbacterial
genomes.Anotherinterestingobservationistheabsenceofimmunological
responseelementsinthehost,asitistheimmunedeficiency(IMD)pathway,
whichispresentinothernon-symbioticinsectsandcontrolstherecognition
ofGram-negativebacteria.Also,thehostlackspeptidoglycanrecognition
proteins(PGRPs),thatdetectcertainpathogensandtriggerimmunological
responses.Inparallelwiththisgenomesanalysistherehasbeenalsogreat
progressinthestudyofmolecularprocessesthatgovernhost-bacteriaphys-
iology,manygeneticstudiesshowtheimportanceandevolutionofcertain
genes.Inconclusiontheareaoftheinsectsymbiologyisanimportantarea
withmostadvancedresearch.

7

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Figure1.2:ThelightorganoftheEuprymnascolopessquidislocatedintheventral
partofthebody.Theinternalcomponentsofthesquidlightorganathatching
haveverywelldevelopedappendagestowhichbacteriaareattracted.Appendages
regresswhenVibriofisherihassuccessfullycolonizedthecryptepithelium.The
imagebelow-rightdepictstheprogressionofthecolonization.(a)Mucusissecreted
inappendagesasapositivefeedbackresponsetobacteriapeptidoglycans.(b)Only
viableGram-negativebacteriaformdenseaggregations.(c)Motileornon-motile
V.fischeriout-competeotherbacteriaandbecomedominantintheaggregation.
(d)V.fischeriaretheonlybacteriaabletomigratethroughtheporesandcolonize
thehosttissue.(e)SymbioticV.fischeribecomenon-motileandinducehost-
epithelialcellswelling.OnlybioluminescentV.fischeriwillsustainlong-term
colonizationofthecryptepithelium.(E.scolopesphoto:E.Roettinger.Schemes
fromNyholmandMcFall-Ngai2004)

8

1.1.2Squid

ODUCTIONINTR

ThesymbioticassociationbetweentheHawaiianbobtailsquidEuprymna
scolopesandthebioluminescentbacteriumVibriofischerihasbeenutilized
asamodelsystemforunderstandingmanysymbiologicallyessentialques-
tions,i.e.,theeffectsofbeneficialbacteriaonanimaldevelopment,thetrans-
missionhypothesis,andtheroleoftheimmunesystemintheacquisitionand
maintenanceofsymbiosis.V.fischeriisaheterotrophanditisfoundinfree-
livingstage.Whenassociatedtothelight-organcrypt(Fig.1.2),itshost
providestothebacteriacarbonandnitrogenintheformofpeptidesand
proteins(GrafandRuby1998).Over-populationofthecryptspacesiscon-
trolledbyadailyventingeventatdawn,involvingtheexpulsionof95%ofthe
cryptcontentsviatheporeseachdawn(LeeandRuby1994).Theremaining
cryptsymbiontswillthenmultiplytorepopulatethecryptsoverthefollow-
ingday,completingtheday-dawncyclethatthesymbiosishas.Thelight
producedbythesymbiontisemitteddownward,andthesquidcanmanip-
ulatetheintensityofthelighttomatchtheintensityofdown-wellingmoon
andstarlight,thusmaskingitssilhouettetoevadebottom-dwellingpreda-
tors(JonesandNishiguchi2004).Wholegenomesequencing(Rubyetal.
2005,Mandeletal.2009)hasbroughtalsomanyinsightsintothepoten-
tialfunctionsofsymbiosis.V.fischeriisanextracellularbacterialsymbiont
anditistransmittedhorizontally(takennewlyfromtheenvironmentineach
generation).Theacquisitionoccursthankstotheactivationofthejuvenile
ciliatedspecialtissuebybacterialpeptidoglycans.Afterhatching,thehost
tissueentersincontactwithmanymicrobemembrane-associatedmolecules
andstartssecretingmucusabundantly.Thismucuspermitsadhesionofbac-
teria,especiallyGramnegative,butattheendof2-3hourstheaggregation
isdominatedbyV.fischeri(McFall-Ngaietal.2010).Fromhereon,sym-
biontsinducemorphologicalchangesinthehosttissueandinthebehaviour
ofhemocytesthatflowthroughthehost’shemolymph.Itisnotclearwhat
thefunctionofthesehemocytesmaybe,butitseemstheyhavesometype
ofmemorythatmakethemdistinguishsymbiontsfromhostcellsandclear
otherbacteriabyphagocytosis(Nyholmetal.2009,McFall-Ngaietal.2010).

9

ODUCTIONINTR

Molecularsignalizationpathwaysarestillunknownbutveryactiveresearch
onthematterisunderway.

1.1.3Gutlessoligochaetes
Astheirnameindicates,gutlessoligochaeteshavenomouthorgut,there-
fore,theseworms(2-50mmlongand0.1-0.3mmthick)dependobligatorily
onsymbioticbacteriafornutrition(Dubilieretal.2008).Thesymbiontsare
extracellularbutoccurendogenouslybetweenthecuticuleandepidermis.An
oligochaetelikeOlaviusalgarvensiscanharborasmanyassixco-occurring
symbiontsthatbelongtotheGamma-,Delta-,orAlphaproteobacteria,anda
spirochaetehasalsobeenfound(Blazejaketal.2006,Ruehlandetal.2008).
Enzymeassays,immunohistochemistry,andlabeledcarbonexperimentsin-
dicatethatatleastsomeofthebacterialsymbiontsarethiotrophic,using
reducedsulfurcompoundsaselectrondonorsandfixingCO2autotrophically
togenerateorganiccarboncompounds(Dubilieretal.2006,Ruehlandetal.
2008).AmetagenomicanalysisperformedintheoligochaeteOlaviusalgar-
vensisshowedthatmostprobablethesymbiontsareengagedinasyntrophic
sulfurcyclewhereDeltaproteobacteriaaresulfatereducersandproducethe
reducedsulfurcompoundsthatthiotrophicgammaproteobacteriaoxidizeas
theirprimaryenergysource(Dubilieretal.2001,Woykeetal.2006).Itis
beenproposedthatsomeofthesymbiontsinthesewormshaveavertical
transmission(Dubilieretal.2006).Furthermore,thegenomeisnotreduced
butcontainsahighnumberoftransposableelements.Thismaymeanthat
symbiontsareverticallytransmittedandtheyareinanearlystageofgenome
2008).al.et(Dubilierreduction

1.1.4Vesicomyidclams
Largevesicomyidclams(e.g.Calyptogenaspp.,“Ectenagena”extenta)have
onlyavestigialdigestivetract,thustheydependnutritionallyontheirintra-
cellulargammaproteobacteriasymbionts.Individualbacteriaarecontained
inamembrane-boundvacuole,andthesearehousedwithinhostbacterio-
cytes.Symbiontsarechemoautotrophsusingenergyfromsulfideoxidation.

10

ODUCTIONINTR

Figure1.3:Gutlessoligochaetesymbiosis.Astheylackadigestivesystem,gutless
oligochaeteshostbacterialsymbiontstogettheirnutrition.(a)Oneofthemodel
gutlessoligochaeteOlaviusalgarvensis(Photo:N.Dubilier).(b)Transmission
electronmicrographofsymbiont-containingregionbelowthewormcuticle(cu).
Smallandlargesymbiontmorphotypesareshownwithsmallerandlargerarrows,
respectively.Scalebar:2μm.(FromDubilieretal.1995).(candd)FISH
identificationofbacterialsymbiontswithspecificprobes.Twoofthesixcontained
phylotypesarelocalized,Gamma1(green)andGamma3(red).Scalebars:20
μmin(c)and10μmin(d).(FromRuehlandetal.2008)

Itseemsthatvesicomyidssynthesizeadi-globular,non-hememoleculethat
runswithinthebloodserumandbindsfreesulfide,perhapsviaZn2+residues
(Childressetal.1993;Francketal.2000),toprovidetheirsymbiontswiththe
requiredelectrondonor.Thetransferofnutritionalcompoundstothehost
isstillnotclear,butdetectionoflysozymesinthegillsoftheventbivalve
Calyptogenamagnifica(Fiala-M´edionietal.1994)couldbeanevidenceof
thedigestionofthesymbiontsbythehost.Symbiontsaretransmittedver-
ticallybetweenhostgenerationsviatheegg(CaryandGiovannoni1993);
thismodelissupportedbythephylogeneticcouplingofmitochondrialwith
symbiontgenes(Hurtadoetal.2003).Assymbiontshavenotbeencultured,

11

ODUCTIONINTR

isFigureshow1n.4:inleftCalyptoimagegena(photo:-thiotrophssymwww.exploretheabbiosis.Alargeyss.com).20IntcmherighCalyptot(A)genaTrans-clam
missionelectronmicrographofgillfilament,showingcoccoid-shapedsymbiotic
ria;bacteriamv:mwithinicrovilliab(ofbacterioothcytecell-tayndpes);innb:tercalarynucleuscellsoflacbkingacteriosymcyte;bionni:ts;nb:ucleusbacte-of
inbacteriatercalaryandcell.p(B)eribacterialHighermemmbraneagnification.(arrow).UScaleltrastructurebars:tA,ypical5μm,B,Gram-negativ0.25μem.
(FromCavanaugh1985).

metagenomicshavebeenusedtosequencethebacterialgenomes.Twodiffer-
entwholegenomesequencingswereperformedbyKuwaharaetal.2007and
Newtonetal.2007:afterasmallprocessoftissuehomogenization,filtration
andhostDNAdigestion,bacterialcellswereseparatedfromthehostwhich
permittedsubmissionofthebacterialDNAtoawholegenomesequenceanal-
ysiswithlittlehostDNAinterference.Thesymbiontgenomessequencing
hasshownthatthesearethesmallestgenomeswithinautotrophicbacteria,
andalsohasgiventhepossibilityoflinkingthesymbiosismetabolismand
transmissionhypothesis,withthepotentialimplicatedgenes,aswellasa
betteroverviewofthediversificationandgenomicevolution.Oneofthelat-
estdescriptionsshowsthatvesicomyidsymbiontshavetwodifferentsulfur
oxidationpathways,oneforthiosulfateandoneforsulfide,whichcouldbe
anadaptationtotheresourcecompetitionbetweentubewormsandbivalves
2009).al.et(Harada

12

1.2Summary:Theroleofsymbioses

ODUCTIONINTR

Symbiosisisawaytoobtainshelter,nutrients(neededcompounds),oren-
ergy.Butthisisnottheonlylevelofimportancethatanassociationbetween
organismshas:italsostampsevolutionarytracesonbothsides,andsome-
timesitbringsaneworganismintoplay.Weknownowmanyoftheroles
thatsymbiontshaveinsomeofthewellstudiedsymbioses,howeverthere
arestillmanyunclearpathwaysandmanymysteriousprocesses,suchashow
nutrientsaretransmittedorhowobligatoryanassociationis.Afterphysio-
logical,stableisotopic,enzymatic,andmolecularstudies,weknownowthat
essentialaminoacids,vitamins,andothercofactorsaretransmittedfrom
symbiontstoinsects;alsothatC1-elementsaretransferredfrommethan-
otrophstomussels,snails,andtubeworms(forareviewofmethanotrophs
seePetersenandDubilier2009)inanorganicsourceform,andthatnew
fixedcarboncompoundsareprovidedbychemoautotrophic(sulfuroxidiz-
ers)symbiontstotheirhoststhatvaryfromciliatestoarthropods,including
nematodes,mollusks,andannelids(seeDubilieretal.2008forareview).

13

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14

Box1.SymbiosisConceptandSymbiology.Symbiosisisagreek
word(συμβιωση)meaning:coexistence.Asthereareinconsistenciesin
thedefinedusehofere.theThistermwosrdymwasbiosis,firstithisntrothesisducedwasillareferbiologicaltothedcescriptoronceptians
thetheneedsecondforhalfaoftermtheto19thdescribceneturyt.heTcoheexistencestudyofoflichensdifferentmadeorganismsexplicit
thatresultina‘”new”’entitywithdistinctmorphological,geneticand
ferenmetabtoliclichencapabilities.biologists,theTheScwiss,onceptSimonwasSchbroughwetndenerinto(useb1829-1919)ytwoanddif-
theGerman,AntondeBary(1831-1888),whowaslikelythefirsttouse
theterm‘”symbiosis”’(deBary1878).Sometimebefore,theGerman
bobiotism”’tanist,(FAlberankrt1877)BernhardbutFrhisankwo(rkwas1839-1900),lesspwidelyroposedreadthethantermthat‘of”sym-de
wereBary’s.coinedAroundbyP.theJ.sVaamenBtime,enedentheterms(1845-1910)mutualismreferringandtoca‘beommensalismnefitto
bothorganisms’ora‘benefittooneoftheassociatedorganisms,withno
beconceptnefitorisuharmsedtonowtheadaoysther’,veryrespoftenectivtely.hankstoSymbiosisthewithhistoricalamcutualisticonnec-
tionendevtooursso(ciologyMargulis,economandy,Fpesterolitics,1991).philosophHowyevaer,ndtosothertrictlyndon-scienefinetifican
associationbetweenorganismsasparasitic,mutualistic,orotherwise,is
notinitionanoefasyabmenefitatter,isasnotscertaintraightassoforwciationsard.Fuarenotrthermore,stabletheandthemoleculardef-
mechanismsenablingtheestablishmentofaparasitic,mutualistic,orany
otherlogicalatssoostudyciation,theseareoassoftenciationssimilar(asHent‘symschelbiosis’etal.sensu2000),latoth:uTsithisismthesisost
shallscriptionbecandonsideredunderstandingasymbiolooftgicwoaldifferenstudy,tcsonystems:tributinganewithndosymnovelbioticde-
andanendonuclearone.Thoughnotcommonlyusedinliterature,the
termsymbiologyisthe‘studyofthesymbioses’(Read1970).Icon-
andsidertsheymmecbiologyhanismstoreferthattogovandernfocusthem.onAthesymdifferenbiologicaltsymsbiotictudysaimsystemsto
describethedifferentmembersofanassociation,theirrole,andtheir
relationship,tomodelthesymbiosisonaholisticsystemlevel.Thiscon-
thetributesorganismstotheinvolvedunderstandingintheaofssothepciation.hysiology,ecologyandevolutionof

2Chapter

Habitats

InthischapterIwillintroducethehabitatsthatareofinteresttothisthesis,
fromageneralviewtothespecifichabitat.Twomainmarineecosystems
arereviewed:deep-seacoldseepsandshallow-watercoastalzone.These
habitatsdiffergreatly,buttheybothhostastablebivalvecommunity.

coldDeep-sea2.1seeps

Asageneraldescription,a‘coldseep’isasitewherethereisseepageof
hydrocarbons(ingasorliquidstate),othergasessuchashydrogensulfide,
carbondioxide,andalsobrines,whichcombinetomaketheenvironmentvery
energy-rich.Themainhydrocarbongasinmostseepsismethane.Thereis
notyetanunequivocalexplanationabouthowtheseepagecompositionin
coldseepsissohighlychargedinmethane,itseemsthatphasepartitioning
andfractionationduringupwardmigrationofhydrocarbons,andinteraction
withwater,minerals,andcatalyticallyactivetransitionmetalsinsedimen-
tarybasinsdeterminethefinalgasandoilcomposition(Seewald2003).This
wouldbetheexplanationforthepetrogenicoriginofmethane.Nevertheless,
thereisactivemicrobialactivityinthesubsurfacethatwouldberespon-
sibleforthebiogenicmethaneandsulfidesupply.Methanogenicarchaea
andsulfatereducers,carryoutdiverseanoxicprocesses:methanegenera-
tion,anaerobicmethaneoxidation(AOM)andsulfideproduction.These
processesdeterminehabitatgeochemistry.

15

INTRODUCTION

Figure2.1:GulfofMexico.Themoststudiedsitesaremappedwiththeirrespec-
tiveBathymodiolusandtubewormfauna.Thetwopicturesintherightshowthe
megafaunainChapopotesite(MARUMCopyright).

MexicoofGulf2.1.1

TheGulfofMexico(GoM)istheninthlargestbodyofwaterintheworld,
withanovalshapeandadiameterof1500Km.ItconnectswiththeAt-
lanticOceanthroughtheFloridaStraitbetweentheU.S.A.andCuba,and
withtheCaribbeanSeaviatheYucatanChannelbetweenMexicoandCuba.
TheGoMseaflooriscomposedprincipallyofevaporates,redsediment,in-
trusive,andmetamorphicrocks.Underneath,afewkilometersbelowthe
surfacefloor,ahugedepositofhydrocarbonsisfound.Thesedepositsdate
fromtheupperJurassicperiodandareconsideredtojointlyrepresentoneof
thebiggestreservesintheworld(Nehring1991).Salinedepositsarefound
towardsthesurfacesediment,creatingaverydynamicfloor.Diapirism,or
saline-densitymovements,commonlyoccurthroughouttheGoM.Coldseeps
(methaneandhydrocarbonseepage)arewidespreadintheGoMasaresult
ofitsspecialtectonicsandgeologicalhistory.Here,hydrocarbonseepage
andgashydratesaretwoofthecommonsettings.Methaneandsulfide,
chemosyntheticlife-sustainingelements,arenormallypresentprovidingrich

16

ODUCTIONINTR

Figure2.2:Saltdomes.Diapirism,orsaline-densitymovements,commonlyoccur
throughouttheGoMallowinghydrocarbonstoseeptothesurface.Sourcerocks
aredeeplyburiedbeneaththeallochthonoussalt.Fluidsmigrateupwardthrough
holesinthesaltthrust(arrows).Withinbasins,saltandrelatedfalutsprovide
conduitsforverticalmigrationoffluidstoreservoirsandtoseafloor.(FromSassen
2004)al.et

energysourcesforchemosyntheticbacteria(Lanoiletal.2001,Orcuttetal.
2005).ThebiologyoftheseepsinthenorthernGulfofMexicoiswellstud-
ied(e.g.Fisher1993,Cavanaughetal.1987,Cavanaugh1993,Carney1994,
Cordesetal.2005,2007),butinthesouthernpartthestudiesarescarcer,
makinginterestingacomparisonbetweenthesymbioticfaunaofthenorthern
andsouthernsites.InthesouthernGoM,offtheMexicanstateofCampeche
thereisaregioncalledCampecheKnollswithadepthofalmost3000m.This
areahasahummocky(manylowridgespresent)topographyderivedfrom
diapirism.Trapsorpathsofhydrocarbonsinthiszonearefoundfrequently
seepingtothesedimentsurfaceandwatercolumn(Ewing1991,Zhaoand
Lerche1993).Averyuniqueareaofthedeepoceanwasdiscoveredhere
inNovember2003:amongthelargedesertsofsoftsedimentamonticuleof
solidifiedasphaltwasfound(MacDonaldetal.2004).Lava-likefluidsand
well-developedmetazoancommunitieslivinginbetweentheasphaltlayers
wereobserved.ThelocalitywascalledChapopote(“tar”inSpanish).

17

ODUCTIONINTR

oteopChap2.1.2Chapopoteisauniquecoldseepwhereasphalt,gashydrates,andhydrocar-
bonsarepresentalltogetherinadeep-seaenvironment(2930metersdepth).
Itseemsthattheasphaltflowsoutfromtimetotime,makingthehabitat
verydynamic.Itissuggestedthatshiftsinthishabitatoccurinrelatively
shorttimeperiods(MacDonaldetal.2004),consequentlythebiologicalcom-
munitywouldhavetore-structureconstantly.Nevertheless,shrimps,tube-
worms,bivalves,andotherfaunaareabundantandcoexistinthisamazing
environment.Whatmakesthissiteaspecialhabitatwithuniqueparameters
isthattheoilhasmoreasphaltenesthatmakeitheavier,withadensity
higherthanwater.Thus,theoilstaysintheseafloor,whileinothersettings
theoilleaksupwardtothewatersurface.Anditisnotjustthatitstaysin
thedepthsbutitstaysinanoxicareaandhydrocarbonscanbeaerobically
oxidized.Also,thisisasystemwithnewsubstrateforthemegafaunato
settle,astherearenotjustcarbonatesbutalsosolidasphaltformations.

2.2Shallow-watercoastalzone

The“coastalzone”isatransitionalareainwhichterrestrialenvironments
influencemarineenvironmentsandmarineinfluenceterrestrialones(Carter
1988).Thisisazoneconformedmainlyofshallowwaterhabitatsthatare
characterizeddependingontheirgeographiclocation,andbiogeochemical
parameters.Importantparametersarethedepth,grainsize(fine-grained
orcoarse-grained),sedimentology(softbottom,carbonateconcretions),and
hydrodynamics.Fivetrophicguildsarerecognizedwithintheshallowwa-
termollusks:suspension-feeders,deposit-feeders,carnivores,woodborersand
chemoautotrophs;andthesearedistributedwithinsixhabitattiers:epifau-
nalcemented,epifaunalbyssate,semi-infaunal,shallowinfaunal,deepinfau-
nalandboring(Stanley1970,GrillandZuschin2001)(Fig.2.3).Incontrast
tothedeepsea,wherethereislackoforganicmatterfromphotosynthetic
primaryproduction,shallowwatersareorganicrichatthepelagicandben-
thiclevel.Additionally,loadsoforganicmatterandnutrientsfromhuman
practicesaredepositedintheseecosystems(Anderssonetal.2005).Forall

18

ODUCTIONINTR

Figure2.3:Bivalvesintheirhabitattiers.(a)epifaunalcemented(e.g.oysters),
(b)epifaunalbyssate(e.g.mytilids),(c)semi-infaunal(e.g.modiolids),(d)shal-
lowinfaunal(e.g.venerids),(e)deepinfaunal(e.g.mactrids,razorclams),(f)
deepinfaunal(lucinids)-thathaveatubesystemwhichisformedwiththeirex-
tendablefoottoobtainhydrogensulfidefromunderlyingsediments,(g)boring
(e.g.lithophagins).(FromGrillandZuschin2001).

oftheabovereasons,lifeofbivalvesinthisecosystembecomesverydynamic,
astheyshouldbeconstantlyadaptingtothechangingconditions.Yet,they
mightnotsucceedanddiseaseswoulddiminishbivalvepopulations(seealso
Section3.3:shallow-waterbivalves).

19

3Chapter

Hosts

3.1Deep-seaBathymodiolusmussels

Deep-seamytilidmusselsofthegenusBathymodiolushavebeenfoundand
studiedallaroundtheworld.Thepresenceofthisgenusislimitedtohy-
drothermalventsandcoldseeps(Disteletal.2000,Miyazakietal.2010).
Bathymodiolinmussels(Bathymodiolusspp.andrelatives)relyfortheirnu-
tritiononendosymbiontsharboredinbacteriocytes,specializedcellsofthe
gilltissue.Somebathymodiolinspecieshostthiotrophicsymbionts,methan-
otrophsorboth(seeSection4.1formoredetailsonsymbionts).Theyretain
theabilitytofilter-feedwhich,incombinationwiththeirsymbioticassoci-
ations,contributestotheirbroadgeographicsuccess(Fisheretal.1987).
IntheGulfofMexicoseeps,fivebathymodiolinspecieshavebeendescribed,
threeofthembelongingtotheBathymodiolusgenus:B.childressi,B.brooksi,
B.heckerae(Gustafsonetal.1998).B.childressimusselshavebeenfound
allalongtheLouisianaslopeincludingtheAlaminosCanyon.B.brooksihas
beenfoundintheAtwaterCanyonandco-existingwithB.childressiinthe
AlaminosCanyon,andwithB.heckeraeintheWestFloridaEscarpment.
B.heckeraemusselshavebeenreportedfromtheWestFloridaEscarpment,
andalsooutoftheGoMinBlakeRidge,offEastFlorida(seeFigure2.1
forspecieslocationintheGoM).Bathymodiolinmusselsharbordifferenten-
dosymbionts.WhereasB.childressihasonlymethanotrophs(Fisheretal.
1987,DistelandCavanaugh1994,Duperronetal.2007),B.brooksiandB.
heckeraepossessadualsymbiosisofthiotrophicandmethanotrophicbacte-

20

ODUCTIONINTR

Figure3.1:PhylogenyofBathymodiolusmusselsbasedonCOIandND4sequences.
veThent;(scalefullcbarircles)indicatescold-w0ater.01sseep;(ubstitutionssquares)pweroodsite./whale(emptboyne;circles)(triangles)hydrothermalshallow.
(FromMiyazakietal.2010).

ria(Cavanaughetal.1987,Fisher1993,Duperronetal.2007).Recently,
morethantwophylotypesofbacteriawereobserved,namelyinB.heckerae
whichharborsfourco-ocurringsymbionts,amethanotroph,twophylogenet-
icallydistinctthiotrophs,andamethylotroph-relatedone(Duperronetal.
2007).Todate,phylogenyanddistributionofBathymodiolusspp.mussels
andtheirsymbiontsfromtheGoMhaveonlybeendescribedinspeciesfrom
northernlocations(seeTable3.1),anditisnotknownhowmusselsandtheir
symbiontsfromthesouthernGoMarerelatedtotheformerones.Mitochon-
drialcytochromecoxidasesubunitI(COI)genehasbeenusedtodetermine
thephylogenywithinBathymodiolusspecies(Miyazakietal.2004,Iwasaki
etal.2006,Jonesetal.2006)withagooddefinition.Howeveranalysiswith
severalconcatenatedgenesasND4and28SrRNA(e.g.Wonetal.2008,

21

ODUCTIONINTR

Miyazakietal.2010)promisetogivebetterphylogenetichistories.
Table3.1:DistributionofBathymodiolusmusselsandtheirsymbionts.Tin-
dicatesthiotrophicandMmethanotrophicandtheirrelativeabundance.HV–
hydrothermalvent;CS–coldseep.(modifiedfromDeChaineandCavanaugh2005
andDuperronetal.2005
ZoneSpeciesSymbHabReference
PACIFIC
EastP.RiseB.thermophiusTHVFiala-M´edionietal.1986
NorthFijiB.breviorTHVDistelandCavanaugh
1998al.etDubilier1994,JapanB.japonicusMHVandCSHashimotoandOkutani
1994B.platifronsMHVandCSFujiwaraetal.2000,Barry
2002al.etB.septemdierumTHVFujiwaraetal.2000
B.sp.THVMcKinessetal.2005
ATLANTIC
Mid-AtlanticB.azoricusT>MHVFiala-Medionietal.2002
RidgeB.puteoserpentisT>MHVDisteletal.1995
GulfofMexicoB.childressiMCSFisheretal.1987Distel
&BlakeRidgeandCavanaugh1994
B.heckeraeM>TCSCavanaughetal.1987,
Salernoetal.2005,Duper-
2007al.etronB.brooksiM>TCSFisher1993,Duperron
2007al.etGabonMarginBathymodiolussp.M+TCSDuperronetal.2005
BarbadosB.boomerangM+TCSvonCoselandOlu1998

B.brooksiM>T
GabonMarginBathymodiolussp.M+T
BarbadosB.boomerangM+T

CSSCCS

3.2Deep-seaEscarpiatubeworms
Adultvestimentiferantubewormslackadigestivetractanddependontheir
chemoautotrophicsymbiontsfornourishment.Theyhosttheirsymbionts
inaspecializedorgan,thetrophosome,ahighlyirrigatedtissuecomplexed
withbacteriocytes.Tubewormtaxonomyhasbeenintensivelyinvestigated
22

0.10

ODUCTIONINTR

8295Ridgeia spp.TS
Oasisia alvinae, U74069, AY646013VEN
Y645989, U74053, ARiftia pachyptila82Y645993, U74075, Aevnia jerichonanaT96Seepiophila jonesiY326303, (Zaire Margin), AEscarpia southwardae Escarpia laminataEscarpia laminata, (Alaminos Canyon, GoM), A, (Florida Escarpment, GoM), AY129128Y129131
Escarpia spicata, (Santa Catalina Basin whale fall), U84262EPS
, (Guaymas vent), U74064Escarpia spicataES , (Guaymas seep), U74065Escarpia spicataEscarpia laminata, (Alaminos Canyon, GoM), AY129129OLD
Cest Florida Esc, GoM), U74063, (WEscarpia laminataY129130, (Alaminos Canyon, GoM), A100Escarpia laminata87, (Chapopote Knoll, GoM)Escarpia laminataY129134 sp., (Louisiana Slope, GoM), A95Escarpiarough, Japan), D50594 cf., (Nanaki TParaescarpia spp.LamellibrachiaWF, AB259569Osedax japonicus

Figure3.2:PhylogenyofvestimentiferantubewormsbasedonCOIsequences.
Tubewormsfromthethreedifferentdeep-seahabitatsareshown:vents(violet),
coldseeps(blue)andwood-fall(W-inbrown).Newspecimenfromthisstudy
appearsinbold.Thetreewasbuiltbasedonallthesequencespubliclyavailable,
usingRAxML,with100bootstrapreplicatesandrootedonOsedaxjaponicus.
Scalebarindicates10%estimatedbasesubstitution.

inrecentyears(e.g.McHugh2000,Halanychetal.2001,Roussetetal.2007,
McMullinetal.2003).Thecurrentclassificationplacesalltubewormsin-
sidethevestimentiferangroupwhichbelongstothefamilySiboglinidae(Mc-
Mullinetal.2003).Theyhavemanymorphologicalandmolecularfeatures
incommon,suchasnomouthorfunctionalgut,atrophosometissuefullof
symbioticbacteriaandcloselyrelatedCOIsequences.Tubewormsarefound
generallyinhighlysulfidichabitatsoncontinentalmargins,hydrothermal
vents,andcoldseeps,withseepsinhabitedmainlybyescarpidsandlamel-
librachids.Bothgroupsarewidelydistributedinalloceanbasinsbutthe
Indian.VestimentiferantubewormsfromthenorthernGulfofMexicohave

23

ODUCTIONINTR

beenwellstudied,forexamplebyMcMullinetal.2003,whomadeanexten-
sivestudyofthephylogenyandbiogeographyofthesetubewormsandtheir
symbiontsusingthe18SrRNA,COI,and16SrRNAgenes.Alotofdata
wasgeneratedfromthisstudywhichshowedthattherewasnocongruence
orclearpatternbetweenbothhostandsymbiontphylogeny.Twoescarpid
speciesEscarpialaminataandSeepiophilajonesi,arecharacteristicinthe
GoMbasin,asisthelamellibrachidLamellibrachialuymesi(Nelsonetal.
1995,McMullinetal.2003).Therefore,weexpecttofindthesetubeworms
orcloselyrelatedspeciesatChapopote.Nomolecularstudies,asfaraswe
know,havebeenperformedwithspeciesinthesouthernGoMandthusitis
ofinteresttocomparethesesoutherntubewormsandtheirsymbiontsfrom
anasphalticlocation,withthenottoodistantnortherntubeworms.Vent
vestimentiferansymbiontsarerelatedandbelongtotheGammaproteobac-
teriagroup.Seepsymbiontsarephylogeneticallymorediverse;nevertheless,
lamellibrachidandescarpidsymbiontsformaclusterwiththesulfide-oxidizer
symbiontbacteriaoftheventvestimentiferanswithintheGammaproteobac-
3.2).Fig.(teria

24

longingebtsbionymsthiotrophicndicatei3GandG21,Gts.bionymstheirndarmsowetub
foistributionD.2:3bleaTdthetoseepCS–coldt;envydrothermalHV–hfall;dooWF–w2003.aletMcMullinybsuggestedroupsgtifferen
rpiaaEsc

CatalinataiteSanSDepth1240abWFH7991namdlFe
RefU77482ccAbymG2S1998ldmaneRefFU84262ccA

ataspicE.sp.HostZoneEasterncfiicPa

asinFTmasyGuaTSmasyGuaFTmasyGuaca,uFdeJuanalleyVMiddleyCantereyMonon
BsuMan1660-190020201000165324001653

ODUCTIONINTR

onyroughCanoughoughasinTrrTTaBwaiaiAlaminosLauOkinaNankNankS.680-1000120022001890300

SCSVCSVCSCS
Cal.etaletekrijenhoV2007lateMeoDi2000aletekrijenhoV2007andNelson2000;Fisher2003etMcMullinMcMullin2003andNelson2000;Fisher2003etMcMullin
G1VG1G1G2VN/DG2
DQ232903AF165908,9DQ232902129113YA129093,4YA129102,8,9YA
U74065U74064U74055129137,8YAN/DardwSouthSCN/D1997jimaKoD50594VN/D1997jimaKoD50595V1997ldmaneFU77481G11997kBlacU74061129128-YA30
N/Dal.al.etet19971997kBlacBlac1997kkBlac19971997kBlacMcMullin20032002McMullin2003
jimaKo50593D

ataspicataspicE.E.

L.arhamibbL.arhami

arhamibL.ahinospicceP.ahinospicceP.ahinospicceP.scarpideNewlumnaocL.laminataE.
nretsWecfiicPaofGulfMexicopage.extnnouedintCon..

25

ODUCTIONINTR

Marginscarp-EFloridatmeneSlopLouisianaoteopChaponyCanGreenonyCanGreenHillBushBanksGarden
ZaireiteS

3300540-640291515001500580540
DepthabCSCSCSCSCSCSCS
Hal.etandNelson2000;FisherMcMullin20032003etMcMullinstudyThis1997ldmaneFandNelson2000FisherandNelson2000FisherandNelson2000Fisher
Ref129106,7YA129089YAU77479129100YA129110YA129092YA
ccAedutinonc–.23ableT
bymG1G3G1G1G3G3G3
S1997kBlacetMcMullin2003studyThis
U74063,129131YA129134YAN/D2004Andersen3263034YA
RefccA

26

sp.laminataHostE.

Zone

sp.sp.escarpidSecondlaminataE.achialibrmelaLuymesilcf.L.achialibrmelaLjonesiS.daesouthwarE.
tictlanAE

3.3Shallow-waterbivalves

INTRODUCTION

Shallowwaterbivalvesarewidespreadalongcoastalhabitats.Theyarefilter-
feedinganimalsthatdrawwaterinovertheirgills,extractingorganicmatter
fromthewaterinwhichtheylive.Anoystercanfilteruptofivelitersof
waterperhour(Prieuretal.1990).Suspendedmatter(phytoplankton,zoo-
plankton,algae,andothernutrientsandparticles)istrappedinthemucusof
agill,andfromthereistransportedtothemouth,whereitiseaten,digested,
andexpelledasfecesorpseudofeces.Duetothisfilter-feedingmechanism,a
highquantityofbacteriaaccumulatesinthegilltissue.Bivalvesharvested
forhumanconsumptionaresubmittedtoadepurationprocess,wherewa-
terisrunthroughtheirgills,toreducetheamountofparticlesandbacteria
presentonthistissue.Bacterialcommunitiesofbivalveshavebeencharacter-
ized,butmainlyfromthehumanhealthstandpointofview,biasingresearch
towardthestudyofthepathogenicbacteriadiversity.Therearealsore-
searcheffortsinanalyzingthepotentialofbivalve-bacteriaassociationsas
producersofmetaboliteswithantimicrobialagents(e.g.Zhengetal.2005,
Lemosetal.1985,Ivanovaetal.1998,Burgess1999).Zhengetal.(2005)
describedthatmorecultivatedbacteriaassociatedwithinvertebrates(20%)
haveantimicrobialactivitythanbacteriaisolatedfromseaweed(11%),water
(7%),orsediment(5%).Thesebacteriaarenotconsidered‘truesymbionts’
butonlyassociatedbacteria.However,dependingonthesymbiosisdefini-
tionused(i.e.thesensulatoconcept)thesebacteriacouldbeconsidered
realsymbiontsastheirnutritionwouldbebasedonthevitamins,polysac-
charides,andfattyacidsfromthehosttissue;andontheotherhandthey
wouldbeexcretingproductssuchasaminoacidsandtoxins,propitiousto
theirhost’sdevelopment(Zhengetal.2005,Armstrongetal.2001).

27

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28

Bonitionx2.sIystemsmmforunologybacteriaofbivarealves.limited.MNolecularevsertheless,tudiesoftherebivialvsealreadyrecog-
spsomeonsesknothatwledgetakeaboplaceutthewhenbivalveconfronimmtingunologicalbacteriasorystemapathogens.ndtheBre-i-
valvespossessvariouslevelsofdefensemechanisms,andingeneral,they
haveveryeffectivehumoralandcellulardefenseresponses.Thefirst
colevceklles,includescuticlesnaturalandmphucus.ysicocThehemicalsecondbarriers,levelofsucdhasefensetheeincludesxoskceletons,ircu-
latinghemocytesandsolublefactorsinthehemolymph.Antimicrobial
pandedptidesefensissecreted(Gestalbyethemoal.cytes2007).haveHboweenever,identhetified:mainmytilins,activitmyofyticins,the
hemocytesisthephagocytosis,asinvertebrateslackleukocytes,mono-
cytes,ormacrophages.Andwhiletheyhavenotevolvedacomplexim-
munology(Canesietal.2002),hemocytescanhavearesponse(chemo-
taxisorchemokinesis)tomoleculesormetabolitesofbacteria(certain
lipopundoubtedlyolysacchinvarides,olvedformincellylatedrcompecognitionounds,bypoeptides,psonization)orlectinsandtthatogetherare
withtheotherhemolymphfactors,triggerawiderangeofdefensemech-
anisms(Canesietal.2002).Afternonself-recognition(byligand-receptor
interactionsnotcharacterizedtodate)theforeignbacteriumorthepar-
ticlewithisthisinternalizedphagosomeinttoofaormptrimaryhesecondaryphagosome.phagosomeLysosomalandgranshortlyulesaffteruse
vinategratecuoles,taondmouldtheseavenzymesacuole.haDvebigestiveenegobservlandsed,proforveidexample,enzymesintotMytilushese
edulistopossessN-acetyl-muramyl-hidrolases,lysozymescapableofde-
gradingbacteriacellwalls(Birkbecketal.1987).Differentbacterial
sensitivitiessuggestthattheroleofsurfaceinteractionsbetweenbacte-
inriavaanddingmicrohemolymphorganismcompinonenthetsistissuecrucial(Prieurindetal.etermining1990,theRinkfateevicohfathend
1996).ullerM¨

4Chapter

ymSBacterialtsbion

4.1Chemosyntheticsymbionts

Chemosyntheticsymbiosiswasdiscoveredalmost30yearsagoinmarinein-
vertebrates,inparticularwithinthemegafaunafromthehydrothermalvents
andcoldseepswhereitwasobservedthatprimaryproductionisnotbased
onphoto-butchemosynthesis(Cavanaughetal.1981).Ithasbeeninferred
thatthesesymbioses(thiotrophicandmethanotrophic)arebasedonamutu-
alisticassociationwherethehostprovidesthesubstratestothesymbiontand
thesymbiontpaysinreturnbyprovidingorganiccarbon.Chemosynthetic
symbiosesarewidespreadinmarineinvertebrates,andtherelationshipvaries
dependingonthehostorganism.Forexample,inshrimpstheassociationis
epibiotic(e.g.Segonzacetal.1993,Petersenetal.2010);withclams(e.g.
VanDoverandTrask2000),mussels(e.g.DeChaineandCavanaugh2005),
andtubeworms(e.g.Cavanaugh1985)itisintracellular;andfinallyextra-
cellularsymbiontsarefoundingutlessoligochaetes(e.gDubilieretal.2001,
Ruehlandetal.2008)andsponges(e.gVaceletandDonadey1977,Friedrich
etal.1999).Intracellularsymbiosisisveryspecificandfewsymbiontphylo-
types,basedonelectronmicroscopy,16SrRNAsequenceanalysis,andFISH,
aredetectedineachhost,meaningthatthediversityislimitedandprobably
species-specific.Therelativeabundanceofeachphylotypeisalsovariable.
Thestudiesinbathymodiolinmusselswherethiotrophic,methanotrophic,or
bothbacterialsymbiontsarepresentwithdifferentrelativeabundancesof
eachphylotypeareshowninTable3.1.

29

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Figure4.1:Bathymodiolinmusselshostintheirgillsthiotrophic,methanotrophic
orbothtypesofbacteria.Top-leftimagedepictsthelocationofgills,howthewater
flowthroughthem(bluearrows)andatransversalcutnormallyusedformicro-
scopicalpreparations(Schemefrom:http://homes.bio.psu.edu).Inthetop-right
atransversalcutofaBathymodiolusthatharboursadualsymbiosisishybridized
withFISHspecificprobesforthiotrophic(green)andformethanotrophicbacterial
symbionts(red).Scalebar:10mum.(Photo:L.Raggi).Bottom-leftimageisa
scanningelectronmicrographshowinganopenedbacteriocyterevealingabundant
intracellularbacteria.(Photo:Fisheretal.1987).Bottom-rightisatransmission
electronmicrographshowingsmallmorphotype(thitrophs)andlargemorphotype
bacteria(methanotrophs).Scalebar:1mum.(FromDuperronetal.2005).

30

ODUCTIONINTR

4.1.1Thiotrophicsymbionts
Thiotrophicorsulfuroxidizerbacteria(alsocalledchemoautotrophic)are
abletogettheirenergyfromsulfideorotherinorganicsulfurcompounds,
oxidizingitwithoxygenornitrate.TheATPthatisproducedfuelsau-
totrophicCO2fixation(Figure4.2).Althoughdifferentphylotypesarefound
ineachhostthemajoritybelongtotheGammaproteobacteriagroup(Figure
4.4).Intherecentlysequencedchemosyntheticendosymbiontgenomesofthe
clamsCalyptogenamagnifica(CandidatusRuthiamagnifica)andC.okutanni
(CandidatusVesicomyosociusokutanii),alargenumberofbiosyntheticpath-
wayswerepresent(Newtonetal.2007,Kuwaharaetal.2007).Thesulfur
oxidationprocesshasbeenanalyzedbymeansofgenesandtheirtranscripts
byHaradaandcollegues(2009)anditseemsoxidationpathwaysfunctionsi-
multaneously.Theyproposedthatthiotrophicsymbiontsoxidizesulfideand
thiosulfate.Sulfideisoxidizedtosulfitebyreversibledissimilatorysulfite
reductase(rdsr).Sulfiteisoxidizedtosulfatebyadenosine5´-phosphosulfate
(APS)reductase(apr)andATPsulfurylase(sat).Bymeansofthesulfur-
oxidizingmultienzymesystem(sox),thiosulfateisoxidizedtoelementalsul-
fur,whichisthenreducedtosulfidebydissimilatorysulfitereductase(dsr).
Inaddition,thiosulfatemayalsobeoxidizedintosulfatebyanothercompo-
nentofsox(Figure4.2).TheenzymeAPRispresentinboththereductive
andtheoxidativesulfurpathways,catalizingthetransformationbetween
APSandsulfite,inbothdirections.TheaprAgeneencodesforthealpha
subunitofthisenzyme,andithasbecomeamarkergenetoidentifythepres-
enceofthiotrophicbacteriainasymbioticsystem.Thiotrophicbacteriaare
potentiallyprovidingtheirhostwiththemajorityofitsnutrition(Newton
etal.2007,Haradaetal.2009).

4.1.2Methanotrophicsymbionts
Aerobicmethanotrophsarebacteriathatusemethaneasbothanenergy
(electrondonor)andacarbonsource(forreviewseeCavanaughetal.2006,
McDonaldetal.2008andPetersenandDubilier2009).Theyareincluded
inthebroaderclassofthemethylotrophs,whicharedefinedasoxidizersof

31

ODUCTIONINTR

S2H+H

Reverse electron flow

Electron flow

NADH

2CONADHfixationey of compounds:KH2S,HS-sulfide
osulfurS2-thiosulfateS O232-SOsulfite3sqrHS-SoAPSadenosine
dsr 5’-phosphosulfate2-rdsrSOsulfate4SO32-H+protons
AMPe-apaprr2S O32-
2 O1/2sorAPSPPisatO2HsoxADPATP2-SO4ATP

Figure(thiotrophs).4.2:OxidationSulfideisofoxidizedreducedtosulfursulfitebcompyrevoundsersiblebysulfurdissimilatorychemolitotrophssulfitere-
ductasereductaserdsrapr.andSulfiteAiTPsoxidizedsulfurylasetosatsulfate.Bbyymaeansdenosineof5the´sulfur-o-phosphosulfatexidizing(mAPS)ul-
tienzymesystemsox,thiosulfateisoxidizedtoelementalsulfur,whichisthen
reducedtosulfidebydissimilatorysulfitereductasedsr.Inaddition,thiosulfate
mayalsobeoxidizedintosulfatebyanothercomponentofsox.Almostallsulfur-
ooxidationxidoreductasepathwaypathsawarey(presengreentainrroaw),whicthiotrophichdoessymnotbionhatveesxceptulfitefaorsinthetsermedi-ulfite
atecompound.Fromallpathwayselectronsfromsulfurcompoundsfeedintoan
electrontransportchain(throughmembraneproteins:flavoprotein(FP),quinone
(Q)andcytochromesbc1,c,aa)anddriveaprotonmotiveforcethatresultsin
ATPproductionandareverseelectronflowthatproducereducingpower(NADH)
forCfixation.(Imagemodifiedfrom:Haradaetal.2009,Newtonetal.2007,
MadiganandMartinko2009).

32

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Figure4.3:Aerobicmethaneoxidationbymethanotrophs.Methane(CH4)iscon-
vertedtomethanol(CH3OH)bytheenzymemethanemonooxygenase.Aproton
motiveforceisestablishedfromelectronflowinthemembrane,andthisfuelsAT-
Pase.Methanotrophsassimilateeitherallorone-halfoftheircarbon(depending
onthepathwayused)attheoxidationstateofformaldehyde(CH2O).(Madigan
2009).oartinkMand

C1compounds,suchasmethanol,formate,andcarbonmonoxide(Bowman
2006).Thegenecodingfortheactivesubunitoftheparticulatemethane
monooxygenase(pmoA)isanindicatoroftheaerobicmethaneoxidation
pathway.Theparticulatemethanemonooxygenase(pMMO)isamembrane
boundcopperandironcontainingenzymeanditisthefirstenzymeinthe
aerobicoxidationofmethanepathway(Figure4.3).Ithasbeenfoundinall
methane-oxidizingbacteriainvestigatedsofar(Elsaiedetal.2006,Nerces-
sianetal.2005)exceptforthegenusMethylocella(Theisenetal.2005).It
catalyzesthetransformationofmethaneintomethanol.Methanolisfurther
convertedtoformaldehyde,andthisiseasilyrecognizedinthebiosynthesis
pathways.Thesymbionttransferstheassimilatedcarbonrapidlytothehost
(Fisheretal.1987,FisherandChildress1992,Streamsetal.1997)andthe
isotopicsignatureofthetissue(principallymembranelipids)becomesvery
negative,closetothevaluesofthebiogenicmethane(Jahnkeetal.1995,

33

ODUCTIONINTR

Pondetal.1998,MacAvoyetal.2002).

4.2Hydrocarbondegraders

Nohydrocarbondegradersymbionthasyetbeendescribed.However79bac-
terialspecieshavebeendescribedthatdegradehydrocarbonsandusethemas
thesolecarbonandenergysource(Prince2005).Crudeoilorpetroleumisa
complexmixture(perhapsthemostcomplexorganicsubstanceonEarth)
ofmorethan17,000compoundsthatcanbeclassifiedintofourgroups:
saturatedandaromatichydrocarbons,andnon-hydrocarboncomponents:
resins,andasphaltenes(Headetal.2006).Therearetwotypesofisolated
bacteriathatusehydrocarbonsalmostexclusivelyastheircarbonsource,
theonesthatuseavarietyofsaturatedhydrocarbons:Alcanivoraxspp.,
Oleiphilusspp.,Oleispiraspp.,Thalassolitusspp.,andPlanomicrobiumspp.;
andCycloclasticusspp.thatusearangeofpolycyclicaromatichydrocarbons
(PAH).However,thereareagoodnumberofbacteriathatdegradePAHbut
notastheironlysourcebelongingtothegenus(Pseudomonas,Aeromonas,
Flavobacterium,Beijerinckia,Alcaligenes,Micrococcus,Vibrio,andMycobac-
terium).Cycloclasticusarethusuniqueandarecommonlyfoundblooming
inoilspills(Kasaietal.2002,Maruyamaetal.2003).ThefirstCycloclasticus
sp.bacteriumwasisolatedin1995beingMethylobacter,Methylomonasand
thesulfur-oxidizingsymbiontsisolatedfrommarineinvertebratesLucinoma
aequizonataandThyasiraflexuosatheclosestrelatives(Dyksterhouseetal.
1995).FattyacidcompositionofisolatedCycloclasticusisnotpeculiaras
theirpredominantfattyacidsare16ω7cisand16:0,whicharecharacteristic
ofgeneralbacteria.Howeveranunidentifiedfattyacidpeakwithacarbon
lengthof11.798wasobservedbyDyksterhouseetal.(1995).Methanehas
notbeenobservedtobedegradedbyCycloclasticusbutbiphenyl,naphtalene,
anthracene,phenanthrene,salicylate,toluene,benzoate,acetate,propionate,
andglutamateweredegradedandutilizedassolecarbonsource,afterobser-
vationsbothincultureandintheenvironment(Dyksterhouseetal.1995,
Kasaietal.2002,2003,Demanecheetal.2004).

34

88

43

1006526313425261007066

ODUCTIONINTR

Escherichia coliHalomonas elongata75Pseudomonas mendocinaHydrogenovibrio marinus100Thiomicrospira thyasirae88C5161Calyptogena sp. Calyptogena fossajaponica Florida symbiontsymbiont
symbiontCalyptogena magnifica 9998Vesicomya sp. Calyptogena elongata Gulf of Mexicosymbiont symbiont
9961Vesicomya gigas Ectenagena extenta symbiontsymbiont
symbiontCalyptogena kilmeri 10098Vesicomya lepta Calyptogena pacifica symbiontsymbiont
2665Vesicomya sp. Calyptogena phaseoliformis Blake Ridge symbiontsymbiont
symbiontIdas sp. 41symbiontGigantidas gladius 3185Adipicola crypta Bathymodiolus brevior symbiontsymbiont
90symbiontBathymodiolus hirtus 2534Bathymodiolus sp. Bathymodiolus septemdierum Juan de Fuca symbiontsymbiont
2697Bathymodiolus azoricus Bathymodiolus puteoserpentis symbiontsymbiont
70100Bathymodiolus brooksi Bathymodiolus thermophilus symbiontsymbiont
uncultured Beggiatoa sp.6643Beggiatoa alba45symbiontThyasira flexuosa 9893Codakia costata gill Lucinoma aequizonata symbiontsymbiont
96symbiontLucina nassula 4339Escarpia spicata Lucina floridana symbiontsymbiont
9296TRidgeia piscesae evnia jerichonana symbiontsymbiont
42100Riftia pachyptila Lamellibrachia columna symbiontsymbiont
100symbiontLamellibrachia barhami 25100Seepiophila jonesi Lamellibrachia cf. luymesi symbiontsymbiont
99Rhabdochromatium marinum100Thiocystis gelatinosa39symbiontOlavius algarvensis 10045Stilbonema sp. Inanidrilus leukodermatus symbiontsymbiont
4664Laxus cosmopolitus Olavius crassitunicatus symbiontsymbiont
3426Olavius ilvae Olavius loisae symbiontsymbiont
10061Bathymodiolus brooksi Bathymodiolus platifrons symbiontsymbiontM
37symbiontBathymodiolus puteoserpentis 7126Bathymodiolus japonicus Bathymodiolus childressi symbiontsymbiont
30100Bathymodiolus sp. Bathymodiolus azoricus symbiontGabon margin symbiont
100symbiontBathymodiolus heckerae 100Methylomonas methanica 99Methylomonas fodinarum94Methylomonas scandinavica56Methylobacter capsulatusMethylobacter luteus0.10Figure4.4:Phylogenyofthiotrophicandmethanotrophicendosymbiontshosted
bymarineinvertebratesandfree-livinggammaproteobacteria.Treeinferredfrom
16SrRNAgenesequencesandbasedon1000maximumparsimonyreplicates.The
twousualphylotypespresentinBathymodiolusspp.areboxedandlettered(Cand
M)forchemoautotrophicandmethanotrophicrespectively.Allsymbioticbacteria
arelabelled‘symbiont’whilefree-livingaredesignatedbytaxonomicnamealone.
(ModifiedfromDeChaineetal.2006).35

ODUCTIONINTR

4.3Intranuclearparasites

Endonuclearorganismshavebeenobservedandstudiedsincethenineteenth
century(reviewedbyG¨ortz1983).Thefirststudiedorganismswereeukary-
otes,suchasflagellatespresentinciliatesnuclei.Nuclearbacteriainciliates
wereobservedlaterandenoughevidencetocorroboratetheywerebacteria
wassummarizedbyPreer(1975).Endonuclearsymbiosiscommonlyoccurs
inciliates(G¨ortz2006).Itwasnotuntil1986thatElston(1986)described
anendonuclearpathogenicbacteriuminthegillsoftherazorclam(Siliqua
patula)asunprecedentedinmetazoans,callingit‘nuclearinclusionx’(NIX).
Kerketal.(1992)isolatedtheRNAofNIXbacteriatoanalyzethe16SrRNA
geneandtheyconcludeditwasanovelgenuswithintheGammaproteobac-
teria.Zielinskietal.(2009)foundanintranuclearbacteriumingilltissuesof
BathymodiolusmusselscallingitCandidatusEndonucleobacterbathymodi-
olin.The16SrRNAgenesequenceswerenotfoundinaregularclonelibrary
withbacterialuniversalprimersbutwithspecificprimers.Aglobaldistribu-
tionstudyshowedthepresenceoftheendonuclearbacteriainB.puteoser-
pentis(fromtheMid-AtlanticRidge),B.azoricus(thecollectionsiteisnot
specified),B.brooksi(fromthenorthernGoM),B.heckerae(fromthesouth-
ernGoM)andotherBathymodiolusspp.fromtheMid-AtlanticRidgeand
PacificAntarcticRidge.All16SrRNAgenesequencesgrouptogetherwithin
theGammaproteobacteriaphylum.Closelyrelatedsequencestotheones
foundinmusselshavebeenfoundinotherinvertebratesassponges,corals,
ascidiansandseaslugs.Itissuggestedthatthisbacteriumishost-specific,
asonlyonesequenceof16SrRNAisfoundineachhostspecies.InZielin-
skietal.(2009)anameforthemusselsphylotypeisproposed:Candidatus
E.bathymodioli.Howpathogenicthesebacteriaareforthemusselsisstill
unknown.Interestingly,theendonuclearbacteriaarenotfoundinthebacte-
riocytenucleibutexclusivelyinintercalarycellnuclei,thatarefreeofother
symbionts.Thissuggeststhatthemethanotrophicandthiotrophicsym-
biontsmightpreventtheinfectioninbacteriocytecellsandtherebyprevent
hostdeath.Insponges,anendonuclearbacteriumhasbeenclearlyobserved
anditsmorphologyisverysimilartotheoneobservedintheBathymodiolus

36

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Figure4.5:‘Ca.E.bathymodioli’invariousmusseltissuesanddevelopmental
stages.BD.Non-ciliatedgilltissuewithintranuclearbacteriuminintercalarycells
whichalternatewithbacteriocytes.E.Guttissue.InimagesBEintranuclear
bacteriaareshowningreenandeukaryotictissueisrepresentedinyellow.Nuclei
andbacterialendosymbioticDNAinbacteriocytesappearinblue.F.Non-ciliated
gilltissuewithintranuclearbacteria;intranuclearbacteriaappearinbrightyel-
low,whereaseukaryotictissueisrepresentedbyayellowishtobrownishcolour.
Chemoautotrophicandmethanotrophicbacterialendosymbiontsinbacteriocytes
areshowningreenandredrespectively.NucleiwerestainedwithDAPIand
appearinblue.GP.DevelopmentalstagesofCa.E.bathymodioliinB.puteoser-
pentisgilltissues.Theintranuclearbacteriumappearsingreen,thenucleusin
blue.ImagesHMresultfromprojectionofastackofseveraltwo-dimensional
layersontoonesinglelayerreflectingtheoverallthree-dimensionalstructureon
atwo-dimensionalplane.GJ.Seriesshowinggrowthfromasingleshortrodto
asinglefilamentinStages1and2.K.Twooverlappingfilamentsorfilamentin
theprocessoflongitudinalbinaryfissionintransitionfromStage2toStage3.L.
Twoseparatefilaments(Stages23).M.Filamentousassemblyconsistingofeither
onesinglelongcoiledfilamentorseveralfilaments(Stage3).N.Stacksofshorter
filaments(Stage4)resultingfromtransversefissionsofcoiledfilaments.O.Long
rodsresultingfromdivisionofStage4filaments.(Zielinskietal.2009).
37

ODUCTIONINTR

mussels(Friedrichetal.1999).Nofurtherstudiesaboutthisbacteriumin

spongeshasbeendoneyet.Zielinskietal.(2009)proposedadevelopmental

cycleforthesenovelbacteriainbathymodiolinmussels(Figure4.5).Ithas

beenreportedthatmusselsinhabitingdifferenthabitatsmightbeinfested

withparasitesindifferentabundancesduetodifferencesintheirphysio-

logicalcondition(Smithetal.2000,Bergquistetal.2004).Also,massive

mortalitieshavebeenreportedwithoutapparentexplanation.Thus,itisof

interesttostudythedistributionandabundanceoftheendonuclearbacteria

inmusselscomingfromhabitatswithdifferentenvironmental

witheconomicalimportanceasaretheshallow-waterbivalves.

38

factors

nda

5Chapter

Methodsofstudy

ationCultiv5.1

Mostofwhatweknowaboutphysiologyoforganismsisbasedonlaboratory
cultures.Cultivationislimitedbecausesolittleisknownabouttheneedsfor
growingaspecificorganism.Eachorganismhasdifferentneeds.Aculture
mediumhastohaveallthenutrients,metals,andextraorganiccompounds
thattheorganismtogrowrequires.Thereare“selective”,“differential”,and
“enriched”mediathatdefinetheisolationofaparticularspecies.Forthe
intranuclearbacteriathatIattemptedtogrowinthispresentwork,wedeal
withbacteriathatgrowinahighorganiccontentenvironment:thebivalve
tissue.Therefore,enrichedmediawerethepreferredonesfortheassayto
growbivalveintranuclearbacteria.Threedifferentmediawereusedinsolid
(with1.5%Agar)andliquidpresentations:MarineMedium2216(Difco),
MinimumMediumwithandwithoutCTAB(0.1%yeast,0.01%peptone,
1.5%agar,100mCTAB,dissolvedinseawater),andWLNutrientMedium
(Difco)in3.5%NaCl.Tostarttheculturesbivalvesamplewashomogenated
withsterileseawaterbyusingahomogenizerandwasseriallydilutedwith
sterileseawater.Then,analiquotofeachdilutionwasspreadontothe
isolationmediumplateorinbottleswithliquidmedium.

5.2Molecularmarkers:16SrRNA,aprA,pmoA

Thestudyofsymbiosisisnotaneasytask,especiallybecausethesystemis
usuallynotseparableandshouldbestudiedasawhole.Thus,independent-

39

METHODSOFSTUDY

culturemethodsareessentialtothestudyofsymbioses.Molecularstudiesof
bacterialdiversityprobablystartedwiththeworkofLaneetal.1985where
aneasymethodwasproposedtorapidlyanalyzethe16SrRNAgenesofa
non-isolatedgroupofbacteria.Thismethodpermitstheanalysisofeach
organism’smoleculesseparately,givingthenthepossibilityofstudyinga
hostandasymbiontsimultaneously.Theinsituhibridization(ISH)method
(Giovannonietal.1988)andthenlaterthefluorescenceinsituhibridiza-
tion(FISH)method(Amannetal.1995)completedtheanalysis(full16S
rRNAcycleanalysis),becausebasedonthesamemoleculeispossibletolo-
calizespecificbacteria,inthiscasethespecificsymbiontinsidethehost(e.g.
Dubilieretal.1999,Blazejaketal.2006,Duperronetal.2007).
Toinvestigatemoreaboutenergysources,theamplificationandsequenc-
ingofmetabolicmarkergeneswasperformed.Thealreadywellinvestigated
genesinmusselandtubewormsymbiontsarethegenesoftheactivesubunit
oftheparticulatemethaneoxigenase(pmoA),markerofthemethanotrophy,
andofthesubunitAoftheadenosyl-phosphatereductase(aprA)markerof
thethiotrophy.Inadditioninthisthesis,assaystoamplifymetabolicmarker
genesofhydrocarbondegradation,likemono-anddi-oxygenaseswereper-
formed(seeManuscriptIformoredetailinmaterialandmethods).

40

41

Aims

Therearestillmanybasicquestionstobeansweredinthemicrobialsymbi-
ologystudies,questionsinheritedfromthemicrobialecologyfundamentals.
Whoisoutthere,orbettersaid:whichsymbiontsareoutthere?Whereand
howmanyarethere?Whatistheirfunction?Thisthesisisastudyaimed
tofindanswerstothesequestions.
Themainobjectiveofthisthesisistodescribeandanalyzethesymbiont-
hostdiversityinwhatitseemstobethreedifferentsymbiosisscenarios:a
mutualistic,aparasitic,andaprobablecommensal.Thefirstcaseisillus-
tratedbythechemosyntheticBathymodiolusmusselsandtubewormssym-
bioses.Thesecond,bytheintranuclearbacteriafoundinBathymodiolusspp.
andshallow-waterbivalves.WhatIcallthethirdsymbiosisconsistsofall
gill-associatedbacteriafoundinallthestudiedbivalvespecies.

iosisbSymtheticChemosyn

Inordertogaingreaterinsightintothisbroadtopicandre-investigateBathy-
modiolussp.andcoldseeptubewormsymbioses,Ihadtheopportunityto
beinvolvedinacollaborationthatmysupervisorDr.NicoleDubilierhad
forgedwithDr.AntjeBoetius,andbeabletoinvestigatethemegafauna
anditsassociatedsymbioticmicrobiotapresentinanewlydiscoveredcold
seepthathastheuniquecharacteristicofpresentingasphaltflows,givinga
seriesofnewparametersthatinfluencethelifeofthementionedorganisms.
ThesenewsettingsthatIhavedescribedinSection2.1.2aretheexplana-
tionforthediscoveryofanewhydrocarbon-degradingsymbiontpresentin
Bathymodiolusheckerae.Theresultsofthisinvestigationarepresentedin
Chapter6andsynthesizedintheManuscriptI.Duringthesearchforthis

42

Aims

newsymbiontmentionedaboveinothersitesandotherBathymodiolusspp.,
IobservedasyetundescribedsofarepibionticEpsilonproteobacterialying
onapicalfilamentsoftheBathymodioluschildressigills.Epsilonproteobacte-
riahavebeendescribedassulfuroxidizingsymbiontsininvertebratespecies
(e.g.Rimicarisexoculata,Alvinoconchahessli).Ihaveincludedtheseresults
inareviewofthebivalvemicrobiotaintheManuscriptIII.

Intranuclearbacteriaandotherassociatedbacteriainbivalves

BasedontheresultsofthestudybyZielinskietal(2009)whereIhadtheop-
portunitytoparticipateinresearchingthepresenceofintranuclearbacteriain
Bathymodiolusspp.(ManuscriptIV)andthestateoftheartthatIdescribed
inSection4.3,Iinvestigatedtheeconomicallymoreimportantshallow-water
bivalves,focusingonthedistributionofintranuclearbacteria.Theobjective
ofthisstudywastodeterminewhetherthesebacteriaarebroadlypresentin
bivalvesandtodevelopamethodtoscreenfortheseintranuclearbacteria.
TheresultsofthisinvestigationareshowninManuscriptII.Aftermicrobi-
ologicalandmolecularstudies,ahighdiversityofbacteriawasfoundinthe
studiedbivalves.Someofthesebacteriawereisolatedandmanyofthem
wereobservedwithFISHmethodsinassociationwiththebivalvegilltissue.
TheresultsoftheseinvestigationsaresummarizedinManuscriptIII,giving
anoverviewofpaststudiesandshowinghowshallow-waterbivalvescouldbe
usedasamodelforstudyingbacteria-invertebrateassociations.

43

Results

trPa

and

44

I

I

iscussionD

6Chapter

Studiesfromanasphaltcoldseep

ChemosyntheticlifewasdiscoveredinChapopote,southernGulfofMex-
ico(GoM)associatedtolava-likeflowsofsolidifiedasphalt,oilseepsand
gashydratedepositswerealsopresent(MacDonaldetal.2004).Thesite
iscolonizedbyanimalswithchemosyntheticsymbiontssuchasvestimen-
tiferantubeworms,mussels,andclams.Morphologicalandmolecularanaly-
ses(COIgene)of4musselindividualsand4tubeworms,twomusselspecies
arepresentatChapopote,BathymodiolusheckeraeandB.brooksi,anda
singleEscarpiatubewormspecies.Comparative16SrRNAsequenceanaly-
sisandFISHshowedthatallthreehostspeciesharborintracellularsulfur-
oxidizingsymbiontsthatarehighlysimilaroridenticaltothesymbionts
foundinthesamehostspeciesfromnorthernGoMsites.Themusselsalso
harbormethane-oxidizingsymbionts,andtheseareidenticaltotheirnorth-
ernGoMconspecifics.Unexpectedly,wediscoveredanovelsymbiontinB.
heckerae,thatiscloselyrelatedtohydrocarbondegradingbacteriaofthe
genusCycloclasticus.WefoundinB.heckeraethephenolhydroxylasegene
andstablecarbonisotopeanalysesoflipidstypicalforheterotrophicbacteria
wereconsistentlyheavierinB.heckeraeby3thaninB.brooksi,indicating
thatthenovelsymbiontmightuseisotopicallyheavyhydrocarbonsfromthe
asphaltseepasanenergyandcarbonsource.Thediscoveryofanovelsym-
biontthatmaybeabletometabolizehydrocarbonsisparticularlyintriguing
becauseuntilnowonlymethaneandreducedsulfurcompoundshavebeen
identifiedasenergysourcesinchemosyntheticsymbioses.Thelargeamounts
ofhydrocarbonsavailableatChapopotewouldprovidethesemusselsym-

45

RESULTSANDDISCUSSION

bioseswitharichsourceofnutrition.InthischapterIpresentalltheresults
obtainedthroughouttheinvestigationofthissubject.

6.1PhylogenyoftubewormsandmusselsfromChapopote
PhylogeneticresolutionwiththeCOIgeneworkedwellforbathymodiolin
mussels,integratingtheChapopoteindividualsintodefinedgroupsofB.
heckeareandB.brooksi(Figure6.2a)species.However,asobservedbefore
(forreviewseeMcMullinetal.2003),theresolutionofthisgeneisnotsuffi-
cientfordeterminingtubewormspecies(Figure6.1a),especiallywithinthe
escarpidswhichhaveaverysimilarCOIsequence.Inspiteofthat,itgives
agooddefinitionofthetubewormgenera,inthiscaseEscarpia.Thephylo-
geneticanalysisofthetubewormsincludingothermolecularmarkers(asthe
18SandND4mitochondrialgene)wouldbeneededtodifferentiatebetween
Escarpiaspecies.However,vestimentiferantubewormshavearemarkable
plasticity(Blacketal.1997)andthereforeE.laminata,E.southwardaeand
E.spicatacouldbethesamespecies.Toresolvethis,apopulationgenetic
studywouldberequired.

6.2PhylogenyofchemosyntheticBathymodiolusandEscarpia
tsionbsymInthetwotubewormindividualsthatwereanalyzedby16SrRNA,thepres-
enceofasinglethiotrophicbacterialphylotypefallingintheGroup1(defined
byMcMullinetal.2003)wasfound(Figure6.1b).TheEscarpiasp.sym-
biont16SrRNAsequenceofthisstudywasidenticaltotheE.laminata
fromtheFloridaEscarpmentinthenorthernGoM(McMullinetal.2003),
andE.spicatafromtheGuaymasseep(Vrijenhoeketal.2007).TwoEs-
carpiatubewormsfromtwocollectionsitesinChapopotewereanalyzedwith
FISHspecificprobesdesignedforthisstudy(seedetailssitesandprobesin
ManuscriptI).Symbiontswerelocalizedinonlyoneoftheanalyzedtube-
worms.Highabundancethroughthewholetransversalwormtissuecould
bedetectedinapatchydistribution(Figure6.3c).DAPIsignalscorrelated
withthesymbiontsignals.TheexplanationforthelackofFISHsignalsin
46

(a) COI

RESULTSANDDISCUSSION

Y326303 (Zaire Margin), AEscarpia southwardaeEscarpia laminataEscarpia laminata (Alaminos Canyon, GoM), A (Florida Escarpment, GoM), AY129128Y129131
(Santa Catalina Basin whale fall), U84262Escarpia spicataTubeworm 4 (Chapopote, GoM) (Guaymas seep), U74065Escarpia spicata (Guaymas vent), U74064Escarpia spicataubeworm 2 (Chapopote, GoM)TY129129, (Alaminos Canyon, GoM), AEscarpia laminataEscarpia laminataEscarpia laminata, (W, (Alaminos Canyon, GoM), Aest Florida Esc, GoM), U74063Y129130
9098TTubeworm 1 (Chapopote, GoM)ubeworm 3 (Chapopote, GoM)
Y129134sp. (Louisiana Slope, GoM), AEscarpia 100Seepiophila jonesi10099100Paraescarpia cf., echinospica (Nanaki Trough, Japan), D50594
spp.Lamellibrachia 100100Riftia pachyptila96100evnia jerichonataT10099Oasisia alvinae1000.10spp.Ridgeia

Y129092endosymbiont (GB, nGoM), A(b) 16S rRNASeepiophila jonesi LamellibrachiaLamellibrachia cf. luymesi sp. endosymbiont (Bush Hill, nGoM), Aendosymbiont (Green Canyon, nGoM), AY129110Y129100Group 3
Y129088unclassified escarpiid symbiont (GB, nGoM), AEscarpia laminataLamellibrachia barhami endosymbiont (Atwater Canyon, nGoM), A endosymbiont (Monterey Canyon, EP), AY129102Y129094Group 2
endosymbiont (Whale fall, SCB), U77482Escarpia spicataendosymbiont (Lau Basin, WP), U77481Lamellibrachia columna endosymbiont (Guaymas seep), DQ232902Lamellibrachia barhami endosymbiont (Guaymas seep), DQ232903Escarpia spicataEscarpia laminataLamellibrachia barhami endosymbiont (Middle V tubeworms endosymbiont (Chapopote, sGoM)alley, NEP), AY129113Group 1
sp. endosymbiont (Green Canyon, nGoM), U77479LamellibrachiaY129106 endosymbiont (Florida Escarpment, nGoM), AEscarpia laminata ent groupV endosymbiont, AF165908Escarpia spicata endosymbiont, AF165909Escarpia spicata gill symbiont, U62131Solemya terraeregina0.10 gill symbiont, L01575Thyasira flexuosa

FIG 2. Phylogenetic affiliation of Escarpia tubeworms and their bacterial symbionts. (a) Tree based on COI gene sequences. Maximum-likelihood tree showing vestimentiferan tubeworm species from vent and seep environments including the 4 individuals of this study (sequences highlighted in gray). Only bootstrap values greater than 70 % are shown. (b) Tree based on 16S rRNA gene sequences. Maximum-likelihood tree shows within the gamma-proteobacteria phylum, thiotrophic symbionts of seep and vent vestimentiferans. Only one phylotype was present in the two investigated tubeworms (in bold), and fell in group 1 (McMullin at al 2003) within Escarpia spicata, Escarpia laminata and Lamellibrachia spp.

Figure6.1:PhylogeneticaffiliationofEscarpiatubewormsandtheirbacterial
symbionts.(a)TreebasedonCOIgenesequences.Maximum-likelihoodtree
showingvestimentiferantubewormspeciesfromventandseepenvironmentsin-
cludingthe4individualsofthisstudy(sequenceshighlightedingray).Onlyboot-
strapvaluesgreaterthan70%areshown.(b)Treebasedon16SrRNAgene
sequences.Maximum-likelihoodtreeshowswithintheGammaproteobacteriaphy-
lum,thiotrophicsymbiontsofseepandventvestimentiferans.Onlyonephylotype
waspresentinthetwoinvestigatedtubewormindividuals(inbold),fallingingroup47
1(McMullinetal.2003)withEscarpiaspicata,EscarpialaminataandLamelli-
brachiaspp.symbionts.

RESULST

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Figure6.2:PhylogeneticaffiliationofBathymodiolusmusselsandtheirbacterial
symbionts.(a)TreebasedonCOIgenesequences.Maximumlikelihoodtreeshow-
ingBathymodiolusspp.fromventandseepenvironmentsincludingthe4individ-
ualsofthisstudy(highlightedingray).Onlybootstrapvaluesgreaterthan70%
areshown.(b)PhylogeneticreconstructionofbacterialsymbiontsofBathymodio-
lusmusselsbasedon16SrRNAgenesequences.Maximum-likelihoodtreeshows
withintheGammaproteobacteriaphylumthiotrophic,Cycloclasticus-related,Psy-
chromonas-relatedandmethanotrophicbacteria.Thephylotypesinvestigatedin
thisstudyareshowninbold.NotethatB.heckeraeindividualshavetwodifferent
thiotrophicphylotypes,oneCycloclasticus-relatedandonemethanotrophicphy-
lotypes,andB.brooksipresentonlyonethiotrophic,onePsychromonas-related,
andonemethanotrophicphylotypes.

thesecondtubewormanalyzedmightbebecausebasedonROVimages,the
samplewascomingfromatubewormcommunitythatlookeddead,therefore
theirsymbiontsmightnotbeveryactiveanymoreortubewormsmightbe
loosingthem.TEMobservationsofthesamplewouldbenecessarytocorrob-
oratetheabsenceofthesymbionts.Wemightevenobservemanysymbionts
digested.ingebThe16SrRNAanalysisofthebathymodiolinmusselsshowedthatin
B.heckerae,twodifferentthiotrophicbacteriaphylotypes(TIandTII)and
onemethanotrophic(M)werepresent(Figure6.2.InB.brooksi,onlyone
thiotrophic(TI)andonemethanotrophic(M)wererecognized.WithFISH
specificprobes,eachphylotypeofthiotrophsandmethanotrophswerelo-
calizedinbothindividualsofeachspecies(Figure6.3).Whencomparing
tothemusselsfromthenorthernGoM(Duperronetal.2007)theyhave
morethiotrophicbacteriabasedontheclonelibrariesandalsowithFISH
observations.InB.heckeraethereisacleardominanceofTIIsymbiontover
TIandmethanotrophs(Figure6.3e-g).Thisisaveryinterestingdiffer-
encewiththeotherGulfofMexicomusselsinvestigatedtodate(Cavanaugh
1993,Fisher1993,Duperronetal.2007)wherethedominantphylotypehas
alwaysbeenthemethanotrophicone.Althoughtherearenotpunctualmea-
surementsforsulfide,orothersulfursources,ormethanecompoundsinthe
collectionsite,weknowthatthereisabsenceofsulfideinthewatercolumn
andthereishighmethaneconcentration(A.Boetius,personalcomm).In

49

RESULTSANDDISCUSSION

consequencewesuspectthatthesulfidemightbediffusingwithdifficulties
frombelowtheasphaltandbeingconsumedbythethiotrophicbacteriaas
soonasitreachesthemussels.Thepresenceofboththiotrophicphylotypes
presentinonebacteriocytes(Figure6.3eandf),supportstheideathat
eachphylotypeconsumesadifferentsulfursource,howeverwecannotdis-
cardthattheycouldbecompetingforthesameresourceandthatiswhyin
somebacteriocytesonephylotypeseemtodominate.Ithasbeenshowna
positivecorrelationbetweentheamountofcompoundspresentintheenvi-
ronmentandthequantityofeachsymbiontinbathymodiolinmussels(Trask
andVanDover1999,Fiala-Medionietal.2002,Salernoetal.2005),however
itcouldalsodependonthehostneeds,ifthesulfurcompoundsprovided
bythiotrophs(i.e.vitamins,aminoacids,carboncompoundsfromCO2as-
similation,etc)arelow,theycouldstimulateaugmentationofthiotrophic
bacterialcontentinthetissuetobeabletoincreasethesulfur-compounds
uptake.Nevertheless,itisclearthatthetotalandrelativeabundanceofthe
differentphylotypesdependstoagreatextentonthebiogeochemistryofthe
environment(Duperronetal.2007).

6.3NovelsymbiontsinBathymodiolusmussels

Inadditiontothesymbiontsdescribedabove,bacterialphylotypesthathave
farneverbeenfoundincloseassociationwithanimalswerefound.APsy-
chromonas-relatedspecieswasfoundinthe16SrRNAclonelibraryofB.
brooksi.WithspecificprobesforthisbacterialgenusthepresenceofPsy-
chromonas-relatedbacteriainthemusseltissuewasconfirmed(Figure6.3
k).Likethepreviouslydescribedthiotrophicandmethanotrophicbacteria,
thePsychromonas-relatedphylotypealsoappearedtobepresentwithinthe
hosttissue,howevertheydonotseemtobeintracellularlikethefirstones
(Figure6.3).Theywerefarlessabundantthanthethiotrophicandmethan-
otrophicsymbionts,andcouldonlybefoundbyFISHinoneofthetwoB.
brooksiindividuals.Psychromonasareheterotrophicgammaproteobacteria
frequentlyfoundincold-watersediments(Ivanovaetal.2004,Xuetal.2003).

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RESULTSANDDISCUSSION

Figure6.3:BathymodiolusmusselsandEscarpiatubewormsfromChapopote.
FISHimagesofbacteriocytesinthemusselgillfilamentsandinthetubeworm
trophosome.(a)BathymodiolusbrooksiandB.heckeraemusselstogetherwithes-
carpidtubewormsontheasphaltbottomatChapopote,southernGulfofMexico.
Eachspeciesharborsitsownspecificbacterialphylotypes.(b)Escarpiatubeworms
fromthisstudybearchemoautotrophictubewormsymbionts.(c)Localizationof
thesymbionts(arrows)withaFISHspecificprobethroughatubewormcross-
section.(d-g)B.heckeraemusselandrespectiveFISHimages:B.heckeraeshell
hasanelongatedshape(d);itsfilamentousgillshousemethanotrophs(notshown
here)andtwodifferentchemoautotrophicbacterialphylotypes.Thehostnuclei
areinblue,thiotrophsTIinred,andthiotrophsTIIinyellow(e,f).Anewhy-
drocarbondegrader(Cycloclasticus-related)symbiontingreen,co-existswiththe
methanotrophicbacteriainblueandthethiotrophsinpink(g).(h-k)B.brooksi
mussel(h)andrespectiveFISHimages:TheshapeoftheB.brooksishellisrounder
anditissmallerthanB.heckerae.B.brooksigillfilaments(autofluorescenceof
thetissueispurple)houseamethanotrophicbacterialphylotyope,inred,anda
thiotrophicone,ingreen(i).Adetailof(i)showshostnucleiinblue,methan-
otrophsinredandthiotrophsingreen(j).APsychromonas-relatedbacteriawas
associatedwithB.brooksigilltissues(k).Scalebars:(c,e,i)=50μm;(d,h)=
5cm;(f,g,k)=5μm;(j)=10μm.

Onlyonce,anotherPsychromonas-relatedphylotypehasbeenobservedas-
sociatedwithananimal,inthebonesofawhalefall(Goffredietal.2004).
Thepresenceofthesebacteriaseemtoberelatedtoahighorganicmatter
contentintheenvironment,whichinthiscaseitexistsattheChapopotesite
andwouldexplaintheirpresenceinB.brooksitissue.
ACycloclasticus-relatedbacteriumwasfoundinB.heckerae16SrRNA
clonelibrary.WithspecificFISHprobesIobservedCycloclasticus-related
bacteriaco-existinginthesamebacteriocytesasthethio-andmethan-
otrophicendosymbiontsinbothB.heckeraeindividuals(Figure6.3g).With
thePHLIPimagesoftwareitwascalculatedthattheCycloclasticus-related
bacteriamakeup6%ofthetotalendosymbiontbiovolume(resultsfromD.
Fink,MPIBremen).Isuggestthatthehydrocarbonsintheenvironment
makeCycloclasticussp.presencepossible,astheyhavebeenfoundalsoin
oilysedimentsfromshallowanddeepwaters(seesection4.2.However,this
isthefirsttimethatCycloclasticussp.isobservedasanintracellularbac-
teriumwithinanimaltissues.Themembranemoleculesofthisbacterium

52

RESULTSANDDISCUSSION

mightbesimilartothesymbiontstothepointthatB.heckeraemusselslet
themgetinsidethem.Theassociationmightprovidethisbacteriumthead-
vantageofbeinginanaerobichabitatwithavailablenutrients(astheflow
ofwaterthroughthegillswouldbringthenecessaryresources)andforthe
musselthiswouldprovideanewnutritionsourcefromthedegradationof
ounds.comparomatic

Host-bacteria6.4yecificitsp

ThetwodifferentsystemsthatIhavebeenworkingwith,Escarpiatube-
wormsandBathymodiolusmussels,showcontrastingpatternsintheirsym-
biontspecificity.Alackofhost-specificitycanbeobservedinEscarpiatube-
worms:thesamesymbiontphylotypeisbroadlydistributedwithindifferent
tubewormspecies.Andthesametubewormspeciesfromdifferenthabitats
orgeographiclocationscanhostadifferentbacterialphylotype(seeFigure
6.1b).Thismightbeexplainedbyahorizontaltransmissionofsymbionts,
thatinothertubewormslikeRidgeiapiscesaeandRiftiapachyptilahasbeen
corroborated(BrightandSorgo2003,Nussbaumeretal.2006).However,
ahorizontalsystemcouldalsohaveaveryspecificsymbiontselection,like
inthesquid-Vibriosymbiosis(seesection1.1.2).Forthetubeworms,this
couldmeanthatthemolecularrecognitionprocessisnothighlyspecific,al-
lowingpromiscuityofbacterialphylotypeswithinthedifferenthosts.This
isincontrasttothemusselswhereeachmusselspecieshostsspecificbacte-
rialphylotypes(Fig7.2b).Itmightcertainlybethatsomesymbionts,like
thethiotrophsTIandmethanotrophsfromthisstudy,aretransmittedver-
ticallyexplainingwhysymbiontsareexactlythesame(basedon16SrRNA)
inmusselsfromdistantplacesasnorthernandsouthernGulfofMexico,
butotherslikethiotrophTII(wherenorthernandsouthernphylotypesare
close-relatedbutnotidentical)areperhapstransmittedenvironmentally.An
alternativeexplanationtotheverticaltransmissionisaveryspecifichost-
symbiontrecognitionsystem,wheremethanotrophsandthiotrophsineach
host-speciesmighthaveparticularmoleculesandtheywouldberecognized
andinteriorized.Futurestudiesfocusinginthestudyofrecognitionmolecules

53

RESULTSANDDISCUSSION

Figure6.4:StablecarbonisotopemeasurementsoflipidsextractedfromtwoB.
heckerae(heck),twoB.brooksi(brook),and4Escarpiasp.(esc)tissue.Carbon
isotopevaluesoflipids(circlesandbars)andbulktissue(reddiamonds).Thevalues
ofmethane(CH4)andheavierhydrocarbons(CnHx)thatarecharacteristicofthe
siteareshonw.

(e.g.lectins)mightrevealthemechanismforthehost-symbiontspecificity,
andmightalsorevealhownovelassociationse.g.B.heckerae-Cyclocasticus
established.ebcan

6.5Metabolismofthesymbioses

GenesaprA(subunitAgeneoftheadenosyl-phosphatereductase[APR])
andpmoA(activesubunitgeneoftheparticulatemethanemonooxygenase
[pMMO])wereanalyzed.AnaprAgenesequencewasfoundineachhost
species(6.5a).Afterthe16SrRNAclonelibrarytherearetwothiotrophic
bacterialphylotypes,thustwodifferentaprAsequenceswereexpected.Most
likelytheaprAclonelibrarywasnotscreenedenoughtofindthesecond
thiotrophicphylotype,thatitisperhapsthelowabundantphylotype(TI).
Basedon16SrRNAandaprAanalysesbothinvestigatedtubewormscontain
onlyoneidenticalthiotrophicphylotype,andeachmusselspeciesalsobear
anownthiotrophicphylotype.OnepmoAsequencewasalsopresentand
uniqueineachmusselspecies(6.5b).

54

RESULTSANDDISCUSSION

Thevaluesofthisstudyfortheδ13CofthefattyacidsoftheEscarpia
tubewormsrangedfrom-27fortheshortchainfattyacidsto-43for
thelongerorcomplexlipids.AscanbeseeninFigure6.4thecompound
specificstablecarbonisotopesofthedifferentEscarpiaspeciesareallvery
similarbetweenindividuals.Thisiscongruentwithformerstudiesasisotopic
measurementswouldshowusavaluecloseto-29afterthefractionation
ofCO2inthechemoautotrophicprocess(Fangetal.1993).Forthemussels
wehaveabitmorecomplexstorybecauseofthedualsymbiosis.Theclose
phylogeneticrelationshipofthethiotrophic-andmethanotrophic-relateden-
dosymbiontsfoundinthisstudywiththepreviouslydescribedones(i.e.in
thenorthernGoMandotherbasins),thepresenceofthepmoAandaprA
genes,andtheirlipidisotopicvalues(seeManuscriptI),suggestthatthese
endosymbiontshavethepotentialtooxidizebothsulfurandmethane.How-
ever,electronmicroscopy,andmetabolicassays,likesulfideandmethane
incorporationsareneededtoconfirmthis.Themainisotopicvaluesofboth
musselspeciesarebetween-40and-60(seeFigure6.4).Thesevalueshave
directcorrelationtotheisotopicvalueofthecarbonsource.Methanevalues
forthethermogenicmethaneinthenorthernGoMarebetween-44and-46
andforbiogenicmethane-64to-65(Sassenetal.1999).
Specificbiomarkersforthenovelsymbionts(6.3)wouldhavebeenidealto
trackisotopicsignatures,howevernospecificlipidmarkersareknownfrom
cultivatedPsychromonasspp.,orCycloclasticusspp.exceptfortheshort
lengthpeakof11.798(see4.2,butnoshortlipidswereanalyzed.Following
thehypothesisthatCycloclasticus-relatedbacteriacouldalsobedegrading
hydrocarbons,Ilookedforthegeneticpresenceofanoxygenase.Ifound
themethyl-toluene-phenolhydroxylase(MTPH)geneinB.heckeraemussels
(Figure6.5).Furthermore,whenanalyzingthelipiddata(byF.Schubotz
andshowwithdetailsinManuscriptI)weobservedthatlipidswereheavier
inB.heckeraethaninB.brooksi(Figure6.4).Infact,bulktissueandcom-
poundspecificstablecarbonisotopesshowedameanaverageenrichmentfor
B.heckeraeincomparisontoB.brooksi.Accordingtothefattyacidcom-
positionandthephylogeneticanalysesitisclearthatbothmusselshosta
dualsymbioses.Ifbothsymbiontswouldutilizethesamecarbonsources

55

RESULTSANDDISCUSSION

wewouldexpectthemtohavesimilarlipidisotopicvalues.Therefore,the
average2.5depletionofB.brooksicomparedtoB.heckeraeismostlikely
explained1)bythepresenceoftheadditionalhydrocarbondegradingsym-
biont,or2)moreactivebacteriainoneofthespecies.AlthoughIhave
showedthegenefortheMTPHenzymetobepresent,bothresultsgiveus
onlytheclueforapotentiallyactiveassociation.Herewegivefirstevidence
forthestillincompletestudyoftheintracellularmicrobialcommunityof
bathymodiolinmusselsthatseemstobemorediversethanpreviouslyrecog-
nized.Infactdifferentmicrobialpopulationscouldcausethepresenceofall
thesedifferentlipidsthatwefindamongthehosts.Clearlymorephysiolog-
icaldataareneededtoexplaintheecologicalroleofthesenewmysterious
bioses.sym

Summary6.6

ThisisthefirsttimethatB.brooksiandB.heckerae)presentedhigher
abundanceofthiotrophicthanmethanotrophicbacteria.Relativeandtotal
symbiontabundancemightdependonenvironmentalgeochemistry,andon
hostandsymbiontmetabolism.Theinhabitationofanewsymbiontmight
beprovokedbythecharacteristicsettingsthatthisasphalticcoldseephas,
providingthisbacteriumtheadvantageofbeinginanaerobichabitatwith
availablenutrients(astheflowofwaterthroughthegillsmightbebringing
thenecessaryresources)andforthemusselthiswouldprovideanewnutrition
sourcecomingfromthedegradationofaromaticcompounds.

56

RESULTSANDDISCUSSION

Figure6.5:Phylogeneticreconstructionofbacterialsymbiontsbasedonmetabolic
markergenes.Thethreesequencesofthisstudyarehighlightedingray.(a)
Maximum-likelihoodtreebasedonthealphasubunitoftheAPSreductasegene
(aprA)sequences.(b)Maximum-likelihoodtreebasedontheactivesubunitof
theparticulateMMOgene(pmoA)sequences.Thisgenewaspresentonlyin
Bathymodiolusspp.Thesequencesofthisstudy(inbold)groupedwithformer
Bathymodiolussequences.(c)Maximum-likelihoodtreebasedontheMTPHgene.
(d)MTPHgenewaspresentonlyinB.heckerae(1.1and1.2)andnotinB.brooksi
(2.1and2.2).Thesequencefellwithinaclusterofsequencesfromhydrocarbon
s.tvironmenen

57

7Chapter

Bacteriaassociatedwithbivalves

Bacteriaassociatedwithbivalvesisaveryextensivetopic.Ifwehavea
quickoverviewofallthebacteriathathavebeendescribedassymbionts,
pathogens,orsimplybacteriaoccasionallyassociatedtoacertainbivalve
tissue,manybranchesofthebacterialkingdomarecovered(seeFigure7.1),
withapreferenceforGram-negativebacteria.Itistruethatbacteriacould
beopportunisticandbeonlyintransit,however,itisinterestingtonotethat
notallandeverygroupofbacteriaarecovered,suggestingthatcertainbac-
teriadonotlivewellinabivalvehabitat.ForexampleDeltaproteobacteria
andPlanctomyceteshaveneverbeenfoundassociatedwithbivalves.How-
ever,thesebacteriahavebeenfoundassociatedwithotherinvertebrateslike
spongesandinthecaseofDeltaproteobacteriaevenaspartofanendosym-
bioticassociation(i.e.asintheoligochaetesymbiosisdescribedinSection
1.1.3).InthenextsectionsIdiscusstheresultsobtainedfrommolecular
analysispresentingtheintranuclearbacteriaandthegeneralobserveddiver-
.ysit

7.1Intranuclearbacteria

Bacteriathatinvadeeukaryoticnucleiarecommonlyfoundinprotistsbut
haverarelybeenobservedinmulticellulareukaryotes.Recently,wedescribed
intranuclearbacteriaindeep-seahydrothermalventandcoldseepmussels
ofthegenusBathymodiolus(Zielinskietal.2009).Phylogeneticanalyses
showedthatthesebacteriabelongtoamonophyleticcladeofGammapro-
teobacteriaassociatedwithmarineanimalsasdiverseassponges,corals,

58

Chloroflexi

1Candidate division OP1Candidate division OD1Cyanobacteria

Deinococcus-ThermusCandidate division OP10SynergistetesAquificalesCandidate division OP2 and OP5ThermodesulfobacteriaCandidate division OP1

RESULTSANDDISCUSSION

Actinobacteria

Firmicutes

Candidate division OP10Candidate division OP8SynergistetesNitrospiraeAquificalesAcidobacteriaCandidate division OP2 and OP5DeferribacteresThermodesulfobacteriaCandidate division OP9Firmicutes-ClostridiaCandidate division OP1PlanctomycetesChlamydiaeCandidate division OP3LentisphaeraeerrucomicrobiaVMagnetococcusFibrobacteresGammaproteobacteria4SpirochaetesChlorobiaGammaproteobacteria1Gammaproteobacteria3FusobacteriaGammaproteobacteria-PiscirickettsiaceaeDeltaproteobacteriaEpsilonproteobacteriaBacteroidetesBetaproteobacteriaGammaproteobacteria2Alphaproteobacteria

1.00

Figure7.1:Phylogenetictreeofthemainbacterialphylabasedon16SrRNAgene.
Mostoftheexistingbacteriaphylaarevisualizedhere.Branchesinpurplearethe
phylaforwhichbacterialspecieshavebeenfoundassociatedtobivalves.

59

RESULTSANDDISCUSSION

bivalves,gastropods,echinoderms,ascidians,andfish.However,exceptfor
thebathymodiolinmusselsandashallowwaterbivalve(thePacificrazor
clamSiliquapatula)noneofthesemetazoa-associatedbacteriahavebeen
showntooccurinsidenuclei.Ithasbeensuggestedthatintranuclearbacte-
riamaycausemassmortalitiesinbivalvesbutacausalrelationshiphasnever
beenestablished.Becauseoftheirpotentiallylethaleffectonbivalvepop-
ulations,Ilookedforthepresenceofintranuclearbacteriaineconomically
importantandcommerciallyavailablebivalvespecies,i.e.oysters(Cras-
sostreagigas),razorclams(SiliquapatulaandEnsisdirectus),bluemussels
(Mytilusedulis),manilaclams(Venerupisphilippinarum),andcommoncock-
les(Cerastodermaedule).Fluorescenceinsituhybridization(FISH)revealed
thepresenceofintranuclearbacteriainallinvestigatedbivalvesexceptoys-
tersandbluemussels(seedetailsinManuscriptII).AFISHprobetargeting
allcurrentlyknownintranucleargammaproteobacteriawasdesignedforfu-
turehigh-throughputanalysesofmarineinvertebrates.Furthermore,primers
weredesignedtoquantifytheabundanceofintranuclearbacteriawithreal
timePCR.Preliminarytestswiththeseprimersshowedmassiveamountsof
intranuclearbacteriainsomebivalvespecies,raisingthequestionifthese
mightsignificantlyaffectnotonlythehealthofthebivalvesbutpossiblyalso
ofthehumansthateatthem.

7.2Diversityofbacteriaassociatedwithbivalves

Ahighdiversityofbacterialphylotypeswasfoundinour16SrRNAsequence
analysis,somearerecurrentbacteriathathaveshownupinpreviousbivalve
studies(Table7.1).Therepresentationofbacteriahereinhasagammapro-
teobacteriadominance,ashasbeenseeninthepreviousmolecularstudiesof
bivalve-associatedbacterialcommunities(Schulzeetal.2006,Cavalloetal.
2009).Bivalvesequencesfellincladeswithbacteriafromorganic-richen-
vironments:oilspills,bone-falls,fecesorinvertebratetissue.Mostofthem
belongtotheGammaproteobacteriagroup(Figure7.2)butalsothereare
Alphaproteobacteria(Figure7.3),Epsilonproteobacteria(Figure7.3),Bac-
teroidetes(Figure7.4),ActinobacteriaandSpirochetes(Figure7.5).Itcan

60

RESULTSANDDISCUSSION

behypothesizedthatthesebacteriaarespecializedonhigh-organicmatter
habitatsandthatmoreanalysismightshowahostspecies-specificbacterial
.yunitcommIntheEpsilonproteobacteriagroupitwasobservedthatthereisapar-
ticulargroupthatencloseshydrothermalventsequences(7.3).Othertwo
sequencesarefrominvertebratetissue,ParalvinellapalmiformisandB.plat-
ifrons.ThislastoneistheclosestrelativetothesequenceofB.childressi
analyzedherein.FISHanalysisrevealedthatEpsilonproteobacteriaareat-
tachedtocilia-likestructuresofthemusselgill(seeFig.2inManuscript
III).OtherEpsilonproteobacteriahavebeendescribedasinvertebrateec-
tosymbionts(e.g.inRimicarisexoculata,whichitisnotsurprisingthatthe
diversityofthisassociationisnotlimitedtocertaininvertebrates(i.e.crus-
taceans)butitmightextendwidelyinthewholeinvertebrategroup.
Associatedspirochaetesstandoutbecausetheyseemtobeastablebac-
terialcommunityinbivalvestyles(Noguchi1921,Bernard1970,Pasteretal.
1996,Prieuretal.1990,MargulisandFester1991).InmyFISHobservations
(seeFig.2inManuscriptIII)Iseethemwellestablishedinbivalvegilltissue.
Also,aspirochetesequencefromDNAgillshasbeendescribedinaLucinoma
sp.cold-seepclambyDuperronetal.(2007).Itmightbeworthwhiletodo
apopulationgeneticsstudytoinvestigatethevariabilityofthesespirochetes
specieswithinthedifferentbivalves.Itisnotclearsofarifspirocheteshave
anecologicalroleorimportanceintheassociationwithbivalves,butthey
mightbeanubiquitousmicroflorawithinthemollusksgroup(Prieuretal.
1990).Margulisetal.(1991)namedassymbiontsthestudiedspirochetesin
oysters.Wemightbeabletostudythemasubiquitoussymbioticbacteria
es.alvbivin

61

RESULTSANDDISCUSSION

0.10

VVibrio sp. Wibrio sp. SOMBO17, -10, DQ923444cuttlelfish egg case, AJ936947
clone 6, Ensis directus, Syltapes semidecussatusIsolate TMytilus edulis, SyltListonella anguillarum, X71821Crassostrea gigas., Syltficinalis, AJ936940ibrio sp. 24A, ANG gland of Sepia ofVibrio splendidus, Pecten maximus, AJ515226VMya sp., Crassostrea gigas., Mytilus edulis, Syltibrio sp. 3d, AF388392Vibrio pomeroyi, AJ560649VVibrio sp. A2, sea dragonmucus bacterium 55, mucus, Oculina patagonica, A, EF467288Y654793
ibrio sp. Mel 34, Crassostrea gigas, AJ582805VVVibrio sp. P90, ibrio sp. LMG 23856, Ruditapes philippinarum, EF599163hydrocarbon polluted sediments, EU195934
ibrio splendidus, AJ515230Vibrio lentus, AM162659VCrassostrea sp., Ensis directus, SyltMytilus edulis, Crassostrea sp., Ensis directus, Sylt, DQ925852Antarctic bacterium PHNZ10C1, Antarctic surface seawateruncultured VAliivibrio logei, Aibrio sp., Y771721hindgut of Mudsuckers, DQ340191
Aliivibrio wodanis, Y17575Y292925Aliivibrio logei, Aibrio sp. 6(2006), diseased sporophyte, Laminaria japonica, DQ642809VEnsis directus clone, Syltclone 17, Crassostrea sp., Sylt, AB304808marine sediment adjacent to sperm whale carcassesPsychromonas ossibalaenae, Siboglinum fiordicum, EU086771gamma proteobacterium endosymbiont of Psychromonas japonica, Psychromonas aquimarina, marine sediment adjacent to sperm whale carcasses,marine sediment adjacent to sperm whale carcasses, AB304804 AB304805
cultivatedPsychromonas spp. gill symbiont, (Chapopote Knoll, southern GoM)Bathymodiolus brooksi finis, AF500080Shewanella afIsolate Cerastoderma edule,SyltShewanella colwelliana, AB205577, AJ936953cuttlelfish egg caseShewanella sp. SOMBO46, uncultured bacterium, JAlteromonadaceae bacterium T1, apan TGerman Wrench sediment adden Sea, AB013824, AY177717
clone 3, Crassostrea sp.., SyltY216447uncultured bacterium, temperate estuarine mud, Aclone 37, Cerastoderma edule, Sylt, EU707315Janssand intertidal sandy sediment, middle flatuncultured bacterium, Alcanivorax sp. P75, uncultured bacterium,hydrocarbon polluted sediments orange microbial mat on a grey whale carcass, Pacific Ocean,, EU195941 AY922230
Alcanivorax borkumensis, Y12579Y258105Alcanivorax sp. DG813, Aapes semidecussatusIsolate 6 T thiotrophic groupBathymodiolus .sppCycloclasticus methanotrophic groupBathymodiolus symbionts groupestimentiferan VNIX cladePseudomonas sp. 47, EU883662Pseudomonas stutzeri, EU531806 EU849665oil contaminated soil,Pseudomonas putida, apes semidecussatusIsolate 4 TPseudomonas antarctica, soil, AM933495Pseudomonas sp. Cam-1, AF098464Pseudomonas sp. Sag-1, AF098467

Figureobtained7.2:inGthisstudyareammaproteobacterialhighlightdedivinersitygreen.inCbivaloneslves.oriThesolatesclonesareandshownisolatesand
namedaftertheirhostorigin.
62

0.10

RESULTSANDDISCUSSION

Sylt, Ensis, clone 133lake sediment enriched with heavy metals, DQ997838Ralstonia pickettii, SA-3, soil, DQ854843Ralstonia sp. Y922181unc. alpha proteobacterium, grey whale bone, Pacific Ocean, A, EU594271alpha proteobacterium STF-07, North Sea oil reservoirSylt, Crasosstrea sp., clone 6erasakiella pusilla, AB006768Terasakiella sp. UST061013-067, EF587999Tuncultured organism, deep-sea octacoral, DQ395939apes semidecussatus, TIsolateSulfitobacter litoralis, DQ097527Y902210Sulfitobacter sp. BR1, A1-B-4, EU016167Sulfitobacter sp. S1, EU365589Arctic seawaterSulfitobacter sp. BSw20064, Y573043Sulfitobacter sp. ARCTIC-P49, AY902203Sulfitobacter sp. B7, Aunidentified bacterium, AJ278782water sample from 10 m depth, AJ534229Sulfitobacter sp. HEL-77, Sulfitobacter sp. GAI-21, AF007257clone uncultured B. childressi, nGoMBathymodiolus platifrons gill symbiont, AB250697
Y531588uncultured bacterium, Indian Ocean hydrothermal vent, A mucus secretions, AJ441206Paralvinella palmiformisuncultured epsilon proteobacterium, uncultured epsilon proteobacterium, deep sea hydrothermal vent, AJ575999uncultured bacterium, iron mat, FJ535279uncultured epsilon proteobacterium, microbial fuel cell fed with marine plankton, EU052242uncultured bacterium,uncultured epsilon proteobacterium KT Montastraea faveolatac1160, AF235116 - diseased tissue, FJ203167
epsilon clone, epsilon clone, Crassostrea Ensis directussp. Mex, Crasosstrea sp., Sylt
epsilon proteobacterium Oy-M7, adult oyster mantle, DQ357825uncultured bacterium, Namibian upwelling system, EF645952epsilon clone, uncultured bacterium, Mya sp., SyltZostera marina roots, EF029017
sp., FranceCrassostreaclone 187, , Svalbard, Arctic, EU050947uncultured bacterium, sediment from the Kings Bay1 sp. BSs20195, DQ51431Arcobacteruncultured bacterium, Pearl River Estuary sediments at 6cm depth, EF999357Y548986uncultured bacterium, marine environment, Arench, AB015256unidentified epsilon proteobacterium, deepest cold-seep area of the Japan T's tube, AF449239Riftia pachyptilauncultured epsilon proteobacterium, Y548997uncultured bacterium, marine environment, Asp., SyltCrassostrea clone 27,

entsV

A

E

Figure7.3:Alpha-andEpsilonproteobacterialdiversityinbivalves.Theclones
dirandeictus,solatesCroassostrbtainedeaginigast,hisTaspestudysareemidehcighlighussatust,edB.inchildrblue.essiCandlonesfMyaromsp.Ensis16S
rRNAlibraries.Hydrothermalventgroupishighlightedalso.

63

TRESULS

DISCUSSIONNDA

Y285943Flavobacteriaceae bacterium G1B2, A, AF493675Polaribacter sp. SW019, coastal seawaterunidentified marine eubacterium, aggregate, L10947uncultured marine bacterium, diatom bloom, DQ372843uncultured marine bacterium ZD0255, algal bloom, AJ400343, SyltCerastoderma eduleclone 196, Y794064uncultured bacterium soil, sub-antarctic, Auncultured bacterium, sponge endosome, uncultured bacterium, sediment from the Kings BayTethya aurantium, AM259819, Svalbard, Arctic, EU050901
uncultured bacterium, oxygen minimum zone in eastern South Pacific, DQ810319Y948376, AHalichondria paniceasponge bacterium Zo9, uncultured bacterium, rural aerosol, EF451601uncultured bacterium, aerobic activated sludge, EF648154uncultured bacterium, compost, AM183001clone, clone, Ensis directusCrassostrea , Syltsp. Mex
Gelidibacter sp. UL5, Marine, Ulva sp., AM180740Olleya marilimosa, EF660466bacterial endosymbiont Idasuncultured bacterium, host egg, sp., AM402958Sepia officinalis, AM162577

0.10

Figure7.4:Bacteroidetesinbivalves.Theclonesobtainedinthisstudyarehigh-
lightedingray.

64

RESULTSANDDISCUSSION

ferenceseRNoguchi 1921Prieur 1981, Sugita et al e et al. 2006, 1981, SchultzThis studyNoguchi 1921, Murchelano & Brown 1968jagopalan &SivaRlingan a1978Noguchi 1921, Prieur 1981, this studylingan ajagopalan &SivaR1978Sugita et al 1981this studyNoguchi 1921, Prieur 1981, e Sugita et al 1981, Schultzet al. 2006, This studyNoguchi 1921Noguchi 1921, Cundell & ung 1975oYNoguchi 1921, Sugita et al 1981Sugita et al 1981Sugita et al 1981e et al. 2006Schultz
bact.Neisseria, CorynobacteriumNeisseria, CorynobacteriumMoraxellaSulfitobacterCorynobacterium, Arthrobacter
Others ermentativeFProteolytic bact.PhotobacteriumCCdiaEpsilonISSCCISS
yChlamSother AlphaadersSCSISother GammaSCCSMSCSCSCSS
degrdrocarbon- HyCSBacteroidetesSSSISSpirochetesMIS EMMMC
VibrioCSCISC 87%C26%MCC31%C 25%CCCCCCCSCSCCCCCCCCCCSCCISSCSCSCMCCCCCCCCCCISISISIS
MicrococcusEnterobacteria.anella spp-ShewAcinetobacterISAchromobacterPseudomonasSCICSISCCC
CSSpeciesOystersOstrea edulisCrassostrea gigasC. virginicaC. cuculataMusselsMytilus edulisMercenaria mercenariaMytilus viridisMytilus coruscusClamsCerastoderma eduliTapes (Venerupis) spp.Siliqua patulaEnsis spp.Mya arenariaMactra veneriformisPhacosoma japonicumScapharca broughtoniiPanopea abrupta

65

cultured(M),icroscopicallymdeobserveenbevahacteriabwheretudiesSes.lvabivintudiessyrsitedivacterialB.1:7bleaTvhastudyneohantoremecausebeatedeprarelettersSome(S).sequencedrRNA16Stheiror(I),isolated(C),aracterizedhce.tudyshistromfulturescandsequenceshetredInecies.psthe

RESULTSANDDISCUSSION

Bacterial7.3ationcultiv

Bacteriabelongingtothesamemonophyleticcladetowhichintranuclearbac-
teriabelong(calledhereNIX-clade,Figure7.6)haverecentlybeenisolated
fromseveralorganisms:atropicalsponge(Nishijimaetalinprep),aseaslug
Elysiaornata(KurahashiandYokota2007),andanechinoidTripneustes
gratilla(Beckeretal.2007).Unluckily,inthesestudiestherehasnotbeen
microscopicalobservations(NishijimaandKurahashi,pers.comm.),andit
isnotknownifbacteriawereinthehostnuclei.Nevertheless,Idecidedto
trythecultivationoftheintranuclearbacteriaassociatedtoTapessemide-
cussatus,Cerastodermaedule,Crasosstreagigas,andEnsisdirectuswiththe
mediausedinthetwopublishedstudiesandanenrichedmedium.Selected
bivalvesweretheonesthatpresentedNIX-relatedbacteriainthe16SrRNA
analysisandglycerolsampleswereavailable.
NoNIX-relatedbacteriawerecultivable.However,manyothertypesof
bacteriawereisolated.NotverysurprisinglyIcouldisolateseveralVibrio
spp.fromT.semidecussatus.Vibriospp.seemtoberecurrentinbivalves
(seeTable7.1forallthecultivatedandobservedbacteriainbivalves)andin
facttheyarecalledfacultativepathogens(Prieuretal.1990,Beaz-Hidalgo
etal.2010).ThestudyofVibriospp.isverybroadbecauseofthehu-
manpathogenicstrainsthatassociatetoandcanbetransmittedthrough
ediblebivalves(e.g.Canesietal.2005,ParanjpyeandStrom2005).Vibrio
strainswerealsoobtainedfromTapessemidecussatus,includinginthemedia
withCTAB,thatwasusedbeforebyPlanteandcoworkers(2008)toisolate
surfactant-resistantbacteriawiththeaimtoobtainbacteriapotentiallyusu-
fulforenvironmentalremediation.Alreadyobservedinpreviousworks(Ra-
jagopalanandSivalingam1978,Sugitaetal.1981)Icouldcultivatebacteria
fromtheActinobacteriaphylum(Kocuriasp.andDermacoccussp.),and
Bacillussp.oftheFirmicutesphylum.Krokinobactersp.andAlcanivorax
sp.isolatesareofspecialinterestbecausethefirstonesarebacteriathat
seemtobespecializedinthedegradationoforganicmatter(Khanetal.
2006)andthesecondonesarehydrocarbon-assolesourcedegradingbacteria
(Headetal.2006).Manytimescoastalbivalvehavebeentakenasabiologi-

66

RESULTSANDDISCUSSION

wall-less spirochete, M87055clone, Crassostrea Spirochaeta coccoidessp. Mex, host hindgut, Neotermes sp., AJ698092
spirochete endosymbiont lucinid bivalve, AM236337uncultured spirochete BHI80-158, uncultured Spirochaeta sp., termite gut homogenate, Alvinella pompejana tubes, AJ431240Reticulitermes speratus, AB088873
Y995150uncultured spirochete sp. anaerobic lake hypersaline sediment, ASpC-2, Auncultured spirochete hypersaline lake, AF454308clone, Borrelia sp. R57, Ensis directus, SyltClethrionomys glareolus, AY626138
sp. CP1, U42638Cristispira uncultured organism deep-sea octacoral, DQ395607clones, Spironema culicisEnsis directus of mosquito , Culex pipiensCerastoderma edule, AF166259., Sylt
Y605160uncultured spirochete, microbial mat, Auncultured bacterium, hypersaline microbial mat, DQ154857uncultured candidate, hypersaline microbial mat: Guerrero Negro, DQ329631sp., SyltCrassostrea clone, uncultured spirochete, termite gut, uncultured spirochete, gut proctodeal, Microcerotermes Nasutitermes sp., EF454267sp. 2, AB191843
uncultured spirochete, biofilm at hydrothermal vent orifice, AM712343, AJ877255Ilyobacter psychrophilus, SyltEnsis directusclone, , Svalbard, Arctic, EU050917uncultured bacterium, sediment from the Kings BayFusobacteria bacterium HAW-EB21, Auncultured bacterium, Amsterdam mud volcano, deep Eastern Mediterranean, AY579753Y592371
sp., cold seep sediment, AB189363Fusobacterium uncultured uncultured Fusobacteria Fusobacteria bacterium Ko71bacterium, 1, basalt, AF550592Paralvinella palmiformis mucus secretions, AJ441229
Y548984uncultured bacterium, marine environment, Auncultured prokaryote, hot fluid vent, AM268765, EF063619uncultured bacterium, anaerobic methanogenic UASB reactorclone, Crassostrea sp. Mexuncultured bacterium, full-scale anaerobic UASB bioreactor, AY426453
sp. HNR05, root, Chinese cabbage, EU373345Kocuria apes semidecussatusTIsolate 1 Dermacoccus sp. Ellin183, AF409025apes semidecussatusTIsolate 2 Y269868Micrococcus sp. 5, Carpodacus mexicanus, A

0.10

Figure7.5:SpirochaeteandFusobacteriadiversityinbivalves.Theclonesand
isolatesobtainedinthisstudyarehighlightedinviolet.Clonesorisolatesare
shownandnamedaftertheirhostorigin.

67

RESULTSANDDISCUSSION

calmarkertoassesspollution(e.g.Nishihamaetal.1998,Bresleretal.1999,
Verlecaretal.2006).ThepresenceofAlcanivoraxbacteriainclamstissue
couldindicateastrongoilinfluenceinNorthSeabeaches,andinfactthis
canbeaneasy-to-evaluatebiologicalmarkerforoilpollution:thepresence
ofAlcanivoraxspp.inbivalvestissue.

7.4SummaryandOutlook

Bivalvegillsprovidetobacteriaanidealhabitat,withprotectionfromgrazers
andconstantfluidofnutrients.Bacteriamightbecomplementingtheirhost
nutritionorcontributingtometaboliteproduction.Degradationofbacteria
bybivalveenzymeshasbeenobservedanditseemsthatthisdegradation
providesdissolvedcompounds(BirkbeckandMcHenery1982,Amouroux
andAmouroux1988,McHeneryandBirkbeck1985)andimprovesbivalve
nutrition(Delaunayetal.1992).Bacteriacouldprovide5%to10%carbon,
and20%nitrogenfromthebivalverequirements(Prieuretal.1990).Astable
microbiotacouldbeprovidingprotectiontobivalvesthankstocompetition
againstotherbacteriapotentiallypathogenic.Also,microbiotacouldbe
secretingantimicrobialsubstancesthathavebeenobservedtobecommunin
bacteriaisolatedfrombivalves(Zhengetal.2005).
Ecologicalstudieswithmoleculartechniquesarescarceandtheycould
helptodisentangletheinteractionpatternsbetweentheendemicmicroflora
andtheinvasiveone,explainingthebenefitsandcontrarietiesthatsymbi-
oticorpathogenicbacteriabringalong.Itisimportanttounderstandthe
distributionofpathogenicbacteriainthemarineenvironmentstopredictpo-
tentialhealthconcernstransmittedbyseafood.Ecologicalparameterssuch
asnutrientavailability,temperature,andsalinityinfluencethepresenceand
persistenceofbacteria.However,IsuggestthatbacterialikeVibrio,Pseu-
domonas,SpirochetesandEpsilonproteobacteria(seeTable7.1fordifferent
bacteriaoccurrence)presentinbivalvesarenotonlyrandomlythere.Itis
inparttheresultofthesurroundingwatercommunitybutitmightalsobe
theresultofacommonevolutionbetweenhostandbacteriathatnormally
associatetoinvertebratesorhighorganicmattercontenthabitats.Wewould

68

RESULTSANDDISCUSSION

Nix-721

Nix-721

90 Endonucleobacter bathymodioli group *Bnix-643/Bnix-64
81100Ensis directusCerastoderma edule razor clam clone, Sylt NIX-I (4) clam clone, Sylt NIX-I (5) **Bnix-643II/Tnix-64Nix-721
98Tripneustes gratillaocean water clone, Bohai Bay echinoid isolate bacteria, AM495252, ocean water, FJ154998
79100Oceanrickettsia ariakensis from Oceanrickettsia ariakensis, from Crassostrea ariakensisCrassostrea ariakensis, DQ1, DQ12391418733
oyster clone, GoMCrassostrea gigas65100Siliqua Patula razor clam clone, full seq*Snix-64/Snix-643Nix-721
fish tract clone, EU884930Pomacanthus sexstriatus ascidian clone, Mediterranean Sea, DQ884170Cystodytes dellechiajei coral clone, DQ312235Alcyonium antarcticum8399100Alcyonium antarcticumAlcyonium antarcticum coral clone, DQ312237 coral clone, DQ312243
coral clone, DQ312244Alcyonium antarcticumNIX clade8396Crassostrea gigasEnsis directus oyster clone, GoM razor clam clone, SyltCnix-64/Cnix-1249
clam clone, Sylt NIX-IICerastoderma edule99, USA, EU799933ocean water clone, Newport Harbour ascidian clone, DQ884169Cystodytes dellechiajei88Endozoicomonas elysicola from Elysia ornata sea slug, AB196667Nix-721
Y494615 fish gill clone, ASalmo salarY700601 coral clone, APocillopora damicornis84Venerupis philippinarum clam clone **Tnix-64/Tnix-643
octocoral clone, DQ889921Erythropodium caribaeorum68 octocoral clone, DQ889931Erythropodium caribaeorumsponge clone, DQ917879Muricea elongata 91bleached sponge clone, DQ917830Muricea elongata 89Muricea elongata Spongiobacter nickelotoleranssponge clone, DQ917877, isolated from marine sponge, AB205011Nix-721
84Chondrilla nuculaPetrosia ficiformis sponge clone MOLA sponge clone, AM259915 531, AM990755
sponge clone HOC2, AB054136Halichondria okadia67 seastar clone KMD001, EU599216Asterias amurensis sponge clone HOC22, AB054156Halichondria okadia100 sponge clone HOC25, AB054159Halichondria okadia95 bivalve gill clone, EF508132Acesta excavata gill clone, seagrass beds, EU487857Ruppia maritima63 gill clone, seagrass beds, EU487858Ruppia maritima, AF084850Bdellovibrio bacteriovorusSequences from intranuclear bacteria confirmed by FISH0.10***Sequences that are found intranuclearly or sorrounding the nuclei (after FISH)Sequences from this study - shallow water bivalvesspp.Bathymodiolus Sequences from deep-sea mussels Cultured strainsProbes used in this study

Nix-721

Nix-805

Nix-805

Figure7.6:16SrRNAphylogenetictreebasedonmaximumlikelihood(RAxML)
analysis.NIX-cladebelongstotheGammaproteobacteria.Sequencesfromthis
study(highlightedingrey)andallcloselyrelatedsequencesfoundintheliterature,
bathincludingymodiolithet(inhreeyellocultivw)areatedshostrainswn.P(inrobesblue)(inandgreentheCsquare)andidatusdesignedEndontotargetuclear
eachspecifichostweredesignedforuseinFISHanalysisandNix-721andNix-805
forrealtimePCR.Sequenceswithastar(*)indicatenuclearphylotypesconfirmed
bysurroundingFISH.ThetheT.nucleiasemidecussatusndveryrarelysequenceinside.(withtwostars**)wasfoundtypically

69

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72

Resultingmanuscriptsfromthisthesiswork
tributions:oncand

ManuscriptI:L.Raggi,F.Schubotz,K.-U.Hinrichs,J.M.
Petersen,J.FeldenandN.Dubilier.Bacterialsymbiontsof
BathymodiolusmusselsandEscarpiatubewormsfromChapopote,
anasphaltseepinthesouthernGulfofMexico.
preparation.inscriptuManL.R.:developedtheconcepttogetherwithN.D.,didthe16SrRNA
sequencingandanalyses,designedFISHprobesandperformed
theFISHexperiments,helpedwiththecarbonisotopeanalyses,
conceivedandwrotethemanuscript.F.S.:didthecarbonisotope
analysisoflipids.K.U.H.:developedthecarbonisotopeanalysis
method.J.M.P.:conceivedandeditedthemanuscriptwithL.R.
J.F.:providedsamples,andinformationaboutthegeochemical
parametersfromtheChapopotesite.N.D.:developedtheconcept
withL.R.,conceivedandeditedthemanuscript.
ManuscriptII:L.Raggi,D.FinkandN.Dubilier.Anintranu-
clearbacterialparasiteinshallowwaterbivalves.
preparation.inscriptuManL.R.:developedtheconcepttogetherwithN.D.,didthe16SrRNA
sequencingandanalysis,designedFISHprobesandperformedthe
FISHexperiments,conceivedandwrotethemanuscript.D.F.:
performedtheqPCR.N.D.:developedtheconceptwithL.R.,con-
ceivedandeditedthemanuscript.

74

MANUSCRIPTS

ManuscriptIII:L.Raggi,andN.Dubilier.Minireview:Bacte-
rialdiversityofshallow-waterbivalves.
preparation.inscriptuManL.R.:developedtheconcept,didthe16SrRNAsequencingand
analyses,performedcultivationexperiments,conceivedandwrote
themanuscript.N.D.:developedtheconceptwithL.R.,conceived
andeditedthemanuscript.

ManuscriptIV:F.U.Zielinski,A.Pernthaler,S.Duperron,
L.Raggi,O.Giere,C.BorowskiandN.Dubilier.(2009).
Widespreadoccurrenceofanintranuclearparasiteinbathymodi-
olinmussels.EnvironmentalMicrobiology11(5):1150-67.
F.U.Z.:developedtheconcept,didthesampling,cloning,sequenc-
ing,andmicroscopyanalyses.A.P.:performedsomeFISHex-
periments.S.D.:providedsupportwithsampling,phylogenetic
reconstruction,andprobedesign.L.R.:did16SrRNAsequenc-
ingandFISHexperimentsonspecimensfromtheGulfofMexico.
O.G.:helpedwithsamplinganddidtheTEM.N.D.:developed
theconcept,conceivedandeditedthemanuscript.

75

:IscriptuMan

BacterialsymbiontsofBathymodiolus

umsselsandrpiaaEsc

tubeormswfromChapopote,anasphaltseepinthesouthern

Luciana

76

ofGulfMexico.

Raggi,FlorenceSchubotz,Kai-UweHinrichs,

JillM.PetersenandNicoleDubilier

In

ationarepPr

ChapopoteEscarpiaandBathymodiolussymbioses

Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms
from Chapopote, an asphaltic seep in the southern Gulf of Mexico
L. Raggi1, F. Schubotz2, K.-U. Hinrichs2, J.M. Petersen1, J. Felden1, N. Dubilier1
1 Max-Planck Institut for Marine Microbiology, Celsiusstr. 1, 28359, Bremen, Germany
2 MARUM, University of Bremen, Leobener Str., 28359 Bremen, Germany
Keywords: endosymbiosis, thiotrophic, methanotrophic, Bathymodiolus mussels, Escarpia
tubeworms, asphalt, cold seep
Running head: Chapopote Escarpia and Bathymodiolus symbioses
Corresponding author: Nicole Dubilier
Max Planck Institut for Marine Microbiology
Celsiusstr. 1
emen r B28359,any mGer 932 Phone: +49 421 2028e-mail: ndubilie@mpi-bremen.de
This article is intended for publication in the journal:
Marine Ecology Progress Series

77

IscriptuMan ractstAb Chemosynthetic life was recently discovered in the southern Gulf of Mexico (sGoM) where
lava-like flows of solidified asphalt cover a large area at 3000 m depth; with oil seeps and gas
hydrate deposits also present (MacDonald et al. 2004). Animals with chemosynthetic
symbionts such as vestimentiferan tubeworms, mussels, and clams colonize this site called
Chapopote. Based on morphological and molecular analyses (COI gene), two mussel species
are present at this site, Bathymodiolus heckerae and B. brooksi, and a single Escarpia
tubeworm species. Comparative 16S rRNA sequence analysis and FISH showed that all three
host species harbor intracellular sulfur-oxidizing symbionts that are highly similar or
identical to the symbionts found in the same host species from northern GoM (nGoM) sites.
The mussels also harbor methane-oxidizing symbionts, and these are identical to their
northern GoM conspecifics. Unexpectedly, we discovered a novel symbiont in B. heckerae
that is closely related to hydrocarbon degrading bacteria of the genus Cycloclasticus. We
found in B. heckerae the Methyl-toluene-phenol hydroxylase (MTPH) gene and stable carbon
isotope analyses of lipids indicative for heterotrophic bacteria were consistently heavier in B.
heckerae by 3‰ than in B. brooksi., indicating that the novel symbiont might use isotopically
heavy hydrocarbons from the asphalt seep as an energy and carbon source. The discovery of a
novel symbiont that may be able to metabolize hydrocarbons is particularly intriguing
because until now only methane and reduced sulfur compounds have been identified as
energy sources in chemosynthetic symbioses. The large amounts of hydrocarbons available at
Chapopote would provide these mussel symbioses with a rich source of nutrition.

78

ChapopoteEscarpiaandBathymodiolussymbioses

Introduction
The discovery of cold seeps was a surprising event showing great communities of big sized
organisms living at these recondite places (Paull et al. 1984). Cold seep ecosystems are some
of the most productive on Earth, and it was initially unclear how, with what it seemed to be
very little input of organic matter from photosynthesis, these new fauna could even survive
down there (Van Dover 2000). Association of these organisms with chemosynthetic bacteria
is the answer. More and more different sites and chemosynthetic-based habitats are been
discovered, as the underwater tools are each time more equipped to stay longer and deeper in
the sea floor. In April 2004 a natural asphalt (bitumen) deposit at 3000 m depth in the
Campeche Knolls, southern Gulf of Mexico (sGoM), was discovered and was named
Chapopote (MacDonald et al. 2004). Cold seeps with methane and hydrocarbon seepage are
widespread in the GoM as a result of its unique tectonics and geological history (Macgregor
1993, Bryant et al. 1991, Ewing 1991), however this is the first time that a classical cold-seep
fauna community was found coexisting with natural asphalt. The biology and microbiology
of the northern GoM seeps is well studied (e.g. Fisher 1993, Cavanaugh et al., 1987,
Cavanaugh 1993, Carney et al. 2006, Cordes et al. 2005, 2007, Duperron et al. 2007), in
contrast with the little studied southern GoM (Fig. 1). The presence of symbiotic bacteria in
Bathymodiolus mussels and in escarpid tubeworms is one of the intensively studied topics in
the nGoM. Three Bathymodiolus species in the northern Gulf of Mexico have been described;
B. childressi, with methanotrophic bacteria, B. heckerae and B. brooksi, with a dual
symbiosis of methanotrophic and thiotrophic bacteria. Also three different tubeworms genera
are recognized and all of them bear thiotrophic symbionts: Escarpia sp., Lamellibrachia sp.
and Sepiophila sp. The distribution of the different species varies along the GoM (Fig 1), in
the Louisiana Slope B. childressi, L. luymesi, and S. jonesi are common and abundant;
Atwater Canyon retain only B. brooksi and Missisipi Canyon only B. childressi. In the
Florida Escarpment B. heckerae, B. brooksi and E. laminata are found. The Alaminos
Canyon holds B. brooksi, B. childressi, and E. laminata. These species seem to be endemic of
the GoM except B. heckerae that is found also in the Blake Ridge diapir (Salerno et al. 2005).
All these species of both tubeworms and mussels get nutrients and benefits from their

79

IscriptuMan chemosynthetic association using only sulfide, methane, or both as energy sources, no further
sources are currently known.
Lipid analysis of mussels in the northern Gulf of Mexico, that only contain methanotrophic
symbionts have been performed in former studies. It has been observed that depending on
whether methane has a biogenic or a thermogenic source, the isotopic values of the tissue
vary between -79 to -80 ‰ and -45 to -40 ‰, respectively (Jahnke et al. 1995, MacAvoy et
al. 2002). Methane values for the thermogenic methane in the northern GoM are between -44
and -46 ‰ and for biogenic methane -64 to -65 ‰ (Sassen et al. 1999). At the Chapopote
Knoll, the stable carbon isotope composition of methane is between -40 ‰ (in the asphalts) to
-60‰ (in the sediments), representing a mixture of thermogenic and biogenic methane
(Schubotz et al. subm.). In this study, we do genetic (phylogenetic and metabolic) and lipid
analyses focusing on the question of whether the asphalt in Chapopote site might be shaping
the community, in particular the bacterial symbiotic community of invertebrates, adding an
extra carbon source. This habitat is a novel setting because of this heavy oil called asphalt.
Asphalt has high amounts of asphaltenes that make it heavier than water. Thus, the oil stays
in the seafloor, while in other settings the oil leaks upward to the water surface. It creates
then an interface where hydrocarbons can be aerobically oxidized. Also, this is a system with
new substrate for the megafauna to settle, as there are not just carbonates but also solid
asphaltic formations.

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ChapopoteEscarpiaandBathymodiolussymbioses

Material and methods
Specimen collection
Mussels and vestimentiferan tubeworms were collected at the base of Chapopote Knoll with
the ROV Quest aboard the RV Meteor during the M67/2 cruise (April 2005). Bathymodiolus
mussels were collected from a mussel bed (21°53.98´N; 93°26.12`W) at a water depth of
2923 m. Four mussels were recovered during dive 83; two were identified as species 1 and
the other two as species 2. The mussel gills were dissected immediately after recovery and
segments were frozen and stored at -20°C for DNA or lipid extraction. Other fragments were
fixed for FISH with 2% PFA and stored at 4°C in 0.5X PBS / 50% ethanol.
Tubeworms were collected in two different dives: dive 82 at 21°53.95´N; 93°26.23`W, 2918
m water depth, and dive 83 at 21°53.94´N; 93°26.25`W and water depth 2915 m. Three
tubeworms from dive 82 (Tbw 1, Tbw 2 and Tbw 3) and one from dive 83 (Tbw 4) were
extracted from their tube and different pieces were frozen for DNA extraction or fixed for
.FISH DNA extraction and PCR amplification
DNA was extracted from frozen tissue according to Zhou et al. (1996) with the following
modifications. Briefly, 2 ml of extraction buffer and 20 µl proteinase K (20 mg/ml) were
added to approximately 100 mg of sample, and incubated for 1.5 hours at 37°C. Then 200 µL
of 20% SDS were added and incubated for 2 hr at 56°C. The liquid phase was recovered after
centrifugation at 14000 g for 20 min and cleaned once with 1 V
phenol/chloroform/isoamylalcohol (25:24:1) and a second time with
chloroform/isoamylalcohol (24:1). DNA was precipitated with 0.6 V isopropanol and
dissolved in Tris-EDTA buffer. The extracted DNA was used for both host and bacterial
symbiont analysis.
Host COI genes were amplified using 36 PCR cycles. For the mussels the primers LCO-
1560 and HCO-2148 (Jones et al. 2006) were used and for the tubeworms LCO1490 and

81

IscriptuMan HCO2198 (Folmer et al. 1994). Bacterial 16S rRNA genes were amplified using 20 PCR
cycles with universal primers GM3 (8F) and GM4 (1492R) (Muyzer et al. 1995). Metabolic
marker genes were amplified using 23 PCR cycles. Primers aps1F and aps4R were used to
amplify aprA gene (Blazejak et al. 2006) and the pmoA gene was amplified with A189F and
MB661R primers (Costello & Lidstrom 1999). We tested all the ring-hydroxylating
monooxygenases primers used by Baldwin et al. (2003), and only the RMO-F and RMO-R
pair gave an amplification product of the expected size (see Table S2 for primer sequences
and annealing temperatures). The expected 500 bp product was cloned and sequenced. We
obtained sequences of 300 bp length which resulted to be a fragment of a
e.oxylasmethane/toluene/phenol hydr Tubeworm genetic analysis
COI PCR products of the four tubeworms were directly sequenced from the PCR product, in
both directions. Symbiont 16S rRNA and aprA genes of tubeworms 1 and 3 were cloned
using the TOPO-TA system (Invitrogen) and analyzed. Genes pmoA and MTPH could not be
amplified in the tubeworms.
Mussel genetic analysis
COI PCR products of the four mussels were directly sequenced in both directions. Symbiont
genes of each mussel (16S rRNA, aprA, pmoA and MTPH were cloned using the TOPO-TA
(Invitrogen) or pGEM-T Easy (Promega) cloning vectors. 16S rRNA clones were partially
sequenced with primer 907RC (Muyzer & Smalla 1998) and representative clones from each
host individual were chosen for full sequencing in both directions. Metabolic marker genes
were sequenced with the respective primers (both strands). aprA and pmoA genes were
analyzed in one individual of each species. MTPH was amplified from both individuals of B.
heckerae but the product of only one individual could be successfully cloned and sequenced.
Sequencing was performed using the BigDye terminator v3.1 Cycle Sequencing Kit along
with the Genetic Analyzer Abiprism 3130 (Applied Biosystems).

82

ChapopoteEscarpiaandBathymodiolussymbioses

isylogenetic analyshP Sequences were analyzed with Sequencher (Genes Codes Corporation) and ARB (Ludwig et
al. 2004) sofwares. 16S rRNA Sequences were aligned within the Ref_96 SILVA database
(Pruesse et al. 2007). Databases for each metabolic marker gene were constructed with
publicly available sequences for each gene, and the alignment was produced with
CLUSTALW implemented in ARB (Ludwig et al. 2004). Phylogenetic trees were calculated
with the ARB software. All sequence comparisons are given as percentage sequence identity
(% similar nucleotides) after calculations of distance matrix. For tree reconstruction, only
long sequences (~1400 bp for 16S rRNA, ~650 bp for COI, ~1200 bp for aprA, ~450 bp for
pmoA) were used, except for the MTPH gene that we used the 300 bp sequence. Phylogenetic
trees of 16S rRNA gene sequences were calculated by neighbor joining, maximum parsimony
and maximum likelihood methods. As the differences in the resulting tree topographies were
not significant we only present the maximum likelihood results. Sequences with more than
99.7% identity (% identical nucleotides) were grouped and are shown as one single sequence.
To assess nodes robustness in the trees, 1000ML bootstrap replicates were run.
Probe design and testing
All probes used in this study were checked for mismatches against our sequences of interest
and the Silva 96 SSU Ref database (Pruesse et al. 2007). The Cycloclasticus probe (Cypu-
829) was originally used without formamide (Maruyama et al. 2003). We performed a
formamide series between 10% and 70%, and the probe hybridized between 10% and 40%
formamide. The probe NON338 (Amann et al. 1990) was used as a negative control and
EUB338 (Wallner et al. 1993) as a positive control. Two new probes were designed (using
the ARB Probe Design tool) to match the Escarpia symbiont, TbwT-643 and TbwT-139,
both of which were tested for FISH at 20% formamide. Since these probes gave low intensity
signals, we tested an HRP-labeled TbwT-643 probe. A 20% to 60% formamide series was

83

IscriptuMan performed and signals were observed at all formamide concentrations (see probes and
formamide concentrations for all probes in Table 1S).
FISH and CARD-FISH
A piece of tubeworms and gills were dehydrated in an ethanol series and embedded in low-
melting temperature polyester wax (Steedman 1957). Wax cubes were cut into 5 or 6 µm
sections with an RM2165 microtome (Leica, Germany) and mounted on Superfrost-Plus
slides (Menzel-Gläser). Polyester wax was removed by washing three times in absolute
ethanol (5 min each), and sections were rehydrated in a 96%-80%-70% ethanol series.
Sections were permeabilized in Tris-HCl (20 mM, pH 8), proteinase K (0.05 mg ml-1 in Tris-
EDTA, pH 8, at 37°C), and washed in MilliQ water (5 min each). For in situ hybridizations
with fluorochrome- (FISH) or horseradish peroxidase (HRP)-labeled probes (CARD-FISH)
and subsequent staining with DAPI, sections were processed as described previously
(Duperron et al. 2007, Lösekann et al. 2008, Pernthaler et al. 2002).
Lipid biomarker/isotopic analysis
Lipids of freeze-dried and homogenized mussel gill and tubeworm tissue were extracted four
times with a modified Bligh and Dyer method described in Sturt et al. (2004). Briefly, in the
first two steps, dichloromethanol (DCM), methanol and a phosphate buffer (2:1:0.8) were
added to the soft tissue and cell lysis was initiated during microwave extraction (15 min at
70°C; Brand), for the last two steps the phosphate buffer was exchanged with 0.5 M
trichloroacetic acid. Total lipid extracts were collected after liquid-liquid extraction with
DCM and deionized Milli-Q water. Aliquots of the total lipid extracts were saponified into
free fatty acids and neutral lipids with aqueous 0.5M KOH in methanol (3 h at 80°C)
following the protocol of Elvert et al. (2003). Fatty acids and neutral lipids were derivatized
with Bis(trimethylsilyl)trifluoroacetamide (BSTFA), yielding trimethylsilyl (TMS) -
derivatives, before analysis by gas chromatography. Structural identification of compounds
was achieved using a GC-MSplus-DSQ system (Finnigan Trace). An injection standard

84

ChapopoteEscarpiaandBathymodiolussymbioses

(squalane) was added for quantification purposes prior to analysis on a Thermo-Finnigan
Trace GC coupled to a FID. Determination of compound specific stable carbon isotopic
compositions was performed on a gas chromatograph coupled to an isotopic ratio mass
spectrometer (GC-IRMS). Intact polar lipids (IPLs) were also analyzed with a HPLC-ESI-
MSn system as described previously (Sturt et al. 2004). All isotopic values are reported in the
delta notation (d13C) and are relative to the Vienna PeeDee Belemnite Standard. The isotopic
compositions of the TMS-derivatives were corrected for the isotopic values of the methyl
groups attached during derivatisation (-47.2‰). The standard deviation of replicates and an
injection standard (hexatriacontan) was <1 ‰.
sticStatis The statistical analyses were performed with the SigmaStat software (version 3.5; Jandell
Scientific, San Rafael, CA). To determine differences in isotopic fatty acid values ANOVA
was performed. As the data were not normally distributed, comparisons were analyzed using
the nonparametric Kruskal-Wallis ANOVA on ranks with Dunn´s method as the post hoc
.tste

85

IscriptuMan s ltuRes Host COI gene phylogeny
Cytochrome oxidase I (COI) gene sequences from the four tubeworms were >99.7%
identical, and shared >99.1% identity with the described species Escarpia laminata from the
northern Gulf of Mexico, E. spicata from the Guaymas and Santa Catalina Basins and with E.
southwardae from the Zaire Margin (McMullin et al. 2003, Black et al. 1997, Feldman et al.
1998, Andersen et al. 2004). Four almost identical haplotypes were found within our
sequences each with one synonymous substitution in three polymorphic sites. They fell
within a well supported clade of Escarpia sequences (98% bootstrap support) and this clade
grouped together with other seep vestimentiferans with 99% bootstrap support (Fig 2).
COI gene sequences of the mussels identified morphologically as Bathymodiolus sp. 1 were
99% identical and clustered with sequences from B. heckerae from the northern Gulf of
Mexico (>98.4% identity). Sequences of the two mussels identified as Bathymodiolus sp. 2
were 99.4% identical and clustered with B. brooksi sequences from the northern Gulf of
Mexico (>98.8% identity). From here on, we call B. heckerae to Bathymodiolus sp.1 and B.
brooksi to Bathymodiolus sp.2.
Tubeworm symbiont 16S rRNA phylogeny and in situ localization
The 9 full sequences obtained from the 16S rRNA clone libraries of both analyzed
tubeworms were identical between each other (a total of 209 partial sequences were analyzed
and also identical) and with the endosymbionts of Escarpia laminata and Lamellibrachia sp.
from the Northern GoM (Nelson & Fisher 2000, McMullin et al. 2003), and E. spicata and
Lamellibrachia barhami from the Guaymas Basin (Vrijenhoek et al. 2007). These sequences
together with the almost identical symbiont sequence of L. columna (99.9%) from Lau Basin,
and L. barhami from Middle Valley (Nelson & Fisher 2000, McMullin et al. 2003) formed a
well supported clade with a 100% value (Fig. 3b). The bacterial symbionts were clearly
observed in sections of the tubeworm trophosome tissue with the specific probe TbwT-643.

86

ChapopoteEscarpiaandBathymodiolussymbioses

They are present in highly abundant groups and patchily distributed in the trophosomal tissue
(Fig. 5a).
Bathymodiolus heckerae symbionts and in situ localization
The B. heckerae 16S rRNA clone library was dominated by sequences close related to
thiotrophic B. heckerae symbionts. Two different thiotrophic-related sequences were found
with a 96.3% identity between them and one of them was identical to a sequence from the
nGoM, and the second one had a 98.4% identity with the closest relative from the nGoM as
well. We also obtained one methanotroph-related phylotype, which matched exactly with
methanotrophic-related sequence from the nGoM (Fig. 3b). In addition to the above-
described bacteria a Cycloclasticus-related species was found in the B. heckerae data-set
which had a 97.9% identity with Cycloclasticus spirillensus and other cultivated
Cycloclasticus sp. (see Fig. 3b). All different bacteria were localized with fluorescence in situ
hybridization (FISH) in both individuals B. heckerae 1 and 2, including the Cycloclasticus-
related species (Fig. 4 and Fig. S2). This last one showed very low fluorescence intensity
with FISH therefore a CARD-FISH probe was used to have a more evident observation. The
different endosymbionts were observed with specific probes for thiotrophic- and
methanotrophic- Bathymodiolus symbiont (Table S1). The methanotrophic- and thiotrophic-
related endosymbionts were co-localizing in the bacteriocytes showing a higher abundance of
thiotrophic ones (4e-f, i-j). It is not clear in our observation if the biovolume of the thiotrophs
is also larger than the methanotrophs due to the small size of the thiotrophs and the big size of
the methanotrophs (Fig 4e). Cycloclasticus-related bacteria were co-localizing in B. heckerae
with the other symbionts and with a triple hybridization it was estimated that the
Cycloclasticus-related bacteria were making 6% of the total endosymbionts. These bacteria
were not detected in other Bathymodiolus tissues, not B. heckerae from nGoM, nor B.
childressi from the nGoM, neither B. brooksi from Chapopote.

87

IscriptuMan Bathymodiolus brooksi symbionts and in situ localization
The B. brooksi 16S rRNA clone library was dominated by sequences close related to
thiotrophic symbionts of B. brooksi from the nGoM. A single thiotrophic- and one
methanotrophic-related phylotypes were obtained in this library (100% and 99.8% identities
to respective sequences from the nGoM). In addition a Psychromonas-related phylotype was
found and was most closely related to Psychromonas profunda (96.5% identity) and to a
whale-fall clone sequence (96.7% identity). All the different endosymbionts were localized in
situ with specific probes in both B. brooksi individuals. Thiotrophic- and methanotrophic-
related symbionts were present both inside bacteriocytes. Psychromonas-related bacteria
were present scarcely also within the mussel tissue; However, they do not seem to be in the
same focus plain as the endosymbionts (Fig. S1).
Metabolic marker genes
The aprA gene coding for the alpha subunit of APS reductase was amplified in B. heckerae,
B. brooksi and Escarpia sp. We analyzed 48 clones from B. heckerae 2 and as all the
sequences were identical we decided to analyze only 2 to 8 clones from each host species.
The sequences were identical between individuals for each species. The comparative
sequence analysis (Fig. 5a) grouped Bathymodiolus spp. aprA sequences with other
bathymodiolin spp. (72.5-93.9% identity). Escarpia sp. aprA grouped with other annelids
(gutless oligochaetes) and Astomonema nematode symbionts (82.5-87.8%). The pmoA gene
was only found in Bathymodiolus sp. No nucleotide differences were found within
individuals of each Bathymodiolus species. Comparative sequence analysis (Fig. 5b) showed
that B. heckerae and B. brooksi pmoA sequences are closely related to each other and to the
other Bathymodiolus spp. (90–100% identity). Methane-oxidazing free-living bacteria
sequences form a clade that is close-related (70-90%) to the Bathymodiolus group. The alpha
subunit of the MTPH gene could be amplified only from both B. heckerae individuals (Fig.
5c-d), suggesting that Cycloclasticus bacteria are only present in this species.

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isLipid analys The main fatty acids in all four investigated Escarpia species were 18:1ω7, 20:5, and 16:1ω7.
Other major fatty acids were 16:0, 18:2, 20:4, 20:2, 20:1, 22:2 and 18:0. 18:1ω7 and 16:1w7
are known biomarkers for sulfur oxidizing bacteria (Conway & Capuzzo 1991). Within all
samples 18:1ω7 and 16:1ω7 fatty acids are always most enriched and the longer and
unsaturated the fatty acids the more depleted they are (Fig. S4). This observation is consistent
with findings by Pond et al. (2002), who described a synthesis of elongated and desaturated
fatty acids from the bacterial starting product. The values of this study for the δ13C fatty
acids of the Escarpia tubeworms range from -27‰ for the short chain fatty acids up to -43‰
for the longer or complex lipids, having no variation between individual compounds. Lower
values might be due to the fractionation during the chain elongation that takes place as the
two carbon acetyl groups are added to the carbon chain (Deniro & Epstein 1977, Monson &
Hayes 1982) or when the desaturating enzymes create monounsaturated acids from saturated
fatty acids (Monson & Hayes 1982, Abrajano et al. 1994). The stable carbon isotope
composition of the weighted average mean of all fatty acids is -31.2 ± 1.5 ‰. When plotting
mean values of any tubeworm fatty acid stable carbon isotopic composition against any other
tubeworm it can be observed that all values plot on the 1:1 slope (see example in Fig. 6c).
Three sterols dominated the neutral fraction, cholesterol, ergosta-dien-ol, and cholesta-dien-ol
(For a complete overview of compounds see table S3). Sterols have the most depleted values,
between -37.7 for cholesterol to -39.2‰ for ergosta-dien-ol.. The intact polar lipid
composition of the Escarpia species is very complex, among the most abundant head groups
are phosphatidylcholines (PC) and of phosphatidyl-ethanolamines (PE) as diacylglycerols
and plasmalogens. Also present are phosphatidyl-serines (PS) and glycosidic ceramides
(sphingolipids) between many other unknown.
The dominating fatty acids in both mussels were 16:1ω7 and 16:0. They also contained large
amounts of 18:0, 16:1ω7, 20:1ω7 and polyunsaturated fatty acids 18:3, 18:2, 20:3, 20:2 and
22:2. 16:1ω7, 16:1ω7 and 20:1ω7, biomarkers that have been used as indicators for
thiotrophy were detected in both mussel species. On the contrary, the methanotrophic lipid
16:1ω8 was only detected in B. brooksi , while diplopterol, which is also a marker for aerobic

89

IscriptuMan methanotrophy (e.g. Hinrichs et al. 2003), was only observed in B. heckerae. Both mussels
contain also 4-methyl sterols, which to date have only been found in methanotrophic bacteria
(Jahnke et al. 1995, Schouten et al. 2000) and indeed they were always most depleted in 13C
(-54.9 to -50.9‰). B. brooksi additionally contained moderate amounts of lanosterol, and
4,4,-dimethylcholesta-dienol. The weighted average stable carbon isotope composition of all
fatty acids for both B. heckerae species was -41.9‰ and -44.4‰ for the two B. brooksi
species (see Table S3 for complete list of fatty acid isotopic values). The bulk gill tissue of
both B. brooksi species is also approximately 2‰ lighter compared to both B. heckerae
species, consisting of -42.9 and -40.8‰ in average, respectively. And as general observation
the carbon isotopic values of the fatty acids compared to the bulk tissue have an average
offset of ca. 2.5 ‰ for all compounds (Fig. 6a). Compound specific stable carbon isotopes of
fatty acids cluster closely together but a general trend can be observed towards the more
unsaturated the fatty acids the more depleted values they have (e.g 18:3 and 20:3 being
approx. 3 ‰ lighter than the 18:1 and 20:1). When plotting the mean B. heckerae fatty acid
stable carbon isotopic composition against the mean B. brooksi fatty acids it can be observed
that all values plot below the 1:1 slope, indicating an average depletion of B. brooksi fatty
acids in comparison to B. heckerae (Fig. 6b). This is supported by ANOVA values with p =
<0.001 (H=19,099 with 3 degrees of freedom) and the Dunn´s method, showing significant
differences between species but not between individuals of the same species. The analysis of
intact polar lipids (IPLs) revealed the fatty acid combinations and head groups of the intact
membrane lipids. Main polar headgroups of both B. heckerae and B. brooksi were composed
PE, PC, phosphatidylphosphonoethanolamines (Phos-phono), and minor amounts of PS and
phosphatidylinositols (PI). Phosphatidyl-glycerols (PG) were only detected in B. heckerae. In
B. brooksi also minor amounts of the Sphingolipid ceramide-PE (PE-Cer) were detected
). S3leb(Ta

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ChapopoteEscarpiaandBathymodiolussymbioses

Discussion
Identity of tubeworms and mussels from the Chapopote asphalt seep
The chemosynthetic fauna present at Chapopote in the sGOM is reminiscent of cold seeps in
the nGOM, dominated by tubeworms and mussels. In the first description of the Chapopote
site by MacDonald et al. (2004), the tubeworms were identified morphologically as
Lamellibrachia sp., and the mussels as Bathymodiolus sp.. Our comparative pylogenetic
analysis of the COI genes of four tubeworms identified these as belonging to the genus
Escarpia. Analysis of the four mussels sampled identified these as belonging to two separate
species, one of which is closely related to B. heckerae from the nGOM, the other to B.
brooski from the nGOM. The phylogenetic resolution of the COI gene worked well for
bathymodiolin mussels, integrating the new species into a defined group, we could name the
two species B. heckerae and B. brooksi. However, as observed before (for rev. see McMullin
et al. 2003), the resolution of this gene is not sufficient for determining tubeworm species,
especially within the escarpids that have a very similar COI sequence. However it gives a
good definition of the tubeworm genera, in this case Escarpia. The phylogeny analysis of the
tubeworms including other molecular markers (as the ND4 mitochondrial gene) would be
needed to differentiate between Escarpia species. However, we can not let drop that
vestimentiferan tubeworms have a remarkable plasticity (Black et al. 1997) and therefore E.
laminata, E. southwardae and E. spicata could be the same species. To resolve this, a
population genetic study would be required.
Phylogeny and in situ distribution of previously described symbionts
The endosymbiotic bacteria of the Bathymodiolus mussels and Escarpia tubeworms
investigated in this study are, like the hosts themselves, closely related or even identical to
their counterparts in the nGOM. Although a clone library might not be representative of the
true abundance because of well-characterized method-biases (Reysenbach et al. 1992, Suzuki
& Giovannoni 1996, Acinas et al. 2005) our clone library analysis showed a dual symbiosis

91

IscriptuMan in both mussel species, with a dominant abundance of sulfur-oxidizers. This was corroborated
with FISH observations (Fig. 4). The high abundance of thiotrophs contrasts with previous
studies of Bathymodiolus mussels from the nGOM. In all nGOM mussels investigated to
date, methanotrophic symbionts had a dominating abundance based on FISH and RNA slot
blot hybridizations (Cavanaugh 1993, Fisher et al. 1993, Duperron et al. 2007). Methane
concentrations were generally high at Chapopote, indicating that this was not the limiting
factor for the presence of methanotrophic symbionts (MacDonald et al. 2004). Unfortunately,
there are no geochemical data available for the site at which the mussels were sampled.
Methane and sulfide availability in the mussel habitat has been shown to directly influence
the relative abundance of thiotrophs and methanotrophs in the mussel gill tissue (Trask and
Van Dover 1999, Fiala-Medioni et al. 2002, Salerno et al. 2005, Duperron et al. 2007, Riou
et al. 2008). If methane is only delivered to the surface in short bursts during asphalt
eruptions, and shorter hydrocarbons are the first to diffuse out of the asphalt, then there may
not have been much methane available in the mussel habitat at the time of sampling. In these
areas, sulfide may be more abundant than methane, which would explain why thiotrophs are
relatively more abundant in the mussels at this site compared to the nGOM. At Chapopote, it
might be important that the hydrocarbons are trapped below the bituminous formations
because anaerobic hydrocarbon degradation (with sulfate) would support higher production
of sulfide (Boetius 2005). It will be important in future studies to carry out punctual
measurements of sulfide and methane concentrations in the different habitats to be able to
link them to the symbiotic microbial community.
Metabolic capabilities of previously described symbioses
Based on the phylogenetic relationship of the Escarpia tubeworm symbionts to the sulfur-
oxidizing symbionts of other tubeworms, we hypothesized that they are also sulfur-oxidizing
chemolithoautotrophs. To investigate this further, we analyzed stable carbon isotopes and
lipid profiles, and the presence of a key gene for sulfur oxidation, aprA, in the tubeworm
tissues. Based on our geochemical analyses on the Escarpia tubeworms and the lipid profile
showing 18:1w7 and 16:1w7 fatty acids presence in all four investigated individuals, we can

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ChapopoteEscarpiaandBathymodiolussymbioses

conclude that they contain thiotrophic symbionts. If the tubeworms rely entirely on their
symbionts for their nutrition, we would expect the tubeworm tissue to have a carbon stable
isotopic composition of around -29‰, based on the fractionation of CO2 during
chemoautotrophy (Fang et al. 1993). The δ13C values of this study for the fatty acids of the
Escarpia tubeworms are indeed around this value, except for the lower values for the longer
fatty acids, which could be explained by host fractionation while synthesizing the longer fatty
acids. The compound-specific stable carbon isotope values of Escarpia sp. show no
significant differences between individuals (Fig. 6), indicating a similar nutritional strategy
for all tubeworm individuals investigated. We were able to amplify and sequence an aprA
gene from tubeworm trophosome tissue (Fig. 5a). Our aprA sequences clustered with those of
free-living sulfur-oxidizing bacteria and the other thiotrophic symbionts, suggesting that this
bacterial phylotype is capable of oxidizing sulfide. Before this study, there was only a single
published aprA sequence available from lamellibrachid or escarpid tubeworms, from a
tubeworm found at mud volcanoes in the Mediterranean (Duperron et al. 2009). The aprA
sequences from other thiotrophic symbionts from deep-sea chemosynthetic ecosystems such
as the clam and mussel symbionts fall into lineage I, as defined by Meyer & Kuever (2007).
In contrast, our sequence from the Chapopote Escarpia tubeworm symbiont falls into lineage
II, and clusters with sequences from symbionts of oligochaete worms found in shallow
marine sediments (Fig. 5).
According to our metabolic marker and lipid analyses, both mussel species contain pmoA,
aprA genes and lipids characteristic of thiotrophic (16:1w7 and 18:1w7) and methanotrophic
bacteria (16:1w8, 16:1w5, and diplopterol). The pmoA gene separates well B. heckerae from
B. brooksi bacterial phylotypes. Thus, the presence of the pmoA gene and the lipid profiles
suggest that symbionts in the mussels have the potential to oxidize methane and use it as a
source of energy and carbon, as was shown in previous studies for methanotrophy in
bathymodiolin mussels (Fisher et al. 1987, 1993, Nelson et al. 1995, Cavanaugh 1993). The
main isotopic values of both species are more enriched than typically observed for dual
symbiont bearing mussels (i.e. methanotrophic and thiotrophic symbionts). Nevertheless, this
is in direct correlation to the isotopic value of the carbon source in the Chapopote site. The
heavier isotopic values for the mussel’s lipids are explained by the combination of the

93

IscriptuMan fractionation by methanotrophic bacteria that deplete by 10 - 20‰ (Barker & Fritz 1981), and
thiotrophic bacteria that provoke a depletion by 24.4‰ (Scott et al. 2004). The δ13C of the
methane at Chapopote is between -41‰ and -70‰, and water column DIC has a δ13C of
approximately 0‰ (Gruber et al. 1999). We could say then, that the isotopic values that we
got in this study could be the result of the uptake of the different carbon sources (CO2, CH4
and heavier hydrocarbons) in this complex symbiosis.
Novel symbionts in Bathymodiolus mussels, an adaptation?
Mussels in this novel environmental setting seem to have adapted well to this rare bituminous
settling surface after their normal association to carbonates. And it could be that
establishment of new symbioses helps this adaptation to happen. Independent of the host
needs, the presence of potential new bacterial symbionts seem to be related to a high organic
matter content in the environment, which in this case it does exist in Chapopote site and
would explain the presence of Psychromonas bacteria in B. brooksi tissue, and of
Cycloclasticus-related bacteria in B. heckerae. Psychromonas bacteria are heterotrophic
organisms frequently found in cold-water sediments. Only once, another Psychromonas-
related phylotype has been observed associated with an animal tissue, in the bones of a whale
fall (Goffredi et al. 2004). Cycloclasticus is a common organism that blooms in oil spills, and
it is commonly found and detected in water analysis (Kasai et al. 2002, Maruyama et al.
2003) . Here we suggest that the hydrocarbon load in the environment makes the presence of
this Cycloclasticus bacterium possible, as they have been found also in oily sediments from
shallow and deep waters. However, this is the first time to our knowledge that Cycloclasticus
sp. is observed as an intracellular bacterium as it has been shown by our FISH analysis. We
have showed here the presence of the gene for the MTPH enzyme in B. heckerae. We suggest
that the presence of this gene is due to the Cycloclasticus-related bacteria. The association
with the mussel host might provide this bacterium the advantage of being in an aerobic
habitat with available nutrients (hydrocarbons flowing through the gills) and for the mussel
this would provide a new nutrition source coming from the degradation of aromatic
compounds, including a detoxification of them. We are not aware of any specific lipid

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ChapopoteEscarpiaandBathymodiolussymbioses

markers that have been found in the so far cultured Cycloclasticus spp. When analyzing the
lipid data we can observe that all lipids are generally heavier in B. heckerae than in B.
brooksi (Fig. 6). In fact, bulk tissue and compound specific stable carbon isotopes showed a
mean average enrichment in δ13C for B. heckerae in comparison to B. brooksi. As we
discussed before it is clear that both mussels host a dual symbioses. Since the mussels co-
occur, it is highly likely that the symbionts of both utilize the same carbon sources. In
addition, the relative abundance of thiotrophs and methanotrophs is comparable for both
species, indicating that the relative contribution of thiotrophy and methanotrophy to the
nutrition of each mussel host is comparable. Based on these observations, we would expect
them to have similar lipid isotopic values. However, B. brooksi is consistently lighter than B.
heckerae, in bulk and compound-specific isotope analysis, and this is most likely explained
by the contribution of the additional hydrocarbon degrading symbiont to host nutrition.
Concluding remarks
The sGOM Chapopote Knoll fauna is similar to that at the West Florida Escarpment, nGoM,
having the presence of B. heckerae, B. brooksi and E. laminata. This gives an interesting
comparison between hosts and symbionts of both places. This study shows that the input of
hydrocarbons, derived probably from the asphalt, has directly influenced the diversity of
symbionts found in the local chemosynthetic mussels. The use of improved molecular
techniques and the discovery and investigation of novel environmental settings may reveal
that this phenomenon is more common than previously assumed. The unique environmental
conditions at Chapopote define not only the free-living microbial communities but also the
symbiotic ones.

95

IscriptuMan Acknowledgements
We thank A. Boetius who endowed the mussel samples; and the officers, crew of the Meteor,
and pilots of the ROV Quest, for expert assistance. We thank H. Sahling for the map
composition and the fruitful discussion. This work was supported by the Max-Plank Society,
the International Max-Plank Research School for Marine Microbiology and the CONACyT-
DAAD scholarship.
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316-322 62:

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Tables and figures
Fig. 1 Gulf of Mexico Basin. The most studied sites are shown with their respective
Bathymodiolus and tube worm fauna.
Fig. 2a Phylogenetic affiliation of Escarpia tubeworms based on COI gene sequences.
Maximum-likelihood tree showing vestimentiferan tubeworm species from vent and seep
environments including the 5 individuals of this study (sequences highlighted in gray). Only
bootstrap values greater than 70 % are shown.
Fig. 2b Phylogenetic reconstruction of bacterial symbionts of vestimentiferan tubeworms
based on 16S rRNA gene sequences. Maximum-likelihood tree shows within the -
proteobacteria phylum, thiotrophic symbionts of seep and vent vestimentiferans. Only one
phylotype was present in the two investigated tubeworms (in bold), and fell in group 1
(McMullin at al 2003) within Escarpia spicata, Escarpia laminata and Lamellibrachia spp.
Fig. 3a Phylogenetic affiliation of Bathymodiolus mussels based on COI gene sequences.
Maximum likelihood tree showing Bathymodiolus spp. from vent and seep environments
including the 4 individuals of this study (highlighted in gray). Only bootstrap values greater
hown.e s arthan 70 % Fig. 3b Phylogenetic reconstruction of bacterial symbionts of Bathymodiolus mussels based
on 16S rRNA gene sequences. Maximum-likelihood tree shows within the γ-proteobacteria
phylum thiotrophic, Cycloclasticus-related, Psychromonas-related and methanotrophic
bacteria. The sequences from this study are shown in bold, Note that B. heckerae individuals
have two different thiotrophic phylotypes, one Cycloclasticus- and one methanotrophic-
related phylotypes. In our B. brooksi datasets we have found one thiotrophic-, one
Psychromonas, and one methanotrophic-related phylotypes.

101

IscriptuMan Fig 4. Bathymodiolus mussels and Escarpia tubeworms from this study, and FISH images of
bacteriocytes in the mussel gill filaments and in the tubeworm trophosome. (a)
Bathymodiolus brooksi and B. heckerae mussels together with escarpid tubeworms settle in
the asphaltic sediment at Chapopote cold seep in the southern Gulf of Mexico. Each
metazoan species harbors its own specific bacterial phylotypes. (b) Escarpia tubeworms from
this study bear chemoautotrophic tubeworm symbionts. (c) Localization of the symbionts
(arrows) with a FISH specific probe through a tubeworm cross-section. (d-g) B. heckerae
mussel and respective FISH images: B. heckerae shell has an elongated shape (d); Its
filamentous gills house methanotrophic (pink) and thiotrophic (green) bacteria (e). Two
different chemoautotrophic bacterial phylotypes have been recognized. The host nuclei are in
blue, thiotrophs I in red, and thiotrophs II in yellow (f). A new hydrocarbon-degrader
symbiont in green, is co-existing with the methanotrophic bacteria in blue and the thiotrophs
in pink (g). (h-k) B. brooksi mussel and respective FISH images: the shape of the B. brooksi
(h) shell is more round and smaller than B. heckerae.. When analyzed with FISH B. brooksi
gill filaments (autofluorescence of the tissue is purple) house a methanotrophic bacterial
phylotyope in red, and a thiotrophic one in green (i). A detail of (i) shows host nuclei in blue,
methanotrophs in red and thiotrophs in green (j). A Psychromonas-related bacteria was found
to be associated with B. brooksi gill tissue (k). Scale bars: (c, i) = 50µm; (d, h) = 5 cm; (e, f,
g, k) = 5 µm; (j) = 10 µm.

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Fig 5. Phylogenetic reconstruction of bacterial symbionts based on metabolic marker genes.
Sequences highlighted in gray. (a) Maximum-likelihood tree based on the alpha subunit of
the APS reductase gene (aprA) sequences. (b) Maximum-likelihood tree based on the alpha
subunit of the particulate MMO gene (pmoA) sequences. We found this gene present only in
Bathymodiolus spp. The sequences of this study grouped with former Bathymodiolus
sequences. (c and d) Maximum-likelihood tree based on the phenol hydroxylase gene. We
could amplify the gene in B. heckerae and not in B.brooksi (see d). The sequence fall within
sequences related to high content hydrocarbon environments.
Fig 6. Stable carbon isotope measurements of lipids extracted from B. heckerae, B. brooksi,
and Escarpia sp. tubeworms tissue. (a) Carbon isotope values of lipids (circles and bars) and
bulk tissue (diamonds) from Bathymodiolus spp. and Escarpia sp. tissues. (b) B. heckerae
carbon isotope values plotted against B. brooksi values. (c) Escarpia tubeworms B and 4
carbon isotope values plotted against A1 and A2.
Table S1. Primers and probes. All the oligonucleotides used in this study are listed and their
anneling temperature or formamide concentration that were applied to them.
Table S2. Clone description of this study. In the case of the 16S rRNA gene the number of
partial sequences are shown and complete ones are in parenthesis. No data means weather we
did not obtained any sequence or we did not amplify from that individual. The dash meaning
we run the PCR but no product was obtained.
Table S3. Isotopic composition and quantity (mg of lipid per g of tissue) of fatty acids from
Escarpia tube worms
Table S4. Isotopic composition and quantity (mg of lipid per g of gill tissue) of fatty acids
from Bathymodiolus mussels.

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Fig. S1 Cross-section of gill filament of B. brooksi. (a) Methanotrophic-related bacteria

(blue) and Psychromonas-related bacteria (orange). In gray the auto fluorescence of the

tissue. Psychromonas-related bacteria are not abundant and seem to be filamentous-like

ia.bacter (Trhiotophic-rlated bactereia are not sohn but they arwe similarly prheckerae tissue). (b) Psychromonas-related bacteria in pink and DAPI in blue.

ent ases in B.

Fig. S2 Cross-section through gill filaments of B. heckerae. (a) Thiotrophic-related bacteria

T1 (in red), and T2 (in yellow). Scale bar in 40 µm. (b) A detail from a, scale bar 10 µm.

104

30˚N

25˚N

20˚N

Mexico

100˚W

ChapopoteEscarpiaandBathymodiolussymbioses

USA

Louisiana Slope “B”. childressi L. luymesiS. jonesiEscarpia sp. Alaminos CanyonB. brooksi“B”. childressiE. laminata

Mississipi Canyon“B“. childressiAtwater CanyonB. brooksi

Chapopote (this study)B. heckeraeB. brooksiYucatanE. laminataPeninsula

90˚W95˚W

Florida EscarpmentB. heckeraeB. brooksiE. laminata

0

85˚W

Fig. 1. Gulf of Mexico Basin. The most studied sites are shown mussels and tubeworm fauna. Bathymodiolus with their respective

km

500

80˚W

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(a) COI

Y326303 (Zaire Margin), AEscarpia southwardaeEscarpia laminataEscarpia laminata (Alaminos Canyon, GoM), A (Florida Escarpment, GoM), AY129128Y129131
(Santa Catalina Basin whale fall), U84262Escarpia spicataubeworm 4 (Chapopote, GoM)T (Guaymas seep), U74065Escarpia spicata (Guaymas vent), U74064Escarpia spicataubeworm 2 (Chapopote, GoM)TY129129, (Alaminos Canyon, GoM), AEscarpia laminataEscarpia laminataEscarpia laminata, (W, (Alaminos Canyon, GoM), Aest Florida Esc, GoM), U74063Y129130
9098TTubeworm 3 (Chapopote, GoM)ubeworm 1 (Chapopote, GoM)
Y129134sp. (Louisiana Slope, GoM), AEscarpia 100Seepiophila jonesi10099100Paraescarpia cf., echinospica (Nanaki Trough, Japan), D50594
spp.Lamellibrachia 100100Riftia pachyptila96100evnia jerichonataT10099Oasisia alvinae1000.10spp.Ridgeia

(b) 16S rRNALamellibrachiaSeepiophila jonesi cf. luymesi endosymbiont (GB, nGoM), Aendosymbiont (Green Canyon, nGoM), AY129092Y129100
Lamellibrachiaunclassified escarpiid symbiont (GB, nGoM), A sp. endosymbiont (Bush Hill, nGoM), AY129088Y129110
Escarpia laminataLamellibrachia barhami endosymbiont (Atwater Canyon, nGoM), A endosymbiont (Monterey Canyon, EP), AY129102Y129094
endosymbiont (Whale fall, SCB), U77482Escarpia spicataendosymbiont (Lau Basin, WP), U77481Lamellibrachia columna endosymbiont (Guaymas seep), DQ232902Lamellibrachia barhami endosymbiont (Guaymas seep), DQ232903Escarpia spicata tubeworms endosymbiont (Chapopote, sGoM)Escarpia laminataLamellibrachiaLamellibrachia barhami endosymbiont (Middle V sp. endosymbiont (Green Canyon, nGoM), U77479alley, NEP), AY129113
Y129106 endosymbiont (Florida Escarpment, nGoM), AEscarpia laminata ent groupV endosymbiont, AF165908Escarpia spicata endosymbiont, AF165909Escarpia spicata gill symbiont, U62131Solemya terraeregina0.10 gill symbiont, L01575Thyasira flexuosa

Group 3Group 2

Group 1

FIG 2. Phylogenetic affiliation of Escarpia tubeworms and their bacterial symbionts. (a) Tree based on COI gene sequences. Maximum-likelihood tree showing vestimentiferan tubeworm species from vent and seep environments including the 4 individuals of this study (sequences highlighted in gray). Only bootstrap values greater than 70 % are shown. (b) Tree based on 16S rRNA gene sequences. Maximum-likelihood tree shows within the gamma-proteobacteria phylum, thiotrophic symbionts of seep and vent vestimentiferans. Only one phylotype was present in the two investigated tubeworms (in bold), and fell in group 1 (McMullin at al 2003) within Escarpia spicata, Escarpia laminata and Lamellibrachia spp.

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(a)

(b)

ChapopoteEscarpiaandBathymodiolussymbioses

, (Florida Escarpment, GoM), DQ513441Bathymodiolus heckerae78Bathymodiolus heckeraeBathymodiolus heckerae, (Blake Ridge, Atl), A, (South Florida, GoM), AY649793Y649794
sp1 Ind1, (Chapopote Knoll, GoM)Bathymodiolus 98sp1 Ind2, (Chapopote Knoll, GoM)Bathymodiolus 82, (Barbados), DQ513449Bathymodiolus boomerang10098Bathymodiolus Bathymodiolus aff. boomerangsp., (Nigeria), EF051242, (Regab, Gulf of Congo), DQ513450
, (Lucky Strike, MAR), AB170061Bathymodiolus azoricus100Bathymodiolus breviorBathymodiolus puteoserpentis, (Lau Back Arc, WP), A, (Snake Pit, MAR), AB170062Y275544
, (Southern Central Indian Ridge), AB170042Bathymodiolus marisindicus, (Eastern Pacific Rise), AF456301Bathymodiolus thermophilusBathymodiolus brooksiBathymodiolus brooksi, (Alaminos Canyon, GoM), A, (Florida Escarpment, GoM), AY649797Y649798
8676Bathymodiolus Bathymodiolus sp2 Ind1, (Chapopote Knoll, GoM)sp2 Ind2, (Chapopote Knoll, GoM)
, (Alaminos Canyon, GoM), EF051244Bathymodiolus brooksi9976Bathymodiolus Bathymodiolus sp., (New Zeland), AB255739sp., (Manus, WP), AB101431
, (Japan margin), AB170054Bathymodiolus aduloides groupjaponicus - B.childressiIdas washingtoniaBenthomodiolus lignicola, (New Zealand), AY275546, (New Zealand), AY275545
0.10

Seep

entVSeepentVentSeep-V

cultured strainsPsychromonas(b)Psychromonas Bathymodiolus brooksiwhale bone clone C3F9, marine environment, A Y548975gill symbiont, (Chapopote Knoll, southern GoM) spp.
Bathymodiolus brooksiB. azoricus and B. puteoserpentisgill symbiont, (Chapopote Knoll, southern GoM) thiotrophic symbionts
thiotroph, (Alaminos Canyon, northern GoM), AM236331Bathymodiolus brooksi thioautotrophic gill symbiont, (vent, Galapagos Rift), DQ321716Bathymodiolus thermophilus sp. symbiont, (vent, Juan de Fuca), DQ077893Bathymodiolus Mytilid associated bacteriaThiotrophsgill symbiont , (Chapopote Knoll, southern GoM) Bathymodiolus heckerae thiotroph, (WFE, northern GoM), AM236328Bathymodiolus heckeraeBathymodiolusBathymodiolus marisindicus aff. brevior thioautotrophic gill symbiont (vent, Central Indian Ridge), DQ077891thiotrophic gill symbiont, (vent, Central Indian Ocean), DQ321715
uncultured bacterium, ridge flank crustal fluid, (crustal fluid), DQ513047gill symbiont, (Chapopote Knoll, southern GoM)Bathymodiolus heckeare thiotroph, (WFE, northern GoM), AM236327Bathymodiolus heckerae Cycloclasticus cultivated strainsBathymodiolus sp. thiotroph, sea water, (seep, Gulf of Guinea), AJ745718
, AB086228dibenzofuran-degrading bacterium DBF-MAK, sea waterCycloclasticusmarine metagenome, AACY023983802 spp.uncultured bacterium, (Newport Harbor), EU799707gill symbiont, (Chapopote Knoll, southern GoM)Bathymodiolus heckerae rough), AB036710 methanotroph, (vent, Okinawa TBathymodiolus platifrons methanotroph, (cold seep, northern GoM), AM236329Bathymodiolus childressi methanotroph, (WFE, northern GoM), AM236325Bathymodiolus heckerae, AJ745717 sp. methanotroph, sea waterBathymodiolusBathymodiolus japonicus Bathymodiolus heckerae methanotroph, (vent, Okinawa Through), AB03671gill symbiont, (Chapopote Knoll, southern GoM)1Methanotrophs
methanotroph, (Menez Gwen, MAR), AM083967Bathymodiolus azoricusgill symbiont, (Chapopote Knoll, southern GoM)Bathymodiolus brooksi methanotroph, (cold seep, northern GoM), AM236330Bathymodiolus brooksi B. azoricus and methanotrophic endosymbiont of Idas sp., AM402955B. puteoserpentis methanotrophic symbionts
0.10FIG 3. (a) Phylogenetic affiliation of based on COI gene sequences. Maximum likelihood tree showing Bathymodiolus Bathymodiolus mussels and their bacterial symbionts. (a) Tspp. from vent and ree
seep environments including the 4 individuals of this study (highlighted in gray). Only bootstrap values Bathymodiolusgreater than 70 % are shown. (b) Phylogenetic reconstruction of bacterial symbionts of gene sequences. Maximum-likelihood tree shows within the Gammaproteo-mussels based on 16S rRNAbacteria phylum thiotrophic, The phylotypes investigated in this study are shown in bold. Note that Cycloclasticus-related, PsychromonasB. heckerae-related and methanotrophic bacteria. individuals have two
difbrooksi ferent thiotrophic phylotypes, one present only one thiotrophic, one CycloclasticusPsychromonas-related and one methanotrophic phylotypes, and -related, and one methanotrophic phylotypes.B.

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ChapopoteEscarpiaandBathymodiolussymbioses

Laxus oneistus symbiont, FJ573249Allochromatium vinosum, U847590.20Olavius algarvensis symbiont 3 AB8871 , AM234051Olavius ilvae symbiont 3 AB19, AM234055Bathymodiolus heckerae symbiont (Chapopote Knoll)Bathymodiolus symbiont, BAB56133Bathymodiolus brooksi symbiont (Chapopote Knoll)B childressi endosymbiont, CAK55530Bathymodiolus thermophilus, EF641960B. heckerae symbiont (Chapopote Knoll, GoM)Bathymodiolus azoricus, EF641959B. childressi bacteria endosymbiont, CAJ85652Candidatus VBathymodiolus brevior, EF641958esicomyosocius okutanii, AP009247B heckerae endosymbiont, CAJ85654B childressi endosymbiont, CAK55529
Candidatus Ruthia, CP000488B brooksi endosymbiont, CAJ85650Candidatus Pelagibacter ubique, CP000084B. brooksi symbiont (Chapopote Knoll, GoM)Escarpia tubeworm symbiont (Chapopote Knoll)B. childressi endosymbiont, CAK55528Astomonema symbiont, DQ890382B azoricus endosymbiont of, AAX48775Olavius algarvensis symbiont 1 AB8895, AM234050Methylosarcina fibrata, AAF04265Olavius ilvae symbiont 1 APS41, AM234053Methylomicrobium pelagicum, AAC61804Inanidrilus makropetalos gamma 1 symbiont, AM228901uncultured methanotroph, AAG16920Inanidrilus leukodermatus gamma 1 symbiont, AM228902Methylobacter psychrophilus, AAX48776Lucinoma afThiobacillus denitrificans, Af. kazani endosymbiont, AM236338Y296750Methylomonas sp., ABD62312Methylobacter sp., ABD62299
0.10103..823079 AE006Chlorobium tepidum, 821 SRB

cduncultured bacterium, Sewage sludge compost, AB286802uncultured bacterium, Sewage sludge compost, AB286772uncultured bacterium, Hydrocarbon contaminated soil, DQ196344uncultured bacterium, Sewage sludge compost, AB2867810.10Bathymodiolus heckerae symbiont (Chapopote Knoll) 1.11.22.12.2
uncultured bacterium, Sewage sludge compost, AB286756uncultured microorganism, Lab-scale activated sludge degrading phenol, EF589029omato rhizoplane, EF151008Burkholderia sp., TY875728, APseudomonas mendocina, Phenolic polluted riverPseudomonas sp., Phenol-degrading bacterium, EF687004Y875742, APseudomonas fluorescens, Phenolic polluted riveruncultured bacterium, Sewage sludge compost, AB286815ariovorax sp., Phenol-stimulated enrichment, AB051715Vuncultured microorganism, Phenol-stimulated enrichment, AB051730uncultured bacterium, Sewage sludge compost, AB286796Alicycliphilus sp., EF596778uncultured bacterium, Sewage sludge compost, AB286777uncultured bacterium, Sewage sludge compost, AB286767

Fig. 5 Phylogenetic reconstruction of bacterial symbionts based on metabolic marker genes. The three sequences of this study are highlighted in gray. (a) Maximum-likelihood tree based on the alpha subunit the particulate MMO gene (of the APS reductase gene (pmoAaprA) sequences. We found this gene present only in ) sequences. (b) Maximum-likelihood tree based on the alpha subunit of Bathymodiolus spp. The
sequences. (c and d) Maximum-Bathymodiolus sequences of this study (in bold) grouped with former in B.brooksilikelihood tree based on the phenol hydroxylase gene. We (see d). The sequence of this study fall within sequences related to high content hydrocarbon could amplify the gene in B. heckerae and not
environments.

Fig 4. Bathymodiolus mussels and Escarpia tubeworms from this study, and FISH images of bacteriocytes in the mussel gill filaments and in the tubeworm trophosome. (a) Bathymodiolus brooksi and B. heckerae mussels together with escarpid tubeworms settle in the asphaltic sediment at Chapopote cold seep in the southern Gulf of Mexico. Each metazoan species harbors its own specific bacterial phylotypes. (b) Escarpia tubeworms from this study bear chemoautotrophic tubeworm symbionts. (c) Localization of the symbionts (arrows) with a FISH specific probe through a tubeworm cross-section. (d-g) B. heckerae mussel and respective FISH images: B. heckerae shell has an elongated shape (d); Its filamentous gills house methanotrophic (pink) and thiotrophic (green) bacteria (e). Two different chemoautotrophic i are in blue, thiotrophs I in red, and bacterial phylotypes have been recognized. The host nuclembiont in green, is co-existing with the thiotrophs II in yellow (f). A new hydrocarbon-degrader symethanotrophic bacteria in blue and the thiotrophs in pink (g). (h-k) B. brooksi mussel and respective FISH images: the shape of the B. brooksi (h) shell is more round and smaller than B. heckerae.. When analyzed with FISH B. brooksi gill filaments (autofluorescence of the tissue is purple) house a methano-in green (i). A detail of (i) shows host nuclei in trophic bacterial phylotyope in red, and a thiotrophic one blue, methanotrophs in red and thiotrophs in green (j). A Psychromonas-related bacteria was found to i) = 50μm; (d, h) = 5 cm; (e, f, g, k) = 5 μm; (j) be associated with B. brooksi gill tissue (k). Scale bars: (c,= 10 μm.109

ManIscriptu

0-20-3-40C13δ0-50-60-7 1 21 2CnHxCH4 sp. 1 sp. 2
sp. 4 sp. 3B. heckeraeB. heckeraeB. brooksi B. brooksiEscarpiaEscarpiaEscarpiaEscarpia

w1:1618:18:1w
w1:2018:16:22:20:
:20:2016:20:1w1
20:18:18:
Cholesta-dien20:-18:
Cholestero
Ergosta-dien-Squalen

heckerae, B. brooksi, and Escarpia Fig. 6 Stable carbon isotope composition of lipids extracted from sp. tubeworms tissue. (a) Carbon B.
isotope values of lipids (circles and bars) and bulk tissue (diamonds) from Bathymodiolus carbon isotope values plotted against spp. and Escarpia B. brooksisp. tissues. (b) values. (c) Escarpia B. heckerae
carbon isotope values tubeworms B and 4 plotted against A1 and A2.

110

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AnnelingRef.Primer sequence (5´-3´)Primers GenetemperatureMuyzer et al., 1995AGAGTTTGATCATGGCGM3 (8F) 4416S rRNAMuyzer et al., 1998TACCTTGTTACGACTTGM4 (1492R)CCGTCAATTCCTTTGAGTTT907RCATRCTDATTCGWATTGALCO-1560Jones et al., 200655COICCYCTAGGRTCATAAAAAGAHCO-2148GGTCAACAAATCATAAAGATATTGLCO-1490Folmer et al., 199455TAAACTTCAGGGTGACCAAAAAAT HCO2198CAaprAAprA-1-FWTGGCAGATCATGATYMAYGG54MeyerandKuever
2007GCGCCAACYGGRCCRTAAprA-5-RVGGNGACTGGGACTTCTGGA189FHolmes et al., 199555pmoACCGGMGCAACGTCYTTACCMB661RTCTCVAGCATYCAGACVGACGRMO-FBaldwin et al., 200353hydroxylaseTTKTCGATGATBACRTCCCARMO-RFormamideProbe sequence (5´-3´)ProbesSpecies% (46°C)heckerae TI B.and B. brooksi TBthio-193CGAAGATCCTCCACTTTA20Duperronetal.,
2007B. heckerae TIIBheck-193AAGAGGGCTCCTTTT20Duperronetal.,
2007spp.BathymodiolusBangM-138ACCAGGTTGTCCCCCACTAA20Duperronetal.,
2005methanotrophsCycloclasticusCypu-829GGA AAC CCG CCC AAC AGT20-50Maruyamaetal.,
2003spp.PsychromonasEilers et al., 200020GGTCATCGCCATCCCCGAPsychr-1453spp.Escarpiasp.Tbw-643TCTACCACACTCTAGTCAGGCAG20-60This study
thitrophsEscarpiasp.Tbw-139TCCGAGTTGTCCCCCACTAC20This study
thitrophs

Table1S.Primersandprobes.Alltheoligonucleotidesusedinthisstudyare
listedandtheirannelingtemperatureorformamideconcentrationthatwere
applied to them.

113

uManIscript

SpeciesSpecimenT1

1119 (5)B. heckerae180 (3)2156 (3)1B. brooksi111 (3)2140 (5)A1Escarpia sp.69 (4)B1

T2

56 (5)3 (1)

M

5 (2)21 (2)63 (3)

C

3 (3)

P

3 (3)

aprA

6474422

hydroxpmoA

32494x49x98 - x - x

Table2S.Clonedescriptionofthisstudy.Inthecaseofthe16SrRNAgenethenumberofpartial
sequence,sequencesaredashthatshownweanddidnotcompletetrytoonesamplifyareinfromthatparenthesis.individual.NodataThexmeansmeanswedidwenotruntheobtainedPCRbutany
no product was obtained.

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B.brook Ind.2B.brook Ind.1B.heck Ind.2B.heck Ind.1Compound?mg lipid/g gill0,12MEANDEVmg lipid/g gillMEANDEVmg lipid/g gill0,23MEANDEVmg lipid/g gill0,05MEANDEV
14:014:10,140,10-39,30,060,07-41,2-44,21,00,60,210,17-43,8-42,50,040,04-42,2-43,6
16:115:080,000,140,0310,690,16-40,6-38,72,92,520,04-49,3-38,04,4
16:116:1570,696,21-41,21,00,082,18-42,5-41,40,20,22,0523,63-45,00,10,485,07-45,0-45,10,70,8
16:216:20,540,32-41,90,30,19-41,60,70,080,02
16:017:1 ?1,970,03-39,51,70,60-41,60,44,870,08-43,91,21,070,01-44,30,6
0,010,050,0617:0 ?17:017:00,040,160,270,06-39,50,1
18:318:32,58-42,12,30,79-44,70,80,291,09-45,10,80,260,09-46,3-44,20,20,0
18:218:20,190,21-40,0-45,20,50,80,090,07-39,4-45,00,91,40,330,24-42,4-42,21,00,90,110,08-44,2-43,90,20,4
18:118:19110,140,12-40,0-41,81,63,10,080,11-41,30,80,361,08-39,3-39,81,71,90,100,24-43,8-41,10,60,2
18:118:1570,140,76-40,51,00,020,32-41,80,30,352,06-42,9-41,90,110,44-54,5-44,70,10,3
18:00,23-36,91,50,15-39,10,70,85-42,50,80,20-42,70,1
??0,240,06-42,71,10,060,06-42,5
0,6-42,50,1020:320:220:30,511,32-42,0-43,83,11,10,110,46-44,0-43,81,30,30,780,05-45,7-46,30,52,10,200,11-43,1-46,72,00,6
20:220:21,75-41,30,40,58-42,00,51,700,32-43,5-44,10,00,40,050,35-45,4-44,40,30,4
20:120:19130,360,50-40,6-41,11,70,50,120,16-44,1-40,92,60,30,800,59-43,3-43,21,00,30,180,13-42,7-44,40,21,1
20:170,95-40,70,90,36-41,00,53,11-43,20,60,63-43,40,0
16:1 MAGE22:20,18-47,21,00,080,19-46,4-42,01,10,70,110,03-43,90,0
-43,90,02C16:1 MAGE?16:0 MAGE0,700,11-41,8-37,80,70,03-49,51,000,12-44,90,40,190,03-44,5-45,20,41,2
18:1 MAGE ?C17 MAGE0,040,010,000,010,00
18:1 MAGE ?18:0 MAGE0,050,050,020,010,140,050,050,01-42,90,0
0,020,14-43,80,09?C20:1 MAGEC20:2 MAGE0,140,13-40,9-42,00,040,02-41,70,60,510,00-43,8-53,51,70,50,170,16-43,9-48,91,11,4
Cholesterol0,08-42,90,01-35,10,50,050,03
4-Methyl-cholesta-N,N-dien-3b-ol4-Methyl-cholesta-N-en-3b-ol3,760,13-50,8-51,30,30,830,02-51,60,53,790,11-53,2-57,01,70,63,370,12-54,21,5
4-Methyl-cholesta-N,N,N-trien-3b-ol?0,120,060,030,060,130,160,12
Stigmasterol?Lanosterol ?0,030,060,140,16-48,8-52,81,20,40,400,45-43,8-46,40,30,5
Diplopterol?0,120,01-42,40,10,030,10-45,60,0
0,3-43,00,25??0,19-41,10,1-41,80,50,01
?16:1 FAME0,06-43,71,00,110,03-41,3-43,70,40,70,0040,02
0,0-45,00,8116:1 FAMEC18:3 FAME16:0 FAME0,330,16-40,9-40,90,80,3
0,02C18:2 FAME0,04C18:2 FAMEC18:1 FAMEC18:1 FAME0,060,13-35,5-42,60,20,1
C20:3 FAMEC18:0 FAME0,080,05-36,3-40,30,00,4
C20:2 FAMEC20:3 FAME0,170,01-43,30,3
C20:2 FAMEC20:1 FAME0,230,05-43,0-39,90,60,5
C20:1 FAMEC20:1 FAME0,170,16-41,7-40,80,70,7
musselsBathymodiolusfatty acids from Table 4S. Isotopic composition and quantity (mg of lipid per g of gill tissue) of 115

ManIscriptu

Tube worm BTube worm A2Tube worm A1Compound13headC (‰)mg/gbody13C (‰)mg/ghead13C (‰)mg/gbody13C (‰)mg/ghead13C (‰)mg/gbody113C (‰)
16:114:0 FAs FAs-28,84,93-27,92,20-29,12,19-38,0-28,10,48-28,2-27,4
16:116:0 FAs FAs-36,53,390,13-34,70,102,03-35,70,081,74-35,50,060,42-34,0-31,6-32,0
18:2 FAs18:2 FAs-37,0-37,03,211,03-36,11,600,51-35,81,330,65-36,40,280,04-35,2-33,2
18:118:1 FAs FAs-28,814,550,35-28,86,000,32-28,75,820,21-28,41,170,05-28,8-28,4
20:4 FAs18:0 FAs-40,0-33,32,981,31-37,3-36,61,720,76-35,8-34,61,821,05-36,5-34,90,430,08-36,1-35,1-34,9-30,7
20:5 FAs20:2 FAs-38,1-33,54,772,14-38,1-34,22,861,03-36,2-32,92,201,16-37,0-31,70,870,26-36,6-33,0-36,1-31,8
20:120:1 FAs FAs-32,1-39,12,752,71-31,5-36,41,481,51-30,1-34,42,621,86-29,0-34,70,110,19-30,7-34,8-30,5-33,2
22:2 FAs1,33-32,10,82-30,60,95-30,80,26-33,6-31,6
16:0 alcohol16:1 alcohol0,19-36,20,030,04-36,20,01-32,60,170,46-29,1-34,7
18:0 alcohol18:1 alcohol-42,00,450,43-36,3-30,20,040,16-36,40,110,19-30,3-34,60,020,02-29,3-35,20,910,18-29,1-36,6
20:1 alcohol0,51-30,50,29-30,80,21-28,50,03-32,60,14-30,4
0,0120:0 alcohol-36,80,0118:1 MAGE18:0 MAGE20:2 MAGE0,180,05-37,1-41,20,15-35,60,140,08-35,3-41,60,020,01-36,1-32,70,09-36,5
Cholesterol20:1 MAGE-41,8-24,75,050,22-38,9-32,85,290,20-36,6-32,32,750,16-36,7-30,70,870,03-38,7-32,40,510,08-37,6-32,0
Ergosta-dien-olCholesta-dien-ol-43,2-42,64,162,24-40,9-40,73,812,03-40,2-38,71,522,94-38,8-39,70,480,53-40,2-38,90,330,37-39,2-38,3
tube wormsEscarpiafatty acids from Table 3S. Isotopic composition and quantity (mg of lipid per g of tissue) of

116

Tube worm 4bodyheadmg/g13C (‰)mg/g13C (‰)
-27,5-26,831,390,101,301,52-31,88-31,85-36,0-33,3
0,24-28,8-27,114,830,13-34,230,252,362,55-36,46-37,33-37,4-36,8
0,710,73-32,31-31,34-34,1-34,5
-32,1-30,770,320,710,040,06-27,70-33,010,160,32-29,0-33,1
0,140,09-29,42-32,260,280,21-34,9-31,7
-28,450,10-37,00,07-31,800,11-32,00,09-37,240,091,933,72-38,43-35,480,810,48-37,9-36,1
-38,10,472,25

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An intranuclear bacterial parasite in shallow water bivalves

Luciana Raggi1, Dennis Fink1, Nicole Dubilier1
1Max Planck Institute for Marine Microbiology, Celsiussr. 1, 28359, Bremen, Germany
Keywords: Intranuclear bacteria, NIX, symbiosis, parasitic association, invertebrates
Running head: Intranuclear bacteria of bivalves.
Corresponding author: Nicole Dubilier
E-mail: ndubilie@mpi-bremen.de
Intended as a Brief report in Environmental Microbiology

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ractstAb Marine intranuclear bacteria have a potentially lethal effect on bivalve populations, thus
we set out to look for the presence of intranuclear bacteria in economically important and
commercially available bivalve species. These included oysters (Crassostrea gigas),
razor clams (Siliqua patula and Ensis directus), blue mussels (Mytilus edulis), manila
clams (Venerupis philippinarum), and common cockles (Cerastoderma edule). 16S rRNA
analysis and fluorescence in situ hybridization (FISH) revealed the presence of
intranuclear bacteria in all investigated bivalves except oysters and blue mussels. A FISH
probe targeting all currently known intranuclear Gammaproteobacteria was designed for
future high-throughput analyses of marine invertebrates. Furthermore, primers were
designed to quantify the abundance of intranuclear-related bacteria with real time PCR.
Preliminary tests showed massive amounts of intranuclear bacteria in some bivalve
species, raising the question as to whether these significantly affect not only the health of
the bivalves, but possibly also of the humans that eat them.
Introduction
Bacteria that invade eukaryotic nuclei are commonly found in protists, but have rarely
been observed in multicellular eukaryotes (Grandi et al. 1997; Arneodo et al. 2008).
Recently, we described intranuclear bacteria in deep-sea hydrothermal vent and cold seep
mussels of the genus Bathymodiolus (Zielinski et al. 2009). This bacterial parasite
appears to infect a nucleus as a single cell, replicate in massive numbers and eventually
cause the nucleus to burst, liberating thousands of bacteria with the possibility to infect
contiguous nuclei. Phylogenetic analyses showed that these bacteria belong to a
monophyletic clade of Gammaproteobacteria associated with marine animals as diverse
as sponges, corals, bivalves, gastropods, echinoderms, ascidians, and fish. However,
except for bathymodiolin mussels and a shallow water bivalve, the Pacific razor clam
Siliqua patula (Kerk et al. 1992), none of these metazoa-associated bacteria have been
shown to occur inside nuclei. When this intranuclear bacterium was first described, it
was named “Nuclear Inclusion X” (NIX) (Elston, 1986), from here on we referrer to the
monophyletic group described in Zielinski et al. (2009) as the NIX clade. Intranuclear
non-Rickettsia-like bacteria have been observed by TEM in marine metazoans, such as
sponges (Vacelet 1970, Friedrich et al. 1999), Ruditapes (Tapes, Venerupis) decussatus
clams (Azevedo 1989), Siliqua patula razor clam (Elston 1986, Kerk et al. 1992) and

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Bathymodiolus spp. mussels (Zielinski et al. 2009). With the exception of Kerk et al. and
Zielinski et al., the observations were not associated with molecular data. It has been
suggested that these intranuclear bacteria may cause massive mortality in bivalves, with
potential impact on hatchery economics (Elston 1986, Ayres et al. 2004). Within this
bacterial group there are cultivated bacteria, the Endozoicomonas sp. (Kurahashi and
Yokota 2007) and surfactant-resistant bacteria (Plante et al. 2008), all of which were
isolated from marine invertebrates. In the current study, we investigated the presence of
these intranuclear bacteria in coastal consumable bivalves with both culture-dependent
and -independent methods. In contrast with deep-sea bivalves, shallow-water bivalves
have a diverse and abundant microflora. Molecular studies of this diversity are limited
(e.g. Cavallo et al 2009), in part due to the fact that research on bacterial-bivalve
interactions is largely focused on known human pathogenic bacteria (e.g Vibrio spp.).
These studies have never reported the presence of bacteria related to the intranuclear
group. We report here that NIX bacteria belonging to the monophyletic group are
commonly present in bivalves and have a widespread distribution.
Material and Methods
Sampling site and processing
Bivalves were collected in April 2008 from intertidal zones in Sylt, Germany. Ensis sp.
were collected in Königs Bay (Königshafen) and Mytilus edulis, Cerastoderma sp. and
Crassostrea gigas at Oddewatt. Collection of Ensis sp. clams is traditionally known to be
difficult due to their burying behavior. However, upon arrival to the beach, a small
community of around 200 clams was observed on the surface of the intertidal sediment.
Siliqua patula clams were collected from Kalaloch Beach on the Pacific coast of
Washington State, USA. Crassostrea gigas (France and Mexico), and Tapes
semidecussatus (Italy) were obtained in local markets in Bremen and Mexico City. The
organisms were kept alive in seawater or on ice until further processing. The gills were
dissected and segments were place in 96% ethanol and stored at -10°C for DNA analysis.
Other fragments were fixed for FISH analysis with 1% PFA and stored at 4°C in 50%
ethanol/PBS, and a third fragment was stored in 6% glycerol for cultivation attempts.
ation ltivuC Three different media were used in solid (with 1.5 % Agar) and liquid presentations:
Marine Medium 2216 (Difco), Minimum Medium with and without CTAB (0.1% yeast,
0.01% peptone, 1.5% agar, 100µm CTAB, dissolved in sea water), and WL Nutrient

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Medium (Difco) in 3.5% NaCl. No NIX were recovered, but many other bacteria were
(results of the cultivated bacteria will be presented in a separate report).
DNA extraction and PCR amplification
DNA was extracted from frozen or ethanol fixed tissue following the Zhou et al. (1996)
protocol with small modifications. Briefly, 2 ml of extraction buffer and 20 µl proteinase
K (20 mg/ml) were added to approximately 100 mg of tissue, and incubated 1.5 hours.
200 µL of 20% SDS were added and incubated 2 hr at 56°C. The liquid phase was
recovered after centrifugation at 14000 g for 20 min and cleaned once with 1 V
phenol/chloroform/isoamilalcohol (25:24:1) and a second time with
chloroform/isoamylalcohol (24:1), precipitated with 0.6 V isopropanol and dissolved in
TE buffer. DNA was extracted from a variety of animals, but we report here only those
hosts for which a positive 16S rRNA sequence analysis confirmed the presence of NIX.
The extracted DNA was used for both host and bacterial symbiont analysis. Hosts COI
genes were amplified using 36 PCR cycles using the LCO1490 (5'-
GGTCAACAAATCATAAAGATATTG-3´) and HCO2198 (5´-
TAAACTTCAGGGTGACCAAAAAATCA-3´) primers (Folmer et al., 1994). Bacterial
16S rRNA genes were amplified using 20 PCR cycles with universal primers GM3 (8F)
and GM4 (1492R) (Muyzer et al., 1995).
Quantification of bacteria: real time PCR
Primers for the amplification of a 100 bp fragment of the 16S rRNA were designed using
the probe design tool of ARB (Ludwig et al., 2004). The primers matched all sequences
in the NIX-clade, with no match to other sequences from bivalve-associated bacteria:
Nix-721F (5'-AGTGGCGAAGGCGACACT-3') and Nix-805R (5'-
GACATCGTTTACGGCGTGG-3'). Oligonucleotides were checked for their potential to
form secondary structures using the analysis tool NetPrimer
(http://www.premierbiosoft.com). For PCR conditions, the Eurogentech (Germany)
protocol was followed. The annealing temperature was 60ºC and 50 cycles were used.
isylogenetic analyshP COI products of the host invertebrates were directly sequenced (both strands). Bacterial
16S rRNA of each organism was cloned using the TOPO-TA system (Invitrogen) and
clones were partially sequenced with primer 907RC (Muyzer et al., 1998). Clone
sequences belonging to the NIX-clade from each host individual were chosen for full

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sequencing in both directions. Sequence data were analyzed with Sequencher (Genes
Codes Corporation), 16S rRNA sequences were aligned with the ARB/SILVA aligner
(Pruesse et al. 2007), and phylogenetic trees were calculated with the ARB software
(Ludwig et al., 2004). All sequence comparisons are given as percentage sequence
identity (% similar nucleotides) after calculations of a Neighbor-Joining distance matrix.
For tree reconstruction, only long sequences (~1400 bp) were used. Maximum likelihood
phylogenetic tree was calculated using RAxML (GTRGAMMA distribution model, 100
bootstrap replicates).
FISH and CARD-FISH
A gill piece from each specimen was dehydrated in an ethanol series and embedded in
low-melting polyester wax (Steedman, 1957). Wax cubes were sectioned with a Leica
microtome (5-6 µm) and mounted on Plus Frosted Slides (Menzel-Gläser). Polyester wax
was removed in three rinses in absolute ethanol (5 min each), and sections were
rehydrated in a short ethanol series (3 concentrations). For better penetration of probes,
sections were subsequently incubated in Tris-HCl (20 mM, pH 8), proteinase K (0.05 mg
ml-1 in Tris-EDTA, pH 8, at 37°C), and washed in MilliQ water (5 min each). For in situ
hybridizations with mono- or horseradish peroxidase (HRP)-labeled probes (CARD-
FISH) and subsequent staining with DAPI, sections were processed as described
previously (Zielinski et al 2009, Duperron et al 2007). The probes used in this study were
designed based on the primer sequences. EUB338 probe was used as a positive control.
Probe NON338 was used as a negative control for background autofluorescence. All
hybridizations were performed using 20% formamide.

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Results and Discussion
16S rRNA sequence analysis
In our 16S rRNA sequence analysis (Fig. 1), we obtained roughly 30 sequences from
each reported host (Table 1), with the exception of M. edulis, C. edule and T.
semidecussatus, with 4, 61 and 51 clone sequences, respectively. We found sequences
belonging to the monophyletic group described in Zielinski et al. (2009), which we call
the NIX-clade, in five of the bivalve species analyzed: Ensis directus, Cerastoderma
eduli, Crassostrea gigas, Siliqua patula, Tapes semidecussatus. The amount and quality
of DNA was limited, due to inefficient DNA extractions for some organisms. Two
different NIX phylotypes were found in some of the bivalves, however the phylotypes of
each host were nearly always different. Among the phylotypes found in this study (Figure
1), there are three cultivated strains isolated from a sponge (Nishijima et al. in prep), a
sea slug (Kurahashi and Yokota 2007) and an echinoid (Becker et al 2007). These
cultivated bacteria were not yet checked for in situ localization (personal communication
from researchers), which, should they be confirmed as intranuclear, could pave the way
towards physiological studies of intranuclear bacteria. All sequences in the NIX-clade
except two (from marine water) belong to animal-associated bacteria (Figure 1). This
suggests these bacteria are specialized at living within animal tissue and may get from
them nutrients, protection and surface for attachment, parameters potentially essential for
their life cycle. This study confirms previous findings that the NIX clade is a
monophyletic group (Zielinski, et al. 2009).
and abundance ation localizituIn s We could localize intranuclear and not intranuclear bacteria belonging to the NIX-clade
with specific probes (Table 2) in S. patula, E. directus, C. edule, and T. semidecussatus,
which corresponded to only one of the phylotypes of each host (Fig. 2). We were not able
to localize the second phylotype that occurred in C. edule and E. directus, as the specific
probes (Cnix-643, Cnix-64, Cnix-1249 for C. edule, and Cnix-1249 for E. directus
phylotype) did not show an intranuclear signal, and any positive signal other than nuclear
could not be discern from background fluorescence. In the two different oysters, no
positive signals with any bacterial probe (i.e. Eub-338, Gamma, and Tnix-64) were
observed. In fact, the oyster tissue looked very clean (no bacteria or particles), which is
likely due to the normal and extensive depuration process that oysters are put through for
commercial use. In contrast, C. edule had a massive amount of particles and high

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background, making it difficult to distinguish a positive signal outside nuclei. The second
phylotype may be present, but could not be observed under these conditions. S. patula
and T. semidecussatus frequently had massive infections, as did C. edule, though to a
lesser degree (see Fig. 2-4). T. semidecussatus was unique in that NIX bacteria were
found normally surrounding the nucleus and rarely inside (Fig. 2). These FISH
observations suggest that NIX-bacteria are not exclusively intranuclear. However, as
observed in T. semidecussatus, they may need to live associated with nuclei, potentially
providing a mechanism for nucleus entry, easier than first thought.
The natural aggregation of NIX bacteria makes their quantification by FISH impossible.
As such, we sought to develop a qPCR (real time PCR) based assay for high-throughput
NIX-specific quantification, as described in the methods section. Preliminary results of
qPCR are consistent with FISH observations (Table 1). S. patula had the highest quantity
of NIXclade bacteria, as observed in both qPCR and in situ analyses (Fig 2X). According
to the NIX life cycle proposed by Zielinski et al. (2009), the bacterial infections observed
here are in the last developmental stage (Fig. 4). With the oligonucleotide probes
designed in this study, it would now be possible to corroborate the presence of
intranuclear-related bacteria in any of the organisms for which there is a published
sequence, such as in the fish Pomacanthus sextriatus, the sponges Petrosia ficiformis,
Chondrilla nucula, Muricea elongata, and the corals Halichondria okadia,
Erythropodium caribaeorum.
Mass mortalities
What we observed in Sylt, a group of ~200 clams lying on the intertidal sediment is a
low-scale mass mortalty. Some clams were dead and some did not perform their usual
burying when feeling contact. The cause of the dying Ensis clams in Sylt coast is unclear.
However, it is highly possible that the mortality was due to the presence of NIX-realted
bacterial parasite. Siliqua patula razor clams have been observed to experience mass
mortality related to a nuclear bacterial parasite (Elston 1986, Ayres et al. 2004) and it
would not be surprising that a species as closely related as E. directus can become
diseased by NIX bacteria as well. Typically, bacteria and parasites are persistent in
certain species and it is a delicate equilibrium, likely dependent on environmental factors
that keeps these organisms under control. An imbalance may cause certain parasites to
bloom and opportunistically damage their host. As such, a NIX disease ecology or
epidemiological study, requires a thorough assessment of environmental parameters,
which, with the use of the qPCR primers designed and tested here, could be correlated
with the emergence of the mass mortalities due to intracellular bacterial infections.

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Conclusions
It is clear that the distribution of these NIX-related bacteria is widespread, not only
biogeographically, as was suggested by Zielinski et al (2009), but also among
phylogenetically diverse host invertebrates. The persistent occurrence of these bacteria
suggests that they have both a parasitic stage and a non-parasitic host stage, some hosts
appear to support high numbers of bacteria without presenting any physical disease. Is the
symbiotic state of this bacteria-bivalve interaction in a host-commensal relationship
rather than host-parasitic? Perhaps the observed association is a transition between these
symbiotic concepts. The intranuclear presence of bacteria is an evolutionary process not
well understood, requiring further ecological and physiological studies. Furthermore, the
issue is of great importance due to the economical repercussions that mass mortality
could have for aquaculture, as well as the consequences the infections of these bivalves
have on human health. Acknowledgements
We are grateful to R. Elston for providing the Siliqua patula samples and helpful
discussions. To M. Duhaime for her constructive revision. This work was supported by
the Max Planck Society, Germany. L.R. was supported by a CONACyT-DAAD
scholarship.

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Tables and Figures
gure 1.iF16S rRNA phylogenetic tree based on maximum likelihood (RAxML) analysis. NIX-
clade belongs to the Gammaproteobacteria. Sequences from this study (highlighted in
grey) and all closely related sequences found in the literature, including the three
cultivated strains (in blue) and the Candidatus Endonuclear bathymodioli (in yellow) are
shown. Probes (in green square) designed to target each specific host were designed for
use in FISH analysis and Nix-721 and Nix-805 for real time PCR. Sequences with a star
(*) indicate nuclear phylotypes confirmed by FISH. The T. semidecussatus sequence
(with two stars **) was found typically surrounding the nuclei and very rarely inside.
4 gure 2-iFFluorescence in situ hybridization (FISH) images of NIX bacteria in the different
bivalves. We used two probes for detecting the precense of NIX bacteria. Only images
with one probe are shown because they gave similar results. 2) Specific probes (Tnix-
1249 and Tnix-64) for NIX in T. semidecussatus. Nuclei stained with DAPI (blue). FISH
signals (with Tnix-1249) are in green. Scale bars: 10 µm. 3) FISH with specific probes
(Tnix-64 and Bnix-643-II) for NIX in C. edule. DNA is stained with DAPI (host nuclei in
blue). FISH signals (with Tnix-64) are in red. Bar scales in a and b: 20 µm, for c: 10 µm.
4) Specific FISH for razor clam sequences (signals in red and host nuclei in blue). (a-c)
specific probes (Tnix-64 and Bnix-643_II) for E. directu, signals with Tnix-64 are shown.
(d-e) and specific probes (Snix-64, Snix-643) for S. patula. FISH with Snix-64 is shown
in images. Scale bars in a: 30 µm, in c: 10 µm, in d: 20 µm.
ble 1.aT16S rRNA analysis. Number of clone sequences obtained from each host. NixI or NixII
are different phylotypes in each host. Two different phylotypes could be found in
E.directus, C.edule and C. gigas. The last line is the number of copies obtained in the
qPCR assay.
e 2lbTaOligonucleotide probes used in this study. Different probes matching each bivalve
(green) and sometimes having a mistmatch (x). Matching squares are labelled with ‘nucl’,
‘not nucl’, or ‘both’ after FISH observation showed nuclear or not nuclear. In the case of
Ensis sp. bacteria were localized both inside and outside nuclei with the same probe.

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Intranuclearbacteriaofbivalves

Nix-721

Nix-721

90 Endonucleobacter bathymodioli group *Bnix-643/Bnix-64
81100Ensis directusCerastoderma edule razor clam clone, Sylt NIX-I (4) clam clone, Sylt NIX-I (5) **Bnix-643II/Tnix-64Nix-721
98Tripneustes gratillaocean water clone, Bohai Bay echinoid isolate bacteria, AM495252, ocean water, FJ154998
79100Oceanrickettsia ariakensis, from Oceanrickettsia ariakensis from Crassostrea ariakensisCrassostrea ariakensis, DQ1, DQ12391418733
oyster clone, GoMCrassostrea gigas65100Siliqua Patula razor clam clone, full seq*Snix-64/Snix-643Nix-721
fish tract clone, EU884930Pomacanthus sexstriatus ascidian clone, Mediterranean Sea, DQ884170Cystodytes dellechiajei coral clone, DQ312235Alcyonium antarcticum8399100Alcyonium antarcticumAlcyonium antarcticum coral clone, DQ312243 coral clone, DQ312237
coral clone, DQ312244Alcyonium antarcticumNIX clade8396Crassostrea gigasEnsis directus oyster clone, GoM razor clam clone, SyltCnix-64/Cnix-1249
clam clone, Sylt NIX-IICerastoderma edule99, USA, EU799933ocean water clone, Newport Harbour ascidian clone, DQ884169Cystodytes dellechiajei88Endozoicomonas elysicola from Elysia ornata sea slug, AB196667Nix-721
Y494615 fish gill clone, ASalmo salarY700601 coral clone, APocillopora damicornis84Venerupis philippinarum clam clone **Tnix-64/Tnix-643
octocoral clone, DQ889921Erythropodium caribaeorum68 octocoral clone, DQ889931Erythropodium caribaeorumsponge clone, DQ917879Muricea elongata 91bleached sponge clone, DQ917830Muricea elongata 89Muricea elongata Spongiobacter nickelotoleranssponge clone, DQ917877, isolated from marine sponge, AB205011Nix-721
84Chondrilla nuculaPetrosia ficiformis sponge clone MOLA sponge clone, AM259915 531, AM990755
sponge clone HOC2, AB054136Halichondria okadia67 seastar clone KMD001, EU599216Asterias amurensis sponge clone HOC22, AB054156Halichondria okadia100 sponge clone HOC25, AB054159Halichondria okadia95 bivalve gill clone, EF508132Acesta excavata gill clone, seagrass beds, EU487857Ruppia maritima63 gill clone, seagrass beds, EU487858Ruppia maritima, AF084850Bdellovibrio bacteriovorusSequences from intranuclear bacteria confirmed by FISH0.10***Sequences that are found intranuclearly or sorrounding the nuclei (after FISH)Sequences from this study - shallow water bivalvesBathymodiolus Sequences from deep-sea mussels spp.Cultured strainsProbes used in this study

Nix-721

Nix-805

Nix-805

Fig.116SrRNAphylogenetictreebasedonmaximumlikelihood(RAxML)anal-
ysis.NIX-cladebelongstotheGammaproteobacteria.Sequencesfromthisstudy
(highlightedingrey)andallcloselyrelatedsequencesfoundintheliterature,in-
cludingthethreecultivatedstrains(inblue)andtheCandidatusEndonuclear
bathymodioli(inyellow)areshown.Probes(ingreensquare)designedtotarget
eachspecifichostweredesignedforuseinFISHanalysisandNix-721andNix-805
forrealtimePCR.Sequenceswithastar(*)indicatenuclearphylotypesconfirmed
byFISH.TheT.semidecussatussequence(withtwostars**)wasfoundtypically
surroundingthenucleiandveryrarelyinside.
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Fig.3FISHwithspecificprobes(Tnix-64andBnix-643-II)inC.edulegilltissue.
Ina-cDNAisstainedwithDAPI(inblue),andFISHsignals(withTnix-64)are
inred.Barscalesforaandb:20μm,forc:10μm.(a)imageiswithoutprobe
andahugebacterialinfectioncanbeobservedlabeledwithDAPI.Background
fluorescenceofhosttissueisobservedinred.

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Fig.4SpecificFISHforNIX-relatedsequencesinrazorclams(i.e.E.directusand
S.patulagilltissue.SignalsareinredandhostnucleilabeledwithDAPIinblue.
(a-c)specificprobes(Tnix-64andBnix-643-II)forE.directus,signalswithTnix-
64areshown.Backgroundfromtissueautofluorescenceisseeningreen.(d-e)
specificprobes(Snix-64,Snix-643)forS.patula.FISHwithSnix-64isshownin
images(red).Autofluorescenceoftissueisblue.Scalebarsina:30μm,inc:10
μm,ind:20μm.

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Table1.16SrRNAanalysis.Numberofclonesequencesobtainedfromeachhost.
beNixIfoundorNixIinIaE.dirreectusdifferen,C.etdphuleylotyandpesC.inegigasach.hTost.helastTwolinedisifferenthetpnuhmylotberypofesccopiesould
obtainedintheqPCRassay.

Table2.Oligonucleotideprobesusedinthisstudy.Differentprobesmatching
eachbivalve(green)andsometimeshavingamistmatch(x).Matchingsquaresare
labelledwithnucl,notnucl,orbothafterFISHobservationshowednuclearornot
nuclear.InthecaseofEnsissp.bacteriawerelocalizedbothinsideandoutside
nucleiwiththesameprobe.

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of

Bacterialdiversity

Minireview:

Luciana

Dubilier

Nicole

s,oyaggi-HoR

In

epPrationar

Bacteriadiversityofbivalves

Minireview: Bacterial diversity in shallow-water bivalves
Luciana Raggi1, Nicole Dubilier1
1Max Planck Institute for Marine Microbiology, Celsiussr. 1, 28359,
Bremen, Germany
Keywords: bacteria-bivalve interaction, symbiosis, invertebrates
Running head: Bacterial diversity in bivalves
Corresponding author: Nicole Dubilier
E-mail: ndubilie@mpi-bremen.de

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Abstract
Bacterial diversity in bivalves is a multidisciplinary topic, from biological
and basic research to economical interests, including aquaculture and
medicine. In this paper we review how these different disciplines have
influenced the few bivalve microbiota studies available. We present new
data from 16S rRNA analysis, including sequences from clone libraries and
isolates, and fluorescence in situ hybridization (FISH) from shallow-water
bivalves: Ensis directus, Cerastoderma edule, Crassostrea gigas, Siliqua
patula, Tapes semidecussatus, Mytilus edulis, and Mya arenaria, localizing
specific groups of bacteria associated with them. We also performed
cultivations from C. edule, and T. semidecussatus. The isolated bacteria
belong to bacteria phyla that are frequently found in organic rich
environments: oil spills, bone-falls, feces or invertebrate tissue. Most of
them belong to the Gammaproteobacteria group, and others to the
Alphaproteobacteria, Bacteroidetes, Actinobacteria and Spirochetes.
These associations might be occurring not only because of the surface
that bivalves provide to bacteria, but also because the association could
provide nutritional or protection benefits for both partners.

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Fig. 1 Phylogentic tree of the main bacterial phyla based on 16S rRNA gene. Almost all existing bacteria phyla
are visualized here. Branches in purple are the phyla for which bacterial species have been found associated to
bivalves.
Bacteria-bivalve associations
Bacteria-bivalve association studies have been enriched by the discovery
of intracellular bacterial symbionts of mussels and clams in the deep-sea.
However, most studies have been focused on species with intracellular
symbionts and there are scarce studies in the other associated bacteria.
Through its history, two topics have inspired the study of bacteria-bivalve
associations in shallow waters, the first one is the human pathogens that
edible bivalves can concentrate (Kueh and Chan 1984, Prieur et al. 1990,
Cabello et al 2005), and the second the economical importance that

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Fig. 2 Mya arenaria transversal sections of gill tissue in left panels. FISH with specific
probes for spirochetes (Spiro-1400 – 5’-CTCGGGTGGTGTGACGGGCG–3’). In right
panels B. childressi, with spirochete specific probe (Spiro-1400) in top image, and in image
below with specific probe for epsilonproteobacteria (Epsy-682 – 5’
CGGATTTTACCCCTACAC-3’). Scale bars: 10 µm in left panels and bottom right image; 5
ght image.itop rµm in

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healthy hatcheries have in aquaculture (Prieur 1987, Romero and Espejo
2001). In fact, because of these two reasons, already more than one
century ago the depuration processes (cleaning based on maximizing the
natural filtering activity of shellfish holding it in tanks of clean seawater)
in particular for oysters were implemented. The study of these
associations to use them as models for studying physiological interactions
between eukarya and bacteria, and the possibility to study evolutionary
pathways of symbiosis has seldom been the focus. With this last
intention, we synthesize all the available information about bacteria and
their association to shallow-water bivalves, and with molecular studies we
contribute to the understanding of bacterial diversity in bivalves. the
bacterial diversity of these living habitats. The first surprising thing that it
is noted when reviewing described bacteria associated with bivalves is
that their diversity is very broad and they cover almost all main branches
of the Bacteria kingdom (see Fig. 1).
Microflora diversity
Pathogenic, commensal and beneficial bacteria have all been described as
members of bivalve microflora. Most of the diversity studies performed
with bivalves are culture-dependent (Lovelace et al. 1968, Murchelano
and Brown 1968, Brisou 1962, Prieur 1981), meaning that the knowledge
that we have about bivalve microflora diversity is limited to the estimated
1% bacterial population that is cultivable (Kjelleberg et al. 1993) or even
less than 0.001% as described for oysters (Romero and Espejo 2001).
Occurrence of bacteria has been studied intensively in the bivalve
digestive tract (Prieur 1981, Minet et al. 1987) and in the crystalline style
(Bernard 1970, Paster et al. 1996, Prieur et al. 1990) Various bacterial
genera have been observed to be commonly

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Table 1 Isolates from T. semidecussatus, C. edule and E. directus. Gill pieces
stored in glycerol were homogenized and cultivated in three different media.
Isolate Host Medium1 Identity2 Closest relative Acc.3
TI-1 T. semidecussatus HNM Kocuria sp. MOLA 658 AM990771
TI-2 T. semidecussatus HNM Dermacoccus sp. Ellin183 AF409025
TI-3 T. semidecussatus HNM Sulfitobacter sp. MOLA 8 AM990784
TI-4 T. semidecussatus HNM Uncultured bacterium clone PL-10B7 AY570580
TI-5 T. semidecussatus HNM Uncultured Vibrionaceae AY627367
TI-6 T. semidecussatus HNM Alcanivorax sp. PA23 EU647559
TI-7 T. semidecussatus HNM Pseudoalteromonas sp. BSs20060 EU433327
CI-1 C. edule HNM Shewanella sp. P117 EU195929
Vm1-1 T. semidecussatus MM+CTAB 1084/1121 (96%) %#&$*)FJ025776
Vm1-2 T. semidecussatus MM &(# '%#!#"&&$%GQ149233
Os1 C. gigas MA %#"#'%$#%(&AB198089
Vm1-3 T. semidecussatus MA %# "'(&&'%"'AY292936
Vm1-4 T. semidecussatus MA %#&$DQ146980
Hs1-1 C. edule MA %#"#'%$#%(&AB198089
Hs1-2 C. edule MA "( '(% '%&$ #"EU375181
E1-1 E.directus MA  (&&$ #"FN395284
E1-2 E.directus MA  (&)"&'$""&&CP000903
Vm1-5 T. semidecussatus MA+CTAB %#&$FJ457601
Vm1-6 T. semidecussatus MA+CTAB %#&$FJ457601
1Media were used in solid (with 1.5 % Agar) and liquid presentations: MA - Marine
Medium 2216 (Difco), MM - Minimum Medium with and without CTAB (0.1% yeast,
0.01% peptone, 1.5% agar, 100µm CTAB, dissolved in sea water), and HNM - WL
Nutrient Medium (Difco) in 3.5% NaCl.
2This identity is with respect to closest relative of next column, it is shown first the number
of base pairs ration of the isolate sequence over the closest relative.
3Accesión numbers correspond to the published closest relatives.

associated to shallow-water bivalves: Vibrio, Pseudomonas, Spirochaetes,
Achromobacter, Flavobacteria, Micrococcus, Bacillus and also anaerobic
bacteria Bacteroidetes and Chlostridium.
Many studies have focused on Vibrio spp. since they are facultative
pathogens of humans and bivalves. Vibrio spp. are a regular component

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of the microflora and they might have an “important ecological niche”
(Prieur et al. 1990). Some have been related with the few described
bivalve diseases like bacillary necrosis or brown ring disease
(Grishkowsky and Liston 1974, Tubiash et al. 1970, Elston et al. 2008).
Recently, a review on the diversity and pathogenesis of Vibrio spp. was
made by Beaz-Hidalgo et al. (2010). With respect to bivalve lethality the
only two associated species are V. aestuarianus and V. splendidus. Other
species that seem to be commonly associated to bivalves are V.
alginolyticus and V. harveyi, and it is been observed that environmental
parameters such as salinity and temperature influence their diversity
(Pujalte et al 1999, Arias et al 1999, Beaz-Hidalgo et al 2010). Using
different media (High Nutrient Media, Minimum Media and Marine Agar,
with or without CTAB [Cetyl trimethylammonium bromide]), we have
been able to cultivate 18 different bacterial strains belonging to a variety
of bacterial phyla (Table 1). As expected, we obtained Vibrio strains from
Tapes semidecussatus, including in the media with CTAB, that was used
before by Plante and coworkers (2008) to isolate surfactant-resistant
bacteria with the aim of obtaining bacteria useful for environmental
remediation. As already observed in previous studies (Rajagopalan &
Sivalingan 1978, Sugita et al 1981) we could cultivate bacteria from the
Actinobacteria phylum (Kocuria sp. and Dermacoccus sp.), and Bacillus
sp. of the Firmicutes phylum. Krokinobacter sp. and Alcanivorax sp.
isolates are of special interest because the first ones are bacteria that
seem to be specialized in the degradation of organic matter (Khan et al.
2006) and the second are bacteria that use hydrocarbons as a sole carbon
and energy source (Head et al. 2006). Many times coastal bivalves have
been taken as a biological marker to assess pollution (e.g. Nishihama et
al 1998, Bresler et al 1999, Verlecar et al 2006). For example, the
presence of Alcanivorax bacteria in clam tissue indicates a strong oil
contamination in North Sea beaches (Brakstad and Lodeng 2005). Many
bacterial phylotypes were found in our 16S rRNA sequence analyses,

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some are recurrent bacteria that have shown up in previous bivalve
studies (Table 2). Most of these sequences are from
gammaproteobacteria, as it has been seen in previous molecular studies
of bivalve-associated bacterial communities (Schulze et al. 2006, Cavallo
et al. 2009). We could detect groups of bacteria that seem to associate
with high-content organic matter: oil spills, bone-falls, feces or
invertebrate tissue. In addition to gammaproteobacteria there were also
Alphaproteobacteria, Bacteroidetes, Actinobacteria and Spirochetes. We
hypothesize that these bacteria are specialized in high-organic matter
habitats and that further analysis of more individuals and more species
will show a characteristic community perhaps host species-specific.
Associated spirochaetes stand out because they seem to be a stable
bacterial community in bivalves (Bernard et al. 1970, Paster et al. 1996,
Prieur 1990, Margulis and Fester 1991). Our FISH observations (Fig 2)
show them well established in bivalve gill tissue. It might be interesting to
study the variability of these spirochetes species within the different
bivalves. It is not clear so far if spirochetes have an ecological role or
importance in the association with bivalves, but they seem to be
ubiquitous microflora within the mollusk group (Prieur 1990) and perhaps
the whole invertebrate group, as other spirochetes have been found in
oligochaete worms (Ruehland et al. 2008). Margulis et al. (1991) named
as symbionts the studied spirochetes in oysters.
Do intracellular symbionts reduce microflora diversity?
Six families of bivalves have been found to be associated with intracellular
symbiotic bacteria: Vesicomyidae (Calyptogena , Vesicomya), Mytilidae
(Bathymodiolus, Idas), Solemyidae (Solemya, Acharax), Lucinidae
(Lucina, Codakia), Thyasiridae (Thyasira), and Teredinidae (Lyrodus). All
are deep-sea water organisms except for Solemya, Thyasira and lucinids.
It is remarkable that these organisms associate with such a restricted

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diversity of bacteria, perhaps due to the presence of the endosymbionts.
However it is still possible that diversity is in fact not reduced and we
have not been able to see it in cultures or in clone library analyses due to
the high abundance of the bacterial symbionts. In our 16S rRNA analysis
(sequencing and FISH) of B. childressi, we observed the presence of
different bacteria associated with the tissue apart from the well
characterized methanotrophic symbiont: an epsilon proteobacterium, a
Candidatus Endonuclear bathymodiolin and a spirochete phylotype. This
could suggest that the B. childressi recognition system is not as specific
as in the other Bathymodiolus spp. In fact B. childressi is one of the
lowest positioned bathymodiolin mussels in the COI phylogenetic analysis
except for the latest described B. sp. from Juan de Fuca (Duperron et al.
2009) and could have an immunological system more similar to the
shallow water bivalves. However, in-depth studies of other species might
reveal that the diversity of bathymodiolin mussels is much higher than
previously assumed.
Nutrition and protection
Bivalve gills provide an ideal habitat for bacteria, with protection from
grazers and constant flow of nutrients. Bacteria might be complementing
their host nutrition or contributing to metabolite production. Degradation
of bacteria by bivalve enzymes has been observed and it seems that this
degradation provide dissolved compounds (Birckbeck & McHenery 1982,
Amouroux & Amouroux 1988) and improves bivalve nutrition (Samain et
al. 1987). Bacteria could provide 5% to 10% of the carbon, and 20% of
the nitrogen from the bivalve requirements (Prieur et al 1990). A stable
microbiota could be providing protection to bivalves thanks to competition
against other, potentially pathogenic bacteria. Also, microbiota could be
secreting antimicrobial substances that have been observed to be
commun in bacteria isolated from bivalves (Zheng et al. 2005).

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Table 2 Bacteria found in the different bivalves. Studies where bacteria have been observed microscopically (M), cultured (C), isolated (I), or
their 16S rRNA sequenced (S). Some letters are repeated because more than one study have characterized the species. In red the sequences
and cultures from this study.

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Summary and conclusions
Bacterial diversity in bivalves is a multidisciplinary topic, with from
researches in biological, medical, aquaculture and basic research.
Ecological studies with molecular techniques are scarce and they could
help to disentangle the interaction patterns between the endemic
microflora and the invasive ones, as well as the benefits that symbiotic or
the harm that pathogenic bacteria bring along. It is important to
understand the distribution of pathogenic bacteria in the marine
environments to predict potential health concerns transmitted by seafood.
Ecological parameters such as nutrient availability, temperature, and
salinity influence the presence and persistence of bacteria. However we
suggest that bacteria present in bivalves are not only randomly
associated with their hosts. It is in part the result of the surrounding
water community but also the result of a common evolution between host
and bacteria that normally associate with invertebrates or occur in
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Widespreadoccurrenceofanintranuclear

parasiteinbathymodiolinmussels

FrankU.Zielinski,AnneliePernthaler,S´ebastien

Duperron,LucianaRaggi,OlavGiere,Christian

148

BorowskiandNicoleDubilier

PublishedinEnvironmentalMicrobiology

(2009)MicrobiologyEnvironmental

Bathymodiolusintranuclearparasite

1/j.1462-2920.2008.01847.x11doi:10.1

Wparasiteidespreadinventoccurrenceandseepofanbathymodiolinintranuclearmusselsbacterial

FrankU.Zielinski,1,2AnneliePernthaler,3†opmentalcycleof“Ca.E.bathymodioli”showedthat
SébastienDuperron,4LucianaRaggi,1OlavGiere,5theinfectionofanucleusbeginswithasinglerod-
ChristianBorowski1andNicoleDubilier1*shapedbacteriumwhichgrowstoanunseptatedfila-
1MaxPlanckInstituteforMarineMicrobiology,Symbiosismentofupto20mmlengthandthendivides
Group,Celsiusstr.1,28359Bremen,Germany.repeatedlyuntilthenucleusisfilledwithupto80000
2HelmholtzCentreforEnvironmentalResearch–UFZ,bacteria.Thegreatlyswollennucleusdestroysits
DepartmentofEnvironmentalMicrobiology,Permoserhostcellandthebacteriaarereleasedafterthe
Str.15,04318Leipzig,Germany.nuclearmembranebursts.Intriguingly,theonlynuclei
3CaliforniaInstituteofTechnology,DivisionofGeologicalthatwereneverinfectedby“Ca.E.bathymodioli”were
andPlanetarySciences,1200ECaliforniaBlvd,thoseofthegillbacteriocytes.Thesecellscontainthe
Pasadena,CA91125,USA.symbioticsulfur-andmethane-oxidizingbacteria,
4UniversitéPierreetMarieCurie,ÉquipeAdaptationauxsuggestingthatthemusselsymbiontscanprotect
MilieuxExtrêmes,7QuaiStBernard,75005Paris,theirhostnucleiagainsttheparasite.Phylogenetic
France.analysesshowedthatthe“Ca.E.bathymodioli”
5UniversityofHamburg,BiozentrumGrindelundbelongstoamonophyleticcladeofGammaproteobac-
ZoologischesMuseum,Martin-Luther-KingPlatz3,teriaassociatedwithmarinemetazoansasdiverseas
20146Hamburg,Germany.sponges,corals,bivalves,gastropods,echinoderms,
ascidiansandfish.Wehypothesizethatmanyofthe
sequencesfromthiscladeoriginatedfromintra-
Summarynuclearbacteria,andthatthesearewidespreadin
Manyparasiticbacterialiveinthecytoplasmofmulti-marineinvertebrates.
cellularanimals,butonlyafewareknowntoregularly
invadetheirnuclei.Inthisstudy,wedescribeIntroduction
thenovelbacterialparasite“CandidatusEndonucleo-
bacterbathymodioli”thatinvadesthenucleiofdeep-Bacteriainhabiteveryimaginableplaceonearth.Withthe
seabathymodiolinmusselsfromhydrothermalventsevolutionoftheeukaryotes,numerousbacteriahave
andcoldseeps.Bathymodiolinmusselsarewellestablishedasymbioticorparasiticrelationshipinside
knownfortheirsymbioticassociationswithsulfur-eukaryoticcells.Mostofthesebacterialiveinthecyto-
andmethane-oxidizingbacteria.Incontrast,thepara-plasm,butsomehavefoundtheirwayintoeukaryoticcell
siticbacteriaofventandseepanimalshavereceivedcompartments,includingmitochondria(ChangandMus-
littleattentiondespitetheirpotentialimportanceforgrave,1970;Episetal.,2008),chloroplasts(Wilcox,
deep-seaecosystems.Wefirstdiscoveredtheintra-1986;Schmid,2003a,b)andnuclei(MailletandFolliot,
nuclearparasite“Ca.E.bathymodioli”inBathymodio-1967;Grandietal.,1997;Arneodoetal.,2008).Although
lusputeoserpentisfromtheLogatchevhydrothermaleukaryoticcellcompartmentshavebeeninvestigatedfor
ventfieldontheMid-AtlanticRidge.Usingprimersdecadeswithlightandelectronmicroscopy,littleisknown
andprobesspecificto“Ca.E.bathymodioli”wefoundaboutintracompartimentalbacteria,particularlythosethat
thisintranuclearparasiteinatleastsixotherbathy-liveinthenucleiofeukaryotes.Mostcommonlydescribed
modiolinspeciesfromventsandseepsaroundthefromprotists(Fokin,2004;Görtz,2006),almostnothingis
world.Fluorescenceinsituhybridizationandtrans-knownaboutintranuclearbacteriaofmetazoans.Rickett-
missionelectronmicroscopyanalysesofthedevel-sialAlphaproteobacteriaoccasionallyinvadenucleiof
theirarthropodormammalianhostsbutoccurmainlyin
thecytoplasm(Burgdorferetal.,1968;Urakamietal.,
Receivedcorrespondence.29July,E-mail2008;acceptedndubilie@mpi-bremen.de;11NovemberT,el.(2008.+49)*For4211982;Pongponratnetal.,1998;Ogataetal.,2006).Apart
fromthesefacultativeintranuclearRickettsia,reportsof
2028Centre932;forFax(Environmental+49)4212028Research580.–†UFZ,PresentDepartmentaddress:ofHelmholtzEnviron-intranuclearbacteriathataremorphologicallyorphy-
mentalMicrobiology,PermoserStr.15,04318Leipzig,Germany.logeneticallydistinctfromtheRickettsialesarerarein
©Journal2009ThecompilationAuthors©2009SocietyforAppliedMicrobiologyandBlackwellPublishingLtd

149

scriptuManVI

2F.U.Zielinskietal.
metazoans.Onlyasingle16SrRNAsequenceiscurrentlypoundsandmethane,areprovidedbythehydrothermal
knownfromanintranuclearbacteriumthatdoesnotfluidsatventsandthroughhydrocarbonseepageatcold
belongtotheRickettsiales(Kerketal.,1992).ThisGam-seeps.Boththemusselsandtheirchemosyntheticbacte-
maproteobacterium,called‘NuclearInclusionX’(NIX),riamutuallybenefitfromtheirsymbiosis:themussel
causesmassmortalitiesinthePacificrazorclamSiliquafacilitatesaccesstothereductantsandoxidantsthat
patula(Elston,1986;Ayresetal.,2004).Afewmorpho-arenecessaryforenergyproduction(suchassulfide,
logicalobservationsofnon-Rickettsiales-likeintranuclearmethaneandoxygen)bysupplyingitssymbiontswitha
bacteriaRuditapeshavedecussatusbeendescribed(Azevedo,from1989),theandveneridtwomarineclamprovideconstantcarbonfluidflow.Incompoundsexchange,thatthesupportbacterialthegrowthsymbiontsand
Aplysinasponges(formerlyVerongia)(Vacelet,1970;maintenanceofhostbiomass(Stewartetal.,2005;
Friedrichetal.,1999).Cavanaughetal.,2006).Inadditiontotheirsymbiotic
beenDeep-seastudiedhydrothermalextensivelyventssinceandcoldtheirseepsdiscoveryhavebeenbacteria,describedsomethatventareandseepcolonizedbymusselsparasiteshavesuchrecentlyas
25–30yearsago.Giventhehighdiversityofmetazoansviruses,Rickettsia-andChlamydia-likebacteria,ciliates,
currentlydescribedfromventsandseeps(Sibuetandfungiandtrematodes,butbeyondtheirmorphological
Olu-LeRoy,2002;Wolff,2005;Desbruyèresetal.,description,almostnothingisknownaboutthesepara-
2006a),remarkablylittleisknownabouttheirparasitessites(Powelletal.,1999;Wardetal.,2004;VanDover
(deBuronandMorand,2004).Todateaboutadozenetal.,2007).
metazoanmacroparasiteshavebeendescribed(deThisstudydescribesanovelbacterialparasiteof
BuronandSegonzac,2006a,b).Afewstudies,using18Sbathymodiolinmussels.Usingcomparative16SrRNA
rRNA-basedmoleculartechniques,havesuggestedthatsequenceanalysis,fluorescenceinsituhybridization
theremaybeahighabundanceofparasiticprotistat(FISH)andtransmissionelectronmicroscopy(TEM),we
vents(Atkinsetal.,2000;Edgcombetal.,2002;López-showthatthisparasitelivesinthenucleiofmusselsfrom
Garcíaetal.,2007),andthemorphologyoffungal,protist,ventsandseepsaroundtheworld.Wedescribethelife
bacterialandviralparasiteshasbeendescribedinmorecycleofthisintranuclearparasitethroughreconstruction
detailinsomeventandseepmussels,clamsandlimpetsofitsdevelopmentalcyclefromasolitarycelltothepro-
(Powelletal.,1999;Terlizzietal.,2004;Wardetal.,2004;liferationofupto80000bacteriawithinasinglegreatly
Millsetal.,2005;VanDoveretal.,2007).Thesestudiesenlargednucleus.Weproposethename“Candidatus
suggestthepotentialimportanceofparasitesforventandEndonucleobacterbathymodioli”forthisbacterium.The
seepecosystems,yetcomprehensiveinvestigationsofgenusname‘Endonucleobacter’translatesfreelyinto
thetaxonomy,phylogeny,andlifecycleofventandseep‘bacteriumlivinginsidethenucleus’andthespeciesname
parasitesarestilllacking.‘bathymodioli’referstothehostgenusofventandseep
MusselsofthegenusBathymodiolus(Bivalvia:Mytil-mussels,Bathymodiolus,inwhichwediscoveredthis
idae)occurworldwideatdeep-seahydrothermalventsparasite.
andCavanaugh,coldseeps2006;(TGénioarasovetetal.,al.,2008).2005;IntheDeChaineabsenceandof
lightandthus,photosyntheticcarbonfixation,theseResults
musselsdependonchemosyntheticbacterialsymbionts
fortheirnutrition(Stewartetal.,2005;Cavanaughetal.,DiscoveryofintranuclearbacteriainBathymodiolus
2006).Theseendosymbiontsoccurinthemussel’sgillputeoserpentis
tissue,inthecytoplasmofbacteriocytesthatregularlyThreegammaproteobacterial16SrRNAphylotypes
alternatewithsymbiont-freeintercalarycells(Fiala-werefoundingilltissuesofB.puteoserpentisfromthe
MédioniandLePennec,1987;Disteletal.,1995).LogatchevhydrothermalventfieldontheMid-Atlantic
BathymodiolinmusselscanharbourtwotypesofRidge(Fig.1A).Inadditiontothesequencesalready
chemosyntheticbacteria:achemoautotrophicsulfuroxi-knownfromthesulfur-andmethane-oxidizingsymbionts
dizer,capableoffixingCO2inthepresenceofsulfideor(Duperronetal.,2006),anovel16SrRNAsequencewas
thiosulfateasenergysources,andamethaneoxidizerdiscovered(“CandidatusEndonucleobacterbathymodioli”
thatsourceuses(Fishermethaneetal.,as1987;bothaNelsonetcarbonal.,and1995;anPimenovenergyofinFig.16S1A).rRNAThisnovelsequencessequencefromfellbacteriainacladeassociatedconsistingwith
etal.,2002).Somemusselspeciesharbouronlymarineanimalsincludingsponges,corals,aseaslug,an
thiotrophicoronlymethanotrophicsymbionts,whileotherascidian,aseaurchinandafish(93–97%identity,
musselspeciesharbourbothtypesandthusliveinadualTableS1).Thiscladealsoincludedthesequencefromthe
sourcessymbiosisforthe(DeChainebacterialandsymbionts,Cavanaugh,reduced2006).Thesulfurenergycom-S.‘Nuclearpatula(KerkInclusionetalX’.,1992).parasiteTheofthemonophylyPacificofrazorthiscladeclam
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Intranuclearbacteriainventandseepmussels3
wassupportedinbothmaximumlikelihoodandBayesianRidge),asingle16SrRNAclonewasfoundwitha
analyses(supportvalues:60and1.00).Thecultivatedsequencethatdifferedby1.9%(26bp)fromallother“Ca.
speciesicomonaswithinelysicolathis,acladestrictlywereallaerobicandheterotrophic:mesophilicEndozo-bac-E.‘minorbathymodioli”phylotypeIII’inTphylotypesableS2).foundThisinthisphylotypespecieswas(calledmost
teriumfromthegastrointestinaltractoftheseaslugElysiacloselyrelatedtothesequencefromB.aff.thermophilus
ornata(KurahashiandYokota,2007),andthreespecies(Pacific-AntarcticRidge)(Fig.1B).Withtheexceptionof
fromspirillum-likemarine(H262)sponges,bacteriumonerod-shaped(Sfanosetal(H425).,2005),andoneandthissequencesminorfellphylotypeintoIII,threeallclustersother“Ca.reflectingE.theirbathymodioli”geo-
Spongiobacternickelotolerans(unpublishedinformationgraphicoriginsfromtheMid-AtlanticRidge,thePacific-
fromGenBank).Theclosestrelativestothecladecon-AntarcticRidge,andtheGulfofMexico(Fig.1B).The
taining“Ca.E.bathymodioli”wereZooshikellagangwhen-correlationbetweengeneticdistancesofthe“Ca.E.
sis,achemoorganotrophic,aerobicandhalophilicisolatebathymodioli”16SrRNAphylotypesandgeographicaldis-
fromidentitytosedimentstheof“aCa.E.Koreantidalbathymodioli”flat(Yietal.,sequence2003)from(91%fortancesallofphylotypessample(P=locations0.04withwasminorstatisticallyphylotypeIIIsignificantand
B.puteoserpentis)andtwosequencesfromRickettsia-P=0.01withoutminorphylotypeIII).
likebacteriacausingmassmortalityintheoysterCras-
undersostreaaccessionariakensisnumbers(unpublishedDQ118733informationundinDQ123914).GenBankDevelopmentalcycleof“Ca.E.bathymodioli”
FluorescenceinsituhybridizationanalyseswithprobesDetailedFISHandTEManalysesof“Ca.E.bathymodioli”
specifictothe“Ca.E.bathymodioli”phylotypeshowedinB.puteoserpentisgilltissuesrevealedsixdistinct
thethatitnucleioriginatedofB.fromputeoserpentisbacteriathatcellsoccurred(Fig.2).Gillexclusivelytissuesinsingledevelopmentalrod-shapedstagesbacterium(Figs2,3(1.8and¥0.45).mInm)isStagepresent1,a
weremostheavilycolonized(Fig.2A–DandF),buttheinsidethenucleus(Fig.2BandG).InStage2,the
bacteriawerealsoobservedinnucleiofthegut(Fig.2E),bacteriumhasgrowntoanunseptatedfilamentofup
digestivegland,labialpalp,mantleandfoot(datanotto18–20mmlength(Fig.2H–J).InStage3,aloosely
shown).intercalaryInthecellsgillweretissues,infected,onlynucleiwhereasoftheintranuclearsymbiont-freebac-(Fig.wrapped2M).Wefilamentouscouldnotcoilisclearlyvisiblediscerninsideifthisthecoilhostconsistsnucleus
teriawereneverobservedinthebacteriocytescontainingofseveralseparatefilamentsoronelongunseptated
thethiotrophicandmethanotrophicsymbionts(Fig.2B–Dfilament.However,onrareoccasionsweobservedtwo
andfilamentsF).Tconfirmedransmissionthattheelectronbacteriamicroscopywereinsideanalysistheofhostgillappearedfilamentstoofbeequalinthelengthprocess(Fig.of2L)andlongitudinalfilamentsdivisionthatin
nuclei(Fig.3).somenuclei(Fig.2K),suggestingthattheStage3coil
consistsofseveralfilaments.ThehostnucleiinStage3
Widespreadoccurrenceof“Ca.E.bathymodioli”inhavereducedbecometoathinmorelayerirregularalongintheshapenuclearandchromatinmembraneis
deep-seamussels(Fig.3AandD).Transversebinaryfissionofthefilamen-
UsingPCRprimersandFISHprobesspecifictothe“Ca.touscoilsleadstoStage4inwhichstacksofshorter
E.bathymodioli”phylotypefoundinB.puteoserpentis,wefilamentsofupto8–10mmlengthfillthehostnucleus
searchedforthesebacteriainotherbathymodiolinhosts(Fig.2EandN).Atthisstage,hostnucleiareatleasttwo
fromOcean,thehydrothermalGulfofventsMexico,andandcoldtheseepsPacificinOceanthe(Fig.Atlantic4,totransversethreetimesbinarythefissionsvolumeofleadtouninfectedtheformationnuclei.ofRepeatednumer-
Table1,TableS2).Wefound“Ca.E.bathymodioli”inallousrodsinStage5withthenucleifurtherenlargedto
hostspeciesexceptB.aff.boomerang(SoutheastAtlan-aboutfivetimesthevolumeofuninfectednuclei.Chroma-
tic)sequencesandB.werebreviorvery(WestcloselyPacific).relatedAll“Cato.E.eachotherbathymodioli”withtinsomeispartsnearlyofthecompletelynuclearreducedmembraneand(Fig.barely3E).visibleInStagealong
98.8%identitybetweensequencesonaverageand6,thebacteriahavereproducedmassivelyforminga
98.1%identitybetweenthetwomostdistantphylotypesroundtoovalaggregateofupto30mmindiameterthatis
(Fig.1B).Inmosthostspecies,asingle16SrRNAphy-stillsurroundedbyamembrane(Fig.2P).Atthisstage,
lotypedominatedtheclonelibraries.Lessdominantphy-theinfectednucleiarenolongervisiblewithinstructurally
lotypesthatdifferedbyatmost2bpfromthedominantintacthostcells,butoccurextracellularlywithinthegill
phylotypeswerealsofoundinseveralhostspecies.epithelium.Ataveragebacterialsizesof1.8¥0.4mm,
Thesephylotypesoftenco-occurredinthesamehostStage6nucleicancontainbetween10000and80000
individualsandweresharedbetweenindividualsbacteria.Theseaggregatesareeventuallydisruptedand
(TableS2).InB.sp.fromWideawake(Mid-Atlanticthebacteriareleasedintothefluidssurroundingthe
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α

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Environmental©2009TheMicrobiologyAuthors

Bathymodiolusintranuclearparasite

Intranuclearbacteriainventandseepmussels5
A.Fig.The1.tree16SshowsrRNAthe“phylogeneticCandidatustreeinferredEndonucleobacterfrommaximumbathymodioli”likelihoodcladeand(greyBayesiantrapezoid)analysis.togetherwithitsclosestrelatives.Coloursof
blue,sequencesandfromfromascidiansmarineinpurpleinvertebrates(seawereslug,seaassignedurchinandaccordingfishtohostbacteriaarephylogenyshown,withinblack).bacteriaAlsofromincludedbivalvesintheshowntreeinarered,(i)fromtheclosestspongesinfree
livingrelativeswithintheOceanospirillalesandAlteromonadales;(ii)themethane-oxidizingendosymbiontsofbathymodiolinmussels(blue
frombox);(iii)ciliatestheandtickssulfur-oxidizing(purplebox).endosymbiontsBdellovibrioofbathymodiolinbacteriovoruswasmusselsusedas(yellowanbox);outgroup.and(iv)TheintranuclearphylogeneticbacteriareconstructionbelongingistobasedtheprimarilyRickettsiaonles
nearlyfullsequences.Partialsequences(508–983bp)aremarked(°).Methane-andsulfur-oxidizingsymbiontsofbathymodiolinmusselsare
labelledMOXandSOXrespectively.Valuesatnodesrepresentmaximumlikelihoodbootstrapvaluesinpercentage(firstvalue)andposterior
probabilities(secondvalue).Scalebarrepresents10%estimatedbasesubstitution.Onlybootstrapvalues60andposteriorprobabilities
>B.T0.80reeareof“Ca.shown.E.MAR,bathymodioli”Mid-AtlanticsequencesRidge;PfromAR,ventandPacific-AntarcticseepmusselsRidge;GoM,(collectionGulfsitesofofMexico.musselsareshown).The“Ca.E.bathymodioli”
aformsequencesclade.monophyletic

musselgills.Three-dimensionalexamplesofthedevelop-Theshiftfromfilamentousgrowthtomassivereproduc-
mentalStages2,3,4and6canbeviewedassupportingtionthroughtransversefissionmaybetriggeredbynutri-
animations(VideoesS1–S4).Inadditiontothedetailedentlimitation.ThechromatinofStage3nucleiisgreatly
FISHandTEManalysesofthe“Ca.E.bathymodioli”reducedtoathinlayeralongthenuclearmembrane
developmentalstagesinB.puteoserpentis,FISHanaly-(Fig.3AandD).Giventhatthisreductiondoesnotappear
sesofB.azoricus,B.sp.(Wideawake),B.aff.thermophi-tobephysicallyinducedbecausethebacteriadonot
lus,B.brooksi,B.heckerae,and“B.”childressigilltissuescompletelyfilloutthenucleusatthisstage,itislikelythat
showedthepresenceofsimilardevelopmentalstagesinthebacteriahaveusedthechromatinfornutrition(see
thesehostspeciesaswell.below).InB.bacteriovorus,transitiontothemultiple
fissionphaseoccurswhenthecytoplasmisconsumed
andthehost’sresourcesareexhausted(Horowitzetal.,
Discussion1974;Angert,2005;Lambertetal.,2006).
Multipleroundsoftransversebinaryfissionbetween
DevelopmentalcycleStages3and6leadtomassiveswellingofthehostnuclei.
Thecolonizationofanucleusrequiresseveralsteps:theGreatlyenlargednucleiaretypicalforprotistsinfected
infectionofthehostcell,passagethroughthecytoplasm,withintranuclearbacteria(TableS3)andhavealsobeen
andpenetrationofthenuclearmembrane.NoneofthesedescribedinthePacificrazorclamS.patulainfectedwith
stageswereobservedinthisstudy,presumablybecausetheintranuclearpathogen‘NIX’(Elston,1986).Itisintrigu-
theseeventsoccuronveryshorttimescalesandareingthatintwoofthehostspeciesshowntobeinfected
thereforeonlyrarelyvisible.Thefirstdiscernibleinfectionwith“Ca.E.bathymodioli”inthisstudy(B.heckeraeand
stageinourstudywasthepresenceofasinglerod-B.puteoserpentis),previouslightmicroscopicalanalyses
shapedbacteriuminthemusselnucleithatgrowstoanindicatedthepresenceofhypertrophiednucleiinsome
unseptatedfilamentofabout20mmandpossiblylongertissues(Wardetal.,2004).Transmissionelectronmicros-
(Stages1and2inFig.2G–J).Thismaybeacharacter-copyandFISHanalysesareneededtoclarifyifthese
isticfeatureofintranuclearbacteria.Inthemarinespongenucleardistortionswerecausedbyviruses,assuggested
Aplysina,intranuclearbacteriacanformfilamentsuptobyWardandcolleagues(2004),orbyintranuclearbacte-
350mminlength(Vacelet,1970;Friedrichetal.,1999),ria.Similarly,bacteriadescribedas‘Rickettsia-like’and
andinfectiousformsofHolosporaobtusacanreach‘Chlamydia-like’basedonlightmicroscopicalanalysesof
20mminlengthinthemacronucleusoftheciliatePara-gilltissuesofventandseepmollusks(Powelletal.,1999;
meciumcaudatum(Görtz,2006).UnseptatedfilamentousTerlizzietal.,2004;Wardetal.,2004;Millsetal.,2005)
growthhasalsobeenobservedinBdellovibriobacterio-mightnotbebacteriabelongingtotheRickettsiaand
vorus,adeltaproteobacterialpredatorofGram-negativeChlamydiabutrather“Ca.E.bathymodioli”parasites.
bacteria(Angert,2005;Dworkin,2006).ThetransitionThecompletionofthe“Ca.E.bathymodioli”cellcycle
fromStage2toStage3appearstotakeplacethroughrequiresthereleaseoftheinfectednucleifromthehost
longitudinalfission(Fig.2K).Longitudinaldivisionisrarecell.Thismostlikelyoccursthroughthedestructionofthe
amongbacteriaandhasonlybeendescribedinthesulfur-hostcellandruptureofthehostcytoplasmicmembrane.
oxidizingsymbiontsofthreemarinehostgenera,theIn‘NIX’-infectedS.patula,thehostcellsarerupturedby
nematodeLaxussp.(Polzetal.,1992;1994),thegutlessthegreatlyenlargednuclei,indicatingagreaterresiliency
oligochaeteOlavius(GiereandKrieger,2001;Brightandofthenuclearoverthecytoplasmicmembrane(Elston,
Giere,2005),andthesand-dwellingciliatesofthegenus1986).Whenthe“Ca.E.bathymodioli”-infectednuclei
Kentrophoros(Fauré-Fremiet,1951;Raikov,1971).becomeextracellular,theyarestillsurroundedbyamem-
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Intranuclearbacteriainventandseepmussels7
Fig.2.“Ca.E.bathymodioli”invariousmusseltissuesanddevelopmentalstages.(ThisfigurewaspreparedasanRGBimageandconverted
toCMYKmodeforprint.TheoriginalRGBimageisprovidedasFigureS1inSupportingInformation).
A.Cross-sectionthroughajuvenilemusselshowingthedistributionoftheintranuclearbacteriumthroughoutthegilltissue.Intranuclear
bacteriaareshowningreenandmusseltissueappearsinorange.Themorphologyresultsfromstainingnucleiandbacterialendosymbiotic
DNAwithDAPIwhichwasassignedanorangecolour.AB,ascendinggillbranch;DB,descendinggillbranch;ID,innerdemibranch;M,
demibranch.outerOD,mantle;B–D.Non-ciliatedgilltissuewithintranuclearbacteriuminintercalarycellswhichalternatewithbacteriocytes.
E.Guttissue.InimagesB–Eintranuclearbacteriaareshowningreenandeukaryotictissueisrepresentedinyellow.Nucleiandbacterial
endosymbioticDNAinbacteriocytesappearinblue.
F.Non-ciliatedgilltissuewithintranuclearbacteria;intranuclearbacteriaappearinbrightyellow,whereaseukaryotictissueisrepresentedbya
yellowishtobrownishcolour.Chemoautotrophicandmethanotrophicbacterialendosymbiontsinbacteriocytesareshowningreenandred
respectively.NucleiwerestainedwithDAPIandappearinblue.
G–P.Developmentalstagesof“Ca.E.bathymodioli”inB.puteoserpentisgilltissues.Theintranuclearbacteriumappearsingreen,thenucleus
inblue.ImagesH–Mresultfromprojectionofastackofseveraltwo-dimensionallayersontoonesinglelayerreflectingtheoverall
three-dimensionalstructureonatwo-dimensionalplane.
G–J.SeriesshowinggrowthfromasingleshortrodtoasinglefilamentinStages1and2.
K.TwooverlappingfilamentsorfilamentintheprocessoflongitudinalbinaryfissionintransitionfromStage2toStage3.
L.Twoseparatefilaments(Stages2–3).
M.Filamentousassemblyconsistingofeitheronesinglelongcoiledfilamentorseveralfilaments(Stage3).
N.Stacksofshorterfilaments(Stage4)resultingfromtransversefissionsofcoiledfilaments.
O.LongrodsresultingfromdivisionofStage4filaments.

brane.Thismembraneismostlikelyofnuclearorigin,DNAsynthesis.Recentstudieshaveshowntheimpor-
becauseitstainedpositivelywith4,6-diamidino-2-tanceofextracellularDNAasanutritionalsourcefor
phenylindole(DAPI),suggestingremnantsofnuclearfree-livingbacteria(Lennon,2007;Pinchuketal.,2008),
DNAalongtheinsideofthemembrane.Finally,thebac-butnothingisknownaboutthemetabolismofintra-
teriamustescapefromthemembrane-surroundedStagenuclearbacteriaandtheirgenomeshavenotyetbeen
6nuclei,buthowtheydothisremainsunclear.Notunex-sequenced.Althoughnotanintranuclearbacteriumbut
pectedly,weonlyveryrarelysawsingleextracellular“Ca.ratherabacterialparasite,B.bacteriovorusefficiently
E.bathymodioli”,asthewashingprocedureforthefixationconsumesitsprey’scellularcontentsincludingnucleic
ofgilltissueswouldhaveremovedmostlooselyattachedmaterial.Ithasbeenstudiedextensively(reviewedin
cells.LittleisknownabouthowotherintranuclearbacteriaJurkevitch,2006)anditsgenomecontains20different
escapetheirhosts.IntheciliateParameciumcaudatum,deoxyribonucleasegenesforDNAhydrolysis(Rendulic
infectiousformsoftheintranuclearbacteriumHolosporaetal.,2004).Afurthersourceofnutritionfor“Ca.E.
obtusamakeuseofthedivisionapparatusofthehostbathymodioli”mayalsobecytosolicsubstrates,particu-
nucleustoescapefromthenucleus(Fokin,2004;Görtz,larlyduringlaterdevelopmentalstagesinwhichthe
2006).InthebacterialpredatorB.bacteriovorus,15extremeenlargementofthenuclearmembranemight
lipaseshavebeenidentifiedthatdissolvetheoutermem-enabletheleakageofsubstratesfromthecytosoltothe
braneofitshostsandenableitsrelease(Rendulicetal.,nucleus.
2004).Wedonotcurrentlyknowif“Ca.E.bathymodioli”Afewbacteriathatfallwithinthemonophyleticcladeto
contributeactivelytotheirreleasefromthemembrane-which“Ca.E.bathymodioli”belongshavebeencultivated:
boundaggregate,forexample,throughtheexcretionofE.elysicola(isolatedfromaseaslug–Kurahashiand
lipases,orifthisprocessispassive,forexample,throughYokota,2007)andthreebacteriaisolatedfrommarine
mechanicaldisruptionofthegreatlyswollenaggregratesponges,Spongiobacternickelotolerans(unpublished
membrane.informationfromGenBank)andtwounnamedspecies
calledH262andH425(Sfanosetal.,2005).Cultivation
ChromatinasanutritionalsourceinformationisonlyavailableforE.elysicolawhichisan
aerobic,mesophilicheterotrophbutdetailsforgrowth
ThedisappearanceofchromatinduringthedevelopmentsubstratesofE.elysicolawerenotdescribedanditisnot
of“Ca.E.bathymodioli”suggeststhatthehostDNAwithclearifthisspeciescangrowonDNAalone(Kurahashi
itssurroundingchromosomalproteinsprovidesthenutri-andYokota,2007).
tionforthegrowingandreproducingparasites.Chroma-
tinreductionhasalsobeenobservedinprotistsinfected
withintranuclearbacteria(TableS3).Clearly,DNApro-Host–parasite–symbiontinteractions
videsarichsourceofsugar,nitrogenandphosphorus.BothourFISHandTEManalysesshowedthatonly
ATPforbiomasssynthesiscouldbeacquiredbybreak-symbiont-freeintercalarygillcellswereinfectedby“Ca.E.
ingdownhostDNAorhostnucleotidescouldbeusedforbathymodioli”whereasthenucleiofbacteriocytescon-
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Fig.3.Transmissionelectronmicroscopyimagesshowingdifferentstagesinthedevelopmentof“Ca.E.bathymodioli”.
A.assemblyOvalofelongatedStage3.nucleusNotethewithfilamentousintranuclearbacteriaappearanceinoflongitudinal,bacteriainthistransversalstageandandthatthecross-sectionfilamentsmostarelikelyunseptated.representingthefilamentous
B.Uninfectednucleus(left)andinfectednucleus(right).Theinfectednucleusrepresentsacross-sectionofeitheratwistedStage2filament
oranearlyStage3filamentouscoil.
D.C.StagePear-shaped4nucleusnucleusshowingwithastackintranuclearofshorterbacteriainfilamentsinlongitudinal,cross-section.transversalandcross-sectionmostlikelyrepresentingthemultifilamentous
E.coilofSwollenStageStage3.The5nucleusnucleus,withnormallybacteriaatinthebasallongitudinalendofandthecellcross-section.whenNoteuninfected,theisreducednowatthelengthapicalofeachend.singlebacteriumascompared
withtheelongatedforminStage3.Alsonoticetheabsenceofchromatinexceptfornarrowremnantsalongthenuclearmembrane.
F.Dividingfilament(middle)showingtransversebinaryfission.
G.Singlebacteriumshowingelectrondenseparticlesdistributedthroughoutthecell.
H.Onesinglebacteriumincross-section.NotetheinnerandoutermembranecharacteristicofGram-negativebacteria.

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Bathymodiolusintranuclearparasite

Intranuclearbacteriainventandseepmussels9
Fig.mussels4.Samplinginvestigatedsitesinofthisstudybathymodiolin.Circles
showhydrothermalvents(red)andcold
seepsLogatchev;(yellow).WA,MG,Wideawake;MenezLI,Gwen;LilliputLO,
Canyon;AlaminosAL,Ridge);(Mid-AtlanticAT,MississippiAtwaterVCanyon;alley;CH,WF,WestChapopote;FloridaMC,
(GabonEscarpmentContinental(GulfofMargin);Mexico);GFRE,,GermanRegab
LadyFlats(NorthFiji(Pacific-AntarcticBack-ArcRidge);Basin).WL,White

tainingthethiotrophicandmethanotrophicendosym-2005).However,mortalitycouldnotbeclearlylinkedto
biontswereneverinfected.Thissuggeststhatthesecellsparasitismasshiftsinseepagemightalsohavecaused
areprotectedagainstinfection,eitherbecausethebacte-musselandclamdeaths(Wardetal.,2004;Millsetal.,
riocytesdifferfromotherhostcellsinamannerthatpre-2005).
ventsparasiteinfectionorbecausethesymbiontsprovide
protection.Howthisprotectionmightbeaffordedisnot
clear,buttheconsequencesaresubstantial.Asthebac-Globaloccurrence
teriocytenucleiarenotinfected,itislikelythatchemosyn-Ourstudiesshowthat“Ca.E.bathymodioli”occursin
theticenergyproductionandcarbonfixationbytheseepandventmusselsfromaroundtheworld.Wecould
endosymbioticprimaryproducersremainsfullyfunctional,notfindtheintranuclearparasiteinonlytwospecies,B.
thusassuringthenutritionalsupplyofthehost.Whiletheaff.boomerangfromtheGaboncontinentalmarginand
infectionmaybedeleterioustotheintercalarycells,theB.breviorfromtheWestPacific.Itispossiblethatsome
overallhealthofthehostdoesnotappeartobesignifi-bathymodiolinspeciesarenotinfectedby“Ca.E.bathy-
cantlyaffectedbecauseitspowerplants,thebacterio-modioli”.However,therearenosharedcharacteristics
cytes,arenotinfected.Thisassumptionissupportedbybetweenthetwomusselspecieslacking“Ca.E.bathy-
thefactthatwehavenotobservedmassmortalityofthemodioli”thatwouldexplainwhythesehostswouldnotbe
B.puteoserpentispopulationattheLogatchevhydrother-infectedwiththisglobalintranuclearparasite:Neitherare
malventfieldduringthefourresearchcruiseswehavetheycloselyrelatedtoeachother,nordotheyoccurinthe
hadtothisfieldbetween2004and2007,despitethesamegeographicregion.Analternativeexplanationisthat
regularpresenceof“Ca.E.bathymodioli”withinthepopu-thesespeciescontainedintranuclearbacteriaatlevelstoo
lationthroughoutthisperiod.ThisisincontrasttothelowtobedetectedwithPCRandFISH.Althoughwedid
massmortalitycausedbytheintranuclearbacteriaofthenotquantifyinfestationlevels,wedidobservedifferences
clamS.patulaandtheRickettsia-likebacteriaoftheintheabundancesof“Ca.E.bathymodioli”bothbetween
oysterCrassostreaariakensisthatdonothavesymbiotichostsfromdifferentfieldsandbetweenindividualsofthe
bacteriaintheirgills(Elston,1986;Ayresetal.,2004;samespecies(TableS2).
unpublishedinformationinGenBankunderaccession
DQ123914).und18733DQ1numbersWhatisnotcurrentlyknownisifchemosyntheticsym-Biogeographicalclusters
biontsprotecttheirhostsagainstotherparasitesbesidesIntheGulfofMexico,B.brooksiandB.heckeraeshare
“Ca.E.bathymodioli”.Massmortalityofsymbiont-thesame“Ca.E.bathymodioli”16SrRNAphylotype
containingmusselsandclamswasobservedattheBlakedespitethefactthatthesehostsarenotcloselyrelatedto
RidgeseepoffthesoutheasterncoastoftheUSAandeachother(Jonesetal.,2006;Cordesetal.,2007).
morphologicalanalysesoftheclamsandmusselsTogetherwiththefactthatthe“Ca.E.bathymodioli”phy-
showedthatbothwereinfestedwitheukaryoticandlotypesfallintothreeregionalclustersthatreflectthe
prokaryoticparasites(Wardetal.,2004;Millsetal.,originoftheirhosts,thissuggeststhathostgeography
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10F.U.Zielinskietal.
WHoleW′W′W′W′W′′EE′E′′E′
W′W′W′W′W′W′
Woods′44°58.7478N′S′10°54.86751
(W31°3112°22.36S12°22.3524S′12°22.3503S′12°22.3515S′1.846213°1S′173°55.029S′173°54.940S′1173°54.91S′9°37.9419S′84°55.03N′93°26.12N′88°29.8N′94°30.12N′89°08.20N′
Alvin′N′submersiblem303538ROV/6-5m303556ROV/6-j6m305366ROV/16-34°48.6293m2987125ROV/1B-24°48.6275m2986125ROV/7-44°48.6231m2987125ROV/12-616°59.449m2009-1266GTV16°59.486m2000-999GTV37°47.4673m2212-1-430GTV5°52.8134m3150M5Ang21°53.98m2915Sp1.2Sp1.1,m2915Sp2.2Sp2.1,26°21.32m2226M29GoMM34GoM
37°51m850105,MG3,(2001)4°48.64m2998109GTV/A-3(2007)9°33.1783m1496200ROV/9-3(2005)16°59.348m1976-639GTV(1999)(2002);(2002)(2005)
-335GTV(2004)14°45.1629m3016al.et
StoffersOndreasGoM–26°02.00m3284M6(2006)27°34.10m1893M24GoM–
LongitudeLatitudeDepthIDSampleReferencescientistChiefyearMonth,
(2005);al.etal.etal.etal.etal.etal.etal.et
al.etStecherStoffers.P2001Jun/Jul-grabVT3FoundationBiozaireDuperronSibuetM.2001NovictorV2andFisherR.C.2003OctAlvin1/I1SeepsDeepBohrmannandSpiessBohrmannG.2006AprQuestM67/2MeteorandFisherR.C.2003OctAlvin1/I1SeepsDeep28°07.40m10504178/mc853-2–FisherR.C.2006MayAlvin15/3Atlantis
CarneyCarneyB.B.sp.sp.aff.aff.mannedtheFrance),,(IfremerictorVandGermany)Bremen,ofUniversity(Marum,Questvehiclesremotely-operatedtheusingrecoveredwereSamplesvents.hydrothermalfromSpecies(2006b).colleaguesandDesbruyèrestoaccordingnamefieldentVseeps.coldfromSpecies
aSarradinSarradinM..P2001Jun/JulictorVOSTAGwenMenezKuhnKuhn.T2004Jan/FebQuestIHydromar2Logatchev/IrinaHaaseHaaseK.2005AprQuestIIMarsuedWideawakeHaaseHaaseK.2005AprQuestIIMarsuedLilliputLilliput/MainHalbachHalbach.P1998Aug/Sep-grabTVIIHyfifluxLady/LHOSWhite(38°S)FlatsGermanContinentalGabonMargin/RegabFloridaestWEscarpmentCampecheKnolls/ChapopoteContinentalLousianaalleyVSlope/AtwaterContinentalLousianaSlope/AlaminosCanyonContinentalLousianaSlope/MississipiCanyon
1.ableTBathymodiolusRecoverynameCruiseLocationspeciesazoricusB.puteoserpentisB.B.B.breviorB.B.B.heckeraeB.heckeraeB.brooksiB.brooksiB.brooksiB.childressi“B.”childressi“B.”a.Germany).Kiel,GmbH(Oktopusgrab-controlledVTaandUSA),Institution,Oceanographicb.c.d.
sites.samplingandstudythisininvestigatedmusselsBathymodiolinbbddd
cbdbddthermophilusddboomerangbbAuthorsThe2009©Journalcompilation©2009SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology

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Intranuclearbacteriainventandseepmussels11

Fig.5.Proposeddevelopmentalcycleof“CandidatusEndonucleobacterbathymodioli”.Theinfectionbeginswithasinglerod-shaped
bybacteriumlongitudinalinsidefission,thenucleusyieldsseveral(Stage1)suchthatfilamentsgrowsintothatanareunseptatedlooselywrappedfilamentinofauptofilamentous18–20mcoilmin(Stagelength3).(StageThese2).subsequentlyMultiplication,formpossiblystacks
5).ofshorterFurtherfilamentsmultiplicationofuptoresults8–10inmaminvoluminouslength(Stage4).membrane-surroundedRepeatedtransversebacterialbinaryaggregatefissionsleadcontainingtouptonumerous80000rod-shapedrod-shapedbacteriabacteria(Stageand
thedestructionofthehostcell(Stage6).Theseaggregatesareeventuallydisruptedandthebacteriareleased.
andthesenotassociations.cospeciationThisplayedahypothesisroleinisthesupportedestablishmentbyourofathesebetterdeep–seaunderstandingassociations.ofhistoricaldispersalpatternsin
statisticalanalysesthatshowedasignificantcorrelation
betweeninvestigationgeneticofandmorehostgeographicspeciesdistances.fromdifferentHowever,geo-theConclusions
beforegraphicthisregions,hypothesisparticularlycanbetheWestsubstantiated.Pacific,isneededThisspreadinstudyshowsbathymodiolinthatmusselsintranuclearfrombacteriahydrothermalarewide-vents
fieldOneonthemusselsouthernspecies,B.Mid-Atlanticsp.fromRidgetheWideawakeharbouredtwoventandbathymodioli”coldseeps.belongTheto16SarRNAmonophyleticsequencescladeofthat“Ca.con-E.
“otherCa.E.by1.9%.bathymodioli”OnefallsphylotypesintothethatdifferedMid-AtlanticfromRidgeeachsistsmetazoansofassequencesdiversefromasbacteriasponges,associatedcorals,withseamarineslugs,
Ridgeclusterclusterwhereas(Fig.the1B).otherThisonefallsindicatesinthethatalthoughPacific-Antarcticmostclams,sequencesascidians,withinthisseacladeurchinsisandknownfish.toOnlyoriginateonefromofanthe
“Ca.E.bathymodioli”bacteriaclusteraccordingtotheirintranuclearbacterium,thepreviouslydescribed‘NIX’
thegeographypast,,intherethismaycasehavefrombeenventsitestransoceaniconthecrossingPacific-inetalparasite.,1992;fromAtheyresetrazoral.,clam2004)S.butpatulamorphological(Elston,1986;descrip-Kerk
AntarcticRidgetothesouthernMid-AtlanticRidge.His-tionsofintranuclearbacteriaexistforotheranimalhosts
fortoricalsomedispersalbathymodiolinacrosshostoceanspeciesbasinshas(Jonesbeenetal.,suggested2006;sinawithinthisspongesclade,(Vforacelet,example,1970;fromFriedrichtwoetalspecies.,1999)ofAply-and
Olu-LeassumeRoythatitetal.,would2007),nothaveandthereoccurredisinnotheirreasonintra-to(Ttheablevenerid2).WeclampostulateRuditapesthatmanydecussatusofthesequences(Azevedo,within1989)
nuclearbacteria.Extensivecomparativeanalysesofthethiscladeoriginatefromintranuclearbacteria,andthat
phylogenyofbathymodiolinhosts,theirsymbiontsandtheseparasitesarewidespreadinmarineanimals,includ-
theirintranuclearbacteriawillprovideanidealdatasetforingclamsandothershellfishconsumedbyhumans.
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12F.U.Zielinskietal.
proceduresExperimental(1999).Samplingsitesandprocessing
alet(1992).NineBathymodiolusspeciesfromsixdeep-seahydrothermal
aletventPacificfieldsOceanandwerefivecoldinvestigatedseepsites(Fig.4,locatedTableinthe1).OneAtlantictofourand
recoveryindividualsofmusselseachwerespeciesimmediatelywereexaminedtransferred(Tableinto1).chilledUpon
Friedrich(1970);aceletVforFISHandTEMinvestigations.
bottomseawaterandprocessedaspreviouslydescribed
Referencefrozen(Duperronandetstoredal.,at2005;-20°C2006;forDNA2007).extractionBriefly,asgillwelltissueasfixedwas
Cloningandsequencing“Ca.E.bathymodioli”from
a(Logatchev)puteoserpentisB.PhylogenymKerk(1986);ElstonGamma16studyThisGamma20toup0.4;gen)transformantswerefromsubsequentlyfourtransformed.individualsA(277totalofclones1108perpositiveindi-
GenomicDNAwasextractedfromgilltissueaccordingto
Zhouuniversalandbacterialcolleaguesprimers(1996).GM3FPCRandwasGM4Rperformed(Muyzerusingetalthe.,
2.71995)¥10-and5;aEppendorf,high-qualityTaHamburg,qDNAGermany).polymerase(errorAmplificationrate
products(Promega).wereOnepurifiedShotTandOP10ligatedcompetentintoE.pGEM-TcolicellsEasy(Invitro-vectors
m)¥¥25nightvidual)inwereV96pickedMicroWbyellPlatesblue/white(Nunc,screeningWiesbaden,andgrownGermany)over-
totalcontainingof1009200clonesmlwereLuria–Bertani/ampicillinscreenedfortherightbrothpersizedwell.insertA
taxonHigher-rankingbacteriaofDesignation(SizeShapenucleiinsideGamma4.5leastat150–350;FilamentousbacteriaIntranuclearrich,membraneMultilayered,(NIX)X’Inclusion‘Nuclear(1989)Azevedond1.3øellipsoidaltoSphericalbacteriaEndonucleobioticCa.bathymodioli”Cyclesequencingaccessories,equipmentandconditions
(252primerpairclones(Yperanisch-Perronindividual)etbyal.,PCR1985)usingandthe854clonesM13F/M13Rhad
an384insertcloneswithwiththetheexpectedrightsizesizedofinsertapproximatelywere1500partiallybp.
cleavedpartiallyfolded,complexly1.8filamentoustoRod-shapedetal.,1995)wasusedassequencingprimer.Theresulting
sequenced(96clonesperindividual).PCRproductswere
G50purifiedinSuperfineMultiScreen-HVresinplates(Amersham(Millipore)usingBiosciences)Sephadexand
ingsequencedKitalongusingwiththetheBigDyeGeneticTerminatorAnalyzerv2.0CycleAbiprismSequenc-3100
(AppliedBiosystems).TheGM3Foligonucleotide(Muyzer
partialsequenceswereanalysedwithBioEditSequence
AlignmentimplementationEditorversion(Thompson7.0etal(Hall,.,1994).1999)usingUniquelytheoccurringClustalW
MetazoaindatetodescribedbacteriaIntranuclearRickettsiae).intranuclearfacultative(excludingotherbathymodiolinmussels
2.ableTtaxonMetazoaspeciesHostPoriferaDemospongiaaerophoba;AplysinacavernicolaA.MolluscaBivalviapatulaSiliquadecussatusRuditapesBathymodiolusavailable.sequencerRNA16Sno.probe,gammaproteobacterialgeneralwithFISHonbasedPhylogenya.determined.notnd,Primersbathymodioli”specifically(Logatchev)targetingwerethe16SdesignedrRNAusinggenetheof“Ca.reverseE.
sequenceswereignored.Repetitivesequenceswere
grouped.sequencedThreebyclonessequencingperbothgrouptheandcodingindividualandwerenon-codingfully
Endonucleobacter(Y518F,anisch-Perron1099Fandetal1.,193R1985),534R(Buchholz-Cleven(Muyzeretetalal.,.,1997).1993),
DNAstrandswiththesequencingprimersM13F,M13R
“wereasdescribedforpartialsequencing.Sequenceswere
assembledusingSequencher(GeneCodesCorporation,
primerhttp://wwwsiteswere.genecodes.com).discarded.Thevectorialremnantsandthe
spp.Cloningandsequencing“Ca.E.bathymodioli”from
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andcomplementarysequenceofprobeBnix64asaforward2008).GeographicaldistanceswereestimatedusingGoogle
primersequenceof(bnix-64F:probeAGCGGTBnix1249asaAACAGGTCTreverseAGC)primerand(bnix-theEarthsamplev.4locations.basedonminimumoceanicdistancesbetween
1267R:amplificationproductGCAGCTTCGCGACCGTCT)(forprobedesignseeresultingbelow).inaA1203combi-bp
nationofthebnix-64FprimertogetherwiththeuniversalDesignofprobestargeting“Ca.E.bathymodioli”
coverbacterial1409bpreverseoftheprimer16SGM4RrRNAwasgene.eventuallyStepsforchosencloning,toBasedonthe16SrRNAfullsequence(1468bp)of“Ca.E.
bathymodioli”fromB.puteoserpentis(Logatchev)three
sequencingandanalysingintranuclearbacteriabelongingtoprobesweredesignedusingtheprobedesigntoolofARB
“Ca.serpentisE.withbathymodiolithe”exceptionwereasthatdescribedonly16abovefortransformantsB.puteo-per(Ludwigetal.,2004).Toverifytheirspecificity,probesBnix64
individualwerepickedandscreenedfortherightsizedinsert,(GCTCACCCA)AGACCTGTTandBnix1249ACCGCT),Bnix643(CCGT(GCAGCTTCGCGACCGTCT)ACTCTAGC
andperaturestheforpositivetheclonesfullybnix-64F/bnix-1267Rsequenced.andTheannealingbnix-64F/GM4Rtem-werecheckedagainstthe16SdatasetoftheRibosomal
primerpairswere56°Cand45°Crespectively.Sequencingetal.,Database2007).ProjectTheXmostusingtherecentonlineprobeProbematchMatchagainsttool(Colethe
wasperformedusingtheBigDyeTerminatorv3.1Cycle
SequencingKitalongwiththeGeneticAnalyzerAbiprismBnix64RDP-Xtargeteddataset10.4also12(October2008)gammaproteobacterialrevealedthatsequencesprobe
3130(AppliedBiosystems).belongingtotheOceanospirillalesandAlteromonadales.
AccessionnumbersfiveProbeunclassifiedBnix643targetedalsogammaproteobacterial13cyanobacterialsequencesandsequences,one
The16SrRNAgenesequencesbelongingto“Candidatuswasconfirmeddeltaproteobacterialtospecificallysequence.targetHowever“Ca.,E.probebathymodioli”Bnix1249
EMBLEndonucleobacterdatabase(Kulikovabathymodioli”etal.,have2007)beenunderregisteredaccessionatthe(Logatchev).Withtheamplificationofmore“Ca.E.bathymo-
dioli”phylotypesfromotherbathymodiolinspeciesitbecame
(TablenumbersS2).FM162182toFM162195,andFM244838evidentthattheprobesBnix1249andBnix643hadonemis-
matcheachtosomeoftheotherintranuclearphylotypes
(TableS2).Allthreeprobesaredepositedintheoligonucle-
PhylogeneticreconstructionotideaccessionprobenumbersdatabasepB-01516‘probeBase’to(LoypB-01518.etal.,2007)under
SequenceswereanalysedusingARB(Ludwigetal.,2004)AllGermany).probesSpecificwerefluorescentlyhybridizationlabelledconditions(biomers.net,forallthreeUlm,
andnucleotidecomparedBLASTwith(McGinnistheNCBIandMadden,nucleotide2004),databasetheRDP-Xusingprobesweredeterminedbyvaryingtheformamideconcen-
databaseusingtheSequenceMatchtool(Coleetal.,2007),probestrationinthehybridizedhybridizationequallywellbufferwith(Pernthalerthetargetetal.,organism2002).Allat
andtheSilvarRNAdatabaseusingtheSINAwebaligner
(Pruesseetal.,2007).Highlysimilarsequenceswere35%formamideconcentration.ProbeEUB338(Amannetal.,
includeddisplayinginthemorethananalysis25%andgapsalignedaswellusingasClustalX.positionsPositionsambigu-and1990)thecoveringNON338mostprobe(Wbacteriaallnerwasetalused.,as1993)aasapositivecontrolcontrolfor
ouslyalignedwereremovedfromtheanalysis.Thefinalbackgroundautofluorescence.
alignmentcomprised1416positions.Phylogeneticanalyses
werehoodanalysis.performedTheusingformerBayesianwasrunasusingwellasMrBayesmaximum3(v3.1.2)likeli-Fluorescenceinsituhybridization
(RonquistReversibleandmodelalongHuelsenbeck,with2003)Gamma-distributedunderaratesGeneralofTevo-imeSubsamplesofB.puteoserpentistissuessuchasgill,gut,
lutionandaproportionofinvariantsites.Analyseswereper-digestivephosphategland,bufferedlabialsalinepalps,(PBS:mantle137andmMfootNaCl,were2.7fixedmMinKCl,1¥
CarloformedforMarkov2000chains.000SamplegenerationstreesusingwerefourtakenparalleleveryMonte100010mMNa2HPO4,2mMKH2PO4)containing2%paraformal-
dehydeat4°Cfor9–18h.Sampleswerewashedthreetimes
treesgenerations.wereusedPosteriorassupportprobabilitiesvaluesforcalculatednodesinoverthe5000tree.Thebestbyplacingtheminfresh1¥PBSfor10mineachtimeand
subsequentlytransferredintocoldPBS/ethanolsolutioncon-
basedmaximumon100likelihoodjumbleanalysisreplicates.wasToassessperformedtheusingrobustnessPHYLIPoftaining1¥PBSandpureethanolinequalparts.Samples
nodes,1000MLbootstrapreplicateswererun.Thealignmentwerekeptat4°Conboardtheresearchvessel,air-freighted
usedforthephylogeneticanalysiscanbeobtainedfromthebacktothelaboratoryat4°Candfinallystoredat-20°C.
EMBLdatabase(accessionnumber:Align_001264).FixedspecimenswereembeddedinSteedman’sWax
(Steedman,1957)andsectionedwithamicrotomeinto
10Frostmmslidesthick(Fishersections.TheScientific),sectionsdewaxedwereinplacedthreeontosuccessiveSuper-
Statisticalanalysesbathsofabsoluteethanolfor5mineachandairdried.Sec-
Totestbathymodioli”thehypothesis16SrRNAthatgeneticsequencesdistanceswerebetweencorrelated“Cawith.E.tions(Pernthalerwereetthenal.,2002)coveredwithcontaining200mloffluorescentlyhybridizationlabelledbufferoli-
1-theirusingthegeographicalprogramdistances,R-packageaMantel(CasgraintestandwasLegendre,performedwithagonucleotideglassprobescoverslip(5andngmlhybridizedfinalat46°Cconcentration),for10mincoveredina
AuthorsThe2009©Journalcompilation©2009SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology

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VIscriptuMan

14F.U.Zielinskietal.
histologicalmicrowaveoven(MicrowaveResearchandAppli-lengthhanddiameter(width)dwasdeterminedforseveral
cations,Laurel,MD,USA)withthepoweroutputsetto20%.intranuclearcellsusingTEMimages(averagesize
Theethanolslidesfor1wereminrinsedeach.inSingle1¥PBS,hybridizationsMQwater,andtargetingabsolutespe-1.8¥0.4mm,itfollowsthath=1.4mm,d=0.4mm).
usingcifically“probeCa.E.Bnix1249bathymodioli”labelledwith(Logatchev)Cy3.ForwerevisualizationperformedofAcknowledgements
allusedbacteria(AmanninetB.al.,1990).puteoserpentisDouble,Cy3-labelledhybridizationsEUB338targetingwasWearegratefultothechiefscientistsandthecaptainsand
crewsoftheinvolvedresearchvessels(Table1)aswellas
EUK516eukaryotic(Amann18SetrRNAal.,were1990).TripleperformedhybridizationsusingCy5-labelledtargetingthechiefengineersandcrewsoftheROV’sQuest4000and
thechemoautotrophicendosymbiont,themethanotrophicVictorexcellent6000supportandthetoobtainmanneddeep-seasubmersiblehydrothermalAlvinforventprovidingand
usingendosymbionttheprobesandtheBMARt-193eukaryotic(Cy3),18SrRNABMARm-845wereperformed(Cy5)coldseepsamples.Weappreciateourlaboratoryassistants
(Duperronetal.,2006)andEUK516(Fluos).Forquadrupleskills.SilkeWWeetzelareandgratefultoSabineVictoriaGaudeOrphanandfortheirlettingusindispensableuseher
hybridizationstargetingadditionally“Ca.E.bathymodioli”
(Logatchev)Fluos-labelledBnix1249wasused.theDeltaVSoftWisionorxRTdeconvolutionRestorationandMicroscopyimageanalysisSystemalongsoftwarewithas
Deconvolution(restoration)microscopywellauthoras(Fthe.U.Z.)ImarisisparticularlyimageanalysisthankfultosoftwareVictoriapackage.OrphanThefor
TheairdriedslideswereembeddedinaDAPI-amendedduringhostingpartshiminofherthislabstudyat.theWethankCaliforniaShanaInstituteGoffrediofTandechnologythree
mountantMicroscopyandSystemevaluated(AppliedonaPrecision,DeltaVisionIssaquah,RTWA,RestorationUSA)anonymousreviewersforhelpfulcommentsandsuggestions
fortheimprovementofthismanuscript.Thisworkwas
usingequippedanwithOlympusappropriateIX71filter(Olympus,setsforCenterCy3,ValleyCy5,,PDAPIA,USA)andsupportedbygrantsfromthePriorityProgram1144of
theGermanScienceFoundation(DFG).Thisispublication
Worxfluorescein.imageForanalysisimagesoftwarecapturewasanduseddeconvolution(AppliedthePrecision,Soft-number24ofthePriorityProgram1144‘FromMantleto
Issaquah,WA,USA).ImageswerefurtherprocessedandOcean:Energy-,Material-andLifecyclesatSpreadingAxes’
DFG.theofwwwanalysed.bitplane.com)usingthethatwasImarisalsosoftwareusedtoassignpackagecolours(http://to
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AuthorsThe2009©Journalcompilation©2009SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology

165

IscriptuManV

18F.U.Zielinskietal.
Fig.S1.Figure2ofmaindocumentinoriginalRGBmode
(forfigurecaption,seeFig.2inmaindocument).
TableS1.Closestrelativesof“CandidatusEndonucleo-
(Logatchev).bathymodioli”bacterTableS2.Nucleotidedifferencesofdominantandminor
“Ca.E.bathymodioli”16SrRNAsequences.
TableS3.Intranuclearbacteriadescribedfromprotists.
Videos.Animationsshowstacksof2Dimagescreatedby
movingthefocalplanethroughthez-axisof10mmthick

166

sectionsat0.2mmintervals.S1:developmentalstage2;S2:
developmentaldevelopmentalstagestage6.3;S3:developmentalstage4;S4:
contentPleaseornote:functionalityWiley-Blackwellofanyaresupportingnotresponsiblematerialsforsuppliedthe
bytheauthors.Anyqueries(otherthanmissingmaterial)
shouldbedirectedtothecorrespondingauthorforthe
article.

AuthorsThe2009©Journalcompilation©2009SocietyforAppliedMicrobiologyandBlackwellPublishingLtd,EnvironmentalMicrobiology

trPa

Concluding

168

VI

remarks

8Chapter

GeneralSummary,ConclusionsandOutlook

8.1SymbiontdiversityinChapopote

MymarinemicrobiologystudiesstartedafterthediscoveryoftheChapopote
SeepintheGulfofMexico.IfinishedmyBachelorstudiesanalyzingthemi-
crobialdiversityofsedimentsamplescomingfromthisunusualsite.Itwas
quiteinterestingtoseethatmicrobialcommunitiesweredifferentintwodif-
ferentbutnearbyspotswithinthesite.Itseemedthatthepresence/absence
ofoilinthesampleswasdeterminingthemicrobialdiversityprofile.Iwas
veryexcitedwhenIwasallowedtoanalyzetheBathymodiolusspp.com-
ingfromthissamesiteaspartofmyPhDthesiswork.Thesemusselshad
beenstudiedallaroundtheworldandinparticularinthenorthernGulf
ofMexico(GOM).Then,itwasveryinterestingtogettocharacterizethis
Bathymodiolus-bacteriasymbiosisinthisnewhabitat.Ifthefree-livingbac-
terialcommunityoftwoveryclosespotswasalreadyverydifferent,Ithought
withmorereasonsthesymbiontsfromtwoverydistantsitesoneachsideof
theGulfofMexicowoulddiffergreatly.Myknowledgeaboutsymbiosiswas
verylimitedatthattimeandInevertookintoaccounttheveryfinesystem
thattheseassociationsare.Symbiosisissuchaspecificsystemsthatwhen
IanalyzedthemusselsfromthesouthernGoM,musselsandtheirbacteria
wereexactlythesame(basedonstandardCOIand16SrRNAanalysis)asthe
onesfromthenorth.1000milesofseparationwasmeaningnothingforthe
symbiosisprofile.However,twobacterialphylotypes,differenttotheclassi-
calsymbionts,seemedtobepresentafterthe16SrRNAsequenceanalysis.

169

CONCLUDINGREMARKS

TheywereindeedpresentafterlocalizationbyFISH.Themostsurprising
onewasthephylotyperelatedtocultivatedCycloclasticussp.bacteria,be-
causethisbacteriumseemedtocheatthehost-symbiontrecognitionsystem
andcouldco-habitwiththechemosyntheticsymbionts.Whetherthisnew
symbiosisdescribedwithdetailinthebodyofthisthesisisastableassocia-
tion,wedonotknow.IhaveanalyzedthetwoB.heckeraeorganismswhere
Cycloclasticusphylotypewasfound,butwewouldneedtohaveabetter
samplecollectiontobeabletodostatisticalanalysisaboutthedistribution
ofthisnewassociation.There-visitofChapopotesiteisessentialtodoa
furthercharacterizationofthissymbiosis.Experimentsinsitucouldbethen
performed,astheinjectionoflabeledhydrocarbonstositeandthecollection
ofthemusselstoanalyzeCincorporation.Alternatively,incubationoffresh
gillsonboardwiththelabeledhydrocarbonscouldbedone.However,ifthis
isnotperformedinapressurizedchamberandwithalltheabioticfactors
controlled,themetabolicmechanismsmightbeveryfarfromtheoriginal
ones.Itmightalsobeinterestingtotrycultivationofbacteriafromhomog-
enizedgillsasnotbeinganobligatesymbiontmightmakeeasiertogrowth
itonculture.Actually,thecurrentknowledgeoftheBathymodiolinmussels
abouttransmissiontellsusthattransmissioninthissystemishorizontal(or
environmental)andbacterialsymbiontsshouldhaveafree-livingstage.With
therightconditionsthesymbioticbacteriashouldbecultivable.However,
cultivationisnotaneasytaskandthereforecultivation-independentmethods
areanalternative.Isolationorenrichmentofsymbiontswithphysicalmech-
anismsareindevelopmentandthisisopeningthedoorstohavecomplete
genomestoanalyzegenomicevolutionandbiochemicalpathwaysimportant
inthedescriptionofsymbioticinteractions.Butforperforminganyofthese
experimentsthatImentionedhereabove,moreBathymodiolusmusselsfrom
Chapopotewouldbeneeded.

8.2TheSandPconcept

TheSconceptisapplicabletomanyofthebacteriaassociatedwiththe
bivalvesstudiedinthisthesis,whilethethiotrophicandmethanotrophicen-

170

REMARKSCONCLUDING

dosymbiontsofBathymodiolusmusselsareclearlyP-symbionts.S-symbionts
fromthisstudycouldbeCycloclasticus-relatedphylotype,theNIXphylo-
typesandpossiblyallbacteriadescribedinshallow-watermussels.Ofcourse
morephysiologicalcharacterizationtodeterminewhetherthesebacteriaare
mutualists,commensalists,orparasitesisstillneeded.Nonetheless,this
studyisoneofthefirstmolecularcharacterizationsaboutbacterialdiversity
inbivalves.ItisclearthatbacterialikeNIX-bacteriaareS-symbiontsasthey
arenotalwayspresentintheorganism,thehostdoesnotneedthemobligato-
rilyandthebacteriahaveaparasiticbehavior.ForCycloclasticus-bacterium
wehaveadifferentsetupasthisbacteriummightbebecomingaP-symbiont.
However,populationecologyandphysiologystudiestoanalysethespecific
recurrenceandactivityofthisphylotypewouldbeneededtodescribethis
speciesasaP-symbiont.Theshallow-waterbivalvesofthisstudyseemto
beassociatedonlytoS-symbionts,however,itisstillanearlyhypothesis
becausenotmanystudiesareathand.Afterthisstudyandtheprevious
onestherearesomenon-pathogenicbacteriathatseemtobepresentregu-
larly:Vibriospp.,Shewanellaspp.andspirochetes.Vibriospeciespresent
inbivalvesaremostofthetimenon-pathogenic.Isuggesttheycouldbe
protectingpathogeniconesofinfectingthehost.Havingthenamutualis-
ticroleasinthesymbiosisofsquid-Vibrio,whereVibriohasafunctionof
protection.Inthiscase,theVibriofunctionwouldbethroughcompetition
andnotthroughluminescence.Shewanellaaremarinebacteriathathave
theabilitytochelatemetals,theymightthenbeprotectingbivalvesfrom
theaccumulationofthem.Spirochetesseemtobealsowelladaptedtobi-
valvestissue.Theyarefrequentlypresentinbivalvesandwithaparticular
distribution.Howevertheroleofanyofthenon-chemosyntheticbacteriain
bivalveshasnotbeenstudiedinaprocess-orientedwayandthereisthelack
ofinformationnotjustinthewholephysiologicaldirectionbutalsointhe
purephylogeneticcharacterizationandbiologicaldistribution.Ithinkthe
cultivationofbacteriaassociatedtobivalvesshouldbefurtherestablishedto
thenbeabletodoexperimentswithsterilebivalves(astreatedwithantibi-
otics)andnotjustobservetheeffectonthebivalvesbutstudytheactive
proteinsandsugarstounveiltheinteractionprocesses.

171

REMARKSCONCLUDING

Conclusions8.3

ThisPhDthesiscontributestotheunderstandingofthediversityofchemosyn-
theticandnon-chemosyntheticbivalvesymbioses.Bivalvesareaworthy
modeltostudysymbiosis:1)theyhaveasimplerimmunologicalsystemthan

vertebrates,2)manyofthespeciesarelargeorganismswhichgivesmorearea

andbiologicalmaterialtoworkwith,3)theyarevectorsfortransmissionof
bacterialandvirusdiseasestohumans.Andfinally,4)inthephylogenetic
evolutionofbivalveswecanobservethedifferentphysiologicalbehaviours
witheverydifferentassociation:fromextracellularheterotrophictothein-
tracellularchemosyntheticones.Thusitisalwaysimportanttoanalyzeboth

bivalveandsymbioticbacteriaphylogeniestobeabletocharacterizetheevo-

lutionofbivalvesymbioses.Bivalvecommunicationsystemmightbesimilar
withinthewholegroup,thennon-chemosyntheticbivalvescouldgiveusan-
swersaboutthecommunicationbetweenbacteriaandtheirbivalvehostsin
achemosyntheticsymbiosis.

172

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195

Glossary

Bacteriocytespecializedcellofaneukaryoticorganismthatbearssymbi-
bacteria.oticEctosymbiontinhabitingoutsidetheorganism.
Endosymbiontinhabitinginsidethehostcell(endocellularly).
Endogenousthatliveinsidetheorganism,butnotnecessarilyendocellu-
.larlyP-symbionttheprimaryandmostabundantsymbiontinahostorganism.
Generallytheyareverticallytransmittedendosymbionts.
S-symbiontthesecondaryandlessabundantsymbiontinahostorganism.
Generallytheyarehorizontallytransmittedandtheirpresenceisfacultative.
Verticaltransmissionsymbiontsaretransmittedfromtheparentstothe
offspring.Horizontaltransmissionsymbiontsaretakenfromtheenvironmentwhether
directorindirectcontactwithhostrelatedorganism.
Facultativesymbiontstheirpresenceisnotobligatoryforhostsurvival.
Chemosynthesis-Chemosyntheticorganismsconvertoneormorecarbon
molecules(usuallycarbondioxideormethane)andnutrientsintoorganic
matterusingmethane(methanotrophs)orinorganicmoleculessuchashy-
drogensulphide(thiotrophs)asasourceofenergy,ratherthansunlight,as
hesis.tphotosyninThiotrophyThiotrophicorganismsorsulphuroxidizers(alsocalledchemoau-
totrophic)usereducedsulphurcompounds(e.g.hydrogensulphide)aselec-
trondonorsandfixCO2togeneratetheirorganicmatter.
Methanotrophy-isaspecialcaseofmethylotrophy,usingsingle-carbon
compoundsthataremorereducedthancarbondioxide,asacarbonand
source.energyAutotroph-organismabletosynthesizeorganiccompoundsfromCO2.
HeterotrophorganismwithanutritionthatisnotbasedinCO2butin
theuptakeoforganiccompounds.

196

197

Actswledgemenkno

AndfinallythemomenttosayThanks!:

TomyreviewersProf.Dr.UliFischerandDr.NicoleDubilier,andthe
membersofmythesiscommitteeinthebeginningandintheend:Dr.Antje
Boetius,Dr.HeikoSahling,Dr.Kai-UweHinrichs,Dr.FrankZielinski
andProf.Dr.BarbaraReinholdfortheirvaluablecommentsandfruitful
discussions.

ToNicole,foryourguidance,sharedknowledgeandcontagiousenthusiasm.
Thanksforlettingmejoinyourgroup.Ithasbeenagreatexperience!

ToallthepeopleIworkedwithandhelpedmeallthetime:Nicole,Jill,
Silke,Frank,Florence,Janine,Dennis,Paola,Cecilia,CarolineV,Stefan,
Rebecca,Betty...

ToHeiko,whogavemehelpwheneverIneededit,almostsincethefirstday
IarrivedtoBremen.

ToElvaandLuisawhoseededthefirstdesireonmetocometoBremen.

Toallsymbiosisgroup,MolecularEcology,andMPIpeople.Inparticular
toChristiane,Karl-Heinz,Rudy,Jens,Bernhard,andKatrinforthewarm
hostinginthiscoldcountry.

ToourlibrarianBerndStickfortforthemanyarticlesthathesearchedfor
me.

198

Tomymarmicfriends:Alexandra,Angela,Angelique,Astrid,Ilaria,Joanna,
Melissa,Paola,Sandra,Daniel,Ivo,Lars,Mohammad,Elmar,Sonja,Dennis
andKarinaforbeingalwaysthere.

Thankstoallourcoupledfriendsthatsharedlotofnicecouplemoments:
KarinaandSven,MohammadandSiham(pluschildren),CeciyFer,Tania
yTona,AndrewandJoe,MinaandDanny,AlbanandDiana,Claudiaand
Sven,RenzoandPetra,MarinaandFrank,ManuelandSybil,Claudiaand
Falk,AstridandSimon,IvoandJessi.

TomycapoeiraMestreandfriends:Flavio-Grilo,Martin-Quase,Andr´es-Los
Andes,Stefan-Aranha,Stefanie-Basurinha,Ole-Koala,Eli-Magrinha,Ula-
Brigadeiro,Esther-Fofinha,Maren-Barriguda,Gunter-Marinheiro,Matthias-
Caoleo,Dennis-Baixista,Heinrich-Girafa,Andreas...andalltheothersbe-
causewithouttheirsupport,joy,andenergythiswouldn’thavebeenpossible.

ToXimenaforbeingalwaysthereandkeepmeupdatedaboutfriendsand
Mexico.nilife

TomyPablo,becausehisimmensepatience,comprehension,andlovetome
andmywork,havemadeofthisahappylife.

Tomyparentsfortheinfinitetrustonme,andtheunconditionalsupport.

ToallthepeopleIamnotmentioningherebutalwaysgavemeahandora
smile.

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