Caenorhabditis elegans as a Model for Microbiome Research

The nematode Caenorhabditis elegans is used as a central model system across biological disciplines. Surprisingly, almost all research with ... DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Supplementaldata totalviews ViewArticleImpact SHAREON RobertBrucker RowlandInstitute,HarvardUniversity,UnitedStates MarkJ.Mandel UniversityofWisconsin-Madison,UnitedStates DavidW.Waite MinistryforPrimaryIndustries,NewZealand Theeditorandreviewer'saffiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction AuthorContributions ConflictofInterestStatement Acknowledgments SupplementaryMaterial References Opensupplementaldata Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex Checkforupdates Peoplealsolookedat HYPOTHESISANDTHEORYarticle Front.Microbiol.,23March2017Sec.MicrobialSymbioses https://doi.org/10.3389/fmicb.2017.00485 CaenorhabditiselegansasaModelforMicrobiomeResearch FanZhang1†,MaureenBerg2†,KatjaDierking3,Marie-AnneFélix4,MichaelShapira2*‡,BuckS.Samuel1*‡andHinrichSchulenburg3*‡ 1AlkekCenterforMetagenomicsandMicrobiomeResearch,BaylorCollegeofMedicine,Houston,TX,USA 2DepartmentofIntegrativeBiology,UniversityofCalifornia,Berkeley,Berkeley,CA,USA 3ZoologicalInstitute,Christian-AlbrechtsUniversityKiel,Kiel,Germany 4CentreNationaldelaRechercheScientifique,InstitutdeBiologiedel'EcoleNormaleSupérieure,InstitutNationaldelaSantéetdelaRechercheMédicale,ENS,PSLResearchUniversity,Paris,France ThenematodeCaenorhabditiselegansisusedasacentralmodelsystemacrossbiologicaldisciplines.Surprisingly,almostallresearchwiththiswormisperformedintheabsenceofitsnativemicrobiome,possiblyaffectinggeneralityoftheobtainedresults.Infact,theC.elegansmicrobiomehadbeenunknownuntilrecently.ThisreviewbringstogetherresultsfromthefirstthreestudiesonC.elegansmicrobiomes,allpublishedin2016.Meta-analysisofthedatademonstratesaconsiderableconservationinthecompositionofthemicrobialcommunities,despitethedistinctgeographicalsampleorigins,studyapproaches,labsinvolvedandperturbationsduringwormprocessing.TheC.elegansmicrobiomeisenrichedandinsomecasesselectivefordistinctphylotypescomparedtocorrespondingsubstratesamples(e.g.,rottingfruits,decomposingplantmatter,andcompostsoil).ThedominantbacterialgroupsincludeseveralGammaproteobacteria(Enterobacteriaceae,Pseudomonaceae,andXanthomonodaceae)andBacteroidetes(Sphingobacteriaceae,Weeksellaceae,Flavobacteriaceae).TheyareconsistentlyjoinedbyseveralrareputativekeystonetaxalikeAcetobacteriaceae.Thebacteriaareabletoenhancegrowthofnematodepopulations,aswellasresistancetobioticandabioticstressors,includinghigh/lowtemperatures,osmoticstress,andpathogenicbacteriaandfungi.Theassociatedmicrobesthusappeartodisplayavarietyofeffectsbeneficialfortheworm.Thecharacteristicsoftheseeffects,theirrelevanceforC.elegansfitness,thepresenceofspecificco-adaptationsbetweenmicrobiomemembersandtheworm,andthemolecularunderpinningsofmicrobiome-hostinteractionsrepresentpromisingareasoffutureresearch,forwhichtheadvantagesofC.elegansasanexperimentalsystemshouldproveofparticularvalue. Introduction TheModelOrganismC.elegansHasBeenStudiedwithoutItsMicrobiome ThenematodeCaenorhabditiselegansisoneofthemainmodelspeciesinthelifesciences,yetasurprisinglylargepercentageofmorethan40%oftheworm'sgenerepertoireisstillwithoutknownfunction(Petersenetal.,2015).Alikelyreasonisthatthisnematodeisalmostexclusivelystudiedunderhighlyartificiallaboratoryconditions,usingasingleisolate,thecanonicalstrainN2,whichshowssubstantialadaptationstothelaboratoryenvironment(Sterkenetal.,2015).Thisstrainisusuallymaintainedinthepresenceofonlyasinglebacterium,itslaboratoryfoodEscherichiacolistrainOP50,whileothermicrobesareroutinelyremovedthroughableachingprotocol(Stiernagle,2006).CurrentstudieslargelyignorethenaturalecologyofC.elegans.Thespeciesshowsaworld-widedistribution,especiallyintemperateregions,whereitiscommonlyfoundinrottingplantmattersuchasdecomposingfruits(e.g.,FrézalandFélix,2015).Initsnaturalhabitat,thenematode'smicrobiome,heredefinedsensulato,includingagutmicrobialcommunityandpossiblyalsomicrobesphysicallyassociatedwiththeC.eleganssurface,islikelyakeydeterminantoflifehistory(Petersenetal.,2015),inanalogytothefundamentalroleofthemicrobiotainthebiologyofallmulticellularorganismsexaminedtodate(McFall-Ngaietal.,2013;e.g.,BoschandMiller,2016).Untilrecently,onlyveryfewstudieshadexploredtheinteractionsbetweenC.elegansandmicrobesfromitsenvironment(Grewal,1991;GrewalandWright,1992;VenetteandFerris,1998;AveryandShtonda,2003;Coolonetal.,2009;MacNeiletal.,2013;Montalvo-Katzetal.,2013). ThecurrentpaucityofmicrobiomestudiesinC.elegansisunexpected,becauseseveralcharacteristicsmakethisnematodeideallysuitedfortheexperimentalanalysisofhost-microbeinteractions.First,C.elegansishighlyamenabletogeneticmanipulation.Second,thepresenceofmicroorganismscanbeefficientlycontrolledusingthebleachingprotocol,whichisonlysurvivedbynematodeeggsbutnomicrobes,thusallowingcultivationofnematodesunderaxenicormonoxenicconditions(Stiernagle,2006).Third,thenematodeistransparentsothatmicrobecolonizationcanbeeasilymonitoredinwholeanimalsusingsimplemicroscopy.Fourth,severallifehistoryreadoutsrelevantforstudyingC.elegans-microbiomeinteractionsarewellestablished:e.g.,thoserelatedtostressresistance,lifespan,populationgrowth,andfecundity.Takentogether,C.elegansisapowerfulexperimentalmodeltosystematicallyanalyzetheeffectsofthemicrobiomeonthehostandviceversa.Duetotheseadvantages,C.eleganshasbeenusedextensivelyforstudyinghost-pathogeninteractions,includingmostlybacterialpathogens,butalsofungi,microsporidiaandviruses.Thisworkhasexpandedourunderstandingofmechanismofinnateimmunity(MeiselandKim,2014;CohenandTroemel,2015;Dierkingetal.,2016;EwbankandPujol,2016;KimandEwbank,2016).Morerecentworkaddressedthenematode'sinteractionswithputativecommensalandprobioticbacteria,suchasComamonas,Bacillussubtilis,Lactobacillus,andBifidobacterium,yieldingnewinsightsintothemechanismsbywhichbacteriaortheirmetabolitesinfluencesignaling,metabolismandlife-historyintheC.eleganshost(reviewedinClarkandHodgkin,2014). In2016,threeindependentstudiesprovidedthefirstdescriptionofthemicrobiomeofC.elegansanditsnaturalenvironment.Takingcomplementaryapproaches(Table1),theyexploredforthefirsttimetheinteractionsofC.eleganswithitsassociatedcommunityofmicrobes(Bergetal.,2016a;Dirksenetal.,2016;Samueletal.,2016).Theaimofthisreviewistoprovideanoverviewoftheunderstandingemergingfromthesethreestudies,andthepotentialofC.eleganstoserveasaninformative,experimentallyaccessiblenewmodelsystemforthedissectionofhost-microbiomeinteractions.Wesummarizethethreestudies,highlightinghowtheyhavestartedtodefinethenaturalmicrobiome,andcombinetheminanewmeta-analysisrevealingasignatureoftheC.elegansmicrobiomethatisrobusttothedistinctstudyapproachesused.Wediscussthelikelybiologicalfunctionsoftheworm'smicrobiomeandconcludebypointingtopromisingavenuesforfutureresearch,whichexploittheadvantagesofC.elegansasanexperimentalandgeneticmodelsystem. TABLE1 Table1.OverviewofthefirstthreesystematicanalysesoftheC.elegansmicrobiome. TheC.elegansNaturalMicrobiome TwoofthethreeC.elegansmicrobiomestudiesexaminedthenaturalmicrobialenvironmentsofwildC.elegans(Table1)(Dirksenetal.,2016;Samueletal.,2016).Usingdeepsequencingofthe16SrDNAV4regionbacterialcontentwasprofiledinanextensivesetofnaturalhabitats(substrates)ofC.elegansfromdifferentsamplingsites(NorthernGermany,Portugal,andFrance)—i.e.,compost,rottingapples,andotherfruits,rottingstems,plusvectorinvertebratesusedfordispersal.CharacterizedenvironmentalmicrobialcommunitieswerecomposedofthousandsofOperationalTaxonomicUnits(OTUs,representingbacterialtaxonomicgroups),demonstratingextensivediversity,dominatedbyProteobacteria,Bacteroidetes,Firmicutes,andActinobacteria.Oftheover250bacterialgenerathatwereidentifiedinrottingapples,forexample,themostabundantwereEnterobacteriaceaeandaceticacid-producingAcetobacteriaceae.Intriguingly,manybacterialphylotypeswereconsistentlyidentifiedfromquitedisparatewormsubstrates(e.g.,compost,snail,rottingappleandrottingorange),suggestingthatthesetaxaaregenerallypartofthenaturalenvironmentofC.elegans. Strikingly,themicrobialcompositionofsomeofthesehabitatscanpredictthesuccessofwildC.eleganspopulationslivinginthem.Samueletal.showedthatlargeproliferatingpopulationsofC.elegansweremorelikelypresentinrottingappleswithsimple,Alphaproteobacteria-rich(Acetobacteriaceae)communities,whilethosewithhighlevelsofBacteroidetesorpotentialpathogenstendedtocontainnon-proliferatingdauers(Samueletal.,2016).Inreconstructionexperimentsoftwocommunitieswithabout20speciesofnaturalbacteria,fastergrowthandreproductionofC.eleganswasalsoobservedwhencommunitycompositionresemblednaturalenvironmentswithproliferatingC.elegans(80%Proteobacteria,Alphaproteobacteria-rich),ratherthanthosecontainingnon-proliferatingdauers(40%Proteobacteria,enrichedforGammaproteobacteriaandBacteroidetes).Machine-learningbasedanalysessuggestthatspecificmicrobialtaxaaredrivingC.eleganspopulationgrowthaswell—i.e.,bothEnterobacteriaceaeandAcetobacteriaceaearepredictiveofproliferatingpopulations,whiletheconversewastrueforaBacteroidetes(Flavobacteriaceae),andtwoGammaproteobacteriafamilies(XanthomonadaceaeandPseudomonadaceae)(Samueletal.,2016).Asoutlinedbelow,variouscombinationsofpairsofdetrimentalandbeneficialbacteriafromthesefamiliessuggestthattheimpactoftheBacteroidetesisonlyobservedathighabundance(>80%ofthecommunity),andthatbothbeneficialandpathogenicbacteriacanexertinfluenceatlowabundance(Samueletal.,2016).Theseobservationssuggestthattheimpactofthemicrobiomeiscontextdependentandinvolvesacomplexinterplaybetweendifferentcommunitymembers. DirksenandcolleaguesadditionallyanalyzedthebacterialcommunitiesinnaturalC.elegansisolates(Table1,Figure1),inordertoexaminewhetherassociatedwormmicrobiomesdifferedfromtheircorrespondingsubstrates(Figure1)(Dirksenetal.,2016).Caenorhabditiselegansfromnaturalhabitatsharboredspecies-richbacterialcommunities,includingalargevarietyofdistincttaxonomicgroups(Dirksenetal.,2016).ThemostcommonOTUswereunclassifiedEnterobacteriaceaeandmembersofthegeneraPseudomonas,Stenotrophomonas,Ochrobactrum,andSphingomonas.Moreover,theidentifiedC.elegansmicrobiomeisdistinctfromthemicrobialcommunityofthecorrespondingsubstratesandofcongenericnematodessuchasC.remanei,possiblysuggestingthepresenceofaspecies-specificmicrobiome,anotionthatwasmorerecentlyproposedbyastudyexaminingdifferencesinthemicrobiotasofdifferentCaenorhabditisspecies(Bergetal.,2016b).Importantly,microbiomesofwormscollectedfromdifferentsamplingsitesandsubstratesresembledeachotherand,additionally,themicrobialcommunityfromsinglewormsimmediatelyafterisolationfromthewildoverlapswiththemicrobiomefromwormpopulationsexpandedinthelabfromoveraperiodofseveralweeks(withoutadditionoflabfood)(Dirksenetal.,2016).TheseobservationsstronglysuggestthatC.elegansharborsacharacteristicmicrobiomethatisdefinedbyitspropertiesasaspeciesandthustheunderlyinggenome,irrespectiveofanyenvironmentaland/orgeographicvariations.ItisyetunclearwhetherthischaracteristicmicrobiomeisactivelyselectedbyC.elegansortheresultofdifferencesinnematodecolonizationefficacyofthevariousbacteriaorboth. FIGURE1 Figure1.CompositemicrographsoftheC.elegansmicrobiome.(A)CompositemicrographofthemouthregionofC.elegans,and(B)ofthemiddlepartoftheworm(anterioristotheleftinbothcases).Nematodeswereraisedonanexperimentalmicrobiomebasedon14abundantbacterialtaxa,followedbymicroscopicanalysis(Dirksenetal.,2016).BacteriaarestainedinredwithaeubacterialFISHprobeandareobservedassmalldotsthroughouttheentiregut.WormnucleiarestainedinbluewithDAPI.Thepicturein(A)istakenfromDirksenetal.(2016),whilethatin(B)isnew,courtesyofPhilippDirksenfromtheSchulenburglab. Tomodelnaturalenvironmentsinthelab,workintheShapiralabestablishedanexperimentalpipeline,inwhichgenetically-homogenouswormpopulations,initiatedfromgerm-freelarvaeofthestandardN2strainareraisedindiverselab-basedenvironmentsthatemulatehabitatsfromwhichC.eleganshasbeenisolatedinthewild(Table1)(Bergetal.,2016a).Comparisonsofmicrobialcommunitiesfromnematodesandtheircorrespondingmicrocosmenvironments(bothanalyzedbyV416SrDNAdeepsequencing)identifiedacharacteristicC.elegansgutmicrobiome,distinctfromtheenvironment,andadditionallysuggestedthatassemblyofthenematodegutmicrobiomewasessentiallyadeterministicprocessundertheseconditions.Thereproducibilityofwormmicrobialcommunitiesenabledidentificationofasharedcoremicrobiome,whichaccountedfor>50%ofallbacterialtaxa.Theanalysisofnematodemicrobiomesfromthesemicrocosmexperimentsadditionallyrevealedthepresenceoftwodistincttypes,whichwereindependentoftechnicalvariables,anddifferedintheabundanceofcorefamilies,aswellasinclusionofauxiliarytaxa.Subsequentexperimentsevaluatingtheeffectsoftemperatureonmicrobiotacompositionfoundthatchangesinmicrobeabundanceinwormswerefrequentlyintheoppositedirectiontochangesintheenvironment,stronglysuggestinghostmediation.Amorerecentstudyconfirmedthat,ontopofenvironmental-dependentvariability,hostgeneticshadasignificantcontributiontoshapingcompositionofthemicrobiome:microbialcommunitiesweremoresimilarinwormsofthesamestrainthanbetweenwormsofdifferentstrainsandspecies(Bergetal.,2016b). SimilarityandDifferencesoftheC.elegansMicrobiomeacrosstheThreeStudyApproaches BringingtogetherthethreestudiesenablesustobetterdefinetheC.elegansgutmicrobiotabycomparingmicrobiomecompositionsbetweenwormsanddifferentsubstrates,ascharacterizedbydifferentlabswithdistinctstudyapproachesandindifferentpartsoftheworld(seemeta-dataforsamplesinSupplementaryTable1).Principlecoordinateanalysisusingphylogenetic-basedunweighteddistancesbetweenallmicrobiotas,fromwormsandfromtheirsubstrates,demonstratedthatinthediversityspacedefinedbythedistributionofsubstratemicrobiotas,wormmicrobiomestookupalimitedsub-space(SeefilledsymbolsinFigure2A).Analyzedwormmicrobiomesincluded(i)singlewormscharacterizedshortlyaftertheirisolation(naturalworms;studybyDirksenetal.,2016),(ii)groupsofwormsmaintainedforapproximately2weekswiththeirnativemicrobiomesunderlaboratoryconditionsbeforemicrobialanalysis(labenrichedworms;studybyDirksenetal.,2016),and(iii)wormsofthelaboratorystrainN2raisedincompostmicrocosms(microcosmworms;studybyBergetal.,2016a).ThestrongoverlapamongthesemicrobiomesandtheirdistinctcompositioncomparedtothecorrespondingsubstratesstronglysuggeststhatC.elegansassemblesfromtheenvironmentadefined,non-randommicrobialcommunity,whichisrobusttovariationsinstudyapproach(i.e.,microcosmsvs.naturalworms),labsinvolved,andtoperturbationsduetomaintenanceofwormsunderlaboratoryconditionsratherthantheirnaturalenvironments(i.e.,naturalvs.labenrichedwormsinthestudybyDirksenetal.,2016).SucharobustsignatureinmicrobiomecommunitycompositionhighlightsthesuitabilityoftheC.elegansmodelfordissectinghost-microbiomeinteractionsandtheunderlyinggeneticsirrespectiveofthestudyapproach. FIGURE2 Figure2.Cross-studycomparisonofC.elegansandsubstratemicrobiomes.(A)PrinciplecoordinateanalysesbasedonunweightedUniFracdistancesshowsdistinctclusteringofC.elegans(filled)fromrottingfruitorcompostsubstrates(open)regardlessofthestudyoforigin.Athree-dimensionalrepresentationoftheresultsisprovidedinSupplementaryVideo1.ThecharacteristicsoftheincludedsamplesispresentedinSupplementaryTable1,whiletheidentifiedOTUsandtheirabundancesaregiveninSupplementaryTable2.Allmicrocosmdatasets(giveningreen)arefromBergetal.(2016a).Allnaturalandlabenrichedwormdatasets(giveninfilledpurpleandmagentasymbols)arefromDirksenetal.(2016).ThesubstratedatasetsforrottingstemareexclusivelyfromSamueletal.(2016),whilethoseforvectorandrottingfruitsincludedatafrombothDirksenetal.(2016)andSamueletal.(2016),andthoseforcompostareexclusivelyfromDirksenetal.(2016).C.elegansmicrobiotasaregenerallylessdiversethansubstratesasassessedbyShannonalphadiversityindices(B),andexhibitmoresimilarcompositionwithineachwormgroupthantosubstratesorbetweensubstrates(C).Non-parametricp-values≤0.002arenoted:a,vs.substrates;b,vs.soilmicrocosm;c,vs.wormgroup. ThepresenceofadistinctsignatureoftheC.elegansmicrobiomeacrossstudiesisconfirmedbyrelatedstatisticalanalyses.Unweighteddistancestakeintoconsiderationonlypresenceoftaxa,disregardingtheirabundance,andthereforerepresenttheoverallrichnessofmicrobiotas,withthoseinwormsappearingtohostasubsetofthebacteriaavailableintheirenvironment.Inagreementwiththis,wormmicrobiotasgenerallyshowsubstantiallylowermicrobialdiversitycomparedtotheirrespectivesubstrates,withtheexceptionofrottingfruitsthatarealreadysimplethemselves(Figure2B).Theyalsoshowagreatersimilarityamongthemselves,asdemonstratedbysmallerinter-microbiotadistances(Figure2C).ThenaturalC.elegansmicrobiomesexhibitedthehighestvariationamongnematodegroups.Interestingly,theidentifiedmicrobialcommunitiesappearedtobedividedintotwodistinctgroups.Oneoftheseclusteredwithalmostallmicrobiomesfromlab-enrichedwormsandsomeofthemicrocosmnematodes,whereasthesecondgroupclusteredwithaseparatesetofmicrobiomesofthemicrocosmnematodes(Figure2A).Whetherthisdivisionrecapitulatesthetwomicrobiometypespreviouslyreportedforthemicrocosmexperiments(Bergetal.,2016a)isyetunclear.Nevertheless,thepresenceofdistincttypesamongnaturalC.eleganssamplessuggeststhatnematodesmayharbordifferent“enterotypes.”Inmicrocosmexperiments,distincttypesmaybeattributedtoenvironmentalmicrobeavailabilityandmicrobialcompetition,assuggestedbyecologicalnetworkanalysis(Bergetal.,2016a).Inwildisolates,variationinhostgenetics,shouldalsobeconsideredasapotentialdeterminantofthepresenceofsuchtwomicrobiometypes.Theimportanceofhostgeneticsinthiscontextissupportedbytwoadditionalfindings:TheanalysisoftheexperimentalmicrobiomebyDirksenetal.identifiedasignificantinfluenceofC.elegansstrainonbacterialcommunitycomposition(Dirksenetal.,2016).Amorerecentsetofmicrocosmexperiments,inwhichdifferentC.elegansstrainsandrelatedspecieswereraisedonthesamesubstrate,showedco-clusteringofnematodegutmicrobiotasaccordingtotheirgenotype(Bergetal.,2016b). ManybacterialtaxawerecommonlyidentifiedamongtheC.elegansmicrobiotas(Figures3A,B;SupplementaryTable2).Strikingly,260bacterialOTUs(operationaltaxonomicunits)wereidentifiedinallofthestudies(Figure3A,inset;SupplementaryTable2).Severalbacterialtaxawereparticularlyabundantinwormmicrobiotas(Figure3C),includingthreeGammaproteobacteria:Enterobacteriaceae,Pseudomonadaceae,andXanthomonadaceae.Commoninnaturalmicrobiotas,butlesssoinmicrocosmexperimentsweretheAlphaproteobacteriamembersSphingomonadaceae,andthreeBacteroidetesfamilies(Sphingobacteriaceae,Flavobacteriaceae,andWeeksellaceae)(Figure3C).Interestingly,Acetobacteriaceae,whichwerefoundtocorrelatewithlargepopulationsofproliferatingC.elegansinrottingapples(Samueletal.,2016),werepresentatlowlevelsinallofthenaturalwormsthatwereexamined(Figure3C).Itisnotlikelythatthislow,yetconsistentpresenceisduetocontamination,asseveralotherclassesofbacteriapresentathighlevelsinsubstrateswerereproduciblyexcludedfromcolonizationoftheworms,includingforexamplePlanctomycetesandmostAcidobacteria(Figure3B).Moreover,althoughAcetobacteriaceaearecommoninfruit,theirabundanceismuchlowerincompost,fromwhichmostofthecharacterizednaturalC.eleganswereisolated.InadditiontoAcetobacteriaceae,severalotherProteobacteria(Moraxellaceae,Comamonadaceae,andRhodobacteraceae)andActinobacteria(MicrobacteriaceaeandActinomycetales)alsofitintothisrare,butcommoncategorywithinthenaturalC.eleganssamples.Thecombinationoflowabundanceinnematodes,yetapparentimportancefortheirfitness,suggeststhatmembersoftheAcetobacteriaceaeandpossiblyalsotheotherabovelistedfamiliesmayserveaskeystonetaxaoftheC.elegans-microbiomeassociationwithcurrentlyunknownfunction.Furtheranalysesareneededtoelucidatethesepotentialroles. FIGURE3 Figure3.IdentificationofacoremicrobiomeofC.elegans.(A)ScatterplotofOTU-levelmeanrelativeabundanceandcommonalityacrossall62C.elegansmicrobiomes.Inset,VenndiagramofthesharedOTUsfromeachofthegroupsofmicrobiotas.(B)ComparisonofmeanrelativeabundanceinallC.elegansand119substratesamples.Thecolorsofcirclesin(A,B)indicatetheOTUsfromdistinctbacterialphyla,whilecirclesizetheirabundance,ashighlightedinthelegendonthefarright.(C)Heatmapof14bacterialfamiliesthatarepresentin100%ofthenaturalwormmicrobiomesshowingabundanceacrosssamples(in%).Redboxeshighlightthosethatareabundantalsoinlab-enrichedandmicrocosmmicrobiotas.Thecolorsoftheverticalcolumnontheleftoftheheatmaparethesameasin(A,B)andindicatethedifferentbacterialphyla.Amoredetailedheatmap,whichadditionallyincludesallsubstratesamples,isprovidedasaSupplementaryFigure1.AlistoftheidentifiedOTUsandtheirabundancesinC.elegansandsubstratesisprovidedasaSupplementaryTable2. TheC.eleganscoremicrobiotaemergingfromthemeta-analysisisnotverydifferentfromthosedefinedbyeachoftheseparatestudies.Furthermore,membersofthetwomoreprominentfamilies,EnterobacteriaceaeandPseudomonadaceae,wereisolatedfromC.elegansinearlierstudies(Grewal,1991;Ladyginaetal.,2009).Together,thisindicatesthatasignificantpartoftheC.elegansmicrobiomeisofareproduciblydefinedcompositionthatisdominatedbyGram-negativebacteria,inparticularfast-growingbacteriawithflexiblemetabolisms.Thesebacteriaaretypicallystrongcompetitors,bothintheenvironment,wheretheyareeffectivecolonizersofrottingfruit,andalsoinsidetheworm(Bergetal.,2016a). PossibleFunctionsoftheWorm'sMicrobiome ConsideringtheconsistentassociationbetweenC.elegansandtheidentifiedbacterialtaxa,itisofinteresttoknowifandwhatadvantagestheymayprovidefortheirhost.Samueletal.demonstratedthatnearly80%ofthemorethan550bacteriaisolatedfromFrenchsubstrates(BIGbandJUbcollections)canindividuallysupportC.elegansgrowth(Samueletal.,2016).Thetestedcollectionscomprised437bacteriafromrottingOrsayapples(orotherhabitatsfromsitesaroundParis)harboringlargepopulationsofC.elegansand128isolatesfromavarietyofsampletypesandlocationswhereC.elegans(and/orC.briggsae)animalswereidentified.Usingacombinationofphysiologicalmeasures,growthratesandinductionofstressandimmunereportergenes,thesecollectionsofbacteriawerecategorizedasbeinggenerally“beneficial”(promotestress-freegrowth),“detrimental”(impairgrowth,kill,activatestress/immunereporters)or“intermediate”(mixedresponses).SeveralProteobacteria,includingEnterobacteriaceae,Gluconobacter,Enterobacter,ProvidenciaandalsomostLactococcusstrainsweremore“beneficial”toC.elegans.MoredetrimentalgeneraincludedBacteroidetes,suchasChryseobacteriumandSphingobacterium,andpotentiallypathogenicGammaproteobacteria(e.g.,XanthomonasandStenotrophomonas).Interestingly,isolateswithingeneravariedininfluenceonC.elegansphysiology(e.g.,measuredwiththehelpofstressreportergenesorgrowthcharacteristics),withtheexceptionofGluconobacter,suggestingtheimportanceofstrain-leveldifferencesingenecontent(Samueletal.,2016). Dirksenetal.alsoestablishedanexperimentalmicrobiome(Figure1),consistingof14bacterialstrainsthatwereisolatedfromwildC.elegansandrepresentedabundantgeneraoftheworm'snativemicrobiome(Dirksenetal.,2016).ThreedifferentC.elegansstrains,thelaboratorystrainN2andtwonaturalisolates(allthreeisogenicandwithdifferentgenotypes,asmeasuredwiththehelpofmicrosatellites;HSunpublisheddata),weregrownontheexperimentalmicrobiomeandbacterialpopulationsinwormswereanalyzedattwodifferentdevelopmentalstages,thefourthlarvalstage(L4)andadults.AnalysisofthebacterialpopulationsofthesewormsrevealedthatthedevelopmentalstageaswellasthehostgenotypecaninfluencethecompositionoftheC.elegansmicrobiome.Intriguingly,certainbacteriaappeartobespecificallyenrichedinworms(whencomparedtotheexperimentalmicrobiotaonagarplates),especiallyOchrobactrumMYb71andStenotrophomonasMYb57.ThisobservationpossiblyindicatesthatthesetaxaareabletocolonizetheC.elegansintestine.AtleastforOchrobactrumMYb71,theabilitytopersistinthenematodeintestinewasdemonstratedinaseparateexperiment(Dirksenetal.,2016).Inaddition,theexperimentalmicrobiomewasfoundtoenhancewormfitnessincomparisontopresenceofonlythestandardlaboratoryfoodE.coliOP50andmeasuredusingpopulationgrowthasproxy.Fitnesswasinthiscaseincreasedunderdifferentstressconditions,includinghigheraswellaslowertemperatures,differentmediaandsalinities.AnalysisofindividualbacterialisolatesfurtherhighlightedthatthepositiveeffectonfitnessislikelycausedbyProteobacteria;especiallyrepresentativesofthegeneraPseudomonas,Achromobacter,Acinetobacter,andComamonasassociatedwithsubstantiallylargerpopulationgrowththanthatobservedundercontrolconditions(Dirksenetal.,2016). Thebest-characterizedcontributionsofgutmicrobesweretohostimmunity.TheShapiralabpreviouslyidentifiedaPseudomonasmendocinagutisolatethatconferredresistancetoinfection.RaisingwormsontheisolateprotectedwormsfromsubsequentexposuretopathogenicP.aeruginosa,slowing-downcolonizationandkilling(Montalvo-Katzetal.,2013).Thisprotectionwasfoundtobeprovidedbylow-levelactivation(orpriming)ofp38signaling,acentralmoduleinC.elegansimmunity(Kimetal.,2002;Troemeletal.,2006;Shiversetal.,2010;Blocketal.,2015).WhiletheabilityofthePseudomonascommensaltoprovideprotectionfromthePseudomonaspathogen,maybeassociatedwiththesimilaritybetweenthem,otherPseudomonasisolateswereunabletoprovideprotection,indicatingagreaterspecificityinrecognitionandimmuneactivation.Inthestandardinfectionprotocol,apriorexposuretotheP.mendocinacommensalwasonlyabletodelaycolonizationanddeath.However,inamorenaturalscenario,inwhichP.aeruginosawasspikedintosoilwithgrowingworms,infectioncouldbecompletelyaverted(MBandMSunpublisheddata),stressingtheimportanceofsuchcommensalsforwormfitnessinitsnaturalhabitat.Morerecently,newisolatesofEnterobactercloacae,obtainedeitherfromC.elegans(1isolate)orC.briggsae(2isolates),werefoundtoprotectthewormfromapathogenicstrainofEnterococcusfaecalis.Interestingly,protectionwasspecifictothehostfromwhichthebacteriawereisolated:TheE.cloacaeisolatefromC.elegansonlyprotecteditsoriginalhost,butdidnotprotectC.briggsae,andviceversaforthetwoC.briggsaeisolates(Bergetal.,2016b).ThesefindingssuggestspecificselectionofprotectivesymbiontsbythehostandpossiblyevensomeformofCaenorhabditis-Enterobacterco-adaptation.Suchapossibilityagreeswitharecentdemonstration,usingcontrolledevolutionexperiments,ofco-adaptationsbetweenC.elegansandadifferentprotectivebacterialstrain,whichreducedinfectionbypathogenicStaphylococcusaureus(Fordetal.,2016;Kingetal.,2016). TwoPseudomonasisolates,obtainedfromwildC.elegansanddistinctfromP.mendocina,wererecentlyshownbyDirksenetal.toinhibitthegrowthofsixfungalstrains,allsimilarlyisolatedfromnaturalC.elegans(Dirksenetal.,2016).Moreover,oneoftheseisolatesprotectedC.elegansfromdeathbyawell-establishedfungalinfectionmodel,theascomyceteDrechmeriaconiospora(Lebrigandetal.,2016;Zugastietal.,2016).FungalinducedmortalitywascompletelypreventedwhennematodeswereexposedtothepathogenicfungusinthepresenceofthePseudomonasisolateMYb11.ItwasstillsignificantlyreducedwhenwormswerefirstgrownonMYb11duringdevelopmentandthenexposedtothefungusasadultsonnewplates,whichonlycontainedthelaboratoryfoodE.coli,butnotMYb11,possiblyindicatingalong-lastingprotectiveeffectfromthelatterbacterium(Dirksenetal.,2016).Thesestudies,addedtothosefromtheShapiralab,assigndiverseanti-pathogeniccontributionsofPseudomonadstoC.elegans,whichmaysuggestasharedhistoryofinteractions,andperhapsofevolution. Samueletal.expandedthespectrumofbacterialcontributionstoC.eleganspathogenresistance(Samueletal.,2016).WhenC.elegansgrowthwasassessedinthecontextofbinarydilutionseriesofthreebeneficial(Gluconobactersp.GRb0611,Enterobacteriasp.JUb54,Providenciasp.JUb39)andthreedetrimentalbacteria(Serratiasp.JUb9,Pseudomonassp.GRb0427andChryseobacteriumsp.JUb44),thenthebeneficialbacteriasignificantlyreducedthenegativeeffectofthedetrimentaltaxaonwormgrowth.Notably,similaramountsofeachequallybeneficialnaturalbacteria(orE.coliOP50)didnothavethesamemitigatingeffectoneachofthepathogens,suggestingthateachwashavingitsownspecificprotectiveimpactratherthanexhibitingasimpledilutionoftheconcentrationofagivenpathogen.Whetherthesemechanismsoccurdirectlyonthepartofthehost(e.g.,immune-boosting),indirectlybyinhibitinggrowthofthepathogens,orviaarelatedmethodremainstobeseen. FutureChallenges C.eleganspossessesamicrobiomewithadefinedsignature,whichcanencompassalargenumberofbacterialtaxaperindividualworm.TheexactpresenceandrelativeabundanceofbacterialtaxacanvarysubstantiallyamongsingleC.elegansisolatesfromthewild(Dirksenetal.,2016)(Figure2A).Aparticularchallengeistodeterminethestabilityofthismicrobialcommunityandthestrengthofassociationofmicrobiotamemberswiththeirhost.Arebacterialstrainsabletopersistoverlongtimeperiodsinnematodehosts,evenifthesemigratebetweensubstrates?Aresuchstrainsabletopersistindauerstages,likelyusedbythehostforlong-distancemigration,andaretheytransmittedverticallybetweenhostgenerations?TowhatextentdoCaenorhabditisspeciesdifferintheirassociatedmicrobiomes,especiallywhenconsideringhoststrainsfromdifferentorigins?Futureeffortswillneedtocatalogthespecificfunctionsofdifferentmembersofthemicrobialcommunity,includingdominanttaxa,butalsothelessabundantkeystonetaxa(i.e.,thosetaxaconsistentlyfoundatlowfrequencyacrosswormsamples).DoindividualbacterialstrainsengageinmutualisticinteractionswithC.elegans—e.g.,byenhancingreproductiveratesoftheirhostsbyamelioratingaccesstonutritionfromanewlycolonizedsubstratewhilethehostenhancesmicrobes'dispersalopportunities?Thesequestionscouldbetestedusingexperimentalevolutionapproaches(e.g.,Masrietal.,2015),includingmulti-generationalpropagationofC.elegans-microbepopulationsondefinedsubstrates,andexaminedbymicroscopicanalysisofbacterialcolonizationandpersistenceaswellasbymeasuringhostandbacterialfitness. ThenatureofinteractionsbetweenhostsandtheirmicrobiotaisanimportantstandingquestionthatcouldbeaddressedintheC.elegansmodel.Ontheonehand,tightassociationbetweenC.elegansandspecificbacterialtaxamaysuggestco-evolution.Inthiscase,weexpectreciprocalgeneticchangesinC.elegansandindividualmicrobiallineages,resultinginco-adaptationsthataremanifestedinthemolecularinteractionsamonghostandthespecificmicrobes(e.g.,theexpressionofspecificmicrobialsignalingmoleculesandcorrespondinghostreceptors).Ontheotherhand,itispossiblethattheworm'smicrobiotaisflexiblyassembledfromtheenvironment,andconsistsofvaryingbacterialstrainsandtaxa,whichhoweverreproduciblyfulfillparticularfunctions.However,wecurrentlylackmoleculardataandalsomoredetailedinformationonthefunctionaleffectsofthebacteriatoassessthetwoalternatives.Someoftheavailabledatastillprovidessupportforeachofthehypotheses.Thatthewormmicrobiotaislargelyreproducibleevenwhenstartingfromdiverseenvironmentsisconsistentwiththefirstpossibility(Bergetal.,2016a;Dirksenetal.,2016;andmeta-analysispresentedhere).Asignificantcontributionofhostgeneticstoshapingofthegutmicrobiotafurtherofferssupport(Bergetal.,2016b;Dirksenetal.,2016).However,astrongcontributionofenvironmentaldiversitytogutmicrobiotacompositionratheragreeswiththesecondpossibility(Bergetal.,2016b).Thatbothalternativesareimportantisconsistentwiththerecentmodel,proposedbyoneofus(Shapira,2016),whichsuggeststhegutmicrobiometobedividedintotwoparts.First,acoremadeofcommensalswithtightassociationswiththehost,potentiallysharingco-evolutionaryhistoryandpossiblymaintainedbyverticaltransmission.Second,aflexiblemicrobialpoolthatdependsonenvironmentalavailabilityandcanprovidefunctionalversatility,possiblyadvantageousinachangingenvironment.Thetwopresentedalternativesmayactuallyrepresentoppositeendsofarangeofinteractions.Asanexampleforassociationsofatypethatmayliefurthertowardthecenterofthisrange,onecanconsidertheacquisitionofbeneficialsymbiontsfromagreaterenvironmentaldiversity,relyingonmechanismspermittingpartnerchoiceorcheckingforpartnerfidelity.Thishasbeenshowntooccurinthecolonizationofthebobtailsquid'slightorganbyVibriosymbiontsfromthemarineenvironment(Kremeretal.,2013;Aschtgenetal.,2016),aswellasintheacquisitionofXenorhabdusgut-residingbacteriabytheSteinernemaentomoparasiticnematode(Murfinetal.,2015).FiguringouthowC.elegansobtainsthedifferentmembersofitscharacteristicgutmicrobiotaremainstobeelucidated. AparticularstrengthoftheC.elegansmodelisitsamenabilitytogeneticmanipulation.Thisstrengthcouldbecomplementedbygeneticanalysisofindividualbacterialtaxa.Forexample,ifacertainbacterialstrainormixtureisfoundtohaveastronginfluenceonaparticularphenotype,thegeneticsoftheinteractioncouldbedissectedbyforwardandreversegeneticanalyses,ideallyinbothpartners.Suchtwo-sidedgeneticanalyseswillopenthepossibilitytocharacterizeindetailhostaswellasmicrobialmolecularprocessesthatcontrolhost-microbiomeinteractions. MethodsUsedforMeta-Analysis Thethreestudiesappliedthesame16SrRNAgeneprimerstargetingvariableregion4(515F/806R)inbacteria(Caporasoetal.,2012).However,goodqualityreadsweresometimesobtainedwiththeforwardprimer,andsometimeswiththereverseprimer.Inordertofacilitatecross-comparisons,forwardreadswereusedforallexperiments[includingre-sequenced(IlluminaMiSeq)samplesfromSamueletal.,2016],sacrificinginsomecasesthenumberofreadspermicrobiota.Additional(previouslyunpublished)sequenceswereincluded,withDNAisolatedfromC.eleganssubstrates,suchasrottingapplesfromOrsay(FR),rottingPetasitesstemsfromIvry(FR)andslug/snailvectorsfromSanteuil(FR).FastqfilesfromthethreestudieswereseparatelyqualitytrimmedandfurtherprocessedusingtheQIIMEsoftwarepackage(v1.9.0)(Caporasoetal.,2010).Sequencereadswithanaveragequalityscorebelow25andmorethan1ambiguousbasewerediscarded.Sequenceswhichpassedqualityfiltersweretruncatedto150bplengthtofacilitatecomparisonswiththeIlluminaHiSeqreadsofBergetal.(2016a),givingrisetoadatasetcontaining15,197,186readstotal,withameanof74,862andmedianof51,932readspersample.Resultingfastafileswereconcatenatedintoonefile,andthe16SrRNAgenesequenceswerefurtheranalyzedusingQIIME.DenovoOTUextractionwasperformedwiththeuclustoptioninQIIME.SingletonswereremovedfromcentroidconsiderationusingtheUSEARCH(Edgar,2010)suite.ResultingreadswereclusteredusingtheUPARSEalgorithmat3.0%(4mismatches)clusteringradius.CentroidsweremappedtotheGreengenes13.8databasefortaxonomicassignmentat97%(3.0%clusteringradius)identity.CentroidsfailingtomaptothedatabasewereevaluatedwithUTAXforadenovotaxonomicassignment.Sequencesthatmatchplantchloroplast,mitochondrial,orarchaeal16SrRNAwereremoved.Sequencesusedforourmeta-analysisareavailablefrompublicdatabases,includingtheEuropeanNucleotideArchivefortheSchulenburglabdata(www.ebi.ac.uk/ena;accessionnumberERP014530);theSequenceReadArchivedatabasefortheSamuellabdata(www.ncbi.nlm.nih.gov/sra;accessionnumberSRS1849345),andtheMG-RASTmetagenomicarchivefortheShapiralabdata(http://metagenomics.anl.gov;accessionnumbersmgp13213andmgp21372).Thesamplenames,accessionnumbers,andadditionalmeta-dataarepresentedinSupplementaryTable1.IdentifiedOTUs,theirabundances,taxonomicclassifications,andthe16SrDNAfragmentconsensussequencesofthemostabundantC.elegans-associatedOTUsaregiveninSupplementaryTable2. DiversityindiceswerecomputedinQIIMEusingcore_diversity_analyses.pywithdefaultparameters.Forestimatesofalpha-diversity(withinsample),sampleswererarefiedto5,000sequences,andthosesampleswithfewerreadswereremoved.AlphadiversitywasdeterminedusingShannonIndex.Beta-diversity(betweensample)distancematricesweregeneratedusingOTUtablesrarefiedto500observationstoincludeasmanysamplespossible.AphylogenetictreeofsequencesrepresentingthecentroidforeachOTU(arepsettree)wasgeneratedusingClustalOmegawithanenhancedversionofmBedanddefaultparameters(SieversandHiggins,2002).Usingthisrepsettree,phylogenetic-basedunweightedUniFrac(LozuponeandKnight,2005)methodswereusedtofacilitatecomparisonsofpresence/absencepatterns(richness)betweensampleandsubstratetypes.PhylogeneticrelatednessoftheOTUsandsimilarityofcompositionbetweensamplesareintegratedtocreateUniFracdistancematricesthatallowthiscomparisonthatwerevisualizedbyprinciple-coordinateanalysesinQIIME.Heatmapsweregeneratedonnon-rarefied,relativeabundanceOTUtablesandplottedinRusingggplot.VenndiagramswerecreatedbasedonsharedOTUsbetweencomposite(pooled)samplesforeachsubstrateorsampletype.Insomecases(i.e.,Shannondiversity,betadiversityboxplotsandheatmap),reverseread-basedassessmentsofmicrocosmsamplesfrom(Bergetal.,2016a)wereincludedtobetterreflecttheconclusionsoftheoriginalstudies. AuthorContributions MS,BS,MF,andHSconceivedthework.FZandBSgeneratednewmicrobiomedata.FZ,MB,MS,BSperformedthemetaanalysis.Allauthorsresearchedtheliteratureandwrotethemanuscript. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WethankthemembersoftheFélix,Samuel,Shapira,andSchulenburglabsfordiscussion.WearegratefulforfundingtoKDandHSfromtheGermanScienceFoundationwithintheCollaborativeResearchCenterCRC1182ontheoriginandfunctionofmetaorganisms(projectsA1.1,A1.2,andA4.3).MBissupportedbytheNationalScienceFoundationGraduateResearchFellowshipProgram(DGE1106400). SupplementaryMaterial TheSupplementaryMaterialforthisarticlecanbefoundonlineat:https://www.frontiersin.org/article/10.3389/fmicb.2017.00485/full#supplementary-material SupplementaryTable1.Overviewofincludeddatasets,sequenceaccessionnumbers,andconsideredmeta-data. SupplementaryTable2.Overviewofidentifiedoperationaltaxonomicunits(OTUs).SheetIshowstheidentifiedOTUsandtheirabundancesintheC.eleganssamples,whilesheetIIthoseforsubstratesamples.SheetIIIpresentsalistofallOTUswiththeirtaxonomicclassification.SheetIVgivesthe260OTUscommonlyfoundamongthenematodesamples,includingthecorresponding16SrDNAfragmentsequences. SupplementaryVideo1.Three-dimensionalvisualizationoftheresultsofthePrincipleCoordinateAnalysis.Figure2Aofthemaintextshowspartofthesameresults.Botharebasedonthesameanalysis.ThecolorcodeissimilartothatofFigure2A:red,rottingstemsubstrates;darkred,compostsubstrates;orange,vectorsubstrates;lightblue,rottingfruitsubstrates;verylightgreen,microcosmsubstrates;purple,naturalwormssamples;pinkt,labenrichedworms;andbrightgreen,microcosmwormsamples.Allthreeprinciplecoordinatesareshownalongthethreeaxes. SupplementaryFigure1.Heatmapoftherelativeabundanceof14bacterialfamiliesthatarepresentin100%ofthenaturalwormmicrobiomes.Seelegendontherightforabundancelevels.Taxaandboxesinredhighlightthosethatareabundantalsoinlab-enrichedandmicrocosmmicrobiotas.TheheatmapforthewormsamplesisalsoshowninFigure3Cofthemaintext,buthereextendedbythesubstratesamples. 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Editedby:RobertBrucker,RowlandInstituteatHarvard,USA Reviewedby:MarkJ.Mandel,NorthwesternUniversity,USADavidWilliamWaite,UniversityofQueensland,Australia Copyright©2017Zhang,Berg,Dierking,Félix,Shapira,SamuelandSchulenburg.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:MichaelShapira,[email protected],[email protected],[email protected] †Sharedfirstauthorship. ‡Sharedseniorauthorship. ThisarticleispartoftheResearchTopic Experimentalmodelsinanimal-associatedmicrobiota Viewall 36Articles Peoplealsolookedat Download