Molecular Analysis of Microbiota Along the Digestive Tract of Juvenile Atlantic Salmon (

Salmo salar L.)

Author(s): P. Navarrete, R. T. Espejo and J. Romero
Source: Microbial Ecology, Vol. 57, No. 3 (April 2009), pp. 550-561
Published by: Springer
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MicrobEcol (2009) 57:550-561
DOI 10.1007/S00248-008-9448-X

HOST MICROBE INTERACTIONS

MolecularAnalysisofMicrobiotaAlongtheDigestiveTract
ofJuvenileAtlanticSalmon(Salmosalar L.)
P. Navarrete• R. T. Espejo • J. Romero

Received: 8 February2008 /Accepted:28 August2008 /Publishedonline: 17 September2008

© SpringerScience + Business Media, LLC 2008

AbstractDominantbacterialmicrobiotaof the gut of tal influenceson the bacterialcompositionof the gut

juvenilefarmedAtlanticsalmonwas investigated usinga microbiota.Overall,theseresultsindicatea homogeneous
combination ofmolecularapproaches. Bacterialcommunity composition of thebacterialcommunity compositionalong
composition fromthestomach,thepyloriccaeca, and the tractof rearedjuvenile salmon.This
the gastrointestinal
intestinewas assessed by extracting DNA directlyfrom community is mainlycomposed of Pseudomonasspp.,
each gutcompartment. Temporaltemperature gradient
gel whichcould be derivedfromwaterinfluent and may be
electrophoresis (TTGE) analysisof 16S ribosomalDNA associatedwithsalmonin thishatchery.
selectively
(rDNA) ampliconsshowedverysimilarbacterialcomposi-
tions throughout the digestivetract.Band sequencing
revealeda narrowdiversity of species witha dominance Introduction
of Pseudomonasin the threecompartments. However,
cloningrevealedmorediversity amongthePseudomonas The gastrointestinal tractis a compositeecosystemcon-
sequences. To confirmthese results,we analyzed the a
taining complex and dynamicconsortiumof micro-
bacterialcommunity by amplifying the variable16S-23S organisms,usually called microbiota,which appear to
rDNA intergenic spacerregion(ITS). SimilarITS profiles play a keyrole in thenutrition and healthof thehost[4,
were observedamong gastrointestinal compartments of 16]. Evidencefortheroleofmicrobiota in fishwas recently
salmon, confirming the TTGE results.Moreover,the revealed [5, 34, 35]. Using germ-free zebrafish,these
dominant ITS bandat 650 bp, identified as Pseudomonas, reports showed that gut microbiota mightbe involvedin
was observedin theITS profilefromfishcollectedin two important processes like stimulation of epithelialprolifera-
seasons(July2003 and 2004). In contrast, aerobicculture tion,promotion ofnutrient metabolism, and innateimmune
analysisrevealedShewanellaspp. as the mostprevalent A
responses. keyaspectof these results was thespecificity
isolate.This discrepancy was resolvedby evaluating16S of the hostresponseat the gene expressionlevel,which
rDNA and ITS polymerasechain reactionamplification dependedon thebacterialcomposition ofthedigestive tract
efficiency fromboth Shewanellaand Pseudomonasiso- [34]. Therefore, itmaybe relevant to knowthecomposition
lates.Verysimilarefficiencies were observedin the two of microbiota of rearedfish,especiallysalmonids,which
bacteria.Hence, this discrepancymay be explainedby constitute an important economicindustry in Chile.
preferential cultivationof Shewanella spp. under the Currentknowledgeof the diversityin the bacterial
experimental conditions.Also, we includedanalysesof composition of salmonmicrobiota is largelybased on the
pelletedfeedandthewaterinfluent to exploreenvironmen- use of classical culture-dependent techniquesand the
contribution of this approachhas been reviewed[3, 10,
P. Navarrete• R. T. Espejo • J. Romero(ISI) 19,36]. However,ithas beenshownthata largeproportion
Laboratoriode Biotecnología, ofbacteriaarenotisolatedon traditional agarsubstrates[1],
Institutode Nutricióny Tecnologíade los Alimentos,
anditis currently accepted thattheseculture-based methods
Universidadde Chile,
El Líbano 5524, Macul, P.O. Box 138-11,Santiago,Chile detectonlya smallfraction of bacteriapresentin thegut
e-mail:jromero@inta.cl [46]. As a possiblealternative, molecularmethodsallow

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BacterialMicrobiotain the Salmon Gut 551

bacterialidentification independent of theirabilityto grow bacterialmicrobiota of theentiregutby molecularanalysis

in synthetic media.Thesemethodshavebeenused to study of 16S rDNA and ITS.
thebacterialcomposition of haddocklarvae [18], halibut
larvae[23], andtheintestinal contents of coho salmon[42]
and rainbow trout [24]. They also allowed for the Methods
identification ofAcinetobacter junii and a novelMycoplas-
ma phylotype as thepredominant bacterialpopulationsin SamplesCollectionand Processing
farmed and wildadultsalmon[20].
The salmon gut is composedof separateanatomical Salmo salar juvenilespecimensof 1-30 g werecollected
compartments: the esophagus,a U-shaped stomach,the froma hatchery(latitude32° S, Chile) in two seasons:
pyloriecaeca, and theintestine (Fig. la) [47]. As in many betweenJulyand October,2003 and duringJuly,2004. In
fish,thepyloriccaeca areblindappendagesattachedto the both seasons, fish were reared in freshwater with a
intestine in the regionnear the stomach[6]. The pH is temperature of 16°C year-round and normal densities were
reported to be between 3.0 and 4.5 in the stomach, between about 50 kg/m3. The fish collected were healthyjuvenile
7.0 and 7.5 in thepyloriccaeca, and a pH of 9.0 has been salmonthathad neverbeen treatedwithantibiotics. They
reportedin the distalpartof the intestine[32, 37]. Our werefedwithconventional pelletedfeedwithout probiotic,
hypothesis was thatanatomicaland physiologicaldiffer- prebiotic,immunomodulatory, or inhibitory agents.The
ences along the gut would favora specificmicrobiotain averagedailyintakeofpelletedfeedfromEWOS was 2.8%
each compartment. Thereare no reportsaboutthemolec- of body weightfor all fish.This was accomplishedby
ular analysisof microbiotaassociatedwiththe different manualfeedingsevento eighttimesthroughout thedayand
compartments of the gastrointestinal tract of salmon, its observationoffish to check diet and
acceptance satiety. The
its
composition, complexity, or its stability,especially fishwere anesthetized using benzocaine and killedby a
followingdietarychangesor treatment with antibiotics, blow to thehead. Collectedsalmonweresize-graded. The
whichare routinepracticesin aquaculture.Since different biggestsalmon(30 g; collectedinJuly2004) wereanalyzed
microorganisms inducedifferent responsesin thehostand individually, meaningthateveryfishwas dissectedsepa-
some conferpotentialbenefits[34], knowledgeof the ratelyfromtheothersandeverygutcompartment (stomach,
bacteriacommonly associatedwithdifferent gastrointestinal pyloric caeca, and intestine) was analyzed separately.
compartments may be usefulformanipulating microbiota Smallerfishwere dividedintoeightgroupscomposedof
as a strategy to improvenutrition or preventpathogenic ten specimenseach, withan averageweightof 1.5, 3, 4,
infection. In thisstudy,we used a ribosomalmarker-based and 14±0.5 g (July-October 2003) and 4, 14, 17, and 23±
approachto determine the bacterialcompositionof the 0.5 g (July2004). The entiregutoftenspecimens pergroup
different compartments of the gastrointestinal tractof was homogenizedand referredto as pooled samples.
juvenileAtlanticsalmon(Salmosalar). Bacterialcomposi- Poolingsampleswas performed because it is a common
tionwas determined by analysis of 16S ribosomal DNA to
practice study the gut microbiota in fish[2, 21] and
(rDNA) ampliconsusingtemporal temperature gradientgel previous studiesshowed thatindividual microbiota arewell
electrophoresis (TTGE) and and
by cloning sequencing the representedbypooledsamples of individualgut microbiota
main amplicons.To assess diversityat the intraspecies [42]. Dead fishwereplacedin sterileplasticcontainers and
level, the main ribosomalintergenic space region(ITS) transportedto the laboratoryon ice. Samples were
ampliconswere identified by sequencing.These results processed immediatelyupon arrivalin the laboratory,
werethencomparedto thoseobtainedby cultureto get a usuallywithin2 h. Gastrointestinal sampleswereobtained
morecomprehensive overviewof thebacterialpopulations by asepticallydissectingthe fishand carefully extracting
present.Since bacterialcommunities were extracted from the entiregastrointestinal tractundera stereomicroscope.
each gutcompartment thatcontainedepithelium and feed For individualanalysisof thebiggestfish(30 g), thethree
digesta,themicrobiota analyzedwas a combination ofboth anatomicalcompartments (U-shapedstomach,a developed
autochthonous (able to colonize the epithelialsurfaceor pyloricregion,and intestine)were carefullyseparated
mucus of the host gut) and allochthonous(transient or duringdissection.Each gut section (stomachs,pyloric
associatedwithdigesta)bacteria.The mainobjectiveofthe caeca, and intestines) was sliced and weighedseparately,
present work was to examine bacterial composition along and an equal amount of cold sterilephosphate-buffered
thedigestivetractof salmonwithspecialemphasison the salinewas added.Forthesmallestfish,theentiregutsfrom
dominant bacterialpopulation presentin each gutcompart- ten specimens of the same weight were sliced and
ment.Also, the analysesof waterinfluent and feedwere homogenized together. Theseslicedgutsweresubsequently
includedto explorethe environmental influences.To our homogenizedin an ice bathwithvigorousvortexing for
knowledge,the presentworkis the firstexaminationof 3 min.Sinceall samplesincludedgutepithelia, mucus, and

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552 P. Navarreteet al.

Figurei TTGE profilesof 16S

rDNA amplification products
fromAtlanticsalmongut.a A
schematicview of the salmon
digestivetract,b TTGE profiles
fromstomach,pyloriccaeca,
and intestineof fiveindividuals
(witha body weightof 30 g).
Each lane correspondsto a
singlecompartment, each de-
rivedfromindividualfishnum-
beredfrom1 to 5. Common
bands showingthe same migra-
tionpatternsare indicated(#7-
B6). c TTGE profilesof the
entiredigestivetractsfromfish
of 4, 14, 17, and 23 g {lanes 4,
14, 17, and 23) comparedto
thosefromseparatedcompart-
mentsobtainedfroma single
individual(lanes S: stomach,
PC: pyloriccaeca, and /:
intestine)

digesta,themicrobiota
analyzedwas a combination ofboth
autochthonous(able to colonizethe epithelialsurfaceor
mucusofthehostgut)andallochthonous bacteria(transient).
Simultaneously,freshwater sampleswere obtaineddi-
rectlyfromthe water influentcorrespondingto the
hatchery'swatersourceand thefeedused at thehatchery
were collected and transported on ice. Samples were
processedimmediately upon arrival
in thelaboratory.
DNA Extraction
and Purification
DNA fromthegastrointestinalcompartments was obtained
fromhomogenates by lysisusingsodiumdodecylsulfate
and incubationat 70°C. The lysateswere subsequently
extractedwith phenol/chloroform and precipitatedwith
ethanolas previouslydescribed[41]. A finalpurification
was carriedout usingWizardDNA Clean Up (Promega,

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BacterialMicrobiotain the Salmon Gut 553

Madison, WI, USA). DNA fromwater influentwas thepresenceof similaramplicons,bandsshowingthesame

5 1 of waterthrough
obtainedby filtering a 0.2-mmfilter. migration in lanes
different were digestedwithAlul and
Bacteriawerethenresuspended in TE buffer
(Tris0.01 M, Haelll as below.
described
EDTA 0.001 M, NaCl 0.15 M, pH 7.8), and lyses were
performedas describedabove.DNA frompelletedfeedwas Restriction Analysis
LengthPolymorphism
Fragment
obtainedby homogenizing 15 g in TE bufferfollowedby
DNA extraction as describedabove. Productsof the 16S rDNA or ITS PCR amplification were
digestedfor 2 h at 37°C in 1.5 U of Alul or Haelll
and Analysisof theProducts
PCR Amplification endonuclease(Invitrogen).
restriction The resultingfrag-
mentswere subsequently analyzedby polyacrylamide gel
Amplification of the V3-V5 regionof 16S rDNA was electrophoresis stainingas described.
and silvernitrate
carriedout to obtainprofilesof thebacterialcommunities
presentin different compartments, water influent,and Cloningand SequenceAnalysis
pelletedfeed. The extracted DNA was polymerasechain
reaction(PCR)-amplifiedusing conserved 16S rDNA PCR productswere cloned into a TOPO TA vector
bacterial
domain-specific primers 341F (5f-GCCTACGGGA accordingto theprocedureindicatedby themanufacturer
GGCAGCAG^' withGC clampsat the5' end) and 907R The resulting
(Invitrogen). plasmidswerepurified usingthe
^'-CCGTCAATTCMTTTGAGTTM') as previouslyde- Wizard Plus SV MiniprepSystem(Promega).Plasmids
scribed[27]. PCR reactionswereperformed as described containingan insertwereselected,and their16S rDNA or
[42] witha reactionmixture (30 'ú) containing 0.2 mM of ITS were PCR-amplified as describedabove and treated
each deoxynucleoside triphosphate, 0.05 U/ml Platinum enzymes characterization.
withrestriction for
Taq DNA polymerase (Invitrogen, Diego,CA, USA), 1x
San 16S rDNA fromthe cloninglibrary,the re-amplified
polymerase reaction
buffer, 2 mM MgCl2,and 0.25 pmol/ml bands,amplifiedribosomalspacers,and bacterialisolates
of eachprimer. werepurified usingWizardPCR Preps(Promega)andthen
Amplificationof the 16S-23S rDNA intergenic regions sequenced with an Applied Biosystems310 automatic
was performed as describedby Espejo and Romero[15] sequencer (Foster City, CA, USA). ABI Prism dye
usingprimersGl and LI [22]. Semi-quantitative amplifi- terminatorsequencingkitswere used withprimers907R
cationwas performed as previously described[14]. Ampli- and LI for 16S rDNA genes and ITS, respectively. Se-
ficationof 16S rRNAtogether withits neighboring 16S- quenceswere depositedin GenBank (EF587700-EF5 87702,
23S rDNA spacerwas performed as described [41], using DQ889968-DQ889982, DQ067264-DQ067274,
primers357F and LI [28]. For purification of this last EU794379-EU794397)and alignedwithreference sequen-
amplicon,theproductwas separatedon a 1% agarosegel, ces using Sequence Match softwarefromthe Ribosomal
and theband migrating at 1.8 kb was excised.The DNA DatabaseProjectII (RDP II) website[12]. Distancematrices
was subsequentlyextractedfromthe agarose gel by wereconstructed fromthealignedsequencesand corrected
centrifugationthrough glass wool at 12,000g for20 s in formultiplebase changesatsinglenucleotide using
positions
The extract
a microcentrifuge. was diluted1:200 in distilled the Jukesand Cantormethodincludedin the TREECON
for
water,and 15 fxlwas used amplification. Productswere program[48]. Usingthesameprogram, a phylogenetic tree
analyzed using polyacrylamide gel electrophoresis and was constructed a
by neighbor-joining method. Bootstrap-
stainingas previously
silvernitrate described[13]. ping was performed using the bootstrapmodus of the
program,and valuesabove40% arereported.
TGGE Analysis
BacterialCountsand Cultivation
PCR productsobtainedfrom34IF and 907R primers were
separatedbyTTGE on a 6% (w/v)polyacrylamide gel 1x
in Totalbacterialcountspresentin salmonand watersamples
TAE running buffer (Tris0.04 M, acetate0.002 M, EDTA were performedby epifluorescencemicroscopyusing
0.001 M, pH 8.5) and a temperature gradientfrom66°C to acridineorange,as previously described[40]. Forbacterial
70°C [26]. Electrophoresiswas runforabout20 h at 65 V serialdilutionsof guthomogenates
cultivation, fromeach
in a D-Code System(Bio-Rad,Hercules,CA, USA). After entirecompartment that includedmucus and epithelial
gels were stainedfor 1 h by incubation
electrophoresis, attachedbacteria(autochthonous bacteria)and bacteria
withSybrGreenat roomtemperature. The dominant bands fromthe digestacontent(allochthonous) were platedon
wererecognized as intensebandsin eachTTGE pattern and soy agar (TSA,
trypticase Difco, Sparks, MD, USA), and
wereexcisedfromthegel and elutedovernight in 50 [ú of theplateswere incubatedfor10 days at 17°C in aerobic
MilliQwater;1 'i' was used forreamplification.To testfor Serialdilutionsof waterand feedsampleswere
conditions.

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554 P. Navarreteet al.

also platedand incubatedas describedabove. Colonies The presenceof Pseudomonaswas examinedin fish
werecountedafter10 days,and the colony-forming unit smallerthan30 g. TheTTGE profiles obtainedfrom thesefish
(CFU) per gramof gastrointestinal tractwas calculated. (4 to 23 g) did notdiffer the
from TTGE of 30 g (Fig. lc).
50-200colonieswereselectedforanalysis.
Platescontaining Restrictionenzymeanalysisconfirmed thatbandBl inthese
A randomselectionof 80 coloniesfromeach compartment smaller fish also correspondedto Pseudomonas. This
(stomach,pyloriccaeca,andintestine)[38] was subcultured assessment also revealedthepresenceof an additionalband
andcharacterizedaccording to thecolonymorphology, 16S (bandB7) thatwas closely to
related Stenotrophomonas sp.
rDNA restrictionfragment lengthpolymorphism (RFLP),
and ITS profiles,revealingcoincidenceamongthe three Assessedby 16S rDNA Cloning
Diversity
approaches.Representativeisolatesfromeach RFLP group
wereidentifiedby 16S rDNA partialsequencing. To studythemoleculardiversity of bacteriapresentin the
salmon gut, the 16S rDNA ampliconsof the stomach
StatisticalAnalysis sample showingthe largercomplexityin TTGE were
cloned.Eightycloneswereanalyzedby RFLP usingAlul
Assuming100% efficiency whenthe DNA templatewas and Haelll, resulting in six RFLP groups(Table 1). The
doubledin eachcycle,thePCR efficiency was calculatedas mostabundant groupcorresponded to soybeanchloroplasts
£=10(~slope)-l,whereE was the PCR efficiency. PCR (35% of the clones). AnotherthreeRFLP groups(I- III),
efficiencieswere expressedas means±SE of triplicate representing 30%, 20%, and9% oftheclonescorresponded
standardcurves.PCR efficiencies were analyzedwitha to the genus Pseudomonas. Two other RFLP groups
paired t test, and differenceswere concluded to be (IV-V), together representing 6% of theclonesanalyzed,
significant when/?<0.05. corresponded to Acinetobacter. Phylogeneticanalysis
shownin Fig. 2 illustrates themoleculardiversity among
Pseudomonassequencesand revealedthatPseudomonas
Results sequencesfromRFLP groupsIII and I were similarto
TTGE bandBl and bandB2, respectively.
Composition of BacterialCommunities Assessed
by PCR-TTGE Composition of BacterialCommunities Assessed
by ITS Analysis
The bacterialcomposition of stomach,pyloriccaeca, and
intestinefromtenjuvenile(30 g) salmonwas analyzedby CoincidentwiththeTTGE analysis,ITS profilesfromgut
16S rDNA PCR-TTGE. This approachrevealeda simple compartments were similaramongindividuals, showinga
and similarbacterialcompositionin each gastrointestinal dominanceof a widespreadband (650 bp) in all gastroin-
compartment forall individuals.
Five bandswereobserved testinalcompartments (Fig. 3). However,thepresenceof
in the stomachsamples,and two to threebands were severalweakerbandsin theITS profiles mayindicatemore
observed in the pyloric caeca and intestinalsamples diversity in microbiota composition. Sequenceanalysisof
(Fig. lb). TTGE bands with the same migrationpattern the dominant band using BlastN indicated thattheclosest
showed the same restriction profile(Alul and Haelll) match- with 98% -
identitycorresponded Pseudomonas
to
indicating thattheycorresponded to the same sequence fluorescens(accessionnumber EF5877001)andshowedthe
(data not shown). At least one band with the same sameorganization oftRNAs(tRNAIle andtRNAAla) andnon-
migration patternwas sequencedforidentification
(Table 1). codingregionsas wholegenome-sequenced Pseudomonas.
The main bands (Bl and B2) obtainedfromstomach, To testifthepresenceofPseudomonascorresponded to
pyloric caeca, and intestinesamples corresponded to a particular or a general observation, ITS analysiswas
Pseudomonasspp. Threebands observedin the stomach performed in gutsamplesfromfishcollectedfromthesame
samplescorresponded to 16S rDNA of chloroplasts from hatchery butduringdifferent seasons(Fig. 4). The presence
Glycine max (bands B3 and B4) and chloroplastsor of the dominant 650 bp band and the similarITS-RFLP
mitochondrion fromZea mays (band B5), derivedfrom profilesamongthefishsuggeststhata dominant bacterial
the vegetalmaterialincludedin the pelletedfeed,which populationrelatedto Pseudomonaswas presentin salmon
containedapproximately 10% soybeanmeal and 5% corn regardless of theseason(Fig. 4).
gluten.Also, intestinal samplesshowedthepresenceof a
weakband(bandB6) witha 16S rDNA sequencerelatedto BacterialCounts
Comamonassp. This bandwas detectedin 70% (7/10)of
the fish,showingthatthis species, thoughnot always The averagetotalbacterialdensityyielded1x 107,8x 106,
present,was a commoninhabitant of theintestine. and 5xlO7 bacteria/gof stomach,pyloriccaeca, and

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BacterialMicrobiotain the Salmon Gut 555

Table 1 Nearest-match of 16S rDNA sequences obtainedfromTTGE bands, cloning approaches,and bacterialisolates from
identification
salmongutcompartments,waterinfluent,and pelletedfeed,withknownsequences in the RDP II database

Name RFLP Accession Percent Affiliation

phylum/class Closest sequence Gram
groupa number identity

TTGE bands fromindividuals

Band 1 (Bl) DQ889976 99 Proteobacteria/y- Pseudomonasfluorescens;BiotypeA; ATCC 17555
Proteobacteria (AJ308303)
Band 2 (B2) DQ889977 97 Proteobacteria/y- Pseudomonas sp. MWH1 (AJ556801)
Proteobacteria
Band 3 (B3) DQ889978 99 Eukaryota ChloroplastGlycinemax soybean(DQ3 17523)
Band4(B4) DQ889979 100 Eukaryota ChloroplastGlycinemax soybean(DQ3 17523)
Band 5 (B5) DQ889980 99 Eukaryota Mitochondrion Zea mays(AY506529)
ChloroplastZea mayscom (Z00028)
Band 6 (B6) DQ889981 99 Proteobacteria/
ß- Comamonasaquatica; LMG 5937. (AJ430346)
Proteobacteria
TTGE bands fromwaterinfluent
Band Sl-1 EU794386 100 Bacteroidetes/ Flavobacteriumsp. (AF493664)
Flavobacteria
Band S 1-2 EU794387 98.1 Bacteroidetes/ Flavobacteriumsp. (AM934673)
Flavobacteria
TTGE bands frompelletedfeed
Band 1-A EU794379 99.7 Fusobacteria/ llyobacterpsychrophilus(AJ877255)
Fusobacteria
99.7 Fusobacteria/ Fusobacteria bacterium(AY579753)
Fusobacteria
Band2-A EU794381 99.4 Fusobacteria/ llyobacterpsychrophilus(AJ87^'255)
Fusobacteria
99.4 Fusobacteria/ Fusobacteria bacterium(AY579753)
Fusobacteria
Band 1-J EU794380 100 Eukaryota Mitochondrion Zea mays(AY506529)
Band 2-J EU794382 100 Eukaryota Mitochondrion Zea mays(AY506529)
Band 2-K EU794383 95 Firmicutes/Bacilli Sporolactobacillusterrae(AJ634662)
Band 2-M EU794384 100 Actinobacteria/ sp. (DQ337529)
Bifidobacterium
Actinobacteria
TTGE bands frompools
Band pool DQ889982 100 Proteobacteria/
ß- maltophilia;e-a21;
Stenotrophomonas
17 (B7) Proteobacteria (AJ293470)
Cloningfromindividuals
Clon 4 I DQ889968 99 Proteobacteria/y- Pseudomonas sp. MFY160 (AY331371)
Proteobacteria
Clon 8 I DQ889970 100 Proteobacteria/y- Pseudomonas sp. MFY 160 (AY33 1371)
Proteobacteria
Clon 28 I DQ889973 100 Proteobacteria/y- Pseudomonas sp. WG7#1(AY263469)
Proteobacteria
Clon 34 II DQ889974 94 Proteobacteria/y- Pseudomonas sp. BW 11M 1 (AY 118 112)
Proteobacteria
Clon 5 III DQ889969 100 Proteobacteria/y- Pseudomonasfluor'escens;biotypeA; ATCC 17555
Proteobacteria (AJ308303)
Clon 26 IV DQ889972 98 Proteobacteria/y- Acinetobactersp. 18III/A01/072(AY576723)
Proteobacteria
Clon 36 V DQ889975 99 Proteobacteria/y- Acinetobacter
johnsonii; ATCC 17909T; Z93440
Proteobacteria
Clon 25 VI DQ889971 100 Eukaryota ChloroplastGlycinemax soybean(DQ3 17523)
Pelletedfeed isolates(relativeabundance%)
Al (18%) EU794388 100 Firmicutes/Bacilli Vagococcussp. (AB2 11029) +
100 Vagococcuslutrae(DQ39528 1)
A2 (22%) EU794389 100 Actinobacteria/ Microbacteriumsp. (AM403722) +
Actinobacteria

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556 P. Navarreteet al.

Table 1 (continued)

Name RFLP Accession Percent phylum/class Closest sequence

Affiliation Gram
groupa number identity

A3 (17%) EU794390 99.9 Firmicutes/Bacilli EnterococcussaccharolyticusATCC 43076 +

(DQ411816)
A4 (20%) EU794391 100 Firmicutes/Bacilli Macrococcus caseolyticus(EU048336) +
A5 (23%) EU794392 100 Firmicutes/Bacilli StaphylococcuslentusATCC29070T(D83370) +
Waterisolates(relativeabundance%)
Wl (25%) EU794393 99.8 Proteobacteria/y- Pseudomonas sp. (DQ219370)
Proteobacteria
W2 (20%) EU794394 99 Proteobacteria/y- Rheinheimerasp. (EU037278)
Proteobacteria
W3(18%) EU794395 100 Proteobacteria/
ß- Ralstonia sp. HAB-01; AB051680
100 Proteobacteria Cupriavidussp. PS12; DQ777727
W4(15%) EU794396 98.6 Bacteroidetes/ Flavobacteriumsp. (AM 177631)
Flavobacteria
W5 (22%) EU794397 97 Bacteroidetes/ Unculturedbacterium(EF632769)
96.1 Flavobacteria Flavobacteriumsp. AM934641
a Abundance
(%) of RFLP group:I: 30%, II: 20%, III: 9%, IV: 3%, V: 3%, and VI: 35%

intestine,respectively.The averagecountsof cultivable

accordingto thesimilarity
of theirpatterns.
Representative
bacteriawere 2.2xlO4, 3.1*103, and 1.9xlO5 CFU per isolatesfromeach groupwere identified by 16S rDNA
stomach, pyloriccaeca,and intestine, The total
respectively. partialsequencing(Table 2). Among them,the isolates
bacterialcountfromthewatersupplywas 1x 106bacteria/clustering withShewanellaweremostfrequently obtained
ml, whereasthe cultivablecountwas 6x 102 CFU/ml.In in thegutcompartments, whereasPseudomonaswas only
pelleted feed samples, 4.3 xlO2 CFU/g was obtained.recoveredfromthe intestineat very low levels. Other
Considering all samplesassessed,thecultivability
was <1%.
generasuchas Microbacterium, Cellulomonas, and Serra-
tici were observed with different relative abundances
Composition of BacterialCommunities
Assessed (Table 2).
by Analysisof CultivableBacteria The ITS ampliconsfromtheseisolateswerecompared
withthepreviousITS profilesobtainedafteramplification
Eightyisolatesfromeach gutcompartment
werecharacter- of DNA extracteddirectlyfromthe gut compartments
ized by 16S rDNA RFLP. Five groupswere obtained (Fig. 3b). ITS bandswithsimilarelectrophoretic migration

Figure 2 Phylogenetictree of Pseudomonas sp BW11M1

16S rDNA sequencesobtained Band1(B1)
fromjuvenileAtlanticsalmon. Clone 5
Pseudomonas sp ED105
Neighbor-joining phylogenetic Pseudomonas cfsynxanthaV4.BP.03
treeshowingtherelationship Pseudomonas fluonscens BiotypeA
y' _ proteobacteria
betweensequencesretrieved . Pseudomonassp. MWH1
88
fromtheTTGE profiles,clones, i-Band2(B2)
and theirclosestrelative U IClone 8
sequencesdepositedin the RDP I SS" Clone 28
II database.The treewas con- gelone 4
structedbased on the341-907 ^ I Clone 34
26
regionof the 16S rDNA genes, j-Clone
usingTREECON version1.3b.
| (Clone 36
"I ATCC 17909T
A bootstrapanalysiswas per- ■- I Acinetobacterjohnsonii
Acinetobactersp. 18 III
10°
formedwith100 repetitions,and loo S35
| Acinetobacterjohnsonii
values greaterthan40% are Band 6 (B6)
shown Stenotrophomonasmaltophiliae-a21
Stenotrophomonassp. Ellin 162
™1 I Band 5 (B5) 7T*
100|Comamonas aquatica LMG 5937 [J,- Proteobacteria
_^^_^_^^-^_^^^_^_^^^^^^^^^^^^^_ Archaeoglobus fulgidus

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BacterialMicrobiotain the Salmon Gut 557

Figure 3 Analysisof ITS

(16S-23S rDNA intergenic
spacer) amplification products
and theirRFLP profilesfrom
Atlanticsalmon(S. salar), a ITS
profilesfromstomach,pyloric
caeca, and intestineof three
individualsfroma totalof ten
analyzed,witha body weightof
30 g each. The numberin each
lane correspondsto each indi-
vidual,b ITS profilesof stom-
ach, pyloriccaeca, and intestine
froma singlefish(30 g; lanes S:
stomach,PC: pyloriccaeca, and
/: intestine)comparedto ITS
profilesof bacterialisolates
(lanes Ps: Pseudomonas,Sw:
Shewanella,Mb: Microbacte-
rium,Ce: Cellulomonas).x
indicatesthepresenceof a
commonband betweenisolates
and the directprofiles

to ITS fromShewanella(500 bp) wereobservedin salmon culturingefficiencyof Pseudomonas or a low PCR

gutITS profiles.PseudomonasisolatesshowedITS bands amplification of Shewanella.In orderto resolve
efficiency
with similarsize (650 bp) and sequence (EF587700- thisissue,we evaluatedthematchingof primersin silico
observedin all compartments and PCR amplification
EF587702) to thatpreviously forbothbacteria.Primers
efficiency
of thesalmongut.Altogether, theseobservationsconfirm 341 and 907 used for16S amplification wererevisedby
that Pseudomonas is abundantand common in these using the Probe Matchtool in the RDP II database.No
salmonguts. preference
amplification was inferredsince bothprimers
matchedcorrectlywith70% of thesequencesforPseudo-
PCR Amplification forPrevailingBacteria
Efficiency monasand Shewanella.PrimersGl and LI used forITS
amplificationwere evaluatedin the GenBank Genome
The discrepancybetweenthe resultsfrommolecularand databaseusing16S and23S rDNApresent inthesequenced
culturalapproachesmay be attributed to eithera low genomes of Pseudomonas and Shewanella (13 strains

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558 P. Navarreteet al.

Originof DominantPseudomonasRetrieved
fromSalmon
DigestiveTracts

To determine thepossiblesourcesof Pseudomonasfound

in the digestivetractof juvenile salmon,bacterialDNA
fromwaterinfluentand pelletedfeed was analyzedby
PCR-TTGE. All bands fromwaterinfluent and pelleted
feed TTGE profiles were excised and identifiedby
sequencing(Table 1). The resultsshowedthatthedominant
bacterialpopulations fromwaterandfeedaredifferent from
thoseinthedigestivetractofsalmonandPseudomonaswas
not detectedin these samples. The water sample was
dominatedby bacteriabelongedto Flavobacteria(Class)
whereaspelletedfeedwas dominated by bacteriabelongto
Fusobacteria, Bacilli, and Actinobacteria
(Class; Table 1).
Bacterialisolatesrecovered frompelletedfeedshowedonly
Gram-positive bacteria,whereasonlyGram-negative bac-
teriawereretrieved fromwaterinfluent (Table 1). Indeed,a
Pseudomonasbacterium was isolatedfromwaterinfluent
showing 97% identity with band 1 retrievedfromthe
salmondigestivetract(Table 1).

Discussion

In thisstudy,we analyzedthebacterialcomposition along

thegastrointestinal tractofjuvenilesalmonusingdifferent
molecularmethods.The emphasiswas placed on the
dominantbacterialpopulationsbecause theymay have a
specificfunction associatedwitheach anatomicalcompart-
ment of the digestivetract.A cultureapproach was
includedto obtaina comprehensive visualizationof the
microbiota.
Figure 4 a RFLP obtained by digestion with AM of the ITS
amplifiedfromDNA extractedfrompooled gastrointestinal tracts The combinedresultsof themolecularfingerprints from
fromfivefishof varyingsizes. Lane Ps correspondsto Pseudomonas PCR-TTGEand ITS andtheresultsofthecloninganalyses
isolate,and lanes PI 4, P4, P3, P1.5, and P4.1 correspondto pools of
fishwithsizes of 14,4.1, 4, 3, and 1.5 g, respectively.
clearlydemonstrated thatbacterialcomposition along the
These fishwere
collectedin different seasons fromthe same location.M corresponds gut of juvenile salmon was similarand dominatedby
to a 100-bpmolecularweightmarker(Invitrogen).b RFLP obtained Pseudomonasspp. In orderto ruleout thepossibility that
by digestionwithAM of theITS amplifiedfromDNA extractedfrom thisnarrowdiversity maybe due to primer bias, 16S rDNA
tractsfromfivefishof varioussizes compared
pooled gastrointestinal
to stomach,pylorie caeca, and intestinefroma single fishof 30 g
primerswere analyzedusing the informatics tool Probe
Match in RDP II. This databasecontainsover 450,000
(lanes S: stomach,PC: pyloriecaeca, and /: intestine).Lanes PI 4, P4,
P3, and PL 5 correspondto pools describedin a. M correspondsto a sequences,whichare distributed in 33 phyla [11]. Each
100-bp molecularweightmarker(Invitrogen) primer matched with over 70% of the total sequences
deposited in RDP all
II, recognizing phyla.Morethan70%
of thesequencesassignedin eachphylumwererecognized
each). Bothprimers had identicalsequencetargetsin both by theseprimers. Therefore, no specificpreference during
bacteria.Simultaneously,
a semi-quantitative
PCR approach amplificationshould be expected, suggestingnarrow
showedsimilaramplification efficiencyforbothbacteria. diversityis nota consequenceof primer bias.
ITS amplificationefficiencywas 53.4±2.05% forPseudo- Cloningshowedgeneticdiversity withinPseudomonas
monasand55.3±3.3% forShewanella,(t test;/?=0.43997), sequenceswith calculatednucleotidedifferences among
whereas16S rDNA amplification was 89.3±
efficiency theseclonesrangingfrom0.4% to 4.8%. Considering that
2.4% for Pseudomonasand 89.7±3.0% forShewanella intergenomic differences in the nine genomes of Pseudo-
(t test;/?=0.85508). monas(availableat MicrobialGenomesNCBI) rangefrom

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BacterialMicrobiotain the Salmon Gut 559

Table 2 Relativeabundanceand nearest-match

identification
of bacterialisolatesobtainedfromsalmongut compartments

Bacterialisolategroups Phylum/class ITS (bp) Relativeabundance(%)

Salmon gut compartment

Genus/accessionnumber Stomach Pyloriccaeca Hindgut

Microbacterium/DQ067265-7 Actinobacteria/Actinobacteria 420 58.0 25.0 6.9

Cellulomonas,Oerskovia/DQ067270-'-3 Actinobacteria/Actinobacteria 480, 450, 380, 370 16.0 0.0 0.5
Serratia/DQ067268 Proteobacteria/gamma 480, 400 12.0 0.0 0.0
Shewanella/DQ067272,DQ067269 Proteobacteria/gamma 500 14.0 75.0 92.2
Pseudomonas/DQ067274 Proteobacteria/gamma 650 0.0 0.0 0.5

0.4% to 5.9% and also thatintragenomic differencesare measuredforbothbacteria.Hence,thesediscrepant results

less than0.2%, the differences foundamongthePseudo- may be betterexplainedby preferential cultivationof
monasclonessuggestthepresenceof severaldistinguish- Shewanellaspp. underthe experimental conditions.It is
able Pseudomonasstrainsin thegutsamples. also important to notethatstrictly anaerobicbacteriawere
The dominanceof a particularbacterialgroup was not investigatedin our study.Ringe et al. [39] have
previouslyobserved in salmonid guts using culture- previously suggestedthatthepredominant bacteriaisolated
independent methods. Holben et al. [20] reported thatsome from the salmonid gutare aerobes or facultative anaerobes.
genera were highly abundant in reared Atlantic salmon In our study, molecular approaches did not reveal the
fromtwo different locations:in the Scottishhatchery, presenceof anaerobes.Although be
theymay present, their
Mycoplasmacorrespondedto 81% of clones retrieved, abundancesare not highenoughto be detectedby these
whereas in the Norwegian hatchery,Acinetobacter methods.
accountedfor 55%. Althoughothergenera were also The salmongastrointestinal tractis made up of a U-
present,theirabundancewas closerto 2%. Interestingly, shapedstomach,pyloriccaeca, and intestine. Thus,it may
in wild salmon(entirelycarnivorous), the abundanceof be expectedthatthe compositionof bacterialmicrobiota
Mycoplasma was 96% of the clones analyzed [20]. along the salmon gastrointestinal tractwould also be
Similarly,Pond et al. [31] described the intestinalmicro- different, showingregionalspecialization.However,the
biota of rainbow trout a
by using cloningapproach.They similar community composition foundalong thedigestive
reportedonly two major groups among 200 clones tractof juvenile salmon suggeststhatconditionsfound
analyzed,whichcorresponded to Clostridium andAeromo- withineach compartment are not sufficientto select
nas. Furthermore, Kim et al. [24] reported thatClostridium different bacterialpopulations.The absence of major
dominated microbiota in rainbowtroutanalyzedby DGGE. differences may be due to several factors,namely,the
The carnivorous dietof salmonmayexplainin partthelow continuous presenceof feedwithhighproteincontent may
number oftaxaobserved,sincea recentstudyindicated that neutralizethe pH along the gut. Feed particlesare not
dietinfluences thebacterialdiversity of thedigestivetract. retarded in thesalmongut:thepyloriccaeca fillwithinthe
Bacterialdiversity increasesfromcarnivory to omnivory to same time course as the intestine,thus limitingfeed
herbivory [25]. fermentation [7-9, 20, 43]. This contrasts withthe caeca
Our knowledgeaboutthefishmicrobiota was obtained in birdsand mammals,whichhave largerretention times
using several culture approaches and these methods for fermentation functions [44]. Holben et al. [20] reported
revealeda wide rangeof microorganisms inhabitingthe low concentrations of acidic bacterialmetabolites (acetic
digestivetractoffish[3, 10, 19, 36]. Our cultureanalysis acid,lacticacid) indicating thata decreasing redoxgradient
showed,however,thatless than1% of themicroscopically is absent in salmon,leading to low levels of bacterial
observed bacteria in salmon gut compartments were fermentation. Furthermore, thepresenceof Pseudomonas,
cultivable,indicatingthat microbiotadetectedwith the which have an aerobic metabolism,suggeststhat the
cultureapproachmayrepresent onlya smallfraction ofthe juvenilesalmongutis predictedto have significant levels
total microbiota,as previouslydescribed[23, 42]. By of oxygenalong the gut, hence limitingthe growthof
cultureanalysis,Shewanellaappearedas themostabundant fermentative bacteria[31, 33].
bacterium in the salmondigestivetract,whichcontrasted Previousculture-based studieshave reported thatbacte-
withthemolecularresults.Since PCR pitfallsmayinclude ria presentin thehatchery environment may influence the
bias
efficiency [50], the PCR amplification efficiencyof of
composition gastrointestinal microbiota [10, 36]. Specif-
16S rDNA and ITS frombothShewanellaand Pseudomo- ically,itwas suggested thatbacteriafromwaterorfeedmay
nas isolates was evaluated. The same efficiencywas surviveand multiplyin the digestivetract[30, 49]. As

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560 P. Navarreteet al.

revealedby the TTGE analysisof thewaterinfluent and acknowledgesa scholarshipfromCONICYT-Chile and Dr. Stekel a
fromINTA-Nestlé.Partial supportwas derived froman
salmonfeed,Pseudomonasdo notrepresent thedominant fellowship
INNOVA CORFO grant(05CT6PPT-09) and FONDECYT 1080480.
bacteriain these samples,probablybecause theywere
absentor presentin verysmallproportions (less than1%)
[29]. However,a bacterialisolateidentified as Pseudomo-
nas was retrieved fromthe watersample showing97% References
identitywithdominant band 1 fromthesalmon.Converse-
ly,dominant bacteriafromwaterandpelletedfeedwerenot 1. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic
detected inthesalmondigestive tract.Theseresultssuggest identificationand in-situdetectionof individualmicrobial-cells
thatthe salmondigestivetractis a favorablehabitatfor without cultivation.MicrobiolRev 59:143-169
2. AndlidT, Vazquez-JuarezR, GustafssonL (1998) Yeasts isolated
harboringsome bacteria (Pseudomonas) that may be fromtheintestine of rainbowtroutadhereto and growin intestinal
derivedfromminorbacteriapresentin thewaterinfluent. mucus. Mol Mar Biol Biotech 7:115-126
This host selectionwas recentlydemonstrated by the 3. Austin B (2006) The bacterial microfloraof fish, revised.
of microbiota between differ- ScientificWorldJournal 6:931-945
reciprocal transplantationgut
4. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A,
entspecies[33]. Moreover, Pseudomonaswas detectedas a SemenkovichCF, Gordon JI (2004) The gut microbiotaas an
commonand dominant component in mostof thesamples environmental factorthatregulatesfatstorage.ProcNati Acad Sci
independent of boththe date of collectionand fishsize, USA 101:15718-15723
thatthesebacteriamaybe selectively associated 5. Bates J,MittgE, KuhlmanJ,Baden K, CheesmanS, Guindulain
suggesting
T (2006) Distinctsignals fromthe microbiotapromotedifferent
with salmon in this hatchery.These data supportthe
aspects of zebrafishgut differentiation.Dev Biol 297:374-386
traditional idea thatthe composition and diversity of gut 6. Bergot P, Solari A, Luquet P (1975) Comparaisondes surfaces
microbiota maydependnotonlyon hostselectionbutalso absorbantesdes caeca pyloriqueset de l'intestinchez la truitearc-
on local conditions.This statement is reinforced our en-ciel(Salmo gairdneriRich). Ann Hydrobiol6:27-43
by
7. Buddington RK, Chen JW, Diamond J (1987) Genetic and
previousfinding, which described Pseudomonas as a
phenotypicadaptationof intestinalnutrienttransportto diet in
dominant bacteriarecoveredfromcoho salmonrearedin fish.J Physiol393:261-281
thesamehatchery [42]. 8. BuddingtonRK, Diamond JM (1987) Pyloric ceca of fish: a
The successful oí Pseudomonaswithinfish "new" absorptiveorgan.Am J Physiol252:G65-G76
permanence
9. BuddingtonRK, KrogdahlA, BakkeMcKellep AM (1997) The
gutcompartments maybe due theeventualattachment of intestinesof carnivorousfish: structureand functionsand the
the bacteriumto digested food particles,mucus-like relationswithdiet.Acta PhysiolScand 161:67-80
material, orto thehostepithelium thatprevent itsmicrobial 10. Cahill MM (1990) Bacterial-flora of fish- a review.Microb Ecol
washout[45]. ThepossibleroleofPseudomonaswithinthe 19:21^1
11. Cole JR,Chai B, Farns RJ,Wang Q, Kulam SA, McGarrellDM,
digestivetractof fishhas been recentlyaddressed:this GarrityGM, Tiedje JM (2005) The Ribosomal Database Project
bacteriamodulates theexpression ofsomegenesofthehost (RDP-II): sequencesand tools forhigh-throughput rRNA analysis.
relatedto nutrient metabolism and innateimmuneresponse Nucleic Acids Res 33:D294-D296
12. Cole JR,Chai B, FarrisRJ,Wang Q, Kulam-Syed-Mohideen AS,
[34, 35].
McGarrellDM, Bandela AM, CardenasE, Garrity GM, Tiedje JM
Pseudomonadshave been reportedas common,often
(2007) The ribosomal database project (RDP-II): introducing
dominant, members of themicrobialcommunities of other myRDP space and qualitycontrolledpublic data. Nucleic Acids
fishandofwater[19, 35, 42]. Recently, itwas demonstrat- Res 35:D169-D172
ed thatstrainsassignedto different Pseudomonasspecies, 13. Espejo RT, Escanilla D (1993) Detectionof HIV1 DNA by a
whichshared>95% in 16S rDNA identity, simple procedureof polymerasechain reaction,using "primer-
mayhave very dimer"formation as an internalcontrolof amplification.Res Virol
dissimilar genomesindicated by low valuesof DNA-DNA 144:243-246
hybridization (7-37%) [17]. The stability of Pseudomonas 14. Espejo RT, Feijoo CG, Romero J, Vasquez M (1998) PAGE
spp. over the time evaluated in thishatchery maybe due to analysis of the heteroduplexesformedbetween PCR-amplified
16S rRNA genes: estimationof sequence similarityand rDNA
thishigh genomeversatility, whichcould providesome
complexity.Microbiology144(Pt 6):1611- 1617
advantagein thegutenvironment. 15. Espejo RT, Romero J (1997) Bacterial communityin copper
Altogether, ourresultssuggestthattheAtlanticsalmon sulfide ores inoculated and leached with solution from a
gutfavors Pseudomonas establishmentandthatthisbacterial commercial-scale copper leachingplant.Appi EnvironMicrobiol
63:1344-1348
population dominated thegutof thesesalmon.However,it 16. GordonJI(2005) A genomicview of our symbiosiswithmembers
shouldbe notedthatthegastrointestinal microbiota described ofthegutmicrobiota.JPediatrGastroenterol Nutr40(Suppl 1):S28
in this studymay not be representative of all Atlantic 17. Goris J, Konstantmidis KT, Klappenbach JA, Coenye T,
VandammeP, Tiedje JM (2007) DNA-DNA hybridization values
salmon,especiallythosein an openenvironment.
and theirrelationshipto whole-genomesequencesimilarities.IntJ
SystEvol Microbiol57:81-91
Acknowledgments This project was supported by a grant 18. GriffithsS, Melville K, Cook M, VincentS, PierreM, Lanteigne
(FONDECYT No. 1061121) from CONIC YT-Chile. P. Navarrete C (2001) Profilingof bacterialspecies associated with haddock

£) Springer

This content downloaded from 62.122.73.177 on Sat, 21 Jun 2014 05:37:56 AM

All use subject to JSTOR Terms and Conditions
BacterialMicrobiotain the Salmon Gut
larvicultureby PCR amplificationof 16S rDNA and denaturing
J Aquat Anim Health 13:355-363
gradientgel electrophoresis.
19. Hansen GH, OlafsenJA (1999) Bacterialinteractions in earlylife
stagesof marinecold waterfish.Microb Ecol 38:1-26
20. Holben WE, WilliamsP, GilbertMA, SaarinenM, SarkilahtiLK,
ApajalahtiJH (2002) Phylogeneticanalysis of intestinalmicro-
floraindicatesa novel Mycoplasmaphylotypein farmedand wild
salmon.Microb Ecol 44:175-185
21. Hovda MB, Lunestad BT, Fontanillas R, Rosnes JT (2007)
Molecular characterisationof the intestinalmicrobiotaof farmed
Atlanticsalmon{Salmo salar L.). Aquaculture272:581-588
22. JensenMA, WebsterJA,StrausN (1993) Rapid identification of
bacteria on the basis of polymerase chain reaction-amplified
ribosomalDNA spacer polymorphisms. Appi EnvironMicrobiol
59:945-952
23. JensenS, 0vreâs L, Bergh O, Torsvik V (2004) Phylogenetic
analysis of bacterialcommunitiesassociated with larvae of the
Atlantichalibutproposesuccessionfroma uniformnormalflora.
SystAppi Microbiol27:728-736
24. Kim DH, Brunt J, Austin B (2007) Microbial diversityof
intestinalcontentsand mucus in rainbow trout{Oncorhynchus
mykiss).J Appi Microbiol 102:1654-1664
25. Ley RE, Hamady M, Lozupone C, TurnbaughPJ, Ramey RR,
BircherJS, Schlegel ML, TuckerTA, SchrenzelMD, KnightR,
GordonJI (2008) Evolutionof mammalsand theirgut microbes.
Science 320:1647-1651
26. Magne F, AbelyM, BoyerF, MorvilleP, PochaitP, Suau A (2006)
Low speciesdiversity and highinterindividual in faeces
variability
of preterminfantsas revealedby sequences of 16S rRNA genes
and PCR-temporal temperaturegradient gel electrophoresis
profiles.FEMS MicrobiolEcol 57:128-138
27. McCracken VJ, Simpson JM, Mackle RI, Gaskins HR (2001)
Molecular ecological analysis of dietaryand antibiotic-induced
ofthemouseintestinal
alterations microbiota.JNutr131:1862-1870
28. Moreno C, Romero J, Espejo RT (2002) Polymorphismin
repeated16S rRNA genes is a commonpropertyof typestrains
and environmentalisolates of the genus Vibrio.Microbiology
148:1233-1239
29. MuyzerG, Dewaal EC, Uitterlinden ofcomplex
AG (1993) Profiling
microbial-populations by denaturinggradientgel-electrophoresis
analysisof polymerasechain reaction-amplified genes-codingfor
16S ribosomal-RNA. Appi EnvironMicrobiol59:695-700
30. OlafsenJA(2001) Interactions betweenfishlarvaeand bacteriain
marineaquaculture.Aquaculture200:223-247
31. Pond MJ, Stone DM, Alderman DJ (2006) Comparison of
conventionaland moleculartechniquesto investigatetheintestinal
microfloraof rainbowtrout{Oncorhynchusmykiss).Aquaculture
261:194-203
32. Ransom D, Lannan C, Rohovec J,FryerJ (1984) Comparisonof
histopathology caused by Vibrioanguillarumand Vibrioordaliiin
threespecies of Pacificsalmon.J Fish Dis 7:107-115
33. Rawls JF,Mahowald MA, Ley RE, GordonJI (2006) Reciprocal
gut microbiotatransplants fromzebrafishand mice to germ-free
recipientsrevealhosthabitatselection.Cell 127:423-433
This content downloaded from 62.122.73.177 on Sat, 21 Jun 2014 05:37:56 AM
All use subject to JSTOR Terms and Conditions
561
34. Rawls JF, Samuel BS, Gordon JI (2004) Gnotobioticzebrafish
reveal evolutionarily conservedresponsesto the gut microbiota.
Proc Nati Acad Sci U S A 101:4596^601
35. Rawls JF,Mahowald MA, Goodman AL, TrentCM, GordonJI
(2007) In vivoimagingand geneticanalysislinkbacterialmotility
and symbiosisin the zebrafishgut. Proc Nati Acad Sci U S A
104:7622-7627
36. Ring0 E, BirkbeckT (1999) Intestinalmicrofloraof fishlarvae
and fry.Aquae Res 30:73-93
37. Ringo E, Olsen RE, Mayhew TM, MyklebustR (2003) Electron
microscopy of the intestinalmicrofloraof fish. Aquaculture
227:395-415
38. Ringo E, SperstadS, MyklebustR, RefstieS, KrogdahlA (2006)
Characterisationof the microbiotaassociated with intestineof
Atlanticcod {Gadus morhuaL.): the effectof fishmeal, standard
soybean meal and a bioprocessed soybean meal. Aquaculture
261:829-841
39. Ringo E, Strom E, Tabachek J (1995) Intestinalmicrofloraof
salmonids:a review.Aquae Res 26:773-789
40. Romero J, Espejo R (2001) The prevalence of noncultivable
bacteriain oysters{Jiostreachilensis,Philippi,1845). J Shellfish
Res 20:1235-1240
41. Romero J, Garcia-Várela M, Laclette JP, Espejo RT (2002)
Bacterial 16S rRNA gene analysis revealed thatbacteriarelated
to Arcobacterspp. constitutean abundantand commoncompo-
nent of the oystermicrobiota{Jiostreachilensis). Microb Ecol
44:365-371
42. Romero J, NavarreteP (2006) 16S rDNA-based analysis of
dominantbacterialpopulationsassociatedwithearlylifestagesof
coho salmon{Oncorhynchus kisutch).Microb Ecol 5 1:422-430
43. R0nnestad I, Rojas-Garcia CR, Skadal J (2000) Retrograde
peristalsis;a possible mechanismforfillingthe pyloriccaeca? J
Fish Biol 56:216-218
44. RustM (2002) Nutritional physiology.In: HalverJ,HardyR (eds)
Fish nutrition.Academic,San Diego, pp 368^46
45. SonnenburgJL,AngenentLT, GordonJI (2004) Gettinga gripon
things: how do communitiesof bacterial symbiontsbecome
establishedin our intestine?Nat Immunol5:569-573
46. Suau A, BonnetR, SurrenM, Godon JJ,Gibson GR, Collins MD,
Dore J (1999) Directanalysisof genes encoding16S rRNA from
complex communitiesreveals many novel molecular species
withinthe humangut.Appi EnvironMicrobiol65:4799-4807
47. SuyehiroY (1942) A studyon the digestivesystemand feeding
habitsoffish. JpnJ Zool 10:1-303
48. vandePeerY, DeWachterR (1997) Constructionof evolutionary
distance trees with TREECON for Windows: accountingfor
variationin nucleotidesubstitutionrateamongsites.ComputAppi
Biosci 13:227-230
49. VerschuereL, Rombaut G, Sorgeloos P, VerstraeteW (2000)
Probioticbacteria as biological control agents in aquaculture.
MicrobiolMol Biol Rev 64:655-671
50. von Wintzingerode F, Göbel U, StackebrandtE (1997) Determi-
nationof microbialdiversityin environmental samples:pitfallsof
PCR-based rRNA analysis.FEMS MicrobiolRev 21:213-229
4y Springer