Chemoautotrophic, Sulfur-Oxidizing Symbiotic Bacteria on Marine Nematodes: Morphological
and Biochemical Characterization
Author(s): Martin F. Polz, Horst Felbeck, Rudolf Novak, Monika Nebelsick, Jörg A. Ott
Source: Microbial Ecology, Vol. 24, No. 3 (Nov. - Dec., 1992), pp. 313-329
Published by: Springer
Stable URL: http://www.jstor.org/stable/4251275
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Microb Ecol (1992) 24:313-329
ECOLOGY
MICROBIAL
(? 1992 Springer-VerlagNew York Inc.
Chemoautotrophic, Sulfur-OxidizingSymbiotic Bacteria on Marine
Nematodes: Morphological and Biochemical Characterization
MartinF. Polz,* HorstFelbeck,2RudolfNovak,' MonikaNebelsick,l and
JorgA. Ott'
1Institutfur Zoologie, Abteilung fur Meeresbiologie und Ultrastrukturforschung,Universitat Wien,
Vienna, Austria; and 2Marine Biology Research Division, Scripps Institution of Oceanography, La
Jolla, California 92093, USA
Received: December 23, 1991; Revised: May 11, 1992
Abstract. The marine,free-livingStilbonematinae(Nematoda:Desmodorida) inhabitthe oxygen sulfidechemoclinein marinesands. They arecharacterizedby an associationwithectosymbioticbacteria.Accordingto theirultrastructurethe bacteriaare Gram-negativeand form morphologicallyuniform
coats that cover the entirebody surfaceof the worms. They are arrangedin
host-genusor host-speciesspecific patterns:cocci form multilayeredsheaths,
rods, and crescent-or filament-shapedbacteriaform monolayers.The detectionof enzymesassociatedwithsulfurmetabolismandof ribulose-1,5 bisphosphatecarboxylaseoxygenase, as well as elementalsulfurin the bacteriaindinatureof the symbionts. Their reproductive
cate a chemolithoautotrophic
patternsappearto optimize space utilizationon the host surface:vertically
standingrodsdivideby longitudinalfission, whereasotherbacteriaformnonseptatefilamentsof up to 100 ,umlength.
Introduction
bacteriaplay an importantrole in the colonizationof
Symbioticchemoautotrophic
extreme habitatsby invertebrates.They are the source of an unusualmode of
nutritionin whichthe animalhosts derivetheircarbonentirelyor to a greatextent
fromthe symbioticprokaryotes(see [19] for a review). This is especiallyremarkable at deep-sea hydrothermalvents where the high populationdensities of the
vestimentiferantubewormRiftiapachyptila [5, 15] and of bivalves [4, 16] are
basedon a symbiosiswith sulfur-oxidizingbacteria.The symbiontsare contained
within specializedcells of the host body and use geothermallyreducedsulfide
emittedfromthe ventsandoxygenfromthe surrounding
waterto fix carbondioxide
(see [3, 19] for a review).
*Present address: The Biological Laboratories, HarvardUniversity, 16 Divinity Avenue, Cambridge,
MA 02138, USA.
314
M. F. Polz et al.
Prokaryote-eukaryote
associationsof this type can, however, also be found in
reducingsedimentswherethe decompositionof organicmattervia sulfatereduction
may producehigh concentrationsof sulfide in the interstitialpore water [30]. In
these habitats,intracellularbacterialsymbiontshave been detectedin bivalves [4,
16] andpogonophorans[44]. In oligochaetesfromcalcareoussands,the bacterialie
betweenthe cells of the epidermis[ 17, 24]. A commonfeatureof all theseanimals,
includingthehot ventspecies, is eitherthe lackof mouthandgutor thereductionof
feedingappendagesanddigestivetract.Aside fromthese endosymbioses,bacteria
have been found growing on the surface of the hydrothermalvent polychaete
Alvinellapompejana[1 1, 22] andthe brackishwaterpriapulidHalicryptusspinulosus [35]. Heavymetalandsulfidedetoxification,respectively,havebeensuggested
as the most likely roles of these symbionts.
A remarkableandin manyways uniquegroupamongtheseectosymbiosesfrom
sulfidic habitatsare the Stilbonematinae,a small subfamilyof marinefree-living
nematodes.They are characterizedby a species-specificcoat of ectosymbiotic
prokaryotes[36, 37, 48]. The morphologyof the bacterialcoat is very specificand
any given stilbonematidspecies can be identifiedby the appearanceof its coat
underthe dissectingmicroscope.However,in contrastto the otherectosymbioses,
the nematodesappearto graze on theirmicrobialsymbionts.This has been suggested by differentauthors[29, 37, 48] based on microscopicobservations.The
wormscan bendandreacheveryregionof theirbodywiththe mouth;theirguts are
frequentlyfilled with bacteriamorphologicallyand ultrastructurally
like the symbionts[37, 38]. This assumednutritionaldependencewas recentlysubstantiated
by
stablecarbonisotope measurements(aI3C) [38]. These a'3C values lie withinthe
rangefor chemoautotrophic,
sulfur-oxidizingsymbiontsandtheirrespectivehosts
observedso far, and differ markedlyfrom non-symbiontbearingnematodesfrom
the same habitat.A chemoautotrophic,
sulfur-dependent
nutritionof the bacterial
symbiontshas been proposedfrom sulfide incubationexperiments[40] and has
subsequentlybeen postulatedfromenvironmentalobservations[37, 38] and from
metabolicstudieson the nematodes[41]. The Stilbonematinae
occurexclusivelyin
sulfidic sands, where they can be found concentratedaroundthe oxygen-sulfide
interface[37]. Due to theirsmallsize the wormscan migrate,like all meiofauna,in
the porespaceof the sedimentwithoutalteringits structureandarethereforeableto
follow changesin the chemicalgradients[37, 38, 41].
This studyaimsto clarifythe natureof the symbiontsandto give furthersupport
to the assumedbenefitthe bacteriamayderivefrombehavioralpatternsobservedin
the hosts. We usedelectronmicroscopyandbiochemicaltests in orderto characterize the symbiontsmorphologicallyandto addresstheirmodeof energygeneration
andsurvivalundertheirnaturalhabitatconditions.
Materials and Methods
Collectionof Material
Stilbonematidswere collectedfromtropical,warm-temperate,
andMediterranean
calcareoussands. The tropicalsite was a shallow subtidalcoralline sandbarat
CarrieBow Cay, Belize BarrierReef (Belize, CentralAmerica;see [37]; the
Symbiotic Bacteria on Marine Nematodes
315
sandswere variousshallowsubtidalsandsin NorthCarolinaand
warm-temperate
Florida (see [36]). Some Catanema sp. and Eubostrichus cf. parasitiferus speci-
mens were collectedin VestarBay, Rovinij, Yugoslavia,in 3 m depth,using the
samemethodas describedin [37].
Scanning Electron Microscopy (SEM)
Selected specimenswere relaxedin MgCl2isotonicto seawaterand fixed in 4%
in 0.1 M sodiumcacodylate,0.05 M NaCl, 0.35 M saccharose,pH
glutaraldehyde
7.2, for 2 hours. Specimenswere washedtwice in sodiumcacodylatebufferand
postfixedin 1%OS04 in 0.2 M sodiumcacodylatebuffer,0.3 M NaCl, pH 7.2 for
1 hour,dehydratedin ethanol,transferred
into 100%acetone,subsequentlycritical
point dried, coated with a gold layer of approximately20 nm thickness, and
examinedwith a JEOLJSM-35CFscanningelectronmicroscope.
Transmission Electron Microscopy (TEM)
Fortransmissionelectronmicroscopy,Stilbonemasp., Catanemasp., andEubostrichuscf. parasitiferusspecimenswerefixed in 3%glutaraldehyde,
2%formaldehyde, 0.1% acrolein,DMSO, and CaCl2in 0.1 M sodiumcacodylatebuffer,pH
7.2, at 4?C for 2 hours. The specimens were subsequentlyrinsed in sodium
cacodylatebuffer,postfixedin 2%OS04 in 0.1 M sodiumcacodylatebuffer,rinsed
again, dehydratedwith ethanol,andembeddedin SPURRepoxy resin. A detailed
descriptionof the fixationprocedureis given in Nebelsicket al. [33]. Leptonemella
sp. specimenswerefixed andprocessedas outlinedin Ottet al. [36]. The sections
wereexaminedwith a Zeiss EM9-S2transmissionelectronmicroscope.
DAPI-Staining
Eubostrichuscf. parasitiferusspecimenswere fixed in 4% formaldehydein artificial seawaterat 4?C overnightand stained in diamidino-phenylindole (DAPI,
Sigma ChemicalCo., St. Louis, MO) (5 [ig pil-' in Mcllvain'scitric acid-phosphatebuffer,pH 4.5) for 60 min, destainedin bufferfor 60 min, and mountedin
50%glycerolin buffer(modifiedfrom[39, 46]). Cells wereexaminedimmediately
afterstainingundera Leitzepifluorescencemicroscopeequippedwith anHBO-100
high pressuremercurylight source.
Frequency of Dividing Cells (FDC)
To gain informationon the state of the symbioticbacterialpopulationwe determinedFDC as a parameterto estimatebacterialgrowth[26]. In the ectosymbiotic
bacteriaof Catanema sp. from Belize, division stages are exceptionallywell
discernibleon SEM micrographs.We thereforeused this species to determinethe
M. F. Polz et al.
316
fractionof dividingcells to the totalbacterialpopulation.Areasof intactcoverwere
discernedfromobviouslydisturbedpartsshowingmoresparsebacterialepigrowth.
A total of 25 SEM photographswere takenat 10,000-foldmagnification,each
coveringan areaof 91.5 pLm2.FDC was determinedfor "intact"and "disturbed"
areasrespectively.
Enzyme Assays
Because individualstilbonematidsweigh only about 25 pLgwet weight each,
specimensof three differentspecies had to be pooled in orderto obtainenough
materialfor enzyme analysis.These batcheshad the following composition:Catanema
(54.6% ? 9.1%),
Stilbonema
(31.1% ? 8.8%)
and
Robbea
(14.3% ? 2.6%). The wormswerecollectedfromthe CarrieBow Cay sandbarand
separatedfromothernematodesandmeiofauna.Batchesof 5-10 mg wet wt were
to the laboratory.
blotteddryandfrozenin liquidnitrogenfor transportation
Extractswere preparedfrom these batchesof whole worms in triethanolamine
buffer (0.2 M, pH 7.3, 1%TritonX-100) in a fourfoldvolume (w/v) in a glass
homogenizer.All furthersteps were carriedout as describedelsewhere[15, 17].
Enzymeactivitywas tested for the enzymes:ribulose-1,5-bisphosphate
carboxylase oxygenase (RuBisCO)(EC 4.1.1.39.), ATP sulfurylase(EC 2.7.7.4.), adenosine 5'-phosphosulfatereductase(APS reductase)(EC 1.8.99.1.), rhodanese
(EC 2.8. 1. 1.), sulfiteoxidase (EC 1.8.3. 1.), andnitratereductase.Exceptfor the
test for sulfiteoxidase, which was done as described[6], the enzymeswere tested
as describedearlier[15, 17]. The use of the artificialelectronacceptorFe(CN)63insteadof the morecommonlyusedcytochromec in the test for sulfiteoxidasewas
necessary because of the small amountof tissue available. The same cuvette
includingthe extractcould be used afterwardsfor the test of APS-reductaseby
addingAMPto the test. In addition,activityof sulfiteoxidasehas beenreportedto
be 14 timeshigherin tests with Fe(CN)63- as comparedto cytochrome[14].
Elemental Sulfur
Elementalsulfur (SO)was determinedby HPLC [31] as described [10]. Two
stilbonematidspecies weretestedseparately.Two batchesconsistingof 100 specimens of Catanemasp. andtwo batchesconsistingof 50 specimensof Stilbonema
sp. were assayed. The wormswere collected from the CarrieBow Cay sandbar,
washed three times in sterile seawater,blotted dry, and stored in 2 ml 100%
ethanol.The vials containingthe wormswere sonicatedpriorto opening,the fluid
transferredto centrifugationvials, centrifuged,and 50-100 Pl aliquotstakenfor
HPLCanalysis. The remainderwas dried, reextractedwith 2 ml hexane, and an
aliquottakenfor anotherHPLCanalysisto assess if the Socontainedin the samples
hadcompletelydissolvedin the ethanol.
To test if S?was confinedto thebacteriaor if it couldalso be foundin the worms
as a possible sulfidedetoxificationproduct,threebatches,each consistingof 100
specimensof Catanemasp., werepurifiedof theirsymbiontsby rinsingin 50 ppm
benzalkoniumchlorideandtreatedas describedabove.
SymbioticBacteriaon MarineNematodes
317
Poly-4-Hydroxy Alkanoic Acids (PHA)
To determinethe polyestercontentof the symbioticbacteria,a mixedbatchof 200
worms,consistingof the threespecies Catanemasp., Stilbonemasp., andRobbea
sp. (in the samecompositionas describedfor the enzymeassays), was subjectedto
methanolysisin the presenceof 3% sulfuricacid (v/v) as describedby Braunegg
acid methyl esters. The
et al. [2] to obtainthe constituent3-hydroxycarboxylic
with a GC-14AShimadzugas
methylesterswere assayedby gas chromatography
chromatograph
equippedwith a capillarycolumn(15 m by 0.53 mm, CarloErba
Instruments)anda flame ionizationdetector.A 2-,u fractionof the organicphase
was analyzed;nitrogen(30 ml min- ) was used as the carriergas. The temperature
of the injectorand detectorwere 180 and 200?C, respectively. A temperature
programwas usedfor efficientseparationof theesters(70?Cfor 1 min, temperature
increaseof 8?C min-', 160?Cfor 5 min). Underthese conditions,the retention
times for standardsof poly-,B-hydroxy
butyricacid (PHB) and of poly-3-hydroxy
valericacid (PHV) were 3.25 min and 4.61 min, respectively.Benzoic acid was
usedas an internalstandardandhada retentiontimeof 7.73 min.
Results
Types and Morphology of Bacterial Coats
Stilbonematidectosymbioticbacteriacan be groupedin three morphologically
distinguishabletypes:cocci, rods, andextremelylarge, eithercrescent-or threadshapedbacteria[36, 37]. Eachsymbionttype is boundto a specific stilbonematid
hostgenusor hostspecies. Accordingto morphologicalcriteria,thebacterialcoatis
monospecific.The mode of attachmentof the bacteriais characteristicfor each
type: Coccoid bacteriaform multilayeredsheaths, whereas rod- and crescentshapedbacteriaarealwaysarrangedin orderedmonolayers.
Multilayers. Several species of the genera Stilbonema and Leptonemella were
associatedwith coccoid bacteria,forminga multilayeredcoat on the wormsurface
(e.g., Stilbonemasp., Fig. lA, B). The bacteriaare embeddedin a gelatinous
matrix,which is clearlyvisible using light microscopy[37]. The bacterialsheath
covered the entire body of the worm (Fig. IA), except the head capsule. On
Stilbonemasp., the bacteriaformeda dense layerup to 10 cells thick;the average
cell was 1.3 pLmlong and 0.6 p.m wide, giving an overall coat thickness of
approximately7.5 pLm[41]. A close-up at a disruptedsite (Fig. iB) revealsthe
morphologyof the individualcells, but gives no indicationof how the bacteria
adhereto the worm'scuticle. In all investigatedspecimens,the bacterialcoverwas
detachedfromthe host surfaceandthe bacteriaappearedto adheremorefirmlyto
one another.The mucousmatrixin which the bacteriawere embeddedprobably
shrankduring the preparationprocess [21], but is still visible as small fibers
interconnectingthe individualcells (Fig. IB). The arrangement
of bacterialcells
lacksan orderedpattern.
Monolayers. Monolayers occur on all Catanema and Eubostrichus species and on
certain Leptonemella species.
318
M. F. Polz et al.
Fig. 1. SEM illustratingthe morphologicaldiversityanddifferentadhesionpatternsof the stilbonematidectosymbioticbacteria.A, B, Stilbonemasp.; C-F, Catanemasp. 1;G, Catanemasp. 2; H, yet
undescribedgenus;I, J, Eubostrichuscf. dianae; K, L, Eubostrichuscf. parasitiferus.A: Midbody
regionof Stilbonemasp. completelycoveredby a multilayerof coccoidbacteria.B: Highermagnification of a disruptedsite in the bacterialcover revealingbacterialmorphology.Note the underlying
cuticle and the small fibers interconnectingthe cells. C: Midbodyregionof Catanemasp. from the
Sea coveredby a monolayerof corn-kernelshapedbacteria.D: Highermagnificationof
Mediterranean
a disruptedsite. Arrowindicatespossiblelongitudinalfission. E: Sharponsetof the bacteriallayerin
the same species. Note reductionof worm's diameterand mucoid layer coveringanteriorend. F:
Bacteria-freeanteriorend is frequentlycoveredby suctorianciliates. G: Elongatedbacteriaon Catanema sp. from the CaribbeanSea; mucus visible as condensedspots between the cells. Arrow
indicatespossible longitudinalfi'ssion.H: Rods on a stilbonematidspecies belongingto a yet unde-
SymbioticBacteriaon MarineNematodes
_-Sa __.*::Ws~'
319
Hs
scribed genus from the MediterraneanSea covered by a dense slime layer. I: Posteriorend of
Eubostrichuscf. dianae with extremelylong bacteriagiving the worma hairyappearance.J: Higher
magnificationrevealingthepresenceof numerousother,smallerepibacteria.K: Anteriorandposterior
end of Eubostrichuscf. parasitiferuswithsymbiontsarrangedin a characteristic
helix;a, anteriorend;
p, posteriorend. L: Highermagnification.Bacteriaattachedwithbothendsto the worn's cuticle.Note
the increasinglengthof the cells fromproximalto distalend fromthe worm's surface.Bar markers
represent10 p.min panelsA, C, E, F, andK, and 1 p.min panelsB, D, G, H, I, J, andL.
320
M. F. Polz et al.
In Catanemasp. 1 from the Mediterranean
Sea, the bacteriawere corn-kernel
shaped(Fig. IC,D), with one end attachedto the worm's cuticle. They were 1.6
urmlong and 1.3 pm wide and stood tightly packed, a characteristicfor all
Catanemamonolayers.The bacterialcoat beganwith a sharponset some distance
fromthe anteriorend of the worm(Fig. lE). Here, the worm'sdiameteris reduced
by exactly the thicknessof the bacteriallayer, so thatthe overalldiameterof the
wormdoes not increase(see Nebelsicket al., [33] for a detaileddiscussion,[37]).
the bacteriacouldbe detected,butthe
No mucuseithercoveringor interconnecting
bacteria-freeanteriorend had a possiblymucoidmatrixoverlyingthe cuticle(Fig.
IE). A peculiarityof this Catanemaspecies is shownin Fig. IF. Suctorianciliates
frequentlygrow on the bacteria-freeanteriorend. The ciliates were, however,
restrictedto the areawhereno bacterialepigrowthoccurs.
Catanemasp. 2 from Belize is associatedwith large rod-shapedbacteria.The
symbiontsaveraged2.1 ,umin lengthand0.6 uLmin widthandstoodupright(Fig.
IG), frequentlycoveredby a thin mucouslayer. The remainsof this layercan be
seen as smallspotsbetweenthe individualcells. On SEMphotographsmostof the
bacteriaappearto have a knob-likethickeningon bothends.
An as yet undescribedgenuswithinthe Stilbonematinae
fromthe Mediterranean
Sea carriesrods, averaging1.8 pLmby 0.7 pLm(Fig. 1H). They resembledthe
symbiontsdescribedabovefor Catanemasp. 2, butlackedtheirexpandedendsand
had a more elliptical shape. They were also usually coveredby a dense mucous
layer.
The genus Eubostrichusis characterizedby an associationwith exceptionally
large bacteria(Fig. 1I-L). They cover the whole body of the worm; only the
mouth-coneremainsfree. No mucoidsubstancecouldbe detectedon SEMmicrographsof membersof this genus. In Eubostrichuscf. dianaethe cells of almostthe
entiresymbiontpopulationwere up to 100 pLmlong. One end was attachedto the
worm'scuticle, whereasthe otherwas free (Fig. 1I,J). At the basis of this fur-like
cover a morphologicallydiversecommunityof othermicroorganisms
was present.
The symbiontsof Eubostrichuscf. parasitiferuswere crescent-shapedand adheredwithbothendsto theirhost, forminga helix in whichthe worm'sbody is the
centralcore (Figs. 1K, L and2E). The individualcells averaged0.63 Pm in width
andwereup to 30 uLm
long, with lengthsincreasingfromthe proximalto the distal
end of the wormbody (Fig. 1K).
Ultrastructure of Bacterial Cells
Generally,the ultrastructure
appearedrelativelyuniformamongthe differenttypes
of symbionts.Theircell envelopes were Gram-negativeand smooth(Fig. 2 and
unpublisheddata). Only the outer layer of the cell wall of the symbiontsof
Leptonemellasp. had a slightlyundulatingappearance(Fig. 2B). This may, however, be an artifact,as described[12], due to the differentfixationprocedureused
for specimensof this species. Two types of sphericalcytoplasmicinclusionswere
generallypresentin bacteriatakenfromfreshlyextractedworms:electrontransparent globulesof variablesize (Fig. 2D and F) and slightlyelectrondense vesicles
usuallylargerthanthe former(Fig. 2D andF). The firsttype resembledthe sulfur
globules found in chemoautotrophicsulfur-oxidizingbacteria, the second was
SymbioticBacteriaon MarineNematodes
321
V4._."'K
,
attachFig. 2. TEMof thin sectionsof stilbonematidectosymbiontsillustratingthe ultrastructure,
ment, and mode of division. A, Stilbonemasp.; B, Leptonemellasp.; C, D, Catanemasp. 2; E F,
Eubostrichuscf. parasitiferus.A: Multilayerof coccoid symbiontsof Stilbonemasp. Bacterialcells
arepackedwiththe two typesof inclusionbodies.c, cuticle. B: Binaryfissionof the symbiontsin the
multilayerof Leptonemellasp. Wormswerekeptin oxygenatedseawater;all globularinclusionshave
disappeared.Note the slightlyundulatingcell wall. C: Highlyorderedarrayof the rod-likesymbionts
in Catanemasp. from the CaribbeanSea. Cells containnumerousinclusionbodies. c, cuticle. D:
fissionin theCatanemasymbiont.g, putativestoragevesicles;s, putativesulfurglobules;
Longitudinal
arrowindicatespossiblecarboxysomes.D: Sagittalsectionthroughthebacterialcoverof Eubostrichus
cf. parasitiferus.Bacteriaare nonseptatefilaments.Note fibrils extendingfrom the bacteriato the
worm's cuticle. c, cuticle. F: Section of the Eubostrichuscf. parasitiferussymbiontat a higher
_.m
2
in
magnification.g, putativePHA vesicles; s, putativesulfurglobules. Bar markersrepresent
panelsA, C, andE, 1 p.min panelD, and200 nm in panelsB andF.
322
M. F. Polz et al.
_______
Fig. 3. DAPI stainingof the
crescent-shapedrods on
Eubostrichuscf. parasitiferus
the presenceof
~~revealing
numerousnucleoidsper bacterial
cell. Bar marker represents 5
Em.
similarto PHA inclusion bodies. Especially the latterappearedmembranesurroundedon some of the micrographs.Whenkeptin oxygenatedseawaterfor more
than 12 hoursthe globulesdisappeared,withoutleaving any visible remains,and
the cytoplasmstainedhomogeneouslydarkexceptforthe nuclearregion(Fig. 2B).
TEM of cross sections of Catanemasp. 2 (Fig. 2C) showedthe highly regular
of the bacterialcells in the monolayeron the worm'scuticle. Polyhearrangement
dral,electrondensebodieswerescatteredthroughoutthe cytoplasmof the microorganisms(Fig. 2D)).They resembledcarboxysomes[41]. We could, however,only
observe them in preparationsof the Catanemasp 2 symbionts(Fig. 2D)). The
symbiontsof both Eubostrichus cf. dianae and E. cf. parasitiferus were not
multicellularfilamentsbut single cells (Fig. 2E). In serialTEM sectionsno cross
wall formationcould be observed.
Bacterial Cell Division
Thebacteriaarrangedin multilayerson Stilbonemasp. andLeptonemellasp. divide
by the binaryfission typicalfor Gram-negativebacteria,formingtwo equaldaughtercells thatfully separate(Fig. 2B).
The uprightsymbiontsof Catanemasp. fromthe Caribbean,however,divideby
longitudinalfission (Fig. 2D). The cell volume doubles, and division startswith
simultaneousinvaginationsof the cell wall on both ends; these extendtowardthe
middleuntiltwo daughtercells of identicalshapeseparate.
No division stagescould be detectedin any of the large symbiontsof Eubostrichus cf. dianae andE. cf. parasitiferusin eitherSEM of TEM. DAPI-stainingof
theepibacteriaof E. cf. parasitiferusdidrevealthe presenceof severalnucleoidsin
each cell (Fig. 3). Up to 16 nucleoids were contained within one single cell of
approximately 30 tim length.
FDC
In all stilbonematids, disturbed areas in the bacterial coat could be frequently found
afterextractionof the wormsfrom the sediment.Althoughthe reasonwhy some
SymbioticBacteriaon MarineNematodes
*'
.L
t j ^k44'
323
jm"
Fig. 4. SEM of (A) a typicalundisturbedand (B) a typicaldisturbedarea in the bacterialcoat of
to theplaneof the figure.Barmarkers
Catanemasp. Thebacterialcells arerodsstandingperpendicular
represent1 pLm.
areas of the cuticle are less densely populatedcannot be deduced from these
observations,a comparisonbetween intactand disturbedspots on Catanemareveals a highly significantdifferencein the FDC (Kolmogorov-Smirnov;
n, = 13,
bacterial
of
?the
<
areas
13.9%
in
intact
P
(SD
5.0)
12;
0.0001).
Although
n2=
cells were dividingat the time of fixation,the fractionwas 34.2% (SD -+ 10.7) in
disturbedareas.Figure4A andB shows a typicalintactanda typicaldisturbedarea
as designatedby the authors.
Enzyme Assays
The activity (in pRmol/gfresh weight/mmn?. SD) for RuBisCO was 0.02 ?- 0.013
(n = 6). ATP-sulfurylase,sulfite-oxidase,and nitratereductaseshowedactivities
of 2.7 ? 1.9 (n = 7), 96 ? 41 (n = 5), and 0.2 (n = 1), respectively.Neither
APS-reductasenorrhodanesecouldbe detected.
Elementalsulfurwas presentin high amountsin all samplescontaininghost and
symbioticbacteria(Table 1). Division of the S0 values for the total sampleby the
numberof wormsusedfor eachassaygives a rangeof 6.2 to 8.3 ng S' perspecimen
for Catanemasp. and 3.8 to 15.6 ng S' per specimen for Stilbonemasp. In
symbiont-freeCatanemasp. So was not detected(Table1), indicatingthatelemental sulfuris confinedto the bacteria.
PHA
Two peaks in the sample
PHB and PHV were detectedby gas chromatography.
elutedwith the same retentiontime as the PHB and PHV standards,respectively.
M. F. Polz et al.
324
determinedby HPLCfor two stilbonematid
genera.
Table 1. TotalS?concentration
S0 (ng/batchof worms)
Assay
Assay 2
Stilbonema with symbionts
Catanema with symbionts
778
827
193
620
Catanemawithoutsymbionts
ND
ND
Genus
Assay 3
ND
One batchof wormsconsistedof 50 and 100 specimensof Stilbonemaandof CatanemafromBelize,
respectively.Threebatchesof Catanemawere strippedfromtheirsymbioticbacteriaandthe worms
wereanalyzedalone. ND, not detected(detectionlimit 1 ng atomS perml).
The first showed a retention time of 3.23 min as compared with 3.25 for the PHB
standard, and the second, 4.59 min as compared with 4.61 min for the PHV
standard.The total amount present for the 200 specimens tested was approximately
15 ,ug for the putative PHB peak and approximately 22 jig for the putative PHV
peak. PHAs represent a carbon storage compound in many bacteria and are usually
formed under conditions where plenty of energy and carbon is available but oxygen
or nitrogen is limiting (see [45] and references therein). It was also present in the
endosymbiont-containing oligochaete Inanidrilus (Phallodrilus) leukodermatus
[25].
Discussion
We have shown microbial epigrowth on a number of different species of the small
nematode subfamily Stilbonematinae. This is unusual because microbial epigrowth
on the nematode cuticle is a relatively rare phenomenon. In most representativesof
this taxon the surface is colonized by microorganisms only when the animal dies.
Only the Stilbonematinae and some species from a few other nematode families are
associated with microorganisms throughouttheir life span. Very rarely did we find
individuals lacking the bacterial coat: even first stage juveniles show the characteristic epigrowth [36]. In some taxonomic investigations, however, the microbial
symbionts have not been mentioned and have probably been overlooked or ignored
by the nematologists [8, 28].
The extreme morphological uniformity of ectosymbiotic bacteria that we have
demonstratedin the stilbonematid symbiosis is to our knowledge unique in invertebrateectosymbiosis. For example, dense coats of bacteria are seen on the surface on
the pompeii worm Alvinella pompejana (Polychaeta) [22], on Halicryptus spinulosus (Priapulida) [35], on Zebra cyathostomes (Nematoda) [13], and on some
representatives within the Desmodoridae (Nematoda) ([1], personal observation).
However, these are composed of diverse bacterial morphotypes. In the Stilbonematinae, morphological diversity of associated bacteria can only be found between
species (Figs. 1, 2). The only exception is the genus Eubostrichus where additional
bacterial morphotypes were frequently lying on the cuticle (Fig. 11, J, L). Their
relation to the conspicous symbionts is not known, and they may representbacteria
trappedin the matrix formed by the larger cells.
SymbioticBacteriaon MarineNematodes
325
characteristics:Their
All stilbonematidsymbiontsshare similarultrastructural
cell wall appearsGram-negativeand the same two types of inclusionbodies are
vesiclesresemble
containedwithinthe cytoplasm(Fig. 2). Theelectrontransparent
sulfur globules found in the chemoautotrophicsymbiontsof clams [47] and of
oligochaetes[25], as well as free-livingsulfur-bacteriasuch as Beggiatoa [32].
the cell shapeof the respectivestilboneAlthoughthey sharesimilarultrastructure,
matid symbiontsis very distinctiveand is host species or host genus specific.
Whetherthe bacterialcoats are monospecific,as theiruniformappearancewould
suggest, must remain unanswered;however, it is difficult to imagine another
mechanismthatwouldresultin suchuniformpopulationsof distinctmorphological
of the symbiontswill be
types. To clarify this questionfurther,characterization
necessaryand shouldinclude 16S rRNAsequencingandidentificationby specific
gene probes.
Several lines of evidence suggest that the symbiontsare sulfur-oxidizingand
chemoautotrophicin nature. We were able to demonstratethe presence of
RuBisCO,the key enzymeuniqueto the Calvin-Bensoncycle, commonlyused to
test for autotrophicCO2 fixation [3, 15, 17, 44]. The activity detected in the
nematodesis comparablewith that obtainedfor similarsized animalscontaining
chemoautotrophic
symbionts:0.02 pimolg-' min-' for the Stilbonematinaeversus, for example, 0.05 for the oligochaete Inanidrilus (Phallodrilus) leukodermatus
[17], and 0.027 to 0.112 and 0.0068 to 0.0217 for the small pogonophorans
Silboglinumfiordicum and S. atlanticum, respectively [44]. On the other hand, the
valuesfor the Stilbonematinaeappearlow if comparedwith macrofaunalsymbioses: RuBisCOactivityof 0.22 to 1.13 Fmol g- 1 min-1 was reportedfor the giant
vent tubewormsand0.01 to 2.4 ,umolg-' min-' for bivalves [15,
hydrothermal
16]. It is, however, difficultto compareenzyme data from differentsymbioses.
First, the portionof the bacteriato the total biomassof the symbiosismay vary
considerably;second, in largehosts, extractswerepreparedfromtissuesharboring
the microorganisms,whereassmallerhosts were usuallyextractedwhole. In the
specificcase of the Stilbonematinae,beingthe smallestmetazoanhostsdetectedso
far, specimensfrom three differentspecies had to be pooled to obtain enough
materialfor enzymeanalysis.This leavesthe possibilitythatone or two of the three
species used in the analysismightbe associatedwith non-autotrophic
symbionts;
similarinclusionsin all threesymbionts
however,we have shownultrastructurally
(Fig. 2 and unpublisheddata)and that the two testedcontainedSo (Table 1), an
intermediaryproductin the oxidationof reducedsulfurspecies. Furtherevidence
for dependenceon chemoautotrophically
fixed CO2 comes from stable carbon
isotoperatiosfor the samethreespecies [38]. The wormsshowedsimilarvaluesto
otherinvertebratesknownto containendosymbioticsulfur-oxidizingbacteriaand
free-livingthiobacteria[38], whereasnon-symbioticnematodesand detritusfrom
the same habitathad much higher values. Accordingto the two-source-mixing
model [9], we wouldexpectthatthe a13Cvaluesobservedfor the Stilbonematinae
would not fall within the range of values observedfor other chemoautotrophic
sulfur-oxidizersif one or more of the worm species were associatedwith heterotrophicbacteria.
The Stilbonematinaeoccur exclusively in reduced,marinesands, concentrated
aroundthe sulfide-oxygeninterface[37]. Althoughthe crescent-shapedsymbionts
of Eubostrichuscf. parasitiferuswereinitiallydescribedas "blue-greenalgae"[23,
326
M. F. Polz et al.
48], the bacteriaare clearlynonphotosynthetic.
We did not observepigmentsand
we regularlyfind the nematodesin sedimentdepthswhereno light penetrationis
possible. Further,the nematodesand their bacteriahave remainedviable for
monthsin sandmesocosmskeptin completedarkness.Whenfreshlyextracted,the
symbiontsgenerallyshow the typicalpurewhiteBeggiatoa-likecoloration,which
is lost uponexposureto oxygenatedseawater[41]. This compareswith the white
colorationof the symbiont-containing
gills of clams [47] andof oligochaeteswith
subcuticularsymbionts[25], whichhas been shownin bothto be correlatedto the
presenceof sulfurglobulesin thebacteria.Uponincubationin Na235S,thebacterial
coat of Eubostrichussp. accumulatedsulfur[40]. In microrespiration
experiments
of Stilbonemasp., Catanemasp. 2, and Robbea sp., overall consumptionof
oxygenof the nematodeandits symbiontscouldbe enhancedfollowingexposureto
either thiosulfate or sulfide, whereas oxygen consumptionof symbiont-freed
wormstreatedthe sameway remainedunchanged[41].
The activitiesof the enzymes we could detect give furthersupportto the proposed natureof the symbionts.ATP-sulfurylaseand sulfite oxidase may be involved in the generationof energy from reducedsulfurcompounds,and nitrate
reductasecould serve eitheras an assimilatoryenzymeor have a respiratoryrole.
Thevaluesfor ATP-sulfurylase
lie in therangefoundin smallhosts [ 17, 44] butare
low if comparedwith large-bodiedhosts [16]. However, the activity of sulfite
oxidaseappearshigh in comparisonto othersymbionts[17] and free-livingsulfur
bacteria[7, 27]. This may be due to the higherefficiency of the artificialelectron
acceptorused in the test as comparedwith the naturalacceptor,cytochromec,
resultingin an apparentlyhigheractivity.AlthoughAPS-reductaseandrhodanese
weredemonstratedin otheranimalscontainingsulfurbacterialsymbionts[ 16, 44],
they were not detectedin the small oligochaeteI. leukodermatus[ 17]. This might
be due to a differentcompositionof the bacterialpopulationin the nematodesand
the oligochaete. Again, with the amountof materialavailablewe regardthese
assays as qualitativefor the presenceof these enzymes. However,the co-occurrenceof enzymesof sulfurmetabolismand S? as well as the evidencementioned
above strongly suggest the capacity for energy generationfrom reducedsulfur
species in the bacteria.
Elementalsulfurmight play a criticalrole in the nutritionand survivalof the
symbiontsunderhabitatconditions.Storageof this compoundrepresentsa common characteristicof certainsulfur-oxidizingautotrophs.It can eitherserve as an
electronacceptoror the energyconservedin it can be mobilizedduringthe temporaryabsenceof externalsourcesof reducedsulfur[34, 42]. The lattercharacteristic
may be of particularsignificance.The sulfideconcentrationin the naturalenvironment of the Stilbonematinaeis highly variablewith time and space [37]. The
nematodeswere shown to react behaviorallyto concentrationgradientsbetween
oxygen andsulfide[37, 38]. In areasof high sulfideconcentration(over200 pLM),
the worms preferentiallydwell just above the sulfide maximum,whereasat low
concentrations(50 FLMand below) they were observedto migrateverticallybetweenthe oxic andthe anoxic, sulfidiczones [38], resultingin two distinctdistribution maxima[37]. Assumingan aerobic,sulfur-dependent
energygenerationin the
stilbonematidsymbionts,as ourdatasuggest,thishostbehavioris beneficialforthe
bacteria.At high sulfideconcentrationsthey are kept in the oxygen-sulfideinterface, whereas at low concentrationsthey are sequentiallyexposed to oxic and
anoxic, sulfidic conditionsby the movementsof theirhosts betweenthe distinct
Symbiotic Bacteria on Marine Nematodes
327
sedimentzones. Respirationexperimentssuggestedfor the symbiontsan abilityto
accumulateSounderanoxic, sulfidicconditions[41]. S?mightthusbe accumulated
from sulfide availablein deep, anoxic sedimentlayers and then subsequentlybe
oxidizedwith molecularoxygen aftermigrationto shallowersedimentzones.
We have observedthatall bacteriain the stilbonematidsymbiosisexhibithighly
specificandconsistentadhesionpatterns(Figs. 1, 2). This is especiallyremarkable
for the monolayers.The attachmentpatternsof boththe Catanemasymbiontsand
the Eubostrichussymbiontsoptimizeutilizationof cuticle space, with each cell
adheringto the worm's surface. We suggest that longitudinalfission, as in the
Catanemamonolayers,could preventloss of cuticle contactduringreproduction,
indicatingdependenceon free adhesionsites. Ourdataon FDC for the Catanema
symbiontdemonstratea strongcorrelationbetweenavailabilityof adhesionsites in
a given regionof the cuticleandthe proportionof dividingcells in thatregion(Fig.
4). This is a propertythathas been describedonly for ectosymbioticbacteriaof the
ciliateKentrophorusfasciola
[ 18] andforbacteriaattachedto cuticularspinesin the
hindgutof the cockroachBlaberusposticus [20].
It is remarkablethatno division stages could be detectedin the extremelylong
symbiontsof Eubostrichus.These are in the shapeof elongatedspindleson E. cf.
parasitiferusattachedat both apices. The bacterialcells differ substantiallyin
lengthalthoughthe distancebetweenattachmentsites is very uniform(Fig. 1L).
This suggeststhatthese bacteriaundergoconsiderablegrowthby elongationwithout apical reattachmentand withoutcell division. Numerousnucleoidsin these
cells (Fig. 3) also indicateextensivegrowthwithoutdivision. In the case of E. cf.
dianaeon whichthe symbiontsare attachedwith one end, only the samephenomenon may lead to symbiontcells being up to 100 [im in length. Two explanations
for the bacteriahavingsuch unusualdimensionsappearreasonable:the extraordinarycell lengthmightbe an inherenttraitof specific bacterialstrainsgrowingon
therespectiveEubostrichusspeciesor it mightbe triggeredby someagent,possibly
producedby the nematodehost. In bothcases thiscouldresultin a maximizationof
bacterialbiomasswitheach cell havingcontactto the host surface.
In conclusion, our data stronglysuggest that the bacteriaare Gram-negative,
The morphologicaluniformityof the respective
sulfur-oxidizingchemoautotrophs.
symbiontsmay point to a highly specific associationbetweenthe two respective
partners.Thusthe symbiosesin the Stilbonematinae
appearto resembleendosymbioses between invertebratesand sulfur-bacteria,in symbiontultrastructure
and
storageproducts,modeof energygenerationandrole in hostnutrition.Whereasthe
bacteriaappearto benefit from the chemical environmentin which the worms
preferentiallydwell, the hosts seem to feed on theirsymbionts.
Acknowledgments. This study was supported by the FFWF (Austria), grant nos. 6576 and 7814 (J. Ott,
principal investigator) and NSF grant no. OCE-901 1835 (H. Felbeck, principal investigator). We
thank Stephanie Kalousek MA for help with the PHA analysis and Dr. Paul Dando for the So
determination (supported by CEC MAST contract 44). M.F.P. thanks Drs. Colleen Cavanaugh and
Daniel Distel for critical comments and Christian Luschnig for many interesting discussions.
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