Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Article (PDF Available)inApplied and Environmental Microbiology 71(4):1694-700 · May 2005with57 Reads
DOI: 10.1128/AEM.71.4.1694-1700.2005 · Source: PubMed
Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2005, p. 1694–1700 Vol. 71, No. 4
0099-2240/05/$08.000 doi:10.1128/AEM.71.4.1694–1700.2005
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Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on
the Gabon Continental Margin (Southeast Atlantic): 16S rRNA
Phylogeny and Distribution of the Symbionts in Gills
Se´bastien Duperron,
Thierry Nadalig,
Jean-Claude Caprais,
Myriam Sibuet,
Aline Fiala-Me´dioni,
Rudolf Amann,
and Nicole Dubilier
IFREMER De´partement Environnement Profond, Centre de Brest, Plouzane´,
and Universite´ Pierre et Marie Curie
(Paris 6), Observatoire Oce´anologique, Banyuls-sur-Mer,
France, and Max Planck Institute
for Marine Microbiology, Bremen, Germany
Received 28 June 2004/Accepted 25 October 2004
Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills
and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed
Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast
Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA
analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis
with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill
epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic
symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or
in the relative abundances of the two symbionts were observed in mussels collected from three different mussel
beds with methane concentrations ranging from 0.7 to 33.7 M. The 16S rRNA sequence of the methanotrophic
symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid
mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophy-
letic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel
species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows
that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a
hydrothermal plume near Japan.
Symbiotic associations with thiotrophic (sulfur-oxidizing)
and methanotrophic (methane-oxidizing) bacteria occur in a
wide array of animal species that live in reducing environments
with high sulfide and methane concentrations, such as hydro-
thermal vents, whale skeletons, sunken wood, and cold seeps
(4, 6, 11, 13, 15, 40). Cold seeps occur worldwide on both active
and passive margins (39), and these ecosystems harbor a high
proportion of invertebrates associated with symbiotic bacteria
(34), including highly specialized annelids (Siboglinidae), as
well as bivalve clams (Thyasiridae, Vesicomyidae, Lucinidae)
and bathymodiolid mussels (Mytilidae).
Mussels of the genus Bathymodiolus are found worldwide in
vents and seeps at depths from 400 to 3,600 m (41). The
bacterial symbionts occur in specialized cells of the gill called
bacteriocytes (13, 15) and have been characterized in about
10 of the 22 known species by using transmission electron
microscopy (TEM), stable isotopes, enzymology, and molecu-
lar analyses (5, 7, 10, 14, 16, 30, 31, 37). Some species, like
Bathymodiolus thermophilus from east Pacific vents, harbor
only thiotrophic bacteria, while others, like Bathymodiolus chil-
dressii from the Gulf of Mexico, have only methanotrophic
symbionts (6). A dual symbiosis, in which a single host harbors
both thiotrophic and methanotrophic bacteria, has been de-
scribed for four species, two from cold seeps in the Gulf of
Mexico (Bathymodiolus brooksii and Bathymodiolus heckerae)
(5, 16) and two from vents along the Mid-Atlantic Ridge (Ba-
thymodiolus azoricus and Bathymodiolus puteoserpentis) (10,
14). The following data have been presented as evidence for
the presence of dual symbionts in these species. Two distinct
morphotypes have been shown to cooccur within the bacterio-
cytes by using TEM (5, 10, 14, 16). Enzyme assays and immu-
nohistochemistry analyses have confirmed the presence of en-
zymes used by thiotrophic and methylotrophic bacteria (5, 14,
16, 30). Stable isotope analyses of gill tissues (5, 7, 16, 37) and
lipid biomarkers (31) indicated that methanotrophy and thio-
trophy are sources of nutrition, with filter feeding a possible
further source, as a functional gut is still present in most
bathymodiolids (29). Phylogenetic evidence for dual symbiosis
so far only exists for B. puteoserpentis from the Mid-Atlantic
Ridge (10). By using comparative 16S rRNA sequence analysis
and fluorescence in situ hybridization (FISH), two distinct
-proteobacterial phylotypes were shown to coexist within the
host bacteriocytes, and the thiotrophic and methanotrophic
symbionts were most closely related to the symbionts of mus-
sels harboring only a single symbiont phylotype (10).
While symbiotic invertebrates obtained from vent and seep
sites in the north Atlantic have been described, symbioses of
vent or seep invertebrates from the south Atlantic have not
* Corresponding author. Mailing address: Max Planck Institute
for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen, Germany.
Phone: 49 (0)4212028932. Fax: 49 (0)4212028580. E-mail: ndubilie
† Present address: Universite´ Louis Pasteur (Strasbourg), Labora-
toire de Dynamique, Evolution et Expression de Ge´nomes de Micro-
organismes, CNRS FRE 2326, Institut de Botanique, Strasbourg,
been described previously. The discovery of a large active
pockmark area (depth, 3,150 m) on the Gabon margin (south-
east Atlantic) (28) provided the opportunity to study a possible
new Bathymodiolus species (R. von Cosel, personal communi-
cation). Mussels up to 175 mm long form dense beds and
dominate the macrofaunal community at this site (3). In this
study, the morphology of symbiotic bacteria in this Bathymo-
diolus sp. was investigated by using TEM, the identities and
phylogenetic relationships of the bacteria were determined by
comparative 16S rRNA analysis, and the distribution of the
bacteria was characterized by FISH. Measurement of methane
and sulfide concentrations within mussel beds prior to speci-
men collection provided an ecological basis for comparing
different habitats.
Sampling and storage. Bathymodiolus sp. individuals were collected with the
remotely operated vehicle VICTOR 6000 during the Biozaire 2 cruise (2001;
IFREMER; sponsored by Total; chief scientist, Myriam Sibuet) to the Congo-
Angola-Gabon margin, close to the Zaire channel (equatorial east Atlantic).
Specimens were collected at a depth of 3,150 m from three mussel beds (mussel
beds M1, M2 and M3) in a pockmark area called Re´gab (05°52.8134S,
009°37.9419E). Mussel beds M2 and M3 were 30 m from each other, while M1
was about 130 m from both M2 and M3. Specimens were collected at the center
of mussel beds M1 to M3 and at the periphery of mussel bed M3. All specimens
were prepared in the following manner. One gill of each mussel was fixed for
transmission electron microscopy (see below). The other gill was divided into two
parts, one of which was immediately frozen in liquid nitrogen and the other of
which was fixed for FISH (see below).
Methane and sulfide measurements. Seawater samples for determination of
methane and sulfide concentrations were collected a few minutes before mussel
samples were collected from the center of each of the three mussel beds (mussel
beds M1, M2, and M3) and at the periphery of mussel bed M3. At each of these
four collection sites, two replicate samples were taken at the bottom of the
mussel bed with a syringe inserted into the bed and positioned 2 to 3 cm above
the seafloor, and two replicate samples were taken just above the top of the
mussel bed, where the mussels were surrounded by seawater. Methane concen-
trations were measured by gas chromatography by using headspace injection
(33), and sulfide concentrations were determined photometrically by the method
described by Fonselius (19).
Transmission electron microscopy. Gill pieces from eight individuals were
fixed in 3% glutaraldehyde in 0.4 M NaCl buffered with 0.1 M cacodylate (pH
7.4) for 2 h and were postfixed in 1% osmium tetroxide for1hinthesame buffer.
Fixed gills were dehydrated in a graded ethanol series and embedded in Araldite.
Semithin sections were stained with toluidine blue. Ultrathin sections were con-
trasted with uranyl acetate and lead citrate and examined with an HITACHI
H-7500 transmission electron microscope.
DNA extraction. DNA was extracted individually from gill tissues of three
mussels, one from each site, by the method described by Zhou et al. (44) by using
proteinase K for cell digestion and a standard chloroform-isoamyl alcohol ex-
traction procedure. DNA was precipitated in isopropanol, washed with ethanol,
resuspended in sterile-filtered water, and stored in aliquots at 20°C.
16S rRNA PCR amplification. Bacterial 16S rRNA was amplified from gill
tissue DNA by using the universal bacterial primers 8F and 1492R (26). The
reaction mixture contained 50 pmol of each primer, 5 g of bovine serum
albumin, 2.5 mol of each deoxynucleoside triphosphate, 1 ExTaq buffer, and
1UofTaq polymerase (TaKaRa, Otsu, Japan), and the volume was adjusted
with sterile water to 50 l. An initial denaturation step (96°C for 5 min) was
followed by 25 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 3 min and
a final elongation step at 72°C for 5 min. PCR bias was minimized by using only
25 amplification cycles (32) and pooling four separate PCRs for each mussel.
Amplified DNA was purified with a QIAquick PCR purification kit (QIAGEN,
Hilden, Germany).
Cloning and sequencing. PCR products of the correct size (1,500 bp) were
cloned with a TOPO-TA kit (Invitrogen, Carlsbad, Calif.). A 16S rRNA clone
library was constructed for each of the three mussels. The insert size of white
Escherichia coli colonies was controlled after lysis of the cells in cracking buffer
(0.1 M NaOH, 10 mM EDTA, 1% sodium dodecyl sulfate [SDS], 10% glycerol)
and analysis of the supernatant by gel electrophoresis. Positive clones were
grown overnight in 1.5 ml of Luria-Bertani medium, and plasmids were prepared
from the pelleted cells with a QIAprep Miniprep kit (QIAGEN). For each of the
three individuals, 51 to 59 clones were sequenced partially (500 bp) in a
variable region of the 16S rRNA (E. coli positions 518 to 1,000). After align-
ment with BioEdit (22), manual correction, elimination of three chimeras by
using ChimeraCheck (8), and visual examination of the alignments, seven rep-
resentative clones were fully sequenced in both directions (Table 1). Sequencing
reactions were performed by using ABI BigDye and an ABI PRISM 3100 genetic
analyzer (Applied Biosystems, Foster City, Calif.).
Phylogenetic reconstruction. Sequences were compared with the database by
using BLAST (1), and highly similar sequences were included in the analysis.
Sequences were aligned with ARB (25) and were manually corrected. Prelimi-
nary analyses of the seven full sequences were performed by distance and par-
simony methods by using the PHYLIP package (12) to select an appropriate data
set (choice and number of sequences and positions). For the selected data set 36
sequences were used, but only 23 of these sequences are shown in Fig. 2 (the
accession numbers of the sequences not shown in Fig. 2 are AF035728,
AF035723, AF035727, AF035721, AB044744, X72767, AY029915, L34955,
U77481, U62131, L40809, AF069959, and U11021). The 36 sequences were
analyzed by the maximum-likelihood method with TREEFINDER (23) by using
a general time reversible model and an eight-category discrete approximation of
a distribution (estimated , 0.209) to account for among-site evolutionary rate
heterogeneity. Maximum-likelihood bootstrap values were obtained from 1,000
replicates analyzed by the same method.
FISH. Two mussels from each collection site (center of mussel beds M1, M2,
and M3 and periphery of mussel bed M3) were fixed for FISH in 2% formalde-
hyde in sterile seawater at 4°C for 2 h. After two washes in 1 phosphate-
buffered saline (10 mM sodium phosphate, 130 mM NaCl), samples were stored
at 20°C in 0.5 phosphate-buffered saline–50% ethanol (1:1). Fixed gill frag-
ments were dehydrated in an increasing ethanol series and xylene before they
were embedded in paraffin. Transverse sections (thickness, 4 m) were cut with
an RM 2165 microtome (Leica, Wetzlar, Germany) and collected on Superfrost
slides (Fisher, Pittsburgh, Pa.) coated with 3-aminopropyltriethyloxysilane. Par-
affin was removed from the sections with xylene (three 10-min treatments), and
the sections were rehydrated in a decreasing ethanol series, permeabilized for 10
min in 0.2 M HCl, rinsed for 10 min in 20 mM Tris buffer (Tris-HCl, pH 8),
permeabilized again for 5 min at 37°C in Tris buffer containing 0.5 gof
proteinase K ml
, and rinsed for 10 min in 20 mM Tris buffer. After air drying,
the sections were circled with a Pap Pen (G. Kisker, Mu¨hlhausen, Germany).
Hybridization was performed in preheated chambers containing tissue wetted
with hybridization buffer (0.9 M NaCl, 0.02 M Tris-HCl, 0.01% SDS, 30%
formamide). The circled sections were covered with a mixture containing 100 ng
of probe in 30 l of hybridization buffer and incubated for3hat46°C. The slides
were washed in a buffer (0.1 M NaCl, 0.02 M Tris-HCl, 0.0001% SDS, 5 mM
EDTA) at 48°C for 15 min, rinsed with MilliQ water, air dried, and covered with
TABLE 1. Numbers of partial sequences (from nucleotide 518 to nucleotide 1,000 based on E. coli numbering) and nearly full sequences
(1,500 nucleotides) obtained from cloned PCR products amplified from gill DNA of three Bathymodiolus sp. specimens
Specimen source
No. of partial sequences No. of full sequences
Thiotrophs Methanotrophs Chimeras Total Thiotrophs Methanotrophs Total
M1 40 19 0 59 1 2 3
M2 57 0 1 58 2 0 2
M3 39 10 2 51 1 1 2
Total 136 29 3 168 4 3 7
VectaShield (Vector, Burlingame, Calif.) and a coverslip. A total of 43 gill
sections obtained from eight mussels collected from the four different sites
(Table 2) were visually examined for differences in relative symbiont abundance
by using an LSM 510 confocal microscope (Zeiss, Jena, Germany).
A specific probe was designed for each of the two 16S rRNA phylotypes found
in Bathymodiolus sp. by using the PROBE_DESIGN function of ARB, and the
specificity was checked by using BLAST. The probe specific for the methanotro-
phic symbiont, BangM-138 (5-ACCAGGTTGTCCCCCACTAA-3), was la-
beled with Cy3 (Thermo, Waltham, Mass.), and the probe specific for the thio-
trophic symbiont, BangT-642 (5-CCTATACTCTAGCTTGCCAG-3), was
labeled with Cy5 (Biomers.net, Ulm, Germany). Gills of the dual-symbiont
mussel B. azoricus were used to test the specificity, as the thiotrophic and
methanotrophic symbionts of this mussel have a 1-bp mismatch in the 16S rRNA
region targeted by the probes designed for this study. Both the BangM-138 probe
and the BangT-642 probe were highly specific with 30% formamide, as no signal
was observed in B. azoricus gill sections, while a strong signal was visible in
Bathymodiolus sp. gill sections. The general Bacteria probe EUB338 (2) was used
as a positive control, and the NON338 (42) probe was used as a negative control
(both were labeled with Cy3).
Nucleotide sequence accession numbers. The EMBL accession numbers for
the two sequences described in this paper are AJ745717 (methanotrophic sym-
biont) and AJ745718 (thiotrophic symbiont).
Methane and sulfide concentrations in the mussel beds. The
methane concentrations measured at the bottom of mussel
beds M2 and M3 close to the seafloor were in the same range
(33.7 and 23.7 M, respectively) and decreased at the top of
the beds by 30% at mussel bed M2 and by 47% at mussel bed
M3 (Table 2). At the bottom of the M1 mussel bed, the meth-
ane concentrations were 33- to 48-fold lower (0.7 M) than
they were at the bottom of the two other beds, and the con-
centration increased slightly at the top of mussel bed M1 to 1.6
M. At the periphery of the M3 mussel bed, the methane
concentrations both at the bottom and at the top of the bed
were 50% lower than the concentration at the center of this
mussel bed (Table 2). The sulfide concentrations were below
the detection limit (1 M) at all sampling sites. No obvious
differences in mussel biomass were observed for mussel beds
M1, M2, and M3.
Transmission electron microscopy. Bacteria were abundant
in the apical half of the gill bacteriocytes, and numerous
phagolysosome-like bodies occurred in the basal region of the
cells (Fig. 1A). Two distinct bacterial morphotypes coexisted
within each bacteriocyte (Fig. 1B). The smaller morphotype
(0.42 0.07 by 0.33 0.06 m) was a rod-shaped or coccoid
bacterium, and the larger morphotype (1.23 0.16 by 1.07
0.10 m) was coccoid with stacked membranes in its cyto-
plasm. In almost all bacteriocytes examined, the smaller mor-
FIG. 1. Transmission electron micrographs of Bathymodiolus sp. gill sections with endosymbiotic bacteria. (A) Transverse section showing an
overview of a bacteriocyte (plb, phagolysosome-like bodies). Scale bar 10 m. (Inset) Large morphotype with stacked internal membranes
(arrow) and a dividing stage of the small morphotype (arrowhead). Scale bar 0.5 m. (B) Apical part of a bacteriocyte showing the distinct
distribution of the two morphotypes. The smaller morphotype (sm) occupies the apical part of the bacteriocyte toward the mantle fluid, while the
larger morphotype (lm) is located more basally. Scale bar 1 m.
TABLE 2. Methane and sulfide concentrations in the water
collected at the bottom and top of three mussel beds and numbers
of individuals and gill filament sections examined at each
sampling site by fluorescence in situ hybridization
Mussel bed
Concn (M) of
No. of
no. of
M1 (center) 0.7 1.6 1 12 10
M2 (center) 33.7 23.2 1 12 5
M3 (center) 23.7 12.5 1 12 4
M3 (periphery) 11.8 6.3 1 12 24
The detection limit for methane was 50 nM, and the detection limit for
sulfide was 1 M.
photype was most abundant in the apical region of the cell
(Fig. 1B), while the larger morphotype occurred more basally.
16S rRNA phylogeny of the symbionts. Comparative 16S
rRNA sequence analysis of 165 clones from three Bathymo-
diolus sp. individuals revealed two distinct bacterial phylotypes
(Table 1). The sequence variation within each phylotype was
very low (0 to 0.2%). Phylogenetic trees constructed from a
variety of data sets and by using a variety of methods displayed
topologies almost identical to that of the maximum-likelihood
tree shown in Fig. 2. Both phylotypes clustered in the -Pro-
teobacteria subdivision. One phylotype belongs to a monophy-
letic group that includes all known sequences of methanotro-
phic symbionts associated with bathymodiolid hosts (from the
Gulf of Mexico, the Mid-Atlantic Ridge, and Japan). Its closest
relative (97.8% identity) is the methanotrophic symbiont of
Bathymodiolus japonicus from vents in the Okinawa Trough,
near Japan (21).
The second Bathymodiolus sp. phylotype belongs to a large
clade that includes thiotrophic symbionts associated with bi-
valves belonging to three families (Vesicomyidae, Thyasiridae,
and Mytilidae), as well as three environmental sequences,
clones ZA2525c and ZA2329c from an upwelling zone off the
coast of Namibia and clone SUP05 from the Suiyo Seamount
hydrothermal plume, south of Japan (36). The second Bathy-
modiolus sp. phylotype is most closely related to the SUP05
clone (99.3% identity). Among symbiotic bacteria, its sequence
is more similar to the sequence of symbiont I from the thyasirid
Maorithyas hadalis (97.8%) (20) than to any of the four previ-
ously published sequences for thiotrophic symbionts of bathy-
modiolid mussels (97.4%). As determined by all three tree-
ing methods, this sequence never fell within the well-supported
monophyletic group of sequences from thiotrophic mussel
symbionts (maximum-likelihood bootstrap value, 97.0). In-
stead, it formed a weakly supported monophyletic group with
clone SUP05, M. hadalis symbiont I, and the symbionts of vesi-
comyids (maximum-likelihood bootstrap value, 60). There-
fore, except for its closest relative, clone SUP05, the exact phy-
logenetic position of the second Bathymodiolus sp. phylotype
remains unclear.
FISH. Hybridization with gill tissue sections by using specific
probes confirmed the coexistence of the two symbionts in the
gill bacteriocytes (Fig. 3). The probe specific for the meth-
anotroph-related sequence (BangM-138) hybridized to large
coccoid bacteria assumed to be the large morphotype observed
by TEM. The probe specific for the thiotroph-related sequence
(BangT-642) hybridized to bacteria similar in size and shape to
the smaller TEM morphotype.
A nonquantitative, visual comparison of gill sections from
two individuals collected from each of the mussel beds (center
of mussel beds M1, M2, and M3 and periphery of mussel bed
M3) revealed a distinct distribution of the two symbiont phy-
lotypes in most sections (34 of 43 sections examined) (Table 1).
The methanotroph-related symbionts occurred in the more
basal region of the bacteriocytes, while the thiotroph-related
FIG. 2. Phylogenetic relationships, based on maximum-likelihood analyses of 16S rRNA sequences, of the thiotrophic and methanotrophic
endosymbionts of Bathymodiolus sp. (boldface type) in the -Proteobacteria (1,266 sites analyzed; L ⫽⫺8,744). Two -proteobacterial species were
used as an outgroup (only A. tumefaciens is shown). Bootstrap percentages were obtained by using 1,000 maximum-likelihood replicates, and values
greater than 60% are indicated at the nodes. Scale bar 10% estimated base substitutions.
symbionts consistently occupied the apical end of the bacterio-
cytes exposed to the environment (Fig. 3B). The most apical
part of the bacteriocytes (width, 1 to 3 m) was often almost
exclusively occupied by the thiotroph-related symbionts. No
obvious differences in the distribution and relative amounts of
the thiotroph- and methanotroph-related symbionts were ob-
served among gill sections of Bathymodiolus sp. collected from
mussel beds M1 to M3.
The Bathymodiolus sp. mussels studied here from the Gabon
margin harbor two morphologically distinct bacteria that re-
semble the methanotrophic (5, 14, 16) and thiotrophic (13, 17)
symbionts of other Bathymodiolus species. Comparative 16S
rRNA sequence analysis and FISH confirmed that Bathymo-
diolus sp. lives in a dual symbiosis with bacteria related to
methane- and sulfide-oxidizing symbionts.
As expected, the Bathymodiolus sp. methanotroph 16S rRNA
sequence falls within the monophyletic clade that includes all
known methanotrophic symbionts of bathymodiolid mussels
(9, 10, 21). In contrast, the phylogenetic position of the Bathy-
modiolus sp. thiotroph 16S rRNA sequence is surprising, be-
cause the sequence does not fall in the monophyletic group of
thiotrophic symbionts from all other Bathymodiolus hosts (9,
10, 21). This monophyletic group consists exclusively of thio-
trophic symbionts from hydrothermal vent mussels (from the
Mid-Atlantic Ridge, the East Pacific Rise, and the western
Pacific), while the thiotroph sequence obtained in this study is
the first thiotroph sequence from a cold-seep mussel. A further
unexpected result is the close relationship of the Bathymodio-
lus sp. thiotrophic symbiont to the environmental clone se-
quence SUP05 (36) and the placement of two environmental
clone sequences obtained from surface waters off the Nami-
bian coast (accession no. AF382104 and AF382101) (Fig. 2) in
the Bathymodiolus-Vesicomya/Calyptogena clade. In all previ-
ous analyses this clade consisted exclusively of clam and mussel
symbionts (9, 10, 20, 21).
One explanation for the lack of monophyly for vent and seep
mussel thiotrophs is that the symbioses may have evolved in-
dependently of each other. It is intriguing that in a recent study
of vent mussels from the Mid-Atlantic Ridge acquisition of the
thiotrophic symbiont from the environment was suggested
(43). Uptake of a free-living bacterial species from the envi-
ronment is one way in which these associations could have
developed independently during convergent evolution. How-
ever, environmental symbiont transmission is not necessarily a
good indication of convergent evolution as some symbioses in
which environmental transmission occurs have clearly been
established through cospeciation, like the symbiosis between
luminescent Vibrio bacteria and squid (27).
FIG. 3. Fluorescence in situ hybridization images of Bathymodiolus sp. symbionts in gill filaments. The thiotrophic symbionts (red) occupy the
apical region of the bacteriocytes, while the methanotrophic symbionts (green) are located more basally. (A) Scale bar 10 m. (B) Scale bar
5 m.
An alternative explanation for the lack of monophyly for the
thiotrophic symbionts of Bathymodiolus sp. and the thiotrophic
symbionts of vent mussels is that there is indeed a sister group
relationship between them, but sequences that could allow
resolution of this relationship are not yet known. In this case a
single acquisition event would have occurred, with consequent
speciation leading to separation of vent and seep mussels,
followed by diversification in the two environments. Clearly,
sequences from additional mussel species and related free-
living bacteria are needed to fully resolve the evolutionary
history of Bathymodiolus symbioses.
Ecology of the Bathymodiolus sp. symbiosis. Sulfide and
methane gradients over time and space are assumed to play a
major role in determining the distribution, biomass, and pro-
ductivity of symbiotic invertebrates at vents and seeps (18, 34,
35). However, little is known about how variations in these
energy sources affect the nutrition of hosts living in dual sym-
bioses with sulfide- and methane-oxidizing bacteria. Previous
studies of Mid-Atlantic Ridge mussels with dual symbionts
indicated that there is a nutritional response to fluid gradients,
with an increase in the relative amounts of sulfide oxidizers and
reliance on thiotrophy in mussels from sites with higher sulfide
concentrations and, correspondingly, an increase in the rela-
tive amounts of methane oxidizers and reliance on methanot-
rophy in mussels exposed to higher methane concentrations (7,
37). In this study, no variations in the relative amounts of
thiotrophic and methanotrophic symbionts were observed in
Bathymodiolus sp., despite a nearly 50-fold difference in meth-
ane concentrations between sample sites. One explanation for
this unexpected result is that even at the lowest concentration
(0.7 M), methane may not be limiting for the growth of the
methanotrophic symbionts. Alternatively, the nonquantitive
methods used here may not have been sufficient to recognize
small differences in relative symbiont amounts. A further im-
portant consideration is that the snapshot quality of the one-
time methane and sulfide measurements may not reflect the
average concentrations over longer times, as some studies have
indicated that seepage fluxes can vary greatly not only over
space but also over time (24, 34, 38).
The site-independent distribution of the Bathymodiolus sp.
symbionts within each bacteriocyte, with the thiotrophs occu-
pying a more apical position than the methanotrophs, has not
been described previously. The closer proximity of the thiotro-
phic symbionts to the apical edge of the bacteriocytes suggests
that these bacteria are more dependent on exchange with the
mantle fluids that contain seawater from the environment.
Intriguingly, the sulfide concentrations in the seawater at the
collection site were much lower than the methane concentra-
tions. Thus, low sulfide concentrations could limit the distri-
bution of the thiotrophs to the regions closest to the circulating
mantle fluids, where sulfide is more readily available. Corre-
spondingly, the methanotrophs are able to inhabit a more basal
region of the bacteriocytes, because diffusive loss of methane
through the bacteriocytes is compensated for by higher meth-
ane concentrations. While FISH has not been used previously
to obtain a general overview of symbiont distribution, ultra-
structural analyses of other Bathymodiolus species have re-
vealed a more regular distribution of methanotrophs and thio-
trophs throughout the bacteriocyte (10, 14, 16, 43).
This study shows the importance of examining physicochem-
ical parameters, such as sulfide and methane concentrations, at
spatial and temporal scales relevant to the organisms living in
cold seeps. Time series measurements of gradients of these
parameters and correlation with symbiont distribution, relative
amounts, and biomass are needed to obtain a better under-
standing of the influence of the environment on the relation-
ships established in nature between hosts and their symbiotic
We thank the pilots and crew of the N/O L’Atalante and the ROV
Victor 6000 for efficient cooperation during the Biozaire 2 cruise (2001;
IFREMER; chief scientist, Myriam Sibuet). We thank Karine Olu,
Alexis Fifis, and Ann Andersen for the maps of macrofaunal commu-
nities, for on-board dissections, and for sample preparation, and R.
Von Cosel is acknowledged for providing unpublished taxonomic re-
sults. The technical assistance of B. Rivie`re and D. Saint-Hilaire with
microscopy and sectioning is gratefully acknowledged. We also thank
Claudia Bergin, Anna Blazejak, and Silke Wetzel for great technical
assistance with molecular methods.
We thank the oil and gas company Total for sponsoring the Biozaire
project. S.D. is a student in the International Max Planck Research
School of Marine Microbiology Ph.D. program, and his grant is co-
funded by IFREMER and MPI. This work was supported by the Max
Planck Society, IFREMER, University Pierre-et-Marie Curie, and
CNRS (UMR 7621).
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    • "Hybridizations were performed according to Duperron et al. (2005) using buffers containing 30 and 40% formamide, and probes were labelled with Cy-3 or Cy-5. Slides were counterstained with DAPI, and observed under an Olympus BX61 epifluorescence microscope (Olympus, Japan). "
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