RESEARCH ARTICLE
Chemosynthetic bacteria found in bivalve species from
mud volcanoes of the Gulf of Cadiz
Clara F. Rodrigues1,2, Gordon Webster3, Marina R. Cunha1, Sébastien Duperron2 &
Andrew J. Weightman3
1
CESAM and Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, Aveiro, Portugal;
Université Pierre-et-Marie-Curie, UMR 7138 (UPMC CNRS IRD MNHN), Systématique, Adaptation, Evolution, Paris, France; and
3
Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK
2
Correspondence: Clara F. Rodrigues,
Université Pierre-et-Marie-Curie, UMR 7138
(UPMC CNRS IRD MNHN), Systématique,
Adaptation, Evolution, 7, quai St Bernard,
bâtiment A, 75005 Paris, France.
Tel.: 133 1 44 27 39 95;
fax: 133 1 44 27 58 01;
e-mail: [email protected]
Received 15 May 2009; revised 10 May 2010;
accepted 11 May 2010.
Final version published online 9 June 2010.
MICROBIOLOGY ECOLOGY
DOI:10.1111/j.1574-6941.2010.00913.x
Editor: Michael Wagner
Keywords
symbiosis; nutrition; bivalves; mud volcanoes;
bacterial 16S rRNA genes.
Abstract
As in other cold seeps, the dominant bivalves in mud volcanoes (MV) from the
Gulf of Cadiz are macrofauna belonging to the families Solemyidae (Acharax sp.,
Petrasma sp.), Lucinidae (Lucinoma sp.), Thyasiridae (Thyasira vulcolutre) and
Mytilidae (Bathymodiolus mauritanicus). The d13C values measured in solemyid,
lucinid and thyasirid specimens support the hypothesis of thiotrophic nutrition,
whereas isotopic signatures of B. mauritanicus suggest methanotrophic nutrition.
The indication by stable isotope analysis that chemosynthetic bacteria make a
substantial contribution to the nutrition of the bivalves led us to investigate their
associated bacteria and their phylogenetic relationships based on comparative 16S
rRNA gene sequence analysis. PCR-denaturing gradient gel electrophoresis analysis and cloning of bacterial 16S rRNA-encoding genes confirmed the presence of
sulfide-oxidizing symbionts within gill tissues of many of the studied specimens.
Phylogenetic analysis of bacterial 16S rRNA gene sequences demonstrated that
most bacteria were related to known sulfide-oxidizing endosymbionts found in
other deep-sea chemosynthetic environments, with the co-occurrence of methaneoxidizing symbionts in Bathymodiolus specimens. This study confirms the
presence of several chemosynthetic bivalves in the Gulf of Cadiz and further
highlights the importance of sulfide- and methane-oxidizing symbionts in the
trophic ecology of macrobenthic communities in MV.
Introduction
Symbiotic relationships between bacteria and marine invertebrates in deep-sea environments derive all or part of their
nutrition from symbiont metabolism (Fisher, 1990; Cavanaugh, 1994; Lee et al., 1999; Petersen & Dubilier, 2009).
Symbiotic associations with thiotrophic (sulfur- and sulfideoxidizing) and methanotrophic (methane-oxidizing) bacteria occur in a wide range of animal species that live in
reducing environments, such as hydrothermal vents, whale
falls, sunken wood, cold seeps and sediments (Cavanaugh
et al., 2006; Duperron et al., 2009). Sulfur-based symbioses
are by far the most commonly reported within these systems
(Distel et al., 1988; Imhoff et al., 2003; Stewart et al., 2005).
In particular, bivalves with sulfide-oxidizing gill symbionts
are frequently found in environments that are inhospitable
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to other invertebrates due to a low oxygen (O2) content and
the presence of free hydrogen sulfide (H2S) (reviewed in
Southward, 1986). Sulfide-oxidizing bacteria are involved in
symbioses with members of at least five bivalve families,
occurring intracellularly in Vesicomyidae, Lucinidae, Mytilidae and Solemyidae, and mostly extracellularly in Thyasiridae. Bivalve symbioses have been studied using molecular
methods and sequences of many host species and their
associated symbionts have been published (e.g. Distel et al.,
1988; Eisen et al., 1992; Imhoff et al., 2003; Duperron et al.,
2007a, b). Bivalve symbionts belong to several related clades
within the Gammaproteobacteria (Dubilier et al., 2008).
Besides sulfide oxidizers, some members of the Mytilidae
have methane-oxidizing gammaproteobacterial symbionts,
and others have multiple symbioses with both sulfide- and
methane-oxidizing bacteria within their gill tissues (Fisher,
FEMS Microbiol Ecol 73 (2010) 486–499
487
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
1990; Cavanaugh et al., 1992; Duperron et al., 2008).
Recently, possible symbionts belonging to other bacterial
phyla such as Spirochaetes (Duperron et al., 2007a) and
Bacteroidetes (Duperron et al., 2008) have also been
reported.
Sulfide- and methane-oxidizing bacterial symbionts play
similar roles in the symbiosis by providing their hosts with a
substantial fraction of their nutritional carbon and energy
needs by utilizing sources otherwise unavailable to metazoans. The hosts, in turn, confer the symbionts with simultaneous access to necessary substrates, notably O2 and H2S
and/or CH4, from oxic and anoxic environments, respectively (Cavanaugh, 1994). This provides the bacteria with a
stable environment and helps to buffer them against
the temporal and spatial variability of electron donors
and acceptors characteristic of a dynamic vent and seep
environment.
The Gulf of Cadiz has a strategic central location in
relation to other known seeps (Nordic margin, Western
Mediterranean, Gulf of Guinea) and vents (Mid-Atlantic
Ridge), and is located west of the Gibraltar Arc, between
Iberia and the African plate (Fig. 1). This area has experienced a complex tectonic history with several episodes of
extension, strike–slip and compression related to the closure
of the Tethys Ocean, the opening of the North Atlantic and
the African–Eurasian convergence since the Cenozoic (Maldonado et al., 1999). Because of this ongoing compression,
rapidly deposited sediments intensely dewater and form mud
volcanoes (MV) and other fluid escape structures (Dı́az-delRı́o et al., 2003) releasing fluids rich in hydrocarbons. Since
the first discovery of MV in the Gulf of Cadiz in 1999 (Baraza
et al., 1999), it has become well established that the whole
area is under compressive deformation (Pinheiro et al., 2003)
and that mud volcanism and processes associated with the
escape of hydrocarbon-rich fluids can sustain a broad
diversity of chemosynthetic assemblages (Niemann et al.,
2006; Rodrigues, 2009). Compared with other marine gas
seepage and methane-rich environments, MV from the Gulf
of Cadiz show a low or a medium range in the methane
turnover rates, reflecting relatively mild fluxes of methane
and sulfide (Niemann et al., 2006). However, gas hydrates
have been collected at a number of MV that are associated
with major tectonic faults and these are considered to be the
most active in the Gulf of Cadiz region (Magalhães, 2007).
The geochemistry of such fluids is highly variable at different
MV and even at different locations of a single MV, implying
that fluids are transported from different depths/sources
through faults that altogether constitute a feeder system
(Stadnitskaia et al., 2006; Hensen et al., 2007). Analysis of
the porewaters of many sediment samples suggests that their
chemical composition consists of a mixture of seawater,
deeper-sourced MV fluid and freshwater from the decomposed gas hydrates (Mazurenko et al., 2002).
FEMS Microbiol Ecol 73 (2010) 486–499
The dominant bivalves, so far, identified at MV in the
Gulf of Cadiz are large chemosymbiotic species belonging to
the families Solemyidae (Acharax sp. and Petrasma sp.),
Lucinidae (Lucinoma sp.; Rodrigues, 2009), Thyasiridae
(Thyasira vulcolutre; Rodrigues et al., 2008) and Mytilidae
(Bathymodiolus mauritanicus; Génio et al., 2008). In this
study, the stable isotopic signature (13C, 15N and 34S) of a
number of the bivalves collected from these Gulf of Cadiz
MV was determined to provide information on the nature of
their nutritional source. The indication that the activities of
autotrophic and methanotrophic bacteria make a substantial contribution to the nutrition of these bivalves led us to
examine the diversity of their bacterial symbionts and
further investigate the phylogenetic relationship with other
bivalve endosymbionts based on a comparative analysis of
16S rRNA genes.
Materials and methods
Study sites and bivalve collection
Bivalve species belonging to the families Solemyidae, Lucinidae, Thyasiridae and Mytilidae have been found at a number
of MV in the Gulf of Cadiz (Fig. 1). In summary, Petrasma
sp. have mostly been found in shallower MV (Mercator MV,
Gemini MV, Kidd MV, Yuma MV, Ginsburg MV and Darwin
MV) from 358 to 1105 m depth, while Acharax sp. have a
deeper bathymetric range (960–3902 m, occurring in Yuma
MV, Ginsburg MV, Jesus Baraza MV, Captain Arutyunov
MV, Carlos Ribeiro MV and Porto MV; Rodrigues, 2009).
Both these species of solemyids are found buried deep in the
sediment. In contrast, live specimens of Lucinoma sp.
(Lucinidae) and B. mauritanicus (Mytilidae) have only been
recorded at single MV (C.F. Rodrigues, unpublished data).
For instance, Lucinoma sp. were identified at the crater of
Mercator MV (358 m) where active methane bubbling was
observed and B. mauritanicus in the fissures of carbonate
slabs covering the crater at Darwin MV (1115 m). Thyasira
vulcolutre (Thyasiridae) was recorded in the craters of
Captain Aruryunov MV (1320 m, where it reached high
densities) and Carlos Ribeiro MV (2200 m) (Génio et al.,
2008; Rodrigues et al., 2008).
In this study, specimens were collected using a
TV-assisted grab or a USNEL box-corer from seven MV
located in the Gulf of Cadiz (Fig. 1): Mercator MV, Gemini
MV (El Arraiche field; Van Rensbergen et al., 2005), Darwin
MV, Ginsburg MV, Meknès MV, Yuma MV (Western
Moroccan field) and Carlos Ribeiro MV (Deep water field)
during several TTR cruises (IOC-UNESCO) and the Microsystems 2007 (NIOZ) cruise onboard the RV Prof. Logachev
and the RV Pelagia, respectively (Table 1, also listing grid
references of sampling sites). Bivalve species were examined
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488
C.F. Rodrigues et al.
(a)
(b)
Fig. 1. Gulf of Cadiz sampling sites showing (a) the distribution and (b) morphology of Acharax sp. (1), Petrasma sp. (2), Lucinoma sp. (3), Thyasira
vulcolutre (4) and Bathymodiolus mauritanicus (5). Indicates bivalve specimens collected for analysis. CA, Captain Arutyunov; CR, Carlos Ribeiro; Dar,
Darwin; Fiu, Fiuza; Gem, Gemini; Gin, Ginsburg; JB, Jesus Baraza; Kid, Kidd; Mek, Meknès; Mer, Mercator; Por, Porto; Yum, Yuma. Photographs by Dr
Graham Oliver (National Museum of Wales, Cardiff, UK).
and species were determined with the aid of the taxonomic
expert Graham Oliver (National Museum of Wales, Cardiff,
UK). One to three specimens of each species from different
MV were dissected and prepared separately for DNA extrac2010 Federation of European Microbiological Societies
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tion. The soft tissues (gills and foot) were removed from the
shells, dissected, washed three times in sterile water and
stored 80 1C for symbiont characterization studies and
stable isotope measurements.
FEMS Microbiol Ecol 73 (2010) 486–499
489
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
Table 1. Summary of the bivalve species collected and type of chemosynthetic bacteria associated with gill tissues of bivalves from mud volcanoes from
the Gulf of Cadiz
Mud volcano
(MV)
Water depth
(m)
Bivalve species
(family)
Carlos Ribeiro MV
Darwin MVw
2200
1115
Thyasira vulcolutre (Thyasiridae)
Bathymodiolus mauritanicus
(Mytilidae)
Petrasma sp. (Solemyidae)
Acharax sp. (Solemyidae)
Petrasma sp. (Solemyidae)
Lucinoma sp. (Lucinidae)
Petrasma sp. (Solemyidae)
Acharax sp. (Solemyidae)
Petrasma sp. (Solemyidae)
Gemini MVz
Ginsburg MV‰
Meknès MVz
Mercator MVk
Yuma MV
418
983
701
358
1030
Number of
specimens
collected
Type of chemosynthetic
process suggested by
stable isotope analysis
Putative endosymbionts
detected by analysis of
bacterial 16S rRNA genes
2
3
Thiotrophy
Methanotrophy
2
1
3
1
3
3
2
Thiotrophy
Thiotrophy
Thiotrophy
Thiotrophy
Thiotrophy
Thiotrophy
Thiotrophy
Unknown
Thiotrophic and
methanotrophic
Thiotrophic
Thiotrophic
Unknown
Thiotrophic
Thiotrophic
Thiotrophic
Thiotrophic
Cruise TTR16 – station AT615Gr (35147.238N, 8125.272W).
w
Cruise TTR16 – station AT608Gr (35123.531N, 7111.475W).
Cruise Microsystems 2007 station M2007_10 (35116.900N, 6145.350W).
‰
Cruise TTR16 – station AT607Gr (35122.677N, 714.979W).
z
Cruise TTR15 – station AT586Gr (34159.146N, 714.380W).
k
Cruise TTR15 – station AT569Gr 35117.917N, 6138.717W).
Cruise TTR16 – station AT604Gr (35125.820N, 716.330W).
z
Stable isotope analysis
In the laboratory, samples were lyophilized and homogenized in a mortar and pestle, and then separated into
batches for 13C, 15N and 34S analyses. The ground sample
for carbon analysis was acidified with HCl (1 M) until no
further bubbling occurred; the sample was then resuspended
in distilled water, centrifuged and the supernatant was
discarded (this procedure was repeated three times); finally,
the sample was dried at 60 1C. The ground sample for sulfur
analysis was resuspended in distilled water, shaken for
5 min., centrifuged and the supernatant was discarded; this
procedure was repeated and the sample was dried at 60 1C.
All samples were analyzed at ISO-Analytical Laboratory
(Cheshire, UK) using the elemental analysis-isotope ratio
MS method.
The isotope compositions are reported relative to standard material and follow the same procedure for all stable
isotopic measurements as follows:
h
i
dx E ¼ ðx E=y EÞsample =ðx E=y EÞstandard 1 100
ð1Þ
where E is the element analyzed (C, N or S), x is the
molecular weight of the heavier isotope and y the lighter
isotope (x = 13, 15, 34 and y = 12, 14 and 32 for C, N and S,
respectively). The standard materials with which the samples
are compared are PDB (Pee Dee Belemnite) for carbon; air
N2 for nitrogen; and CDT (Canon Diablo toilite) for sulfur.
DNA extraction
DNA was extracted from freeze-dried gill tissue of bivalves
using the DNeasys Blood and Tissue kit (Qiagen) using the
FEMS Microbiol Ecol 73 (2010) 486–499
manufacturer’s protocol, with some modifications. In summary, to facilitate DNA extraction, the tissue was initially
placed in a FastPrep Lysing Matrix Tubes E (MP Biomedicals) and homogenized for 40 s at speed 6 using a FastPrep
instrument (QBiogene) in lysis buffer ATL and proteinase
qK supplied with the DNeasys Blood and Tissue kit
(Qiagen). Subsequently, samples were then incubated at
56 1C until the tissue was completely lysed and the remaining procedure was followed as recommended by the manufacturer. DNA extracts were visualized by standard agarose
gel electrophoresis, and the DNA was quantified against the
Hyperladder I DNA marker (Bioline) using the Gene Genus
Imaging System (Syngene). It should be noted that before
the above DNA extraction procedure was performed, preliminary experiments carried out on ethanol-preserved
tissue were not successful. This was thought to be due to
DNA degradation during ethanol storage as reported previously for mammalian tissues (Kilpatrick, 2002).
PCR amplification of bacterial 16S rRNA genes
Bacterial 16S rRNA genes were amplified from tissue
DNA samples using the primer combinations 27F-1492R
(Lane, 1991) under the following PCR conditions. PCR
mixtures contained (total 50 mL, molecular-grade water)
0.4 pmol mL1 of primers, 1 mL of tissue DNA template,
1 reaction buffer (Bioline), 1.5 mM MgCl2, 1.5 U BioTaq
DNA polymerase (Bioline), 0.25 mM each dNTP and 10 mg
bovine serum albumin (BSA). Reaction mixtures were held
at 95 1C for 2 min, followed by 30 cycles of 94 1C for 30 s,
52 1C for 30 s and 72 1C for 90 s plus 1 s per cycle, with a final
extension step of 5 min at 72 1C. PCR products analyzed by
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490
denaturing gradient gel electrophoresis (DGGE) were then
reamplified in a nested PCR reaction using the reaction
mixture described above without BSA. This reamplification
step was added after initial observations that some samples
did not amplify by direct PCR and products were only
observed after nested PCR. Therefore, for consistency, all
samples were amplified by nested PCR before DGGE, which
had the advantage that the same PCR products that could be
cloned were also screened by PCR-DGGE. Bacterial 16S
rRNA genes were reamplified with the primers 357FGC518R (Muyzer et al., 1993) at 95 1C for 5 min, followed by 10
cycles of 94 1C for 30 s, 55 1C for 30 s, 72 1C for 60 s, and 20
cycles of 92 1C for 30 s, 52 1C for 30 s and 72 1C for 60 s, with
a final step of 10 min at 72 1C.
DGGE analysis
DGGE was performed using a DCodeTM Universal Mutation
Detection System (Bio-Rad Laboratories). The PCR samples
were loaded onto 8% w/v polyacrylamide (37.5 : 1 acrylamide : bisacrylamide) gels in a 1 Tris–acetate–EDTA (TAE)
buffer with a denaturing gradient ranging from 30% to 60%
(denaturation of 100% corresponds to 7 M urea and 40% v/v
deionized formamide). Electrophoresis was run for 10 min
at 80 V and then 290 min at 200 V in 1 TAE buffer at 60 1C
(Webster et al., 2003, 2006). The gel was stained with SYBRGold (Molecular Probes). Dominant DGGE bands were
excised and sequenced with the 518R primer, and partial
bacterial 16S rRNA gene sequences using NCBI BLAST (http://
www.ncbi.nlm.nih.gov) to identify sequences with highest
sequence identity.
Bacterial 16S rRNA gene libraries
Only products assessed by DGGE and without a PCR
product in the negative controls were cloned. Each 16S
rRNA gene library was constructed from five independent
PCR products that were pooled and cleaned using the
Wizard PCR Preps DNA Purification System (Promega)
according to the manufacturer’s instructions. Cloning was
with pGEM-T Easy (Promega) according to the manufacturer’s instructions, using optimized insert : vector ratios
and overnight ligation at 4 1C. Libraries were screened by
PCR with M13 primers. All 16S rRNA gene clones with
verified inserts were randomly selected and sequenced using
an ABI 3130xl 16 capillary Genetic Analyzer.
Phylogenetic analysis
Sequence chromatographs were analyzed using the CHROMAS
software package version 1.45 (http://www.technelysium.
com.au/chromas.html). Partial sequences were checked for
chimeras with CHIMERA CHECK from the Ribosomal Database
Project II (http://rdp.cme.msu.edu/), and their closest rela2010 Federation of European Microbiological Societies
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C.F. Rodrigues et al.
tives were identified by NCBI BLAST (http://www.ncbi.nlm.
nih.gov). All nucleotide sequences were aligned using CLUSTALX (Thompson et al., 1997) with sequences retrieved from
the database. Alignments were edited manually using BIOEDIT
SEQUENCE ALIGNMENT EDITOR version 5.0.9 (Hall, 1999) and
regions of ambiguous alignment were removed. The phylogenetic relationships between pairs of 16S rRNA gene
sequences were determined using both distance and maximum parsimony and implemented in MEGA4 (Tamura et al.,
2007). The LogDet distance analysis (Lockhart et al., 1994)
was used as the primary tool for estimating phylogenetic
relationships, but other methods including p-distance and
Jukes–Cantor were also carried out, which yielded similar
tree topologies. All LogDet distances trees were constructed
using the minimum evolution criterion and the data were
bootstrapped 1000 times to assess support for nodes.
All 16S rRNA gene sequences retrieved during this study
were deposited in the EMBL database (http://www.ebi.ac.
uk/embl/) under accession numbers FM213420–FM213421
and FN822778–FN822779.
Results
Stable isotope analysis
In this study, different bivalve tissues (gills, foot and mantle)
were analyzed separately, but isotopic values from different
tissue types showed no significant difference. However, clear
differences in the average d13C, d15N and d34S isotopic
values of tissues from different bivalve species were observed. The carbon isotopic signature of B. mauritanicus
specimens was significantly depleted ( 52.4 1.0%) than
solemyid, lucinid and thyasirid species (average d13C values
between 35.6 1.3% and 28.8 0.6%) (Fig. 2).
The average d15N values (Fig. 2) varied between 3.4 0.3% (Acharax sp. from Ginsburg MV) and 16.1 0.5%
(Petrasma sp. from Gemini MV). The average d34S results
showed a wide range of values from 16.5 3.1% (Petrasma
sp. from Meknès MV) to 116.8 0.2% (B. mauritanicus
from Darwin MV). Specifically for the thiotrophic species
of solemyids, lucinids and thyasirids, the values varied
between 16.5 3.1% (Petrasma sp. from Meknès MV)
and 11.5 0.9% (T. vulcolutre).
Molecular analysis
DNA from foot and gill tissues were analyzed separately, and
no bacterial symbiont sequences were amplified in any of the
foot tissue samples analyzed. DGGE profiles of bacterial 16S
rRNA genes derived from the gill tissue from several bivalves
are shown in Fig. 3, and a summary of the DGGE bands
excised and sequenced is presented in Table 2. High 16S
rRNA gene sequence similarity ( 4 98%) was found between
the bacteria identified in this study and endosymbionts from
FEMS Microbiol Ecol 73 (2010) 486–499
491
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
MV_1) only revealed bacterial 16S rRNA gene sequences
related to Bathymodiolus puteoserpentis thioautotrophic
symbionts (Figs 3 and 4a, Table 2). However, further PCR
and cloning analysis on another specimen (B. mauritanicus
Darwin MV TTR17_1) identified the co-occurrence of
thiotrophic and methanotrophic sequences in this species
(Fig. 4a; C.F. Rodrigues & S. Duperron, ongoing work), and
methantrophic sequences were related to other Bathymodiolus sp. symbionts. In addition, Fig. 3 also suggests that more
bacterial phylotypes (DGGE bands) were present within the
B. mauritanicus specimens and it is possible that some of
these bands may represent methanotrophic endosymbionts.
Discussion
Fig. 2. Dual isotope plots summarizing the mean ( 1SE) d13C vs. d15N
and d13C vs. d34S values for bivalves in each MV. CR, Carlos Ribeiro; Dar,
Darwin; Gem, Gemini; Gin, Ginsburg; Mek, Meknès; Mer, Mercator;
Yum, Yuma.
other similar bivalve species. Most of the excised DGGE
band sequences belonged to the Gammaproteobacteria,
although some sequences were related to members of other
bacterial phyla including Alphaproteobacteria, Betaproteobacteria, Epsilonproteobacteria, Chlamydiae and Spirochaetes.
Bacterial 16S rRNA gene libraries constructed from the
same gill tissue samples as analyzed by DGGE were in
agreement. The majority of bacterial sequences obtained by
PCR cloning from different specimens [e.g. Acharax sp.
(from Yuma MV and Ginsburg MV), Petrasma sp. (from
Gemini MV, Mercator MV and Yuma MV), Lucinoma sp.
(from Mercator MV) and B. mauritanicus (from Darwin
MV)] were assigned within a Gammaproteobacteria clade
(Fig. 4a) in which many thiotrophic and methanotrophic
symbionts of marine invertebrates are included. Similarly,
bacterial phylotypes from other phylogenetic groups were
also obtained and are shown in Fig. 4b.
Interestingly, despite the evidence for methanotrophic
nutrition provided by stable isotopic data, the first molecular analysis of the mytilid specimen (B. mauritanicus Darwin
FEMS Microbiol Ecol 73 (2010) 486–499
Analysis of the natural stable isotopic compositions of
tissues (d13C, d15N and d34S) has been widely used as a
means to determine food sources in organisms inhabiting
reducing environments (Levin et al., 2000; MacAvoy et al.,
2003; Van Dover et al., 2003). The isotopic values reported
here are within the range of values described previously for
bivalves harboring symbionts (Fisher, 1990; Petersen &
Dubilier, 2009). Solemyid, lucinid and thyasirid species in
the Gulf of Cadiz exhibited d13C values typical of seep and
vent symbiotic bivalves that only bear sulfur-oxidizing
bacteria and use a carbon source less depleted in 13C, such
as inorganic carbon (Brooks et al., 1987). Stable isotope
values are known to be influenced by the RubisCO form
used by the symbiont (Robinson & Cavanaugh, 1995), and
the values presented here would fit with the use of a type I
RubisCO, as documented in most chemoautotrophic bivalves (Elsaied & Naganuma, 2001; Scott et al., 2004).
The d13C value determined for the mytilid species was
within the range reported previously in cold seep methanotrophic mussels ( 40% to 70%) (Kennicutt et al., 1992;
Conway et al., 1994; MacAvoy et al., 2005). Previous studies
suggest that tissue carbon isotopic values of mytilids reflect
the relative input of thermogenic and biogenic methane
seepage at a given location (Kennicutt et al., 1992; Duperron
et al., 2007b), and the values reported here seem to indicate
the use of thermogenic methane by mytilid mussels at
Darwin MV, although no d13C values for methane have
been published for this site. In practice, however, the d13C
values for animals having symbiotic methane oxidizers can
be difficult to interpret, as their tissues rarely have d13C
values that would be expected through fractionation of the
source methane or inorganic carbon (Petersen & Dubilier,
2009). Other factors can influence the d13C values in B.
mauritanicus, such as the co-occurrence of sulfide- and
methane-oxidizing symbionts, which will be sensitive to the
relative activities of each of the symbionts.
The observed d15N discrepancy between bivalve species
from the Gulf of Cadiz suggests that they are either using
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492
1
13
4
5
6
T. vulcolutre Carlos Ribeiro MV_1
Marker
B. maurita nicus Darwin MV_3
B. maurita nicus Darwin MV_1
B. maurita nicus Darwin MV_2
Petrasma sp. Meknes MV_3
Petrasma sp. Meknes MV_1
Petrasma sp. Meknes MV_2
Acharax sp. Ginsburg MV_1
Petrasma sp. Yuma MV_8
Petrasma sp. Yuma MV_7
Acharax sp . Yuma MV_6
Acharax sp. Yuma MV_2
Acharax sp. Yuma MV_1
Marker
3
Marker
2
Petrasma sp. Mercator MV_3
Petrasma sp. Mercator MV_1
Petrasma sp. Mercator MV_2
Lucinoma sp. Mercator MV_1
Marker
Marker
C.F. Rodrigues et al.
14
10
7
8
11
9
12
15
Fig. 3. DGGE profile of bacterial 16S rRNA genes from the gills of several chemosynthetic bivalves collected in MV from the Gulf of Cadiz. Numbers
indicate the bands that were excised and sequenced and boxes indicate groups of bands with similar sequences (see Table 2). Marker, DGGE marker as
described in Webster et al. (2003).
different chemical species of nitrogen, tapping into different
pools of nitrogen or discriminating differently after acquisition of their nitrogen source (MacAvoy et al., 2005).
The interspecific d15N variations may be due to the speciesspecific types of symbionts characterized by different
fractionation factors occurring during the assimilation of
dissolved inorganic nitrogen and/or due to the location of
the symbionts. Another hypothesis could be the relative
availability of reduced compounds for these co-occurring
bivalves depending on the depth at which they live in the
sediment. The relatively high d15N values ( 4 5%) measured for Petrasma specimens from Meknès MV and Gemini
MV may indicate symbiont sparseness, since Levin &
Michener (2002) suggested that this value may be at the
limit for species with symbionts, although it also could be
due to the shallower location of these sites and the utilization of organic matter from photosynthetic origin as was
shown for Bathymodiolus azoricus at different depths (Riou
et al., 2010).
The d34S values ( o 5%) in solemyids, lucinids and
thyasirids clearly indicate a thiotrophic mode of nutrition
with reliance on sulfide from seeps because such depleted
d34S values of tissues indicate a sulfur source other than
seawater sulfate (seawater sulfate d34S = 121%; Trust & Fry,
1992; Vetter & Fry, 1998). Intraspecies differences may be
caused by varying dependence on their symbionts with
changing sulfide content in the sediment as was noted for
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Thyasira sarsi and Thyasira equalis (Dando & Spiro, 1993).
Moreover, the highly depleted d34S value found in Petrasma
sp. from Mercator MV and Meknès MV, and Lucinoma sp.
from Mercator MV could be derived from the utilization of
biogenically produced H2S. On the other hand, the less
depleted d34S value of the mytilid (B. mauritanicus) tissue,
combined with the highly depleted carbon signature, reinforces the evidence that these organisms rely on methane
oxidation for their energy production, but does not exclude
the possibility of some reliance on the assimilatory uptake of
inorganic sulfur from seep fluids for the biosynthesis of
organic sulfur compounds (Vetter & Fry, 1998). Because the
isotopic fractionation associated with sulfate reduction to
H2S by bacteria is large and variable, the primary sulfur
source is often difficult to identify based on d34S values alone
(Kennicutt et al., 1992). Nevertheless, the analysis of stable
isotope ratios does support the hypothesis for chemoautotrophic-sourced host carbon nutrition for several species in
the Gulf of Cadiz and provides strong evidence to warrant
further investigation using molecular microbiological
methods.
DGGE analysis of bacterial 16S rRNA genes was used
initially to obtain an overview of the bacterial diversity
present in the gill tissues of all the bivalve species. For most
of the specimens, a dominant brightly stained band was
found and this was considered to represent the main
bacterial symbiont. This was further supported by sequence
FEMS Microbiol Ecol 73 (2010) 486–499
493
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
Table 2. Closest bacterial 16S rRNA gene sequence matches to excised DGGE bands using the nucleotide BLAST program
DGGE band
(accession number)
Closest sequence match
by NCBI nucleotide
BLAST (accession number)
Phylogenetic
group
% Sequence similarity
(alignment length, bp)
1 (FM213436)
Lucinoma annulata symbiont
(M99449)
Gammaproteobacteria
98 (172)
2 (FM213438–FM213441)
Solemya velum symbiont
(M90415)
Gammaproteobacteria
98–99 (165)
3 (FM213442)
Solemya velum symbiont
(M90415)
Gammaproteobacteria
98 (169)
4 (FM213443)
Uncultured bacterium clone
TAG_368_2CF3 (FN393030)
Epsilonproteobacteria
99 (150)
5 (FM213444)
Uncultured bacterium clone
FW1023-058 (EF693294)
Acharax sp. endosymbiont
‘Oregon 56’ (AJ441197)
Chlamydiae
91 (174)
Gammaproteobacteria
100 (194)
7 (FM213446, FM213447)
Solemya velum symbiont
(M90415)
Gammaproteobacteria
98–99 (194)
8 (FM213448)
Solemya velum symbiont
(M90415)
Gammaproteobacteria
98 (194)
9 (FM213449)
Uncultured bacterium clone
C2-E05 (FJ930196)
Acharax sp. endosymbiont
‘Oregon 56’ (AJ441197)
Betaproteobacteria
100 (194)
Gammaproteobacteria
100 (194)
Uncultured bacterium clone
C2-E05 (FJ930196)
Bradyrhizobium elkanii strain
NBRC 14791 (AB509378)
Bathymodiolus puteoserpentis
thioautotrophic gill symbiont
(DQ321712)
Betaproteobacteria
99 ((94)
Alphaproteobacteria
100 (169)
Gammaproteobacteria
98 (194)
Uncultured bacterium clone
Kwat47 (EU035868)
Uncultured bacterium clone
SPG12_461_471_B37
(FJ746172)
Uncultured bacterium clone
GHIV10 (EU857749)
Gammaproteobacteria
85 (198)
Alphaproteobacteria
94 (170)
Spirochaetes
95 (165)
6 (FM213445)
10 (FM213450)
11 (FM213451)
12 (FM213452)
13 (FM213453)
14 (FM213455)
15 (FM213454)
X (FM213437)w
Environmental location
of the closest sequence match
Lucinoma annulata gill tissue,
sediment, Santa Monica Basin,
CA
Solemya velum gill tissue,
eelgrass beds, Woods Hole,
MA
Solemya velum gill tissue,
eelgrass beds, Woods Hole,
MA
Rimicaris exoculata
Hydrothermal vent field, MidAtlantic Ridge
Groundwater, Oak Ridge, TN
Acharax sp. gill tissue, Hydrate
Ridge cold seep sediment,
Cascadia Margin
Solemya velum gill tissue,
eelgrass beds, Woods Hole,
MA
Solemya velum gill tissue,
eelgrass beds, Woods Hole,
MA
Porites compressa coral
mucus, Hawaii
Acharax sp. gill tissue, Hydrate
Ridge cold seep sediment,
Cascadia Margin
Porites compressa coral
mucus, Hawaii
Glycine max root nodule
Bathymodiolus puteoserpentis
gill tissue, Snake Pit
hydrothermal vent, Atlantic
Ocean
Seawater from a deep-sea
coral reef, Norway
Deep-sea sediment, South
Pacific Gyre
Cerastoderma edule
crystalline style
DGGE bands were excised from the DGGE gels shown in Fig. 3.
w
DGGE band X was excised from replicate PCR-DGGE analysis of 16S rRNA genes derived from Lucinoma sp. specimen 1 found at Mercator MV (analysis
not shown in Fig. 3).
analysis, as dominant DGGE bands and sequences obtained
by PCR cloning from the gills of bivalves (Petrasma sp.,
Acharax sp., Lucinoma sp. and B. mauritanicus) were often
related to known thiotrophic symbionts belonging to the
Gammaproteobacteria (Figs 3 and 4; Table 2), and in all
FEMS Microbiol Ecol 73 (2010) 486–499
cases, grouped phylogenetically with bacterial symbionts
from related bivalve species. However, in some instances,
other bacterial taxa were also identified including members
of the Spirochaetes, Epsilonproteobacteria and Alphaproteobacteria, inferring the possibility of other novel symbioses.
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494
C.F. Rodrigues et al.
(a)
Idas sp. symbiont T1 (AM402956)
Bathymodiolus puteoserpentis symbiont (DQ321712)
Bathymodiolus azoricus symbiont (AY235676)
Bathymodiolus brooksi symbiont (AM236331)
Vesicomya gigas symbiont (AF035726)
Calyptogena elongata symbiont (AF035719)
Bathymodiolus sp. symbiont (AJ745718)
Maorithyas hadalis symbiont I (AB042413)
Conchocele sp. symbiont (AJ441190)
Methylobacter marinus A45 (AF304197)
Methylomicrobium agile ATCC35068 (X72767)
Crenothrix polyspora clone 23 (DQ295890)
Bathymodiolus puteoserpentis symbiont (U29164)
Bathymodiolus brooksi symbiont (AM236330)
Bathymodiolus japonicus symbiont (AB036711)
Bathymodiolus sp. symbiont (AJ745717)
Bathymodiolus childressi symbiont (U05595)
Bathymodiolus platifrons symbiont (AB036710)
Thiotrophic
Methanotrophic
Thyasira flexuosa symbiont (L01575)
Solemya pusilla symbiont (U62130)
Anodontia phillipiana symbiont (L25711)
Solemya terraeregina symbiont (U62131)
Lucina pectinata symbiont (X84980)
Lucina nassula symbiont (X95229)
Codakia orbicularis symbiont (X84979)
Lucinoma aequizonata symbiont (M99448)
Lucinoma kazani symbiont (AM236336)
Lucinoma annulata symbiont (M99449)
Lucinoma sp. associated clone Mercator MV_1_1 (FM213432) 12/14 clones
Lucina floridana symbiont (L25707)
Codakia costata symbiont (L25712)
Lucina nassula associated clone CK_G1_5 (EU487789)
Beggiatoa alba ATCC33555 (AF110274)
Thioploca ingrica (L40998)
Captain Arutyunov MV sediment clone CpA BacB18 (FN397816)
Acharax sp. ‘Oregon 56’ symbiont (AJ441197)
Acharax sp. associated clone Yuma MV_1_1 (FM213423) 8/8 clones
Acharax sp. associated clone Ginsburg MV_1_1 (FM213424) 10/10 clones
Acharax sp. ‘Pakistan’ symbiont (AJ441188)
Thiobacillus prosperus DSM5130 (AY034139)
Solemya occidentalis symbiont (U41049)
Petrasma sp. associated clone Yuma MV_8 _1 (FM213425) 2/2 clones
Solemya velum symbiont (M90415)
Petrasma sp. associated clone Mercator MV_1_1 (FM213426) 13/15 clones
Petrasma sp. associated clone Gemini MV_1_1 (FM213435) 10/10 clones
Thiorhodovibrio winogradskyi MBIC2776T (AB016986)
Solemya reidi symbiont (L07864)
Thiotrophic
0.02
Fig. 4. Phylogenetic trees of bacterial 16S rRNA gene sequences associated with the gills of some bivalve species from the Gulf of Cadiz belonging to
(a) Gammaproteobacteria and (b) other bacterial taxa. Minimum evolution trees derived from LogDet distance analysis. The trees were constructed
from 1030 (a) and 754 (b) bases of aligned 16S rRNA gene sequences. Bootstrap support values over 50% (1000 replicates) are shown. First value,
bootstrap derived by LogDet distance; second value, derived by maximum parsimony. Representative sequences of Deltaproteobacteria (Desulfosarcina
variabilis M34407, Desulfobacter postgatei AF418180 and Desulfovibrio desulfuricans ssp. desulfuricans AF192153) and Deinococcus-Thermus
(Thermus aquaticus L09663, Meiothermus ruber L09672 and Deinococcus radiodurans M21413) were used as outgroups in (a) and (b), respectively.
For comparison, preliminary data from a more extensive study on Bathymodiolus mauritanicus (Cruise TTR17 station AT664GR; 35123.520N,
7111.485W) are also included to confirm the presence of methanotrophic symbionts in these specimens (C.F. Rodrigues & S. Duperron, unpublished
data).
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FEMS Microbiol Ecol 73 (2010) 486–499
495
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
Bay of Cadiz sediment clone D30 (GQ249572)
Eel River Basin seep clone Mn3b-B6 (FJ264597)
Bay of Cadiz sediment clone A19 (GQ249483)
Petrasma sp. associated clone Mercat or MV_3_2 (FM213430) 3/7 clones
Sulfurovum sp. NBC37-1 (AP009179)
Lucinid bivalve collection site (Cedar Key, FL) clone CK_1C2_11 (EU487914)
Kazan MV sediment clone KZNMV-25 B15 (FJ712566)
Epsilonproteobacteria
Milano MV microbial mat clone Milano-WF2B-31 (AY592919)
Alviniconcha hessleri symbiont (AB205405)
Rimicaris exoculata symbiont (U29081)
Arcobacter sp. CpA_a5 (FN397893)
Hydrogenimonas thermophila EP1-55-1% (AB105048)
Sulfurospirillum arcachonense DSM9755 (Y11561)
Nitrosospira multiformis ATCC25196 (AY123807)
Nitrosomonas europaea ATCC25978 (AF353160)
Acidovorax avenae ssp. citrulli ATCC29625 (AF078761)
Delftia acidovorans B (AB074256)
Betaproteobacteria
Acidovorax temperans CCUG11779 (AF078766)
Panama seawater clone 6C233393 (EU805385)
Bering Sea seawater clone t8 (GQ452890)
Deep-sea octacoral clone ctg_NISA211 (DQ396042)
Petrasma sp. associated clone Mercator MV_1_2 (FM213427) 1/15 clones
Radopholus similis symbiont Wolbachia sp. (EU833482)
Stenobothrus lineatus symbiont Wolbachia sp. (EU727131)
Kytorhinus sharpianus symbiont Rickettsia sp. (AB021128)
Branchipolynoe sp. associated clone HPf-28 (AB244979)
Alphaproteobacteria
Ophiopholis aculeata symbiont (U63548)
Thyasira vulcolutre associated clone Carlos Ribeiro MV_1_1 8/8 clones
Great Barrier Reef sponge isolate NW001 (AF295099)
Kalahari Shield subsurface water clone DR938CH110701SACH95 (DQ230971)
Apis cerana japonica gut isolate Mesorhizobium sp. Acj104 (AB480752)
Mesorhizobium amorphae SEMIA849 (FJ025123)
Oak Ridge uranium-contaminated site clone FW 1023-058 (EF693294)
Xenoturbella westbladi symbiont (EF177461)
(b)
Petrasma sp. associated clone Mercator MV_3_1 (FM213429) 3/7 clones
0.05
Rhabdochlamydia crassificans CRIB01 (AY928092)
Chlamydiae
Chlamydophila caviae ATCCVR813 (D85708)
Loch Duich sediment clone LD1-PA25 (AY114316)
Waddlia sp. G817 (AY184804)
Parachlamydia acanthamoebae Seine (DQ309029)
Hawaii Ocean seawater clone 40998AA_96 (FJ189959)
Firmicutes
Lactococcus lactis ssp. lactis Pic6 (EU337109)
Petrasma sp. associated clone Mercat or MV_1_3 (FM213428) 1/15 clones
Fusibacter paucivorans DSM12116 (NR_024886)
Clostridium propionicum DSM1682 (X77841)
Cerastoderma edule crystalline style clone GHIV10 (EU857749)
Lucinoma sp. associated clone Mercator MV_1_3 (FM213434) 1/14 clones
Deep-sea octacoral clone ctg_CGOGA33 (DQ395980)
Japan Sea seep sediment clone JS624-21 (AB121108)
Nautilus macromphalus symbiont (AM399077)
Spirochaetes
Alvinella pompejana isolate Spirochaeta sp. BHI80-158 (AJ431240)
Spirochaeta halophila (M88722)
Spirochaeta coccoides SPN1T (AJ698092)
Olavius loisae symbiont (AF104475)
Lucinoma kazani associated clone L2.22 (AM236337)
Svenzea zeai associated clone E145 (FJ529346)
Svenzea zeai associated clone A85 (FJ529295)
Bay of Cadiz sediment clone D32 (GQ249574)
Lucinoma sp. associated clone Mercat or MV_1_2 (FM213433) 1/14 clones
Jiangella gansuensis YIM 002 (AY631071)
Ornithinimicrobium sp. CNJ824 PL04 (DQ448703)
Actinobacteria
Streptomyces coelicolor A3(2) (X60514)
Petrasma sp. associated clone Mercator MV_3_3 (FM213431) 1/7 clones
Lophelia pertusa associated clone A02_CR02_full`(AM911348)
Kalahari Shield subsurface water clone EV818CFSSAHH49 (DQ336995)
Propionibacterium sp. 215(113zx) (AM410900)
Propionibacterium acnes ATCC6919 (AB042288)
Hydrocarbon seep clone BPC009 (AF154099)
Fig. 4. Continued.
In contrast to other symbiotic thyasirids (Distel & Wood,
1992), no known thiotrophic Gammaproteobacteria symbionts were found in the gills of T. vulcolutre. Instead,
bacterial phylotypes belonged to uncultured members of
FEMS Microbiol Ecol 73 (2010) 486–499
the Alphaproteobacteria and a distantly related group of
Gammaproteobacteria with unknown metabolisms. Because
only a fraction of thyasirid species are symbiotic, and the
symbionts are mostly extracellular, this group of bivalves has
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c
496
been described as representative of an early stage in the
evolution of the bacterium–bivalve symbiosis (Dufour,
2005). It may be that T. vulcolutre is asymbiotic and the
observed stable isotope values suggestive of thiotrophy
could be due to ingestion of free-living sulfur-oxidizing
bacteria.
Consistent with the DGGE analysis, a number of other
bacterial phylotypes were also present in the 16S rRNA gene
libraries (Fig. 4b), supporting the possibility that members
of the genera Petrasma, Acharax, Lucinoma and Thyasira
may have more than one known symbiont as reported
previously (Distel & Wood, 1992; Krueger & Cavanaugh,
1997; Imhoff et al., 2003; Duperron et al., 2007a, b). This is
further supported by multiple symbioses in Bathymodiolus
sp. that have two metabolically distinct symbionts (Duperron et al., 2009) and up to six bacterial phylotypes in Idas sp.
(Duperron et al., 2008). It is possible that, as more work is
undertaken to understand bacterium–bivalve symbioses,
many more bacterial phylotypes with symbiotic properties
will be discovered. In our study, one 16S rRNA gene clone
from Lucinoma sp. was related to Spirochaetes symbionts
found in deep-sea octocorals (Penn et al., 2006); corals are
known to harbor several bacterial groups (Webster &
Bourne, 2007). Spirochaetes have been found in a number
of other marine invertebrates as symbionts (e.g. gutless
marine oligochaetes, Blazejak et al., 2005; Ruehland et al.,
2008), but also not necessarily as symbionts (e.g. Lucinoma
kazani, Duperron et al., 2007a). Phylotypes belonging to
Betaproteobacteria, Epsilonproteobacteria and Actinobacteria
were also found in Petrasma sp. specimens and, again,
suggest greater symbiont diversity than thought previously.
Epsilonproteobacteria are increasingly being recognized as an
ecologically significant group of bacteria in deep-sea hydrothermal environments (Nakagawa et al., 2005) and cold
seeps, where they occur as sulfur-oxidizing species (Takai
et al., 2005). Alternatively, it has also been suggested that
Epsilonproteobacteria may act as sulfide detoxifiers, as was
shown for an epsilonproteobacterial phylotype found in two
endemic hydrothermal vent fauna (Campbell et al., 2006).
In addition to symbiotic relationships, the presence of
other bacteria such as Chlamydia-like bacteria in a Petrasma
sp. from Mercator MV (Fig. 3 and Table 2) may indicate the
occurrence of parasitism, similar to the Chlamydia-like
inclusions described in the digestive tissue of Bathymodiolus
heckerae from Blake Ridge (Ward et al., 2004). Parasitic
infections can impair the growth, reproduction, competitive
ability, stress tolerance and survival of host species (reviewed
by Ward et al., 2004). Similarly, the sparseness of symbionts
suggested by the 15N values in some Petrasma sp. specimens
is reinforced by the absence of a recognized symbiont in the
Petrasma sp. from Meknès MV. This observation may
indicate that this species of Petrasma does not rely exclusively on chemoautotrophic nutrition and may be mixo2010 Federation of European Microbiological Societies
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C.F. Rodrigues et al.
trophic. However, suspension feeding in the congener clam
species, Solemya velum, is limited (Scott et al., 2004). It may
also be possible that symbiont density may vary in these
bivalve species according to the availability of metabolic
substrates, such as H2S.
The occurrence of almost identical symbiont phylotypes
in specimens of Acharax sp. from geographically very distant
localities (Gulf of Cadiz, NE Pacific Ocean and Indian
Ocean; Imhoff et al., 2003) is quite remarkable, although
identical sulfur-oxidizing bacterial ectosymbionts have been
reported for geographically distinct ciliates (Rinke et al.,
2009). This high sequence similarity between Acharax sp.
symbionts may indicate that these bacteria are relatively
modern descendants of a much more ancient phylogenetic
line (Imhoff et al., 2003). The large phylogenetic distances
between symbionts of different Solemyidae species support
an ancient evolutionary history because all solemyid genera
Petrasma, Solemya and Acharax have a particularly deep
branching point compared with other clusters of symbiotic
Gammaproteobacteria (Fig. 4a) as suggested previously
(Imhoff et al., 2003). In fact, the association between
endosymbionts and host animals can be highly specific.
Because none of these phylotypes show a close relationship
with free-living sulfur bacteria, it can be concluded that
symbiotic and free-living sulfur bacteria represented separate lineages that have undergone a long period of divergence (Imhoff et al., 2003).
The majority of the analyses on the bacteria associated
with mytilid specimens in the present study revealed phylotypes with 100% sequence similarity to B. puteoserpentis
thioautotrophic symbionts (Won et al., 2003). However,
preliminary results from a more extensive study (included in
Fig. 4a for comparison) have identified the presence of a
bacterial phylotype closely related to methanotrophic endosymbionts of Bathymodiolus sp. (C.F. Rodrigues &
S. Duperron, unpublished data), confirming a dual symbiosis with sulfur and methane oxidizers as known for other
species (e.g. B. azoricus and B. puteoserpentis, Distel et al.,
1995; Fiala-Médioni et al., 2002). Nevertheless, more investigation needs to be carried out using FISH and/or electron
microscopy techniques to confirm the presence, localization
and relative abundance of methanotrophic bacteria in the
gills of B. mauritanicus.
Chemosynthetic bacteria found in bivalves from
the Gulf of Cadiz -- an overview
Molecular analysis (PCR-DGGE and cloning of 16S rRNA
genes) of the bacterial symbionts supported assumptions
based on stable isotope data that Petrasma sp., Acharax sp.
and Lucinoma sp. have dominant thiotrophic bacterial
symbionts. However, in T. vulcolutre, no known thiotrophic
symbionts were detected despite stable isotope evidence
FEMS Microbiol Ecol 73 (2010) 486–499
497
Chemosynthetic bacteria in bivalves from the Gulf of Cadiz
suggesting thiotrophy. Bathymodiolus mauritanicus was
shown to possess a dual symbiosis with thio- and methanotrophic symbionts, although methanotrophs were only
detected in new specimens after further investigation.
Therefore, the Gulf of Cadiz MV symbiotic community
appears to be similar to other vent and seeps symbiosisdominated communities and that thiotrophy or sulfur
oxidation is the dominant bacterial chemoautotrophic activity in most bivalves (Fisher, 1990), although, based on
stable isotope data, methane does seem to be the main
source of carbon for Bathymodiolus.
The solemyids, Acharax sp. and Petrasma sp., have a
widespread distribution in the Gulf of Cadiz, and despite
the range of d13C, d15N and d34S values suggesting different
uptake and fractionation processes, their symbionts are
highly species specific. The sparseness of symbionts in some
Petrasma sp. specimens (Meknès MV) points to a status of
nonobligatory symbiosis for these species. However, of the
five bivalve species studied in the Gulf of Cadiz, Acharax sp.
and Petrasma sp. are the only hosts whose family is known to
contain vertically transmitted endosymbionts (e.g. Solemya,
Krueger & Cavanaugh, 1997) and, therefore, the apparent
worldwide distribution of these symbionts may result from
an ancestral distribution of their primordial hosts (Imhoff
et al., 2003). In addition, this study also suggests the
possibility that additional symbionts occur in some bivalve
species, which needs further investigation.
This study clearly confirms the occurrence of several
chemosynthetic species in the Gulf of Cadiz. However, the
possible benefits to the partners in chemosynthetic symbioses seem to be of varying importance in these symbioses
and more studies are needed to understand the role of the
symbionts in these bivalves in detail. FISH techniques
should be applied to confirm the symbiont presence,
localization and abundances.
Acknowledgements
This research was supported by the HERMES project EC
contract GOCE-CT-2005-511234, funded by the European
Commission Sixth Framework Programme under the priority ‘Sustainable Development, Global Change and Ecosystems,’ and C.F.R. was also supported by a PhD grant (SFRH/
BD/17085/2004) from Fundação para a Ciência e Tecnologia. In addition, new analyses were also funded by the
European Commission Seventh Framework Programme
(FP7/2007–2013) under the HERMIONE project contract
number 226354. Thanks are due to the co-chief-scientists
Luı́s Pinheiro (Departamento de Geociências, Universidade
de Aveiro) and Michael Ivanov (Moscow State University)
for the invitation to participate in the TTR cruises (Training
Through Research Programme, IOC-UNESCO) and Henk
de Haas for the invitation to participate in the Microsystems
FEMS Microbiol Ecol 73 (2010) 486–499
2007. We are indebted to Graham Oliver of the National
Museum of Wales (Cardiff, UK) for species taxonomic
identification. Finally, we would also like to thank the
reviewers for their comments that helped improve the
manuscript.
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