ICES Journal of Marine Science (2011), 68(2), 349 –356. doi:10.1093/icesjms/fsq120
LabHorta: a controlled aquarium system for monitoring
physiological characteristics of the hydrothermal
vent mussel Bathymodiolus azoricus
Ana Colaço*, Raul Bettencourt, Valentina Costa, Silvia Lino, Humberto Lopes, Inês Martins,
Luis Pires, Catarina Prieto, and Ricardo Serrão Santos
IMAR and Department of Oceanography and Fisheries of the University of the Azores, Horta, Azores 9001-382, Portugal
*Corresponding Author: tel: +351 292 200436; fax: +351 292 200411; e-mail: [email protected].
Colaço, A., Bettencourt, R., Costa, V., Lino, S., Lopes, H., Martins, I., Pires, L., Prieto, C., and Serrão Santos, R. 2011. LabHorta: a controlled
aquarium system for monitoring physiological characteristics of the hydrothermal vent mussel Bathymodiolus azoricus. – ICES Journal of
Marine Science, 68: 349 –356.
Received 31 August 2009; accepted 11 May 2010; advance access publication 16 August 2010.
LabHorta is a facility composed of laboratories and retrievable deep-sea cages created to support and expand the capabilities of
research cruises. It also enhances the ability to conduct experimental studies with organisms from deep-sea hydrothermal vents
and other deep-sea environments, while keeping them under controlled conditions of pressure and water chemistry. This paper presents a case study with the vent mussel Bathymodiolus azoricus (which harbours a dual symbiosis) collected at the Menez Gwen
hydrothermal vent field at 840-m depth, transported to experimental aquaria at atmospheric pressure and maintained under four
different controlled experimental conditions to study their comparative condition index (CI). Environmental parameters were monitored daily and efforts were made to keep these constant. During the first few months, there were differences between the CI scores of
mussels kept under the various conditions. After 6 months, the differences are not so clear but mussels still had sulphur-oxidizing
bacteria when fed with sulphide. The methane oxidizer bacteria disappear even in the presence of methane. A range of CI scores
appeared as a function of the culture type. The LabHorta facility is a good tool for performing long-term physiological studies of
deep-sea organisms, simulating possible changes in the natural environmental where they normally thrive.
Keywords: condition index, experimental conditions, hydrothermal vents, Menez Gwen, mussels, stable isotopes, symbionts.
Introduction
The Menez Gwen hydrothermal vent field (840-m depth, 37.518N
32.318W) is the shallowest vent field known with chemosynthetic
fauna on the Mid-Atlantic Ridge, and it is the closest to the Azores
Triple Junction (ATJ; Colaço et al., 1998; Desbruyères et al., 2001).
Hydrothermal vents were first discovered at the Galapagos Rift in
the eastern Pacific Ocean in 1977 (Lonsdale, 1977). Warm water containing hydrogen sulphide, carbon dioxide, methane, and other
chemicals seeps through fractures on the seabed and sustains invertebrate communities at the hydrothermal vent sites. Free-living and
symbiotic chemoautotrophic bacteria oxidize the hydrogen sulphide
and methane to generate the energy used for synthesis of the organic
compounds that serve as the basis of the foodweb. Each vent site is
characterized by its own unique mixture of fauna that varies in
density and diversity of species. From an applied science perspective,
the hydrothermal environment provides a useful analogue of anthropogenically polluted marine environments (Ventox, 2003), with the
notable and important difference that the complex biological communities that live around deep-sea vents can be traced back in the
fossil record to at least the Mesozoic era (McArthur and
Tunnicliffe, 1998), allowing sufficient time for the evolution to
develop specific adaptations enabling them to face their environmental toxicity (Ventox, 2003). These novel biochemical and
# 2010
molecular adaptations have the potential for important biotechnological discoveries (Deming, 1998).
The hydrothermal vent mussel Bathymodiolus azoricus dominates the ATJ hydrothermal vent fields, forming large mussel
beds on the seafloor and on chimneys (Colaço et al., 1998;
Desbruyères et al., 2001). Studies have shown that similar
mussels can quickly adapt to a wide range of environmental conditions (Smith, 1985) and can filter-feed to supplement their diet
(Page et al., 1991). These mussels are usually the last survivors in a
hydrothermal vent field where there is no longer any active venting
(Van Dover, 2000). Mussel populations undergo a natural biological succession, with their length and tissue dry weight increasing as
the individuals get older; they eventually die with diminishing
hydrothermal vent activity (Van Dover, 2002).
Bathymodiolus azoricus and the related species to the south,
Bathymodiolus puteoserpentis, are unusual in that they host both
thio- and methanotrophic bacterial endosymbionts (Distel et al.,
1995; Fiala-Médioni et al., 2002; Won et al., 2003). Based on fatty
acid analysis, Pond et al. (1998) concluded that the two types of endosymbiont were equally important in the nutrition of B. azoricus from
the Menez Gwen vent field, whereas Colaço et al. (2007) did not find
methanotrophic fatty acid biomarkers on the mussels from Menez
Gwen. According to Fiala-Médioni et al. (2002), there is evidence
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350
A. Colaço et al.
for a greater dependence on methanotrophy in the Menez Gwen
mussel population than in the Lucky Strike population. Cell counts
from electron micrographs of gills showed larger methanotroph
numbers at Menez Gwen; the gills of mussels from Menez Gwen
showed lower activity of enzymes characteristic of thioautotrophic
bacteria and also lower d13C ratios than those from Lucky Strike
(Fiala-Médioni et al., 2002). These findings suggest that environmental conditions may regulate a balance between the different symbiont populations associated with B. azoricus (Duperron et al., 2009).
Stable isotope studies suggest that B. azoricus from different
sites within the Lucky Strike vent field (Trask and Van Dover,
1999; Colaço et al., 2002a) showed a predominance of nutritional
input from the thiotrophs at one site (Eiffel Tower) and a predominance of methanotrophy at another (Sintra). Bathymodiolus
puteoserpentis from the other Logatchev vent field derived most
of their nutrition from their methanotrophic endosymbionts
(Southward et al., 2001), whereas those from the Snake Pit vent
field were more heavily dependent on their thiotrophic endosymbionts (Robinson et al., 1998). The existence of a dual symbiosis
could thus confer greater environmental tolerance and increased
niche space to the mytilid host in the stochastic hydrothermal
vent habitat (Colaço et al., 2002b; Fiala-Médioni et al., 2002).
Water temperature, food availability, and the reproductive cycle
of mussels may influence the meat yield and biochemical composition of mussels (Fernandez-Reiriz et al., 1996; Okumus and
Stirling, 1998). Changes in the symbiont–host relationship and
the feeding capabilities of the mussel might be expected to affect
its physiological state. This can be evaluated using the condition
index (CI). The CI of animals is governed by seasonal factors
(including sexual maturity) and food availability (Romeo et al.,
2003). It is a good tool for summarizing physiological parameters
such as growth and reproduction, as well as the health and condition of animals (Lucas and Beninger, 1985). The plasticity of
B. azoricus conferred by its dual symbiosis makes this species a
very good model to work with under controlled experimental conditions to perform nutritional, physiological, ecotoxicological, and
immunological experiments (Bettencourt et al., 2008; Company
et al., 2008; Riou et al., 2008), and LabHorta (Colaço et al.,
2002c) is the ideal large-scale facility for undertaking in vivo
studies under experimental conditions.
At LabHorta, we conducted a series of experiments in four
experimental settings with the goal of exploring how individuals
of this species tolerate their physiological conditions and how
they maintain and depend on their host symbionts.
Material and methods
Specimen collection
Specimens of B. azoricus (Von Cosel et al., 1999) were collected
from Menez Gwen, 37.518N 32.318W, 840 m, using either the
French ROV “Victor 6000” operated from the RV “L’Atalante”
or in acoustically retrieved cages measuring 1.25 m2 and covered
on the sides and base with 2-cm plastic mesh (Dixon et al.,
2001, 2002). These cages had been previously deployed over the
diffuse vents and filled with 200 mussels per cage by the ROV.
Mussel collection and cage deployment by the ROV were made
during the Portuguese-funded Sehama cruise in August 2002.
A small research vessel, the RV “Arquipelago”, was used to retrieve
the cages in January and April 2003.
Vent mussels were brought to the sea surface in August (also
considered summer), either in a closed, unpressurized, and insulated container, at 78C, or in an open basket where they were
subjected to both pressure and temperature variation. On board
the ship, the soft tissues were removed and frozen to be transported back to the laboratory for the characterization of T0. In
January (also considered winter) and April (also considered
spring), the cages were recovered 20 min after release and the
mussels for the experiments were kept in chilled seawater at
6–78C to match their natural environmental temperature,
during transit to the Azorean island of Faial.
Experimental design conditions
At LabHorta (Colaço et al., 2002c), the mussels from the April cage
were kept under different experimental conditions at atmospheric
pressure. Four maintenance conditions were used. The mussels
(around 50 individuals with similar sizes) were placed in 30-l seawater aquaria at 88C and supplied with different combinations of
sulphide and methane (Table 1). They were kept under conditions
similar to those at the vent sites, except pressure (Sarradin et al.,
1998, 1999). The water supplied to the aquaria was oceanic and
replaced daily during the first 30 days and at weekly intervals thereafter. Sulphide (0–90 mmol l21 in the aquaria) was supplied discontinuously, using a peristaltic pump, at 2 ml min21 for
15 min every hour, in the form of a 20 mmol l21 sodium sulphide
solution in seawater adjusted to between pH 8.6 and 9.2. Methane
(50 mmol l21 in the aquaria) was supplied continuously as a
bubble stream. A colorimetric method (Cline, 1989) was used to
monitor the sulphide concentrations. A GMI Gasurveyor 524 gas
sensor, was modified and calibrated to measure methane in seawater that had been extracted into a head space in 1-l bottles.
Oxygen, pH, and temperature were measured using WTW Oxi
340i and pH 340i probes.
Mussel measurements
Mussels from the cage collected in April were used in the experiment. Week 0 was the week these mussels were recovered.
During the course of each experiment, three mussels were
sampled at a time. Shell length was measured to 0.1 mm for all
the mussels. Tissues were dissected and frozen at 2808C.
Table 1. Physical – chemical parameters of the experimental conditions and mussel sizes.
Experimental
conditions
H2S
H2S + CH4
CH4
Seawater
Menez
Gwen
Mussel size (mm)
53.4 + 3.7 (45.9; 60.2)
51.5 + 4.0 (45.3; 60.1)
50.8 + 4.8 (43; 57.1)
49.5 + 4.3 (40.2; 56.6)
–
Oxygen (%)
49.3 + 16.8 (8.2; 83.3)
50.2 + 13.3 (9.9; 71.0)
62.7 + 10.9 (28.3; 83.6)
63.7 + 11.5 (30.4; 85.3)
30% ≤ O2 ≤ 60%
pH
7.5 + 0.4 (6.7; 8.4)
7.4 + 0.4 (6.1; 9.0)
7.6 + 0.4 (6.3; 9.0)
7.7 + 0.4 (6.4; 9.0)
6.2 ≤ pH ≤ 8
Temperature (88 C)
7.9 + 0.5 (6.6; 9.4)
8.1 + 0.6 (6.7; 10.4)
7.9 + 0.7 (6.5; 9.7)
7.6 + 0.7 (6,2; 9.8)
7.58 ≤ T ≤ 8.28
H2S (mM)
25.4 + 18.36 (0; 90)
22.0 + 20.2 (0; 97.2)
–
–
0 ≤ [H2S] ≤ 62
Menez Gwen conditions are as published by Sarradin et al. (1998, 1999). The values are the mean + s.d. (minimum; maximum).
CH4 (mM)
–
47.3 + 31.4 (2.9; 379.4)
35.6 + 20.1 (2.9; 120.6)
–
[CH4] , 100 mM
351
LabHorta: a controlled aquarium system
Tissues (gill, foot, and the rest of the body) were dissected, frozen
at –808C, and dried to determine the dry weight. The CI used for
the hydrothermal vents mussels was adapted from Smith (1985).
CI is the ratio of total soft tissue dry weight (g) to shell length
(mm). The gill condition index (GI) is the relation of dry weight
of gill tissue to shell length:
CI =
total soft tissue dry weight
× 100,
shell length
gill tissue dry weight
GI =
× 100.
shell length
and GI, and stable isotope signal. ATukey high significant difference
(HSD) test was used to perform post hoc mean comparisons for significant effects. Tests were performed with STATISTICA 6.0
(StatSoft). Differences were considered significant when p , 0.05.
Statistical methods were selected in accordance with Sokal and
Rohlf (1995) and Zar (1999).
Results
CI and GI in the wild population
The sizes of the mussels used in the different seasons (Table 2)
were not statistically different (H ¼ 1.209, d.f. ¼ 2, p ¼ 0.546,
n ¼ 10). No influence is expected on the indices as a result of
size. Variations in the CI and GI from mussels dissected immediately after collection are presented in Table 2. Mussel GI varied significantly (p , 0.05) between seasons/months, but CI did not.
Tissue preparation for light and electron microscopy
Gill tissue pieces were fixed in modified Trump’s fixative (3% glutaraldehyde and 3% paraformaldehyde formulated with a fixation
buffer containing 0.15 M Na-cacodylate, 0.3 M sucrose, 0.2 M
NaCl, and 0.008 M CaCl) according to Distel and Felbeck (1987).
Following primary fixation, samples were washed in 0.1 M cacodylate buffer (pH 7.8), post-fixed in 1% osmium tetroxide in cacodylate buffer for 1 h, dehydrated in ethanol, and embedded in Spurr
resin (Sigma). Ultra-thin sections were mounted on copper grids
and were double stained with uranyl acetate and lead citrate.
Stable isotope analyses
Automated d13C and d15N were performed using a Thermo
Finnigan DeltaPLUSXL IRMS connected to an elemental analyser
(EA; Flash Series 1112) by a continuous flow interface (Finnigan
Conflo III), using the following working procedure: for the
measurements of d15N, reference materials used were IAEA-N1:
d15N ¼ 0.43 + 0.7‰; IEAE-N2: d15N ¼ 20.41 + 0.12‰. For
d13C measurements IAEA-C6 (sucrose): d13C ¼ 210.4 + 0.1‰
was used as a reference material.
Biochemical analyses
Carbohydrate content was measured in NaCl extract as described by
Dubois et al. (1956) in the presence of 5% phenol and concentrated
H2SO4 and deduced from a glucose calibration curve. The levels of
carbohydrate were expressed as milligrammes of dry weight.
Tissue samples were ground and homogenized in 100 mM Tris
buffer, pH 8.1. The homogenates were centrifuged for supernatant
separation for 30 min at 25 000g, 48C. Total proteins were determined using the BioRad DC protein assay kit based on the
Lowry method (Lowry et al., 1951). Bovine serum albumin was
used as the reference standard. Results were expressed as milligrammes of dry weight.
CI and GI from experimental mussels
There were no significant differences in the sizes of the mussels
(Table 1) between experiments, which we interpret as implying
that CI and GI were not affected by the size. The CI and GI of
mussels from the different experimental conditions evolved differently during the 40 weeks of the experiment (Figures 1 and 2), but
there was no significant treatment effect on the indices for all the
tanks during the first 20 weeks. During the first 3 months (12
weeks), all the tanks showed a similar pattern, with mussel CIs
rising and falling around the spring CI value. This period was considered an adaptation period. After that, CI decreased in all tanks,
reaching values close to those obtained for the specimens caught
in summer. Tanks in which methane was the energy source were
an exception and presented a higher CI. After the summer period,
there was a tendency for the CI to rise again. They were unable to
Table 2. CI and GI, together with mussel sizes, for the B. azoricus
dissected immediately after collection at different times of year.
January (winter; n ¼ 3)
April (spring; n ¼ 3)
August (summer; n ¼ 4)
Mussel size (mm)
CI
GI
51.54 + 5.96
1.09 + 0.14 0.31 + 0.04
46.30 + 2.23
1.08 + 0.13 0.43 + 0.05
49.13 + 7.77
0.73 + 0.17 0.17 + 0.12
Results presented as the mean + s.d.
Statistical analyses
Data were first tested for normality by normal probability plots, and
the homogeneity of variances was checked using Bartlett’s test.
Differences in the sizes, CI, and GI between different seasons,
protein, and carbohydrate concentrations were analysed using the
Kruskal –Wallis test. The Dunn test was used for a posteriori analysis
(Zar, 1999). The significance level was set at 0.05. Differences in the
sizes of the experimental animals were tested through a one-way
analysis of variance (ANOVA). Tukey’s test was used as post hoc
comparison of means. A factorial ANOVA was used to test for the
effects of treatments and time, and their interaction with the CI
Figure 1. Variation in CI over time under the four different
experimental conditions: with plain seawater; with added methane;
with added methane and sulphide; with added sulphide. 0 week,
April; 4 weeks, May; 8 weeks, June; 12 weeks, July; 20 weeks,
September; 24 weeks, October; 28 weeks, November; 32 weeks,
December; 36 weeks, January; 40 weeks, February. Scale bars
represent the standard errors.
352
A. Colaço et al.
reach, however, the CI levels of the wild mussels caught in winter,
which showed the same values as the spring mussels.
Mussel GIs showed the same pattern as CIs, but in summer
under some experimental conditions, GIs reached higher values
than those recorded in mussels collected in summer with the
ROV (T0). The mussels kept in seawater, however, and those
kept with sulphide showed lower GIs than the wild summer
mussels after week 20. Mussels in the other tanks reached that
GI at the end of the experiment.
Presence of symbionts
Figure 2. Variation in GI over time under the four different
experimental conditions: with plain seawater; with added methane;
with added methane and sulphide; with added sulphide. 0 week,
April; 4 weeks, May; 8 weeks, June; 12 weeks, July; 20 weeks,
September; 24 weeks, October; 28 weeks, November; 32 weeks,
December; 36 weeks, January; 40 weeks, February. Scale bars
represent the standard errors.
Gill sections were analysed under TEM microscopy. Fresh mussels
presented both types of bacteria (Kadar et al., 2005). After 30
weeks, the mussels kept in sulphide or sulphide with methane
still had sulphide-oxidizing bacteria in their gills (Figure 3),
whereas those kept in seawater or in methane had no endosymbiotic bacteria.
After 1 year (46 weeks), the gill tissue of the mussels kept in sulphide with methane were in poor condition with no membrane,
microvilli, or bacteria visible (Figure 3).
Figure 3. TEM images from the gill filament sections and bacteriocytes from mussels kept at LabHorta under different experimental
conditions: (a) after 30 weeks in sulphide; (b) after 30 weeks in sulphide and methane; (c) after 30 weeks in methane; (d) after 30 weeks in
plain seawater; (e) after 46 weeks in sulphide and methane; (f) after 54 weeks in seawater. See Table 1 for the detail of each experiment. Scale
bar 1 mm. Sb, sulphur-oxidizing bacteria; N, nucleus; L, lysosome.
LabHorta: a controlled aquarium system
Figure 4. Total carbohydrate concentration (mg g21 dry weight)
in the gills and foot from B. azoricus over time under different
experimental conditions. Markers are the mean values and the
bars are the standard error bars. 0 week, April; 4 weeks, May; 12
weeks, July; 24 weeks, October; 36 weeks, January.
353
Figure 5. Total protein concentration (mg g21 dry weight) in the
gills and foot from B. azoricus over time under different experimental
conditions. Markers are the mean values and the bars are the
standard error bars. 0 week, April; 4 weeks, May; 12 weeks, July;
24 weeks, October; 36 weeks, January.
Carbohydrate and protein content
There was an overall decrease in the carbohydrate content of the
foot with time (Figure 4). The carbohydrates in the gills initially
increased, then decreased after a few months. In the gills of the
mussels kept with both sulphide and methane, the amount of
total carbohydrate stabilized after an initial increase. Protein
content of the tissues (gill and foot; Figure 5) initially maintained
their levels until July (week 12), then decreased until the 30th week
(January). Although there was no significant difference in carbohydrate and protein content between experimental conditions,
there were significant differences between time, with October
and January being different from April until July.
Stable isotopes
The d13C signal changes over time in the different experimental
conditions (Figure 6), despite there being no significant differences over time (Table 3). Significant differences are, however,
present between experiments. The d13C signal of the mussels
kept in seawater is different from that of the mussels kept with
different energy sources.
Discussion
Studies of deep-sea hydrothermal bivalves have revealed that the
species are strictly dependent on interstitial fluid emissions and
derive their food indirectly via symbiotic relationships with chemosynthetic bacteria present in their gill tissues (Le Pennec
et al., 1990). Bathymodiolus azoricus presents a dual symbiosis in
which both sulphur- and methane-oxidizing metabolic pathways
have been found (Fiala-Médioni et al., 2002). The CI of coastal
mussels is governed by seasonal factors (including sexual maturity) and food availability (Romeo et al., 2003). We examined
how the presence or the absence of both or either symbionts
could affect B. azoricus physiological status. This was possible
because we knew that: (i) the digestion of symbionts is a significant
mechanism of carbon transfer from symbiont to the host (Streams
et al., 1997); (ii) B. azoricus hosts both symbionts inside bacteriocytes in its gills; and (iii) our experimental conditions provided
selected nutriments for the symbiotic bacteria. We used the CI
as an indicator of B. azoricus nutritional status and the GI as an
indicator of symbiont status. A decrease in both indices was
observed in mussels after 12 weeks of maintenance. A similar
decrease was observed between wild mussels collected in April
(T0) and later in August (T12 weeks). This similarity suggests
that such a decrease is natural. We observed an increase in CI
for the wild mussels between August and January. This increase
was not observed, however, during our experiments, demonstrating some dysfunction in mussel metabolism. Differences in CI and
GI variations were observed between the various experimental
conditions tested. Mussels kept with two energy sources presented
the highest CI and GI, followed by mussels kept with methane.
Mussels kept with sulphide presented good CI and GI values compared with wild mussels, but lower CI and GI values than mussels
with methane. Mussels kept in plain seawater were starving and
presented the lowest CI and GI values.
354
A. Colaço et al.
Figure 6. Variation in the d13C levels in the gills and foot of B. azoricus over time and under different experimental conditions. Middle point is
the mean; the box is the standard error value and the whiskers are the standard deviation value.
Table 3. Results of the factorial ANOVA for the effects of treatment and time, and their interaction with the CI and GI and with the d13C
signal (emboldened values reflect statistically significant effects).
CI
Effect
Weeks
Experiment
Weeks × experiments
SS
7.244
0.508
1.091
d.f.
10
3
30
MS
0.724
0.169
0.036
GI
F-value
11.46
2.68
0.058
p-value
<0.001
0.052
0.956
SS
0.731
0.113
0.156
d.f.
10
3
30
d 13C gill
Weeks
Experiment
MS
0.073
0.038
0.005
F-value
12.45
6.41
0.89
p-value
<0.001
0.001
0.637
d 13C rest
SS
d.f.
MS
F-value
p-value
SS
d.f.
MS
F-value
p-value
7.35
38.52
4
3
1.84
12.84
1.55
10.86
0.202
<0.001
4.64
11.51
4
3
1.159
3.835
1.474
4.878
0.225
<0.001
The CI and GI values we observed in our experimental tank
conditions, combined with the observations of Fisher et al.
(1988) and Raulfs et al. (2004), support the hypothesis that
mussel bacterial population dynamics vary according to the availability of chemical resources. The higher CI and GI values of
mussels with access to both energy sources or to methane alone
allowed us to hypothesize that methanotrophic bacteria are very
important for mussels in terms of nutrition. In contrast, mussels
kept with sulphide alone always showed lower CI and GI values.
Two hypotheses can be derived from these observations. First,
according to Fiala-Médioni et al. (2002), there is evidence of a
greater dependence on methanotrophy in the Menez Gwen
mussel population based on the analysis of cell counts from
TEMs, with larger methanotroph numbers. Second, because of
the wide variations in sulphide concentrations in our experimental
tanks, the thiotrophic bacteria population was not stable and
started to decline. Knowing that host-driven digestion of endosymbionts provides a direct mechanism for symbiont contribution
to host nutrition (Fisher and Childress, 1992), the variations
observed during the first few weeks are undoubtedly caused by
the establishment of an equilibrium between bacterial cell division
and host digestion. Sulphide variations in the tanks might also
impair bacterial cell division and consequently lead to gaps in
host-driven nutrition, or an increase in thiotrophic symbiont
digestion by mussels, leading to the cessation of bacterial activity.
The ability of mussels to stay alive in plain seawater for a year is
remarkable. Fiala-Médioni et al. (1986) stated that vent mussels
were able to absorb and incorporate dissolved amino acids and
that heterotrophic processes involving dissolved organic matter
may interfere with autotrophic pathways. The survival of these
mussels, despite their low CI and GI values, led the authors to
state that they developed an ability to use any dissolved organic
matter as a food energy source to stay alive and even develop
gonads (Colaço et al., 2006). The data from TEM, biochemical
analyses, and the CI are not very well correlated. It may be possible,
however, that in the gills of mussels from the methane experiments
LabHorta: a controlled aquarium system
selected for microscopic analysis, the bacteria were digested more
quickly than they multiply. The increase in the carbohydrate concentration in the gills is in line with studies conducted by
Bettencourt et al. (2008) that observed a prevalence of carbohydrate granules in the gills after up to 3 months in an aquarium,
a decrease after 6 months, and a later disappearance. From analysing the stable isotope data, it can be said that the d13C signal
depends on the presence of bacteria, because the animals kept in
plain seawater are the ones with a statistically different d13C level
relative to the other treatments. We also infer that animals kept
in the presence of sulphide were using the carbon produced by
sulphur-oxidizing bacteria, as demonstrated by the depletion of
13
C. The delay in the 13C response relative to other indices is
most likely the result of the lower turnover rate of the tissues, a
situation that has been observed in nature (Dattagupta et al.,
2004) and that was also observed by Riou et al. (2008). Some
authors state that thio- and methanotrophic bacterial endosymbionts are equally important in the nutrition of the vent mussel
at the Menez Gwen vent field (Pond et al., 1998). The better CI
and GI values over time in animals fed with methane is an important factor to be taken into account.
The lowest survival tolerance of mussels was observed in late
winter, which corresponds to the recovery period of energy expenditure in gonad formation. According to Boucart and Lubet
(1965), spawning induces weight loss and a lower CI. This suggests
that environmental conditions may regulate a balance between the
physiological activities of the different symbiont populations
associated with these mussels. The existence of a dual symbiosis
could therefore confer a greater environmental tolerance and a
broader niche space to the mytilid host in the stochastic hydrothermal vent habitat (Fiala-Médioni et al., 2002).
The results from this experiment indicate that the LabHorta facility provides excellent conditions for conducting long-term physiological studies of deep-sea organisms, allowing simulation of the
variations in the environment in which they normally thrive.
Acknowledgements
We thank the following people and organizations for their help and
support during this study: the captain and crew of the RV
“l’Atalante”, the ROV “Victor 6000” team, the captain and crew
of the RV “Arquipélago”; the EU Framework Contract No
EVK3-CT1999-00003 (Ventox), which funded the cage system
and partially funded LabHorta; Sérgio Gulbenkian and Marisa
Pardal from the Instituto Gulbenkian de Ciência, who allowed us
to use their facilities for the TEM slides; and the Azorean
Regional Directorate for Science and Technology, which funded
the LabHorta infrastructure. The research was funded by the
SEAHMA project (FCT/PDCTM 1999/MAR/15281) and
REEQ/953/MAR/2005 project. IMAR-DOP/UAç research activities are additionally supported through the pluri-annual and programmatic funding schemes of FCT (Portugal) and Azorean
Regional Directorate for Science and Technology (DRCT, Azores,
Portugal) as Research Unit no. 531 and Associate Laboratory no.
9 (ISR-Lisboa).
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