Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts
1 Monterey Bay Aquarium Research Institute (MBARI), 7700 Sandholdt Road,
Moss Landing, CA 95039, USA
2 Department of Ecology, Evolution and Marine Biology, University of
California at Santa Barbara, CA 93106, USA
3 Department of Biology, Pennsylvania State University, University Park, PA
16802, USA
4 Department of Physics, University of California Santa Barbara, Santa
Barbara, CA 93106, USA
* Author for correspondence (e-mail: girguis{at}mbari.org)
Accepted 9 July 2002
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Summary |
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Key words: metabolite uptake, proton-equivalent, tubeworm, Riftia pachyptila, Lamellibrachia cf luymesi, Urechis caupo, sulfide, oxidation, chemoautotrophy, symbiosis, vestimentiferan, hydrothermal vent
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Introduction |
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Both vent and seep vestimentiferan symbionts are sulfide specialists that
specifically utilize hydrogen sulfide to produce energy
(Wilmot and Vetter, 1990).
This metabolic process yields two primary end-products, an oxidized sulfur
compound (such as thiosulfate or sulfate) and protons
(Nelson and Hagen, 1995
).
Although a small proportion of these protons may be utilized in other
microbial reductive metabolic processes, e.g. the reduction of inorganic
carbon and nitrate (Girguis et al.,
2000
), the surplus protons must be eliminated from the symbionts
and their host into the environment.
In a previous paper, we described extremely high rates of net proton
elimination into the environment by the tubeworm R. pachyptila, the
dominant tubeworm at the hydrothermal vent communities along the East Pacific
Rise (Girguis and Childress,
1998). However, we did not measure the quantitative relationship
between proton elimination and host or symbiont metabolism. In the present
study, we address these relationships, in particular the effects of sulfide,
inorganic carbon and oxygen uptake on net proton elimination by the deep-sea
vestimentiferan tubeworms R. pachyptila and Lamellibrachia
cf luymesi (Kennicutt et al.,
1985
), and by the echiuran worm Urechis caupo. L. cf
luymesi flourishes at the hydrocarbon seeps in the Gulf of Mexico
(Macdonald et al., 1989
).
U. caupo is an echiuran worm that inhabits sulfide-rich environments
and possesses mechanisms for oxidizing sulfide to prevent metabolic poisoning
(Menon and Arp, 1998
). Because
U. caupo does not possess symbionts to which it is metabolically
coupled, any observed proton elimination by U. caupo during sulfide
exposure should be the result of sulfide detoxification. We chose to study
U. caupo as a means of examining proton elimination that does not
result from intracellular symbiont metabolism (note that vestimentiferans
cannot survive without their symbionts, so they cannot be used for such
experiments). In addition, we used four ATPase inhibitors to examine the
mechanisms of proton elimination by R. pachyptila and L. cf
luymesi would correlate primarily with symbiont metabolic processes
and that any disruption to proton elimination (e.g. by the use of these
inhibitors) would have negative repercussions on the metabolism of both the
host and the symbiont.
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Materials and methods |
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A clump of Lamellibrachia cf luymesi (van de Land and
Nørrevang) tubeworms were collected by the DSV Johnson Sea
Link from the outskirts of the Brine Pool NR1 hydrocarbon seep site
(27°43'24''N, 91°16'30''W) at a depth of
approximately 650 m during an expedition to the Gulf of Mexico in July 1998.
L. cf luymesi were brought to the surface in a
temperature-insulated box. Upon reaching the surface, they were immediately
transferred to large buckets containing seawater chilled to 7°C and later
maintained at atmospheric pressure in a large cooler that contained
circulating, aerated seawater chilled to 7°C. Upon return to port, an
intact clump of L. cf luymesi was transported in a large
cooler containing ice-cold seawater to the University of California at Santa
Barbara. The worms were immediately placed in a flow-through aquarium
containing seawater at 5°C (pumped from offshore, coarse-filtered and
chilled prior to flowing into the aquarium) and a layer of anoxic mud on the
bottom of the aquarium. Worms were maintained in this aquarium for several
days prior to experimentation. Care was taken to use healthy, active,
undamaged worms that had intact `roots' for experimentation
(Julian et al., 1999). For
experimentation, L. cf luymesi were placed into specially
built two-compartment respiration chambers that allowed the posterior and
anterior halves of the tubeworms to be isolated into different streams of
flowing water (Freytag et al.,
2001
). All experiments were conducted within 17 days of
collection.
Urechis caupo (Fisher and MacGinitie) were collected in November 1996 from the Morro Bay mudflats (35°40'12''N, 120°79'93''W) by a suction gun, consisting of a polyvinylchloride (PVC) tube with an [UNK]-ring-sealed plunger designed to extract worms from their burrows. Worms were transported to Santa Barbara in ice-cold seawater and, upon arrival, immediately placed into flowing seawater at 15° (pumped from offshore to our seawater tables). Worms were allowed to acclimate to the water tables for 3 days before experiments began. All experiments on U. caupo were conducted within 8 days of collection.
Measuring metabolite flux by R. pachyptila, L. luymesi
and U. caupo
In all experiments, a worm or worms were placed into two respirometry
aquaria. A third aquarium always served as a control and was devoid of
worms.
In all experiments, seawater was filtered (0.2 µm diameter) and pumped via a metering pump (Cole-Parmer, Inc.) into an acrylic gas equilibration column and bubbled with a combination of CO2, 5% H2S/95% N2, O2 and N2 or He to achieve in situ dissolved gas concentrations (conditions used in experiments are described below). Mass flow controllers (Sierra Instruments, Inc.) regulated the gas flow into the equilibration column. A proportional pH controller and two metering pumps were used to regulate seawater pH (Prominent Industries, Inc.). A sodium nitrate solution (5 mmol l-1) in 0.2µm filter-sterilized seawater was pumped into the equilibration column at a rate that produced in situ seawater nitrate concentrations. The resulting seawater was then pumped from the equilibration column into each aquarium using three high-pressure pumps (for R. pachyptila experiments; Lewa America, Inc.) or three metering pumps (for L. cf luymesi and U. caupo experiments; Prominent Industries). Aquarium temperature was maintained by immersion in a circulating waterbath (Fisher Inc.). R. pachyptila aquarium pressure was maintained at 27.5 MPa via pneumatically charged or spring-loaded backpressure valves (Circle Seal, Inc.). Aquarium effluents passed through computer-driven stream selection valves (Valco, Inc.), allowing automated control of effluent analysis.
To determine metabolite flux rates, one seawater stream at a time was
directed towards a gas extractor (Fig.
1) that stripped the gases dissolved in the seawater and routed
them for analysis by a membrane-inlet mass spectrometer. The extractor is
based on the principles of our gas chromatograph seawater inlet
(Childress et al., 1984) and
allows us to run dissolved gas analyses continuously with higher resolution
(3-5 times the sensitivity of analyzing seawater directly; data not shown) and
with reduced maintenance of the membrane inlet. To our knowledge, this device
is unique in both its design and application in mass spectrometry. In the
extractor, seawater was bubbled with helium while being mixed with
helium-sparged 20% o-phosphoric acid/80% deionized water. The
addition of the degassed phosphoric acid mixture dramatically reduced the pH,
converting both inorganic carbon and sulfide species to carbon dioxide and
hydrogen sulfide, respectively. A quartz-tipped optical level controller
(Levelite Inc.) maintained the fluid level in the extractor. As dissolved
gases are extracted from the seawater, they are carried to the membrane-inlet
mass spectrometer (Hiden Analytical Inc.) to measure changes in the partial
pressures of CO2, H2S, O2 and N2
(Kochevar et al., 1992
). The
mass spectrometer is capable of detecting extremely small changes in partial
pressure but, for quantitative determination of metabolite flux, these data
were converted into changes in concentration by calibrating the mass
spectrometer with a Hewlett-Packard 5890A gas chromatograph
(Childress et al., 1984
). For
the calibrations, 500 µl gas-tight syringes with 30 gauge sideport needles
(Hamilton, Inc.) were used to collect seawater samples from water- and
gas-tight septa just before the extractor. Regression plots of partial
pressure versus total concentration were used to produce standard
curves. In all cases, the concentrations used for calibration spanned at least
one order of magnitude and encompassed the range of concentrations used in our
experiments. In addition, at least 25 samples were collected and used for each
chemical parameter; in all cases, r2
0.90. Calibrated
data, as well as the flow rate of the effluent and the total mass of the
organisms, were used to determine mass-specific metabolite flux rates.
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For all shipboard experiments, R. pachyptila tubeworms were
maintained in respirometer aquaria
(Kochevar et al., 1992) under
in situ conditions (
CO2=5-6 mmoll-1,
H2S=250-300 µmoll-1, [O2]=100-210
µmoll-1, [NO3-]=40-50,
µmoll-1, pH 6.5, 12°C, 27.5 MPa) until autotrophy was
established. Autotrophy describes a worm that exhibits net inorganic carbon,
oxygen and sulfide uptake from the environment, and net elimination of protons
into the environment. Autotrophy typically commenced after 12-24 h.
For all experiments with L. cf luymesi, tubeworms were
placed in the split-vessel respirometer aquaria
(Freytag et al., 2001) and
maintained under in situ conditions until autotrophy was established
(
CO2=2 mmoll-1 in both top and bottom chambers,
H2S=500 µmoll-1 in the bottom chamber,
[O2] in the top chamber=100-210 µmoll-1,
[NO3-]=40-50 µmoll-1 in both top and
bottom chambers, pH 6.5 in the bottom chamber, pH 8.0 in the top chamber,
12°C, 206 Pa). Autotrophy typically commenced after 48-72 h.
For all experiments with U. caupo, two worms were placed into 38 cm inner diameter Tygon tubing. Both ends of the tubing were fitted with reducing connectors to allow coupling to 0.3 cm diameter polyethylene tubing. Polypropylene plastic mesh was used to prevent the worms from occluding the incurrent and excurrent openings. Metering pumps (Prominent Industries) were used to flush the tubing with chilled, air-saturated seawater from the equilibration column. The entire assembly was placed into a circulating waterbath to maintain the temperature at 15°C. Worms were kept in this tubing (hereafter described as tubing aquaria) with flowing seawater for 12 h prior to experimental manipulations. One tubing aquarium was maintained without worms and served as our control.
To calculate changes in oxygen and sulfide concentration in the seawater
caused by U. caupo pre- and post-sulfide exposure, 500 µl
gas-tight glass syringes with 30 gauge sideport needles (Hamilton, Inc.) were
used to collect seawater samples through gas-tight septa from both
experimental and control tubing aquaria. Total dissolved oxygen and sulfide
concentrations in each sample were determined by gas chromatography using a
Hewlett Packard 5890 gas chromatograph modified for analyzing dissolved gases
in seawater (Childress et al.,
1984).
After completing in vivo experiments, R. pachyptila, L.
cf luymesi or U. caupo were removed from the aquaria,
weighed on a motion-compensated shipboard balance when at sea
(Childress and Mickel, 1980) or
an electronic balance (Mettler, Inc.) when on shore, quickly dissected on ice
and frozen in liquid nitrogen for further analyses. In most cases, the empty
worm tubes were left in the pressure aquaria for several hours and subjected
to the same experimental conditions to determine the fraction of the observed
flux rates attributable to bacterial growth or to other phenomena associated
with the tubes. In this study, as well as previous reports, we have
demonstrated no significant contribution of free-living bacteria to our
observed metabolite flux rates (Girguis et
al., 2000
).
Determination of proton elimination rates by R. pachyptila,
L. luymesi and U. caupo
To determine the proton elimination rates by R. pachyptila, L. cf
luymesi or U. caupo, the seawater pH of the excurrent flows
of the experimental and control aquaria was measured by a double-junction pH
electrode resistant to interference from sulfide (Broadley-James, Inc.) and an
Orion (model 920A) or Radiometer PHM 93 pH meter. In the R.
pachyptila and L. cf luymesi experiments, the electrode
was housed in an O-ring-sealed acrylic flow-through cell (volume 1.35 ml) with
offset inlet and outlet ports to aid in clearing gas bubbles. The effluent
stream in the flow-through cell was maintained at 206 kPa to reduce
off-gassing, and the assembly was positioned after our automated stream
selection valves. pH was measured every 0.25 s, recorded by a computer and
averaged over 7.5 min.
In the U. caupo experiments, two worms were placed into the tubing aquaria, one per aquarium, as described above. The entire assembly was placed into a circulating waterbath to maintain the temperature at 15°C. Worms were kept in these tubing aquaria with flowing seawater for 12 h prior to sulfide exposure. One tubing aquarium was maintained without worms and served as our control. For the sulfide exposure experiments, seawater in the equilibration column was bubbled with hydrogen sulfide, to bring the dissolved sulfide concentration up to 100 µmoll-1, and was pumped into the tubing aquaria. Exposure to sulfide continued for 7 h. During this time, the pH of the seawater from the control and experimental aquaria was measured by collecting samples of control and experimental effluent seawater in 60 ml disposable gas-tight syringes every 8-10 min. The effluent was transferred to 125 ml beakers, and the pH was measured with the aforementioned pH electrode and meter.
In seawater, there is considerable buffering by bicarbonate and other
inorganic anions. To calculate the organisms' proton elimination rates
accurately during the experiments, the buffering of protons by inorganic acid
anions had to be considered (because of their relatively low abundance in our
seawater, organic acid anions were not considered;
Johnson et al., 1988).
Equations for the dissociation of carbonic acid, water, boric acid and
hydrogen sulfide in seawater as a function of temperature, salinity and
pressure were used with our effluent pH measurements, temperature and gas
chromatographic measurements of
CO2 and
H2S (as described above) to calculate total alkalinity. The
general form of this expression is:
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Total alkalinity was then used to calculate the hydrogen ion concentration required to produce the observed differences in the pH between experimental and control aquaria effluents. Total proton elimination rates were then calculated from the hydrogen ion concentrations, the effluent flow rates and the mass of the worms.
Sulfide uptake and proton elimination by R. pachyptila
and L. luymesi
During the HOT 96 expedition, three autotrophic R. pachyptila
tubeworms, weighing 7-16 g each, were placed into two of the high-pressure
respirometry aquaria (one in one aquarium, two in the other aquarium).
Dissolved gaseous hydrogen sulfide concentration was changed in the aquarium
seawater at specific times over several hours to achieve a series of final
seawater sulfide concentrations between 0 and 700 µmol l-1.
Worms were kept at each incremental hydrogen sulfide concentration until their
sulfide and proton flux rates stabilized (typically 4-7 h). Other than
experimental variation in external sulfide concentrations, worms were kept
under constant in situ conditions for the duration of the experiment
(CO2=5 mmol l-1, [O2]=150 µmol
l-1, [NO3-]=40-65 µmol l-1, pH
6.2, 12°C, 27.5 MPa). The above experiment was repeated during the HOT 97
expedition using four freshly collected worms (two in each aquarium).
To determine the effect of sulfide uptake on rates of proton elimination by
L. cf luymesi, two tubeworms, weighing 4-6 g each, were
placed into one two-chamber respirometry vessel. Although simulating the
conditions found in situ utilized the same equipment as in the R.
pachyptila experiments, the seawater physico-chemical conditions differed
(CO2=1.8 mmol l-1,
O2=300
µmol l-1,
H2S=0 µmol l-1,
temperature, 5°C, pressure, 200 Pa). When the oxygen uptake of L.
cf luymesi stabilized, dissolved gaseous hydrogen sulfide was added
to the posterior chamber fluid of the respirometer vessels in two increments
over several hours to achieve final seawater sulfide concentrations of first
238 µmol l-1 and then 515 µmol l-1. Aquaria
conditions were kept at each incremental sulfide concentration until the
worms' sulfide and proton flux rates stabilized, typically 2-5 h. Other than
experimental variation in external sulfide concentrations, worms were kept
under constant in situ conditions for the duration of the
experiment.
Inorganic carbon uptake and proton elimination by R.
pachyptila
During the HOT 97 expedition, four autotrophic R. pachyptila
tubeworms, weighing 4-15 g each, were placed into two high-pressure aquaria
(two per aquarium). Inorganic carbon concentration in the seawater was
increased from 3 to 10 mmol l-1 over 6 h. The inorganic carbon
concentration was then reduced to 4.4±0.12 mmol l-1 and
maintained for 11 h to accustom the tubeworms to the lower environmental
inorganic carbon concentration. Finally, the inorganic carbon concentration in
the seawater was increased to 8.7 mmol l-1, kept at this
concentration for 10 h and then decreased to 2.1 mmol l-1 over 4
h.
Oxygen uptake and proton elimination by R. pachyptila
During the HOT 96 expedition, four autotrophic R. pachyptila
tubeworms, weighing 6-8 g each, were placed into two high-pressure aquaria
(two per aquarium). Sulfide concentration in the aquaria was maintained at
210-250 µmol l-1, while dissolved seawater oxygen concentration
was decreased from 350 to 78 µmol l-1 over 8 h. During the HOT
97 and LARVE 98 expeditions, three autotrophic R. pachyptila
tubeworms were placed into two aquaria (one in one aquarium, two in the other)
and the dissolved oxygen concentration was then decreased from 394 and 314
µmol l-1, respectively, to <3 µmol l-1
(Childress et al., 1984) over
11 and 13 h, respectively.
The effects of transport inhibitors on proton elimination by
R. pachyptila
During the HOT 96, HOT 97 and LARVE 98 expeditions, four inhibitors of
ATPases, N-ethylmaleimide (NEM), amiloride, lansoprazole and
vanadate, were used in separate treatments on R. pachyptila to assess
the mechanism by which protons are being eliminated into the environment. NEM
non-selectively inhibits ATPases and is a general metabolic poison
(Hilden and Madias, 1991).
amiloride inhibits Na+ exchange
(Kleyman and Cragoe, 1988
).
Vanadate inhibits P-type ATPases
(Chatterjee et al., 1992
).
Lansoprazole is highly specific to and inhibits P-type
K+/H+-ATPases
(Tomiyama et al., 1994
). In
each treatment, 2-5 autotrophic R. pachyptila worms, weighing 4-14 g
each, were placed into two of the three high-pressure aquaria (one, two or
three worms per aquarium). In all treatments, inhibitors were introduced into
the system before the high-pressure pumps to prevent depressurization of the
respirometer chambers. Inhibitors were dissolved in 10 ml of deionized water,
except for amiloride, which was dissolved in 10% dimethylsulfoxide (DMSO) in
deionized water. The seawater concentrations of NEM, amiloride, lansoprazole
and vanadate in these respirometer chambers were 2 mmol l-1, 1 mmol
l-1, 2 mmol l-1 and 750 µmol l-1,
respectively (Goffredi, 1998
).
After addition of the inhibitor, the worms were left uninterrupted for at
least 6 h while seawater dissolved gas concentrations and pH were being
measured. The aquaria were continuously flushed by seawater under in
situ conditions.
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Results |
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In our experiments on L. cf luymesi, exposure to hydrogen sulfide induced proton elimination from nearly undetectable rates to 11.90±0.94 µequiv g-1 h-1 (mean ± S.D., N=14) (Table 1). Increasing the total sulfide concentration in the bottom chamber water from 238±44 µmol l-1 to 515±71 µmol l-1 total H2S resulted in a concomitant increase in proton elimination in the top chamber from 5.31±1.39 µmol g-1 h-1 to 11.90±0.94 µmol g-1 h-1, respectively. In addition, the increased total sulfide in the bottom chamber led to a corresponding increase in sulfide uptake by L. cf luymesi in the bottom chamber (from 0.91±1.2 µmol g-1 h-1 to 2.6±0.2 µmol g-1 h-1, respectively). Due to technical difficulties, proton elimination by L. cf luymesi into the bottom chamber of the aquaria was not measured.
Prior to sulfide exposure, U. caupo exhibited no proton elimination (Table 1). Upon exposure to 100 µmol l-1 sulfide, U. caupo exhibited a modest rate of proton elimination into the environment for approximately 45 min (2.17±1.06 µmol g-1 h-1; mean ± S.D., N=5) (Table 1). The rate of proton elimination was five- and 20-fold lower than that of L. cf luymesi and R. pachyptila respectively. The sulfide oxidation rate by the worm, more specifically the quantity of sulfide detoxified by the worm, was not determined. A comparison of oxygen uptake rates for the two U. caupo groups, one group maintained in seawater and the other exposed to 100 µmol l-1 sulfide in seawater, showed a significant difference: 3.12±0.19 and 4.55±0.61 µmol g-1 h-1, respectively (means ± S.D., P<0.0001; N=4 worms per group; MannWhitney U-test).
The effects of inorganic carbon uptake on proton elimination
by R. pachyptila
In our experiments, proton elimination rates correlated with changes in
environmental inorganic carbon concentrations only while environmental
inorganic carbon concentrations were in flux
(Fig. 4). Continuously
increasing the inorganic carbon concentration in the respirometer aquaria
resulted in short-lived, but continuously increasing, proton elimination rates
(Fig. 4A), while continuously
decreasing the environmental inorganic carbon concentration decreased proton
elimination rates by R. pachyptila
(Fig. 4C). When environmental
inorganic carbon concentrations were held constant, there was no correlation
between inorganic carbon uptake and proton elimination rates
(Fig. 4B). During all three
inorganic carbon regimes, seawater sulfide concentrations and R.
pachyptila sulfide uptake rates remained constant.
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The effects of oxygen uptake on proton elimination by R.
pachyptila
One of three experiments demonstrated a near-cessation of proton
elimination when environmental oxygen tensions decreased from 350 to 78
µmol l-1 (Table
2). In this experiment, oxygen uptake was diminished from
12.4±3.6 µmol g-1 h-1 to 1.9±4.3
µmol g-1 h-1 (means ± S.D., N=17-19).
In two later experiments, proton elimination rates nearly ceased when oxygen
tension was reduced to 5 µmol l-1 or below
(Childress et al., 1984). In
these two experiments, the reduced oxygen tensions eliminated sulfide uptake
by R. pachyptila (Table
2).
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The effects of transport inhibitors on proton elimination by
R. pachyptila
With the exception of lansoprazole, exposure to all ATPase inhibitors
reduced proton elimination rates by at least 96%
(Table 3). Amiloride, vanadate
and NEM all led to the cessation of proton elimination by R.
pachyptila, as well as eliminating inorganic carbon uptake and
drastically reducing sulfide and oxygen uptake rates (for example, see
Fig. 5). Exposure of R.
pachyptila to lansoprazole reduced proton elimination rate by 17.8%
(Table 3), and reduced sulfide
uptake rates by 26%. Lansoprazole did not, however, affect the inorganic
carbon or oxygen uptake rates of R. pachyptila.
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Discussion |
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R. pachyptila, however, does experience anoxia in situ,
and we chose to examine the contribution of anaerobic metabolism to proton
elimination. In two of our hypoxia experiments, R. pachyptila
maintained at an oxygen concentration below 5 µmol l-1
demonstrated drastically reduced proton elimination rates and undetectable
inorganic carbon and sulfide uptake rates
(Table 2). Protons eliminated
by R. pachyptila during these experiments are probably the result of
anaerobic metabolism, i.e. glycolysis, and are 1-14% of proton elimination
rates observed during aerobic sulfide oxidation. Although previous studies
have shown that R. pachyptila can survive anoxic conditions for 60 h
(Arndt et al., 1998;
Goffredi, 1998
), our
experiments demonstrate that symbiont function ceases very quickly and that
symbiont metabolism cannot be sustained in the absence of oxygen
(Fig. 3).
In one of our oxygen experiments, lowering environmental oxygen
concentrations to 78 µmol l-1 resulted in the cessation of
proton elimination, inorganic carbon uptake and sulfide uptake. It is possible
that net proton elimination is an oxygen-dependent process (such as a redox
proton pump; Steinmetz and Andersen,
1982). However, we suggest that it is more likely that proton
elimination is indirectly dependent on energy from aerobic metabolism. Proton
elimination by the intertidal worm Sipunculus nudus was induced by
anaerobic conditions, but the subsequent proton elimination correlated with
overall metabolic rate, in particular with aerobic respiration rate
(Portner et al., 1991
). We
agree with these authors that there is a correlation between energy-consuming
ion translocation and energy availability, which is primarily derived from
aerobic metabolism.
In situ, R. pachyptila will also encounter large fluctuations in
environmental CO2 concentrations (from 2.1 to 7.1 mmol
l-1; Childress et al.,
1993; Goffredi et al.,
1997
; Johnson et al.,
1988
). Inorganic carbon acquisition by R. pachyptila
occurs by diffusion of CO2 from the environment into the vascular
blood, so maintaining the internal pH alkaline relative to the environment is
paramount to sustaining inorganic carbon acquisition
(Goffredi et al., 1997
). Our
experiments showed that proton elimination by R. pachyptila was not
correlated with inorganic carbon concentrations when all environmental
metabolite concentrations were maintained at constant levels
(Fig. 4). Under these
conditions, there is no net production of protons from the acquisition and
transport of inorganic carbon by R. pachyptila because CO2
is converted to bicarbonate in the vascular blood and is reconverted to
CO2 for use by the symbionts
(Scott et al., 1999
). However,
transient increases in CO2 concentration induced proton elimination
(Fig. 4), andR.
pachyptila appeared to eliminate protons while the internal and
environmental pools of inorganic carbon pools were equilibrating. We suggest
that protons are eliminated to control pH as the bicarbonate concentration
increases in the vascular blood. This response is similar to that of organisms
experiencing hypercapnia (Arndt et al.,
1998
; Goffredi et al.,
1999
; Kochevar et al.,
1991
).
Interestingly, proton elimination by R. pachyptila will also
effectively reduce the pH of the seawater in contact with the gill, further
favoring the passive influx of carbon dioxide. In addition to the large
surface area and the presence of abundant carbonic anhydrase
(Goffredi et al., 1999;
Kochevar et al., 1991
), this
mechanism may further enhance the ability of R. pachyptila to acquire
inorganic carbon for its symbionts.
In the low-pH vent environment (Johnson
et al., 1988), proton elimination by R. pachyptila occurs
against a concentration gradient, so the process must be coupled to ATP
hydrolysis. The transport of protons may occur via a cation
exchanger, e.g. a Na+/H+ exchanger, or via a
proton-translocating ATPase (Tomiyama et
al., 1994
). During our inhibitor experiments, both vanadate and
amiloride (Fig. 5) were very
effective at inhibiting proton elimination, suggesting that P-type ATPases and
possibly Na+/H+-ATPases are involved in net proton
elimination. The rates of inorganic carbon and sulfide uptake were also
reduced, suggesting that symbiont metabolic processes are disrupted by the
cessation of proton elimination (Fig.
5). However, the potential of these inhibitors to inhibit
Na+/K+-ATPases and disrupt cellular Na+
concentrations is a confounding factor. Lansoprazole, a highly specific
K+/H+-ATPase inhibitor
(Sachs et al., 1995
),
inhibited 17.8% of proton elimination, demonstrating the role of
K+/H+-ATPases in proton elimination
(Table 3). These are the first
live-animal experiments detailing the rates and mechanisms of proton exchange
by any deep-sea organism, and they suggest that R. pachyptila
possesses both K+/H+-ATPases and
Na+/H+-ATPases that are involved in sulfide-driven
proton elimination. A recent in vitro study of frozen R.
pachyptila tissues has shown high activities of ATPases in the plume, as
well as activities of both K+/H+-ATPases and
Na+/H+-ATPases
(Goffredi and Childress,
2001
). Although that study estimated that 2-6% of the total
ATPases are K+/H+-ATPases, the present results suggest
that a larger percentage of the ATPases of R. pachyptila are
K+/H+-ATPases. It is difficult to determine whether the
discrepancy is due to incomplete efficacy of inhibitors in the whole animal or
in vitro experiments. In addition, proton elimination by
H+-ATPases, e.g. electrogenic proton pumps, cannot be ruled out
because they may also account for a large fraction of proton elimination.
The protons generated by symbiont sulfide oxidation are not coupled to
oxidative phosphorylation (as are the proton by-products of anaerobic
metabolism; Hochachka and Somero,
1984) and are primarily a waste product of symbiont metabolism.
Disposing of these protons may represent a large fraction of the energetic
costs of R. pachyptila and L. cf luymesi.
H+-translocating ATPases usually translocate 1-3 protons per ATP
hydrolyzed (Steinmetz and Andersen,
1982
) and at typical R. pachyptila proton elimination
rates (Table 3), between 12 and
15 µmol g-1 h-1 ATP should be utilized in proton
exchange. Using a ratio of 6.2 ATP per mole of O2 for aerobic
metabolism (Hochachka and Somero,
1984
), 2.4µmol O2 g-1 h-1 is
involved in ATP synthesis for proton elimination. This accounts for
approximately 25% of the oxygen taken up by R. pachyptila and 60% of
the host's oxygen consumption (determined by eliminating sulfide from the
environment and stopping symbiont autotrophic metabolism).
Proton elimination and sulfide uptake rates by R. pachyptila are
4-7 times higher than the corresponding rates by L. cf
luymesi when both are exposed to comparable environmental levels of
sulfide (approximately 500 µmol l-1; Figs
3,
4). The conservation in the
stoichiometry of protons produced per sulfide oxidized by R.
pachyptila and L. cf luymesi (5.82±0.17 and
3.84±0.59 protons per sulfide, respectively) suggests that the symbiont
pathways of sulfide oxidation may be similar. Our experimental results fall
within the range of theoretical and experimental models of the number of
protons generated per sulfur oxidized (see
fig. 3 in
Nelson and Hagen, 1995).
Individual variation in these values may result from differences in the rates
of reduction of inorganic carbon and nitrogen
(Girguis et al., 2000
).
Our experiments with U. caupo illustrate the pronounced difference
in proton elimination rates between chemoautotrophic symbioses and
non-symbiotic metazoans. Although we did not quantify the rate of sulfide
oxidation of U. caupo, we have demonstrated that exposure to sulfide
induced proton elimination as well as a significant (P<0.05)
change in oxygen consumption rates (Table
1). However, proton elimination by U. caupo was
short-lived, lasting less than 45 min, and was typically 5-20 times less rapid
than in L. cf luymesi and R. pachyptila
(Table 1). Non-symbiotic
organisms that reside in chemically reduced habitats may exhibit ephemeral
proton elimination resulting from the oxidative detoxification of reduced
substrates and environmentally induced hypercapnia (e.g. sipunculid worms;
Pörtner et al., 1991) but
do not require high sustained rates of proton elimination. A previous study
found that proton elimination by Sipunculus audus averaged 0.08-0.32
µmol g-1 h-1 and lasted for nearly 3 days
(Portner et al., 1991
). U.
caupo, however, exhibited much higher, albeit shorter-lived, proton
elimination rates. Neither S. audus nor U. caupo exhibited
rates comparable with those of R. pachyptila or L. cf
luymesi.
Although proton concentrations in organisms are typically 3-5 orders of
magnitude lower than those of the most prevalent cytoplasmic ions
(Hochachka and Somero, 1984),
the ability to regulate intracellular and extracellular pH is ubiquitous
amongst organisms. Chemoautotrophic symbioses, in particular those of R.
pachyptila and others with a relatively high metabolite flux, must
contend with the continuous net production of protons by the symbionts and of
protons produced by their own metabolism. As the principal waste product of
sulfide oxidation, the ability of the host rapidly and efficiently to
eliminate protons produced by sulfur metabolism is a necessary adaptation to
this mode of symbiosis. While the incurred metabolic costs of proton
elimination by R. pachyptila and L. cf luymesi
appear to be tremendous, protons are the primary end-product of sulfur
oxidation. The rapid and copious elimination of protons by vestimentiferans is
an essential adaptation to symbiosis with sulfide-oxidizing bacteria, the
absence of which would result in the rapid acidification and eventual
metabolic dysfunction of both host and symbiont.
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