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First published online September 19, 2006
Journal of Experimental Biology 209, 3795-3805 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02413
The hydrocarbon seep tubeworm Lamellibrachia luymesi primarily eliminates sulfate and hydrogen ions across its roots to conserve energy and ensure sulfide supply
Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
* Author for correspondence (e-mail: szd103{at}psu.edu)
Accepted 27 June 2006
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Summary |
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Key words: Lamellibrachia luymesi, hydrocarbon seep, tubeworm, root, sulfate, proton
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Introduction |
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). However, obligate symbioses, including vestimentiferans,
have the additional burden of eliminating waste products generated by their
symbionts. Vestimentiferan tubeworms lack a mouth or gut, and derive their
nutrition from intracellular endosymbiotic bacteria housed in an organ called
the trophosome (Cavanaugh et al.,
1981; Jones,
1981). These bacteria oxidize hydrogen sulfide and produce sulfate
and hydrogen ions as byproducts, and the tubeworm's mechanism for eliminating
these wastes is among its suite of adaptations crucial for this symbiosis
(Girguis et al., 2002;
Goffredi et al., 1998;
Goffredi et al., 1999).
Lamellibrachia luymesi van der Land and Nørrevang 1975
(Gardiner and Hourdez, 2003)
is a vestimentiferan tubeworm associated with hydrocarbon seepage at 400-1000
m depths on the upper Louisiana slope of the Gulf of Mexico. It forms 1-2 m
tall bush-like aggregations that can contain over one thousand individuals,
and provide habitat for a wide variety of heterotrophic fauna
(Bergquist et al., 2003;
Cordes et al., 2005a;
Cordes et al., 2005b). L.
luymesi grows extremely slowly, and has a lifespan of at least 170-250
years (Bergquist et al., 2000;
Fisher et al., 1997). Like all
vestimentiferans, it requires sulfide for its survival and growth. Sulfide is
produced within sediments at hydrocarbon seeps by microbial sulfate reduction
coupled with oxidation of hydrocarbons such as methane and oil
(Aharon and Fu, 2003;
Boetius et al., 2000;
Joye et al., 2004).
Whereas sulfide levels around the anterior gill-like plumes of adult
tubeworms are generally less than 0.1 µmol l-1
(Freytag et al., 2001), levels
in the sediment underlying them are typically greater than 1.5 mmol
l-1 (Julian et al.,
1999). L. luymesi obtains sulfide from the sediment using
long, root-like, posterior extensions of its body
(Freytag et al., 2001;
Julian et al., 1999). Large
L. luymesi aggregations can have extensive networks of roots more
than a meter deep in the underlying sediment
(Fig. 1). These roots extend
below the point at which the tubeworm tubes are attached to a carbonate rock
substrate (Bergquist et al.,
2002; Fisher et al.,
1997).
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;
Goffredi et al., 1998;
Goffredi et al., 1999), L.
luymesi must possess mechanisms for eliminating sulfate and hydrogen
ions. The hydrothermal vent tubeworm Riftia pachyptila has been
relatively well studied, and has become a model for understanding
vestimentiferan physiology. R. pachyptila does not grow a root, but
uses its vascular plume to obtain sulfide and exchange other nutrients with
its external environment (Arp et al.,
1985). Its blood sulfate concentration is 22-25 mmol
l-1 (Goffredi et al.,
1999), which is lower than the seawater sulfate concentration of
approximately 29 mmol l-1. Thus R. pachyptila is thought
to eliminate sulfate ions across its plume surface using membrane transporters
that pump sulfate against the concentration gradient, consuming energy in the
process (Goffredi et al.,
1999). Likewise, R. pachyptila eliminates protons using a
high concentration of proton-specific ATPases in its plume membrane, and this
process represents a substantial metabolic cost for the tubeworm
(Girguis et al., 2002;
Goffredi and Childress,
2001). Internal pH and sulfate concentrations of L. luymesi have not been previously reported. If they were similar to those of R. pachyptila, L. luymesi would also have to utilize active membrane transport to eliminate sulfate and hydrogen ions across its plume into the surrounding seawater. However, unlike R. pachyptila, L. luymesi could conceivably use its root for waste elimination. Microbial sulfate reduction depletes sulfate and hydrogen ions from the sediment pore-water surrounding L. luymesi roots, creating a favorable gradient for these ions to diffuse out of the roots. L. luymesi could avoid the high energetic demands of sulfate and proton elimination across its plume by passive transport of these ions across its roots.
L. luymesi could derive another important benefit from eliminating
sulfate across its roots. In hydrocarbon rich sediments where L.
luymesi are found, microbial sulfide production can become limited by
sulfate availability (Arvidson et al.,
2004; Joye et al.,
2004). A number of authors have speculated that L.
luymesi might sustain microbial sulfide production by `irrigating' the
sediments with sulfate (Cordes et al.,
2005a; Cordes et al.,
2003; Freytag et al.,
2001; Julian et al.,
1999). Cordes et al. (Cordes
et al., 2005a) modeled the sulfide sources and demands of mature
tubeworm aggregations and concluded that their demands could be satisfied over
their entire lifespan only if individuals supplied sulfate across their roots
into the sediment.
In this study, we measured the sulfate concentration and pH of L. luymesi body fluids and confirmed that elimination of these ions across the root surface would be energetically favorable in their natural habitat. We conducted laboratory experiments with live L. luymesi to measure sulfate and hydrogen ion elimination rates across their plume and root surfaces, and used anion transport inhibitors to begin to examine the molecular mechanism by which sulfate elimination occurs.
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Materials and methods |
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The tubeworms were transported in chilled seawater to the laboratory at the
Pennsylvania State University where they were maintained at ambient pressure
in specially designed aquaria inside cold rooms (6°C). The base of each
aquarium was fitted with a polyvinylchloride (PVC) grating with holes drilled
into it, which was connected to a hose that could be used to introduce sulfide
under the sediment in the aquarium (Fig.
2). The grating was covered with a layer of crushed corals, above
which was a 
10 cm layer of sediment collected from the seafloor of the
Gulf of Mexico. The remainder of the aquarium was filled with synthetic
seawater (SSW), made using Reef Crystals® (Aquarium Systems Inc., Mentor,
OH, USA). The SSW in the aquariums was filtered and aerated using a
flow-through aquarium filtration system, and about 10% of it was replaced with
freshly made SSW once a week. The tubeworms were `fed' twice a week by adding
a stock solution containing 6 g of sodium sulfide dissolved in about 7 l of
SSW through the PVC hose directly into the sediment below the tubeworms, at a
rate of approximately 3 l h-1.
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). The
pellet was discarded and the supernatant was diluted to 25 ml using
double-distilled deionized water. The sulfate concentration in the supernatant
was determined using turbidimetric analysis, details of which are described
below.
Live tubeworm experiments
Sulfide spontaneously oxidizes to form sulfate in the presence of oxygen,
so sulfate excretion by tubeworms could not be measured while simultaneously
exposing the animals to sulfide. Instead, tubeworms were first exposed to
sulfide so that they accumulated bound sulfide in their blood, and then
transferred to experimental chambers without sulfide to measure sulfate and
proton excretion rates. The tubeworms used for our experiments were typically
30-50 cm long, with wet tissue masses of between 2 and 5 g. Assuming that
these tubeworms contained 14 ml of blood, and the bound sulfide concentrations
in their blood were 150-170 µmol l-1
(Freytag et al., 2001),
sulfide oxidation would produce approximately 0.15-0.8 µmoles of sulfate in
total. This amount of sulfate would be undetectable in seawater, which has a
sulfate concentration of 29 mmol l-1. Thus a sulfate-free
artificial seawater (SF-ASW) medium was used when measuring tubeworm sulfate
elimination rates. The SF-ASW contained 500 mmol l-1 sodium
chloride, 9 mmol l-1 potassium chloride, 9.3 mmol l-1
calcium chloride, 48.5 mmol l-1 magnesium chloride, and 2.5 mmol
l-1 sodium bicarbonate. The SF-ASW had a salinity of 36
and
its pH was adjusted to 8.0 using a 0.5 mmol l-1 sodium hydroxide
solution.
Sulfate elimination rates of freshly collected tubeworms were determined as a reference for comparison with rates of tubeworms maintained in aquariums. These rates were measured in a cold room (6-8°C) on board the ship using tubeworms that had been collected from the seafloor less than 24 h previously. Seawater was drained from their tubes, and they were soaked in SF-ASW for 20 min to minimize the sulfate carried over by their tubes into the experimental chambers. One or two animals were then inserted into a polycarbonate respirometer, which was filled with SF-ASW and re-circulated continuously using a peristaltic pump. At the end of 48 h, the solution from the respirometer was collected and its sulfate concentration was determined. The tissue wet masses of these tubeworms were estimated from the volume of seawater their soft tissues displaced in a 100 ml graduated cylinder, as there was no shipboard balance available.
The remaining experiments were performed with animals maintained in aquaria
for a period of 1-15 months. Before a tubeworm was used for an experiment, it
was transferred from the maintenance aquarium to a 2-l glass graduated
cylinder containing SSW, where its plume coloration and reflexes were observed
during a period of 15-20 h to ensure that it appeared to be in good
physiological condition. A tubeworm was selected for an experiment if it
routinely extended its plume outside its tube, its plume was bright red in
color, and if it reacted to a sharp knock on the glass cylinder by rapidly
withdrawing its plume back into its tube. Before an experiment, tubeworms were
`fed' by incubating them with a 500 µmol l-1 solution of sodium
sulfide in SSW for 48 h. SSW was drained from their tubes, and they were
soaked in SF-ASW for 20 min. They were then inserted into a `split-chamber'
polycarbonate respirometer, which enabled separation of their anterior and
posterior halves into distinct, watertight chambers
(Freytag et al., 2001;
Girguis et al., 2002). These
chambers contained SF-ASW that was continuously re-circulated using a
peristaltic pump. A headspace of air supplemented the oxygen dissolved in the
SF-ASW in the anterior chamber. Since the concentration of oxygen in air is
about 25 times higher than that dissolved in seawater at 6°C
(Carpenter, 1966), circulation
of the SF-ASW through the headspace of air was intended to prevent hypoxic
conditions. The volume of the headspace was approximately 50 ml for small
worms (up to 3.5 g wet mass) and approximately 100 ml for larger worms. At the
end of 48 h, the solutions from the plume and root chambers were collected and
their sulfate concentrations and pH were determined. The worm was then removed
from its tube, and its wet mass was measured.
`Fed' tubeworms were exposed to a sulfate-free medium during the above experiments. As a control for sulfate loss from the animal driven by the large sulfate gradient between their tissues and the SF-ASW, we conducted experiments with `starved' tubeworms. These experiments were performed using the same procedure described above for the `fed' tubeworms, except the tubeworms were incubated in SSW without sulfide for at least 96 h prior to measuring their sulfate and proton elimination rates.
The effects of inhibitors on tubeworm sulfate and proton elimination rates
were measured in a three-part experiment. The inhibitors we used were the
general anion exchange inhibitors DIDS
(4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and SITS
(4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid), and
the potent chloride transport inhibitor NPPB (5-nitro-2-(3-phenylpropylamino)
benzoic acid) (Cabantchik and Greger,
1992). In these experiments, the procedures used for `feeding'
tubeworms and measuring elimination rates were the same as those described
above with `fed' tubeworms. First, a tubeworm was `fed' sulfide for 48 h and
its `baseline' sulfate and proton elimination rates were measured. It was then
removed from the experimental chamber and `fed' for another 48 h, following
which it was incubated with an inhibitor solution for 3 h. Its
`inhibitor-exposed' sulfate and proton elimination rates were then determined.
Finally, it was `fed' for another 48 h, after which its `post-exposure'
sulfate and proton elimination rates were measured. The wet mass of the
tubeworm was then determined as above. The various inhibitor solutions used
for these experiments were 0.2 mmol l-1 DIDS, 0.4 mmol
l-1 SITS and 0.1 mmol l-1 NPPB. To make an inhibitor
solution, either DIDS, SITS or NPPB was first dissolved in 250 µl of
dimethyl sulfoxide (DMSO) and then mixed with 250 ml of SSW so that the final
concentration of DMSO in the solution was 0.1% by volume. Control experiments
were performed in which tubeworms were exposed to a 0.1% solution of DMSO in
SSW instead of an inhibitor solution. DIDS, SITS, NPPB and DMSO were all
purchased from Sigma Chemical Company, St Louis, MO, USA.
Measurement of sulfate elimination rates
Sulfate concentrations of solutions collected from plume and root chambers
in the experiments described above were measured using barium chloride
turbidimetry, and all samples were analyzed in duplicate. Specifically, one
SulfaVer® reagent packet (Hach Chemical Company, Loveland, CO, USA) was
added to 25 ml of the sample, and the solution was vortexed. The reaction was
allowed to proceed for exactly 5 min, after which the absorbance of the sample
was measured at 450 nm using a Beckman DU-64 spectrophotometer (Beckman
Coulter Inc., Fullerton, CA, USA). The sulfate concentration was calculated
from a standard curve generated using serial dilutions of a 2500 mg
l-1 sulfate standard (Hach Chemical Company) diluted using SF-ASW.
As the sulfate concentration in the SF-ASW introduced into the plume and root
chambers at the beginning of each experiment was empirically determined to be
zero, the entire sulfate measured in the final samples was assumed to have
been released by the tubeworms. Sulfate elimination rates were calculated from
sulfate measurements, incubation times in chambers with SF-ASW and tubeworm
mass.
Measurement of proton elimination rates
pH of solutions collected from experimental chambers were measured to
estimate the amount of protons released by tubeworms. The SF-ASW was first
equilibrated with air by stirring it continuously for 48 h at 6°C with a
headspace of air above it, to ensure that it reached a steady pH value. A 50
ml sub-sample of the SF-ASW was kept aside in a sealed bottle for subsequent
analysis of initial pH and buffering capacity. The SF-ASW was circulated with
tubeworms inside split-respirometers for a period of 48 h, at the end of which
pH of the final plume and root chamber samples were measured. The pH of the
initial SF-ASW was measured at the same time, and its buffering capacity was
determined empirically by titration with a 0.1 mol l-1 hydrochloric
acid solution. The titration curve was used to determine the proton release
into the plume or root chambers necessary to cause the observed differences in
pH between the initial and final SF-ASW solutions. Control experiments in
which SF-ASW equilibrated as above was circulated in split-respirometers
without tubeworms showed that there was no change in pH of SF-ASW in the
absence of tubeworms. Proton elimination rates of tubeworms were calculated
from proton measurements, incubation times in chambers with SF-ASW, and
tubeworm mass.
Data analysis and statistics
The program MINITAB (Minitab Inc., State College, PA, USA) was used for all
statistical analyses. Student's t-tests were used to compare sulfate
and proton excretion rates between `fed' and `starved' animals, and also for
inhibitor studies. When the a priori expectation was that a treatment
would reduce the rate of sulfate or proton release, one-sided t-tests
were used. For inhibitor experiments where the same individual was used for
more than one treatment, paired t-tests for were used for
comparisons. Correlations between parameters were analyzed using the Pearson
method.
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Laboratory maintenance of tubeworms
Tubeworms were successfully maintained alive in aquaria in the laboratory
at 6°C and under atmospheric pressure. Most of the tubeworms extended
their plumes outside their tubes when sulfide-rich SSW was being introduced
into their aquarium. The trophosomes of tubeworms maintained in aquaria were
observed to be light to dark green in color, indicating the presence of
elemental sulfur reserves (Pflugfelder et
al., 2005). Some tubeworms incurred damage to their roots during
collection from the seafloor, and a majority of these worms added new root
tube material to the posterior ends at which their root tubes had been broken.
Laboratory-maintained tubeworms eliminated sulfate at a rate of
0.457±0.176 µmoles g-1 wet mass h-1 (mean
± s.d., N=32), which was not significantly lower
(P=0.304, one-sided t-test) than the rate at which freshly
collected tubeworms eliminated sulfate (mean ± s.d. =
0.509±0.144 µmoles g-1 wet mass h-1,
N=3).
`Fed' tubeworms: sulfate and proton elimination rates
L. luymesi eliminated both sulfate and hydrogen ions at
significantly higher rates across their roots than across their plumes
(Table 1; sulfate:
P=0.0001, N=32; protons: P=0.0003, N=12;
two-sided paired t-tests). Plume sulfate elimination rates were
weakly correlated with root sulfate elimination rates
(Fig. 3A; R=0.31,
P=0.066). Conversely, plume proton elimination rates were strongly
correlated with root proton elimination rates
(Fig. 3B; R=0.91,
P<0.0001). Proton elimination rates were negatively correlated
with sulfate elimination rates, when measured across the plumes
(Fig. 4A; R=-0.69,
P=0.012) and across the roots
(Fig. 4B; R=-0.67,
P=0.017). Total proton elimination rates (plume and root rates
combined) were also negatively correlated with total sulfate elimination rates
(Fig. 4C; R=-0.78,
P=0.003).
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Comparison between `starved' and `fed' tubeworms
`Starved' tubeworms eliminated sulfate across their plumes at a slightly
lower average rate than `fed' tubeworms, but this difference was not
statistically significant (Table
1; P=0.143, one-sided t-test, d.f.=26). However,
`starved' tubeworms eliminated sulfate across their roots at a substantially
lower rate than `fed' tubeworms, and this difference was highly significant
(Table 1; P<0.0001,
one-sided t-test, d.f.=26). `Starved' tubeworms had similar levels of
proton elimination as `fed' tubeworms, across their plumes
(Table 1; P=0.771,
one-sided t-test, d.f.=9) as well as across their roots
(P=0.530, one-sided t-test, d.f.=9).
The effect of inhibitors on sulfate and proton elimination rates
Control experiments in which tubeworms were exposed to 0.1% DMSO showed
that this treatment did not significantly decrease sulfate elimination rates
either across the plumes or roots of tubeworms
(Fig. 5A; plume:
P=0.447; root: P=0.945; one-sided paired t-tests;
N=5). However, exposure to DIDS or SITS caused a significant decrease
in the sulfate elimination rates across roots of L. luymesi, as
compared to `baseline' values (Fig.
5B,C; DIDS: P=0.045, one-sided paired t-test,
N=7; SITS: P=0.040, one-sided paired t-test,
N=6). Neither inhibitor significantly decreased sulfate elimination
rates across plumes of these animals (DIDS: P= 0.229, one-sided
paired t-test, N=7; SITS: P=0.188, one-sided paired
t-test, N=6). DIDS appeared to have an irreversible effect
on sulfate transport across roots, as sulfate elimination rates of
`post-exposure' animals were significantly lower than that of `baseline'
values (P=0.015; one-sided paired t-test, N=7).
However, `post-exposure' sulfate elimination of SITS-exposed tubeworms was not
significantly lower than the `baseline values' (P=0.779, one-sided
paired t-test, N=6). Exposure to NPPB did not cause a
significant decrease in sulfate elimination across either roots or plumes of
tubeworms (Fig. 5D; plume:
P=0.317; root: P=0.327, one-sided paired t-tests;
N=4). None of the above inhibitors had a significant effect on proton
release across either plumes or roots of tubeworms (P>0.1 for all
inhibitors).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Cavanaugh et al.,
1981; Childress et al.,
1984; Freytag et al.,
2001). In addition, they ensure that bacterial sulfide oxidation
is not inhibited by a build-up of end products, namely, sulfate and hydrogen
ions. The hydrothermal vent tubeworm R. pachyptila eliminates these
waste ions across its plume surface
(Girguis et al., 2002;
Goffredi et al., 1999). By
contrast, we found that the hydrocarbon seep tubeworm L. luymesi
primarily used its roots to excrete these waste ions
(Table 1). The main goal of
this study was to identify the mechanism and location of sulfate and proton
elimination. Owing to experimental constraints, we did not simultaneously
expose tubeworms to sulfide while measuring their sulfate and proton
elimination rates. Thus the rates we report are probably lower than the rates
characteristic of L. luymesi in their natural habitat.
The plume is considered to be the primary exchange organ for hydrothermal
vent vestimentiferans because of its large surface area, extensive
vascularization, and short diffusion distances between blood vessels and the
external environment (Arp et al.,
1985; Gardiner and Jones,
1993). Sulfate and hydrogen ions are produced by symbionts located
in the trophosome (Cavanaugh et al.,
1981) and in the case of R. pachyptila are carried by the
vascular blood to the plume, where they are probably eliminated across the
plume epithelium (Goffredi et al.,
1999). Unlike R. pachyptila, vascular connections between
the trophosome and the body wall have been reported for L. luymesi
(Gardiner and Jones, 1993;
van der Land and Nørrevang,
1977), which might provide a direct route for transfer of ions
between the two tissues in the seep species. Additionally, waste ions might be
transferred to the coelomic fluid, which is in equilibrium with the vascular
blood for most ions, and is contained within two cavities between the
trophosome and the body wall (Childress et
al., 1984; Jones,
1981). The coelomic fluid might mediate transfer of ions from the
trophosome to the body wall, where they might be eliminated across the body
wall epithelium. In the case of L. luymesi, the root body wall might
comprise a significant exchange surface. For example, a mature L.
luymesi tubeworm that is about 1 m tall above its point of attachment to
the carbonate substrate can have a root that is about the same length (1 m)
(Cordes et al., 2005a). The
root can be approximated as a cylinder with 1.5 mm diameter, having a surface
area of 47 cm2. The mass of a 1 m tall L. luymesi
individual is about 6 g, based on the mass to length conversion described by
Cordes et al. (Cordes et al.,
2003). The plume surface area of L. luymesi has not been
measured, but using measurements made on a vestimentiferan of similar
morphology, the long-skinny morphotype of Ridgeia piscesae, the plume
surface area of a 6 g worm is approximately 53 cm2 (A. C. Anderson,
J. F. Flores, S. Hourdez, manuscript submitted). Thus the surface areas of the
root and plume are of the same order of magnitude in L. luymesi and
both are probably important gas and ion exchange surfaces.
From an energetic standpoint, the root rather than the plume of L.
luymesi might be the favorable exchange surface for sulfate and hydrogen
ion elimination. Passive facilitated diffusion can mediate membrane transport
of an ion in the direction of its electrochemical gradient, whereas
energetically expensive active transport is required to transport an ion
against its gradient (Byrne and Schultz,
1994). In this study, we found that L. luymesi body
fluids have an average sulfate concentration of 23 mmol l-1 and an
average pH of 7.12. We did not measure intracellular pH and sulfate
concentrations of plume or root epithelial cells. However, R.
pachyptila intracellular pH was very similar to that of its body fluids
(Goffredi et al., 1999), and
we assumed the same for L. luymesi. L. luymesi cells
probably have a resting membrane potential of -70 mV, which is typical for
most animal cells (Lodish et al.,
2000). Based on the Nernst equation
(Hille, 1992), the
electrochemical gradient would favor proton efflux from L. luymesi
only when external pH values were greater than 8.4. Similarly, sulfate efflux
would be favorable if the extracellular sulfate concentration were lower than
the intracellular concentration. In their natural habitat, L. luymesi
plumes are bathed in seawater that has a sulfate concentration of about 29
mmol l-1 and a pH of about 7.7, whereas their roots are surrounded
by sediment pore-water in which sulfate and hydrogen ions are depleted as a
result of microbial sulfate reduction
(Aharon and Fu, 2000;
Arvidson et al., 2004). For
example, at sediment depths greater than 20 cm at hydrocarbon-rich sites,
pore-water sulfate concentrations can vary between 0 and 18 mmol
l-1 and pH can vary between 7.9 and 9.0
(Aharon and Fu, 2000).
Therefore, L. luymesi could possibly eliminate sulfate and hydrogen
ions using passive facilitated diffusion across its root epithelial membrane,
whereas it would require energetically expensive ion pumps to eliminate these
ions across its plume membrane.
Sulfate elimination from experimental animals
In order to measure sulfate elimination rates across plume and root
surfaces of L. luymesi, we incubated them for 48 h with a
sulfate-free medium inside split-chamber respirometers
(Freytag et al., 2001;
Girguis et al., 2002). We
performed our experiments with either `fed' tubeworms that had previously been
exposed to sulfide for 48 h, or `starved' tubeworms that had previously been
deprived of sulfide for at least 96 h. During the experiments, `fed' tubeworms
probably eliminated sulfate derived from oxidation of sulfide carried in their
blood. Conversely, `starved' tubeworms had minimal bound sulfide in their
blood (Freytag et al., 2001)
and were unlikely to eliminate sulfate derived from sulfide oxidation.
Moreover, since we exposed these tubeworms to a sulfate-free external medium,
a portion of the sulfate they eliminated during the experimental period was
probably driven by diffusion not mediated by specific membrane transporters.
Goffredi et al. (Goffredi et al.,
1999) found that the sulfate level in the coelomic fluid of R.
pachyptila deprived of sulfide for 48-72 h was just 5 mmol l-1
lower than that of sulfide-exposed tubeworms. Based on this, we could assume
that `fed' and `starved' tubeworms faced similar gradients when exposed to
SF-ASW, and eliminated similar levels of sulfate by unmediated diffusion. This
allowed us to estimate the rate of sulfate elimination derived from sulfide
oxidation alone, by subtracting sulfate elimination rates of `starved'
tubeworms from those of `fed' tubeworms. In doing so, we found that 85% of the
total sulfate derived from sulfide oxidation was eliminated across the root
surface. There was a substantial and statistically significant difference
(0.182 µmol h-1 g-1 wet mass) between the average
sulfate elimination rates across roots of `fed' and `starved' tubeworms
(Table 1). Conversely, there
was a small and statistically insignificant difference (0.033 µmol
h-1 g-1 wet mass) between the average sulfate
elimination rates across plumes of `fed' and `starved' animals.
In their natural habitat, tubeworms might occasionally experience
sulfate-free conditions across their roots
(Arvidson et al., 2004).
However, their plumes are always exposed to seawater containing 29 mmol
l-1. It appears that under our experimental conditions L.
luymesi eliminated sulfate across their plumes primarily by unmediated
diffusion, while they eliminated most of the sulfate derived from sulfide
oxidation across their roots. The extent of plume sulfate elimination would
depend on the gill surface area and the sulfate concentration gradient,
whereas the extent of root sulfate elimination would depend on the amount of
sulfide oxidation the tubeworm underwent during the experimental time period.
Plume surface area may differ between individual tubeworms and is not likely
to correlate with the rate of sulfide oxidation within the animal. This may
explain the relatively weak correlation we found between plume and root
sulfate elimination rates of individual tubeworms
(Fig. 3A).
The mechanism of sulfate release
We treated live L. luymesi with inhibitors of membrane anion
transport in order to deduce the mechanism of sulfate elimination. The anion
exchange inhibitors, DIDS and SITS significantly inhibited root sulfate
elimination (Fig. 5B,C), but
had no significant effect on plume sulfate elimination. Both DIDS and SITS can
bind reversibly, but are also known to have covalent binding capacities
leading to irreversible effects
(Cabantchik and Greger, 1992).
We found that with respect to L. luymesi sulfate transport, DIDS was
the more potent of the two inhibitors and had an irreversible effect, whereas
SITS was less potent and appeared to bind reversibly
(Fig. 5B,C). Probes of anion
transport, such as DIDS and SITS often have broad specificities
(Cabantchik and Greger, 1992),
but sensitivity to these inhibitors indicates the presence of an anion
antiport system (Gerencser et al.,
1996). Thus L. luymesi roots most probably contain
sulfate exchangers through which they mediate the excretion of this ion,
whereas their plumes do not appear to have this mechanism. Similar to our
findings, Goffredi et al. (Goffredi et
al., 1999) did not find evidence of DIDS or SITS-sensitive sulfate
exchangers in R. pachyptila plumes.
DIDS and SITS-sensitive sulfate transporters are found in a variety of
taxonomic groups including invertebrates
(Gerencser et al., 1996;
Gerencser et al., 1999;
Shimuzu and Bradley, 1994),
fish (Renfro, 1999;
Renfro and Pritchard, 1983)
and mammals (Markovich, 2001;
Pritchard and Renfro, 1983).
DIDS and SITS inhibit the well-studied mammalian band-3 anion exchanger from
red blood cell membranes that can mediate transport of chloride, bicarbonate
and sulfate (Markovich, 2001).
In marine organisms, sulfate is commonly exchanged with chloride and
bicarbonate that are abundant in seawater
(Gerencser et al., 1996;
Gerencser et al., 1999;
Renfro, 1999). Thus, it is
plausible that L. luymesi roots have an anion transporter that
exchanges sulfate ions for either chloride or bicarbonate ions. To examine
whether L. luymesi roots eliminate sulfate using sulfate-chloride
antiports, we analyzed sensitivity of root sulfate transport to NPPB, a potent
chloride transport inhibitor (Cabantchik
and Greger, 1992; Culliford et
al., 2002; Gelband et al.,
1996). Interestingly, NPPB significantly affects chloride
transport across the bacteriocyte membrane in the closely related tubeworm
R. pachyptila (de Cian et al.,
2003). We found that NPPB did not have a significant effect on
sulfate transport across L. luymesi roots
(Fig. 5D), indicating that
sulfate transport across the L. luymesi root membrane might not occur
via a sulfate-chloride exchanger.
Alternately, sulfate elimination across L. luymesi roots might
occur via sulfate-bicarbonate exchangers. Sulfate-bicarbonate
antiports are found in several organisms including rats
(Pritchard and Renfro, 1983),
teleosts (Renfro, 1999) and
lobsters (Gerencser et al.,
1999), and are often sensitive to DIDS and SITS. For L.
luymesi, uptake of bicarbonate in lieu of sulfate elimination across its
roots is reasonable in light of several facts. Bicarbonate levels in sediment
pore-water surrounding tubeworms roots are high
(Joye et al., 2004;
MacDonald, 1998). Bicarbonate
is produced in the sediments as a byproduct of sulfate reduction coupled with
hydrocarbon oxidation, the same process that produces sulfide
(Sassen et al., 1994;
Valentine, 2002). Therefore,
bicarbonate uptake by tubeworms from the sediment could enhance sulfide
production due to end-product removal. Further, tubeworms could utilize the
bicarbonate they take up across their roots for carbon fixation by their
symbionts. R. pachyptila takes up inorganic carbon in the form of
carbon dioxide by diffusion across its plume surface, facilitated by the high
partial pressures of CO2 in acidic vent waters
(Childress et al., 1993;
Goffredi et al., 1997).
However, L. luymesi plumes are bathed in seawater with pH of about
7.7 (Aharon and Fu, 2000), at
which pCO2 levels are negligible. L. luymesi might obtain
at least part of its inorganic carbon from the sediment pore-water across its
roots. This is consistent with the fact that L. luymesi tissues often
have depleted stable carbon isotope values that reflect incorporation of
inorganic carbon derived from oxidized methane and crude oil
(Kennicutt, II et al., 1992;
Roberts and Aharon, 1994).
Finally, carbonate encrustation of tubeworm root tubes could reduce their
permeability to sulfate and sulfide. None of the several thousand L.
luymesi that have been collected to date had carbonate deposited directly
on the root tube surface, although root-balls of the aggregations are often
partially embedded in carbonate (Fig.
1) (Cordes et al.,
2005a). It is plausible that L. luymesi limit carbonate
precipitation directly onto their root tubes by taking up bicarbonate and
releasing protons across their roots, thereby decreasing pore-water
bicarbonate concentrations and pH (Cordes
et al., 2005a).
Proton elimination from experimental animals
We found that `fed' tubeworms eliminated protons across their roots on
average three times faster than across their plumes
(Table 1). The root proton
elimination rates of individual tubeworms were strongly correlated with plume
elimination rates (Fig. 4B),
and the relationship had a slope of approximately 2.5. This indicates that on
an average, for every proton eliminated across the plume, 2.5-3 protons were
eliminated across the root. Overall, approximately 67% of the total proton
elimination occurred across the roots.
In our study, we found that `fed' and `starved' tubeworms eliminated
protons at very similar rates (Table
1). Owing to experimental constraints, we were unable to expose
the tubeworms to sulfide during the measurement of proton flux. Thus our
proton flux values were rather low, and in the same order of magnitude as
`pre-sulfide' exposure rates of L. luymesi measured by Girguis et al.
(Girguis et al., 2002).
Girguis and co-workers found that proton elimination by R. pachyptila
ceased just 1-2 h after exposure to sulfide was terminated. Therefore, it is
likely that in our experiments neither `fed' nor `starved' tubeworms
eliminated a significant amount of protons derived from sulfide oxidation.
Instead, the proton flux we measured may have been derived from heterotrophic
processes.
Moreover, we included a headspace of air in the anterior chamber to prevent
hypoxic conditions during our experiments. However, post-hoc
calculations based on heterotrophic oxygen consumption rates of L.
luymesi (Freytag et al.,
2001) indicated that 3-3.5 g worms might have consumed all the
oxygen available to them in the first 30-35 h of the 48 h experiment, and
therefore might have produced protons as a result of anaerobic metabolism. In
Figs 3 and
4, we have indicated animals
that might have experienced hypoxia using different symbols. We observed no
apparent differences with respect to the proton elimination patterns of these
animals compared to those that did not experience hypoxic conditions, and the
trends we report here do not change if we omit these animals from the
analyses.
The relation between proton and sulfate elimination
Sulfate transport is dependent on proton gradients in a number of different
organisms (Gerencser et al.,
1996; Yildiz et al.,
1994). Sulfate transport could also be directly coupled with
proton transport through proton-sulfate symports
(Leustek and Saito, 1999;
Renfro and Pritchard, 1983).
If this type of transporter were used by L. luymesi for sulfate
elimination, we would expect to see a positive correlation between sulfate and
proton elimination rates of individual tubeworms. By contrast, we observed a
significant negative correlation between these rates
(Fig. 4). Moreover, inhibition
of sulfate elimination by DIDS and SITS did not affect proton elimination
across L. luymesi roots. This combined evidence suggests that proton
and sulfate elimination might not be coupled in L. luymesi. The
negative correlation between proton and sulfate elimination rates is difficult
to explain in terms of known metabolic or membrane processes. Further studies
that examine membrane transport in L. luymesi in more detail are
needed before a viable explanation can be provided.
|
).
Moreover, if bicarbonate uptake across the roots does occur, it might
supplement L. luymesi inorganic carbon uptake and help to prevent
carbonate precipitation on its root tubes. |
Acknowledgments |
|---|
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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