The sulfide binding characteristics of blood serum were studied in vitro in two deep-sea vesicomyid clams, Calyptogena pacifica and Vesicomya gigas. Both the C. pacifica and the V. gigas serum concentrated sulfide at least an order of magnitude above ambient levels. V. gigas accumulated sulfide faster than C. pacifica, reaching saturation at 5000 M after an hour. C. pacifica bound sulfide at half the rate of V. gigas, reaching saturation in about two hours at a substantially higher concentration of sulfide. The observed distribution of the animals near cold seeps in the Monterey Submarine Canyon can be explained by their different sulfide binding abilities. The hypothesis that cold seeps are actually much more unstable sources of sulfide than previously assumed is explored

Snively, Gillian

Aquatic Commons

Sulfide Binding Characteristics
of Blood Serum in
Calyptogena pacifica and
Vesicomya gigas
Gillian Snively
Rt. 2 Box 619F
Morgan Hill, CA 95037
(408)779-4321
Advisor: Dr. R. E. Kochevar
Hopkins Marine Station
Stanford University
June 3, 1994
Sulfide Binding Characteristics of ......
Abstract
The sulfide binding characteristics of blood serum were studied in-
vitro in two deep-sea vesicomyid clams, Calyptogena pacifica and
Vesicomya gigas. Both the C. pacifica and the V. gigas serum
concentrated sulfide at least an order of magnitude above ambient
levels. V~ gigas accumulated sulfide faster than C. pacifica,
reaching saturation at 5000 M after an hour. C. pacifica bound
sulfide at half the rate of V. gigas, reaching saturation in about two
hours at a substantially higher concentration of sulfide. The
observed distribution of the animals near cold seeps in the Monterey
Submarine Canyon can be explained by their different sulfide binding
abilities. The hypothesis that cold seeps are actually much more
unstable sources of sulfide than previously assumed is explored.
2
(Sulfide Binding Characteristics of ...it
Introduction
Calyptogena pacifica and Vesicomya gigas are members of the
deep-sea clam family Vesicomyidae. Both burrow in the soft,
sulfide-rich benthic substrate surrounding hydrothermal vents or
cold seeps. In the last four years, they. have been discovered in the
Monterey Submarine Canyon near coid seeps between 550 and 900
meters depth. The large sulfur-rich gills of the animals contain
intracellular symbiotic chemoautotrophic bacteria. The clams'
feeding grooves and guts are simple and reduced in size, suggesting
that they rely on nutritional supplements from their symbionts.
This is corroborated by isotopically light carbon isotope levels, as
are typical of chemosynthetically derived carbon (J. P. Barry, pers.
comm.).
The clams are oriented with their feet buried in the reducing
substrate and their siphons exposed to the relatively sulfide-free
bottom water. It has been hypothesized that they take up sulfide
through the foot and obtain carbon dioxide and oxygen from overlying
ambient seawater (Arp et al. 1984). This would require a system to
transport the sulfide through the clams' circulatory system from the
foot to the bacteria in the gills without poisoning the surrounding
tissues or oxidizing the reduced, energy-rich form of sulfur.
Sulfide, even at nanomolar concentrations, is a potent inhibitor of
aereobic respiration through the cytochrome-c oxidase system
(National Research Council 1979). Sulfide inhibition of cytochrome-
c oxidase from the giant hydrothermal vent clam C. magnifica has
been shown to be offset by the re-addition of the clam's blood to the
system (Powell and Somero 1986), suggesting that the binding
agents serve to protect the animals' tissues as well as provide
3
lSulfide Binding Characteristics of .....
sulfide for their symbionts.
The circulatory systems of vesicomyid clams contain ample
quantities of blood with hemoglobin containing red blood cells.
Studies of C. magnifica and C. elongata have found a sulfide-specific
binding moiety in the animals' blood serum (Childress et. al 1993,
Arp et. al 1984); preliminary research on C. ponderosa and V.
cordata indicates that their blood has a similar capacity for sulfide
accumulation (Fisher 1990). set out to explore the binding
characteristics, over different concentrations and times of
incubation, of C. pacifica and V. gigas blood serum.
Materials and Methods
Experimental Animals
Organisms were collected using Monterey Bay Aquarium Research
Institute's (MBARI) remote operated. vehicle ROV Ventana from cold
seep locations around the Monterey Canyon. Collection sites ranged
from 550-900 meters in depth. The two clams studied, Vesicomya
gigas and Calyptogena pacifica, are associated with sulfide-rich
seeps. Animals were maintained from one to ninety days in 4° C
reduced oxygen sea water at atmospheric pressure until they were
sacrificed. Animal shells were scrubbed clean and shaken to
evacuate water before dissection. All blood was aspirated in
syringes, pooled, and stored at 4° C.
Sample Preparation
Initial studies concluded that whole blood and extracted serum
displayed the same binding sensitivity, so experiments were
4
Sulfide Binding Characteristics Of ...~
performed using clam serum. Whole blood was centrifuged for ten
minutes at 6200G. The serum was extracted and diluted ten fold
with 0.2 Jlm filtered, UV-sterilized sea water. The dilutions were
then aliquoted in 1 ml quantities in dialysis membrane tubing (MWCO
12-14,000, SpectralPor #2). All visible air bubbles were evacuated
and both ends of the dialysis tUbing were clamped.
A 0.4 M hydrogen sulfide stock solution was made with de-
oxygenated filtered sea water and sodium sulfide crystals. Ten
percent HCI was added to re-establish a pH of 8. This stock was
used for all subsequent working concentrations of sulfide.
Sulfide Incubations
For each measurement of sulfide binding as a function of external
concentration, 750 ml of filtered sea water was bubbled with
nitrogen for ten minutes to remove all oxygen. The appropriate
amount of sulfide stock was then added to achieve the desired
sulfide concentration. One dialysis bag each of C. pacifica and V.
gigas dilute serum was added and the jars were sealed with
Parafilm and screw caps. After at least six hours of incubation at
4° C, the serum samples and the dialysate were analyzed for sulfide
content using a HP5890 Series II gas chromatograph modified for
quantitation of dissolved gases in liquid samples (Childress et aI.,
1984) as descibed below.
250 JlI of each sample was injected using a gas-tight syringe
(Hamilton) into a heated, teflon-sealed, glass extraction chamber
pre-loaded with dilute, gas stripped phosphoric acid and silicon
anti-foam. As the sample entered the extraction chamber, dissolved
carbonates (C032-, HC03-, H2C03) and sulfides (S2., HS-) were
5
Sulfide Binding Characteristics of ... .. ::
converted to C02 and H2S (respectively), and were stripped, along
with other dissolved gases (e.g. 02, N2, CH4, CO), by helium carrier
gas flowing through the chamber. This gas mixure was then carried
directly to a packed column (6' x 1/8" teflon-lined stainless steel,
packed with Unibeads 1S 80/100) where C02 and H2S were separated
from the remaining gases, which passed through an automatically-
actuated valve onto a second column (18' x 1/8" stainless steel,
packed with Mol Seive 5A 60/80) for further separation. Upon
elution from the columns, concentrations of all gases were
determined using a thermal conductivity detector.
To determine the time course of sulfide uptake, several serum
samples from both species were placed in a 1-liter volume of
300±50 J.lM sulfide solution. The dialysate was stirred continuously
to ensure uniform sulfide distribution. At each time point, one pair
of serum samples was removed and immediately placed in air tight
syringes to prevent the sulfide from oXidizing. The initial and final
dialysate and the samples were then analyized for sulfide content.
Serum bound sulfide concentrations were calculated as follows:
bound sulfide = (serum sample sulfide content - dialysate sulfide content) * 10
This compensated for both the ten fold dilution of blood serum and
for the unbound sulfide present in the serum due to free diffusion
across the dialysis membrane.
Results
Both the C. pacifica and the V. gigas serum concentrated sulfide
substantially above ambient levels (Fig. 1). At low external sulfide
concentrations (below 500 J.lM), all bound sulfide concentrations
6
Sulfide Binding Characteristics of ...!!:L
were at least an order of magnitude above the ambient levels. V.
gigas appeared to reach saturation at about 5000 j..lM, but three
outlying data points prohibit conclusive assertions. C. pacifica
consistently bound higher levels of sulfide than V. gigas. Again,
quantitative comparison between the two clams is difficult as there
appear to be some outlying. points. The range of external sulfide
concentrations used in these trials may not have saturated the
serum of C. pacifica, as the upward trend did not approach an
asymptote similar to that evident in V. gigas.
V. gigas accumulated sulfide faster than C. pacifica, but reached
saturation at a lower sulfide concentration than C. pacifica (Fig. 2).
V. gigas initially bound sulfide at a rate of 350 j.1mol*1 serum- 1*min-
1, while C. pacifica concentrated it a little less than half the speed,
at 156 j.1mol*1 serum- 1*min- 1 . V. gigas appeared to reach saturation
at a concentration of approximately 5000 j.1M, which is consistant
with the trend seen in the study of. sulfide binding as a function of
external concentration. Again, C. pacifica did not clearly reach
saturation, as the serum sulfide concentrations at the highest
external sulfide levels still show an upward trend.
Discussion
Sulfide binding in vesicomyid clams serves three vital functions.
It prevents sulfide poisoning of aerobic metabolism, concentrates
sulfide and transports it from the uptake site in the foot to the
symbiotic bacteria in the gill, and preserves the most reduced,
energy-rich form of sulfur by preventing the spontaneous oxidation
of sulfide during transport. As animals sacrificed the day after
collection displayed the same sulfide binding curve as those
7
Sulfide Binding Characteristics of ...!li
maintained for 90 days, I suggest that sulfide binding levels may be
constitutive and species-specific rather than highly regulated,
although this is certainly an area for further experimentation. The
two aspects of sulfide binding discussed in this paper--absolute
level bound and rate of binding--deseNe further exploration.
My data suggest that the blood of both species binds suifide at
least an order of magnitude above ambient levels. However, the two
species differ considerably in binding characteristics. C. pacifica
consistently bound more sulfide than V. gigas when both were
allowed to reach equilibrium at comparable external sulfide
concentrations. In situ, V. gigas inhabits areas of high sulfide
concentration, while C. pacifica is more abundant at sites with
lower sulfide levels (R. E. Kochevar, pers. comm.). As blood binding
of sulfide may be an important defense against sulfide toxicity
(Powell and Somero 1986), elevated sulfide levels may inhibit
aerobic respiration in C. pacifica, while the lower sulfide levels on
which C. pacifica thrives may not be sufficient for V. gigas to
supply adequate sulfide to its symbionts. C. pacifica also survives
better and longer in the laboratory. This may be partially linked to
its ability to bind the lower levels of sulfide characteristic of
aquarium maintenance.
Saturation was evident in V. gigas after an hour and in C. pacifica
after approximately two hours. Rate studies on the blood serum C.
magnifica suggest that it binds sulfide more slowly than the C.
pacifica and V. gigas. Arp et al. (1984) discovered that C. magnifca,
which wedges its foot into hydrothermal vent fissures and is in .
close contact with warm, sulfide rich water, appears to accumulate
sulfide in a linear fashion over at least 20 hours. Timed binding
8
Sulfide Binding Characteristics of ...~
studies performed on the sulfide binding hemoglobin of the
vestimentiferan Riftia pachytila suggest continuous sulfide
accumulation until saturation, which occurs after about 16 hours
(Arp and Childress 1983). The sulfide concentrations surrounding
the animals are intermittant, but their vascular blood levels
indicate that substantiai quantities of sulfide are avaiiibie in their
environments (Arp and Childress 1983). Sulfide binding rates in
both Riftia and C. magnifica are an order of magnitude slower than
the rates observed in C. pacifica and V. gigas.
It has been assumed that the cold seep environment has a low,
steady, continuous flow of sulfide in comparison to the
concentrated, capricious sulfide in hydrothermal vents. In this
scenario it seems that selection would favor high sulfide affinities
with little regard for rapid binding rates. In light of the fact that
the sulfide binding rates in C. pacifica and V. gigas are much higher
than those of the aforementioned hydrothermal vent species, this
may be called into question. One possible explanation of the fast
binding rates of seep animals may be that the sulfide levels of cold
seeps, as well as being lower than those of vents, are highly
variable. Selective pressure would then favor clams that could
concentrate sulfide in large quantities as quickly as possible. This
reconsideration of the seep environment is supported by time lapse
videos of clam fields in which it has been observed that
occasionally, a patch of· mud within a clam field will suddenly turn
black and then fade to its normal color over several hours (J. P.
Barry, pers. comm.). These data suggest that the cold seeps may be
more dynamic systems then previously recognized.
C. ponderosa and V. cordata are found associated with
9
Sulfide Binding Characteristics Of ...tt
hydrocarbon seeps in the Gulf of Mexico. Both animals leave
characteristic trails of up to 205 em (Rosman et al. 1987). It has
been suggested that this burrowing behavior is necessary to provide
a constant source of sulfide to the clams' symbionts (Fisher 1990).
As this foraging behavior is not evident in C. pacifica and V. gigas,
the coid seeps must provide sufficient net amounts of sulfide, but
sulfide levels may fluctuate considerably over short temporal
scales.
It is hypothesized that the distribution of C. pacifica and V. gigas
may be in part controlled by their sulfide binding characteristics.
The comparatively rapid sulfide binding evident in the clam serum
suggests that the cold seeps may have ephemeral bursts of sulfide.
This is a radical deviation from current dogma. Further observation
of animals with chemotrophic symbionts in conjunction with studies
of their environments may support the hypothesis that the sulfide
binding characteristics of the animals may be indicative of the
surrounding sulfide availibilities.
Acknowledgements
I would like to give a hearty thanks to all the people who made my
research possible:
Dr. Randy Kochevar, for his guidance, assistance, and patience
Dr. Jim Barry and the crew of the Pt. Lobos and the ROV Ventana for
animal retrevial
MBARI for facilities, reagents, and equipment
Dr. Jim Watanabe for his tireless cheer and editing assistance
and
10
Sulfide Binding Characteristics Of ...~
Connie Kim, Alan Lam, and Edwardo Martinez for their unflagging
support.
11
Sulfide Binding Characteristics af ...!it
Literature Cited
Arp, AJ., J.J. Childress, and C.R. Fisher Jr. 1984. Metabolic and
blood gas transport characteristics of the hydrothermal vent
bivalve, Ca/yptagena magnifica. Physiological Zoology. 57:648-662.
Arp, A.J. and J.J. Childress. 1983. Sulfide binding by the blood of the
hydrothermal vent tube worm Riftia pachyti/a. Science. 219:295-
297.
Childress, J.J., C.R. Fisher Jr., J.A Favuzzi, A.J. Arp and D.R. Gros.
1993. The role of a zinc-based, serum-borne sulphide-binding
component in the uptake and transport of dissolved sulphide by the
chemoautotrophic symbiont-containing clam Ca/yptagena e/angata.
Journal of Experimental Biology. 179:131-158.
Fisher, Charles R. Jr. 1990. Chemoautotrophic and methanotrophic
symbioses in marine invertebrates. Reviews in Aquatic Sciences.
2:399-436.
National Research Council: Hydrogen Sulfide. Baltimore: University
Park Press 1979. (Committee on Medical and Biological Effects of
Environmental Pollutants. Subcommittee on Hydrogen Sulfide:
Division of Medical Sciences. Assembly of Life Sciences).
Powell, M.A. and G.N. Somero. 1986. Adaptations to sulfide by
hydrothermal vent animals: sites and mechanisms of detoxification
and metabolism. Biological Bulletin. 171 :274-290.
Rosman, I., G.S. Boland and J.S. Baker. 1987. Epifaunal aggregations
of Vesicomyidae on the continental slope off Louisiana. Deep-Sea
Research. 34: 1811.
12
Sulfide Binding Characteristics of ...il.
Figure Legends
Figure 1: Serum bound sulfide as a function of external sulfide
concentration
Figure 2: Time dependence of sulfide binding. Experiment performed
with dialysate concentration of 300±50 ~M sulfide solution.
13
Figure 1
15000~-------------------------,
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h. V. gigas
•
10000
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•
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600 800 1000
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Figure 2
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Sulfide binding characteristics of blood serum in Calyptogena pacifica and Vesicomya gigas

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Sulfide binding characteristics of blood serum in Calyptogena pacifica and Vesicomya gigas

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