COMPARISON OF METABOLIC POTENTIALS OF COLD SEEP
CLAMS (GENUS CALYPTOGENA) OCCURRING IN VARYING
SULFIDE ENVIRONMENTS
Justin Dodds Holl
Abstract.- The metabolic potentials of two Monterey submarine canyon cold
seep clam species, Calyptogena kilmeri and C. pacifica, were compared using
two enzymes involved in ATP generation: citrate synthase (CS), used in
aerobiosis, and malate dehydrogenase (MDH), primarily used in anaerobiosis.
Although both species live in high sulfide environments, C. kilmeri experiences
a significantly higher sulfide concentration than C. pacifica (86 mM sulfide vs.
O.1mM sulfide). MDH and CS assays were conducted on foot, gill, mantle, and
adductor tissues from both species. Calyptogena kilmeri was found to have
lower CS activity and higher MDH activity than C. pacifica. The lower CS activity
in C. kilmeri suggests that the near anoxic environment caused by high sulfide
concentrations suppresses aerobic metabolism, favoring a high anaerobic
metabolic potential in this species.
Introduction
Sulfide-rich cold seep sites in the Monterey Submarine Canyon support
a chemosynthetic community that includes vesicomyid clams of the genus
Calyptogena. Living 1000 m below the water's surface in a highly reduced
environment devoid of sunlight, these clams depend upon endosymbiotic
bacteria, concentrated in their gills, to oxidize sulfide, and to use the energy to
drive the net fixation of CO, (Felbeck et al. 1985). This study focuses on
Calyptogena kilmeri and C. pacifica, the most abundant clam species in the
cold seep community. In a recent study, Barry et al. (Barry et al., accepted
1996) demonstrated that the distribution of these clams is closely related to
sulfide concentrations which range from »10 mM HøS at the source of the seep
to -O mM at the periphery of the seep site. Calyptogena kilmeri is found in areas
of high sulfide concentration towards the center of seepage sites, whereas C.
pacifica is found at lower sulfide concentrations towards the fringe of seepage
sites (see Figure 1). Interestingly, there are distinct limits to these distributions.
If sulfide levels exceed - 8 mM in the center of the seep, even C. kilmeri cannot
survive (Powell and Somero 1983), leaving a barren zone (Barry et al.,
submitted 1996). At places where sulfide levels are - O mM, no clams can
exist because there is not a sufficient source of energy. The gradient of sulfide
concentrations from the center of the seep (sulfide source) to the fringe of the
seep is so severe that the sulfide levels experienced by clams can vary from 10
mM to O mM in as short a distance as 3 m (Barry et al., accepted 1996).
This study was designed to determine whether different sulfide exposure
levels in the two species were correlated with differences in metabolic potential.
Because C. pacifica lives in a zone of lower sulfide concentration than C.
kilmeri, and because sulfide is the primary source of energy for these species,
hypothesized that C. pacifica would have a lower metabolic potential than
would C. kilmeri. To test this hypothesis, two metabolic enzymes were used as
indicators of metabolic activity.
This study shows that sulfide concentrations play only a partial role in the
zonation of these species in the Monterey Submarine Canyon, and indicates
that oxygen may be a key regulating factor in the distribution of the two
congeners.
METHODS
Samples were taken from the cold seep site known as Clam Field in the
Monterey Bay Submarine Canyon (see Figure 2) using the remotely operated
vehicle Ventana, operated by the Monterey Bay Aquarium Research Institute
(MBARI). The clams were collected from the canyon floor at a depth of
-1000 m by the mechanical arm of the Ventana. Once the clams were brought
to the surface, the height, length, and width of each were measured, and they
were held at 0°C until transported to a -70°C freezer at Hopkins Marine Station.
Six C. kilmeri and four C. pacifica were assayed for both MDH and CS
activity in the adductor, foot, gill, and mantle tissues. The protocol for the
preparation of the homogenate for each enzyme assay was similar. The
adductor, foot, gill, and mantle tissues were dissected on ice to retard protein
denaturation and subsequent loss of enzyme activity. Approximately 0.1 g of
each tissue were placed in separate Duall glass homogenizers. Each tissue
was diluted 10x by adding 20mM imidazole/CI (pH of 7.1 at 20°0) to each
sample. Once the tissues were homogenized, approximately 10 mL of each
homogenate were placed in an Eppendorf centrifuge tube and spun in a
Eppendorf Centrifuge 5402 at 4°C at 1400 rpm for 15 minutes. The supernatant
was decanted from each homogenate and stored on ice until assayed.
MDH activity measurements were made by adding 10 ul of enzyme
supernatant to 2 ml of assay solution (20.0 ml of 200 mM imidazole/CL (pH 7.1
at 20°0), 0.0015 g oxaloacetate, 0.0053 g NADH, deionized water to bring
volume to 50.0 ml) in a Varian or Perkin Elmer Lambda 3B model
spectrophotometer. Decrease in NADH concentration was recorded at 340 nm
on a Soltec Chart Model 1241 at a speed of 15 cm/min.
CS activity measurements were made by adding 50 ul of enzyme
supernatant to 2 ml of assay solution (25.5 ml of 50 mM imidazole/CI (pH 8.2 at
20°0), 3.0 ml of MgCl, in 50 mM imidazole/CI:, 0.012 g of 0.1 mM DTNB, 0.003
g of acetyl-Coenzyme A, total volume 30 ml; oxaloacetate solution: 0.053 g
oxaloacetate, 10 ml of 50 mM imidazole/CI (pH 8.2 at 20°0)) in a Varian or
Perkin Elmer Lambda 3B model spectrophotometer. Increase in DTNB
concentration was recorded at 412 nm on a Soltec Chart Model 1241 at a
speed of 10 mm/min.
Using known chart speeds and chart recordings, absorbance change per
minute was calculated and converted to IU/ g wet weight.
Results
To account for the disparity in adult size between spp. (mean length of
81mm for C. kilmeri, mean length of 37 mm for C. pacifica and no overlap of
sizes of the two populations) enzyme activity vs. size were plotted for each
tissue (adductor, foot, gill, and mantle) to test for a significant correlation
between size and enzyme activity. Linear regression analysis for these data
indicated no significant correlation between enzyme activity and size (data not
shown).
Enzyme activity data for MDH from the four tissues sampled in C kilmeri
and C. pacifica are shown in Figure 3. For MDH, the differences in enzyme
activity between C. kilmeri and C. pacifica, as measured by a model 1 anova
test followed by a Tukey HSD test, are significant at a-0.05 for the foot
(p=0.021) but not for the adductor, gill, or mantle (p=0.961, p=0.71, p=0.906).
As can be seen in Figure 3, MDH activity is higher in the foot of C. kilmeri than in
that of C. pacifica.
Enzyme activity data for CS from the four tissues sampled in C. kilmeri
and C. pacifica are shown in Figure 4. For CS, the differences in enzyme
activity between C. kilmeri and C. pacifica are significant for the adductor and
the mantle (p20.001, p=0.001), but not for foot or gill (p-0.992, p-0.501). As
can be seen in Figure 4, CS activity is higher in the adductor and mantle of C.
pacifica than in that of C. kilmeri.
Discussion
The hypothesis put forth in the introduction, that sulfide concentrations
control Calyptogena distribution, leads to the prediction that C. kilmeri should
have higher enzyme activity for both MDH and CS. However, the results of this
study show that this relationship does not hold. The inverse relationship
between CS and MDH activity in Calyptogena spp. suggests that the
relationship between sulfide concentration and metabolic potential is more
complex than initially thought, and indicates that C. kilmeri and C. pacifica may
pursue different metabolic strategies. Although MDH and CS are both
metabolic enzymes, they play different roles. Malate dehydrogenase is
primarily involved in anaerobiosis, whereas CS is involved in aerobiosis. This
difference in position in the metabolic pathway, combined with their different
activity patterns in the two spp. suggests that variability in environmental oxygen
levels plays a key role in species zonation.
Oxygen levels at the cold seep sites are very low (20.5 ml of Ö2/L sea
water vs. surface water oxygen levels of 6-7 ml of Ö2/L sea water) because the
cold seeps are within the oxygen minimum zone (ÖMZ). In addition, the high
levels of sulfide at the seep sites reduce the already low oxygen levels to even
lower concentrations. The combined effect of the ÖMZ and the reducing power
of sulfide results in O2 concentrations that vary from 0 ml of Ö2/L sea water in
the center of the seep to 0.5 ml of Ö2/L sea water where the concentration of
hydrogen sulfide is O mM.
Calyptogena kilmeri lives in a higher sulfide and lower oxygen
environment than C. pacifica, and has higher MDH activity indicative of the
anaerobic pathway and lower CS activity indicative of the aerobic pathway than
does C. pacifica. This suggests that C. kilmeri can out-compete C. pacifica in a
reduced environment, but that in a less reduced environment, the opposite is
true. Calyptogena kilmeri may be unable to survive at the low sulfide
concentrations that C. pacifica prefers, because C. pacifica has the ability to
elevate sulfide levels in blood serum by a factor of 10 to 60 times ambient
levels, while C. kilmeri can elevate sulfide levels by a factor of only 5 to 10
(Kochevar and Barry 1994). In other words, C. pacifica exploits the higher
oxygen environment by adapting to low sulfide levels. Similarly, one may
hypothesize that C. kilmeri has a more efficient mechanism for elevating internal
oxygen levels.
It may be simplistic to suggest that the only two factors controlling
Calyptogena distribution are sulfide and oxygen concentrations. This study fits
into the context of a theory suggested by Barry et al. regarding the relatively low
abundance of C. pacifica at seeps with high levels of sulfide. Barry suggests
that, assuming larval supply is sufficient, C. pacifica must either be 1) intolerant
of sulfide levels greater than -1mM, 2) inhibited by low levels of oxygen
postulated for central seep locations, or 3) excluded from high sulfide sites via
competition with C. kilmeri (Barry et al., accepted 1996). This study provides
evidence that C. pacifica is indeed inhibited by low levels of oxygen, and is not
able to compete with its congener, C. kilmeri, in near-anoxic environments.
Acknowledgments
Tam very grateful to the George Somero lab at Hopkins Marine Station
for their patience and zest for science, Jim Barry and Patrick Whaling for their
cooperation and interest in this study, Jim Watanabe for his statistical
assistance, and Peter Fields for the revision of this manuscript.
References
Barry, J.P., Greene, H.G., Orange, D.L., Baxter, C.H., Robison, B.H., Kochevar,
R.E., Nybakken, J.W., Reed, D. L., and C. M. McHugh. 1995. Biologic
geologic characteristics of cold seeps in Monterey Bay, California. Deep¬
sea Research (submitted).
Barry, J.P, Kochevar, R.E., and C.H. Baxter. 1996a. The influence of pore¬
water chemistry and physiology in the distribution of vesicomyid clams at
cold seeps in Monterey Bay: Implications for patterns of chemosynthetic
community organization. Limnology and Oceanography, (accepted).
Felbeck, H, Powell, M.A., Hand, S.C., and Somero, G.N. 1985. Metabolic
adaptations of hydrothermal vent animals. Biological Society of
Washington.., 6:263.
Kochevar, R.E. and Barry J.P. (1994) Physiology of vesicomyid clams from
Monterey Canyon cold seeps .Transactions, American Geophysical
Union „,75(3):203.
Powell, M.A., and Somero, G. N. 1983. Blood components prevent sulfide
poisoning of respiration of the hydrothermal vent tube worm Riftia
pachyptila -Science 219: 297-299.
Figure Legends
Schematic representation of a cold seep at the Clam Field site
Figure 1
showing spp. distribution relative to sulfide levels. High sulfide
levels are depicted as dark and low sulfide levels are depicted as
light.
Figure 2
Digital image of the Monterey Submarine Canyon showing the
location of the Clam Field site, where the samples were collected.
lmage from Jim Barry (MBARI).
Figure 3
Comparison of MDH activity of C. kilmeri and C. pacifica for
adductor, foot, gill, and mantle tissues including standard error
bars.
Figure 4
Comparison of CS activity of C. kilmeri and C. pacifica for
adductor, foot, gill, and mantle tissues including standard error
bars.
-3m
C. kilmeri
C. pacifica
+


Barren
Zone
Om

C. kilmeri
C. pacifca
*



High sulfide
Low sulfide

3m
2 7
Activity (I.U./g wet weight)


O
0
(
0.
(D
—:
O
D
2.0
16
0.5
O.O
Comparison of Citrate Synthase for
C. kilmeri and C. pacifica
C. pacifica 4
S. kilmeri


ADDUCTOR FOOT GILL MANTLE
Tissue
F