Abstract
In this study I examined metabolic rates of Mytilus californianus located at the
upper and lower extremes of the Mytilus distribution zone in an exposed rocky intertidal
area at Hopkins Marine Station in Pacific Grove, CA. Mussels high in the zone spend less
time exposed to normoxic conditions as a result of longer emersion periods. They also
experience greater temperature fluctuations. Such abiotic stresses were expected to make
metabolic rates among high and low mussels different. Respiration (O2 consumption) and
malate dehydrogenase activities of gill tissue were used as indicators of metabolism. The
design of this experiment led to mussel acclimation over a period of 25 days. When
plotted against time, enzyme activities of low mussels showed a significant (p = 0.048
decrease in activity. Additionally, a significant (p = 0.014) decrease was observed in O,
consumption of high mussels. The unexpectedly rapid physiological plasticity shown by
M. californianus resulted in small sample sizes (enzyme n = 10, respiration n = 4) of
freshly collected individuals, which may have contributed to the non-significant
differences seen in high and low mussel MDH activities (p = 0.059) and respiration rates
(p = 0.74). It was also concluded from this experiment that MDH activity is not a good
predictor of respiration rate, possibly due to its role in both aerobic and anaerobic
metabolism.
1. Introduction
The rocky intertidal forms a variable environment where many specialized and
diverse species thrive. Abiotic stresses such as tides, wave splash and impact, desiccation,
food availability, and temperature establish steep gradients that affect how and where
different organisms can survive in this highly variable macro-habitat (Denny 1988). As a
result, intertidal species inhabit discrete vertical zones. Biochemical and physiological
processes have been shown to vary between congeneric species and even between distinct
conspecific populations residing at different heights (Pickens 1965, Hofmann & Somero
1995, Stillman & Somero 1996, Tomanek & Somero 2000).
The purpose of this experiment was to further explore the relationship between
physiology and vertical zonation in intertidal organisms. The experimental subject chosen
was a sessile species, Mytilus californianus. It is a common prey for predatory animals
(e.g. Pisaster ochraceous), making it a central species in community dynamics. Its range
extends from the Aleutian Islands southward to Baja California and the species is usually
found in the mid-intertidal zone of exposed coasts. Because of its mid-intertidal location,
populations of M. californianus living at the lower extreme of the vertical zone are
usually emersed only once a day during low tide. Conversely, individuals inhabiting the
upper region experience long emersion periods, rarely being entirely submerged. Thus,
upper mussels are more likely exposed to abiotic stresses such as desiccation, high
temperature, and hypoxia.
The physiology of high and low mussels was expected to differ as a result of the
different environmental conditions in their microhabitats. One possible scenario was that
high individuals, which have less time to feed and respire as a result of longer emersion
periods, compensate by having bursts of aerobic metabolism when exposed to normoxic
conditions (submerged). A second possibility is that low mussels are exposed to a richer
source of nutrients and maintain a metabolic rate that is higher than that of mussels living
higher in the distribution zone. Lack of literature and similar studies, however, made it
difficult to give either scenario, or any other, more credibility. Consequently, the
hypothesis here states only that metabolic rates of high and low mussels would be
different. The indices of metabolic rate measured were respiration (O2 consumption) and
activity of the enzyme malate dehydrogenase (MDH).
2. Materials & Methods
2.1. Animals
Mussels (Mytilus californianus) were first collected on May 8", 2001 from a
northwest facing rocky intertidal site at Hopkins Marine Station in Pacific Grove, CA.
Approximately fifty mussels, 50 to 60 mm in length, were obtained from the upper and
lower extreme at which M. californianus is found at that particular location. A second set
of 8 samples of the same size was taken on the 26" of May from the same location.
Shortly after sampling, the organisms were placed in large holding tanks with
continuously flowing fresh seawater.
2.2. Experimental procedure
Oxygen consumption rate was chosen because it is a good indicator of
metabolism and has been used in previous studies (Babarro et al. 2000). A modification
in this study was that dissected gill tissue was used instead of the entire organism. This
was done because mussel gills have been found to continue respiring after removal. It is
also a quicker and more efficient method because one does not have to wait for mussels
to gape.
Respiration was measured in a sealed, water-jacketed respirometer at 15°C using
excised gill tissue. An oxygen sensitive electrode was attached to a Powerlab unit that
allowed for collection and analyses of data on a computer.
Each respiration run was began by assembling the chamber, which consisted of a
stirbar, platform, and mesh pieces (used to hold gill tissue in place). The chamber was
rinsed with filtered seawater (FSW) containing 15 mg of Penicillin G and 15 mg
Streptomyocin per liter and then filled to capacity with more FSW. The system was
allowed to thermally equilibrate. Meanwhile, calibration of the electrode took place.
Dissection of the mussel gill was done as follows: the bivalve was opened by cutting
the anterior and posterior adductor muscles, followed by carefully cutting and removing
the gills from both valves. These were then placed in a petri dish filled with FSW
containing antibiotics and washed twice with FSW. One gill was transferred to a beaker
of FSW and placed in a water bath at 15°C. The other gill was kept in FSW on ice for use
later in the enzymatic analysis.
Once the chamber had reached 15°C it was purged of any air bubbles present. The
gill tissue was carefully placed inside the mesh attached to the platform, making sure that
the gill was securely in place. Not doing so resulted in the gill floating up and touching
the electrode tip, which interfered with the electrode's measuring capability. The
chamber was filled to the point of overflow and any newly introduced bubbles were
removed. The lid was then placed on the chamber and screwed in place. It was important
to make sure no air bubbles were present because they could have oxygenated the water
resulting in erred measurements.
The electrode was secured in place and recording was begun. Each gill tissue was
measured for 30 minutes and then the wet weight of the gill was determined. Äfter the
gill was removed, the chamber was refilled with FSW and resealed to quantify
background respiration by bacteria. Control run measurements were recorded for 20
minutes.
Every day the chamber was cleaned with a 10% bleach solution for 10 minutes.
Any remaining bleach was deactivated with a 1% sodium thiosulfate solution. All parts
were rinsed with deionized water and laid out to dry overnight.
2.3. Analysis
Activities of MDH were measured because it is a simple enzyme to assay. This
was an important consideration due to time constraints. There are limitations, however, to
the use of MDH because it is an indicator of aerobic and anaerobic ATP generating
capacity. See Figures 1 and 2 for the specific role of MDH in both metabolic pathways
Therefore, the results obtained from MDH were closely examined and plotted against
respiration rates to see if indeed this enzyme was a good aerobic metabolic indicator.
The reaction MDH catalyzes is:
Oxaloacetatic acid + NADH + H — Malate + NAD
Although the substrate is oxaloacetic acid (OAA), the conversion of NADH to NAD is
actually what was measured over time. The reason for this is that these two compounds
have very different absorbances at 340 nm. Therefore, they provide a good means by
which to perform a colorimetric assay.
Malate dehydrogenase activity was measured spectrophotometrically at 340 nm at
15°C. The first step involved preparing a cocktail containing 50 ml of 200 mM imidazole
buffer (pH 7.0 % 20°C), 0.0054 g NADH (final conc. = 0.15 mM), and 0.0014 g OAA
(final conc. = 0.2 mM). This cocktail was prepared daily and kept on ice throughout the
experiment because NADH and OAA degrade over time. The gill tissue was then
weighed wet, minced, and homogenized in a five-fold dilution of 200 mM imidazole
buffer. Finally, the homogenate was transferred to an eppendorf tube and centrifuged at
14,000 RPM for 5 minutes at 4°C.
Spectrophotometer readings were taken using cuvettes filled with 2 mL of
cocktail. To initiate the reactions, homogenate samples were transferred into cuvettes
with a 25 uL Lang-Levy pipette. Reaction data were recorded for 30 seconds. Five
replicates were taken for each sample of tissue.
2.4. Calculations and statistics
Respirometry readings were converted from slopes describing decreases in O2
concentration over time to umol O2s g" wet wt. However, only the set of data points
from 10 to 30 minutes were used. The reason for using only these data was that the first
10 minutes usually gave high values that were inconsistent with the rest of the data.
Control slopes (10 to 20 minute data points) were first subtracted from experimental
slopes and then converted using the equation:
(chamber volume) * (A O2 concentration/100) * (O2 solubility) = X umol O2;s
This value was then divided by the wet weight of the gill tissue.
MDH results were standardized from A Absorbance/min to umoles OAA
converted to malate per minute per gram of wet tissue. The first step was to convert A
Absorbance/min to units of activity, which equals (A Absorbance/min * 2.0)/6.22. The
value of 2.0 represents the amount of cocktail placed in the cuvette and 6.22 is the
micromolar extinction coefficient for NADH. This value was then divided by the wet
weight of the gill tissue, which had been homogenized in a 5X dilution of buffer. This
meant that in every mL of homogenate there was 0.2 g of tissue (it was assumed that a
gram of tissue had a volume of 1 mL). Because 25 uL of homogenate were added to each
cuvette, every reaction had a total of 0.005 g of tissue. Thus, activity values were divided
by this value. To obtain final values for a given gill, the five replicate values were
averaged.
Respiration rates and enzymatic activities of freshly collected high and low
mussels were compared using one-way ANÖVA tests. Values of mussels that had been
placed in holding tanks for more than one day were graphed over time and the resulting
slopes were tested to see if they were significantly different from a slope of O. All
calculations were done using Microsoft Excel.
3. Results
3.1. Respiration rate
Freshly collected low M. californianus (n = 2) had oxygen consumption rates with
an average of 0.00141 + 0.00027 umol O2 sec" g" wet wt. (mean + S.E.M.), while
freshly collected high mussels (n = 2) had an average of 0.00126 + 0.00029 umol O2 sec
gwet wt. These averages were not significantly different (p = 0.74). (Fig. 3)
During the 25 days of the experiment, low and high mussels held in tanks with
continuous flowing seawater exhibited a decrease in oxygen consumption, as can be seen
in Fig. 4. The change observed in low M. californianus was significant (p = 0.014)
whereas the change observed in high mussels was not (p = 0.126)
3.2. Malate dehydrogenase activity
The activity average for freshly collected high mussels (n = 5) was 40.088 + 1.44
umoles OAA min" g wet wt. Average for fresh low mussels (n = 5) was 34.032 + 2.35
umol OAA min" g’ wet wt. Similar to respiration rate averages, these were not
significantly different (p = 0.059). (Fig. 5)
Over time, MDH activities also showed acclimatory changes. High mussels
showed a significant (p = 0.048) decrease in activity during the 25 days they were held.
An insignificant (p = 0.147) increase in activity, however, was noted in low mussels. See
Fig. 6.
3.3. Respiration rates vs. MDH Activity
When plotted against each other (Fig. 7) oxygen consumption rates and enzyme
activity results showed that MDH was not a good predictor of aerobic metabolic rate.
4. Discussion
The design of this experiment led to acclimation by Mytilus californianus, which
confounded the results and did not allow me to conclusively answer the question of
whether or not a metabolic difference exists between low and high intertidal mussel
populations. Rather, this experiment strongly suggested that M. californianus is a plastic
organism capable of modifying its physiology over a period of 25 days in response to
environmental factors. This experimental design artifact resulted in small sample sizes of
freshly collected organisms, which may explain the non-significant differences between
low and high mussels. The results for fresh high (n = 5) and low (n = 5) mussel MDH
activity were very close to being significant (p = 0.059). It is important to note, however,
that because both indicators of metabolic activity showed significant convergences over
time it is only logical that an initial difference exists between low and high M.
californianus populations.
The observed decrease in oxygen consumption rate of low mussels indicates a
decline in metabolic rate. A speculative, yet logical, explanation for this observation is
that low M. californianus were removed from their natural habitats, where they are
normally submerged and exposed to a rich source of nutrients, and placed in holding
tanks where food may have been less. This conjecture is supported by Dahlhoff and
Menge’s (1996) finding that near shore food availability is strongly correlated to
metabolic activity. Their results showed that mussels living at a wave-exposed site with
relatively high phytoplankton concentrations had higher citrate synthase and MDH
activities than organisms living at a wave-protected site with low phytoplankton
concentrations.
As explained in the introduction, MDH is involved in aerobic as well as anaerobic
metabolism. The results attained in this experiment seem to be better explained by the
enzyme's role in anaerobiosis. Because high mussels have longer emersion periods they
also have longer time spans in which they must close their valves to prevent desiccation.
This forces high organisms to switch over to anaerobic metabolism for extended periods
of time, resulting in a reliance on MDH for producing ATP under oxygen-limited
conditions. Thus, when high M. californianus are placed in an environment where they
are continuously submerged, as it was in the holding tanks, one would expect to see a
decrease in the enzyme’s activity because the need for generating ATP anaerobically is
reduced. This is what was indeed observed.
The preceding argument that MDH activities measured in this experiment were
largely activities of anaerobic metabolism would also explain why such poor correlation
was seen between respiration rates and enzyme activities.
In the future I think it would be interesting to repeat this experiment using a larger
number of freshly collected samples from more than one site to control for any possible
location factors. It may also be interesting to use entire mussels for respiration
measurements as opposed to only gill tissue. Perhaps doing a long-term study would
reveal seasonal variation in these two M. californianus populations. Another possible
experiment would be to use an enzyme involved only in aerobic metabolism, such as
citrate synthase. Finally, I think it is worth designing an experiment in which mussels are
exposed to abnormally long periods of emersion and heat stress and then tested for MDH
activities. This would show whether or not MDH activity measurements in Mytilus
californianus are actually from anaerobic metabolism.
5. Conclusions
This experiment illustrated that Mytilus californianus is a physiologically plastic
organism capable of altering its metabolic rate over a period of 25 days, as shown by a
decreasing respiration rate in laboratory-acclimated mussels from low sites in the
intertidal zone, and a decrease in MDH activity in laboratory-acclimated high organisms
The results did not, however, indicate metabolic rate differences between freshly
collected high and low populations of M. californianus. Additionally, the two indices
used to measure metabolic rate, MDH activity and respiration, showed no correlation.
6. Acknowledgements
1 am grateful to Peter Fields for his help with the MDH assays and overall support
and advice; Karen Braby and Eric Sanford for their help with the respirometry
measurements; George Somero for all the guidance, advice, and the idea of this project;
Jim Watanabe for help in analyzing data; and Jason Podrabsky for his help editing this
manuscript. In closing I would like to thank the rest of the Somero lab for making this
experience enjoyable and all the 175H Professors for the introduction into the world of
research.
7. Literature Cited
Babarro, J.M.F., Fernandez-Reiriz, M.J., and Labarta, U. 2000. Metabolism of the mussel
Mytilus galloprovincialis from two origins in the Ria de Arousa (north-west
Spain). Journal of the Marine Biological Association of the United Kingdom. 80:
865-872.
Dahlhoff, E.P. and Menge, B.A. 1996. Influence of phytoplankton concentration and
wave exposure on the ecophysiology of Mytilus californianus. Marine Ecology
Progress Series. 144: 97-107.
Denny, M.W. 1988. Biology and the Mechanics of the Wave-Swept Environment,
Princeton University Press, Princeton, NJ
Hofmann, G.E. and Somero, G.N. 1995. Evidence for protein damage at environmental
temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the
intertidal mussel Mytilus trossulus. The Journal of Experimental Biology. 198:
1509-1518.
Pickens, P. E. 1965. Heart rate of mussels as a function of latitude, intertidal height, and
acclimation temperature. Physiological Zoology. 38: 390-405.
Stillman, J.H. and Somero, G.N. 1996. Adaptation to temperature stress and aerial
exposure in congeneric species of intertidal porcelain crabs (genus Petrolisthes):
correlation of physiology, biochemistry and morphology with vertical
distribution. The Journal of Experimental Biology. 199: 1845-1855.
Tomanek, L. and Somero, G.N. 2000. Time course and magnitude of synthesis of heat-
shock proteins in congeneric marine snails (genus Tegula) from different tidal
heights. Physiological and Biochemical Zoology. 73(2): 249-256.
8. Figure Legend
Fig. 1. Aerobic metabolism, indicating role of MDH.
Fig. 2. Anaerobic metabolism, indicating role of MDH.
Fig. 3. Average respiration rates of high and low freshly collected mussels. Error bars
are standard errors of the means.
Fig. 4.
Respiration rates of laboratory-acclimated mussels plotted against the number
of the day the sample was tested.
Fig. 5. Average enzyme activities of high and low freshly collected mussels. Error bars
are standard errors of the means.
Fig. 6.
Enzyme activities of laboratory-acclimated mussels plotted against the number
of the day the sample was tested.
Fig. 7. Respiration rates of high and low mussels plotted against enzyme activities of
high and low mussels.
Fig. 1.
GoP
pyluvate
----.
AcetylCoA
oxoloocelote
citrate
cis-ôconitate
malate
isodit

fumarate
a-ketoglutarate,
A
0
succingte
succinylCoA
21 12 21 124
24
S
NV
NAD
ADP-P;—
LAIEI
flovoprotein
cytochrome b
ADP-P,—
cytochrome c
cylochrome a
ADP.PAT
2H++0,
HO
ELECTRON TRANSFER CHAIN
C
Fig. 2.
GLYCOGEN
P.
GP
GLUCOSEG6P
ATP ADP
ATP¬
ADP—
FBP
GAP
-NAO+ 4-----
-----
---
NA
NADHA
—NADH
3DPG
— ADP
— ATP
3964
2P6A
PEF
— ADP
— ATP
osportote
pyruvote
oetycoa
NADH
oxoloocetate
aloniney
NADH
ethono
alanopine
NAD.
strombine
ocetote
molote
lysopine
octopine
fumorgte
-ADP
loctote
succinoreATP
— ADP
— ATP
propionote
NAD
Fig. 3.
0.0018
0.0016
0.0014
5 0.0012
0.001
0.0008
5 0.0006
O.0004
0.0002
Mean Respiration Rates of Fresh Mussels
p=0.74
(2)
(2)
High
Low
Fig. 4.
0.0018
0.0016
0.0014
0.0012
0.001
0.0008
0.0006
0.0004
0.0002
Respiration vs. Day Äfter Collection
Low R° = 0.7299
P=0.014
...
..

High Mussels
High R“ = 0.403
Low Mussels
p-0.126
Linear (Low Mussels)
- - - Linear (High Mussels
10
20
30
Day
Fig. 5.
45
40
o 35
5 30
20
01
Mean Fresh Mussel Enzyme Activity
p=0.059
(5)
Low
High
Fig. 6.
40
38
36
- 34
§ 32
§ 30
5 28
26
5 24
+ 22
20
Enzyme Activity vs. Day after Collection
High R“ = 0.574

p-0.048
High Mussels

Low Mussels
- - - Linear (High Mussels
—Linear (Low Mussels)
LOW R2 = 0.3704
p=0.147
20
30
Day
Fig. 7.
0.0018
50.0016
3 0.0014
NO.0012
0
5 0.001
0.0008
50.0006
8 0.0002
Respiration vs. Enzyme Activity
P2 5 00518

R?=0.4753

30
Activity (umol OAA min g*)
High Mussels
Low Mussels
Linear (Low Mussels)
- - - Linear (High Mussels)
20
40