The Effect of Acclimation to Intertidal and Subtidal Conditions
on the Thermal Stability of Supernatant Lactate
Dehydrogenase From Two Species of Porcelain Crab (Genus
Petrolithes)
Rebekah Harrison
ABSTRACT:
In Monterey Bay, Porcelain crabs inhabit discrete vertical zones: Petrolithes
cinctipes is found intertidally and P. manimaculis is found subtidally. Differences in
maximal temperature and emersion time for each species are associated with their individual
vertical zonation. The thermal stability of lactate dehydrogenase (LDH) in a homogenate
supernatant from P. cinctipes is greater than that from P. manimaculis. Purification of LDH
removes this difference, causing the thermal stability of purified LDH from P. cinctipes to
decrease to that of P. manimaculis. Thus some extrinsic factor may influence the proteins
thermal stability. To determine whether the enzyme thermal stability could be altered,
acclimated crabs to various conditions of temperature and emersion. Temperature rose
between 8-15°C above ambient seawater temperature during the 5.5 hour emersion time in
one treatment, but did not change in the constantly immersed treatment. High levels of
mortality were only observed for P. manimaculis in the emersion treatment. Thermal
stabilities of LDH in homogenate supernatants did not change over a 36 day acclimation
period. Differences in LDH thermal stability between species persisted. These data suggest
that the mechanism causing the difference in stability between supernatant and purified
enzyme is a highly regulated and conserved trait which was not affected by conditions of the
experiment.
INTRODUCTION
The global distribution of marine organisms is influenced by a wide
variety of physical factors, with temperature playing a dominant role (Dalhoff
and Somero, 1993). In many areas of the marine environment temperatures
remain fairly constant over significant distances and periods of time. Along
the rocky intertidal, however, temperatures can vary drastically within
narrow spatial and temporal ranges. These temperature gradients, in
conjunction with a variety of other factors, have been shown to cause discrete
zonation patterns for numerous organisms (Stillman and Somero, 1996;
Jensen, 1995
Crabs of the genus Petrolisthes (Anomura: Porcellamidae) are a highly
speciose group of intertidal and subtidal organisms which have been found
all along the Pacific coast (Haig, 1960). Different species within this genus
have been shown to inhabit separate microhabitats based on both their
longitudinal and vertical distributions (Jensen, 1995). In the rocky intertidal
areas of Monterey Bay, two species of Petrolithes can be easily found: P
cinctipes, an intertidal species, and P. manimaculis, a subtidal species. Based
in their vertical distribution patterns, these species experience different
temperature and emersion patterns. Individuals of P. cinctipes have been
shown to encounter maximal temperatures ranging from 24-32°C and
significant periods of emersion (maximum of 6-8 hours, two times per day).
The more subtidal species, P. manimaculis, on the other hand, sees maximal
temperatures within the range of 18-19°C and experiences less emersion time
(maximum 3-5 hours once per day, and only during spring tides) (Stillman,
personal communication).
The difference in habitat conditions between these two species has
raised some interesting questions. Temperature can affect numerous aspects
of an organism’s physiology from its motility to its ability to reproduce. Many
of the physiological effects caused by temperature occur due to changes at the
level of protein structure and function. In order to better understand
temperature effects, research has focused on trying to elucidate some of the
factors that establish thermal tolerance differences between organisms.
Results have shown that small changes in both the cellular environment of
the protein as well as subtle differences in protein structure can have
significant effects on thermal stability. For example, Dahlhoff and Somero
(1993) demonstrated that molluscs of the genus Haliotis show protein
adaptations correlated with differences in habitat temperatures (Dahlhoff and
Somero, 1993). Previous studies have also investigated the thermal tolerance
of two species of Porcelain crab, P. cinctipes and P. manimaculis, as indexed by
the thermal stability of lactate dehydrogenase (LDH), a glycolytic enzyme. The
results of this work have shown two interesting phenomena (J.H. Stillman,
unpublished data) (Figure 1). First the thermal stability of LDH from P.
manimaculis is consistently lower than that from P. cinctipes. Second, there
is a difference in thermal stability of LDH between homogenate supernatants
and the purified enzyme. This difference was most pronounced for P.
cinctipes. The thermal stability of purified LDH from both species was
practically identical. (J.H. Stillman, unpublished data). These data suggest that
a change in the protein’s cellular surroundings, rather than a change in the
protein sequence itself, is responsible for the increased thermal stability of
LDH from P. cinctipes.
If a difference between the two species in the milieu surrounding LDH
is in fact causing the increased thermal stability of LDH from P. cinctipes,
perhaps this is a trait which is able to be manipulated by the organism based
on the conditions of its natural habitat. Because P. cinctipes and P.
manimaculis live in different habitats, studying the effect of their
environment on the thermal stability of LDH could provide some insight
into the method of stabilization found in P. cinctipes. We hypothesized that if
exposed to experimental environments similar to those seen in the natural
environment, that is P. cinctipes in simulated intertidal conditions and P.
manimaculis in simulated subtidal conditions, the thermal stability of LDH
from both species would remain unchanged by experimental manipulation.
Similarly, the thermal stability of LDH for P. cinctipes exposed to simulated
subtidal conditions would remain unchanged since the experimental
conditions would be less thermally stressful than the conditions it
experiences in its natural environment. However, for P. manimaculis
exposed to the increased stress of the simulated intertidal conditions, it is
possible that the thermal stability of supernatant LDH would increase. To
investigate this hypothesis, we examined the acclimation of thermal stability
of LDH in claw muscle supernatant from both species, by exposing specimens
to either subtidal conditions (continual immersion) or intertidal conditions
(periods of emersion).
MATERIALS AND METHODS
Specimen Collection and Acclimation
Crabs used in the study were collected during low tide on 9 April 1997
from Fisherman’s Wharf in Monterey Bay, California, and immediately
transported to Hopkins Marine Station, where they were held in flow
through aquaria until being placed into the experimental pools at 4:00 pm on
14 April, 1997. The pools were constructed from two hard plastic "kiddy
pools" (K-Mart). Each tank was supplied with an inflow of local seawater, and
a drainage site was constructed from PVC tubing. A two inch layer of gravel (2
cm in diameter) was placed on the floor to insure the retention of moisture
during emersion. To simulate natural conditions, rocks collected from the
intertidal region of Hopkins Marine Station were placed in each pool, fully
covering the gravel. Water level was regulated by valves controlling both
inflow and drainage. Overflow was prevented by drilling holes around the
upper rim of each pool.
Specimens were divided into the two experimental pools with 182 P.
cinctipes and 94 P. manimaculis per pool. An effort was made to maintain a
similar size distribution for each species. We simulated intertidal conditions
by draining one pool on a daily basis from 10:00 am until 3:30 pm, during
which time it was exposed to full sunlight. To simulate subtidal conditions,
water levels remained high in the other experimental pool. The crabs in each
pool were fed in the evenings on a semi-daily basis by dissolving two frozen
cubes consisting of a one to one ratio of seawater and fish pellet homogenate
(10.3 grams/tub)(Moor-Clark, Washington).
Specimens for analysis were collected, wrapped in aluminum
foil, and immediately frozen at -80°C and kept at this temperature until use.
Collection of specimens occurred on the following days of the experiment:
Collection (9 April), Day 1 (14 April), Day 4 (17 April), Day 8 (21 April), Day 15
(28 April), Day 22 (5 May), Day 29 (12 May), Day 36 (19 May). Five individuals
from each treatment pool were collected at each time point except for Day 29
and Day 36 when eight crabs from each pool were collected.
Temperature
Ten temperature measurements were made daily for each pool at 10:00
am and 3:30 pm with a hand-held electronic thermocouple (Omega). Under
rock temperatures were measured by attaching the themocouple to a thin
probe.
Thermal Stability of LDH
Frozen specimens were removed from the -80°C freezer and a single
claw was removed. Tissue from each claw was dissected out, weighed, and
homogenized in a ground glass homogenizer (Kontes-Duall) with six
volumes of 50 mM potassium-phosphate buffer pH 6.8 (K-Phos). All
procedures were carried out on ice. The homogenate was then centrifuged at
4°C for 20 minutes at 16000 g. The supernatant was removed, placed in a new
tube on ice, and its activity was assayed at 20°C in 2 ml of 150 uM NADH
(beta- nicotinamide adenine dinucleotide, reduced form) and 5 mM pyruvate
in 80 mM imidazole-Cl, pH=6.9 at 20°C (following Childress and Somero,
1979). Activity was assayed by measuring the change in absorbance of NADH
at 340 nm in a spectrophotometer coupled to a chart recorder. Change in
optical density per minute (ÖD/min) was calculated by measuring the first
linear portion of the recording for each assay (approximately the first 30-45
seconds). Samples with activities over .250 ÖD/min. were diluted to
approximately .200 ÖD/min. with K-Phos. Supernatant samples were then
aliquoted into 200 ul thin-walled tubes in volumes ranging from 45 ul to 100
ul, depending on the amount of supernatant available. Denaturation was
performed by placing the samples in a pre-heated thermal cycler at 70°
removing them at specific time intervals, and placing them on ice. For P.
manimaculis the denaturation time points were 0, 10, 20, 30, 40, and 60
minutes. For P. cinctipes the denaturation time points were 0, 10, 20, 30, 40,
60, 80, and 100 minutes. All samples besides the 0 time point were respun in
the refrigerated microfuge for 2 minutes after heating to remove the
precipitate that formed during heating. The thermal stabilities of each sample
were then measured in duplicate. Half-lives were calculated from regression
analysis of log % activity vs. denaturation time.(Figure 2) Analysis of variance
(ANOVA) tests using the half-life data were carried out using Systat 6.0. For
P. cinctipes the number of individuals for which thermal stabilities were
assayed varied (n=5: for the day of collection and Day 1 of the experiment, n-3
for Day 4 subtidal, Day 8 intertidal and subtidal, Day 22 intertidal and subtidal
and n-4: for Day 4 intertidal, Day 36 intertidal and subtidal), but in the study
of P. manimaculis the number of sampled individuals was always four.
Survivorship
Survivorship was determined by collecting the crabs remaining in the
experimental pools at the end of the 36 day treatment period. The number of
crabs remaining was compared to the number expected to remain from the
original numbers once the 36 specimens collected throughout the duration of
the experiment were accounted for. Data were analyzed by a G-test for
independence (Sokal and Rolf, 1995)
Dialysis
Crabs collected from Fisherman’s Wharf in Monterey at low tide on 10
May 1997 and frozen at -80wC were used. Tissue was dissected from multiple
individuals of each species (six individuals of P. manimaculis and three of P
cinctipes), combined, and homogenized in six volumes of K-Phos. The
homogenates were spun in the refrigerated centrifuge at 16000 g for 30
minutes and the activities of the samples were then measured. One ml of
each species' supernatant was transferred to an Eppendorf tube and placed in
the refrigerator. The remaining supernatant from each species,
approximately three ml total, was aliquoted into three separate lengths of
dialysis tubing, and dialyzed 2, 4, or 17 hours against two liters of K-Phosphate
buffer pH 6.8. Buffer was replaced with fresh buffer at the end of the two and
four hour time points. After dialysis, the supernatant was removed from the
dialysis tubing, transferred to an Eppendorf tube, and held on ice until LDH
activity was assayed.
After the removal of the final dialysis sample at 17 hours, all of the
supernatant solutions (0, 2, 4, and 17 hours time points) were aliquoted into
80ul volumes and denatured at 70°C in the thermal cycler, as outlined above,
for the tests of thermal stability. Comparisons of the half-life for each time
point of a given species against the half-life for 0 hours of dialysis for that
species were made.
RESULTS
Temperature
Temperatures measured at 10:00 am in both pools ranged between 10-
14°C throughout the duration of the experiment (Figure 3). In the subtidal
pool mean temperatures at 3:30 pm were extremely similar to those at 10:00
am. Mean temperatures in the intertidal pool, however, were greatly
elevated at 3:30 pm, after the 5.5 hour emersion period, as compared to 10:00
am temperatures (Figure 3). On four days maximal temperatures recorded in
the intertidal pool were above 28°C. This has been shown to be the
temperature at which 50% of P. manimaculis die (LT 50 for P. manimaculis)
(Stillman, unpublished data). On Day 33, the mean temperature at 3:30 pm
exceeded 28°C, but on no day did the mean temperature recorded reach 33°C
the LT 50 for P. cinctipes . Maximal temperatures exceeded 33°C only once
during the duration of the experiment. It should be noted, that the
temperatures recorded are characteristic of those that have been found in
natural subtidal and intertidal conditions (Stillman and Somero, 1996,
Hoffmann and Somero, 1995
Thermal Stability of LDH
The thermal stability of LDH from P. cinctipes was consistently higher
than for P. manimaculis throughout the experiment (Figure 4). Statistical
analysis (three factor ANÖVA of species, time, and treatment) demonstrated a
significant difference between species with a p value of less than 0.0001.
There was, however, no significant effect of acclimation treatments or
acclimation time on the thermal stability of LDH for either P. cinctipes or P.
manimaculis (Figure 5, 6). Similarly, comparisons made within species,
using two factor ANÖVAs, yielded no significant difference with either
acclimation treatment or time for either P. cinctipes or P. manimaculis
(Table 1).
Survivorship
A G-test of independence for recovery showed a significant interaction
between species and treatment (G-value after Williams' correction was equal
to 38.147, p=6.56x10-10). Examination of the data for non-proportionality
demonstrated that this significance could be attributed to a recovery of 15.52%
for intertidal P. manimaculis as compared to all other recoveries which were
approximately 90% or above. (Table 2)
Dialysis
The thermal stability of LDH in a homogenate supernatant for P.
cinctipes showed the highest stability after 2 hours of dialysis, as measured by
half-life relative to 0 hours (Figure 7). For both 2 and 4 hours of dialysis the
stabilities were elevated above that measured at 0 hours of dialysis. For P.
manimaculis all three (2, 4, and 17 hour) time points assayed showed an
increase in half-life relative to 0 hours of dialysis. None of the increases
noted for these assays, however, were to the same extent as that seen for
either the two or four hour time points of P. cinctipes. The final thermal
stability of LDH from P. manimaculis (6.96 minutes)was lower than that from
P. cinctipes (24.73 minutes).
DISCUSSION
Thermal Stabilities
Studies on proteins have demonstrated the existence of several
different mechanisms capable of causing changes in protein thermal stability.
On an evolutionary time scale, small changes in amino acid sequence of a
protein have been shown to affect protein thermal stability (Somero, 1995
Differences in the kinetic properties of proteins from congeners occurring in
different thermal habitats have provided evidence for variation of protein
structure based in adaptive response to environmental temperature. On a
much shorter time scale, animals have been shown to display differential
production of specific isozymes with acclimation conditions (Lin and Somero,
1994). Both of these mechanisms of adaptation, however, are at the level of
protein structure. Protein stabilization has also been shown to be affected by
the presence of other molecules within the proteins’ cellular environment
such as inorganic ions (Voordouw et al., 1976; Lara et al., 1990) and organic
solutes, as well as other proteins (e.g. heat shock proteins) (Somero, 1995)
We hypothesize that it is a difference in the cellular environments
surrounding LDH between the two species that is the mechanism causing the
thermal stability of LDH from P. cinctipes to be higher than that of P.
manimaculis . The inability to generate a change in thermal stability upon
acclimation does not discredit stabilizing solutes as a possible mechanism.
Instead, it suggests that whatever is used to elevate the thermal stability of
LDH was not affected by the organisms within the 36 day time scale of our
experiment. The consistency of our data throughout the duration of the
experiment seems to imply that thermal stability is a fairly conserved and
regulated feature. Whatever causes the difference in LDH thermal stability
between species is maintained despite changes in environmental conditions.
There is also the possibility that other factors of our experimental
design had significant effects on our results. It should be noted that each
condition (subtidal and intertidal) was found in only one experimental pool.
Without replication we were unable to use a nested ANÖVA analysis to
account for any underlying variation within our tanks. Furthermore, the
lack of replication meant that both species of crab were found together in a
single experimental tank. Use of a two factor ANOVA within species is,
therefore, not entirely applicable. However, one of the three factors, species
accounted for most of the variation present (Mean Square = 3500.364) which
might have overwhelmed the ability to note any variation attributed to the
other two factors of time and treatment. We ran two factor ANÖVAs despite
the issue mentioned above in order to determine how the variance was
partitioned between the remaining factors, treatment and time.
One factor that we hypothesized might have affected our ability to note
any acclimation affect was the experimental duration. While previous
studies of temperature acclimation suggest that 36 days (Lin and Somero,
1994) should be long enough to see an effect, there is the possibility that the
extension of the experiment would have demonstrated some change in
thermal stability of the LDH supernatant. We also noted during our
dissection procedures that the specimens we used were in different stages of
both reproductive and molt cycles. It is not known what effect molting or
reproduction might play in changing the intracellular environment of the
crabs. Another factor which might have had an effect on our ability to see any
significant change with acclimation was the fact that the crabs in this study
were fed a diet that was different from what they receive in their natural
environment. Porcelain crabs are not scavengers or predators, but rather are
filter feeders which use their maxillipeds to strain material from the water
(Jensen, 1995). If the crabs in the study were lacking a constituent in their
presence of a small stabilizing molecule in the supernatant of P. cinctipes.
The increase in thermal stability relative to 0 hours for both species is a point
of question and could possibly be attributed to experimental error. If the zero
time point is ignored, however, the noticeably larger decrease in thermal
stability of LDH for P. cinctipes over the course of dialysis is what would be
expected if some small molecule providing stabilization was being dialyzed
away. The final half-life for P. cinctipes is still noticeably higher after 17
hours of dialysis than that of P. manimaculis (6.96 minutes for P.
manimaculis compared to 24.73 for P. cinctipes) despite the similarity in
thermal stability of purified LDH from both species. This suggests that
repetition of the dialysis experiment should follow a longer time course.
Studies have also implied that some stabilizing molecules may bind tightly to
the protein itself and would, therefore, take longer to dialyze away (J.
Carpenter, personal communication.). Running the experiment with both a
larger sample size and for a longer duration will help to elucidate the effects
of other cellular constituents on the thermal stability of LDH from Petrolithes
specie:
In summary, the data that we gathered suggest that acclimation did not
occur within the time and duration of our experiment. Rather we noted a
conservation of protein stability throughout the time course of the
experiment. This high degree of regulation, however, does not seem to be
linked to a difference in protein sequence, but implies the use of some other
mechanism. What this mechanism is remains unclear. Although the basic
trend of our dialysis experiment (excluding the zero time point) suggests the
possible involvement of some low-molecular-weight solute, the change in
stability over time for P. cinctipes was hardly considerable. This absolute
stability measurement tentatively suggest that the use of low-molecular¬
weight molecules may not be the mechanism responsible for the differences
in stability between species. Despite the uncertainty of the underlying
mechanism, significant difference in the survivorship between species
among the simulated intertidal and subtidal pools suggests that P. cinctipes is
e
5
more suited for survival in areas with frequent emersion than P.
manimaculis.
LITERATURE CITED
Childress, J.J. and Somero, G.N. (1979). Depth-related enzymatic activities in
muscle, brain and heart of deep-living pelagic marine teleosts. Mol.
Biol. 52: 273:283
Conejero-Cara, F., Mateo, P., Aviles, F., and Sanchez-Ruiz, J. (1991). Effect of
Zn“2 on the thermal denaturation of carboxypeptidase B. Biochemistry.
30: 2067:2072.
Dahlhoff, E. and Somero, G.N. (1993). Kinetic and structural adaptations of
cytoplasmic malate dehyrogenases of eastern pacific abalone (genus
Haliotis) from different thermal habitats: biochemical correlates of
biogeographical patterning. J. Exp. Bio. 187: 137-150
Haig, J. (1960) The Porcellanidae (Brustacea Anomura) of the Eastern Pacific.
pp. 77-79, 90-94. Los Angeles: University of Southern California Press.
Jensen, G. (1995). To each his zone. Natural History. July: 27-30.
Lin, J.J. and Somero, G.N. (1995). Temperature-dependent changes in
expression of thermostable and thermolabile isozymes of cytosolic
malate dehydrogenase in the eurythermal goby fish Gillichthys
mirabilis. Physiol. Zool. 68 (1): 114-128.
Sillman, 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. J. Exp. Bio. 199: 1845-1855.
Stillman, J.H. personal communication. 199
Sokal, R. and Rohlf, F. (1995) Biometry. pp. 731-732. New York: W.H. Freeman
and Company.
Somero, G.N. (1995). Proteins and temperature. Annu. Rev. Physiol. 57: 43-68.
Voordouw, G, Milo C., and Roche, R. (1976). Role of bound calcium ions in
thermostable, proteolytic enzymes. Sepration of intrinsic and calcium
ion contributions to kinetic thermal stability. Biochemistry. 15:
3716:3723.
28

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FIGURE LEGENDS
Figure 1: Log percent average activity of purified versus supernatant LDH
from P. cinctipes and P. manimaculis . For supernatants, thermal stability of
LDH from P. cinctipes is significantly higher than that from P. manimaculis.
Thermal stability of LDH in a supernatant is greater than that of the purified
enzyme for both species. Error bars represent standard deviations. (P.
cinctipes n=2;P. manimaculis n-4)
Figure 2: The method behind the derivation for the half-life of an enzyme.
The line is representative of a linear regression of thermal stability data
plotted as log percent activity versus denaturation time in minutes.
Figure 3: Mean temperatures for both the intertidal and subtidal pools
recorded at 10:00 am, when both pools were immersed, and 3:30 pm, the end
of the emersion period for the intertidal pool. Temperatures in the subtidal
pool remained constant across time points, while the intertidal pool showed a
increase in temperature after emersion.
Figure 4: Thermal stability of LDH in a supernatant for P. cinctipes and P.
manimaculis represented as percent average activity versus time in minutes
that the supernatant was incubated at 70°C. The thermal stability of LDH
from P. cinctipes was consistently higher than forP. manimaculis. Error bars
represent standard deviations. (P. cinctipes n=5;P. manimaculis n-4)
Figure 5: Average half-life in minutes for LDH in supernatants of claw
muscle from P. cinctipes in both subtidal and intertidal conditions for all
time points assayed in the experiment (Days 1, 4, 8, 22, and 36). Error bars
represent standard deviations. (Day 1 n-5; Day 4 subtidal, Day 8 and Day 22 n-
3; Day 4 intertidal and Day 36 n-4)
Figure 6: Average half-life in minutes for LDH in supernatants of claw
muscle from P. manimaculis in both subtidal and intertidal conditions for
all time points assayed in the experiment (Days 1, 4, 8, 22, and 36). Error bars
represent standard deviations. (n-4 for all time points)
': Thermal stability of LDH in a supernatant of claw muscle after
gure 7
dialysis for both P. cinctipes and P. manimaculis. Thermal stability is
represented b
y half-life relative to 0 hours of dialysis for all assayed time
points (0, 2, 4, and 17 hours of dialysis)
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