Heat-Shock Proteins in T. californicus • 2
Abstract
The supralittoral harpacticoid copepod Tigriopus californicus is
subject to a wide variety of external stressors. Animals were
collected from splash pools at Mussel Point, Hopkins Marine Station,
Pacific Grove, CA during May 1994 and flash frozen at poolside to
examine the expression of HSP60 heat-shock proteins. No correlation
was found between HSP60 expression in animals and splash pool
salinity, suggesting acclimation through osmoregulation. Differential
expression of HSP60 between T. californicus populations, however,
was noted.
Introduction
The harpacticoid copepod Tigriopus californicus, an inhabitant
of splash pools at or above the high-tide mark, is subject to wide
and largely unpredictable variations in temperature, pH, salinity and
water loss. As the high splash pool environment is influenced by
erratic meteorological and tidal disturbances, T. californicus must
exhibit great tolerance to withstand the daily and seasonal
variability of its habitat. The organism is thought employ
homeostatic, or at least enantiostatic, mechanisms to enhance its
survivability. Recently, the induction of heat shock proteins has been
shown to occur in T. californicus in response to extremes of
temperature and salinity in the laboratory, suggesting that such
proteins may increase the organism's tolerance to adverse
environmental conditions in its natural habitat (Ha, 1993; McEvoy,
1993)
The heat-shock response, despite its somewhat limiting name,
may be elicited by a wide variety of stressors, including anoxia,
ethanol, heavy metals, amino acid analogues, inhibitors of oxidative
Heat-Shock Proteins in T. californicus
phosphorylation, thermal and osmotic fluctuations, and even viral
infection (Craig, 1985; Morimoto, et al., 1990). Upon application of a
stressor, cultured cells and tissues have been shown to respond by
preferentially synthesizing a set of proteins, known as the heat-
shock proteins (HSPs), while the synthesis of most other proteins is
inhibited or significantly decreased. Production of HSPs may be
greatly intensified or even induced de novo, with physiological
stress, and thus the heat-shock proteins are identified by their
markedly enhanced synthesis, compared to controls, after
perturbation (Lindquist, 1986).
Furthermore, it appears that the induction of HSPs coincides
with the acquisition of resistance to severe stress. In yeast,
Saccharomyces cerevisiae, a rapid shift in culture temperature from
23 to 36°C resulted in protection of cells from death at 52°C, a
normally lethal temperature for yeast cells (Craig, 1985). Moreover,
studies have found that the kinetics of thermotolerance induction
and decay are closely correlated with the kinetics of heat-shock
protein synthesis and degradation, suggesting that the synthesis of
HSPs is the crucial element in the acquisition of tolerance (Lindquist
1986). In the harpacticoid copepod Tigriopus brevicornis, high
salinity acclimation was found to enhance the survivability of
individuals in a broader range of temperatures (Damgaard and
Davenport, 1994). Though the physiological basis for this
heterologous tolerance has not been elucidated, it is reminiscent of
the numerous stressors capable of inducing the heat-shock response.
The stress protein response has been observed in all organisms
investigated thus far. Immunological studies have shown heat shock
proteins to be among the most highly conserved proteins in nature
Though differences exist
(Morimoto, et al., 1990; Lindquist, 1986).
amongst various organisms, for example in the number of heat-
shock proteins and their molecular weights, such conservation in
protein sequence suggests that either stress is a universal experience
of living creatures, or that the proteins play an essential role in
normal physiological processes (Morimoto, et al., 1990; Huey and
Bennett, 1990). Yet, while it generally assumed that the HSPs protect
• 3
Heat-Shock Proteins in T. californicus
cells from adverse environmental effects, the mechanism of their
action remains unclear
Given the demonstrated ability of T. californicus to tolerate a
wide range of adverse environment conditions, it seems appropriate
to examine the role the heat-shock response plays in situ. Variation
in the heat-shock response was examined at the level of protein
synthesis for several unique populations of T. californicus, collected
from high tide pools along Mussel Point, Hopkins Marine Station in
Pacific Grove, California to provide further insight into the ecological
and physiological significance of the stress protein response.
Materials and Methods
Collection of Tigriopus californicus. Animals were collected with a
small strainer consisting of 100um nylon mesh and PVC pipe from
high tide pools along the west side of Mussel Point at Hopkins Marine
Station in Pacific Grove, California. Animal samplings from each pool
were placed in 17 X 100mm polypropylene test tubes, capped, and
dropped into a Dewar flask filled with liquid nitrogen for flash
freezing. Rock pool temperatures were measured with a Yellow
Springs TeleThermometer. Salinity measurements in parts per
thousand (%/00) were performed with a American Optics portable
refractometer. Water samples from each pool were collected in 35 X
45 mm containers with lids and pH was measured with a Beckman
940 pH meter within 30 minutes.
Collection and maintenance of control animals. Animals were
collected with a small strainer (see description, supra.), placed in a
large beaker containing sea water and carried back to the laboratory
Animals were maintained in Instant Ocean artificial seawater
(Aquarium Systems, Mentor, ÖH) at 32%/00 or 60%00 and kept in a
laboratory incubator with a 12hr light: 12hr dark cycle at
approximately 23°C. Animals were fed ground Tetramin (Tetra Sales,
Morris Plains, NJ) three times per week and were acclimated for at
least 48hr prior to experiments.
• 4
Heat-Shock Proteins in T. californicus • 5
Isolation of soluble protein. Prior to protein extraction, flash frozen
animals were thawed on ice. Animals were then washed with 2L
UV-filtered sea water. Protein samples from each T. californicus
population were obtained by grinding whole animals, without regard
to sex, in a 2ml Teflon-glass homogenizer (Wheaton Instruments,
Millville, NJ) with 200ul homogenization buffer (10ml PBS in lOmM
EDTA, 50ul PMSF, 1Oul pepstatin, 1Oul leupeptin, 100ul chymostatin,
50ul NP-40). Homogenate was centrifuged for 10 minutes at 15000
rpm and supernatant removed. Protein concentrations were
determined by the method of Bradford using the MicroAssay
protocol developed by Pierce Laboratories, Inc. (Rockford, IL).
Quantifications were calculated from readings at 595nm from a
Beckman DU-7 spectrophotometer. Proteins samples were then
mixed 1:1 in 2x solubilizing buffer (8M urea, 4% SDS, 100mM tris,
0.01% bromophenol blue) with dithiothreitol and boiled for 3
minutes. Samples were either used immediately or stored at -20°C.
Separation of proteins. Proteins were separated by one¬
dimensional sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) on 10% minigels (8 X 7 cm) of 1.5mm
thickness according to the procedure described by Laemmli (1970)
Resolution was at a constant current of 25mA per gel at room
temperature. Pre-stained molecular weight standards (Sigma, St.
Louis, MO.) were loaded onto each gel for the determination of
molecular weights of proteins. Gels not destined for electrophoretic
transfer were subsequently stained with Coomassie blue for at least
3 hours and destained overnight in 10% isopropanol and 10% acetic
acid.
Electrophoretic transfer. Electroblotting of proteins to Immobilon-P
PVDF transfer membranes (Millipore, Bedford, MA) was performed
according to the procedure described by Burnette (1981) with slight
modifications. Following electrophoresis, stacking gel was removed
and the resolving gel and two pieces filter paper (Whatman,
Hillsboro, OR) were soaked for approximately 5 minutes in transfer
Heat-Shock Proteins in T. californicus • 6
buffer (49.6 mM tris, 384mM glycine, 20% methanol, 0.01% SDS )
Immobilon was soaked approximately 5 minutes in methanol. Gel
and Immobilon were mounted in a Hoeffer TE22 transfer apparatus
between the two pieces of Whatman filter paper. Transfer was
carried out at 25mA constant current for 12 hours at 4°C.
Immunostaining. Äfter transfer, Immobilon membranes were
removed from the gel and blocked with TBS/Milk/Tween (60ml 5X
TBS, 15g Carnation nonfat dry milk, 0.3ml Tween20) for 1 hour at
37°C. Blots were subsequently washed with TBS/Tween (IX TBS, 0.5%
Tween) for 15 minutes. Probing occurred with Rabbit Anti-HSP60
polyclonal antibody, developed against Synechococcus sp.
(cyanobacteria) (StressGen, Victoria, B.C., Canada) in 1:1000 dilution
in TBS/Milk/Tween at room temperature for 1 hour with rapid
rocking. Following primary antibody incubation, blots were rinsed
for 10 min in IX TBS, 10 min in TBS/Tween and 10 min in IX TBS
Secondary antibody probing occurred with anti-rabbit IgG (whole
molecule) peroxidase conjugate (Sigma, St. Louis, MO) diluted 1:1000
in IX TBS for 1 hour with rapid rocking. Äfter rinsing, blots were
incubated in Renaissance wester blot chemiluminescence reagents
(DuPont NEN, Boston, MA) for 1 minute and imaged on Kodak X¬
OMAT AR film.
Densitometric analysis. Wet gels and X-ray photographs of
immunoblots were scanned by transmittance with a green filter
using a Pharmacia ImageMaster DTS and analyzed with Pharmacia
ImageMaster Software (Version 1.0) according to prescribed
methods.
Results
In the field at Mussel Point, salinity in the high tide pools
inhabited by Tigriopus californicus varied from 15 to 88%00 during
the period of experiments. The most saline rock pool (88%0) in
which T. californicus was found completely evaporated after
Heat-Shock Proteins in T. californicus
approximately 1 week of observation. An independent population of
T. californicus from this pool did not revive after the next rainfall, as
the subject pool became networked with a neighboring pool of lesser
salinity.
Temperatures recorded in pools inhabited by T. californicus
ranged from 12.1 to 27.2°C. Low pool temperatures generally were
produced rapidly by rare periods of high wave activity, which
happened to largely wash out high rock pools long detached from the
sea. During these periods of high tides, salinity in the various pools
was noted to return rather quickly to that of the ocean (ca. 33%00)
High tides also had the effect of depleting the population of T.
californicus in some pools, but most populations appeared to recover
within several days.
Figure 1 shows a immunoblot, probed for HSP60, for several
unique populations of T. californicus after equal loading for total
protein (20ug). Laboratory controls at 32%00 and 60%0 appear in
lanes 1 and 2, respectively, numbered left to right. Lanes 3 through
8 represent T. californicus flash frozen at poolside for various splash
pools, with salinities ranging 33 to 78.7%0, increasing left to right
along the blot (see Table 1). Note that neither temperatures nor pHs
were constant during these samplings, but these parameters were
deemed not significantly different as salinities. Prominent reaction
of the anti-HSP60 polyclonal antibody occurs with protein(s) of
approximately 58kD. Note some anti-HSP60 cross-reactivity occurs
with lower molecular weight proteins in certain populations,
particularly in lanes 3 and 6.
Densitometric analysis of the 58kD bands is shown in Figure 2.
Trace densities (O.D. X mm) for these bands are listed in Table II.
Trace densities are linearly proportional to quantity of antigen
present, provided antigen-antibody interactions are quantitative in
the presence of excess antibody (Fernández and Kopchick, 1990).
Protein in lanes 6 and 8 were obtained from T. californicus in the
same rock pool, sampled at different times—when salinities were
measured at 60% and 78.7°/00, respectively. It appears that
significantly more HSP6O is produced in the T. californicus population
from this pool, over control levels and over levels obtained for other
Heat-Shock Proteins in T. californicus • 8
populations. Lanes 4 (43.2%0), 5 (51.9%0), and 7 (78.7%00),
representing animal samples obtained from unique splash pools,
have approximately equal levels of HSP60 protein(s) at 58kD. Lane
3, corresponding to a sampling from a 33%00 rock pool, appears to be
somewhat anomalous, perhaps due to underloading.
Discussion
The levels of HSP60 production under increased salinities in in
situ populations of Tigriopus californicus were found to be different
than those determined by acute salinity shock experiments
performed in the laboratory by Ha (1993) and McEvoy (1993). Ha
found that when T. californicus was acclimated at 16.5%0 artificial
seawater for 24hrs and then immediately transferred to 66%0
seawater, dramatic induction of HSP60 and HSP7O occurred. Ha
further noted that the level of heat-shock proteins in individuals
declined to "pre-shock" levels after approximately four hours at
66%o. In concordance, Hakimzadeh and Bradley (1990) found a
gradual return to normal (non heat-shock) proteins after severe heat
shock in the copepod Eurytemora affinis. Since the heat-shock
response appears to be transient, differences in findings may be
related to acclimation time. In the wild, animals are rarely, if ever
subjected such rapid hyperosmotic shock, as evaporative processes
occur over a much greater time scale.
Since approximately equal
amounts of HSP6O at 58kD were found among the different
populations at varying salinities (Pool Z, 43%0; Pool D, 51%00; Pool Y,
72%/0), it may be that T. californicus copes with a hypersaline
environment without maintaining higher basal levels of HSPs. An
extension of this finding is the prediction that species which live and
function in more stringent habitats (such as hypersaline
environments) have higher thresholds for HSP induction.
Though studies have shown that the kinetics of
thermotolerance induction and decay closely coincide with the
kinetics of heat-shock protein synthesis and degradation (Lindquist,
1986), there is some evidence that tolerance is independent of HSP
Heat-Shock Proteins in T. californicus
induction. Easton, et al. (1987) found in the salamander Eurycea
bislineata that heat hardening (the ability to withstand higher
maximum temperatures with repetitive treatments) occurred
regardless of whether HSP synthesis was induced. Thus it appears
that acclimation to stringent conditions may be independent of heat
shock protein synthesis.
The source of T. californicus euryhalinity may then be related
to its ability to osmoregulate its intracellular solute pool. Burton and
Feldman (1982) found that during hyposmotic stress, the
concentration of free amino acids within the body homogenate of
individuals significantly decreased in 3 to 5hrs. Under hyperosmotic
stress, free amino acid concentration was found to increase within
3hrs. Remarkably, as little an increase as 2%/00 has been shown to be
sufficient to induce proline synthesis in animals acclimated to
seawater at 16.5%0 (Burton, 1991). Thus in the wild, where gradual
salinity changes are commonplace, it appears that T. californicus may
maintain homeostasis by osmoregulation rather than by exhibiting a
stress protein response.
This does not explain, however, why some populations of T.
californicus exhibit consistently greater concentrations of heat-shock
Animal populations in Pool X, sampled during
proteins than others.
two distinct periods in this study (at 60%00 and 78.7°/00), appeared to
express more HSP60 than other pools. The reasons for this are
unclear. Perhaps the inhabitants of this pool, as a distinct population
with unique genetic characteristics, simply express higher basal
HSP60 expression. Additionally, we may not rule out the possibility
that increased synthesis is the result of a stressor for which we did
not account. However, this latter possibility seems unlikely, given
Variation in
Pool X’s proximity to other pools included in this study.
the level of HSP60 production was also observed between control
animals at 32%0 and 60%/00, obtained from different populations.
Comparison of stained gels for the various populations indicates
that protein composition varied somewhat from group to group.
Certain bands appeared more intense in some populations compared
to others. In some cases, unique bands were observed for a given
population. The explanation for this remains unclear. Perhaps true
• 9
Heat-Shock Proteins in T. californicus
genetic differences exist between populations. Variation in protein
composition may also be related to the unique physico-chemical
environment of each pool. Alternatively, differences may be
artificially exaggerated due to fallible sampling techniques. Nimkin
(1977) found a significant positive association of salinity and percent
females in high saline pools. Female copepods apparently are more
resistant to stressors than males (Hakimzadeh, et al., 1990;
Damgaard, et al., 1994). If sex ratios are truly different amongst the
various splash pools tested, then a sampling error may have
occurred, as no attempt was made to separate males and females
prior to homogenization. This possibly accounts for observed protein
differences across populations.
In any case, if different populations of T. californicus have
different protein compositions, it may be unwise to weigh animals
based on total protein concentration. For example, suppose two
hypothetical populations (« and B) of T. californicus produced
identical amounts of HSP6O. But suppose population a had twice as
much blunderin (a hypothetical 116kD protein that serves no
purpose) as population ß. If we load the lanes of our gel for equal
total protein, transfer protein to a nylon membrane and then probe
for HSP60, population B would appear to have more HSP60 than
population a. A new method of weighing the animals must
therefore be developed. Counting individual animals may be the
best alternative. However, the problem of variability in chemical
composition amongst individuals would remain. We would also be
introducing a new factor—individual variability in body size.
Another obstacle to unambiguous, definitive results is the fact
that this study was conducted in the field. Thus it was not possible
to easily measure the effect of one variable while holding all others
constant. In the future, it may be more productive to devise splash
pool models in which variables may be controlled.
• 10
Heat-Shock Proteins in T. californicus° 11
Conclusion
The degree of conservation of HSPs and the heat-shock
response across species implies strong natural selection. Since
natural selection can only act on the stress response when it is
evoked, stress which induces HSPs must be somewhat common
among individual organisms. We have sampled Tigriopus
californicus from rock pools of various salinities and measured the
expression of HSP60 in animals. No correlation was found between
HSP60 expression in animals and pool salinity, apparently because of
the ability to osmoregulate. However, differential production of
HSP60 was noted among distinct T. californicus populations.
Whether these differences are genetic or phenotypic remains to be
elucidated.
Acknowledgments
I am grateful to Dr. R. Paul Levine for his guidance and
everlasting commitment to this project. Additional thanks go out to
Dr. Mark Denny and Dr. Jim Watanabe for providing me with the
means to carry about the field work associated with this study.
Thank you to Dr. Jan von Kampen, Sunny Sanders, and Dr. Virginia
Weis for taking time out of their busy schedules to assist me and for
their steadfast patience and support.
Literature Cited
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to unmodified nitrocellulose and radiographic detection with
antibody and radioiodinated protein A. Analytical
Biochemistry, 112:195-203.
Heat-Shock Proteins in T. californicus° 12
Burton, R.S. (1991). "Regulation of Proline Synthesis During Osmotic
Stress in the Copepod Tigriopus californicus:" Journal of
Experimental Zoology, 259: 166-173.
Burton, R.S. and M.W. Feldman. (1982). "Changes in Free Amino
Acid Concentrations During Osmotic Response in the Intertidal
Copepod Tigriopus californicus. Comparative Biochemistry
and Physiology, 73A: 441-445.
Craig, E.A. (1985). "The Heat Shock Response." CRC Critical
Reviews in Biochemistry. Volume 18, Issue 3.
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salinity preference and temperature tolerance in the high-
shore harpacticoid copepod Tigriopus brevicornis. Marine
Biology, 118:443-449.
Easton, D.P., P.S. Rutledge, and J.R. Spotila. (1987). "Heat shock
protein induction and induced thermal tolerance are
independent in adult salamanders." Journal of Experimental
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Fernández, E. and J.J. Kopchick. (1990) "Quantitative Determination
of Growth Hormone by Immunoblotting. Analytical
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Ha, C.M. (1993). "Stress Induced Heat Shock Proteins in Tigriopus
californicus. Unpublished manuscript on file at Hopkins
Marine Station Library, Stanford University, in Final Papers,
Biology 175H.
Hakimzadeh, R. and B.P. Bradley. (1990). "The Heat Shock
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Fluctuating Thermal Environments: An Ecological and
Evolutionary Perspective." In R.I. Morimoto, A. Tissières, and C.
Georgopoulos (Eds.), Stress Proteins in Biology and Medicine
(pp. 37-59). New York: Cold Spring Harbor Laboratory Press.
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Laemmli, U.K. (1970). "Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227: 680-
685.
Lindquist, S. (1986). "The Heat-Shock Response", Annual Review of
Biochemistry, 55:1151-91.
MCEvoy, T.A. (1993). "Identification and Induction of Heat Shock
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file at Hopkins Marine Station Library, Stanford University, in
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Morimoto, R.I., A. Tissières, and C. Georgopoulos. (1990). "The
Stress Response, Function of the Proteins, and Perspectives." In
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Nimkin, K. (1977). "Differential Mortality to Salinity Stress and its
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Table I
Sampled Pools
MNNNNNNNNNN
Salinity (
Sa
) Temperature (C)
8.00
Laboratory Control
32.0
23.0
8.00
60.0
Laboratory Control
23.0
Pool A
17.0
8.76
9.01
43.2
17.0
Pool Z
51.9
8.74
Pool D
18.2
60.0
8.87
Pool X
9.11
19.5
Pool Y
Politn
ad
Table II
HSP60 Trace Densities for T. californicus Populations
(Band at 58 KD)
soscoceosesssssossesesesseeesssesssssssseessessocosoeeeesesssoooesse
Sample Trace Density (OD X mm)

1.958
Laboratory Control
1.226
Laboratory Control
0.279
Pool A
1.602
Pool Z
1.429
Pool D
2.459
Pool X
Pool Y
1.503
mnen
e: Band quantification is performed as follows: Average optical density of each row of pixels is
calculated across sample width (horizontally). Average optical density for each pixel row is then integrated
over the band height (vertically). This gives the dimensions OD X mm.
List of Figures
Figure 1. Western blot with anti-HSP60 polyclonal antibody probe.
Samples obtained from various Tigriopus californicus populations at
Mussel Point. Twenty (20) ug samples of soluble protein derived
from laboratory animals maintained in 32%0 and 60%00 seawater
were loaded in lanes 1 and 2, respectively (numbered left to right).
Soluble protein (20ug) derived from in situ populations was loaded
in lanes 3 through 8. Order of these lanes, left to right, is as follows:
Pool A (33%/0), Pool Z (43.2%/0), Pool D(51.9%0), Pool X(60%/00), Pool
Y(72.8%0), and Pool X(78.7%/0). Prominent reaction occurs with
protein(s) at 58kD. Note cross-reactivities in lanes 3 and 6.
Figure 2. Histogram representing densitometric analysis of 58kD
band from Western blot. Units along the ordinate are O.D. X mm
(trace density). Animals of Pool X (lanes 6 and 8) apparently
produce higher levels of HSP60.

8

a
3

8