ABSTRACT
1 examined the changes in levels of endogenous heat shock protein 70 (Hsp 72 and Hsp
74) in the Tegula congeners T. brunnea, T. pulligo, and T.funebralis in response to cold shock. I
also monitored behavioral changes caused by cold exposure. To examine Hsp levels, snails were
first placed in either 1° or 4° C (40.5° C) seawater for either 2 or 10 h, then placed back in
ambient temperature (13° C) seawater. Levels of Hsp expression 2 and 10 h after the end of the
shock were quantified via western analysis. These levels were compared to those of snails not
exposed to cold stress. In individuals facing 10 h cold exposures, levels of Hsp 72 after 2 h of
recovery in general did not vary substantially from pre-exposure levels, but decreased
significantly after 10 h of recovery. These data suggest that different exposure lengths may
confer different signals that activate distinct physiological responses to cold shock. The
behavioral study found that following cold (1-3° C) exposure, the subtidal congeners T. brunnea
and T. pulligo were more likely to enter a chill coma (foot retracted, operculum firmly closed)
than T. funebralis. These behavioral differences could indicate that the subtidal species would be
less capable than T. funebralis of responding to environmental cues and conditions following
cold shock.
INTRODUCTION
In an age of increasingly varying weather patterns and global climate change, the
mechanisms by which organisms cope with thermal stress merit study. While much work has
been devoted to the ability of organisms to withstand heat stress, relatively little has looked at the
lower thermal tolerances of species. In this study, I examine the cold stress response of the
Tegula congeners T. funebralis, T, brunnea, and T. pulligo.
The Tegula species used provide an ideal study system for examining important aspects
of thermal tolerance and cold stress. As they are closely related (though there are some concerns
about T. pulligo, see Hellberg 1998), any comparisons between their thermal biology are unlikely
to be confounded by differences in phylogeny. Also, one would expect them to experience
different ranges of temperatures as they live at different tidal heights and have different northern
distributions. T. funebralis inhabits the low- to mid- intertidal zone, and has a northern
distribution that extends to Vancouver Island, British Columbia (Abbott and Haderlie 1980). T.
brunnea lives in the low intertidal to subtidal zone, and ranges northward to Cape Arago, Oregon
(Abbott and Haderlie 1980). T. pulligo lives exclusively in the subtidal zone, and ranges as far
north as Sitka, Alaska (Abbott and Haderlie 1980).
T. funebralis has been shown previously to be more capable of handling heat stress than
T. brunnea (Tomanek 2002). However, at the northern extreme of its range T. funebralis should
also experience colder temperatures than either T. brunnea or T. pulligo during emersion. To
date, little research has been conducted to explain this aspect of the thermal biology of the
Tegula congeners. This study looks at the patterns of Hsp 70 expression in response to different
cold stresses in the different species, as well as the variation in behavioral response of the species
to cold shock.
MATERIALS AND METHODS
Chill Coma Experiment
This study subjected snails to four different temperature treatments. In two of the
treatments snails were placed in containers filled with ambient temperature (13° + 1° C)
seawater. These containers were placed in either an ice bath (1" treatment) or a cold room (air
temperature 4° C, 2“ treatment), and the seawater was allowed to equilibrate thermally with its
surroundings for 12 hours. Seawater in the cold room reached temperatures of 8° C(+ 1° C) and
6° C(+1° C) after 6 and 12 hours, respectively. Seawater in the ice bath reached temperatures of
6°C (+1° C) and 3° C (+ 1° C) after 6 and 12 hours, respectively. In the other two treatments,
snails were exposed directly to either 1° C (+1° C) or 4° C (40.1° C) air for 6 hours. Five large
adults of each species were used for each treatment. Snails were returned to ambient temperature
seawater at the end of the treatments. Individuals were observed 20 minutes and 24 hours
following the end of the cold exposures. During each observation, individuals were categorized
as being dead, active, or in a chill coma. For the purposes of the experiment, animals were dead
if they did not exhibit a foot withdrawal response, active if their operculum was not closed and
they exhibited a foot withdrawal response, and in a chill coma if their operculum was closed
firmly.
Analysis of Hsp 70 Expression
All snails to be used for the experiment were placed in an acclimation tank with ambient
temperature (13° C + 1° C) seawater for five days prior to cold exposure. Individuals were
subjected to four different exposure treatments: 1° C seawater (40.5° C) for 2 hours. 1° C
seawater (40.5° C) for 10 hours, 4° C seawater (40.5° C) for 2 hours, and 4° C seawater (40.5° C)
for 10 hours. Ten large adults of each species were used for each treatment. Following
exposure, the animals were placed in ambient temperature seawater. To ensure that individuals
stayed fully immersed in water during the entire experiment, snails were placed inside small
mesh-enclosed containers inside larger seawater tanks for all of the incubations. The gill tissue
of five individuals of each species was removed 2 hours and 10 hours following the end of each
cold exposure treatment. For a control sample, the gill tissue of five animals of each species was
taken before any cold exposure.
The gill tissues obtained by this method were used to quantify Hsp72 and Hsp74
expression via solid phase immunochemical analysis (western analysis). The analysis was run
according to the protocol described by Tomanek and Somero (2002) with the following
exceptions and additions: 300ul of homogenization buffer was added to the gill tissue of T.
pulligo for homogenization, and total protein concentration was determined using the BCA
protein (Pierce) assay rather than the micro-BCA (Pierce) protein assay. The resulting data for
each species and heat-shock protein were analyzed separately using an asymmetrical, 3-factor
ANOVA model with exposure temperature, exposure period, and recovery period as the three
factors. Each factor was treated as being fixed and orthogonal to the other factors. Significant
results from the models were investigated using the Student Newmann-Keuls (SNK) test.
RESULTS
Chill Coma Experiment
All snails were active one day following their exposure to cold treatments. However, 20
minutes following their exposure, all individuals of T. brunnea in both the 1° C air and the
seawater in the ice bath were inactive. All of the T. pulligo exposed to 1° C air were also inactive
at the 20 minute time point. All T. funebralis individuals in each treatment group were active
after 20 minutes.
Analysis of Hsp70 Expression
The ANOVAs for levels of Hsp 72 in T. brunnea and T. funebralis and levels of Hsp 74
in T. pulligo and T. funebralis all showed highly significant (p«0.01) three way interactions
between the factors. The ANÖVA for Hsp 74 in T. brunnea displayed significant interactions
between exposure length and exposure temperature as well as between recovery period and
exposure temperature, and the ANÖVA for Hsp 72 in T. pulligo showed significant two way
interaction between all pairs of factors.
The most consistent pattern in the data was a significant reduction in Hsp 72 levels
relative to control values after 10 hours of recovery in groups with 10-hour cold exposure times
(Figs. 1-3). This pattern was present in all 10-hour exposure treatments. In all of these groups
except for T. pulligo exposed to 4° C water, Hsp 72 levels after 2 hours of recovery were similar
to control values and significantly greater than the levels after 10 hours of recovery. Groups
subjected to 2-hour exposures did not show any difference in Hsp 72 levels after 10 hours of
recovery relative to control, with the exception of T. pulligo exposed to 4° C water (Fig. 3). Of
these 2-hour exposure treatments, only T. pulligo exposed to 1° C and 4° C water and T. brunnea
exposed to 4° C water showed levels of Hsp 72 after 2 hours of recovery that were significantly
different from pre-exposure levels (Figs. 2, 3).
Expression of Hsp 74 proved difficult to quantify accurately, as the bands on the gel were
generally weak and often overrun by the Hsp 72 signal. No consistent patterns of expression
were found either within or among species. As previous studies have indicated that Hsp 74 is
constitutively expressed and does not usually respond strongly to thermal stress (Tomanek
2002), we assume the same is occurring in this case.
DISCUSSION
The highly significant interaction terms from the ANÖVAs suggest that none of the
factors are acting independently of each other. In particular, it seems that a combination of a 10-
hour exposure time and a 10-hour recovery time alone cause a significant down regulation of
Hsp 72, and to a lesser extent Hsp 74. This would seem to indicate that snails exposed to
prolonged cold stress exhibit a different response profile than those experiencing only transient
exposure to cold.
These results differ from studies of cold shock responses of other organisms, which
generally noted transient increases in levels of inducible Hsp 70 or its mRNA precursor (Ju et al.
2002; Martinez et al. 2001). In their study of gene expression in catfish, Ju et al. (2002) noted a
transient increase of inducible Hsp 70 2 hours following initial exposure to reduced temperature.
This increase in the level of inducible Hsp 70 had subsided after 24 hours of exposure (Ju et al.
2002). A study of Hsp production in nematodes also showed a transient increase in inducible
Hsp 70 (Martinez et al. 2001). The increased levels were noticeable after 2 hours, but had
subsided after 24 hours (Martinez et al. 2001). Both of these studies maintained their
experimental organisms at the cold exposure temperature and did not bring animals back to non¬
stress inducing temperature (Ju et al. 2002; Martinez et al. 2001). In our experiment, no
samples were taken until the organisms had been in ambient temperature seawater for at least 2
hours. These differences in sampling procedures could explain the discrepancies between the
experiments. Any increase in endogenous levels of inducible Hsp 70 during cold exposure in this
experiment could have already subsided during the 2-hour recovery period after which the first
sample was taken. Similarly, the decrease in Hsp 72 seen in the 10-hour exposure treatments in
this experiment might not have occurred in the other studies because the animals were not
allowed a recovery period. Alternatively, the differences between the results of the studies might
simply reflect taxonomic differences of the study animals chosen for each experiment.
Although there was relatively little variation among the different species in patterns of
Hsp 70 expression following cold shock, evidence from the chill coma experiment indicates that
T. funebralis may be less severely stressed by exposure to cold than its two subtidal congeners.
While cold exposure may not directly kill T. brunnea or T. pulligo, it may render them incapable
of responding to other cues and conditions in the environment, putting them at a significant
disadvantage relative to T. funebralis. Transplant studies similar to those done for heat stress
(Tomanek 2002) could indicate whether the observed differences have any effect in situ.
Since T. funebralis seem more capable of tolerating cold treatment, but no interspecific
variation of patterns of Hsp 70 expression were found, T. funebralis may have some other
method of coping with cold stress. This could be investigated more directly in further studies by
tracing radioactively labeled amino acids or using DNA micro-array technology.
CONCLUSIONS
All species exhibited a significant decrease in endogenous Hsp 72 10 h following the end
of a 10 h cold exposure. This suggests that different lengths of exposure to cold elicit different
physiological responses to the cold shock. While patterns of Hsp 72 expression were similar
among species, there were differences in behavior of individuals exposed to 1° C air and 3° C
water. The chill comas exhibited by T. brunnea in the 1° C air and 3° C water and T. pulligo in
the 1° C air indicate that these species could be less capable than T. funebralis of responding to
changes in the environment following cold shock. Since patterns of Hsp 72 expression were
similar among species, it may be that T. funebralis has some other mechanism for dealing with
cold stress not possessed by its subtidal congeners.
ACKNOWLEDGEMENTS
The author wishes to thank George Somero and Lars Tomanek for their continual guidance on all
aspects of the project. Thanks also to Jim Watanabe for his aid with statistical analysis of the
data, as well as James Lopez and Anne Schwedt for their assistance with lab techniques and
protocols.
LITERATURE CITED
Ju, Z., R. A. Dunham, R. A., and Liu, Z. 2002. Differential gene expression in the brain of
channel catfish (Ictalurus punctatus) in response to cold acclimation. Molecular Genetics and
Genomics. 268:87-95
Martinez, J., J. Perez-Serrano, W. E. Bernadina, and F. Rodriguez-Caabeiro 2001. Stress
response to cold in Trichinella species. Cryobiology. 43:293-302.
Morris, R. H., D. P. Abbott, and E. C. Haderlie. Intertidal Invertebrates of California. Stanford
University Press: Stanford, 1980.
Tomanek, L. 2002. The heat shock response: its variation, regulation, and ecological
importance in intertidal gastropods (genus Tegula). Integrative and Comparative Biology
:797-807.
Tomanek, L. and G. Somero. 2002. Interspecific- and acclimation-induced variation in levels of
heat-shock proteins 70 (Hsp 70) and 90 (Hsp 90) and heat-shock transcription factor 1 (HSF 1) in
congeneric marine snails (genus Tegula): implications for regulation of hsp gene expression.
Journal of Experimental Biology. 205:667-685.
FIGURE LEGENDS
Fig. 1. Levels of Hsp 72 in T. funebralis relative to bovine Hsc 70 standard. Error bars are
standard errors of the mean (n=5). Asterisks denote significant difference from pre-exposure
values.
Fig. 2. Levels of Hsp 72 in T. brunnea relative to bovine Hsc 70 standard. Error bars are
standard errors of the mean (n=5). Asterisks denote significant difference from pre-exposure
values.
Fig. 3. Levels of Hsp 72 in T. pulligo relative to bovine Hsc 70 standard. Error bars are standard
errors of the mean(n=5 for control, 2-hour exposures, n—4 for 10-hour exposures). Asterisks
denote significant difference from pre-exposure values.
0.1

001
10 10h
— pre-exposure
2h recovery
Oh recovery
Fig. 1


40 10h
OC 2h
Exposure Regime

40 2h
10
0.1

0.01
1C 10h
— Pre-exposure
2h recovery
1Oh recovery
Fig. 2
L
40 10h
OC 2h
Exposure Regime

402h
0.1
0.01
— pre-exposure
2h recovery
Oh recover
4
10 10h
Fig. 3

40 10h
OC 2h
Exposure Regime

40 2h