ABSTRACT:
Previous studies have demonstrated that a suite of physiological and biochemical factors
contribute to the establishment of a species’ thermal tolerance limits and vertical distribution
patterns in the marine intertidal. In this study, the thermal limits of heart function were
determined for three congeneric marine snail species of the genus Tegula, all inhabiting discrete
vertical zones in the intertidal. T. funebralis is found in the low- to mid-intertidal, T. brunnea in
the subtidal to low-intertidal, and T. montereyi in the subtidal zone. Using impedance electrodes,
changes in heart rate were monitored in field-acclimatized and lab-acclimated specimens of each
species, in response to thermal stresses in water. Significant interspecific differences in
Arrhenius break temperature values (ABT, the temperature at which heart rate began a sharp
decline) were observed. Average ABTs for field-acclimatized T. funebralis, T. brunnea, and T.
montereyi were 31°C, 25°C and 24°C, respectively, establishing a positive correlation between
cardiac upper thermal tolerance limits and maximum habitat temperatures. Flatline
temperatures, defined as those at which hearts ceased to beat, followed the same trend—T.
funebralis hearts stopped beating at significantly higher temperatures than the two subtidal
species. In response to cold stress, T. funebralis maintained cardiac function at lower
temperatures than its congeners, demonstrating that it is more eurythermal. In all three species,
ABTs of specimens lab-acclimated to 22°C were higher than those of 14°C lab-acclimated snails.
T. funebralis, however, showed the smallest ABT difference between acclimation temperatures,
indicating that it may have a lower capacity for thermal acclimation. The results of this study
suggest that the mid-intertidal species T. funebralis is living closer to its upper thermal tolerance
limits. Thus T. funebralis populations, and perhaps warm-adapted intertidal species in general,
may be at highest risk in the event of global climate change.
INTRODUCTION:
The marine intertidal is one of the harshest environments on earth. Organisms inhabiting
the transition from land to sea face a host of environmental stresses, including temperature
variation, desiccation stress, wave force, and salinity (Vernberg and Vernberg 1972; Denny
1988). The daily tidal cycle is largely responsible for the drastic, rapid temperature fluctuations
typical of the intertidal, exposing organisms to alternating terrestrial and aquatic conditions.
Many species inhabit discrete vertical zones in the marine intertidal (Connell 1961).
These zonation patterns are established by a host of abiotic factors, such as temperature and
aerial exposure, as well as biotic factors like competition and predation (Connell 1961; Edney.
1961; Paine 1969). Studies have shown that species inhabiting mid- to high-intertidal zones
experience more extreme environmental stresses than subtidal species. For instance, some high-
dwelling gastropods can undergo body temperature increases of 20-25°C during low tide
emersions (Hofmann and Somero 1995), whereas many subtidal animals never face temperatures
above that of the ambient seawater.
These drastic differences in thermal habitats are possible only because intertidal
organisms have differentially evolved adaptations for thermal tolerance. Numerous studies have
shown that the upper vertical limits of species distributions are correlated with thermal tolerance
limits (Wethey 1983; Jensen and Armstrong 1991; Stillman and Somero 2000). A suite of
physiological, biochemical, and morphological factors contribute to the establishment of these
limits- mitochondrial respiration, nerve function, action potential generation, heart function,
protein stability, and heat-shock protein response are some such traits (Somero 2002; Stillman
and Somero 1996).
One of these thermal tolerance parameters, heart function, is the subject of this study.
Considering that a functioning circulatory system is critical for oxygen delivery to animal
tissues, the limits of heart function surely contribute to the establishment of thermal optima.
Marine snails of the Genus Tegula were chosen for this cardiac study. Tegula is an ideal genus
for heat stress research because of the abundance of closely related species that occupy diverse
thermal habitats and discrete vertical zones (Watanabe 1984; Hellberg 1998). Three
differentially distributed temperate Tegula congeners were selected. T. funebralis inhabits the
low- to mid-intertidal, and can encounter temperatures over 33°C when emersed. T. brunnea is
found in the low-intertidal to subtidal zone. It is exposed to air only during low low tides and
experiences maximum habitat temperatures of approximately 23°C. Finally, T. montereyi is an
exclusively subtidal species, whose body temperature never exceeds ambient seawater
temperatures.
The Tegula congeners selected for this experiment have been studied extensively with
respect to thermal tolerance. Whole animal thermal limits have been determined—the LTso
values (the temperature at which 50% mortality is observed) for T. funebralis, T. brunnea, and T.
montereyi are 42.5°C, 36°C and 36°C, respectively (Tomanek and Somero 1999). Interspecific
differences in enzyme thermal stabilities have also been characterized in these congeners. For
example, the thermal stabilities of malate dehydrogenases from five Tegula congeners increase
with increasing vertical position (Somero 2002). Results of Tegula heat shock protein (hsp)
experiments correspond with these findings (Tomanek and Somero 1999, 2000; Tomanek and
Sanford 2003). The synthesis of hsps in T. funebralis is initiated, maximized, and terminated at
higher temperatures than in T. brunnea and T. montereyi (Tomanek and Somero 1999).
These Tegula studies demonstrate clear correlations between maximum habitat
temperatures and thermal tolerance limits. It is apparent that T. funebralis, the higher-occurring
intertidal species, has many physiological and biochemical adaptations allowing it to endure
warmer temperatures than its subtidal relatives. The purpose of this investigation was to
elucidate the role of heart function among these thermal adaptations, by determining and
comparing the upper and lower cardiac thermal tolerance limits of Tegula congeners from
different tidal heights.
MATERIALS AND METHODS:
Collection and care of specimens
Tegula specimens were collected from the intertidal at Hopkins Marine Station in Pacific
Grove, California (36° 36’N, 121° 54’W). Snails of the lowest-dwelling species, T. montereyi,
were collected while SCUBA diving, and T. funebralis and T. brunnea were gathered on foot,
the latter during minus low tides. All specimens were adults of medium to large body size (20-
25 mm basal diameter), found in the upper region of their species’ vertical distribution. Äfter
collection, snails were held in re-circulating water baths of ambient seawater for 24 hours before
experimentation. These specimens were deemed ’field-acclimatized’.
Two aquaria were set up for the laboratory acclimation experiment—one at 14°C and one
at 22°C. Fifteen snails of each species were held in each tank for 15-19 days and fed fresh kelp
every three days. The water level of the tanks was kept high enough so that the animals could
never fully emerse themselves. At the end of the acclimation period heat ramps were run,
identical in design to those performed on the field-acclimatized specimens (see below).
Heart Rate Measurements
Using a small hand drill, two holes were drilled into the shell of each snail, adjacent to
the pericardial space. Prime drilling locations were determined initially by dissecting specimens
of each species and locating the heart in relation to shell coils. Ceramic-coated copper electrodes
were inserted into these holes and secured in place with SuperGlue"", positioned as close to the
heart as possible. To prevent the snails from emerging during experimentation, the outer lip of
each shell was glued to a clean glass microscope slide. The snails were suspended using metal
clamps that held cork pieces glued to the top of each shell. The two impedance wires in each
animal were connected to an impedance pneumograph, and the resulting signals were amplified
and recorded using PowerLab““ Data Acquistion System (Fig. 1). Heart rate measurements
were performed in filtered, aerated seawater, temperature-controlled by a Lauda"" waterbath
system. This setup accommodated a maximum of 6 animals per run.
For heat stress experiments, the waterbath temperature was held constant at 13°C for 1
hour, then increased at a rate that was determined to be environmentally realistic (1°C every 15
minutes). Äfter reaching 40°C, or once all specimens’ hearts had failed, whichever came first,
water temperature was decreased as rapidly as possible, to assess recovery of cardiac function.
Heart rate measurements were made every 7 minutes for the duration of the run.
Cold stress experiments were also performed. Animals were held at 13°C for one hour,
and then water temperature was decreased to nearly 0°C at a rate of 1°C every 10 minutes. Äfter
heart failure, temperature was returned to ambient. Following every run, the snails were
weighed and placed in a re-circulating water bath at 13-15°C for 72 hours. Each animal was
examined every 24 hours following experimentation to determine recovery and mortality rates.
In addition to temperature ramps, control runs were performed, to ensure that the stress of
the experimental set-up (which included drilling, invasive electrode placement, and gluing) was
not significantly contributing to the heart failure and mortality observed. In these runs, T.
funebralis and T. brunnea heart rates were monitored for 7 hours in the absence of heat stress—
temperature was held constant at 13°C. In the week following the experiment, the control snails
were closely monitored, to track recovery.
Snail body temperature was approximated using a Knox-gelatin-filled snail shell, since
gelatin has been shown to approximate the conductive properties of snail tissue (Tomanek and
Somero 1999). The artificial snail was prepared for a run, just like a living animal, and a
thermocouple probe was inserted into the gelatin. During a heat ramp, the gelatin-temperature
was recorded, in order to estimate snail body temperature in relation to water temperature.
Data Analysis
Heart rates were expressed as beats per minute and plotted over time for every run, to
visually assess cardiac responses to temperature change. Arrhenius plots were generated for
each animal by converting heart rate values to the natural logarithm of beats per minute and
plotting these values against a temperature transformation (1000/K). Arrhenius break
temperatures (ABTs) were determined using regression analyses to generate the best fitting line
on both sides of an inflection point on the Arrhenius plot. The intersection of these two
regression lines was taken as the ABT. Flatline temperatures (FLTs) were also determined as the
temperature at which the last the measurable heart beat occurred. Critical temperatures, both
ABTs and FLTs, were averaged for each species and acclimation condition, and statistical
significance of comparisons was assessed using ANOVA.
RESULTS:
Heart rates of snails from all three Tegula species increased in response to heat stress, up
to the ABT, after which they declined to zero (Fig. 2). Once temperature was reduced, all
species recovered cardiac function. Within 24 to 48 hours after experimentation, however, 92%
of T. funebralis specimens and 100% of T. brunnea and T. montereyi specimens had died. T.
brunnea had significantly slower heart rates, on average, than T. funebralis and T. montereyi, and
thus their break points were less obvious upon visual inspection (Fig. 2B). ABTs were easily
determined, however, by generating Arrhenius plots for each specimen (Fig. 3).
Significant interspecific differences in break temperatures were observed (pS.001). The
average ABTs for field-acclimatized T. funebralis, T. brunnea, and T. montereyi were
31.1+0.7°C, 25.0+0.5°C, and 24.2-0.7°C, respectively (Fig. 4). The differences between T.
funebralis and both subtidal congeners were statistically significant, but T brunnea and T.
montereyi did not have significantly different ABTs. These results establish a positive correlation
between upper cardiac limits and maximum habitat temperatures in Tegula congeners. Flatline
temperatures followed this trend as well. Average FLTs for T. funebralis, T. brunnea, and T.
montereyi were 39.4+0.2°C, 32.4+0.2°C, and 33.1+0.1°C, respectively (Fig. 4). Flatline
temperatures showed much less intraspecific variation than Arrhenius break temperatures, and all
interspecific FLT differences were statistically significant (pr.0001).
In response to cold stress, heart rates of all species showed gradual declines to zero. No
clear break points could be identified, so only FLTs were determined and compared. T.
funebralis maintained cardiac function down to 2.1+0.2°C, while T. brunnea and T. montereyi
hearts failed at 3.5+0.3°C and 4.8+0.5°C, respectively. T. funebralis can therefore endure
significantly warmer and colder temperatures than its subtidal relatives (Fig. 5). Äfter all
specimens had flat-lined, temperature was rapidly returned to ambient, and all species recovered
heart function. Unlike the heat-stressed specimens, however, none of the cold-stressed snails
died during the three-day holding period following experimentation.
ABTs and FLTs were determined for 14°C and 22°C lab-acclimated snails of each species
in response to heat stress. The two subtidal congeners, T. brunnea and T. montereyi, showed
significantly higher ABTs after 22°C-acclimation than after 14°C-acclimation (Table 1). T.
funebralis ABTs, however, did not significantly increase in response to warm-acclimation. The
2°C-acclimated T. funebralis specimens had only slightly higher ABTs, on average, than the
14°C-acclimated snails (Fig. 6A). For all species, flatline temperatures appeared to be less
plastic than Arrhenius break temperatures. The difference between 14°C and 22°C-acclimated
FLTs was only approximately 1°C for all congeners (Fig. 6B).
Heart failure was not observed in the constant-temperature control run. Some heart rate
fluctuation occurred in the first hour, but after this initial acclimation, cardiac function remained
fairly stable in all the animals (Fig. 7). During the 7 day holding period following the run, all
control specimens were alive and functioning normally. These results indicate that the
experimental procedure was not a confounding variable.
Tegula body temperature was approximated using a gelatin-filled shell. As shown in Fig.
8, the gelatin temperature consistently lagged only about 0.5°C behind that of the water. This
suggests that, when immersed, Tegula tissue temperature approaches water temperature.
DISCUSSION:
This study found a strong positive correlation between the cardiac thermal tolerance
limits of Tegula congeners and their vertical position. In the two subtidal species, heart failure
occurred at significantly lower temperatures than in T. funebralis, the higher-dwelling congener.
A positive correlation was also found between vertical position and eurythermality. T. funebralis
hearts proved capable of enduring the warmest and coldest temperatures, while T. brunnea and
T. montereyi, the lower-dwelling species, had narrower cardiac thermal tolerance ranges.
Comparing the average ABTs of field-acclimatized species to their maximum habitat
temperatures exposes a critical finding. T. funebralis appears to be living much closer to its
upper cardiac limits than do its subtidal relatives. In their mid-intertidal habitats, T. funebralis
individuals regularly reach temperatures above 30°C. Body temperatures as high as 33°C have
even been reported (Tomanek and Somero 1999). Therefore, maximum habitat temperature of T.
funebralis is actually higher than its ABT (31.5°), meaning this species may experience
compromised heart function on a regular basis. This is certainly not the case for the subtidal
S, well below its
congeners. Maximum habitat temperature for T. brunnea is approximately 2
ABT of 25.0°C. Similarly, T. montereyi is unlikely to exceed 20°C, which is far from its ABT of
24.2°.
This finding- that the higher-occurring Tegula species is living closer to its thermal
limits- is not unprecedented. Previous studies of Tegula thermal tolerance have made parallel
conclusions. For example, heat shock experiments have shown that mid-to high intertidal
species such as T. funebralis and the subtropical species T. rugosa are likely to induce heat shock
response more frequently than subtidal species such as T. brunnea and T. montereyi (Tomanek
and Somero 1999). Activation of the heat-shock proteins occurs in response to thermal
denaturation of proteins during heat stress (Feder and Hofmann 2000). Therefore, the higher
incidence of heat-shock response in warm-adapted Tegula congeners indicates more frequent
protein denaturation.
Facing the consequences of frequent heat shock may require significant energy
expenditures- it has been suggested that mid- to high-intertidal organisms may have higher
costs of living’ as a result (Somero 2002). Differential growth rates of Tegula congeners are
consistent with this assertion. T. montereyi grows the fastest, T. brunnea is intermediate, and T.
funebralis, the highest-occurring species, grows most slowly (Frank, 1965; Watanabe, 1982).
Differences in heart rate observed in this study may also be related to energetic costs. T.
funebralis heart rates were significantly higher than the two subtidal species, even when
corrected for differences in body size (Fig. 9). Perhaps an elevated cardiac output is necessary in
T. funebralis in order to fuel the cellular processes associated with its frequent heat shock
responses.
The results of the acclimation experiment may have great ecological significance, since
thermal acclimation capacity can be used to predict a species’ susceptibility to global climate
change (Stillman 2003). In this study, T. funebralis heart function showed the least acclimatory
plasticity of all three congeners. Acclimation to 22°C failed to induce a significant ABT increase
in T. funebralis. Considering this result, and the finding that T. funebralis is living closer to its
cardiac limits, it seems that T. funebralis may be at greater risk in the event of global warming
than its subtidal congeners. Even a 1 or 2°C increase in average global temperatures could
physiologically compromise these animals.
All of my major findings on Tegula thermal tolerance are congruent with results of heat
stress experiments on porcelain crabs of the genus Petrolisthes. Many physiological aspects of
thermal tolerance, such as heart function and nerve function have been studied comparatively in
Petrolisthes congeners, and numerous significant correlations between vertical distribution and
thermal limits have been found (Stillman and Somero 1996, 2000; Stillman 2002, 2003). As for
cardiac function, the highest-occurring species, P. cinctipes and P. gracilis, have ABTs that are
higher and closer to maximum habitat temperatures than the ABTs of low-intertidal and subtidal
species. Also, cardiac limits and whole animal thermal limits of high-intertidal Petrolisthes
species show lower acclimation capacities than lower-occurring congeners (Stillman 2003).
Thus, warm-adapted Tegula species as well as warm-adapted Petrolisthes species appear
to be at highest risk in the event of global climate change. Although only two genera are
currently implicated, it does seem possible that this may be a broader trend in the marine
intertidal zone: high-occurring intertidal organisms, in general, could be more susceptible to the
effects of global warming.
ACKNOWLEDGEMENTS:
I would like to thank my advisor, Geroge Somero, for all of his guidance, support, and
enthusiasm. Members of the Somero Lab have been extremely helpful as well—special thanks
go to Caren Braby and Maxine Chaney for sharing their invaluable heart rate expertise, and to
Brad Buckley for his help with equipment for my acclimation experiment. l’m also very grateful
to Jim Watanabe, Freya Sommer, and Will Tyburczy, who collected many T. montereyi
specimens for me during their SCUBA dives.
LITERATURE CITED:
Connell, J. H. 1961. The influence of interspecific competition and other factors on the
distribution of the barnacle Chthamalus stellatus. Ecology 42, 710-723.
Denny, M. W. 1988. Biology and the mechanics of the wave-swept environment. Princeton
University Press, Princeton.
Edney, E. B. 1961. The water and heat relationships of fiddler crab (Uca spp). Trans. Roy. Soc.
South Africa 34, 71-91.
Frank, P.W. 1965. Shell growth in a natural population of the turban snail, Tegula funebralis.
Growth 29: 395-403.
Hellberg, M. E. 1998. Sympatric sea shells along the sea’s shore: the geography of
speciation in the marine gastropod Tegula. Evolution. 52, 1311-1324.
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. J. Exp. Biol. 198, 1509-1518.
Jensen, G. C. and Armstrong, D. A. 1991. Intertidal zonation among congeners: factors
regulating distribution of porcelain crabs Petrolisthes spp. (Anomura: Porcelanidae). Mar.
Ecol. Prog. Ser. 73: 47-60.
Paine, R. T. 1969. The Pisaster-Tegula interaction: prey patches, predator food preference, and
intertidal community structure. Ecology 50, 950-961.
Stillman, J. H. 2002. Causes and consequences of thermal tolerance limits in rocky intertidal
Porcelain crabs, genus Petrolisthes. Integ. Comp. Biol. 42, 790-796.
Stillman, J. H. 2003. Acclimation capacity underlies susceptibility to climate change. Science
301,65.
Stillman, J. H., 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 distrubtion. J. Exp. Biol. 199,
1845-1855.
Stillman, J. H., Somero, G. N. 2000. A comparative analysis of the upper thermal tolerance
limits of eastern pacific porcelain crabs, genus Petrolisthes: Influences of latitude, vertical
zonation, acclimation, and phylogeny. Physiol. Biochem. Zool. 73, 200-208.
Somero, G. N. 2002. Thermal physiology and vertical zonation of intertidal animals: optima,
limits, and costs of living. Integ. Comp. Biol. 42, 780-789.
Tomanek, L., Somero, G. N. 1999. Evolutionary and acclimation-induced variation in the heat-
Shock responses of congeneric marine snails (genus Tegula) from different thermal
Habitats: implications fo limits of thermoterlance and biogeography. J. Exp. Biol. 202,
2925-2936.
Tomanek, L., Somero, G. N. 2000. Time course and magnitude of synthesis of heat-shock
Proteins in congeneric marine snails (genus Tegula) from different tidal heights. Physiol.
and Biochem. Zool. 73, 249-256.
Tomanek, L., Sanford, E. 2003. Heat-shock protein 70 (hsp70) as a biochemical stress
Indicator: an experimental field test in two congeneric intertidal gastropods (genus
Tegula). Biol. Bull. 205, 276-284.
Vernberg W. B. and Vernberg, F. J. 1972. Environmental physiology of marine organisms.
Springer-Verlag, New York.
Watanabe, J. M. 1982. Aspects of community organization in a kelp forest habitat: Factors
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Table 1: Critical temperatures of lab-acclimated Tegula congeners in response to heat stress.
Average Arrhenius break temperatures (ABTs) and flatline temperatures (FLTs) are listed, with
associated standard errors of mean.
T. montereyi
T. brunnea
T. funebralis
(N=6)
(N=5)
28.5 +0.500
20.2 40.800
21.7 40.8°
14°C-acclimated
ABT
30.1 40.500
26.8 40.700
22°C-acclimated
25.7 40.600
39.8 40.200
31.7 40.400
33.6 40.300
14°C-acclimated
FLT
40.7 40.400
32.84 0.300
22°C-acclimated
34.1 +40.200
FIGURE LEGEND:
Fig. 1. Snail heart rate traces from Chart Recorder software for a T. funebralis specimen (A) and
a T. montereyi specimen (B). Time and voltage scales are provided.
Fig. 2. Heart rate profiles of Tegula congeners in response to heat stress. Representative
specimens of each species were chosen, with T. funebralis, T. brunnea, and T. montereyi shown
in A, B, and C, respectively.
Fig. 3. Arrehnius plots of the Tegula congener heart rate profiles shown in Fig. 1. In each plot,
linear regressions were performed on both sides of the inflection point, and the intersection of the
two lines was taken as the Arrhenius Break Temperature (ABT). T. funebralis, T. brunnea, and
T. montereyi specimens are represented in A, B, and C, respectively.
Fig. 4. Critical Temperatures of field-acclimatized Tegula congeners. Average Arrhenius Break
Temperatures and flatline temperatures are shown for each species, and error bars are standard
errors of the mean (n=13 for T. funebralis, n=11 for T. brunnea, and n=10 for T. montereyi).
Fig. 5. Cardiac thermal tolerance ranges of Tegula congeners. Lower limits are average flat-line
temperatures from cold ramps, and upper limits are average flatline temperatures from heat
ramps. Error bars are standard errors of the mean (for upper limits n=13 for T. funebralis, n=11
for T. brunnea, and n=10 for T. montereyi; for lower limits, n-6 for all species).
Fig. 6. Effects of thermal acclimation on Tegula congener critical temperatures. Average
Arrhenius break temperatures are shown in A, and average flatline temperatures are shown in B.
Error bars are standard errors of the mean (n=6 for T. funebralis, n-5 for T. brunnea, and n-6 for
T. montereyi).
Fig. 7. Constant-temperature control runs. Heart rate profiles of T funebralis (A) and T.
brunnea (B) specimens over 7 hours with constant temperature. Control runs were designed to
assess the potentially confounding effects of the experimental conditions on heart function.
Fig. 8. Estimation of snail body temperature. A gelatin-filled snail shell was used to
approximate the thermal properties of snail tissue. Thermocouple probe measurements of the
gelatin are shown in relation to the temperature of the water bath.
Fig. 9 Average resting heart rates of Tegula congeners. "Resting" were measured as the heart
rate after an hour of acclimation to 13°C, for all specimens subjected to heat ramps. Error bars
are standard errors of the mean (n-24 for T. funebralis, n-20 for T. brunnea, n-21 for T.
montereyi).
A
Fig. 1



A

1 minute

i

...
..
1 minute
0.25 V
0.5 V
Fig. 2
45 -
5 40

3 35
o
o0
00
50
gos gonsood
0000

0
0:00:00 1:12:00 2:24:00 3:36:00 4:48:00 6:00:00 7:12:00 8:24:00
Time
35
30

20
egeounge en o o   oonn 0.
0:00:00 1:12:00 2:24:00 3:36:00 4:48:00 6:00:00 7:12:00 8:24:00
Time

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e
.
000
0 0o 9 6
o%00

0:00:00 1:12:00 2:24:00 3:36:00 4:48:00 6:00:00 7:12:00 8:24:00
Time
19
• Temperature
o T. funebralis
Heart Rate
• Temperature
o T. brunnea
Heart Rate
• Temperature
o T. montereyi
Heart Rate
C
Fig. 3
ABT
y = 6.1276x + 22.96
R2 = 0.8077
4
y = 31.819x —100.69
R2 = 0.8251
s
25
3.55 3.5 3.45 3.4 3.35 3.3 3.25 312 3.15
Temperature 11000/K
y = -5.5019x + 19.925
y = 24.657X -81.24
R? = 0.6556
R2 - 0.7389


3.55 3.5 3.45 3.4 3.35 3.34 3.25 3.2 3.15
Temperature 11000/K
ABT
V =-13.652x + 48.457
y = 21.307x - 69.024

R? = 0.8036
Rå= 0.7459


3.55 3.5 3.45 3.4 3.35 3.3 3.25 3.2 3.115
Temperature [1000/KJ
20
20
T. funebralis
T. brunnea
T. montereyi
Fig. 4
□ Arrhenius Break Temperature
Flatline Temperature
45
40
T. funebralis
T. brunnea
T. montereyi
Fig. 5
Lower limit
EUpper limit
32
3 28
26
24
5 22
- 20
41
39
5 35
5 33
29
Fig. 6


22°
14°
Acclimation Temperature

22°
14°
Acclimation Temperature
——T. funebralis
—T. brunnea
T. monterey
—8—T. funebralis
—+T. brunnea
4T. montereyi
22
517
5 12
7
Fig. 7
APTA AMA
A A4
A AAAA
AAAA AAA a

90 0 9odoargo

o 0
O
o
Q .

0:00
1:12 2:24 3:36 4:48 6:00 7:12
Time
22
17
N
2P8



3:36 4:48 6:00 7:12
1:12 2:24
0:po
Time
24
Snail 1
Snail 2
A Snail 3
XTemperature
O Snail 1
Snail 2
A Snail 3
XTemperature
5 30
Fig. 8

338588888850
10
0:00:00 1:12:00 2:24:00 3:36:00 4:48:00 6:00:00 7:12:00 8:24:00
Time
* Water Temperature
o Gelatin-filled Snail
Temperattre
7
26
0 5
T. funebralis
T. brunnea
T. montereyi
Fig. 9