Shannon Taylor
Abstract
Tegula funebralis, the black turban snail, is an intertidal gastropod with a distinctive intertidal
size gradient; large snails tend to live in the lower portion of the snail's range and small snails in
the upper portion. Many factors have been suggested to explain this phenomenon, including
differential larval settlement, juvenile mortality, response to light, and, most commonly,
predation pressure. I investigated the potential contribution of thermal stress on the size gradient
in a study area that has been shown to have very little predation on Tegula. I performed two
types of experiments: temperature measurements of gelatin-filled snails and heat shock protein
analysis of gill tissue from large and small snails that had been caged in the intertidal. Although
my heat shock analysis was inconclusive, temperatures were consistently lower in small snails
than in large snails, both in a controlled environment and in the field, a disparity possibly caused
by differences in the absorptivity of large and small snail shells. Heat shock protein analysis
could verify that larger snails experience this increased heat stress. Finally, a vertical size
gradient also appears to occur between different intertidal gastropod species, another indication
that size-dependent thermal stress could be important in determining intertidal gastropod
zonation.
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Introduction
Tegula funebralis, the black turban snail, is a common intertidal gastropod with a wide
vertical distribution in the intertidal zone; it occurs from the very low zone to the high-mid zone.
This wide range results in very different microhabitats for individual snails at opposite edges of
the spread—higher snails are emersed for longer during low tides and therefore experience
higher levels of thermal stress and desiccation rates. However, the snails are not uniformly
distributed by size within this range. Several studies both in the Pacific Northwest and at
Hopkins Marines Station (HMS) have established that larger Tegula are more abundant in the
low end of the snails' range, while the upper portion is occupied almost exclusively by smaller
snails (Pacific NW: Paine 1969, Markowitz, 1980, Doering and Phillips 1983; HMS: Vara and
Wright 1964, Polk 1998).
There have been many conjectured explanations for the size gradient phenomenon.
These include differences in larval settlement (Paine 1969), gradients in juvenile mortality
(Vermeij 1972), and reactions to light (Doering and Phillips 1983). However, the most common
suggestion is predation pressure, primarily by the starfish Pisaster ochraceus. Paine (1969) was
the first to suggest this possibility, hypothesizing that large snails would move into the predator-
infested low intertidal environment because the benefits of more abundant food resources
outweighed the heightened predation risk. Markowitz (1980), who found that seastars prefer
large snails to small snails and that small snails had a more effective escape response, suggested
that the gradient could also be due to small snails moving upward to escape predation. However,
large snails were found to have faster escape speeds than small snails, and the only effect of
Pisaster would be to create an opposite gradient (Doering and Phillips 1983). These authors
concluded that "responses to the sea star are not necessary for size gradient formation.'
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This is the case at HMS, where there is a size gradient but little predation of any sort on
Tegula (Rocca 1995). After verifying that the size gradient existed in my experimental area, I
investigated the importance of thermal stress on its formation. First, I measured the temperatures
of gelatin-filled snail shells over a short time course in a dry environment and over a long time
course in the field at high and low intertidal sites. In addition to these temperature-monitoring
experiments, lattempted to quantify the levels of heat stress experienced by large and small
snails by measuring their levels of heat shock proteins (hsps).
Hsps are induced during the heat shock response, which occurs in almost all cells (Parsell
and Lindquist 1993). Hsps are molecular chaperones that help renature denatured proteins,
reduce their aggregation, and dispose of irreversibly denatured proteins. The synthesis of hsps is
thought to be proportional to the amount of stress the cell experiences (DiDomenico et. al. 1982).
Unfortunately, the hsp quantification experiments did not produce any conclusive results.
However, I found that, under equal conditions, large snails consistently reach higher
temperatures than small snails. Because earlier work indicates that small temperature increases
such as the ones I found can have negative physiological consequences, heat stress could be an
important factor in establishing the vertical size gradient of Tegula funebralis.
Materials and Methods
Definition of "large," "medium," and 'small
For all but the size distribution experiment, large snails are those between 17 mm
maximum basal diameter (mbd) and 22 mm mbd, and small snails are those between 7 mm mbd
and 12 mm mbd. In the size distribution study large snails were 17 or more mm mbd, medium
snails were between 12.1 and 16.9 mm mbd, and small snails were 12 or less mm mbd.
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Size Distribution
Sampling was conducted using a 0.25 m* quadrat and a measuring tape. I lay my transects
perpendicular to the shoreline and measured all the snails that fell within the quadrat at each
meter mark. Ithen used a surveyor’s level and stadia rod to determine the height of each of my
measurement areas.
Lethal Temperature
Snails were placed in a water bath at 13°C and heated at approximately 0.4°C per minute.
At regular time intervals, ten individuals of both large and small snails were removed and tested
for withdrawal response to tactile foot stimulation.
Controlled Heating Experiment
Tremoved four large and four small snails from their shells, then filled the shells with
gelatin, inserted a thermistor, and sealed the shell with silicone to prevent evaporation of the
water in the gelatin. The thermistor was attached to a Stow Away brand datalogger, which was
set to record the snail’s temperature every 5 seconds. The datalogger was enclosed in an Icalite
brand waterproof case in preparation for the field experiment described below.
All eight snails with dataloggers attached were placed on a cement surface in direct
sunlight on a clear day. I oriented all the shells in the same direction relative to the sun. The
snails were left undisturbed, but under constant observation, for one hour.
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Field Heating Experiment
The same gelatin-filled shells described above (minus one small shell that proved
unreliable in the above experiment) were attached to rocks in the intertidal zone: one small and
two large in the low intertidal (at the bottom of Tegula's range), and two each of large and small
in the high-mid intertidal (at the top of Tegula's range). The shells were secured to the rock by a
piece of fiberglass securing the thermistor cord and Z-spar securing the shell itself (Fig. 1). The
dataloggers were set to record the temperature every five minutes and the shells were left
attached for three weeks.
Heat Transfer Analysis
Tused the following equations to analyze the results of the heating experiments:
dU
Qs-Qe -Qe where
U= internal energy
Qs= solar energy gain
Oc = heat loss by convection to ambient air
OR = heat loss by radiation to adjacent bodies or to the sky
dU = me dT where
m = mass = Vp (volume times density) = 4/3 mo
c = specific heat
= Al where
a = absorptivity of snail surface to solar radiation
A = area exposed to sun. I approximated the snail as a sphere of radius r, so this would
be approximately ar
1 = Solar insolation
Qc - h.A.(T.-T,) where
h. = convective heat transfer coefficient
A. = effective area across which convective heat transfer occurs. Again I assumed a
sphere, so this would be the surface area, 4m
Ts = surface temperature of the snail
T= ambient air temperature
Tassumed radiant heat loss to be negligible.
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Field Setup for Heat Shock Experiment
Adjacent to the dataloggers, 1 attached four wire mesh cages: one cage each for large and
small snails in both the high and low intertidal areas. Twenty snails that had been acclimating in
13°C seawater for one week were put in each cage. I attached the cages four days before the
beginning of a nine-day low tide cycle (Day 0). I took a five-snail sample from each cage on
days 6, 8, and 12 of the experiment. These snails were placed immediately on dry ice and kept
frozen until dissection.
Heat Shock Analysis
Ifirst dissected the gill tissue of the snails under non-heat-shock producing conditions (in
ice-cold sea water). I immediately added 80 uL of homogenization buffer (32 mmol/L Tris-Cl
pH 7.5 at 4°C, 2% (w/v) SDS, 1 mmol/L EDTA, 1 mmol/L Pefabloc, 10 ug/mL pepstatin,
lOug/mL leupeptin) to small snail gill tissue and 200 uL to large snail tissue, then boiled the
mixture for five minutes. Next, I homogenized the tissue with a silicone pestle and boiled the
homogenate again for five minutes. Finally, I centrifuged the homogenate at 15,800 g for 15
minutes and removed the supernatant, discarding the tissue pellet. I determined the protein
concentration of each sample using the Pierce Micro BCA assay according to the manufacturer's
instructions.
separated the protein samples by SDS-PAGE (200V for approximately 45 minutes; 5 ug
of protein in each lane) on a BioRad mini-gel. Then I transferred the protein to a nitrocellulose
membrane (1 hour 15 minutes at 80 mA) and let the membranes dry overnight.
1 put each membrane in its own 45 mL tube and incubated the tubes with constant
agitation. First, I blocked the membranes with blocking buffer (0.025M Tris-Cl pH 7.5, O.15M
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Nacl, 0.1% (v/v) Tween, 0.02% (w/v) Thimerosal, 5% (w/v) non-fat dried milk) for one hour,
then washed them twice with tris-buffered saline (TBS; 0.025M Tris-Cl pH7.5, O.15M NaCl). I
then incubated the membranes for one hour with a 1:2,500 solution of Buffer A (TBS, 2.5%
(w/v) bovine serum albumin, 0.02% (w/v) Thimerosal) and a monoclonal rat antibody (IgG)
against hsp70 (clone 7.10; affinity BioReagent MA3-001). I next washed the membranes for
five minutes with TBS, twice for five minutes each with TBS containing 0.1% (v/v) Tween, then
a final five minutes with plain TBS. After that I incubated the membranes for 30 minutes with a
1:2000 solution of Buffer A and a rabbit-anti-rat bridging antibody (IgG; vector AI-4000),
followed by four washes as before (TBS/TBS-Tween/TBS-Tween/TBS). Finally, I incubated
the membranes for 30 minutes in a 1:5000 solution of Buffer A and horseradish-peroxidase
Protein A (BioRad), then washed the membranes for five minutes in TBS, three times for ten
minutes each in TBS with Tween, and, lastly, five more minutes with TBS.
These membranes were soaked in enhanced chemiluminescent reagent (ECL, Amersham)
for one minute and sealed in plastic wrap in an autoradiography cassette. I then exposed them, in
a darkroom, to pre-flashed hyperfilm (Amersham) for varying times (between 10 and 30
seconds) to obtain the most ideal exposures.
Results
As expected, the percentage of the Tegula population composed of large snails decreased
with intertidal height (Fig. 2). In the low zone, large animals composed up to 100 percent of the
population, while small snails composed almost 100 percent of the higher populations.
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The lethal temperature experiment revealed that there is no difference between the
temperature of heat death in large and small snails; all snails died during the interval between 43
and 44 degrees Celsius.
During the lab heating rate experiment, one datalogger was eliminated from the analysis
and from the field due to extremely anomalous results. The other seven dataloggers showed that
although small snails heat up more quickly at first, large snails reach higher temperatures (Fig.
3). This same result was seen for snails in the low intertidal, although only two dataloggers
survived the three weeks intact (Fig. 4). Unfortunately, no dataloggers from the large snails and
only one datalogger from the small snails returned usable data in the high intertidal, preventing
further analysis.
Only one run of the heat shock analysis was successful, so the data were insufficient for
analysis.
Discussion
Although the faster heating and cooling rates of smaller snails were expected, given that
they have a higher ratio of surface area to volume, the consistently higher temperatures of larger
snails are more difficult to explain. Using the heat transfer equations given in the Materials and
Methods, the ratio of the heating rate of a small snail to a large snail with twice the radius (e.g., a
small snail with radius 5 mm and a large snail with radius 10 mm) is:
sml ()
-(sml)
a'1 - h. (4mr2)(T. -7.)
g Gg) ar(2r)1-h(Ar(2r))Ts-T.)
which simplifies to:
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2
di
af
Urg) «1 -4h. (T,-T.)
dt
Given that he, 1 and 74 are equal, and that the snails start out with equivalent surface
temperatures (Ts), the only possible explanation for the observed temperature difference is that
large snail shells have a higher absorptivity than small ones. Since absorptivity is closely related
to color, this indicates that the ratio of black shell to white shell for large snails may be greater
than for small snails. Simple field measurements should be done to determine if this hypothesis
is true.
Given two to five degree C warmer body temperatures for large snails and the equivalent
heat tolerance of snails of different sizes, living higher in the intertidal would be more difficult
for large snails over time. Tomanek and Somero (1999) found that at 36 degrees—a temperature
often reached by the large gelatin-filled snails but never by the small ones—Tegula funebralis
gill tissue stopped producing any non-hsp protein, a sign of extreme heat stress. An analysis,
like the one 1 attempted, of heat shock proteins in both large and small snails kept under
equivalent environmental conditions would verify whether living large snails are truly under
greater heat stress, and the magnitude of the stress if it exists.
The advantage of small size appears not to be restricted simply to the species Tegula
funebralis. There also seems to be an interspecies intertidal size gradient that warrants further
investigation. For example, the subtidal species Tegula brunnea is generally larger than the mid¬
intertidal Tegula funebralis, while the high-intertidal species of genus Littorina are generally
smaller than both species of Tegula (Shannon Taylor, unpublished observations). This gradient
may not be due entirely to temperature (for instance, small snails may be better able to exploit
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macroalgae and rocks for protection for the sun and predators (Garrity 1984)), but heat stress is
likely to be involved.
Conclusion
Although the intertidal size gradient of Tegula funebralis is multifactorial, heat stress seems to
play an important role in maintaining the upper limits. Because something— possibly their
lowered absorptivity— helps small snails avoid the high temperatures of large snails, the small
snails live in a less stressful microenvironment than their large counterparts and, therefore, are
likely to have a higher chance of survival. This small-size advantage may also be an interspecies
phenomenon
Acknowledgements
I would like to thank my advisors, Prof. George Somero and Dr. Lars Tomanek, for helping me
conceive and run my experiments. I would also like to thank my father, Steve Taylor, for
helping me to understand heat transfer physics.
Shannon Taylor
Works Cited
DiDomenico, B. J., G. E. Bugaisky, and S. Lindquist. 1982. The heat shock response is self-
regulated at both the transcriptional and posttranscriptional levels. Cell 31: 593-603.
Doering, P. H. and D. W. Phillips. 1983. Maintenance of the shore-level size gradient of Tegula
funebralis: Importance of behavioral responses to light and sea star predators. J. Exp.
Mar. Biol. Ecol. 67: 159-173.
Garrity, S. D. 1984. Some adaptations of gastropods to physical stress on a tropical rocky shore.
Ecology 65(2): 559-574.
Markowitz, D. V. 1980. Predator influence on shore-level size gradients in Tegula funebralis.
J. Exp. Mar. Biol. Ecol. 45: 1-13.
Paine, R. T. 1969. The Pisaster-Tegula interaction: Prey patches, predator food preference, and
intertidal community structure. Ecology 50(6): 950-961.
Parsell, D. A. and S. Lindquist. 1993. The function of heat-shock proteins in stress tolerance:
degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27: 437-496.
Polk, R. E. 1998. The Tegula funebralis shore-level size gradient: Implications of susceptibility
to predation, and settlement. Unpublished student paper from Hopkins Marine Station,
Stanford University.
Rocca, M. E. 1995. Factors affecting the intertidal zonation of Tegula funebralis and T.
brunnea. Unpublished student paper from Hopkins Marine Station, Stanford University.
Tomanek, L. and G. N. Somero. 1999. Evolutionary and acclimation-induced variation in the
heat shock responses of congeneric marine snails (genus Tegula) from different thermal
habitats: Implications for limits of thermotolerance and biogeography. J. Exp. Biol. 202:
2925-2936.
Vermeij, G. J. 1972. Intraspecific shore-level size gradients in intertidal molluscs. Ecology 53:
693-700.
Wara, W. M. and B. B. Wright. 1964. The distribution and movement of Tegula funebralis in
the intertidal region, Monterey Bay, California. Veliger (suppl.) 6: 30-37.
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Figure Captions
Fig. I Close-up photo of a large snail attached with Z-spar and fiberglass to an intertidal rock.
Fig. 2 The size distribution (by percentage of population) of Tegula funebralis in the
experimental region. Error bars are 95% confidence intervals.
Fig. 3 Temperatures of gelatin-filled snails on concrete in direct sunlight. Both lines are
averages (small snails n=3, large snails n—4).
Fig. 4 Temperatures of two gelatin-filled snails attached in the field (low intertidal) as per Fig. 1,
Each data set is from a single datalogger.
Figure 1
13

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120%
100%
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Figure 2
error bar for small snails at
1.7 meters extends to 148%


-0.2 0.0 0.3 0.6 0.7 1.7
1.8 1.9 2.2 2.3 2.5 2.6 2.7 2.9 4,3
Height above Zero Tide
Large Snails (169 mm) MMedum Snals (12.1 t0 16.9 mm) ESmall Snais (612.1 mm).
14
5 32
.
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Figure 3
Average Temperature of Gelatin-filled Snail Shells Heated in Direct Sunlight
f
e

F



10.1200 101812 102824 10.3338 104048 104800 105512 110224 110938 1848 112400 1312 113824
Time
— Small Snais — Large Snais
25
4/29/00
5/4/00
Figure 4

5/9/00
5/14/00
Date
— Small Snail — Large Snail
5/19/00
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