Body Temperature of the Intertidal Snail Chlorostoma Funebralis

Home , Bulla gouldiana, Tegula funebralis

Preference Versus Performance: Body Temperature of the Intertidal Snail Chlorostoma funebralis

SARAH TEPLER, KATHARINE MACH, AND MARK DENNY* Hopkins Marine Station of Stanford University, Paciﬁc Grove, California 93950

Abstract. Evolutionary theory predicts that, in variable intertidal shores. In general, animals more tolerant of ther- environments, it is advantageous for ectothermic organisms mal extremes extend higher on the shore (reviewed in to prefer a body temperature slightly below the physiolog- Newell, 1970, pp. 451–468; Somero, 2002, 2010). Because ical optimum. This theory works well for many terrestrial shifts in intertidal zonation can be a sensitive bellwether of organisms but has not been tested for animals inhabiting the ecological response to changes in the thermal environment, hypervariable physical environment of intertidal shores. In it has been suggested that rocky intertidal shores can serve laboratory experiments, we allowed the intertidal snail as a model system for exploring the consequences of global Chlorostoma funebralis to position itself on a temperature warming (Helmuth et al., 2006; Benedetti-Cecchi et al., gradient, then measured its thermal preference and deter- 2006; Somero, 2010). To this end, considerable effort has mined an index of how its performance varied with temper- been expended to understand the genetic and biochemical ature. Snails performed a biased random walk along the basis for tolerance of extreme temperatures in intertidal temperature gradient, which, contrary to expectations, animals (reviewed by Somero, 2002, 2010). caused them to aggregate where body temperature was 15 to Have intertidal animals evolved according to the same 17 °C below their temperature of optimum performance and rules as terrestrial animals? Studies have measured thermal near the species’ lower thermal limit. This “cold-biased” preferences in a few nearshore organisms (e.g., snails: New- behavioral response may guide snails to refuges in shaded ell, 1970; Casterlin and Reynolds, 1980; Herrera et al., cracks and crevices, but potentially precludes C. funebralis 1996; Munõz et al., 2005; abalone: Hecht, 1994; a crab: from taking full advantage of its physiological capabilities. McGaw, 2003; other crustacea: Lagerspetz and Vainio, 2006). However, because these measurements of behavioral Introduction preference have not been accompanied by measurements of The body temperatures of intertidal ectotherms can vary physiological optima, they are difficult to compare to ter- drastically when low tides subject them to the exigencies of restrial results. the terrestrial environment, making the intertidal zone of In this study, we take a tentative step toward comparing wave-washed rocky shores one of the most thermally stress- the evolution of thermal preference in terrestrial and inter- ful on Earth. Winter cooling and summer heating can cause tidal organisms by both measuring behavior and determin- intertidal animals to exceed their lethal limits (e.g., Kan- ing an index of performance in the black turban snail wisher, 1955; Newell, 1970; Sutherland, 1970; Murphy and Chlorostoma (formerly Tegula) funebralis (A. Adams, Johnson, 1980; Helmuth, 1998; Helmuth and Hofmann, 1855). C. funebralis is common in the mid- to low intertidal 2001; Wethey, 2002; Stillman, 2003; Somero, 2005; Harley, zone (0 to ϩ1.8 m relative to mean lower low water) on 2008; Miller et al., 2009), and interspecific differences in rocky shores of the west coast of North America (Abbott tolerance of the thermal environment have been implicated and Haderlie, 1980; Doering and Phillips, 1983). It fre- in helping to establish the vertical zonation characteristic of quently experiences high temperatures when exposed during daytime low tides, with a maximum recorded body temperature of 34.5 °C (Tomanek and Sanford, 2003). In Received 1 April 2010; accepted 25 January 2011. * To whom correspondence should be addressed. E-mail: mwdenny@ comparison, Stenseng et al. (2005) demonstrated that heart stanford.edu failure does not occur until 39.4 °C, and Tomanek and

107 108 S. TEPLER ET AL.

Somero (1999) measured a median lethal temperature of Grove, California (36° 36ЈN, 121° 54ЈW), the same site 42.5 °C. Studies of the expression of heat-shock proteins used by Tomanek and Somero (1999, 2000) and Stenseng et yield similarly high thresholds: increased synthesis of heat- al. (2005). Snails were collected immediately before exper- shock proteins—indicating the onset of thermal stress— imentation and tested without acclimation. begins at 27 °C in C. funebralis, and protein synthesis does not cease entirely until 39 °C (Tomanek and Somero, 1999). Thermal limits Furthermore, in contrast to its lower intertidal congeners (C. montereyi and C. brunnea), C. funebralis constitutively To design our temperature-preference experiments, we produces two major types of heat-shock proteins (Tomanek, ﬁrst needed to ascertain C. funebralis’s thermal limits. In 2005) and is able to up- and down-regulate production typical laboratory evaluations of thermal tolerance, snails within a single tidal cycle (Tomanek and Somero, 2000). are immersed in water initially at ambient sea-surface tem- Thus, C. funebralis appears well adapted to cope with high perature, and temperature is then increased (Tomanek and temperatures. C. funebralis is tolerant of low temperatures Somero, 1999; Stenseng et al., 2005). Samples of snails are as well— heart function ceases only when body temperature periodically removed from elevated temperatures and im- falls below 3 °C (Stenseng et al., 2005). mediately tested for viability. The process is continued until If C. funebralis follows the pattern typical of terrestrial all snails are dead, and the temperature required to kill half organisms, physiological optimum body temperature should the snails in a sample group is reported (Tomanek and be intermediate between the upper and lower temperatures Somero, 1999; Stenseng et al., 2005). In this protocol, snails at which its heart stops (3 and 39.4 °C); a simple average are exposed to elevated temperatures for only short periods, suggests an optimum temperature near 21 °C. Similarly, if providing little information about tolerance for extended terrestrial theory applies to C. funebralis, its preferred tem- exposure to high temperatures. However, animals in the perature should be lower than, but within a few degrees of, ﬁeld may be subjected to elevated temperatures throughout this optimum. Shell color can affect body temperature by a low tide (Ͼ12 h), and it is in response to this period that determining the fraction of solar irradiance absorbed by a they would be most likely to express a thermal preference. snail (e.g., Heath, 1975; McQuaid and Scherman, 1982; To understand the relationship between thermal tolerance

Etter, 1988; Phifer-Rixy et al., 2008). C. funebralis’s black and time of exposure, we determined Lt50 (the time required shell could thereby help it maintain a preferred temperature to kill 50% of snails) for a range of temperatures. For each near 21 °C even if air temperature is lower. trial, 20 snails were placed in a sealed 1-liter jar along with It would be difficult to test these predictions unambigu- a seawater-saturated paper towel to maintain 100% relative ously in the field where confounding effects (such as the humidity. Jars were then submersed in a water bath for presence or scent of predators) could obscure any behav- intervals of 15, 30, 60, and 120 min at each of five temper- ioral response to temperature. Instead, we tested our predic- atures (37.2, 38.4, 39.3, 40.2, and 41.6 °C). In repeat trials, tions in the laboratory where confounding effects could be the range of exposure periods was adjusted for each tem- minimized. We allowed C. funebralis to position itself on a perature to more precisely determine Lt50. Additional trials temperature gradient, thereby expressing its preferred body to determine Lt50 of C. funebralis at 34 °C required longer temperature. By video-recording movement on the thermal periods of exposure, from 7 to 14 h; a similar procedure was gradient, we quantified an index of physiological perfor- followed, but with netting replacing the jar lid to provide air mance as a function of temperature, allowing us to estimate circulation. the snail’s optimum temperature. C. funebralis’s preferred Death was defined by a total lack of response to tactile temperature is far below its optimum temperature—indeed, stimuli. Responsiveness was determined by poking the pro- near the low temperature at which its heart stops—suggest- truded feet of snails or gently tugging on the edge of the ing that different rules apply to at least this intertidal animal operculum with forceps. For the high-temperature trials, if than apply to many terrestrial species. the snail retracted its foot or was completely retracted prior to stimulation, it was deemed responsive. For the longer, 34 Materials and Methods °C trials, the status of a completely retracted snail was deemed indeterminate, and these snails were not included in Collection of test organisms further analyses. Survivorship was initially evaluated im- Chlorostoma funebralis is common on rocky shores from mediately after treatment; snails were then transferred to Vancouver Island (British Columbia) to central Baja Cali- larger perforated containers and placed in running filtered fornia (Mexico) (Abbott and Haderlie, 1980). Specimens seawater at ambient temperature (Ϸ14 °C) in outdoor sea were collected (October to December 2004, 2005, and 2008 tables. Survivorship was subsequently evaluated at 1 hour, 1 for thermal-limit trials, and January to March 2009 and day, 2 days, and 3 days after treatment. To control for the November to December 2010 for thermal-preference trials) possibility that snail mortality during the recovery period from a protected site at Hopkins Marine Station in Pacific was due to starvation rather than heat stress, we fed sea- CHLOROSTOMA FUNEBRALIS THERMAL PREFERENCE 109 weeds (Macrocystis pyrifera and Chondrocanthus exas- lated) and attachment to the substratum (foot extended/foot peratus) to recovering snails in half of the trials. The Lt50 of retracted) were also recorded, and snails were returned to C. funebralis at each temperature was approximated by the shore. The experimental area was then emptied and fitting a sigmoidal curve to the data using the following scrubbed to remove residual chemical cues and mucous equation, in which f is the fraction of snails killed, T ϭ trails. After seven trials, the temperatures at the ends of the temperature (°C), t ϭ time of exposure to temperature T block were reversed to control for local substratum effects, (minutes), and a is a coefficient (Miller et al., 2009): and a second set of seven trials was conducted. Note that, because of the extreme temperatures at the ends of the 1 ͑ ͒ ϭ Ϫ thermal gradient, the end bins of the arena could not be f T 1 Ϫ (Eq. 1.) t Lt50 approached by crawling snails. In this respect, the arena was 1 ϩ expͩ Ϫ ͪ a effectively endless. At temperatures lower than 2 °C and higher than 28 °C, Thermal preference trials more than 50% of the snails retracted the foot into the shell, a behavior we have not observed in the field but which has In a standard technique for evaluating thermal preference been reported for other trochid gastropods (Newell, 1970, (Licht et al., 1966; Dillon et al., 2009), we placed specimens pg. 468). Because these unattached snails could not move, of C. funebralis on a substratum with a temperature gradient they could not express a temperature preference. Therefore, and allowed them to wander freely. The substratum was a when assessing temperature preference, we treated them horizontal aluminum block (15-cm wide by 1.8-m long) separately from snails whose feet were attached to the within which a stable temperature gradient (–1.7 °C to 34 substratum, and we analyzed the preference of attached °C) had been created by cooling one end and heating the snails in the 14 bins in which they formed the majority. For other. A temperature of 34 °C was chosen as the upper statistical analyses, snails in this portion of the temperature boundary because our measurements of thermal tolerance gradient were categorized as falling into one of seven equal- showed it to be lethal to C. funebralis in a time period sized temperature treatments, each two bins wide. We then slightly less than the duration of a long low tide (12 h), our carried out a multivariate analysis of preference on the experimental period. The experimental area was defined by seven temperature treatments (Lockwood, 1998), with the a 165-cm by 13-cm rectangular boundary of 1-cm-high null hypothesis that snails express no temperature prefer- acrylic plastic strips on the surface of the block, made ence and are therefore uniformly distributed across the watertight with silicone aquarium sealant. The plastic strips seven treatments. were topped with copper foil, which deters many marine As a control for any effects of the arena other than snails and prevented escape. The experimental area was temperature, a second set of 12 trials was conducted with divided along its length into 33 “bins” by drawing hash the entire aluminum block at room temperature. Each trial marks across the aluminum substratum every 5 cm along the was carried out as described above, and the orientation of temperature gradient. The block was surrounded by foam the arena was rotated 180° after the sixth trial to control for insulation on its bottom and sides to stabilize the tempera- any bias from lighting or visual cues. Analysis was con- ture gradient. ducted as described above, but with 11 “treatments,” each For each trial, the experimental area was filled to a depth three bins wide. These experiments are less than perfect as of about 0.5 cm with fresh seawater, which quickly attained a control because, in the absence of the thermal gradient, the same horizontal temperature gradient as that of the snails had access to the ends of the arena, and these end bins aluminum block. Two freshly collected specimens of C. have 2.32 times the wall area of other bins. When not funebralis were placed aperture-down in each bin, and to crawling, individuals of C. funebralis prefer to be in contact minimize visual cues, the block was covered at a height of with a wall or another snail; thus the end bins are biased 2 cm above the substratum with translucent white acrylic toward the accumulation of snails. To cope with this prob- plastic sheets. The snails were then left overnight (12ϩ h) lem, in the analysis of snail distributions in the constant- under constant illumination (2.80 ␮mol photons m–2 s–1). temperature arena, we used the number of snails per bin per All light-level measurements were made with a LiCor LI- area of wall as our measure of abundance at locations within 250A quantum radiometer. At the end of each trial, the the arena. temperature of the water (which equaled the temperature of the substratum) at the center of each bin was measured to Thermal preference behavior video trials the nearest 0.1 °C with a thermocouple thermometer (HH23, Omega Engineering). The small size and slow movement of The experiments outlined above measure the final posi- snails ensured that their body temperatures were equal to the tion (and hence, temperature) of snails, but provide no temperature of the water and substratum at their location. information about the movement of snails leading to those Snail positions (from which temperature could be calcu- positions. To quantify movement, we repeated the experi- 110 S. TEPLER ET AL. ments on the temperature gradient while filming snails as they crawled. This required alteration of the experimental visual field. Many intertidal snails have excellent vision (Cronin, 1986), and C. funebralis has been shown to possess both directional vision (eyestalks) and general somatic pho- toreceptors as well as the ability to detect substratum color (Kosin, 1964; Marchetti and Geller, 1987). In our thermal preference trials, the ability of snails’ vision to provide cues to their location on the experimental substratum was minimized by covering the apparatus with a uniform, translucent white ceiling. However, this surface precluded observing Figure 1. A “leg” of a snail’s trail is the distance traveled along the snails while they moved. To both film snails and minimize axis of the temperature gradient between changes of direction. visual cues, we placed the temperature gradient apparatus in a dark box 1.8-m long, 0.6-m wide, and 1.2-m high. Two infrared L.E.D. strips and a Watec AD-502A video camera of snails would be Gaussian (ϭ normal) with mean and fitted with a Fujinon YF2.8A-2 wide-angle lens were skew of 0 (Berg, 1984; Denny and Gaines, 2000). Any mounted in the top lid of the box. An opaque door (1.2 ϫ nonrandom behavior would result in a shift of the mean 0.3 m) in the front of the box provided access to the displacement away from 0, and possibly in the introduction temperature gradient. The inside of the box was painted flat of skew. Here, skew was assessed with the index g1 (Zar, black, all seams were sealed with gaffer’s tape, and the box 1999), and the probability of obtaining skew of a given was ventilated with a fan attached to a series of light- magnitude was assessed using standard bootstrap proce- precluding baffles. C. funebralis exhibits no shadow avoid- dures (Efron and Tibshirani, 1993). We analyzed 194 indi- ance under the far red of a darkroom light (Kosin, 1964) and vidual snail trails. presumably cannot see infrared light. Within the dark box, the temperature gradient was cov- Performance ered with clear acrylic plastic sheets, replacing the translu- A variety of metrics can be used to gauge physiological cent sheets from the previous experiments. Snails seldom performance. Some—such as growth rate and reproductive move in complete darkness (Doering and Phillips, 1983); to output—have direct ties to the fitness of an organism but induce normal movement it was necessary to crack the top cannot be measured in short-term experiments. Others— lid to allow diffuse ambient light (Ͻ0.01 ␮mol photons/m2/ such as jumping or sprinting performance, the standard s), which is too faint to induce shadow-avoidance in C. measures used in studies on lizards and frogs (Angilleta, funebralis (Kosin, 1964). Trials were recorded as stop- 2009)—are only indirectly tied to fitness but are easily motion video with a Panasonic time lapse VCR AG-6730, 1 measured. In this study, we quantified the rate at which frame every 6.44 s, and the direction of the gradient was snails initiated legs of travel (defined above) as an index of reversed after half the experiments (5 trials). intensity of activity, and used this index as our measure of Trails were tracked by manually following snails’ images physiological performance. As with jumping and sprinting, with permanent marker on acetate sheets placed against the this index is only indirectly related to fitness, but it is readily video screen. Trails of 9–10 randomly chosen snails were measured from video records and has the advantage of tracked at the start of each trial, and 9–10 more were providing information directly applicable to the mathemat- tracked starting halfway through the trial. The labeled ace- ical simulation of snail movements described below. tate sheets were then scanned into digital format; ImageJ, ver. 1.42q (NIH, Bethesda, Maryland) was used to deter- Simulations mine the position of the snail over time. Position data were then used to find the average crawling speed and the length Because C. funebralis withdraws into its shell at extreme and direction of legs of travel within single trails. A leg is temperatures (see above), it is possible that random motion defined as the displacement between two subsequent could mimic thermal preference in the final distribution of changes in direction along the long axis of the temperature snails on our temperature gradient: given sufficient time, a gradient (Fig. 1). ImageJ was also used to translate the snail moving entirely at random on the gradient would position of snails on the experimental substratum (observed eventually encounter a temperature extreme enough to in our video images) to the corresponding temperatures. cause it to withdraw into its shell. Random motion could Movement of snails was assessed relative to the null thus lead to a final distribution in which snails are aggre- hypothesis that snails moved randomly. That is, if snails gated at extreme temperatures, the same distribution that moved along the temperature axis of the substratum in an would be obtained if snails preferred these extreme temper- unbiased random walk, the distribution of net displacement atures. To explore this possibility, we simulated several CHLOROSTOMA FUNEBRALIS THERMAL PREFERENCE 111 aspects of the behavior of C. funebralis on a temperature gradient. Using a standard approach to a random walk (Berg, 1984; Denny and Gaines, 2000), a “snail” was allowed to take steps of length d along the x-axis representing the temperature gradient. For each step, a random number was chosen from a uniform distribution between 0 and 1. If that number exceeded a threshold p, the snail took a step in the positive x direction toward higher temperatures. If the random number was less than or equal to p, the step was taken in the negative x direction toward cooler temperatures. If p equaled 0.5, the snail’s random walk was unbiased; that is, it had equal chance of moving in each direction at each step. If p was greater than 0.5, the walk was biased toward movement to the cold end of the axis, if p was less than 0.5, the walk was biased toward movement to the hot end of the axis. Figure 2. In each simulation, we placed 66 hypothetical snails on In an unbiased random walk, the probability that a snail will travel distance L (here measured in °C) before changing direction (L ϭ leg the x-axis in positions corresponding to the center of the length) is 1/2n where n is the number of steps in the leg. For example, a bins defined by hashmarks on the actual gradient. Each snail snail initially takes a step toward the cold end of the temperature gradient. was then tracked through N steps (see below). If the snail The probability that it will step back to its initial position (for a leg length moved to a simulated temperature below 2 °C or above 28 of 1 step) is 1/2. The probability that it takes two steps in the same direction 2 etc. n ϭ °C, it was assumed to withdraw into its shell, and no further before reversing course is 1/2 , Now, the number of steps taken is L/d, where d is the length of each step. Thus, of all legs of all lengths, the motion was allowed. The final position of each snail and its fraction of legs having length L is state (attached or unattached) were recorded. This procedure 1 Prob ͑L͒ ϭ (Eq. 3) was repeated 100 times, and the fraction of snails occupying 2L/d each bin was averaged across trials. From our video measurements of L we can determine the fraction having certain lengths, as shown in this figure. The value of d is then chosen to A step length d of 0.918 °C was chosen on the basis of minimize the deviation of Eq. 3 from these empirical data. data from our video trials. With this step length, the distribution of probabilities of multiple steps in the same direction best matched the distribution of the lengths of individ- by its maximum value provided an appropriately scaled ual legs of travel in observed snail trails (Fig. 2). N was set estimate of s (see Fig. 4 in the Results). to 73, the number of steps of length d a snail would take in 12 h, crawling at the median speed recorded in our trials Results (6.12 °C h–1). Two experiments were conducted using this simulation. Thermal limits In the first, p was varied to determine what level of bias (if Survivorship was high in the first 2 days after snails were any) would be required to yield a final distribution of exposed to an elevated temperature, with a notable die-off hypothetical snails similar to that observed for real snails. on the 3rd day. Trials carried out up to 5 days post-exper- The second experiment was designed to explore potential iment showed no further decrease in survivorship. Conse- effects of body temperature on the likelihood that a snail quently, we used survivorship at 3 days as our index for would initiate a step. The movement of real snails revealed thermal tolerance. There was no significant difference in a temperature-dependence: frequency of step initiation survivorship between fed and unfed snails held for as long peaked at a temperature of about 20 °C. We implemented as 5 days after treatment (Student’s t-test,Pϭ 0.99). The this effect in the simulation as follows. At each time incre- log of median survival time (Lt ) decreased linearly with ment, we chose a random number from a uniform distribu- 50 increasing temperature (Fig. 3) tion between 0 and 1. If this number was less than a threshold s, the snail then proceeded to take a step in the Thermal optimum direction determined by the choice of a second random number and the threshold p (as described above). By vary- The relative probability of snails initiating a leg as a ing s between 0 and 1 according to the temperature of the function of body temperature is approximated by a third- snail (set by its location on the temperature gradient), we order polynomial (Fig. 4): could vary the stepping rate of our hypothetical snails. We ϭϪ 3 ϩ 2 Ϫ ϩ fit a third-order polynomial curve to the observed frequency s 0.000375T 0.0131T 0.0645T 0.0559 of leg initiation at each temperature. Dividing this equation (Eq. 2) 112 S. TEPLER ET AL. ϭ Ͻ had a negative skew (g1 –0.88, P 0.05), indicating that there were more long displacements toward low temperatures than toward high temperatures. When displacement data were normalized by total possible displacement in the direction of movement (i.e., the distance to the end of the gradient in that direction), the distribution of this ratio had a negative mean (–0.11, Student’s t-test, P ϽϽ 0.001) and ϭ Ͻ was negatively skewed (g1 –0.33, P 0.05, data not shown). The interaction between net displacement and starting temperature was significant. The warmer the starting temperature, the more negative (cold) the net displacement (P ϭ 0.004, r2 ϭ 0.042, Fig. 7). To quantify the difference between types of movement (a few long legs vs. many short legs), the longest leg of each snail’s trail was divided by the total displacement of the Figure 3. As temperature increases, the time required to kill 50% of ϭ trail. Large absolute values of this leg-length ratio (those snails decreases. Log10 Lt50 9.78–0.204T, where T is experimental temperature (r2 ϭ 0.995, P ϽϽ 0.001). approaching 1) identify snails that made one large step that dwarfed otherwise small movements, whereas small absolute values identify snails for whom even the longest step where T is temperature (°C). This index of performance is was not a large component of total displacement (Fig. 8). high for temperatures between 16 °C and 24 °C, with a peak More trails had large absolute values in the cold-ward at 20 °C. (negative) than in the warm-ward direction, particularly for leg-length ratios close to 1.0; however, the means of abso- Thermal preference lute value for cold-ward and hot-ward travel are not significantly different (Student’s t-test, P Ͼ 0.1). On a surface of uniform temperature, all snails remained attached to the substratum. Average temperature among trials was 20.3 °C with a standard deviation of 1.26 °C. Simulations Multivariate analysis of their final location does not reject Simulations implementing a non-biased random walk the null hypothesis that C. funebralis has no thermal pref- (P ϭ 0.5) and no temperature-dependent variation in the 2 erence (Hotelling’s T ϭ 294.84, n ϭ 12, P Ͼ 0.16). In probability of movement yielded a final distribution with other words, when offered no temperature cues, the distri- equally large sharp peaks of unattached snails at both ex- bution of snails cannot be distinguished from random (Fig. tremes of the temperature gradient (Ͼ21% of all snails), 5A). In contrast, for snails offered a thermal gradient, analysis of attached snails rejects the null hypothesis (Hotell- ing’s T2 ϭ 27.264, n ϭ 7, P Ͻ 0.001) (Fig. 5B). Owing primarily to the small number of trials, post hoc tests are not powerful enough to definitively locate the preferred temperature, but examination of 95% confidence intervals of snails’ locations suggests that the fraction of snails attached to the substratum at temperatures between 3 and 5 °C was greater than the fraction seen in other temperature treatments (Fig. 5B). The apparent aggregation of attached snails at temperatures of 3 to 5 °C—temperatures at which unattached snails are common (Fig. 5C)—indicates that these aggregated snails moved as far as possible toward the lower thermal limit set by cessation of heart function.

Thermal preference behavior The distribution of total displacement of individual trails ϽϽ had a negative mean of –2.15 °C (Student’s t-test, P Figure 4. Variation in the probability of step initiation as a function 0.001), indicating that on average snails moved toward of body temperature. The solid line is the third-order polynomial ﬁt to the lower temperatures (Fig. 6). This displacement distribution data shown (Eq. 2). CHLOROSTOMA FUNEBRALIS THERMAL PREFERENCE 113

Figure 5. Location of Chlorostoma funebralis individuals on the temperature gradient at the end of a 12-h experiment. (A) All snails on the constant-temperature arena. The horizontal line is the fraction of snails that would be found in each of the 33 bins if snails distributed themselves uniformly. Note that bins were lumped in groups of three for statistical analysis. (B) Attached individuals of C. funebralis; the horizontal line is the fraction of snails that would be found in each of the 14 temperature treatments containing attached snails if snails distributed themselves uniformly. (C) Unattached individuals of C. funebralis; the horizontal line is the fraction of snails that would be found in each of the 11 treatments containing unattached snails if snails distributed themselves uniformly. All error bars are 95% conﬁdence limits. with few snails remaining on the central area of the gradient temperature-dependent probability of movement yielded a (Fig. 9A). Simulations in which walks were again unbiased distribution with a large gradually tapering peak (12%) at but the probability of taking a step varied with temperature Ϸ4 °C (Fig. 9D). While this last simulation did not perfectly resulted in ﬁnal distributions with a large sharp peak of mimic our distributional results (Fig. 9E), it was the only nonviable snails (20%) at the hottest extreme of the gradient simulation that possessed a dominant and tapering peak of (Fig. 9B). If the random walk was biased toward cold-ward attached snails at the cold end of the gradient with compar- movement (P ϭ 0.63) with no temperature-dependent vari- atively reduced peaks of unattached snails at the extremes. ation in the probability of movement, most snails (nearly In summary, temperature-dependent rates of movement 60%) ended up unattached at very low temperatures (Fig. could not re-create actual results for attached and unat- 9C). Lastly, the simulation of movement incorporating a tached C. funebralis distributions without the incorporation slight bias toward cold-ward movement (P ϭ 0.63) and of bias. 114 S. TEPLER ET AL.

Figure 6. The distribution of snail displacements. The mean displace- Figure 8. The distribution of normalized maximum step lengths for ment, –2.15 °C, is significantly less than 0. steps toward the hot end of the gradient (open circles) and toward the cold end of the gradient (filled circles). Normalized maximum step length is the longest step in each snail’s trail divided by the longest possible step. Discussion Chlorostoma funebralis exhibits a thermal optimum at Ϸ20 °C, near that expected by analogy to terrestrial organ- brates prefer (Micallef, 1966, cited in Newell, 1970; Hecht, isms and similar to the optima measured for other trochid 1994; Munõz et al., 2005). snails (Micallef, 1966, as cited in Newell, 1970, pg. 471). In Why might C. funebralis have evolved a preference for a contrast, the snail’s apparent thermal preference is near (or low body temperature? Martin and Huey (2008) hypothe- even at) the species’ lower thermal limit, the temperature at sized that, for most ectotherms, environmental variability which its heart stops (Stenseng et al., 2005). Although C. (such as that typical of the intertidal zone) decouples pre- funebralis lacks a strictly directional response to the tem- ferred body temperatures from physiologically optimal perature of the substratum, there is a measurable bias to its body temperatures. Animals “choose” the relatively benign wandering that results in net displacement to body temper- detriment of having a body temperature slightly below the atures as cold as possible. There is no indication in our data optimum in order to avoid the potentially serious negative that C. funebralis prefers the elevated body temperatures effects of slightly exceeding the optimum. That is, in a that its physiology can withstand, that its dark coloration variable thermal environment, there is an evolutionary ad- could potentially provide, and that other nearshore inverte- vantage to erring on the cooler, and thus “safe,” side of temperature preference. However, the model of Martin and Huey (2008)—when applied to a variety of insects and lizards—predicts, on average, a preferred body temperature only 1.8 °C below the physiological optimum. In contrast, C. funebralis’s “preferred” body temperature of 3 to 5 °C is 15–17 °C below its apparent physiological optimum. To fit the Martin and Huey model, a shift this large would require a grossly asymmetric performance curve, but C. funebralis’s performance curve (Fig. 4) closely resembles that of lizards and insects. In a recent modification of the theory of Martin and Huey (2008), Asbury and Angilletta (2010) incorporated the ef-

fects of thermodynamics (essentially, a Q10 effect) on thermal preference, and predict a slightly larger gap between preferred and optimum temperatures, but not to the extent that preferred temperature approaches a species’ lower thermal limit. Figure 7. The warmer the temperature at which a snail began its Interpretation of C. funebralis’s behavior in an evolution- travels, the more negative (toward the cold) its displacement. Net displace- ary context is further complicated by the fact that it is ment (°C) ϭ 2.21 – [0.245 ϫ starting temperature (°C)]. unlikely that the snails’ “preference” for near-freezing tem- CHLOROSTOMA FUNEBRALIS THERMAL PREFERENCE 115

Figure 9. Results from simulated random walks. (A) An unbiased random walk (P ϭ 0.50) with equal probability of stepping across all temperatures. (B) As in A, but with probability of stepping set according to Eq. 2. (C) A biased random walk (P ϭ 0.63) with equal probability of stepping across all temperatures. Note the large fraction of unattached snails at 3 °C. (D) As in C, but with probability of stepping set according to Eq. 2. (E) Empirical results from our experiments. peratures—as observed in the laboratory—could reliably be evolved in response to the temperatures found in other expressed in the intertidal zone. On the Monterey Peninsula, portions of the snails’ geographic range. where the experimental snails were collected, temperatures A similar disconnect between thermal preference and are seldom this cold. Measurements of minimum body physiological optima has been observed in some reptiles temperature have not been conducted at Hopkins Marine (e.g., nocturnal lizards; Huey and Bennett, 1987) and is Station (HMS), but the lowest sea-surface temperature re- strongly associated with habitat-seeking behavior that con- corded during a 7-year period preceding our study was 8.98 veys a competitive or anti-predation advantage. Studies of °C, well above the temperature preferred by the snails in the these animals have shown that while they possess a high laboratory. The lowest air temperature recorded at HMS thermal optimum and thermal preference in the laboratory, during this period was 1.50 °C, near the preferred temper- their habitat selection in the field restricts them to low ature, but temperatures this low are rare: air temperatures temperatures, sometimes to physiological detriment (Ang- below 5 °C occurred only 0.28% of the time in the 7-year illetta et al., 2006). Although C. funebralis displays a dif- record. It is possible that on clear nights C. funebralis’s ferent pattern, with thermal preference in the laboratory body temperature could be lower than either water or air diverging sharply from thermal optimum, a similar situation temperature (Gates, 1980), but clear nights are themselves may apply: cold-biased wandering enables C. funebralis to rare in notoriously foggy Monterey. It is thus difficult to travel toward safe, but metabolically suboptimal, habitats. imagine how a preference for a body temperature of 3 to 5 We hypothesize that temperature serves as a cue for em- °C could be advantageous to C. funebralis at HMS, al- ersed individuals of C. funebralis to navigate to cool, dark though the possibility remains that temperature preference crevices in the mid- and low intertidal zones, habitats in 116 S. TEPLER ET AL. which they are buffered from desiccation (Marchetti and We used snails of a wide range of sizes in our preference Geller, 1987) and perhaps predation. Indeed, during poten- trials. If, as with light, the response of juveniles to temper- tially stressful, midday low tides, these snails are often ature is different from that of adults, it could have been a found aggregated in crevices (pers. obs.). This temperature- source of noise in our study. Our estimation of the physio- cued behavior of C. funebralis may be augmented by visual logically optimum temperature is based on an index of cues: adults of the species have been shown to be photo- locomotory activity rather than on actual fitness, and inter- negative (Doering and Phillips, 1983). pretation of our behavioral data would benefit from addi- The hypothesis that C. funebralis uses temperature as a tional, and more direct, measurements of the temperature at cue to find suitable habitat is bolstered by our finding that which C. funebralis’s fitness is optimized. The results of body temperature is correlated with the extent and direction this study, therefore, should be treated as only a step toward of snails’ movement. Body temperatures above average a full understanding of how C. funebralis interacts with its ocean temperature (11.7 °C) are associated with relatively environment, and an even smaller step toward an evolution- long, “purposeful” movements to colder areas (Figs. 7, 8). ary theory that explains why C. funebralis interacts with its In summary, our findings suggest that the prevailing environment in this fashion. theory for the evolution of thermal preference, which works well for many terrestrial species, cannot be applied directly Literature Cited to C. funebralis. We propose that C. funebralis differs from Abbott, D. P., and E. C. Haderlie. 1980. Prosobranchia: marine snails. expectations due to selection for behavior that guides snails Pp. 230–307 in Intertidal Invertebrates of California, R. H. Morris, to safe, but metabolically suboptimal, habitats. However, D. P. Abbott, and E. C. Haderlie, eds. Stanford University Press, the mechanism of this selection, and a theoretical account- Stanford, CA. ing of its evolutionary effects, await future research. Angilletta, M. J., Jr. 2009. Thermal Adaptation: A Theoretical and Empirical Synthesis. Oxford University Press, Oxford. Angilletta, M. J., Jr., A. F. Bennett, H. Guderly, C. A. Navas, F. Thermal tolerance Seebacher, and R. S. Wilson. 2006. Coadaptation: a unifying prin- ciple in evolutionary thermal biology. Physiol. Biochem. Zool. 79: The logarithmic relationship between median survival 282–294. time and body temperature found for C. funebralis (Fig. 2) Asbury, D. A., and M. J. Angilletta. 2010. Thermodynamic effects on has been noted in other invertebrates (Newell, 1970, pp. the evolution of performance curves. Am. Nat. 176: E40–E49. 454–468), fish (Brett, 1956), and fungi (Metarhizium spp., Benedetti-Cecchi, L., I. Bertocci, S. Vaselli, and E. Maggi. 2006. Li and Feng, 2009). To date, no mechanism has been Temporal variance reverses the impact of high mean intensity of stress in climate change experiments. Ecology 87: 2489–2499. suggested to account for this relationship, but our data Berg, H. 1984. Random Walks in Biology. Princeton University Press, indicate a possible approach to elucidating a mechanism. Princeton, NJ. After thermal stress, most of the snails became responsive Brett, J. R. 1956. Some principles in the thermal requirements of fishes. (e.g., recovered cardiac function; Stenseng et al., 2005) Q. Rev. Biol. 31: 75–87. within an hour of returning to ambient temperature, and Casterlin, M. E., and W. W. Reynolds. 1980. Behavioral response of the New England dogwhelk, Nassarius trivittatus, to a temperature death occurred gradually over the next 72 h. Investigation of gradient. Hydrobiologia 69: 79–81. protein, cell, and organ function in these three “die-off Chapman, M. G. 2000. Poor design of behavioural experiments gets days” could help explain what actually kills snails, perhaps poor results: examples from intertidal habitats. J. Exp. Mar. Biol. Ecol. providing some insight into the logarithmic relationship 250: 77–95. between exposure time and body temperature. Cronin, T. W. 1986. Photoreception in marine invertebrates. Am. Zool. 26: 403–415. Denny, M. W., and S. Gaines. 2000. Chance in Biology. Princeton Caveats University Press, Princeton, NJ. Dillon, M. E., G. Wang, P. A. Garrity, and R. B. Huey. 2009. Thermal It is difficult to isolate the effect of a single environmental preference in Drosophila. J. Therm. Biol. 34: 109–119. factor (such as temperature) on an organism, and studies of Doering, P. H., and D. W. Phillips. 1983. Maintenance of the shore- preference in response to a single cue are impossible (Chap- level size gradient in the marine snail Tegula funebralis (A. Adams): man, 2000). Our laboratory experiments were designed to importance of behavioral responses to light and sea star predators. J. maximize the influence of temperature while eliminating as Exp. Mar. Biol. Ecol. 67: 159–173. Efron, B., and R. J. Tibshirani. 1993. An Introduction to the Bootstrap. many external factors as practical, but there is no doubt that Chapman and Hall, New York. external factors remained. For example, we have not tested Etter, R. T. 1988. Physiological stress and color polymorphism in the for possible effects of circadian or tidal rhythms or the intertidal snail Nucella lapillus. Evolution 43: 660–680. effects of artificial as opposed to ambient light. Further- Gates, D. M. 1980. Biophysical Ecology. Springer-Verlag, New York. more, Doering and Phillips (1983) found that size (age) is a Harley, C. D. G. 2008. Tidal dynamics, topographic orientation, and temperature-mediated mass mortalities on rocky shores. Mar. Ecol. factor in the distributional gradient of C. funebralis, with Progr. Ser. 371: 37–46. larger snails using light as a cue to move downward in the Heath, D. J. 1975. Colour, sunlight and internal temperatures in the intertidal zone while juveniles remained higher on the shore. land-snail Cepaea nemoralis. Oecologia 19: 29–38. CHLOROSTOMA FUNEBRALIS THERMAL PREFERENCE 117

Hecht, T. 1994. Behavioural thermoregulation of the abalone, Haliotis Munõz, J. L. P., G. R. Finke, P. A. Camus, and F. Bozinovic. 2005. midae, and the implications for intensive culture. Aquaculture 126: Thermoregulatory behavior, heat gain and thermal tolerance in the 171–181. periwinkle Echinolittorina peruviana in central Chile. Comp. Biochem. Helmuth, B., N. Mieszkowska, P. Moore, and S. J. Hawkins. 2006. Physiol. A 14: 92–98. Living on the edge of two changing worlds: forecasting the responses Murphy, D. J., and L. C. Johnson. 1980. Physical and temporal factors of rocky intertidal ecosystems to climate change. Annu. Rev. Ecol. influencing the freezing tolerance of the marine snail Littorina littorea Evol. Syst. 37: 373–404. (L.). Biol. Bull. 158: 220–232. Helmuth, B. S. T. 1998. Intertidal mussel microclimates: predicting the Newell, R. C. 1970. Biology of Intertidal Animals. Marine Ecological body temperature of a sessile invertebrate. Ecol. Monogr. 68: 51–74. Surveys, Faversham, Kent, UK. Helmuth, B. S. T., and G. E. Hofmann. 2001. Microhabitats, thermal Phifer-Rixy, M., M. Heckman, G. C. Trussell, and P. S. Schmidt. 2008. heterogeneity, and patterns of physiological stress in the rocky inter- Maintenance of clinal variation for shell colour phenotype in the flat tidal zone. Biol. Bull. 201: 374–384. periwinkle Littorina obtusata. J. Evol. Biol. 21: 966–978. Herrera, F. D., F. B Ramirez, B. B. Sevilla, and C. Farfań. 1996. Somero, G. N. 2002. Thermal physiology and vertical zonation of Behavioral thermoregulation of Bulla gouldiana (Gastropoda: Opistho- intertidal animals: optima, limits, and costs of living. Integr. Comp. branchia: Cephalaspidea). J. Therm. Biol. 21: 319–322. Biol. 42: 780–789. Huey, R. B., and A. F. Bennett. 1987. Phylogenetic studies of co- Somero, G. N. 2005. Linking biogeography to physiology: evolutionary adaptation: preferred temperatures versus optimal performance temper- and acclimatory adjustments of thermal limits. Front. Zool. 2: 1–9. atures of lizards. Evolution 41: 1098–1115. Somero, G. N. 2010. The physiology of climate change: how potentials Kanwisher, J. W. 1955. Freezing in intertidal animals. Biol. Bull. 109: for acclimation and genetic adaptation will determine ‘winners’ and 56–63. ‘losers.’ J. Exp. Biol. 213: 912–920. Kosin, D. F. 1964. The light responses of Tegula funebralis. Veliger 6 Stenseng, E., C. E. Braby, and G. N. Somero. 2005. Evolutionary and (Suppl.): 46–50. acclimation-induced variation in the thermal limits of heart function in Lagerspetz, K. Y. H., and L. A. Vainio. 2006. Thermal behavior of congeneric marine snails (genus Tegula): implications for vertical crustaceans. Biol. Rev. 81: 237–258. zonation. Biol. Bull. 208:138–144. Li, J., and M. Feng. 2009. Intraspecific tolerance of Metarhizium Stillman, J. H. 2003. Acclimation capacity underlies susceptibility to anisopliae conidia to the upper thermal limits of summer with a climate change. Science 301: 65. description of a quantitative assay system. Mycol. Res. 113: 93–99. Sutherland, J. P. 1970. Dynamics of high and low populations of the Licht, P. 1966. The relationship between preferred body temperature limpet Acmaea scabra (Gould). Ecol. Mongr. 40: 169–188. and testicular heat sensitivity in lizards. Copeia 1965: 428–436. Tomanek, L. 2005. Two-dimensional gel analysis of the heat-shock Lockwood, J. R. 1998. On the statistical analysis of multiple-choice response in marine snails (genus Tegula): interspecific variation in feeding preference experiments. Oecologia 116: 475– 481. protein expression and acclimation ability. J. Exp. Biol. 208: 3133– McGaw, I. J. 2003. Behavioral thermoregulation in Hemigrapsus nu- 3143. dus, the amphibious purple shore crab. Biol. Bull. 204: 38–49. Tomanek, L., and E. Sanford. 2003. Heat-shock protein 70 (Hsp70) as McQuaid, C. D., and P. A. Scherman. 1982. Thermal stress in a high a biochemical stress indicator: an experimental field test in two con- shore intertidal environment: morphological and behavioural adapta- generic intertidal gastropods (genus: Tegula). Biol. Bull. 205: 276–284. tions of the gastropod Littorina africana. Pp. 213–224 in Behavioral Tomanek L., and G. N. Somero. 1999. Evolutionary and acclimation- Adaptation to Intertidal Life, G. Chelazzi and M. Vannini, eds. Plenum induced variation in the heat-shock responses of congeneric marine Press, New York. snails (genus Tegula) from different thermal habitats: implications for Marchetti, K. E., and J. B. Geller. 1987. The effects of aggregation limits of thermotolerance and biogeography. J. Exp. Biol. 202: 2925– and microhabitat on desiccation and body temperature of the black 2936. turban snail, Tegula funebralis (A. Adams, 1855). Veliger 30: 127–133. Tomanek L., and G. N. Somero. 2000. Time course and magnitude of Martin, T. L., and R. B. Huey. 2008. Why “suboptimal” is optimal: synthesis of heat-shock proteins in congeneric marine snails (genus Jensen’s inequality and ectotherm thermal preferences. Am. Nat. 171: Tegula) from different tidal heights. Physiol. Biochem. Zool. 73: 249– E102–E118. 256. Micallef, H. 1966. The ecology and behaviour of selected intertidal Wethey, D. S. 2002. Biogeography, competition, and microclimate: the gastropods. Ph.D. thesis, University of London. barnacle Chthamalus fragilis in New England. Integr. Comp. Biol. 42: Miller, L. P., C. D. G. Harley, and M. W. Denny. 2009. The role of 872–880. temperature and desiccation stress in limiting the local-scale distribu- Zar, J. H. 1999. Biostatistical Analysis, 4th ed. Prentice Hall, Upper tion of the owl limpet, Lottia gigantea. Funct. Ecol. 23: 756–767. Saddle River, NJ. P. 68.