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Comparative Biochemistry and Physio

Thermoregulatory behavior, heat gain and thermal tolerance in the

periwinkle Echinolittorina peruviana in central Chile

Jose L.P. Munoz a,c, G. Randall Finke a,b, Patricio A. Camus a,c, Francisco Bozinovic a,b,*

aCenter for Advanced Studies in Ecology and Biodiversity, Santiago 6513677, ChilebDepartamento de Ecologıa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago 6513677, Chile

cDepartamento de Ecologıa Costera, Facultad de Ciencias, Universidad Catolica de la Ssma. Concepcion, Casilla 297, Concepcion,Chile

Received 21 April 2005; received in revised form 25 July 2005; accepted 3 August 2005

Abstract

The amount of solar radiation absorbed by an organism is a function of the intensity of the radiation and the area of the organism exposed

to the source of the radiation. Since the prosobranch gastropod Echinolittorina peruviana is longer than it is wide, its areas of the lateral sides

are approximately twice as large as the areas of the frontal and dorsal faces. We quantified the orientation of the intertidal prosobranch E.

peruviana with respect to the position of the sun and solar heat gain in the different orientations. In the field, 80.9% of the E. peruviana

monitored on sunny summer days tended to face the sun frontally or dorsally while only 19.1% faced the sun with the larger lateral sides. On

overcast summer or on winter days, this trend was not observed. We then show that the body temperature of individuals increases more

rapidly and reaches higher equilibriums when the lateral sides are facing the sun than when they face the sun with either of the smaller frontal

or dorsal sides. These results therefore show that the orientation behavior of E. peruviana is thermoregulatory and that it permits the

organisms to maintain lower temperatures on hot summer days.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Behavioral thermoregulation; Thermal ecology; Gastropod; Littorinid; Rocky intertidal; Central Chile

1. Introduction

Variations in the thermal regime are particularly relevant

for ectothermic animals, since temperature has both direct

and indirect effects upon physiological and ecological

processes (e.g. Huey and Stevenson, 1979; Johnston and

Bennett, 1996; Portner, 2002; Zippay et al., 2004). In

invertebrates of small body size the primary means of

thermoregulation — defined here as any means used by an

organism to maintain body temperature at or as near to

optimal levels as possible — are behavioral (Kingsolver and

Watt, 1983), in that thermal patches of different quality are

selected by the animal that as best as possible maintain

homeostatic temperatures for physiological processes (John-

1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpa.2005.08.002

* Corresponding author. Departamento de Ecologıa, Facultad de Ciencias

Biologicas, Pontificia Universidad Catolica de Chile, Santiago 6513677,

Chile.

E-mail address: fbozinov@bio.puc.cl (F. Bozinovic).

ston and Bennett, 1996). In this respect, thermal physiology

and heat constraints are significant elements in determining

the activity and behavior of animals. This is particularly

important for marine invertebrates inhabiting intertidal

environments, where the animals are periodically exposed

to terrestrial conditions with the cyclic rise and fall of the

tides (e.g. Widdows et al., 1979). Thus, depending on the

latitude and time of year, air temperature may drop below

freezing or rise up to 20 -C more than in seawater (Finke,

2003). The mechanisms available to the animals for dealing

with these extreme conditions are related to their mobility

and can be classified as either ‘‘fight’’ or ‘‘flight’’, in the

sense that organisms can either ‘‘sit tight’’ and tolerate the

conditions, or attempt to seek out a more amenable thermal

microhabitat. For sessile organisms, the former is the only

option available, as their immobile nature precludes the

possibility of relocating to more amenable habitats. Mobile

organisms, on the other hand, have the ability to move

around and select different microhabitats, while at the same

logy, Part A 142 (2005) 92 – 98

J.L.P. Munoz et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 92–98 93

time attempting to avoid predators, and adjust their location

and bodies to seek suitable environmental conditions. To do

this, intertidal animals may compensate or adjust their

behavior and/or physiology, even changing the duration,

frequency and/or the form of body exposures to thermally

extreme conditions (Warburton, 1973; McMahon, 1990).

Several studies have investigated the role of biotic and

abiotic factors in determining the patterns of distribution and

abundance of prosobranch gastropods from the family

Littorinidae on rocky intertidal shores (Gendron, 1977;

Boulding and Van Alstyne, 1993; Chapman and Underwood,

1996; Soto and Bozinovic, 1998; Sokolova and Portner,

2003). Of these factors, the most predominant ones affecting

Littorinid distribution include predators, dislodgement by

waves, and heat load (Atkinson and Newbury, 1984; Garrity,

1984; Chapman and Underwood, 1992; Chapman and

Underwood, 1994; Rochette and Dill, 2000). However, lit-

torinids must experience long periods of emersion, and their

behavior and physiology are thus especially affected by tem-

perature and desiccation. In one species (Littorina saxatilis)

differences in mean body temperature between high- and low-

shore individuals of 10 -C were recorded during low tides

(Sokolova et al., 2000). Consequently, thermal conditions

appear to be closely related with the patterns of movement,

aggregation, habitat selection, morphology and pigmentation

of many littorinid species (e.g., McMahon, 1990; Britton,

1995; Chapman and Underwood, 1996; Jones and Boulding,

1999; Sokolova and Berger, 2000).

In the southeastern Pacific, the periwinkle Echinolittorina

peruviana is distributed between Panama and central Chile

(Guzman et al., 1998), facing a wide variety of thermal

regimes from tropical to temperate conditions. In central

Chile, E. peruviana is one of the most conspicuous species of

the rocky intertidal shore (Santelices, 1980), occurring at high

tidal levels and in the splash zone. The species is known to

resist direct exposure to the sun for long periods, although

juveniles tend to be restricted to protected microhabitats and

adults show characteristic seasonal variations in abundance

and vertical distribution (Santelices et al., 1986; Guzman et

al., 1998). Soto and Bozinovic (1998) and Rojas et al. (2000)

demonstrated that the preferential body temperature of E.

peruviana in the field showed a close relationship with

environmental temperature (substratum and air). Thermal

preference tests showed that this species prefers substratum

temperatures between 12 and 20 -C (extreme rock temper-

atures measured regularly exceeded 30 -C and occasionally

reached 45 -C). However, comparisons between body and

environmental temperatures revealed that during summer,

body temperatures were higher than surrounding air temper-

ature and lower than substratum temperatures. These authors

suggested that this pattern indicates a strong behavioral role

in the thermoregulation of this species via the selection of

substrate in a particular temperature range and the formation

of aggregations (see Soto and Bozinovic (1998) and Rojas et

al., 2000 for details). It has been suggested by Garrity (1984)

that littorinids from tropical zones orient themselves such that

the apices of the shells point to the sun, a pattern that was also

observed in E. peruviana in central Chile (J.L. Munoz, pers.

obs.). Such orientation is expected to reduce the surface area

of the body exposed to the sun (as opposed to orienting itself

broadside), thereby reducing the heat gain via absorption of

solar radiation (Finke, 2003).

Thus, our aim is to quantify and evaluate the behavioral

plasticity of E. peruviana in response to direct solar radiation

and the importance of these behavioral changes on heat gain

and thermal tolerance in an extreme experimental system.

Specifically, we attempt to understand the effects of thermo-

regulatory constraints on the frequency of body exposition to

direct sun (via orientation) and their implications for thermal

homeostasis. We first describe the seasonal patterns of body

orientation and exposure to direct solar heat gain in the field and

then we study their behavioral mechanistic basis through heat

gain curves and thermal tolerance under laboratory conditions.

2. Materials and methods

This study of a population of E. peruviana, was conducted

in the Coastal Station for Marine Research of the P.

Universidad Catolica de Chile at Las Cruces, central Chile

(33-35VS; 71-38VW), during austral winter and summer 2004.

Las Cruces is located near the southern distribution limit of E.

peruviana, in a temperate region where thermal conditions

are presumably less stressful than those at lower latitudes.

The study site consisted in a series of rocky outcroppings

adjacent to a coarse sand/broken shell beach protected from

the direct exposure of incoming waves. Relatively flat,

horizontal platforms were selected from among the out-

croppings based on the natural presence of E. peruviana

during low tides. The tidal range at the site was approximately

1.8 m and the outcroppings used were all above the high tide

mark and only wetted during high tide by the splash and spray

of waves.

2.1. Field observations

During three consecutive sunny in both summer and

winter, we recorded the activity of individual E. peruviana

based on visual observations made in the field. Animals of

similar size (mean maximum length of approximately

1.00T0.25cm) were collected from a single platform approx-

imately 3 m above chart datum (lower low water). A

minimum of 45 individuals were then haphazardly distrib-

uted on the platform and gently splashed with sea water. A

similar number of individuals were treated in the same

manner, but placed beneath a roof providing shade (in

summer only). After 20 min the apical orientation of each

snail was recorded using a sun dial (0.5�0.5�20 cm

wooden dowel mounted perpendicularly on a protractor) to

determine the angle of orientation of each animal in reference

to the sun (Fig. 1). Shell orientations were divided into four

groups. If the major axes of the shells were perpendicular to

Fig. 1. Orientation of E. peruviana shells recorded in field experiments during summer and winter 2004. A: summer, B: summer shaded, C=winter.

J.L.P. Munoz et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 92–9894

the sun, with their apices oriented from between 45- and 135-(90-T45-) or 225- to 315- (270-T45-) were considered as

broadside to the sun. Alternatively, if the snails oriented their

spires to the sun, with the apices pointing to 315- to 45-(0-T45-) or 135- to 225- (180-T45-) the response was

considered facing the sun inline.

Concurrent measurements of the shaded air temperature

and solar radiation of the study area during observation

periods weremade using a thermistor, placed 20 cm above the

surface in a shaded white hood, and a LI-COR global solar

radiation pyranometer, respectively. Signal conditioning and

data recording for both instruments were provided by a LI-

COR LI-1400 datalogger programmed to register radiation

and temperature every 20 min. Data were processed by taking

the mean of 2 h windows for each day (10:00–12:00, 12:01–

14:00; 14:01–16:00) followed by the mean of each window

across three days.

2.2. Laboratory experiments

Heating curves were generated by placing operative

temperature (To) models in a small wind tunnel equipped

with infrared heat-lamps. To models were made as originally

defined by Chappell and Bartholomew (1981) and subse-

quently applied by Helmuth (2002) to intertidal invertebrates.

A Cu-constantan thermocouple was inserted inside a hol-

lowed out E. peruviana shell and mounted in a natural

posture on a sheet of granite with dental cement. To

standardize and maximize the influence of heat load on To,

we used a single model maintained in different orientations to

the infrared lamp. To was read from a Thermocouple

CardAcp (Nomadics TC6 Card Acq) connected to a Compaq

laptop computer and recorded every 10 s. A digital photo-

graph of the To model from the perspective of the heat source

was then taken with a digital camera and the area exposed to

the heat source in the different orientations was then

determined using the program UTHSCSA Image Tool v. 2.0.

In addition, the thermal tolerance of E. peruviana was

assessed in the laboratory simulating low tide conditions in

the wind tunnel. Three groups of 30 individuals each were

placed on a sheet of granite and subjected to a 2.5 h period of

aerial exposure under infrared lamps (1027 W m�2) at three

different temperatures: 37, 42, and 47 -C. Every 30 min, six

individuals from each group and temperature were taken to

aquaria at 16 -C and maintained for 24 h to record their

survival. The experiment was repeated three times under the

same conditions. Experimental animals were collected from

vertical rocky walls in the field, as thermal tolerances may be

highly variable among individuals inhabiting horizontal

substrata (e.g., Williams and Morritt, 1995).

Table 2

Variation in exposed area and thermal kinetics for operative temperature

models oriented at two different angles with respect to the heat source

Orientation 0- Orientation 90-

Exposed area 64.6 mm2 77.1 mm2

Tbmax. 46.9 -C 54.4 -C

t0.5 0:02:40 min 0:03:33 min

R2 for adjusted equations 0.95 0.89

Table 1

Average (TS.D.) environmental conditions recorded at Las Cruces, central

Chile, during experimental periods in summer and winter 2004

Time

(h)

Solar

radiation

(W m�2)

Relative

humidity (%)

Air

temperature

(-C)

Summer 10:00–12:00 842.20T83.65 75.03T8.49 19.17T1.70

12:01–14:00 984.73T18.47 70.96T8.92 20.06T1.58

14:01–16:00 742.84T164.9 69.85T9.02 20.33T1.56

Winter 10:00–12:00 159.80T37.19 84.90T3.04 14.27T1.1312:01–14:00 138.07T38.18 84.17T6.08 12.14T1.47

14:01–16:00 122.68T40.11 78.96T6.05 13.38T1.20

J.L.P. Munoz et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 92–98 95

2.3. Statistics

Statistical analyses were performed using the Statistica\(2001) version 6.0 statistical package for Windows\ operat-

ing system. Field data were analyzed byChi-squared tests, and

survival data from thermal tolerance experiments by two-way

ANOVA. Heating curves were fit using Michaelis–Menten

curve fit in the Enzyme Kinetics module version 1.1 for

SigmaPlot\ (2004) version 9.0, fromwhichmaximum To and

t0.5 — i.e. the time to obtain the half of maximum To — were

obtained as a function of shell orientation to the heat source.

3. Results

Environmental conditions at the study site showed the

characteristic seasonal variation of Mediterranean climates

(Table 1). Overall, average levels of radiation and temper-

ature in winter reached about 16% and 66% of those in

summer, respectively, while relative humidity was little

variable and increased ca. 15% in winter. In both seasons,

average daily variations in air temperature and humidity were

small and did not exactly follow the fluctuation in solar

radiation, which tended to peak earlier in winter than in

Time (min)

00:00:00 00:10:00 00:20:00 00:30:00

Ope

rativ

e T

empe

ratu

re (

°C)

25

30

35

40

45

Fig. 2. Heating curves obtained in laboratory for snail models oriented at

angles of 0- (=inline in text; filled circles) and 90- (=broadside in text;

open circles) with respect to the heat source.

summer. Snail orientation varied significantly over the

seasons and treatments (Fig. 1). An outstanding difference,

revealed by chi-squared test (v2 = 18.571, g.l. = 3,

P=0.0003), was that in summer over 70% of individuals

showed an orientation with their spires facing the sun inline in

comparison to snails maintained under shade which did not

show this trend. During winter no significant pattern of sun

orientation was recorded (v2=1.16, g.l.=3, P=0.761; seeFig. 1) meaning that individuals did not show the pattern of

orienting themselves with their spires facing the sun inline.

No significant differences in maximum length (measured in

27 individuals from each experimental level) were detected

between the three sampling levels (one-way ANOVA

F2, 75=0.1458, P=0.865).

In the laboratory, heating was faster when the major axes

of the models were perpendicular (broadside) to the infrared

lamp, and slower if models were oriented with their spires

inline to the heat source (Fig. 2). The former orientation

exhibited a greater area exposed to the heat source, a higher

Tmax, and a longer t0.5 than the model in the inline position

(Table 2).

In thermal tolerance experiments, we recorded a signifi-

cant interactive effect between duration of exposure and

temperature on survival (Table 3). The three selected

temperatures were stressful for E. peruviana, and all

individuals were motionless after 4 to 6 min. However, at

37- C survival was 100% independent of duration (Fig. 3),

while it showed a rapid decrease at higher temperatures and

with longer exposures. From 30 to 60 min of exposure,

survival decreased stronger at 47 -C than at 42 -C, and less

than 10% after 120 or 150 min at both temperatures.

4. Discussion

Our field experiments demonstrate that E. peruviana has

the ability to display a rapid photokinetic response oriented

Table 3

ANOVA results for the survival of E. peruviana exposed for different time

periods ( P) at three temperatures (T)

Source df M.S. F P

P 4 0.56846 11.51961 0.002111

T 2 1.43838 29.14811 0.000212

P *T 8 0.04935 2.70288 0.022802

Error 30 0.01826

Time (min)

0 20 40 60 80 100 120 140 160 180

Surv

ival

(%

)

0

20

40

60

80

100

120

140Ta = 37°CTa = 42°CTa = 47°C

Fig. 3. Survival of E. peruviana after varying exposure (duration) to three

experimental temperatures (TS.D.).

J.L.P. Munoz et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 92–9896

to the sun (within 20 min). Although there are no optical

studies in this species, some littorinids are known to possess

a lens-type eye with lower sensitivity but higher resolution

than other prosobranchs, allowing them to form images and

perceive polarization planes of sunlight (Hamilton and

Winter, 1982; Seyer, 1992, 1998). We cannot assure that E.

peruviana possesses identical capabilities, but it certainly

may use visual stimuli to aid it in its orientation in order to

facilitate thermoregulation. Nevertheless, our results suggest

that sun-orientation behavior would be a functional response

dependent on the level of thermal load. The thermal kinetics

of E. peruviana models showed that aligning the longitudinal

axis of the body with the heat source is an effective way to

reduce the heat gain. Since orientation responses were

significant only in summer, when average radiation and

temperature increased ca. 6 and 1.5 times above winter levels,

respectively, and only when snails were directly exposed to

the sun, these responses appear to be largely driven by the

potential for thermal stress. Thus, seasonal and geographic

variability in factors affecting thermal stress (primarily solar

radiation) may modulate this behavior in populations from

temperate regions, and it might bemore frequent in tropical or

subtropical regions.

It has been suggested by some authors that manipulation

of Littorinid gastropods produces confounding effects in

studies of their locomotory responses (Chapman, 1999,

Petraitis, 1982). In our study, the manipulation of the animals

was performed in order to evaluate their response and how

they reoriented with respect to the position of the sun. Our

observations in the field indicate that after the 20 min the

snails had reoriented and ceased to move. We therefore feel

that while the manipulation may have accelerated the

response of the E. peruviana, the response does not seem to

be an artifact of manipulation.

The above suggests that, at least in E. peruviana,

orientation responses are displayed as a supplementary

thermoregulatory mechanism when environmental condi-

tions exceed critical levels for one or more factors. Some

works (e.g., Markel, 1971) have documented a positive

correlation between thermal resistance and intertidal height

for both temperate and tropical littorinid species. E.

peruviana also exhibits a variety of other behaviors which

would contribute to effective thermoregulation. For the E.

peruviana population of Las Cruces, Soto and Bozinovic

(1998) showed that body temperatures were maintained

above and below rock-surface temperatures during winter

and summer, respectively, as a result of active microhabitat

selection. In addition, Rojas et al. (2000) compared the same

population used by Soto and Bozinovic (1998) and in this

study with another population inhabiting a subtropical

environment. In both cases these authors found a negative

relationship between the size of aggregations and the rate of

water loss. On the other hand, our field and laboratory

observations showed that snails rapidly withdrew into shells

and sealed up the operculum as substratum temperature

became critical, a characteristic response of littorinids under

thermal stress (Jones and Boulding, 1999). In this regard, the

effects of environmental humidity on the physiological

condition of littorinids may be higher (e.g., Jones and

Boulding, 1999) or lower (e.g., Pardo and Johnson, 2004)

depending on the species and study conditions. In our field

experiments, humidity was relatively high (around 80%) and

similar during winter and summer, suggesting that thermal

stress could be more important for E. peruviana than

desiccation. Our thermal tolerance assessment showed that

100% of snails became motionless at 37 -C, the lowest

experimental temperature, which points to the importance of

thermal stress. It is possible that temperatures lower than 37

-C may also induce coma in this species, although heat coma

temperature (HCT) in littorinids may be highly variable.

While HCT was about 43 -C in tropical species (Britton,

1992), it fluctuated around 30 -C in species from the British

Isles (Clarke et al., 2000a,b), and 32 -C in temperate and sub-

artic populations of L. saxatilis (Sokolova and Portner, 2003).

Likewise, although HCT values may be stable within

populations (Clarke et al., 2000b), they may also depend on

the previous thermal history, acclimation to seasonal varia-

tions in thermal conditions and body size of animals (Clarke

et al., 2000a).

From the above, it is clear that intra- and inter-specific

generalizations on behavioral thermoregulation in littorinids

are not straightforward. Oriented responses to the sun in E.

peruviana would be a case in point, which can be assessed on

a wider latitudinal scope in association with alternative

mechanisms. Sokolova et al. (2000) showed that upper and

lower shore littorinids of the same species may have different

mechanisms of thermal resistance, although the former have a

greater ability to resist temperature and desiccation stress. In

fact, Rojas et al. (2000), in comparing two populations of E.

peruviana separated by 10- of latitude, concluded that local

environmental differences determine the effectiveness of

thermoregulatory mechanisms. Whatever the case, behavior

can play a key role in the thermal physiological ecology of

littorinids, although some authors reasonably disagree with

J.L.P. Munoz et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 92–98 97

this conclusion in particular cases. For instance, Chapman

(1995) found that spatio-temporal aggregations of Littorina

unifasciata were irregular and related to small scale processes

suggesting that explanations based on desiccation and

temperature would not be reliable for this species. Indeed,

behaviors showing thermoregulatory benefits such as climb-

ing (e.g.,Williams andAppel, 1989) can also be interpreted in

very different terms (e.g., predation escape; Vaughn and

Fisher, 1988). We therefore suggest that the behavioral scope

displayed by littorinids must not be considered a priori an

arsenal of thermoregulatory mechanisms, and when such a

role is demonstrated in a given case it does not necessarily

apply to any other case without proper evaluation.

On the other hand, littorinid behavior may interact with a

number of factors and processes related in different ways with

responses to thermal and desiccation stress, which deserve

more attention. For instance, relationships between temper-

ature, rates of aerial and aquatic respiration, proportion of the

tidal cycle exposed to air, and vertical distribution (McMahon

and Russell-Hunter, 1977); metabolic compensation related

with variations in enzymatic activity at different tidal levels

and temperatures (Sokolova and Portner, 2001); genetic

responses related to energetic constraints and recovery from

metabolic depression under anoxia (Larade and Storey,

2002); or local genetic differences between high and low

shore snails related with enzymatic activity involved in

anaerobic energy production (Panova and Johanesson, 2004).

None of these aspects have been studied in E. peruviana,

which normally occur at higher tidal levels than other species

from the Chilen intertidal and often remain emersed during

high tides, facing potentially stressful conditions continu-

ously. Based on this paper and prior works (Soto and

Bozinovic, 1998; Rojas et al., 2000), we conclude that E.

peruviana can make facultative use of microhabitat selection,

aggregation, opercular closing, and sun-oriented responses in

its arsenal mechanisms for combating thermal stress. How-

ever, we can only speculate so far in regard to the precise

conditions under which such behaviors are displayed and

their use will relate to metabolic and other physiological

processes at different levels.

Acknowledgments

This study was funded by FONDAP 1501-0001 (pro-

gram 1) to FB. JLPM acknowledges P. Manrıquez, R. Soto

and D. Naya for valuable help. This paper is part of the

undergraduate dissertation of JLPM.

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