Heat-Shock Response of the Upper Intertidal Barnacle Balanus glandula: Thermal Stress and Acclimation

Heat-Shock Response of the Upper Intertidal Barnacle Balanus glandula: Thermal Stress andAcclimationAuthor(s): Michael S. Berger and Richard B. EmletSource: Biological Bulletin, Vol. 212, No. 3 (Jun., 2007), pp. 232-241Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/25066605 .

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Reference: Biol. Bull. 212: 232-241. (June 2007) ? 2007 Marine Biological Laboratory

Heat-Shock Response of the Upper Intertidal Barnacle Balanus gl?ndula: Thermal Stress and Acclimation

MICHAEL S. BERGER*1 AND RICHARD B. EMLET

Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon 97420

Abstract. In the intertidal zone in the Pacific Northwest,

body temperatures of sessile marine organisms can reach 35

?C for an extended time during low tide, resulting in poten tial physiological stress. We used immunochemical assays to examine the effects of thermal stress on endogenous

Hsp70 levels in the intertidal barnacle Balanus gl?ndula. After thermal stress, endogenous Hsp70 levels did not in crease above control levels in B. gl?ndula exposed to 20 and

28 ?C. In a separate experiment, endogenous Hsp70 levels were higher than control levels when B. gl?ndula was

exposed to 34 ?C for 8.5 h. Although an induced heat-shock

response was observed, levels of conjugated ubiquitin failed to indicate irreversible protein damage at temperatures up to

34 ?C. With metabolic labeling, we examined temperature acclimation and thermally induced heat-shock proteins in B.

gl?ndula. An induced heat-shock response of proteins in the

70-kDa region (Hsp70) occurred in B. gl?ndula above 23

?C. This heat-shock response was similar in molting and

non-molting barnacles. Acclimation of B. gl?ndula to rela

tively higher temperatures resulted in higher levels of pro tein synthesis in the 70-kDa region and lack of an upward shift in the induction temperature for heat-shock proteins.

Our results suggest that B. gl?ndula may be well adapted to

life in the high intertidal zone but may lack the plasticity to

acclimate to higher temperatures.

Introduction

Intertidal organisms inhabit an interface between aquatic and terrestrial habitats where they are exposed to extreme

physical conditions during low tides (Lewis, 1978; Ne well,

1979). These organisms experience body temperatures that

exceed the temperature of the surrounding air and regularly

approach sublethal thermal limits (Helmuth, 1999; Helmuth

and Hofmann, 2001; Tomanek and Sanford, 2003). Organ isms residing higher in the intertidal are more likely to

experience prolonged thermal and desiccation stresses than are organisms lower in the intertidal (Wolcott, 1973; Hof

mann and Somero, 1995; Roberts et al, 1997; Halpin et al,

2002). One physiological adaptation to living in a stressful

habitat like the intertidal is the synthesis of molecular chap erones (Feder and Hofmann, 1999; Hochachka and Somero,

2002). Molecular chaperones such as heat-shock proteins (Hsps)

act to rescue damaged proteins and prevent them from

aggregating, thereby helping conserve the pool of existing

proteins from irreversible damage (Parsell and Lindquist, 1993; B?chner, 1996; Fink, 1999). Though energetically

expensive, the expression of molecular chaperones may

protect existing protein pools during periods of acute and

chronic stress and thus reduce the subsequent higher cost of

de novo protein synthesis (Somero, 2002; Hartl and Hayer Hartl, 2002; Hofmann et al, 2002).

Elevated levels of Hsps may not completely repair pro teins denatured by thermal stress; some irreversible protein

damage can occur. When a protein is irreversibly damaged,

ubiquitin, a low-molecular-mass protein, is bound to the

damaged protein, marking it for degradation by cytoplasmic

proteases (Hershko and Ciechanover, 1992; Hochstrasser,

1995). In studies that measured the level of ubiquitin con

jugate, the amount of irreversibly damaged protein in

creased as the level of stress increased in both field and

laboratory studies (Lee et al, 1988; Hofmann and Somero,

1996a; Halpin et al, 2002; Spees et al, 2002). A series of influential studies have been published on the

physiological response of rocky intertidal organisms to eco

logically relevant environmental stresses (Sanders et al,

Received 14 March 2006; accepted 20 February 2007. * To whom correspondence should be addressed. E-mail: msberger@

uci.edu 1 Current address: Ecology and Evolution, University of California,

Irvine, 321 Steinhaus Hall, Irvine, CA 92697.

232



HEAT-SHOCK OF BALANUS GL?NDULA 233

1991; Sharp et al, 1994; Hofmann and Somero, 1995; Tomanek and Somero, 1999; Hamdoun et al, 2003). Most

studies have focused on the responses of molluscs, and

information for other abundant intertidal organisms, such as

algae, urchins, or barnacles, is limited. The acorn barnacle

Balanus gl?ndula Darwin 1854, a sessile, thermotolerant,

ubiquitous, and important member of the middle-to-high

rocky intertidal zone along the eastern Pacific Ocean (New

ell, 1979; Morris et al, 1980), is an ideal organism in which

to examine the physiological response to ecologically rele

vant stress in a high intertidal habitat where organisms are

exposed to sublethal conditions for extended times.

Molting distinguishes crustaceans from other organisms

(e.g., molluscs) in the middle-to-high rocky intertidal. Molt

ing in a crustacean involves cuticular reorganization, limb

regeneration, and protein degradation (Skinner et al, 1992;

Hopkins, 1993; Roer and Dillaman, 1993). Since molecular

chaperones have multiple cellular functions (Lindquist, 1986; Parsell and Lindquist, 1993), the process of molting could potentially elicit an increase in Hsp levels and either

confound or interact with the effects of temperature. The heat-shock response is plastic and can be adjusted on

the basis of an organism's past experience. For example, the

induction temperature at which Hsp expression is turned on

can be adjusted by long-term seasonal acclimatization or

short-term acclimation (reviewed in Hofmann et al, 2002).

However, some intertidal organisms exist at the boundary of

their physiological limit to thermal stress and do not have

the capacity to acclimate to higher temperatures (Stillman and Somero, 2000; Stillman, 2003; Tomanek, 2005).

We explored the heat-shock response of B. gl?ndula after

thermal stress, by addressing the following questions: (1) At

what temperature does B. gl?ndula respond physiologically to thermal stress by expressing a heat-shock response? (2)

Do specimens experiencing different thermal environments

in the intertidal zone differ in their responses to physiolog ical stress? (3) Does the molt cycle affect the heat-shock

response? (4) Does thermal acclimation affect the pattern of

expression of heat-shock proteins in a thermally tolerant

intertidal barnacle?

Materials and Methods

Collection site

Unless otherwise noted, adult specimens of Balanus

gl?ndula Darwin 1854 were collected from the intertidal

zone at the mouth of the South Slough Estuary in Charles

ton, Oregon (43?20.4'N, 124? 19.4'W). The barnacles were

removed from pilings under the Charleston bridge or col

lected from an adjacent cobble field.

Endogenous Hsp70 and ubiquitin-conjugate levels in the

laboratory

In two laboratory experiments designed to examine the

heat-shock response of B. gl?ndula, we used an immuno

chemical assay to measure levels of endogenous Hsp70 and

ubiquitin conjugate during a 16-h recovery period after

thermal incubation. Field-collected barnacles were main

tained in a flowing seawater table at ambient temperature for longer than one week at an average temperature (range) of 11.2 ?C (11.0 to 11.3 ?C) in experiment 1 and an average

temperature (range) of 12.0 ?C (10.8 to 13.8 ?C) in exper iment 2. In experiment 1, barnacles were exposed to 11, 20, and 28 ?C for 5 h, which approximated low-tide emersion.

In experiment 2, B. gl?ndula was exposed to 14 ?C (max imum temperature during acclimation) and 34 ?C for 8.5 h.

The temperature and exposure period were raised in exper

iment 2 in an effort to induce a heat-shock response. To

mimic conditions in the intertidal zone during low tide, barnacles were not submerged in water, but the humidity was maintained at 100%. Immediately after thermal incu

bation, barnacles were placed in seawater at 11 ?C (exper iment 1) and 14 ?C (experiment 2). At 0, 2, 4, 10, and 16 h

after thermal incubation was terminated, the prosomas of

five barnacles from each temperature treatment were dis

sected, frozen with liquid nitrogen, and stored at -80 ?C for

analyses of endogenous Hsp70 and ubiquitin conjugate (see methods outlined below).


field

Two field experiments were performed to examine the

endogenous levels of Hsp70 and ubiquitin conjugate in B.

gl?ndula. In the first experiment, barnacles were collected on similarly sized cobbles (?10 X 10 X 5 cm, L X W X H) from the intertidal zone at 1.7 and 0.4 m above mean lower

low water (MLLW); the 0.4-m site was partially shaded by a thin layer of dried algae (Ulva sp.). The mean surface

temperature (?1 SD) on three cobbles encrusted with B.

gl?ndula at the time of collection was 29.5 ?C (0.4) at 1.7 m

above MLLW and 23.7 ?C (0.1) at 0.4 m above MLLW. In the second field experiment, similarly sized cobbles covered

with barnacles were collected at 1.7 m above MLLW to measure Hsp70 levels from B. gl?ndula on the warmer

upper surface compared to the cooler underside of the

cobbles. The mean surface temperature (?1 SD) on four

cobbles was 29.0 ?C (0.8) on the upper surface and 24.7 ?C

(0.8) on the shaded underside. After collection, barnacles were placed in a flowing seawater table at an ambient

temperature of 13.6 ?C for 4 h. After this time, the prosoma was removed from each barnacle, immediately frozen with

liquid nitrogen, and stored at -80 ?C for analyses of en

dogenous Hsp70 and ubiquitin conjugate (see methods out

lined below).



234 M. S. BERGER AND R. B. EMLET

Effect of molt cycle on temperature-induced protein

expression

An independent experiment was performed to examine

the effect of molting on the heat-shock response. Barnacles were collected in the field during an evening low tide in

December 2002 and maintained in a seawater table at 11 ?C

for about 12 h. After 12 h, barnacles in distinctly recognized molt stages (Davis et al, 1973) of interecdysis (stage C) or

proecdysis (stage D2_3) were assayed, using the metabolic

labeling assay outlined below, for thermally induced protein

synthesis.

Acclimation and field acclimatization

To examine the effects of acclimation to temperature, B.

gl?ndula was collected on pilings in the intertidal at the

mouth of the South Slough Estuary, Charleston, Oregon, and from a rocky intertidal bench on the open coast in

Sunset Bay State Park, Charleston, Oregon, during August 2003; the two collection sites were separated by about 6 km.

Barnacles were returned to the laboratory and acclimated in

aquaria at a constant temperature of 10, 16, or 22 ?C for 8

weeks. Water was changed every 4 days, and barnacles

were fed the diatom Skeletonema costatum in excess. After

acclimation, metabolic labeling assays were performed (see

below). To test the effects of thermal stress on field-accli

matized barnacles, specimens were collected in the field

during an evening low tide in August 2003, maintained in a

seawater table at about 10 ?C for about 12 h, and then

assayed for thermally induced protein synthesis. A meta

bolic labeling assay was used, following the procedures outlined below.

Endogenous Hsp70 analysis (Western-blot immunochemical assay)

Endogenous Hsp70 tissue preparation and Western-blot

analysis of heat-shock protein (Hsp70) followed methods

and used reagents outlined by Hofmann and Somero (1995) with the following modifications. Samples were homoge nized and centrifuged at 16,000 X g for 10 min at 4 ?C.

Sample protein concentration was determined by a Coomas

sie blue assay (BioRad). The supernatant was diluted 1:1

(v/v) with sodium dodecyl sulfate (SDS) sample buffer, heated for 5 min at 100 ?C, and centrifuged at 16,000 X g for 30 s. An aliquot of the homogenate was stored at ?80

?C for ubiquitin-conjugate analysis. From the remaining

supernatant, 10 /xg of total protein per sample was electro

phoretically separated on a 7.5% SDS-polyacrylamide gel

(Laemmli, 1970). Separated proteins were transferred to a

presoaked nitrocellulose membrane (0.45 jmm, Schleicher &

Schuell) with a Mini Trans-Blot cell (BioRad), and then

dried in an oven at 50 ?C for 20 min. The membrane was

blocked, incubated for 1.5 h with a primary antibody diluted

1:2500 (anti-Hsp70, rat monoclonal, MA3-001, Affinity

Bioreagents; binds to constitutive and induced isoforms), incubated for 30 min with a secondary antibody diluted

1:2000 (rabbit anti-rat, AI-4000, Vector Laboratories), and

then incubated for 1 h with protein-A horseradish peroxi dase conjugate diluted 1:5000 (BioRad, 170-6522). To vi

sualize the proteins, an enhanced chemiluminescence detec

tion method (ECL reagents, Amersham) was used, followed

by exposing the membrane to X-ray film (Kodak

X-OMAT). Optical density of each visualized band was

quantified using Gel-Pro Analyzer software (ver. 4.0, Media

Cybernetics). When multiple gels were compared within an

experiment, all bands on each gel were normalized to a

positive control prepared from heat-shocked mussel (Myti lus californianus) so relative comparisons could be made.

Ubiquitin-conjugate analysis

Ubiquitin-conjugated proteins were quantified following the methods of Hofmann and Somero (1995) with the fol

lowing exceptions. Samples were diluted to a concentration

of 5 /xg ml-1 in a saline solution, loaded in triplicate 100-uJ

volumes onto a presoaked nitrocellulose membrane (0.45

/xm, Schleicher & Schuell) in a dot-blot vacuum apparatus

(Bio-Rad), gravity-fed through the membrane, and dried at

50 ?C for 20 min.

The membrane was blocked, incubated for 1.5 h with a

polyclonal rabbit anti-ubiquitin-conjugate primary antibody diluted 1:2500 (provided by G. Hofmann, University of

California, Santa Barbara), and then incubated for 1 h with

protein-A horseradish-peroxidase-conjugate diluted 1:5000

(Vector Laboratories, PI-1000). Visual detection and anal

ysis was identical to methods previously outlined for

Hsp70.

Metabolic labeling assay

To quantify thermally induced protein synthesis, a series

of laboratory experiments were conducted using the follow

ing general methods. Specific experimental manipulations were described in previous sections. Two right and two left

depressor muscles (e.g., scutorum rostralis and scutorum

lateralis) from B. gl?ndula specimens with a basal diameter

greater than 15 mm were dissected in a cold room at 10 ?C.

Muscle fibers were placed in a barnacle "Ringer's" solution

(Hoyle and Smyth, 1963), with 10 mmol 1_1 glucose added.

Each depressor muscle was assigned to one of four temper

ature treatments of 10, 23, 28, or 33 ?C for 2.5 h. This

dissection procedure allowed the in vitro exposure of mus

cle tissue from one individual to four temperatures. After

thermal incubation, muscle tissue was incubated in 500 /xl of the Ringer's solution with 50 /xCi of 35S-labeled methi

onine/cysteine (NEG-772, Perkin Elmer) at 10 ?C for 4 h to

allow adequate time for uptake of the label, washed three




times in the Ringer's solution, frozen with liquid nitrogen, and stored at ?20 ?C for no longer than 2 weeks.

Frozen samples were homogenized in buffer consisting of

32 mmol F1 Tris-HCL, pH 6.8, 2% SDS, and 1 mmol F1

phenylmethylsulfonyl fluoride, heated to 100 ?C for 5 min, and then centrifuged at 16,000 X g for 10 min. Determina

tion of the 35S-methionine/cysteine incorporated into pro tein was by liquid scintillation counting (see Tomanek and

Somero, 1999). Equivalent amounts of radiolabeled protein were separated electrophoretically on 7.5% SDS-polyacryl amide gels (Laemmli, 1970). The radioactivity of labeled

protein loaded into gels varied between experiments, but never within an experiment. Typically, 60,000 counts

min-1 (cpm) were loaded, but in a few cases it was neces

sary to load 40,000 or 50,000 cpm. Gels were fixed in 60%

distilled water, 30% methanol, and 10% acetic acid for 1 h, dried under vacuum, and then exposed to X-ray film (Kodak

X-OMAT) with an intensifying screen (Kodak BioMax

Transcreen LE) for 12 h (60,000 cpm), 14.5 h (50,000 cpm), or 18 h (40,000 cpm) at -80 ?C. Recognizable bands in the

70-kDa and 90-kDa regions of each gel were quantified

using Gel-Pro Analyzer software (Media Cybernetics). The

proteins (heat-shock proteins) quantified in this series of

experiments were referred to by the range of their molecular mass. Proteins in the 70-80-kDa region were referred to as

Hsp70, and those in the 89-95-kDa region were referred to as Hsp90.

Statistical analysis

Data examined in this study were analyzed with Systat (ver. 9.0, SPSS Inc.) and Statistica (ver. 6.0, Statsoft). The

assumptions of normality and homoscedasticity were tested

with a Kolmogorov-Smirnov test with Lilliefors option and a Cochran's C test, respectively. The assumption of normal

ity was violated a few times; however, ANOVAs are robust to departures from normality (Sokal and Rohlf, 1995; Un

derwood, 1997). All repeated-measures ANOVAs were

subjected to Mauchly's test of sphericity (Crowder and

Hand, 1990). Violations of sphericity were interpreted by

using the Greenhouse-Geisser epsilon to adjust the appro

priate degrees of freedom.

To test the effects of incubation temperature on endoge nous Hsp70 and ubiquitin-conjugate levels (experiments 1

and 2), separate ANOVAs were performed. All dependent variables were natural-log-transformed prior to analysis.

To determine differences between endogenous Hsp70 and ubiquitin levels in field-collected barnacles, separate r-tests were performed. Data were natural-log-transformed

for endogenous Hsp70 levels in both field experiments prior to analysis.

Because muscle tissue from a single barnacle was tested

across all incubation temperatures in all metabolic labeling experiments, a repeated-measures ANOVA was performed

with incubation temperature as the repeated measure. The

analysis was performed separately for Hsp70 and Hsp90. All data were natural-log-transformed prior to analyses. In

acclimation experiments, band densities from incubation

temperatures of 23, 28, and 33 ?C were normalized to the mean of the 10 ?C control treatment so comparisons could

be made between acclimations (Tomanek and Somero,

1999). Comparisons between treatments within an experi ment were determined from visual observation of the data.

Results


laboratory

Only one resolvable band (75-kDa isoform) was observed

for Balanus gl?ndula in all immunochemical assays (Fig. 1). Two bands (77-kDa and 72-kDa isoforms) were resolv

able in the positive control prepared from heat-shocked

tissue from Mytilus californianus. Over a 16-h period during experiment 1, Hsp70 levels did

not significantly increase when B. gl?ndula was exposed to

elevated temperatures of 20 and 28 ?C compared to the

control temperature of 11?C (Fig. 2A, ANOVA, F2 72 =

0.93, P = 0.40). Levels of Hsp70 did vary significantly over

time (ANOVA, F5 72 =

3.7, P = 0.005); however, in

creased or decreased levels occurred across all tempera

tures, including the control. A nonsignificant interaction

between temperature and time further indicated that levels

of Hsp70 did not vary between temperature treatments

11 ?C 20 ?C 28 ?C + control

Figure 1. Western blot image of Hsp70 isoforms in Balanus gl?ndula from experiment 1. All subsequent immunochemical assays displayed a similar response. Captions above each lane correspond to incubation

temperature of B. gl?ndula and Mytilus californianus positive control (+ control).




SD.

> >s ? (/) o c

Q. "D </> ?

1 s > Q.

d)

0.7 n

0.6

0.5

0.4

0.3 -\

0.2

0.1 ^

0.0

0.35

0.30

U) & ^ (fi c c O <D o -o

il O" Q.

s?

0.25 H

0.20

0.15

0.10

J5 & (D ce S

"" 0.05

0.00

Incubation temperature

11 ?C

n 20 ?c

28 ?C

t?a

i

i j. i i i X

I

0 2 4 6 10

Hours following thermal incubation

16

Figure 2. Levels of (A) endogenous Hsp70 and (B) ubiquitin conju

gate in Balanus gl?ndula after recovery from thermal incubation (experi ment 1 ). Each bar represents the mean of five individuals; error bars are 1

standard error.

during the 16-h recovery period (ANOVA, F10 72 =

0.50, P =

0.892). In experiment 2, significantly higher levels of

Hsp70 were observed in the 34 ?C treatment compared to

the 14 ?C control (Fig. 3A, ANOVA, Fx, 48 =

8.10, P =

0.006). During the recovery period in experiment 2, endog enous Hsp70 levels did not change significantly (ANOVA,

F5 48 =

0.84, P = 0.53). An interaction of temperature and

time was not observed (ANOVA, F5 48 =

0.56, P = 0.73).

Levels of ubiquitin conjugate were measured in experi ments 1 and 2 to determine whether irreversible protein

damage occurred in response to thermal stress during lab

oratory experiments. Ubiquitin-conjugate levels did not

vary significantly as a result of incubation temperature

(ANOVA, F2 72 = 1.42, P =

0.25) or over time (ANOVA,

F5 72 =

0.35, P = 0.88) throughout the entire 16-h recovery

period after thermal incubation during experiment 1 (Fig.

2B). Ubiquitin-conjugate levels also did not vary signifi

cantly as an effect of temperature (ANOVA, F, 48 =

0.84, P =

0.36) or over the course of recovery (ANOVA, F5 48 =

0.80, P = 0.55) after exposure to 34 ?C for 8.5 h during

experiment 2 (Fig. 3B).

Endogenous Hsp70 and ubiquitin-conjugate levels in the field

Endogenous levels of Hsp70 from specimens of B. gl?n dula collected on cobbles from 1.7 m and 0.4 m above mean

lower low water (MLLW) were not significantly different

(t =

1.54, P = 0.17). In addition, there was no significant

difference between ubiquitin-conjugate levels in B. gl?n dula collected from the high intertidal and mid-intertidal

zones (t = ?0.42, P ?

0.68). In the second field experi ment, endogenous Hsp70 levels in B. gl?ndula attached to

the warmer top and cooler underside of the cobbles were not

significantly different (t =

-1.40, P = 0.22).

Effect of molt cycle on temperature-induced protein expression

Comparisons of Hsp70 and Hsp90 levels between two

prominent molt-cycle stages (i.e., interecdysis and pro

ecdysis) suggest that molt stage did not affect the levels of

induced heat-shock protein expression across incubation

temperatures (Fig. 4, Table 1). A significant increase in

Hsp70 levels was observed as incubation temperature in

creased (Fig. 4A, Table 1). Levels of Hsp90 also signifi

Incubation

| temperature

14 C

D 34 ?C

0 2 4 6 10 16

Hours following thermal incubation

Figure 3. Levels of (A) endogenous Hsp70 and (B) ubiquitin conju

gate in Balanus gl?ndula after recovery from thermal incubation (experi ment 2). Each bar represents the mean of five individuals; error bars are 1

standard error.




c 0 TJ

O

?L

>

C

o

o o o

o c

3000

2500

2000

1500

1000 -

500 -

0

600

500

400

300

200 -\

100

0

molt-stage

| interecdysis

proecdysis

Hsp70

Hsp90 B

L? Mi 10 23 28 33

Incubation temperature (?C)

Figure 4. The effect of molt stage on temperature-induced protein

synthesis in isolated muscle tissue from Balanus gl?ndula for (A) Hsp70 and (B) Hsp90. Each bar represents the mean of six individuals; error bars

are 1 standard error. See Table 1 for statistics.

cantly varied over incubation temperature (Table 1). How

ever, unlike the Hsp70 pattern in which levels continued to

increase as incubation temperature increased, Hsp90 levels

reached a maximum at 28 ?C and then decreased at 33 ?C

(Fig. 4B). The autoradiograph image in Figure 5 represents a typical response of B. gl?ndula muscle tissue to thermal

stress in this and other metabolic labeling assays.

Acclimation and field acclimatization

The effects of acclimation and acclimatization on the

heat-shock response differed between the two populations of B. gl?ndula examined during August 2003. For the

Charleston (CH) population of B. gl?ndula, Hsp70 levels

varied significantly as a result of acclimation temperature

(Table 2). Barnacles acclimated to 16 ?C and 22 ?C dis

played higher levels of Hsp70 across all incubation temper atures compared to barnacles that were field-acclimatized or

acclimated to 10 ?C (Fig. 6A). In addition, the field-accli

matized barnacles from the CH population displayed pat

terns of Hsp70 synthesis that were similar to those in the 10

?C acclimation treatment (Fig. 6A).

Although levels of Hsp70 in the Sunset Bay (SB) popu lation did significantly increase as incubation temperature increased, those levels were not affected by acclimation

temperature alone (Fig. 6B, Table 2). Acclimation temper ature interacted significantly with incubation temperature such that levels of Hsp70 for barnacles acclimated to 10 ?C

did not increase as incubation temperature increased, whereas Hsp70 levels in all other acclimatization and accli

mation treatments did increase with incubation temperature

(Fig. 6B, Table 2). At an incubation temperature of 28 ?C, field-acclimatized barnacles from the SB population dis

played a pattern similar to those in the 10 and 16 ?C

acclimation treatment; but at an incubation temperature of

33 ?C, the field-acclimatized Hsp70 level was similar to that

in the 22 ?C acclimation treatment (Fig. 6B). Levels of Hsp90 did not vary significantly as a function

of acclimation or incubation temperature in the CH popu lation; no recognizable patterns were observed (Fig. 6C,

Table 2). In contrast, the effect of acclimation on Hsp90 levels did vary significantly in the SB population; Hsp90 levels tended to decrease as acclimation temperature in

creased (Fig. 6D, Table 2). However, Hsp90 levels in the

SB population did not vary significantly across incubation

temperatures (Table 2).

Table 1

Repeated-measures ANOVA on the effect of molt stage on temperature induced Hsp70 and Hsp90 levels in Balanus gl?ndula (see Fig. 4)

Sum of Mean

Source* squares df square F-ratio P

Hsp70 Between subjects

Molt stage 0.816 1 0.816 0.984 0.350

Error 8.300 10 0.830

Within subjects1

Temperature 61.132 3 20.377 20.337 0.000

Temperature X molt-stage 0.735 3 0.245 0.245 0.740

Error 30.060 30 1.002

Hsp90 Between subjects

Molt stage 0.003 1 0.003 0.001 0.970

Error 28.079 10 2.808

Within subjects2

Temperature 46.058 3 15.353 10.783 0.001

Temperature X molt-stage 7.024 3 2.341 1.645 0.220

Error_42.712 30

1.424_

*The assumption of sphericity (Mauchly's test) was violated in all

repeated-measures ANOVAs.

The degrees of freedom were multiplied by the Greenhouse-Geisser

epsilon, and an adjusted P value was calculated. The adjusted P values are

reported. Greenhouse-Geisser epsilon values for within subjects: * 0.5275

and 2

0.6006.




10 ?C 23 ?C 28 ?C 33 ?C

Figure 5. Autoradiograph of protein expression patterns from Balanus

gl?ndula muscle tissue. Captions above each lane correspond to incubation

temperature of the barnacles.

Discussion

Intertidal organisms in the Pacific Northwest regularly

experience body temperatures from 20 to 35 ?C during low

tides (Tomanek and Somero, 1999; Hamdoun et al, 2003;

Fitzhenry et al, 2004). Metabolie labeling experiments in

this study (Figs. 4 and 6) indicated that Hsp70 is induced in

Balanus gl?ndula at 23 ?C, regardless of acclimatization or

acclimation, indicating that heat-shock proteins are upregu lated in this species on a regular basis.

In B. gl?ndula, the pattern of Hsp90 synthesis differed

from that of Hsp70. For specimens examined in December

2002, maximum expression of Hsp90 was between 23 and

28 ?C and then decreased at 33 ?C; in comparison, Hsp70 remained at maximum expression levels above 28 ?C (Fig. 4). Presumably, at temperatures above 33 ?C the expression of Hsp90 approached a negligible level. A difference in the

patterns of expression of the two heat-shock proteins is not

surprising, as different molecular chaperones perform dif

ferent functions within a cell (Lindquist, 1986; Parsell and

Lindquist, 1993). Levels of molecular chaperones have been shown to

increase in some intertidal molluscs during the recovery

period after thermal stress (Hofmann and Somero, 1996b;

Tomanek and Somero, 2000). A reduction in metabolic rate

during aerial exposure and a resultant decrease in protein

synthesis have been suggested as the mechanism regulating the lag time in Hsp synthesis in bivalve molluscs (see

Hofmann and Somero, 1996b). No lag time for maximal

expression of Hsp70 was seen in B. gl?ndula in the 16 h

after thermal stress. Our results suggest that levels of en

dogenous Hsp70 were either high before thermal stress or

were synthesized during the stress period. Immunochemical assays are very effective for measuring

heat-shock response in a variety of molluscs (Hofmann and

Somero, 1995; Dahlhoff et al, 2001; Tomanek and Sanford,

2003; Sorte and Hofmann, 2005), but they may be a less

effective measurement method in the crustacean B. gl?n

dula. In Mytilus edulis, immunochemical assays that used

Table 2

Repeated-measures ANOVA on the effect of acclimation on temperature induced Hsp70 and Hsp90 levels from Balanus gl?ndula collected during the summer from Charleston population and Sunset Bay population (see

Fig. 6)_

Sum of Mean

Source squares df square F-ratio P

Hsp70?Charleston population Between subjects

Acclimation 24.742 3 8.247 3.704 0.023

Error 62.344 28 2.227

Within subjects

Temperature 27.806 2 13.903 33.763 0.000

Temperature X acclimation 1.001 6 0.168 0.408 0.870

Error 23.060 56 0.412

Hsp70?Sunset Bay population Between subjects

Acclimation 12.236 3 4.079 1.230 0.320

Error 98.811 28 3.315

Within subjects

Temperature 39.011 2 19.506 28.266 0.000


Error 38.645 56 0.690

Hsp90?Charleston population Between subjects

Acclimation 30.069 3 10.023 0.480 0.700

Error 584.965 28 20.891

Within subjects

Temperature 2.720 2 1.364 0.113 0.890


Error 678.624 56 12.118

Hsp90?Sunset Bay population Between subjects

Acclimation 9.218 3 3.073 3.291 0.035

Error 26.142 28 0.934

Within subjects

Temperature 0.123 2 0.061 0.551 0.580


Error 6.243 56 0.111




Charleston population

24

20

16

12

Hsp70 ' Field acclimatized 10 ?C acclimated

I 16 ?C acclimated : 22 ?C acclimated

Sunset Bay population

Hsp70 t ?

Ifi l M Hsp90

?^ J 1^ Jli, iM?L. 28 33

Incubation temperature (?C)

Figure 6. The effect of thermal acclimation on relative temperature-induced protein synthesis in isolated

muscle tissue from Balanus gl?ndula. Thermally induced Hsp70 levels in B. gl?ndula from (A) Charleston

population and (B) Sunset Bay population. Thermally induced Hsp90 levels in B. gl?ndula from (C) Charleston

population and (D) Sunset Bay population. Each bar represents the mean of eight individuals normalized to the

10 ?C control treatment; 10 ?C control levels equal one and are not displayed. Error bars are 1 standard error.

See Table 2 for statistics.

the same antibody as in the present study identified two

obvious Hsp70 isoforms, presumably constitutive and in duced isoforms (Hofmann and Somero, 1995; Halpin et al,

2002). Typically, a cell will synthesize both constitutive and inducible isoforms of molecular chaperones (Sanders, 1993;

Feder and Hofmann, 1999). Only one resolvable band,

possibly consisting of both constitutive and inducible

Hsp70, was visible in B. gl?ndula. Therefore, relatively high constitutive levels may have made the resolution of

induced levels within the same protein band difficult to

observe. This would explain why a heat-shock response was

observed with the immunochemical method only after pro

longed exposure to 34 ?C (presumably an extreme level of

stress). An alternative is that the antibody we used was not

specific to the inducible Hsp70 isoform. However, this is an

unlikely explanation since an induced response was ob served during experiment 2 (Fig. 3). If an immunochemical

assay is to be employed for future work on B. gl?ndula, two-dimensional gel electrophoresis should be used to sep arate the constitutive from the induced isoform.

Two desert ant species that regularly experience temper atures above 50 ?C while foraging express heat-shock pro teins at a temperature as low as 25 ?C (Gehring and Wehner,

1995). Gehring and Wehner (1995) suggest that the ants

synthesize Hsps at relatively low temperatures prior to

exposure to high temperatures as an adaptation to foraging in a high-temperature habitat. Immunochemical assays on

B. gl?ndula indicated high levels of endogenous Hsp70 at

control temperatures. A correlation between thermotoler

ance and constitutive Hsp70 levels has been observed in

marine snails (Sorte and Hof mann, 2004). Therefore, high levels of endogenous Hsp70 might suggest that B. gl?ndula is preparing for the physiological stress it will potentially encounter during low tide.

Endogenous levels of Hsp70 were not significantly dif

ferent for B. gl?ndula individuals collected from intertidal

elevations of 1.7 m and 0.4 m above mean lower low water

(MLLW) or for those from the tops and undersides of cobbles at 1.7 m above MLLW. A higher intertidal height is

usually more stressful because the intensity of the thermal stress is in part a function of the change in temperature

multiplied by the duration of exposure (Hochachka and

Somero, 2002). We expected higher levels of endogenous Hsp70 expression in habitats presumed to be more stressful

(1.7 m vs. 0.4 m and top vs. bottom of cobbles). For Mytilus

californianus, Roberts et al (1997) reported higher levels of

endogenous Hsp70 in individuals collected higher in the

intertidal zone than in those collected lower in the zone.

Similarly, for M. trossulus, Hsp70 levels were higher for

individuals from a more stressful intertidal habitat com

pared to a subtidal habitat (Hofmann and Somero, 1995). In both field experiments with barnacles, recorded tempera tures were high enough to elicit a heat-shock response in the

more stressful habitats of the high intertidal zone (1.7 m

above MLLW) or cobble topside. Constitutively expressed

Hsp70 isoforms may have masked any heat-induced iso




forms in barnacles from the high intertidal and on the top of

the cobble.

When B. gl?ndula experienced temperatures as high as 34

?C for 8.5 h in the laboratory or when specimens encoun

tered high temperatures in the field, they did not show

evidence of irreversible protein damage. These results differ

from other studies that have examined levels of ubiquitin

conjugate after thermal stress. In multiple cases, an increase

in ubiquitin conjugate levels and subsequent irreversible

protein damage occurred for Drosophila (Lee et al, 1988), intertidal mussels (Hofmann and Somero, 1995, 1996a;

Buckley et al, 2001; Halpin et al, 2002), and lobster (Spees et al, 2002) when they experienced physiologically stress

ful conditions and showed a heat-shock response. Addition

ally, levels of ubiquitin conjugate were elevated when Bala

nus crenatus, a. subtidal congener of B. gl?ndula, was

exposed to a temperature of 26 ?C (M. Berger, unpubl. data). These results from organisms other than B. gl?ndula

suggest that although heat-shock proteins were expressed at

elevated levels, some irreversible protein damage occurred.

A heat-shock response occurred in B. gl?ndula above 23 ?C,

yet there was no indication of irreversible protein damage. This result implies that B. gl?ndula is well adapted to living in a stressful habitat and may have a concentration of

heat-shock proteins adequate to remediate protein denatur

ation. Further work correlating the relationship between

Hsp expression and irreversible protein damage in both

laboratory and field-collected specimens is necessary. We found that the expression of Hsp70 and Hsp90 varied

as a function of temperature, but not as a function of the two

molt stages examined. This result differs from other work

showing an increase in levels of Hsp90 mRNA in lobster

(Homarus americanus) claw muscle tissue during stage D2 of molting (Spees et al, 2003) and in lobster midgut tissue

injected with a molting hormone (Chang et al, 1999). Our

finding that molt stage did not affect levels of heat-shock

protein in B. gl?ndula suggests that molting barnacles will

not be more susceptible than non-molting barnacles to del

eterious physiological effects of thermal stress.

Increases in acclimation or acclimatization temperature

are typically associated with upward shifts in the induction

temperature of heat-shock proteins (Dietz and Somero,

1992; Tomanek and Somero, 1999; Buckley et al, 2001;

Buckley and Hofmann, 2002). This was not the case for

acclimated or summer-acclimatized specimens of B. gl?n

dula; an upward shift in the Hsp induction temperature did

not occur. In fact, barnacles acclimated to higher tempera tures (see Fig. 6) showed higher levels of Hsp70, which was

contrary to the expectation that an organism acclimated to

lower temperatures will be more sensitive to increased

thermal stress. Recent studies suggest that some thermotol

erant intertidal organisms may be the least plastic in their

ability to acclimate to higher temperatures (Stillman, 2003;

Stenseng et al, 2005; Tomanek, 2005). Because B. glan

dula regularly experiences high temperatures for an ex

tended time, it may be operating at a maximal level without

the ability to further modify a physiological response. If the

predication is correct, thermally tolerant species like B.

gl?ndula may be more sensitive to increased global temper atures than previously expected, especially at the southern

limit of their geographic distributions.

Acknowledgments

We thank G. Hofmann and M. Ryan for technical help in

the laboratory. We also thank A. Helms for help in main

taining organisms in the laboratory. Earlier drafts of this

manuscript were improved from comments by L. Burnett, I.

McGaw, P. Phillips, A. Shanks, N. Terwilliger, and two

anonymous reviewers. This research was supported by NSF

grant OCE-9911682 to R. Emlet, NSF-IGERT grant 9972830 to J. Postlethwait, and ERD-OCRM-NOAA grant

NA170R1172 to M. Berger. This research was performed in partial fulfillment of a Ph.D. in the Department of Biol

ogy, University of Oregon.

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Heat-Shock Response of the Upper Intertidal Barnacle Balanus glandula: Thermal Stress and Acclimation