Thermal history-dependent expression of the hsp70 gene in purple sea urchins: Biogeographic patterns and the effect of temperature acclimation

Journal of Experimental Marine Biology and Ecology 327 (2005) 134 – 143 www.elsevier.com/locate/jembe

Thermal history-dependent expression of the hsp70 gene in purple sea urchins: Biogeographic patterns and the effect of temperature acclimation Christopher J. Osovitz a, Gretchen E. Hofmann a,b,* a

Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA 93106-9610, United States b Marine Science Institute, University of California, Santa Barbara, CA 93106-9610, United States Received 11 February 2005; received in revised form 31 May 2005; accepted 10 June 2005

Abstract Thermal variation associated with large spatial scales is thought to significantly affect the physiology of organisms in marine systems and consequently limit their distribution. To examine this phenomenon, this study compared the heat shock protein (hsp70) mRNA expression of purple sea urchins (Strongylocentrotus purpuratus) collected from different geographic locations and those acclimated to different temperatures. Tube feet were removed from S. purpuratus freshly collected from Fogarty Creek, Oregon (FC-OR) and Carpinteria, California (CRP-CA) and from urchins acclimated to 5 8C and 20 8C in the laboratory. Tissue samples were then immediately frozen or incubated at various temperatures between 5 8C and 36 8C for 30 min in vitro. Real-time PCR was used to measure the hsp70 mRNA levels of each sample. The results showed evidence for differential regulation of hsp70 between S. purpuratus in both field and laboratory studies. For example, the incubation temperature that induced maximum hsp70 mRNA expression (T max) was higher in CRP-CA urchins (26 8C) than those from FC-OR (23 8C). Additionally, although the T max of the two acclimation groups was identical (26 8C), S. purpuratus acclimated to 5 8C failed to induce hsp70 mRNA after incubation at 32 8C, while tube feet of the 20 8C-acclimated urchins remained transcriptionally active. In general, the extent of hsp70 mRNA induction was substantial, exceeding 250-fold in the 5 8C-acclimated urchin tissues after incubation at 26 8C. These data suggest that the thermal variation encountered across S. purpuratus’ range is sufficient to effect the regulation of thermally sensitive genes, like hsp70. D 2005 Elsevier B.V. All rights reserved. Keywords: Biogeography; Heat shock response; hsp70; Strongylocentrotus purpuratus; Thermal acclimation

1. Introduction * Corresponding author. Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA 93106-9610, United States. Tel.: +1 805 965 6175; fax: +1 805 965 4724. E-mail address: [email protected] (G.E. Hofmann). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.06.011

In marine systems, environmental factors such as temperature, salinity, and pO2 are thought to limit species ranges (e.g. Dunson and Travis, 1991; Hutch-

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ins, 1947; Somero, 2005). The effect of temperature on community assemblages in particular has recently become very relevant, as many studies have shown that the Earth’s recent increase in average temperature has coincided with a pole-ward shift of many species ranges (Root et al., 2003). However, this trend should only hold true if temperature is setting species borders, and some studies have suggested that temperature is not the primary factor determining marine species’ ranges (e.g. Gaylord and Gaines, 2000; Hummel et al., 2000). Additionally, though physiological studies of performance across large spatial scales are prevalent in terrestrial systems (Chown et al., 2004; Gaston, 2003), very few have been conducted in marine systems. For example, Sorte and Hofmann (2004) found that an intertidal snail Nucella canaliculata living near the southern edge of its range in California was less abundant and contained higher levels of stress proteins in their tissues than those living near the center of their range in Oregon. Also, Henderson and Seaby (1999) found that the growth rates of the sea snail Liparis liparis individuals at their southern range boundary were highest during the coldest months. Overall, the physiological effects of temperature on marine communities are unclear, but their understanding would greatly enhance our power to predict the biological consequences of future climate change. In order to study the physiological effects of geographic thermal variation in marine systems, this study compared the expression of a molecular chaperone, heat shock protein 70 (hsp70), in purple sea urchins (Strongylocentrotus purpuratus) collected from subtidal populations in Oregon and southern California and after laboratory acclimation to 5 and 20 8C. Heat shock protein (Hsp) expression is a good metric for estimating the physiological effects of thermal variation because their expression is extremely sensitive to temperature and reflects the thermal history of the organism (Feder and Hofmann, 1999). Hsps are expressed in response to acute increases in some environmental factors (e.g. temperature, pO2, salinity, etc.) and the presence of high amounts of these proteins is often used as a proxy for cell stress (Dahlhoff, 2004). Mechanistically, as cellular proteins, Hsps are molecular chaperones and play an important role in basic protein biosynthesis and protection from thermally induced protein denaturation

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and aggregation across virtually every taxa (Parsell et al., 1993; Hartl and Hayer-Hartl, 2002). In marine systems, intertidal ectotherms are known to induce Hsps in response to short-term increases in environmental temperature (e.g. Hofmann and Somero, 1995). However, Hsp expression also responds to long-term changes in temperature, suggesting that it plays a role in the adjustment to less stressful gradual temperature changes as well. Specifically, the thermal set-points of Hsp expression are altered by long-term thermal acclimation (Barua and Heckathorn, 2004). Thermal acclimation alters the minimum temperature required to induce Hsp expression (Ton), the temperature at which maximum Hsp expression occurs (T max), and the upper thermal limit of Hsp synthesis (Toff) (Barua and Heckathorn, 2004). Although the effects of seasonal acclimatization on the thermal set-points of Hsp expression have been investigated (e.g. Dietz and Somero, 1992; Buckley et al., 2001; Hamdoun et al., 2003), their relationship to geographic thermal variation has never been studied. Geographic differences in the thermal set-points of hsp70 expression may be an important physiological response to environmental variation, because Hsp synthesis can be very costly (e.g. Krebs and Holbrook, 2001). Therefore, it may be advantageous for marine organisms living in the warmer regions of their geographic range to set their thresholds of Hsp expression to warmer temperatures in order to avoid unnecessary Hsp expression. Here, we chose to measure the mRNA levels of a principal inducible hsp gene, hsp70, using quantitative real-time PCR. Quantitative real-time PCR is currently the most sensitive method to quantify mRNA levels (Bustin, 2000). Studies that have measured the transcription of hsp mRNA after thermal acclimation have commonly used less quantitative measures, like northern analysis (e.g. Currie et al., 2000; Spees et al., 2002). Due to its extremely high sensitivity, real-time PCR is an ideal method to accurately quantify hsp70 induction profiles. The present study employs real-time PCR to measure hsp70 levels in S. purpuratus tube feet in response to thermal variation in both field and laboratory settings. S. purpuratus was chosen as a model because of its large geographic range, which extends from Baja California to southern Alaska, across which, there is thought to be no genetic breaks (e.g. Flowers et al.,

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2002). In the field portion of this study, the thermal setpoints of hsp70 mRNA induction were compared between S. purpuratus collected from Fogarty Creek, OR (FC-OR) and those collected from Carpinteria, CA (CRP-CA). In the laboratory portion of this study, the thermal set-points of hsp70 mRNA induction were compared between S. purpuratus acclimated to 5 and 20 8C, temperatures that bracket those of the most extreme sustained temperatures experienced by S. purpuratus in their natural habitat. S. purpuratus’ Alaskan habitat routinely reaches temperatures near 4 8C during the winter, while its southern California habitat commonly reaches temperatures above 20 8C during the summer (Coastal Data Information Program at SCRIPPS Institute of Oceanography; http://cdip. ucsd.edu/). Geographic and acclimatory differences in the thermal set-points of hsp70 expression would suggest that environmental temperature variation significantly affects S. purpuratus’ physiology. Such variation in gene expression is thought to be a very important mechanism by which organisms deal with environmental variation (Schulte, 2001; Oleksiak et al., 2002; Podrabsky and Somero, 2004). Our prediction was that real-time PCR would elucidate differential hsp70 mRNA expression between S. purpuratus with different thermal histories.

to the urchin habitat (~ 20 ft depth). At FC-OR, temperature data was collected from a 1 m depth approximately 250 m off shore from FC-OR. The FC-OR temperature data logger was lost, so only data acquired prior to 8/5/04 is reported here. For the laboratory acclimation, S. purpuratus were collected from the Santa Barbara Channel in late January 2004. Seawater temperature was approximately 14 8C at the time of collection. Four urchins with test sizes of 7–8 cm were placed into each of two recirculating aquaria filled with unfiltered freshly collected seawater. The temperatures of the two aquaria were adjusted at the rate of 2–3 8C every 3 days toward 5 8C and 20 8C. The animals were kept at those two temperatures for 4 weeks and were fed kelp (Macrocystis) weekly. 2.2. Tissue collection Prior to temperature incubation, multiple 25–50 mg portions of tube feet tissue were excised from each urchin. From each urchin, one tissue aliquot was frozen immediately upon dissection (referred to as t 0 samples), and the remaining tissue samples were placed into microcentrifuge tubes containing 0.5 ml of seawater. Those tubes were incubated at one of several temperatures for 30 min and frozen immediately afterward. The incubation temperatures for each experimental group are shown in Table 1.

2. Materials and methods 2.3. Total RNA extractions and cDNA synthesis 2.1. Sea urchin collections and field temperature data For the field comparisons, S. purpuratus specimens were collected from the shallow subtidal zone at Fogarty Creek, OR (FC-OR) (latitude: 44851VN; longitude 124800VW) and Carpinteria, CA (CRPCA) (latitude: 34823VN; longitude: 119831VW) in mid-August 2004. The urchins collected from FCOR had test sizes of 7–8 cm and were shipped to laboratory facilities at UC, Santa Barbara immediately after collection, then kept at 10 8C in a recirculating aquarium for 48 h prior to tissue sampling (see below). The urchins from CRP-CA had test sizes of 4–5 cm and were kept at 16 8C in a recirculating aquarium for 24 h after collection. Ocean temperature was collected from data loggers deployed in the field several weeks earlier. At CRP-CA, temperature was collected from a benthic data logger directly adjacent

Frozen tissues were homogenized in TRIzolR (Invitrogen) reagent and processed according to manufacturer’s instructions with the following adjustments. A second organic extraction (100% chloroform) was performed directly after the initial TRIzol extraction. Total RNA was precipitated in the recommended voTable 1 Temperature incubation regimes for the four S. purpuratus experimental groups Group 5 8C

Temperatures of 30 min in vitro incubation

5 8C, 11 8C, 13.5 8C8C, 16 8C, 18.5 8C, 20 8C, 23 8C, 26 8C, 28.5 8C, 36.5 8C 20 8C 5 8C, 16 8C, 20.5 8C, 23.5 8C, 26 8C, 29 8C, 32 8C, 36 8C FC-OR 10 8C, 20 8C, 23 8C, 26 8C, 29 8C, 32 8C, 36 8C CRP-OR 16 8C, 20 8C, 23 8C, 26 8C, 29 8C, 32 8C, 36 8C

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lume of 100% isopropanol plus an equal volume of salt solution (initial concentration: 1.2 M NaCl, 0.8 M disodium citrate). After the RNA was re-suspended in nuclease free water, the total RNA extraction was further purified using the Aquapure RNA isolation kit (Bio-Rad) according to manufacturer’s protocol, except that the RNA was precipitated in the recommended volume of 100% isopropanol plus an equal volume of salt solution (1.2 M NaCl, 0.8 M disodium citrate). Final RNA concentrations were determined using an ultraspec UV/visible spectrometer (Amersham Biosciences). For the reverse transcription step, 100 ng of total RNA was transcribed using oligo dT and random priming (iscript cDNA synthesis kit, Bio-Rad). 2.4. mRNA quantification The relative levels of mRNA for the hsp70 and actin genes in the tube feet samples were determined using quantitative real-time PCR. Primers for realtime PCR were designed using Bio-Rad Beacon designer software. The hsp70 primers were designed against an 89-base pair region EST (Caltech; EST no. PM990802-01-0471-.y1) published on the Sea Urchin Genome Project website (http://sugp.caltech. edu/) that showed great homology to hsp70 genes in BLAST searches of NCBI’s database and displayed temperature sensitivity in preliminary experiments. The actin primers were designed against an 89-base pair region (nucleotides 344 to 432) of a partial sequence of the actin 1 gene in S. purpuratus (Genbank accession no. j01166.1; Schuler et al., 1983). Quantitative real-time PCR reactions were performed with 2 Al of cDNA synthesis product in 20 Al SYBR green supermix (Bio-Rad) reactions run in duplicate. The PCR was run for 40 cycles with the following cycle parameters: 10 s at 94 8C; 10 s at 58 8C. Ct, the number of cycles at which the fluorescence of the reaction exceeds the threshold, values greater than 30 were not included in analysis. The primer concentrations for hsp70 (1.0 AM) and actin (0.5 AM) were empirically determined based on lowest Ct values and highest efficiencies. Fluorescence threshold values were set at levels to maximize PCR efficiency, and only reactions with efficiencies within 90%–110% were analyzed. Four 10-fold serial dilutions of a single experimental sample from each PCR plate

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were used as a standard curve to calculate PCR efficiency. No template controls and one of three cDNA standards were run on each plate to allow comparisons among plates. Melt curve analysis was performed following each PCR to confirm that only a single product was amplified. To control for genomic DNA (gDNA) contamination, non-transcribed RNA from each sample was also used as template for real-time PCR. Amplification using the actin primers showed that gDNA contamination constituted no more than 1/ 1000 of the amplification observed with transcribed cDNA samples. Using the hsp70 primers, amplification of gDNA from the samples incubated between 20 and 29 8C showed amplification no greater than 1/100 of the amplification observed with transcribed cDNA samples. However, in a few other samples, gDNA amplification totaled almost 1/3 of the amplification observed of the remainder of the cDNA samples, so the levels of hsp70 mRNA in the samples with very low expression may be slightly over-estimated. Reported relative mRNA levels were calculated in the following fashion: (1) hsp70 and actin Ct values were first normalized to that of the corresponding product of the cDNA standard from each plate. (2) Resultant hsp70 values were then normalized to the resultant actin values for the each sample. The actin gene was deemed to be an appropriate internal control because the actin mRNA levels did not differ significantly across heat incubations. To calculate the fold inductions, the final hsp70 mRNA level was normalized to the t 0 expression level for each specimen. 2.5. Data analysis Significance of the effect of incubation temperature within acclimation and field groups was determined using one-way analysis of variance (ANOVA), and significance of effect of acclimation temperature and collection site was determined using two-way ANOVA with incubation temperature and acclimation temperature or collection site as factors. One-way ANOVA was necessary or within experimental group comparisons in order to analyze all of the data points that were not common to both experimental groups. Data were transformed to meet assumptions of ANOVA using y = x 0.2, chosen according to the method of Box and Cox (1964). O’brien’s test for homogeneity of variances and the Kolmogorov–Smir-

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nov goodness-of-fit test were used to check for homoscedasticity and approximate normality, respectively. Tukey–Kramer Honestly Significant Difference tests were used to conduct post-hoc comparisons among group means.

3. Results

20 18

Temperature ( C)

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16 14 12 10

3.1. General observations

8

FC-OR

6

CRP-CA

7/12/04 7/19/04 7/26/04

On average, between 0.1 and 3 Ag of RNA was recovered from each ~ 50 Ag sample of S. purpuratus tube feet tissue, which proved sufficient for real-time analysis. Table 2 shows the relative levels of hsp70 and actin mRNA in selected samples from the four experimental groups after the 30 min in vitro incubations. Both one-way ANOVA of all data points and two-way ANOVA among incubation temperatures only common to both experimental groups indicated that incubation temperature significantly affected hsp70 mRNA levels within each experimental group ( P b 0.0001). Additionally, acclimation temperature in the laboratory study significantly affected hsp70 expression ( P N 0.0001), although biogeographic site did not ( P = 0.585). In general, the hsp70 mRNA transcription profiles resembled a bell curve, with the majority of hsp70 mRNA induction after incubations between 20 8C and 30 8C and maximum induction after incubation at 26 8C (see Figs. 2 and 3). 3.2. Latitudinal comparison: Oregon vs. California sea urchins Seawater temperature at FC-OR was similar to that of CRP-CA from 7/15/04 to 7/21/04, then remained approximately 5 8C lower than that of CRP-CA from 7/22/04 to 8/5/04 (the last day of data collection from

8/2/04

8/9/04

8/16/04

Date Fig. 1. Seawater temperature at the two study sites: Fogarty Creek, OR (FC-OR) and Carpenteria, CA (CRP-CA) for 1 month prior to specimen collection. The temperature logger from FC-OR was compromised on 8/5/04.

FC-OR) (Fig. 1). The seawater temperature at CRPCA was approximately 16 8C at the time of collection while that of FC-OR was approximately 11 8C on 8/5/ 04 (Fig. 1). The level of hsp70 mRNA expression between specimens collected from the two sites was comparable after most temperature incubations, but there was evidence for a shift in the thermal set-points (Fig. 2A). The most striking difference between the hsp70 expression profiles of the two groups of urchins was that the T max of the FC-OR urchins was 3 8C lower (23 8C) than that of the CRP-CA urchins (26 8C) (Fig. 2A). Additionally, the FC-OR urchins displayed 2.7fold greater expression of hsp70 mRNA after the 20 8C treatment and 3.1-fold lower expression after the 32 8C treatment (Table 2). Also, the untreated (t 0) samples from the FC-OR urchins displayed 2.9-fold greater expression of actin mRNA ( P b 0.05) (Table 2). Conversely, after normalization to the expression levels of the t 0 samples, the T max of both urchin groups became 26 8C (Fig. 2B). Also, because the hsp70 levels of the t 0 samples from the FC-OR urchins were greater than those of the CRP-OR urchins, the CRP-OR urchins

Table 2 Relative levels of hsp70 and actin mRNA from S. purpuratus tube feet after 30 min temperature incubations in vitro Experimental group

t0

t0

20 8C

23 8C

26 8C

29 8C

32 8C

5 8C 20 8C FC-OR CRP-CA

hsp70/actin 0.003 0.014 0.025 0.009

actin 1.274 1.340 1.044 0.358

hsp70/actin 0.115 0.033 0.113 0.043

hsp70/actin 0.459 0.382 0.706 0.446

hsp70/actin 0.934 1.378 0.587 1.037

hsp70/actin 0.356 0.669 0.220 0.589

hsp70/actin 0.008 0.139 0.041 0.128

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3.3. Laboratory comparison: 5 8C- vs. 20 8C-acclimated sea urchins

*

Like the field groups, the magnitude of hsp70 mRNA expression between the 5 8C and 20 8C accli-

* *

A

160 FC-OR CRP-CA

140

1.0 0.5

* * **

0.0 t0

120 100

* * * *

*

Incubation temperature ( C)

80

B

60

400 5 C

exhibited much higher hsp70 inductions after normalization to the expression levels of the t 0 samples (Fig. 2B). The average fold induction of the CRP-CA urchins after the 26 8C incubation was 132, while that of the FC-OR urchins was 22.

100

36

32

29

26

20 23

0 18

Fig. 2. Relative levels of hsp70 mRNA within tube feet isolated from S. purpuratus recently collected from Fogarty Creek, OR (FCOR) and Carpinteria, CA (CRP-CA) after incubation at one of several temperatures in vitro for 30 min. Error bars indicate standard error. Incubations at 10 8C and 16 8C were unique to the FC-OR and CRP-CA groups, respectively. (A) Values are reported relative to a common standard. and indicate value is significantly different from t 0 within the FC-OR and CRP-OR groups, respectively. The Ton, T max, and Toff for the FC-OR urchins were 20 8C, 23 8C and 36 8C, respectively, while the Ton, T max, and Toff for the CRP-CA urchins were 20 8C, 26 8C, and 36 8C, respectively. (B) Values normalized to the t 0 values for each specimen. RNA from a t 0 sample from one 5 8C-acclimated urchin was lost during extraction, so the values were normalized to the level of the 5 8C, which was comparable in all other individuals. N = 4, except 5 8C-acclimated, t 0 sample, where N = 3.

200

13 16

Incubation temperature ( C)

11

36

32

29

26

23

20

16

10

T-

0

0

20 C

300

5

20

t0

40

hsp70 mRNA level retative to t0 sample

hsp70 mRNA level relative to t0 sample

B

1.5

36

Incubation temperature ( C)

* *

32

36

32

29

26

23

20

16

T-

0

0.0

20 C

2.0

29

*

5 C

26

*

0.2

2.5

23

0.4

20

*

16 18

0.6

13

*

5 11

0.8

Relative level of hsp70 mRNA

FC-OR CRP-CA

1.0

10

Relative level of hsp70 mRNA

A 1.2

139

Incubation temperature ( C) Fig. 3. Relative levels of hsp70 mRNA within tube feet isolated from S. purpuratus acclimated to 5 8C and 20 8C after incubation at one of several temperatures in vitro for 30 min. Error bars indicate standard error. Incubations at 11 8C, 13 8C, and 18 8C were unique to the 5 8C acclimation group, while the incubation at 36 8C was unique to the 208C acclimation group. (A) Values are reported relative to a common standard. and indicate value is significantly different from t 0 within the 5 and 20 8C acclimation groups, respectively. indicates a significant difference between the mean expression of the two acclimation groups after incubation at that temperature. The Ton, T max, and Toff for the 5 8C acclimated urchins were 20 8C, 26 8C and 32 8C, respectively, while the Ton, T max, and Toff for the 20 8C acclimated urchins were 20 8C, 26 8C, and 36 8C, respectively. (B) Values were normalized to the t 0 values for each specimen. N = 4, except FC-OR, 23 8C sample and CRP-CA, 36 8C sample, where N = 3.

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mation groups was comparable after incubation at most temperatures between 5 and 36 8C. The majority of induction of hsp70 occurred between 20 8C and 32 8C, with maximum induction after incubation at 26 8C (Fig. 3A). However, expression near the extreme temperatures of the incubation treatments differed between the acclimation temperatures. For example, the 5 8C-acclimated urchins had 3.5-fold higher expression of hsp70 mRNA after the 20 8C incubation, while the 20 8C-acclimated urchins exhibited 16.8fold higher hsp70 inductions after the 32 8C incubation ( P b 0.05; Table 2). Additionally, the hsp70 mRNA level from the t 0 samples of the 20 8C-acclimated urchins was 5.4-fold higher than those of the 5 8C-acclimated urchins ( P b 0.05) (Table 2). The magnitude of induction was very pronounced in both acclimation groups, but due to the higher levels of hsp70 mRNA in the t 0 samples in the 20 8C acclimation group, the expression of the 5 8C-acclimated urchins was higher, with the average T max exceeding 250-fold. (Table 2, Fig. 3B).

4. Discussion Results of this study provide definitive evidence that thermal history of purple sea urchins modified the expression pattern of a thermally sensitive gene, hsp70, which encodes for the molecular chaperone Hsp70. Importantly, we were able to detect differences in hsp70 expression patterns that mapped onto the biogeographic distribution of S. purpuratus and, further, that these patterns correlated with expected differences in temperature exposure at the two sites that are 108 latitude apart. Overall, there were four salient findings of this study: (1) S. purpuratus from Fogarty Creek, OR displayed lower thresholds for hsp70 mRNA expression than those from Carpinteria, CA, (2) S. purpuratus laboratory-acclimated to 5 8C showed similar decreases in the thresholds of hsp70 mRNA induction when compared to those acclimated to 20 8C, (3) even without heat stress, there were differences in the hsp70 levels between both the laboratory and field groups, and (4) overall, the magnitude of hsp70 mRNA induction was very high, ranging to a mean 250-fold above baseline expression levels for the 5 8C-acclimated S. purpuratus.

4.1. Differences in the thermal set-points of hsp70 expression between urchins collected from Oregon and California We found evidence for a difference in the thermal set-points of hsp70 mRNA induction between S. purpuratus freshly collected from Fogarty Creek, Oregon (FC-OR) and those from Carpinteria, California (CRP-CA). Most definitively, T max of hsp70 expression in the tube feet of the FCOR S. purpuratus was 38 lower (23 8C) than that of the CRP-CA urchins (26 8C). Additionally, the FC-OR urchins displayed much lower levels of hsp70 mRNA after incubation at the higher incubation temperatures, 29 8C and 32 8C, and higher expression at a lower incubation temperature, 20 8C (Fig. 2). This result suggests that urchins living in more northern, cooler waters, maintain a lower Ton and Toff of hsp70 mRNA expression. Similar differences in Hsp expression have been reported extensively in the literature in response to thermal acclimation (Barua and Heckathorn, 2004 for review) or seasonal acclimatization (e.g. Buckley et al., 2001; Hamdoun et al., 2003). Although this is the first study to investigate thermal set-points of Hsp expression across large spatial scales, such differential expression could be fundamental for marine ectotherms in tolerating substantial environmental variation associated with large spatial distances. Differences in the thermal set-points of Hsps between individuals living at different latitudes are likely to be advantageous, as over-expression of Hsps is known to be costly (e.g. Krebs and Holbrook, 2001). Therefore, alterations in the thresholds of Hsp expression across large spatial scales appear to reduce the probability of over-expression in the tissues of the northern populations and under-expression in tissues of the southern populations. One consideration that should be noted in the field comparison is that the shifts in threshold of expression could be caused by the differences in average test size between the two sites (4–5 cm for the CRP-CA urchins; 7–8 cm for the FC-OR urchins). However, although body size may affect the standing stocks of Hsps (G. Somero, pers. comm.), the authors can find no study linking body size to threshold of Hsp expression.

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4.2. Differences in the thermal set-points of hsp70 expression between S. purpuratus acclimated to 5 8C and 20 8C in the laboratory The thermal set-points of hsp70 expression were also compared between S. purpuratus acclimated to 5 8C and 20 8C in the laboratory in order to investigate to what extent the geographic trends were a result of differential thermal history. Although the T max of the two laboratory acclimation groups was the same (26 8C), differences similar to those discovered in the field study regarding the thermal setpoints of hsp70 mRNA expression were discovered near Ton and Toff of hsp70 induction. A substantial difference in the Toff of hsp70 was observed, as the 5 8C-acclimated urchins also showed higher hsp70 levels at the lower stress temperatures, like 20 8C (Fig. 3). Additionally, the 20 8C urchins contained significantly higher levels of hsp70 mRNA after incubation at 32 8C, while the 5 8C-acclimated urchins showed no induction at that temperature (Fig 3). These results indicate that thermal acclimation to 5 8C and 20 8C was sufficient to induce a shift in the Ton and Toff of hsp70 mRNA expression in S. purpuratus. The pattern of the shift is very similar to that of the differences in hsp70 expression between the field-collected sea urchins, suggesting that thermal variation played a central role in the differential hsp70 expression observed between the S. purpuratus collected from Oregon and California. The results regarding both the differences in the T max and the Toff of hsp70 mRNA expression between the experimental groups have additional implications. First, a comparison of the T max of each urchin group suggests that recent thermal history may not be the only influence on the threshold of hsp70 induction. The average difference in temperature between FCOR and CRP-CA was ~ 5 8C during the 3 weeks prior to collection, and the T max of the FC-OR urchins was 3 8C lower than that of the CRP-CA urchins. However, acclimation to 5 8C and 20 8C for 4 weeks did not shift the T max in S. purpuratus. The inability of the laboratory-acclimated urchins to adjust their T max in response to thermal acclimation may be due to permanent physiological adjustments in response to environmental variation during early ontogenesis (e.g. Zamer and Mangum, 1979). Alternatively, genetic distance between the two urchin populations could

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be the cause, although most studies have failed to find a geographic genetic structure in S. purpuratus (e.g. Flowers et al., 2002; Palumbi and Wilson, 1990). Additionally, the finding that thermal acclimation results in a shift in the Toff of hsp70 mRNA expression has implications regarding the mechanism behind the upper thermal synthesis limit of Hsps. Although studies have shown that thermal acclimation results in a shift in the Toff of Hsps at the protein level (Barua and Heckathorn, 2004), no study has reported a thermal shift in the Toff of hsp transcription levels in response to acclimation. The shift in hsp70 mRNA expression found in the current study suggests that the urchins’ failure to induce Hsps at high temperatures is to some extent caused by a reduction in transcriptional ability. 4.3. Initial stock of mRNAs Differences were discovered in the hsp70 mRNA levels in the untreated (t 0) samples between both the acclimation groups and the field groups. The standing level of hsp70 mRNA was 5.4-fold higher in the tissue of the 20 8C-acclimated urchins than that of the 5 8Cacclimated urchins (Table 2), suggesting that living at such a high constant temperature results in the need for thermal protection of S. purpuratus’ tissues. This increased level in standing stock of inducible hsp70 has been reported in other organisms and is usually attributed to thermal stress associated with the acclimation temperature (e.g. Dietz and Somero, 1992). However, the opposite trend was observed in the field comparison, as the colder acclimatized FC-OR urchins had 2.9-fold higher hsp70 mRNA levels than the CRP-CA urchins. Although the specimens were kept in 10 8C aquaria for 2 days prior to tissue collection, this trend was possibly due to stress associated with overnight transport from Oregon to California. Additionally, the t 0 samples from the FC-OR urchins contained approximately 3 times the level of actin mRNA than those of the CRP-CA urchins. However, the significance of the difference in actin mRNA levels between the California and Oregon S. purpuratus was unclear. All in all, these data show that in order to cope with environmental variation, S. purpuratus separated by large spatial scales employed differential gene expression not only under stressful conditions, but under everyday conditions as well.

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4.4. Magnitude of hsp70 inductions The magnitude of the hsp70 mRNA inductions observed in this study was very high, exceeding 500-fold after incubation at 26 8C in a single 5 8Cacclimated urchin. Conversely, most studies report an average magnitude of hsp70 mRNA induction at T max to be less than 20-fold (e.g. Spees et al., 2002). This discrepancy is likely due to the relatively high sensitivity of quantitative real-time PCR, as S. purpuratus hsp70 mRNA expression quantified by northern analysis did not reveal an induction nearly as robust (C. Osovitz, unpublished data). The most intense hsp70 inductions in this study were seen in the 5 8C-acclimated urchins and those collected from CRP-CA (Figs. 2B and 3B). However, these relatively high inductions were not a product of relatively high transcript abundance in the incubation samples, but of relatively low transcript abundance in the t 0 samples in these groups (Table 2). Therefore, the relatively high hsp70 inductions observed in this study may primarily owe to real-time PCR’s superior ability to accurately quantify extremely low levels of transcript.

5. Conclusions The results of this study show that hsp70 mRNA expression in the tube feet of S. purpuratus increased dramatically in response to temperatures that emulate the maximum of its natural environment and that this response is plastic. The Ton, Toff, and T max of hsp70 mRNA expression were lower in field acclimatized S. purpuratus collected from Fogarty Creek, OR compared to those collected from a latitude 108 farther south at Carpinteria, CA, where the seawater temperature was approximately 5 8C warmer. Furthermore, hsp70 mRNA expression within the tube feet of S. purpuratus acclimated to 5 8C in the laboratory showed similar decreases in Ton and Toff as compared to those acclimated to 20 8C, suggesting that thermal variation was a central cause of the differential expression observed in the geographic comparison. Differential expression of both hsp70 and actin mRNA was also discovered between the untreated (t 0) tissues of the FCOR and CRP-CA urchins, indicating that alterations in gene expression are used by S. purpuratus to cope with environmental variation under non-stressful conditions

as well. Overall, these results suggest that the thermal variation encountered across S. purpuratus’ biogeographic range is physiologically significant and alteration of gene expression is one of the ways in which this species copes with this variation.

Acknowledgements The authors would like to acknowledge PISCO for temperature data from Fogarty Creek, OR, and the Santa Barbara Coastal LTER (Director: Dr. Dan Reed) for collection of sea urchins and temperature data from Carpinteria, CA and Santa Barbara, CA. We also thank Dr. Jeff Richards (University of British Columbia) for assistance developing real-time PCR, Jessica Dutton for excellent laboratory assistance, and Brian Kinlan for consultations on statistical analysis. This research was supported by an NSF Graduate Research Fellowship to CJO and NSF grant OCE0425107 to GEH. This is contribution number 188 from PISCO, the Partnership for Interdisciplinary Studies of Coastal Oceans funded primarily by the Gordon and Betty Moore Foundation and David and Lucile Packard Foundation. [SS]

References Barua, D., Heckathorn, S.A.S., 2004. Acclimation of the temperature set-points of the heat-shock response. J. Therm. Biol. 29, 185 – 193. Box, G.E.P., Cox, D.R., 1964. An analysis of transformations. J. R. Stat. Soc., B 26, 211 – 252. Buckley, B.A., Owen, M.E., Hofmann, G.E., 2001. Adjusting the thermostat: the threshold induction temperature for the heatshock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204, 3571 – 3579. Bustin, S.A., 2000. Absolute quantification of mRNA using realtime reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169 – 193. Chown, S.L., Gaston, K.J., Robinson, D., 2004. Macrophysiology: large-scale patterns in physiological traits and their ecological implications. Funct. Ecol. 18, 159 – 167. Currie, S., Moyes, C.D., Tufts, B.L., 2000. The effects of heat shock and acclimation temperature on Hsp70 and Hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons. J. Fish Biol. 56, 398 – 408. Dahlhoff, E.P., 2004. Biochemical indicators of stress and metabolism: applications for marine ecological studies. Annu. Rev. Physiol. 66, 183 – 207.

C.J. Osovitz, G.E. Hofmann / J. Exp. Mar. Biol. Ecol. 327 (2005) 134–143 Dietz, T.J., Somero, G.N., 1992. The threshold induction temperature of the 90-kda heat-shock protein is subject to acclimatization in eurythermal goby fishes (genus Gillichthys). Proc. Natl. Acad. Sci. U. S. A. 89, 3389 – 3393. Dunson, W.A., Travis, J., 1991. The role of abiotic factors in community organization. Am. Nat. 138, 1067 – 1091. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243 – 282. Flowers, J.M., Schroeter, S.C., Burton, R.S., 2002. The recruitment sweepstakes has many winners: genetic evidence from the sea urchin Strongylocentrotus purpuratus. Evolution 56, 1445 – 1453. Gaston, K.J., 2003. The Structure and Dynamics of Geographic Ranges. Oxford University Press, Oxford. Gaylord, B., Gaines, S.D., 2000. Temperature or transport? Range limits in marine species mediated solely by flow. Am. Nat. 155, 769 – 789. Hamdoun, A.M., Cheney, D.P., Cherr, G.N., 2003. Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance. Biol. Bull. 205, 160 – 169. Hartl, F.U., Hayer-Hartl, M., 2002. Protein folding—molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852 – 1858. Henderson, P.A., Seaby, R.M., 1999. Population stability of the sea snail at the southern edge of its range. J. Fish Biol. 54, 1161 – 1176. Hofmann, G.E., Somero, G.N., 1995. Evidence for protein damage at environmental temperatures–seasonal changes in levels of ubiquitin conjugates and Hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198, 1509 – 1518. Hummel, H., Bogaards, R.H., Bachelet, G., Caron, F., Sola, J.C., Amiard-Triquet, C., 2000. The respiratory performance and survival of the bivalve Macoma balthica (L.) at the southern limit of its distribution area: a translocation experiment. J. Exp. Mar. Biol. Ecol. 251, 85 – 102. Hutchins, L.W., 1947. The bases for temperature zonation in geographical distribution. Ecol. Monogr. 17, 325 – 335.

143

Krebs, R.A., Holbrook, S.H., 2001. Reduced enzyme activity following Hsp70 overexpression in Drosophila melanogaster. Biochem. Genet. 39, 73 – 82. Oleksiak, M.F., Churchill, G.A., Crawford, D.L., 2002. Variation in gene expression within and among natural populations. Nat. Genet. 32, 261 – 266. Palumbi, S.R., Wilson, A.C., 1990. Mitochondrial-DNA diversity in the sea-urchins Strongylocentrotus purpuratus and Strongylocentrotus droebachiensis. Evolution 44, 403 – 415. Parsell, D.A., Taulien, J., Lindquist, S., 1993. The role of heatshock proteins in thermotolerance. Philos. Trans. R. Soc., B 339, 279 – 286. Podrabsky, J.E., Somero, G.N., 2004. Changes in gene expression associated with acclimation to constant temperatures and fluctuating daily temperatures in an annual killifish Austrofundulus limnaeus. J. Exp. Biol. 207, 2237 – 2254. Root, T.L., Price, J.T., Hall, K.R., Schneider, S.H., Rosenzweig, C., Pounds, J.A., 2003. Fingerprints of global warming on wild animals and plants. Nature 421, 57 – 60. Schuler, M.A., Mcosker, P., Keller, E.B., 1983. DNA-sequence of 2 linked actin genes of sea-urchin. Mol. Cell. Biol. 3, 448 – 456. Schulte, P.M., 2001. Environmental adaptations as windows on molecular evolution. Comp. Biochem. Physiol., B 128, 597 – 611. Somero, G.N., 2005. Linking biogeography to physiology: evolutionary and acclimatory adjustments of thermal limits. Front. Zool. 2 (1) (http://www.frontiersinzoology.com/content/2-1/1). Sorte, C.J.B., Hofmann, G.E., 2004. Changes in latitudes, changes in aptitudes: Nucella canaliculata (Mollusca: Gastropoda) is more stressed at its range edge. Mar. Ecol. Prog. Ser. 274, 263 – 268. Spees, J.L., Chang, S.A., Synder, M.J., Chang, E.S., 2002. Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin. Cell Stress Chaperones 7, 97 – 106. Zamer, W.E., Mangum, C.P., 1979. Irreversible non-genetic temperature adaptation of oxygen-uptake in clones of the sea-anemone Haliplanella luciae (Verrill). Biol. Bull. 157, 536 – 547.