Heat Shock Proteins in Juvenile Steelhead ... - Wiley Online Library

Transactions of the American Fisheries Society 134:399–410, 2005 q Copyright by the American Fisheries Society 2005 DOI: 10.1577/T03-181.1

[Article]

Heat Shock Proteins in Juvenile Steelhead Reflect Thermal Conditions in the Navarro River Watershed, California INGEBORG WERNER* Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616, USA

TIMOTHY B. SMITH Center for Aquatic Ecology, Illinois Natural History Survey, 606 East Peabody Street, Champaign, Illinois 61820, USA

JOAQUIN FELICIANO Department of Wildlife, Fish, and Conservation Biology, University of California, One Shields Avenue, Davis, California 95616, USA

MICHAEL L. JOHNSON John Muir Institute of the Environment, University of California, One Shields Avenue, Davis, California 95616, USA Abstract.—This study examined expression levels of two heat shock proteins, hsp72 and hsp78, in white muscle of steelhead (anadromous rainbow trout Oncorhynchus mykiss) parr collected between 25 July and 4 August 2000 at 11 sites in the Navarro River watershed, California. The goal was to determine whether site-specific thermal conditions were causing cellular stress responses in resident fish. The results demonstrated a highly significant sigmoidal relationship of the inducible isoform hsp72 and a linear relationship of the constitutive isoform hsp78 with water temperatures. Laboratory experiments showed that hsp72 was induced in fish from coldwater as well as warmwater sites by exposure to water at 258C. Stream temperatures above which significantly elevated hsp72 levels were detected in field-collected fish were 18–198C in terms of both short- and long-term averages and 20–22.58C in terms of daily maximum averages. The highest hsp72 levels were measured at warmwater sites with the largest diurnal temperature fluctuations ($6.58C). The two dominant factors influencing water temperature were air temperature and the degree of shade provided by riparian vegetation. Our data suggest that elevated hsp72 expression levels in steelhead parr from several tributaries are indicative of cellular stress caused by thermal conditions during summer months.

The heat shock protein response is one of the most important mechanisms to repair and prevent the damaging effects of thermal cellular stress and is indicative of the denaturation of cellular proteins during exposure to elevated temperatures (Feige et al. 1996; Somero 2002). This response manifests itself in the rapid induction and expression of heat shock proteins (hsps). Among the hsp protein families, hsp70 is the most prominent group, present in all cell types and localized in the cytosol, mitochondria, and endoplasmic reticulum. It is composed of multiple constitutively expressed (hsc70, grp78) and inducible members (hsp72) whose functions include the correct folding, repair, and

* Corresponding author: [email protected] Received October 20, 2003; accepted September 2, 2004 Published online April 15, 2005

translocation of intracellular proteins, suppressing protein aggregation, and reactivating denatured proteins (Feige et al. 1996). Increased synthesis of hsps in response to thermal cellular stress has been reported for many species of teleosts (Iwama et al. 1998) and other organisms ranging from bacteria to humans (Morimoto et al. 1990). Modifications of hsp induction and expression have also been associated with adaptive responses to environmental conditions in closely related species (Dietz and Somero 1992; Somero 2002). Recent research shows that hsps are involved in immune system responses (Zuegel and Kaufmann 1999) and also interact with multiple key components of signaling pathways that regulate growth and development (Nollen and Morimoto 2002). Water temperature is among the most important environmental variables influencing the distribution and success of steelhead (anadromous rain-

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bow trout Oncorhynchus mykiss) and other salmonid populations (Baltz et al. 1987; Sullivan et al. 2000; Dunham et al. 2001). Structural modifications along rivers and changes in land use patterns have led to altered flow and temperature regimes with associated increases in sedimentation and water temperatures in numerous streams along the Pacific Coast. Juvenile steelhead rear for 1–3 years (Moyle 2002) in freshwater streams before smolting and migrating to the ocean. They are most vulnerable to increases in stream water temperatures, especially during summer months in reaches with low flows, high air temperatures, and little or no riparian vegetation. Optimal temperature ranges vary depending on life stage and acclimation temperature. Steelhead fry show highest survival rates when eggs are exposed to temperatures between 78C and 108C. For juveniles and adults, reported chronic lethal limits range from 22.8–278C (Threader and Houston 1983) and critical (acute) thermal limits from 27.5–328C, depending on acclimation temperature (10–258C) and body size, with large fish being less tolerant than small fish (Myrick and Cech 2001). The thermal heterogeneity of streams (particularly small streams) significantly complicates the task of identifying and quantifying thermal stress in resident fish, and the available, mostly laboratory-derived information on temperature tolerance is of limited value. Diurnal temperature fluctuations can be substantial, and the availability of thermal refuges, such as deepwater pools or shaded areas, determines if the fish experience harmful temperatures (Nielsen et al. 1994) or not. It is often unclear if fish are able to avoid the areas of higher temperature and find thermal refuges, or if they are exposed to physiologically stressful temperatures. If exposed, it is not clear how individual fish respond to elevated water temperatures. The physiological consequences of chronic and sublethal thermal stress are difficult to identify and measure, and are likely to vary with acclimation history of resident fish. They can, nevertheless, have serious implications for the survival and growth of steelhead populations (Brett 1995). The objective of this study was to detect and quantify the exposure and sublethal, cellular response to stressful thermal conditions in resident West Coast steelhead parr (age-0) by measuring cellular levels of hsp70 proteins in white muscle tissue. Hsp70 profiles in field-collected fish were later compared with induction profiles obtained under controlled laboratory conditions. The field study area, the Navarro River watershed, is rep-

resentative of numerous streams along the Pacific Coast of North America where elevated water temperature has become a major stress factor for aquatic ecosystems. West Coast steelhead, whose distribution ranges from Alaska to Southern California (Moyle 2002), are endangered south of Point Conception, California, and numerous runs north of there are either classified as threatened (Point Conception to Russian River, including Central Valley) or have been proposed for listing (U.S. National Marine Fisheries Service 2005). Efforts to conserve steelhead stocks include a variety of regulatory efforts targeting dominant environmental stressors, in particular thermal loading and sedimentation. Methods Study area.—The Navarro River watershed is a 785-km2 drainage located in the Coast Range of Mendocino County, California (398109200N, 1238409060W; Figure 1). It is composed of five subwatersheds that are oriented from west to east: the North Fork of the Navarro River, Flynn Creek, Indian Creek, Anderson Creek, and Rancheria Creek. Precipitation in the watershed falls primarily between October and May. Average annual discharge is 14.5 m3/s. Air temperatures in the coastal (western) drainages range from 38C to 308C, and coastal fog is common during the summer months. Temperatures in the eastern portion of the watershed can range from near 08C to 408C. Climate as well as land use influence the density and type of riparian vegetation. There are no potential point sources (industry, agriculture, road crossings) of toxic chemicals, and anthropogenic input of such chemicals into the river system is minimal. The Navarro River has been placed on the 1998 State of California 303-d list for impaired water bodies with a medium priority rating for elevated temperature and sedimentation–siltation. Fish sampling.—Fish were collected between 25 July and 4 August 2000 at 11 sites located in the watershed. Sampling sites covered all major subdrainages, including second-, third-, and fourthorder streams (Figure 1; Table 1). Steelhead parr (age-0) were collected from both riffles and pools with beach seines at all sites except Upper Anderson, Lower Indian, and Middle Rancheria creeks. Fish from those sites were collected using a Smith-Root Model 12B backpack electrofisher. With the exception of Lower North Fork and Lower Flynn Creek (both coldwater sites), collections were performed in the mornings before water temperatures reached 208C and completed within 2 h.

THERMAL CONDITION REFLECTED IN TROUT HEAT SHOCK PROTEINS

Collected fish were transferred to plastic 150-L flow-through tubs situated in the respective stream. A random sample of 10 steelhead parr was removed from flow-through tubs, decapitated, and frozen on dry ice within one-half hour of collection. Only five fish were collected from Middle Flynn Creek and only one from Middle Anderson Creek due to the small size of the steelhead populations there. Frozen samples were returned to the laboratory on dry ice, measured, weighed, and dissected. Tissue samples were stored at 2808C. Water quality parameters.—Temperature was continuously recorded in 30-min intervals at each site with a HOBO data logger fastened to a small piece of iron reinforcing bar and left throughout the summer on the substrate of a shaded run within each site. No temperature data were collected for Upper Rancheria Creek because the site became dewatered early in the year. Fish at this site were collected from isolated pools in the streambed. We have summarized temperature data as average and maximum temperatures for 3 h, 24 h, 7 d, and 1 month prior to fish collection. Monthly temperature averages were measurements from 1 July 2000 to the date when fish were sampled at individual sites (Table 1). Weekly and monthly average and maximum temperatures are commonly used by regulatory agencies to characterize temperature regimes in streams, and reflect the short- and longterm thermal history of resident fish. Conductivity and pH measured in June and September 2000 across the watershed ranged from 181 to 299 S/ cm, and pH 6.6–7.9, respectively. Ionized ammonia concentrations were measured monthly, and average concentrations for 25 April–27 July 2000 ranged from 2.25 mg/L NH14 at Lower Rancheria Creek to 34.9 g/L NH14 at Middle North Fork, with a maximum value of 95.9 mg/L NH14 at MNF in July. Physical site conditions.—For site characterization, flow, percent shade, and width-to-depth ratio were measured for sampling sites on Anderson, Indian, and Rancheria creeks and the North Fork of the Navarro River between 4 and 10 September 2001 (Table 2). Percent shade was measured using a convex spherical densitometer held 0.3 m above the surface of the water in the middle of the channel. The mean percent of shade was calculated from three measurements taken at the downstream end, upstream end, and middle of the reach. To obtain the wetted channel width-to-depth ratio, 11 transects were spaced evenly along the length of the reach. The wetted width of the channel and the depth of the water were measured at three locations

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spaced evenly along each transect. The means of 11 width measurements and 33 depth measurements were used to calculate wetted channel width-to-depth ratio. Flow was measured using an electronic flowmeter (Marsh-McBirney; Flo-Mate 2000) and the velocity–area method for measuring volumetric flow. Laboratory temperature exposure.—Fish were collected with beach seines on 8 October 2001 from one coldwater (‘‘coastal,’’ namely, Lower North Fork) and one warmwater (‘‘inland,’’ namely, Lower Rancheria Creek) site. Lower Rancheria Creek is close to our Middle Rancheria Creek (MRC) field site (see above), and there is a less than 0.58C difference in air and water temperatures. Water temperatures at Lower North Fork and Middle Rancheria Creek on 7–8 October 2001 were 13.48C and 16.78C (24-h averages) and 14.08C and 19.98C (24-h maxima), respectively. Fork length was 70.3 6 16.0 mm (average 6 SD; range, 49–109 mm) for ‘‘coastal,’’ and 62.1 6 13.7 mm (41–90 mm) for ‘‘inland’’ fish. Fish were placed in aerated water from the collection sites and transported in plastic bags to the Center for Aquatic Biology and Aquaculture (CABA) at the University of California, Davis. Upon arrival, fish were placed in separate circular flow-through tanks (158C) and fed daily. Fish received combined formalin (25 mg/L) and malachite green (0.1 mg/L) treatments (45 min/d static bath for 5 d) for external parasites, plus a nitrofurazone antibiotic (25 mg/L) treatment (45 min/d static bath for 10 d). Until initiation of the temperature experiments they were fed ad libidum daily using a semimoist fish chow (Rangen, Inc., Buhl, Idaho; 3/320 pellet). Two weeks after arrival at CABA, two subgroups of 12 fish were transferred to flow-through tanks containing water heated to 25 6 0.28C. Control groups were caught by nets, transferred to buckets, and returned to 158C tanks to serve as handling controls. Conductivity of the water at the time of our experiments was 730 mS, the pH was 8.0–8.2, and oxygen concentration was 8.9–11.2 mg/L. After 2 h at 258C fish were transferred to 158C tanks, and four fish from both exposed and control groups were sacrificed by decapitation (time 5 0 h). White (axial) muscle samples were dissected from the left lateral flank and flash frozen in liquid nitrogen for hsp analysis. Additional samples (n 5 4 per treatment) were taken 6 h and 24 h thereafter and preserved at 2808C until analysis. Fish were not fed for the duration of the experiment. Heat shock protein analysis.—We analyzed

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TABLE 1.—Geographic location and temperature data (8C) for steelhead sampling sites in the Navarro River watershed in July–August 2000. All weekly and hourly temperature averages are for the period preceding the sampling date and time. All monthly temperature averages are for the period from 1 July 2000 to the date of fish collection. The following abbreviations are used: MMAT 5 mean monthly average temperature; MMTmax 5 mean monthly maximum temperature; MWAT 5 mean weekly average temperature; MWMT 5 mean weekly maximum temperature; MMTmin 5 mean monthly minimum temperature; and MDTR 5 mean daily temperature range from July 1 to the date of fish collection. Sampling site Lower Flynn Creek Middle Flynn Creek Lower North Fork Middle North Fork Lower Indian Creek Upper Indian Creek Middle Rancheria Creek Upper Rancheria Creekb Lower Anderson Creek Middle Anderson Creek Upper Anderson Creek a b

Sampling date 1 3 1 4 25 26 28 31 2 2 1

Aug Aug Aug Aug Jul Jul Jul Jul Aug Aug Aug

GPS coordinatesa 39.1615, 39.1847, 39.1545, 39.1735, 39.0590, 39.0776, 38.9483, 38.8503, 39.0538, 39.0140, 38.9904,

123.5823 123.5981 123.6197 123.5602 123.4397 123.3748 123.3242 123.2392 123.4330 123.3724 123.3146

3-h average 24-h average

MWAT

MMAT

24-h maximum

15.2 15.1 17.7 17.5 18.0 18.1 19.0

16.1 16.2 18.7 18.2 19.2 21.6 20.3

15.6 15.6 18.2 18.3 18.2 18.4 21.2

15.2 14.6 17.6 18.0 18.7 19.1 20.5

17.3 17.3 20.3 19.3 23.7 27.9 23.7

18.9 20.1 18.1

21.4 23.3 21.6

20.3 22.5 20.4

19.4 21.4 19.7

25.3 27.0 28.6

The first value is degrees north latitude, the second degrees west longitude. Not measured (see Methods); fish collected from isolated pools.

hsp70 proteins using Western blot techniques described in Werner et al. (2001). Briefly, white (axial) muscle samples were homogenized on ice in a hypotonic solution containing 66 mM of trisHCl (pH 5 7.5), 0.1% Nonidet, 10 mM of EDTA, 10 mM of dithiothreitol, and protease inhibitors. Following centrifugation at 48C, supernatants were collected, mixed with sample buffer (Laemmli

1970), and then heated (958C for 2 min). Total protein concentration was determined by means of the Biorad DC protein assay based on Lowry et al. (1951). Subsamples of equal total protein content (25 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12.5% polyacrylamide gels with 5% stacking gels (Blattler et al. 1972) using the buffer system de-

FIGURE 1.—Map of steelhead sampling sites in the Navarro River watershed, Mendocino County, California.

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TABLE 1.—Extended.

Sampling site

MWMT

MMTmax

MMTmin

Mean 24-h range

MDTR

Lower Flynn Creek Middle Flynn Creek Lower North Fork Middle North Fork Lower Indian Creek Upper Indian Creek Middle Rancheria Creek Upper Rancheria Creek Lower Anderson Creek Middle Anderson Creek Upper Anderson Creek

17.0 16.6 19.7 19.9 22.3 23.3 25.4

16.7 15.4 19.0 19.6 22.8 23.8 24.4

14.3 13.7 16.4 16.8 16.3 16.4 17.6

2.1 2.4 3.0 1.8 7.0 10.7 7.0

2.4 1.7 2.6 2.8 6.5 7.4 6.8

24.4 26.5 27.4

22.9 25.5 26.6

17.3 18.1 16.0

6.8 7.0 11.1

5.6 7.4 10.6

scribed by Laemmli (1970). Calibrated molecular weight (MW) markers and 50-ng recombinant human hsp70 (Stressgen Biotechnologies Corp., Victoria, British Columbia; NSP-555) were applied to each gel to serve as internal standards for MW determination and blotting efficiency. Measured band density of 50-ng hsp70 antigen ranged from 3.5 to 5.8 on blots of samples from the field study and from 3.4–5.1 on blots of samples from laboratory experiments. No adjustments were made to band density measurements. Proteins were separated and then electroblotted onto Immobilon-P membranes, which were blocked for 30 min. A monoclonal antibody for hsp70 (Affinity Bioreagents, Golden, Colorado; dilution 1:500, MA3001) was used as probe. This antibody recognizes constitutive (hsp70, hsc70, and grp78) and inducible (hsp72) members of the hsp70 protein family in steelhead and other fish species (Werner et al. TABLE 2.—Physical site characteristics of sampling sites in the Navarro River watershed.

Sampling site Lower Flynn Creeka Middle Flynn Creeka Lower North Fork Middle North Fork Lower Indian Creek Upper Indian Creek Middle Rancheria Creek Upper Rancheria Creek Lower Anderson Creek Middle Anderson Creek Upper Anderson Creek a

Not measured.

Channel width–depth Shade (%) ratio Flow (m3/s)

81.73 65.00 49.40 35.19 10.75

15.17 15.86 65.79 46.44 26.28

0.004 0 0.014 0.018 0.003

63.44 39.35 26.17

17.82 60.06 20.34

0.007 0.005 0.008

2001; Washburn et al. 2002; Viant et al. 2004). In steelhead, this antibody recognizes at least two hsp70 isoforms of approximately 72 and 78 kDa (Figure 2). While hsp78 was constitutively expressed and possibly corresponds to mammalian grp78/BiP, we have not established homologies with hsps from other species. Using onedimensional electrophoresis, we cannot exclude the possibility that the hsp bands that were assigned to a specific size-class contain more than one hsp homologue. Blots were incubated for 90 min with primary antibody, then washed in trisbuffered saline solution containing 0.05% Tween20. Alkaline phosphatase-conjugated goat-anti-rat IgG (Sigma, St. Louis, Missouri; 1:30,000) was used to detect the hsp70 probe. Bound antibody was visualized by a chemiluminescent substrate (Tropix, Inc., Bedford, Massachusetts; CDP-Star), and protein bands were quantified by densitometry (Biorad GS710). Protein band intensity is expressed as band density minus background. Pellets resulting from the centrifugation of homogenized muscle samples were analyzed for hsp70 proteins following methods described by Werner et al. (2001). Very little hsp70 was detectable and band intensities reflected the pattern seen in hsp70 levels of the supernatant fraction (data not shown). Initial analysis of 22 liver samples identified at least two constitutive proteins of MW 71 and 78 kDa, but differences between sites were less pronounced and data were more variable than in muscle tissues. We were not sure if the constitutive 71 kDa protein was homologous to the hsp72 detected in white muscle, and therefore focused further analyses on the supernatant fraction of muscle tissues.

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FIGURE 2.—Representative Western blot of hsp72 and hsp78 detected in white muscle tissue of steelhead parr. See Figure 1 for site codes. Blots contain three replicate samples from each field site except for MAC). An hsp70 antigen (Stressgen Biotechnologies, Inc.) was used to track blotting efficiency. Band density readings were adjusted for background.

Statistical analyses.—All hsp data were tested for normality and equality of variance. If data were normally distributed, they were further analyzed by one-way analysis of variance (ANOVA) and subsequent pairwise multiple comparison by the Fisher least-significant-difference method. If normality tests failed, we used Kruskal–Wallis oneway ANOVA and Dunn’s test for pairwise multiple comparisons between groups (sampling sites). Data derived from our laboratory experiment was additionally compared by t-test or Mann–Whitney rank-sum test. Significance level was p less than 0.05. All regression analyses were conducted with the statistical software packages SPSS 7.0 and SigmaStat 2.0 (SPSS, Inc., Chicago, Illinois). Since only one fish was obtained at MAC, hsp results from this site were excluded from statistical analysis. Results Water Temperature and Physical Site Characteristics Temperature curves at four representative sites for the period from 21 June 2000 to the dates of fish collection are shown in Figure 3, and sitespecific temperature data are presented in Table 1. Measured average and maximum water temperatures (T) at field sites during the 24 h prior to fish sampling were 16.1–23.68C and 17.3–28.68C, respectively. Temperatures as well as daily T ranges were closely tied to geographic location and significantly correlated with longitude in a linear fashion, reflecting the distance from the Pacific Ocean and a corresponding increase in air temperature (24-h average T: P 5 0.006; 24-h maximum T: P 5 0.003; mean 24-h T range: P 5 0.002). Weekly and monthly T averages closely followed these patterns. Correlations between 3 h, 24 h, weekly, and monthly T averages were both linear and highly significant (24 h versus 3 h T-average:

P 5 0.0002; 24 h versus weekly average T (MWAT): P 5 0.0005; 24 h versus monthly average T (MMAT): P , 0.0001), as were correlations between 24 h, weekly, and monthly maximum T (24 h versus mean weekly maximum T (MWMT): P 5 0.0001; 24 h versus mean monthly maximum T (MMTmax): P , 0.0001) and respective daily T ranges (24-h T range versus mean daily T-range in July 2000 (MDTR): P , 0.0001). Among physical site characteristics, only the percentage of shade was significantly correlated with water temperature at sampling sites (Table 2). Percent shade was highest near the coast (ca. 80%) and decreased steadily to ca. 20% inland (percent shade versus longitude: P 5 0.004). Shade was inversely correlated with monthly Tmax and average T (MMTmax: P 5 0.010; MMAT: P 5 0.045), and MDTR (p 5 0.020), but not significantly correlated with weekly or 24-h T measurements. Average minimum T (MMTmin) for the same time period were not significantly correlated with geographic location (P 5 0.1) or percent shade (P 5 0.7). Neither the wetted channel width-to-depth ratio nor flow was significantly correlated with site water temperature. Expression of hsp70 in Field-Collected Fish Differences in hsp72 expression levels between sampling sites were highly significant (Figure 4a). Levels of hsp72 showed a significant third-order sigmoid (threshold) relationship with T averages, T maxima, and daily T ranges (Table 3). The strongest correlation was observed with the average daily maximum T for the month of July (P , 0.0001). Temperature thresholds (i.e., temperature between minimum and significantly increased hsp72 levels) were 18–198C for average T and 20–22.58C for maximum T (Table 3). Standard errors for the upper plateaus of the sigmoid hsp72 curves were relatively large (8.03–8.76%), reflecting the de-


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FIGURE 4.—Differential expression of (a) hsp72 and (b) hsp78 in steelhead parr (see location codes in Figure 1). The letters A, B, C indicate significantly different groups (P , 0.05). The values of hsp72 and hsp78 represent mean densitometer readings of protein bands detected by Western blotting (6SE); n 5 10, except for MFC (n 5 5) and MAC (n 5 1).

FIGURE 3.—Representative graphs of daily maximum (solid lines) and minimum (dotted lines) water temperatures from 21 June to the fish collection date at a coldwater site (Lower North Fork), two moderately warm sites (Lower Indian Creek and Middle Rancheria Creek), and a warmwater site (Upper Anderson Creek).

gree of variation for these maximum values. For weekly averages (MWAT), the threshold temperature was approximately 188C, but MWAT values overlapped between several coldwater and warmwater sites, and were therefore poorly associated

with hsp72 levels, which meant that no curve fit was possible (Table 3). For example, MWAT values at sites Lower Indian Creek and Upper Indian Creek (18.2–18.48C) were similar to MWAT at Lower North Fork and Middle North Fork (18.2– 18.38C), but there was a significant difference in hsp72 levels likely due to the much larger daily temperature fluctuations at Lower Indian Creek and Upper Indian Creek (Table 1). Average weekly T, therefore, appear to be of limited value for the characterization of site-specific temperature regimes. Site-specific differences in hsp78 expression levels were mostly not significant (Figure 4b), but means showed a linear correlation with maximum T and daily T-ranges (Table 3). Neither hsp72 nor hsp78 protein levels were correlated with minimum T (MMTmin: P . 0.05). Expression of hsp70 in Laboratory-Exposed Fish Exposure to 258C for 2 h significantly induced hsp72 expression in steelhead collected from cold-

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TABLE 3.—Statistical relationships of site water temperature averages preceding fish collection with hsp72 and hsp78 expression in skeletal muscle and the resulting estimated threshold temperatures for hsp72 overexpression; NA 5 not applicable. Temperature measurement 3-h average

11.36/{1 1 exp[2(x 2 17.9)/0.01]} 0.96; P 5 0.0004 11.4/{1 1 exp[2(x 2 19)/0.07]} 0.96; P 5 0.0004 NA ya 5 11.44/{1 1 exp[2(x 2 18.44)/0.13]} P 5 0.0004 r 5 0.96; y 5 11.2/{1 1 exp[2(x 2 21)/0.17]} P 5 0.0005 r 5 0.96; y 5 11.47/{1 1 exp[2(x 2 21.38)/0.43]} P , 0.0004 r 5 0.96; y 5 14.03/{1 1 exp[2(x 2 22.31)/0.98]} P 5 0.0001 r 5 0.99; y 5 12.03/{1 1 exp[2(x 2 6.75)/0.07]} P 5 0.0001 r 5 0.98; y 5 13.29/{1 1 exp[2(x 2 5.56)/0.88]} P 5 0.0002 r 5 0.97; y 5 12.98/{1 1 exp[2(x 2 5.21)/0.77]} P , 0.0001 r 5 0.98;

y r y r

24-h average 7-d average Monthly average 24-h maximum 7-d mean of daily maxima Monthly mean of daily maxima 24-h range 7-d mean of daily ranges Monthly mean of daily ranges a

Correlation with hsp72 5 5 5 5

Estimated threshold temperature (8C)

Correlation with hsp78

18

r 5 0.57;

P 5 0.106

19

r 5 0.61;

P 5 0.081

18.25 18.0–18.5

r 5 0.56; r 5 0.66;

P 5 0.116 P 5 0.051

21 20–22 20–22.5 6–6.5 4–6 4–6

y r y r y r y r y r y r

5 5 5 5 5 5 5 5 5 5 5 5

2.14 1 0.67; 1.21 1 0.69; 0.73 1 0.76; 7.29 1 0.70; 7.02 1 0.71; 6.89 1 0.76;

0.033x P 5 0.04 0.39x P 5 0.039 0.42x P 5 0.019 0.41x P 5 0.035 0.49x P 5 0.033 0.53x P 5 0.019

Sigmoidal curve fit not possible (see text).

water (coastal) as well as warmwater (inland) sites (Figure 5a). Despite maintenance of fish at a constant temperature of 158C for 2 weeks before the experiment, basal hsp72 levels in inland fish (controls) were higher than in coastal fish. Expression levels of hsp78 in coastal as well as inland fish were significantly higher than respective controls 24 h after exposure to 258C (Figure 5b). Discussion Most fish, as ectotherms, have body temperatures close to that of their surrounding water temperature (Moyle and Cech 2000), and expression levels of temperature-inducible hsps can therefore be excellent monitors of an organism’s exposure to elevated water temperatures. Temperatures requiring the organism to raise this protective cellular response are considered ‘‘stressful’’ in the sense that metabolic energy needs to be diverted for increased hsp expression in order to maintain cellular homeostasis (Parsell and Lindquist 1993; Somero 2002). This cellular stress response is species, organ, and stressor specific (Iwama et al. 2004), and is reflective of an organism’s sensitivity to temperature (Somero 2002). Certain chemicals can induce hsps (Bierkens 2000), but given the absence of chemical stressors in the watershed and the strong relationship of hsp72 levels with water temperatures, we conclude that the appearance of elevated levels of this inducible hsp in muscle of juvenile steelhead from Anderson, Indian, and

Rancheria creeks is the consequence of a protective response to cellular stress caused by sitespecific thermal conditions. Relatively few studies have examined the hsp response in fish populations in the field. Fader et al. (1994) showed that hsc/hsp70 levels in four fish species collected from the wild and a hatchery were significantly lower in winter and fall than in summer and spring, with the highest levels occurring in spring. The observed pattern was explained by water temperature as well as the relative rise in water temperature from winter to spring. The study described here also showed that water temperature, as well as daily temperature range, were important factors influencing cellular hsp72 levels. Lund et al. (2002) concluded that parr of Atlantic salmon Salmo salar experienced thermal stress at 238C and above based on the analysis of hsp70 and hsc70 mRNA levels measured in whole body torsos. Levels of mRNA generally change within minutes after a temperature shock, while changes in protein expression are detectable after approximately 30–60 min, reaching peak levels after 12–24 h. Unlike Lund et al. (2002), our study attempted to link hsp70 protein levels to the thermal history of field-collected fish, and to identify field temperature conditions under which fish were experiencing apparent thermal cellular stress indicated by the increased expression of hsp72. The water temperatures above which significantly elevated hsp72 expression levels were ob-


FIGURE 5.—Expression of (a) hsp72 and (b) hsp78 in the white muscle tissue of steelhead parr from a coldwater site (‘‘coastal’’) and a warmwater site (‘‘inland’’) exposed to water at 258C for 2 h (HS) and 158C (controls [C]). The values of hsp72 and hsp78 represent mean densitometer readings of protein bands detected by Western blotting (6SE); n 5 4. Samples were taken immediately (0 h), 6 h, and 24 h after the 258C treatment. The letters A and B indicate significant differences between fish groups (P , 0.05); the asterisks indicate significant differences from the controls.

served in this study concur with what is known about the sublethal consequences of thermal stress in steelhead. ‘‘Threshold’’ temperatures determined in this study were 18–198C for short- and long-term T averages, and 20–22.58C for maximum T during the 24 h preceding fish collection. Sublethal effects (such as decreased ovulation, egg production, and embryo survival) were observed in rainbow trout exposed to temperatures of 188C and above for 3 months (Pankhurst et al. 1996). In experiments on thermal preferences of steelhead (Myrick and Cech 2000), hatchery fish acclimated to constant (168C) or diel cycling temperature regimes (16 6 28C) selected temperatures between 188C and 198C. The selected temperatures closely matched the temperature where growth rates were highest.

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The fact that fish were collected from geographically distinct areas within the Navarro River watershed raises the question whether high levels of hsp72 in fish from the warmwater, inland sites are reflective of acclimatization or genetic adaptation of subpopulations to thermal conditions. For example, closely related species can exhibit significant differences in temperature tolerance, and it has been shown that this tolerance is tied to induction patterns of hsp70 proteins (Nakano and Iwama 2002). Our laboratory study revealed that basal hsp72 levels were higher in steelhead parr from a warmwater, inland site than in fish from a coldwater, coastal site, even 14 d after collection and maintenance at 158C. It is possible that hsp72 is relatively stable in these fish and decreased slowly during the 14-d holding period. It is also possible that fish from warmwater sites have adapted to their thermal environment by overexpressing hsp72. However, exposure to 258C led to significant increases in hsp72 expression in fish from both warmwater and coldwater sites, and peak hsp72 levels were similar between the two groups. This suggests that fish from coastal as well as inland sites respond to high water temperatures in a similar manner. Genetic adaptation within the same species is only possible if thermal conditions have been perennially different among sites and there was no interference with the homing fidelity of these steelhead populations. This is unlikely here, since steelhead populations within the Navarro River watershed can interbreed. However, further study of subpopulations is warranted to conclusively answer the question if the observed differential expression of hsp72 between warmwater and coldwater sites was due to genetic adaptation or not. In addition to thermal cellular stress, a number of factors can influence hsp70 expression levels in wild fish, and it is important to take these into consideration when interpreting data from this study. Several hsps, among them hsp70, can be induced by certain chemical toxicants (Sanders 1993; Bierkens 2000) and pathogens (Forsyth et al. 1997; Cho et al. 1997). In the Navarro River watershed chemical toxicants are unlikely to play a significant role. The area is remote and sparsely populated, and water samples collected during storm events in the 1999–2000 and 2000–2001 rainfall seasons from the tributaries of the Navarro showed that no constituent exceeded federal and state water quality standards. Standard U.S. Environmental Protection Agency three-species toxicity tests (U.S. Environmental Protection Agency

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1994) performed on water samples collected in 1999–2000 did not indicate the presence of toxic compounds, and steelhead parr liver burdens of heavy metals did not differ significantly among sites (Johnson et al. 2002). We have no data evaluating the prevalence of disease among Navarro River fish. However, the synthesis of hsp70 is much greater in response to thermal cellular stress than to other known inducers, and any response to pathogens or chemicals would likely be masked by high levels of hsps induced by the thermal environment of the organism (Bierkens 2000). Additional potentially confounding factors for the interpretation of hsp results are the influence of some stress hormones on hsp expression. Adrenaline increases in response to temperature stress in sockeye salmon O. nerka (Mazeaud et al. 1977), and it can induce hsp70 in fish and mammals (Ackerman et al. 2000; Maloyan and Horowitz 2002). In contrast, handling stress and the associated increase in blood cortisol do not induce hsp70 expression in rainbow trout (Vijayan et al. 1997; Washburn et al. 2002). Fish collected for this study were sacrificed within 30 min of capture, which is insufficient time to result in significant modification of hsp70 protein expression due to physiological stress. Finally, increased muscle activity can induce hsp70 expression in the muscle tissue and blood of mammals (Milne and Noble 2002), but it is presently unclear if this also occurs in ectothermic organisms such as fish. Fish activity does tend to increase under thermally stressful conditions, and the potential existence of a combined effect of increased muscle activity and temperature on hsp72 expression cannot be excluded. It is difficult to escape these potential colinearities in field studies, and the common cause of such secondary effects would ultimately be temperature stress. Clearly, expression levels of hsp70 proteins are not a specific, but rather an integrative cellular stress indicator signaling the level of cellular injury inflicted by the impact of a single stressor or a combination of stressors. The physiological consequences of maintaining elevated hsp levels for long periods of time are presently unknown. Protein synthesis and repair are energy intensive processes (Roberts et al. 1997; Hofmann and Somero 1995; Somero 2002), and it is possible that the continued production of hsps confers a fitness cost (Krebs and Loeschcke 1994; Feder et al. 1992). Viant et al. (2004) showed that increased expression of hsp72 and hsp89 in steelhead parr in response to chronic exposure to 208C (from 158C) was associated with a

decrease in high-energy compounds (ATP, phosphocreatine, glycogen) in liver and muscle tissues. In addition, several hsps are associated with cellular signaling molecules and receptors, which regulate growth and development (Nollen and Morimoto 2002; Queitsch et al. 2002). Although we solely analyzed hsp70 isoforms in white muscle tissue, other studies have demonstrated that most other organs and tissues overexpress a number of hsps in response to thermal cellular stress (Iwama et al. 1998; Smith et al. 1999; Palmisano et al. 2000; Currie et al. 2000; Washburn et al. 2002). Given the extent and potential duration of the thermal cellular stress response in juvenile steelhead from inland tributaries of the Navarro River, it is therefore possible that organisms exposed to prolonged or repeated periods of elevated temperature may experience metabolic energy deficits. In summary, based on the pattern of increased hsp72 expression in fish at warmer sites, the existing information on thermal tolerance of steelhead, and the overall lack of toxicological stressors in the Navarro watershed, we conclude that the juvenile fish caught at Lower, Middle, and Upper Anderson Creek; Lower and Upper Indian Creek; and Middle and Upper Rancheria Creek were experiencing and responding to thermal conditions that caused cellular stress. All of these sites are located inland and are characterized by a lack of riparian vegetation, high water temperatures, and large daily temperature fluctuations. Although increased synthesis of hsp72 signals a disruption of cellular protein homeostasis, the capacity of organisms to compensate through adaptive mechanisms does not yet allow a full understanding of the physiological and ecological implications. A better understanding of the functional mechanisms of thermotolerance, the role of hsp72 in adaptation, and the consequences of maintaining high expression levels of hsps is needed to allow predictions about potentially harmful effects at the organism or population level. Acknowledgments We thank Rick Bush, Henry Calanchini, Drew Gantner, Davy Lu, Melissa Turner, Joe Kiernan Garrett Cotham, Julie Vu, and Katie Woodside for help in the field and in generating and processing the data. We very much appreciate comments by Peter Moyle and Mark Viant, University of California–Davis. Funding was provided by a grant from the California Department of Transportation (contracts 43A0014 and 43A0073) to M.L.J.


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