Marine Biology (2005) 146: 985–993 DOI 10.1007/s00227-004-1508-2
R ES E AR C H A RT I C L E
Cascade J. B. Sorte Æ Gretchen E. Hofmann
Thermotolerance and heat-shock protein expression in Northeastern Pacific Nucella species with different biogeographical ranges
Received: 22 August 2003 / Accepted: 2 November 2004 / Published online: 22 December 2004 Springer-Verlag 2004
Abstract We investigated physiological traits responsible for determining the tide-height and latitudinal distributions of Northeastern Pacific Nucella congeners. First, we determined the thermotolerances of two species of intertidal dogwhelks, N. ostrina and N. canaliculata, which co-occur on the Oregon coast. We found that N. ostrina, which are distributed higher on the shore, and thus experience higher habitat temperatures, than N. canaliculata, had correspondingly higher heat-coma temperatures. Second, we acclimated individuals of all five Northeastern Pacific Nucella congeners to a common temperature and determined their thermotolerances, measured as recovery from thermal exposure, after a 5-day, 3-week, and 7-week acclimation period. The south-latitude (N. emarginata) and mid-latitude (N. ostrina) high-intertidal species were more thermotolerant than the mid-latitude low-intertidal (N. canaliculata and N. lamellosa) and north-latitude high-intertidal (N. lima) species. The results of these two experiments suggest that temperature plays a role in determining the tide-height and latitudinal distributions of these Nucella species. Finally, we measured total and inducible levels of an evolutionarily conserved and ecologically relevant protein, the 70-kDa heat-shock protein (Hsp70), which has been found to confer thermotolerance in model laboratory organisms. The results showed that the level of total, not stress inducible, Hsp70 was a better predictor of thermotolerance and that there were species-specific differences in the relationship between Hsp70 expression and thermotolerance. We suggest that Hsp70 expression may be important in conferring thermotolerance in Nucella species in nature and that higher levels of Communicated by P.W. Sammarco, Chauvin C. J. B. Sorte Æ G. E. Hofmann (&) Department of Ecology, Evolution and Marine Biology and the Marine Science Institute, University of California, Santa Barbara, CA 93106, USA E-mail:
[email protected] Tel.: +1-805-8936175 Fax: +1-805-8934724
molecular chaperones may underlie increased thermotolerance between conspecifics.
Introduction Although species’ geographic ranges are generally distinct and fixed, the factors determining species’ range boundaries are largely unknown. An organism’s fitness, or ability to survive and reproduce, in a particular location is affected by a combination of abiotic (e.g. temperature, salinity, and substrate availability) and biotic factors (e.g. predation, competition, and food availability; see Brown 1984). If organisms’ distributions are related to abiotic factors, then environmental conditions may define physiological and geographical boundaries outside of which individuals are unable to survive (Menge and Sutherland 1987; Huey 1991). Traits responsible for species’ distributions can be identified by comparing closely related species with different distributions and matching environmental stress resistances to their distributions (Hoffmann and Blows 1994). In our study, we used this approach to investigate the role of temperature in determining the distinct latitudinal and tide-height distribution patterns of five intertidal snail congeners. Environmental factors, such as temperature, influence organismal condition at several levels of organization (Somero 2002), including organismal levels (e.g. feeding rate, Sanford 1999, 2002) and subcellular levels (e.g. protein synthesis and functioning, Somero 1995). Here, we investigate the expression of heat-shock proteins (Hsps), which have been shown to confer thermotolerance in model laboratory organisms (for reviews, see Lindquist 1986; Sanders 1993; and Feder and Hofmann 1999), as a potential mechanism linking environmental stress and organisms’ distributions. Hsps are molecular chaperones, produced as part of the heatshock response, which assist in the refolding of stress-
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denatured cellular proteins and prevent these proteins from aggregating in the cell (Parsell and Lindquist 1993; Fink 1999). Such protein aggregations are essentially toxic in the cells, and they increase organisms’ energy costs by requiring degradation and replacement (Hartl and Hayer-Hartl 2002). Recent studies in natural systems have shown that Hsp expression patterns exhibit plasticity based on an organism’s thermal history (Dietz and Somero 1992; Hofmann and Somero 1995, 1996a; Roberts et al. 1997; Tomanek and Somero 1999; Buckley et al. 2001). The direct effect of the heat-shock response on the energy costs and environmental tolerance of organisms suggests that it may be involved in determining species’ distribution patterns (Feder and Hofmann 1999; Somero 2002). Rocky intertidal habitats are ideal for biogeographical studies of environmental stress effects for two reasons. Due to emersion during the tidal cycle, their poikilothermic inhabitants experience temporally predictable bouts of temperature and desiccation stress (Bertness et al. 1999). In addition, intertidal organisms are restricted to the shoreline and have one-dimensional latitudinal distributions with two (a north and a south) range boundaries (Sagarin and Gaines 2002). A second dimension to intertidal organisms’ ranges lies perpendicular to the shore along a tide-height gradient, and, due to differences in emersion time, the intensity of temperature stress increases directly with tide height (Hofmann 1999). We chose intertidal dogwhelks in the genus Nucella as our study organisms because five congeneric species with distinctive latitudinal distributions occur along the North American West Coast (Table 1, Fig. 1; Palmer et al. 1990; Collins et al. 1996; Sorte and Hofmann 2004). Within a site, species’ distributions also differ with respect to tide height, with N. lamellosa and N. canaliculata restricted to low tide heights and N. ostrina, N. emarginata, and N. lima found at high tide heights (Table 1; Palmer 1980). Thus, the distributions of these species may be determined by environmental factors that vary with latitude and tide height. In this study, we address two specific questions about the physiological basis of the distributions of NorthTable 1 Nucella spp. Distributions of Northeastern Pacific Nucellaspecies (Palmer 1980; Collins et al. 1996; Sorte and Hofmann 2004) Species
Maximum tide heighta
Latitudinal range
N. lima N. lamellosa
6 4
N. canaliculata
5
N. ostrina
6
N. emarginata
6
Japan to Vancouver Island, B.C. Aleutian Island, Alaska to Santa Cruz, Calif. Aleutian Island, Alaska to Piedras Blancas, Calif. Yakutat, Alaska to Pt. Conception, Calif. Half Moon Bay, Calif. to Baja California, Mexico
Fig. 1 Nucella spp. Latitudinal distributions of Northeastern Pacific Nucella species (Collins et al. 1996; Sorte and Hofmann 2004)
eastern Pacific Nucella species: (1) Are there speciesspecific differences in thermotolerance that correlate with the distinct temperature ranges of Nucella habitats, and (2) Are Hsp expression patterns correlated with Nucella thermotolerances? We examined two measures of organismal thermotolerance—recovery from thermal exposure and heat-coma temperature—and compared thermotolerances to distributions and habitat temperatures. We then measured cellular levels of a 70-kDa Hsp, Hsp70, in the same individuals to determine whether Hsp expression was correlated with organismal thermotolerance.
Materials and methods Comparison of Nucella congeners Collection and acclimation conditions
a On a 0–6 scale with 0 and 6 representing low and high tide heights, respectively (Palmer 1980)
Individuals of Nucella congeners used for the thermotolerance studies were transported to the laboratory in open bags, containing seawater-soaked paper towels, and were kept cold on blue ice packs. In the laboratory, dogwhelks were kept in either running seawater tables or chilled aquaria. Dogwhelks were fed ad libitum with the mussels Mytilus trossulus or M. californianus. In order to distinguish between the effects of thermotolerance acquired in the field and species-specific genetic differences,
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thermotolerance was determined after 5-day, 3-week, and 7-week laboratory acclimations at a common temperature (10C). The three mid-latitude species, N. ostrina, N. canaliculata, and N. lamellosa were collected at two Oregon sites, Seal Rock (4430¢N, 12405¢W, for the 5-day acclimation group) and Cape Blanco (4250¢N, 12433¢W, for the 3- and 7-week acclimation groups), on 28 and 18 May 2002, respectively. Dogwhelks were laboratory acclimated in running seawater tables until their thermotolerances were assayed. Individuals of the low-latitude species, N. emarginata, were collected at Coal Oil Point (3424¢N, 11952¢W), near Santa Barbara, California on 5 October 2002. Individuals of N. lima, the high-latitude species, were collected at Sitka, Alaska (5703¢N, 13520¢W) on 27 October 2002 and were shipped overnight to Santa Barbara. N. emarginata and N. lima were maintained in aquaria until their thermotolerances were assayed. Determination of thermotolerance Using methods adapted from Bertness and Schneider (1976), the thermotolerance assay consisted of a 2-h thermal exposure and a 90-min recovery period. For each trial, dogwhelks were randomly assigned to 1 of 10 plastic chambers. The assay was performed on emersed dogwhelks at 100% humidity, achieved by adding 1 ml seawater to each chamber. The chamber tops were open, and the chambers were half-immersed in a large beaker of water. The water temperature in the beaker was raised from 12 to 32C, at a rate of 1C every 5 min, by an aquarium heater suspended in the beaker. Uniform heating was accomplished by setting the beaker on a magnetic stir-plate and was routinely checked by measuring water and chamber temperatures with a handheld digital thermometer (Omega HH21). After the 2-h thermal exposure, dogwhelks were transferred to new individual chambers of cold (10C), aerated seawater for a 90-min recovery period. At the end of the recovery period, the aeration (which created turbulence) was stopped to allow dogwhelks, if able, to right themselves and attach to the chambers. Recovery was determined by probing the dogwhelks’ feet with dissecting forceps, and a ‘score’ was assigned based on their responses as follows: 0 = dead (did not respond to probe), 1 = moribund (responded only after repeated probing), 2 = alive (responded immediately by withdrawing from probe), and 3 = recovered (reattached to chamber). Immediately upon scoring, the dogwhelks from the 3week acclimation group were flash-frozen on dry ice and stored at 70C for use in the following biochemical assays. Immunodetection of heat-shock proteins Samples of Nucella foot tissue were subsequently dissected and homogenized. Foot tissue was used because it
is metabolically active and sufficient quantities are readily isolated from surrounding tissues and food items. Foot tissue samples were homogenized in approximately five volumes of a buffer containing 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 1 mM PMSF, and 4% SDS. The homogenates were boiled at 100C for 5 min and then centrifuged at 14,000·g for 15 min. The supernatant was removed and stored at 70C, and its protein concentration was determined by the Bradford method using Coomassie plus protein assay reagent (Pierce). Gel electrophoresis and electrophoretic transfer were performed as described by Hofmann and Somero (1995) and Buckley et al. (2001), except that 5 lg of total protein were loaded on all gels, and protein transfer was run for either 15 h at 30 V or 1 h at 100 V. For immunodetection of total Hsp/Hsc70 (including both inducible and constitutive isoforms), we used an anti-Hsp70 rat monoclonal antibody (Affinity Bioreagents; MA3–001). Western blotting was performed as described by Halpin et al. (2002; after Hofmann and Somero 1995), except that 0.1% Tween-20 in PBS was used for all washes, and 0.1 lg of purified Hsc70 (70kDa heat-shock cognate protein) was used as a standard and positive control (bovine Hsc70; StressGen). All total Hsp/Hsc70 levels are reported relative to the purified bovine Hsc70 standard. The inducible form of Hsp70 was detected by an antiHsp70 rabbit polyclonal antibody (StressGen; SPA-812) using the same protocol as for total Hsp/Hsc70, with the following exceptions. The secondary antibody used was a goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (Bio-Rad; 170–6515), and the blot was washed 3·15 min with 0.3% Tween-20 in PBS. A supernatant was prepared from heat-shocked mussel (Mytilus californianus) gill tissue, and 5 ll were loaded on each gel as a standard and positive control. All inducible Hsp70 levels are reported relative to the Hsp70 present in this heat-shocked mussel standard. SuperSignal chemiluminescent substrate (Pierce), a Versadoc imaging system (Bio-Rad), and Quantity One software (Bio-Rad) were used for detection and densitometric analysis of the Hsp bands.
Comparison of Nucella canaliculata and N. ostrina Determination of heat-coma temperature In addition to the thermotolerance assay described above, heat-coma temperatures were determined for the two Nucella species that regularly co-occur on the Oregon coast N. canaliculata and N. ostrina. Dogwhelks were collected from Fogarty Creek (4450¢N, 12403¢W), a site on the central Oregon coast, on 29 January 2002. Dogwhelks were collected along a ‘wave-exposed’ and a ‘wave-protected’ vertical transect at ‘low’ and ‘high’ tide height locations. On the wave-exposed transect, low and high corresponded to actual tide heights of 1.19 and 2.94 m above mean lower low water (MLLW), respec-
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tively (tide heights were not surveyed for the wave-protected transect). Dogwhelks were maintained in aquaria at 13C until the assays were performed on 6 and 7 February 2002. Each dogwhelk was placed in a separate plastic container (n=20). As with the Nucella congener comparison, dogwhelks were emersed in 100% humidity conditions. The containers were open via holes in the lid and immersed at the bottom in an aquarium filled with water. The aquarium water temperature was raised, at a rate of 1C every 5 min, by an aquarium heater, and the aquarium water was stirred manually. Uniform heating was monitored by measuring water and container temperatures with a hand-held digital thermometer (Omega HH82). Every 10 min, the containers were tipped on their side to identify which dogwhelks were still attached to the container and which had undergone heat coma. Some dogwhelks reattached to the container following initial signs of heat coma, therefore the times at final detachment were used to determine the heat-coma temperature. Data analysis Analyses were performed using general linear models (analyses of variance; ANOVA) in the SAS System for Windows v. 8 (SAS Institute). ANOVA assumptions of normality and homogeneity of variance were evaluated by visually examining residual plots. Multiple comparisons were made of least-squares means after Bonferroni adjustment. Least-squares means were appropriate due to unequal sample sizes. Data are expressed as mean±standard error, and statistical significance was accepted at P
