The 70-kDa heat shock protein response in two intertidal sculpins,Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance

The role that hsp70 plays in influencing thermal tolerance of a whole animal is not clearly understood. We explored this question by examining liver hsp70 response in the tidepool sculpin (Oligocottus maculosus) and fluffy sculpin (O. snyderi), which have distinct distribution patterns in the intertidal zone. The tidepool sculpin is in upper and lower tidepools, while the fluffy sculpin is exclusively in lower tidepools during a low tide. We conducted experiments in order to investigate: (1) habitat water temperatures; (2) upper thermal tolerance limits; (3) the cellular hsp70 response to changes in water temperature in nature; (4) induction temperatures for hepatic hsp70 and hsp70 mRNA; and (5) effects of long-term heat stress on liver hsp70 levels, in these sculpins. Accordingly, we found: (1) the tidepool sculpin was exposed to a wider temperature range in nature; (2) the tidepool sculpin had higher lethal and induction temperatures for hsp70; (3) the liver hsp70 level of the tidepool sculpin was less sensitive to changes in water temperatures; and (4) the tidepool sculpin had higher constitutive hsp70 levels in nature, compared with the fluffy sculpin. From these results, we proposed that the less thermally sensitive tidepool sculpin may enhance its thermal tolerance by having a large pool of cellular hsp70, thus allowing it to inhabit the upper intertidal zone with relatively large and unpredictable fluctuations in environmental variables.     

The onset temperature of the heat-shock response and whole-organism thermal tolerance are tightly correlated in both laboratory-acclimated and field-acclimatized tidepool sculpins (Oligocottus maculosus)

We examined the relationship between thermal tolerance, measured as critical thermal maximum (CT(max)), and aspects of the heat-shock response in tidepool sculpins (Oligocottus maculosus) acclimated to constant laboratory temperatures or acclimatized to field conditions. The CT(max) of fish laboratory acclimated to 6°, 13°, and 20°C were 27.6° ± 0.1°C, 29.5° ± 0.1°C, and 30.8° ± 0.1°C, respectively, increasing linearly by 0.2°C for each 1°C increase in acclimation temperature. The CT(max) of field-acclimatized fish from the low intertidal (29.9° ± 0.1°C) was significantly lower than that of fish from the mid- (30.5° ± 0.1°C) and high (30.4° ± 0.1°C) intertidal. CT(max) and the onset temperature of hsp70 induction in gill (T(on)) were highly correlated in both laboratory-acclimated and field-acclimatized sculpins, with T(on) occurring at 2°C below CT(max) in all cases. However, there was no consistent relationship between CT(max) and the maximum levels of gill hsp70 mRNA. Predicted "acclimation" temperature (15.9° ± 0.3°C) and mean habitat temperature (15.9° ± 1.6°C) were similar for sculpins from low intertidal pools, but this relationship was not apparent in mid- and high intertidal fish. Mark-recapture experiments indicated that approximately 80% of fish from low intertidal pools were residents of that pool, but residency rates were less than 50% in mid- and high intertidal pools, which may explain the lack of correlation between CT(max) and habitat variables in these groups. These data indicate that gill hsp70 T(on) and CT(max) are highly correlated indicators of the thermal performance of tidepool sculpins in both laboratory and field settings.     

Cross-tolerance in the tidepool sculpin: the role of heat shock proteins

Cross-tolerance, or the ability of one stressor to transiently increase tolerance to a second heterologous stressor, is thought to involve the induction of heat shock proteins (Hsp). We thus investigated the boundaries of cross-tolerance in tidepool sculpins (Oligocottus maculosus) and their relationship to Hsp70 levels. Survival of sculpins exposed to severe osmotic (90 ppt, 2 h) and hypoxic (0.33 mg O(2)/L, 2 h) stressors increased from 68% to 96%, and from 47% to 76%, respectively, following a +12 degrees C heat shock. The magnitude of this heat shock was critical for protection. A +10 degrees C heat shock did not confer cross-tolerance, while a +15 degrees C heat shock was deleterious. Sculpins required between 8 and 48 h of recovery following the +12 degrees C heat shock to develop cross-tolerance. There was no association between Hsp70 levels before the onset of the secondary stressor and cross-tolerance. However, branchial Hsp70 levels following osmotic shock were highly correlated with the time frame of cross-tolerance. Thus, Hsp70 induction by the priming stressor may be less important than the ability of the cell to mount an Hsp response to subsequent stressors. The time frame of cross-tolerance is similar to the interval between low tides, suggesting the possible relevance of this response in nature.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Expression of 70 kDa heat shock proteins in antarctic and New Zealand notothenioid fish

The cold and constant water temperature of the Southern Ocean surrounding Antarctica provides a natural laboratory to address questions of temperature adaptation in marine organisms. In this study, endogenous levels and the number of isoforms of the 70 kDa heat shock protein multigene family (hsp70) of Antarctic and cold temperate notothenioid fishes were determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Tissues from three Antarctic Trematomus congeners had significantly lower levels of 70 kDa Hsp isoforms than their temperate confamilial from New Zealand waters. However, these two thermally disparate sets of fish did not differ in number or pattern of 70 kDa Hsp isoforms expressed under normal physiological conditions. Additionally, levels of 70 kDa Hsp isoforms in specimens of one Antarctic species, Trematomus bernacchii, acclimated to 4 degrees C were significantly higher than non-acclimated conspecifics, indicating a direct effect of temperature on Hsp expression in this species. This study shows that constitutive expression of some members of the 70 kDa Hsp multigene family have been maintained, despite the absence of environmental heat stress for at least 2.5 million years.     

Daily stress protein (hsp70) cycle in chitons (Acanthopleura granulata Gmelin, 1791) which inhabit the rocky intertidal shoreline in a tropical ecosystem

Stress protein (heat shock protein, hsp70) response is involved in protecting organisms from the detrimental effects of environmental stressors, such as radiation and high temperatures. Tropical chitons can briefly tolerate high temperatures. However, they minimize the effects of elevated temperature during daylight hours and periods of tidal air exposure by remaining in rocky intertidal microhabitats along the shoreline of tropical waters. To study the natural variability of the hsp70 level, individuals of the polyplacophoran species Acanthopleura granulata Gmelin, 1791 were sampled every 4 h on two days in spring of 1999. Hsp70 levels were separately measured in the supernatant of the intestinal tract and foot muscle homogenates with a standardized immunoassay. The hsp70 level in the intestinal tract was highest in the early morning, decreased during the mid-morning hours and dropped to a comparatively low level in the afternoon, before increasing again during the night. The stress protein level in the foot muscle followed the daily air temperature curve with a time delay of a few hours, reaching the highest level in the afternoon and the lowest level in the early morning. The stress protein response can be interpreted as a sign of heat tolerance development and may play a role in allowing A. granulata to tolerate the temperature variability typical of its intertidal habitat.     

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus)

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.     

Diurnal regulation of Hsp70s in leaf tissue

Steady-state mRNA levels for three Hsp70s were found to be regulated by a distinctive light/dark mechanism in spinach leaves. Messenger RNAs for the chloroplast stromal and two cytosolic forms displayed a diurnal expression pattern under isothermal conditions that appeared to be independent of circadian control. While protein blot data showed relatively constant Hsp70 protein levels, the higher Hsp70 mRNA levels in the light paralleled the diurnal cycle of total cell protein synthesis. Fractionation studies showed that the major cytosolic Hsp70 cognate group was associated with polysomes. Therefore, the variation of Hsp70 mRNAs is consistent with the diurnal metabolic activity of plant photosynthetic cells in which the demand of protein biogenesis for chaperone function and tissue temperature are highest during the day.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.