Field and laboratory investigations of the thermal influence on tissue-specific Hsp70 levels in common carp (Cyprinus carpio).

Thermal discharge from power stations can affect normal environmental conditions and change in heat shock proteins expression of native fish with increasing temperature. In this study, we investigated levels of Hsp70 in the heart, kidney, brain and gill of the common carp Cyprinus carpio both in long-term heat discharge environment and after 24 h acute heat shock exposure. In laboratory exposure experiments, fish acclimated at 10 degrees C were exposed to various elevated temperatures (20, 24 and 28 degrees C). Hsp70 concentrations were determined in tissues by Western blotting analysis after one dimensional SDS-PAGE separation. In the field study, the level of Hsp70 in the gill of the carp remained at control values, and Hsp70 expression in the heart, kidney and brain underwent a 2.8 to 3.7-fold increase. A lower thermal sensitivity of the Hsp70 response of the brain, compared with the heart, kidney and gill, was observed in the laboratory experiments. Our data show that these tissues had different levels of Hsp70 responses to thermal influence both in acute exposure and long-term acclimation. The pattern of tissue Hsp70 expression may have a close relationship with the thermal tolerance of the carp and allows the fish to survive long-term thermal pollution.     

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.     

Core temperature is regulated, although at a lower temperature, in rats exposed to hypergravic fields

1. In rats acclimated to 23 degrees C (RT rats) or 5 degrees C (CA rats), core temperature (Tc), tail temperature (Tt) and oxygen consumption (VO2) were measured during exposure to a hypergravic field. 2. Rats were exposed for 5.5 h to a 3 g field while ambient temperature (Ta) was varied. For the first 2 h, Ta was 25 degrees C; then Ta was raised to 34 degrees C for 1.5 h. During this period of warm exposure, Tc increased 4 degrees C in both RT and CA rats. Finally, Ta was returned to 25 degrees C for 2 h, and Tc decreased toward the levels measured prior to warm exposure. 3. In a second experiment at 3 g, RT and CA rats were exposed to cold (12 degrees C) after two hours at 25 degrees C. During the one hour cold exposure, Tc fell 1.5 degrees C in RT and 0.5 degree C in CA rats. After cold exposure, when ambient temperature was again 25 degrees C, Tc of RT and CA rats returned toward the levels measured prior to the thermal disturbance. 4. Rats appear to regulate their temperature, albeit at a lower level, in a 3 g field.     

Beta-adrenergic stimulation enhances the heat-shock response in fish

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that beta-adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat-induced induction of heat-shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this beta-adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100-fold and noradrenaline increased 50-fold in trout after a 1-h heat shock at 25 degrees C, approximately 12 degrees C above acclimation temperature. This is the first time the adrenergic heat-shock response has been described for a temperate fish species. We conclude that beta-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.     

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.     

Field and laboratory investigations of the thermal influence on tissue-specific Hsp70 levels in common carp (Cyprinus carpio)

Thermal discharge from power stations can affect normal environmental conditions and change in heat shock proteins expression of native fish with increasing temperature. In this study, we investigated levels of Hsp70 in the heart, kidney, brain and gill of the common carp Cyprinus carpio both in long-term heat discharge environment and after 24 h acute heat shock exposure. In laboratory exposure experiments, fish acclimated at 10 °C were exposed to various elevated temperatures (20, 24 and 28 °C). Hsp70 concentrations were determined in tissues by Western blotting analysis after one dimensional SDS-PAGE separation. In the field study, the level of Hsp70 in the gill of the carp remained at control values, and Hsp70 expression in the heart, kidney and brain underwent a 2.8 to 3.7-fold increase. A lower thermal sensitivity of the Hsp70 response of the brain, compared with the heart, kidney and gill, was observed in the laboratory experiments. Our data show that these tissues had different levels of Hsp70 responses to thermal influence both in acute exposure and long-term acclimation. The pattern of tissue Hsp70 expression may have a close relationship with the thermal tolerance of the carp and allows the fish to survive long-term thermal pollution.     

Relationship between rapid cold-hardening and cold acclimation in the eggs of the yellow-spotted longicorn beetle, Psacothea hilaris

Rapid cold-hardening (RCH) and cold acclimation (ACC) were examined in eggs of the yellow-spotted longicorn beetle, Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae). When eggs incubated at 25 degrees C were transferred directly to conditions of -22 degrees C for 2h, less than 30% survived, whereas exposure to 0 degrees C for 4h prior to transfer to -22 degrees C increased survival to nearly 60%. The rapidly enhanced cold tolerance (RCH) was transient and lost rapidly after 1h at 25 degrees C. Incubation at 15.5 degrees C for 9 days (ACC) also enhanced cold tolerance. Comparison of the cold tolerance of non-treated eggs and eggs pre-treated to give RCH, ACC, or ACC+RCH allowed the relationship between the two hardening processes to be determined. At a mild subzero temperature (-10 degrees C) an RCH effect was not detected, whereas only RCH is effective at the severest subzero temperature just above the SCP (-26 degrees C). At intermediate temperatures (-16, -22 and -25 degrees C), ACC and RCH enhanced survival in combination. Therefore, the two hardening processes have different physiological bases but operate concomitantly over a wide temperature range.     

Cold acclimation of Arabidopsis thaliana results in incomplete recovery of photosynthetic capacity, associated with an increased reduction of the chloroplast stroma

The effects of short-term cold stress and long-term cold acclimation on the light reactions of photosynthesis were examined in vivo to assess their contributions to photosynthetic acclimation to low temperature in Arabidopsis thaliana (L.) Heynh.. All photosynthetic measurements were made at the temperature of exposure: 23 degrees C for non-acclimated plants and 5 degrees C for cold-stressed and cold-acclimated plants. Three-day cold-stress treatments at 5 degrees C inhibited light-saturated rates of CO2 assimilation and O2 evolution by approximately 75%. The 3-day exposure to 5 degrees C also increased the proportion of reduced QA by 50%, decreased the yield of PSII electron transport by 65% and decreased PSI activity by 31%. In contrast, long-term cold acclimation resulted in a strong but incomplete recovery of light-saturated photosynthesis at 5 degrees C. The rates of light-saturated CO2 and O2 gas exchange and the in vivo yield of PSII activity under light-saturating conditions were only 35-40% lower, and the relative redox state of QA only 20% lower, at 5 degrees C after cold acclimation than in controls at 23 degrees C. PSI activity showed full recovery during long-term cold acclimation. Neither short-term cold stress nor long-term cold acclimation of Arabidopsis was associated with a limitation in ATP, and both treatments resulted in an increase in the ATP/NADPH ratio. This increase in ATP/NADPH was associated with an inhibition of PSI cyclic electron transport but there was no apparent change in the Mehler reaction activity in either cold-stressed or cold-acclimated leaves. Cold acclimation also resulted in an increase in the reduction state of the stroma, as indicated by an increased total activity and activation state of NADP-dependent malate dehydrogenase, and increased light-dependent activities of the major regulatory enzymes of the oxidative pentose-phosphate pathway. We suggest that the photosynthetic capacity during cold stress as well as cold acclimation is altered by limitations at the level of consumption of reducing power in carbon metabolism.     

Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55.

The psychrotrophic bacterium Arthrobacter globiformis SI55 was grown at 4 and 25 degrees C, and the cell protein contents were analyzed by two-dimensional electrophoresis. Cells subjected to cold shocks of increasing magnitude were also analyzed. Correspondence analysis of protein appearance distinguished four groups of physiological significance. Group I contained cold shock proteins (Csps) overexpressed only after a large temperature downshift. Group II contained Csps with optimal expression after mild shocks. Group III contained proteins overexpressed after all cold shocks. These last proteins were also overexpressed in cells growing at 4 degrees C and were considered to be early cold acclimation proteins (Caps). Group IV contained proteins which were present at high concentrations only in 4 degrees C steady-state cells and appeared to be late Caps. A portion of a gene very similar to the Escherichia coli cspA gene (encoding protein CS7.4) was identified. A synthetic peptide was used to produce an antibody which detected a CS7.4-like protein (A9) by immunoblotting two-dimensional electrophoresis gels of A. globiformis SI55 total proteins. Unlike mesophilic microorganisms, this CS7.4-like protein was still produced during prolonged growth at low temperature, and it might have a particular adaptive function needed for balanced growth under harsh conditions. However, A9 was induced at high temperature by chloramphenicol, suggesting that CS7.4-like proteins have a more general role than their sole implication in cold acclimation processes.     

Expression of mt2 and smt-B upon cadmium exposure and cold shock in zebrafish (Danio rerio)

Metallothionein-2 (mt2) and similar to metallothionein-B (smt-B) are included in the MT gene family. The objective of this study was to compare mt2 and smt-B messenger (m)RNA expressions after cadmium exposure and cold shock with whole-mount in situ hybridization in immature zebrafish (Danio rerio) and with a semi-quantitative RT-PCR in mature zebrafish. Three-day post-fertilization (dpf) larvae were treated with 0, 0.08, 0.26, and 0.89 microM cadmium for 24 and 48 h, and some larvae were challenged with a normal (28.5 degrees C) or low temperature (12 degrees C) for 12, 24, and 48 h. Results were obtained. (1) During embryonic and larval development, mt2 mRNA existed at 6 h post-fertilization (hpf), and the level rapidly increased to 24 hpf, then it gradually increased with further larval growth. smt-B was found at 12 hpf, and it also rapidly increased to 24 hpf, but remained constant during further larval development. (2) The mt2 mRNA signals and whole-body Cd contents displayed dose- and time-dependent responses after Cd exposure. After cold shock, mt2 mRNA signals also showed time-dependent expression. But smt-B mRNA signals were not appeared by either challenge. Besides, mature zebrafish were treated with 1.78 microM Cd and found that the highest levels of smt-B mRNA (smt-B/beta-actin) appeared in brain, and seems a reverse expression between smt-B mRNA and mt2 in brain after Cd exposure. Apparently, mt2 is possibly more relevant to Cd detoxification and cold shock adaptation in zebrafish larvae compared to smt-B, but smt-B might be related to certain physiological functions in neural (or brain) of mature zebrafish.     

Rapid metabolic adaptation in European sea bass (Dicentrarchus labrax) juveniles fed different carbohydrate sources after heat shock stress

A study was conducted to evaluate the effect of two dietary carbohydrate sources (waxy maize starch and glucose) on the metabolic adaptation of sea bass juveniles (initial weight: 24 g) to a heat shock treatment (temperature rise from 18 degrees C to 25 degrees C within 24 h). Two isonitrogenous and isolipidic diets were formulated to contain 20% waxy maize starch (WS diet) or 20% glucose (GLU diet). Triplicate groups of fish were fed to near satiation for 4 weeks at both temperatures (18 degrees C and 25 degrees C). Then, fish previously maintained at 18 degrees C were submitted to a heat shock (18 degrees C to 25 degrees C) and continued to be fed with the same diets during 1 more week. The higher water temperature significantly improved growth performance, feed efficiency, as well as protein efficiency ratio, independently of diet. At 25 degrees C, but not at 18 degrees C, growth of fish fed the WS diet was higher than that of fish fed the GLU diet. Plasma glucose levels were higher in sea bass fed the GLU diet and not influenced by water temperature. Fish fed a glucose diet or reared at high temperatures (25 degrees C) showed enhanced liver glycolytic, lipogenic and gluconeogenic capacities compared to fish fed a starch diet or reared at low temperatures (18 degrees C). For the majority of the enzymes studied, 1 week seemed to be enough time for metabolic adaptation in sea bass submitted to an acute heat shock. Irrespective of carbohydrate source, HSP70 gene expression was similar in both cold water (18 degrees C) and warm water (25 degrees C) acclimated sea bass. A weak down regulation was observed after heat shock only in fish fed the GLU diet. This suggests that HSP70 gene expression is not affected by the rearing temperature per se.     

Rapid cold hardening in the western flower thrips Frankliniella occidentalis

A rapid cold hardening process is reported in first instar larvae of Frankliniella occidentalis. When larvae are transferred directly from 20 degrees C to -11.5 degrees C for 2h there is 78% mortality, whereas exposure to 0 degrees C for 4h prior to transfer to -11.5 degrees C reduces mortality to 10%. The response can also be induced by exposure to 5 degrees C for 4h or by gradual cooling at rates between 0.1 and 0.5 degrees C min(-1.) The acquired cold tolerance is transient and is rapidly lost (after 1h at 20 degrees C). Rapid cold hardening extends survival times at -11.5 degrees C and depresses lethal temperatures in short (2h) exposures. Rearing at 15 degrees C (12L:12D), (a cold acclimation regime for F. occidentalis), does not protect against the cold shock induced by direct transfer to -11.5 degrees C (which rapid cold hardening does) but does extend survival time at -5 degrees C (i.e. increased chill tolerance) whilst rapid cold hardening does not. The rapid and longer term cold hardening responses in F. occidentalis therefore appear to have different underlying mechanisms.     

Cold Stress-Induced Acclimation in Rice is Mediated by Root-Specific Aquaporins

Cold acclimation process plays a vital role in the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. However, it remains elusive whether a cold acclimation process enhances root water uptake (a component of chilling tolerance) in chilling-sensitive crops such as rice. By analyzing the root hydraulic conductivity under cold stress for a prolonged time, we found that cold stress induced a gradual increase in root osmotic hydraulic conductivity [Lp(r(os))]. Compared with the control treatment (roots and shoots at 25°C), low root temperature (LRT) treatment (roots at 10°C; shoots at 25°C) dramatically reduced Lp(r(os)) within 1 h. However, Lp(r(os)) gradually increased during prolonged LRT treatment and it reached 10-fold higher values at day 5. Moreover, a coordinated up-regulation of root aquaporin gene expression, particularly OsPIP2;5, was observed during LRT treatment. Further, comparison of aquaporin gene expression under root-only chilling (LRT) and whole-plant chilling conditions, and in the roots of intact plants vs. shootless plants, suggests that a shoot to root signal is necessary for inducing the expression of aquaporin genes in the root. Collectively, these results demonstrate that a cold acclimation process for root water uptake functions in rice and is possibly regulated through aquaporins.     

Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance

The level of expression of the group 3 late embryogenesis abundant abscisic acid-regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2°C exposure (cold acclimation), followed by a return to 25°C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt).     

Effect of two distinct stressors on gene expression of the type 1 IP3 receptors

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers, which triggers calcium release from intracellular pools via IP3 receptors. Previously we have shown that single immobilization stress increased gene expression of both, the type 1 and type 2 IP3 receptors (IP3R1 and IP3R2, respectively). In this study we evaluated whether long-term exposure to softer stressor (cold exposure to 4 degrees C) can affect the response to single immobilization stress. We examined modulation of the type 1 IP3 receptor gene expression by each stressor separately, and then in their combination. Rats were immobilized for 30 min and 120 min and were decapitated immediately or 3 h after immobilization. Cold stress was performed by exposure of animals to 4 degrees C temperature for 1, 7 and 28 days. To determine the effect of both stressors in combination, animals exposed to cold for 28 days were afterwards exposed to immobilization for 120 min and decapitated 3 h after the end of stressful stimulus. Our results verify that single immobilization increases the IP3R1 gene expression in left atria of rat heart, while cold stress elevates the level of gene expression only after the exposure to cold for 7 days. The exposure to cold for 28 days did not increase the gene expression of the type 1 IP3 receptor compared to control. Application of both stressors (28 days of cold exposure followed by 120 min of immobilization with subsequent 3 h rest) showed the tendency of increased IP3R1 gene expression compared to absolute, nonstressed control, but level of the type 1 IP3 receptor mRNA was significantly lower compared to mRNA levels of solely immobilized animals. Thus, cold exposure affects the response of the gene expression of the type 1 IP3 receptor to immobilization stress.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.