A comparison of Hsp70 expression and thermotolerance in adults and larvae of three Drosophila species

Heat shock proteins (Hsps) and other molecular chaperones perform diverse physiological roles. One is to facilitate, in part, organismal thermotolerance, of which the functional consequences depend on Hsp70 concentration and developmental stage in Drosophila melanogaster. To test whether an Hsp70-thermotolerance relationship is a general phenomenon within Drosophila, I assayed Hsp70 concentration at a range of temperatures in intact larvae and adults of three species, D. melanogaster, D. simulans, and D. mojavensis, and compared those results to the increase in survival to heat shock that occurs after an Hsp70 inducing pretreatment. Larvae of D. melanogaster and D. simulans responded similarly to heat; they expressed Hsp70 maximally at 36-37 degrees C, and their tolerance of 1 h heat shocks increased by 1.5-2 degrees C. By contrast, D. mojavensis, which tolerates higher temperatures than do D. melanogaster and D. simulans, expressed Hsp70 only at higher temperatures, although the 36 degrees C pretreatment still increased thermotolerance. Critically, the temperature that maximally induced Hsp70 was a poor inducer of thermotolerance in D. mojavensis and may have harmed larvae. Results for Drosophila adults, which tolerated heat poorly compared to larvae, likewise suggest that a close link between peak Hsp70 expression and maximal induction of thermotolerance is a feature of D. melanogaster, and not of the other species. Neither D. simulans nor D. mojavensis adults increased tolerance after exposure to the temperatures that maximally induced Hsp70.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: Effect of temperature variability

It is considered that extreme environmental temperature, rather than mean temperatures exert a selective pressure in ectotherms. Consequently, it is important to understand how the predicted increase in temperature variance with a higher frequency of extreme events in climate change is likely to impact on organisms. Thermal tolerance traits (i.e. chill-coma, recovery time, Hsp70 expression) are directly linked with performance in ectotherms and have consequences in life-history traits. We examined the effects of temperature variability on thermal tolerance and life-history traits through ontogeny of an insect with a complex life-cycle: the yellow mealworm beetle Tenebrio molitor. We established two common gardens with 100 recently ovoposited eggs each. Larvae were reared from hatching to adult on either a variable (mean=18 °C and a variance of 6.8 °C) or constant (18±1 °C) thermal environment. Development rate and growth rate were similar between thermal environments. Results indicate that larvae reared in a variable environment are more cold-tolerant than larvae of a constant environment. Interestingly, these results are reversed in the adult stage, outlining an inter-stage physiological cost. Gene expression pattern of an Hsp70 gene was well correlated with larval thermotolerance to cold in the variable environment but higher gene expression in adults is not correlated with individual's thermotolerance. We conclude that chill-coma, recovery time and Hsp70 gene expression are plastic in response to a thermal environment but also change significantly their responses depending on the ontogenetic stage, implying that the response of adult individuals is linked to early stages of the life-cycle.     

Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis

The 70-kilodalton heat shock protein family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. While the role of the constitutively expressed stress proteins in thermotolerance is largely unknown, de novo expression stress proteins in response to elevated temperatures has been associated with increased thermotolerance in many cell lines, developing embryos and adult organisms. Distinct, hemiclonal hybrids between the livebearing fish species Poeciliopsis monacha and P. lucida varied in their abilities to survive temperature stress, with survival being greatest when rates of temperature increase to 40°C were slowest and when P. monacha genomes were combined with a sympatric P. lucida genome. Quantification of Hsp70 under heat shock conditions and Hsc70 under normal physiological conditions indicated that variation in survival among hemiclones was best explained by the combined effects of these two proteins. Similar complex interactions between maternal and paternal genomes and rate of temperature increase were found to underline patterns of survival, Hsp70 accumulation and Hsc70 abundance. These data suggest that the relationship between Hsps and thermotolerance is more intricate than previously thought and that Hsps contribute to thermal adaptation in these fishes through genetic interactions specific to particular environments.     

The role of the heat shock proteins (HSP70 and sHSP) in the thermotolerance of freshwater amphipods from contrasting habitats

The aim of this study was to clarify the significance of HSP70 and sHSP for thermotolerance in freshwater amphipods. We compared four amphipod species from different freshwater habitats and biogeographical regions (Central Europe vs. Lake Baikal). Test individuals were exposed to thermal stress generated by a water temperature of 25 °C. The thermotolerance of the species, determined by median lethal time (LT50), followed in decreasing order by Gmelinoides fasciatus, Echinogammarus berilloni, Gammarus pulex, Eulimnogammarus verrucosus. HSP70 and sHSP base level concentrations for the species were determined at control (i.e. non-stress) conditions. For HSP70, the base levels were positively correlated to the species' thermotolerances. For sHSP, however, only thermotolerant G. fasciatus showed a high level. Thermal stress at 25 °C water temperature caused a deferred onset of HSP70 and sHSP expression followed by a subsequent offset, delineating a unimodal response curve. The time lag to the expression onset of HSP70 was shorter in the thermosensitive species, compared to thermotolerant ones. Conversely, the time span until the maximum level of HSP70 was variable, not showing a dependence on the thermotolerance properties of the species. The peak concentration in G. pulex was distinctly higher than in the other species, whereas E. verrucosus did not develop a well-defined response maximum at all. In sHSP, the temporal pattern of expression was even more variable than in HSP70. However, the thermosensitive species E. verrucosus showed a time lag of expression onset significantly shorter than the other species and thermotolerant G. fasciatus developed the most pronounced response maximum. Basing on these results, the cellular response to thermal stress in amphipods is more consistently reflected by HSP70, compared to sHSP.     

Evolvability of Hsp70 expression under artificial election for inducible thermotolerance in independent populations of Drosophila melanogaster

To test whether expression of the inducible heat-shock protein Hsp70 increases under selection for inducible thermotolerance in Drosophila melanogaster, we performed artificial selection on replicate sets of Drosophila lines founded from two independent populations. Selection entailed pretreatment at 36 degrees C to induce thermotolerance and Hsp70 expression, followed by a more severe heat shock, whose temperature varied between sexes and among generations to achieve 50% mortality. Inducible thermotolerance increased slowly and continuously in selected lines and was 37%-50% greater than in controls after 10-11 generations. Lines founded from the two populations differed in their coevolution of Hsp70 expression. In lines founded from Evolution Canyon, Israel, Hsp70 level initially increased and thereafter was unchanged; replicate lines exhibited two temporal patterns of response to selection. In lines founded from Australia, Hsp70 levels increased throughout selection. In both cases, however, the increase in Hsp70 level averaged only 15%, suggesting that pleiotropy in Hsp70 function constrains evolutionary increase in its expression.     

Mild thermotolerance induced at 40 °C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 °C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 °C, 3 h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H₂O₂. Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and γ-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H₂O₂. This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H₂O₂-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.     

NATURAL VARIATION IN THE EXPRESSION OF THE HEAT-SHOCK PROTEIN HSP70 IN A POPULATION OF DROSOPHILA MELANOGASTER AND ITS CORRELATION WITH TOLERANCE OF ECOLOGICALLY RELEVANT THERMAL STRESS

Although Hsp70, the principal inducible heat-shock protein of Drosophila melanogaster, has received intense scrutiny in laboratory strains, its variation within natural populations and the consequences of such variation for thermotolerance are unknown. We have characterized variation in first-instar larvae of 20 isofemale lines isolated from a single natural population of D. melanogaster, in which larvae are prone to thermal stress in nature. Hsp70 expression varied more than twofold among lines after induction by exposure to 36°C for one hour, with an estimated proportion of the variation due to genetic differences of 0.24 ± 0.08. Thermotolerance with and without a Hsp70-inducing pretreatment, survival at 25°C, and developmental time also varied significantly. As expected, expression of Hsp70 correlated positively with larval thermotolerance. By contrast, lines in which larval survival was high in the absence of heat stress showed lower than average Hsp70 expression and lower than average inducible thermotolerance. This conditional performance suggests an evolutionary trade-off between thermotolerance and the ability to produce higher concentrations of Hsp70, and survival in a benign environment.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris

The mannan endo-1,4-β-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPzαA. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60 °C. The optimal pH value and temperature for activity were 5.0 and 45 °C, respectively. It was highly thermotolerant with a half-life time of 15 min at 90 °C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069 U/mL after cultivation for 56 h in 50 L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels.     

Thermotolerance is developmentally dependent in germinating wheat seed

During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38-42°C) prior to imposition of a 2 hour heat shock (51-53°C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a `thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5-triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition.     

Non-Lethal Heat Shock of the Asian Green Mussel,Perna viridis,Promotes Hsp70 Synthesis,Induces Thermotolerance and Protects Against Vibrio Infection

Mild heat stress promotes thermotolerance and protection against several different stresses in aquatic animals, consequences correlated with the accumulation of heat shock protein 70 (Hsp70). The purpose of this study was to determine if non-lethal heat shock (NLHS) of the Asian green mussel, Perna viridis, an aquatic species of commercial value, promoted the production of Hsp70 and enhanced its resistance to stresses. Initially, the LT₅₀ and LHT for P. viridis were determined to be 42°C and 44°C, respectively, with no heat shock induced death of mussels at 40°C or less. Immunoprobing of western blots revealed augmentation of constitutive (PvHsp70-1) and inducible (PvHsp70-2) Hsp70 in tissue from adductor muscle, foot, gill and mantel of P. viridis exposed to 38°C for 30 min followed by 6 h recovery, NLHS conditions for this organism. Characterization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that PvHsp70-1 and PvHsp70-2 respectively corresponded most closely to Hsp70 from P. viridis and Mytilus galloprovincialis. Priming of adult mussels with NLHS promoted thermotolerance and increased resistance to V. alginolyticus. The induction of Hsp70 in parallel with enhanced thermotolerance and improved protection against V. alginolyticus, suggests Hsp70 functions in P. viridis as a molecular chaperone and as a stimulator of the immune system.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.     

Distinct chemical contaminants induce the synthesis of Hsp70 proteins in green microalgae Desmodesmus subspicatus: Heat pretreatment increases cadmium resistance

Using western-blotting techniques, we examined the effect of differently acting contaminants, such as anthracene (PAH), cadmium (heavy metal) and chloridazone (herbicide), as well as heat shock on the production of two Hsp70 proteins (cytoplasmic and stromal) in planktonic algae Desmodesmus subspicatus. All contaminants applied stimulated production of both Hsp70s in a concentration-dependent manner, but heat shock treatment turned out to be the most effective. Heat shock pretreatment (for 1 h at 40 °C) induced tolerance to cadmium in algal cells (measured by changes in growth rate), but not to anthracene or chloridazone. Two Hsp70s from D. subspicatus cells representing cytoplasmic and stromal proteins were purified by ATP-affinity chromatography.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

The migratory locust Locusta migratoria L., which is widely distributed throughout the world, exhibits within- and between-population variation in cold tolerance. To understand physiological adaptation in populations, we studied the genetic basis of thermotolerance in Hainan (tropical) and Liaoning (temperate) populations and measured expression of Hsp70 and Hsp90 mRNA in both populations at low (0 degrees C) and high temperatures (40 degrees C). Phenotypic variation of thermotolerance is heritable. Heritable characteristics differed among different stages of locust egg development, as well as among different measures of thermotolerance. Nuclear genetic factors, rather than cytoplasmic factors, contribute to differences in cold tolerance between the tropical and temperate populations of the migratory locust; for heat tolerance, maternal effects were involved in three stages of egg development. Expression of Hsp90 mRNA was induced in temperate population after heat shock (40 degrees C x 12h), whereas expression of Hsp70 and 90 was induced in tropical population after cold shock (0 degrees C x 12h). We suggest that thermotolerance of locust eggs has a complex genetic basis and heat shock proteins may be involved in differences of thermotolerance between locust populations.     

HERITABILITY OF EXPRESSION OF THE 70KD HEAT-SHOCK PROTEIN IN DROSOPHILA MELANOGASTER AND ITS RELEVANCE TO THE EVOLUTION OF THERMOTOLERANCE

The principle inducible heat-shock protein of Drosophila melanogaster, Hsp70, contributes to thermotolerance throughout the entire life cycle of the species but may also reduce fitness in some life stages. In principle, selection might maximize the benefits of Hsp70 expression relative to its costs by adjusting the magnitude of Hsp70 expression for each life-cycle stage independently. Therefore we examined whether the magnitude of Hsp70 expression varied during the life cycle and the relationship of this variation to several life-history traits. For 28 isofemale lines derived from a single natural population, estimates of heritable variation in Hsp70 expression ranged between 0.25 and 0.49, and the association among variation in first- and third-instar larvae and in adults correlated highly. Thus, Hsp70 expression is genetically coupled at these developmental stages. A line engineered with extra copies of the hsp70 gene produced more Hsp70 and survived heat shock much better than did a control strain. Among natural lines, Hsp70 expression was only weakly related to tolerance of heat shock and to larva-to-adult survival and developmental time at permissive temperatures. Additionally, lines with high adult survival developed slowly as larvae, which is a possible trade-off. These and other findings suggest that trade-offs may maintain quantitative variation both in heat-shock protein expression and in life-history traits that associate with thermotolerance.