Heat shock proteins and survival strategies in congeneric land snails (Sphincterochila) from different habitats

Polmunate land snails are subject to stress conditions in their terrestrial habitat, and depend on a range of behavioural, physiological and biochemical adaptations for coping with problems of maintaining water, ionic and thermal balance. The involvement of the heat shock protein (HSP) machinery in land snails was demonstrated following short-term experimental aestivation and heat stress, suggesting that land snails use HSPs as part of their survival strategy. As climatic variation was found to be associated with HSP expression, we tested whether adaptation of land snails to different habitats affects HSP expression in two closely related Sphincterochila snail species, a desert species Sphincterochila zonata and a Mediterranean-type species Sphincterochila cariosa. Our study suggests that Sphincterochila species use HSPs as part of their survival strategy following desiccation and heat stress, and as part of the natural annual cycle of activity and aestivation. Our studies also indicate that adaptation to different habitats results in the development of distinct strategies of HSP expression in response to stress, namely the reduced expression of HSPs in the desert-inhabiting species. We suggest that these different strategies reflect the difference in heat and aridity encountered in the natural habitats, and that the desert species S. zonata relies on mechanisms and adaptations other than HSP induction thus avoiding the fitness consequences of continuous HSP upregulation.     

Heat shock protein expression in relation to reproductive cycle in land snails: Implications for survival

Land snails are subject to daily and seasonal variations in temperature and in water availability and use heat shock proteins (HSPs) as part of their survival strategy. We tested whether the reproductive cycle of land snails affects the endogenous levels of HSPs, and their involvement in the reproductive process. We examined HSP levels in the foot tissue of two Sphincterochila species, S. cariosa and S. zonata, before and after laying eggs, and analyzed the albumen gland (reproductive organ) of both species and eggs of S. cariosa for the presence and quantity of various HSPs. Our study shows reduction in the expression level of Hsp70 isoforms and Hsp90 in S. zonata foot and of Hsp74 in S. cariosa foot during the period preceding egg laying compared to the post-reproductive stage. Hsp70 isoforms and Hsp25 were highly expressed in both large albumen glands and in freshly laid eggs of S. cariosa, whereas large albumen glands of S. zonata expressed mainly Hsp70 isoforms. We conclude that a trade-off between survival and fertility is responsible for the expression level of HSPs in the foot tissue of Sphincterochila snails. Our study shows that HSPs are involved in the reproductive process. We propose that parental provision of HSPs may be part of a "be prepared" strategy of Sphincterochila snails, and that HSPs may play important roles in the survival strategy of land snails during the early life stages. Our observations also highlight the importance of the reproductive status in study of whole organisms, especially when assessing the HSP response to stress.     

Heat shock proteins and resistance to desiccation in congeneric land snails

Land snails are subject to daily and seasonal variations in temperature and in water availability and depend on a range of behavioral and physiological adaptations for coping with problems of maintaining water, ionic, and thermal balance. Heat shock proteins (HSPs) are a multigene family of proteins whose expression is induced by a variety of stress agents. We used experimental desiccation to test whether adaptation to different habitats affects HSP expression in two closely related Sphincterochila snail species, a desiccation-resistant, desert species Sphincterochila zonata, and a Mediterranean-type, desiccation-sensitive species Sphincterochila cariosa. We examined the HSP response in the foot, hepatopancreas, and kidney tissues of snails exposed to normothermic desiccation. Our findings show variations in the HSP response in both timing and magnitude between the two species. The levels of endogenous Hsp72 in S. cariosa were higher in all the examined tissues, and the induction of Hsp72, Hsp74, and Hsp90 developed earlier than in S. zonata. In contrary, the induction of sHSPs (Hsp25 and Hsp30) was more pronounced in S. zonata compared to S. cariosa. Our results suggest that land snails use HSPs as part of their survival strategy during desiccation and as important components of the aestivation mechanism in the transition from activity to dormancy. Our study underscores the distinct strategy of HSP expression in response to desiccation, namely the delayed induction of Hsp70 and Hsp90 together with enhanced induction of sHSPs in the desert-dwelling species, and suggests that evolution in harsh environments will result in selection for reduced Hsp70 expression.     

Application of heat shock proteins as stress markers in aquatic organisms using endemic Baikal amphipods as an example

When heat shock proteins (HSPs) are used as biomarkers in monitoring studies of aquatic ecosystems, it is necessary to take into account the specificity of synthesis of these proteins in various organisms. This especially applies to endemic species and species with narrow ranges of adaptation for specific conditions in certain water bodies. In this study, we assessed the possibility to use HSPs as molecular stress markers in species with a narrow niche breadth using endemic Baikal amphipods (Crustacea, Amphipoda) as an example. The effect of stress induced by toxicants and temperature has been assessed. Proteins of families HSP70 and low-molecular-weight HSP related to alpha-crystallins were used as biomarkers. Temperature- and toxicant-induced stresses induced low-molecular-weight HSP synthesis in the endemic amphipod species studied. However, induction of HSP70 synthesis in the same species after temperature stress has not been detected. The specificity of synthesis of HSP70 is discussed. The results obtained in this study suggest that low-molecular-weight HSPs can be used as stress markers in Baikal species and species with a narrow niche breadth.     

Application of heat shock proteins as stress markers in aquatic organisms using endemic Baikal amphipods as an example

When heat shock proteins (HSPs) are used as biomarkers in monitoring studies of aquatic ecosystems, it is necessary to take into account the specificity of synthesis of these proteins in various organisms. This especially applies to endemic species and species with narrow ranges of adaptation for specific conditions in certain water bodies. In this study, we assessed the possibility to use HSPs as molecular stress markers in species with a narrow niche breadth using endemic Baikal amphipods (Crustacea, Amphipoda) as an example. The effect of stress induced by toxicants and temperature has been assessed. Proteins of families HSP70 and lowmolecular-weight HSP related to α-crystallins were used as biomarkers. Temperature-and toxicant-induced stresses induced low-molecular-weight HSP synthesis in the endemic amphipod species studied. However, induction of HSP70 synthesis in the same species after temperature stress has not been detected. The specificity of synthesis of HSP70 is discussed. The results obtained in this study suggest that low-molecular-weight HSPs can be used as stress markers in Baikal species and species with a narrow niche breadth.     

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.     

Resistance to desiccation and distribution patterns in the land snail Sphincterochila

The land snail Sphincterochila is represented in Israel by five species that replace one another along a climatic gradient that ranges from the Mediterranean areas receiving up to 1000 mm down to arid areas receiving only 70 mm of rain per year.
The resistance to three weeks of desiccation was studied in all five species. Total water loss was lowest in the desert-dwelling species S. zonata and highest in the Mediterranean S. aharonii and the desert-dwelling S. prophetarum , while the steppe-dwelling S. cariosa and S. fimbriata were intermediate. Sphincterochila zonata had a significantly thicker epiphragm, the lowest area specific water vapour conductance, the quickest response to desiccation (secretion of epiphragm), and a favourable surface-to-volume ratio compared to all other species. The Mediterranean-type water economy of S. prophetarum , despite its desert distribution, is related to its being an under-rock-dweller while S. zonata , which has the best adaptions to desiccating conditions, thrives in exposed areas. It is concluded that the differences in resistance to desiccation among the five species of one genus correspond to the gross distribution patterns and reflect the abiotic regime and the microhabitat of each species.     

Evolution of the response to heat shock in genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophila species and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilis group exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogaster Oregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters of Drosophila and variation in the numbers of gene copies and hsp70 isoforms in group virilis were found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genus Drosophila is discussed.     

Expression of heat shock proteins in adult honey bee (Apis mellifera L.) workers under hot-arid subtropical ecosystems

Heat stress elicits the expression of heat shock proteins (HSPs) in honey bee subspecies. These highly conserved proteins have significant role in protecting cells from thermal-induced stresses. Honey bees in subtropical regions face extremely dry and hot environment. The expression of HSPs in the nurses and foragers of indigenous (Apis mellifera jemenitica) and imported European (Apis mellifera ligustica and Apis mellifera carnica) honey bee subspecies after heat shock treatment were compared using SDS-PAGE. Hsp70 and Hsp82 were equally expressed in the nurses of all tested bee subspecies when exposed to 40 °C and 45 °C for 4 h. The forager bees exhibited differential expression of HSPs after heat stress. No HSPs was expressed in the foragers of A. m. jemenitica, and Hsp70 was expressed only in the foragers of A. m. ligustica and A. m. carnica at 40 °C. A prominent diversity in HSPs expression was also exhibited in the foragers at 45 °C with one HSP (Hsp70) in A. m. jemenitica, two HSPs (Hsp40 and Hsp70) in A. m. carnica, and three HSPs (Hsp40, Hsp60 and Hsp70) in A. m. ligustica. No HSPs was expressed in the control nurse and forager bees at any of the tested temperatures. These findings illustrated the differences in HSP expression among nurse and forager bees. It is obvious that the native foragers are more heat tolerant with least HSPs expression than exotic bee races. Further investigations will help to understand the potential role of HSPs in the adaptability, survival, and performance of bee subspecies in harsh climate of the subtropical regions.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.     

Nitrogen availability alters patterns of accumulation of heat stress-induced proteins in plants

Mounting evidence suggests that heat-shock proteins (HSPs) play a vital role in enhancing survival at high temperature. There is, however, considerable variation in patterns of HSP production among species, and even among and within individuals of a species. It is not known why this variation exists and to what extent variation in HSPs among organisms might be related to differences in thermotolerance. One possibility is that production of HSPs confers costs and natural selection has worked towards optimizing the cost-to-benefits of HSP synthesis and accumulation. However, the costs of this production have not been determined. If HSP production confers significant nitrogen (N) costs, then we reasoned that plants grown under low-N conditions might accumulate less HSP than high-N plants. Furthermore, if HSPs are related to thermotolerance, then variation in HSPs induced by N (or other factors) might correlate with variation in thermotolerance, here measured as short-term effects of heat stress on net CO₂ assimilation and photosystem II (PSII) function. To test these predictions, we grew individuals of a single variety of corn (Zea mays L.) under different N levels and then exposed the plants to acute heat stress. We found that: (1) high-N plants produced greater amounts of mitochondrial Hsp60 and chloroplastic Hsp24 per unit protein than their low-N counterparts; and (2) patterns of HSP production were related to PSII efficiency, as measured by F _v/F _m. Thus, our results indicate that N availability influences HSP production in higher plants suggesting that HSP production might be resource-limited, and that among other benefits, chloroplast HSPs (e.g., Hsp24) may in some way limit damage to PSII function during heat stress.     

A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus.

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Heat shock proteins Hsp70-1 and Hsp70-3 Are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos.

Heat Shock Proteins (HSPs) represent a variety of protein families that are induced by stressors such as heat and toxicants, and the induction of HSPs in the organogenesis stage rodent embryo is well established. It has been proposed that thermotolerance and chemotolerance result from expression of the HSPs. However, whether these proteins function to prevent dysmorphogenesis and which family members serve this function are unknown. Therefore, we evaluated the specific ability of stress-inducible Hsp70-1 and Hsp70-3 to prevent arsenite-induced dysmorphology in the cultured mouse embryo using gain- and loss-of-function models. Loss of HSP function was accomplished by injecting antisense oligonucleotides directed against hsp70-1 and hsp 70-3 mRNAs into the amniotic cavity of cultured Day 9 mouse embryos. Suppression of hsp70-1 and hsp70-3 expression resulted in an up to six-fold increase in the incidence of arsenite-induced neural tube defects. Gain of HSP function was accomplished by microinjecting a transgene with a constitutive promotor driving expression of the hsp70-1 coding region, and resulted in a decreased incidence of arsenite-induced neural tube defects. These results indicate that Hsp70-1 and Hsp70-3 are both necessary and sufficient for preventing arsenite-induced dysmorphology in early-somite staged mouse embryos. Mol. Reprod. Dev. 59:285-293, 2001.     

Evolution of the Response to Heat Shock in Genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophilaspecies and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilisgroup exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogasterOregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters ofDrosophila and variation in the numbers of gene copies andhsp70 isoforms in group viriliswere found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genusDrosophilais discussed.