Heat Shock Proteins and Adaptation to Variable and Extreme Environments

Evgen’ev, Michael B.; Garbuz, David G.; Zatsepina, Olga G.

doi:10.1007/978-94-017-9235-6_4

Michael B. Evgen’ev⁴,
David G. Garbuz⁴ &
Olga G. Zatsepina⁴

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2 Citations

Abstract

Eukaryotic organisms evolved sophisticated sensing mechanisms and generic responses at the cellular level to various forms of mild, chronic or acute stresses including HS. Exposure to elevated temperature as well as many other stressful factors disturbs normal folding of proteins while preexisting proteins may undergo denaturation and aggregation which is harmful for the cell (Hightower 1991; Szalay et al. 2007). Gene expression changes are a major component of various stress responses, along with alterations in protein-lipids ratio, rate of metabolism, fragmentation of Golgi complex and endoplasmic reticulum and other multiple disturbances (Kruuv et al. 1983; Vigh et al. 2007; Welch and Suhan 1985). Drastic changes in cytoskeleton structure (Toivola et al. 2010; Welch and Suhan 1986) and drop in ATP level in the cell represent important landmarks of stress response observed in different organisms (Lambowitz et al. 1983; Patriarca and Maresca 1990). Specifically, HS inhibits process of splicing of cellular mRNAs and may lead to complete blockade of cell cycle (Yost and Lindquist 1986). Acute HS usually resulted in the accumulation of high concentration of denatured proteins may also induce cell death by apoptosis or necrosis. Notably, adaptive responses to stress definitely depend on the organism and the natural environments in which it has been evolved and underwent selection.

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Proteostasis and Adaptation to High Temperature Stress

Role of Heat Shock Proteins in Oxidative Stress and Stress Tolerance

The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats

Article 01 January 2017

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4.1 General Response to Heat Shock and Other Forms of Stress

Eukaryotic organisms evolved sophisticated sensing mechanisms and generic responses at the cellular level to various forms of mild, chronic or acute stresses including HS. Exposure to elevated temperature as well as many other stressful factors disturbs normal folding of proteins while preexisting proteins may undergo denaturation and aggregation which is harmful for the cell (Hightower 1991; Szalay et al. 2007). Gene expression changes are a major component of various stress responses, along with alterations in protein-lipids ratio, rate of metabolism, fragmentation of Golgi complex and endoplasmic reticulum and other multiple disturbances (Kruuv et al. 1983; Vigh et al. 2007; Welch and Suhan 1985). Drastic changes in cytoskeleton structure (Toivola et al. 2010; Welch and Suhan 1986) and drop in ATP level in the cell represent important landmarks of stress response observed in different organisms (Lambowitz et al. 1983; Patriarca and Maresca 1990). Specifically, HS inhibits process of splicing of cellular mRNAs and may lead to complete blockade of cell cycle (Yost and Lindquist 1986). Acute HS usually resulted in the accumulation of high concentration of denatured proteins may also induce cell death by apoptosis or necrosis. Notably, adaptive responses to stress definitely depend on the organism and the natural environments in which it has been evolved and underwent selection.

Live organisms exhibit amazing plasticity in the regulation of genes involved in response to different forms of stress. Up- and down regulation of the expression of multiple genes in response to stress play central role in the adaptation of organisms to both short-term and long-term pronounced changes of environment. The regulation of adaptive response may be realized at the level of transcription induction, translation efficacy and/or stability of resulted mRNAs or proteins.

Stress response usually represents a choice between two antagonistic programs such as induction of adaptogenes including various Hsps or switching on the genes underlying cells death by apoptosis (Anckar and Sistonen 2007; López-Maury et al. 2008; He et al. 1998; Chen and Brandizzi 2013; Chu et al. 1996). Moderate heat shock predominantly leads to the induction of heat shock genes playing chaperoning function to prevent proteins misfolding and degradation and, hence, provide adaptive response. More severe heat stress inhibits the expression of Hsp genes and activate the expression of genes responsible for apoptosis (Fig. 4.1) (Chen and Brandizzi 2013; Murray et al. 2004).

At the present time during genomic era, new approaches including High-throughput Sequencing and DNA microchip technologies have been developed and used to monitor the consequences of heat shock and other forms of stress at the genome level. These studies performed in yeast, fruit flies and mammals including humans enabled one to describe complete transcriptome after various forms of stress including HS. The large-scale transcriptomic study of many species showed that organisms respond to drastic temperature changes with a multitude of transcriptional changes: up to 40 % of genes showed an altered expression after temperature exposure experiments depending on the species. High temperature has usually stronger effects on gene expression than low temperature, reflected by the higher number of temperature-responsive genes. Such studies help to gain insights into molecular processes underlying induced thermotolerance and acclimation and allowed to describe a lot of genes besides “classical” Hsp genes that dramatically changed their expression after temperature elevation in yeast (Gasch et al. 2000; Lyne et al. 2003), Drosophila (Girardot et al. 2004), human cells (Murray et al. 2004), E. сoli (Riehle et al. 2005), fishes (Buckley et al. 2006) etc. Such studies revealed hundreds of stress-related genes and demonstrated that the level of transcription and translation modulation depends on the severity and duration of stress applied. It is of note that phylogenetically distant forms express functionally similar groups of genes including heat shock genes, antioxidative genes, genes involved in the metabolism of hydrocarbons, and genes involved in ubiquitin-dependent degradation of proteins (Buckley et al. 2006). The role of many genes with temperature-dependent expression is not clear yet. For example, it was shown in Drosophila that heat shock leads to significant induction of transcription of genes shark and TotA (from turandot family), however, functions of these genes in the cells after HS were not revealed (Ekengren and Hultmark 2001).

We are well aware that Hsps discussed in this review is not the only protective molecules providing heat-resistance in various organisms. Thus, in other strains and species, mechanisms of basal thermotolerance may include synthesis of thermoprotective osmolites such as trehalose in the yeast (Saccharomyces cerevisiae), (Hottiger et al. 1987) and homeoviscious adaptation (i.e. adjustment in the lipid constituents of cells). Besides, thermotolerance may be due to enhanced stability of cellular proteins through modification of their primary structure and regulated depression of normal house-keeping cellular functions to moderate energy requirements under stressful conditions. Thus, in yeast the production of the disaccharide trehalose is induced by temperature elevation more that 20-fold (Hottiger et al. 1987). Transgenic strain of D. melanogaster, transformed with overexpressed trehalose-6-phosphate synthase gene (tps1), exhibits high tolerance to hypoxia. It was shown that the increase of trehalose concentration in the cells prevents aggregation of various proteins such as Na⁺/K⁺-ATPase and tubulin and participates in proteins folding in cooperation with chaperones (Chen et al. 2002; Diamant et al. 2001). Generally speaking, adaptive stress responses may be divided into generic responses shared by many stresses and specific responses evolved to cope with a particular challenge (Feder 2007; Lindquist 1986; Nadal et al. 2011). Below we shall discuss in detail the presumptive adaptive roles of heat shock proteins synthesized in response to HS and many other stresses in various eukaryotic organisms, although we clearly understand that stress tolerance results from numerous physiological and molecular mechanisms, of which Hsps are collectively only one.

All proteins and other compounds synthesized in response to temperature elevation may be roughly divided into the following major groups:

1.
Molecular chaperones comprising Hsps per se and their co-factors involved in restoration of misfolded cellular proteins and, hence, prevention of harmful aggregates formation (Ellis et al. 1989; Lindquist 1986).
2.
Components of ubiquitin-proteasome system (UPS) responsible for degradation of irreversibly damaged and denatured proteins (Raboy et al. 1991).
3.
RNA- and DNA-modifying enzymes necessary for DNA reparation and restoration of normal RNA-processing mechanisms (Bügl et al. 2000; Jantschitsch and Trautinger 2003).
4.
Carbohydrates metabolism enzymes (Buckley et al. 2006; Malmendal et al. 2006; Voit and Radivoyevitch 2000).
5.
Regulatory proteins including transcriptional factors and protein kinases.
6.
Transport and antioxidant proteins (e.g. glutathion S-transferase) as well as enzymes involved in toxin inactivation (cytochrome p450 etc.).
7.
Proteins involved in cellular membranes stabilization (Welker et al. 2010).
8.
Non-coding RNAs, e.g. hsr-ω (Arya et al. 2007; Jolly and Lakhotia 2006).

Therefore, rapid and abundant synthesis of various Hsps represents the most generic cellular response after temperature elevation, hypoxia, high concentrations of heavy metals, oxidative stress and other multiple forms of stresses. The synthesis of similar but not identical groups of Hsps has been repeatedly demonstrated for organisms from different unrelated phyla including yeast (S. cerevisiae), mollusks (Mytilus), insects (Cataglyphis, Drosophila, Bombyx mori), fishes (Gillichthys), amphibians (Xenopus), reptiles (Phrynocephalus, Gymnodactylus, Lacerta) etc. (reviewed by Feder and Hofmann 1999). Although it was shown by different groups that Hsps are actively synthesized after stress in the laboratory, it was necessary to prove by field work that their synthesis represents adaptive response evolved in nature to maximize cell and whole organism survival in response to drastic changes in the environment that put them at risk. It was also important to dissect the mechanisms underlying general HS response and define specific roles of individual Hsps families in ecological context in buffering extracellular challenges to minimize intracellular damage.

In general, all organisms may be divided into two groups: stenothermal and eurythermal species. Stenotherms inhabit areas with narrow temperature ranges and may be further subdivided into thermophilic and cold-adapted organisms. Endoparasites of mammalians represent good examples of thermophilic stenothermal organisms. On the other hand, Antarctic animals that have evolved for many millions of years under highly stable, cold temperature represent extreme cold-adapted stenotherms (Somero 2005). Eurythermal species dwell in habitats with highly variable temperature and intertidal organisms that experience every day fluctuations represent an excellent example of this particular group. Notably, from the very beginning Hsp70 was the main object of the investigations on adaptive role of Hsps in aggressive and highly variable environments. Such attention is quite understandable taking into account the key role of Hsp70 family members in proteins folding and transport under normal conditions and after stress (see Chap. 2). Besides, this family of Hsps is highly conserved and abundant synthesis of Hsp70 is easy to detect after HS in various organisms from yeast to humans and flies by Western blotting technique even using heterologous anti-bodies (see Craig et al. 1983; Schlesinger 1990; Feder and Hofmann 1999 for reviews). Therefore, many attempts were performed to correlate the synthesis of Hsp70 with adaptation of various organisms to fluctuating or extreme environmental conditions (Hightower 1991; Hoffmann 2010; Lyashko et al. 1994; Tomanek and Somero 2000; Ulmasov et al. 1993). However, it becomes evident that the unambiguous attribution of stress resistance to Hsp70 or other Hsps groups requires more than correlate evidence (Feder 2007; Jensen et al. 2010; Somero 2005).

4.2 Role of Hsps in Adaptation to Fluctuating Environmental Conditions of Terrestrial Organisms

4.2.1 Interspecific Comparisons (Distant Taxa)

The modes of thermal adaptations certainly greatly depend on the present state of environment and the evolution the species underwent. Many organisms successfully cope with extreme environmental conditions including heat shock by multiple behavioral adaptations such as hiding and escaping heat stress while others live in habitats with comparatively constant temperature regimes (Feder 2007; Feder and Hofmann 1999; Somero 2005).

Sometimes, different life stages e.g. larvae and adults routinely encounter strikingly different environmental conditions in terms of stress. Thus, while larvae and pupae of Drosophila and many other insect species are often heated to stressful temperatures in their natural medium (e.g. rotten fruits) the adults may easily escape high temperature areas and rapidly move to microhabitats with temperatures close to the physiological ones (Feder 1997). Thus, Hamada et al. (2008) identified a thermal sensing pathway in D. melanogaster that is tuned on to avoid non-preferred temperatures. The ion channel of the transient receptor potential (TRP) family dTrpA1 functions as a molecular sensor of temperature and activates a small set of anterior cell neurons, the function of which is crucial for selection of preferred temperatures.

Besides, many species from various taxonomic groups including insects and mammals evolved diapauses, hibernation or occasionally desiccation to survive adverse periods of life (Gusev et al. 2011; King et al. 2013; Rinehart et al. 2006).

The comparison of Hsps levels in geographical populations of the same species or different species inhabiting environments with contrasting temperature regimes represents the most widely approach to implicate expression of various Hsps and in particular Hsp70 in adaptation. Such integral ecological approach exploring field studies of non-model organisms may not only shed light on physiological functions of individual Hsps but also permits to evaluate their role in evolution of adaptations to adverse environmental conditions.

Historically, this ecology-based approach with the goal to investigate Hsps specifically in thermally adapted organisms was originally applied in 1980s by several groups including ourselves (Evgen’ev et al. 1987; Feder and Hofmann 1999; Kee and Nobel 1986; Steinert and Pickwell, 1988).

For our large-scale analysis lasted for more than 30 years we used different organisms from leishmania and lizards to mammalian species including camel and human tribes inhabiting thermally contrasting landscapes such as deserts and cold or temperate climatic zones and active at different time during the day.

Thus, we began our studies by comparing cell lines of silk worm (Bombyx mori) and gypsy moth (Lymantria dispar) (Evgen’ev et al. 1987). It is believed that silk worm (B. mori) originated from warm regions of South-East Asia while gypsy moth (L. dispar) dwells in cold and moderate climate areas. To our surprise the cells of B. mori survive and synthesize all groups of Hsps at extreme temperatures up to 49 °С, while L. dispar cells abolish all protein synthesis and die at temperatures exceeding 40 °С. In other words, they behave exactly as Drosophila cells and most of the investigated insect species (Dehghani et al. 2011; Feder et al. 1996; Feder and Hofmann 1999; Lindquist 1986; Lozovskaya and Evgen’ev 1984). Subsequently, we performed a comparative analysis of the larval and adult forms of the two above-mentioned moth species and corroborated our results obtained using cell lines. In all such experiments B. mori specimens or isolated organs actively synthesized Hsps at higher temperatures and exhibited much higher thermotolerance in comparison with L. dispar. After this discovery we decided to check whether high thermotolerance coupled with the ability to synthesize Hsps at extreme temperatures represents a common feature of organisms from warm climate zones and deserts.

In the frame of these large-scale studies we compared heat-shock protein expression in several lizard species from Middle Asia sand deserts (Turkmenistan) and common lizard (Zootoca vivipara by modern nomenclature or Lacerta vivipara in old papers), a species inhabiting temperate areas up to the latitude of the Polar Circle and higher. In the deserts of Turkmenistan the day temperature in summer may exceed 45 °С, while the sand surface is heated up to 70 °С. However, even most resistant lizards (e.g. toad headed sand lizards Phrynocephalus interscapularis) cannot survive such extreme temperatures and try to escape over-heating by digging into the sand or by other behavioral adaptations.

We assumed that xeric species inhabiting for a long time extremely hot and dry desert environments should have evolved adaptive behavioral and physiological mechanisms to cope with such adverse conditions and possibly peculiarities of Hsps synthesis represent an essential component of these adaptations. Therefore, we investigated in detail heat shock response (HSR) in nine lizard species from thermally contrasting climate zones including deserts, and these studies in general corroborated our earlier conclusions made when studying Lepidoptera species (Evgen’ev et al. 1987; Ulmasov et al. 1992). Thus, we demonstrated that desert lizard species are able to synthesize Hsps at much higher temperatures in comparison with the species from cold or moderate areas. In our investigation we used an extremely thermotolerant xeric species Chinese toad-headed sand lizard (P. interscapularis) active at the day time, a desert species Caspian gecko (Caspian gecko) which is active at night, and temperate climate common lizard (L. vivipara). The comparison of ³⁵S-methionine incorporation into proteins of the compared species indicated that in highly eurythermal Chinese toad-headed sand lizard (P. interscapularis) proteins are still synthesized at 50 °C and higher while in the common lizard L. vivipara inhabiting cold and temperate latitudes all protein synthesis is abolished at temperatures higher than 42 °C and the animals rapidly die. Furthermore, there was a clear-cut positive correlation between the constitutive level of Hsp70 in the cells and thermotolerance of all studied species. The analysis of constitutive level of Hsp70 detected in the cells of the above species indicated that toad-headed lizards (P. interscapularis) exhibits a maximum concentration of Hsp70, Caspian gecko also exhibits a lower but significant level of Hsp70, while in common lizard the presence of Hsp70 under normal physiological conditions is hardly detectable (Fig. 4.2).

The presence of two isoforms of Hsp70 family that are synthesized at different temperature regimes in P. interscapularis represents another important observation made in this study (Fig. 4.3). We designated these proteins Hsp68(+) and Hsp68(−) and demonstrated that while the first protein is synthesized predominantly in response to moderate HS, the latter form Hsp68(−) is constitutively synthesized under normal temperature conditions and after acute HS when the synthesis of all other cellular proteins is completely inhibited (Ulmasov et al. 1992). Subsequently, differential synthesis of various Hsp70 family members depending on the severity of HS was corroborated in many other studies (Feder and Hofmann 1999; Mizrahi et al. 2012; Tomanek and Somero 1999; Tomanek 2005).

It is known that all desert animals regularly encounter drastic fluctuations in temperature and, hence, the body temperature of these poikilothermal animals should also change significantly depending on these fluctuations. Along these lines, Cherlin and Muzichenko (1983) demonstrated that daily fluctuations of the body temperature of Middle Asia desert lizard range from 20 to 44 °С.

It was interesting to find out whether the observed fluctuations in the environmental and body temperature, somehow, influence the synthesis of Hsps and specifically Hsp70 in the body of the animal subjected to such drastic changes of the temperature in the field conditions. In the course of these field studies the lizards (P. interscapularis) were collected in the desert throughout the day starting from 6 a. m. and in vivo injected with ³⁵S-methionine. The labeled proteins were isolated from the livers of injected animals at different time intervals, run on two-dimensional (2D) gels, and labeled Hsp70 members, hsp68(+) and Hsp68(−), were excised and quantified. These experiments demonstrated clear-cut positive correlation between the body (and soil) temperature observed during the day and the levels of Hsp70 synthesis in the cells of P. interscapularis (Fig. 4.4). Notably, comparatively high level of Hsps synthesis continues for some time after the body temperature drops to 30–35 °С (Ulmasov et al. 1999).

Therefore, it was suggested that high constitutive level of Hsp70 observed in desert lizard species probably represents the major adaptation of xeric organisms in general to environmental challenges such as drastic fluctuation in temperature regime and provides extremely high thermotolerance characteristic for various xeric animals.

High content of Hsp70 and other chaperones in the cells under normal physiological conditions apparently represents “preparative defense strategy” which enables xeric organisms to normally function in the broad range of environmental temperatures without de novo induction of stress proteins and switching off the synthesis of normal cellular house-keeping genes.

Thus, when comparing ³⁵S-methionine incorporation into proteins of thermotolerant desert species Caspian gecko and common lizard (L. vivipara) we demonstrated that the intensity of proteins synthesis after heat shock (39 and 42 °C) is significantly higher in the latter species. Moreover, the temperature of Hsp70 synthesis induction (T-ON) in L. vivipara is significantly lower in comparison with gecko. In other words, as a rule in Northern species the threshold of Hsps induction is lower while the intensity of Hsps synthesis is significantly higher after moderate HS in comparison with desert thermoresistant species with high constitutive levels of Hsps.

The observed high thermoresistance of desert lizard species may be in principle explained by either high stability of Hsps present in their cells after they encounter stressful conditions or by constitutive presence of high concentration of correspondent mRNA. Northern blot analysis demonstrated that the observed differences in the constitutive levels of Hsp70 between P. interscapularis and other xeric lizard species and common lizard (L. vivipara) are most likely regulated at the level of transcription, and thermoresistant species are usually characterized by significantly higher levels of Hsp70 mRNAs under non-stress conditions (Ulmasov et al. 1992) (Fig. 4.2).

Later, similar differences in HSR were revealed when the Cataglyphis ant, which is one of the most thermotolerant land insect known, was compared with a few other insect species. The two species of Cataglyphis used in the study forages at body temperature above 50 °C and their critical thermal maxima are equal to 54–55 °C (Gehring and Wehner 1995). The synthesis and accumulation of heat shock proteins were analyzed in these highly thermotolerant ants and compared with another ant species Formica polyctena, an ant living in more temperate climates and to two Drosophila species. Similar to B. mori and desert lizards (P. interscapularis) protein synthesis in Cataglyphis ant species continues at temperatures higher than 45 °C as compared to 39 °C determined for Formica ants and studied Drosophila species. In contrast to Drosophila species, desert ant species (Cataglyphis) were characterized by significant constitutive levels of Hsps in their cells prior to HS. These findings were interpreted by the authors as preadaptation to extreme temperatures encountered by this species (Gehring and Wehner 1995).

The observed pronounced differences in the constitutive levels of Hsp70 between species from habitats with contrasting thermal regimes may be also explained by different quantity or/and structure of HSF1 in the compared organisms. In order to explore this possibility we investigated by Northern and Western blot analysis the concentration of HSF1 and correspondent mRNA in the cells of P. interscapularis and L. vivipara (Zatsepina et al. 2000). The experiments clearly demonstrated that the level of HSF1 and correspondent mRNA is significantly higher in the cells of L. vivipara in comparison with P. interscapularis. Therefore, there is a clear-cut negative correlation between the concentration of Hsp70 and HSF1 in the cells of the species studied. Southern heat-adapted species are characterized by high level of Hsp70 and low level of HSF1 in the cells under normal conditions while Northern species (L. vivipara) exhibits the reverse relationship between these two values (Fig. 4.2). It is plausible to speculate that high constitutive concentration of Hsps in the cells of xeric species allows them to cope with temperature fluctuations without additional induction of Hsps. On the other hand, Northern species rarely exposed to HS normally do not contain high concentration of Hsps in the cells but are able to rapidly respond to temperature increase by intensive synthesis of Hsps due to high constitutive content of HSF1 in the cells. In fact the observed negative correlation between Hsps and HSF1 in the compared lizard species perfectly corresponds to the so called “cellular thermometer” model (see the Chap. 3).

Furthermore, the intensity of Hsp genes transcription strongly depends on the efficiency of transcription factors (e.g. HSF1) binding with promoters of stress genes which may include competition between negative and positive regulators of transcription. We performed in vitro binding experiments to monitor HSF1 binding efficiency in the studied lizard species and demonstrated that thermotolerant species characteristically differ from the more high-latitude species L. vivipara by the presence of certain amount of activated HSF1 bound to Hsp genes promoters under normal physiological conditions.

On the other hand, activation (trimerization) of HSF1 and its detectable binding with HSEs in common lizards occurs at 34 °С, and only at 39 °С in P. interscapularis. Interestingly, dissociation of HSF1 from DNA in the course of recovery after HS is much longer in L. vivipara in comparison with xeric P. interscapularis (6 and 1 h, respectively). In addition, competition experiments demonstrated significantly higher binding affinity of HSF1 with Hsp promoters of xeric lizard species in comparison with that of L. vivipara (Zatsepina et al. 2000).

Therefore, high constitutive level of Hsp70 and other Hsps in the cells of desert lizards is likely controlled at the transcription level and may be due to the presence of certain amount of active HSF1 at their Hsp genes promoters providing their “leakage” under normal temperature conditions (Zatsepina et al. 2000). Likewise, subsequently, it was shown in goby (Gillichthys mirabilis) that the temperature of HSF1 activation positively correlated with acclimation temperature, which suggests that apparent plasticity in HSF1 activation may play an important role in the heat shock response in various eurythermal organisms and, hence, participate in general environmental control of Hsps gene expression (Buckley and Hofmann 2002).

Generally speaking, in the case of phylogenetically distant forms discussed above the observed variability in Hsps levels may resemble their different evolutionary history, while the differences in Hsps constitutive synthesis and induction patterns after HS detected in geographical populations of the same species or phylogenetically close forms from contrasting ecological conditions may rather resemble their adaptation to specific thermal conditions including extreme ones.

In fact, any comparison of phylogenetically distant forms dwelling in contrasting environments and looking for correlations is apt to severe critics because unrelated species should be by definition quite different in many respects due to the divergence process (Garland and Adolph 1994). On the other hand, the investigation of multiple species from contrasting habitats (e.g. multiple species of lizards from different taxa) may provide some useful information which should be, however, accepted with caution. Therefore, the most direct way to detect correlation between the manifestation of certain trait (e.g. Hsp70 levels) and adaptation to adverse conditions is to study closely related species or, ideally, several geographical populations of the same species from contrasting thermal environments.

4.2.2 The Comparative Analysis of Closely Related Forms

The comparison of close species belonging to the same group or genus but differing by distribution, latitude and consequently average temperature of their habitats represents another and probably more reliable and widely used approach to look for correlation between thermal adaptation and pattern of Hsps synthesis.

Along these lines, during the last two decades 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 as expected usually have a higher thermotolerance but exhibited various patterns of HSR (Dehghani et al. 2011; Garbuz et al. 2008; Feder and Hofmann 1999; Feder and Krebs 1997; Lindquist 1986; Somero 2005; Ulmasov et al. 1992).

It was shown, that in Drosophila Hsp70 genes are practically not active in most tissues under normal temperature conditions but can be rapidly induced by temperature elevation or other forms of stress producing huge amounts of chaperones which can reach about 1 % from total amount of cellular proteins (Velazquez et al. 1983). Notably, under non-stress conditions certain copies of Hsp70 family are characterized by low tissue-specific expression in spermatogonia of second instar larvae and prepupa (Lakhotia and Prasanth 2002). In Drosophila, constitutive members of Hsp70 family (e.g. Hsc70) apparently serve as major chaperones at normal temperature while the induction of Hsp70 genes likely serves as last defense line under critic and subcritic conditions.

Therefore, in this respect Drosophila species differ drastically from most other insect species studied so far where usually significant constitutive levels of Hsps are observed under normal temperature conditions (Dehghani et al. 2011; Garbuz et al. 2008; Gehring and Wehner 1995).

In our studies we explored several closely related Drosophila species belonging to the virilis group and several other species of the same genus as outgroups.

D. virilis is the most karyotipically primitive species of the virilis phylad and is probably ancestral to it if not to the entire virilis group (Patterson and Stone 1952). D. virilis is distributed throughout the Northern Hemisphere primarily South to 40°N latitude. D. lummei, considered the closest relative of D. virilis, occurs from just above 40°N to just above 65°N latitude and from Sweden West to the Pacific coast of Asia (Fig. 4.5). These species are separated by at least five to six millions years of divergence and can be even crossed in the laboratory to provide fertile progeny (Morales-Hojas et al. 2006; Patterson and Stone 1952; Spicer and Bell 2002). Many aspects of the thermal phenotypes of the species under study exhibit countergradient variation similar to that in other taxa (Feder and Hofmann 1999; Ulmasov et al. 1992; Zatsepina et al. 2001); i.e. the magnitude and threshold for traits correspond to the thermal environments in which the species occur. Thus, the lower-latitude species D. virilis exceeds the higher-latitude species D. lummei in basal thermotolerance (see also Garbuz et al. 2003), and temperature threshold for HSF activation. Amazingly, we failed to observe induced thermotolerance in D. lummei and pretreatment with mild temperatures even decreased the rate of survival after acute HS (Fig. 4.6). As also shown previously (Garbuz et al. 2003), D. virilis and D. lummei express similar amounts of heat-shock mRNA and protein at low to moderate heat-shock temperatures. On the other hand intense heat shock (e.g. 40–41 °C) almost abolishes heat-shock mRNA and protein expression in D. lummei, whereas considerable expression persists after such heat shock in D. virilis (Fig. 4.7). For example, Hsp70 concentration is similar in D. virilis and D. lummei after 37.5 °C heat shock but is markedly greater in D. virilis than in D. lummei after more severe heat shocks (Fig. 4.7). Notably, in D. lummei the reduction in protein level after acute heat shock is even greater for the small Hsps and Hsp40 than for Hsp70. Northern hybridization data with total RNA demonstrated that in D. lummei Hsp70 genes transcription is completely inhibited at 40 °C and restored only after 1 h of recovery at normal temperature. In the latter species the Hsp70 accumulation after acute HS is detectable only after 3 h recovery period. On the other hand, HSF in D. lummei efficiently binds with promoter DNA as revealed in vitro assay even at 41 °C which implies that acute HS (40 °C and higher) does not block HSF activation in this species but apparently interferes with other components of transcription machinery. In contrast, in D. virilis significant synthesis of Hsp70 mRNA efficiently occurs at 40° and even 41 °C. Most species of the virilis group studied exhibited positive correlation between the Hsp70 accumulation after heat exposure and thermotolerance. Thus, D. montana species from comparatively cold climatic zones was significantly less thermotolerant and was able to synthesized Hsps only after moderate HS in contrast to xeric D. novamexicana species belonging to the same species group (Garbuz et al. 2003).

Different levels of Hsp70 after HS in species of the virilis group are correlated with the structure of Hsp70 cluster and Hsp70 copy number comprising the cluster (see Chap. 5 for the details). Thus, 2D electrophoresis revealed more Hsp70 isoforms in D. virilis geographical strains with higher Hsp70 copy number (Garbuz et al. 2003; Evgen’ev et al. 2004).

However, while the relationship between species thermoresistance and Hsp70 synthesis was evident when species and strains of the virilis species group were compared, this correlation was lacking when a species from other Drosophila groups were included into the analysis (Fig. 4.7). Thus, adults of D. melanogaster (Oregon R strain) found the most thermosensitive strain in our studies synthesize maximal quantity of Hsp70 at 36–37 °C exceeding in this parameters all other Drosophila strains and species taken for comparison (Fig. 4.7a) (Krebs 1999).

Likewise, D. mojavensis, a highly eurythermal species from repleta group, with critic temperature equal to 43.5 °C synthesize significantly less Hsp70 after mild HS in comparison with D. melanogaster flies (Krebs 1999). Probably in desert flies that often encounter temperature fluctuations induction of intensive Hsps synthesis after mild HS will be deleterious for their growth and development (Feder and Krebs 1997; Zatsepina et al. 2001).

It is of note that all xeric Drosophila species used in our studies (e.g. D. virilis, D. novamexicana and D. mojavensis) were characterized by comparatively high temperature for maximal Hsp70 induction (38.5 °C) which was 1 °C higher than that of D. lummei and D. montana and 1.5 °C higher than in D. melanogaster (Garbuz et al. 2002, 2003). Remarkably, at higher temperatures after severe HS all studied thermotolerant species accumulate significantly more Hsp70 in comparison with D. melanogaster and other relatively thermosensitive species (Garbuz et al. 2002).

It is noteworthy that according to diverse genetic and biochemical evidence, the inducible Hsp70 proteins play key roles in basic and inducible tolerance specifically to extreme temperatures close to critic ones for the given species. Both the magnitude and threshold of inducible Hsp70 expression are correlated with the natural thermal regime in many species in many taxa (Feder and Hofmann 1999; Norris and Hightower 2000). Our results and data accumulated by other groups corroborated this conclusion showing that in Drosophila species inducible members of Hsp70 family play important protective role particularly in the organisms from extreme environments or after acute HS (Garbuz et al. 2003). The investigation of D. melanogaster strain with deleted Hsp70 copies which is not able to survive acute HS (Bettencourt et al. 2008; Gong and Golic 2006) provided independent proof for this statement (see Chap. 8 for details).

Since it is known that Hsp70 is not the only Hsps responsible for thermotolerance the patterns of induction of other Hsps families after heat exposure in a wide spectrum of Drosophila species were also compared. Thus, significantly higher thermotolerance observed in D. virilis and other xeric species of the group coupled with lower levels of Hsp70 accumulation in comparison with D. melanogaster strains after moderate HS suggests possible involvement of other chaperones providing D. virilis thermotolerant phenotype. It was demonstrated that D. virilis and xeric Drosophila mojavensis from repleta group are characterized by higher levels of Hsp40 and small Hsps synthesis after moderate and acute HS in comparison to D. lummei, and certain strains of D. melanogaster (Garbuz et al. 2003; Shilova et al. 2006). It is known that sHsps prevent denatured proteins from aggregation (Morrow et al. 2006), while Hsp40 belonging to J-proteins family serves as a cofactor for Hsp70 (See Chap. 2). Therefore, in principle, significant accumulation of Hsp40 may enhance the Hsp70 efficiency without the increase in Hsp70 concentration. The results obtained suggest that the Hsp40 and small heat shock proteins may play a significant role in thermotolerance and adaptation to temperature fluctuations in thermophilic Drosophila species.

It is of note that there are a few exceptions from the general pattern implying the important role of Hsps in thermotolerance in desert animals. Thus, when comparing Hsp expression in two closely related snail species of the genus Sphincterochila, a desert species S. zonata and a Mediterranean species S. cariosa, it was demonstrated 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 desert-inhabiting species (S. zonata). The authors speculated that the observed different strategies reflect the difference in heat and humidity encountered in the natural habitats, and that the desert species S. zonata relies on mechanisms and adaptations other than Hsps induction and constitutive Hsp70 synthesis thus avoiding the fitness consequences of continuous Hsp upregulation (Mizrahi et al. 2012).

Differential induction of individual Hsps in snail organs represent another HSR peculiarity described by this group, thus, in S. cariosa, heat stress caused rapid induction of Hsp70 and Hsp90 in the foot and kidney tissues, whereas the desert-inhabiting xeric species S. zonata displayed delayed induction of Hsp70 proteins in the foot and upregulation of Hsp90 alone in the kidney. Interestingly, similarly to highly eurythermal lizard P. interscapularis the investigated snail species contain two isoforms of Hsp70 that are synthesized with different kinetics and in response to different forms of HS (Mizrahi et al. 2012).

Another pertinent investigation has been recently performed using a well-studied model object, migratory locust (Locusta migratoria), which is native to the semiarid regions of the World and excellent for study adaptation to fluctuating temperature conditions. In this organism previous exposure to sublethal high temperatures effectively protects neuronal function against future hyperthermia, but unlike many other organisms described above, the deep physiological adaptations are not accompanied by a robust increase of Hsp70 transcript or protein in the nervous system. It was supposed that the observed lack of Hsp70 induction after HS may be explained by very high level of the constitutive level of Hsps in the tissues and the thermoprotective effect of pre-HS on the nervous system might be mediated not by Hsp70 induction but rather by post-translational modifications or protein trafficking (Dehghani et al. 2011).

In contrast to flies, locusts and other highly mobile organisms that may rapidly escape high temperature zones (Feder and Hofmann 1999), many sessile organisms, as well certain ontogenetic stages such as eggs, larvae and pupae dwelling in periodically heated substrate are subject to daily or seasonal variations in temperature and water availability and, hence, apparently use Hsps synthesis as major component of their survival strategy.

4.2.3 Intraspecific Comparison

Although multiple interspecific comparisons in ecological aspect provided rather reliable correlations between the levels and patterns of Hsps synthesis and species thermotolerance in ecological context (Ulmasov et al. 1992; Feder and Hofmann 1999; Tomanek 2005), in the case of intraspecific studies the situation is not so unequivocal.

In D. melanogaster, a number of physiological traits related to thermal extremes including adult cold resistance and heat resistance show clinal patterns (reviewed in Hoffmann et al. 2003). There is also clinal variation in this species for the incidence of reproductive diapause, thorax length and cold and heat resistance (Hoffmann and Weeks 2007; Mitrovski and Hoffmann 2001; Schmidt et al. 2005) that correlates with levels of resistance to thermal extremes (Schmidt and Paaby 2008). In India, highland populations of D. melanogaster have higher levels of desiccation resistance and melanism compared with lowland populations, which matches the hotter and drier conditions experienced in highlands (Parkash et al. 2008a, b).

However, within D. virilis and D. lummei geographical strains, little if any countergradient in terms of thermotolerance and Hsps synthesis variation is evident (Figs. 4.6 and 4.7). Possibly, the extensive gene flow among populations swamps incipient adaptation to local conditions, a situation that may not be universal in Drosophila species (Michalak et al. 2001). Likewise, the investigation of latitudinal clines for stress resistance in D. simulans from eastern Australia did not reveal any clinal pattern for desiccation or heat resistance (Arthur et al. 2008).

Furthermore, as in the case of D. melanogaster (Krebs et al. 1996), adaptation to laboratory conditions of D. virilis wild type strains caught in nature does not seem to significantly contribute to the observed intraspecific HSR patterns. Thus, two strains caught at the same location near Tashkent, Uzbekistan (T53 and T61) exhibit identical patterns of thermotolerance and Hsp70 induction despite the fact that T61 was captured recently and the T53 more than 30 years ago. The only conspicuous departure from this pattern is for D. virilis strain 160, a marker strain with at least one known recessive mutation on each autosome. The basal thermotolerance for this strain is considerably lower than for all other D. virilis strains studied so far probably due to multiple mutations present (Garbuz et al. 2003).

Likewise, Jensen et al. (2010) were not able to correlate heat-induced Hsp70 synthesis and several measurements of adult heat tolerance in three independent populations of D. melanogaster, estimated independently in three laboratories and using slightly different protocols. Although these exceptionally thorough studies revealed substantial within population variation in both Hsp70 and heat tolerance, the authors were not able to detect any significant correlation between these parameters in adults. It was concluded that variation in Hsp70 expression is only likely to explain a small proportion of the observed variation in adult thermoresistance (Jensen et al. 2010).

Probably highly coordinated heat-induced synthesis of Hsp70 encoded by several conserved copies comprising a cluster in Drosophila species provides an optimal response to different microclimatic conditions within the species distribution range and the observed intraspecific variations in adult heat tolerance are not directly connected with Hsps system.

However, constant pressure of natural selection is able to modulate HSR within the species.

“Evolution Canyons” intensively studied in recent years represents a perfect example of such natural selection in D. melanogaster (Korol et al. 2006; Rashkovetsky et al. 2006). Micropopulations of this species occur on North- and South-facing slopes of the canyon with greatly differing climatic regimes and in the intervening region; the slopes are 400 m apart at the top and 100 m apart at the bottom. Although adult Drosophila can traverse several kilometers in a single day, populations on each slope have diverged in body size, heat and desiccation tolerance, thermal preference for oviposition, fluctuating asymmetry in morphological traits, rates of mutation and recombination, and mate preference (Hübner et al. 2013; Rashkovetsky et al. 2006). The investigators monitored the divergence of the slopes using neutral microsatellite markers and a candidate gene (Hsp70) strongly linked to resistance to environmental stress. The frequencies of putatively neutral and non-neutral (Hsp70Ba, a heat shock gene) markers in D. melanogaster collected from the middle elevation of each slope were monitored. Luckily it was demonstrated that D. melanogaster from the Evolution Canyon in Israel are polymorphic for 1.2-kb P element insertion that interrupts a regulatory region of one of Hsp70 genes and, hence, should interfere with transcription and decrease thermotolerance and resistance to other stresses. The analysis performed demonstrated that there are remarkable differences in the frequencies of both microsatellites and P element bearing Hsp70Ba allele in micropopulations inhabiting the two slopes (Michalak et al. 2001). Characteristically, flies dwelling in the Southern slope and often exposed to heat contain many folds less frequently the Hsp70Ba allele with disrupted promoter.

Notably, when the authors directly investigated the role of small Hsps and Hsp40 in thermoadaptation of flies collected from both slopes they were able to demonstrate more pronounced expression of Hsp40 in the flies collected from the Southern slope after mild HS implying a significant contribution of Hsp40 to thermoadaptation under local microclimatic conditions (Carmel et al. 2011). Therefore, it was possible to conclude that the observed divergence in thermoresistance and local adaptation is consistent with certain degree of genetic isolation despite the contiguity of the D. melanogaster populations in the canyon (Hübner et al. 2013; Korol et al. 2006; Rashkovetsky et al. 2006).

D. melanogaster flies (T strain) collected in sub-equatorial semiarid tropical zone of Africa in the 1970s (J.R. David, personal communication 1978) represents another example of pronounced differences in heat shock response observed in geographical populations of the same species. T strain was remarkably tolerant of sustained laboratory culture above 30 °C previously considered to be the limit for continuous culture of this species (Parsons 1973). Furthermore, the basal thermotolerance of T strain adults significantly exceeded that of standard wild-type (Oregon R) strain used as a control. In this exceptional ability T strain outperformed other D. melanogaster strains and compares favorably with many cactophilic desert Drosophila species (Krebs and Loeschcke 1999; Stratman and Markow 1998). However, surprisingly, induced thermotolerance of high temperatures, which in D. melanogaster is due in part to the inducible molecular chaperone Hsp70, is only modest in T strain. Expression of Hsp70 protein and Hsp70 mRNA is likewise reduced and has slower kinetics in this strain (T) than in a standard wild-type Oregon R strain. T strain has higher critic temperature (i.e. the temperature of heat shock when less than 1 % of treated flies survive) in comparison with Oregon R flies (41 °C vs. 40 °C). Notably, maximal level of Hsp70 synthesis was observed in both compared strains at 37 °C but in the case of T strain the level of Hsp70 accumulation was two to threefold lower (Zatsepina et al. 2001). On the other hand, at higher temperatures close to critic value (e.g. 39 °C) flies of T strain still synthesized Hsp70 while in Oregon R flies the synthesis of this chaperone was completely inhibited although Hsp70 mRNA was detected in both strains. This observation suggests higher thermostability of translational machinery in strain T (Fig. 4.8). The compared strains also differ in a similar way in the patterns of constitutive and heat-inducible levels of other molecular chaperones. Thus, T strain is characterized by higher levels of Hsp40 and small heat shock proteins after HS in comparison with D. melanogaster Oregon R strain (Zatsepina et al. 2001).

It was shown that the lower Hsps expression in the T strain after moderate HS apparently has no basis in the compromised activation of the heat-shock transcription factor (HSF), which is similar in T and Oregon R flies. Subsequently, it was demonstrated that the observed reduced expression of Hsp70 likely stems from insertion of two transposable elements, H.M.S. Beagle in the intergenic region of the 87A7 Hsp70 gene cluster and Jockey in the Hsp70Ba gene promoter (Zatsepina et al. 2001) (see Chap. 6 on the role of transposable elements in HSR).

The data accumulated when studying D. melanogaster micropopulations from the “Evolution Canyon” and T strain versus Oregon R strain imply strong variation within a single species. Evidently, adaptation via natural selection is sufficiently strong to overcome even the immense phylogenetic inertia of the heat shock response.

4.2.4 Adaptive Role of Hsps in Homothermal Thermophilic Organisms

Most of the studies on the possible role of Hsps in thermoresistance were performed using insects and other poikilothermal organisms, because homothermal animals preserve constant body temperature under various temperature regimes and may only slightly modulate it predominantly in skin or other external tissues subjected to temperature challenge. However, in some special cases significant changes in external tissues subjected to environmental challenges were observed, thus, in camels often experiencing high temperature in arid areas the skin may reach 40 °C and even more. The camel (Camelus dromedarius) represents a homothermal organism perfectly adapted to extreme conditions of arid zones and is capable of tolerating extreme heat accompanied by a significant elevation (up to 41 °C) of the whole body temperature (Schmidt-Nilsen 1972). Western blot analysis demonstrated that camel lymphocytes constitutively express Hsp73 protein which can be significantly induced by heat shock (Ulmasov et al. 1993). Surprisingly, another member of Hsp70 family (Hsp72) is not induced in lymphocytes but can be induced in skin fibroblasts. Probably skin fibroblasts of this animal, which are often exposed to an extremely high temperature challenge, required the synthesis of additional protective protein (Hsp72). Notably, based on labeled methionine incorporation analysis, it was concluded that the general synthesis of proteins in camel cells is more resistant to heat than that seen in human cells (Ulmasov et al. 1993). Recently, inducible and constitutive camel Hsp70 genes have been sequenced and characteristic differences in the structure of human and camel Hsp70 regulatory regions were detected (Garbuz et al. 2011a).

Similarly, when two ethnic human groups inhabiting for many generations contrasting thermal environments (Russians vs Turkmens, inhabiting desert Turkmenistan, Middle Asia) were investigated it was demonstrated that while fibroblasts isolated from Turkmens after severe heat shock exhibited intensive synthesis of all Hsps in parallel with synthesis of many other cellular proteins, only trace synthesis of Hsps was observed in the second group, Russians (Fig. 4.9) (Lyashko et al. 1994).

When survival of fibroblasts after severe heat shock treatment was assessed by colony formation assay, the cells of the first group (Russians) exhibited significantly lower survival rates (Lyashko et al. 1994).

Another way to explore the role of Hsps in whole body adaptation in mammals is to monitor the allele frequency of specific SNPs in different heat shock genes in different populations of the same breed. It was shown by several authors that nucleotide changes occurring naturally in the flanking regions of the HSPA1A gene might affect the inducibility, level of expression, and stability of Hsp70 mRNA and, hence, contribute to stress tolerance. Thus, in human peripheral mononuclear cells, polymorphisms in the coding region of HSPA1A gene were associated with an increased ability to respond to heat stress (Singh et al. 2006). In pigs, an Hsp70 gene polymorphism located in the promoter and 3’-UTR was associated with mRNA stability and stress response (Schwerin et al. 2002). Recently haplotype analysis of the eight SNPs of the HSPA1A gene revealed their involvement in heat resistance in Chinese Holstein cattle (heat sensitive breed of cattle that originated in Europe). The analysis demonstrated the presence of significant differences between individuals carrying different haplotypes for most of the heat-tolerance traits studied (Xiong et al. 2013). Such SNPs may be useful in the future as molecular markers to assist selection for heat tolerance in various domestic animals and maybe used as genetic tools to experimentally improve tolerance to high temperature and other forms of stress.

4.3 Comparative Data on HSR in Aquatic Organisms

It is evident that aquatic species due to physical properties of water which includes high thermal conductivity should have body temperatures close to that of their surrounding which, however, can change very rapidly in the intertidal zone or in shallow water, which is heated by the sun.

Various intertidal and littoral aquatic (mostly marine) organisms represent another good object for investigation of diverse behavioral and molecular mechanisms, including Hsps synthesis, evolved to cope with fluctuating temperature conditions.

One of the most variable and unpredictable habitats on Earth is the marine rocky intertidal zone located at the boundary between the terrestrial and marine environments. Mussels dominate rocky intertidal habitats throughout the world and, being sessile, endure wide variations in temperature, salinity, oxygen, and food availability due to daily, tidal, seasonal and climatic cycles. Analysis of gene-expression changes in the mussels, fishes and crustaceans species dwelling in different intertidal habitats were summarised in numerous publications (Buckley et al. 2001, 2006; Dong et al. 2008; Podrabsky and Somero 2007; Somero 2005; Stillman and Somero 2000; Tomanek 2008).

In general, when studying various marine organisms from contrasting environments it was demonstrated that similar to terrestrial organisms, threshold of HSF activation and, hence, Hsps induction in the cold-adapted species is significantly lower that in more thermophilic forms. Thus cold-adapted clams Mytilus trossulus begin to synthesize Hsp70 at lower temperature than a more Southern species M. galloprovincialis (Buckley et al. 2001). In a study of Hsp70 in a genus of outbred subtropical fish, a direct correlation between Hsp70 levels and survival at elevated temperature as a test of phenotypic diversity in acquired thermotolerance was found (Norris and Hightower 2000).

Moreover, the threshold of Hsps induction in snails, mussels, crabs and fishes characteristically varies depending on the climatic cycle and in summer the temperature of Hsps induction is significantly higher than in winter (Buckley and Hofmann 2002; Dong et al. 2008; Tomanek 2008).

Characteristically, as a rule acclimation procedure increases the threshold of Hsps induction in relatively thermophylic or eurythermic species to less extent in comparison with cold-adapted organisms and the latter can usually survive larger temperature increase in comparisons with their physiological range than thermophylic related species. Thus, the investigation of consequences of acclimation process in snails of the genus Tegula demonstrated that a more thermoresistant species T. funebralis exhibits lower ability to respond to acclimation in terms of modulation of Hsps threshold temperature etc. in comparison with closely-related cold-adapted species T. brunnei and T. montereyi (Tomanek 2005; Tomanek and Somero 2000). As a rule, species subjected to daily temperature changes are characterized by maximal thermoresistance. Multiple experiments exploring various thermally contrasting intertidal species of mussels and other marine organisms enable one to conclude that acclimation procedure may be highly efficient and can enhance thermoresistance of organisms occupying moderately variable thermal environments (range <10 °C), like the subtidal zone. In such organisms acclimation may activate the HSR at temperatures significantly above those they normally experience in their habitats. In contrast, species from highly variable thermal environments (range <20 °C) or certain eurythermal species have a limited acclimatory plasticity because such species probably “exhausted” genetic components of their thermal adaptation. Thus, they may live close to their thermal limits and any further increase in temperature is probably going to push them beyond those limits (Tomanek 2005; Tomanek and Somero 1999).

It is evident that the ability to acclimate to variable environmental conditions may affect the biogeographic range of species, their success in colonizing new habitats, and their likelihood of surviving rapid anthropogenic climate change. The responses to temperature acclimation (4 weeks at 7, 13 and 19 °C) in gill tissue of the warm-adapted intertidal blue mussel Mytilus galloprovincialis, an invasive species in the Northeastern Pacific, and the cold-adapted M. trossulus, the native congener in the region, were compared (Tomanek 2005). Using two-dimensional gel electrophoresis and tandem mass spectrometry, the authors demonstrated that the cold-adapted M. trossulus showed increased abundances of molecular chaperones after acclimation at 19 °C, but M. galloprovincialis did not, suggesting that the two species differ in their long-term upper thermal limits. In contrast, the warm-adapted M. galloprovincialis showed a stronger response to cold acclimation than M. trossulus, including changes in abundance in higher number of proteins and differing protein expression profiles between 7 and 13 °C, a pattern absent in M. trossulus. It is necessary to keep in mind that consequences of the acclimation process by far are not restricted by the increase of certain Hsps synthesis but lead to profound changes in the general transcription pattern. Thus, acute heat shock exposure (24, 28 and 32 °C) after acclimation to 13 °C for 4 weeks enabled to identify 47 and 61 distinct proteins that changed abundance in M. galloprovincialis and M. trossulus, respectively. As expected the onset temperatures of greater abundance of some members of the heat shock protein Hsp70 and small Hsps families were lower in the cold-adapted M. trossulus.

In the work of Hamer et al. (2004) seasonal changes of the Hsp70 level in mussels were registered. Maximal levels of Hsp72 and Hsp70 were observed in mussels after summer (September), and minimal levels in winter (December). The observed small changes in sea salinity could not cause significant Hsp70 proteins induction.

Overall, these results help to explain why M. galloprovincialis has replaced M. trossulus in Southern California over the last century, but also suggest that M. trossulus may maintain a competitive advantage at colder temperatures. It was also speculated that anthropogenic global warming may reinforce the advantage M. galloprovincialis has over M. trossulus in the warmer parts of the latter’s historical range (Tomanek 2005).

In general, intertidal organisms exhibit patterns of Hsps synthesis similar to those of lizard species inhabiting thermally contrasting biotopes (see above).

Thus, four limpet species of the genus Lottia, exhibiting a broad vertical distribution on wave-exposed rocky shores and subjected to dramatic temperature changes during the low tide apparently have distinct strategy of Hsp70 expression depending on the height and orientation in the rocky intertidal zone. In the high-intertidal zone, L. scabra typically occupies horizontal surfaces fully exposed to the sun, L. austrodigitalis primarily occupies vertical or overhanging surfaces, while L. pelta and L. scutum are restricted to the low- and mid-intertidal zones. Western blot analysis demonstrated that field-collected as well as acclimated specimens of the two high-intertidal species had significantly higher constitutive levels of Hsp70 than the low- and mid-intertidal species. Therefore, their cells are preadapted to dramatic temperature changes occurring in these microhabitat conditions. Species dwelling in moderate and low-intertidal zones do not normally experience such temperature gaps and begin to actively synthesize Hsps only after temperature elevation exactly like was observed in Lacerta lizard (Dong et al. 2008).

Very similar pattern of Hsps synthesis was demonstrated for three species of snails in the genus Tegula. In these species daily temperature fluctuations for relatively thermophylic and eurythermic T. funebralis range from 13 to 36 °С, while in cold-adapted species T. brunnei and T. montereyi inhabiting low-tide zone the temperature varies from 13 to 19 °C and these organisms are rarely subjected to HS in their natural habitats. As expected, the Hsps synthesis in these cold-adapted species is strictly inducible in contrast to T. funebris specimens which constitutively synthesize Hsp70 under normal non-stress conditions exhibiting preparative defense strategy (Tomanek 2005; Tomanek and Somero 2000). Interestingly, the development rate of the latter species is significantly lower in comparison with that of congeneric cold-adapted species possibly as a trade off for high constitutive concentration of Hsp70 which was shown to hamper the development in Drosophila (Krebs and Feder 1997).

Although most studies of HS and acclimation in aquatic organisms were conducted using marine organisms virtually the same regularities were determined in freshwater species. Thus, the investigation of HSR in two congeneric species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal (Fig. 4.10) that strikingly differ in their vertical distribution and thermal tolerance was performed.

E. verrucosus is a stenobiotic species that preferred temperature 5–6 °С (Timofeyev and Shatilina 2007). E. cyaneus is a representative species of the upper littoral zone, the most thermally fluctuating environment in the lake. E. cyaneus is significantly more thermoresistant than E. verrucosus, and its preferred temperatures are 11–12 °C (Timofeyev and Kirichenko 2004). It was demonstrated that the basal level of Hsp70 synthesis is five times higher in E. cyaneus inhabiting the upper littoral compared to the thermosensitive E. verrucosus from the more thermally stable lower littoral habitat. The high basal level of Hsp70 likely contributes to the thermotolerant phenotype of E. cyaneus and is possibly evolved in response to drastic temperature fluctuations characteristic for the upper littoral zone of Lake Baikal (Votintsev 1961). In addition to the high constitutive expression of Hsp70, E. cyaneus also showed a relatively high temperature threshold necessary for Hsp70 induction (27 °C) and relatively low level of Hsp70 accumulation in response to acute HS (only two-fold). Thus, high basal level of Hsp70 obviously allows E. cyaneus to overcome temperature fluctuations without additional synthesis, since both short-term acute HS (25 °C) and the mild stress (16 °C) with following recovery does not induce a significant increase in the level of Hsp70 in this species (Bedulina et al. 2013).

In contrast, the more thermosensitive E. verrucosus is characterized by several times lower basal level of Hsp70, and much more pronounced induction of Hsp70 concentration in response to the acute HS, HS with recovery, mild HS. Thus, significant Hsp70 induction in E. verrucosus already takes place at 13 °C, which is only 6 °C above the acclimation temperature, and Hsp70 expression reaches higher levels (eight-fold) within 24 h after the acute HS. Along these lines, studies of the Baltic Sea amphipods also showed that thermally resistant semi-terrestrial intertidal species Orchestia gammarellus has a higher basal level of Hsp70 compared to the thermosensitive subtidal Gammarus oceanicus as well as less pronounced Hsps induction in response to HS (Bedulina et al. 2010).

4.4 Cold-Adapted Stenothermal Organisms

In the previous section we report about high constitutive concentrations of several Hsps in several aquatic species which often experience dramatic changes in the temperature of their habitats. It is evident that the aquatic environment can be extremely stressful to its sessile inhabitants and to other organisms which cannot rapidly escape the stressful conditions.

In this respect, stenothermal marine high-latitude (in particular, Antarctic) animals that thrive at temperatures down to −1.9 °C are of special interest for understanding molecular mechanisms underlying resistance to stable and extreme conditions. Usually seawater temperatures at 15 m depth range from −1.8 to 1.8 °C. The species dwelling under such conditions are usually highly endemic, they have been geographically isolated in a constantly cold environment for around 25–22 MYA (reviewed in Clarke and Johnston 1996) and are extremely stenothermal (Somero and DeVries 1967) The investigation of heat shock response of Antarctic marine species is very important, because the Southern Ocean has a very stable and narrow temperature range, where sudden temperature rises of 5 °C have not occurred for millions of years. Antarctic species do not survive even modest temperature rise and die even after short temperature exposure at 5–10 °C (Somero and DeVries 1967). In Antarctic notothenioids as well as several invertebrate species the loss of the heat-shock response was discovered (Carpenter and Hofmann 2000; Clark and Peck 2009; Hofmann et al. 2000). Thus, in the notothenioid fish Trematomus bernacchii, acute heat stress and cadmium exposure failed to induce Hsp synthesis and increase transcription of any mRNAs for Hsps (except for the cochaperone Hsp40) (Hofmann et al. 2000; Buckley and Somero 2009). Surprisingly, closely related forms sharing the same ancestor and dwelling in cold waters (8–12 °C) of the New Zealand area are able to effectively synthesize Hsps after the temperature elevation (Petricorena and Somero 2007). To elucidate the mechanism responsible for the lack of Hsps induction in T. bernacchii, HSF1 activity, Hsp70 mRNA production and protein synthesis patterns, hepatocytes of this species were examined (Buckley et al. 2004). Interestingly, in the course of these studies the presence of activated HSF and Hsp70 mRNA were detected in this organism in non-stress conditions. However, HS and chemical inducers of the heat shock response failed to increase Hsp70 mRNA levels, HSF1 activity or the levels of any Hsp.

It was also shown that inducible Hsp70 is expressed permanently in the cold-adapted T. bernacchii and related Antarctic species, Pagothenia borchgrevinki (Nototheniidae), Harpagifer antarcticus (Harpagiferidae) and Lycodichthys dearborni (Zoarcidae) (Buckley et al. 2004; Clark et al. 2008; Place and Hofmann 2005). It is likely that certain level of Hsp70 in the cells under non-stress conditions may be necessary to cope with aggregated and misfolded proteins occurring under extremely low temperature (Privalov 1990).

Sequence analysis of the 5′-region of Hsp70 gene of the Antarctic fish E. focardii, which also lack the heat shock response, revealed the presence of potentially functional HSEs and also StREs (Stress response elements), both of which confer the ability to respond to stress (La Terza et al. 2001). One may speculate that because of the presence of permanently active HSF and resulted constitutive Hsps synthesis, the further HSF activation after heat treatment is complicated or requires the induction temperature higher than lethal temperature for the examined Antarctic organisms.

Indeed, investigation of Hsp70 induction in clam (Laternula elliptica) revealed up-regulation of two isoforms of Hsp70 genes at 10–15 °C in digestive gland and gill tissue (Clark et al. 2008). The induction temperature for these genes was between 8 and 10 °C, while permanent expression of Hsp70 and Hsp78 isoforms was detected in this species.

The investigation of the limpet (Nacella concinna) also revealed constitutive expression of all members of Hsp70 family. The induction temperature was 15 °C higher than this species can experience in Antarctic waters (seawater temperatures at 15 m depth range from −1.8 to 1.8 °C). Furthermore, three other investigated Antarctic species, a sea star (Odontaster validus), a gammarid (Paraceradocus gibber) (Clark et al. 2008) and a ciliate (Euplotes focardii) (La Terza et al. 2001) completely lost heat shock response. In the case of the sea star and the gammarid several Hsp70 gene family members were identified in genomes, but they did not show either inducible or significant permanent expression. Larvae of a terrestrial Antarctic flightless midge species Belgica antarctica (Diptera, Chironomidae), are also not able to synthesize Hsps after temperature elevation (Rinehart et al. 2006). Interestingly, this midge species also exhibits high level of constitutive Hsps which can be further increased by UV-radiation. Conversely, adults of this species exhibit no constitutive up-regulation of their Hsps synthesis while Hsps induction can be thermally activated (Rinehart et al. 2006). Another well-known model species Hydra оligactis, dwelling in extremely cold and stable aquatic conditions also lost the ability to synthesize Hsps in response to temperature elevation despite the presence of correspondent genes in the genome. However, in this particular case the absence of Hsp70 after HS results from very low stability of correspondent mRNA (Brennecke et al. 1998). Surprisingly, close Hydra species (H. vulgaris) characterized by the virtual absence of senescence is able in response to HS synthesize Hsps that are implicated in this extraordinary phenomenon (Martínez and Bridge 2012).

In the previous section we described a few species which are not able to respond to temperature elevation by Hsps synthesis induction. Therefore, stenothermal organisms (mostly aquatic) inhabiting stable cold environments for many million years which practically never encounter any significant fluctuations in temperature, salinity and other challenges may lose to different degree their ability to respond to various forms of stress by Hsps induction. The enhanced lipid membrane densities (e.g. higher concentrations of mitochondria), characteristic changes in enzyme kinetic properties and, even more so, loss of genetic information (e.g. myoglobin and hemoglobin in notothenoid fishes) reflect the specialization of Antarctic organisms to constant low temperature conditions (Pörtner et al. 2007).

Generally speaking, at all levels analyzed, the functional specialization to permanently low temperatures evolved in aquatic organisms implies reduced tolerance to high temperatures, as a trade-off (Petricorena and Somero 2007; Pörtner et al. 2007). Probably high constitutive synthesis of Hsp70 and other chaperones common for such stenothermal forms represents the major compensatory molecular adaptation to deal with elevated levels of protein damage constantly occurring at low temperature conditions which are far from optimum for normal protein folding (Fraser et al. 2007; Fraser and Rogers 2007; Privalov 1990; Place et al. 2004; Todgham et al. 2007). The upregulation of Hsps demonstrated in many Antarctic organisms suggested that low-temperature conditions may be significantly denaturing to cellular proteins, an observation that was supported by elevated levels of ubiquitin-conjugated protein detected in Antarctic notothenoid fish (Place et al. 2004).

4.5 Organisms from High Temperature and Salinity Areas (Extremophiles) and Related Forms

Along with described above desert organisms and aquatic (mostly marine) species that evolved to cope with thermally highly variable environments, there are animal species thriving in highly aggressive extreme areas under conditions of constant high temperature and/or salinity.

During the expedition to Kunashir (Kuril Islands) in 2005 insect larvae of different sizes were collected in the hot mineralized sulphur volcanic spring Fig. 4.11.

The larvae were determined to belong to Diptera species Stratiomys japonica van der Wulp (Diptera: Stratiomyidae), common name “soldier flies”. The collected larvae were consequently compared with a few other species belonging to the same family (Stratiomyidae). The recent phylogenetic analysis of Stratiomyidae based on molecular characters was performed (Brammer and von Dohlen 2007).

The larvae of Stratiomyomorpha thriving in the hot springs are collectors-gatherers of fine organic particles. They have strong larval cuticle characteristically shagreened and encrusted with plates or “warts” of CaCO₃, which is unique among the Diptera (Rozkošný 1982, 1997; Woodley 1989). These features might facilitate colonization of extreme semiaquatic habitats such as hot volcanic springs of Kunashir Island (the Kuril Islands).

Stratiomyid larvae belonging to different lineages are known to inhabit extreme aquatic conditions, such as hypersaline and thermal waters, and demonstrate a remarkable resistance to different chemicals (reviewed in McFadden 1967 and Rozkošný 1982, 1997).

The aim of our study was to compare and try to correlate thermotolerance and the patterns of Hsps expression in (semi)aquatic larvae of several stratiomyid species belonging to different evolutionary lineages and inhabiting extreme habitats with contrasting temperatures and chemistry.

In this work we have investigated four stratiomyids: Stratiomys japonica from Stolbovskoi hot spring on Kunashir Island; two species, Stratiomys singularior and Nemotelus bipunctatus from hypersaline lakes in the Crimea; and Oxycera pardalina from a cold spring near St.-Petersburg.

S. singularior is a very common and widely-distributed Transpalaearctic typical eurythermal species. In the Crimea, the larvae of this species were abundant in shallow pools saturated with H₂S, with mineralization approximately 80 g/l. Nemotelus bipunctatus was also collected in Crimea in the water margin zone of coastal lagoon-derived Lake Koyashskoe where mineralization of water near the shore was about 280 g/l (Golubkov et al. 2007). The measured temperature in the habitats of the two Crimean stratiomyids used in our studies varied from 15 to 30 °C, but apparently it can be higher in summer and much lower in winter.

For comparison with the above described three thermoresistant species we took cold-adapted species O. pardalina from the same family which is widely distributed in Western and Central Europe. The larvae of O. pardalina are confined to clear-water cold carbonate springs near St.-Petersburg with a year-round stable low water temperature (5.5–7.5 °C) and low level of total mineralization (0.38–0.42 g/l).

The larvae of the four species were exposed to different temperatures and assessed for survival and Hsps synthesis. The LT₅₀ and critical temperature (i.e. the temperature of HS treatment resulting in less than 1 % of survival) were also determined in their larvae.

The LT₅₀ and critical temperatures of S. japonica larvae did not depend on the temperature of maintaining in the laboratory (31 or 37 °C). Apparently, maintenance of these larvae at different temperatures before HS did not significantly affect their thermotolerance. Therefore, the acclimation procedure was not effective in the studied species. This observation is not surprising because it was previously demonstrated in other highly eurythermal organisms which also did not respond to acclimation procedure (Tomanek 2005; Somero 2005). It was speculated that such forms exhausted genetic components of their thermal adaptation and, hence, live close to their thermal limit. Due to this characteristic feature in the HSR, species from extreme and highly variable environments are likely to be more affected by climate change than species from moderately variable environments.

It is of note, that D. lummei flies (see above) were also characterized by the absence of induced thermotolerance probably because in this case the representatives of this cold-adapted species having a diapause rarely (if ever) encounter acute HS in nature.

In general, our experimental data corroborate our field observations on Kunashir Island, in which S. japonica larvae died within a few minutes after being swept or transferred into areas of the hot spring where water temperature exceeded 50 °C (Fig. 4.11). For the two Crimean species, LT₅₀ for a 30-min heat shock were 45.5 °C for S. singularior 46.5 °C for N. bipunctatus, while critical temperatures for these species were 47 and 48 °C, respectively (Fig. 4.5). For O. pardalina larvae from the cold spring, critical temperature was 43 °С, while LT₅₀ was 41.2 °С. Although heat tolerances were in general correlated with the typical habitat temperatures of these larvae, most surprising is the huge gap (38 °C) between the natural habitat temperatures (6–10 °C) and critical temperatures for O. pardalina larvae and in this respect O. pardalina apparently greatly exceeds the other three highly eurythermal Stratiomyidae species (Fig. 4.12).

Exploring antibodies recognizing inducible members of Hsp70 family we performed broad-scale analysis of HSR in the Stratiomyidae species studied after different HS treatments. High levels of Hsp70 were present in the larvae of all four species at the temperatures of their habitats, and also at much lower temperatures (ca. 25 °C in the case of S. japonica larvae kept several weeks at this temperature in the laboratory). The constitutive concentrations of Hsp70 were approximately the same irrespective of the habitat temperatures of the studied species in nature. A 30-min heat shock treatment increased Hsp70 synthesis two to three-fold in S. japonica, maximally after 43 °C treatment. Accompanying the Hsp70 induction in this species are pronounced increases in small Hsps and Hsp68, and a small but significant increase in hsc70 concentration. By contrast, Hsp induction immediately after heat shock treatment is comparatively less in the two Crimean species, and hardly detectable in O. pardalina (Fig. 4.13). Notably, the accumulation of Hsp70 continues in the cells of the treated larvae for many hours after heat shock and plateaus approximately 24–36 h after the treatment.

Therefore, the pattern of Hsps in all studied Stratiomyidae species drastically differs from that of D. melanogaster and other Drosophila species where virtually no inducible Hsp70 is present at normal temperature while even mild HS induced dramatic increase in the synthesis of all groups of Hsps. Furthermore, the synthesis of all Hsps in Stratiomydae species in contrast to Drosophila species, lasts for many hours and reaches plateaus only after 24–36 h following HS. Intriguely, trypsin proteolysis and subsequent mass spectrometry showed that Stratiomyidae Hsp70 is much closer to the correspondent protein from the thermotolerant mosquito species Aedes aegypti than to Hsp70 from known species of Drosophila (Garbuz et al. 2008). Surprisingly, experiments exploring (³⁵S)L-methionine labeling failed to detect significant synthesis of Hsps in the control untreated larvae of the species studied.

Northern blot hybridization surprisingly failed to reveal any significant levels of either Hsp70 mRNA or small Hsps transcripts under control conditions in any Stratiomyidae species studied. Only RT-PCR studies were able to reveal weak leakage of Hsp70 genes in S. japonica under control conditions (Fig. 4.14). Heat shock, however, resulted in strong and rapid induction of corresponding mRNAs, with a maximum at 43–45 °C and an apparent drop of transcription at 47 °C. The latter temperature represents the LT₅₀ for this highly thermoresistant species and probably damages the transcription machinery. The Hsp70 mRNA synthesized in response to heat shock persists in the cells for many hours and is still detectable in trace amounts even 48 h after HS.

As indicated by electrophoretic mobility-shift assays, in contrast to certain desert lizards mentioned above (e.g. P. interscapularis), the active form of HSF is not detectable under control conditions in Stratiomys species studied. In response to temperature elevation, however, HSF as expected binds specifically to heat-shock element (HSE) and is able to transactivate the Hsp genes. Therefore, the surprisingly high concentration of Hsps observed in the cells of Stratiomyidae species without heat shock is not accompanied by significant transcription of corresponding genes under non-stress conditions. The phenomenon may be probably explained by very low but continuous expression of heat shock genes demonstrated by RT-PCR analysis or, less probably, by the synthesis of Hsps at some earlier stages of larval development when the larvae have been subjected to more severe shock. The last speculation seems less probable since it implies extremely high stability of Hsps in Stratiomyidae species. Furthermore, high concentration of Hsps has been detected in Stratiomyidae larvae kept for a month in the laboratory at 25, 31 or 37 °C.

Apparently, critical temperatures for (semi)aquatic larvae of this family are close to or slightly lower than 50 °C, and the larvae are unable to tolerate higher temperatures. According to Pritchard (1991), no insects were proved to live in hot springs above 50 °C, and very few, mostly Diptera, above 40 °C. Our results confirm this general conclusion.

The data obtained in our studies indicate that the inducible member of the Hsp70 family and other Hsps are continuously present at high concentrations in the larvae of four species of Stratiomyidae family, even in the absence of heat shock.

Characteristically, in contrast to the larvae, in the adult flies of the species investigated, immunoblotting revealed only trace amounts of Hsp70 under non-heat shock conditions.

The magnitude of thermotolerance observed in the investigated Stratiomydae species, in fact, exceeds that of most insect species studied, with Bombyx mori being a prominent exception (Evgen’ev et al. 1987). This comparative uniformity in the Stratiomydae species is exhibited despite differing natural thermal regimes, and includes LT₅₀ and critical temperatures. Mild heat shock (37 °C) pretreatment did not significantly improve tolerance for extreme temperatures in S. japonica larvae which, hence, seem not to have induced thermotolerance.

It is evident that, molecular mechanisms underlying up-regulation of heat shock proteins may differ characteristically depending on the organism. Thus, in desert lizards Phrynocephalus interscapularis, high constitutive levels of Hsps and correspondent mRNA are correlated with activation of HSF under control conditions, and may be further dramatically induced by heat shock treatment (Zatsepina et al. 2000). Apparently animals constantly subject to extreme temperatures have elaborated molecular mechanisms allowing them to maintain a relatively high Hsps level under normal conditions. In this way, they are preadapted for life under the conditions of constant stress without drastic changes in the house-keeping and Hsp genes activity.

The observed constitutive expression of Hsps in the cells of the Stratiomyidae larvae may help them to survive the diapause period spent at the larval stage.

Interestingly, the one Stratiomyidae species investigated that naturally occurs in a stable albeit cold thermal environment, O. pardalina, although exhibits significant increase in heat shock RNAs synthesis after temperature elevation, synthesized less Hsps than other thermotolerant species after high temperature challenge. The constitutive Hsps synthesis observed in this particular species suggests that extremely cold habitats, may be as challenging for proteins synthesis and homeostasis as in the case of cellular proteins functioning in aggressive extreme conditions of hot volcanic springs (see above Sect. 4.4).

In contrast to the larvae, adults (flies) of all Stratiomyidae species in our study which are able to escape from high temperature areas and, hence, are not subject to the severe conditions of hot or highly mineralized aquatic environments, and their survival apparently does not depend on maintaining high continuous levels of chaperones. The difference in the environment microhabitats probably accounts for the apparent dichotomy between larvae and adults in Hsp70 expression. It is of note, that such dichotomy in the synthesis of chaperones is even more pronounced in the cold-adapted Antarctic animals where larvae in contrast to adults completely lost the ability to activate their Hsps (see Sect. 4.4).

Regardless of the exact mechanism, the functionally extended presence of Hsps in the larvae of all Stratiomyidae species investigated may meet the increased chaperoning needs that these animals experience in response to adverse conditions of hot volcanic streams, highly mineralized Crimean lakes or cold springs in the environs of St.-Petersburg. This maintenance prevents cell injury and irreversible protein aggregation that may occur in response to these types of stress.

Interspecific comparison is a common approach in physiological ecology and other related fields of biology. However, several authors justly underlined many logical and statistical problems associated with using interspecific and in particular two-species comparisons for studying adaptations (Garland and Adolph 1994). In the case of Stratiomyidae species used in the present investigation we cannot state with certainty that exceptionally high thermotolerance and developmental dichotomy in terms of Hsps synthesis represent genetic adaptations in response to natural selection operated in the species studied (high and low temperatures and salt concentration). It is much more likely for this particular group that the observed adaptations to extreme conditions may be a characteristic feature of the ancestor of the whole Stratiomyidae family enabling the descendants successfully colonize various inhospitable environments. There are data suggesting that Mezozoic stratiomyids were already preadapted to colonize different types of extreme habitats, and such colonization occurred repeatedly in different genera (Rasnitsyn and Quicke 2002; Rozkošný 1997; Whalley and Jarzembowski 1985).

The performed investigation of a broader spectrum of Stratiomyidae and other Diptera species helped to check this preferred hypothesis.

For our analysis Diptera species belonging to different families and inhabiting highly variable environments were taken into analysis. Besides additional Stratiomyidae species i.e. Oplodonta viridula, Beris chalybata and Odontomia sp., we used two species of horse flies (Tabanidae) Haematopota pluvialis and Chrysops relictus and three species of biting midges belonging to Ceratopogonidae family (Dasyhelea modesta, Dasyhelea flavoscutellata and Dasyhelea sp. ex. gr. cinсta). The investigation of HSR response at the molecular level including synthesis and stability of Hsps and corresponding mRNAs in several Diptera species enables to describe features probably common to all Diptera as well as molecular adaptations restricted to certain families.

Oplodonta viridula, Beris chalybata, Chrysops relictus and Dasyhelea flavoscutellata belong to typical cold-adapted stenothermal species, Odontomia sp. and Haematopota pluvialis were collected in the Crimea in the same place as S. singularior and N. bipunctatus, while biting midges (Dasyhelea modesta and Dasyhelea sp. ex. gr. cinсta) dwell at the littoral area of Northern freshwater lakes. It was shown that all studied species are characterized by significant constitutive level of Hsp70 under physiological conditions of their correspondent niches. Characteristically, the horse flies (C. relictus) differ from the investigated representatives of Stratiomyidae and Drosophila by significantly higher stability of Hsp70 mRNA (Garbuz, Przhiboro, personal communication 2014).

Therefore, numerous Drosophila species characterized by completely repressed expression of inducible Hsps under normal conditions and strong activation of HSPs synthesis after temperature elevation rather represent exceptions when compared with other Diptera.

Notably, constitutive or chronic expression of Hsps, is not uniformly beneficial. For example, experiments on transgenic D. melanogaster strains with extra copies of Hsp70 genes (see Chap. 8) have demonstrated that overexpression of Hsp70 could be harmful and may increase lethality during development (Krebs and Feder 1997). Evidently the expression level of Hsps in each species and geographical population is a balance between benefits (high thermotolerance) and costs (e.g. negative impact of Hsps overexpression on growth, fertility and other characteristics). It is necessary to keep in mind that Drosophila species and other Diptera species used for comparison e.g. Stratiomyidae species greatly (many folds) differ by duration of life-span and this dramatic difference may be the major factor in the observed contrasting patterns of their heat shock response and apparent different sensibility to the constitutive presence of high concentration of Hsp70 in the cells.

In the studies exploring several phylogenetically distant Diptera species we described different patterns of Hsps expression in the families belonging to the order. Thus, while in all Drosophila species, the level of Hsps is not detectable under normal physiological conditions and increased dramatically as temperature increased, in the Stratiomyidae species studied we detected high constitutive level of inducible member of Hsp70 in the cells at normal temperature and HS treatment was able to induce only moderate increase in concentration of Hsps including Hsp70. Surprisingly, horse-flies (C. relictus) are characterized by high stability of Hsp70 mRNA after HS.

Finally, larvae of two species of the Chironomidae family dwelling in Antarctic regions and in North Russia exhibit very peculiar pattern of Hsp70 expression. Thus, in contrast to all species of other Diptera families mentioned above, in these species high constitutive level of Hsp70 mRNA was detected in the larvae under normal physiological conditions and HS treatment was ineffective in the induction of Hsp70 at either transcription or translation levels (Rinehart et al. 2006; Garbuz, Przhiboro personal communication 2014). Therefore, even within the same order (Diptera) different species have strikingly different patterns of heat shock response summarized in Table 4.1.

Table 4.1 Various levels of Hsp70 regulation in different Diptera species

Full size table

4.6 Special Cases

4.6.1 Stress Proteins in the Hibernating and Desiccating Organisms

Numerous studies demonstrated important protective role of various Hsps in preserving the integrity of transcription and translation machinery under stressful conditions including extreme ones. Naturally, soon after the discovery of Hsps their concentrations were monitored in “special cases” of adaptations, where organisms enter a diapause, anoxia or severe desiccation. Indeed, various groups of Hsps in many cases dramatically up-regulated in such organisms, at certain stages of their life-cycle associated with long-term anoxia or severe desiccation. Thus, cells of encysted embryos of the brine shrimp (Artemia franciscana) survive continuous anoxia for periods of years, during which their metabolic rate is undetectable (King et al. 2013). It was shown that abundant electron-dense granules consisting of the small heat shock/α-crystallin protein (p26) are present in the nuclei of anoxic embryos. Diapause-destined embryos of Artemia accumulate massive amounts of Hsp26 (up to 15 % of total cellular proteins) during the periods of continuous anoxia and this chaperone is not detected or induced in any other life-cycle stages (Beristain et al. 2010). Contrary to expectation, proteins of Hsp70 and Hsp90 families did not undergo anoxia-induced nuclear translocation in the cells of Artemia encysted embryos, an unusual result since induction of these Hsps and such translocations were often observed in cells from a variety of organisms experiencing adverse, stressful conditions. For example, Atlantic copepods of genus Calanus (Calanus finmarchicus) accumulate high quantities of small Hsps during diapause (Aruda et al. 2011). Furthermore, Tardigrade species (“slow steppers”) which have unique stress-adaptations that allow them to survive extreme heat, cold and radiation accumulate Hsps including several isoforms of Hsp70 together with glycogen, glycolipids and other specific agents during desiccation (Schill et al. 2004). It is known that low molecular Hsps at high concentration are able to convert the cytoplasm into gel-like matter which effectively preserves water and prevents proteins aggregation.

Furthermore, embryos of annual killifish Austrofundulus limnaeus, dwelling in the ephemeral lakes of Northern regions of South America accumulate large quantities of Hsp70 before entering the diapause during the periods of drying of their habitats. These diapausing embryos are highly resistant to a number of environmental insults such as high temperature, dehydration, anoxia, and increased salinity (Podrabsky et al. 2007). Interestingly, in this species embryo-specific form of inducible Hsp70 is expressed during embryonic development and is elevated during diapause. It was suggested that constitutive expression of Hsp70 during development may afford these embryos protection from environmental stresses during development more quickly than relying on the induction of a classic heat shock response.

The sleeping chironomid Polypedilum vanderplanki, inhabiting temporary water pools in semi-arid regions of Africa represents another example of possible involvement of Hsps in surviving severe water loss (Fig. 4.15). This chironomid species one of very few insects able to survive prolonged complete desiccation at larval stage, entering a state called anhydrobiosis. Surprisingly, even after years in a dry state, larvae are able to revive within a short period of time, completely restoring metabolism. In order to survive such extreme environments, organisms need to develop special adaptations both at physiological and molecular levels. The analysis performed demonstrated quite different regulation of Hsps in the course of dehydration and rehydration in the larvae. Heat-shock-responsive Hsp70 and Hsp60 genes of this organism showed a two-peak expression in dehydrating and rehydrating larvae. Both small alpha-crystallin heat shock proteins (sHsps) transcripts were accumulated in the desiccated larvae, but showed different expression profiles. Transcripts of Hsp23 was limited to the late stages of desiccation, suggesting possible involvement of this protein in the glass-state formation observed in anhydrobiotic larvae (Gusev et al. 2011). Furthermore, analysis of the sleeping chironomid genome showed intensive clustering-associated increase in the number of sHsps-coding genes associated with anhydrobiosis. The expression of the sHsps in these anhydrobiotic chironomid-specific clusters are tightly linked to the larvae stage and highly induced by desiccation (Gusev, personal communications).

Similarly, the important role of Hsps in maintaining cellular integrity and enzyme activity during desiccation and rehydration processes has been postulated in the flesh fly (Sarcophaga crassipalpis) (Hayward et al. 2004). The patterns of the observed Hsps induction characteristically differed in diapausing and nondiapausing pupae. Thus, in nondiapausing pupae, the expression of inducible Hsp23 and Hsp70 was upregulated by desiccation, while in diapausing pupae, the transcripts of these chaperones are already highly expressed and not further upregulated by desiccation.

The obtained results indicate distinct role for the different Hsps during desiccation stress and rehydration recovery (Hayward et al. 2004).

Up-regulation of various Hsps in response to hypothermy and various forms of oxidative stress usually observed at different stages of hibernation is not restricted to arthropods but was also detected in vertebrates (Sills et al. 1998). The investigation of the heat shock response in anoxia-tolerant turtle (Trachemys scripta elegans) demonstrated a strong fivefold increase in the amount of HSF1 under anoxic conditions and upregulation of five Hsps including Hsp70. The data demonstrate organ-specific regulation of Hsps induction during anoxia exposure and aerobic recovery in this turtle species which is probably represents an important aspect of cytoprotection with regard to underwater hibernation of these turtles in cold water (Krivoruchko and Story, 2010).

Mammalian species also explore modulation in the intensity of heat shock proteins synthesis in different phases of hibernation. Thus, compared with summer-active ground squirrels (Spermophilus tridecemlineatus), levels of the mitochondrial stress protein GRP75 were consistently higher in certain organs (e.g. intestinal mucosa) of hibernators in each hibernation state (Carey et al. 2000). Furthermore, ground squirrels increase Hsp70 family members as well as ubiquitin-protein conjugates during hibernation (Sills et al. 1998; Feder and Hofmann 1999). Interestingly, different organs of hibernating animal may exhibit a peculiar pattern of Hsps synthesis. Thus it was demonstrated that in the bat (Murina leucogaster) certain muscles did not show any sign of atrophy or tension reduction and this phenomenon correlates with elevated level of Hsp70 in the muscles of the hibernating bats (Lee et al. 2008).

4.6.2 The Role of Hsps in the Life-Cycle of Parasites

Parasites which represent another “special case” may synthesize various Hsps as cellular defense mechanism to survive drastic temperature changes they encounter during their life-cycle. Induction of certain groups of Hsps usually accompanies the infection of mammalian or avian hosts from ectothermal hosts such as insects or by free-living stages. In the case of free-living stages of parasites that do not involve an animal vector sometimes induction of Hsps accompanies the transition from environment to the homothermal host.

The transition from environment with low or temperate temperatures to homothermal host with internal temperature of 37 °C or above represents a characteristic life history feature of many parasitic organisms.

Multiple studies demonstrated developmentally regulated synthesis of Hsps during the life-cycle of parasites which may respond to profound temperature changes by synthesizing different quantities of certain Hsps depending on the variations in their host temperature and life stage. It was shown that different species of intracellular parasites in the genus Eimeria (Apicomplexa: Coccidiida) synthesize Hsp70 in the process of infection of mammalian host (mouse) (Clark et al. 1996). The levels of Hsp70 expression in sporozoites of a wild-type parent strain and two precocious lines of Eimeria tenella, were compared to investigate the relationship between the heat shock proteins expressed by the parasite and virulence of the strain. The synthesis of Hsp70, which was observed in the entire sporozoites of the wild strain, was drastically reduced to the anterior portion in the precocious lines. The level of Hsps may be dramatically increased in the course of host change, thus in Trypanosoma brucei transcription of Hsp70 and Hsp83 homologues is enhanced more than 100-fold after the parasite leaves the tsetse fly and enters the mammalian host likely due to cluster organization of Hsp70 genes providing rapid and high induction of these genes in the species (See Chap. 5) (Muhich and Boothroyd 1989).

Thus, Schistosoma mansoni actively synthesized Hsp70 after entering human skin from the water (Neumann et al. 1993). It was also shown that phagocytosis induce Hsp70 expression in Toxoplasma gondii. Interestingly, the induction of this chaperone is observed only in virulent strains of this parasite while avirulent forms either do not synthesize Hsp70 in the course of phagocytosis or synthesize low amounts of this protein (Lyons and Johnson 1995).

The malarial organism Plasmodium falciparum begins to synthesize Hsp70 at 39 °С, which correlates with periodical body temperature changes usually observed in the malaria patients (Biswas and Sharma 1994). Sometimes the pattern of the Hsps in parasite is organ- or tissue-specific. Thus, when the helminth Trichinella spiralis infects rodents, an enhanced expression of Hsp25 and Hsp60 and of these plus Hsp70 was observed at certain, yet different, time-points during infection in rat spleen and rat brain, respectively (Martinez et al. 1999). Multiple examples of developmentally regulated expression of different classes of Hsps in parasitic organisms during the transition to their hosts include parasitic nematodes, protists, cestode parasites and many other species (Biswas and Sharma 1994; Neumann et al. 1993).

It is evident that heat stress severity experienced by the parasites infected poikilothermal organisms such as reptiles or fish or insects should predominantly depend on the temperature of the host body which can vary dramatically.

Along these lines, it was shown that Leishmania species dwelling in the blood of thermoresistant desert toad-headed agama actively synthesize Hsps at 40 °C while in the nocturnal gecko all protein synthesis in the parasite cells is completely inhabited at 38 °C (Ul’masov et al. 1988). These experiments exploring different lizard species from thermally contrasting niches for the first time revealed coevolution of parasite-host system with respect to such vital trait as molecular response to temperature elevation (HS).

Notably, large quantities of Hsps usually produced by bacteria and parasites in the host cells after infection often elicit an immune response and may be useful in generating vaccines. Along these lines, it was shown that Leishmania infantum deletion mutant, lacking both Hsp70-II alleles (ΔHsp70-II), provided protection against Leishmania infection in the L. major-BALB/c infection model and can be a safe live vaccine as immunodeficient SCID mice, and hamsters, infected with mutant parasites did not develop any sign of pathology (Carrión et al. 2011; Folgueira et al. 2008). It was shown that tumour necrosis factor-alpha and phagocytosis induces expression of stress proteins in virulent promastigotes of Leishmania donovani(Salotra et al. 1995).

In general, parasites evolved to respond to temperature changes either by high constitutive synthesis of certain Hsps or by their rapid induction following the infection.

Likewise, the heat-induced disaggregase Hsp104 of Candida albicans plays a role in biofilm formation and pathogenicity in a worm infection model. Biofilm formation by cells lacking Hsp104 gene proved to be defective in different in vitro models. Thus, biofilms formed by the wild-type strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the Hsp104 mutant showed multiple structural defects and appeared patchy and loose. Apparently these defects result in decreased virulence of the Hsp104 mutant observed in the C. elegans infection model, hence, providing an indication of a role for Hsp104 in C. albicans virulence, in addition to its key role in the thermotolerance (Fiori et al. 2012).

Pathogenic bacteria such as Salmonella typhimurium or Listeria monocytogenes also actively synthesize various chaperones such as GroEL, GroES, DnaK and HtpG in response to phagocytosis and reactive oxygen species and certain stress proteins e.g. GroEL or DnaK may reach high concentration (Hanawa et al. 1999; Morgan et al. 1986; Wiesgigl and Clos 2001). Similarly, certain bacterial endosymbionts that infect various insect species such as bacterium Buchnera constitutively express a protein, symbionin belonging to Hsp60 (GroEL) family at very high levels as well as several other bacterial chaperones (Sato and Ishikawa 1997).

On the other hand, high conservatism exhibited by Hsps even between very distant phyla sometimes results in cross-reaction of the anti-bodies produced against bacterial Hsps with epitopes of orthologous host’s Hsps which may lead to severe autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (Tsoulfa et al. 1989; Panchapakesan et al. 1992).

4.6.3 Heat Shock Proteins in Defense

It is known that giant hornets (Vespa mandarinia japonica) represent natural enemies of the honeybees and specifically Japanese honeybees (Apis cerana japonica). Giant hornets often attack the hives, kill the bees and consume honey stored in the honeycomb. The bees are practically defenseless when attacked by hornets because they cannot pierce their thick chitin. However, the Japanese bees developed an effective defensive strategy for such cases. When the first hornets (“scouts”) appear, numerous bees (more than 500) form a ball around the intruder (Fig. 4.16). The temperature inside the bee balls reaches 46 °C and the giant hornets are killed in less than 10 min when they are trapped in a bee ball created by the Japanese honeybees. It was found that the CO₂ concentration inside the bee ball also reaches a maximum in the initial 0–5 min phase after bee ball formation. The lethal temperature of the bees is significantly higher than that of hornets (up to 49–50 °C) and it was concluded that CO₂ produced inside the bee ball by honeybees is a major factor together with the temperature involved in defense against giant hornets (Ono et al. 1995; Sugahara and Sakamoto 2009).

In experimental ecologically relevant episodes of hyperthermia between 33 and 50 °C, Hsp70 expression and Hsp70/Hsc70 activity in brains of nonflying laboratory-held bees increased by only two to three times at temperatures 46–50 °C. In this feature honey bee resembles other heat-resistant organisms with low heat shock mediated Hsp70 induction relatively to the basic Hsp70 level. Moreover, Hsp70 induction after such HS was practically undetectable in thoracic-flight muscles. These data suggest that certain honey bee tissues, especially flight muscles, are extremely thermotolerant. Furthermore, Hsp70 expression in the thoraxes of flight-capable bees is probably flight-induced by oxidative and mechanical damage to flight muscle proteins rather than temperature (Elekonich 2009).

A unique case of exploring high temperature for defense was described by Wehner et al. (1992) for desert ants Cataglyphis that are able to tolerate body temperature of 50 °C for at least 10 min. These ants presumably voluntarily undergo body temperature of 50 °C and more, probably to escape predators (Wehner et al. 1992).

Furthermore, it was shown in goldfish (Carassius auratus) that the expression of Hsp70 can be induced by the presence of a predator. Remarkably, when the fishes were exposed to a potential predator, bluegills (Lepomos macrochirus), they experienced an increase in Hsp70 mRNA level in the brain. When goldfish individuals were placed in a transparent tank around which bluegills were swimming, Hsp70 mRNA expression was significantly increased after 6 and 12 h. Notably, goldfish exposed to water circulating through a tank with several bluegills showed no sign of changes in Hsp70 mRNA expression. These results suggest that psychological stress may enhance Hsp70 mRNA expression in the brain just via visual perception and such presumably defensive response may somehow modulate the consequences of the stress (Kagawa and Mugiya 2000).

4.6.4 Hsps as Biomarkers of Environmental Pollution

Large scale field studies of stress proteins in various non model organisms indicated that heat shock proteins may be induced in response to abiotic contamination of the environment, in particular to different chemical pollutants. Basing on these data it was suggested to use Hsps as sensitive natural biomarkers providing information about current state of the environment, which may help to estimate the biological impact of toxic chemicals to live organisms and predict adverse consequences of such exposure. Such biological approach based on quantitative analysis of Hsps is often more sensitive and reproducible than direct monitoring of environmental contamination. There are three aspects of stress proteins that are essential for their use as biomarkers of pollution:

1.
Hsps are the important components of the cellular protective and adaptive response;
2.
synthesis of Hsps is likely to be induced by a large number of xenobiotics;
3.
different Hsps are highly conserved in all organisms from bacteria and plants to man and in this regard the same sets of antibodies (kits) can be used for their detection and measurement in various organisms (Bierkens 2000). The estimation of Hsps concentrations as biomarkers for understandable reasons is applied predominantly in marine and soil toxicology (Feder and Hofmann 1999).

It is of note, that measurements of Hsps level as a biomarker of pollution were already applied in a variety of organisms including: plants (algae) (Enteromorpha intestinalis); reef coral Montastraea franksi; clamps (Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus); planarian Dugesia (Girardia) schubarti; copepods (Tigriopus japonicas); amphipods (Gammarus roeseli); insect larvae (Chironomus tentans); fishes Cyprinodon nevadensis amargosa, Limanda limanda, Carassius carassius, zebrafish; ascidian Pseudodistoma crucigaster and many others (Bierkens 2000; Hallare et al. 2005; Lee et al. 2006; Lewis et al. 2001; Mićović et al. 2009; Rios-Sicairos et al. 2010; Venn et al. 2009; Wu et al. 1996).

In several cases, such field studies clearly demonstrated correlation between levels of different pollutants (especially heavy metals) and Hsps levels.

Thus, in Enteromorpha intestinalis Hsp70 expression was increased with copper exposure but was not unaltered following exposure to the herbicide Irgarol 1051 (Lewis et al. 2001). Strong induction of Hsp70 occurred in the blue mussels Mytilus edulis from Southern Baltic at high concentration of heavy metals. Combination of metals (Cd + Cu) was found to increase the Hsp70 level more intensely than related concentrations of singularly applied metals (Radłowska and Pempkowiak 2002). In Mytilus trossulus Hsp70 induction roughly correlated with arsenic and cadmium levels in water (La Porte 2005; Radłowska and Pempkowiak 1998). In the Mytilus galloprovincialis Hsp70 and metallothioneins were induced by heavy metals, Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution (Mićović et al. 2009).

To identify a sensitive biomarker of freshwater monitoring, pollutant-induced expression of heat shock proteins (Hsps) in the larvae of the aquatic midge Chironomus tentans (Diptera, Chironomidae) was evaluated. Different substances, such as nonylphenol, bisphenol-A, 17-alpha-ethynyl estradiol, bis(2-ethylhexyl)phthalate, endosulfan, paraquat dichloride, chloropyriphos, fenitrothion, cadmium chloride, lead nitrate, potassium dichromate, benzo[a]pyrene and carbon tetrachloride were used. The response of the Hsps gene expression to chemical exposure was rapid and sensitive to even very low chemical concentrations but it was not stressor specific. Interestingly, an increase in the expression of Hsps genes was observed not only for a stress inducible form (Hsp70), but also for a constitutively (HSC70) expressed form (Lee et al. 2006).

Furthermore, zebrafish embryos can be used for water quality assessing with detecting of Hsp70 levels as a biomarker. To evaluate the suitability of these tests for environmental screening, fertilized zebrafish eggs were exposed to water samples collected from five sites of varying levels of pollution from Laguna Lake, Philippines. Reconstituted water was used as laboratory control while water samples from a highly polluted freshwater source were used as a positive control. The levels of Hsp70 in embryos, exposed in water from two sites located closest to Manila, the Philippine capital (Northern West Bay and Central West Bay), showed a pronounced Hsp70 elevation relative to the control probes (Hallare et al. 2005).

Similarly, it was shown that kidney Hsp30 and liver Hsp70 expression can serve as sensitive biomarkers for the presence of field environmental stressors in wild crucian carp populations (An et al. 2014). In field populations of the dab (Limanda limanda) Hsp70 levels correlated with DNA damage level caused by stress conditions (Schröder et al. 2000).

At the present time highly sensitive and cheap RT-PCR approach is commonly used to estimate the levels of Hsp genes expression under various environmental conditions. Thus, hepatic expression of Hsp70 and cytochrome P450 1A mRNAs in white mullet (Mugil curema) monitored by RT-PCR from July, 2005 until July, 2006 in three coastal lagoons located in the Southern Gulf of California, Mexico, was higher for both genes in the Urias Estuary, which was considered the most polluted among the three systems, especially during the rainy season (summer to fall). These results indicate that fishes Mugil curema is a good candidate species to implement biomonitoring programs in tropical coastal environments (Rios-Sicairos et al. 2010).

It is well known that the impacts of marine pollutants on reef corals and their symbiotic algae are an important element of global coral reef decline. It was shown that Hsp70, and to a lesser extent Hsp90 expression increases significantly following exposure to two major marine toxic pollutants (copper and dispertant Corexit 9527) and, hence, their levels may serve as a good indicator of specific coral reef state (Venn et al. 2009).

Hsps as biomarkers in field bioassays were sometimes used in terrestrial invertebrates. Specifically, the Hsp70 response was investigated in isopods Oniscus asellus exposed to a heavy metal gradient around smelters near Avonmouth, UK. In the sites closest to the smelter, O. asellus showed highest Hsp70 levels. The authors conclude that Hsp70 level in isopod species can be a suitable biomarker of heavy metal pollution in metal-contaminated field sites (Arts et al. 2004).

Likewise, small heat shock protein 20 of the intertidal copepod Tigriopus japonicus can be used as a possible biomarker for exposure to endocrine disruptors (Seo et al. 2006).

Robert Tanguay and his group were the first to use the levels of autoantibodies against Hsp27, Hsp60 and Hsp70 for evaluation of deleterious effects of various chemicals and heat on the health state of workers of harmful manufactures, such as chemical industry or steelmaking enterprise (Wu et al. 1996, 1998; Yang et al. 2007).

The goal of these studies was to evaluate lymphocyte and/or plasma Hsp70 levels and levels of autoantibodies against Hsp70 as biomarkers for assessing exposure response of steel workers to complex coke oven emissions (COEs). It was shown that lymphocyte Hsp70 levels increased in the group with intermediate exposure to emissions but decreased in the high-exposure group (Yang et al. 2007). Presence of autoantibodies to heat stress proteins was detected in workers exposed to high temperature and carbon monoxide. Antibodies to Hsp27 and Hsp71 were found more frequently in the high temperature and carbon monoxide-exposed groups than in controls. Anti-Hsp60 antibodies were only detected in workers exposed to high temperature or carbon monoxide (Wu et al. 1996). Exposure to benzene induces autoantibodies to Hsp72 (Wu et al. 1998).

Despite the obvious benefits the wide use of Hsps levels measurements as biomarkers of environmental pollution is by hindered for several reasons. First of all since the induction of Hsps is as a rule is unspecific for different stresses, sometimes it is difficult or impossible to explain the observed increase to a definite stressor. Second, usually organisms in nature undergo multistress challenges. For example, stress from herbicides may be influenced by the temperature or humidity elevation. Furthermore, sometimes Hsps expression may be modified by certain uncontrolled factors such as parasites contamination. For example, in amphipod Gammarus roeseli Hsp70 level increases significantly at response to heavy metals (Pd), but in the case of infection with cystacanths of the acanthocephalan (Polymorphus minutus) infected individuals did not exhibit Hsp70 induction (Sures and Radszuweit 2007). Therefore, the monitoring of Hsps levels in field populations should be always carried out in parallel with measurements of many other environmental parameters that may strongly influence the results of such measurements. Specifically, in articles cited above the changes in Hsps concentrations in response to pollution were shown to correlate with: rate of growth (Lewis et al. 2001); cytochrome P450 level (Rios-Sicairos et al. 2010); P-glycoprotein level (Venn et al. 2009); metallothioneins level (Mićović et al. 2009); hemoglobin gene expression (Lee et al. 2006); embryonic lethality (Hallare et al. 2005); alanine aminotransferase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) activity (Wu et al. 1996).

4.7 Peculiar Structure and Functions of Cellular Proteins in Animals from Thermally Contrasting Environments

Apart from exploring a very ancient and universal system of heat shock genes found in all organisms studied so far, in the species dwelling under extremely cold or xeric conditions many cellular house-keeping proteins underwent adaptive evolution and acquired specific traits combining molecular stability on the one hand and structural flexibility on the other. The investigation of many enzymes from the organisms inhabiting thermally contrasting ecological niches established the correlation between the average temperature of environment and proteins stability. Thus, blue mussel (Mytilus galloprovincialis), a native of the Mediterranean Sea, is displacing the native blue mussel (Mytilus trossulus) from its habitat in central and Southern California apparently due to physiological adaptations. These adaptations enabling the former species to perform better at high temperatures include the structure and function of the enzyme malate dehydrogenase (cMDH) (Fields et al. 2001). The demonstrated relative warm adaptation of M. galloprovincialis cMDH may be one of physiological features that increase the competitive ability of the invasive species in warm habitats (Fields et al. 2001). Interestingly, these differences consistent with warm adaptation result from minor changes in sequence: the M. trossulus ortholog differs from M. galloprovincialis ortholog by only two substitutions.

Likewise, studies of orthologs of lactate dehydrogenase (LDH) and other vital enzymes performed in different laboratories have revealed the importance of conserving kinetic properties and structural stability during adaptation to temperature, and recently identified the types of amino acid substitutions causing such adaptive variations of proteins in ecological aspect (see below).

There are also a number of examples of qualitative adaptive strategies, when expression of distinctly different isoenzymes contributes to seasonal thermal adaptation by adjusting a particular metabolic node to new environmental conditions. Such isoforms were described for acetylcholine esterase, ferritin, choline esterase, LDH and many other enzymes (see Zakhartsev et al. 2007).

It was also shown that stabilization of proteins tertiary structure preventing their heat denaturation in thermoresistant species was usually achieved by means of specific amino acid substitutions resulting in the formation of additional intramolecular links in the vicinity of active center which stabilize the enzyme structure (Fields 2001).

As a result, in organisms resistant to heat proteins denaturation and misfolding begins at significantly higher temperatures and, hence, the activation of HSF after the disruption of HSF-Hsp90 complexes and beginning of abundant Hsps synthesis occur at much higher temperatures in comparison with related thermosensitive species.

The investigation of various (mostly fungi) thermophiles helps to understand major trend in proteins evolution to cope with constant high temperature conditions. Although proteomes of highly thermophilic prokaryotes provided a lot of valuable information in this respect and were successfully exploited in biotechnology, many proteins required for eukaryotic cell functioning under high temperature conditions are absent from bacteria or archaea. Recently, the comparative analysis of the first genome of the thermophilic fungi (Chaetomium thermophilum) with the genomes of two mesophilic species (Thielavia terrestris and Thielavia heterothallica) revealed consistent amino acid substitutions apparently associated to thermophily. Importantly, the same substitutions were also found in an independent lineage of thermophilic fungi (Van Noort 2013). The most consistent pattern observed in almost all fungal lineages of the thermophilic genomes is the substitutions of lysine by arginine and tryptophane (Van Noort 2013).

Furthermore, whole-genome analysis allowed to conclude that in the case of hyperthermophilic archaea the encoded proteins are characterized by an increase in frequency of charged amino acid residues and a decrease in that of polar uncharged residues as compared to the mesophilic counterparts. However, a discrepancy is noticeable in the overrepresentation of positively and negatively charged residues in hyperthermophilic proteins. The percentage of positively charged amino acids residues is significantly higher in hyperthermophiles compared to mesophiles. Furthermore, there are significant increases in aromaticity and average hydrophobicity and a decrease in the usage of polar uncharged amino acid residues (Ser, Thr, Gln and Asn) in thermophilic proteins. Examination of surface charge distribution reveals a marked increase of positive charge in the surfaces of thermophilic proteins as compared to their mesophilic counterparts (Das et al. 2006; Haney et al. 1999; Nakashima et al. 2003). These features apparently determine the extremely high thermal stability of thermophilic arhaea proteins and their ability to function at high temperatures up to near 100 °C. In general, thermophilic archaea demonstrate many adaptive differences from other organisms living in ordinary conditions, non-connected with protein structure and chaperone functions, in particular unusual structure of membrane phospholipids, DNA supercoiling and many others (Mirambeau et al. 1984; van de Vossenberg et al. 1998).

Although Hsps and in particular Hsp70 are highly conserved in close species there are data implying that temperature selection may modulate the structure and function of the chaperones depending on specific thermal habitats. It was shown in a few studies that orthologous variants of molecular chaperone from differential thermally adapted species significantly vary in their thermal responses. Thus, comparison of Hsc70 orthologs from polar and temperate notothenoid fishes differ in their ability to prevent thermal aggregation of lactate dehydrogenase (Place and Hofmann 2005). Hsc70 from the Antarctic species lost the ability to completely refold denatured LDH at a lower temperature in comparison with Hsc70 from the temperate species. Likewise, Hsc70 from highly eurythermal marine goby (Gillichthys mirabilis) exhibits temperature insensitivity across the range of temperatures that are ecologically relevant for this species during luciferase refolding assays (Place and Hofmann 2001).

These data suggest that structure and functions of chaperones maps onto the thermal history of host species and that temperature selection acted on their molecular structure.

4.8 Conclusions

The broad scale analysis performed herein includes the data obtained in various organisms belonging to different taxa from mollusks and insects to mammalian species that dwell under highly variable and sometimes aggressive habitats. The accumulated evidence enables to conclude that certain chaperone-dependent molecular adaptations may be common for highly phylogenetically diverse organisms while others may be restricted to a specific group or environment. The major established regularities are summarized as follows:

1.
Most thermoresistant organisms are characterized by comparatively high constitutive level of Hsp70 and other Hsps in relation to close forms from temperate regions. Such preparative defense strategy provides certain degree of preadaptation at the cellular and organism levels and usually does not require massive Hsps induction under conditions of moderate heat stress in comparison with thermosensitive forms (Fig. 4.17).
Fig. 4.17
Typical patterns of Hsp70 induction in thermosensitive and thermoresistant organisms. HSR of two thermally contrasting mollusk species are given as example. Two species of limpets (L. pelta and L. scabra) dwelling in thermally contrasting biotopes were chosen for comparison. Thermoresistant species (i.e. L. scabra) usually exhibit high constitutive Hsp70 level and modest induction after HS. In contrast, thermosensitive species (i.e. L. pelta) are characterized by low Hsp70 levels and strong induction after temperature elevation (Adapted from Dong et al. 2008 with permission)
Full size image
2.
High constitutive levels of Hsp70 and other chaperones often observed in highly eurythermic and/or thermophilic forms may be regulated at different levels that include transcription efficacy; Hsp genes copy number, and stability of correspondent mRNAs or chaperones themselves. Specific mechanisms underlying constitutive and inducible levels of Hsps may vary within the same order (e.g. Diptera).
3.
Different organisms explore members of specific Hsps families or their combinations (Hsp70, small Hsps or Hsp40) to respond to stressful conditions and relative role of particular chaperones or their isoforms strongly depends on the severity of the challenge
4.
Activation of HSF and induction of Hsps synthesis (threshold) in heat tolerant organisms occurs at higher temperatures in comparison with related thermosensitive forms, which implies that cellular proteins from thermophilic organisms as well as transcriptional and translational machinery in general are more stable than their mesophilic counterparts.
5.
Insect life stages (e.g. larvae versus adults) often differ significantly in the heat shock response and thermoresistance. Thus, larvae of certain stenothermal cold-adapted insect species which constitutively expressing Hsp70, partially or completely lost the ability to activate Hsps synthesis after HS, while their adults preserved this ability.
6.
Organisms entering certain life-stages to survive extreme conditions (desiccation, anabiosis, diapause etc.) usually accumulate large amounts of certain Hsps necessary for survival and subsequent recovery.
7.
Hsps may be involved in pathogenicity of certain parasites species.
8.
Different Hsps may be used as biomarkers of environmental pollution.

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Michael B. Evgen’ev, David G. Garbuz & Olga G. Zatsepina

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Evgen’ev, M.B., Garbuz, D.G., Zatsepina, O.G. (2014). Heat Shock Proteins and Adaptation to Variable and Extreme Environments. In: Heat Shock Proteins and Whole Body Adaptation to Extreme Environments. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-9235-6_4

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