Heat shock proteins in the mechanisms of stress adaptation in Baikal amphipods and Palaearctic <Emphasis Type="Italic">Gammarus lacustris</Emphasis> Sars II. Small HSP family

The role of low-molecular-weight (small) heat shock proteins (sHSPs) in the mechanisms of thermal and toxic resistance in fresh-water organisms was investigated. The four endemic species of Lake Baikal: Gmelinoides fasciatus (Stebb.), Eulimnogammarus cyaneus (Dyb.), E. vittatus (Dyb.), and Ommatogammarus flavus (Dyb.), and Gammarus lacustris Sars, a representative of the Palaearctic fauna, were studied. The effect of the temperature factor was evaluated by exposure of amphipods to temperatures of 20, 25, and 30°C, and the effect of the toxic factor, by exposure to cadmium chloride solutions with concentrations of 50, 10, 5, 0.5, and 0.05 mg/l. All the species showed a common trend of increasing content of sHSPs; species-specific features were observed in the synthesis of the proteins. It was concluded that sHSPs participate in the mechanisms of thermal and toxic resistance in the investigated amphipod species.     

Attitude of Baikal and Palearctic amphipods to oxygen as a factor of environment and mechanism of adaptation to its lowered level

The paper presents the materials of a comparative study of characteristics of anaerobic mechanisms use under the conditions of lowered oxygen content by Baikal amphipods Eulimnogammarus vittatus (Dyb.), E. cyaneus (Dyb.), as well as by Palearctic Gammarus lacustris Sars. It is shown that the system of anaerobic metabolism works most effectively for Palearctic G. lacustris. This species is characterized by the least degree of use of low-efficient way of energy production—anaerobic lipolysis, and the highest degree of use of the most efficient way of energy production under the conditions of low oxygen content-anaerobic succinate production. For the Baikal species that are less resistant to the lowered level of oxygen we show more expressed induction of anaerobic lipolysis and lower degree of engagement of anaerobic succinate production.     

Antioxidant response to natural organic matter (NOM) exposure in three Baikalean amphipod species from contrasting habitats

The aim of the present work is to comparatively evaluate the oxidative stress response on exposure to natural organic matter (NOM) in three amphipod (Crustacea, Amphipoda) species from different taxonomic groups and different habitats of Lake Baikal. Endemic species from Lake Baikal were used: the shallow-water dwelling Gmelinoides fasciatus (Dyb.), Pallasea cancelloides (Gerstf.), and the deep-layer inhabitant Ommatogammarus flavus (Dyb.). Three key enzymes, catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST), were studied. The applied NOM from Lake Schwarzer (Germany) directly impacts the two littoral species which quickly respond. The response is characterized by a significant decrease of POD and an increase of CAT activities. GST activity remains stable or decreased slightly. In contrast to the littoral amphipods, the deep-layer inhabitant O. flavus showed no significant reaction to NOM exposure, probably due to decreased adaptive ability of this species. The stable environment of the Baikalean deep zones obviously does not provide triggers for the development of flexible antioxidant or general defense systems.     

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal

We studied various aspects of heat‐shock response with special emphasis on the expression of heat‐shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5‐fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.     

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

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

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

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

A transcriptome-wide assessment of differentially expressed genes among two highly divergent,yet sympatric,lineages of the freshwater Atyid shrimp, <Emphasis Type="Italic">Paratya australiensis</Emphasis>

The current study evaluated the possible toxic effects of the water-soluble fraction of crude oil on the general cellular stress-response mechanisms of two dominant representatives of Lake Baikal’s littoral community, the endemic amphipod species Eulimnogammarus verrucosus and E. cyaneus. The acute toxicity effects on the cellular stress-response mechanisms of amphipods were studied in the laboratory by exposing amphipods in water from Lake Baikal to addition of a water-soluble fraction of crude oil at concentrations considered safe for the aquatic environment. The present study found that even short-term exposure to a water-soluble fraction of crude oil at concentration of 50 µg/L, established as the threshold limit for fishery and aquaculture water reservoirs in the Russian Federation, directly affected the general stress-response markers HSP70 and lipid peroxidation and significantly changed the activity of antioxidant enzymes in both studied species. This result confirms the high sensitivity of Baikal endemics to crude oil. Thus, it also indicates that established standards and threshold limit values of oil concentrations estimated for ecological monitoring of general water reservoirs cannot be applied directly to the unique Lake Baikal ecosystem.     

Changes in concentrations of circulating heat‐shock proteins in House Finches in response to different environmental stressors

Heat‐shock proteins (HSP) are molecular chaperones that play key roles in the maintenance of cellular homeostasis under variable environmental conditions. Although HSP are frequently used in studies of wild vertebrates as indicators of stress, no one to date has assessed responses of HSP60, HSP70, and HSP90 in the same species to different environmental stressors. We studied changes in the circulating concentrations of HSP60, HSP70, and HSP90 in wild‐caught House Finches (Haemorhous mexicanus) in response to multiple and sequential stressful environments, including high temperatures, transportation, and pathogen exposure. House Finches sampled during a period of low‐environmental stress with moderate ambient temperatures had low levels of HSP60 and modest levels of HSP70 and HSP90 compared to birds sampled during a presumably more stressful period with high temperatures. After exposure to high‐ambient temperatures, transportation in a vehicle, and exposure to Mycoplasma gallisepticum, captive finches were found to have increasingly higher levels of HSP60. HSP70 tended to rise in response to each stressor, but to drop in the weeks between stress challenges. HSP90 levels increased significantly only in response to pathogen challenge. Our observations suggest that HSP60 and HSP70 are indices of a range of stressors in House Finches, whereas HSP90 primarily reflects health state.     

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

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

Mechanisms of antioxidant system activation in freshwater amphipoda: II. Baikal littoral <Emphasis Type="Italic">Eulimnogammarus cyaneus</Emphasis> (Dyb.)

The goal of the work is to understand the mechanisms of activation of the antioxidant system under the oxidative action of endogenous character in the Baikal endemic species Eulimnogammarus cyaneus (Dyb.). The oxidative action was induced by exposing the amphipoda in the solutions of a laboratory humin-containing preparation. Experiments showed that two different mechanisms can trigger the antioxidant system in the Baikal amphipoda of E. cyaneus. Depending on the conditions and duration of preliminary acclimatization, a change of the activation mechanism of antioxidant protection or its separate elements is observed. For the organisms with the duration of preliminary acclimatization within three days, we observed the involvement of a specific mechanism allowing efficient elimination of the formed AFK in the case of low concentrations of humin-containing preparation. This mechanism is characterized by the inhibition of enzymatic activity of peroxidase and glutathione-S-transferase, as well as by a decrease in the level of hydrogen peroxide. In the Baikal amphipoda kept for a long time in an artificial environment, the oxidant activates the mechanism of protection similar to that observed previously for the Palearctic species. This mechanism is characterized by an increase of the enzymatic activity of peroxidase, a decrease of the activity of catalase, and the absence of inhibition of glutathione-S-transferase.     

A new species of grayling <Emphasis Type="Italic">Thymallus svetovidovi</Emphasis> sp. nova (Thymallidae) from the Yenisei basin and its position in the genus <Emphasis Type="Italic">Thymallus</Emphasis>

The Upper Yenisei grayling Thymallus svetovidovi sp. nova that inhabits the headwaters of the Yenisei River in Mongolia is described. From the other representatives of the genus Thymallus, the species differs in the elements of body coloration, dorsal fin pattern, some morphometric characters, and genetic characteristics. Besides this species, most of the Yenisei basin is inhabited by Baikal grayling T. baicalensis Dyb., and its low reaches, by the Arctic grayling T. arcticus (Pall.).     

Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791)

For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.     

Metabolic regulation of polyamines and γ‐aminobutyric acid in relation to spermidine‐induced heat tolerance in white clover

• Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance.
• Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ‐aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT‐PCR, were applied.
• Results showed that exogenous Spd significantly alleviated heat‐induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up‐regulating genes encoding Chl synthesis (PBGD and Mg‐CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up‐regulated HSP70, HSP70B and HSP70‐5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress.
• In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

     

Two Radix spp. (Gastropoda: Lymnaeidae) endemic to thermal springs around Lake Baikal represent ecotypes of the widespread Radix auricularia

In this study, we re‐examine two species of freshwater gastropods of the genus Radix Montfort, 1810 (family Lymnaeidae), endemic to the geothermal springs in the Lake Baikal region in the southern part of eastern Siberia — Lymnaea (Radix) hakusyensis Kruglov et Starobogatov, 1989, and Lymnaea (Radix) thermobaicalica Kruglov et Starobogatov, 1989. The alleged species status of these endemics has been re‐assessed by means of an integrative approach combining molecular genetic taxonomy techniques with the traditional methods based on shell and soft body morphology. Phylogenetic reconstructions were made using both mitochondrial (COI) and nuclear (ITS2) DNA markers. We used topotypic samples of both species and specimens sampled from other sites around Lake Baikal. The results demonstrate that the two endemic species are only synonyms of a widespread Holarctic species, Radix auricularia (Linnaeus, 1758), and represent its intraspecific morph (ecotype) adapted to living in thermal springs. A new synonymy is proposed: Thermoradix Kruglov et Starobogatov, 1989 = Radix Montfort, 1810 (syn. n.).     

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms.     

Evolution of the Response to Heat Shock in Genus Drosophila

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