Eulimnogammarus Cyaneus Research Articles

Cold-adapted amphipod species upon heat stress: Proteomic responses and their correlation with transcriptomic responses

The cellular heat shock response (HSR) comprises transcriptomic and proteomic reactions to thermal stress. It was here addressed, how the proteomic, together with the transcriptomic HSR, relate to the thermal sensitivities of three cold-adapted but differently thermo-sensitive freshwater amphipod species. The proteomes of thermosensitive Eulimnogammarus verrucosus and thermotolerant Eulimnogammarus cyaneus, both endemic to Lake Baikal, and of thermotolerant Holarctic Gammarus lacustris were investigated upon 24 h exposure to the species-specific 10 % lethal temperatures (LT10). Furthermore, correlations of heat stress induced changes in proteomes (this study) and transcriptomes (previous study with identical experimental design) were examined. Proteomes indicated that the HSR activated processes encompassed (i) proteostasis maintenance, (ii) maintenance of cell adhesion, (iii) oxygen transport, (iv) antioxidant response, and (v) regulation of protein synthesis. Thermo-sensitive E. verrucosus showed the most pronounced proteomic HSR and the lowest correlation of transcriptomic and proteomic HSRs. For proteins related to translation (ribosomal proteins, elongation factors), transcriptomic and proteomic changes were inconsistent: transcripts were downregulated in many cases, with levels of corresponding proteins remaining unchanged. In the Eulimnogammarus species, levels of hemocyanin protein but not transcript were increased upon heat stress, suggesting a HSR also directed to enhance oxygen transport. Thermosensitive E. verrucosus showed the most pronounced relocation of transcription/translation activity to proteostasis maintenance, which may indicate that the general species-specific stability of protein structure could be a fundamental determinant of thermotolerance. By combining transcriptomic and proteomic response data, this study provides a comprehensive picture of the cellular HSR components in the studied amphipods.

Diet affects body color and energy metabolism in the Baikal endemic amphipod <em>Eulimnogammarus cyaneus</em> maintained in laboratory conditions

Proper diet is critical for laboratory-reared animals, as it may affect not only their welfare, but also experimental results. Amphipods (Crustacea: Amphipoda) play important roles in ecosystems and are often used in environmental research. Endemic amphipods from the ancient Lake Baikal are promising for laboratory bioassays; however, there are currently no laboratory cultures. In this work, we determine how different diets affect the color and metabolism of a laboratory-reared Baikal amphipod, Eulimnogammarus cyaneus. We found that in freshly collected blue-colored animals, body color correlated with total carotenoid content. Total carotenoid levels did not differ after long-term (two months) feeding with a close to natural carotenoid-enriched, or even a carotenoid-depleted diet. Nevertheless, antennae color was closer to red in the natural-like diet group. It is likely that the carotenoids from the commercial diet are not properly metabolized in E. cyaneus. The animals fed commercial diets had a higher glycogen content, which may signify a higher metabolic rate. Overall, we show that a carotenoid-enriched diet optimized for decapods is not optimal for amphipods, likely due to different carotenoid compositions, and the diet for long-term rearing of E. cyaneus and other Baikal amphipods requires supplementation.

Different ways to play it cool: Transcriptomic analysis sheds light on different activity patterns of three amphipod species under long-term cold exposure.

Species of littoral freshwater environments in regions with continental climate experience pronounced seasonal temperature changes. Coping with long cold winters and hot summers requires specific physiological and behavioural adaptations. Endemic amphipods of Lake Baikal, Eulimnogammarus verrucosus and Eulimnogammaruscyaneus, show high metabolic activity throughout the year; E.verrucosus even reproduces in winter. In contrast, the widespread Holarctic amphipod Gammarus lacustris overwinters in torpor. This study investigated the transcriptomic hallmarks of E.verrucosus, E.cyaneus and G.lacustris exposed to low water temperatures. Amphipods were exposed to 1.5°C and 12°C (corresponding to the mean winter and summer water temperatures, respectively, in the Baikal littoral) for one month. At 1.5°C, G.lacustris showed upregulation of ribosome biogenesis and mRNA processing genes, as well as downregulation of genes related to growth, reproduction and locomotor activity, indicating enhanced energy allocation to somatic maintenance. Our results suggest that the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the preparation for hibernation; downregulation of the actin cytoskeleton pathway genes could relate to the observed low locomotor activity of G.lacustris at 1.5°C. The differences between the transcriptomes of E.verrucosus and E.cyaneus from the 1.5°C and 12°C exposures were considerably smaller than for G.lacustris. In E.verrucosus, cold-exposure triggered reproductive activity was indicated by upregulation of respective genes, whereas in E.cyaneus, genes related to mitochondria functioning were upregulated, indicating cold compensation in this species. Our data elucidate the molecular characteristics behind the different adaptations of amphipod species from the Lake Baikal area to winter conditions.

Low annual temperature likely prevents the Holarctic amphipod Gammarus lacustris from invading Lake Baikal

Species with effective thermal adaptation mechanisms allowing them to thrive within a wide temperature range can benefit from climatic changes as they can displace highly specialized species. Here, we studied the adaptive capabilities of the Baikal endemic amphipods Eulimnogammarus verrucosus (Gerstfeld, 1858) and Eulimnogammarus cyaneus (Dybowsky, 1874) compared to the potential Holarctic Baikal invader Gammarus lacustris Sars, 1863 at the cellular level including the energy metabolism and the antioxidant system. All species were long-term exposed to a range of temperatures (1.5 °C to mimic winter conditions and the three species-specific preferred temperatures (i.e., 6 °C for E. verrucosus, 12 °C for E. cyaneus and 15 °C for G. lacustris). At 1.5 °C, we found species-specific metabolic alterations (i.e., significantly reduced ATP content and lactate dehydrogenase activity) indicating limitations on the activity level in the Holarctic G. lacustris. Although the two Baikal endemic amphipod species largely differ in thermal tolerance, no such limitations were found at 1.5 °C. However, the cold-stenothermal Baikal endemic E. verrucosus showed changes indicating a higher involvement of anaerobic metabolism at 12 °C and 15 °C, while the metabolic responses of the more eurythermal Baikal endemic E. cyaneus may support aerobic metabolism and an active lifestyle at all exposure temperatures. Rising temperatures in summer may provide a competitive advantage for G. lacustris compared to the Baikal species but the inactive lifestyle in the cold is likely preventing G. lacustris from establishing a stable population in Lake Baikal.

Thermal reaction norms of key metabolic enzymes reflect divergent physiological and behavioral adaptations of closely related amphipod species

Lake Baikal is inhabited by more than 300 endemic amphipod species, which are narrowly adapted to certain thermal niches due to the high interspecific competition. In contrast, the surrounding freshwater fauna is commonly represented by species with large-scale distribution and high phenotypic thermal plasticity. Here, we investigated the thermal plasticity of the energy metabolism in two closely-related endemic amphipod species from Lake Baikal (Eulimnogammarus verrucosus; stenothermal and Eulimnogammarus cyaneus; eurythermal) and the ubiquitous Holarctic amphipod Gammarus lacustris (eurythermal) by exposure to a summer warming scenario (6–23.6 °C; 0.8 °C d−1). In concert with routine metabolic rates, activities of key metabolic enzymes increased strongly with temperature up to 15 °C in E. verrucosus, whereupon they leveled off (except for lactate dehydrogenase). In contrast, exponential increases were seen in E. cyaneus and G. lacustris throughout the thermal trial (Q10-values: 1.6–3.7). Cytochrome-c-oxidase, lactate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase activities were found to be higher in G. lacustris than in E. cyaneus, especially at the highest experimental temperature (23.6 °C). Decreasing gene expression levels revealed some thermal compensation in E. cyaneus but not in G. lacustris. In all species, shifts in enzyme activities favored glycolytic energy generation in the warmth. The congruent temperature-dependencies of enzyme activities and routine metabolism in E. verrucosus indicate a strong feedback-regulation of enzymatic activities by whole organism responses. The species-specific thermal reaction norms reflect the different ecological niches, including the spatial distribution, distinct thermal behavior such as temperature-dependent migration, movement activity, and mating season.

Proteomics reveals sex-specific heat shock response of Baikal amphipod Eulimnogammarus cyaneus

The ancient Lake Baikal is the largest source of liquid freshwater on Earth and home to a unique fauna. Several hundred mostly cold-adapted endemic amphipod species inhabit Baikal, an ecosystem that is already being influenced by global change. In this study, we characterized the core proteome and heat stress-induced changes in a temperature-tolerant endemic amphipod, Eulimnogammarus cyaneus, using a proteogenomic approach (PRIDE dataset PXD013237) to unravel the molecular mechanisms of the observed adverse effects. As males were previously found to be much more tolerant to thermal stress, we placed special emphasis on differences between the sexes. For both sexes, we observed adaption of energy metabolism, cytoskeleton, lipid, and carbohydrate metabolism upon heat stress. In contrast, significant differences were determined in the molecular chaperone response. Females from the control conditions possessed significantly higher levels of heat shock proteins (HSP70, HSPb1, Hsc70-3), which, in contrast to males, were not further increased in response to heat stress. The inability of females to further increase heat shock protein synthesis in response to temperature stress may be due to sex-specific processes, such as egg production, requiring a large proportion of the available energy. As ovigerous females synthesize generally higher amounts of protein, they also need higher levels of molecular chaperones for the folding of these new proteins. Thus, the higher sensitivity of females to heat shock may be due to the lack of molecular chaperone molecules to counteract the heat-induced protein denaturation.

The level of putative carotenoid-binding proteins determines the body color in two species of endemic Lake Baikal amphipods.

Color is an essential clue for intra- and interspecies communication, playing a role in selection and speciation. Coloration can be based on nanostructures and pigments; carotenoids and carotenoproteins are among the most widespread pigments in animals. Over 350 species and subspecies of amphipods (Crustacea: Amphipoda) endemic to Lake Baikal exhibit an impressive variability of colors and coloration patterns, including intraspecific color morphs. However, the mechanisms forming this diversity are underexplored, as while the carotenoid composition of several transparent, green, and red species was investigated, there have been no reports on the corresponding carotenoid-binding proteins. In this work, we analyze the coloration of two brightly colored Baikal amphipods characterized by intraspecific color variability, Eulimnogammarus cyaneus and E. vittatus. We showed that the color of either species is defined by the level of putative carotenoid-binding proteins similar to the pheromone/odorant-binding protein family, as the concentration of these putative crustacyanin analogs was higher in blue or teal-colored animals than in the orange- or yellow-colored ones. At the same time, the color did not depend on the total carotenoid content, as it was similar between animals of contrasting color morphs. By exploring the diversity of these sequences within a larger phylogeny of invertebrate crustacyanins, we show that amphipods lack orthologs of the well-studied crustacyanins A and C, even though they possess some crustacyanin-like sequences. The analysis of expression levels in E. cyaneus showed that the transcripts encoding crustacyanin analogs had much higher expression than the crustacyanin-like sequences, suggesting that the former indeed contribute to the color of these brightly colored animals. The crustacyanin analogs seem to act in a similar way to the well-studied crustacyanins in body color formation, but the details of their action are still to be revealed.

Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal concentration range.

The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl2, the sensitivities of the different species to CdCl2 were ranked (24 hr LC50 in mg/L CdCl2 (mean/95% confidence interval)): Gammarus lacustris (1.7/1.3–2.4) < Eulimnogammarus cyaneus (2.9/2.1–4.0) < Eulimnogammarus verrucosus (5.7/3.8–8.7) < Eulimnogammarus vittatus (18.1/12.4–26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl2 only in the more CdCl2-sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl2 for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl2-sensitive species. Relating the CdCl2 exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) at CdCl2 concentrations ≤LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl2 (n}{}hat {=}LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl2 (n}{}hat {=}LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl2, increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl2 n}{}hat {=} LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl2 n}{}hat {=} LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl2exposed individuals of the different amphipod species varied up to 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl2 exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl2 in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl2 stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress.

Spectral sensitivity of the visual system of endemic Baikal amphipods

In this work, we attempted to explore the visual system of Baikal endemic amphipods with behavioral experiments. These data show that a littoral species Eulimnogammarus cyaneus can sense most wavelengths in the human-visible range, while in the case of a deep-water species Ommatogammarus flavus we did not see any response to light.

Transcriptome-level effects of the model organic pollutant phenanthrene and its solvent acetone in three amphipod species

Polyaromatic hydrocarbons (PAH) are common pollutants of water ecosystems originating from incineration processes and contamination with mineral oil. Water solubility of PAHs is generally low; for toxicity tests with aquatic organisms, they are therefore usually dissolved in organic solvents. Here we examined the effects of a typical model PAH, phenanthrene, and a solvent, acetone, on amphipods as relevant aquatic invertebrate models. Two of these species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, are common endemics of the oligotrophic and pristine Lake Baikal, while one, Gammarus lacustris, is widespread throughout the Holarctic and inhabits smaller and more eutrophic water bodies in the Baikal area. Neither solvent nor phenanthrene caused mortality at the applied concentrations, but both substances affected gene expression in all species. Differential gene expression was more profound in the species from Lake Baikal than in the Holarctic species. Moreover, in one of the Baikal species, E. cyaneus, we found that many known components of the cellular xenobiotic detoxification system reacted to the treatments. Finally, we detected a negative relationship between changes in transcript abundances in response to the solvent and phenanthrene. This mixture effect, weaker than the impact by a single mixture component, needs further exploration.

Description of strongly heat-inducible heat shock protein 70 transcripts from Baikal endemic amphipods

Heat shock proteins/cognates 70 are chaperones essential for proper protein folding. This protein family comprises inducible members (Hsp70s) with expression triggered by the increased concentration of misfolded proteins due to protein-destabilizing conditions, as well as constitutively expressed cognate members (Hsc70s). Previous works on non-model amphipod species Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, both endemic to Lake Baikal in Eastern Siberia, have only revealed a constitutively expressed form, expression of which was moderately further induced by protein-destabilizing conditions. Here we describe heat-inducible hsp70s in these species. Contrary to the common approach of using sequence similarity with hsp/hsc70 of a wide spectrum of organisms and some characteristic features, such as absence of introns within genes and presence of heat shock elements in their promoter areas, the present study is based on next-generation sequencing for the studied or related species followed by differential expression analysis, quantitative PCR validation and detailed investigation of the predicted polypeptide sequences. This approach allowed us to describe a novel type of hsp70 transcripts that overexpress in response to heat shock. Moreover, we propose diagnostic sequence features of this Hsp70 type for amphipods. Phylogenetic comparisons with different types of Hsp/Hsc70s allowed us to suggest that the hsp/hsc70 gene family in Amphipoda diversified into cognate and heat-inducible paralogs independently from other crustaceans. Thus, the cognate and inducible hsp70 types in distant taxa may not be recognized by sequence similarity.

Genetic diversity of Microsporidia in the circulatory system of endemic amphipods from different locations and depths of ancient Lake Baikal.

Endemic amphipods (Amphipoda, Crustacea) of the most ancient and large freshwater Lake Baikal (Siberia, Russia) are a highly diverse group comprising >15% of all known species of continental amphipods. The extensive endemic biodiversity of Baikal amphipods provides the unique opportunity to study interactions and possible coevolution of this group and their parasites, such as Microsporidia. In this study, we investigated microsporidian diversity in the circulatory system of 22 endemic species of amphipods inhabiting littoral, sublittoral and deep-water zones in all three basins of Lake Baikal. Using molecular genetic techniques, we found microsporidian DNA in two littoral (Eulimnogammarus verrucosus, Eulimnogammarus cyaneus), two littoral/sublittoral (Pallasea cancellus, Eulimnogammarus marituji) and two sublittoral/deep-water (Acanthogammarus lappaceus longispinus, Acanthogammarus victorii maculosus) endemic species. Twenty sequences of the small subunit ribosomal (SSU) rDNA were obtained from the haemolymph of the six endemic amphipod species sampled from 0–60 m depths at the Southern Lake Baikal’s basin (only the Western shore) and at the Central Baikal. They form clusters with similarity to Enterocytospora, Cucumispora, Dictyocoela, and several unassigned Microsporidia sequences, respectively. Our sequence data show similarity to previously identified microsporidian DNA from inhabitants of both Lake Baikal and other water reservoirs. The results of our study suggest that the genetic diversity of Microsporidia in haemolymph of endemic amphipods from Lake Baikal does not correlate with host species, geographic location or depth factors but is homogeneously diverse.

Investigation of proteome of Baikal endemic amphipod Eulimnogammarus cyaneus (Dybowsky, 1874) using LC-MS

Investigation of proteome of Baikal endemic amphipod Eulimnogammarus cyaneus (Dybowsky, 1874) using LC-MS

Uptake Kinetics and Subcellular Compartmentalization Explain Lethal but Not Sublethal Effects of Cadmium in Two Closely Related Amphipod Species.

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 μg L-1); E. verrucosus: 115 nM (12.9 μg L-1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 μg g-1; E. verrucosus: 0.26 ± 0.07 μg g-1), whereas relative Cd amounts in the biologically detoxified heat stable protein fraction were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size appears to mainly explain species-related differences in Cd uptake and sensitivities. When exposed to Cd at LC1 over 4 weeks, only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates indicating metabolic depression and pointing to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

Lake Baikal amphipods under climate change: thermal constraints and ecological consequences

AbstractLake Baikal, the world's most voluminous freshwater lake, has experienced unprecedented warming during the last decades. A uniquely diverse amphipod fauna inhabits the littoral zone and can serve as a model system to identify the role of thermal tolerance under climate change. This study aimed to identify sublethal thermal constraints in two of the most abundant endemic Baikal amphipods, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, and Gammarus lacustris, a ubiquitous gammarid of the Holarctic. As the latter is only found in some shallow isolated bays of the lake, we further addressed the question whether rising temperatures could promote the widespread invasion of this non‐endemic species into the littoral zone. Animals were exposed to gradual temperature increases (4 week, 0.8 °C/d; 24 h, 1 °C/h) starting from the reported annual mean temperature of the Baikal littoral (6 °C). Within the framework of oxygen‐ and capacity‐limited thermal tolerance (OCLTT), we used a nonlinear regression approach to determine the points at which the changing temperature‐dependence of relevant physiological processes indicates the onset of limitation. Limitations in ventilation representing the first limits of thermal tolerance (pejus (= “getting worse”) temperatures (Tp)) were recorded at 10.6 (95% confidence interval; 9.5, 11.7), 19.1 (17.9, 20.2), and 21.1 (19.8, 22.4) °C in E. verrucosus, E. cyaneus, and G. lacustris, respectively. Field observations revealed that E. verrucosus retreated from the upper littoral to deeper and cooler waters once its Tp was surpassed, identifying Tp as the ecological thermal boundary. Constraints in oxygen consumption at higher than critical temperatures (Tc) led to an exponential increase in mortality in all species. Exposure to short‐term warming resulted in higher threshold values, consistent with a time dependence of thermal tolerance. In conclusion, species‐specific limits to oxygen supply capacity are likely key in the onset of constraining (beyond pejus) and then life‐threatening (beyond critical) conditions. Ecological consequences of these limits are mediated through behavioral plasticity in E. verrucosus. However, similar upper thermal limits in E. cyaneus (endemic, Baikal) and G. lacustris (ubiquitous, Holarctic) indicate that the potential invader G. lacustris would not necessarily benefit from rising temperatures. Secondary effects of increasing temperatures remain to be investigated.

Contrasting cellular stress responses of Baikalian and Palearctic amphipods upon exposure to humic substances: environmental implications

The species-rich, endemic amphipod fauna of Lake Baikal does not overlap with the common Palearctic fauna; however, the underlying mechanisms for this are poorly understood. Considering that Palearctic lakes have a higher relative input of natural organic compounds with a dominance of humic substances (HSs) than Lake Baikal, we addressed the question whether HSs are candidate factors that affect the different species compositions in these water bodies. We hypothesized that interspecies differences in stress defense might reveal that Baikalian amphipods are inferior to Palearctic amphipods in dealing with HS-mediated stress. In this study, two key mechanisms of general stress response were examined: heat-shock protein 70 (HSP70) and multixenobiotic resistance-associated transporters (ABCB1). The results of quantitative polymerase chain reaction (qPCR) showed that the basal levels (in 3-day acclimated animals) of hsp70 and abcb1 transcripts were lower in Baikalian species (Eulimnogammarus cyaneus, Eulimnogammarus verrucosus, Eulimnogammarus vittatus-the most typical littoral species) than in the Palearctic amphipod (Gammarus lacustris-the only Palearctic species distributed in the Baikalian region). In the amphipods, the stress response was induced using HSs at 10 mg L(-1) dissolved organic carbon, which was higher than in sampling sites of the studied species, but well within the range (3-10 mg L(-1)) in the surrounding water bodies populated by G. lacustris. The results of qPCR and western blotting (n = 5) showed that HS exposure led to increased hsp70/abcb1 transcripts and HSP70 protein levels in G. lacustris, whereas these transcript levels remained constant or decreased in the Baikalian species. The decreased level of stress transcripts is probably not able to confer an effective tolerance to Baikalian species against further environmental stressors in conditions with elevated HS levels. Thus, our results suggest a greater robustness of Palearctic amphipods and a higher sensitivity of Baikalian amphipods to HS challenge, which might prevent most endemic species from migrating to habitats outside Lake Baikal.

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.

Heat shock proteins in the mechanisms of stress adaptation in Baikal amphipods and Palaearctic Gammarus lacustris 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.

ОПРЕДЕЛЕНИЕ рН-ПРЕФЕРЕНДУМА И ЧУВСТВИТЕЛЬНОСТИ К УРОВНЮ РАСТВОРЕННОГО КИСЛОРОДА У БАЙКАЛЬСКИХ ЭНДЕМИЧНЫХ И ПАЛЕАРКТИЧЕСКИХ АМФИПОД

6 species of amphipods were studied for the detection of oxygen- and рН preferences, among them 5 Baikalian endemic species: Gmelinoides fasciatus (Stebb.), Eulimnogammarus cyaneus (Dyb.), E. verrucosus (Gerstf), E. vittatus (Dyb.), Brandtiaparasitica (Dyb.) and Palearctic Gammarus lacustris Sars. All investigated species possessed similar рН preferences which varied from neutral to alkalescent values of a hydrogen indicator. According to the decreasing level of oxygen-preferences the investigated species could be arranged in the following order: E. verrucosus = E. vittatus &lt; E. cyaneus &lt; G. fasciatus = G. lacustris. There was not an expressed reaction to the change of oxygen level in Baikalian B. parasitica; that probably testifies the full (or partial) reduction of the reaction in connection with their symbiotic way of life.

Thermal stress defense in freshwater amphipods from contrasting habitats with emphasis on small heat shock proteins (sHSPs)

This study comparatively evaluated small heat shock proteins (sHSP) (related to α-crystallin) and antioxidant enzymes such as peroxidase (POD), catalase (CAT), glutathione S-transferase (GST) as anti-thermal stress response in two contrasting freshwater amphipods, the stenoecious Baikalean endemic Eulimnogammarus cyaneus and the Palearctic Gammarus lacustris. The thermal stress modulated the activity of antioxidant enzymes as well as the sHSP synthesis in both species. In both species, only the declining POD activity showed a clear dependency on exposure time. The most expressed response to elevated temperatures has been the activation of sHSP synthesis, with clear differences in the patterns: in G. lacustris, sHSP concentrations peaked after 12 h with a subsequent decline, while they increased steadily in E. cyaneus. Hence, the stenoecious species did not acclimate to the thermal stress within the given exposure time as the euryecious did.