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.

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 Eulimnogammarus cyaneus, 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.

Comparative Analysis of Proteins of Functionally Different Body Parts of the Fish Parasites Triaenophorus nodulosus and Triaenophorus crassus.

PURPOSE: Studies of proteins expressed in the morphological structures of the parasite are necessary for elucidating the biological functions of unknown proteins and understanding the molecular basis of parasitism. The research aim was to investigate the spatial distribution of major proteins in scolex, immature and gravid proglottids of Triaenophorus nodulosus and Triaenophorus crassus. METHODS: Protein extracts of worm body parts were analyzed using two-dimensional difference gel electrophoresis (DIGE) and mass spectrometry. RESULTS: Comparison of the protein repertoire of the adult worm and the encysted plerocercoid revealed differences between the worm body parts, life stages and parasite species. The content of proteins associated with the cytoskeleton and musculature (actin, myosin regulatory light chain, and tropomyosin 2) decreased with distance from the scolex. Mature proglottids were rich in transforming growth factor-beta-induced protein, propionyl-CoA carboxylase, glutamate dehydrogenase and beta-tubulin. Interspecific variation in T. nodulosus and T. crassus was found in the content of the myosin, paramyosin, the major vault protein and an uncharacterized secreted protein TRINITY_DN24645. Differential expression of TRINITY_DN24645, paramyosin and tropomyosin 2 was found between plerocercoids and adult worms. CONCLUSION: The present study provides the first characteristics of the spatial distribution of the major proteins of T. crassus and T. nodulosus. Comparison of the protein composition of plerocercoids and adult parasites indicates a significant similarity in the proteomic organization of Triaenophorus sp. in the second intermediate and final hosts. The gradual change in the morphological organization of tapeworms in the longitudinal direction coincided with the expression of some structural and metabolic proteins.

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.

Excessive Parallelism in Protein Evolution of Lake Baikal Amphipod Species Flock.

Repeated emergence of similar adaptations is often explained by parallel evolution of underlying genes. However, evidence of parallel evolution at amino acid level is limited. When the analyzed species are highly divergent, this can be due to epistatic interactions underlying the dynamic nature of the amino acid preferences: The same amino acid substitution may have different phenotypic effects on different genetic backgrounds. Distantly related species also often inhabit radically different environments, which makes the emergence of parallel adaptations less likely. Here, we hypothesize that parallel molecular adaptations are more prevalent between closely related species. We analyze the rate of parallel evolution in genome-size sets of orthologous genes in three groups of species with widely ranging levels of divergence: 46 species of the relatively recent lake Baikal amphipod radiation, a species flock of very closely related cichlids, and a set of significantly more divergent vertebrates. Strikingly, in genes of amphipods, the rate of parallel substitutions at nonsynonymous sites exceeded that at synonymous sites, suggesting rampant selection driving parallel adaptation. At sites of parallel substitutions, the intraspecies polymorphism is low, suggesting that parallelism has been driven by positive selection and is therefore adaptive. By contrast, in cichlids, the rate of nonsynonymous parallel evolution was similar to that at synonymous sites, whereas in vertebrates, this rate was lower than that at synonymous sites, indicating that in these groups of species, parallel substitutions are mainly fixed by drift.

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.

Comparison between transcriptomic responses to short-term stress exposures of a common Holarctic and endemic Lake Baikal amphipods.

BACKGROUND: Lake Baikal is one of the oldest freshwater lakes and has constituted a stable environment for millions of years, in stark contrast to small, transient bodies of water in its immediate vicinity. A highly diverse endemic endemic amphipod fauna is found in one, but not the other habitat. We ask here whether differences in stress response can explain the immiscibility barrier between Lake Baikal and non-Baikal faunas. To this end, we conducted exposure experiments to increased temperature and the toxic heavy metal cadmium as stressors. RESULTS: Here we obtained high-quality de novo transcriptome assemblies, covering mutiple conditions, of three amphipod species, and compared their transcriptomic stress responses. Two of these species, Eulimnogammarus verrucosus and E. cyaneus, are endemic to Lake Baikal, while the Holarctic Gammarus lacustris is a potential invader. CONCLUSIONS: Both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles. G. lacustris reacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions.

Indication of ongoing amphipod speciation in Lake Baikal by genetic structures within endemic species.

BACKGROUND: The ancient Lake Baikal is characterized by an outstanding diversity of endemic faunas with more than 350 amphipod species and subspecies. We determined the genetic diversity within the endemic littoral amphipod species Eulimnogammarus verrucosus, E. cyaneus and E. vittatus and investigated whether within those species genetically separate populations occur across Lake Baikal. Gammarus lacustris from water bodies in the Baikal area was examined for comparison. RESULTS: Genetic diversities within a species were determined based on fragments of cytochrome c oxidase I (COI) and for E. verrucosus additionally of 18S rDNA. Highly location-specific haplogroups of E. verrucosus and E. vittatus were found at the southern and western shores of Baikal that are separated by the Angara River outflow; E. verrucosus from the eastern shore formed a further, clearly distinct haplotype cluster possibly confined by the Selenga River and Angarskiy Sor deltas. The genetic diversities within these haplogroups were lower than between the different haplogroups. Intraspecific genetic diversities within E. verrucosus and E. vittatus with 13 and 10%, respectively, were similar to interspecies differences indicating the occurrence of cryptic, morphologically highly similar species; for E. verrucosus this was confirmed with 18S rDNA. The haplotypes of E. cyaneus and G. lacustris specimens were with intraspecific genetic distances of 3 and 2%, respectively, more homogeneous indicating no or only recent disruption of gene flow of E. cyaneus across Baikal and recent colonization of water bodies around Baikal by G. lacustris. CONCLUSIONS: Our finding of separation of subgroups of Baikal endemic amphipods to different degrees points to a species-specific ability of dispersal across areas with adverse conditions and to potential geographical dispersal barriers in Lake Baikal.

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.

Nonspecific stress response to temperature increase in Gammarus lacustris Sars with respect to oxygen-limited thermal tolerance concept.

The previously undescribed dynamics of the heat shock protein HSP70 and subsequent lipid peroxidation products have been assessed alongside lactate dehydrogenase activity for Gammarus lacustris Sars, an amphipod species from the saltwater Lake Shira (Republic of Khakassia). Individuals were exposed to a gradual temperature increase of 1 °C/hour (total exposure duration of 26 hours) starting from the mean annual temperature of their habitat (7 °C) up to 33 °C. A complex of biochemical reactions occurred when saltwater G. lactustris was exposed to the gradual changes in temperature. This was characterized by a decrease in lactate dehydrogenase activity and the launching of lipid peroxidation. The HSP70 level did not change significantly during the entire experiment. In agreement with the concept of oxygen-limited thermal tolerance, an accumulation of the most toxic lipid peroxides (triene conjugates and Schiff bases) in phospholipids occurred at the same time and temperature as the accumulation of lactate. The main criterion overriding the temperature threshold was, therefore, the transition to anaerobiosis, confirmed by the elevated lactate levels as observed in our previous associated study, and by the development of cellular stress, which was expressed by an accumulation of lipid peroxidation products. An earlier hypothesis, based on freshwater individuals of the same species, has been confirmed whereby the increased thermotolerance of G. lacustris from the saltwater lake was caused by differences in energy metabolism and energy supply of nonspecific cellular stress-response mechanisms. With the development of global climate change, these reactions could be advantageous for saltwater G. lacustris. The studied biochemical reactions can be used as biomarkers for the stress status of aquatic organisms when their habitat temperature changes.

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.

Intersexual differences of heat shock response between two amphipods (Eulimnogammarus verrucosus and Eulimnogammarus cyaneus) in Lake Baikal.

Acute temperature fluctuations are common in surface waters, and aquatic organisms may manifest physiological responses to punctuated temperature spikes long before behavioral responses. Ectotherms, especially cryophilic stenotherms such as those endemic to Lake Baikal (Siberia), may demonstrate specialized physiological responses to acute temperature increases because their proteomes have evolved to function most efficiently at lower temperatures (e.g., <10 °C). Therefore, our study questioned the nature and degree of variation in physiological response to acute thermal stress in two congenerous, endemic Baikal amphipod species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus. We hypothesized that because interspecific and intersexual thermosensitivity varies significantly among ectotherms, there would be divergent intersexual and interspecific strategies to withstand acute thermal stress, manifested in different protein compositions and concentrations. We exposed individuals to the species' respective LT50 for one hour followed by a three-hour recovery period. We then performed 1D-PAGE, Western blotting, 2D-PAGE, and Mass Spectrometry techniques and assessed relative intersexual and interspecific changes in proteomic composition and heat shock protein 70 level. Our results demonstrate that females tend to be more sensitive to an acute thermal stimulus than males, most likely because females allocate significant energy to reproduction and less to heat shock response, evidenced by females' significantly lower LT50time. Lower level of Hsp70 was found in females of the thermosensitive E. verrucosus compared to males of this species. No intersexual differences were found in Hsp70 level in thermotolerant E. cyaneus. Higher levels of hemocyanin subunits and arginine kinase were found in E. cyaneus females after heat shock and recovery compared to males, which was not found for E. verrucosus, suggesting interspecific mechanisms for E. cyaneus's higher thermotolerance. These differing responses between species and sexes of Baikal amphipods may reflect more general strategies for maintaining homeostatic conditions during acute thermal stress. As mean surface water temperatures increase worldwide, the net efficiency and efficacy of these strategies could give rise to long term changes in physiology, behavior, and interactions with other species, potentially precipitating population and community level alterations.

Salinity modulates thermotolerance, energy metabolism and stress response in amphipods Gammarus lacustris.

Temperature and salinity are important abiotic factors for aquatic invertebrates. We investigated the influence of different salinity regimes on thermotolerance, energy metabolism and cellular stress defense mechanisms in amphipods Gammarus lacustris Sars from two populations. We exposed amphipods to different thermal scenarios and determined their survival as well as activity of major antioxidant enzymes (peroxidase, catalase, glutathione S-transferase) and parameters of energy metabolism (content of glucose, glycogen, ATP, ADP, AMP and lactate). Amphipods from a freshwater population were more sensitive to the thermal challenge, showing higher mortality during acute and gradual temperature change compared to their counterparts from a saline lake. A more thermotolerant population from a saline lake had high activity of antioxidant enzymes. The energy limitations of the freshwater population (indicated by low baseline glucose levels, downward shift of the critical temperature of aerobic metabolism and inability to maintain steady-state ATP levels during warming) was observed, possibly reflecting a trade-off between the energy demands for osmoregulation under the hypo-osmotic condition of a freshwater environment and protection against temperature stress.

Remote in vivo stress assessment of aquatic animals with microencapsulated biomarkers for environmental monitoring.

Remote in vivo scanning of physiological parameters is a major trend in the development of new tools for the fields of medicine and animal physiology. For this purpose, a variety of implantable optical micro- and nanosensors have been designed for potential medical applications. At the same time, the important area of environmental sciences has been neglected in the development of techniques for remote physiological measurements. In the field of environmental monitoring and related research, there is a constant demand for new effective and quick techniques for the stress assessment of aquatic animals, and the development of proper methods for remote physiological measurements in vivo may significantly increase the precision and throughput of analyses in this field. In the present study, we apply pH-sensitive microencapsulated biomarkers to remotely monitor the pH of haemolymph in vivo in endemic amphipods from Lake Baikal, and we compare the suitability of this technique for stress assessment with that of common biochemical methods. For the first time, we demonstrate the possibility of remotely detecting a change in a physiological parameter in an aquatic organism under ecologically relevant stressful conditions and show the applicability of techniques using microencapsulated biomarkers for remote physiological measurements in environmental monitoring.

Thermal Preference Ranges Correlate with Stable Signals of Universal Stress Markers in Lake Baikal Endemic and Holarctic Amphipods.

Temperature is the most pervasive abiotic environmental factor for aquatic organisms. Fluctuations in temperature range lead to changes in metabolic performance. Here, we aimed to identify whether surpassing the thermal preference zones is correlated with shifts in universal cellular stress markers of protein integrity, responses to oxidative stress and lactate content, as indicators of anaerobic metabolism. Exposure of the Lake Baikal endemic amphipod species Eulimnogammarus verrucosus (Gerstfeldt, 1858), Ommatogammarus flavus (Dybowski, 1874) and of the Holarctic amphipod Gammarus lacustris Sars 1863 (Amphipoda, Crustacea) to increasing temperatures resulted in elevated heat shock protein 70 (Hsp70) and lactate content, elevated antioxidant enzyme activities (i.e., catalase and peroxidase), and reduced lactate dehydrogenase and glutathione S-transferase activities. Thus, the zone of stability (absence of any significant changes) of the studied molecular and biochemical markers correlated with the behaviorally preferred temperatures. We conclude that the thermal behavioral responses of the studied amphipods are directly related to metabolic processes at the cellular level. Thus, the determined thermal ranges may possibly correspond to the thermal optima. This relationship between species-specific behavioral reactions and stress response metabolism may have significant ecological consequences that result in a thermal zone-specific distribution (i.e., depths, feed spectrum, etc.) of species. As a consequence, by separating species with different temperature preferences, interspecific competition is reduced, which, in turn, increases a species' Darwinian fitness in its environment.

On Eulimnogammarus messerschmidtii, sp. n. (Amphipoda: Gammaridea) from Lake Baikal, Siberia, with redescription of E. cyanoides (Sowinsky) and remarks on taxonomy of the genus Eulimnogammarus.

A new amphipod species of the endemic fauna of Lake Baikal (East Siberia, Russia), Eulimnogammarus messerschmidtii sp. n., from the littoral zone of the northern part of the lake is described. The species is characterized by the presence of a group of spines with dense setae on the last 4 body segments. The basal peduncular segment of antenna 1 bears bunches of dense setae without spines, uropods 3 are covered by dense simple setae without plumose setae and the outer ramus has a second small article. The body length of sampled specimens ranges from 7.5 to 18 mm. Population analysis at one of the sampling points revealed a spring-summer reproduction period for this species. This species was previously erroneously identified as E. cyanoides. E. cyanoides is here redescribed in details based on the lectotype. The differences between E. messerschmidtii sp. n., E. cyanoides and other closely related Eulimnogammarus species are described. The taxonomy of the genus Eulimnogammarus is discussed.

A first glimpse at the genome of the Baikalian amphipod Eulimnogammarus verrucosus.

Eulimnogammarus verrucosus is an amphipod endemic to the unique ecosystem of Lake Baikal and serves as an emerging model in ecotoxicological studies. We report here on a survey sequencing of its genome as a first step to establish sequence resources for this species. From a single lane of paired-end sequencing data, we estimated the genome size as nearly 10 Gb and we obtained an overview of the repeat content. At least two-thirds of the genome are non-unique DNA, and a third of the genomic DNA is composed of just five families of repetitive elements, including low-complexity sequences. Attempts to use off-the-shelf assembly tools failed on the available low-coverage data both before and after removal of highly repetitive components. Using a seed-based approach we nevertheless assembled short contigs covering 33 pre-microRNAs and the homeodomain-containing exon of nine Hox genes. The absence of clear evidence for paralogs implies that a genome duplication did not contribute to the large genome size. We furthermore report the assembly of the mitochondrial genome using a new, guided "crystallization" procedure. The initial results presented here set the stage for a more complete sequencing and analysis of this large genome.

A cellular and metabolic assessment of the thermal stress responses in the endemic gastropod Benedictia limnaeoides ongurensis from Lake Baikal.

Our objective was to determine if the Lake Baikal endemic gastropod Benedictia limnaeoides ongurensis, which inhabits in stable cold waters expresses a thermal stress response. We hypothesized that the evolution of this species in the stable cold waters of Lake Baikal resulted in a reduction of its thermal stress-response mechanisms at the biochemical and cellular levels. Contrary to our hypothesis, our results show that exposure to a thermal challenge activates the cellular and biochemical mechanisms of thermal resistance, such as heat shock proteins and antioxidative enzymes, and alters energetic metabolism in B. limnaeoides ongurensis. Thermal stress caused the elevation of heat shock protein 70 and the products of anaerobic glycolysis together with the depletion of glucose and phosphagens in the studied species. Thus, a temperature increase activates the complex biochemical system of stress response and alters the energetic metabolism in this endemic Baikal gastropod. It is concluded that the deepwater Lake Baikal endemic gastropod B. limnaeoides ongurensis retains the ability to activate well-developed biochemical stress-response mechanisms when exposed to a thermal challenge.