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Abstract: 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 12h 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. [Copyright &y& Elsevier]

Thermopreference reactions and avoidance sensitivity to dissolved oxygen levels were measured in three endemic amphipod species from Lake Baikal: Eulimnogammarus verrucosus and E. vittatus, as well as Brandtia parasitica, which is a symbiont of the Baikalean sponge, Lubomirskia baikalensis. The benthic E. vittatus and E. verrucosus demonstrated highly developed thermopreference reactions, whereas B. parasitica did not show thermopreference behaviour. The experimental results also showed that E. vittatus and E. verrucosus avoid water with decreased dissolved oxygen levels, whereas behavioural reactions of B. parasitica did not depend on oxygen concentration. In the presence of sponge fragments, individual B. parasitica moved actively towards them, and concentrated on those fragments. Possible reasons for reduction of behavioural preference in B. parasitica are discussed with a focus on the species' symbiotic status. [ABSTRACT FROM AUTHOR]

Baikal amphipods are one of the most unique freshwater species flocks of over 350 morphological species and subspecies playing multiple ecological roles in this ancient lake. However, molecular phylogeny studies accumulate evidence that this number is underestimated. Previously, we showed that an abundant littoral species Eulimnogammarus verrucosus comprised at least three genetic lineages: southern (S), western (W), and eastern (E), inhabiting corresponding parts of Baikal shore. The Angara River is the only outflow of the lake and is inhabited by amphipods originating from Baikal, which were previously analysed only morphologically. The river has parts with strong current or closed bays as opposed to sea‐like conditions of Lake Baikal, which raises the question of which species were able to adapt here. Here we studied the phylogeny of E. verrucosus from the Angara River, which could be reached by two lineages, S and W. We analysed 17 sites from 0.6 to 650 km from the outflow. Cytochrome c oxidase subunit I (COI) and 18S rRNA analysis showed that the sites closest to Baikal were inhabited by the S and W lineages sympatrically. The other sites were inhabited by the S lineage at both banks and also by the novel A (Angara) lineage. The A lineage was the first to split up from the last common ancestor of all E. verrucosus. Our data confirm the importance of considering cryptic diversity in ecological monitoring, and in order to simplify the identification of the genetic lineage, we developed a web‐app speCOIdent. [ABSTRACT FROM AUTHOR]

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies. [ABSTRACT FROM AUTHOR]

Temperature and mineralization are among the most important environmental factors affecting all processes of aquatic ecosystems, including geographical distribution of water animals. Previously we showed that a brackish water population of Gammarus lacustris , a widespread amphipod, demonstrates substantially higher thermotolerance than a freshwater population. A possible reason for this difference is the fact that brackish water conditions are closer to internal media mineralization. Here we aimed to test this hypothesis and relate the observed effects in animal survival under the heat shock to the status of cellular defence systems. We acclimated four groups of amphipods from the same freshwater population to 0.5 ‰ and 15 ‰ at the temperatures of 6°С or 15°С. Acclimation at 6°С, but not at 15°С, to 15 ‰ significantly increased resistance of the amphipods to heat shock at 30°C. At 6°С activities of antioxidant enzymes and levels of the lipid peroxidation products in G. lacustris did not react to the increase in mineralization and the heat shock, while the level of HSP70 elevated two-fold in amphipods acclimated to mineralization of 15 ‰ compared to animals acclimated to 0.5 ‰. Thus, the observed increase in thermotolerance could be explained by the higher initial level of HSP70 and potentially other heat shock proteins caused by a less energy-demanding, more isotonic level. [Display omitted] • Acclimation at 6 °С to 15 ‰ increased resistance of G. lacustris to heat shock. • HSP70 level elevated in amphipods acclimated to higher mineralization. • Higher initial level of HSPs potentially caused increased thermotolerance. [ABSTRACT FROM AUTHOR]

Simple Summary: Deep-water habitats are the largest ecosystem on the planet: over half of the Earth's surface is covered with a water layer deeper than 200 m and remains poorly explored. Lake Baikal is the only freshwater body inhabited by animals adapted to the deep-water zone independently from their marine counterparts. Comparing these convergently evolved freshwater and marine animals is invaluable for revealing the basic mechanisms of adaptation to high hydrostatic pressure. However, laboratory experiments on deep-water organisms still usually require lifting them to the water's surface and exposing them to potentially hazardous decompression, while endemics from Lake Baikal are poorly studied in this regard. Here, we compared metabolic reactions to such pressure decreases in two Baikal deep-water amphipods (shrimp-like crustaceans) from the genus Ommatogammarus: one species is known to tolerate pressures close to atmospheric levels, while the second was only observed at the pressures from 5 atm and above. We expected that the energy metabolism of the shallower-dwelling species would function better under the atmospheric pressure but found no substantial differences. Thus, despite some difference in long-term survival at atmospheric pressure, both species are suitable for laboratory studies as freshwater model objects adapted to large pressure variations. Lake Baikal is the only freshwater reservoir inhabited by deep-water fauna, which originated mostly from shallow-water ancestors. Ommatogammarus flavus and O. albinus are endemic scavenger amphipods (Amphipoda, Crustacea) dwelling in wide depth ranges of the lake covering over 1300 m. O. flavus had been previously collected close to the surface, while O. albinus has never been found above the depth of 47 m. Since O. albinus is a promising model species for various research, here we tested whether O. albinus is less metabolically adapted to atmospheric pressure than O. flavus. We analyzed a number of energy-related traits (contents of glucose, glycogen and adenylates, as well as lactate dehydrogenase activity) and oxidative stress markers (activities of antioxidant enzymes and levels of lipid peroxidation products) after sampling from different depths and after both species' acclimation to atmospheric pressure. The analyses were repeated in two independent sampling campaigns. We found no consistent signs of metabolic disturbances or oxidative stress in both species right after lifting. Despite O. flavus surviving slightly better in laboratory conditions, during long-term acclimation, both species showed comparable reactions without critical changes. Thus, the obtained data favor using O. albinus along with O. flavus for physiological research under laboratory conditions. [ABSTRACT FROM AUTHOR]

The lipid and fatty acid content of benthopelagic scavenging amphipods Ommatogammarus flavus and O. albinus from different depths of Lake Baikal were studied. These amphipods, known to move across a wide range of depths in Lake Baikal, had a relatively high level of total lipids (up to 46% dry weight) in comparison to other aquatic amphipods. This high level of total lipids resulted from a high concentration of storage lipids (triacylglycerols). The monounsaturated 18:1(n-9) acid in combination with 16:1(n-7), and 18:1(n-7) fatty acids were dominant in the amphipods, reflecting their deep-dwelling scavenging mode of life. Local groups of amphipods were distinguished along a Lake Baikal depth gradient based on the primarily diet-associated differences in fatty acid composition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The multixenobiotic resistance (MXR) mechanism has been demonstrated to be present in a wide range of species, including aquatic organisms. However, amphipods (Crustacea: Malacostraca: Amphipoda), which constitute a large order of arthropods, are extremely poorly studied in this regard. Information on MXR proteins in these animals would be highly relevant, as some amphipods are important models in ecotoxicology due to their roles in many freshwater environments, including the ancient Lake Baikal. In this work, we studied the diversity of ABC transporters in the available transcriptomes of over 60 endemic Baikal amphipods in comparison to other related species. This showed that most classes of ABC transporters are present in all analyzed species and that most Baikal amphipods detectably express no more than one complete ABCB full transporter. We also showed that these sequences were conservative across different species, and their phylogeny was congruent with the species phylogeny. Thus, we chose the abcb1 coding sequence from Eulimnogammarus verrucosus , a widespread species playing an important role in the lake ecosystem, to establish the first heterologous expression system for an amphipod Abcb1/P-glycoprotein based on the Drosophila melanogaster S2 cell line. The resulting stably transfected S2 cell line was expressing the abcb1 of E. verrucosus about 1000 times higher than the homologous fly genes, and the target protein, Abcb1, showed to confer a high MXR-related efflux activity. Our results indicate the suitability of the S2-based expression systems for the study of arthropod ABCB1 homologs. [Display omitted] • The previously available studies on the multixenobiotic resistance mechanism (MXR) in Lake Baikal amphipods are scarce. • Typically, Baikal amphipods possess most ABC transporter subfamilies and express one ABCB full transporter in normal conditions. • ABCB full transporters of amphipods have longer intracellular N-termini than those of well-studied invertebrates. • We stably expressed abcb1 from a Baikal endemic amphipod Eulimnogammarus verrucosus in S2 cells from Drosophila melanogaster. [ABSTRACT FROM AUTHOR]

During physiological studies, it is often required to restrain a small animal for an experiment, as it is typically challenging to perform in vivo imaging or measurements on freely moving individuals. In this article we describe two widely applicable approaches for repeated restraint of small (approx. 0.5-4 cm) decapods and amphipods that are established in our laboratory: immobilization using gentle gluing and suction. Application of both these approaches as well as their advantages and disadvantages are discussed in detail. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

At present, approximately 187 genera and over 1300 species of Microsporidia have been described, among which almost half infect aquatic species and approximately 50 genera potentially infect aquatic arthropods. Lake Baikal is the deepest and one of the oldest lakes in the world, and it has a rich endemic fauna with a predominance of arthropods. Among the arthropods living in this lake, amphipods (Crustacea) are the most dominant group and are represented by more than 350 endemic species. Baikalian amphipods inhabit almost all depths and all types of substrates. The age and geographical isolation of this group creates excellent opportunities for studying the diversity, evolution and genetics of host-parasite relationships. However, despite more than 150 years of study, data investigating the microsporidia of Lake Baikal remain incomplete. In this study, we used molecular genetic analyses to detect microsporidia in the hemolymph of several endemic species of amphipods from Lake Baikal. We provide the first evidence that microsporidian species belonging to three genera (Microsporidium, Dictyocoela and Nosema) are present in the hemolymph of Baikalian endemic amphipods. In the hemolymph of Eulimnogammarus verrucosus, we detected SSU rDNA of microsporidia belonging to the genus Nozema. In the hemolymph of Pallasea cancellous, we found the DNA of Microsporidium sp. similar to that in other Baikalian endemic amphipods; Dictyocoela sp. was found in the hemolymph of Eulimnogammarus marituji and Acanthogammarus lappaceus longispinus. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. Unlabelled Image • Endemic amphipods are extremely important in benthic communities of Lake Baikal. • We studied the proteomes of males and females of Eulimnogammarus cyaneus. • High-throughput mass spectrometry allowed us to find almost 1000 protein groups. • Females possess higher levels of heat shock proteins in control conditions. • Males activate heat shock proteins in heat stress conditions, while females do not. [ABSTRACT FROM AUTHOR]