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.

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.

The diversity and antibiotic properties of actinobacteria associated with endemic deepwater amphipods of Lake Baikal.

The emergence of pathogenic bacteria resistant to antibiotics increases the need for discovery of new effective antimicrobials. Unique habitats such as marine deposits, wetlands and caves or unexplored biological communities are promising sources for the isolation of actinobacteria, which are among the major antibiotic producers. The present study aimed at examining cultivated actinobacteria strains associated with endemic Lake Baikal deepwater amphipods and estimating their antibiotic activity. We isolated 42 actinobacterial strains from crustaceans belonging to Ommatogammarus albinus and Ommatogammarus flavus. To our knowledge, this is the first report describing the isolation and initial characterization of representatives of Micromonospora and Pseudonocardia genera from Baikal deepwater invertebrates. Also, as expected, representatives of the genus Streptomyces were the dominant group among the isolated species. Some correlations could be observed between the number of actinobacterial isolates, the depth of sampling and the source of the strains. Nevertheless, >70% of isolated strains demonstrated antifungal activity. The dereplication analysis of extract of one of the isolated strains resulted in annotation of several known compounds that can help to explain the observed biological activities. The characteristics of ecological niche and lifestyle of deepwater amphipods suggests that the observed associations between crustaceans and isolated actinobacteria are not random and might represent long-term symbiotic interactions.

Metabolic Tolerance to Atmospheric Pressure of Two Freshwater Endemic Amphipods Mostly Inhabiting the Deep-Water Zone of the Ancient Lake Baikal.

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.

Cultivable Actinobacteria First Found in Baikal Endemic Algae Is a New Source of Natural Products with Antibiotic Activity.

Inadequate use of antibiotics has led to spread of microorganisms resistant to effective antimicrobial compounds for humans and animals. This study was aimed to isolate cultivable strains of actinobacteria associated with Baikal endemic alga Draparnaldioides baicalensis and estimate their antibiotic properties. During this study, we isolated both widespread and dominant strains related to the genus Streptomyces and representatives of the genera Saccharopolyspora, Nonomuraea, Rhodococcus, and Micromonospora. For the first time, actinobacteria belonging to the genera Nonomuraea and Saccharopolyspora were isolated from Baikal ecosystem. Also, it was the first time when actinobacteria of the genus Nonomuraea were isolated from freshwater algae. Some rare strains demonstrated activity inhibiting growth of bacteria and yeasts. Also, it has been shown that the strains associated with Baikal alga D. baicalensis are active against both Gram-positive and Gram-negative bacteria. According to this study and previously published materials, diversity of cultivable actinobacteria and rare strains isolated from D. baicalensis is comparable to that of cultivable actinobacteria previously isolated from plant sources of Lake Baikal. Also, it exceeds the cultivable actinobacteria diversity previously described for macroinvertebrates, water, or sediments of Lake Baikal. The large number of rare and active strains associated with the endemic alga D. baicalensis could be the promising sources for biopharmaceutical and biotechnological developments and discovery of new natural compounds.

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.

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.

Parallel in vivo monitoring of pH in gill capillaries and muscles of fishes using microencapsulated biomarkers.

Tracking physiological parameters in different organs within the same organism simultaneously and in real time can provide an outstanding representation of the organism's physiological status. The state-of-the-art technique of using encapsulated fluorescent molecular probes (microencapsulated biomarkers) is a unique tool that can serve as a platform for the development of new methods to obtain in vivo physiological measurements and is applicable to a broad range of organisms. Here, we describe a novel technique to monitor the pH of blood inside the gill capillaries and interstitial fluid of muscles by using microencapsulated biomarkers in a zebrafish model. The functionality of the proposed technique is shown by the identification of acidification under anesthesia-induced coma and after death. The pH in muscles reacts to hypoxia faster than that in the gill bloodstream, which makes both parameters applicable as markers of either local or bodily reactions.

Draft Genome Sequence of Streptomyces sp. Strain IB2014011-1, Isolated from Trichoptera sp. Larvae of Lake Baikal.

Unique ecosystems with specific environmental conditions have been proven to be a promising source for isolation of new actinobacterial strains. Ancient Lake Baikal is one of the greatest examples of an ecosystem with high species biodiversity and endemicity caused by long-lasting isolated evolution and stable environmental conditions. Herein we report the draft genome sequence of Streptomyces sp. strain IB2014011-1, which was isolated from insect Trichoptera sp. larvae collected at the bottom of Lake Baikal.

Natural organic matter (NOM) induces oxidative stress in freshwater amphipods Gammarus lacustris Sars and Gammarus tigrinus (Sexton).

Humic substances comprise the majority of natural organic matter (NOM) on Earth, including dissolved organic matter in freshwater systems. Recent studies show that these substances directly interact with aquatic organisms as chemical stressors. The aim of the present study was to investigate the mode of action of dissolved NOM on the freshwater amphipods Gammarus lacustris Sars and Gammarus tigrinus (Sexton), and in particular, to determine if NOM induces or promotes internal oxidative stress. NOM was isolated by reverse osmosis from a brown-water lake in Brandenburg State, Germany. Oxidative stress markers, such as lipid peroxidation, cell internal hydrogen peroxide concentration, as well as peroxidase, catalase and glutathione S-transferase activities, were quantified. Exposure of both amphipod species to NOM caused a significant increase in lipid peroxidation, hydrogen peroxide concentration, catalase, peroxidase and glutathione S-transferase activities. Both species showed a two-stage antioxidant response: the first stage allowed the organisms to effectively eliminate ROS and to protect cells from damage, whereas the second stage leads to H2O2 accumulation in combination with destruction of lipid structures in the cells and, finally, functional damage or even death of the organism.

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.

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.

Impact of natural organic matter (NOM) on freshwater amphipods.

Natural organic matter (NOM) isolated from the eutrophic Sanctuary Pond (Point Pelee National Park, Canada) has an adverse impact on amphipod species (Gammarus tigrinus and Chaetogammarus ischnus from Lake Müggelsee, Germany, and Eulimnogammarus cyaneus, from Lake Baikal, Russia). Increases in amphipod mortality, changes in peroxidase activity and increases of heat shock protein (hsp70) expression were observed upon exposure to NOM. The highest resistance to the adverse impact of NOM was observed with the endemic Baikalian amphipod E. cyaneus. However, the mechanisms behind this finding remains obscure. If differences in the sensitivity of the hsp70 antibody may be excluded, different modes of action may be postulated: because the adverse impact of NOM may be caused by reactive oxygen species (ROS) and the NOM itself, the observed differences may be due to the action of ROS alone (with E. cyaneus) and a combination of both adverse modes of action (European species).

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.

Specific antioxidant reactions to oxidative stress promoted by natural organic matter in two amphipod species from Lake Baikal.

Aquatic organisms are exposed to a variety of natural chemical stressors such as humic substances. The aim of this study was to investigate the mode of action of natural organic matter (NOM, roughly 80% of which is humic substances) on two freshwater amphipods from Lake Baikal, Eulimnogammarus verrucosus (Gerstf.) and Eulimnogammarus cyaneus (Dyb.), in order to assess the potential oxidative stress of NOM impact. Chosen as oxidative stress markers were lipid peroxidation and cell internal hydrogen peroxide level as well as peroxidase, catalase, and glutathione S-transferase activities. Exposure of amphipods to NOM caused a significant increase in lipid peroxidation but a concomitant decrease in hydrogen peroxide concentration, and peroxidase and (to a lesser degree) glutathione S-transferase activities. An interim increase of catalase activity was observed. A possible reason for the decrease in major antioxidant enzyme activity is exhaustion of the reservoir of reduced substrates in the first stage of the antioxidant defense reaction. Despite the inhibition of major antioxidant enzymes, the studied amphipods were able to successfully resist the NOM oxidative impact and, at low NOM concentrations, to combat lipid peroxidation processes.