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 ABCs of the amphipod P-glycoprotein: Heterologous production of the Abcb1 protein of a model species Eulimnogammarus verrucosus (Amphipoda: Gammaridae) from Lake Baikal.

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.

Tissue Reaction to Low-Density Polyacrylamide Gel as a Carrier for Microimplants in the Adipose Fin of Rainbow Trout.

The implantation of optical sensors is a promising method for monitoring physiological parameters of organisms in vivo. For this, suitable hydrogels are required that can provide a biocompatible interface with the organism's tissues. Amorphous hydrogel is advantageous for administration in animal organs due to its ease of injection compared to resilient analogs. In this study, we investigated the applicability of a semi-liquid 2.5% polyacrylamide hydrogel (PAAH) as a scaffold for fluorescent polyelectrolyte microcapsules (PMs) in rainbow trout. The hydrogel was injected subcutaneously into the adipose fin, which is a small, highly translucent fold of skin in salmonids that is convenient for implanting optical sensors. Using histological methods, we compared tissue organization and in vivo stability of the applied hydrogel at the injection site after administration of uncoated PMs or PMs coated with 2.5% PAAH (PMs-PAAH) for a period of 3 to 14 days. Our results showed that the introduction of PMs into the gel did not have a masking effect, as they were recognized, engulfed, and carried away by phagocytes from the injection site. However, both PMs and PMs-PAAH were found to provoke chronic inflammation at the injection site, although according to cytokine expression in the fish spleen, the irritating effect was local and did not affect the systemic immunity of the fish. Therefore, our study suggests low applicability of 2.5% polyacrylamide as a scaffold for injectable sensors within a timeframe of days.

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.

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.

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.

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.

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.

Microsporidian Parasites Found in the Hemolymph of Four Baikalian Endemic Amphipods.

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.

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.

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.