Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Transcriptional response of Saccharomyces cerevisiae to lactic acid enantiomers.

The model yeast, Saccharomyces cerevisiae, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring S. cerevisiae transcriptional response to various substances, this response is unknown for some substances produced in yeast, such as D-lactic acid (DLA). Here, we explore the transcriptional response of the BY4742 strain to a wide range of DLA concentrations (from 0.05 to 45 mM), and compare it to the response to 45 mM L-lactic acid (LLA). We recorded a response to 5 and 45 mM DLA (125 and 113 differentially expressed genes (DEGs), respectively; > 50% shared) and a less pronounced response to 45 mM LLA (63 DEGs; > 30% shared with at least one DLA treatment). Our data did not reveal natural yeast promoters quantitatively sensing DLA but provide the first description of the transcriptome-wide response to DLA and enrich our understanding of the LLA response. Some DLA-activated genes were indeed related to lactate metabolism, as well as iron uptake and cell wall structure. Additional analyses showed that at least some of these genes were activated only by acidic form of DLA but not its salt, revealing the role of pH. The list of LLA-responsive genes was similar to those published previously and also included iron uptake and cell wall genes, as well as genes responding to other weak acids. These data might be instrumental for optimization of lactate production in yeast and yeast co-cultivation with lactic acid bacteria. KEY POINTS: • We present the first dataset on yeast transcriptional response to DLA. • Differential gene expression was correlated with yeast growth inhibition. • The transcriptome response to DLA was richer in comparison to LLA.

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.

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.

Experimental Crossing Confirms Reproductive Isolation between Cryptic Species within Eulimnogammarus verrucosus (Crustacea: Amphipoda) from Lake Baikal.

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.

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.

Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host.

The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

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.

LazyB: fast and cheap genome assembly.

BACKGROUND: Advances in genome sequencing over the last years have lead to a fundamental paradigm shift in the field. With steadily decreasing sequencing costs, genome projects are no longer limited by the cost of raw sequencing data, but rather by computational problems associated with genome assembly. There is an urgent demand for more efficient and and more accurate methods is particular with regard to the highly complex and often very large genomes of animals and plants. Most recently, "hybrid" methods that integrate short and long read data have been devised to address this need. RESULTS: LazyB is such a hybrid genome assembler. It has been designed specificially with an emphasis on utilizing low-coverage short and long reads. LazyB starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs. This graph is translated into a long-read overlap graph G. Instead of the more conventional approach of removing tips, bubbles, and other local features, LazyB stepwisely extracts subgraphs whose global properties approach a disjoint union of paths. First, a consistently oriented subgraph is extracted, which in a second step is reduced to a directed acyclic graph. In the next step, properties of proper interval graphs are used to extract contigs as maximum weight paths. These path are translated into genomic sequences only in the final step. A prototype implementation of LazyB, entirely written in python, not only yields significantly more accurate assemblies of the yeast and fruit fly genomes compared to state-of-the-art pipelines but also requires much less computational effort. CONCLUSIONS: LazyB is new low-cost genome assembler that copes well with large genomes and low coverage. It is based on a novel approach for reducing the overlap graph to a collection of paths, thus opening new avenues for future improvements. AVAILABILITY: The LazyB prototype is available at https://github.com/TGatter/LazyB .

The diversity of opsins in Lake Baikal amphipods (Amphipoda: Gammaridae).

BACKGROUND: Vision is a crucial sense for the evolutionary success of many animal groups. Here we explore the diversity of visual pigments (opsins) in the transcriptomes of amphipods (Crustacea: Amphipoda) and conclude that it is restricted to middle (MWS) and long wavelength-sensitive (LWS) opsins in the overwhelming majority of examined species. RESULTS: We evidenced (i) parallel loss of MWS opsin expression in multiple species (including two independently evolved lineages from the deep and ancient Lake Baikal) and (ii) LWS opsin amplification (up to five transcripts) in both Baikal lineages. The number of LWS opsins negatively correlated with habitat depth in Baikal amphipods. Some LWS opsins in Baikal amphipods contained MWS-like substitutions, suggesting that they might have undergone spectral tuning. CONCLUSIONS: This repeating two-step evolutionary scenario suggests common triggers, possibly the lack of light during the periods when Baikal was permanently covered with thick ice and its subsequent melting. Overall, this observation demonstrates the possibility of revealing climate history by following the evolutionary changes in protein families.

Chromosome-level genome assembly and structural variant analysis of two laboratory yeast strains from the Peterhof Genetic Collection lineage.

Thousands of yeast genomes have been sequenced with both traditional and long-read technologies, and multiple observations about modes of genome evolution for both wild and laboratory strains have been drawn from these sequences. In our study, we applied Oxford Nanopore and Illumina technologies to assemble complete genomes of two widely used members of a distinct laboratory yeast lineage, the Peterhof Genetic Collection (PGC), and investigate the structural features of these genomes including transposable element content, copy number alterations, and structural rearrangements. We identified numerous notable structural differences between genomes of PGC strains and the reference S288C strain. We discovered a substantial enrichment of mid-length insertions and deletions within repetitive coding sequences, such as in the SCH9 gene or the NUP100 gene, with possible impact of these variants on protein amyloidogenicity. High contiguity of the final assemblies allowed us to trace back the history of reciprocal unbalanced translocations between chromosomes I, VIII, IX, XI, and XVI of the PGC strains. We show that formation of hybrid alleles of the FLO genes during such chromosomal rearrangements is likely responsible for the lack of invasive growth of yeast strains. Taken together, our results highlight important features of laboratory yeast strain evolution using the power of long-read sequencing.

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

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

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

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

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

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

Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations.

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

The diversity of microsporidian parasites infecting the Holarctic amphipod Gammarus lacustris from the Baikal region is dominated by the genus Dictyocoela.

Microsporidia are a highly diverse group of single-celled eukaryotic parasites related to fungi and infecting hosts belonging to all groups of eukaryotes, including some protists, invertebrate and vertebrate animals. We investigated the diversity of microsporidia in the Holarctic amphipod species Gammarus lacustris from mostly, but not limited to, water bodies in the Lake Baikal region. Ribosomal DNA sequencing and host transcriptome sequencing data from various works show that this species is predominantly infected by representatives of the genus Dictyocoela and probably has some features underlying this specific interaction.

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.