Lake Baikal amphipods and their genomes, great and small.

Endemic amphipods (Crustacea: Amphipoda) of Lake Baikal represent an outstanding example of large species flocks occupying a wide range of ecological niches and originating from a handful of ancestor species. Their development took place at a restricted territory and is thus open for comprehensive research. Such examples provide unique opportunities for studying behavioral, anatomic, or physiological adaptations in multiple combinations of environmental conditions and thus attract considerable attention. The existing taxonomies of this group list over 350 species and subspecies, which, according to the molecular phylogenetic studies of marker genes, full transcriptomes and mitochondrial genomes, originated from at least two introductions into the lake. The studies of allozymes and marker genes have revealed a significant cryptic diversity in Baikal amphipods, as well as a large variance in genetic diversity within some morphological species. Crossing experiments conducted so far for two morphological species suggest that the differences in the mitochondrial marker (cytochrome c oxidase subunit I gene) can potentially be applied for making predictions about reproductive isolation. For about one-tenth of the Baikal amphipod species, nuclear genome sizes and chromosome numbers are known. While genome sizes vary within one order of magnitude, the karyotypes are relatively stable (2n = 52 for most species studied). Moreover, analysis of the diversity of repeated sequences in nuclear genomes showed significant between-species differences. Studies of mitochondrial genomes revealed some unusual features, such as variation in length and gene order, as well as duplications of tRNA genes, some of which also underwent remolding (change in anticodon specificity due to point mutations). The next important steps should be (i) the assembly of whole genomes for different species of Baikal amphipods, which is at the moment hampered by complicated genome structures with high repeat content, and (ii) updating species taxonomy taking into account all the data.

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal.

We studied various aspects of heat-shock response with special emphasis on the expression of heat-shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5-fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.

Nanocrystalline graphite films nucleation by the radio frequency bias pretreatment.

New method for nucleation of different nanocrystalline carbon films upon monocrystalline Si substrate was proposed. The process is based on a combination of microwave and radio frequency plasma assisted chemical vapor deposition methods. Potential of the method for nucleation was demonstrated by deposition of nanocrystalline diamond film in pure microwave plasma in one process, immediately after "seeding" procedure. The method was also used for growth of nanocrystalline graphite (NCG) films, which are currently under intensive investigation due to their exceptional electronic properties, particularly fine electron emission characteristics. Deposited NCG films have demonstrated remarkable electron field emission properties having current density of up to 10 A/cm2. The films have also possessed good adhesion to silicon substrate. Carbon films and nucleation layer were characterized by scanning electron microscopy, transmission electron microscopy and Raman spectroscopy.

Sub-littoral and supra-littoral amphipods respond differently to acute thermal stress.

Thermal tolerance was determined in two closely related amphipod species from contrasting environments (sub-littoral and supra-littoral zones of the sea) using HSP expression and the activity of antioxidant enzymes. The levels of HSP70 and small HSPs present in untreated control animals were higher in the supra-littoral Orchestia gammarellus than in the sub-littoral Gammarus oceanicus. Under the acute thermal stress, HSP levels increased less strongly in O. gammarellus than in G. oceanicus. Activities of antioxidant enzymes peroxidase, catalase and glutathione S-transferase, were more pronounced in the supra-littoral O. gammarellus then in the sub-littoral G. oceanicus. We conclude that the environmental temperature regime modifies key cellular defense mechanisms in amphipods. Higher levels of constitutive HSP synthesis and higher levels of antioxidant enzymes in the supra-littoral species likely reflects adaptation to this highly thermally variable environment.

Evaluation of biochemical responses in Palearctic and Lake Baikal endemic amphipod species exposed to CdCl2.

This study evaluated small heat shock proteins (sHSP) (related to alpha-crystallin) and antioxidant enzymes (POD, peroxidase and CAT, catalase) as possible biomarkers for use in toxicological studies. Biochemical responses to cadmium chloride in two Lake Baikal endemic amphipods (Eulimnogammarus verrucosus, Eulimnogammarus cyaneus) and Palearctic species (Gammarus lacustris) were compared. Our findings showed that cadmium chloride toxicity directly influenced POD activity and sHSP synthesis in all amphipod species. The Baikalean endemic and the Palearctic amphipod species responded by decreasing activity of POD and they exhibited a dose-dependent activation of sHSP synthesis. All measured parameters differed among species and depended on the species' ability to resist cadmium chloride toxicity. CAT activity in the Palearctic species responded significantly to cadmium chloride exposure; however, responses were negligible for both Baikalean species. We suggest that synthesis of sHSP, together with changes in POD activity, could be used as biomarkers for further studies of amphipod species including endemics from Lake Baikal.

Natural organic matter (NOM) has the potential to modify the multixenobiotic resistance (MXR) activity in freshwater amphipods Eulimnogammarus cyaneus and E. verrucosus.

Based on the chemical features of natural organic matter (NOM) with its variety of functional groups, we hypothesized that NOM will modify the multixenobiotic-resistance (MXR) of an organism as xenobiotic chemicals do. The MXR system is a general first rather non-specific line of defense against environmental contaminants. The aim of this study was to compare the impacts on MXR activity in amphipod species (Eulimnogammarus cyaneus and E. verrucosus, from Lake Baikal) stressed by cadmium chloride or dissolved NOM for 24 h. NOM exposure concentrations were environmentally realistic. MXR activity was assessed based on rhodamine B efflux; its specificity was proven by a verapamil inhibition assay. It was shown that both NOM and CdCl(2) lead to substantial reduction of the rhodamine B efflux. This suggests that NOM may be regarded as a chemosensor which is able to reduce the efficiency of the MXR system. Possible mechanisms of direct NOM impact on MXR processes are discussed, such as peroxidation of the membranes (including P-glycoproteins) or internal blockage of the MXR pump by bioconcentrated NOM. In general, our results show that well-developed depuration pathways of freshwater organisms in contaminated environments may be impaired by strong chemical stressors and, more important, by natural biogeochemical matrices such as humic substances--humic substances are present in all freshwater systems.

Antioxidant response to natural organic matter (NOM) exposure in three Baikalean amphipod species from contrasting habitats.

The aim of the present work is to comparatively evaluate the oxidative stress response on exposure to natural organic matter (NOM) in three amphipod (Crustacea, Amphipoda) species from different taxonomic groups and different habitats of Lake Baikal. Endemic species from Lake Baikal were used: the shallow-water dwelling Gmelinoides fasciatus (Dyb.), Pallasea cancelloides (Gerstf.), and the deep-layer inhabitant Ommatogammarus flavus (Dyb.). Three key enzymes, catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST), were studied. The applied NOM from Lake Schwarzer (Germany) directly impacts the two littoral species which quickly respond. The response is characterized by a significant decrease of POD and an increase of CAT activities. GST activity remains stable or decreased slightly. In contrast to the littoral amphipods, the deep-layer inhabitant O. flavus showed no significant reaction to NOM exposure, probably due to decreased adaptive ability of this species. The stable environment of the Baikalean deep zones obviously does not provide triggers for the development of flexible antioxidant or general defense systems.

Antioxidant enzyme activity in endemic Baikalean versus Palaearctic amphipods: tagma- and size-related changes.

The activities of key antioxidant enzymes in two endemic Baikalean amphipod species: Pallasea cancelloides (Gerstf), Eulimnogammarus verrucosus (Gerstf) and the widely distributed Palearctic species Gammarus lacustris (Sars) were studied. This work was done to prove or disprove the hypothesis that Baikalean endemics have specifics in antioxidants system different from Palearctic species. The activities of antioxidant enzymes peroxidase, catalase and glutathione-S-transferase were measured in different sections (tagmata) of the amphipods' bodies as well as in different size groups. Well expressed tagma-related differences in peroxidase activity as well as smaller differences in catalase activity were shown in all studied species. There were no measured differences in glutathione-S-transferase activity among body sections. The existence of size-related changes in some antioxidant enzymes and the difference in such changes between Baikalean and Palearctic amphipods were noted. A significant increase in peroxidase activity with the size was found in both Baikalean species while a significant decrease in peroxidase activity was observed in the Palearctic G. lacustris. In Baikalean P. cancelloides, a significant decrease of catalase activity with the increase in age of crustaceans was noted, while in E. verrucosus no such relationship was found. In the Palearctic G. lacustris, a significant increase in catalase activity with the increase in size was noted. All species are shown to have no size-related differences in glutathione-S-transferase activity. The differences between species as well as between both different tagmata and size-classes within a particular species were estimated. It was assumed that the estimated differences in enzymes activity most likely depend on interspecific variation, rather than on conditional specifics in Lake Baikal.