Molecular genetic identification and seasonal succession of toxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir (Russia)

Data on the hepato- and neurotoxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir have been obtained for the first time. Different methods for revealing toxigenic cyanobacteria (light microscopy, PCR analysis, and enzyme-linked immunosorbent assay) demonstrate the same results. Hepatotoxins microcystins and for the first time neurotoxins saxitoxins were detected in the reservoir, whereas cylindrospermopsin and anatoxin-a were not revealed. The presence of mcyE and stxA genes responsible for microcystin and saxitoxin biosynthesis in total phytoplanktonic DNA is demonstrated. The following three genera of cyanobacteria containing mcyE gene are identified: Microcystis (M. aeruginosa, M. viridis), Planktothrix (P. agardhii), and Dolichospermum (Anabaena). It is hypothesized that saxitoxin-producing cyanobacteria Dolichospermum (Anabaena) inhabit the Rybinsk Reservoir.     

Identification of two cyanobacterial strains isolated from the Kotel’nikovskii hot spring of the Baikal rift

Two cyanobacterial strains, Pseudanabaena sp. 0411 and Synechococcus sp. 0431, were isolated from a sample collected in the Kotel’nikovskii hot spring of the Baikal rift. According to the results of light and transmission electron microscopy, as well as of the phylogenetic analysis of the 16S rRNA gene, these cyanobacteria were classified as Pseudanabaena sp. nov. and Synechococcus bigranulatus Skuja. The constructed phylogenetic tree shows that the studied strains are positioned in the clades of cyanobacteria isolated from hydrothermal vents of Asia and New Zealand, separately from marine and freshwater members of these genera, including those isolated from Lake Baikal.     

New host species and new distribution areas of <Emphasis Type="Italic">Salmincola lavaretus</Emphasis> (Copepoda: Lernaeopodidae): A parasite of coregonid fishes of Siberia

Data on the infestation of certain fish species by the parasitic copepod Salmincola lavaretus are presented for the first time. The infested fish species included Bauntovsky whitefish (Coregonus baunti), an endemic species from the Maloye and Bolshoye Kapylyushi lakes; Siberian whitefish (C. pidschian) and Siberian cisco (C. sardinella) from Bolshoye Kapylyushi Lake and Baunt Lake, which belong to the Tsypo-Tsypikan lake system (the Lena River basin, Transbaikalia); and Teletsky whitefish (C. lavaretus natio smitti) from Teletskoe Lake (the Ob River basin). Previously, S. lavaretus had been described as a parasite of Baikal omul (C. migratorius) and Baikal whitefish (C. baicalensis) from Baikal. The taxons of S. longimanus complex (S. longimanus, S. l. sibirica, S. svetlanovi, and S. lavaretus)—parasites of nasal fossae of grayling and coregonid fishes—were registered in the lakes of the Lake Baikal basin (Lake Baikal and Khovsgol Lake), the Lena River, the Yenisei and the Ob rivers (the largest rivers of the Arctic zoogeographic province), and the Kobdo River (the Western Mongolian province). It was assumed that S. longimanus complex is more widely distributed in the water bodies of the Arctic zoogeographic province of the Palearctic.     

The effect of bloom of filamentous green algae on the reproduction of yellowfin sculpin <Emphasis Type="Italic">Cottocomephorus grewingkii</Emphasis> (Dybowski, 1874) (Cottoidae) during ecological crisis in Lake Baikal

In shallow water areas of open Lake Baikal, filamentous green alga of the genus Spirogyra grows abundantly. Together with alga of the genus Ulothrix, it forms algal mats. According to our observations from 2010 to 2013, the spawning habitat conditions for the yellowfin sculpin Cottocomephorus grewingkii (Dybowski, 1874) (Cottidae) proved to be significantly disturbed in the littoral zone of Listvennichnyi Bay (southern Baikal), which, in turn, reduced the number of egg layings. With a 100% projective cover of the floor and a high density of green filamentous algae, the shallow-water stony substrate becomes completely inaccessible for spawning of the August population.     

Aquatic oligochaete fauna (Annelida,Clitellata) in Lake Tonle Sap and adjacent waters in Cambodia

In surveys conducted during 2000–2005, 39 taxa of aquatic oligochaetes belonging to the families Enchytraeidae and Naididae were found in Lake Tonle Sap in the Mekong River Basin, Cambodia. Dominated by naidines and pristinines (29 taxa), they mainly comprised widely distributed species as well as South Asian and Southeast Asian species. Among the four areas studied, the littoral regions of the lake—where inundated forests and aquatic vegetation developed during the flooded seasons—presented the highest number of species. Submerged vegetation in the littoral Lake Tonle Sap harbored abundant epiphytic oligochaetes, especially Stylaria fossularis. In contrast to the rich abundance of naidine and pristinine fauna, tubificines and ryhacodrilines were scarce in and around the lake, irrespective of the vegetation in their habitats. Several Aulodrilus species and Branchiura sowerbyi were the main representatives of the benthic oligochaete assemblages throughout the offshore zone of the lake without vegetation. It is noteworthy that the widely distributed tubificines Tubifex tubifex and Limnodrilus hoffmeisteri were not found in any surveyed locality.     

Consortia in Aquatic Ecosystems of the Transbaikalia

A survey in the basin of the Khilok River (a right tributary of the Selenga River) in 1999–2002 allowed us to reveal and describe the consortia of cyanobacteria Stratonostoc linckia f. linckia and Stratonostoc verrucosum, green alga Cladophora aegagropila, stonewort Nitella opaca, water moss Fontinalis sp., and duckweed Lemna trisulca. The relationship between the consort organisms and edificator plants can become the limiting factor in these communities. The long-term studies of the benthic communities of the Arakhlei Lake demonstrate the significance of the consortium approach in the long-term prediction of changes in the lake ecosystem.     

Colorless sulfur bacteria <Emphasis Type="Italic">Thioploca</Emphasis> from different sites in Lake Baikal

The colorless sulfur bacteria Thioploca spp. found in Lake Baikal are probably a marker for the influx of subterranean mineralized fluids. Bacteria act as a biological filter; by consuming sulfide in their metabolism, they detoxicate it and maintain the purity of Lake Baikal’s water. The bacteria were investigated by various techniques. According to analysis of the 16S rRNA gene fragment, Thioploca sp. from Frolikha Bay, Baikal belongs to the clade of freshwater species found in Lake Biwa and Lake Constance; it is most closely related to Thioploca ingrica.     

Adaptations to the hyporheic in Aloninae (Crustacea: Cladocera): allocation of <Emphasis Type="Italic">Alona protzi</Emphasis> Hartwig, 1900 and related species to <Emphasis Type="Italic">Phreatalona</Emphasis> gen. nov.

Morphological study of Alona protzi Hartwig, 1900, Alona phreatica Dumont, 1983 and Alona smirnovi Petkovski &; Flößner, 1972 reveals close affinities with Alona labrosa Vasiljeva &; Smirnov, 1969. We separate these four species from the polyphyletic Alona Baird, 1843 (Anomopoda: Chydoridae). United under Phreatalona gen. nov., these taxa share primitive features on the limbs, together with specializations to a rheic life mode. Phreatalona contains some of the only true hyporheic taxa within the Cladocera. Endemism in two ancient lakes (P. smirnovi and P. labrosa) and a preference for river sediments in Europe (P. phreatica and P. protzi) suggest a long isolation from typical littoral/benthic biotopes. We discuss close links with southern vicariant Nicsmirnovius, the position of these (hypo)rheic chydorids within the subfamily and their affinities with Acroperus. We remark an independent evolution of external (habitus, postabdomen) vs. internal (limb) morphology in the protzi-complex. Phreatalona is likely tertiary in origin, evolving from a littoral alonine adapting to rheic and finally hyporheic environments. Baikal endemic P. labrosa is likely the most primitive species of the genus. We discuss adaptations and evolution in the hyporheic and the effect on dispersal and biogeography of Phreatalona.     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

Original data on the diversity,ecology, and chemical composition of aquatic and semiaquatic lichens (Lichenes) of the stony littoral of Lake Baikal

Data on semiaquatic epilithic and aquatic lichens predominant in the surf and littoral zones of the test site Cape Berezovyi (Southern Baikal) obtained in 2002–2005 are presented. This is the first report on the distribution of rare and unfamiliar aquatic lichen species, Collema ramenskii Elenk. and Verrucaria spp. on the stony littoral of the western Baikal shore. The lichens form dense aggregations at depths from 1–1.5 to 3–3.5 m, where Verrucaria spp. thalli densely cover 60–70% of the rock debris. Only occasional lichens are observed below 7 m. Features of the penetration of aquatic Verrucaria spp. thalli into the rock debris were examined by electron microscopy. The chemical composition of aquatic lichens was analyzed for the first time. It was shown that aquatic lichens are capable of accumulating Ti, Ga, Y, and rare earths poorly soluble in the water of the stony littoral. The original data point to the active role of aquatic lichens in biogeochemical destruction of rocks of the Baikal stony littoral.     

Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR

Arthrospira species, under the name ‘Spirulina’, are used as food supplement for its protein, vitamins, and minerals which have several health benefits. Cyanobacterial toxins including microcystins can possibly contaminate these dietary supplements causing hepatotoxicity, tumour formation, and other disorders. The safe use of dietary supplements necessitates the need to assess such toxins in the algal food supplement. The methods which evaluate these dietary supplements should be highly sensitive, cost-effective, and rapid. In this study, multiplex HRM qPCR analysis was used to detect microcystin (MC)-producing cyanobacteria in Spirulina dietary supplements. The multiplex HRM qPCR detection limit was found to be 25 ag of mcyB spiked in a standard concentration of pcb (25 pg). Two distinct melt curves characteristic of pcb (Tm 82.8 ± 0.07 °C) and mcyB (Tm 77.9 ± 0.05 °C) were observed. Microcystin contamination was detected only in the fish food supplements and not in human dietary supplements of Spirulina. Liquid chromatography–high-resolution mass spectrometry analysis further confirmed the presence of the congeners of microcystin in the identified positive samples.     

Dependence of the weight on linear dimensions of the body in three chironomid species larvae (Diptera,Chironomidae) from the Lake Baikal littoral

The body size of larvae and the age structure of populations of three chironomid species (Chironomidae) from the Lake Baikal littoral was studied. Young larvae 1.0–2.6 mm long were abundant during the whole period investigated (August 2000–August 2001). Parameters of the equation that allows determining the weight of midge larvae by the width of their head capsule or by their body length were calculated for Paratanytarsus baicalensis, Orthocladius nitidoscutellatus, and Orthocladius sp.     

The Mechanisms for Genetic Diversity of Baikal Endemic Amphipod <Emphasis Type="Italic">Gmelinoides fasciatus</Emphasis>: Relationships between the Population Processes and Paleoclimatic History of the Lake

Amphipods of the species Gmelinoides fasciatus are one of the dominant species in the littoral zone community of Lake Baikal. Earlier, on the basis of the analysis of the mitochondrial COX1 gene, four isolated populations within the species range were identified, between which there were no distinct geographical barriers. In the present study, a phylogenetic reconstruction of the evolutionary history of G. fasciatus with the molecular clock hypothesis is carried out. The population divergence time is dated back to the onset of global climate changes on the Pleistocene–Pliocene boundary at 1.8–2.3 Ma. As a result, the estimated substitution rate in the COX1 gene for G. fasciatus was 1.2–2.4% of substitutions per Myr, which corresponded to the values determined for other groups of invertebrates. It was demonstrated that geographical barriers that formed the populations of G. fasciatus periodically appeared and disappeared in the past, resulting from climate changes associated with the periods of global cooling and warming.     

Experimental investigation of natural populations of <Emphasis Type="Italic">Daphnia galeata</Emphasis> G.O. Sars from the Curonian Lagoon feeding on potentially toxigenic cyanobacteria

The results of laboratory experiments on studying the characteristics of feeding of natural populations of Daphnia galeata from the freshwater Curonian Lagoon on cyanobacteria using the counting and radioisotope methods are discussed. The consumption and assimilation of the cyanobacterium Aphanizomenon flos-aquae by Daphnia have been revealed, as has the inhibition of feeding in Daphnia by species of the genus Microcystis. The results of PCR analysis of the total DNA of plankton from the Curonian Lagoon and enzyme-linked immunosorbent assay have proved the ability of Microcystis populations to produce microcystins, while no genes involved in the synthesis of cyanotoxins have been found in A. flos-aquae.     

Phytoplankton of Lake Bol’shie Shvakshty (Belarus) during the Shift of the Ecosystem from a Macrophyte–Weakly Eutrophic to a Phytoplankton–Hypereutrophic State

Changes in the quantitative characteristics and functioning of phytoplankton in Lake Bol’shie Shvakshty have been assessed. The changes are evoked by the introduction of herbivorous fishes into the lake and the resulting disturbance of ecological balance in the ecosystem and the shift of the lake into a hypertrophic state from a weakly eutrophic state. Human interference has caused the cyanobacteria density (abundance) and biomass values in the overall phytoplankton composition to strongly exceed (3.5 × 10⁹ cells/L and above 68 μg/L chlorophyll-а, respectively) the threshold value for safe recreational use of water bodies (20 million cells/L and 10 μg/L chlorophyll-а) established by the World Health Organization (WHO). The lake can be assigned to the third level of hazard to human health within the classification proposed by the WHO, as the cyanobacteria density is higher than 100 million cells/L and chlorophyll-а content is higher than 50 μg/L. MC-producing Microcystis species were identified among the cyanobacteria that has propagated in the lake in recent years, and five microcystin variants, including the highly toxic MC-LR, have been detected in the water.     

<Emphasis Type="Italic">Cocconeis nanoburyatica</Emphasis> sp. nov.—a new monoraphid diatom species from Lake Baikal

The new monoraphid diatom species C. nanoburyatica sp. nov. is described from Lake Baikal. This taxon differs from closely related taxa C. neodiminuta Krammer, C. neothumensis Krammer, and C. pseudothumensis Reichard by less developed striae, a very large axial area, and broader rounded ends.     

Diversity of Polyketide Synthase Genes in the Genomes of Heterotrophic Microorganisms Isolated from Epilithic Biofilms of Lake Baikal

Many bacterial secondary metabolites, including pharmacologically promising compounds, are synthesized by polyketide synthases (PKS) enzyme complexes. In the present work, nucleotide sequences of the genes encoding 16S rRNA and PKS of heterotrophic bacterial strains isolated from epilithic biofilms of the littoral zone of Lake Baikal were determined. On the basis of molecular phylogenetic analysis of the 16S rRNA genes, six heterotrophic strains were identified: Serratia fonticola 1А and 10А, Pseudomonas umsongensis K10-2 and K10-3, Rheinheimera tilapiae K18, and Flavobacterium sp. 43-09. Sequencing of cloned amplification products for PKS gene cluster revealed 33 sequences. Genes involved in biosynthesis of antibiotics (difficidine, erythromycin, curacin, mixalamide, corallopyronin, and myxothiazol) and cytostatics (romidepsin, spiruchostatin, and disorazol) were found among homologous sequences. The low homology (50–83%) of the PKS amino acid sequences of Baikal bacteria with sequences from GenBank attests to the potential capability of strains to produce new, not yet studied bioactive compounds. The obtained results show that the studied strains may be of practical interest for biotechnological application.     

A transcriptome-wide assessment of differentially expressed genes among two highly divergent,yet sympatric,lineages of the freshwater Atyid shrimp, <Emphasis Type="Italic">Paratya australiensis</Emphasis>

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.     

Detection and Genetic Characterization of Bacteria of the Genus <Emphasis Type="Italic">Pseudomonas</Emphasis> from Microbial Communities of Lake Baikal

The genus Pseudomonas is one of the most diverse and ecologically important groups of bacteria. Numerous representatives of the genus are found in microbial communities of all natural environments, including those closely associated with plants and animals. This ubiquitous distribution determines a necessity of their physiological and genetic adaptations. Molecular methods revealed that bacteria of the genus Pseudomonas were predominant in ulcerative lesions on the skin of Baikal yellowfin Cottocomephorus grewingkii (Dybowski, 1874). According to ribosomal phylogeny, cultivated Pseudomonas spp. isolated from both ulcerative lesions and the water column of Lake Baikal were grouped into the intrageneric cluster IG P. fluorescens. The topology of the phylogenetic tree based on the gene for outer membrane porin OprF generally coincided with that based on the 16S rRNA genes at the intrageneric level; however, it reflected ecological features of the strains of the genus Pseudomonas at the subgroup level. Screening of pathogenicity determinants detected the oprL, ecfX, fliC, and algD genes in the genomes of Pseudomonas spp. isolated from the ulcerative lesions of fish, whereas oprL and gyrB genes were determined in the strains isolated from the water column.