Bacterial and Archaeal Diversity in Hypersaline Cyanobacterial Mats Along a Transect in the Intertidal Flats of the Sultanate of Oman.

Hypersaline intertidal zones are highly dynamic ecosystems that are exposed to multiple extreme environmental conditions including rapidly and frequently changing parameters (water, nutrients, temperature) as well as highly elevated salinity levels often caused by high temperatures and evaporation rates. Microbial mats in most extreme settings, as found at the coastline of the subtropical-arid Arabian Peninsula, have been relatively less studied compared to their counterparts around the world. We report, here, for the first time on the diversity of the bacterial and archaeal communities of marine microbial mats along an intertidal transect in a wide salt flat with strongly increased salinity employing Illumina MiSeq technology for amplicon sequencing of 16S rRNA gene fragments. Microbial communities were dominated by typical halotolerant to halophilic microorganisms, with clear shifts in community composition, richness, and diversity along the transect. Highly adapted specialists (e.g., Euhalothece, Salinibacter, Nanohaloarchaeota) were mainly found at the most extreme, upper tidal sites and less specialized organisms with wide tolerance ranges (e.g., Lyngbya, Rhodovibrio, Salisaeta, Halobacteria) in intermediate sites of the transect. The dominating taxa in the lower tidal sites were typical members of well-stabilized mats (e.g., Coleofasciculus, Anaerolineaceae, Thaumarchaeota). Up to 40% of the archaeal sequences per sample represented so far unknown phyla. In conclusion, the bacterial richness and diversity increased from upper towards lower tidal sites in line with increasing mat stabilization and functional diversity, opposed to that of cyanobacteria only and archaea, which showed their highest richness and diversity in upper tidal samples.

Supratidal Extremophiles--Cyanobacterial Diversity in the Rock Pools of the Croatian Adria.

Hardly any molecular studies have been done on euendoliths of marine coastal environments, especially along the supratidal ranges of carbonate coasts. In our study, we provide a comparative sequence analysis using 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene combined with extensive microscopy of the endolithic community from rock pools of the Croatian Adria. Molecular diversity indices and richness estimates showed high level of diversity, particularly in high-salinity samples. The most common cyanobacteria belong to the order Pleurocapsales, similar to observations on limestone coasts in other parts of the world. Using single-cell amplification sequences of Hormathonema spp., Hyella caespitosa, and Kyrtuthrix dalmatica was for the first time introduced to the public GenBank.Microscopic investigations did not show qualitative variances in diversity among sites with different salinity but clear differences in prevalent organisms from similar environments suggesting that most of them are adapted to inhabit extreme, marine endolithic habitats and that similar species inhabit geographically separated but ecologically similar environments. This is a remarkable concordance rather seldom seen in molecular community studies in support of the hypothesis that endolithic ecosystems are seeded from a global meta-community.The relative diversity of the community is greater than those described from harsh endolithic habitats of cold and hot deserts. The maximum likelihood phylogenetic tree consisting of 166 operational taxonomic units (OTUs) at 96 % sequence similarity revealed 11 distinct clusters. Three clusters contained only epilithic or endolithic taxa, and five clusters contained mixed epilithic and endolithic taxa. Organisms clustered according to their taxonomic affiliations and not to their preferences to salt concentrations.

Ovarian alterations in wild northern pike Esox lucius females.

The aim of the present study was to analyse the occurrence of macroscopically visible ovary alterations in 2 populations of northern pike Esox lucius L. originating from lakes in the Mazurian Lake District (NE Poland). The alterations were characterised by ovary tissue that was morphologically malformed, in part or in whole, and contained immature oocytes, i.e. trophoplastic or previtellogenic oocytes instead of vitellogenic oocytes. These alterations were found only in the ovaries, and no morphological alterations of the testes were noted. Macroscopic and histological analyses were carried out in order to classify the observed alterations in the ovaries. Three types of alterations were identified in which morphological malformations as well as histological investigation of the ovaries were considered. An analysis of the size and age of the fish in relation to the occurrence of alterations as well as of the macroscopic and histological nature of the alteration types was made. The data obtained revealed no lake or age dependency of the observed alterations. Based on the results obtained, we suggest that the presence of endocrine disruptors in the environment or/and genetic factors could be responsible for these kinds of gonad anomalies. However, our results did not allow us to determine the aetiology of the alterations.

Cortical reaction as an egg quality indicator in artificial reproduction of pikeperch, Sander lucioperca.

The aim of this study was to investigate the process of the cortical reaction in eggs of pikeperch, Sander lucioperca (L.), as well as the application of microscopic assessment of this process in egg quality evaluation. The analysis was carried out with eggs obtained from 10 females by artificial reproduction, in which hormonal stimulation with hCG was applied. Subsequently, each sample of eggs (separately from each female fish) was analysed. The analysis included observation of the cortical reaction and the process of egg swelling, and determination of the effect of temperature (12, 14 and 16°C) and the presence of spermatozoa on the cortical reaction. The results indicate that the cortical reaction in pikeperch eggs is quite violent, resulting in visible deformation of eggs between 3 and 5 min after activation. No effect of temperature or the presence of spermatozoa on the cortical reaction was observed. A strong correlation was recorded for the percentage of egg deformations observed and embryo survival rate. The described method of determination of pikeperch egg quality (based on egg deformation rate between 3 and 5 min after activation) may be highly useful, both in scientific research (where high-quality eggs are required) and in hatchery practice.

Dinitrogen-fixing cyanobacteria in microbial mats of two shallow coral reef ecosystems.

Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using (15)N(2) and (13)C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C(2)H(2) reduced microg(-1) Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N(2) microg(-1) Chl day(-1) (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C microg(-1) Chl day(-1) (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.

Body form of leeches (Hirudinida: Piscicolidae) parasitizing fishes.

The body form of leeches, which becomes a modeling object, is a valid ectosomatic feature. It describes borders, in which, we can find internal organs of an individual. The range of realization of the body form, proposed for different groups of leeches, characterize their life spaces. These life spaces are distinct in term of position of body of leeches to the host and to the environment. Individuals originated from the Caspian Sea, Baikal Lake, and from fresh waters of Italy, Finland, Russia, Germany and Poland. Over 1200 individuals, belonging to 11 genera were measured. The measurements have been made according to the model of the leech body form.

Latitudinal gradient of cyanobacterial diversity in tidal flats.

Latitudinal diversity gradients are well-known for plants and animals, but only recently similar patterns have been described for some specific microbial communities in distinct habitats. Although microbial diversity is well-investigated worldwide, most of the studies are spatially too restricted to allow general statements about global diversity patterns. Additionally, methodological differences make it hard and often impossible to compare several studies. This study investigated the cyanobacterial diversity in tidal flats along geographical and ecological gradients based on high-throughput sequencing of 16S rRNA gene fragments (Illumina MiSeq) and environmental data on a large spatial scale from the subtropics to the Arctic. Latitude and strongly correlated environmental parameters (e.g. temperature) were identified as important drivers of cyanobacterial diversity on global scale resulting in a latitudinal diversity gradient similar to that known from plants and animals. Other non-correlated parameters (e.g. grain size) were shown to be more important on local scales, although no consistent pattern occurred across different locations. Among a total number of 989 operational taxonomic units (OTUs) only one cosmopolitan (classified as Coleofasciculus chthonoplastes), but many location-specific and putative endemic ones (78%) were detected. High proportions of rare members of the community (up to 86%) were found in all samples. Phylogenetic beta diversity was shown to be influenced by the developmental stage of the mat community becoming increasingly similar with increasing stabilization.

Lipid biomarkers, pigments and cyanobacterial diversity of microbial mats across intertidal flats of the arid coast of the Arabian Gulf (Abu Dhabi, UAE).

Variations in morphology, fatty acids, pigments and cyanobacterial community composition were studied in microbial mats across intertidal flats of the arid Arabian Gulf coast. These mats experience combined extreme conditions of salinity, temperature, UV radiation and desiccation depending on their tidal position. Different mat forms were observed depending on the topology of the coast and location. The mats contained 63 fatty acids in different proportions. The increased amounts of unsaturated fatty acids (12-39%) and the trans/cis ratio (0.6-1.6%) of the cyanobacterial fatty acid n-18:1omega9 in the higher tidal mats suggested an adaptation of the mat microorganisms to environmental stress. Chlorophyll a concentrations suggested lower cyanobacterial abundance in the higher than in the lower intertidal mats. Scytonemin concentrations were dependent on the increase in solar irradiation, salinity and desiccation. The mats showed richness in cyanobacterial species, with Microcoleus chthonoplastes and Lyngbya aestuarii morphotypes as the dominant cyanobacteria. Denaturing gradient gel electrophoresis patterns suggested shifts in the cyanobacterial community dependent on drainage efficiency and salinity from lower to higher tidal zones. We conclude that the topology of the coast and the variable extreme environmental conditions across the tidal flat determine the distribution of microbial mats as well as the presence or absence of different microorganisms.

Common evolutionary origin of planktonic and benthic nitrogen-fixing oscillatoriacean cyanobacteria from tropical oceans.

The filamentous cyanobacteria belonging to the genus Hydrocoleum (Blennothrix) are among the most common mat-forming cyanobacteria in tropical oceans. We present here the evidence that these benthic cyanobacteria are morphologically and phylogenetically very close to the planktonic species of Trichodesmium. Genetic relationship was established independently with regard to sequences of the 16S rRNA gene, nifH gene, and phycocyanin and phycoerythrin intergenic spacers. The species of both genera formed a common distinct branch in phylogenetically reconstructed cyanobacterial trees, suggesting that the main constituents of cyanobacterial benthos and plankton have an early common origin and both represent major contributors to nitrogen budget of tropical oceans today as in the distant geological past.

A new classification of a preovulatory oocyte maturation stage suitable for the synchronization of ovulation in controlled reproduction of Eurasian perch Perca fluviatilis L.

To improve controlled reproduction of Eurasian perch Perca fluviatilis, the criteria for the evaluation of final oocyte maturation stages were revised. The new classification covers six preovulatory maturational stages (I -VI) from the end of vitellogenesis to germinal vesicle breakdown (GVBD) and was based on macroscopic changes of preovulatory oocytes (position of the germinal vesicle, GVBD, oil droplets coalescence). The observation was performed during out-of-season artificial reproduction with the use of a single hCG injection (500 IU/kg). The classification was subsequently verified with the controlled reproduction of wild female perch with the use of hormonal stimulation (500 IU hCG/kg of body weight at 12°C). The females were at different maturational stages and constituted respective experimental groups (I-VI). During the experiment, ovulation appeared to be considerably synchronized within particular groups. Statistical differences in latency time (time between hormonal treatment and ovulation) were found between experimental groups (mean latency time: 110, 92, 68, 49, 29 and 18 h in groups representing VI, V, IV, III, II and I stage of the proposed classification, respectively). The proposed classification and the results presented in the study allowed for effective synchronisation of ovulation. The use of our new oocyte maturation classification may positively influence the effectiveness of Eurasian perch production.

Marine toxic cyanobacteria: diversity, environmental responses and hazards.

Toxic cyanobacterial blooms have been a primary concern predominantly in the plankton of freshwater bodies. Recently, however, the toxicity of benthic cyanobacteria is increasingly attracting attention of the scientific community and environmental agencies. The occurrence of toxic strains in benthic cyanobacteria is intimately linked to our understanding of the diversity and ecological responses of these organisms under field conditions. To that effect, we are engaged in combined morphotypic and genotypic characterization (polyphasic) of benthic natural populations of cyanobacteria in tropical lagoons and coral reefs, with the objective to provide a reliable reference for further comparative work. The methods of identification based on phenotypic properties and those based on molecular tools for genotypic identification are correlated. The approach is based on identifying the occurrences of cyanobacterial benthic blooms, tested for purity and analyzed by application of molecular tools. The questions addressed include the distinction between marine and freshwater taxa, between populations in geographically separate regions as well as between their potential vs. expressed toxicity.

Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies Phormidium autumnale (Oscillatoriales).

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus Phormidium (Oscillatoriales) from different habitats and geographical sites.

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.

Phylogenetic evaluation of cyanobacteria preserved as historic herbarium exsiccata.

Dried herbarium specimens of cyanobacteria (exsiccata) deposited over 100 years ago were analysed and characterized using combined morphological and molecular approaches. Six representative coccoid and filamentous cyanobacteria from two historic collections and a 15-year-old air-dried environmental sample were studied. Morphological features observed by light and electron microscopy were correlated with the results of 16S rRNA gene sequencing. Historic identifications achieved by means of classical morphology could thus be confirmed by extracted, amplified and sequenced 16S rRNA gene fragments. The results of this study open the possibility of providing genotypic characterizations to botanical type specimens, thus reconciling the botanical and bacteriological approaches to the taxonomic treatment of these micro-organisms.

Microbial spheres: a novel cyanobacterial-diatom symbiosis.

Cyanobacteria, algae and bacteria are widespread inhabitants of North Sea microbial mats. Our studies of these populations showed uncommon modes of living and extraordinary structures, which have not been described before. The structures are spherical objects covering a community of cyanobacteria, diatoms and bacteria. The cultivation of these communities in the laboratory and intensive observations of their exceptional movement has led to some spectacular findings. The sphere formations go through different phases with variation in the dominance of different microorganisms. The role of the bacteria is the most important in the first phase, and can be increased by the addition of signal substances. Spheres surrounded by envelopes of unknown composition and permeability appear, with numerous bacteria and sporadic diatoms inside. Then the cyanobacteria penetrate the spheres and arrange themselves at the surface. The communities proliferate over some weeks and are finally released. Laboratory expositions of the microbial communities to different parameters pinpoint the limits of sphere formation. The metabolic products of the sphere communities are concentrated in the spheres and lead to a different kind of compound compared with the surrounding environment. In this way, the microbial communities strongly influence the structure of the sediments. Uncommon circular structures, which develop into spheres between 0.08 and 3 mm in size were found in subcultures of non-axenic filamentous cyanobacteria enrichments from North Sea microbial mats. These filamentous cyanobacteria ( Phormidium sp.) together with associated benthic diatoms of the genus Navicula and associated heterotrophic bacteria were held as reproducible synergistic cultures. Phormidium sp. filaments tightly intertwined with each other, formed the surface of the spheres, trapping diatoms inside. The formation of "spheres" was the result of radial and synchronous movements of the cyanobacteria. In old cultures, the direction of the cyanobacterial movement has turned in the opposite direction, away from the sphere. The integrity of large "spheres" was influenced by chemotactic phenomena and maintained by some type of trichome-trichome interaction. This suggests the presence of metabolic secondary products, which attract cyanobacteria and influence their movement in a form of chemotactic response.

Prochlorococcus marinus strain PCC 9511, a picoplanktonic cyanobacterium, synthesizes the smallest urease.

The urease from the picoplanktonic oceanic Prochlorococcus marinus sp. strain PCC 9511 was purified 900-fold to a specific activity of 94.6 micromol urea min(-1) (mg protein)(-1) by heat treatment and liquid chromatography methods. The enzyme, with a molecular mass of 168 kDa as determined by gel filtration, is the smallest urease known to date. Three different subunits with apparent molecular masses of 11 kDa (gamma or UreA; predicted molecular mass 11 kDa), 13 kDa (ss or UreB; predicted molecular mass 12 kDa) and 63 kDa (alpha or UreC; predicted molecular mass 62 kDa) were detected in the native enzyme, suggesting a quaternary structure of (alphassgamma)(2). The K:(m) of the purified enzyme was determined as being 0.23 mM urea. The urease activity was inhibited by HgCl(2), acetohydroxamic acid and EDTA but neither by boric acid nor by L-methionine-DL-sulfoximine. Degenerate primers were designed to amplify a conserved region of the ureC gene. The amplification product was then used as a probe to clone a 5.7 kbp fragment of the P. marinus sp. strain PCC 9511 genome. The nucleotide sequence of this DNA fragment revealed two divergently orientated gene clusters, ureDABC and ureEFG, encoding the urease subunits, UreA, UreB and UreC, and the urease accessory molecules UreD, UreE, UreF and UreG. A putative NtcA-binding site was found upstream from ureEFG, indicating that this gene cluster might be under nitrogen control.

Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-'Synechococcus' clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon.

PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of 'Synechococcus' (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511(T), although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511(T), PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-'Synechococcus' strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.

The signal transducer P(II) and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation.

The amino acid sequence of the signal transducer P(II) (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria. P(II) of P. marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942. Moreover, P. marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite. This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (C(i); K(m,app) 240 and 4 micro M, respectively), a result consistent with the unphosphorylated form of P(II) acting as a sensor for the control of C(i) acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803. The present data are discussed in relation to the genetic information provided by the P. marinus MED4 genome sequence.