Benthic Anabaena Research Articles

Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: Buoyancy and dispersal

Benthic cyanobacteria in rivers produce cyanotoxins and affect aquatic food webs, but knowledge of their ecology lags behind planktonic cyanobacteria. The buoyancy of benthic Anabaena spp. mats was studied to understand implications for Anabaena dispersal in the Eel River, California. Field experiments were used to investigate the effects of oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4days, while in low light mats begin to sink in <24h. Floating Anabaena samples were collected from five sites in the watershed and found to contain the cyanotoxins anatoxin-a and microcystin, with higher concentrations of anatoxin-a (median 560, max 30,693ng/gDW) than microcystin (median 30, max 37ng/gDW). The ability of Anabaena mats to maintain their buoyancy will markedly increase their downstream dispersal distances. Increased buoyancy also allows toxin-containing mats to collect along shorelines, increasing threats to human and animal public health.

A detailed morphological, phylogenetic and ecophysiological analysis of four benthic Anabaena (Nostocales, Cyanobacteria) strains confirms deep heterogeneity within the genus

Four benthic Anabaena strains isolated from different localities in the Czech Republic were examined from the morphological, phylogenetic, and physiological points of view. The results of combined analysis showed distinct morphological dissimilarity between the studied strains, which were further found to belong to different phylogenetic groups based on the 16S rRNA gene phylogeny. To assess the temperature and light (irradiance) optima of the strains, we exposed them to various combinations of these two parameters. The experiment revealed unexpectedly high temperature and light optima for some of the strains, while others showed optima that were similar to those of previously studied planktic species of related heterocytous genera. Our study is the first of its kind to be applied to benthic Anabaena strains. Our results indicate that the benthic Anabaena genus is much more complex than previously thought and provide novel insights into the biology and ecology of benthic Anabaena species. With the collection of more data, we expect the genus Anabaena will be split into several new monophyletic taxa, each covering distinct morpho- and ecotypes.

Anabaenolysins, Novel Cytolytic Lipopeptides from Benthic Anabaena Cyanobacteria

Two novel cyclic lipopeptides, anabaenolysin A and anabaenolysin B, were isolated from two benthic cyanobacterial strains of the genus Anabaena. This novel class of cyanobacterial lipopeptides has a general structure of a small peptide ring consisting of four amino acids from which two are proteinogenic and two unusual; glycine1, glycine2, 2-(3-amino-5-oxytetrahydrofuran-2-yl)-2-hydroxyacetic acid3 and a long unsaturated C18 β-amino acid4 with a conjugated triene structure. They are distinguished by the presence of a conjugated dienic structure in the C18 β-amino acid present in anabaenolysin A but not in anabaenolysin B. Conjugated triene structure generates a typical UV spectrum for anabaenolysins for easy recognition. Anabaenolysin A constituted up to 400 ppm of the cyanobacterial dry weight. We found evidence of thirteen variants of anabaenolysins in one cyanobacterial strain. This suggests that the anabaenolysins are an important class of secondary metabolites in benthic Anabaena cyanobacteria. Both anabaenolysin A and B had cytolytic activity on a number of mammalian cell lines.

Phylogenetic and morphological evaluation of Wollea saccata (Nostocales, Cyanobacteria) isolated from the Yenissei River basin (Eastern Siberia, Russia).

For the first time the genus Wollea Bornet et Flahault was recorded in Eastern Siberia (Yenissei River, Karskoe See Basin) and isolated to monoclonal axenic strain ACCS 045. The strain was characterized by a combination of morphological and molecular approaches. It shares the diacritical features of the type-species W. saccata (Wolle) Bornet et Flahault and W. bharadwajae R.N. Singh such as macroscopic gelatinous colonies, tube-like, trichomes more or less straight or slightly curved, sheaths around trichomes are absent, heterocytes intercalary, and paraheterocytic akinete development at both sides of heteocytes in short series. A morphological comparison shows that it differs from W. saccata in the form of colonies being saccule, the form of vegetative cells and the heterocytes being barrel-shaped, the form of terminal cells being conical-rounded, and from W. bharadwajae in the form of akinetes being cylindrical or ellipsoid. The first available 16S rRNA gene sequence of Wollea appeared poorly related to other cyanobacterial sequences. According to the traditional classification systems based on morphological and ecological observations genus Wollea appears closest to Anabaena and Nostoc. However, the phylogenetic placement of Wollea based on 16S rRNA gene sequence was distant from Nostoc and the most closely related to taxa in benthic Anabaena, Sphaerospermopsis, Cylindrospermopsis, and Raphidiopsis.&nbsp;

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea)

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.

The cyanobacterial genus Macrospermum.

This small tropical cyanobacterial group, containing Anabaena volzii LEMMERMANN and a few related species (A. fuellebornii SCHMIDLE, A. unispora GARDNER, A. mysorensis GONZALVES &amp; KAMAT), differs substantially phenotypically from all other planktic or benthic Anabaena types, mainly by the subsymmetric structure of the trichomes, type of akinete formation and restricted ecology. The taxonomic uniformity of all other Anabaenalike clusters (typical benthic Anabaena, planktic Anabaena subg. Dolichospermum, Trichormus, Aphanizomenon, Cuspidothrix) was already supported by molecular analyses. All of them also have their typical morphological markers, which are clearly different from the "Anabaena volzii - cluster". Therefore, this group can not be classified in any of the mentioned revised genera and must be described as a separate generic entity of heterocytous cyanobacteria (although they have not been sequenced to date). The new genus Macrospermum is therefore defined in my article with 4 related, morphologically distinguishable species. The generic name is selected according to the unusually large akinetes.

Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum - 2. part: straight types.

This small tropical cyanobacterial group, containing Anabaena volzii Lemmermann and a few related species (A. fuellebornii SCHMIDLE, A. unispora GARDNER, A. mysorensis GONZALVES &amp; KAMAT), differs substantially phenotypically from all other planktic or benthic Anabaena types, mainly by the subsymmetric structure of the trichomes, type of akinete formation and restricted ecology. The taxonomic uniformity of all other Anabaenalike clusters (typical benthic Anabaena, planktic Anabaena subg. Dolichospermum, Trichormus, Aphanizomenon, Cuspidothrix) was already supported by molecular analyses. All of them also have their typical morphological markers, which are clearly different from the "Anabaena volzii - cluster". Therefore, this group can not be classified in any of the mentioned revised genera and must be described as a separate generic entity of heterocytous cyanobacteria (although they have not been sequenced to date). The new genus Macrospermum is therefore defined in my article with 4 related, morphologically distinguishable species. The generic name is selected according to the unusually large akinetes.

Differential patterns of evolution and distribution of the symbiotic behaviour in nostocacean cyanobacteria

Many cyanobacteria commonly identified as belonging to the genus Nostoc are well-known cyanobionts (symbionts) of a wide variety of plants and fungi. They form symbioses with bryophytes, pteridophytes, gymnosperms and angiosperms that are considerably different in the type of reciprocal interaction between the host and the cyanobiont. The phylogenetic and taxonomic relationships among cyanobionts isolated from different hosts and Nostoc strains isolated from free-living conditions are still not well understood. We compared phylogeny and morphology of symbiotic cyanobacteria originating from different host plants (genera Gunnera, Azolla, Cycas, Dioon, Encephalartos, Macrozamia and Anthoceros) with free-living Nostoc isolates originating from different habitats. After preliminary clustering with ARDRA (amplified rDNA restriction analysis), phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with morphological characterization, obtaining several supported clusters. Two main Nostoc clusters harboured almost all cyanobionts of Gunnera, Anthoceros and of several cycads, together with free-living strains of the species Nostoc muscorum, Nostoc calcicola, Nostoc edaphicum, Nostoc ellipsosporum and strains related to Nostoc commune. We suggest that the frequent occurrence of symbiotic strains within these clusters is explained by the intensive hormogonia production that was observed in many of the strains studied. However, no evidence for discrimination between symbiotic and free-living strains, either by molecular or morphological approaches, could be found. Sequences of Azolla cyanobiont filaments, taken directly from leaf cavities, clustered tightly with sequences from the planktic cyanobacterium Cylindrospermopsis raciborskii, from the benthic Anabaena cylindrica 133 and from Anabaena oscillarioides HINDAK 1984/43, with high bootstrap values. The phylogenetic analysis showed that two distinct patterns of evolution of symbiotic behaviour might exist for the nostocacean cyanobacteria, one leading to symbioses of Nostoc species with a wide variety of plants, the other leading to the association of a unique cyanobacterial type with the water fern Azolla.

Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum - 1. part: coiled types.

This small tropical cyanobacterial group, containing volzii Lemmermann and a few related species (A. fuellebornii SCHMIDLE, A. unispora GARDNER, A. mysorensis GONZALVES & KAMAT), differs substantially phenotypically from all other planktic or benthic types, mainly by the subsymmetric structure of the trichomes, type of akinete formation and restricted ecology. The taxonomic uniformity of all other Anabaenalike clusters (typical benthic Anabaena, planktic subg. Dolichospermum, Trichormus, Aphanizomenon, Cuspidothrix) was already supported by molecular analyses. All of them also have their typical morphological markers, which are clearly different from the Anabaena volzii - cluster. Therefore, this group can not be classified in any of the mentioned revised genera and must be described as a separate generic entity of heterocytous cyanobacteria (although they have not been sequenced to date). The new genus Macrospermum is therefore defined in my article with 4 related, morphologically distinguishable species. The generic name is selected according to the unusually large akinetes.

A high proportion of Baltic Sea benthic cyanobacterial isolates contain apoptogens able to induce rapid death of isolated rat hepatocytes

To assess the potential hepatotoxicity of benthic cyanobacteria, we isolated 41 strains from the Baltic Sea. The bacteria were differentially extracted with solvents of decreasing polarity. The extracts were tested for ability to induce death of primary rat hepatocytes in suspension culture. Mainly morphological criteria were used to discriminate between cell death with apoptotic features (shrinkage, chromatin hypercondensation, budding) or necrotic features (swelling, loss of plasma membrane integrity). The 24 isolates containing hepatotoxic compounds were of the genus Anabaena. The non-toxic isolates were mainly Nostoc and Calothrix. The toxicity was not due to the known hepatotoxic cyanobacterial protein phosphatase inhibitors microcystin or nodularin, as demonstrated by lacking competition with microcystin for PP2A binding. Apoptotic cell death was rapid, being evident from 10 to 60 min after the addition of extract. Sometimes the initial apoptosis was followed by secondary necrosis. Three cyanobacterial extracts produced apoptosis with unusual cell morphology including actin rearrangements. It will be of interest to know if they contain substance(s) acting through novel death pathways. We conclude that benthic Anabaena cyanobacteria represent a rich source of apoptogenic toxins, presumably directed against competitors or predators in the aquatic environment, but obviously able also to induce cell death in mammalian parenchymal liver cells.

Benthic cyanobacteria from the Baltic Sea contain cytotoxic Anabaena, Nodularia, and Nostoc strains and an apoptosis‐inducing Phormidium strain

Benthic cyanobacteria from aquatic environments have been reported to produce biologically active metabolites. However, the toxicity and other biological activities of benthic cyanobacteria from the Baltic Sea are not well known. We determined the biological activities of 21 Anabaena, Calothrix, Nodularia, Nostoc, and Phormidium strains isolated from benthic littoral habitats of the Baltic Sea. We studied whether benthic cyanobacterial extracts caused cytotoxicity by necrosis or induced apoptosis in two mammalian cell lines, a human leukemia cell line (HL-60) and a mouse fibroblast cell line (3T3 Swiss), and examined potential hepatotoxin (microcystin and nodularin) production. Five of the six benthic Anabaena strains, one of the two Nostoc strains, and two of the three Nodularia strains were highly cytotoxic to human leukemia cells. The Calothrix and Phormidium strains did not cause LDH leakage, but the extract of Phormidium strain BECID15 induced apoptosis in the HL-60 cells. Neither the microcystin synthetase E (mcyE) nor the nodularin synthetase F (ndaF) gene was amplified by PCR, and no microcystins or nodularins were detected by the protein phosphatase inhibition assay from the cyanobacterial strains included in this study. This indicates that benthic Baltic cyanobacteria contain potentially harmful cytotoxic compounds even though they do not produce microcystins or nodularins. These cytotoxic compounds remain to be characterized, and the mechanisms of cytotoxicity need to be studied further.

Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (Nostocales, Cyanobacteria)

The heterocytous cyanobacteria form a monophyletic group according to 16S rRNA gene sequence data. Within this group, phylogenetic and morphological studies have shown that genera such as Anabaena and Aphanizomenon are intermixed. Moreover, the phylogeny of the genus Trichormus, which was recently separated from Anabaena, has not been investigated. The aim was to study the taxonomy of the genera Anabaena, Aphanizomenon, Nostoc and Trichormus belonging to the family Nostocaceae (subsection IV.I) by morphological and phylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences. New strains were isolated to avoid identification problems caused by morphological changes of strains during cultivation. Morphological and phylogenetic data showed that benthic and planktic Anabaena strains were intermixed. In addition, the present study confirmed that Anabaena and Aphanizomenon strains were not monophyletic, as previously demonstrated. The evolutionary distances between the strains indicated that the planktic Anabaena and Aphanizomenon strains as well as five benthic Anabaena strains in cluster 1 could be assigned to a single genus. On the basis of the 16S rRNA, rpoB and rbcLX gene sequences, the Anabaena/Aphanizomenon strains (cluster 1) were divided into nine supported subclusters which could also be separated morphologically, and which therefore might represent different species. Trichormus strains were morphologically and phylogenetically heterogeneous and did not form a monophyletic cluster. These Trichormus strains, which were representatives of three distinct species, might actually belong to three genera according to the evolutionary distances. Nostoc strains were also heterogeneous and seemed to form a monophyletic cluster, which may contain more than one genus. It was found that certain morphological features were stable and could be used to separate different phylogenetic clusters. For example, the width and the length of akinetes were useful features for classification of the Anabaena/Aphanizomenon strains in cluster 1. This morphological and phylogenetic study with fresh isolates showed that the current classification of these anabaenoid genera needs to be revised.