Molecular Detection and Ecological Significance of the Cyanobacterial Genera Geitlerinema and Leptolyngbya in Black Band Disease of Corals

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.     

Adaptation of cyanobacteria to the sulfide-rich microenvironment of black band disease of coral

Black band disease (BBD) is a cyanobacteria-dominated microbial mat that migrates across living coral colonies lysing coral tissue and leaving behind exposed coral skeleton. The mat is sulfide-rich due to the presence of sulfate-reducing bacteria, integral members of the BBD microbial community, and the sulfide they produce is lethal to corals. The effect of sulfide, normally toxic to cyanobacteria, on the photosynthetic capabilities of five BBD cyanobacterial isolates of the genera Geitlerinema (3), Leptolyngbya (1), and Oscillatoria (1) and six non-BBD cyanobacteria of the genera Leptolyngbya (3), Pseudanabaena (2), and Phormidium (1) was examined. Photosynthetic experiments were performed by measuring the photoincorporation of [¹⁴C] NaHCO₃ under the following conditions: (1) aerobic (no sulfide), (2) anaerobic with 0.5 mM sulfide, and (3) anaerobic with 0.5 mM sulfide and 10 μM 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). All five BBD cyanobacterial isolates tolerated sulfide by conducting sulfide-resistant oxygenic photosynthesis. Five of the non-BBD cyanobacterial isolates did not tolerate sulfide, although one Pseudanabaena isolate continued to photosynthesize in the presence of sulfide at a considerably reduced rate. None of the isolates conducted anoxygenic photosynthesis with sulfide as an electron donor. This is the first report on the physiology of a culture of Oscillatoria sp. found globally in BBD.     

Microcystin production and ecological physiology of Caribbean black band disease cyanobacteria

Molecular studies of black band disease (BBD), a coral disease found on tropical and subtropical reefs worldwide, have shown that one 16S rRNA gene sequence is ubiquitous. This sequence has been reported to be a member of the cyanobacterial genus Oscillatoria. In this study, extracts of two cultured laboratory strains of BBD Oscillatoria, and for comparison two strains of BBD Geitlerinema, all isolated from reefs of the wider Caribbean, were analysed using Ultra-Performance Liquid Chromatography-Tandem Quad Mass Spectrometry (UPLC-MS/MS). The cyanotoxin microcystin-LR (MC-LR) was found in all strains, and one Geitlerinema strain additionally produced MC-YR. Growth experiments that monitored toxin production using enzyme-linked immunosorbent assay (ELISA) showed that BBD Oscillatoria produced yields of MC-LR equivalent (0.02-0.04 mg g(-1)) independent of biomass and culture conditions (varying temperature, pH, light and organic carbon). This pattern is different from BBD Geitlerinema, which increased production of MC-LR equivalent in the presence of organic carbon in the light and dark and at a relatively lower temperature. These results indicate that different species and strains of BBD cyanobacteria, which can occur in the same BBD infection, may contribute to BBD pathobiology by producing different toxins and different amounts of toxin at different stages in the disease process. This is the first detailed study of laboratory cultures of the ubiquitous BBD cyanobacterium Oscillatoria sp. isolated from Caribbean reefs.     

Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.     

The presence of the cyanobacterial toxin microcystin in black band disease of corals

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.     

Novel diversity within Roseofilum (Desertifilaceae,Cyanobacteria) from marine benthic mats with description of four new species

Benthic cyanobacterial mats (BCMs) are natural phenomena in marine environments. Reports of BCMs occurring across coastal marine environments have increased, partly driven by nutrient loading and climate change; thus, there is a need to understand the diversity involved in the proliferations and potential toxicity of the BCMs. Furthermore, marine cyanobacterial mats are observed growing on and affecting the health of corals with one specific cyanobacterial genus, Roseofilum, dominating the microbial mats associated with black band disease (BBD), a destructive polymicrobial disease that affects corals. To explore the diversity of Roseofilum, cyanobacterial mats from various marine habitats were sampled, and individual isolates were identified based on morphology, 16S rRNA gene phylogenies, 16S–23S ITS rRNA region sequence dissimilarities, and phylogenomics. Four novel species of Roseofilum were isolated from benthic marine mats, three from the coasts of Florida, United States (R. capinflatum sp. nov., R. casamattae sp. nov., and R. acuticapitatum sp. nov.) and one from the coast of France (R. halophilum sp. nov.). Our analyses revealed that Roseofilum associated with coral BBD and those not associated with corals but rather from coastal benthic mats are systematically distinct based on both phylogenetic and phylogenomic analyses. Enzyme-linked immunosorbent assay (ELISA) and LC–MS data indicated that microcystin production was found in one of the four species.     

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature. Electronic Publication     

Fine structure analysis of black band disease (BBD) infected coral and coral exposed to the BBD toxins microcystin and sulfide

Black band disease (BBD) of corals is a complex pathogenic polymicrobial mat community that lyses coral tissue as it migrates over an infected colony. Two known toxins are produced by BBD microorganisms - sulfide, produced by sulfate-reducing bacteria, and microcystin, produced by cyanobacteria. Experiments were carried out to determine the effects of exposing healthy coral fragments to variable concentrations of purified microcystin, sulfide at a concentration known to exist in BBD, and a combination of the two. Healthy fragments of the coral Montastraea annularis were placed into experimental chambers with known toxin/s for 18-22.5 h. Fine structural analysis using scanning electron microscopy (SEM) showed that toxin exposure resulted in thinning or removal of the coral epidermal layer coupled with degradation of the gastrodermis. These effects were exacerbated when both toxins were used in combination. Exposure to sulfide and the highest concentration of microcystin caused zooxanthellae to dissociate from the coral tissue and to form clusters on the coral surface. Examination of coral fragments infected with BBD was carried out for comparison. It was determined that the effects of exposure to sulfide and microcystin on coral fine structure were consistent, both quantitatively and qualitatively, with the effects of artificially induced and naturally occurring BBD on M. annularis.     

Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.     

A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau

Filamentous cyanobacteria forming red and black bands (black band disease, BBD) on 3 scleractinian corals from Palau were molecularly identified as belonging to a single ribotype. Red cyanobacterial mats sampled from infections on Pachyseris speciosa and a massive Porites sp. yielded red strains RMS1 and RMS2 respectively; the black cyanobacterial mat sampled from an infection on Montipora sp. yielded black strain BMS1. Following trials of a range of specialized media and culture conditions, 2 media, Grund and ASN-III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rDNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD-infected corals in the Caribbean. This is the first report of the successful isolation and culture of cyanobacterial strains derived from both red bands and BBD. Based on these findings, it is suggested that the classification of these 2 syndromes as separate coral diseases be postponed until further evidence is collected.     

Cyanotoxin production and phylogeny of benthic cyanobacterial strains isolated from the northeast of Brazil

Most of the knowledge about cyanobacteria toxin production is traditionally associated with planktonic cyanobacterial blooms. However, some studies have been showing that benthic cyanobacteria can produce cyanotoxins. According to this, we aimed to evaluate the production of microcystins and saxitoxins in benthic cyanobacteria isolated from aquatic ecosystems in the Northeast of Brazil and to use a polyphasic approach for their identification. Forty-five clonal strains were isolated from rivers and water supply reservoirs, and identified using morphological and molecular phylogenetic characteristics. In order to evaluate the toxins production, the strains were screened for genes involved in the biosynthesis of microcystins and saxitoxins, positive results were confirmed and cyanotoxins quantified using HPLC. Eight species were identified belonging to the Phormidiaceae, Pseudanabaenaceae and Nostocaceae families. This is the first study in Brazil that shows that strains from the Geitlerinema genus correspond to at least three phylogenetic lineages, which possibly correspond to three distinct species to be subsequently reclassified. The strains that showed one of the genes involved in the cyanotoxins production were analyzed by HPLC and Geitlerinema amphibium, Geitlerinema lemmermannii, Cylindrospermum stagnale and Phormidium uncinatum were identified as producing one or more saxitoxins variants. Thus, this is the first report of saxitoxins production for those first three species and the first report in Brazil for P. uncinatum.     

Fine-structural analysis of black band disease-infected coral reveals boring cyanobacteria and novel bacteria

Examination of coral fragments infected with black band disease (BBD) at the fine- and ultrastructural levels using scanning (SEM) and transmission electron microscopy (TEM) revealed novel features of the disease. SEM images of the skeleton from the host coral investigated (Montastraea annularis species complex) revealed extensive boring underneath the BBD mat, with cyanobacterial filaments present within some of the bore holes. Cyanobacteria were observed to penetrate into the overlying coral tissue from within the skeleton and were present throughout the mesoglea between tissue layers (coral epidermis and gastrodermis). A population of novel, as yet unidentified, small filamentous bacteria was found at the leading edge of the migrating band. This population increased in number within the band and was present within degrading coral epithelium, suggesting a role in disease etiology. In coral tissue in front of the leading edge of the band, cyanobacterial filaments were observed to be emerging from bundles of sloughed-off epidermal tissue. Degraded gastrodermis that contained actively dividing zooxanthellae was observed using both TEM and SEM. The BBD mat contained cyanobacterial filaments that were twisted, characteristic of negative-tactic responses. Some evidence of boring was found in apparently healthy control coral fragments; however, unlike in BBD-infected fragments, there were no associated cyanobacteria. These results suggest the coral skeleton as a possible source of pathogenic BBD cyanobacteria. Additionally, SEM revealed the presence of a potentially important group of small, filamentous BBD-associated bacteria yet to be identified.     

A 4700-Year History of Cyanobacteria Toxin Production in a Shallow Subtropical Lake

Cyanobacterial toxins or cyanotoxins are secondary metabolites produced by cyanobacteria and are found in water bodies around the world causing negative effects on aquatic ecosystems and human health. There are numerous environmental and biological triggers for toxin production, and the ecological role of most toxins is still being determined. Whereas cyanobacterial toxin occurrence appears to be expanding and monitoring efforts have increased in recent years, the history of toxin existence in lakes is poorly understood. Here, I report the history of the cyanotoxin, cylindrospermopsin (CYN), in sediments of hypereutrophic Lake Griffin, Florida, USA, from approximately 4700 years ago to present. The record includes three periods of toxin abundance: one associated with recent, European settlement in the watershed, and the other two during the middle to late Holocene, prior to human impacts on the lake. Each period corresponds to changes in different paleolimnological measurements suggesting drivers of CYN production have varied through time. This CYN record demonstrates the use of sediment toxin concentrations as a tool to reconstruct historic cyanobacterial toxin occurrence and shows that toxin production can occur independent of anthropogenic stressors.     

Metaproteomics reveals metabolic transitions between healthy and diseased stony coral Mussismilia braziliensis

Infectious diseases such as white plague syndrome (WPS) and black band disease (BBD) have caused massive coral loss worldwide. We performed a metaproteomic study on the Abrolhos coral Mussismilia braziliensis to define the types of proteins expressed in healthy corals compared to WPS‐ and BBD‐affected corals. A total of 6363 MS/MS spectra were identified as 361 different proteins. Healthy corals had a set of proteins that may be considered markers of holobiont homoeostasis, including tubulin, histone, Rab family, ribosomal, peridinin–chlorophyll a‐binding protein, F0F1‐type ATP synthase, alpha‐iG protein, calmodulin and ADP‐ribosylation factor. Cnidaria proteins found in healthy M. braziliensis were associated with Cnidaria–Symbiodinium endosymbiosis and included chaperones (hsp70, hsp90 and calreticulin), structural and membrane modelling proteins (actin) and proteins with functions related to intracellular vesicular traffic (Rab7 and ADP‐ribosylation factor 1) and signal transduction (14‐3‐3 protein and calmodulin). WPS resulted in a clear shift in the predominance of proteins, from those related to aerobic nitrogen‐fixing bacteria (i.e. Rhizobiales, Sphingomonadales and Actinomycetales) in healthy corals to those produced by facultative/anaerobic sulphate‐reducing bacteria (i.e. Enterobacteriales, Alteromonadales, Clostridiales and Bacteroidetes) in WPS corals. BBD corals developed a diverse community dominated by cyanobacteria and sulphur cycle bacteria. Hsp60, hsp90 and adenosylhomocysteinase proteins were produced mainly by cyanobacteria in BBD corals, which is consistent with elevated oxidative stress in hydrogen sulphide‐ and cyanotoxin‐rich environments. This study demonstrates the usefulness of metaproteomics for gaining better comprehension of coral metabolic status in health and disease, especially in reef systems such as the Abrolhos that are suffering from the increase in global and local threatening events.     

The cellular stress response of the scleractinian coral <Emphasis Type="Italic">Goniopora columna</Emphasis> during the progression of the black band disease

Black band disease (BBD) is a widespread coral pathology caused by a microbial consortium dominated by cyanobacteria, which is significantly contributing to the loss of coral cover and diversity worldwide. Since the effects of the BBD pathogens on the physiology and cellular stress response of coral polyps appear almost unknown, the expression of some molecular biomarkers, such as Hsp70, Hsp60, HO-1, and MnSOD, was analyzed in the apparently healthy tissues of Goniopora columna located at different distances from the infection and during two disease development stages. All the biomarkers displayed different levels of expression between healthy and diseased colonies. In the healthy corals, low basal levels were found stable over time in different parts of the same colony. On the contrary, in the diseased colonies, a strong up-regulation of all the biomarkers was observed in all the tissues surrounding the infection, which suffered an oxidative stress probably generated by the alternation, at the progression front of the disease, of conditions of oxygen supersaturation and hypoxia/anoxia, and by the production of the cyanotoxin microcystin by the BBD cyanobacteria. Furthermore, in the infected colonies, the expression of all the biomarkers appeared significantly affected by the development stage of the disease. In conclusion, our approach may constitute a useful diagnostic tool, since the cellular stress response of corals is activated before the pathogens colonize the tissues, and expands the current knowledge of the mechanisms controlling the host responses to infection in corals.     

Thermostablility of phycobiliproteins and antioxidant activity from four thermotolerant cyanobacteria

Four cyanobacterial strains including Cyanosarcina sp. SK40, Phormidium sp. PD40‐1, Scytonema sp. TP40 and Leptolyngbya sp. KC45 were selected and investigated for the phycobiliprotein (PBP) content and thermostable antioxidant activity of their cell‐free extracts. The highest content of 181.63 mg/g dry weight phycobiliprotein was found in Leptolyngbya sp. KC45 with phycoerythrin (PE) as the main phycobiliprotein. Among the PBPs of four thermotolerant cyanobacteria, PE from Leptolyngbya sp. KC45 exhibited the highest thermal stability as 80% of the original level remained after being heated at 60°C for 30 min. Antioxidant activities were detected in the cell‐free extracts of all cyanobacteria and that of Leptolyngbya sp. KC45 was also found in the highest value of 7.44 ± 0.14 and 3.89 ± 0.08 mg gallic acid equivalent (GAE) g^?1 dry weights determined by 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH) and reducing power assay, respectively. This also corresponded to the phenolic compound content. Based on DPPH and reducing power assay, antioxidant activities of all cyanobacterial extracts showed the high thermostability as approximately 80% remained after being heated at 80°C for 30 min. However, it clearly indicated that the thermostability of antioxidant activity from the hot spring cyanobacterial cell‐free extract was not contributed only by the PE, but also came from phenolic compounds and other oxidative substances.     

Emergence of toxic cyanobacterial species in the Ganga River,India, due to excessive nutrient loading

Despite of several efforts by the Government of India, pollution in National River Ganga is rising. The aim of the present study is to investigate the pollution in Ganga River in relation to appearance of toxic cyanobacterial strains. Jajmau area of Kanpur city is the industrial hub of Uttar Pradesh and is the main source of adding unwanted discharge into Ganga River. Water samples were randomly collected from the most polluted stretch of Ganga River (Kanpur, Uttar Pradesh, India). Samples were also collected from other major water of Uttar Pradesh to compare their water chemistry with Ganga River. Physico-chemical parameters of water bodies were estimated periodically for three years 2013–2015. Pearson product-mean correlation showed strong correlation between water parameters of sampling sites. Regression analysis showed seasonal variation in water parameters of Ganga River. Cyanobacteria prevalence in Ganga River was highest in May while lowest in August month. Fourteen cultivable cyanobacteria were isolated from Ganga River. Two new isolates, Oscillatoria sp. RBD01 and Leptolyngbya sp. RBD05 were found to be toxic and showed the presence of algal toxin (microcystin). Phylogenetic relatedness of toxic cyanobacterial isolates with their close homologues was established using 16S rRNA sequence analysis. Microcystin content in water samples (extracellular release) and in cyanobacterial isolates (intracellular content) was estimated by enzyme linked immunosorbent assay. Ganga River was found to be positive for microcystin with concentration ≥2 ppb which is above the permissible limit of WHO. Toxic cyanobacteria Oscillatoria sp. RBD01 and Leptolyngbya sp. RBD05 showed the presence of 23 and 17 ppb of microcystin in cells. Growth of the toxic cyanobacteria Oscillatoria sp. RBD01 showed very strong correlation with phosphate (0.834) and nitrate (0.761) content of water. Toxic Oscillatoria sp. RBD01 growing in moderate combination of nitrate (16x) and phosphate (4x) showed optimum growth and protein content. Periodic assessment of water quality and monitoring of toxic cyanobacteria would be helpful in identification and regulation of toxins which are responsible for destroying its sanctity and making it unsafe for human consumption.     

Phylogeny of culturable cyanobacteria from Brazilian mangroves

The cyanobacterial community from Brazilian mangrove ecosystems was examined using a culture-dependent method. Fifty cyanobacterial strains were isolated from soil, water and periphytic samples collected from Cardoso Island and Bertioga mangroves using specific cyanobacterial culture media. Unicellular, homocytous and heterocytous morphotypes were recovered, representing five orders, seven families and eight genera (Synechococcus, Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya, Phormidium, Nostoc and Microchaete). All of these novel mangrove strains had their 16S rRNA gene sequenced and BLAST analysis revealed sequence identities ranging from 92.5 to 99.7% when they were compared with other strains available in GenBank. The results showed a high variability of the 16S rRNA gene sequences among the genotypes that was not associated with the morphologies observed. Phylogenetic analyses showed several branches formed exclusively by some of these novel 16S rRNA gene sequences. BLAST and phylogeny analyses allowed for the identification of Nodosilinea and Oxynema strains, genera already known to exhibit poor morphological diacritic traits. In addition, several Nostoc and Leptolyngbya morphotypes of the mangrove strains may represent new generic entities, as they were distantly affiliated with true genera clades. The presence of non-ribosomal peptide synthetase, polyketide synthase, microcystin and saxitoxin genes were detected in 20.5%, 100%, 37.5% and 33.3%, respectively, of the 44 tested isolates. A total of 134 organic extracts obtained from 44 strains were tested against microorganisms, and 26% of the extracts showed some antimicrobial activity. This is the first polyphasic study of cultured cyanobacteria from Brazilian mangrove ecosystems using morphological, genetic and biological approaches.     

Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments

Three coccoid and two filamentous cyanobacterial strains were isolated from phototrophic biofilms exposed to intense solar radiation on lithic surfaces of the Parasurameswar Temple and Khandagiri caves, located in Orissa State, India. Based on to their morphological features, the three coccoid strains were assigned to the genera Gloeocapsosis and Gloeocapsa, while the two filamentous strains were assigned to the genera Leptolyngbya and Plectonema. Eleven to 12 neutral and acidic sugars were detected in the slime secreted by the five strains. The secretions showed a high affinity for bivalent metal cations, suggesting their ability to actively contribute to weakening the mineral substrata. The secretion of protective pigments in the polysaccharide layers, namely mycosporine amino acid-like substances (MAAs) and scytonemins, under exposure to UV radiation showed how the acclimation response contributes to the persistence of cyanobacteria on exposed lithoid surfaces in tropical areas.     

Potent toxins in Arctic environments – Presence of saxitoxins and an unusual microcystin variant in Arctic freshwater ecosystems

Cyanobacteria are the predominant phototrophs in freshwater ecosystems of the polar regions where they commonly form extensive benthic mats. Despite their major biological role in these ecosystems, little attention has been paid to their physiology and biochemistry. An important feature of cyanobacteria from the temperate and tropical regions is the production of a large variety of toxic secondary metabolites. In Antarctica, and more recently in the Arctic, the cyanobacterial toxins microcystin and nodularin (Antarctic only) have been detected in freshwater microbial mats. To date other cyanobacterial toxins have not been reported from these locations. Five Arctic cyanobacterial communities were screened for saxitoxin, another common cyanobacterial toxin, and microcystins using immunological, spectroscopic and molecular methods. Saxitoxin was detected for the first time in cyanobacteria from the Arctic. In addition, an unusual microcystin variant was identified using liquid chromatography–mass spectrometry. Gene expression analyses confirmed the analytical findings, whereby parts of the sxt and mcy operon involved in saxitoxin and microcystin synthesis, were detected and sequenced in one and five of the Arctic cyanobacterial samples, respectively. The detection of these compounds in the cryosphere improves the understanding of the biogeography and distribution of toxic cyanobacteria globally. The sequences of sxt and mcy genes provided from this habitat for the first time may help to clarify the evolutionary origin of toxin production in cyanobacteria.