Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain.     

Estimating nodularin content of cyanobacterial blooms from abundance of Nodularia spumigena and its characteristic pigments—a case study from the Baltic entrance area

The presence of toxin-producing cyanobacteria is of concern for the management of recreational waters. The abundance of toxic cyanobacteria and environmental concentrations of their toxins may fluctuate substantially within a short time emphasising the need for rapid methods to estimate toxin concentrations in the water. During late June-early September 2002 the abundance, biomass and characteristic pigments of Nodularia spumigena Mertens and the hepatotoxin nodularin produced by N. spumigena were analysed in water samples collected from the Baltic entrance area. Significant relationships were found between cell-bound concentrations of nodularin and the abundance and biomass of N. spumigena with a relationship of approximately 1 pg nodularin per Nodularia-cell. It is suggested that simple counts of Nodularia under the microscope may be used as a rapid on-site technique to estimate potential nodularin concentrations in recreational waters. Comparison with data from Australia shows that cell-bound concentration of nodularin per Nodularia-cell differs between geographically distant areas and therefore such relationships should be established for individual areas. The carotenoids echinenone, canthaxanthin and a cis-canthaxanthin-like caroteniod, identified from a laboratory culture of N. spumigena isolated from the Baltic Sea, were also significantly correlated with concentrations of cell-bound nodularin. However, Aphanizomenon and Anabaena, two genera commonly co-occurring with Nodularia, also contain these pigments and thus the significant correlations obtained presumably originate from Nodularia being the dominant cyanobacterium in all samples collected in 2002.     

Associations of Cyanobacterial Toxin,Nodularin, with Environmental Factors and Zooplankton in the Baltic Sea

Concentrations of a cyanobacterial toxin, nodularin, were measured in the Baltic Sea in 1998 and 1999. Statistical associations of nodularin concentrations with environmental factors were tested by multiple regression analysis. To reveal the toxin-producing organism, colonies of Aphanizomenon and filaments of Nodularia were picked and analyzed for peptide toxins. It was also investigated whether there was an association with zooplankton and Nodularia. All the measured seston samples contained nodularin, but other toxins were not detected by the HPLC analysis. In both years, the highest nodularin concentrations were found at the surface water layer. The nodularin concentrations were positively correlated with silicate concentrations in water. High concentrations of silica in surface water may indicate recent upwelling, which in turn renders surface water rich in nutrients. This upwelling is likely to intensify cyanobacterial growth and toxin production, which may explain this rather unexpected result. The picked Aphanizomenon colonies did not contain nodularin and the dissolved nodularin concentrations were below detection limit. Thus it was concluded that most of the nodularin was bound to Nodularia cells. The abundances of zooplankton (copepods, rotifers, and cladocerans) were unrelated to Nodularia, but were positively associated with Aphanizomenon.     

Toxic and non-toxic Nodularia strains can be distinguished from each other and from eukaryotic algae with chlorophyll fluorescence fingerprinting

Chlorophyll fluorescence induction curves of toxic and non-toxic strains of the cyanobacterium Nodularia were measured and compared with fluorescence curves measured from four species of eukaryotic algae. Both cyanobacteria and algae were isolated from the Baltic Sea. The results show that Nodularia strains can be distinguished from the eukaryotes by applying a pattern recognition procedure to the fluorescence induction curves, suggesting that the fluorescence fingerprinting technique might be useful in environmental monitoring of marine algae. The six studied Nodularia strains could not be distinguished from each other from their fluorescence induction kinetics. However, their fluorescence curves fell into two clear categories, the toxic and the non-toxic Nodularia. Emission spectroscopy and differences in the fluorescence induction curves showed that the ratio of the intensity of the Photosystem I emission peak to the Photosystem II peak is higher in non-toxic Nodularia than in the toxic strains, suggesting that the toxicity affects the structure of the photosynthesis machinery. The effect on photosynthesis may be related to the ability of the microcystins to chelate iron.     

First report of cyanobacterial diversity and microcystins in a Microcystis strain from Sidi Boughaba,a Moroccan coastal lagoon

The cyanobacterial diversity of Sidi Boughaba, a Moroccan coastal lagoon and Ramsar site, was evaluated and its potentially toxic species were isolated and characterised. This study was the first time that cyanobacterial diversity and cyanotoxin production have been characterised in a Moroccan coastal lagoon. Samples collected in June 2004, July 2008 and August 2013 contained 41 species. Three strains of cyanobacteria were isolated, cultured and assessed for their potentially toxic levels of microcystins. Only Microcystis flos-aquae exhibited the presence of microcystins, at a concentration of 823.41 µg g?1 as MC-LR equivalents. MC-RR and MC-WR were also detected. The presence of toxic Microcystis and other potentially toxic strains, especially in periods of bloom proliferations, could pose environmental and health hazards. Cyanotoxin monitoring of this lagoon is highly recommended.     

Toxicity to Daphnia of a compound extracted from laboratory and natural Microcystis spp., and the role of microcystins

1. The microcystin content of a variety of Microcystis spp., from both laboratory strains and natural blooms, was analysed by HPLC. The microcystin content of laboratory strains ranged from 1.6 to 4.3μgmg^?1 dry weight. Yearly and seasonal variation was detected in an analysis of bloom material collected from Bautzen Reservoir over a 3-year period. The microcystin concentration in bloom material ranged from undetectable to 1.16 μg ml^?1 dry weight. 2. Toxicity of laboratory and natural Microcystis to Daphnia pulicaria was determined using an established LC₅₀ technique. Partially purified water extracts from different Microcystis samples exhibited a wide range of toxicity. The highest activity was found in natural Microcystis samples, with an LC₅₀ of 36 μgm^?1 dry weight of Microcystis, whereas one strain did not appear toxic at 1600 μg ml^?1. 3. No correlation was found between the concentrations of microcystins of different laboratory and natural Microcystis strains and the toxicity of extracts to Daphnia pulicaria from the same strains. Therefore, we discriminated between hepatotoxic microcystins and the compound(s) that is toxic to Daphnia, here termed DTC (Daphnia-toxic compound), which is independent of microcystins.     

Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml⁻¹ of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.     

The effect of salinity on the growth,toxin production,and morphology of <Emphasis Type="BoldItalic">Nodularia spumigena</Emphasis> isolated from the Gulf of Gdańsk,southern Baltic Sea

Culture experiments on the toxic Nodularia spumigena strain NSGG-1 isolated from the Gulf of Gdańsk showed a significant effect of salinity on growth and nodularin production. Growth of the NSGG-1 strain, was optimal between 7 and 18 psu, lower at 3 and 24 psu and was significantly inhibited at the extreme salinities of 0 and 35 psu. Nodularin (NOD) content of N. spumigena, estimated by the NOD/Chla ratios, correlated positively with salinity and increased from 0 to 35 psu. The NOD/Chla ratio on day 10 of growth was high, and, reached the maximum at day 30. A sudden increase in salinity from 7 to 18 and 35 psu resulted in plasmolysis of Nodularia cells. Salinity was also observed to have other effects on NSGG-1; the filaments were longest at 7 psu, while an increased number of akinetes were formed at 35 psu. The number of heterocytes was markedly reduced at the extreme salinities. This latter finding might explain why Nodularia blooms do not occur outside a certain salinity range in nitrogen-deficient waters.     

The influence of environmental conditions on the seasonal variation of <Emphasis Type="Italic">Microcystis</Emphasis> cell density and microcystins concentration in San Francisco Estuary

A bloom of the cyanobacteria Microcystis aeruginosa was sampled over the summer and fall in order to determine if the spatial and temporal patterns in cell density, chlorophyll a (chl a) concentration, total microcystins concentration, and percent microcystins composition varied with environmental conditions in San Francisco Estuary. It was hypothesized that the seasonal variation in Microcystis cell density and microcystin concentration was ecologically important because it could influence the transfer of toxic microcystins into the aquatic food web. Sampling for Microcystis cell density, chl a concentration, total microcystins concentration and a suite of environmental conditions was conducted biweekly at nine stations throughout the freshwater tidal and brackish water regions of the estuary between July and November 2004. Total microcystins in zooplankton and clam tissue was also sampled in August and October. Microcystis cell density, chl a concentration and total microcystins concentration varied by an order of magnitude and peaked during August and September when and α^B were high. Low streamflow and high water temperature were strongly correlated with the seasonal variation of Microcystis cell density, total microcystins concentration (cell)⁻¹ and total microcystins concentration (chl a)⁻¹ in canonical correlation analyses. Nutrient concentrations and ratios were of secondary importance in the analysis and may be of lesser importance to seasonal variation of the bloom in this nutrient rich estuary. The seasonal variation of Microcystis density and biomass was potentially important for the structure and function of the estuarine aquatic food web, because total microcystins concentration was high at the base of the food web in mesozooplankton, amphipod, clam, and worm tissue during the peak of the bloom. Handling editor: D. Hamilton     

Phenotypic and toxicological characterization of toxic Nodularia spumigena from a freshwater lake in Turkey

Cyanobacterial blooms have been occasionally observed in Iznik lake, a freshwater body (salinity = 0.5) located in the western part of Turkey. Nodularia spumigena (Mertens in Juergens) was recorded in the lake in the summer months of 2005. Maximum filament concentration of the species (1.3 × 10⁵ fil L^?1) was measured in August and constituted 60% of total cyanobacteria abundance. Trichomes were solitary, straight and had cells containing gas vesicles. Heterocysts were regularly spaced throughout the filaments. In the isolated filaments nodularin was detected by HPLC, ELISA and PPIA as well as LC–MS. HPLC analysis showed that gravimetric nodularin concentration in cultured N. spumigena cells was 578 μg of nodularin per gram dry weight (d.w.). Apart from nodularin, demethylated nodularin variant was also found in Nodularia cell extract. This is the first report of toxic N. spumigena in a European freshwater lake.     

Determination of Oligopeptide Diversity within a Natural Population of Microcystis spp. (Cyanobacteria) by Typing Single Colonies by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Besides the most prominent peptide toxin, microcystin, the cyanobacteria Microcystis spp. have been shown to produce a large variety of other bioactive oligopeptides. We investigated for the first time the oligopeptide diversity within a natural Microcystis population by analyzing single colonies directly with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The results demonstrate a high diversity of known cyanobacterial peptides such as microcystins, anabaenopeptins, microginins, aeruginosins, and cyanopeptolins, but also many unknown substances in the Microcystis colonies. Oligopeptide patterns were mostly related to specific Microcystis taxa. Microcystis aeruginosa (Kütz.) Kütz. colonies contained mainly microcystins, occasionally accompanied by aeruginosins. In contrast, microcystins were not detected in Microcystis ichthyoblabe Kütz.; instead, colonies of this species contained anabaenopeptins and/or microginins or unknown peptides. Within a third group, Microcystis wesenbergii (Kom.) Kom. in Kondr., chiefly a cyanopeptolin and an unknown peptide were found. Similar patterns, however, were also found in colonies which could not be identified to species level. The significance of oligopeptides as a chemotaxonomic tool within the genus Microcystis is discussed. It could be demonstrated that the typing of single colonies by MALDI-TOF MS may be a valuable tool for ecological studies of the genus Microcystis as well as in early warning of toxic cyanobacterial blooms.     

Microcystins derived from lysing Microcystis cells do not cause negative effects on crustacean zooplankton in Lake Taihu, China

Microcystins (MCs) have a toxic effect on crustacean zooplankton in the laboratory, but there is little or no unequivocal evidence in the literature of their lethal effects on crustacean zooplankton in the field. We used the natural microcystins extracted from Microcystis spp. to test if they could cause any negative effects on crustacean zooplankton. We conducted three experiments in enclosures with water from Lake Taihu, China, and microcystins derived by extraction from Microcystis spp. collected from the lake when the species was in bloom conditions. Initial concentrations of extracellular microcystins (EMCs = MC-RR + MC-LR + MC-YR) ranged from 9.7 to 44.9 μg/L in treatments with microcystin addition. Microcystin concentrations sharply decreased on second day in all the three experiments. EMCs at the end of the experiments varied from only 2.7 to 14.2 % of the levels at the start of the experiments. The dominant species of crustacean zooplankton in the lake were Bosmina longirotris, Ceriodaphnia cornuta, Mesocyclops spp., Limnoithona sinensis, Sinocalanus dorrii and Schmackeria inopinus. ANOVA analysis showed that the density and biomass of cladoceran and copepod did not significantly differ between treatments with microcystin addition and controls. Our results indicate that microcystins derived from lysing Microcystis do not cause any negative effects on crustacean zooplankton.     

Unravelling the systematics of Nodularia (Bivalvia,Unionidae) species from eastern Russia

Conservation efforts have been hindered by data deficient conservation status assessments, especially due to taxonomic problems. This is especially true for many eastern Russian species of freshwater mussels, where distinct classification systems have complicated their delimitation and identification. Nodularia is a widespread eastern Asian freshwater mussel genus, present from Vietnam in the south to the Magadan region in eastern Russia in the north. The number of recognized species in the genus Nodularia in eastern Russia has been inflated over the last several decades due to the use of a typological species concept, the so-called 'Comparatory Method'. This method uses a single diagnostic character for species delimitation, i.e., the arc of maximal convexity of the shell's outline. Using this classification system, 10 species were recognized for far eastern Russia under the genus Nodularia Conrad, 1853, divided into three subgenera: Nodularia s. str., Amurunio and Magadaninaia. Since it is not supported by any other classification methods, the current comparatory classification is rejected by many Russian and international scientists, who only recognize a single species for that region, i.e., Nodularia douglasiae. Therefore, the aim of the present study is to clarify the taxonomy and systematics of the Nodularia genus in far eastern Russia and adjacent territories, using a multiple dataset approach that combines distribution with detailed conchological and anatomical analyses with morphometry and COI barcoding molecular techniques. All analyses performed in this study support the existence of a single Nodularia species in eastern Russia, i.e., N. douglasiae.     

Morphology,phylogeny, growth rate and nodularin production of Nodularia spumigena from Brazil

Blooms of the toxic cyanobacterium Nodularia spumigena occur in various locations worldwide, but have not been observed in Brazil until recently. Three Nodularia strains were isolated from summer blooms in experimental shrimp production ponds of Penaeus vannamei in Rio Grande, in southern Brazil; these strains were characterized by morphology, phylogeny, growth rate and toxicity. The strains were identified as N. spumigena based on the size of vegetative cells, heterocytes and akinetes under a light microscope and based on the number of gas vesicles per μm² under a transmission electron microscope. The 16S rRNA gene sequences of the three strains showed high identity (> 99%) with N. spumigena sequences available on the NCBI database but were grouped closer in the phylogenetic tree with N. spumigena strains from Australia and USA than those from the Baltic Sea. The growth rate in batch culture varied between 0.2 and 0.6 μ?d^?1 based on cell density, optical density and chlorophyll-a content. The three strains produced the hepatotoxin nodularin (ELISA plate kit) with similar toxicity values (4.8–4.9?µg?l^?1). We conclude that the three isolated strains are N. spumigena with similar rates of growth and nodularin production. The presence of N. spumigena now represents a potential problem in aquaculture and estuarine environments in Brazil.     

LOCALIZATION OF MICROCYSTIN SYNTHETASE GENES IN COLONIES OF THE CYANOBACTERIUM MICROCYSTIS USING FLUORESCENCE IN SITU HYBRIDIZATION1

To better understand the production of microcystins (MCs) in Microcystis colonies, fluorescence in situ hybridization (FISH) methods were developed to detect DNA involved in the synthesis of these cyanobacterial hepatotoxins. Using colonies of Microcystis aeruginosa (Kütz.) Kütz. isolated from environmental blooms of cyanobacteria and from a colony‐forming, MC‐producing laboratory strain of Microcystis, amplified PCR products were observed, coincident with positive controls. The total MC content of individual colonies of Microcystis, determined by ELISA, showed a positive correlation with colony cross‐sectional area. FISH analysis of Microcystis colonies gave high fluorescence in comparison to negative controls, indicating the presence of MC synthetase DNA (mcyA) in situ. FISH analysis for MC synthetase genes has the potential to be developed into an effective early warning tool for drinking and recreational water management.     

Quantitative assessment of toxic and nontoxic <Emphasis Type="Italic">Microcystis</Emphasis> colonies in natural environments using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization and flow cytometry

Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.     

Initial impacts of <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> blooms on the aquatic food web in the San Francisco Estuary

The impact of the toxic cyanobacterium Microcystis aeruginosa on estuarine food web production in San Francisco Estuary is unknown. It is hypothesized that Microcystis contributed to a recent decline in pelagic organisms directly through its toxicity or indirectly through its impact on the food web after 1999. In order to evaluate this hypothesis, phytoplankton, cyanobacteria, zooplankton, and fish were collected biweekly at stations throughout the estuary in 2005. Concentrations of the tumor-promoting Microcystis toxin, microcystin, were measured in water, plankton, zooplankton, and fish by a protein phosphatase inhibition assay, and fish health was assessed by histopathology. Microcystis abundance was elevated in the surface layer of the western and central delta and reached a maximum of 32 × 10⁹ cells l⁻¹ at Old River in August. Its distribution across the estuary was correlated with a suite of phytoplankton and cyanobacteria species in the surface layer and 1 m depth including Aphanizomenon spp., Aulacoseira granulata, Bacillaria paradoxa, Rhodomonas spp., and Cryptomonas spp. Shifts in the phytoplankton community composition coincided with a decrease in the percentage of diatom and green algal carbon and increase in the percentage of cryptophyte carbon at 1 m depth. Maximum calanoid and cyclopoid copepod carbon coincided with elevated Microcystis abundance, but it was accompanied by a low cladocera to calanoid copepod ratio. Total microcystins were present at all levels of the food web and the greater total microcystins concentration in striped bass than their prey suggested toxins accumulated at higher trophic levels. Histopathology of fish liver tissue suggested the health of two common fish in the estuary, striped bass (Morone saxatilis), and Mississippi silversides (Menidia audens), was impacted by tumor-promoting substances, particularly at stations where total microcystins concentration was elevated. This study suggests that even at low abundance, Microcystis may impact estuarine fishery production through toxic and food web impacts at multiple trophic levels.     

Microcystin-Degrading Activity of an Indigenous Bacterial Strain Stenotrophomonas acidaminiphila MC-LTH2 Isolated from Lake Taihu

Microcystin-LR (MC-LR) and microcystin-RR (MC-RR) produced by harmful cyanobacterial blooms (HCBs) pose substantial threats to the ecosystem and public health due to their potential hepatotoxicity. Degradation of microcystins (MCs) by indigenous bacteria represents a promising method for removing MCs from fresh water without harming the aquatic environment, but only a few microcystin (MC)-degrading bacteria have been isolated and had their mechanisms reported. This study aimed to isolate indigenous bacteria from Lake Taihu, and investigate the capability and mechanism of MC degradation by these bacteria. During a Microcystis bloom, an indigenous MC-degrading bacterium designated MC-LTH2 was successfully isolated from Lake Taihu, and identified as Stenotrophomonas acidaminiphila based on phylogenetic analysis. In the presence of MC-LR together with MC-RR, the strain MC-LTH2 was capable of totally degrading both simultaneously in 8 days, at rates of 3.0 mg/(L⋅d) and 5.6 mg/(L⋅d), respectively. The degradation rates of MCs were dependent on temperature, pH, and initial MC concentration. Adda (3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid) was detected as an intermediate degradation product of MCs using high performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF-MS). To the best of our knowledge, this is the first report of Stenotrophomonas acidaminiphila capable of degrading two MC analogues and other compounds containing Adda residue completely under various conditions, although the mlrA gene in the strain was not detected. These results indicate the Stenotrophomonas acidaminiphila strain MC-LTH2 possesses a significant potential to be used in bioremediation of water bodies contaminated by MC-LR and MC-RR, and is potentially involved in the degradation of MCs during the disappearance of the HCBs in Lake Taihu.     

FATE OF THE TOXIC CYCLIC HEPTAPEPTIDES,THE MICROCYSTINS,FROM BLOOMS OF MICROCYSTIS (CYANOBACTERIA) IN A HYPERTROPHIC LAKE1

The in situ fate of the toxic cyclic heptapeptides, the microcystins, produced by blooms of Microcystis was examined at two stations in a hypertrophic Japanese lake. Microcystins were detected in all samples of Microcystis with quantities varying seasonally and spatially (230–950 μg · g dry wt^?1 at St. 1 and 160–746 μg · g dry wt^?1 at St. 2) and composed of microcystin-LR, -RR, and-YR. Microcystin-RR was the dominant toxin in most samples. A large amount of microcystin (1.1 μg · L^?1) was detected in only one sample of filtered lake water. Accumulation of microcystin in zooplankton was indirectly estimated from a newly developed equation model. Large amounts of microcystin (75–1387 μg · g dry wt^?1) were accumulated in the zooplankton community, which consisted of two cladocerans, Bosmina fatalis Burckhardt and Diaphanosoma brachyurum Lieve, and a copepod, Cyclops vicinus Uljanin, that co-occurred with the toxic Microcystis blooms. The maximum percent of microcystin content in zooplankton to that in Microcystis was 202%. Among the three species of zooplankton, only B. fatalis seemed to be responsible for accumulation of the microcystins because C. vicinus appeared to avoid contact with Microcystis cells and D. brachyurum did not consume colonies of Microcystis. Microcystins may be transferred to higher trophic levels through B. fatalis.