Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

First report of a toxic Nodularia spumigena (Nostocales/ Cyanobacteria) bloom in sub-tropical Australia. I. Phycological and public health investigations.

Cyanobacterial blooms represent one of the most conspicuous and widespread waterborne microbial hazards to human and ecosystem health. Investigation of a cyanobacterial bloom in a shallow brackish water recreational cable ski lake in south-eastern Queensland, Australia revealed the dominance of the toxigenic species Nodularia spumigena. The bloom spanned three months, during which time cell concentrations exceeded human guideline thresholds for recreational risk, and concentrations of the hepatotoxic cyanotoxin nodularin exceeded 200 µg L(-1). Cyanotoxin origin and identification was confirmed by amplification of the ndaF-specific PCR product and sequencing of the 16S rRNA gene. From the limited data available leading up to, and throughout the bloom, it was not possible to establish the set of causative factors responsible for its occurrence. However a combination of factors including salinity, hydraulic retention time and nutrient status associated with an extended period of drought are likely to have contributed. This was the first known occurrence of this species in bloom proportions from sub-tropical Australia and as such represents a hitherto uncharacterized risk to human and ecosystem health. It highlights the need for adaptive monitoring regimes to ensure a comprehensive understanding of the potentially toxic cyanobacteria likely to inhabit any given region. Such monitoring needs to recognize that cyanobacteria have a significant capacity for range expansion that has been facilitated by recent changes in global climate.

First report of a toxic Nodularia spumigena (Nostocales/ Cyanobacteria) bloom in sub-tropical Australia. II. Bioaccumulation of nodularin in isolated populations of mullet (Mugilidae).

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8-47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 µg/kg, range 32.3-56.8 µg/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

Human and rat hepatocyte toxicity and protein phosphatase 1 and 2A inhibitory activity of naturally occurring desmethyl-microcystins and nodularins.

Contamination of water, foods and food supplements by various genera of cyanobacteria is a serious health problem worldwide for humans and animals, largely due to the toxic effects of microcystins (MCs) and nodularin (NOD), a group of hepatotoxic cyclic peptides. The toxins occur in variable structures resulting in more than 90 different MCs and 8 different NODs, many of them not having been investigated for their toxic potency. Potent MCs such as MC-LR have been shown to elicit their hepatotoxic potency via inhibition of hepatic protein phosphatases (PP) 1 and 2A leading to over-phosphorylation of vital cellular proteins. This mechanism of action is also thought to be responsible for the long term tumor promoting action of certain MCs and NOD in the liver. Here, we report on the isolation of certain MCs and NOD as well as a number of their desmethylated derivatives from algae bloom. Subsequently, we determined the cytotoxicity of these compounds in isolated primary human and rat hepatocytes in culture. In parallel experiments, we analyzed the inhibitory potency of these congeners on PP1 and 2A using commercially available enzymes. We found in primary rat hepatocytes that MC-LR, -YR and NOD were cytotoxic, namely in the 10 to >50 nM range, while MC-RR was not. The desmethylated congeners of MC-LR, -YR, and NOD were equally or more-toxic as/than their fully methylated counterparts. In primary human hepatocytes we could show that MC-LR, NOD and the desmethylated variants [³Asp]MC-LR, [7Dha]MC-LR and [¹Asp]NOD were cytotoxic in the 20 to >600 nM range. Inhibition data with human, bovine and rabbit protein phosphatases 1 and 2A were roughly in accordance with the cytotoxicity findings in human and rat hepatocytes, i.e. desmethylation had no pronounced effects on the inhibitory potencies. Thus, a variety of naturally occurring desmethylated MC and NOD congeners have to be considered as being at least as toxic as the corresponding fully methylated derivatives.

Diversity of peptides produced by Nodularia spumigena from various geographical regions.

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.

A new quantitative PCR assay for the detection of hepatotoxigenic cyanobacteria.

Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml⁻¹. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality.

Morphological, toxicological and molecular characterization of a benthic Nodularia isolated from Atlantic estuarine environments.

A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 microm and from 3.71 to 4.25 microm, respectively, while heterocyts were 3.91-4.89 microm long and 4.20-4.74 microm wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC(50) values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp.

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena.

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena. We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.

Removal of surface blooms of the cyanobacteria Nodularia spumigena: a pilot project conducted in the Baltic Sea.

Blooms of Cyanobacteria are a major concern during the summer period in the Baltic Sea Proper. The nitrogen-fixing Nodularia spumigena forms massive toxic blooms in the surface layers, with a concentration of biomass in the uppermost 1-m water layer. This pilot study describes the construction and test of a Nodularia collecting device during the summer of 2006. Oil booms were modified so that their dragging skirt was replaced with a water-permeable forming fabric used in the pulp and paper industry. The results showed that the modified oil booms worked and operated in an effective way when towed in the sea. Calculations showed that the collecting device used in this study has a theoretical capacity of cleaning 0.055 km2 (5.5 ha) of sea surface hr(-1), compared with the 6600 km2 of the Baltic Sea that were covered by Nodularia blooms during the summer of 2005. Future possibilities for Nodularia harvesting are discussed.

Photosynthetic response of Nodularia spumigena to UV and photosynthetically active radiation depends on nutrient (N and P) availability.

Biomass of N. spumigena is distributed within the dynamic photic zone that changes in both light quantity and quality. This study was designed to determine whether nutrient status can mitigate the negative impacts of experimental radiation treatments on the photosynthetic performance of N. spumigena. Cyanobacterial suspensions were exposed to radiation consisting of photosynthetically active radiation (PAR=400-700 nm), PAR+UV-A (=PA, 320-700 nm), and PAR+UV-A+UV-B (=PAB, 280-700 nm) under different nutrient media either replete with external dissolved nitrate (N) and orthophosphate (P; designated as +N/+P), replete with P only (-N/+P), or replete with N only (+N/-P). Under low PAR (75 micromol photons m(-2) s(-1)), nutrient status had no significant effect on the photosynthetic performance of N. spumigena in terms of rETRmax, alpha, and E(k). Nodularia spumigena was able to acclimate to high PAR (300 micromol photons m(-2) s(-1)), with a corresponding increase in rETRmax and E(k). The photosynthetic performance of N. spumigena cultured with supplemental nitrogen was more susceptible to experimental PAR irradiance. Under UVR, P-enrichment in the absence of additional external N (-N/+P) induced lower photoinhibition of photosynthesis compared with +N/-P cultures. However, the induction of NPQ may have provided PSII protection under P-deplete and PAR+UVR conditions. Because N. spumigena are able to fix nitrogen, access to available P can render them less susceptible to photoinhibition, effectively promoting blooms. Under a P-deficient condition, N. spumigena were more susceptible to radiation but were capable of photosynthetic recovery immediately after removal of radiation stress. In the presence of an internal P pool in the Baltic Sea, which may be seasonally available to the diazotrophic cyanobacteria, summer blooms of the resilient N. spumigena will persist.

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.

Microcystins associated with Microcystis dominated blooms in the Southwest wetlands, Western Australia.

Potentially toxic cyanobacterial blooms are becoming common in the freshwater wetlands on the Swan Coastal Plain, Western Australia. During summer the dominant bloom-causing species belong to the genera Microcystis and Anabaena and to a lesser extent Aphanizomenon and Nodularia. Although toxic cyanobacteria have been recorded in the Swan-Canning and Peel-Harvey estuaries in Western Australia, very little is known about the blooms in the surrounding freshwater lakes. In this study, a total of 32 natural bloom samples representing 13 lakes were analyzed by HPLC for microcystin (MC)-LR, -RR, and -YR. Twenty-eight samples proved to be toxic. The highest total microcystin concentration ranged from 1645 to 8428.6 microg L(-1), and the lowest concentrations were less than 10 microg L(-1) with some below the detection limit (< 0.05 microg L(-1)). MC-LR (100%) was the predominant microcystin, followed by MC-YR (71.4%) and MC-RR (60.7%). The presence of a Nodularia spumigena bloom in the freshwater Lake Yangebup was associated with the detection of nodularins (1664 microg L(-1)). This is the first study to demonstrate the presence of microcystins and nodularins in urban lakes on the Swan Coastal Plain, Western Australia.