Deposit-feeders accumulate the cyanobacterial toxin nodularin

Blooms of toxic cyanobacteria may potentially affect food web productivity and even be a human health hazard. In the Baltic Sea, regularly occurring summer blooms of nitrogen-fixing cyanobacteria are often dominated by Nodularia spumigena, which produces the potent hepatotoxin nodularin. Evidence of sedimentation of these blooms indicates that benthic fauna can be exposed to nodularin. In a one month experiment, we simulated the settling of a summer bloom dominated by N. spumigena in sediment microcosms with three species of sediment-dwelling, deposit-feeding macrofauna, the amphipods Monoporeia affinis and Pontoporeia femorata and the bivalve Macoma balthica, and analyzed nodularin in the animals by HPLC–ESI–MS (high-performance liquid chromatography–electrospray ionization–mass spectrometry). We found nodularin in quantities of 50–120 ng g⁻¹ DW. The results show that deposit-feeding macrofauna in the Baltic Sea may contribute to trophic transfer of nodularin.     

Reduction of cyanobacterial toxins through coprophagy in Mytilus edulis

An experiment was conducted to follow the fate of the cyanobacterial toxin, nodularin, produced by Nodularia spumigena through ingestion by Mytilus edulis and re-ingestion of faecal material (coprophagy). Mussels were fed with cultures of N. spumigena, and the faeces that were produced were fed to other mussels not previously exposed to N. spumigena. Concentrations of nodularin were measured in the food (N. spumigena), the mussels and in the faeces in order to make a toxin budget. High concentrations of nodularin were found in the mussels and their faeces after 48 h incubation with N. spumigena. When the toxic faeces were fed to new mussels, the toxin content of faeces was reduced from 95 μg nod g⁻¹ dry weight (DW) to 1 μg nod g⁻¹ DW through the process of coprophagy. Hence, when toxic faeces were fed to mussels, the nodularin concentration of the resulting faecal material was reduced by 99%. Pseudofaeces were produced when the mussels were grazing on N. spumigena, but not when grazing on faeces. The pseudofaeces contained high concentrations of nodularin and apparently intact N. spumigena cells. However, these cells were growth-inhibited and their potential contribution to seeding a bloom is probably limited. Our data indicate that a large fraction of ingested nodularin in M. edulis is egested with the faeces, and that the concentration of nodularin in the faeces is reduced when faeces are re-ingested.     

Detection of nodularin in European flounder (Platichthys flesus) in the west coast of Sweden: Evidence of nodularin mediated oxidative stress

The brackish, bloom-forming cyanobacterium Nodularia spumigena produces a peptide called nodularin, which may induce liver damage in fish. In the summer of 2007, nodularin was detected in liver tissue of European flounder caught in Swedish waters of Öresund, within the upper salinity limit for N. spumigena. Nodularin concentrations ranging between 22 and 557 μg kg⁻¹ liver (d.w.) were detected in fish liver. Nodularin was not detected in blue mussels (Mytilus edulis). Although N. spumigena blooms can occur in the area, the cyanobacteria were only present in very small amounts in 2007. Results suggested that nodularin accumulated in flounder livers during the summer of 2006, when vast N. spumigena blooms were observed in Öresund, and persisted over several months. Nodularin has previously been shown to induce oxidative stress in mice, crustaceans and mollusks but work on the potential negative effects of nodularin on fish is still scarce. To examine the dynamics of nodularin induced oxidative stress in liver tissue of flounder, the differential responses of the antioxidant enzymes glutathione-S-transferase catalase (CAT) and the formation of malondialdehyde (MDA) were monitored during 14 days in flounder exposed to an intraperitoneal injection of nodularin (0, 2, 10 and 50 μg nodularin kg⁻¹ body weight). The activities of GST and CAT in the liver decreased significantly in the 50 μg nodularin kg⁻¹ exposure after 7 days, but were restored to control levels after an additional 10 days of recovery. The results suggested that nodularin induced oxidative stress in terms of decreased GST and CAT activity, which can result in increased vulnerability of the cell to reactive oxygen species (ROS). No significant changes could be found in MDA levels between the treatments. Thus, the antioxidant defense system presumably managed to prevent oxygen mediated toxicity as seen by the unchanged levels of MDA. Alteration of the enzymatic defense system may increase energetic costs, thus reducing fish growth and survival. The present study also suggests that oxidative stress biomarkers can be used in fish to detect early responses to nodularin.     

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.     

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.     

Proteomic profiling of the Baltic Sea cyanobacterium Nodularia spumigena strain AV1 during ammonium supplementation

The cyanobacterium Nodularia spumigena dominates the annual, toxic summer blooms in the Baltic Sea. Although Nodularia has been receiving attention due to its production of the hepatotoxin nodularin, molecular data regarding the regulation of nitrogen fixation is lacking. We have previously reported that N. spumigena strain AV1, unlike model filamentous cyanobacteria, differentiates heterocysts in the absence of detectable nitrogen fixation activity. To further analyze the uncoupling between these two linked processes, we assessed the impact of ammonium ions on the N. spumigena metabolism using a proteomic approach. Proteomic profiling was performed at three different times during ammonium supplementation using quantitative 2-dimensional gel electrophoresis followed by MS/MS analysis. Using this approach, we identified 34 proteins, 28 of which were unique proteins that changed successively in abundance during growth on ammonium. Our results indicate that N. spumigena generally exhibits lower energy production and carbon fixation in the presence of ammonium and seems to be inefficient in utilizing ammonium as an external nitrogen source. The possibility of ammonium toxicity due to PSII damage was investigated and the results are discussed. Our findings have implications in regard to the strategies considered to manage the cyanobacterial blooms in the Baltic Sea.     

Benthic fauna affects recruitment from sediments of the harmful cyanobacterium Nodularia spumigena

Physical disturbance and feeding by macrofauna in the sediment can potentially affect bloom initiation of phytoplankton species that have benthic stages in their life cycle. In this experimental study, we investigated how different species of macrozoobenthos can affect the recruitment of Nodularia spumigena from the sediment to the water column. N. spumigena is a toxic, nitrogen-fixing filamentous cyanobacterium, which forms large summer blooms in the Baltic Sea. Benthic recruitment from resting stages (akinetes) and vegetative cells deposited on the seafloor have long been suspected to initiate the blooms. We found that, depending on species-specific traits, deposit-feeding macrofauna (an amphipod, Monoporeia affinis, a bivalve, Macoma balthica and an invasive polychaete, Marenzelleria cf. arctia) has the potential to either reduce or facilitate recruitment of this cyanobacterium. Shorter filament length in treatments with fauna than in the treatment without indicates feeding on or mechanical destruction of N. spumigena by the animals. Our results show the importance of an often overlooked aspect of phytoplankton bloom initiation, the role of macrozoobenthos.     

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.     

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.     

Allelopathy of Baltic Sea cyanobacteria: no evidence for the role of nodularin

Extracts of Aphanizomenon flos-aquae and Nodularia spumigena,the two most common cyanobacteria forming recurrent blooms inthe Baltic Sea, decrease the abundance of some phytoplanktonspecies via the release of allelopathic substances. We investigatedhow cell-free filtrates of the two cyanobacteria, as well aspurified hepatotoxin nodularin, produced by N. spumigena affectedcell numbers, chlorophyll a content and ¹⁴CO₂ uptake of thecryptophyte Rhodomonas sp. Both cyanobacterial filtrates significantlyretarded the growth of Rhodomonas sp., A. flos-aquae filtrateup to 46%, whereas purified nodularin showed no significanteffect on any of the growth parameters of the cryptophyte. Theseresults suggest that the allelopathic effect of N. spumigenais most probably due to metabolite(s) other than nodularin,possibly acting via the damage of the target cells.     

Long term nutrient loads and chlorophyll dynamics in a large temperate Australian lagoon system affected by recurring blooms of cyanobacteria

While rare globally, blooms of the toxic cyanobacteria Nodularia spumigena are a recurring problem in a few estuaries, such as the Baltic Sea and several southern Australian estuaries. Here, we document recurring Nodularia spumigena Mertens blooms in the Gippsland Lakes, S.E. Australia; a temperate lagoon system with episodic, winter-spring dominated catchment inflows. Physico-chemical conditions exerted a strong influence over bloom development, with blooms consistently occurring at surface water salinities between 9 and 20 (average?=?15), inorganic nitrogen concentrations <0.4?μM, and inorganic nitrogen to reactive phosphorus ratios <5. There was a positive correlation between average annual chlorophyll a and total phosphorus (TP) load in years when there was no Nodularia bloom, but this relationship broke down in Nodularia bloom years, even though there was a strong correlation between in-lake TP and chlorophyll a during these years; this highlights the importance of internal sources of phosphorus to bloom development. Large catchment derived nitrate and nitrite (NO_x) inputs following wildfires and floods in 2007, led to high concentrations of NO_x within the surface waters of the Gippsland Lakes through the second half of 2007 and the start of 2008. We hypothesise that these high NO_x concentrations were a key factor leading to an unprecedented Synechococcus sp. bloom that developed in the austral summer of 2007–2008, despite conditions that would otherwise favour a Nodularia bloom.     

Correlation of the content of hepatotoxin nodularin and glycosidic carotenoids, 4-ketomyxol-2′-fucoside and novel 1′-O-methyl-4-ketomyxol-2′-fucoside,in 20 strains of the cyanobacterium Nodularia spumigena

The carotenoids of 19 different strains of Nodularia spumigena and one Nodularia sphaerocarpa from different global locations were investigated. The molecular structure of the diagnostic pigment in N. spumigena of the Baltic Sea, tentatively named ‘4-keto-myxoxanthophyll-like pigment’ in Schlüter, L., Garde, K., Kaas, H., [2004. A 4-keto-myxoxanthophyll-like pigment is a diagnostic pigment for the toxic cyanobacteria Nodularia spumigena in the Baltic Sea. Mar. Ecol. Prog. Ser. 275, 69–78.] was determined to be a 4-ketomyxol-2′-fucoside. In most of the strains an additional carotenoid was found, identified as the novel 1′-O-methyl-4-ketomyxol-2′-fucoside by 2D NMR. This glycosidic carotenoid methyl ether was found to be a more important diagnostic pigment than the 4-ketomyxol-2′-fucoside for the toxic N. spumigena in the Baltic Sea. Out of the 20 strains 15 were found to produce the hepatotoxin nodularin. The content of carotenoids and nodularin was found to increase relative to chlorophyll a at increasing light intensity and at stationary growth, and nodularin was significantly correlated to both 4-ketomyxol-2′-fucoside and 1′-O-methyl-4-ketomyxol-2′-fucoside, and particular to the sum of these two pigments.     

Occurrence of cyanobacteria and cyanotoxin in the Southern Baltic Proper. Filamentous cyanobacteria versus single-celled picocyanobacteria

In the Baltic Sea, cyanobacterial community is mainly composed of filamentous nitrogen-fixing forms, including the toxic Nodularia spumigena, and single-celled picocyanobacteria (Pcy), represented by Synechococcus spp. The main aim of the work was to test the hypothesis that the picocyanobacteria dependend on the presence of the nitrogen-fixing cyanobacteria. In addition, the contamination of blue mussels and fish with nodularin (NOD), the N.?spumigena toxin, was examined. In years 2008?C2011, the samples for the study were collected in the Southern Baltic Proper using FerryBox system and, occasionally, during research cruises. The analyses showed no correlation between the growth of the nitrogen-fixing cyanobacteria and Synechococcus. Compared with the previously published data, a shift in the composition of Pcy phenotypes was observed. This shift might be an indication of the proceeding changes induced by the reduced nutrient loading and/or climate change. Analyses of NOD revealed differences in the cyanotoxin concentrations between mussels of different shell size. The highest concentration of NOD was detected in the liver of round goby. However, temporarily, also the fish muscles were significantly contaminated with the toxin.     

Occurrence of cyanobacterial blooms in the baltic sea in relation to environmental conditions

Cyanobacterial blooms occur regularly in summer in central parts of the Baltic Sea. They are mainly composed of Aphanizomenon sp. and Nodularia spumigena. Both species have almost similar ecological requirements and can roughly be considered a uniform functional group. In order to identify factors that might favour bloom development, water quality data from monitoring programmes were compared with bloom distribution. A salinity from 3.8 to 11.5 PSU proved important for the spatial distribution of the bloom development. The bloom's onset was triggered by temperatures approximating 16°C provided that global radiation was > 120 W/m² (daily mean) and wind speed was < 6 m/s. Nutrient concentrations decreased immediately before the bloom. The bloom's development ceased with poor weather conditions characterized by low irradiation or high wind speed.     

Effects of Cyanobacteria on Survival and Reproduction of the Littoral Crustacean Gammarus zaddachi (Amphipoda)

Toxic cyanobacteria can have harmful or fatal impacts on aquatic organisms. In the archipelagos of the northern Baltic Sea, the open sea blooms often drift into littoral areas, where they decompose and release toxins and other chemical compounds in the water. However, the effects of cyanobacteria on the littoral organisms have not previously been investigated. We studied the effects of three cyanobacteria species (toxic Nodularia spumigena, non-toxic N. sphaerocarpa and non-toxic Aphanizomenon flos-aquae) and purified dissolved nodularin (produced by N. spumigena) on a common littoral amphipod Gammarus zaddachi. Nodularin was transferred to eggs, juveniles and adults of G. zaddachi, but no significant negative effects of dissolved nodularin were detected on adults, eggs or juveniles. However, survival of adults decreased by the exposure to toxic N. spumigena cells. The egg hatching rate and juvenile survival were not affected when exposed to the three cyanobacteria species. In contrast, a weak decrease in the egg production and an increased abortion of embryos from the brood pouch of females was observed, the later indicating a failure in parental care. Further, a decrease in grazing rate on the filamentous green alga Enteromorpha intestinalis was observed. The results suggest that toxic cyanobacteria blooms are not extremely fatal, but may have, in high concentrations, negative effects on the adult survival, fecundity, and feeding behaviour of gammarids inhabiting the littoral zone.     

Nitrogen Fixation During Blooms of Nodularia in Coastal Waters and Backwaters of the Arkona Sea (Baltic Sea) in 1974

Nitrogen fixation was investigated by means of the acetylene reduction method during the development of a water bloom of Nodularia in coastal waters of the Baltic Sea west of the island of Hiddensee and in backwaters showing different degrees of eutrophication. Depending on plankton density, the values found varied greatly. The maximum of nitrogen fixation values found in extremely dense water blooms under special conditions (Baltic Sea, 2250 μg N₂/l · h; Kleiner Jasmunder Bodden, 374 μg N₂/l · h) are up to 10³ times higher than from other parts of the Baltic Sea or from inland waters. The average nitrogenase activity determined for coastal water populations of the Baltic Sea is 2.15 pg N₂/heterocyst · h and that of the inmost backwaters 0.77 pg N₂/heterocyst · h. The relationship between N₂-fixation and nutrient content in water is discussed.     

Spatial-temporal variation of Aureococcus anophagefferens blooms in relation to environmental factors in the coastal waters of Qinhuangdao,China

The brown tides occurring in the coastal scallop cultivation area of Qinhuangdao, China, in recent years are caused by Aureococcus anophagefferens and significantly impact the scallop industry and the marine ecosystem in this region. Long-term investigations of phytoplankton and hydrological variables in the Qinhuangdao sea area were conducted in this study to understand the spatial-temporal variations of A. anophagefferens in relation to environmental factors. Samples were collected during twelve cruises from July 2011 to December 2013 and were analyzed for the temperature, salinity, dissolved oxygen (DO), nutrients and phytoplankton pigments. All diagnostic pigments of A. anophagefferens, such as chlorophyll c₃ (Chl c₃), Chl c₂, 19′-butanoyloxyfucoxanthin (But-fuco), fucoxanthin (Fuco), and diadinoxanthin (Diad), were detected in the surface water by using high-performance liquid chromatography (HPLC). The highest concentrations of But-fuco (5.64 μg L⁻¹), Fuco (37.94 μg L⁻¹) and chlorophyll a (Chl a, 17.25 μg L⁻¹) occurred in different seasons and sampling sites. The A. anophagefferens bloom (as indicated by But-fuco) usually expanded from the south to the north of the Qinhuangdao sea area, close to scallop-culturing regions. The bloom unusually starts in May, reaches its peak in June and almost disappears in August, with the temperature ranging from ca. 19 °C to 23 °C. The redundancy analysis (RDA) indicated that relatively high salinity (>29) and low inorganic nutrients were suitable for the development of the A. anophagefferens bloom. The ratios of diagnostic pigments to Chl a were not constant during different cruises and generally obeyed two different linear relationships, thus indicating the co-occurrence of the blooms of A. anophagefferens and other species, such as Minutocellus polymorphus. In summary, our work reports the long-term variation of A. anophagefferens blooms based on diagnostic pigments and environmental controls, which may provide more insights into the formation mechanisms of the brown tide in this region.     

Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986

Three water bloom samples were collected in August 1986 from the southern Baltic Sea. Acute toxicity of the samples was determined by mouse bioassay and the toxins were further studied by HPLC. The bloom samples contained equal amounts of cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae and were hepatotoxic. Two hepatotoxic Nodularia spumigena strains were isolated from the samples. The isolates produce a toxic peak indistinguishable from the bloom material in the HPLC analysis. The toxicity of the fractions was verified by mouse bioassay. Thus the toxicity of the bloom samples was in all likelihood caused by Nodularia spumigena.     

Abiotic factors influencing cyanobacterial bloom development in the Gulf of Finland (Baltic Sea)

Blooms of cyanobacteria are a recurrent phenomenon in the Baltic Sea, including the Gulf of Finland. The spatial extension, duration, intensity and species composition of these blooms varies widely between years. Alg@line data collected regularly from ferries as well as weather service and marine monitoring data from 1997 to 2005 are analysed to determine the main abiotic factors influencing the intensity and species composition of cyanobacterial blooms in the Gulf of Finland. It is demonstrated that the development of the Nodularia spumigena Mertens bloom is highly dependent on weather conditions such as photosynthetically active radiation and water temperature. Nutrient conditions, especially the surplus of phosphorus (according to Redfield ratio) related to the pre-bloom upwelling events in the Gulf, affect the intensity of Aphanizomenon sp. (L.) Ralfs blooms. Differences in bloom timing and duration indicate that, if the preconditions (like nutrient ratio/concentration and weather conditions) for bloom formation are favourable, then the Aphanizomenon bloom starts earlier, the overall bloom period is longer and the Nodularia peak might appear in a wider time window. Handling editor: K. Martens