Variations in the Microcystin Production of Planktothrix rubescens (Cyanobacteria) Assessed from a Four-Year Survey of Lac du Bourget (France) and from Laboratory Experiments

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([d-Asp³] and [d-Asp³, Dhb⁷] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 μg L⁻¹. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell⁻¹. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P–PO₄³⁻ concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [d-Asp³] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3–0.7 pg cell⁻¹) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.     

Seasonal variation of cyanobacteria and microcystins in the Nui Coc Reservoir, Northern Vietnam

In order to understand the environmental variables which promote the proliferation of cyanobacteria and variation in microcystin concentrations in the Nui Coc reservoir, Vietnam, physicochemical parameters, the occurrence, and abundance of phytoplankton, cyanobacteria, and microcystin concentration were monitored monthly through the year 2009–2010. The relationships between these parameters were explored using principal component analysis (PCA) and Pearson correlation analysis. The phytoplankton community was mainly dominated by the cyanobacterium Microcystis with higher cyanobacteria abundance during summer and autumn season. PCA and Pearson correlation results showed that water temperature and phosphate concentration were the most important variables accounting for cyanobacteria, Microcystis, and microcystin occurrence. Analysis of the toxins by high-performance liquid chromatography demonstrated the presence of two microcystin variants: microcystin-LR (MC-RR) and microcystin-ddRR (MC-ddRR) with total concentrations of the toxins in filtered samples from surface water ranging from 0.11 to 1.52 μg MC-LR equiv L^?1. The high concentrations of microcystin in the Nui Coc reservoir highlighted the potential risk for human health in the basin. Our study underlined the need for regular monitoring of cyanobacteria and toxins in lakes and reservoirs, which are used for drinking water supplies, not only in Vietnam but also in tropical countries.     

Abundance and toxicity of Planktothrix rubescens in the pre-alpine Lake Ammersee,Germany

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m^?2 were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml^?1 (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion, possibly additionally benefiting from high nitrogen loads.Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb⁷]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.     

Toxin-Producing Anabaena flos-aquae Induces Settling of Chlamydomonas reinhardtii,a Competing Motile Alga

Toxin production is an adaptation that allows cyanobacteria in resource-limiting environments to ameliorate the effects of herbivory and competition with other phototrophs. We demonstrate that the cyanobacterial toxins anatoxin-a and microcystin-LR paralyze the motile green alga Chlamydomonas reinhardtii. In addition, both purified toxins and cyanobacterial extracellular products containing these toxins cause the alga to settle faster than in nontoxic media. In microcosm experiments, the presence of either the cyanobacterium or its extracellular products induce settling in the alga, similar to the response observed with the addition of both anatoxin-a and microcystin-LR. The cyanobacterial production of paralyzing toxins represents a novel mechanism for phytoplankton settling. This prokaryotic/eukaryotic chemical interaction may create a competitor-free zone for cyanobacteria in lake environments, predicating optimal conditions for a toxic cyanobacterial bloom.     

Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya

The phytoplankton communities and the production of cyanobacterial toxins were investigated in two alkaline Kenyan crater lakes, Lake Sonachi and Lake Simbi. Lake Sonachi was mainly dominated by the cyanobacterium Arthrospira fusiformis, Lake Simbi by A. fusiformis and Anabaenopsis abijatae. The phytoplankton biomasses measured were high, reaching up to 3159 mg l⁻¹ in L. Sonachi and up to 348 mg l⁻¹ in L. Simbi. Using HPLC techniques, one structural variant of the hepatotoxin microcystin (microcystin-RR) was found in L. Sonachi and four variants (microcystin-LR, -RR, -LA and -YR) were identified in L. Simbi. The neurotoxin anatoxin-a was found in both lakes. To our knowledge this is the first evidence of cyanobacterial toxins in L. Sonachi and L. Simbi. Total microcystin concentrations varied from 1.6 to 12.0 μg microcystin-LR equivalents g⁻¹ DW in L. Sonachi and from 19.7 to 39.0 μg microcystin-LR equivalents g⁻¹ DW in L. Simbi. Anatoxin-a concentrations ranged from 0.5 to 2.0 μg g⁻¹ DW in L. Sonachi and from 0 to 1.4 μg g⁻¹ DW in L. Simbi. In a monocyanobacterial strain of A. fusiformis, isolated from L. Sonachi, microcystin-YR and anatoxin-a were produced. The concentrations found were 2.2 μg microcystin g⁻¹ DW and 0.3 μg anatoxin-a g⁻¹ DW. This is the first study showing A. fusiformis as producer of microcystins and anatoxin-a. Since A. fusiformis occurs in mass developments in both lakes, a health risk for wildlife can be expected.     

Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152.

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.     

Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.     

Distribution of Microcystins in a Lake Foodweb: No Evidence for Biomagnification

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.     

Coupling high-resolution measurements to a three-dimensional lake model to assess the spatial and temporal dynamics of the cyanobacterium Planktothrix rubescens in a medium-sized lake

In a medium-sized pre-alpine lake (North Italy) the cyanobacterium Planktothrix rubescens has strongly dominated the phytoplankton assemblage since 2000, similar to many pre-alpine lakes, despite improvements in water quality. The objective of this study was to determine the factors governing the spatial distribution of P. rubescens, including the major hydrodynamic processes and the influence of long-term reduction in nutrient concentrations during a period of climate warming. We used an intensive field campaign conducted from February 2010 to January 2011, to evaluate distributions of phytoplankton phyla, as well as P. rubescens, using spectrally resolved fluorescence measurements. These data provided highly spatially and temporally resolved phytoplankton population data suitable to calibrate and validate a coupled three-dimensional hydrodynamic (ELCOM) and ecological model (CAEDYM) of the lake ecosystem. The simulations revealed the fundamental role of physiological features of P. rubescens that led to observed vertical patterns of distribution, notably a deep chlorophyll maximum, and a strong influence of lake hydrodynamic processes, particularly during high-discharge inflows in summer stratification. The simulations are used to examine growth-limiting factors that help to explain the increased prevalence of P. rubescens during re-oligotrophication.     

Isolation and characterization of microcystin producing Microcystis from a Central Indian water bloom

Microcystis aeruginosa Kütz, a well-known microcystin (hepatotoxin) producing cyanobacterium was the dominant bloom-forming organism in a mesotrophic lake at Nagpur in Central India, which was isolated and characterized for morphospecies and microcystin content. Compact spherical colonies, formation of daughter colonies, and clathration of older colonies leading to release of solitary cells, were characteristics of laboratory grown M. aeruginosa. Its growth, monitored as increase in optical density (OD) measured at 678 nm (the wavelength selected using dilution curve technique), exhibited a maximum specific growth rate (μ_max) of 0.34 day⁻¹ which, was attained on the 5th day of the experiment with a doubling time of 3.25 days. Though the morphological characters of the M. aeruginosa under field conditions were not retained under laboratory conditions, the microcystin content and type of variants did match with bloom samples. Reverse phase high performance liquid chromatography (RP-HPLC) analyses revealed that the laboratory grown isolate of Microcystis produced microcystin-RR (732 μg g⁻¹ dry weight biomass) and demethylated microcystin-RR (165 μg g⁻¹ dry weight biomass) variants, which are reported to be less toxic when compared to microcystin-LR. LC/ESI/MS further confirmed the presence of these two variants. Geographical distribution of microcystin variants and their prevailing concentrations need to be considered during formulation of guideline values for drinking and recreational waters.     

Environmental effects of introducing whitefish,Coregonus lavaretus (L.), in Lake Ring

The impact of whitefish (Coregonus lavaretus (L.)) on the trophic structure of eutrophic lakes was studied in Lake Ring, a small eutrophic Danish lake (22.5 ha, mean depth 2.9 m) in which the natural fish fauna is dominated by pike (Esox lucius L.), perch (Perca fluviatilis L.), and eel (Anguilla anguilla (L.)), roach (Rutilus rutilus (L.)) and burbot (Lota lota (L.)) being the only other fish species present. A total of 10993 0+ whitefish were stocked in the lake from October 1989 to July 1990 and the structure of the fish, zooplankton and benthic invertebrate communities studied during the period 1989–1991. Stomach contents analysis revealed that the whitefish mainly ate Daphnia and copepods in 1990–1991, the proportion of copepods decreasing with increasing size of the fish and Daphnia being the overall most important food source. The density of Daphnia in the lake decreased from 72 ind. 1^-1 in 1989 to 9 ind. 1^-1 in 1991; concomitantly the large species Daphnia magna and D. pulex almost disappeared and the density of cyclopoid copepods increased from 72 to 101 ind. 1^-1, presumably because of improved food conditions, while that of calanoid copepods remained virtually unchanged. As a result chl-a increased from 19 to 47 µg 1^-1 and Secchi depth decreased from 2.4 m to 1.7 m, despite there being no change in total P and total N (0.6 mg P 1^-1 and 1.3 mg N 1^-1, respectively). Changes were also observed in the benthic invertebrates; Chaoborus, oligochaetes, and chironomids all decreased, whereas Pisidium increased. It is concluded that the stocking of whitefish in eutrophic lakes for commercial purposes may delay their recovery, or even lead to enhanced eutrophication.     

Presence and genetic diversity of microcystin-producing cyanobacteria (<Emphasis Type="Italic">Anabaena</Emphasis> and <Emphasis Type="Italic">Microcystis</Emphasis>) in Lake Kotokel (Russia,Lake Baikal Region)

A survey was conducted for the presence of cyanobacteria toxins in Lake Kotokel due to a few cases of Haff disease registered in 2008–2009 caused by consumption of fish from Lake Kotokel, and wildlife mortality including large fish kill. The aims of this study were to determine what cyanotoxins (if any) were present in the lake, to describe phytoplankton composition including morphology, density, and species diversity of cyanobacteria, as well as to evaluate the trophic state of the lake. Samples were collected from both nearshore and central sites in August of 2009. Aphanocapsa holsatica dominated the phytoplankton. The presence of toxigenic genotypes of Microcystis spp. and Anabaena lemmermannii was detected by sequencing of PCR-amplified aminotransferase domain of microcystin synthetase gene. LR, RR, and YR microcystin (MC) variants were detected with liquid chromatography-UV mass spectrometry. The data do not shed light on the etiology of Haff disease in Lake Kotokel region, nevertheless taking into account the recreational importance of the lake and its direct connection to Lake Baikal, a necessity to monitor cyanobacteria in these water bodies is evident. This is the first report on simultaneous detection of MC-producing genotypes and MCs in the Lake Baikal region.     

Ontogenetic changes in habitat use by whitefish,Coregonus lavaretus

Synopsis The whitefish, Coregonus lavaretus, in the lake Mj?sa exhibited two niche changes during their life cycle. Juveniles (< 25 cm body length) were confined to the shallow (0–30 m) epibenthic zone. Medium sized whitefish (25–35 cm body length) expanded their habitat use to include the deep (30–90 m) epibenthic zone as well as the pelagic zone. From a body length of 35 cm, habitat use was restricted to the deep epibenthic zone. Small fish in the shallow epibenthic zone ate small and medium-sized prey (zooplankton, insect larvae and surface insects). Medium-sized fish in this zone were in addition feeding on the larger amphipod, Pallasea quadrispinosa. In the pelagic zone, the diet of medium-sized whitefish was dominated by zooplankton, although some larger prey like surface insects and age-0 smelt, Osmerus eperlanus, were also eaten. In the deep epibenthic zone, the diet of both medium-sized and large (< 35 cm) whitefish consisted mainly of the large prey P. quadrispinosa.     

Molecular confirmation of Planktothrix rubescens as the cause of intense,microcystin—Synthesizing cyanobacterial bloom in Lake Ziros,Greece

Cyanobacterial blooms occur increasingly often and raise ecological concerns worldwide. In Mediterranean freshwater ecosystems algal blooms are commonly attributed to Microcystis, Anabaena, and Aphanizomenon genera while Planktothrix is the most common bloom forming cyanobacterium in deep Northern and prealpine European oligotrophic to mesotrophic lakes. In the framework of an undertaken study of cyanobacterial species in lakes of Northwestern Greece we investigated the cyanobacterial diversity in Lake Ziros throughout a 15-month period (January 2006–March 2007) by using molecular methods. Surprisingly, a severe cyanobacterial bloom occurred during the study period, which upon microscopic examination and detailed molecular characterization found to be caused by Planktothrix rubescens species. The appearance of P. rubescens from November 2006 coincided with poor cyanobacterial diversity and resulted in a thick epilimnetic bloom in March 2007 (3.1 × 10⁸ cells/l and microcystin concentration 199 μg/l). Genotype composition of the total cyanobacterial community of the lake was analyzed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rnn operon (rRNA-ITS). A P. rubescens strain closely related to Kpr strain from Lake Klinckenberg, The Netherlands, was found to dominate. The importance of this observation is expanded by the fact that microcystin concentrations recorded in Lake Ziros were the highest measured ever in Greek aquatic ecosystems examined so far and also found amongst the highest recorded worldwide.     

Energy Density of Lake Whitefish Coregonus clupeaformis in Lakes Huron and Michigan

We collected lake whitefish Coregonus clupeaformis off Alpena and Tawas City, Michigan, USA in Lake Huron and off Muskegon, Michigan USA in Lake Michigan during 2002–2004. We determined energy density and percent dry weight for lake whitefish from both lakes and lipid content for Lake Michigan fish. Energy density increased with increasing fish weight up to 800 g, and then remained relatively constant with further increases in fish weight. Energy density, adjusted for weight, was lower in Lake Huron than in Lake Michigan for both small (≤800 g) and large fish (>800 g). Energy density did not differ seasonally for small or large lake whitefish or between adult male and female fish. Energy density was strongly correlated with percent dry weight and percent lipid content. Based on data from commercially caught lake whitefish, body condition was lower in Lake Huron than Lake Michigan during 1981–2003, indicating that the dissimilarity in body condition between the lakes could be long standing. Energy density and lipid content in 2002–2004 in Lake Michigan were lower than data for comparable sized fish collected in 1969–1971. Differences in energy density between lakes were attributed to variation in diet and prey energy content as well as factors that affect feeding rates such as lake whitefish density and prey abundance.     

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.     

Effects of nutrients and warming on Planktothrix dynamics and diversity: a palaeolimnological view based on sedimentary DNA and RNA

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Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.     

First report of microcystins from a Brazilian isolate of the cyanobacteriumMicrocystis aeruginosa

This is the first report on microcystins, cyclic heptapeptide hepatotoxins, from Brazilian water supplies. A colony isolate (NPJB-1) of the colonial cyanobacteriumMicrocystis aeruginosa from Lagoa das Garças, São Paulo, was cultured under non-axenic conditions. Exponential phase cells were harvested, concentrated and lyophilized for mouse bioassays and toxin extraction. The LD₁₀₀ of lyophilized cell suspensions was approximately 31 mg kg^–1 (dry cell weight/animal weight). Isolation, purification and characterization of the toxins were carried out by reversed phase HPLC, HPLC amino acid analysis and fast atom bombardment mass spectrometry. Strain NPJB-1 produces two different hepatotoxic heptapeptide microcystins. The main one was microcystin-LR, the most commonly reported microcystin from cyanobacteria. The other was microcystin-LF, the phenylalanine variant of microcystin-LR. This is the first published report for microcystin-LF.     

D-Leu1]Microcystin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil

[D-Leu1]Microcystin-LR was identified as the most abundant microcystin from a laboratory strain of the cyanobacterium Microcystis sp. isolated from a hepatotoxic Microcystis bloom from brackish waters in the Patos Lagoon estuary, southern Brazil. Toxicity of [D-Leu1]microcystin-LR, according to bioassay and protein phosphatase inhibition assay, was similar to that of the commonly-occurring microcystin-LR, which was not detectable in the Patos Lagoon laboratory isolate. This is the first report of a microcystin containing [D-Leu1] in the cyclic heptapeptide structure of these potent cyanobacterial toxins.