Anabaenopeptins are commonly occurring bioactive peptides of cyanobacterial origin. Cyanobacteria (blue-green algae) are known to be capable of producing a large number of biologically active peptides, but the widespread occurrence of anabaenopeptins in particular, makes them ideal candidates for investigating the reasons that cyanobacteria produce such a complex spectrum of peptides and the wider implications of their natural function(s). Despite the identification of these peptides in cyanobacterial samples, little is known about the concentrations produced. For this reason, methods for the quantitative extraction of anabaenopeptins from lyophilized cyanobacterial cells were optimized. Higher yields of anabaenopeptins were obtained using aqueous methanol extraction than using water alone. However, repeat extractions using 50, 70 or 90% aqueous methanol did not result in significantly different total yields of the anabaenopeptin variants, ABPN-A and -B. Similarly, little difference was found in the quantification of purified ABPN-A and -B by high performance liquid chromatography with photodiode array detection (HPLC-PDA) when analyzed in methanol solutions of different concentrations. The effects of solvent concentration on the laboratory handling of ABPN-A and -B in glass and plastic containers were also investigated. Significantly lower concentrations of dissolved ABPN-A and -B were found when aqueous solutions came into contact with plastics, but not 50 or 100% methanol.

Cyanotoxins are now recognised by international and national health and environment agencies as significant health hazards. These toxins, and the cells which produce them, are also vulnerable to exploitation for illegitimate purposes. Cyanotoxins are increasingly being subjected to national and international guidelines and regulations governing their production, storage, packaging and transportation. In all of these respects, cyanotoxins are coming under the types of controls imposed on a wide range of chemicals and other biotoxins of microbial, plant and animal origin. These controls apply whether cyanotoxins are supplied on a commercial basis, or stored and transported in non-commercial research collaborations and programmes. Included are requirements concerning the transportation of these toxins as documented by the United Nations, the International Air Transport Association (IATA) and national government regulations. The transportation regulations for "dangerous goods", which by definition include cyanotoxins, cover air mail, air freight, and goods checked in and carried on flights. Substances include those of determined toxicity and others of suspected or undetermined toxicity, covering purified cyanotoxins, cyanotoxin-producing laboratory strains and environmental samples of cyanobacteria. Implications of the regulations for the packaging and air-transport of dangerous goods, as they apply to cyanotoxins and toxigenic cyanobacteria, are discussed.

(2005). Contribution of toxic cyanobacteria to massive deaths of Lesser Flamingos at saline-alkaline lakes of Kenya. SIL Proceedings, 1922-2010: Vol. 29, No. 2, pp. 783-786.

Microcystins produced by cyanobacterial ‘blooms’ in reservoirs and lakes pose significant public health problems because they are highly toxic due to potent inhibition of protein serine/threonine phosphatases in the PPP family. A dehydrobutyrine (Dhb)-containing microcystin variant [Asp3, ADMAdda5, Dhb7]microcystin-HtyR isolated from Nostoc sp. was found to potently inhibit PP1, PP2A, PPP4 and PPP5 with IC50 values similar to those of microcystin-LR. However, in contrast to microcystin-LR, which forms a covalent bond with a cysteine residue in these protein phosphatases, Asp,ADMAdda,Dhb-microcystin-HtyR did not form any covalent interaction with PP2A. Since the LD50 for Asp,ADMAdda,Dhb-microcystin-HtyR was 100 μg kg−1 compared to 50 μg kg−1 for microcystin-LR, the data indicate that the non-covalent inhibition of protein phosphatases accounts for most of the harmful effects of microcystins in vivo. A 3-amino-6-hydroxy-2-piperidone containing cyclic peptide, nostocyclin, also isolated from Nostoc sp., was non-toxic and exhibited more than 500-fold less inhibitory potency towards PP1, PP2A, PPP4 and PPP5, consistent with the conclusion that potent inhibition of one or more these protein phosphatases underlies the toxicity of microcystins, both lacking and containing Dhb.

Two leucine aminopeptidase M inhibitors, cyanostatin A and B, were isolated from cyanobacterial water blooms at Loch Rescobie in Scotland, and specifically from a Microcystis species. Both inhibitors were lipopeptides containing 3-amino-2-hydroxydecanoic acid and weak inhibitors of protein phosphatase (PP2A). Both strongly inhibited the activity of leucine aminopeptidase M with IC50 values of 40 and 12 ng/ml, respectively.

Cyanobacteria can generate molecules hazardous to human health, but production of the known cyanotoxins is taxonomically sporadic. For example, members of a few genera produce hepatotoxic microcystins, whereas production of hepatotoxic nodularins appears to be limited to a single genus. Production of known neurotoxins has also been considered phylogenetically unpredictable. We report here that a single neurotoxin, beta-N-methylamino-L-alanine, may be produced by all known groups of cyanobacteria, including cyanobacterial symbionts and free-living cyanobacteria. The ubiquity of cyanobacteria in terrestrial, as well as freshwater, brackish, and marine environments, suggests a potential for widespread human exposure.

A bloom of the non-heterocystous diazotrophic cyanobacterium. Tiichodesmium erythraeurn Ehrenberg is reported in the Canary Islands Archipelago during August of 2004, the warmest period of a meteorological series recorded by the National Institute of Meteorology (Spain) since 1912. Samples showed massive occurrences of T erythraeurn (1000 filaments ml(-1)) in different sectors of northern and southern waters off the central Canary Islands. Water analyses also showed a relatively low presence of dinoflagellates and diatoms. Quasi-true colour satellite images of dust storms, elevated sea surface temperature (the warmest satellite-derived record), chlorophyll a and geostrophic current fields showed satellite-derived optical positives of Trichodesmium in an African upwelling advective, jet-drifting westward current off the south Canary Islands. Analyses for cyanotoxins using HPLC found microcystins, which was confirmed by immunoassay, at concentrations from 0.1 to 1.0 mu g microcystin-LR equivalents (g(-1) dry weight of bloom material). A T. erythraeum bloom such as that observed in August 2004 in the NW African Upwelling does not appear to have been recorded for the area previously. The bloom may have developed due to the exceptionally warm weather and/or to the massive dust storms from the Sahara Desert observed in the NE Atlantic in August 2004.

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC+) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC−) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells' microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC+); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC+); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.

Cylindrospermopsin, a cyanobacterial guanidine alkaloid hepatotoxin and protein synthesis-inhibitor, was assayed for its effects on the germination of pollen from tobacco (Nicotiana tabacum cv Samsun NN). Pollen germination, measured by Alcian Blue dye-binding, was inhibited by cylindrospermopsin between 5 and 1000 μg ml−1. As a protein synthesis-inhibitor, cylindrospermopsin did not inhibit pollen germination to the same extent as cycloheximide on a gravimetric basis, but significantly reduced the amount of 14C-(U)-l-leucine labelling in pollen tubes. The inhibition of tobacco pollen germination may be amenable for development as a bioassay for cylindrospermopsin, although this would require a pre-concentration step for the monitoring of environmental samples. Implications of these observations for current spray-irrigation practices are discussed.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several genera of freshwater cyanobacteria. This compound has been implicated in outbreaks of human sickness and the death of domestic and wild animals. Given that several of the cyanobacterial genera known to produce CYN are common components of the phytoplankton of freshwaters including aquaculture facilities, we studied the accumulation of CYN in the freshwater mussel (swan mussel) Anodonta cygnea.Anodonta were exposed to CYN-producing cultures of the cyanobacterium Cylindrospermopsis raciborskii for 16 days and were found to accumulate the toxin to concentrations up to 2.52 mug g tissue dry weight. There was considerable variation in the concentrations of CYN detected in different parts of the body. At the end of a 2-week accumulation period the distribution of CYN in the body of Anodonta was as follows: haemolymph (68.1%), viscera (23.3%), foot and gonad (7.7%) and mantle (0.9%). No CYN was detected in the gills or adductor muscle of any animals. Following a 2-week depuration period, approximately 50% of the toxin remained in the tissues. Based on the recently derived guideline value for CYN in human drinking water (1 mug l(-1)) and the concentrations of this compound in animal tissues reported here, there is a clear need for the increased monitoring of this compound in organisms grown for human and animal consumption. (C) 2003 Elsevier Ltd. All rights reserved.

For decades frequent mass mortalities of Lesser Flamingos (Phoeniconaias minor Geoffroy) have been observed at alkaline-saline Kenyan Rift Valley lakes. To estimate the potential influence of toxic cyanobacteria on these mass deaths, the phytoplankton communities were investigated in Lakes Bogoria, Nakuru and Elmenteita. Cyanobacterial toxins were analyzed both in the phytoplankton from the three lakes and in isolated monocyanobacterial strains of Arthrospira fusiformis, Anabaenopsis abijatae, Spirulina subsalsa and Phormidium terebriformis. Lake Bogoria was dominated by the cyanobacterium A. fusiformis. In L. Nakuru and L. Elmenteita the phytoplankton mainly consisted of A. fusiformis, A. abijatae and Anabaenopsis arnoldii, and in L. Nakuru an unknown Anabaena sp. was also found. Furthermore, this is the first time A. abijatae and the unknown Anabaena sp. have been found in Kenyan lakes. Phytoplankton wet weight biomass was found to be high, reaching 777 mg L-1 in L. Bogoria, 104 mg L-1 in L. Nakuru and 202 mg L-1 in L. Elmenteita. Using HPLC, the cyanobacterial hepatotoxins microcystin-LR, -RR -YR, -LF and -LA and the neurotoxin anatoxin-a were detected in phytoplankton samples from L. Bogoria and L. Nakuru. Total microcystin concentrations amounted to 155 mug microcystin-LR equivalents g(-1) DW in L. Bogoria, and 4593 mug microcystin-LR equivalents g(-1) DW in L. Nakuru, with anatoxin-a concentrations at 9 mug g(-1) DW in L. Bogoria and 223 mug g(-1) DW in L. Nakuru. In L. Elmenteita phytoplankton, no cyanobacterial toxins were found. A. fusiformis was identified as one source of the toxins. The isolated strain of A. fusiformis from L. Bogoria was found to produce both microcystin-YR (15.0 mug g(-1) DW) and anatoxin-a (10.4 mug g(-1) DW), whilst the A. fusiformis strain from L. Nakuru was found to produce anatoxin-a (0.14 mug g(-1) DW). Since A. fusiformis mass developments are characteristic of alkaline-saline lakes, health risks to wildlife, especially the Arthrospira-consuming Lesser Flamingo, may be expected.

We investigated the mechanisms behind the negative effects of cyanobacteria on zooplankton by comparing the effects of Microcystis aeruginosa single cells, colonies, and toxins on the feeding behavior of Daphnia pulicaria in three independent experiments. The animals were fed a mixture of Scenedesmus supplemented by increasing proportions (0, 20, 50, 80, 100%) of Microcystis or concentrations (0, 50, 500, 5,000 ng ml−1) of purified microcystin‐LR. The changes in feeding behavior, as indicated by the appendage beat, mandible or labrum movement rates were evaluated by a direct observation method that coupled video recording and computerized image analysis. Daphnia responded in a different manner to the presence of single cells and colonies. In the case of the single cells, the mandibular movement rate (MMR) declined more than appendage beat rate (ABR), suggesting that Daphnia have the ability to discriminate between Microcystis and Scenedesmus. Colonies, on the other hand, produced a typical feeding interference response: the animals increased their labral rejection rate (LRR) and showed starvation signs. LRR increased in the presence of both unicellular and colonial Microcystis. In both cases, the changes in MMR and ABR were rapidly reversible and hence unlikely to be caused by intoxication from the presence of cellbound microcystins. In contrast, the addition of purified microcystin‐LR at the concentration of 5,000 ng ml−1 produced a nonreversible impairment of Daphnia feeding behavior.

The hepatotoxin cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis in mammalian cells. It is produced by freshwater cyanobacterial blooms in countries such as Australia, the United States, Israel, Thailand, and Brazil. An interlaboratory comparison was organized as a first step to evaluate the measurement of CYN in lyophilized cyanobacterial cells. Six laboratories from Europe, Israel, and Australia participated in the trial. All of the methods used for extraction of the toxin and the high-performance liquid chromatography (HPLC) analysis were satisfactory on the basis of statistical evaluation, according to ISO standards 5725-1 and -2. Further comparison of all the extraction methods by the organizer indicated that the most effective extraction procedure used 5% formic acid to prevent interference in chromatograms by contaminant compounds when analyzed using HPLC employing isocratic conditions of 5% (v/v) aqueous methanol plus 0.1% (v/v) trifluoroacetic acid as the mobile phase.

Cyanobacterial mats at hot springs on the shore of the alkaline Lake Bogoria, Kenya, were investigated regarding species community and cyanobacterial toxin content. The hepatotoxins microcystin-LR, -RR, -LF and -YR, and the neurotoxin anatoxin-a were present. The mats were dominated by Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. The concentration of microcystins in mat samples, ranged from 221 to 845 microg microcystin-LR equivalents g(-1) DW of mat. Anatoxin-a concentrations ranged from 10 to 18 microg g(-1) DW of mat. A contribution of the cyanobacterial toxins from the hot spring mats to the mass mortalities of Lesser Flamingos is suggested by: (a), the presence of hot spring cyanobacterial cells and cell fragments, and high concentrations of the cyanobacterial hepato- and neurotoxins in flamingo stomach contents and faecal pellets; (b), observations of neurological signs of bird poisoning at the lake. Cyanobacterial toxins in stomach contents, intestine and fecal pellets were 0.196 microg g(-1) fresh weight (FW) for the microcystins and 4.34 microg g(-1) FW for anatoxin-a. Intoxication with cyanobacterial toxins could occur by uptake of detached cyanobacterial cells from the mats, as the flamingos need to drink fresh or brackish water, and to wash their feathers daily, which they do in the vicinity of the hot springs, where salinity is lower than in the main body of water of the lake.

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.

ADVERTISEMENT RETURN TO ISSUEReviewNEXTAnalysis of Cyanobacterial Toxins by Immunological MethodsJ. S. Metcalf and G. A. CoddView Author Information Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee, DD1 4HN, United Kingdom Cite this: Chem. Res. Toxicol. 2003, 16, 2, 103–112Publication Date (Web):February 17, 2003Publication History Received24 June 2002Published online17 February 2003Published inissue 1 February 2003https://pubs.acs.org/doi/10.1021/tx0200562https://doi.org/10.1021/tx0200562review-articleACS PublicationsCopyright © 2003 American Chemical SocietyRequest reuse permissionsArticle Views965Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Biopolymers,Enzyme-linked immunosorbent assays,Immunology,Peptides and proteins,Toxins Get e-Alerts

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 micro mol of photons m(-2) s(-1) on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell(-1) and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 micro mol of photons m(-2) s(-1), the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 micro mol of photons m(-2) s(-1) than at 10 micro mol of photons m(-2) s(-1) and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.

The fate of dissolved microcystin-LR was studied in laboratory experiments using surface water taken from a eutrophic lake. Based on initial range finding, a concentration of 50 microg l(-1) dissolved 14C-microcystin-LR was selected for subsequent time-course experiments. The first was performed in May before the cyanobacterial bloom season and low increases in the radioactivity of particulate fractions occurred with an approx. halving of the cyano-toxin during 4 days. The radioactivity of the dissolved fraction remained stable and there was no significant formation of radiolabelled inorganic carbon. A second time-course experiment was performed in September during the cyanobacterial bloom season. At the end of the four-day incubation period, the microcystin-LR concentration had decreased to an undetectable level and 24% of the added radiolabelled substance was found in different particulate fractions. The study demonstrated that biodegradation of dissolved microcystin-LR occurred in water collected at a lake surface with carbon dioxide as a major end-product.

Liver mass (hepatosomatic index, HSI) increased by approximately 18% and water content in the gut by approximately 13 ml kg−1 in freshwater rainbow trout exposed for 24 h to intact cells of a microcystin-producing cyanobacterium (Microcystis PCC 7813) together with administration of heterotrophic bacterial LPS. Exposure to broken (ultrasonicated) cyanobacterial cells together with administration of bacterial LPS increased HSI by approximately 50% and water content in the gut by almost 30 ml kg−1. Exposure to broken or unbroken Microcystis cells without administration of bacterial LPS resulted in increased water content of the gut (by approximately 13 ml kg−1) with insignificant changes in HIS. Drinking rate increased with increasing dosage of bacterial LPS alone. The increased volume of water in the gut potentially increases the opportunity for uptake of waterborne toxins, including microcystins, and increased liver mass is a symptom consistent with the toxic effects of microcystins. It is concluded that exposure of fish to the cell contents of cyanobacteria (e.g. Microcystis PCC 7813) promotes osmoregulatory imbalance resulting from stimulation of the drinking response, increased volume of fluid in the gut and inability to remove excess water.