Microcystin Synthetase Gene Research Articles

Application of molecular tools for microbial source tracking and public health risk assessment of a Microcystis bloom traversing 300 km of the Klamath River

Microcystis is a globally distributed cyanobacterium that forms dense surface scums in eutrophic freshwater bodies and is also capable of producing potent liver toxins (microcystins). Although it is not commonly observed in riverine environments, high concentrations of Microcystis cells – and microcystins – have been observed on a recurring basis in recent years throughout the Klamath River system (Oregon/California). In this study, a variety of genetic approaches were used to assess the connectivity of Microcystis populations found throughout the Klamath River. In 2012, samples were collected bi-weekly from 16 sites spanning the entire system, including all five reservoirs and Upper Klamath Lake. A newly designed QPCR assay targeting a conserved region within the c-phycocyanin β-subunit gene (cpcB) was used along with a microcystin synthetase gene (mcyE) targeting QPCR assay to quantify the spatiotemporal patterns of total and toxigenic Microcystis. These data were compared with traditional metrics, such as microscopic cell counts and analytical toxin measurements, and the public health implications are discussed. Overall, Microcystis was a minor constituent of the phytoplankton community above Copco and Iron Gate Reservoirs, although it was highly prolific within these reservoirs and our data indicate that most of these populations originate internally. Spatiotemporal variations in the proportional abundances of a single nucleotide polymorphism (SNP), identified by 454 deep sequencing of the cpcBA genes, was used to fingerprint Iron Gate Reservoir as the source of downriver Microcystis assemblages. Throughout the study period, the Microcystis populations remained highly toxic, with total microcystin concentrations ranging from 165μg/L in Copco Reservoir to 3.6μg/L within the lower estuary (0.8km from the Pacific Ocean). These results demonstrate that large quantities of intact and toxic Microcystis cells can withstand passage through hydroelectric installations and transport over distances exceeding 300km. This finding emphasizes that public health risk assessments should consider the impact of cyanobacterial blooms even when they originate in distant upstream locations within a watershed.

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI- MS Identification of Microcystins

Treated water effluent from Phakalane waste water secondary maturation ponds in Gaborone City (Botswana) enters the Limpopo River via Notwane River. Effluent samples from these ponds were collected and investigated using molecular and analytical methods to determine presence of mcy genes and microcystins (MCs), respectively. It was observed that, potentially toxic algal blooms were present in this effluent and therefore algal toxins. Using Polymerase Chain Reaction (PCR) method; cyanobacterial 16S-rRNA and mcyA, -B, -C, -D, -E and -G genes were amplified and PCR products separated by gel electrophoresis and visualized after ethidium bromide staining. DNA sequence for mcyA gene was obtained using PCR products from a newly designed primer pair for the amplification of mcyA gene. BLAST results of the obtained DNA sequence were evaluated and aligned to NCBI database for species identification. The alignment gave the highest similarity (100%) in nucleotide sequence that was aligned to the DNA sequence of a toxigenic M. novacekii T20-3 based on the published data. Microcystin-RR, -YR, -LR and -WR were chromatographed, identified and quantified from M. novacekii T20-3 cell extracts using LC-ESI-MS technique after liquid-partitioning (LP) and solid-phase extraction (SPE) steps complementing PCR findings. Higher quantities of MC-RR, -LR and YR: 53.620 ± 0.063, 12.114 ± 0.024 and 5.280 ± 0.035 μg/g DW were observed, respectively.

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI-MS Identification of Microcystins

Toxic Microcystis novacekii T20-3 from Phakalane Ponds, Botswana: PCR Amplifications of Microcystin Synthetase (mcy) Genes, Extraction and LCESI-MS Identification of Microcystins

The toxicity and enzyme activity of a chlorine and sulfate containing aeruginosin isolated from a non-microcystin-producing Planktothrix strain

The toxicity of six different Planktothrix strains was examined in acute toxicity assays with the crustacean Thamnocephalus platyurus. The presence of toxicity in two strains could be explained by the occurrence of microcystins. The other four Planktothrix strains were not able to produce microcystins due to different mutations in the microcystin synthetase (mcy) gene cluster. In these strains, toxicity was attributed to the presence of chlorine and sulfate containing compounds. The main representative, called aeruginosin 828A, of such a compound in the Planktothrix strain 91/1 was isolated, and structure elucidation by 2D NMR and MS methods revealed the presence of phenyllactic acid (Pla), chloroleucine (Cleu), 2-carboxy-6-(4′-sulfo-xylosyl)-octahydroindole (Choi), and 3-aminoethyl-1-N-amidino-Δ-3-pyrroline (Aeap) residues. Aeruginosin 828A was found to be toxic for T. platyurus with a LC50 value of 22.4μM, which is only slightly higher than the toxicity found for microcystins. Additionally, very potent inhibition values for thrombin (IC50=21.8nM) and for trypsin (IC50=112nM) have been determined for aeruginosin 828A. These data support the hypothesis that aeruginosins containing chlorine and sulfate groups, which were found in microcystin-deficient Planktothrix strains, can be considered as another class of toxins.

Analysis of cylindrospermopsin- and microcystin-producing genotypes and cyanotoxin concentrations in the Macau storage reservoir

The Macau Storage Reservoir (MSR) has experienced algal blooms in recent years, with high levels of Cylindrospermopsis and Microcystis and detectable concentrations of cyanotoxins. To analyze the cyanotoxin-producing genotypes and relate the corresponding cyanotoxins to the water quality parameters, a quantitative real-time polymerase chain reaction (qPCR) was developed and applied to the water samples in three locations of MSR. Cylindrospermopsin synthetase gene (pks) and a series of microcystin synthetase genes (mcys) were used for identifying and quantifying cylindrospermopsin- and microcystin-producing genes, and the corresponding water parameters were measured accordingly.

Global transcriptional responses of the toxic cyanobacterium, Microcystis aeruginosa, to nitrogen stress, phosphorus stress, and growth on organic matter.

Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller (5–22% of genes differentially expressed), transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.

The Genetic Basis for O-Acetylation of the Microcystin Toxin in Cyanobacteria

Microcystins are a family of cyclic peptide toxins produced by cyanobacteria. They are responsible for the toxicosis and death of wild and domestic animals throughout the world. They display extensive variation in amino acid composition and functional group chemistry. O-acetylated microcystins are frequently produced by free-living and symbiotic strains of the genus Nostoc. Here, we show that the production of acetylated microcystins is catalyzed by an acetyl-coenzyme A-dependent O-acetyltransferase (McyL) encoded in the 57 kb microcystin synthetase gene cluster of Nostoc sp. 152. Phylogenetic analysis demonstrates that McyL belongs to a family of enzymes that inactivate antibiotics through O-acetylation. The McyL enzyme has a relaxed substrate specificity, allowing the preparation of semisynthetic microcystins. This study sheds light on the evolutionary origins and genetic diversity of an important class of enzymes involved in antibiotic resistance.

Genotypic composition and the relationship between genotypic composition and geographical proximity of the cyanobacterium Microcystis aeruginosa in western Japan

Microcystis aeruginosa is one of the bloom-forming harmful algae in freshwater ecosystems. We genetically characterized Microcystis populations during bloom-forming periods in various reservoirs, lakes, and ponds in Japan during 2009. Using phylogenetic analysis, we evaluated the relationship between current genotype expansions and geographic location within western Japan and intraspecific variation. Microcystis aeruginosa colonies were isolated at 15 sites and were analyzed by sequencing the 16S-23S internal transcribed spacer (ITS) region of the ribosomal operon, and the potential to produce toxins was assessed by PCR-based detection of the microcystin synthetase gene mcyG. In total, 171 colonies were separated into 41 genotypes. The highest genotypic composition was detected in the south basin of Lake Biwa and the lowest in Lagoon Iba. Cluster analysis indicated no obvious association between genotypic composition and geographic distance. Thus, clear genetic differentiation accompanied by geographic origins was not found in western Japan. The resulting neighbor-joining tree revealed 3 clusters, 2 of which contained strains that showed both nonamplification and amplification of the mcyG gene.

Quantification of toxigenic Microcystis spp. in freshwaters by quantitative real-time PCR based on the microcystin synthetase A gene

A method to estimate the abundance of toxigenic Microcystis in environmental samples by using quantitative real-time PCR was developed and optimized. The basis of this method is the amplification of a highly conserved region of the mcyA gene within the microcystin synthetase gene cluster. Using this method, the average copy number of mcyA gene per cell in toxigenic Microcystis strains was estimated. The molecular markers and method developed in this study can be used to monitor toxigenic strains of Microcystis in Korean freshwaters, in which harmful cyanobacterial blooms are routinely found.

Rapid Detection and Quantitation of Microcystin-Producing Microcystis Using Real-Time PCR

This study is to develop and validate a real-time quantitative PCR (rt-QPCR) assay for rapid quantitation of microcystin-producing Microcystis using unprocessed surface water samples collected from Alberta lakes. Microcystin synthetase gene E (mcyE) was targeted for microcystin-producing Microcystis and 16S rRNA was used to measure blooming level of total cyanobacteria. The assay was optimized and validated with 20 reference samples collected in 2011. The limit of detection (LOD) of rt-QPCR was 50 copies/ml for both mcyE and 16S rRNA. An excellent precision was observed in 24 replicates [coefficient of variation (cv)=1.12% for 1.0E+05 and = 0.79% for 1.0E+03 copies]. The rt-QPCR assay was applied for detection of mcyE in 527 water samples collected from 45 lakes during the open-water season of 2012 in Alberta and 369 samples were mcyE positive. Microcystin-producing Microcystis was detected in 41 out of the 45 lakes in which, relatively high copy numbers of mcyE (≥ 1.0E+05 copies/ ml) were determined in 9 lakes. Cyanobacteria were present in all 45 lakes determined by 16S rRNA. The rt-QPCR assay developed with specific target to mcyE is sensitive, specific and robust for rapid detection and differentiation of toxic Microcystis from non-toxic cyanobacteria in surface water.

Potential use of dissolved cyanobacterial DNA for monitoring toxic Microcystis cyanobacteria in filtered water

Toxic and non-toxic Microcystis sp. are morphologically indistinguishable cyanobacteria that are increasingly posing health problems in fresh water systems by producing odours and/or toxins. Toxic Microcystis sp. produces toxicologically stable water soluble toxic compounds called microcystins (MCs) that have been associated with cases of aquatic life and wildlife poisoning and kills including some cases of human illnesses/deaths around the world. Thus, the need for rapid detection of toxic Microcystis sp. in surface water is imperatively a necessity for early mitigation purposes. Genomic DNA from potentially toxic Microcystis sp. comprises of ten microcystin synthetase (mcy) genes of which six major ones are directly involved in MCs biosynthesis. In Polymerase Chain Reaction (PCR) methodsmcy genes can be amplified from intracellular/extracellular genomic DNA using PCR primers. However, little is known about the limitations of sourcing genomic DNA templates from extracellular DNA dissolved in water. In this work, filtered water (0.45μM) from a Microcystis infested Dam (South Africa) was re-filtered on 0.22μM syringe filters followed by genomic DNA isolation and purification from micro-filtrates (9mL). Six major mcy genes (mcyABCDEG) from the isolated DNA were amplified using newly designed as well as existing primers identified from literature. PCR products were separated by gel electrophoresis and visualized after staining with ethidium bromide. The limitation of using dissolved DNA for amplification of mcy genes was qualitatively studied by establishing the relationship between input DNA concentrations (10.0–0.001ng/μL) and the formation of respective PCR products. The amplification of mcyA gene using new primers with as little as 0.001ng/μL of DNA was possible. Other mcy gene sensitivities reached 0.1ng/μL DNA dilution limits. These results demonstrated that with appropriately optimized PCR conditions the method can provide accurate cost-effective tools for rapid detection of toxic Microcystis sp. in water giving early information for water quality monitoring against MC producing cyanobacteria.

Estimating spatial variation in the abundance of potential microcystin-producing Microcystis spp. using real-time PCR during summer bloom in Lake Taihu

Lake Taihu, which is the third largest freshwater lakes in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, quantitative real-time PCR assays targeting on phycocyanin intergenic spacer (PC-IGS) and a microcystin synthetase gene mcyD were established, respectively. Water samples collected from eight sampling sites (including Zhushan Bay (N5), Meiliang Bay (N2), Gonghu Bay (N4), West lake areas (W2 and W4), south-middle lake areas (S2, S4 and S5)) in August of 2009 and 2010 were analyzed using real time PCR for the distribution and abundance of toxic and total Microcystis populations. The results showed that Microcystis exists as a mixed population of potential toxic and non-toxic genotypes, and there was significant spatial changes in the abundance of potential toxic Microcystis on the basis of quantification by quantitative real-time PCR analysis: the abundance of toxic Microcystis population in 2009 and 2010 varied from 4.08×104 to 8.28×106 copies mL−1, from 4.45×105 to 5.22×107 copies mL−1, respectively. Meanwhile the ratio of the mcyD subpopulation to the total Microcystis varied considerably, from 5.7% to 41.1% in 2009 and from 10.3% to 65.8% in 2010 in all sampling sites, and the value is high in Zhushan Bay and Meiliang Bay with the high level of eutrophication. Correlation analysis showed the abundance of toxic and total Microcystis being strongly related (P<0.01). However, there is different effects of environmental factors on the abundance of toxic and non-toxic Microcystis populations. The abundance of toxic and total Microcystis populations were positively correlated with chlorophyll-a (Chl-a) concentration (P<0.01) suggesting that Microcystis is dominated genera of cyanobacterial bloom in Lake Taihu. It was also found that the abundance of toxic Microcystis and the proportion of toxic subpopulation to the total Microcystis were positively correlated with total phosphorus and orthophosphate concentrations (P<0.01), whereas there was no significant correlation with total nitrogen and nitrate concentration (P>0.05). All data suggest that phosphorus concentration is a critical factor for determining the abundance of toxic Microcystis population.

Toxin content and cytotoxicity of algal dietary supplements

Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC–MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤1μg MC-LR equivalents g−1 dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable.

Identification and analysis of whole microcystin synthetase genes from two Korean strains of the cyanobacterium Microcystis aeruginosa

Microcystins are cyanobacterial hepatotoxins, and are produced by nonribosomal enzyme complexes, mcy gene cluster. In this study, we report on whole mcy gene clusters from two Korean strains of M. aeruginosa that were blooming in Lake Paldang (FCY-26) and Geum river (FCY-28). Their specific gene locus, amino acid information, and sub-cluster orientation were also characterized in both strains. Both gene clusters are of 55 kb, and also each length, number and the arrangement are identical. Their sequence analysis revealed a cluster of 10 genes (mcyA, B, C, D, E, F, G, H, I, and J) involved in the biosynthesis of microcystin, and mcyABC and mcyDEFGHIJ formed two polycistronic operon structures that are transcribed bidirectionally from a central promoter region between mcyA and mcyD. The analysis of SNPs provided different nucleotide composition and amino acid variations in two Korean strains of M. aeruginosa. This approach is useful to develop genetic indicators identifying toxic cyanobacteria and their cyanotoxins, and helpful for a better understanding of the diversities of mcy gene clusters, the biosynthesis of microcystin, and the mediation of environmental parameters causing algal blooming and HABs.

Monitoring toxigenic Microcystis strains in the Missisquoi bay, Quebec, by PCR targeting multiple toxic gene loci

The increasing incidence of mixed assemblages of toxic and nontoxic cyanobacterial blooms in Quebec's freshwater bodies over the last decade, coupled with inherent inadequacies of current monitoring approaches, warrants development of sensitive and reliable tools for assessing the toxigenic potential of these water blooms. In this study, we applied three independent polymerase chain reaction (PCR) assays that simultaneously target the microcystin synthetase (mcy) genes A, E, and G to rapidly and reliably detect and quantify potentially toxic Microcystis genotypes in the Missisquoi bay, Quebec, Canada. Linear regressions of quantitative PCR threshold cycles (Ct ) against the logarithm of their respective Microcystis cell number equivalents resulted in highly significant linear curves with coefficients of determination (R(2) ) greater than 0.99 (p < 0.0001, n = 6) and reaction efficiencies of 91.0, 95.8, and 92.7%, respectively, for the mcyA, mcyE, and mcyG-based quantitative real-time PCR (qPCR) assays. The three assays successfully estimated potential microcystin-producing Microcystis genotypes from all field samples. The proportions of MicrocystismcyA, mcyE, and mcyG genotypes to total Microcystis cell counts showed substantial spatial variability ranging between 1.7-21.6%, 1.9-11.2%, and 2.2-22.6%, respectively. Correlation of microscopically determined total Microcystis counts to qPCR-based MicrocystismcyA, mcyE, or mcyG cell number equivalents resulted in highly significant associations with R(2) > 0.90. Thus, PCR-based assays targeting the mcyA, mcyG, and/or mcyE genes can serve as powerful screening tools for rapid and sensitive estimation of microcystin-producing Microcystis genotypes in freshwater water bodies.

Quantification of microcystin-producing and non-microcystin producing Microcystis populations during the 2009 and 2010 blooms in Lake Taihu using quantitative real-time PCR

AbstractLake Taihu, a large, shallow hypertrophic freshwater lake in eastern China, has experienced lake-wide toxic cyanobacterial blooms annually during summer season in the past decades. Spatial changes in the abundance of hepatotoxin microcystin-producing and non-microcystin producing Microcystis populations were investigated in the lake in August of 2009 and 2010. To monitor the densities of the total Microcystis population and the potential microcystin-producing subpopulation, we used a quantitative real-time PCR assay targeting the phycocyanin intergenic spacer (PC-IGS) and the microcystin synthetase gene (mcyD), respectively. On the basis of quantification by real-time PCR analysis, the abundance of potential toxic Microcystis genotypes and the ratio of the mcyD subpopulation to the total Microcystis varied significantly, from 4.08×104 to 5.22×107 copies/mL, from 5.7% to 65.8%, respectively. Correlation analysis showed a strong positive relationship between chlorophyll-a, toxic Microcystis and total Microcystis; the abundance of toxic Microcystis correlated positively with total phosphorus and ortho-phosphate concentrations, but negatively with TN:TP ratio and nitrate concentrations. Meanwhile the proportion of potential toxic genotypes within Microcystis population showed positive correlation with total phosphorus and ortho-phosphate concentrations. Our data suggest that increased phosphorus loading may be a significant factor promoting the occurrence of toxic Microcystis bloom in Lake Taihu.

Detection and identification of potentially toxic cyanobacteria: Ubiquitous distribution of Microcystis aeruginosa and Cuspidothrix issatschenkoi in Japanese lakes

We studied the frequency and composition of potentially toxic cyanobacteria in 30 samples from 25 Japanese lakes using automated rRNA intergenic spacer analysis (ARISA) and eight primer sets for nodularin, microcystin, cylindrospermopsin, anatoxin-a, and saxitoxin synthetase genes. Potential microcystin- and anatoxin-a-producers were detected in 25 and 7 samples, respectively. Cylindrospermopsin- or saxitoxin-producers were not detected. PCR and clone library analyses indicated that Microcystis was the sole microcystin-producing genus. Moreover, potential microcystin-producing Microcystis strains were detected in 25 of 26 samples which included Microcystis ARISA fragments, suggesting that toxic Microcystis is ubiquitous. Potential anatoxin-a-producers detected in the samples were estimated to be Cuspidothrix issatschenkoi (synonym: Aphanizomenon issatschenkoi) from clone library analyses of the anatoxin-a biosynthesis gene cluster and 16S–23S rDNA intergenic spacer region. Anatoxin-a-producing strains of C. issatschenkoi are known from two lakes in New Zealand and Germany. In contrast, C. issatschenkoi was the second most common toxic cyanobacteria in our study. This is the first report of potential anatoxin-a-producing C. issatschenkoi strains in Asia.

Microcystin producing cyanobacterial communities in Amvrakikos Gulf (Mediterranean Sea, NW Greece) and toxin accumulation in mussels (Mytilus galloprovincialis)

Various cyanobacterial species have the capacity to produce different types of toxins. Microcystins, the most prominent cyanotoxins are considered health hazards because of their potential hepatotoxic effects. They are well known to contaminate freshwater ecosystems but their presence in marine ecosystems has been reported only occasionally. We investigated seasonal changes of microcystin concentrations both in water and in the edible species of mussels Mytilus galloprovincialis collected from Amvrakikos Gulf (salinity ranging from 30‰ to 34‰), the biggest semi-enclosed basin in Greece. The microcystin concentrations in the water ranging from 0.003 to 19.8ngl−1, were below the World Health Organization (WHO) upper limit for recreational activities. In contrast, we found that microcystin concentrations in M. galloprovincialis mussels (ranging from 45±2 to 141.5±13.5ngg−1ww) exceeded the upper limit of the tolerable daily intake (TDI) of microcystin as determined by WHO.Genotype composition of the total cyanobacterial community of the Gulf was analyzed by using denaturing gradient gel electrophoresis (DGGE) profiling of the rRNA internal transcribed spacer region (rRNA-ITS). The cyanobacterial community was found to be dominated almost exclusively by the cosmopolitan species Synechococcus –Synechocystis. In order to determine genes involved in the production of microcystins, a range of both specific and degenerate molecular primers against microcystin synthetase gene cluster (mcyS) was used.To our knowledge this is the first report of the presence of the hepatotoxic microcystins in the Mediterranean Sea, the first study on the accumulation of these toxins in mussels from a Mediterranean marine ecosystem and one of the few published works suggesting a potential association of microcystins with Synechococcus and/or Synechocystis cyanobacteria.The importance of our study is strengthened by the fact that Amvrakikos Gulf is among the most productive Greek “seafood” areas and a Mediterranean wetland of international significance according to Ramsar Convention.

Detection, identification and toxigenicity of cyanobacteria in New Zealand lakes using PCR-based methods

The aims of this study were to investigate the population dynamics and distribution of cyanobacteria in three New Zealand lakes, to detect the presence of microcystin-producing mcyE genes in potentially toxigenic strains and to correlate the finding with microcystin production. A total of 18 samples collected over a 6-week period from Lakes Rotorua, Rotoiti and Rotoehu were found to be predominantly composed of Microcystis spp., Anabaena spp., Coelosphaerium spp. and Aphanocapsa spp. The 820-bp-long region of the microcystin synthetase gene (mcyE) was successfully amplified for 11 out of the 18 water samples in genera of Microcystis and Anabaena. This provides the first documented evidence of Anabaena sp. carrying the potential to produce microcystins in New Zealand. Microcystin-LR was the only variant detected by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis and was present in all samples from Lake Rotoehu.

Analysis of microcystins and microcystin genes in 60–170-year-old dried herbarium specimens of cyanobacteria

Subsamples of cyanobacteria which had been stored as herbarium specimens at the Natural History Museum, London (BM), were analysed for the presence of microcystins and for genes involved in their biosynthesis using PCR. The samples had been collected worldwide between 1839 and 1950 and stored as dried specimens on paper, or between thin sheets of mica, under ambient conditions in the dark. Specimens for analysis were selected on the assumption that the chosen genera or species identified at the time of collection would have had a high potential for microcystin production based on current knowledge of the phylogeny of microcystin biosynthesis among the cyanobacteria. Of the 30 specimens analysed, 46% were positive for microcystins by high performance liquid chromatography with photodiode array detection and 83% were positive for the toxins according to microcystin immunoassay. Ninety seven percent of the specimens had ions which corresponded with known microcystins according to matrix-assisted laser desorption ionization time of flight mass spectrometry and 17% of the samples showed positive PCR bands for the mcyD gene for microcystin synthetase. These results demonstrate the potential for long-term survival of microcystins, and to a lesser extent of microcystin synthetase genes in herbarium specimens of dried cyanobacteria. They also offer the possibility for comparative studies on cyanotoxin occurrence at identifiable sites then and now, and of the use of archived cyanobacterial specimens in retrospective studies in the case of ecotoxicological investigations at the sampling locations.