Benthic Phormidium mats can contain high concentrations of the neurotoxins anatoxin-a and homoanatoxin-a. However, little is known about the co-occurrence of anatoxin-producing and non-anatoxin-producing strains within mats. There is also no data on variation in anatoxin content among toxic genotypes isolated from the same mat. In this study, 30 Phormidium strains were isolated from 1 cm2 sections of Phormidium-dominated mats collected from three different sites. Strains were grown to stationary phase and their anatoxin-a, homoanatoxin-a, dihydroanatoxin-a and dihydrohomoanatoxin-a concentrations determined using liquid chromatography-mass spectrometry. Each strain was characterized using morphological and molecular (16S rRNA gene sequences) techniques. Eighteen strains produced anatoxin-a, dihydroanatoxin-a or homoanatoxin-a. Strains isolated from each mat either all produced toxins, or were a mixture of anatoxin and non-anatoxin-producing genotypes. Based on morphology these genotypes could not be separated. The 16S rRNA gene sequence comparisons showed a difference of at least 17 nucleotides among anatoxin and non-anatoxin-producing strains and these formed two separate sub-clades during phylogenetic analysis. The total anatoxin concentration among toxic strains varied from 2.21 to 211.88 mg kg−1 (freeze dried weight), representing a 100 fold variation in toxin content. These data indicate that both the relative abundance of anatoxin and non-anatoxin-producing genotypes, and variations in anatoxin producing capability, can influence the overall toxin concentration of benthic Phormidium mat samples.

Mat-forming benthic cyanobacteria are widespread throughout New Zealand rivers, and their ingestion has been linked to animal poisonings. In this study, potentially toxic benthic cyanobacterial proliferations were collected from 21 rivers and lakes throughout New Zealand. Each environmental sample was screened for anatoxins using liquid chromatography-MS (LC-MS). Thirty-six cyanobacterial strains were isolated and cultured from these samples. A polyphasic approach was used to identify each isolate; this included genotypic analyses [16S rRNA gene sequences and intergenic spacer (ITS)] and morphological characterization. Each culture was analysed for anatoxins using LC-MS and screened for microcystin production potential using targeted PCR. The morphospecies Phormidium autumnale was found to be the dominant cyanobacterium in mat samples. Polyphasic analyses revealed multiple slight morphological variants within the P. autumnale clade and highlighted the difficulties in identifying Oscillatoriaceae. Only one morphospecies (comprising the two strains CYN52 and CYN53) of P. autumnale was found to produce anatoxins. These strains formed their own clade based on partial 16S rRNA gene sequences. These data indicate that benthic P. autumnale mats are composed of multiple morphospecies and toxin production is dependent on the presence of toxin-producing genotypes. Further cyanobacteria are also characterized, including Phormidium murrayi, which was identified for the first time outside of Antarctica.

Toxic benthic cyanobacterial proliferations in freshwater are becoming increasingly prevalent, and associated animal poisonings are being reported with greater regularity. Despite this, few studies have investigated spatial and temporal variations in freshwater mat-forming Cyanobacteria and their associated toxin production. Some sections of the Hutt and Wainuiomata rivers (lower North Island, New Zealand) contain extensive Phormidium sp. proliferations that sometimes produce anatoxin-a (ATX) and homoanatoxin-a (HTX). The percentage coverage of Phormidium sp., ATX and/or HTX concentrations and a suite of physicochemical parameters were monitored at 8 sites for 12 mo. The percentage coverage of Phormidium mats was greater in the summer months and these correlated with warmer water temperatures and stable river flows. Flows in excess of 3 times the mean resulted in the removal of Phormidium mats. There was no correlation between the presence/absence of Phormidium mats and water-soluble nutrients. The presence and concentration of ATX and/or HTX and their degradation products, dihydroanatoxin-a and dihydrohomoanatoxin-a, was highly variable across all sites and over time. Anatoxin-a and HTX occurrence was restricted to periods of warm water temperatures (above 13.4°C) and below average river flows.

Aims:  The purpose of this study was to determine the variability in anatoxin‐a (ATX) and homoanatoxin‐a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR‐based approach to determine ATX‐ and HTX‐production potential. Methods and Results:  ATX and HTX variability was investigated by collecting 15 samples from 10 × 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography–mass spectrometry (LC–MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co‐occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX‐and‐HTX‐ and non‐ATX‐and‐HTX‐producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC–MS data in only 58% of samples. Conclusions:  Fine‐scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. Significance and Impact of the Study:  Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR‐based technique offers the potential to be a useful early warning technique.

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Benthic cyanobacterial blooms are increasing worldwide and can be harmful to human and animal health if they contain toxin-producing species. Microbial interactions are important in the formation of benthic biofilms and can lead to increased dominance and/or toxin production of one or few taxa. This study investigated how microbial interactions contribute to proliferation of benthic blooms dominated by the neurotoxin-producing Phormidium autumnale. Following a rainfall event that cleared the substrate, biofilm succession was characterised at a site on the Hutt River (New Zealand) by sampling every 2-3 days over 32 days. A combination of morphological and molecular community analyses (automated ribosomal intergenic spacer analysis and Illumina™ MiSeq sequencing) identified three distinct phases of succession in both the micro-algal and bacterial communities within P. autumnale-dominated biofilms. Bacterial composition shifted between the phases, and these changes occurred several days before those of the micro-algal community. Alphaproteobacteria and Betaproteobacteria dominate in the early phase; Alphaproteobacteria, Betaproteobacteria, Sphingobacteria and Flavobacteria in the mid-phase; and Sphingobacteria and Flavobacteria in the late phase. Collectively, the results suggest that succession is driven by bacteria in the early stages but becomes dependent on micro-algae in the mid- and late stages of biofilm formation.

Benthic proliferations of the cyanobacteria Phormidium can cover many kilometres of riverbed. Phormidium can produce neurotoxic anatoxins and ingestion of benthic mats has resulted in numerous animal poisonings in the last decade. Despite this, there is a poor understanding of the environmental factors regulating growth and anatoxin production. In this study, the effects of nitrogen and phosphorus on the growth of two Phormidium strains (anatoxin-producing and non-anatoxin-producing) were examined in batch monocultures. Cell concentrations were significantly reduced under reduced nitrogen (ca. <0.100 mM) and phosphorus conditions (ca. <0.003 mM). Cell concentrations and maximum growth rates were higher for the non-anatoxin-producing strain in all treatments, suggesting there may be an energetic cost to toxin production. Cellular anatoxin concentrations were lowest (169 fg cell(-1)) under the high-nitrogen and high-phosphorus treatment. This supports the growth-differentiation balance hypothesis that suggests actively dividing and expanding cells are less likely to produce secondary-metabolites. Anatoxin quota was highest (>407 fg cell(-1)) in the reduced phosphorus treatments, possibly suggesting that it is produced as a stress response to growth limiting conditions. In all treatments there was a 4-5-fold increase in anatoxin quota in the lag growth phase, possibly indicating it may provide a physiological benefit during initial substrate colonization.

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