Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River.

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

A review of the phylogeny, ecology and toxin production of bloom-forming Aphanizomenon spp. and related species within the Nostocales (cyanobacteria).

The traditional genus Aphanizomenon comprises a group of filamentous nitrogen-fixing cyanobacteria of which several memebers are able to develop blooms and to produce toxic metabolites (cyanotoxins), including hepatotoxins (microcystins), neurotoxins (anatoxins and saxitoxins) and cytotoxins (cylindrospermopsin). This genus, representing geographically widespread and extensively studied cyanobacteria, is in fact heterogeneous and composed of at least five phylogenetically distant groups (Aphanizomenon, Anabaena/Aphanizomenon like cluster A, Cuspidothrix, Sphaerospermopsis and Chrysosporum) whose taxonomy is still under revision. This review provides a thorough insight into the phylogeny, ecology, biogeography and toxicogenomics (cyr, sxt, and ana genes) of the five best documented "Aphanizomenon" species with special relevance for water risk assessment: Aphanizomenon flos-aquae, Aphanizomenon gracile, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Chrysosporum ovalisporum. Aph. flos-aquae, Aph. gracile and C. issatschenkoi have been reported from temperate areas only whereas S. aphanizomenoides shows the widest distribution from the tropics to temperate areas. Ch. ovalisporum is found in tropical, subtropical and Mediterranean areas. While all five species show moderate growth rates (0.1-0.4day-1) within a wide range of temperatures (15-30°C), Aph. gracile and A. flos-aquae can grow from around (or below) 10°C, whereas Ch. ovalisporum and S. aphanizomenoides are much better competitors at high temperatures over 30°C or even close to 35°C. A. gracile has been confirmed as the producer of saxitoxins and cylindrospermopsin, C. issatschenkoi of anatoxins and saxitoxins and Ch. ovalisporum of cylindrospermopsin. The suspected cylindrospermopsin or anatoxin-a production of A. flos-aquae or microcystin production of S. aphanizomenoides is still uncertain. This review includes a critical discussion on the the reliability of toxicity reports and on the invasive potential of "Aphanizomenon" species in a climate change scenario, together with derived knowledge gaps and research needs. As a whole, this work is intended to represent a key reference for scientists and water managers involved in the major challenges of identifying, preventing and mitigating toxic Aphanizomenon blooms.

Temperature-dependent dispersal strategies of Aphanizomenon ovalisporum (Nostocales, Cyanobacteria): implications for the annual life cycle.

Aphanizomenon ovalisporum is a planktonic nostocalean cyanobacterium with increasing research interest due to its ability to produce the potent cytotoxin cylindrospermopsin and its potential invasiveness under the global warming scenario. The present study provides novel data on the potential dispersal strategies of A. ovalisporum by analyzing the influence of temperature (10-40 °C) on akinete differentiation and cell morphometry in cultures of A. ovalisporum UAM 290 isolated from a Spanish pond. Our results confirmed a temperature-dependent akinete differentiation, with the maximum akinete production reached at 20 °C (15 % of the cells), a low basal production at 25-30 °C (<0.4 % of the cells) and no detectable production at 35 °C. Furthermore, we reported the fragmentation of A. ovalisporum filaments at temperatures of 25 °C and above. Additionally, we observed that the morphology of vegetative cells varied under different temperature scenarios. Indeed, a strong negative correlation was found between temperature and the width, length and biovolume of vegetative cells, whereas akinete dimensions remained stable along the temperature gradient. Therefore, linear regressions between temperature and the cell size parameters are herein presented aiming to facilitate the identification of A. ovalisporum in the field throughout the course of the year. This is the first study evidencing that akinete production is triggered by temperatures between 20 and 25 °C in A. ovalisporum and reporting the existence of filament fragmentation as a potential dispersal strategy of this species. The importance of these findings for understanding the annual life cycle and invasive potential of A. ovalisporum is further discussed herein.

The influence of ultrasound frequency and power, on the algal species Microcystis aeruginosa, Aphanizomenon flos-aquae, Scenedesmus subspicatus and Melosira sp.

We report on the effectiveness of sonication on controlling the growth of four problematic algal species which are morphologically different and from three algal divisions. Two cyanobacterial species Microcystis aeruginosa (unicellular) and Aphanizomenon flos-aquae (filamentous), one green alga Scenedesmus subspicatus (colonial) and lastly a diatom species Melosira sp. (filamentous) were subjected to ultrasound of selected low to high frequencies ranging from 20 to 1144 kHz. Microcystis aeruginosa and Scenedesmus subspicatus highest cell removal rates were 16 +/- 2% and 20 +/- 3% when treated with the same ultrasound frequency of 862 kHz but differing energy levels of 133 and 67 kWh m(-3), respectively. Aphanizomenon flos-aquae best removal rate was 99 +/- 1% after 862 kHz and 133 kWh m(-3) of energy, with Melosira sp. achieving its highest cell removal at 83% subsequent to ultrasound of 20 kHz and 19 kWh m(-3). Microcystis aeruginosa and Scenedesmus subspicatus are considered non-susceptible species to ultrasound treatment from a water treatment perspective due to their low cell removal rates; however, photosynthetic activity reduction of 65% for Microcystis aeruginosa does indicate the possible utilization of ultrasound to control bloom growth, rather than bloom elimination. Conversely, Aphanizomenon flos-aquae and Melosira sp. are deemed species highly susceptible to ultrasound. Morphological differences in shape (filamentous/non-filamentous) and cell wall structure (silica/peptidoglycan), and presence of gas vacuoles are probable reasons for these differing levels of susceptibility to ultrasound.

Cyanotoxins: a poison that frees phosphate.

Autotrophic organisms obtain phosphorus from the environment by secreting alkaline phosphatases that act on esters, resulting in inorganic phosphate that is then taken up. New work shows that the cyanobacterium Aphanizomenon ovalisporum obtains inorganic phosphate by secreting the cyanotoxin cylindrospermopsin, which induces alkaline phosphatase in other phytoplankton species.

First report of anatoxin-a-producing cyanobacterium Aphanizomenon issatschenkoi in northeastern Germany.

The neurotoxin anatoxin-a (ATX), has been detected in several northeast German lakes during the last two decades, but no ATX producers have been identified in German water bodies so far. In 2007 and 2008, we analyzed phytoplankton composition and ATX concentration in Lake Stolpsee (NE Germany) in order to identify ATX producers. Sixty-one Aphanizomenon spp. strains were isolated, morphologically and phylogenetically characterized, and tested for ATX production potential by liquid chromatography-tandem mass spectrometry (LC-MS/MS). New primers were specifically designed to identify a fragment of a polyketide synthase gene putatively involved in ATX synthesis and tested on all 61 Aphanizomenon spp. strains from L. Stolpsee and 92 non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from German lakes Langersee, Melangsee and Scharmützelsee. As demonstrated by LC-MS/MS, ATX concentrations in L. Stolpsee were undetectable in 2007 and ranged from 0.01 to 0.12 microg l(-1) in 2008. Fifty-nine of the 61 strains isolated were classified as Aphanizomenon gracile and two as Aphanizomenon issatschenkoi. One A. issatschenkoi strain was found to produce ATX at concentrations of 2354+/-273 microg g(-1) fresh weight, whereas the other A. issatschenkoi strain and A. gracile strains tested negative. The polyketide synthase gene putatively involved in ATX biosynthesis was found in the ATX-producing A. issatschenkoi strain from L. Stolpsee but not in the non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from lakes Stolpsee, Langersee, Melangsee, and Scharmützelsee. This study is the first confirming A. issatschenkoi as an ATX producer in German water bodies.