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Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken toget

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken toget

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species.

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species.

Cylindrospermopsis raciborskii is an invasive, toxin producing cyanobacterium of great ecological significance. This thesis explores the genomic plasticity and adaptive strategies of C. raciborskii strains isolated from different locations worldwide. It further explores the conservation of essential primary metabolic machinery of these strains, and discusses the potential causes of gene loss in these organisms. While strains isolated from Australia exhibited over 97% genomic similarity and 100% similarity within the cylindrospermopsin (CYN) biosynthesis (cyr) gene cluster, comparison of strains from China, Australia and Brazil highlighted diversity within the species, suggesting that geographic location could possibly play important roles in shaping the C. raciborskii genome. Nucleotide sequence based analyses exhibited strain clustering as per geographic location. Toxigenicity and taxonomic identity were not found to impact strain divergence. We report for the first time a varying spatial arrangement of the cyr biosynthesis gene cluster isolated from Chinese strains, compared to Australian strains. We, therefore hypothesize, that the arrangement of this cluster is influenced by the geographic location of isolates and not by overall strain relatedness as considered earlier. Additionally, we examined the effect of nitrogen (N) and phosphorus (P) on growth and ecotype-related changes in production of cylindrospermopsins by C. raciborskii. Significantly higher growth rates were observed, only when N and P were co-supplied. In contrast, toxin cell quotas increased significantly in treatments irrespective of whether N was supplied. These increased toxin quotas correlated with an increase in the proportion of cyrA to 16S genes in the C. raciborskii population, therefore, attributing changes in ecotype dominance as the most likely factor driving changes in toxin production between treatments. Finally, studies to gauge the changes in the transcriptional regulation of the cy

The hypothesis that cyanobacteria can be controlled by commercially available ultrasound transducers was tested in laboratory experiments with cultures of the cyanobacteria Anabaena sp., Cylindrospermopsis raciborskii and Microcystis aeruginosa and the green alga Scenedesmus obliquus that were grown in the absence or presence of ultrasound (mix of 20, 28 and 44 kHz). The Scenedesmus experiment also included a treatment with the zooplankton grazer Daphnia magna. Chlorophyll-a and biovolume-based growth of Anabaena was significantly lower in ultrasound exposed cultures than in controls. Particle based growth rates were higher in ultrasound treatments. Filaments were significantly shorter in ultrasound exposed cultures reflecting filament breakage. Photosystem II efficiency was not affected by ultrasound. In Cylindrospermopsis chlorophyll-a based growth rates and photosystem II efficiencies were similar in controls and ultrasound treatments, but biovolume-based growth was significantly lower in ultrasound exposed cultures compared to controls. Despite biovolume growth rates of the filamentous cyanobacteria were reduced in ultrasound treatments compared to controls, growth remained positive implying still a population increase. In Microcystis and Scenedesmus growth rates were similar in controls and ultrasound treatments. Hence, no effect of ultrasound on these phytoplankton species was found. Ultrasound should not be viewed ``environmental friendly'' as it killed all Daphnia within 15 mm, releasing Scenedesmus from grazing control in the cultures. Based on our experiments and critical literature review, we conclude that there is no music in controlling cyanobacteria in situ with the commercially available ultrasound transducers we have tested. (C) 2014 Elsevier Ltd. All rights reserved.

The hypothesis that cyanobacteria can be controlled by commercially available ultrasound transducers was tested in laboratory experiments with cultures of the cyanobacteria Anabaena sp., Cylindrospermopsis raciborskii and Microcystis aeruginosa and the green alga Scenedesmus obliquus that were grown in the absence or presence of ultrasound (mix of 20, 28 and 44 kHz). The Scenedesmus experiment also included a treatment with the zooplankton grazer Daphnia magna. Chlorophyll-a and biovolume-based growth of Anabaena was significantly lower in ultrasound exposed cultures than in controls. Particle based growth rates were higher in ultrasound treatments. Filaments were significantly shorter in ultrasound exposed cultures reflecting filament breakage. Photosystem II efficiency was not affected by ultrasound. In Cylindrospermopsis chlorophyll-a based growth rates and photosystem II efficiencies were similar in controls and ultrasound treatments, but biovolume-based growth was significantly lower in ultrasound exposed cultures compared to controls. Despite biovolume growth rates of the filamentous cyanobacteria were reduced in ultrasound treatments compared to controls, growth remained positive implying still a population increase. In Microcystis and Scenedesmus growth rates were similar in controls and ultrasound treatments. Hence, no effect of ultrasound on these phytoplankton species was found. Ultrasound should not be viewed ``environmental friendly'' as it killed all Daphnia within 15 mm, releasing Scenedesmus from grazing control in the cultures. Based on our experiments and critical literature review, we conclude that there is no music in controlling cyanobacteria in situ with the commercially available ultrasound transducers we have tested. (C) 2014 Elsevier Ltd. All rights reserved.

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of t

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX tr

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.