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Raphidiopsis (=Cylindrospermopsis) raciborskii was described as a subtropical-tropical cyanobacterium, later reported expanding into temperate regions. Heterocyte presence used to distinguish Cylindrospermopsis from the very similar Raphidiopsis, but recently the two genera were recognized as one and unified. This study aimed to investigate how heterocyte production is related to nitrogen (N) limitation in heterocytous and non-heterocytous strains of R.raciborskii. High N-concentrations did not inhibit heterocyte development in some strains, while prolonged N-starvation periods never stimulated production in others. RT-qPCR was used to examine the genetic background, through the expression patterns of nifH, ntcA and hetR. While gene expression increased under N-restriction, N-sufficiency did not suppress nifH transcripts as previously observed in other diazotrophyc cyanobacteria, suggesting that heterocyte production in R. raciborskii is not regulated by N-availability. Heterocytous and non-heterocytous strains were genotypically characterized to assess their phylogenetic relationships. In the phylogenetic tree, clusters were intermixed and confirmed Raphidiopsis and Cylindrospermopsis as the same genus. The tree supported previous findings of earlier splitting of American strains, while contesting the African origin hypothesis. The existence of two lines of Chinese strains, with distinct evolutionary patterns, is a significant addition that could lead to new hypotheses of the species biogeography.

Several cyanobacteria, including diazotrophic Raphidiopsis raciborskii, can form harmful blooms when dissolved inorganic phosphorus concentrations are very low. We hypothesized that R. raciborskii strains would vary in phosphorus (P) allocations to cell growth and storage, providing resilience of populations to continuously low or variable P supplies. We tested this hypothesis using six toxic strains (producing cylindrospermopsins) isolated from a field population using batch monocultures with and without P and dissolved inorganic nitrogen (DIN). Treatments replete with DIN, irrespective of P addition, had similar exponential growth rates for individual strains. P storage capacity varied 4-fold among strains and was significantly higher in DIN-free treatments than in replete treatments. P was stored by all R. raciborskii strains, in preference to allocation to increase growth rates. P stores decreased with increased growth rate across strains, but weeere not related to the time to P starvation in P-free treatments. The storage capacity of R. raciborskii, combined with strategies to efficiently uptake P, means that P controls may not control R. raciborskii populations in the short term. Intra-population strain variation in P storage capacity will need to be reflected in process-based models to predict blooms of R. raciborskii and other cyanobacteria adapted to low-P conditions.

Cyanobacterial blooms are characterized by intense growth of one or few species that will dominate the phytoplankton community for periods of few months to an entire year or more. However, even during persistent blooms, important seasonal changes among dominant species can be observed. Pampulha reservoir is a tropical eutrophic reservoir presenting permanent blooms. To identify the main species in this environment, a closer analysis performed by microscopy and 16S-rRNA DGGE revealed Cylindrospermopsis raciborskii as highly dominant throughout the year. The second most abundant group comprised species belonging to the Microcystis genus. They followed a well-defined seasonal pattern described by interesting species-specific ecological trends. During thermal stratification in the rainy/warmer season, C. raciborskii dominated in the water column, while Microcystis spp. were abundant at the end of the dry season, a period characterized by higher total phosphorus concentrations. Phylogenetic analyses confirmed the two dominant taxa and their seasonal trends. The results showed that cyanobacteria major controlling factors were strongly species dependent, shifting from physical/climate related (stratification) to more chemical driven (nutrients/eutrophication). Identifying these drivers is therefore essential for the understanding of the bloom dynamics and the real risks associated with each species, and to eventually adopt the most appropriate and effective management strategies.

Mechanisms behind expansion of an invasive cyanobacterium Cylindrospermopsis raciborskii have not been fully resolved, and different hypotheses, such as global warming, are suggested. In the East-Central Europe, it is widely occurring in western part of Poland but only in single locations in the East due to some limiting factors. Therefore, broad-scale phytoplankton survey including 117 randomly selected lakes in Poland and Lithuania was conducted. The results showed that C. raciborskii occurred widely in western part of Poland but was absent from other regions and Lithuania except one lake. The regions in which C. raciborskii was present had higher annual mean air temperature, higher maximum air temperature of the warmest month and higher minimum temperature of the coldest month, demonstrating that average air temperature, and indirectly, the duration of growing season might be more important factor driving C. raciborskii distribution than measured in situ water temperature. In turn, the presence of C. raciborskii in single localities may be more related to physiological adaptations of separated ecotype. Collectively, these results provide novel evidence on the influence of temperature on C. raciborskii distribution in East-European regions but also indicate high ecological plasticity of this species.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Relative fitness of three bloom-forming and potentially toxic cyanobacteria from the subtropical St. John's River, Florida was investigated under a range of nutrient conditions, during a bloom dominated by Cylindrospermopsis raciborskii. Nitrogen (N) was the primary nutrient limiting phytoplankton primary productivity and biomass. Phytoplankton biomass was also enhanced by phosphorus (P) added either alone or jointly with N, suggesting different components of the phytoplankton experienced distinct nutrient limitations. Based on quantitative PCR, the diazotrophic cyanobacteria Anabaena sp. and C. raciborskii were responsible for the primary production response to P additions, while the nondiazotrophic Microcystis aeruginosa appeared to benefit from N released from the diazotrophs. Cylindrospermopsis raciborskii maintained high net growth rates under diazotrophic and nondiazotrophic conditions, while Anabaena sp. growth was significantly reduced under DIN enrichment. C. raciborskii appears to be a generalist with regard to N source, a lifestyle traditionally not considered a viable ecological strategy among diazotrophs. Using facultative diazotrophy, C. raciborskii gains a growth advantage under fluctuating DIN conditions, such as systems that are under the influence of anthropogenic N loading events. The described niche differentiation may be a key factor explaining the recent global expansion of C. raciborskii.

Recent studies indicate genetic diversity of cyanobacteria in eutrophic lakes is not represented well by culture collections or morphology. Yet, few studies have investigated genetic richness and evenness of cyanobacteria using culture-independent methods. We compared the genetic structure of cyanobacteria supported by four neighboring eutrophic lakes during the ice-free season. The partial phycobilincpcB/A genes plus intergenic spacer (PC-IGS) was used as a genetic marker.Sequences were phylogeneticallygrouped by maximum likelihood into genotypes representing sub-genera of the major taxa. Genotypes fell into genera commonly observed by microscopy in these lakes including Microcystis, Aphanizomenon, Chroococcus, Anabaena, and Cylindrospermopsis . Only three genotypes were shared among all four lakes, despite significant water flowage between lakes.A Parsimony P-test indicated lakes were significantly (p=0.01) clustered on the maximum likelihood tree. Pairwise differences using Unifrac distance were moderately or not significant. Analysis of molecular variance (AMOVA) indicated genetic variation among all genotypes (φ=0.06, p

Aims: To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Methods and Results: The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4–7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS). Conclusions: The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp. Significance and Impact of the Study: We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.

Cylindrospermopsis raciborskii, a potentially toxic and highly adaptable freshwater cyanobacterium, was believed to have been misidentified in the Nile at the end of the 19th century. This study reports the presence of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea for the first time in Egyptian fresh waters since that time. Cylindrospermopsis raciborskii appeared in the El-Dowyrat fish pond during May 2002, when bottom waters reached, as a result of climatic change, sufficiently high temperatures to allow the germination of its akinetes in the sediments. Both C. raciborskii and R. mediterranea showed seasonal variations, with highest densities recorded in August of each year. The count of the two species correlated positively with pH, temperature and conductance, and negatively with nutrients, during the study period. The densities of C. raciborskii and R. mediterranea varied significantly along the depth profile of this pond, with peaks obtained at 1 and 0.5m, respectively. Isolates of C. raciborskii and R. mediterranea from this pond exhibited toxicity to Artemia salina, Daphnia magna and mice. Cylindrospermopsis raciborskii extracts had hepatotoxic effects on mice, but R. mediterranea extracts showed neurotoxic effects on mice. The identification of toxic C. raciborskii and R. mediterranea in this pond should be considered during the monitoring of cyanobacteria in drinking and recreational water sources in Egypt.

The Fitzroy impoundment is a long slender water body (10 m deep) formed by the regulation of the Fitzroy River in tropical Australia. Large, monsoonally driven discharges in late summer flush the impoundment repeatedly leaving, after 2 months, a longitudinally uniform, well-mixed water column, rich in dissolved nutrients and with high turbidity. For the rest of the year flows are negligible. Paradoxically, two sites with initially identical nutrient and stratification characteristics, and located only 30 km apart, develop quite different patterns of cyanobacterial succession. The upstream site is initially dominated by Anabaena circinalis which appears in early spring and collapses within the month. A mixed population of Anabaenopsis elenkinii and Aphanizomenon issatschenkoi then develops at both sites. This is followed by a mixture of small cyanobacteria (consisting of Cylindrospermopsis, Planktolyngbya and Limnothrix) which develops mainly at the downstream site and persists for 3 months until flushed away by flood flows. We report on data covering an 8 month period of investigation of the stratification, light climate, temperature and nutrient dynamics at these two sites. We show that large-scale climatic conditions and the local weather pattern set the physical and chemical conditions which determine the cyanobacterial response.

Cylindrospermopsis raciborskii is a planktonic, nostocalean cyanobacterium, which produces an alkaloid heptatoxin, cylindrospermopsin. We performed morphological observations, 16S rDNA sequence analysis, PCR fingerprint analysis of short tandemly repeated repetitive (STRR) sequences, temperature tolerances and toxin analysis to characterize 24 strains of this toxic cyanobacterium isolated from Thailand and Japan. All strains shared common morphological traits characteristic of C. raciborskii and showed high 16S rDNA sequence similarity, forming a defined cluster together with the reference strains from Australia. In particular, some of the Thai strains shared 99.9% to 100% similarity with the Australian strains. Various combinations of STRR primers revealed different and unique DNA band patterns among strains of C. raciborskii. The phylogenetic tree revealed two main clusters of C. raciborskii strains, the Thai/Japan-Shinobazugaike cluster (cluster I) and the Japan-Gonoike cluster (cluster II). Cluster I was further divided into two subclusters, A (only Thai strains) and B (one Thai strain and the Japan-Shinobazugaike strains). Thus, the results from 16S rDNA and STRR analyses showed no clear geographical distinction between Japanese and Thai strains and between Thai and Australian strains. Thai strains were separated into adaptive and non-adaptive groups to low temperature (15 and 17.5 degrees C) and Japanese strains were composed of only low-temperature-adaptive ones. The toxin cylindrospermopsin was detected in some strains of cluster I-A and in one strain of cluster II. We conclude that C. raciborskii is a species that has recently begun to invade, and a species with different physiological strains or ecotypes in temperature tolerance; the toxin is synthesized without any relation to phylogenetic or genetic clusters and to geography.