Microbial community structures and bacteria-Cylindrospermopsis raciborskii interactions in Yilong Lake.

Cylindrospermopsis raciborskii-dominated harmful algae blooms have been reported globally in recent years. However, our understanding of the ecology of C. raciborskii in natural conditions is still poor. In this study, we collected the water samples from a C. raciborskii-blooming lake, Yilong Lake, in Yunnan province, China, and used both culture-dependent and culture-independent approaches to investigate their microbial communities and the interactions between C. raciborskii and the other bacteria. The composition and diversity of microbial communities were revealed with 16S rRNA gene high-throughput sequencing data analysis. Microbial co-occurrences analysis suggests C. raciborskii may have complex associations with other bacteria. Based on co-inoculation tests, we obtained 14 strains of bacterial strains from the water samples that exhibited either algicidal or promoting effects on a strain of C. raciborskii. Two bacterial isolates exhibited a consistent performance between co-occurrence analysis and experimental results. Effects of these bacteria-algae interspecies interactions on the bloom event are discussed. All these results may provide new insights into the C. raciborskii-dominated blooms and how its interspecies relationships with other bacteria may influence the bloom events in eutrophic waters throughout the world.

The Raphidiopsis (= Cylindrospermopsis) raciborskii pangenome updated: Two new metagenome-assembled genomes from the South American clade.

Two Raphidiopsis (=Cylindrospermopsis) raciborskii metagenome-assembled genomes (MAGs) were recovered from two freshwater metagenomic datasets sampled in 2011 and 2012 in Pampulha Lake, a hypereutrophic, artificial, shallow reservoir, located in the city of Belo Horizonte (MG), Brazil. Since the late 1970s, the lake has undergone increasing eutrophication pressure, due to wastewater input, leading to the occurrence of frequent cyanobacterial blooms. The major difference observed between PAMP2011 and PAMP2012 MAGs was the lack of the saxitoxin gene cluster in PAMP2012, which also presented a smaller genome, while PAMP2011 presented the complete sxt cluster and all essential proteins and clusters. The pangenome analysis was performed with all Raphidiopsis/Cylindrospermopsis genomes available at NCBI to date, with the addition of PAMP2011 and PAMP2012 MAGs (All33 subset), but also without the South American strains (noSA subset), and only among the South American strains (SA10 and SA8 subsets). We observed a substantial increase in the core genome size for the 'noSA' subset, in comparison to 'All33' subset, and since the core genome reflects the closeness among the pangenome members, the results strongly suggest that the conservation level of the essential gene repertoire seems to be affected by the geographic origin of the strains being analyzed, supporting the existence of a distinct SA clade. The Raphidiopsis pangenome comprised a total of 7943 orthologous protein clusters, and the two new MAGs increased the pangenome size by 11%. The pangenome based phylogenetic relationships among the 33 analyzed genomes showed that the SA genomes clustered together with 99% bootstrap support, reinforcing the metabolic particularity of the Raphidiopsis South American clade, related to its saxitoxin producing unique ability, while also indicating a different evolutionary history due to its geographic isolation.

Recent Advances in the Ecology of Bloom-Forming Raphidiopsis (Cylindrospermopsis) raciborskii: Expansion in China, Intraspecific Heterogeneity and Critical Factors for Invasion.

Water blooms caused by the invasive cyanobacterium Raphidiopsis raciborskii occur in many reservoirs in the tropical and subtropical regions of China. In recent decades, this species has spread rapidly to temperate regions. Phenotypic plasticity and climate warming are thought to promote the worldwide dispersion of R. raciborskii. However, investigations into the genetic and phenotypic diversities of this species have revealed significant intraspecific heterogeneity. In particular, competition between R. raciborskii and Microcystis aeruginosa was highly strain dependent. Although the concept of an ecotype was proposed to explain the heterogeneity of R. raciborskii strains with different geographic origins, microevolution is more reasonable for understanding the coexistence of different phenotypes and genotypes in the same environment. It has been suggested that intraspecific heterogeneity derived from microevolution is a strong driving force for the expansion of R. raciborskii. Additionally, temperature, nutrient fluctuations, and grazer disturbance are critical environmental factors that affect the population establishment of R. raciborskii in new environments. The present review provides new insights into the ecological mechanisms underlying the invasion of R. raciborskii in Chinese freshwater ecosystems.

Interaction between Raphidiopsis raciborskii and rare bacterial species revealed by dilution-to-extinction experiments.

Interactions between heterotrophic bacteria and cyanobacteria regulate the structure and function of aquatic ecosystems and are thus crucial for the prediction and management of cyanobacterial blooms in relation to water security. Currently, abundant bacterial species are of primary concern, while the role of more diverse and copious rare species remains largely unknown. Using a dilution-to-extinction approach, rare bacterial species from reservoir water were co-cultured with the bloom-forming cyanobacterium Raphidiopsis raciborskii in the lab to explore their interactions by using Phyto-PAM and 16S rRNA gene high-throughput sequencing. We found that a ≤1000-fold bacterial dilution led to bacteria control of the growth and photosynthesis of R. raciborskii. Moreover, the bacterial community compositions in the low-dilution groups were clearly diverged from the high-dilution groups. Importantly, rare species changed dramatically in the low-dilution groups, resulting in lower phylogenetic diversity and narrower niche width. The network complexity and compositional stability of bacterial communities decreased in the low-dilution groups. Collectively, our results suggest that rare bacterial species inhibit R. raciborskii growth and photosynthesis through microbial interactions mediated by species coexistence and interaction mechanisms. Our study provides new knowledge of the ecological role of rare bacteria and offers new perspectives for understanding the outbreak and regression of R. raciborskii blooms.

Isolation and characterization of a novel Lambda-like phage infecting the bloom-forming cyanobacteria Cylindrospermopsis raciborskii.

Cylindrospermopsis raciborskii is a central bloom-forming cyanobacteria. However, despite its ecological significance, little is known of its interactions with the phages that infect it. Currently, only a single sequenced genome of a Cylindrospermopsis-infecting phage is publicly available. Here we describe the isolation and characterization of Cr-LKS3, a second phage infecting Cylindrospermopsis. Cr-LKS3 is a siphovirus with a higher genome similarity to prophages within heterotrophic bacteria genomes than to any other cyanophage/cyano-prophage, suggesting that it represents a novel cyanophage group. The function, order and orientation of the 72 genes in the Cr-LKS3 genome are highly similar to those of Escherichia virus Lambda (hereafter Lambda), despite the very low sequence similarity between these phages, showing high evolutionary convergence despite the substantial difference in host characteristics. Similarly to Lambda, the genome of Cr-LKS3 contains various genes that are known to be central to lysogeny, suggesting it can enter a lysogenic cycle. Cr-LKS3 has a unique ability to infect a host with a dramatically different GC content, without carrying any tRNA genes to compensate for this difference. This ability, together with its potential lysogenic lifestyle shed light on the complex interactions between C. raciborskii and its phages.

Ecophysiological Aspects and sxt Genes Expression Underlying Induced Chemical Defense in STX-Producing Raphidiopsis raciborskii (Cyanobacteria) against the Zooplankter Daphnia gessneri.

Cyanobacteria stand out among phytoplankton when they form massive blooms and produce toxins. Because cyanotoxin genes date to the origin of metazoans, the hypothesis that cyanotoxins function as a defense against herbivory is still debated. Although their primary cellular function might vary, these metabolites could have evolved as an anti-predator response. Here we evaluated the physiological and molecular responses of a saxitoxin-producing Raphidiopsis raciborskii to infochemicals released by the grazer Daphnia gessneri. Induced chemical defenses were evidenced in R. raciborskii as a significant increase in the transcription level of sxt genes, followed by an increase in saxitoxin content when exposed to predator cues. Moreover, cyanobacterial growth decreased, and no significant effects on photosynthesis or morphology were observed. Overall, the induced defense response was accompanied by a trade-off between toxin production and growth. These results shed light on the mechanisms underlying zooplankton-cyanobacteria interactions in aquatic food webs. The widespread occurrence of the cyanobacterium R. raciborskii in freshwater bodies has been attributed to its phenotypic plasticity. Assessing the potential of this species to thrive over interaction filters such as zooplankton grazing pressure can enhance our understanding of its adaptive success.

Heterologous Expression of an Unusual Ketosynthase, SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli.

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15 N2 ] arginine and [1,2-13 C2 ] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.

Comparative proteomics of the toxigenic diazotroph Raphidiopsis raciborskii (cyanobacteria) in response to iron.

Raphidiopsis raciborskii is an invasive bloom-forming cyanobacteria with the flexibility to utilize atmospheric and fixed nitrogen. Since nitrogen-fixation has a high requirement for iron as an ezyme cofactor, we hypothesize that iron availability would determine the success of the species under nitrogen-fixing conditions. This study compares the proteomic response of cylindrospermopsin-producing and non-toxic strains of R. racibroskii to reduced iron concentrations, under nitrogen-fixing conditions, to examine any strain-specific adaptations that might increase fitness under these conditions. We also compared their proteomic responses at exponential and stationary growth phases to capture the changes throughout the growth cycle. Overall, the toxic strain was more competitive under Fe-starved conditions during exponential phase, with upregulated growth and transport-related proteins. The non-toxic strain showed reduced protein expression across multiple primary metabolism pathways. We propose that the increased expression of porin proteins during the exponential growth phase enables toxic strains to persist under Fe-starved conditions with this ability providing a potential explanation for the increased fitness of cylindrospermoipsin-producing strains during unfavourable environmental conditions.

Intra-population strain variation in phosphorus storage strategies of the freshwater cyanobacterium Raphidiopsis raciborskii.

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.

Biogeography of the cyanobacterium Raphidiopsis (Cylindrospermopsis) raciborskii: Integrating genomics, phylogenetic and toxicity data.

Raphidiopsis (Cylindrospermopsis) raciborskii, a globally distributed bloom-forming cyanobacterium, produces either the cytotoxin cylindrospermopsin (CYL) in Oceania, Asia and Europe or the neurotoxin saxitoxin (STX) and analogues (paralytic shellfish poison, PSP) in South America (encoded by sxt genetic cluster) and none of them in Africa. Nevertheless, this particular geographic pattern is usually overlooked in current hypotheses about the species dispersal routes. Here, we combined genomics, phylogenetic analyses, toxicity data and a literature survey to unveil the evolutionary history and spread of the species. Phylogenies based on 354 orthologous genes from all the available genomes and ribosomal ITS sequences of the taxon showed two well-defined clades: the American, having the PSP producers; and the Oceania/Europe/Asia, including the CYL producers. We propose central Africa as the original dispersion center (non-toxic populations), reaching North Africa and North America (in former Laurasia continent). The ability to produce CYL probably took place in populations that advanced to sub-Saharan Africa and then to Oceania and South America. According to the genomic context of the sxt cluster found in PSP-producer strains, this trait was acquired once by horizontal transfer in South America, where the ability to produce CYL was lost.

Cylindrospermopsin- and Deoxycylindrospermopsin-Producing Raphidiopsis raciborskii and Microcystin-Producing Microcystis spp. in Meiktila Lake, Myanmar.

Meiktila Lake is a shallow reservoir located close to Meiktila city in central Myanmar. Its water is used for irrigation, domestic purposes and drinking water. No detailed study of the presence of cyanobacteria and their potential toxin production has been conducted so far. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Meiktila Lake, water samples were collected in March and November 2017 and investigated for physico-chemical and biological parameters. Phytoplankton composition and biomass determination revealed that most of the samples were dominated by the cyanobacterium Raphidiopsis raciborskii. In a polyphasic approach, seven isolated cyanobacterial strains were classified morphologically and phylogenetically as R. raciborskii, and Microcystis spp. and tested for microcystins (MCs), cylindrospermopsins (CYNs), saxitoxins and anatoxins by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). ELISA and LC-MS analyses confirmed CYNs in three of the five Raphidiopsis strains between 1.8 and 9.8 µg mg-1 fresh weight. Both Microcystis strains produced MCs, one strain 52 congeners and the other strain 20 congeners, including 22 previously unreported variants. Due to the presence of CYN- and MC-producing cyanobacteria, harmful effects on humans, domestic and wild animals cannot be excluded in Meiktila Lake.

A Greek Cylindrospermopsis raciborskii strain: Missing link in tropic invader's phylogeography tale.

The cyanobacterium Cylindrospermopsis raciborskii represents a challenge for researchers and it is extensively studied for its toxicity and invasive behaviour, which is presumably enhanced by global warming. Biogeography studies indicate a tropical origin for this species, with Greece considered as the expansion route of C. raciborskii in Europe. The widening of its geographic distribution and the isolation of strains showing high optimum growth temperature underline its ecological heterogeneity, suggesting the existence of different ecotypes. The dominance of species like C. raciborskii along with their ecotoxicology and potential human risk related problems, render the establishment of a clear phylogeography model essential. In the context of the present study, the characterization of Cylindrospermopsis raciborskii TAU-MAC 1414 strain, isolated from Lake Karla, with respect to its phylogeography and toxic potential, is attempted. Our research provides new insights on the origin of C. raciborskii in the Mediterranean region; C. raciborskii expanded in Mediterranean from North America, whilst the rest of the European strains may originate from Asia and Australia. Microcystin synthetase genes, phylogenetic closely related with Microcystis strains, were also present in C. raciborskii TAU-MAC 1414. We were unable to unambiguously confirm the presence of MC-LR, using LC-MS/MS. Our results are shedding light on the expansion and distribution of C. raciborskii, whilst they pose further questions on the toxic capacity of this species.

Variability in a permanent cyanobacterial bloom: species-specific responses to environmental drivers.

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.

Chlorophyll a fluorescence and transcriptome reveal the toxicological effects of bisphenol A on an invasive cyanobacterium, Cylindrospermopsis raciborskii.

Bisphenol A has attracted worldwide attention due to its harmful effects on humans, animals and plants. In this study, the toxicological effects of BPA on Cylindrospermopsis raciborskii were assessed based on chlorophyll a fluorescence and transcriptome analyses. The results showed that the growth of C. raciborskii was significantly inhibited when BPA exceeded 0.1 mg L-1. A marked rise of phase J was observed at a concentration greater than 0.1 mg L-1, while a K phase appeared at 20 mg L-1. The chlorophyll a fluorescence parameters of RC/CS0, F0, φP0, φE0, and ψ0, underwent a significant decline under all treatments of BPA, whereas a significant increase in both VJ and M0 occurred under all concentrations of BPA. Additionally, ABS/RC and DIo/RC markedly increased at 10 mg L-1 and 20 mg L-1. The transcriptome analysis revealed that the genes of photosynthesis, including psbA, psbB, psbC, psbD, apcA, apcB, cpcA, and cpcB, as well as those of chlorophyll and carotenoid biosynthesis, namely hemN, acsF, chlL, chlN, chlP, crtB, pds, were all down-regulated. Moreover, BPA also inhibited the oxidative phosphorylation, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), and fatty acid metabolism in C. raciborskii. Taken together, these results suggest BPA can negatively affect the expression of multiple genes and the vital energy metabolism process to arrest the growth and photosynthesis of C. raciborskii.

The occurrence of toxic cyanobacterium Cylindrospermopsis raciborskii and its toxin cylindrospermopsin in the Huong River, Thua Thien Hue province, Vietnam.

This research reports the presence of species Cylindrospermopsis raciborskii and cylindrospermopsin (CYN) in the Huong River and the relationship between species with environmental factors to find a scientific basis for predicting the risk of pollution of the species and CYN in waters. Strains of C. raciborskii isolated from the river were also identified as potentially toxin-producing through the determination of the presence of toxins in the cultures by ELISA; the presence of the genes involved by PCR confirms the CYN-producing ability of species C. raciborskii from this water body. Our results have confirmed the presence of toxic cyanobacteria C. raciborskii in the Huong River. C. raciborskii from the Huong River are mostly solitary, straight trichomes. Analyses of all C. raciborskii strains from the Huong River by ELISA for cylindrospermopsin were positive. The contents of cylindrospermopsin (CYN) in each strain were different, ranging from 5.25 ng mg-1 wet weight in CR1DD to 70.83 ng mg-1 wet weight in CR1NY. PCR analysis confirmed that the genes involved in the production of this cyanotoxin were present in C. raciborskii. The relationship between densities and toxicity showed a correlation coefficient R of 0.88. This was a relatively high positive correlation index, indicating the close links between densities and toxins: toxin CYN concentrations increased when C. raciborskii densities increased.

Differences in cyanobacterial strain responses to light and temperature reflect species plasticity.

Microcystis aeruginosa and Cylindrospermopsis raciborskii are two cyanobacterial species that dominate freshwaters globally. Multiple strains of each species with different physiology occur, however, many studies have focused only on one or two strains, limiting our understanding of both strain variation and characterisation of the species. Therefore, in this study we examined the variation in growth and morphology of multiple isolates of both species, isolated from two adjacent Australian reservoirs. Four M. aeruginosa strains (=isolates) (one colony-forming, three single-celled morphology) and eight C. raciborskii isolates (five with straight trichomes, three with coiled trichomes) were cultured individually in a factorial designed experiment with four light intensities (L: 10, 30, 50 and 100µmol photons m-2s-1) and two temperatures (T: 20 and 28°C). The specific growth rate (µ), cell volume, and final cell concentration was measured. The light attenuation coefficient (kj), a measure of self-shading, was calculated. The results showed that the intraspecific variation was greater than the interspecific variation. The µ of all isolates of M. aeruginosa and C. raciborskii ranged from 0.16 to 0.55d-1 and 0.15 to 0.70d-1, respectively. However, at a specific light and temperature the mean µ of all M. aeruginosa isolates and C. raciborskii isolates were similar. At the species level, M. aeruginosa had higher growth rates at higher light intensity but lower temperature (L100T20), while straight C. raciborskii had higher growth rates at lower light intensity but higher temperature (L50T28), and coiled C. raciborskii had higher growth rates at higher light intensity and higher temperature (L100T28). The final cell concentrations of M. aeruginosa were higher than C. raciborskii. However, C. raciborskii isolates had greater variation in µ, kj and cell volume than M. aeruginosa. kj varied with light and temperature, and decreased with surface-to-volume ratio within each species. kj was lower for M. aeruginosa compared to C. raciborskii as expected based on cell size, but interestingly, C. raciborskii coiled isolates had lower kj than the straight isolates suggesting lower effect of self-shading. This study highlights the extent of strain variation to environmental conditions and to species variability.

Variations of Growth and Toxin Yield in Cylindrospermopsis raciborskii under Different Phosphorus Concentrations.

The bloom-forming cyanobacteria, Cylindrospermopsis raciborskii, is a producer of the cytotoxic cylindrospermopsin (CYN). In this study, the growth, toxin yield, and expression of CYN biosynthesis genes of C. raciborskii were examined under varying phosphorus (P) concentrations. The results show the cell number at 0.00 and 0.01 mg·L-1 P was significantly lower than that at higher P concentrations (≥0.5 mg·L-1). The chlorophyll a content, filament length, heterocyst, and akinete numbers at P ≤ 0.05 mg·L-1 were also significantly reduced. The intracellular and extracellular CYN concentrations and the extracellular proportions increased during the culture period, and larger values were observed at higher P concentrations. Total CYN content reached 45.34-63.83 fg·cell-1 and extracellular CYN proportion reached 11.49%-20.44% at the stationary growth phase. A significantly positive correlation was observed between CYN production and cell growth rate. Three cyr genes were expressed constantly even at P-deficient conditions. The transcription of cyr genes at P-replete conditions or after P supplementation increased from 1.18-fold to 8.33-fold. In conclusion, C. raciborskii may rapidly reorganize metabolic processes as an adaptive response to environmental P fluctuations. CYN production and cyr gene expression were constitutive metabolic processes in toxic C. raciborskii.

Influence of nitrogen availability on the expression of genes involved in the biosynthesis of saxitoxin and analogs in Cylindrospermopsis raciborskii.

The development of cyanobacterial blooms in inland aquatic ecosystems is greatly promoted by nutrient availability, especially nitrogen and phosphorous. When blooms are dominated by toxigenic species the harmful effects of nutrient loading becomes particularly relevant. Among toxic species, Cylindrospermopsis raciborskii found in South American ecosystems is characterized by the production of saxitoxin and analogs (Paralytic Shellfish Poisoning, PSP), for which the factors that trigger their production have not been elucidated. In this study, the effect of nitrate availability on the relative transcript abundance of two genes (sxtU and sxtI), both involved in different steps of PSP biosynthetic pathway, was addressed in C. raciborskii MVCC19 by qPCR. The relative transcript abundance of both genes significantly increased from the beginning to the end of growth, independent of nitrate availability in the culture medium. Differences between the genes in terms of the levels of relative expression were also found, implying that during growth in nitrate-rich or nitrate-deprived conditions C. raciborskii MVCC19 has the ability to produce different kind of PSP molecules. The presence of nifH transcripts in the nitrogen-depleted treatment confirmed that in the absence of nitrate C. raciborskii fixed atmospheric N2. Moreover, after transferring filaments to nitrate-rich conditions the synthesis of nifH mRNA continued for few hours, suggesting that cell adjustments enabling the utilization of soluble nitrogen sources are not immediate. Our results show that biosynthesis of saxitoxin and analogs in C. raciborskii is not related to nitrate availability, but rather is linked to cyanobacteria growth rate.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

Constitutive cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2 partial pressure conditions.

Cylindrospermopsin (CYN) and 7-deoxy-cylindrospermopsin (dCYN) are potent hepatotoxic alkaloids produced by numerous species of cyanobacteria, including the freshwater Cylindrospermopsis raciborskii. C. raciborskii is an invasive cyanobacterium, and the study of how environmental parameters drive CYN production has received significant interest from water managers and health authorities. Light and CO2 affect cell growth and physiology in photoautotrophs, and these are potential regulators of cyanotoxin biosynthesis. In this study, we investigated how light and CO2 affect CYN and dCYN pool size as well as the expression of the key genes, cyrA and cyrK, involved in CYN biosynthesis in a toxic C. raciborskii strain. For cells growing at different light intensities (10 and 100 µmol photons m(-2) s(-1)), we observed that the rate of CYN pool size production (µCYN) was coupled to the cell division rate (µc) during batch culture. This indicated that CYN pool size under our experimental conditions is constant and cell quotas of CYN (QCYN) and dCYN (QdCYN) are fixed. Moreover, a lack of correlation between expression of cyrA and total CYN cell quotas (QCYNs) suggests that the CYN biosynthesis is regulated posttranscriptionally. Under elevated CO2 (1,300 ppm), we observed minor effects on QCYN and no effects on expression of cyrA and cyrK. We conclude that the CYN pool size is constitutive and not affected by light and CO2 conditions. Thus, C. raciborskii bloom toxicity is determined by the absolute abundance of C. raciborskii cells within the water column and the relative abundance of toxic and nontoxic strains.