Current status and prognosis of Raphidiopsis raciborskii distribution in Bulgaria as part of the southeastern region of Europe.

The cyanobacterial species Raphidiopsis raciborskii (Woloszynska) Aguilera et al. has a high invasiveness potential, which in less than a century leads to its cosmopolitan spread. In the temperate climate of Europe, R. raciborskii has been reported in many countries, but there is still a lack of detailed information about the current status of its distribution in lakes of Bulgaria, as a part of the southeastern range of its spread in Europe. We investigated the distribution of the species using data on the phytoplankton of 122 lakes surveyed during 13-years period (2009-2022). The species was found in 33 lakes (up to 324 m asl), and 14 new localities were registered during the studied period. The results reveal that the number of lakes with the presence of R. raciborskii (27 % of all research lakes) and its contribution to the total phytoplankton biomass, has increased significantly over the last decade. The species has successfully adapted and dominated the phytoplankton in 9 lakes, forming a bloom in 8 of them. The dominant position of R. raciborskii causes loss of species and functional diversity of phytoplankton and displaces the native bloom-forming cyanobacteria. Lakes with and without the species were compared based on the available data on bioclimatic and local environmental variables. Statistically significant differences were established with respect to water transparency, conductivity, maximum depth and maximum air temperature in the warmest month. Species distribution models (SDMs) were used to identify lakes in high risk of future invasion by R. raciborskii. The results of the SDMs implementation confirmed the high maximum air temperature and low water transparency to be important predictors of the occurrence of R. raciborskii in freshwater lakes in Bulgaria. In the areas with high summer temperatures the most suitable for R. raciborskii development were found to be shallow polymictic or medium deep lakes with small surface area and low water transparency. In areas with a suitable climate, the large, deep reservoirs with high transparency as well as macrophyte dominated lakes have a low probability of occurrence of R. raciborskii. Future colonization of lakes above 500 m asl (but most likely below 700 m asl) is also possible, especially in the conditions of global warming. SDMs account for climatic and biogeographic differences of lakes and could help in elucidating the underlying factors that control the occurrence and adaptation of R. raciborskii in a given area.

Unveiling the link between Raphidiopsis raciborskii blooms and saxitoxin levels: Evaluating water quality in tropical reservoirs, Brazil.

The proliferation of Raphidiopsis raciborskii blooms has sparked concerns regarding potential human exposure to heightened saxitoxins (STXs) levels. Thus, comprehending how environmental elements drive the proliferation of this STXs-producing species can aid in predicting human exposure risks. This study aimed to explore the link between cyanobacteria R. raciborskii, STXs cyanotoxins, and environmental factors in 37 public supply reservoirs in the tropical region and assess potential health hazards these toxins pose in the reservoir waters. A Structural Equation Model was used to assess the impact of environmental factors (water volume and physical and chemical variables) on R. raciborskii biomass and STXs levels. Furthermore, the potential risk of STXs exposure from consuming untreated reservoir water was evaluated. Lastly, the cumulative distribution function (CDF) of STXs across the reservoirs was computed. Our findings revealed a correlation between R. raciborskii biomass and STXs concentrations. Total phosphorus emerged as a critical environmental factor positively influencing species biomass and indirectly affecting STXs levels. pH significantly influenced STXs concentrations, indicating different factors influencing R. raciborskii biomass and STXs. Significantly, for the first time, the risk of STXs exposure was gauged using the risk quotient (HQ) for untreated water consumption from public supply reservoirs in Brazil's semi-arid region. Although the exposure risks were generally low to moderate, the CDF underscored the risk of chronic exposure due to low toxin concentrations in over 90% of samples. These outcomes emphasize the potential expansion of R. raciborskii in tropical settings due to increased phosphorus, amplifying waterborne STXs levels and associated intoxication risks. Thus, this study reinforces the importance of nutrient control, particularly phosphorus regulation, as a mitigation strategy against R. raciborskii blooms and reducing STXs intoxication hazards.

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.

Does temperature favour the spread of Raphidiopsis raciborskii, an invasive bloom-forming cyanobacterium, by altering cellular trade-offs?

As a tropical filamentous cyanobacterium, Raphidiopsis raciborskii has attracted much attention due to its expansion and toxin production. However, the mechanisms of its expansion to temperate regions have not been studied in detail. To address the potential strategies, the physiological and metabolomic profiles of R. raciborskii FACHB 1096 isolated from a temperate lake in China were determined and measured at different temperatures (10 °C, 15 °C, 20 °C, 25 °C, and 32 °C). The results demonstrated that temperature significantly changed cell viability, chlorophyll a content, specific growth rate, Chl a fluorescence, and filamentous shape of R. raciborskii. Low temperature decreased cell viability, specific growth rate, and photosynthetic efficiency, while the proportion of akinete and carbon fixation per unit cell were significantly increased compared with high temperature (32 °C). A constructed unimodal model indicated that filament length, cell volume, and cell length/width of R. raciborskii were significantly reduced in both high and low temperature environments. Under low-temperature conditions, R. raciborskii suffered different degrees of oxidative damage and produced corresponding antioxidant substances to resist oxidative stress, suggesting that low temperature changes the metabolic level of the cells, causing the cells to gradually switch from development to defense. Metabolomic data further confirmed that temperature change induced shifts in metabolic pathways in R. raciborskii, including starch and sucrose metabolic pathways, glutathione metabolic pathways, and the pentose phosphate pathways (PPP), as well as metabolic pathways related to the tricarboxylic acid (TCA) cycle. Our results indicated that the trade-offs of R. raciborskii cells among the growth, cell size, and metabolites can be significantly regulated by temperature, with broad implications for its global expansion in temperate waterbodies.

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.

Effects of terrestrial dissolved organic matter on a bloom of the toxic cyanobacteria, Raphidiopsis raciborskii.

The concentration of coloured terrestrial dissolved organic matter (tDOM) from vegetation appears to be increasing in lakes in some regions of the world, leading to the term brownification. The light attenuating effect of coloured tDOM on phytoplankton growth has been a major focus of attention, but the phytotoxic effects of tDOM, particularly on cyanobacterial blooms, are less well understood. This mesocosm study tested whether coloured tDOM, leached from the leaves of a Eucalyptus tree species, inhibited a naturally occurring bloom of the toxic cyanobacterium, Raphidiopsis raciborskii, in a reservoir over a 10 day period. The study found that tDOM leachate, measured as dissolved organic carbon (DOC), inhibited photosynthesis and growth of both R. raciborskii, as well as species present at lower densities, i.e. other cyanobacteria and diatoms. However, the effect was greater at higher tDOM input loads. The photosynthetic yield (Fv/Fm) of cyanobacteria decreased rapidly in treatments with 5.9 and 25 mg L-1 DOC addition, compared to the control (reservoir water with background DOC concentration of 6.85 ± 1.09 mg L-1). tDOM had no measurable effect in the 2 and 3.3 mg L-1 DOC addition treatments. By day 5, cell densities of cyanobacteria, including R. raciborskii, and diatoms, in treatments with 5.9 and 25 mg L-1 DOC addition were significantly lower than the control with no tDOM addition, and this effect continued throughout the experiment. This is despite the leachate addition increasing phosphate concentrations which counteracted the low background concentrations of phosphate. Light attenuation and dissolved oxygen (DO) levels were also affected by the tDOM addition, but were only significantly lower in the 25 mg L-1 DOC treatment compared with the control. DOC, dissolved organic nitrogen (DON) and dissolved organic phosphorus (DOP) concentrations all decreased in the tDOM addition treatments over the first 3 days, as the microbial cell densities increased. The components of the tDOM that decreased over time were determined by 1H NMR spectroscopy in the 25 mg L-1 DOC treatment. After 5 d, the relative concentrations of fatty acids, sugars and gallic acid decreased by around 60%, while concentrations of flavonoids and myo-inositol decreased by 45 and 35% respectively. This study suggests that phytotoxic compounds in tDOM can suppress cyanobacterial blooms, despite the increased nutrient inputs. This has implications for predicting the future likelihood of cyanobacterial blooms in lakes and reservoirs with climate-change driven changes in flow events, and other changes in the amount and types of vegetation cover. Revegetation of riparian zones, resulting in increased tDOM into waterways, may also be beneficial in reducing cyanobacterial blooms.

Bioremoval of Cylindrospermopsis raciborskii cells and cylindrospermopsin toxin in batch culture by the yeast Aureobasidium pullulans.

This study describes the ability of a yeast strain, Aureobasidium pullulans KKUY0701 isolated from eutrophic lake to eliminate Cylindrospermopsis raciborskii and cylindrospermopsin (CYN) toxin. The anti-cyanobacterial activity of this yeast strain was evaluated by growing with living cells and filtrate of C. raciborskii. CYN bioremoval was assayed using living and heat-inactivated yeast cells. Both living cells and filtrate of this yeast strain were able to suppress the growth of C. raciborskii, with total cell death occurring at day 2 and day 3, respectively. Living and inactivated yeast cells, but not yeast filtrate, reduced CYN concentrations released into cyanobacterial cultures, indicating that this toxin might be removed from the culture medium via absorption onto yeast surface rather than enzymatic biodegradation. The adsorption experiments also confirmed the elimination of CYN by living and heat-inactivated yeast. Nevertheless, inactivated yeast exhibited higher capacity (K = 3.3) and intensity (n = 1.4) than living yeast (K = 1.9, n = 1) for CYN adsorption. The study suggests that this yeast strain could be employed for bioremediation of Cylindrospermopsis blooms in freshwaters. Additionally, heat-inactivated yeast biomass could be used in slow sand filters for elimination of CYN in drinking water treatment plants.

Temporal heterogeneity of bacterial communities and their responses to Raphidiopsis raciborskii blooms.

To elucidate the interspecies connectivity between cyanobacteria and other bacteria (noncyanobacteria), microbial diversity and composition were investigated through high-throughput sequencing (HTS) in a drinking water reservoir in Chongqing city, Southwest China, during Raphidiopsis raciborskii blooms. Significant temporal changes were observed in microbial community composition during the sampling period, primarily reflected by variations in relative bacterial abundance. The modularity analysis of the network demonstrated that the bacterial community forms co-occurrence/exclusion patterns in response to variations in environmental factors. Moreover, five modules involved in the dynamic phases of the R. raciborskii bloom were categorized into the Pre-Bloom, Bloom, Post-Bloom, and Non-Bloom Groups. The reservoir was eutrophic (i.e., the average concentrations of total nitrogen (TN) and total phosphorus (TP) were 2.32 and 0.07 mg L-1, respectively) during the investigation; however, Pearson's correlation coefficient showed that R. raciborskii was not significantly correlated with nitrogen and phosphorus. However, other environmental factors, such as water temperature, pH, and the permanganate index, were positively correlated with R. raciborskii. Importantly, Proteobacteria (α-, γ-Proteobacteria), Acidobacteria, Chloroflexi, and Firmicutes were preferentially associated with increased R. raciborskii blooms. These results suggested that the transition of R. raciborskii bloom-related microbial modules and their keystone species could be crucial in the development and collapse of R. raciborskii blooms and could provide a fundamental basis for understanding the linkage between the structure and function of the microbial community during bloom dynamics.

Assessment of the Appearance and Toxin Production Potential of Invasive Nostocalean Cyanobacteria Using Quantitative Gene Analysis in Nakdong River, Korea.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL-1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL-1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

Iron enrichment from hypoxic hypolimnion supports the blooming of Raphidiopsis raciborskii in a tropical reservoir.

Occurring worldwide, blooms of Raphidiopsis raciborskii threaten the use of water resources especially in tropical and subtropical waterbodies. Its high flexibility in the uses of light and macronutrients (C, N, P) frustrates any bloom prediction and control based on macronutrients regulation. To identify the critical factors promoting periodic blooms of R. raciborskii, the trends of meteorological, hydrodynamic, physical, and chemical variables (including macro- and micronutrients: N, P, Fe) were analyzed in a Chinese tropical large reservoir (Dashahe reservoir) over five years. It was hypothesized that Fe availability, mediated by the mixing pattern of the reservoir, played a crucial role in the periodic blooms of the cyanobacterium. To have a more complete understanding, the effects of Fe on growth of a local R. raciborskii strain were tested in a monoculture experiment. The biomass and relative abundance of R. raciborskii in the reservoir showed a clear seasonal trend, with relative abundance > 50% in summer/autumn (July to October). Three habitat types along a dominance gradient were identified in the reservoir and 17 variables were used to compare them. Statistical analysis and habitat comparison showed that temperature and stratification, dissolved Fe and N concentrations in the epilimnion, and dissolved Fe and oxygen concentrations in the hypolimnion were the critical factors driving the dynamics of R. raciborskii in the study reservoir. The habitat dominated by R. raciborskii was characterized by a relatively low availability of macro resources (Zeu/Zm < 1, SRP < 0.01 mg/L, DIN < 0.3 mg/L) and by a high Fe availability supplemented from hypoxic hypolimnion. The dependence of growth on Fe concentration increase was confirmed in culture where the maximum was reached at 0.689 mg Fe /L. Our results suggest that a high Fe bioavailability, also originating from the hypoxic hypolimnion, influences the dynamics R. raciborskii and favors the blooms of the species. As a consequence, Fe concentrations in the water column as well as oxygen measurements along the water column should be routinely included in the monitoring programs aimed at predicting and controlling R. raciborskii blooms.

Heterocyte production, gene expression, and phylogeography in Raphidiopsis (=Cylindrospermopsis) raciborskii.

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.

Species distribution models as a tool for early detection of the invasive Raphidiopsis raciborskii in European lakes.

In freshwater habitats, invasive species and the increase of cyanobacterial blooms have been identified as a major cause of biodiversity loss. The invasive cyanobacteria Raphidiopsis raciborskii a toxin-producing and bloom-forming species affecting local biodiversity and ecosystem services is currently expanding its range across Europe. We used species distribution models (SDMs) and regional bioclimatic environmental variables, such as temperature and precipitation, to identify suitable areas for the colonization and survival of R. raciborskii, with special focus on the geographic extent of potential habitats in Northern Europe. SDMs predictions uncovered areas of high occurrence probability of R. raciborskii in locations where it has not been recorded yet, e.g. some areas in Central and Northern Europe. In the southeastern part of Sweden, areas of suitable climate for R. raciborskii corresponded with lakes of high concentrations of total phosphorus, increasing the risk of the species to thrive. To our knowledge, this is the first attempt to predict areas at high risk of R. raciborskii colonization in Europe. The results from this study suggest several areas across Europe that would need monitoring programs to determine if the species is present or not, to be able to prevent its potential colonization and population growth. Regarding an undesirable microorganism like R. raciborskii, authorities may need to start information campaigns to avoid or minimize the spread.

Transcriptomic responses to phosphorus in an invasive cyanobacterium, Raphidiopsis raciborskii: Implications for nutrient management.

Phosphorus (P) is a vital macronutrient associated with the growth and proliferation of Raphidiopsis raciborskii, an invasive and notorious bloom-forming cyanobacterium. However, the molecular mechanisms involved in P acclimation remain largely unexplored for Raphidiopsis raciborskii. Here, transcriptome sequencing of Raphidiopsis raciborskii was conducted to reveal multifaceted mechanisms involved in mimicking dipotassium phosphate (DIP), ß-glycerol phosphate (Gly), 2-aminoethylphosphonic acid (AEP), and P-free conditions (NP). Chlorophyll a fluorescence parameters showed significant differences in the NP and AEP groups compared with the DIP and Gly-groups. Expression levels of genes related to phosphate transportation and uptake, organic P utilization, nitrogen metabolism, urea cycling, carbon fixation, amino acid metabolism, environmental information, the ATP-synthesis process in glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway were remarkably upregulated, while those related to photosynthesis, phycobiliproteins, respiration, oxidative phosphorylation, sulfur metabolism, and genetic information were markedly downregulated in the NP group relative to the DIP group. However, the expression of genes involved in organic P utilization, the urea cycle, and genetic information in the Gly-group, and carbon-phosphorus lyase, genetic information and environmental information in the AEP group were significantly increased compared to the DIP group. Together, these results indicate that Raphidiopsis raciborskii exhibits the evolution of coordination of multiple metabolic pathways and certain key genes to adapt to ambient P changes, which implies that if P is reduced to control Raphidiopsis raciborskii bloom, there is a risk that external nutrients (such as nitrogen, amino acids, and urea) will stimulate the growth or metabolism of Raphidiopsis.

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.

Permanent occurrence of Raphidiopsis raciborskii and cyanotoxins in a subtropical reservoir polluted by domestic effluents (Itupararanga reservoir, São Paulo, Brazil).

Toxic cyanobacteria blooms are a frequent problem in subtropical reservoirs and freshwater systems. The purpose of this study was to investigate the occurrence of potentially toxic cyanobacteria and the environmental conditions associated with the presence of cyanotoxins in a Brazilian subtropical reservoir. Five collections were carried out at seven sampling locations in the reservoir, during the rainy and dry seasons, between the years 2016 and 2017. There was permanent occurrence of Raphidiopsis raciborskii (Woloszynska) Aguilera, Berrendero Gómez, Kastovsky, Echenique & Salerno (Phycologia 57(2):130-146, 2018), ranging between dominant and abundant, with an average biomass of 38.8 ± 29.9 mg L-1. Also abundant were Dolichospermum solitarium, D. planctonicum, Planktothrix isothrix, and Aphanizomenon gracile. Saxitoxin (STX) was detected in all the collected samples (0.11 ± 0.05 µg L-1). Microcystin (MC) was also detected, but at lower concentrations (0.01 ± 0.0 µg L-1). Low availability of NO3- and phosphorus limitation had significant effects on the R. raciborskii biomass and the levels of STX and MC. It was observed that R. raciborskii was sensitive to thermal stratification, at the same time that STX levels were higher. This suggested that STX was produced under conditions that restricted the growth of R. raciborskii. These are important findings, because they add information about the permanent occurrence of STX and R. raciborskii in an aquatic ecosystem limited by phosphorus, vulnerable to climatic variations, and polluted by domestic effluents.

Simultaneous biodegradation of harmful Cylindrospermopsis raciborskii and cylindrospermopsin toxin in batch culture by single Bacillus strain.

This study investigates the capability of a Bacillus flexus strain isolated from decayed cyanobacterial blooms for the bioremediation of Cylindrospermopsis raciborskii and cylindrospermopsin (CYN) toxin. The algicidal activity of this strain was tested by co-cultivation with C. raciborskii cultures. CYN biodegradation was investigated in the presence of living and heat-inactivated bacterial cells or bacterial filtrate. Living bacterial cells inhibited C. raciborskii growth after 2 days of incubation with complete cell death at day 5. Bacterial filtrate caused a rapid reduction in C. raciborskii growth at the first day, with complete cell lysis at day 3. Only living cells of SSZ01 caused reduction in CYN released into the medium during the bacterial decay of C. raciborskii cells. The biodegradation rate of CYN by SSZ01 relied on initial toxin concentrations. The highest rate (42 µg CYN L-1 day-1) was obtained at the higher initial concentration (300 µg L-1), and the lowest (4µg CYN L-1 day-1) was at lower concentration (50 µg L-1). These results suggest that this bacterial strain could be employed to bioremediate cyanobacterial blooms in freshwaters. Also, the application of this bacterium in slow sand filters would give possibilities for degradation and bioremediation of cyanotoxins in drinking water treatment plants.

Precision early detection of invasive and toxic cyanobacteria: A case study of Raphidiopsis raciborskii.

Blooms of the toxic cyanobacterium, Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii), are becoming a major environmental issue in freshwater ecosystems globally. Our precision prevention and early detection of R. raciborskii blooms rely upon the accuracy and speed of the monitoring method. A duplex digital PCR (dPCR) monitoring approach was developed and validated to detect the abundance and toxin-producing potential of R. raciborskii simultaneously in both laboratory spiked and environmental samples. Results of dPCR were strongly correlated with traditional real time quantitative PCR (qPCR) and microscopy for both laboratory and environmental samples. However, discrepancies between methods were observed when measuring R. raciborskii at low abundance (1 - 105 cells L - 1), with dPCR showing a higher precision compared to qPCR at low cell concentration. Furthermore, the dPCR assay had the highest detection rate for over two hundred environmental samples especially under low abundance conditions, followed by microscopy and qPCR. dPCR assay had the advantages of simple operation, time-saving, high sensitivity and excellent reproducibility. Therefore, dPCR would be a fast and precise monitoring method for the early warning of toxic bloom-forming cyanobacterial species and assessment of water quality risks, which can improve prediction and prevention of the impacts of harmful cyanobacterial bloom events in inland waters.

Phosphorus deficiency stimulates dominance of Cylindrospermopsis through facilitating cylindrospermopsin-induced alkaline phosphatase secretion: Integrating field and laboratory-based evidences.

Potentially toxic Cylindrospermopsis raciborskii blooms are of emerging concerns, as its scale is spreading from tropical regions to high latitudes, increasing the risk of aquatic biota being exposed to cylindrospermopsin (CYN). So far, CYN-producing C. raciborskii strains have only been reported in tropical waters which are commonly phosphorus (P)-deficient, where they can dominate phytoplankton communities. However, the influence of CYN on phytoplankton communities under different P status remains unclear. In this study, we first analyzed the summer observations of 120 tropical reservoirs in Guangdong Province. The proportion of potential CYN-producers was significantly higher in P-deficient and CYN-present reservoirs than that in P-sufficient or CYN-absent ones. This suggested that in P-deficient condition, the potential CYN producers might gain more advantages by the help of CYN. Then, in laboratory experiments we found that upon P deprivation, CYN did not inhibit the cell growth of other algal cells, but significantly stimulates them to secret more alkaline phosphatase (ALP) than in P-sufficient condition. Through transcriptomics, we further revealed that under such P-deficient condition, CYN remarkably induced intracellular nitrogen allocation and protein export system by activating the PIK3/Akt-cGMP/PKG signaling pathways in Scenedesmus bijugatus, thus enhancing its ALP secretion. Our study implies that CYN-induced ALP secretion is facilitated upon P deficiency, thus supporting the dominance of its producers C. raciborskii.

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.