Cylindrospermopsin Production Research Articles

Grazer‐induced toxin production is energetically costly and significantly reduces growth of cylindrospermopsin‐producing cyanobacteria

AbstractToxins have been proposed as a defense mechanism for phytoplankton against zooplankton grazing. However, the associated costs are not understood. Here, we aim to reveal the costs in terms of both physiological and molecular responses in the cylindrospermopsin (CYN)‐producing cyanobacterium Raphidiopsis raciborskii. Exposure of R. raciborskii to Daphnia magna resulted in a significant increase in its CYN cell quota which was grazer cell density‐dependent, indicating an inducible effect on CYN production. A negative and significant relationship between cellular growth rate and CYN production suggested a growth‐defense trade‐off, highlighting the costly nature of toxin production. Despite strong transcriptional responses (121 differentially expressed genes) in R. raciborskii under grazing pressure, the CYN gene cluster remained largely unaffected, implying post‐transcriptional regulation of CYN biosynthesis. Significant variation was observed in transcripts for phosphorus metabolism and photosynthesis‐related genes, and evidenced that energy (ATP) metabolic processes were stimulated. This study is the first to demonstrate the high energy requirements for CYN production and provide insight into the molecular basis underlying the costs of toxin production.

Community Structure and Toxicity Potential of Cyanobacteria during Summer and Winter in a Temperate-Zone Lake Susceptible to Phytoplankton Blooms.

Cyanobacterial blooms are increasingly common during winters, especially when they are mild. The goal of this study was to determine the summer and winter phytoplankton community structure, cyanotoxin presence, and toxigenicity in a eutrophic lake susceptible to cyanobacterial blooms throughout the year, using classical microscopy, an analysis of toxic cyanometabolites, and an analysis of genes involved in biosynthesis of cyanotoxins. We also assessed whether cyanobacterial diversity in the studied lake has changed compared to what was reported in previous reports conducted several years ago. Moreover, the bloom-forming cyanobacterial strains were isolated from the lake and screened for cyanotoxin presence and toxigenicity. Cyanobacteria were the main component of the phytoplankton community in both sampling times, and, in particular, Oscillatoriales were predominant in both summer (Planktothrix/Limnothrix) and winter (Limnothrix) sampling. Compared to the winter community, the summer community was denser; richer in species; and contained alien and invasive Nostocales, including Sphaerospermopsis aphanizomenoides, Raphidiopsis raciborskii, and Raphidiopsis mediterranea. In both sampling times, the blooms contained toxigenic species with genetic determinants for the production of cylindrospermopsin and microcystins. Toxicological screening revealed the presence of microcystins in the lake in summer but no cyanotoxins in the winter period of sampling. However, several cyanobacterial strains isolated from the lake during winter and summer produced anabaenopeptins and microcystins. This study indicates that summer and winter blooms of cyanobacteria in the temperate zone can differ in biomass, structure, and toxicity, and that the toxic hazards associated with cyanobacterial blooms may potentially exist during winter.

Molecular Screening for Cyanobacteria and Their Cyanotoxin Potential in Diverse Habitats.

Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer-PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins-mcyE, saxitoxins-sxtA, cylindrospermopsins-cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.

A cylindrospermopsin-producing cyanobacterium isolated from a microbial mat in the Baltic Sea

Toxic benthic mats of cyanobacteria are associated with water quality problems and animal poisonings around the world. A strain of the filamentous cyanobacterial genus Kamptonema was isolated from a water bloom in the Baltic Sea four decades ago and later shown to produce cylindrospermopsins. However, the exact habitat of this strain remains unclear and cylindrospermopsins have not yet been reported from water blooms in the Baltic Sea. Here, we report the isolation of Kamptonema sp. UHCC 0994 from a benthic microbial mat collected in shallow water on the coast of Helsinki. We obtained draft genome sequences for the Kamptonema spp. PCC 7926 and UHCC 0994 strains that were isolated from the Baltic Sea. These genomes were 90–96% similar to previously studied Kamptonema sp. PCC 6506 and Kamptonema formosum PCC 6407, which were isolated from benthic and North American freshwater environments, respectively. The genomes of all four Kamptonema strains encode complete cylindrospermopsin biosynthetic gene clusters. We detected the production of cylindrospermopsin and 7-epi-cylindrospermopsin in the four Kamptonema strains using high-resolution liquid chromatography mass spectrometry. The four strains encode genes for producing gas vesicles distributed in two to three different regions of their genomes. Kamptonema spp. UHCC 0994 and PCC 7926 have both retained the ability to regulate their buoyancy when grown in liquid culture. Together this suggests that these toxic cyanobacteria may exhibit a tychoplanktic lifestyle in the Baltic Sea. This study suggests that microbial mats containing cyanobacteria could be a source of environmental toxins in the Baltic Sea.

An innovative passive sampling approach for the detection of cyanobacterial gene targets in freshwater sources

Cyanotoxins pose significant human health risks, but traditional monitoring approaches can be expensive, time consuming, and require analytical equipment or expertise that may not be readily available. Quantitative polymerase chain reaction (qPCR) is becoming an increasingly common monitoring strategy as detection of the genes responsible for cyanotoxin synthesis can be used as an early warning signal. Here we tested passive sampling of cyanobacterial DNA as an alternative to grab sampling in a freshwater drinking supply lake with a known history of microcystin-LR. DNA extracted from grab and passive samples was analyzed via a multiplex qPCR assay that included gene targets for four common cyanotoxins. Passive samples captured similar trends in total cyanobacteria and the mcyE/ndaF gene responsible for microcystin production when compared to traditional grab samples. Passive samples also detected genes associated with the production of cylindrospermopsin and saxitoxin that were not detected in grab samples. This sampling approach proved a viable alternative to grab sampling when used as an early warning monitoring tool. In addition to the logistical benefits of passive sampling, the detection of gene targets not detected by grab samples indicates that passive sampling may allow for a more complete profile of potential cyanotoxin risk.

Cytotoxic Effects and Oxidative Stress Produced by a Cyanobacterial Cylindrospermopsin Producer Extract versus a Cylindrospermopsin Non-Producing Extract on the Neuroblastoma SH-SY5Y Cell Line.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN-), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN-. Furthermore, the reactive oxygen species (ROS) increased with time (0-24 h) and CYN concentration (0-1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN-, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN-, which highlights the importance of studying toxic features in their natural scenario.

Diazotrophy modulates cyanobacteria stoichiometry through functional traits that determine bloom magnitude and toxin production.

Harmful cyanobacterial blooms are an increasing threat to water quality. The interactions between two eco-physiological functional traits of cyanobacteria, diazotrophy (nitrogen (N)-fixation) and N-rich cyanotoxin synthesis, have never been examined in a stoichiometric explicit manner. We explored how a gradient of resource N:phosphorus (P) affects the biomass, N, P stoichiometry, light-harvesting pigments, and cylindrospermopsin production in a N-fixing cyanobacterium, Aphanizomenon. Low N:P Aphanizomenon cultures produced the same biomass as populations grown in high N:P cultures. The biomass accumulation determined by carbon, indicated low N:P Aphanizomenon cultures did not have a N-fixation growth tradeoff, in contrast to some other diazotrophs that maintain stoichiometric N homeostasis at the expense of growth. However, N-fixing Aphanizomenon populations produced less particulate cylindrospermopsin and had undetectable dissolved cylindrospermopsin compared to non-N-fixing populations. The pattern of low to high cyanotoxin cell quotas across an N:P gradient in the diazotrophic cylindrospermopsin producer is similar to the cyanotoxin cell quota response in non-diazotrophic cyanobacteria. We suggest that diazotrophic cyanobacteria may be characterized into two broad functional groups, the N-storage-strategists and the growth-strategists, which use N-fixation differently and may determine patterns of bloom magnitude and toxin production in nature.

The effects of salinity and N:P on N-rich toxins by both an N-fixing and non-N-fixing cyanobacteria.

Freshwater ecosystems are experiencing increased salinization. Adaptive management of harmful algal blooms (HABs) contribute to eutrophication/salinization interactions through the hydrologic transport of blooms to coastal environments. We examined how nutrients and salinity interact to affect growth, elemental composition, and cyanotoxin production/release in two common HAB genera. Microcystis aeruginosa (non-nitrogen (N)-fixer and microcystin-LR producer; MC-LR) and Aphanizomenon flos-aquae (N-fixer and cylindrospermopsin producer; CYN) were grown in N:phosphorus (N:P) 4 and 50 (by atom) for 21 and 33 days, respectively, then dosed with a salinity gradient (0 - 10.5 g L-1). Both total MC-LR and CYN were correlated with particulate N. We found Microcystis MC-LR production and release was affected by salinity only in the N:P 50 treatment. However, Aphanizomenon CYN production and release was affected by salinity regardless of N availability. Our results highlight how cyanotoxin production and release across the freshwater - marine continuum are controlled by eco-physiological differences between N-acquisition traits.

Nutrient Regulation of Relative Dominance of Cylindrospermopsin-Producing and Non-cylindrospermopsin-Producing Raphidiopsis raciborskii.

Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) can produce cylindrospermopsin (CYN) which is of great concern due to its considerable toxicity to human and animals. Its CYN-producing (toxic) and non-CYN-producing (non-toxic) strains co-exist commonly in natural water bodies, while how their relative dominance is regulated has not been addressed. In this study, we combined field investigation with laboratory experiments to assessed the relationship between toxic and non-toxic R. raciborskii abundances under different nutrient levels. The rpoC1- and cyrJ-based qPCR was applied for quantifying total and toxic R. raciborskii abundances, respectively. The field survey showed that toxic R. raciborskii was detected in 97 of 115 reservoirs where its proportion ranged from 0.3% to 39.7% within the R. raciborskii population. Both total and toxic R. raciborskii abundances increased significantly with trophic level of these reservoirs, consistent with our monoculture and co-culture experiments showing in an increase in R. raciborskii growth with increasing nitrogen (N) or phosphorus (P) concentrations. In the monoculture experiments, growth rates of non-toxic and toxic strains from Australia or China were not significantly different under the same culture conditions. On the other hand, in the co-culture experiments, the toxic strains displayed a significantly faster growth than non-toxic strains under nutrient-replete conditions, resulting in an obvious shift toward the dominance by toxic strains from day 3 to the end of the experiments, regardless of the strain originating from Australia or China. The reverse was found under N- or P-limited conditions. Our results indicated that the toxic strains of R. raciborskii have a competitive advantage relative to the non-toxic strains in a more eutrophic world. In parallel to an increase in dominance, both toxic strains grown in the mixed population significantly increased CYN production under nutrient-replete conditions as compared to nutrient-limited conditions, suggesting that CYN may be of significance for ecological advantage of toxic R. raciborskii. These results highlight the importance of nutrient availability in regulating abundances and strain dominance of two genotypes of R. raciborskii. Our findings demonstrated that elevated nutrients would favor the growth of CYN-producing R. raciborskii and CYN production, leading to more blooms with higher toxicity at global scale.

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.

Biodiversity, phylogeny and toxin production profile of cyanobacterial strains isolated from lake Latyan in Iran

Monitoring toxigenic cyanobacteria in freshwaters is of great importance due to the adverse health impacts on humans and aquatic organisms. Here we studied cyanobacterial occurrence and biodiversity in a drinking water reservoir in Tehran province, Iran. In total, nine different species representing three orders of Synechococcales, Oscillatoriales and Nostocales were isolated and classified into six families and seven genera ranging from 92.3% to 99.0% similarities in their partial 16S rDNA with GenBank sequences. The cultures were analyzed for cyanotoxins production by the Artemia salina bioassay, ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and also screened for the presence of marker genes involved in toxins production. Ethyl acetate extracts of three strains showed more than 50% mortality on A. salina larvae after 24 h at a concentration of 500 µg/ml. Production of at least one of the cyanotoxins, microcystin (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a), was detected in 6 of the strains. Seven MC variants with a total concentration of 130.6 ng/mg of biomass dry weight were detected for the strain Phormidium sp. UTMC6001 and molecular screening of the mcyE gene also confirmed the presence of this biomarker in its genome. Our study also revealed the production of CYN in a novel picocyanobacterial strain Cyanobium sp. UTMC6007 at 1.0 ng/mg of biomass dry weight. Considering the limited information on freshwater toxic cyanobacteria taxonomy in the Middle East, these findings will expand our knowledge and consequently aid in development of new water management policies in future.

<p><strong>True branching and phenotypic plasticity in the planktonic cyanobacterium <em>Dolichospermum brachiatum sp. nov.</em> (Nostocales, Aphanizomenonaceae), from south-eastern Australia</strong></p>

Phenotypic plasticity in the cyanobacteria provides adaptive strategies to cope with changes in prevailing environmental conditions. Plasticity may manifest in morphological, physiological, or behavioural responses. Determining if variable phenotypes are truly novel expressions evolved via adaptive genetic change, rather than intrinsic characteristics within the spectrum of intraspecific diversity has rarely been studied. Poorly characterised or unrecognised intraspecific variability is as problematic as cryptic diversity in terms of imposing limitations on the use of morphology-based identifications to recognise algal diversity. We isolated strains from a population of filamentous cyanobacteria from the plankton of a temperate lake in Western Victoria, Australia putatively identified as the cylindrospermopsin producer Chrysosporum ovalisporum which displayed aberrant vegetative and apical cells and akinete morphology, and T-type true branching. In culture, strains grown in phosphorus-free media showed morphology and true branching consistent with the wild types suggesting that these features are a phenotypic response to P-limitation. Results of the phylogenetic analyses of these strains using the 16S-23S rRNA, and the secondary structure of the ITS region of the 16S-23S rRNA operon confirmed that the isolated strains were a novel species, here designated Dolichospermum brachiatum. This is the first record of true branching in the genus Dolichospermum and only the second observation from the Aphanizomenonaceae. These findings support our current understanding that true branching in cyanobacteria is a polyphyletic trait, having arisen several times in the evolution of this Phylum.

Different Gene Expression Response of Polish and Australian Raphidiopsis raciborskii Strains to the Chill/Light Stress

R. raciborskii is known for growing under wide ranges of temperature and light. In temperate regions, however, low temperature and high light may serve as a stressful condition for invading tropical populations. The genetic basis of R. raciborskii’s adaptation to this combination of stresses are unknown. In this study, the growth rate and the expression of genes that may be crucial in the response to the chill/light stress of two R. raciborskii strains (differing in their climatic origin and toxicity) exposed to low temperature and high light were examined. Results showed that AMU-DH-30, a non CYN (cylindrospermopsin) producing strain isolated from the temperate region, exhibited under stress the upregulation of genes involved in the protein translation (rbp1, nusG, hflX), membrane fluidity (desA), photosynthetic activity (ccr2 and ftsH), and the accumulation of compatible solutes (asd). In contrast, a CYN producing Australian strain CS-505 was not able to adapt quickly and to continue growth during stress conditions. Intriguingly, CS-505 and AMU-DH-30 had a similar ability to resume growth when the stress conditions subsided. Moreover, in strain CS-505 the cyrB gene was significantly upregulated under the stress conditions. The presented results shed new light on the possible mechanisms involved in the response of R.raciborskii to chill/light stress.

Limited Microcystin, Anatoxin and Cylindrospermopsin Production by Cyanobacteria from Microbial Mats in Cold Deserts.

Toxic metabolites are produced by many cyanobacterial species. There are limited data on toxigenic benthic, mat-forming cyanobacteria, and information on toxic cyanobacteria from Central Asia is even more scarce. In the present study, we examined cyanobacterial diversity and community structure, the presence of genes involved in toxin production and the occurrence of cyanotoxins in cyanobacterial mats from small water bodies in a cold high-mountain desert of Eastern Pamir. Diversity was explored using amplicon-based sequencing targeting the V3-V4 region of the 16S rRNA gene, toxin potential using PCR-based methods (mcy, nda, ana, sxt), and toxins by enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular identification of cyanobacteria showed a high similarity of abundant taxa to Nostoc PCC-73102, Nostoc PCC-7524, Nodularia PCC-935 and Leptolyngbya CYN68. The PCRs revealed the presence of mcyE and/or ndaF genes in 11 samples and mcyD in six. The partial sequences of the mcyE gene showed high sequence similarity to Nostoc, Planktothrix and uncultured cyanobacteria. LC-MS/MS analysis identified six microcystin congeners in two samples and unknown peptides in one. These results suggest that, in this extreme environment, cyanobacteria do not commonly produce microcystins, anatoxins and cylindrospermopsins, despite the high diversity and widespread occurrence of potentially toxic taxa.

Pilot search for cylindrospermopsin-producers in nine shallow Bulgarian waterbodies reveals nontoxic strains of Raphidiopsis raciborskii, R. mediterranea and Chrysosporum bergii

This paper presents results from the first application of molecular-genetic analysis combined with conventional light microscopy (LM) for identification of cylindrospermopsin (CYN) producers in field phytoplankton samples from Bulgaria. In total 68 cyanoprokaryotes were found by LM, out of which 18 were considered potential CYN-producers according to the literature. They occurred in different abundance (0-40%) in five waterbodies. The presence of CYN-producers was sought by application of the primer pair cynsulfF/cylnamR specific for the sulfotransferase gene (cyrJ) considered the best genetic marker for detection of CYN-toxigenic strains. Although CYN was detected by enzyme-linked immunosorbent assay (ELISA) in two waterbodies (lake Vaya and reservoir Mandra), we did not find cyanoprokaryotes with cyrJ gene. Therefore, Raphidiopsis raciborskii, despite being identified by LM in both waterbodies where CYN occurred, was not the producer of this toxin and belonged to the European nontoxic population of the species. The same was true for the less widely spread Raphidiopsis mediterranea and Chrysosporum bergii (both found in small amounts only in lake Vaya). Our results on CYN occurrence without the assignment of its producer in the collected samples suggest that: 1) CYN remains stable in water after disappearance of the toxigenic species, or, more probably, 2) besides the already known species, there are others that are associated with the production of CYN. Because revealing of novel CYN producers is of ultimate importance for health risk assessment and water management, we propose more analyses to be performed in future studies.

Effect of nutrient availability on cylindrospermopsin gene expression and toxin production in Cylindrospermopsis raciborskii

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 82:105-110 (2018) - DOI: https://doi.org/10.3354/ame01877 NOTE Effect of nutrient availability on cylindrospermopsin gene expression and toxin production in Cylindrospermopsis raciborskii Natalia Rigamonti1,4, Luis Aubriot2,3, Fatima Martigani2,3, Sylvia Bonilla2,3, Claudia Piccini1,3,* 1Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, 11600 Montevideo, Uruguay 2Sección Limnología, Instituto de Ecología y Ciencias Ambientales, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay 3Physiology and Ecology Phytoplankton Group, Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República, Uruguay 4Departamento de Aguas y Productos Químicos, Laboratorio Tecnológico del Uruguay. Avenida Italia 6201, 11500 Montevideo, Uruguay *Corresponding author: cpiccini@iibce.edu.uy ABSTRACT: Cylindrospermopsis raciborskii is a bloom-forming diazotrophic cyanobacterium, the worldwide success of which has been attributed to its phenotypic plasticity and to the presence of ecotypes with different environmental preferences. C. raciborskii strains from Oceania and Asia are able to produce cylindrospermopsin, a potent nitrogen-rich cytotoxin. There is no agreement about the biological role of cylindrospermopsin or which environmental factors trigger its production in this species. In the present study, we analyzed the combined effect of nitrate and phosphate availability on the relative expression of a gene involved in cylindrospermopsin synthesis (cyrA) and on toxin production in C. raciborskii CYP011K. The strain was grown under different combinations of nitrate and phosphate availability. We found that, besides cyrA expression being detected in all treatments, the combination of nitrate deprivation and phosphate repletion was the condition significantly inducing cyrA expression and toxin production, concomitant with a higher growth rate. Thus, we demonstrated that cylindrospermopsin production in C. raciborskii is positively related to nitrogen fixation. We hypothesize that under nitrogen-fixing conditions, the production and export of cylindrospermopsin would be a functional strategy for competition with other phytoplankters via a mechanism involving the inhibition of reduced glutathione synthesis and induction of oxidative damage. KEY WORDS: Cylindrospermopsis raciborskii · cyrA · Cylindrospermopsin · Cyanotoxins · Nitrogen availability Full text in pdf format PreviousCite this article as: Rigamonti N, Aubriot L, Martigani F, Bonilla S, Piccini C (2018) Effect of nutrient availability on cylindrospermopsin gene expression and toxin production in Cylindrospermopsis raciborskii. Aquat Microb Ecol 82:105-110. https://doi.org/10.3354/ame01877 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 82, No. 1. Online publication date: September 20, 2018 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2018 Inter-Research.

Toxicity-associated changes in the invasive cyanobacterium Cylindrospermopsis raciborskii in response to nitrogen fluctuations

The cyanobacterium Cylindrospermopsis raciborskii is of particular concern due to its ability to fix nitrogen (N), sporadic bloom, potential toxicity and apparent invasiveness. However, the toxicity associated behavior and response of toxic C. raciborskii under N fluctuations in water have been poorly investigated. The present study initiated based on the field survey in which Cylindrospermopsis species was found to have a high fitness under nitrate concentrations fluctuating from 0.02 mg L−1 to 2.90 mg L−1 in Chinese freshwater lakes. Examination on the role of short-term N fluctuations was conducted in two C. raciborskii strains which were exposed to a range of N concentrations supplied in two patterns, namely one-time pattern and ten-time pattern in which the equal amount of N was divided into ten-time accretions. The results showed the growth of both strains were not vulnerable to the transient nutrient fluctuations. The toxic strain showed considerable toxicological flexibility with the highest yield of cylindrospermopsin (CYN) obtained in the absence of N and the lowest in full medium. Generally, larger amounts of total CYN were observed at lower N levels, indicating that N deficiency promoted the intracellular accumulation and simultaneously restrained the extracellular release of CYN. Furthermore, CYN production was significantly different in two N supply patterns. The maximum quotas of intracellular and extracellular CYN in one-time pattern were respectively 2.79–3.53 and 3.94–7.20 times higher compared to the ten-time pattern. To our knowledge, our results are the first evidence of toxicity variations of C. raciborskii to the impermanent N fluctuations, shedding new light on its toxicological plasticity.

The morphological and molecular detection for the presence of toxic Cylindrospermopsis (Nostocales, Cyanobacteria) in Beijing city, China

Cylindrospermopsis raciborskii and its highly similar relatives Raphidiopsis species have been recognized as globally invasive and expansive filamentous cyanobacteria causing water blooms. Reports on C. raciborskii/Raphidiopsis species and their harmful metabolites such as hepatotoxic cylindrospermopsins (CYNs) in Chinese waters have been increasing, but mostly restricted to the southern regions of China. To further explore the existence and distribution of C. raciborskii in China, six water samples from Beijing city were morphologically and molecularly examined. Five samples of the six were shown to have Cylindrospermopsis filaments with straight and spiral morphotypes. PCR detection targeting on Cylindrospermopsis/Raphidiopsis specific 16S rRNA gene region also showed the positive amplification, and such amplifications were confirmed by sequencing and phylogenetic analysis. As well, three of the five Cylindrospermopsis containing samples were shown to have cyrJ — a gene of CYN synthesis gene cluster. The results represented the presence of toxic Cylindrospermopsis at the most northern line in China so far, indicating rapid expansion of this harmful invasive cyanobacterium. It is strongly suggested that the monitoring on C. raciborskii/Raphidiopsis species and their production of cylindrospermopsin should be emphasized in Beijing and even more northern parts of China.

Use of three monitoring approaches to manage a major Chrysosporum ovalisporum bloom in the Murray River, Australia, 2016.

An unusual bloom of Chrysosporum ovalisporum (basionym Aphanizomenon ovalisporum) occurred for the first time in the Murray River and distributary rivers in New South Wales, Australia, from mid-February to early June 2016. At its greatest extent, it contaminated a combined river length of ca. 2360km. Chrysosporum ovalisporum usually comprised >99% of the total bloom biovolume at most locations sampled, which at times exceeded 40mm3l-1. The origins of the bloom were most likely reservoirs on the upper Murray River, with cyanobacterial-infested water released from them contaminating the river systems downstream. An integrated approach using three analytical methods: (1) identification and enumeration by microscopy, (2) multiplex quantitative polymerase chain reaction (qPCR), and (3) toxin analysis, was used to obtain data for the assessment of risk to water users and management of the bloom. qPCR indicated some cyrA and stxA genes responsible for cylindrospermopsin and saxitoxin biosynthesis respectively were present, but mostly below the level of quantification. No mcyE genes for microcystin biosynthesis were detected. Toxin analysis also revealed that cylindrospermopsin, saxitoxin and microcystin were all below detection. Lack of measurable toxicity in a species usually considered a cylindrospermopsin producer elsewhere meant the possibility of relaxing management guidelines; however, high (Red) alerts needed to be maintained due to risk to water users from other biohazards potentially produced by the cyanobacteria such as contact irritants. A three-tiered monitoring strategy is suggested for monitoring cyanobacterial blooms to provide enhanced data for bloom management.

Dynamics of Cylindrospermopsin Production and Toxin Gene Expression in <i>Aphanizomenon ovalisporum</i>

Aphanizomenon ovalisporum is a cylindrospermopsin (CYN)-producing cyanobacteria species that due to its increasing worldwide distribution has become an important health alarm in the last few years. Several clusters of genes involved in CYN production have been described in different CYN+ cyanobacteria genera, named aoa for Aphanizomenon and cyr for Cylindrospermopsis and others strains. The sequences of those genes are highly similar, but a rearrangement in gene order is also observed. The information on the control of CYN production by gene expression is still scarce, especially in Aphanizomenon. To obtain further information about the control of CYN production in A. ovalisporum, we have quantified the intra and extracellular CYN content, during nine days in BG11 batch cultures under optimal conditions. In parallel, the expression of four genes related to CYN synthesis, aoaA-C and cyrJ, has been analyzed by real time q-PCR. The results show a similar pattern of total CYN accumulation and gene expression. Most of the CYN is found intracellularly. Considering the high nitrogen content in the CYN molecule, we have explored if nitrogen assimilation could be related to CYN synthesis. We found inside the aoaA and aoaC sequences several putative binding domains for the global nitrogen regulator NtcA. The pattern of the ntcA expression along the culture is similar to that of CYN accumulation. Our data suggest that CYN production in A. ovalisporum seems to be controlled both by the expression of genesaoa and ntcA, this last one suggesting the influence of available nitrogen; however, other regulation mechanisms of CYN synthesis cannot be discarded.