Isolation and Characterization of Cylindrospermopsis raciborskii Strains from Finished Drinking Water.

In the summer of 2015, an intense cyanobacterial bloom producing geosmin/2-methylisoborneol (MIB) occurred in the Roxo freshwater reservoir in Alentejo, Portugal. The drinking water supplied from the Roxo water treatment plant (WTP) exhibited an unpleasant odor/taste and a significant cyanobacteria density was detected in the finished water at the exit of the WTP. Cyanobacteria were not evaluated downstream of the WTP, namely, at the city reservoir. The aim of this work was to isolate and characterize viable cyanobacteria present in finished water (exit of the WTP and city reservoir) that withstand conventional water treatment. Treated water samples collected at both sites were inoculated in Z8 culture medium to provide the conditions for putative cyanobacterial growth. After 30 days, filamentous cyanobacteria were observed in cultures inoculated with samples from the exit point of the WTP. Viable trichomes were isolated and identified as Cylindrospermopsis raciborskii by morphometric and molecular analysis. None of the isolates were cylindrospermopsin/microcystin producers, as confirmed by ELISA and amplification of corresponding genes (PS/PKS and mcyA-cd/mcyAB/mcyB). ELISA results were positive for saxitoxin, but saxitoxin and derivatives were not detected by liquid chromatography with fluorescence detection (LC-FLD), nor were their related genes (sxtA/sxtA4/sxtB/sxtM/sxtPer/sxtI). To our knowledge, this is the first report on the establishment of cultures of C. raciborskii that resisted water treatment processes.

A report of Cylindrospermopsis raciborskii and other cyanobacteria in the water reservoirs of power plants in Ukraine.

The occurrence of cyanobacteria in freshwaters attracts much attention due to its associated health threats and ecological implications. Yet data on the composition of cyanobacteria taxa and toxigenicity in some regions is still scarce. Here, we explored the occurrence of cyanobacteria and cyanotoxins in three locations in Ukraine (reservoir for Kasperivtsi Hydrothermal Power Plant and outflowing River Seret, and cooling pond of Khmelnytsky Atomic Power Plant) in summer 2017. Cyanobacteria were a dominant fraction at all stations. A number of potent-toxin producers were identified including Cylindrospermopsis raciborskii, Aphanizomenon gracile, Dolichospermum flos-aquae, and Planktothrix agardhii. Screening for the presence of dissolved and particulate content of microcystins (-LR, -YR, and -RR), cylindrospermopsin, and anatoxin-a yielded negative results. The studied waters displayed no toxicity in human platelets in vitro. Further toxicological and ecological studies are necessary to evaluate the potential presence of cyanotoxin producers in Ukraine.

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.

Distribution of invasive Cylindrospermopsis raciborskii in the East-Central Europe is driven by climatic and local environmental variables.

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

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.

Impact of nitrogen sources on gene expression and toxin production in the diazotroph Cylindrospermopsis raciborskii CS-505 and non-diazotroph Raphidiopsis brookii D9.

Different environmental nitrogen sources play selective roles in the development of cyanobacterial blooms and noxious effects are often exacerbated when toxic cyanobacteria are dominant. Cylindrospermopsis raciborskii CS-505 (heterocystous, nitrogen fixing) and Raphidiopsis brookii D9 (non-N2 fixing) produce the nitrogenous toxins cylindrospermopsin (CYN) and paralytic shellfish toxins (PSTs), respectively. These toxin groups are biosynthesized constitutively by two independent putative gene clusters, whose flanking genes are target for nitrogen (N) regulation. It is not yet known how or if toxin biosynthetic genes are regulated, particularly by N-source dependency. Here we show that binding boxes for NtcA, the master regulator of N metabolism, are located within both gene clusters as potential regulators of toxin biosynthesis. Quantification of intra- and extracellular toxin content in cultures at early stages of growth under nitrate, ammonium, urea and N-free media showed that N-sources influence neither CYN nor PST production. However, CYN and PST profiles were altered under N-free medium resulting in a decrease in the predicted precursor toxins (doCYN and STX, respectively). Reduced STX amounts were also observed under growth in ammonium. Quantification of toxin biosynthesis and transport gene transcripts revealed a constitutive transcription under all tested N-sources. Our data support the hypothesis that PSTs and CYN are constitutive metabolites whose biosynthesis is correlated to cyanobacterial growth rather than directly to specific environmental conditions. Overall, the constant biosynthesis of toxins and expression of the putative toxin-biosynthesis genes supports the usage of qPCR probes in water quality monitoring of toxic cyanobacteria.

Cyanobacteria, microcystins and cylindrospermopsin in public drinking supply reservoirs of Brazil.

Brazil has a history of blooms and contamination of freshwater systems by cyanobacterial toxins. The monitoring relevance of toxins from cyanobacteria in reservoirs for public supply is notorious given its high toxicity to mammals, included humans beings. The most recurrent toxins in Brazilian water bodies are microcystins (MC). However, the recent record of cylindrospermopsin (CYN) in northeastern Brazil, Pernambuco state, alerts us to the possibility that this could be escalating. This study reports occurrence of MC and CYN, quantified with ELISA, in 10 reservoirs, devoted to public drinking supply in northeastern Brazil. The composition and quantification of the cyanobacteria community associated with these water bodies is also presented. From 23 samples investigated for the presence of MC, and CYN, 22 and 8 out were positive, respectively. Considering the similarity of the cyanobacteria communities found in reservoirs from Pernambuco, including toxin-producing species associated to MC and CYN, we suggest that geographic spreading can be favored by these factors. These issues emphasize the need for increased monitoring of MC and CYN in drinking supply reservoirs in Brazil.

Effects of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming behavior of Daphnia (cladocera).

The present study aimed to test the effects of raw water samples from a eutrophic reservoir and of a saxitoxin-producing strain of Cylindrospermopsis raciborskii on the swimming behavior of 2 key herbivore species of Daphnia. Two complementary approaches were used, acute bioassays and behavioral assays using an automated movement tracking system for measuring the following activity parameters: swimming time, resting time, distance traveled, and mean velocity. In both assays, animals were exposed to field samples or to toxic filaments in different concentrations and observed for 2 h to 3 h. In the acute bioassays, there was a decrease in the number of swimming individuals during the exposure period and a recovery following removal from toxic algae. A significant relationship was found between median effective concentration and the saxitoxin content of seston (r(2) = 0.998; p = 0.025) in the acute bioassays with raw water samples. Behavioral assays also showed significant effects in the activity parameters with both field samples and the strain of C. raciborskii, with some recovery during the exposure period. Both approaches corroborated previous research on the effects of neurotoxic C. raciborskii on the swimming activity of Daphnia, and these effects are compatible with the mechanism of action of saxitoxins. The present study showed that activity parameters of aquatic organisms may be a useful tool in the evaluation of sublethal toxicity and detection of neurotoxins in raw water.

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening.

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria. Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower. Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development. Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

Aphanizomenon gracile (Nostocales), a cylindrospermopsin-producing cyanobacterium in Polish lakes.

The cyanobacterial cytotoxin cylindrospermopsin (CYN) has become increasingly common in fresh waters worldwide. It was originally isolated from Cylindrospermopsis raciborskii in Australia; however, in European waters, its occurrence is associated with other cyanobacterial species belonging to the genera Aphanizomenon and Anabaena. Moreover, cylindrospermopsin-producing strains of widely distributed C. raciborskii have not yet been observed in European waters. The aims of this work were to assess the occurrence of CYN in lakes of western Poland and to identify the CYN producers. The ELISA tests, high-performance liquid chromatography (HPLC)-DAD, and HPLC-mass spectrometry (MS)/MS were conducted to assess the occurrence of CYN in 36 lakes. The cyrJ, cyrA, and pks genes were amplified to identify toxigenic genotypes of cyanobacteria that are capable of producing CYN. The toxicity and toxigenicity of the C. raciborskii and Aphanizomenon gracile strains isolated from the studied lakes were examined. Overall, CYN was detected in 13 lakes using HPLC-MS/MS, and its concentrations varied from trace levels to 3.0 µg L(-1). CYN was widely observed in lakes of western Poland during the whole summer under different environmental conditions. Mineral forms of nutrients and temperature were related to CYN production. The molecular studies confirmed the presence of toxigenic cyanobacterial populations in all of the samples where CYN was detected. The toxicity and toxigenicity analyses of isolated cyanobacteria strains revealed that A. gracile was the major producer of CYN.

Hydroxyl radical oxidation of cylindrospermopsin (cyanobacterial toxin) and its role in the photochemical transformation.

Cylindrospermopsin (CYN), an alkaloid guanidinium sulfated toxin, is produced by a number of cyanobacteria regularly found in lakes, rivers, and reservoirs. Steady-state and time-resolved radiolysis methods were used to determine reaction pathways and kinetic parameters for the reactions of hydroxyl radical with CYN. The absolute bimolecular reaction rate constant for the reaction of hydroxyl radical with CYN is (5.08 ± 0.16) × 10(9) M(-1) s(-1). Comparison of the overall reaction rate of CYN with hydroxyl radical with the individual reaction rate for addition to the uracil ring in CYN indicate the majority of the hydroxyl radicals (84%) react at the uracil functionality of CYN. Product analyses using liquid chromatography-mass spectrometry indicate the major products from the reaction of hydroxyl radical with CYN involve attack of hydroxyl radical at the uracil ring and hydrogen abstraction from the hydroxy-methine bridge linking the uracil ring to the tricyclic guanidine functionality. The role of hydroxyl radical initiated pathways in the natural organic matter (NOM) photosensitized transformation of CYN were evaluated. Scavenger and trapping experiments indicate that hydroxyl radical mediated transformations account for approximately ~70% of CYN destruction in surface waters under solar irradiation in the presence of NOM. The absence of solvent isotope effect indicates singlet oxygen does not play a significant role in the NOM sensitized transformation of CYN. The primary degradation pathways for HO• mediated and NOM photosensitized destruction of CYN involve destruction of the uracil ring. The fundamental kinetic parameters determined from these studies are critical for the accurate evaluation of hydroxyl-radical based technologies for the remediation of this problematic cyanotoxin in drinking water and important in the assessment of the environmental oxidative transformation of uracil based compounds.

A CARD-FISH protocol for the identification and enumeration of cyanobacterial akinetes in lake sediments.

Akinetes are the dormant cells of Nostocales (cyanobacteria) that enable the organisms to survive harsh environmental conditions while resting in bottom sediments. The germination of akinetes assists the dispersal and persistence of the species. The assessment of the akinete pool in lake sediments is essential to predict the bloom formation of the Nostocales population. We present here the implementation of an improved catalysed reporter deposition (CARD)-fluorescence in situ hybridization (FISH) protocol to assist the identification and quantification of akinetes in sediment samples. Several 16S rRNA gene oligonucleotide probes were evaluated for labelling akinetes of various species of Anabaena, Aphanizomenon and Cylindrospermopsis. Akinetes of all the taxa studied were successfully labelled and could be easily detected by their bright fluorescence signal. The probes' specificity was tested with 32 strains of different taxa. All six Cylindrospermopsis raciborskii strains were labelled with a specific probe for its 16S rRNA gene. A more general probe labelled 73% of the Anabaena and Aphanizomenon strains. The counting data of field samples obtained with CARD-FISH and the regular light microscopy approach did not differ significantly, confirming the suitability of both methods. The CARD-FISH approach was found to be less time-consuming because of better visibility of akinetes.

Preliminary molecular identification of cylindrospermopsin-producing Cyanobacteria in two Polish lakes (Central Europe).

The presence of toxigenic cyanobacteria capable of biosynthesis of cylindrospermopsin (CYN) was measured in 24 water samples collected from the lakes Bytynskie (BY) and Bninskie (BN) in the Western Poland. The study also covered analysis of toxigenicity and production of CYN by the culture of Cylindrospermopsis raciborskii isolated from BY. The cyrJ gene associated with CYN production was identified in 22 water samples collected in the summer seasons of 2006 and 2007. The presence of CYN was confirmed in 16 samples. The homology searches revealed that amplified sequences of four water samples, which were selected from among all the samples, displayed a strong 99% homology to cyrJ gene of Aphanizomenon sp. 10E6. The culture of C. raciborskii did not contain the cyrJ gene nor the CYN. The specificity of C. raciborskii was confirmed by application of a fragment of the rpoC1. These first genetic analyses have shown that Aphanizomenon seems to be the main cyanobacterial genus responsible for the production of CYN in the Polish lakes. The lack of toxigenicity of the isolated C. raciborskii suggests that it is possible that this invasive species does not demonstrate toxigenic activity in Polish water bodies.

The inhibitory effect of calcium on Cylindrospermopsis raciborskii (cyanobacteria) metabolism

Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca2+) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.

Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential.

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.

Genetic variability of the invasive cyanobacteria Cylindrospermopsis raciborskii from Bir M'cherga reservoir (Tunisia).

Cylindrospermopsis raciborskii is an invasive freshwater cyanobacteria of tropical origin, also found in temperate regions. Due to its known ability to produce potent toxins, such as cylindrospermopsin and the paralytic shellfish poisoning, this species is of major concern from a water quality perspective. This study presents a genetic characterization of four C. raciborskii strains isolated from the Bir M'cherga Tunisian reservoir. The toxicity assessment was investigated via molecular biology tools, which suggested that all the isolated strains were not producing cylindrospermopsin, saxitoxin, or microcystin. This result was further confirmed by HPLC and MALDI-TOF analyses. However, we report for the first time in C. raciborskii the presence of mcyA and mcyE, two segments of the microcystin synthetase mcy cluster. All the strains were identified taxonomically based on the 16S rRNA sequences, and their phylogenetic relationships were assessed using the rpoC1 region. Tunisian strains formed a distinct clade separated from the other African strains.

Genetic diversity and structure of the invasive toxic cyanobacterium Cylindrospermopsis raciborskii.

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.

Investigation of toxin content in Cylindrospermopsis raciborski (Woloszynska) Seenaya and Subba Raju and Aphanizomenon ovalisporum (Forti) strains isolated from shallow lakes of Hungary.

Cylindrospermopsin (CYN) is an alkaloid type cytotoxic metabolite produced by several cyanobacterial species, which caused human illnesses. The occurrence of CYN has been mostly associated with tropical and subtropical cyanobacteria, but recently it is appearing in several countries, all over the world. We analyzed CYN concentration and polyketide synthase/peptide synthetase (PKS /PS) genes, important parts of the gene cluster responsible for the CYN biosynthesis, in 14 isolated/collected Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum strains originated mostly from Hungary. CYN and PKS /PS genes were detected in Aphanizomenon ovalisporum strains isolated from Spain (of our isolation) and isolated in Israel (IL C-164), but the Hungarian isolate from the hyposaline Lake Szelidi had a lack of production capacity. In the Hungarian samples of C. raciborskii, we found no CYN and PKS /PS genes content comparing to CYN producer C. raciborskii AQS originated from Australia.

Toxic cyanobacteria in Florida waters.

The occurrence of toxic cyanobacterial blooms in Florida waters have become more prominent following increased growth, declining groundwater supplies, and identification of impaired surface waters as future drinking water sources. Cyanobacterial toxins have been identified in source waters used for drinking water supply and in post-treated drinking water during algal bloom events. Algal toxin concentrations in post-treated drinking water have exceeded existing and proposed World Health Organization guidelines for the oral consumption of microcystin and cylindrospermopsin. Severe dermatitis has also been reported by swimmers in Florida springs where Lyngbya mats have expanded. The prevalence and toxicity of cyanobacteria should be considered when developing appropriate Total Maximum Daily Loads for impaired Florida waters that do not currently meet their designated use. It could also support further efforts to characterize potential ecological and human health risks due to toxic cyanobacterial blooms. Identification of algal toxins in finished drinking water and reports of severe skin irritation following contact with toxic cyanobacteria should be utilized for justification and implementation of increased monitoring of potentially toxic cyanobacterial blooms by surface water managers and water utilities. Epidemiological studies may also be required in Florida to assess potential human health risks due to algal toxin consumption at the tap and for those exposed to cyanotoxic blooms during recreational use of lakes, springs and rivers. Without adequate water treatment and coordinated state-wide monitoring efforts, it is anticipated that the likelihood for human exposure to cyanobacteria and their toxins will increase as Florida becomes more dependent upon surface waters to supply a growing population and an expanding urban environment. Coordination and communication between surface water managers and public health officials at the local level will be critical to the overall protection of the environment and public health during toxic cyanobacterial bloom events.

First report of toxic Cylindrospermopsis raciborskii and Raphidiopsis mediterranea (Cyanoprokaryota) in Egyptian fresh waters.

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