Cylindrospermopsis Raciborskii Strains Research Articles

Allelopathic activity of cyanobacteria on green microalgae at low cell densities

Allelopathic interactions are thought to play an important role in phytoplankton ecology. Many cyanobacterial genera have been shown to produce compounds with allelopathic activity; however, most studies have used high cyanobacterial cell densities, typical of bloom situations. We investigated whether low cell densities of cyanobacteria exhibit allelopathic activity. Twenty-two cyanobacterial strains were cultured in laboratory conditions, and the effects of their exudates were tested on the microalgae Ankistrodesmus falcatus and Chlorella vulgaris. We found that the exudates from one strain of Cylindrospermopsis raciborskii caused an enhancement of A. falcatus growth, followed by a marked decrease in growth rates, and that exudates from one strain of Oscillatoria sp. strongly inhibited C. vulgaris, as confirmed by dose–response assays. After the removal of the stressors, the microalgae grew at normal rates. The responsible compounds showed low sensitivity to heat and proteinase treatments. However, the majority of the strains did not exhibit appreciable allelopathic activity. Allelopathy may thus be a less frequent process in cyanobacteria at low cell-densities.

Isolated strains of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) produce anatoxin-a like neurotoxins

Isolated strains of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) produce anatoxin-a like neurotoxins

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium . C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.

A rapid microbiotest for the detection of cyanobacterial toxins

Cyanobacteria occur widely in lakes, reservoirs, ponds, and slow flowing rivers. Many species are known to produce toxins (cyanotoxins), a number of which are of concern for health. Cyanotoxins vary in chemical structure and may be found intracellular or released into water. There is not only a wide variation in the toxicity of known cyanotoxins but a substantial number of toxins have to date not been identified chemically. Chemical analysis of cyanotoxins is nowadays not used for routine monitoring because it is time consuming, it requires specialized equipment and expertise, and is hence expensive. There is hence an urgent need for rapid tests in surface waters to detect cyanobacterial toxins because of the need for safe drinking water and safe natural bathing waters, which may be burdened by cyanobacterial blooms or scums. Previous investigations have already shown that larvae of the anostracan crustacean Thamnocephalus platyurus are quite sensitive to neurotoxic and hepatotoxic cyanotoxins. The present paper reports on the sensitivity comparison of the (1 h) Rapidtoxkit (based on a sublethal endpoint) and the (24 h) Thamnotoxkit microbiotest (based on mortality). Both assays make use of larvae of T. platyurus. The Rapidtoxkit is a new microbiotest that determines the decrease of ingestion of colored particles by the crustacean larvae, which are stressed by a short exposure to toxicants. Fifteen cyanobacterial samples composed of laboratory strains and natural bloom samples were tested by both microbiotests. All samples were also analyzed concurrently by HPLC for microcystins and cylindrospermopsin. The correlation coefficient between the two microbiotests (r = 0.82) showed the very good correspondence between the sublethal and the lethal effects. No known toxins could be detected in some samples, although the latter were found highly toxic to the test organisms in both bioassays. These results point to the presence of unknown toxin(s) produced by some cyanobacteria such as e.g., the Cylindrospermopsis raciborskii strain isolated from Lake Balaton in Hungary. This comparative study clearly showed that the 1 h Rapidtoxkit is an attractive rapid alternative to the Thamnotoxkit microbiotest.

Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP). In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods. Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.

Toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii (Cyanophyceae) isolated from Tabocas reservoir in Caruaru, Brazil, including demonstration of a new saxitoxin analogue

Cyanobacteria can produce biotoxins that are significant hazards to humans. After the intoxication incident in 1996 at the city of Caruaru, Brazil, a phytoplankton-monitoring programme was established at its main water supply, the Tabocas reservoir. Data obtained during 1997 and 1998 revealed the dominant species at Tabocas to be Cylindrospermopsis raciborskii, which was responsible for a massive bloom observed in July–October 1998. Laboratory cultures of isolate ITEP-018 demonstrated highly toxic properties, mice inoculated with this strain exhibited the same symptoms as those of paralytic shellfish poisoning, with an acute lethal effect of 9.3 mouse units mg−1 of dry cells. Several saxitoxin analogues were identified in these cultures, specifically saxitoxin (3.3 mol% total toxin content), gonyautoxin 6 (6.4 mol%), decarbamoyl-saxitoxin (8.5 mol%), neosaxitoxin (17.1 mol%), and a new saxitoxin analogue, which proved to be the major product of the Tabocas strain, accounting for 64.6 mol% of the toxin present in the sample analysed. Additionally, decarbamoylneo-saxitoxin was detected by liquid chromatography-mass spectrometry. Cylindrospermopsis raciborskii strain ITEP-018 thus produces at least five saxitoxin analogues, including the most toxic ones as assessed by mouse bioassay.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 μg l −1.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction.

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 micro g l(-1).

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria)

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250mg dry weight cells/kg body weight within 24h and 125mg/kg at 72h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262nm. HPLC-MS/MS confirmed the presence of CYN (M+H 416) and deoxy-CYN (M+H 400). The CYN content in strain CY-Thai was estimated at 1.02mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii.

Hepatic and renal toxicity of the blue–green alga (cyanobacterium) Cylindrospermopsis raciborskii in male Swiss albino mice

When administered to mice, either orally or intraperitoneally, extracts of the cyanobacterium Cylindrospermopsis raciborskii strain AWT 205 induced dose-dependent liver and kidney damage. Liver damage was generally centrilobular, becoming more severe and generalized as the dose increased. Damaged hepatocytes were characterized by increased cellular vacuolation, intercellular spaces, and darker nuclear and cytoplasmic staining. Kidney damage was characterized by a reduction in the number of erythrocytes in the glomerulus and an increase in the space around the glomerulus, increased diameter of the tubule lumina, proximal tubule epithelial necrosis, and the presence of proteinaceous material in the distal tubules. Transmission electron microscopy of the same tissue revealed epithelial cell necrosis in the proximal tubules, suggesting the material accumulating in the distal tubules was in part cell debris from this necrosis. The nature, location, and time course of histological damage were similar for oral and intraperitoneal administration, with maximum damage being observed 2–3 days after treatment. The LD50 (24 h) for intraperitoneally administered Cylindrospermopsis preparations ranged from 50 to 110 mg dry weight of lysed cells per kilogram, whereas the LD50 (7 days) ranged between 20 and 65 mg/kg depending upon the batch examined. In contrast, oral administration of 1400 mg/kg, while inducing clear histological damage, was not fatal to any of the animals used in the study. The extent of comparative severity of damage to the liver and kidneys caused by different batches of Cylindrospermopsis of similar cylindrospermopsin content varied considerably, implying the presence of more than one toxin. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 143–150, 1999

Hepatic and renal toxicity of the blue-green alga (cyanobacterium)Cylindrospermopsis raciborskii in male Swiss albino mice

When administered to mice, either orally or intraperitoneally, extracts of the cyanobacterium Cylindrospermopsis raciborskii strain AWT 205 induced dose-dependent liver and kidney damage. Liver damage was generally centrilobular, becoming more severe and generalized as the dose increased. Damaged hepatocytes were characterized by increased cellular vacuolation, intercellular spaces, and darker nuclear and cytoplasmic staining. Kidney damage was characterized by a reduction in the number of erythrocytes in the glomerulus and an increase in the space around the glomerulus, increased diameter of the tubule lumina, proximal tubule epithelial necrosis, and the presence of proteinaceous material in the distal tubules. Transmission electron microscopy of the same tissue revealed epithelial cell necrosis in the proximal tubules, suggesting the material accumulating in the distal tubules was in part cell debris from this necrosis. The nature, location, and time course of histological damage were similar for oral and intraperitoneal administration, with maximum damage being observed 2–3 days after treatment. The LD50 (24 h) for intraperitoneally administered Cylindrospermopsis preparations ranged from 50 to 110 mg dry weight of lysed cells per kilogram, whereas the LD50 (7 days) ranged between 20 and 65 mg/kg depending upon the batch examined. In contrast, oral administration of 1400 mg/kg, while inducing clear histological damage, was not fatal to any of the animals used in the study. The extent of comparative severity of damage to the liver and kidneys caused by different batches of Cylindrospermopsis of similar cylindrospermopsin content varied considerably, implying the presence of more than one toxin. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 143–150, 1999

Toxicity associated with commonly occurring cyanobacteria in surface waters of the Murray-Darling Basin, Australia

Surveys of cyanobacterial blooms were made over four consecutive summer seasons (1990-93) in surface waters of the Murray-Darling Basin in south-eastern Australia to determine the incidence and geographic distribution of toxicity associated with a range of recognized taxa.<P. In all, 231 field samples and 143 cultured isolates, representing 13 genera, were tested for toxicity by intra-peritoneal mouse bioassay. Toxicity was recorded in 42% of all field samples and was expressed quantitatively on the basis of both dry weight and cell number. Anabaena was the most abundant genus in blooms occurring in riverine and wetland habitats, and Anabaena circinalis was prominent in all field samples that were neurotoxic. Neurotoxicity was not demonstrated in any other species of Anabaena, or in any other genus, in both field and cultured material. Assays for anatoxin-a were negative, and symptoms of neurotoxicity in mice were not consistent with those reported elsewhere for anatoxin-a(s). Hepatotoxic blooms occurred predominantly in standing waters and were invariably caused by Microcystis aeruginosa f. aeruginosa throughout the greater part of the Basin. Toxic blooms of Nodularia spumigena were recorded only in Lake Aiexandrina and Lake Albert at the mouth of the River Murray. Hepatotoxicity was also demonstrated in strains of Cylindrospermopsis raciborskii, which was previously recognized in Australia only as a subtropical cyanobacterium.