Raciborskii Cells Research Articles

Differential protein expression in two bivalve species; Mytilus galloprovincialis and Corbicula fluminea; exposed to Cylindrospermopsis raciborskii cells

The cyanobacteria Cylindrospermopsis raciborskii is considered a threat to aquatic organisms due to the production of the toxin cylindrospermopsin (CYN). Despite the numerous reports evidencing the toxic effects of C. raciborskii cells and CYN in different species, not much is known regarding the toxicity mechanisms associated with this toxin and the cyanobacteria. In this work, a proteomics approach based in the two-dimensional gel electrophoresis and mass spectrometry was used to study the effects of the exposure of two bivalve species, Mytilus galloprovincialis and Corbicula fluminea, to CYN producing (CYN+) and non-producing (CYN−) C. raciborskii cells. Additionally the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) were determined. Alterations in actin and tubulin isoforms were detected in gills of both bivalve species and digestive gland of M. galloprovincialis when exposed to CYN− and CYN+ cells. Moreover, GST and GPx activities changed in gills and digestive tract of bivalves exposed to both C. raciborskii freeze dried cells, in comparison to control animals exposed to the green alga Chlorella vulgaris. These results suggest the induction of physiological stress and tissue injury in bivalves by C. raciborskii. This condition is supported by the changes observed in GPx and GST activities which indicate alterations in the oxidative stress defense mechanisms. The results also evidence the capacity of CYN non-producing C. raciborskii to induce biochemical responses and therefore its toxicity potential to bivalves. The heat shock protein 60 (HSP60), extrapallial (EP) fluid protein and triosephosphate isomerase homologous proteins from gills of M. galloprovincialis were down-regulated specifically with the presence of CYN+ C. raciborskii cells. The presence of CYN may lead to additional toxic effects in M. galloprovincialis. This work demonstrates that proteomics is a powerful approach to characterize the biochemical effects of C. raciborskii and to investigate the physiological condition of the exposed organisms.

Evaluation of quantitative real-time PCR to characterise spatial and temporal variations in cyanobacteria, Cylindrospermopsis raciborskii (Woloszynska) Seenaya et Subba Raju and cylindrospermopsin concentrations in three subtropical Australian reservoirs

Three drinking water storage reservoirs in subtropical southeast Queensland, Australia have regular blooms of the toxic cyanobacterium Cylindrospermopsis raciborskii that can produce cylindrospermopsins. We tested water samples from 16 sites in 3 reservoirs on 2 sampling occasions during a bloom of C. raciborskii in the austral summer and autumn of 2007. Using a range of parameters including quantitative real-time PCR, microscope cell counts and HPLC–MS/MS we correlated the 16S ribosomal RNA gene with total cyanobacteria, the rpoC1 gene with C. raciborskii cell concentrations, and the cyrC gene with cylindrospermopsin concentrations to assess spatial and temporal variability within and between reservoirs. While the correlation between cyrC and cylindrospermopsin cell quotas was good (mean r 2 = 0.61 for February samples and 0.75 for March samples), the correlation between total cyanobacteria and the 16S ribosomal RNA gene, and between C. raciborskii and the rpoC1 gene were poor indicating that further work is needed to develop these novel molecular methods. Spatial and temporal analysis of the distribution of rpoC1, cylindrospermopsin cell quotas, and a range of physical and chemical water quality parameters showed the greatest variation occurred between reservoirs, and within the largest and most spatially diverse reservoir. This suggests that populations of C. raciborskii strains with inherently different cylindrospermopsin cell quotas may be an important driver of toxicity in these reservoirs. An outcome of this study was the observation that deoxycylindrospermopsin always exceeded the cylindrospermopsin cell quota by up to 5-fold, and that a peak cell quota of 60 fg (cylindrospermopsin + deoxycylindrospermopsin) cell −1 was measured.

Lake viruses lyse cyanobacteria, Cylindrospermopsis raciborskii, enhances filamentous-host dispersal in Australia

Abstract Globally, cyanobacterial blooms are increasing along with observations of the controlling influence of viruses. Our aim here was to test whether viruses from an Australian freshwater lake could lyse the cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju. C. raciborskii was selectively isolated from Lake Samsonvale southeast Queensland Australia using a Modified Jaworski Medium (without any form of inorganic nitrogen). Microscopy confirmed the resulting culture of a single cyanobacterial species. Natural viral-like particles (VLPs) were incubated with C. raciborskii cells, the host abundance decreased by 86% in 5 days, while the number of VLPs increased stepwise. As a cell lysed, the filaments of cells split into smaller, but viable, fragments. This process may help disperse the cyanobacterium in the wild. Hence the use of this virus to control blooms may inadvertently encourage the dispersal of toxic filamentous cyanobacteria. The cyanophage (virus infecting cyanobacteria) replication time was 21 h, with an average burst size of 64 viruses cell−1. Transmission Electron Microscopy showed this cyanophage for C. raciborskii, with its long, non-contractile tail and a capsid diameter of 70 nm, belongs to the Siphoviridae family of viruses. This cyanophage can affect the abundance and distribution of the cyanobacterium C. raciborskii in this Australian freshwater lake.

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml(-1) for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml(-1) and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.

Variable Growth Responses of Water Thyme (Hydrilla verticillata) to Whole-Cell Extracts of Cylindrospermopsis raciborskii

Static-renewal ecotoxicity trials monitored growth of Hydrilla verticillata in conjunction with exposure to Cylindrospermopsis raciborskii whole-cell extracts containing the cyanotoxin cylindrospermopsin (CYN). Maximum exposure concentrations were 400 microg L(-1) CYN over 14 days. The responses of Hydrilla to the treatments were variable according to the toxin concentrations and lengths of exposure. Plant deaths, chlorosis, and necrosis were not recorded from treated plants. However, Hydrilla experienced significant growth stimulation and redistribution of plant resources in conjunction with exposure to the whole-cell extracts. Root production was particularly impacted. The results of this study imply that root production could aid in reducing C. raciborskii cell concentrations and CYN toxicity. Results of chlorophyll analyses differed, indicating that CYN in whole-cell extracts might exert complex effects on photosynthesis. This is the first study to describe the responses of an aquatic macrophyte following exposure to C. raciborskii whole-cell extracts containing the cyanotoxin cylindrospermopsin.

Mortality and toxin bioaccumulation in Bufo marinus following exposure to Cylindrospermopsis raciborskii cell extracts and live cultures

Cylindrospermopsis raciborskii is a cyanobacterium responsible for the production of the toxin, cylindrospermopsin (CYN). Tadpoles of the cane toad Bufo marinus were exposed to freeze-thawed whole cell extracts or live cultures of C. raciborskii containing maximum CYN concentrations of 400 μg L −1 or 232 μg L −1, respectively. Exposure to live culture treatment solutions resulted in up to 66% mortality of B. marinus, whereas tadpoles exposed to whole cell extracts containing similar toxin concentrations survived. Decreases in relative growth rates and time spent for swimming were recorded from tadpoles during both types of exposure regimes. Bioconcentration of CYN was not evident following exposure to whole cell extracts containing extracellular toxin. In contrast exposure to live cultures, which contained cell-bound toxin, resulted in maximum average tissue concentrations of 895 μg free-CYN kg −1 fresh weight. This is the first investigation of C. raciborskii exposure effects and toxin bioaccumulation in the developmental stages of an amphibian.

Phenotypical variation in a toxic strain of the phytoplankter, Cylindrospermopsis raciborskii (nostocales, cyanophyceae) during batch culture

A nonaxenic strain of Cylindrospermopsis raciborskii Woloszynska (AWT 205) was grown in batch culture, with and without nitrate as the primary N source. Rapid log-phase growth with nitrate was 1.0 doubling/day versus 0.3 doubling/day without nitrate. Cylindrospermopsin (CYN) production was measured by HPLC. The rate of intracellular CYN production matched cell division rate for both the diazotrophies at cell densities less than 10(7) cell/ml. At cell density > 10(7) cell/ml, additional resource limitation in batch culture slowed log-phase growth to 0.04 division/day and cell division and CYN production decoupled. Intracellular CYN concentration increased at a rate of 0.08 doubling/day, twice the cell division rate. Extracellular CYN as a proportion of the total CYN increased from 20% during the rapid growth phase, to 50% during the slow growth phase. The total CYN yield from cultures grown out to stationary phase (55 days) exceeded 2 mg CYN/I. C. raciborskii cells in log-phase growth, exposed to 1 ppm copper (as copper sulphate), lysed within 24 hours. After copper treatment, all CYN was in the filterable fraction. These findings imply that in naturally occurring blooms of C. raciborskii, the movement of intracellular CYN into solution will be the greatest during stationary phase, when intracellular concentrations are highest and cell lysis is more frequent. The application of algicides that promote cell lysis will exacerbate this effect.

The tolerance of cyanobacterium Cylindrospermopsis raciborskii to low-temperature photo-inhibition affected by the induction of polyunsaturated fatty acid synthesis

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation patterns of the glycerolipids of Cylindrospermopsis raciborskii, a filamentous cyanobacterium, were determined in cells grown at 35 degrees C and 25 degrees C. The lowering of the growth temperature from 35 degrees C to 25 degrees C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. The tolerance to low-temperature photo-inhibition of the C. raciborskii cells grown at 25 degrees C and 35 degrees C was also compared. The lower growth temperature increased the tolerance of C. raciborskii cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii in algal blooming in Lake Balaton (Hungary).