Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C, raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Potential triggers for akinete differentiation in an Australian strain of the cyanobacterium Cylindrospermopsis raciborskii (AWT 205/1)

Understanding the triggers for some cyanobacteria of the Nostocales and Stigonematales orders to produce specialised reproductive cells termed akinetes, is very important to gain further insights into their ecology. By improving our understanding of their life cycle, appropriate management options may be devised to control the formation of these cells, and therefore the potential bloom inoculum which they are thought to provide, may be reduced. This study investigated the effect of chemical (phosphorus limitation), and environmental variables (temperature shock) on akinete differentiation in the freshwater cyanobacterium Cylindrospermopsis raciborskii (AWT 205/1). From the preliminary results, it is suggested that the availability of phosphorus and changes in temperature were a necessary requirement for the formation of akinetes in this particular strain of C. raciborskii. In the four phosphorus treatments investigated (0, 3, 38 and 75 mug l(-1) P), only the two higher treatments produced akinetes (approximately 220 ml(-1)). When the first akinetes were observed in the 38 and 75 mug l(-1) P treatments, filterable reactive phosphorus (FRP) concentrations in the medium were approximately 22 and 52 mug l(-1) P, respectively, indicating that there was no phosphorus limitation. In the temperature shock experiment, akinetes were observed in the 15 and 20degreesC treatments. However, akinetes were degraded (pale yellow colour, limited swelling and shrivelled edges) and in much lower concentrations, which was thought to be a result of the daily temperature shock. We suggest that the formation of akinetes in C. raciborskii (AWT 205/1) can be triggered by an initial temperature shock and that phosphorus is a necessary requirement to allow further growth and full development of akinetes.

First report of the cyanotoxins cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curvata (Cyantobacteria)

A strain of Raphidiopsis (Cyanobacteria) isolated from a fish pond in Wuhan, P. R. China was examined for its taxonomy and production of the alkaloidal hepatotoxins cylindrospermopsin (CYN) and deoxy-cylindrospermopsin (deoxy-CYN). Strain HB1 was identified as R. curvata Fritsch et Rich based on morphological examination of the laboratory culture. HB1 produced mainly deoxy-CYN at a concentration of 1.3 mg(.)g(-1) (dry ut cells) by HPLC and HPLC-MS/MS. CYN was also detected in trace amounts (0.56 mug(.)g(-1)). A mouse bioassay did not show lethal toxicity when tested at doses up to 1500 mg dry weight cells(.)kg(-1) body weight within 96 h, demonstrating that production of primarily deoxy CYN does not lead to significant mouse toxicity by strain BB I. The presence of deoxy-CYN and CYN in R curvata suggests that Raphidiopsis belongs to the Nostocaceae, but this requires confirmation by molecular systematic studies. Production of these cyanotoxins by Raphidiopsis adds another genus, in addition to Cylindrospemopsis, Aphanizomenon, and Umezakia, now known to produce this group of hepatotoxic cyanotoxins. This is also the first report from China of a CYN and deoxy-CYN producing cyanobacterium.

Toxicity and uptake mechanism of cylindrospermopsin and lophyrotomin in primary rat hepatocytes

The toxicities and uptake mechanisms of two hepatotoxins, namely cylindrospermopsin and lophyrotomin, were investigated on primary rat hepatocytes by using microcystin-LIZ (a well-known hepatotoxin produced by cyanobacteria) as a comparison. Isolated rat hepatocytes were incubated with different concentrations of hepatotoxins for 0, 24, 48 and 72 h. The cell viability was assayed by the tetrazolium-based (MTT) assay. Microcystin-LR, cylindrospermopsin and lophyrotomin all exhibited toxic effects on the primary rat hepatocytes with 72-h LC50 of 8, 40 and 560 ng/ml, respectively. The involvement of the bile acid transport system in the hepatotoxin-induced toxicities was tested in the presence of two bile acids, cholate and taurocholate. Results showed that the bile acid transport system was responsible for the uptake, and facilitated the subsequent toxicities of lophyrotomin on hepatocytes. This occurred to a much lesser extent with cylindrospermopsin. With its smaller molecular weight, passive diffusion might be one of the possible mechanisms for cylindrospermopsin uptake into hepatocytes. This was supported by incubating a permanent cell line, KB (devoid of bile acid transport system), with cylindrospermopsin which showed cytotoxic effects. No inhibition of protein phosphatase 2A by cylindrospermopsin or lophyrotomin was found. This indicated that other toxic mechanisms besides protein phosphatase inhibition were producing the toxicities of cylindrospermopsin and lophyrotomin, and that they were unlikely to be potential tumor promoters. (C) 2001 Elsevier Science Ltd. All rights reserved.

Managing the Brisbane River and Moreton Bay: an integrated research/management program to reduce impacts on an Australian estuary

The Brisbane River and Moreton Bay Study, an interdisciplinary study of Moreton Bay and its major tributaries, was initiated to address water quality issues which link sewage and diffuse loading with environmental degradation. Runoff and deposition of fine-grained sediments into Moreton Bay, followed by resuspension, have been linked with increased turbidity and significant loss of seagrass habitat. Sewage-derived nutrient enrichment, particularly nitrogen (N), has been linked to algal blooms by sewage plume maps. Blooms of a marine cyanobacterium, Lyngbya majuscula, in Moreton Bay have resulted in significant impacts on human health (e.g., contact dermatitis) and ecological health (e.g., seagrass loss), and the availability of dissolved iron from acid sulfate soil runoff has been hypothesised. The impacts of catchment activities resulting in runoff of sediments, nutrients and dissolved iron on the health of the Moreton Bay waterways are addressed. The Study, established by 6 local councils in association with two state departments in 1994, forms a regional component of a national and state program to achieve ecologically sustainable use of the waterways by protecting and enhancing their health, while maintaining economic and social development. The Study framework illustrates a unique integrated approach to water quality management whereby scientific research, community participation and the strategy development were done in parallel with each other. This collaborative effort resulted in a water quality management strategy which focuses on the integration of socioeconomic and ecological values of the waterways. This work has led to significant cost savings in infrastructure by providing a clear focus on initiatives towards achieving healthy waterways. The Study's Stage 2 initiatives form the basis for this paper.

Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. In this study, Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 0.2 mg/kg CYN. Animals were sacrificed at 6, 12, 24, 48 and 72 It. DNA was isolated from the mouse livers, and examined for strand breakage by alkaline gel electrophoresis (pH 12). Significant DNA strand breakage was observed in the mouse liver exposed to CYN, suggesting that induction of DNA strand breakage is probably one of the key mechanisms for CYN genotoxicity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Toxicological aspects of treatment to remove cyanobacterial toxins from drinking water determined using the heterozygous P53 transgenic mouse model

The presence of toxic cyanobacteria in drinking water reservoirs renders the need to develop treatment methods for the 'safe' removal of their associated toxins. Chlorine has been shown to successfully remove a range of cyanotoxins including microcystins, cylindrospermopsin and saxitoxins. Each cyanotoxin requires specific treatment parameters, particularly solution pH and free chlorine residual. However, currently there has not been any investigation into the toxicological effect of solutions treated for the removal of these cyanotoxins by chlorine. Using the P53(def) transgenic mouse model mate and female C57BL/6J hybrid mice were used to investigate potential cancer inducing effects from such oral dosing solutions. Both purified cyanotoxins and toxic cell-free extract cyanobacterial solutions were chlorinated and administered over 90 and 170 days (respectively) in drinking water. No increase in cancer was found in any treatment. The parent cyanotoxins, microcystins, cylindrospermopsin and saxitoxins were readily removed by chlorine. There was no significant increase in the disinfection byproducts trihalomethanes or haloacetic acids, levels found were well below guideline values. Histological examination identified no effect of treatment solutions except male mice treated with chlorinated cylindrospermopsin (as a cell free extract). In this instance 40% of males were found to have fatty vacuolation in their livers, cause unknown. It is recommended that further toxicology be undertaken on chlorinated cyanobacterial solutions, particularly for non-genotoxic carcinogenic compounds, for example the Tg. AC transgenic mouse model. (C) 2003 Elsevier Science Ltd. All rights reserved.