Gary J. Jones

Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recreational lake, as determined by HPLC and protein phosphatase inhibition assay

Abstract Algicide treatment of an hepatotoxic Microcystis aeruginosa Kuetzing emend. Elenkin bloom on Lake Centenary caused cell lysis and the release of microcystin into the surrounding water. Where M. aeruginosa was confined to accumulations along the leeward shore of the lake, dissolved microcystin was detected for only 24 hours after spraying. It was presumed that the toxin was rapidly diluted by uncontaminated water from the main body of the lake. At an enclosed site in the south-west corner of the lake, microcystin persisted at high levels (1300–1800 μg l−1) for 9 days before degradation commenced. Microcystin degradation kinetics following this 9 day lag phase were bi-phasic with a rapid phase lasting 3 days (90–95% loss), and a slower phase which continued until a flash flood on day 21. HPLC analysis and protein phosphatase assay revealed the same overall trend of microcystin release, persistence and then degradation. However, there was a rapid increase in protein phosphatase inhibition from day 5 to day 9 before degradation commenced. These results suggest that the initial bacterial transformation of microcystin resulted in a product more inhibitory to protein phosphatase than the parent toxin.

Isolation and toxicity of Cylindrospermopsis raciborskii from an ornamental lake

In Australia, the tropical/subtropical cyanobacterium Cylindrospermopsis raciborskii forms substantial blooms in some drinking water supply reservoirs, rivers and recreational water bodies during the warmer months of the year. This paper describes the isolation, culture and toxicity characterisation of Cylindrospermopsis from a water bloom in a small lake in NSW, Australia. The cyanobacterium grew as straight trichomes terminating with a characteristic heterocyst. The toxic alkaloid cylindrospermopsin was separated and identified by high-performance liquid chromatography at a concentration of 5.5 mg/g dry cells, 0.026 pg/cell. Intraperitoneal injection of sonicated cells caused liver, kidney, intestinal and lung damage, with an LD50 of 52 mg cells/kg mouse body weight at 24 hr, and 32 mg/kg at 7 days. The 24 hr mouse toxicity is not consistent with previous studies using pure cylindrospermopsin, and is suggestive of other toxic compounds in this isolate.

Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate.

ABSTRACT Cell quotas of microcystin (QMCYST; femtomoles of MCYST per cell), protein, and chlorophyll a(Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship betweenQMCYST and specific growth rate (μ), from which we propose a generalized model that enablesQMCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts QMCYST from μ, μmax(maximum specific growth rate), QMCYSTmax(maximum cell quota), and QMCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict aQMCYSTmax of 0.129 fmol cell−1 at μmax and a QMCYSTmin of 0.050 fmol cell−1 at μ = 0. Net MCYST production rate (RMCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell−1 day−1at μmax. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chla ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.

Bioaccumulation of paralytic shellfish poisoning (PSP) toxins from the cyanobacterium Anabaena circinalis by the freshwater mussel Alathyria condola

The Australian freshwater mussel Alathyria condola accumulated high levels of paralytic shellfish poisoning (PSP) toxins when fed the neurotoxic cyanobacterium Anabaena circinalis, shown recently to contain high concentrations of C-toxins and gonyautoxins. Significant accumulation (>80 mu g/100 g of mussel flesh) was detected following 2-3 exposure to water containing 2 x 105 cells/ml A. circinalis. Only trace accumulation of PSP toxins was demonstrated over long-term (5 week) exposure at low concentration (c. 104 cells/ml). The relative abundance of C-toxins, gonyautoxins and saxitoxins in mussels generally matched the toxin profiles of the dietary A. circinalis, although there were differences in the GTX2/3 and C1/2 ratios with time, and an increase in abundance of decarbamoylgonyautoxins. Analysis of mussel tissues after 7 days, exposure to A. circinalis revealed that 96% of the toxins were accumulated in the viscera. As in marine waters, the bioaccumulation of PSP toxins in freshwater mussels may pose a health risk to humans and animals, especially in areas where seasonally decreasing water levels expose mussel beds to surface scums of toxic cyanobacteria.

Sheep mortality associated with paralytic shellfish poisons from the cyanobacterium Anabaena circinalis

This is the first report of sheep mortalities associated with paralytic shellfish poisons (PSPs) from the cyanobacterium Anabaena circinalis Rabenhorst. Fourteen sheep died within 150 m of a farm dam containing a dense bloom of A. circinalis. Extracts from both the cyanobacterium and small intestine from a dead ewe were analysed by high-performance liquid chromtography (HPLC) and found to contain PSPs. The toxin profiles of the cyanobacterium contained a high proportion of C-toxins (70%), whereas toxins in the small intestine content were dominated by gonyautoxin 5 (87%). This observation could be explained by desulfation of the C-toxins in the gut of the sheep. The LD100 of bloom material calculated from HPLC data was consistent with mouse bioassay data (12-25 mg/kg). The symptoms of affected sheep, mouse bioassay data, coupled with HPLC analysis of toxins from the bloom samples and the intestine contents, and the absence of other cyanobacterial alkaloid toxins, indicate that PSPs were responsible for the deaths of the sheep.

GENETIC, MORPHOLOGICAL, AND TOXICOLOGICAL VARIATION AMONG GLOBALLY DISTRIBUTED STRAINS OF NODULARIA (CYANOBACTERIA)

Morphological, toxicological, and genetic variation was examined among 19 strains of Nodularia. The strains examined could be morphologically discriminated into four groups corresponding to N. spumigena Mertens, N. sphaerocarpa Bornet et Flahault, and two strains that did not clearly correspond to currently accepted Nodularia species. Genetic variation was examined using nucleotide sequencing of the phycocyanin intergenic spacer region (cpcBA‐IGS) and RAPD‐PCR. The PCR‐RFLP of the cpcBA‐IGS differentiated four genotypes corresponding to the four morphological groups. However, nucleotide sequencing of 598 bp of the 690‐bp fragment showed that one of the three strains corresponding to N. sphaerocarpa (PCC 7804) was genetically divergent from the other two, suggesting that it constitutes a distinct species. Nucleotide variation within the morphospecies groups was limited (<1%), and all 14 Australian strains of N. spumigena possessed identical cpcBA‐IGS sequences. The RAPD‐PCR differentiated the same groups as the cpcBA sequencing and discriminated each of the seven different Australian populations of N. spumigena. Strains from within a bloom appeared genetically identical; however, strains isolated from different blooms could be separated into either a western or a southeastern Australian cluster, with one strain from western Australia showing considerable genetic divergence. The pattern of variation suggests that individual blooms of N. spumigena are clonal but also that Australian N. spumigena populations are genetically distinct from each other. Examination of genetic distance within and between blooms and within and between morphological groups showed clear genetic dicontinuities that, in combination with the cpcBA‐IGS data, suggest that Nodularia contains genetically distinct morphospecies rather than a continuous cline of genetic variation. Furthermore, these morphospecies are genetically variable, exhibiting hierarchical patterns of genetic variation on regional and global scales. Production of the hepatotoxin nodularin was not restricted to one genetic lineage but was distributed across three of the five genotypic groups. A strain of N. spumigena from a nontoxic Australian population was found to fall within the range of genetic variation for other toxic Australian strains and appears to be a unique nontoxic strain that might have arisen by loss of toxin production capacity.

EFFECT OF CULTURE AND BLOOM DEVELOPMENT AND OF SAMPLE STORAGE ON PARALYTIC SHELLFISH POISONS IN THE CYANOBACTERIUM ANABAENA CIRCINALIS

Paralytic shellfish poisons (PSPs) were detected in 24 of 31 bloom samples dominated by the cyanobacterium Anabaena circinalis Rabenhorst, collected from across Austraia. The ability to produce PSPs has been maintained in everal non‐axenic strains of A. circinalis kept in culture, whereas strains that were non‐toxin‐producing when isolated have remained as such. PSPs were detected and quantified by high‐performance liquid chromatography (HPLC), and the structures were confirmed by electrospray mass spectroscopy. The concentration of toxins in PSP positive samples ranged from 50 to 3400 μg.g‐1 dry weight. Toxin profiles were always dominated by the N‐sulfocarbamoyl‐11‐hydroxysulfate C toxins, C1 and C2 (44–85 mol%), with the remainder consisting of gonyautoxins‐2, −3, and −5, decarbamoylgonyautoxins‐2 and −3, saxitoxin, and decarbamoylraxitoxin. N1‐‐hydroxy PSPs, commonly found in marine dinoflagellates, were absent, suggesting that A. circinalis lacks the enzyme responsible for N1‐‐hydroxylation. On a dry weight basis, the amount of toxin in cultured Anabaena circinalis (strain ACMB06)rose significantly (P < 0.05)over time from 570 to 3400 μg.g.‐1 cells in late stationary phase. However, there was no significant trend in cellular toxin quota (toxin per cell) over the life of the culture; this may be explained by variation in cell mass. On average, batch cultures of Anabaena circinalis contained 19% extracellular toxin, which increased slightly over the growth cycle and had a composition similar to that of the intracellular toxins. As cultures aged, the formation of decarbamoyl toxins and increases in theα‐/β‐epimer ratios of C toxins and gonyautoxins were observed. The variation in these components during stationary phase in culture was sufficient to explain the variation in relative PSP composition observed among natural bloom samples. Because decarbamoylgonyautoxins are much more toxic than C toxins on a molar basis, these transformations also lead to an increase in toxicity of the sample or bloom over time. The transformations of PSPs, which occur during aging and sample storage, render the comparison of PSPs by HPLC unreliable for phenotyping Anabaena circinalis, unless strains are cultured, harvested, and analyzed under standard conditions.

Persistence and degradation of cyanobacterial paralytic shellfish poisons (PSPs) in freshwaters

Paralytic shellfish poisons (PSPs) extracted from the cyanobacterium Anabaena circinalis persisted for over 90 days when incubated in freshwater. The primary transformation reaction was desulfocarbamoylation of the predominant, low toxicity C-toxins, to the more potent decarbamoylgonyautoxins (dc-GTXs). This transformation caused an initial increase in sample toxicity, in spite of an overall decrease in total toxin concentration (on a molar basis) with time. Incubation of PSPs in irrigation drainage water resulted in a six-fold increase in toxicity after 10 days, with the solution remaining more toxic than the original solution for over 60 days. The dc-GTXs thus formed, and the gonyautoxins (GTXs) present in the original sample, degraded slowly for the duration of the 90 day incubations. Similar results were recorded when PSPs were incubated with sterile deionised water, and with sterile deionised, protein precipitated water, but the build-up of dc-GTXs proceeded at a slower rate than with the drainage water. The other reaction noted to be significant was the slow epimerisation of the less stable β-epimers (C2, GTX3) to α-epimers (C1, GTX2). The degradation of C-toxins, gonyautoxins (GTXs) and dc-GTXs could be modelled by simple first-order loss kinetics. When PSPs were incubated at 90°C, the same loss processes and reaction order was observed. The failure of protein precipitation and high temperature to alter the reaction processes, indicates that desulfocarbamoylation of C-toxins, and degradation off GTXs and dc-GTXs is chemically, and not enzymatically, mediated. Upon the collapse of PSP producing A. circinalis blooms, and the subsequent release of toxins into the water column, contaminated water may become more toxic in the short term before the eventual degradation of PSPs to non-toxic products. Unless significant dilution occurs, PSP contaminated waters may contain toxins for more than 3 months.

Response of cultured Microcystis aeruginosa from the Swan River, Australia, to elevated salt concentration and consequences for bloom and toxin management in estuaries

A mixed bloom of Microcystis aeruginosa forma aeruginosa and forma flos-aquae from the Swan River, Western Australia, was confirmed toxic by HPLC analysis. At least four, and possibly 11, microcystins were detected in cell-free extracts. Live bloom material was cultured at salt concentrations up to 21.2 g L–1 (total salts). The cultures were salt tolerant up to 9.8 g L–1. Reduction in the total cell concentration in the first 23 h was only observed in the highest salt treatment and first-order rate constants for cell lysis were higher than the rates for reduction of the intracellular microcystin pool size for that treatment. This suggests preferential lysis of genotypes with lower salinity tolerance and toxigenicity. This increased the toxicity of the mixed bloom population and the apparent microcystin cell quota without any change to the intracellular microcystin pool size. Therefore, the toxicity of bloom material may change through preferential lysis of cells with lower tolerances to changing environmental conditions, including salinity. Managers should be aware that the World Health Organization alert levels of 105 cells mL–1 for human contact exposure to cyanobacteria may not be a suitable prima facie test during these periods.

Effect of irradiance on fatty acid, carotenoid, total protein composition and growth of Microcystis aeruginosa

Abstract The cyanobacterium, Microcystis aeruginosa , was grown under low, standard and high irradiance intensities (20, 40 and 70 μmol m −1 s −1 ) to determine whether irradiance affected pigment, lipid and protein composition, growth yields and the total dry weights of the cell cultures. The components detected in the saponified lipid extracts included C 14 to C 20 fatty acids with various levels of saturation, odd chain-length fatty acids and long-chain alcohols. 16:0 was the main fatty acid detected in all samples and its percentage abundance was significantly higher in cells from the lower and standard irradiance intensities compared with the high irradiance cells. Conversely, the proportions of the 14:0, 15:0, 17:0, 18:0 and 20:0 fatty acids were higher in the high irradiance cells compared with the low and standard irradiance cells. Polyunsaturated fatty acid concentrations were reduced and those of monounsaturated fatty acids were increased in the high irradiance cells compared with the low and standard irradiance cells. Chlorophyll a , zeaxanthin and β-carotene were the most abundant pigments detected. Cells exposed to the standard irradiance treatment had substantially higher amounts of carotenoid, chlorophyll a and total protein after 15 days of growth compared with cells exposed to either the low or high irradiance intensities. The ratios of the zeaxanthin, echinenone and β-carotene with respect to chlorophyll a from the high irradiance cells were approximately double those observed in the low and standard irradiance regimes. It was concluded that the changes in the fatty acid composition occurred as a cellular response to reduce the susceptibility of the cyanobacterial membranes to photo-oxidation.