Effects of hydrology and river management on the distribution,abundance and persistence of cyanobacterial blooms in the Murray River,Australia

Major cyanobacterial blooms (biovolume > 4 mm³ L⁻¹) occurred in the main water reservoirs on the upper Murray River, Australia during February and March 2010. Cyanobacterial-infested water was released and contaminated rivers downstream. River flow velocities were sufficiently high that in-stream bloom development was unlikely. The location has a temperate climate but experienced drought in 2010, causing river flows that were well below the long-term median values. This coupled with very low bed gradients meant turbulence was insufficient to destroy the cyanobacteria in-stream. Blooms in the upper 500 km of the Murray and Edward Rivers persisted for 5 weeks, but in the mid and lower Murray blooms were confined to a small package of water that moved progressively downstream for another 650 km. Anabaena circinalis was the dominant species present, confirmed by 16S rRNA gene sequencing, but other potentially toxic species were also present in smaller amounts. Saxitoxin (sxtA), microcystin (mcyE) and cylindrospermopsin (aoaA) biosynthesis genes were also detected, although water sample analysis rarely detected these toxins. River water temperature and nutrient concentrations were optimal for bloom survival. The operational design of weirs and retention times within weir pools, as well as tributary inflows to and diversions from the Murray River all influenced the distribution and persistence of the blooms. Similar flow, water quality and river regulation factors were underlying causes of another bloom in these rivers in 2009. Global climate change is likely to promote future blooms in this and other lowland rivers.     

Evidence of the existence of a toxic form of Cylindrospermopsis raciborskii (Nostocales,Cyanobacteria) in Japan

Cylindrospermopsis raciborskii is a common, bloom‐forming, planktonic, freshwater cyanobacterium. Toxic populations producing cylindrospermopsin can cause water‐safety problems. Although C. raciborskii is distributed worldwide, the presence of cylindrospermopsin‐producing strains of C. raciborskii was initially reported only in Australia and recently in Thailand. Here, we report the isolation of a toxic strain of C. raciborskii (ISG9) from a freshwater sample collected in Okinawa in 2008. This is the first report describing toxin expression in this species in Japan, detected from a subtropical area. The C. raciborskii species is known to produce cylindrospermopsin as a dominant toxin; however, in this new isolate, the dominant toxin expressed was deoxy‐cylindrospermopsin. The discovery of a toxic strain of C. raciborskii in southern Japan emphasizes the need for basic monitoring schemes for this species in water supplies located in the temperate regions of Japan because of its possible expansion and distribution to other geographic areas.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.     

FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.     

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the beta and alpha subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin.

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cylindrospermopsin is not degraded by co-occurring natural bacterial communities during a 40-day study

Biodegradation of cylindrospermopsin produced by Aphanizomenon ovalisporum UAM 290 was studied. In the 40-day degradation experiment conducted, bacterial communities from two waterbodies with and without previous exposure to the toxin were used. Further, and in order to study the potential effect of other organic substrates on the degradation of cylindrospermopsin, three different sources of cylindrospermopsin were used: toxic extracts obtained by methanolic extraction and by ultrasonication in water with 5% formic acid and 0.9% NaCl and toxin naturally present in the spent media of an Aphanizomenon ovalisporum culture. Despite active growth of the bacterial population and consumption of DOC in presence of the toxic extracts, no degradation of cylindrospermopsin could be observed during the 40-day period. Considering that cylindrospermopsin is abundant in the extracellular fraction and that photodegradation in the field seems to be limited, a lack of efficient biodegradation as observed in our study could be of greatest importance and further explain the accumulation of this toxin in the dissolved fraction of the waterbodies investigated.     

Seasonal dynamics and toxicity of Cylindrospermopsis raciborskii in Lake Guiers (Senegal, West Africa)

Cylindrospermopsis raciborskii is a toxic bloom-forming cyanobacterium that occurs at tropical and temperate latitudes. Despite several reports from Africa, no data were previously available about its dynamics or toxic potential there. We therefore carried out a 1-year survey of the dynamics of C. raciborskii in the main water reservoir in Senegal, Lake Guiers. Cylindrospermopsis raciborskii never formed a bloom in this lake during the period studied, but was dominant during the dry season. The only observed bloom-forming species was a diatom, Fragilaria sp., which displayed a seasonal pattern contrary to that exhibited by C. raciborskii. Principal component analysis applied to environmental and phytoplankton data showed that high C. raciborskii biomasses were mainly related to high temperature and water column stability. Tests for C. raciborskii species-related toxicity and/or toxin synthesis were performed on 21 isolated clones. All the strains isolated tested negative in mouse toxicity bioassays, toxin analysis (MS/MS) and tests for known cylindrospermopsin genes (ps, pks). The limited number of isolates studied, and the occurrence of toxic and nontoxic clones in natural cyanobacterial populations, mean that we cannot conclude that there is no C. raciborskii-associated health risk in this drinking water reservoir.     

Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120.

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.     

Development of Anabaena blooms in a small reservoir with dense sediment akinete population, with special reference to temperature and irradiance

The development of Anabaena ucrainica blooms in a small agriculturalreservoir was monitored in 1998 and 1999. In the reservoir,numerous Anabaena akinetes were found in all regions of thesediment analyzed, with an average cell density in the uppermostlayer (0–2 cm) of 1.5 x 10⁴ cm^-3. Anabaena ucrainica filamentnumbers began to increase exponentially in mid-May 1998 andin late April 1999, when the water temperature exceeded 15°C.The average in situ net growth rate was 0.18 day^-1 as measuredby filament numbers. The effect of temperature on germinationof the akinetes was investigated using Anabaena akinetes takenfrom the reservoir sediment. High germination percentages wereobserved at temperatures between 14 and 23°C; however, theAnabaena akinetes did not germinate without irradiance. Growthexperiments using an axenic culture of A. ucrainica isolatedfrom the reservoir showed that an increase in incubation temperatureto 26°C resulted in a rise in the specific growth rate.Consequently, it was hypothesized that temperature increasescould similarly enhance the growth rate of A. ucrainica duringbloom development. Furthermore, judging from the in situ growthrate of A. ucrainica, initial inocula arising from dense akinetepopulations in the sediment would advance bloom formation andcould enhance the relative probability of Anabaena bloom formation.     

Occurrence of anatoxin-a(s) during a bloom of <Emphasis Type="Italic">Anabaena crassa</Emphasis> in a water-supply reservoir in southern Brazil

Cyanobacterial blooms and the accompanying production of cyanotoxins are a serious global problem. Toxic blooms of Anabaena species are common in lagoons and reservoirs of southern Brazil. Worldwide, species of the genus Anabaena produce the majority of the known hepatotoxins (microcystins) and neurotoxins [anatoxin-a, anatoxin-a(s), and saxitoxins]. This report links a bloom of Anabaena crassa in the Faxinal Reservoir, the main water supply for the city of Caxias do Sul (400,000 inhabitants) in southern Brazil, to the occurrence of anatoxin-a(s) in the water. During the bloom period, the reservoir was strongly stratified, with higher temperatures and a deep anoxic hypolimnion. Two methods for sample concentration (direct and complete extraction) were tested, and direct extraction of samples proved to be more efficient. Water samples collected during the bloom showed 9% acetylcholinesterase inhibition at 50 mg mL⁻¹, corresponding to 0.61 μg of anatoxin-a(s) per gram of lyophilized powder. At these concentrations, symptoms of neurotoxicity and mortality were not observed in tests with Swiss albino mice. Although the concentrations of anatoxin-a(s) in the Faxinal Reservoir were low, these results are important because this is the first record of the toxin for A. crassa. Furthermore, this cyanotoxin is not yet included in Brazilian legislation for drinking-water monitoring, because of the lack of information about toxicity levels and risk calculation for oral doses. The data presented here contribute to the basis for the future inclusion of this toxin in Brazilian legislation for drinking-water quality control, and for the development of analytical methods for this toxin.     

A novel gene encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum

The hepatotoxin cylindrospermopsin is produced by several cyanobacteria species, which may flourish in tropical and sub-tropical lakes. Biosynthesis of cylindrospermopsin is poorly understood but its chemical nature, and feeding experiments with stable isotopes, suggested that guanidinoacetic acid is the starter unit and indicated involvement of a polyketide synthase. We have identified a gene encoding an amidinotransferase from the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum. This is the first report on an amidinotransferase gene in cyanobacteria. It is likely to be involved in the formation of guanidinoacetic acid. The aoaA is located in a genomic region bearing genes encoding a polyketide synthase and a peptide synthetase, further supporting its putative role in cylindrospermopsin biosynthesis.     

Microcystin-LR equivalent concentrations in fish tissue during a postbloom Microcystis exposure in Loskop Dam,South Africa

The effects of a decomposing cyanobacteria bloom on water quality and the accumulation of microcystin-LR equivalent toxin in fish at Loskop Dam were studied in May 2012. Enzyme-linked immunosorbent assay [ELISA] was used to confirm the presence of microcystin-LR equivalent in the water and to determine the microcystin (MCYST) concentration in the liver and muscle of fish. The lowest concentration of extracellular MCYST-LR equivalent was recorded in the lacustrine zone, where no cyanobacterial cells were observed, while the highest concentration (3.25 µg l^?1), 3.25 higher than World Health Organization standard, was observed in the riverine zone. Extremely high MCYST-LR equivalent concentrations of 1.72 µg MCYST-LReq kg^?1 in the liver and 0.19 µg kg^?1 in muscles of Labeo rosae, and 2.14 µg MCYST-LReq kg^?1 in the liver and 0.17 µg kg^?1 in muscles of Oreochromis mossambicus, indicate that the consumption of sufficient fish biomass might cause severe adverse effects in humans. Microscopic analyses of the stomach content of both fish species revealed low numbers of cyanobacterial Microcystis aeruginosa cells in comparison to other phytoplankton. The extracellular MCYST-LR equivalent of the decomposing bloom may have played a major role in the high levels observed in the livers of the two fish species. These findings are important for all downstream water users.     

Pseudo‐nitzschia blooms,domoic acid,and related California sea lion strandings in Monterey Bay,California

Blooms of the toxin‐producing diatom Pseudo‐nitzschia commonly occur in Monterey Bay, California, resulting in sea lion mortality events. The links between strandings of California sea lions suffering from domoic acid (DA) toxicity, toxic cell numbers, and their associated DA concentration in Monterey Bay and in sea lion feces were examined from 2004 to 2007. While Pseudo‐nitzschia toxic cells and DA concentrations were detectable in the water column most of the time, they were often at low levels. A total of 82 California sea lions were found stranded in the Bay between 2004 and 2007 with acute or chronic signs associated with DA poisoning. The highest number with detectable DA in feces occurred in April 2007 and corresponded with the presence of a highly toxic bloom in the Bay. Higher DA levels occurred in feces from sea lions stranding with acute toxicosis and lower concentrations in feces of sea lions exhibiting signs of chronic DA poisoning or not exhibiting any neurologic signs. Results indicated that sea lions are likely exposed to varying levels of DA through their prey throughout the year, often at sublethal doses that may contribute to a continued increase in the development of chronic neurologic sequelae.     

Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.     

Biosynthesis of the tropane-related cyanobacterial toxin anatoxin-a: role of ornithine decarboxylase.

A study was made of the biosynthesis by Anabaena flos-aquae of the tropane-related alkaloid anatoxin-a. Evidence is presented that the toxin arises from ornithine via putrescine (1,4-diaminobutane) and that ornithine decarboxylase (EC 4.1.1.17) is involved. An ornithine decarboxylase preparation, with optimal activity at pH 8, was obtained from Anabaena flos-aquae and partially purified by gel-filtration chromatography on DEAE-cellulose. One major and one minor peak of enzymic activity were obtained with Km values of 1.25 and 2.5 mM, respectively. Plasmid DNA (10 Kb; Mr 6.5 x 10(6] was detected in the toxic strain of Anabaena flos-aquae but not in a non-toxic strain. DNA from the toxin-producing strain of Anabaena flos-aquae transforms the non-toxic into a toxic strain.     

An overview of diversity,occurrence, genetics and toxin production of bloom-forming Dolichospermum (Anabaena) species

The new genus name Dolichospermum, for most of the planktonic former members of the genus Anabaena, is one of the most ubiquitous bloom-forming cyanobacterial genera. Its dominance and persistence have increased in recent years, due to eutrophication from anthropogenic activities and global climate change. Blooms of Dolichospermum species, with their production of secondary metabolites that commonly include toxins, present a worldwide threat to environmental and public health. In this review, recent advances of the genus Dolichospermum are summarized, including taxonomy, genetics, bloom occurrence, and production of toxin and taste-and-odor compounds. The recent and continuing acquisition of genome sequences is ushering in new methods for monitoring and understanding the factors regulating bloom dynamics.     

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.