Disaggregation of colonies of Microcystis (Cyanobacteria) : efficiency of two techniques assessed using an image analysis system

Disaggregation of colonies of Microcystis (Cyanobacteria) into smaller aggregates or single cells is a useful process for improving the accuracy and precision of cell abundance estimates in natural populations. An image analysis system was therefore used to assess the efficiency of two techniques, heating/vortexing and grinding, for disaggregating fixed and live colonies. Eight environmental samples, consisting of either colonial or non-colonial strains, were tested in time course experiments using each technique by analysing the number and total area of the objects (i.e., cells, clumps of cells, colonies) acquired. The results show that i) image analysis is an efficient tool to compare the efficiency of disaggregation techniques on colonies of cyanobacteria, ii) the grinding technique is more efficient in disaggregating all types of Microcystis colony, without significant loss, and iii) the heating technique does not work for all types of colonies, in particular, compact colonies fixed with Lugol’s iodine solution

Improved morphometric and genetic tools for better identification and management of blue green algae

1 ABSTRACTA combination of modern genetics techniques and microscopy were used to identify which species of blue-green algae (cyanobacteria) can produce the toxin cylindrospermopsin in water storages used by the coal mining industry. Prior to this research project, the only known toxin producing cyanobacterium found in relation to industrial sites in Central Queensland was Cylindrospermopsis raciborskii. Large culture collections were established in both Adelaide (AWQC) and Rockhampton (CQU) to facilitate detailed research on problematic species from the Central Queensland region. However, the species isolated are known to occur throughout Australia, including the Hunter Valley. During this project, the toxins cylindrospermopsin and oxycylindrospermopsin were always identified when Cylindrospermopsis raciborskii was present. Total cylindrospermopsin concentrations ranged from 0.2 to 22.1 μg L-1. Aphanizomenon ovalisporum was also found to produce this group of toxins. A highly toxic strain of A. ovalisporum was identified from the Isaacs River region. During intensive genetic survey in the field, the toxin microcystin was also identified and this was associated with the presence of Microcystis. Microcystin concentrations ranged between 1.7 to 3200 μg L-1. The species present was genetically not Microcystis aeruginosa but wasmorphologically similar to Microcystis panniformis.Following unusual genetic results associated with Aphanizomenon and Limnothrix- like material early in the research, additional testing for unknown protein synthesis inhibiting toxins was added to theprotocol. The presence of new toxins was identified as a result. This report details the new toxin producing species and includes a photographic guide to their identification

Application of an image analysis system to enumerate and measure cyanobacteria

"Cyanobacteria, also known as blue-green algae, are the most notorious bloom formers in freshwater environments. Their success is due to their ability to adapt their physiological capacities to compete with other phytoplankton species (e.g., for light and nutrients, Padisák 1997), and the fact that they seem less edible for zooplankton and fish than other non-blooming algae (Gilbert 1996; Reynolds 1998). Cyanobacteria are photosynthetic, oxygen-producing and nitrogen-fixing prokaryotic organisms. They are characterized by the presence of the accessory pigment phycobilin. There are three basic morphological groups: 1) unicells, which may be solitary or aggregated in colonies; 2) undifferentiated non-heterocyst filaments, which also may be solitary or aggregated; and 3) filamentous forms with differentiated cells called heterocysts (Paerl et al. 2001). As a result of increasing environmental stress on freshwater ecosystems (e.g., eutrophication), blooms are increasing in number and frequency world-wide. Besides health problems due to their toxicity (such as the Caruaru tragedy in Pernambuco state, Brazil, Azevedo 1996), and environmental problems (e.g. fish kills and food web alterations), such blooms may also give rise to water quality and engineering problems such as clogging of filters in water treatment works, coloration of the water or production of tastes and odours (Paerl et al. 2001)."--p. 10

Limnothrix and Sphaerospermopsis from Central Queensland, Australia

A new toxin producing freshwater Limnothrix from Central Queensland, Australia has been detected. The features of this cyanobacterium in both field and pure cultures were consistent with the cyanobacterial species Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo and Geitlerinema amphibium (Agardh ex Gomont) Anagnostidis as well as with the earlier Australian descriptions of Limnothrix cf. planctonica Baker and Fabbro. However, genetic analyses closely group this cyanobacterium with the commonly occurring temperate species Limnothrix redekei (Van Goor) Meffert. The Limnothrix may occur as solitary planktonic trichomes, mats on the bottom or within the water column, or in balls floating on the surface. A fine mucilage often confused with a sheath may cover older trichomes. The long, thin trichomes may glide into coils, may flex and are not attenuated at the ends. The cylindrical cells are 1.6-2.0 μm wide and 5.0-6.5 μm long. Constriction at the cross walls is generally not observed and cross walls are often indistinct. Refractile granules are present in the cells, - near the cell walls and sometimes distributed within the cells. A new species of straight Sphaerospermopsis closely aligning with Sphaerospermopsis reniforme (Forti) Zapomĕlová, Jezberová, Hrouzek, Hisem, Rĕháková et Komárková comb. nov. has also been detected. Trichomes are normally solitary, straight or slightly curved often with a metameric structure. Trichhome are found with or without heterocytes and may be constricted at the cross walls. Vegetative cells are 2.5 to 4.9 µm wide and 4.0 to 10.4 µm long. Heterocytes are ovate to sub-cylindrical 3.0 to 3.6 µm wide and 6.8 to 10.3 µm long. The cell contents have aerotopes and the apical cells are tapered or bluntly rounded

Novel toxicity associated with a Limnothrix-like species : mouse bioassay results

Background & Aims: A search for novel cylindrospermopsin (CYN) producers found a Limnothrix strain that inhibited protein synthesis but did not contain detectable CYN or analogues (HPLC, LC-MS, ELISA), nor the CYN gene (Bernard et al 2010). Methods: Mouse bioassays (time-course up to seven days, and set times up to 24h) were performed followed by detailed post-mortem (PM) dissections and histopathology. By 3h, these reduced but clear mucoid diarrhoea (containing sloughed endothelium and blood cells) was observed. Body weights were reduced by 24h and remained below controls for 7d. Liver and spleen weights (% body weight) increased by 4h and remained high until at least 24h. Histopathology (10-24h) indicated cell death in the small intestine (widespread), liver and kidneys. White blood cell numbers increased in the circulation, spleen and lungs. Conclusions: Limnothrix produces a potent in vivo cytotoxin. It is water-extractable and produced by a commonly occurring genus. Hence it is a potential risk to drinking water sources. We are currently (1) clarifying the genetic identity of the toxic strain, (2) determining its Australian distribution, (3) purifying and characterising the active compound, and (4) investigating its mechanism of action post-mortem (PM) dissections and histopathology. Results: By 0.5h post-injection, signs included extreme sensitivity to touch and sudden noises, and hind limb weakness

Toxicity of the cyanobacterium Limnothrix AC0243 to male Balb/c mice

A growing list of freshwater cyanobacteria are known to produce toxic agents, a fact which makes these organisms of concern to water authorities. A cultured strain of Limnothrix (AC0243) was recently shown to have toxic effects in in vitro bioassays. It did not produce any of the known cyanobacterial toxins. The intrapertoneal toxicity of aqueous extracts of the material was therefore tested in mice to determine whether the observed effects might be of public health relevance to drinking water supplies. The results indicate that Limnothrix AC0243 is acutely toxic to mice, causing widespread cellular necrosis in the liver, kidneys and gastrointestinal tract within 24 h of exposure. Sub-lethal effects lasted at least 7 d. These results suggest that Limnothrix AC0243 produces a novel toxin ("Limnothrixin") and that further work is therefore urgently required to quantify the potential public health implications

Novel toxic activity associated with the cyanobacteria Limnothrix : use of screening assays for detection

Development of biochemical screening assays for cyanobacterial toxins allows identification of toxins based on known activity. While this may be diagnostic for the toxin or toxin class that the assay has been designed for (e.g. microcystins, cylindrospermopsins) there is also potential to pick up new, unidentified toxins. In this example, screening cyanobacterial samples for the toxin cylindrospermopsin led to the identification of novel toxic activity from the cyanobacterium Limnothrix.The cell-free protein synthesis assay was inhibited by the Limnothrix extract as would be expected when cylindrospermopsin is present. However analysis by immunoassay (ELISA) and analytical techniques (HPLC, LC-MS) could not detect cylindrospermopsin itself. PCR amplification of genes associated with cylindrospermopsin production was also absent. Further characterisation revealed that the toxic responses induced by the Limnothrix extract could not be attributed to other known cyanobacterial toxins. Distinctive toxic effects of the Limnothrix extract in mammalian cells include significant ATP depletion and markedgranulation of cells as observed by both microscopy and flow cytometry. Current work aimsto identify and characterise the toxic agent present. This case highlights how biochemical screening assays can complement analytical techniques for the identification of toxic activity in cyanobacterial samples. Adequate validation of such techniques is required

Novel toxic effects associated with a tropical Limnothrix/Geitlerinema-like cyanobacterium

The presence of a toxic strain of a fine filamentous cyanobacterium belonging to the Oscillatorialean family Pseudanabaenacea was detected during a survey of cyanobacterial taxa associated with the presence of cylindrospermopsin in dams in Central Queensland (Australia). The strain, AC0243, was isolated and cultured, its genomic DNA extracted and 16S RNA gene sequenced. Phylogenetic analysis placed AC0243 with Limnothrix species, although this genus appears polyphyletic. Moreover, not all morphological characters are consistent with this genus but more closely fit the description of Geitlerinema unigranulatum (R.N. Singh) Komarek and Azevedo. The potential toxic effects of AC0243 extract were assessed chemically and biologically. Cell free protein synthesis was inhibited by the extract. Exposure of Vero cells to the extract resulted in a significant reduction in cellular ATP levels following 24–72 h incubation.The presence of cylindrospermopsin was excluded based on the nature of responses obtained in cell and cell-free assays; in addition, (i) it could not be detected by HPLC, LC-MS, or immunological assay, and (ii) no genes currently associated with the production of cylindrospermopsin were found in the genome.Other known cyanobacterial toxins were not detected. The apparent novelty of this toxin is discussed

Taxonomy and ecology of toxin producing Limnothrix

A new toxin produced by a Limnothrix was discovered while screening cyanobacterial samples for the presence of known and unknown toxins as part of an Australian Coal Association Research Project. It is the first time that a toxin-producing freshwater Limnothrix has been described. The features of this cyanobacterium in both field and pure cultures were consistent with the cyanobacterial species Geitlerinema unigranulatum as well as with the earlier Australian descriptions of Limnothrix cf. planctonica. However, genetic analyses closely group this cyanobacterium with the commonly occurring temperate species Limnothrix redekei. Limnothrix may occur as solitary planktonic trichomes, mats on the bottom or within the water column, or in balls floating on the surface. A fine mucilage, often confused with a sheath, may cover older trichomes. The long, thin trichomes may glide into coils, may flex and are not attenuated at the ends. The cylindrical cells are 1.6-2.0 μm wide and 5.0-6.5 μm long. Constriction at the cross walls is generally not observed and cross walls are often indistinct. Refractile granules are present in the cells - near the cell walls and sometimes distributed within the cells. Limnothrix often co-dominates with Cylindrospermopsis raciborskii in both temperate and tropical environments. In the Fitzroy River system, it is often found just above the level of the thermocline. However, toxin producing material has also been isolated from water sampled from major pipelines within the Central Queensland region

The ACARP project : a search for new toxin producing cyanobacteria from Australia

Abstract: Background & Aims: Evidence from animal deaths suggested the possible presence of unidentified cylindrospermopsin producers in Central Queensland, Australia. As the water supplies in this region are used by the mining industry for drinking and industrial usage, the ACARP project was designed to identify new toxin producing cyanobacteria that may pose a previously unrecognized human health risk. Methods: A combination of microscopy, chemical analyses, bioassays and molecular techniques was systematically used to identify the cyanobacteria sampled and rapidly assess the presence/absence of genes indicative of known toxins and potential new toxin producers. Results: One hundred and fifty-two strains from thirteen genera were isolated and examined in detail. Of these, eleven strains were recorded as showing toxic effects. One of these strains, Limnothrix, demonstrated novel toxic cellular effects and mammalian toxicity. Various strains with a mix of diacritical morphological characteristics used in the identification of Cylindrospermopsis and Aphanizomenon were isolated. Some of these were also associated with novel toxic effects and belong to the newly created genus Sphaerospermopsis. Conclusions: The diversity of toxin producing species within Australian waters is greater than previously recognized. This research identified a new suite of potentially toxic cyanobacteria for inclusion into health and safety protocols. We are currently investigating the genetics, ecology, toxicity and environmental impacts of these organisms