Glenn B. McGregor

Occupational and environmental hazard assessments for the isolation, purification and toxicity testing of cyanobacterial toxins

Cyanobacteria can produce groups of structurally and functionally unrelated but highly potent toxins. Cyanotoxins are used in multiple research endeavours, either for direct investigation of their toxicologic properties, or as functional analogues for various biochemical and physiological processes. This paper presents occupational safety guidelines and recommendations for personnel working in field, laboratory or industrial settings to produce and use purified cyanotoxins and toxic cyanobacteria, from bulk harvesting of bloom material, mass culture of laboratory isolates, through routine extraction, isolation and purification. Oral, inhalational, dermal and parenteral routes are all potential occupational exposure pathways during the various stages of cyanotoxin production and application. Investigation of toxicologic or pharmacologic properties using in vivo models may present specific risks if radiolabelled cyanotoxins are employed, and the potential for occupational exposure via the dermal route is heightened with the use of organic solvents as vehicles. Inter- and intra-national transport of living cyanobacteria for research purposes risks establishing feral microalgal populations, so disinfection of culture equipment and destruction of cells by autoclaving, incineration and/or chlorination is recommended in order to prevent viable cyanobacteria from escaping research or production facilities.

Spatial and temporal variation in algal-assemblage structure in isolated dryland river waterholes, Cooper Creek and Warrego River, Australia

The scale at which algal biodiversity is partitioned across the landscape, and the biophysical processes and biotic interactions which shape these communities in dryland river refugia was studied on two occasions from 30 sites in two Australian dryland rivers. Despite the waterholes studied having characteristically high levels of abiogenic turbidity, a total of 186 planktonic microalgae, 253 benthic diatom and 62 macroalgal species were recorded. The phytoplankton communities were dominated by flagellated cryptophytes, euglenophytes and chlorophytes, the diatom communities by cosmopolitan taxa known to tolerate wide environmental conditions, and the macroalgal communities by filamentous cyanobacteria. All algal communities showed significant differences between catchments and sampling times, with a suite of between 5 and 12 taxa responsible for similar to 50% of the observed change. In general, algal assemblage patterns were poorly correlated with the measured environmental variables. Phytoplankton and diatom assemblage patterns were weakly correlated with several waterhole geomorphic measures, whereas macroalgal assemblage patterns showed some association with variability in ionic concentration.

First Report of a Toxic Nodularia spumigena (Nostocales/ Cyanobacteria) Bloom in Sub-Tropical Australia. I. Phycological and Public Health Investigations

Cyanobacterial blooms represent one of the most conspicuous and widespread waterborne microbial hazards to human and ecosystem health. Investigation of a cyanobacterial bloom in a shallow brackish water recreational cable ski lake in south-eastern Queensland, Australia revealed the dominance of the toxigenic species Nodularia spumigena. The bloom spanned three months, during which time cell concentrations exceeded human guideline thresholds for recreational risk, and concentrations of the hepatotoxic cyanotoxin nodularin exceeded 200 µg L−1. Cyanotoxin origin and identification was confirmed by amplification of the ndaF-specific PCR product and sequencing of the 16S rRNA gene. From the limited data available leading up to, and throughout the bloom, it was not possible to establish the set of causative factors responsible for its occurrence. However a combination of factors including salinity, hydraulic retention time and nutrient status associated with an extended period of drought are likely to have contributed. This was the first known occurrence of this species in bloom proportions from sub-tropical Australia and as such represents a hitherto uncharacterized risk to human and ecosystem health. It highlights the need for adaptive monitoring regimes to ensure a comprehensive understanding of the potentially toxic cyanobacteria likely to inhabit any given region. Such monitoring needs to recognize that cyanobacteria have a significant capacity for range expansion that has been facilitated by recent changes in global climate.

First Report of a Toxic Nodularia spumigena (Nostocales/ Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8–47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 μg/kg, range 32.3–56.8 μg/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

Phylogeny and toxicology of Lyngbya wollei (Cyanobacteria, Oscillatoriales) from north-eastern Australia, with a description of Microseira gen. nov.

Three populations of the freshwater filamentous cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck have been putatively identified from north‐eastern Australia and found to produce the potent cyanotoxin cylindrospermopsin (CYN) and its analog deoxy‐cylindrospermopsin (deoxy‐CYN). We investigated the phylogeny and toxicology of strains and mats isolated from two of these populations using a combination of molecular and morphological techniques. Morphologically the strains corresponded to the type description, however, the frequency of false‐branching was low, and variable over time. Strains and mat samples from both sites were positive for the cyrF and cyrJ genes associated with CYN biosynthesis. Phylogenetic analysis of these genes from Australian L. wollei sequences and comparable cyanobacterial sequences revealed that the genes in L. wollei were more closely related to homologous genes in Oscillatoria sp. PCC 6506 than to homologs in Nostocalean CYN‐producers. These data suggest a common evolutionary origin of CYN biosynthesis in L. wollei and Oscillatoria. In both the 16S rRNA and nifH phylogenies, the Australian L. wollei strains formed well‐supported clades with United States L. wollei (= Plectonema wollei) strains. Pair‐wise sequence similarities within the 16S rRNA clade containing all eleven L. wollei strains were high, ranging from 97% to 100%. This group was distantly related (<92% nucleotide similarity) to other taxa within the group previously considered under the genus Lyngbya sensu lato (C. Agardh ex Gomont). Collectively, these results suggest that this toxigenic group is evolutionarily distinct and sufficiently distant as to be considered a separate genus, which we have described as Microseira gen. nov. and hence transfer to it the type M. wollei comb. nov.

Iningainema pulvinus gen nov., sp nov. (Cyanobacteria, Scytonemataceae) a new nodularin producer from Edgbaston Reserve, north-eastern Australia

A new nodularin producing benthic cyanobacterium Iningainema pulvinus gen nov., sp nov. was isolated from a freshwater ambient spring wetland in tropical, north-eastern Australia and characterised using combined morphological and phylogenetic attributes. It formed conspicuous irregularly spherical to discoid, blue-green to olive-green cyanobacterial colonies across the substratum of shallow pools. Morphologically Iningainema is most similar to Scytonematopsis Kiseleva and Scytonema Agardh ex Bornet & Flahault. All three genera have isopolar filaments enveloped by a firm, often layered and coloured sheath; false branching is typically geminate, less commonly singly. Phylogenetic analyses using partial 16S rRNA sequences of three clones of Iningainema pulvinus strain ES0614 showed that it formed a well-supported monophyletic clade. All three clones were 99.7-99.9% similar, however they shared less than 93.9% nucleotide similarity with other cyanobacterial sequences including putatively related taxa within the Scytonemataceae. Amplification of a fragment of the ndaF gene involved in nodularin biosynthesis from Iningainema pulvinus confirmed that it has this genetic determinant. Consistent with these results, analysis of two extracts from strain ES0614 by HPLC-MS/MS confirmed the presence of nodularin at concentrations of 796 and 1096μgg-1 dry weight. This is the third genus of cyanobacteria shown to produce the cyanotoxin nodularin and the first report of nodularin synthesis from the cyanobacterial family Scytonemataceae. These new findings may have implications for the aquatic biota at Edgbaston Reserve, a spring complex which has been identified as a priority conservation area in the central Australian arid and semiarid zones, based on patterns of endemicity.

Komvophoron kgarii sp. nov. (Oscillatoriales), a new epipelic cyanobacterium from subtropical eastern Australia

McGregor G.B. and Sendall B.C. 2013. Komvophoron kgarii sp. nov. (Oscillatoriales), a new epipelic cyanobacterium from subtropical eastern Australia. Phycologia 52: 472–480. DOI: 10.2216/13-143.1 A new epipelic freshwater cyanobacterium, Komvophoron kgarii sp. nov., was identified from a perennial wallum stream on Fraser Island, northeastern Australia. Komvophoron kgarii was the first species in Komvophoron that was described on the basis of combined morphological, ultrastructural and phylogenetic attributes. The new taxon was distinguished by virtue of its obtuse-conical apical cells that became conically elongated and narrowed toward the ends as they matured. Additionally, trichome separation into hormogonia was preceded by preapical cells beginning to differentiate on either side of the fragmentation point before separation, a feature not previously recorded in this genus. Ultrastructural examination showed that thylakoids formed irregularly arranged blocks of lamellae in both longitudinal and cross-sections. Phylogenetic analysis revealed that the isolated material shared less than 91% 16S ribosomal RNA nucleotide sequence similarity with any other cyanobacterial sequence and formed a novel branch.

Ewamiania thermalis gen. et sp. nov. (Cyanobacteria, Scytonemataceae), a new cyanobacterium from Talaroo thermal springs, north-eastern Australia

Abstract. A new subaerophytic cyanobacterium, Ewamiania thermalis gen. et sp. nov., was isolated from a thermal spring complex in tropical, north-eastern Australia and characterised using combined morphological and phylogenetic attributes. It formed blackish-green hemispherical caespitose mats that began as small circular tufts, maturing to form dense mats up to several metres long. It grew along the crests of the minidams just above the thermal waters as well as along some of the shallow unconfined areas of vent-discharge aprons. Morphologically, Ewamiania is most similar to members of the Scytonemataceae. Filaments were isopolar, cylindrical, straight or flexuous, densely arranged and erect, often parallely fasciculate, with tolypotrichoid false-branching, rarely with scytonematoid false-branching. Vegetative cells were short barrel-shaped or isodiametric, slightly constricted at the cross-walls, with granulated contents. Sheaths were firm, thick, lamellated, uncoloured to yellowish or darkly yellow–brown in colour, cylindrical and closed at the apex. Heterocytes were spherical or ovoid in shape, and occurred in both basal and intercalary positions, generally solitary, but sometimes up to two or three in a series, developing particularly at the base of branches. Reproduction occurred by the production of hormogonia by the formation of necridic cells; hormogonia were not constricted at cross-walls and often included terminal heterocytes. Phylogenetic analyses using partial 16S rRNA sequences obtained from a strain of E. thermalis showed that it formed a well supported monophyletic clade, sharing less than 94.3% nucleotide similarity with other cyanobacterial sequences, including putatively related taxa within the Scytonemataceae. It also formed a novel clade in the nifH phylogeny, which was associated with members of the genus Brasilonema M.F.Fiore, Sant’Anna, M.T.P.Azevedo, Komárek, Kastovsky, Sulek & Lorenzi.