Cryptic toxicity: non-planktonic cyanobacteria represent a

Water Planning Ecology 1Department of Science, Information Technology, Innovation and the Arts

Cryptic toxicity: non-planktonic cyanobacteria represent a significant potential source of cyanotoxins in the freshwater environment

Glenn McGregor1 & Barbara Sendall2

2Queensland Health

Department of Science, Information Technology, Innovation and the Arts Cryptic toxicity: non planktonic cyanobacteria

Background


Pilot study

•  Screened for genes associated with cyanotoxin biosynthesis in samples from a variety of lake and riverine habitats from twelve sites throughout Queensland

•  Cyanotoxins from the four major toxin groups known to occur in Australia was assessed using multiplex tandem real-time PCR


Pilot study – results

•  Metaphyton lake shore – ndaF gene for nodularin production + sxtI gene for PST–production

•  Metaphyton lake shore – cyrC gene for cylindrospermopsin production + mcyE gene for production of microcystins

•  Epipelon stream bed – sxtI gene for PST–production

•  All samples dominated by Oscillatoriales


Lyngbya wollei – background

•  A large benthic cyanobacteria known from a range of freshwater habitats

•  Populations from the southern USA are known to form massive infestations of both benthic and free floating mats in shallow waterways, lakes and reservoirs

•  USA populations are also known to produce neurotoxic saxitoxins

Lyngbya wollei infestation in a Florida spring



•  Lyngbya wollei is also known from riverine systems throughout eastern Australia

•  Following the identification of two south-east Queensland populations, we screened environmental samples by HPLC-MS/MS for a suite of common cyanotoxins –  cylindrospermopsin, deoxy-cylindrospermopsin –  anatoxin-a –  debromoaplysiatoxin –  lyngbya toxin-a –  microcystins (and derivatives) –  saxitoxin (and derivatives)



•  CYN and deoxy-CYN were the only cyanotoxins detected in the field and cultured material (CYN 0–33 µg g-1 dry weight, deoxy-CYN 0.5–547 µg g-1 dry weight)

•  CYN or deoxy-CYN was not detected in any environmental water samples (+ Corbiculidae bivalve)

Seifert, M., McGregor, G., Eaglesham, G., Wickramasinghe, W. & Shaw, G. (2007) First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck. Harmful Algae 6: 73-80.


Lyngbya wollei – additional questions

•  Confirmation of identifications based on morphological characteristics using supporting molecular information

•  How related were Australian populations of L.wollei to populations in the US and elsewhere?

•  Are the molecular mechanisms for CYN and deoxy-CYN production similar to other CYN-producing cyanobacteria?


Methods

•  Strain isolation and purification •  Identification and phylogeny

–  16S rRNA gene –  nitrogenase reductase gene (nifH)

•  Toxicology –  genes associated with cyanotoxin biosynthesis (pks,

mcyE, sxt1)


Identification and phylogeny

•  Two strains successfully isolated –  Yabba Creek at Stirling's Crossing

–  Awoonga Dam

Yabba Creek Awoonga Dam L. wollei strain YC0404

Qld

Tropic of Capricorn


Phylogenetic tree showing neighbour-joining analysis of 15 Lyngbya strains based on partial 16S rRNA sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.

Freshwater Lyngbya wollei clade

Freshwater estuarine clade

Marine clade



Phylogenetic tree showing neighbour-joining analysis based on partial nifH sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.



Toxicology – cyrF pks gene

Evolutionary relationships of 5 taxa based on the partial sequence of the cyrF gene (447 bp) determined by the NJ method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.

Lyngbya wollei YC0404

Lyngbya wollei AW0709

Oscillatoria sp. PCC 6506

Aphanizomenon sp. 10E6

Cylindrospermopsis raciborskii AWT205

100

100

0.01


Significance – Lyngbya wollei

•  Results provide additional support for the original identifications of Australian material based on morphology

•  Both Australian strains were closely related to US strains in both 16S rRNA and nifH phylogenies

•  Australian and US Lyngbya wollei strains form a discrete cluster within the polyphyletic Lyngbya complex

•  Results suggest the need for a further revision of the genus Lyngbya


Significance – Lyngbya wollei

•  Confirms the CYN biosynthesis pathway in Australian Lyngbya wollei strains is similar to that previously identified in C. raciborskii, A. ovalisporum and other CYN producing cyanobacteria

•  Highlights the utility of molecular methods for screening for gene sequences associated with cyanotoxin biosynthesis


Significance – non-planktonic cyanobacteria

•  Potential freshwater HAB events involving non-planktonic cyanobacteria may go undetected because subsurface mats and periphyton are easily missed by conventional sampling and monitoring methods

•  For this reason, it is critical that sampling be conducted using appropriate techniques at scales relevant to resolving cyanobacterial biomass from these habitats


Acknowledgements

•  DSITIA and Queensland Health for supporting this research

•  Steve Carter, Queensland Health Forensic and Scientific Services for chemical analysis of cyanotoxins

•  Wasa Wickramasinghe and Ian Stewart for collection and provision of some of the Lyngbya material used in this project

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Cryptic toxicity: non-planktonic cyanobacteria represent a