CyanoSurvey Project. A National Update on Toxic Cyanobacteria and their Distribution. Andrew Humpage, Virginie Gaget, Suzanne Froscio

Transcription

1 CyanoSurvey Project A National Update on Toxic Cyanobacteria and their Distribution Andrew Humpage, Virginie Gaget, Suzanne Froscio Water Quality Research Australia, United Water, Sydney Water $400k cash ($1.4m including in kind) Dec 09 Mar 12

2 Project Team Research Team: AWQC: Andrew Humpage, Virginie Gaget, Suzanne Froscio, Melody Lau, Julie Culbert QHSS: Barbara Sendall DERM: Glenn McGregor CQU: Larelle Fabbro & Catherine Bernard-Pattinson Technical Advisory Team: Chris Saint, Mike Burch, Peter Baker, Paul Monis, Stephanie Rinck-Pfeiffer, Michele Akeroyd. Industry Partners: SA Water, Sydney Water, United Water International, Water Corp, Melbourne Water, Power & Water, Goulburn Valley Water, Barwon Water, Hunter Water, Wannon Water, Sydney Catchment Authority, Lower Murray Water, North East Water, Ballina Shire Council, Wakool Shire Council, Dept Sustainability & Environment (Vic), Dept Environment & Resource Management (Qld)

3 Project Aims How reliable are our assumptions about: Which species are toxic Which species are species Where various species occur and why What is the best means of quantifying toxin risk? How might the distributions change in a changing climate?

4 CyanoSurvey subprojects: Link to toxicity (2) 1) Toxicity in common but untested strains Picocyanobacteria, Aphanocapsa, Synechocystis, Pseudanabaena, Limnothrix, Coelosphaerium, Planktolyngbya, Anabaenopsis, Aphanizomenon ovalisporum, Cylindrospermum, Raphidiopsis, Trichodesmium, Tolypothrix, Nostoc, Calothrix, Arthrospira, Rivularia, Phormidium. 2) Toxins in main targeted strains Anabaena, Aphanizomenon, Cylindrospermopsis, Microcystis, Planktothrix 3) Taxonomy study Anabaena / Aphanizomenon, Cylindrospermopsis Microcystis aeruginosa / Microcystis flos-aquae Planktothrix 4) Methods comparison Toxin assessment methods: morphology vs genetic vs toxins vs toxicity Link to taxonomy (3) and toxins (2) 5) Phylogeography Link all data to environmental aspects. See if strains are really different all around the country and if toxic ones grow better in specific types of water. Multivariable statistics and mapping. 6) Murray river survey Genetic comparison of new isolates from the Murray river to those collected in 1991 and still in the AWQC collection.

5 1) Toxicity in common but untested strains List of organisms to track down Picocyanobacteria, Aphanocapsa, Synechocystis, Pseudanabaena, Limnothrix, Coelosphaerium, Planktolyngbya, Anabaenopsis, Aphanizomenon ovalisporum, Cylindrospermum, Isolation Raphidiopsis, Trichodesmium, Tolypothrix, Nostoc, Calothrix, Arthrospira, Rivularia, Phormidium. Select samples with cell numbers of target species > 1,000 cells/ml Test sample for known toxins using ELISA, LC-MS/MS and ACE, and unknown toxins using mammalian cell-based assays Pure isolates cultured in 1L of liquid medium Retest

6 2) Toxins in main targeted strains List of organisms to track down Anabaena sp. / Aphanizomenon sp. Cylindrospermopsis sp. Microcystis aeruginosa / Microcystis flos-aquae Planktothrix sp. Isolation & Purification Pure isolates cultured in 1L of liquid medium Testing for known toxins by ELISA/ACE/LC- MS/MS and toxin genes by PCR Relate toxicity to taxonomy

7 3) Taxonomy study 16S rrna gene sequences of cyanobacterial strains collection from the Genbank database Retrieve sequences available for all the targeted genera Build up a general tree Choose 3 markers that are the most available for all the targeted genera and that have different level of resolution (mutation rates) Isolate groups and subgroups Check if primers are published for all of them (if not design them) Choose a reference strain for each group and each subgroup (preferably from the AWQC collection) Inclusion to the study Sequence the 5 markers (including 16S rrna and ITS)

8 3) Taxonomy study List of organisms to track down Anabaena / Aphanizomenon, Cylindrospermopsis Microcystis aeruginosa / Microcystis flos aquae Planktothrix. Isolation Isolates from CS samples + ad hoc samples (from all around Australia) Purification Pure isolates cultured in 1L of liquid medium + reference strains for each sub-group of strains Amplification of 4 coding genes including the 16S rrna gene sequence Amplification of the ITS Sequencing Cloning of multiple copies Build phylogenetic trees for each marker Comparison Build phylogenetic trees with computed data (concatenated sequences)

9 3) Taxonomy study List of organisms to track down Anabaena / Aphanizomenon, Cylindrospermopsis Microcystis aeruginosa / Microcystis flos aquae Planktothrix. Isolation Isolates from CS samples + ad hoc samples (from all around Australia) Purification Pure isolates cultured in 1L of liquid medium + reference strains for each sub-group of strains Amplification of 4 coding genes including the 16S rrna gene sequence MLST Sequencing Amplification of the ITS Cloning of multiple copies Build phylogenetic trees for each marker Comparison Build phylogenetic trees with computed data (concatenated sequences)

10 5) Methods Comparison Select a range of frozen samples containing toxic cyanobacteria (supplement with cultures if necessary) Toxins by ELISA/Field Tests/LC-MS/MS Toxin Genes by qpcr Toxicity by PPIA, PSIA, ACE, Neuro-2A Cell counts and ID on fresh samples Genetic Taxonomy

11 6) Phylogeography All CyanoSurvey samples from project partners Cell counts and ID on fresh samples Water Chemistry on all samples Lat/Long plus historical monitoring data Genetic Taxonomy & Toxicity species and strain preferences Climate change models

12 Potential Sampling Locations

13 Potential Sampling Sampling Locations Locations So Far

14 Thanks to: Lee Bowling & staff, Office of Water, NSW Andrew Wyber, Water Corp. of WA Louisa Davis, DSE, Victoria Robyn Grey-Gardner & Stephen Purvis, CAT, Alice Springs Simon Townsend, Charles Darwin Uni., Darwin Staff of Sydney Water Labs Staff of AWQC Algal Labs Funding: