Toxigenicity Assessment of the Murray River Bloom Using a Rapid and Automated Multiplex Tandem- Polymerase Chain Reaction.

Nijoy John Jex Lab; WEHI Murray River Bloom- 2016

• History of bloom events: 1991, 2007, 2009, 2010 • 194 samples from 18 sites along the Murray River (Victoria)- upstream of Wahgunyah to Wemen • Samples collected from March 21 to May 4th 2016 • Multiplex Tandem-PCR conducted on all samples (retested by conventional PCR) • Bio volume, microscopic ID / cell count, Toxin concentration (HPLC/ELISA)

(source: http://www.theage.com.au/victoria/bluegreen-algal-bloom-chokes-murray-cuts-water-to-farmers-20160308-gndihv.html)

AIMS

• To understand the community composition of the bloom • Assess the toxigenicity • Understand toxin gene expression

(source: http://www.theage.com.au/victoria/bluegreen-algal-bloom-chokes-murray-cuts-water-to-farmers-20160308-gndihv.html)

Toxic in Australia

• Major water quality issue • Threat to public health, animals, water ecosystem

Cyanotoxins Toxicity Genera/species

Microcystins Hepatotoxic Microcystis sp., sp.

Nodularins Hepatotoxic spumigena

Saxitoxins Neurotoxic Dolichospermum circinalis

Cylindrospermopsin Hepatotoxic/Neurotoxic/Cytotoxic raciborskii; Chrysosporum ovalisporum

M. aeruginosa N. spumigena C. raciborskii D. circinalis Methods of detection

• Cannot differentiate toxic from non-toxic blooms • Microbiology/taxonomic expertise

• Less sensitive, effective when toxin is present • Cross reactivity of antibodies against other toxin variants

• Quantitative and sensitive • Expensive, lengthy analytical time

HPLC Molecular techniques: Multiplex-Tandem PCR

• Simultaneous amplification • Automated, rapid and sensitive • 72 individual tests

MT-PCR data

• Diagnostic specificity and sensitivity: 100% and 97.7% respectively (Baker et.al., 2013)- WaterRA project 1043

• Simultaneous amplification of 6 gene targets WaterRA project 1043 Microscopy data

• Predominant species: Chrysosporum ovalisporum • Subdominant species: Planktolyngbya, Chroococcales, Cyanogranis sp., Aphanocapsa sp. • Small quantities : Cylindrospermopsis raciborskii, Microcystis sp., Dolichospermum sp. • Highest Bio volume: 73 mm3/L at Picola Raw inlet • Total BGA count: more than drinking and recreational water guideline

• Cylindrospermopsin producer • Aphanizomenonaceae family • Nitrogen fixing cyanobacteria

C. ovalisporum Murray bloom: MT-PCR Data

Prevalence Overall Abundance

7%

19% 31% 39%

74% 30%

• > 60,000 copies/ml for Microcystis like McyE at Piangil river Abundance (>1000 gene copies/mL)

• > 6,000 copies/ml for CyrC at Numurkah 7%

Saxitoxin

31% Cylindrospermopsin Which species is producing microcystin? McyE Microcystis 62% Progression of the bloom

Wahgunyah

Pental

Conventional PCR , ELISA and HPLC data

• ELISA and HPLC: Low level production MCY and CYNs • Overall toxin levels were low (never rising about 1µg/L), but trace levels were detected by ELISA and by HPLC

Murray bloom -summary

• Two different bloom patterns

• Bloom was a mixture of toxic and non-toxic C. ovalisporum ??

• Microcystin producers other than Microcystis sp. and Dolichospermum sp. ??

• Propose that the gene was not expressed or transcribed

• Environmental factors regulating toxin production- possibly Nitrogen

Murray Bloom-Next steps

• Assess toxin gene expression vs toxin production • Study the activity of NtcA on toxin biosynthesis

• Effect of environmental factor- Nitrogen Acknowledgements

Supervisors Water Research Australia • Assoc. Prof Aaron Jex (Primary supervisor) • Carolyn Bellamy • Dr. Nick Crosbie • Dr. Judy Blackbeard

Mentor Jex Lab, WEHI • Dr. Daniel Hoefel (SA Water)

• Dr. Louise Baker • Dr. Najoua Lalaoui (WEHI) • Dr. Sammantha Emery • Brendan Ansell • Katharina Stracke Industry Partners • Cammille Awburn • Goulburn Murray Water • Goulburn Valley Water ALS • Grampians Wimmera • Lisa Synnott • North East Water • Sussan Cassar • Coliban Water