Genetic and environmental factors influencing Microcystis bloom toxicity Juli Dyble NOAA Great Lakes Environmental Research Lab Ann Arbor, MI

Great Lakes Saginaw Bay western Lake Erie

Great Lakes as an aquatic resource  Largest supply of freshwater in the world  80% of US freshwater supply  Drinking water supply for 40 million US and Canadian citizens citizens  Over 500 beaches for swimming and recreation

Common cyanobacterial HAB genera in the Great Lakes Cylindrospermopsis Anabaena Microcystis Oscillatoria Aphanizomenon

Lake Erie, South Bass Island, Sept 2006 Lake Erie, Put-In-Bay, Sept 2006 Microcystis in the Great Lakes

Present Dominant member of phytoplankton community Blooms frequent and abundant High P input to system (detergents, fertilizers, septic) P abatement programs (Great Lakes Water Quality Agreement) Decrease in chlorophyll, increased water clarity Blooms rare Dreissenid mussel introduction Return of Microcystis blooms up to 90% phytoplankton community Abundant Microcystis blooms, July - Sept

 Zebra mussel (Dreissena polymorpha)  Quagga mussel (Dreissena bugensis)  Introduced by ballast water from eastern Europe

Impacts of zebra mussels on Microcystis  promote growth of toxic Microcystis strains Grazing pressure selective rejection of toxic Microcystis strains consume more palatable species Nutrient excretion provide sufficient energy for growth of toxic strains change N:P ratios Zebra mussels

 Map microcystin concentrations and Microcystis cell numbers in Saginaw Bay and western Lake Erie  Identify environmental factors promoting microcystin production  Develop rapid methods for detection of toxic Microcystis  Accumulation in fish Current projects Microcystis sp.

What makes a cyanobacterial bloom toxic?  Shift in community composition Mostly non-toxic Mostly toxic strains Not producing toxin Light Nutrients Temperature Trace metals Producing toxin  Stimulation of toxin production by environmental factors

What makes a cyanobacterial bloom toxic?  Shift in community composition Not producing toxin Producing toxin  Stimulation of toxin production by environmental factors Mostly non-toxic Mostly toxic strains fold change in toxicity 2-10 fold change in toxicity (Zurawell et al 2005) 2005)

Intracellular total microcystin by HPLC Saginaw Bay August 2004 western Lake Erie Put-In-Bay 58 µg L -1

Distribution of microcystin congeners Relative toxicity: - LR and –LA = 4x more toxic than –YR 10x more toxic than -RR

 Differentiate morphologically identical strains  toxic and non-toxic strains  Identify geographic origin of strains and genetic diversity of populations  Rapid detection Advantages of molecular techniques

 All toxin-producing strains of Microcystis contain genes for microcystin production: mcyA-J  Presence of mcyB = strain able to produce toxin Absence of mcyB = non-toxic Absence of mcyB = non-toxic Identifying toxic strains of Microcystis Pearson et al 2004

Multiplex PCR for toxic Microcystis M Saginaw Bay western Lake Erie mcyB ITS M = molecular weight marker Number of colonies Basin# mcyB total (ITS) Saginaw Erie 4 16 % microcystin producers 90% 25%

Distribution of toxic Microcystis km Stations with mcyB

Distribution of C. raciborskii in the US L. Yale L. Eustis Silver L. L. Griffin L. Harris Little L. Harris Trout L. L. Dora L. Beauclair L. Ola L. Carlton L. Monroe L. Jesup Cylindrospermopsis specific nifH primers

Quantitative PCR for enumerating toxic Microcystis colonies Applications  measure temporal variation in proportion of toxic strains  biweekly sampling at 3 locations in western Lake Erie  identifying conditions under which cells are actively producing toxin (expressing mcyB) producing toxin (expressing mcyB)  zebra mussel grazing  changes in nutrients and light  Tie into circulation models to predict distribution of toxic Microcystis strains and forecast water quality Microcystis strains and forecast water quality western Lake Erie Maumee Bay

Goal  Develop predictive capabilities for presence of toxic cyanobacterial blooms in Great Lakes toxic cyanobacterial blooms in Great Lakes recreational and drinking water supplies recreational and drinking water supplies

  Center of Excellence for Great Lakes and Human Health (Oceans and Human Health Initiative)   Gary Fahnenstiel (NOAA-GLERL)   Hank Vanderploeg (NOAA-GLERL)   Pat Tester, Wayne Litaker (NOAA-Beaufort)   Dave Millie (Florida Institute of Oceanography)   Crew of the R/V Laurentian Thanks …….

Microcystin concentration of concern for routine fish consumption = 7.7 ng g -1 Microcystin concentrations in Perch Lake Erie, summer 2006 ng toxin (g dry mass) -1

Phylogenetic tree based on mcyB Designations according to Mikalsen et al 2003 mcyB1(C) cluster mcyB1(B) cluster  MC-RR  MC-LR

Purpose of cyanotoxins?  Secondary metabolites  no role in primary metabolism, growth or reproduction, but have somehow evolved to benefit the organism  includes alkaloids, polyketides and nonribosomal peptides  Anti-grazer, antibacterial, antifungal  Chemoattractant for microbial community  Unlikely that production of such a major cellular constituent would be retained through evolution if not of biological significance

HAB GROWTH NUTRIENTS GRAZING WIND SPEED WATER TEMPERATURE LIGHT MICROCYSTIN PRODUCTION Environmental factors influencing growth and toxin production in Microcystis

 Microcystin concentrations above the WHO drinking water standards are common in Saginaw Bay and western Lake Erie  Highest close to lake edges where increased human exposure  Multiple strains of Microcystis are present and toxicity may be related to genetic composition of community  Designed assays for detecting toxic strains  Currently working to identify environmental conditions that regulate microcystin production Summary

 HPLC (high performance liquid chromatography)  Distinguishes between variants  Based on retention times  Dependent on availability of standards  Lab-based  Detection limit: ~0.1 µ g/L Measuring microcystins Peak # 1 - RR 2 - LA 3 - YR 6 - LR Harada et al 1999