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
 Over 500 beaches for swimming and recreation
Common cyanobacterial HAB genera
in the Great Lakes

Microcystis Aphanizomenon Oscillatoria

Anabaena Cylindrospermopsis
Microcystis in the Great Lakes

Lake Erie, Put-In-Bay, Lake Erie, South Bass Island,

Sept 2006 Sept 2006
 Microcystis in the Great Lakes
1970 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)

1980

Decrease in chlorophyll, increased water clarity

Blooms rare
1990
Dreissenid mussel introduction

Return of Microcystis blooms

up to 90% phytoplankton community
2000

Present 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 Nutrient excretion

• selective rejection of • provide sufficient energy
toxic Microcystis strains for growth of toxic strains
• consume more palatable • change N:P ratios
species

Zebra mussels
 Current projects
 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
Microcystis sp.
What makes a cyanobacterial bloom toxic?
 Shift in community composition

Mostly non-toxic Mostly toxic strains

 Stimulation of toxin production by environmental factors

Light
Nutrients
Temperature
Trace metals

Not producing toxin Producing toxin

What makes a cyanobacterial bloom toxic?
 Shift in community composition
10-1000 fold
change in toxicity

Mostly non-toxic Mostly toxic strains

 Stimulation of toxin production by environmental factors

2-10 fold
change in toxicity

(Zurawell et al
Not producing toxin Producing toxin 2005)
Intracellular total microcystin by HPLC

August
2004

Saginaw Bay

Put-In-Bay 58 µg L-1
western Lake Erie
 Distribution of microcystin congeners

Relative toxicity:
- LR and –LA = 4x more toxic than –YR
10x more toxic than -RR
Advantages of molecular techniques

 Differentiate morphologically identical strains

 toxic and non-toxic strains

 Identify geographic origin of strains and

genetic diversity of populations

 Rapid detection
 Identifying toxic strains of
Microcystis
 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

Pearson et al 2004
 Multiplex PCR for toxic Microcystis

Saginaw Bay western Lake Erie

M

mcyB
ITS

M = molecular
weight marker

Number of colonies % microcystin

Basin # mcyB total (ITS) producers
Saginaw 36 40 90%
Erie 4 16 25%
Distribution of toxic Microcystis

Stations with
mcyB

km

0 10 20
 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
Cylindrospermopsis specific nifH primers

L. Jesup
+
Distribution of C. raciborskii in the US
 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

 western
identifying conditions under Lake
which Erie
cells are actively
producing toxin (expressing mcyB)
 zebra mussel grazing
 changes in nutrients and light

Maumee Bay
 Tie into circulation models to predict distribution of toxic
Microcystis strains and forecast water quality
 Goal
 Develop predictive capabilities for presence of
toxic cyanobacterial blooms in Great Lakes
recreational and drinking water supplies
Thanks …….
 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
 Microcystin concentrations in Perch
Lake Erie, summer 2006
ng toxin (g dry mass)-1

Monthly averages Monthly averages

2 1000

Toxins (ng g-1)

Toxins (ng g-1)

800

600
1
400

200
22 24 22 9 22 24 22 99
0 0
22 24 22
Jun Jul Aug Oct Jun Jul Aug Oct

Muscle Liver

Microcystin concentration of concern

for routine fish consumption = 7.7 ng g-1
Phylogenetic tree Erie D7

based on mcyB
99 Erie F2

56
Erie F2B
Erie D2
Microcystis botrys
93
Microcystis aeruginosa)
69 Saginaw C3E
91
79
Saginaw C3E
Saginaw A4W
mcyB1(C)
100 Erie D2 cluster
Microcystis aeruginosa
Microcystis viridis  MC-RR
Erie F2B
54
Microcystis aeruginosa
87
Saginaw A4W
Erie F4
98 Erie E2
Microcystis aeruginosa
100 Microcystis botrys
64
Erie D2
100
Microcystis aeruginosa
70 Microcystis sp.
Saginaw C5 mcyB1(B)
Saginaw C3E
Saginaw B1
cluster
Saginaw B1
Saginaw A4W
 MC-LR
Saginaw B1
Saginaw C5
100 Erie D7
Erie F2B
0.1 substitutions per site
Designations according to Mikalsen et al 2003
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
Environmental factors influencing growth and
toxin production in Microcystis

NUTRIENTS

MICROCYSTIN
LIGHT
PRODUCTION
HAB
WATER GROWTH
TEMPERATURE

WIND SPEED

GRAZING
 Summary
 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

 Measuring microcystins
 HPLC (high performance liquid chromatography)
 Distinguishes between variants
 Based on retention times
 Dependent on availability of standards

 Lab-based
 Detection limit: ~0.1 µg/L

Peak #
1 - RR
2 - LA
3 - YR
6 - LR

Harada et al 1999