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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
пЃ¶
Largest supply of freshwater in the world
пЃµ
80% of US freshwater supply
Drinking water supply for 40 million US and Canadian
citizens
пЃµ Over 500 beaches for swimming and recreation
пЃµ
Common cyanobacterial HAB genera
in the Great Lakes
Aphanizomenon
Microcystis
Anabaena
Oscillatoria
Cylindrospermopsis
Microcystis in the Great Lakes
Lake Erie, Put-In-Bay,
Sept 2006
Lake Erie, South Bass Island,
Sept 2006
Microcystis in the Great Lakes
1970
1980
1990
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
2000
Present
Abundant Microcystis blooms, July - Sept
пЃ¶Zebra
mussel (Dreissena polymorpha)
пЃ¶Quagga mussel (Dreissena bugensis)
пЃ¶Introduced by ballast water from eastern Europe
Impacts of zebra mussels on
Microcystis
пЃ¶
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
Current projects
пЃ¶
Map microcystin concentrations and
Microcystis cell numbers in Saginaw Bay
and western Lake Erie
пЃ¶
Identify environmental factors promoting
microcystin production
пЃ¶
Develop rapid methods for detection of toxic
Microcystis
пЃ¶
Accumulation in fish
Microcystis sp.
What makes a cyanobacterial bloom toxic?
пЃ¶
Shift in community composition
Mostly toxic strains
Mostly non-toxic
пЃ¶
Stimulation of toxin production by environmental factors
Light
Nutrients
Temperature
Trace metals
Not producing toxin
Producing toxin
What makes a cyanobacterial bloom toxic?
пЃ¶
Shift in community composition
10-1000 fold
change in toxicity
Mostly non-toxic
пЃ¶
Mostly toxic strains
Stimulation of toxin production by environmental factors
2-10 fold
change in toxicity
Not producing toxin
Producing toxin
(Zurawell et al
2005)
Intracellular total microcystin by HPLC
August
2004
Saginaw Bay
western Lake Erie
Put-In-Bay 58 Вµg L-1
100%100%
-YR -YR
% of total HPLC microcystin
% of total HPLC microcystin concentration
Distribution of microcystin congeners
90% 90%
80% 80%
-RR -RR
70% 70%
-LA -LA
60% 60%
-LR -LR
50% 50%
40% 40%
30% 30%
20% 20%
10% 10%
0% 0%
Saginaw
Saginaw
Erie Erie
Relative toxicity:
- LR and –LA = 4x more toxic than –YR
10x more toxic than -RR
Advantages of molecular techniques
пЃ¶
Differentiate morphologically identical strains
пЃµ toxic and non-toxic strains
пЃ¶
Identify geographic origin of strains and
genetic diversity of populations
пЃ¶
Rapid detection
Identifying toxic strains of
Microcystis
пЃ¶
All toxin-producing strains of Microcystis contain
genes for microcystin production: mcyA-J
пЃ¶
Presence of mcyB = strain able to produce toxin
Absence of mcyB = non-toxic
Pearson et al 2004
Multiplex PCR for toxic Microcystis
M
Saginaw Bay
western Lake Erie
mcyB
ITS
M = molecular
weight marker
Basin
Saginaw
Erie
Number of colonies
# mcyB
total (ITS)
36
40
4
16
% microcystin
producers
90%
25%
Distribution of toxic Microcystis
Stations with
mcyB
km
0
10
20
+
Cylindrospermopsis specific nifH primers
L. Jesup
L. Monroe
+ +
L. Carlton
L. Ola
L. Beauclair
+ + ++ + +
L. Dora
L. Harris
Little L. Harris
Trout L.
Silver L.
L. Griffin
L. Yale
L. Eustis
Distribution of C. raciborskii in the US
+
Quantitative PCR for enumerating
toxic Microcystis colonies
Applications
пЃ¶
measure temporal variation in proportion of toxic strains
пЃµ
пЃ¶
biweekly sampling at 3 locations in western Lake Erie
identifying conditions under
which
cells
are actively
western
Lake
Erie
producing toxin (expressing mcyB)
zebra mussel grazing
пЃµ changes in nutrients and light
пЃµ
пЃ¶
Maumee Bay models to predict distribution of toxic
Tie into circulation
Microcystis strains and forecast water quality
Goal
пЃ¶
Develop predictive capabilities for presence of
toxic cyanobacterial blooms in Great Lakes
recreational and drinking water supplies
Thanks …….
пЃ¶
Center of Excellence for Great Lakes and Human
Health (Oceans and Human Health Initiative)
пЃ¶
пЃ¶
Gary Fahnenstiel (NOAA-GLERL)
Hank Vanderploeg (NOAA-GLERL)
Pat Tester, Wayne Litaker (NOAA-Beaufort)
Dave Millie (Florida Institute of Oceanography)
пЃ¶
Crew of the R/V Laurentian
пЃ¶
пЃ¶
Microcystin concentrations in Perch
Lake Erie, summer 2006
ng toxin (g dry mass)-1
Monthly averages
Monthly averages
1000
Toxins (ng g-1)
Toxins (ng g-1)
2
1
22
24
22
9
Jun
Jul
Aug
Oct
0
800
600
400
200
0
Muscle
22
22
24
24
22
22
99
Jun
Jul
Aug
Oct
Liver
Microcystin concentration of concern
for routine fish consumption = 7.7 ng g-1
Phylogenetic tree
based on mcyB
Erie D7
Erie F2
99
Erie F2B
56
Erie D2
Microcystis botrys
93
69
Saginaw C3E
mcyB1(C)
cluster
Saginaw C3E
91
79
100
Microcystis aeruginosa)
Saginaw A4W
Erie D2
Microcystis aeruginosa
пѓ MC-RR
Microcystis viridis
Erie F2B
54
87
Microcystis aeruginosa
Saginaw A4W
Erie F4
98
Erie E2
Microcystis aeruginosa
100
64
100
Microcystis botrys
Erie D2
Microcystis aeruginosa
70
Microcystis sp.
Saginaw C5
Saginaw C3E
Saginaw B1
Saginaw B1
Saginaw A4W
mcyB1(B)
cluster
пѓ 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?
пЃ¶
Secondary metabolites
пЃµ
пЃµ
no role in primary metabolism, growth or reproduction,
but have somehow evolved to benefit the organism
includes alkaloids, polyketides and nonribosomal
peptides
пЃ¶
Anti-grazer, antibacterial, antifungal
пЃ¶
Chemoattractant for microbial community
пЃ¶
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
LIGHT
HAB
GROWTH
WATER
TEMPERATURE
WIND SPEED
GRAZING
MICROCYSTIN
PRODUCTION
Summary
пЃ¶
Microcystin concentrations above the WHO
drinking water standards are common in
Saginaw Bay and western Lake Erie
пЃµ
пЃ¶
Multiple strains of Microcystis are present and
toxicity may be related to genetic composition of
community
пЃµ
пЃ¶
Highest close to lake edges where increased human
exposure
Designed assays for detecting toxic strains
Currently working to identify environmental
conditions that regulate microcystin production
Measuring microcystins
пЃ¶ HPLC (high performance liquid chromatography)
пЃµ
Distinguishes between variants
Based on retention times
пЃ¶ Dependent on availability of standards
пЃ¶
Lab-based
пЃµ Detection limit: ~0.1 Вµg/L
пЃµ
Peak #
1 - RR
2 - LA
3 - YR
6 - LR
Harada et al 1999
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