genetic and environmental factors influencing microcystis bloom toxicity juli dyble noaa great lakes...
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Genetic and environmental factors influencing Microcystis bloom toxicity
Juli DybleNOAA
Great Lakes Environmental Research LabAnn Arbor, MI
Great Lakes
Saginaw Bay
western Lake Erie
Great Lakes as an aquatic resource
Largest supply of freshwater in the worldLargest supply of freshwater in the world 80% of US freshwater supply 80% of US freshwater supply Drinking water supply for 40 million US and Canadian Drinking water supply for 40 million US and Canadian citizenscitizens Over 500 beaches for swimming and recreation Over 500 beaches for swimming and recreation
Common cyanobacterial HAB genera in the Great Lakes
Cylindrospermopsis Anabaena
Microcystis OscillatoriaAphanizomenon
Lake Erie, South Bass Island, Sept 2006
Lake Erie, Put-In-Bay, Sept 2006
Microcystis in the Great Lakes
MicrocystisMicrocystis in the Great Lakes in the Great Lakes
Present
1970
1980
1990
2000
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 clarityBlooms rare
Dreissenid mussel introduction
Return of Microcystis blooms up to 90% phytoplankton community
Abundant Microcystis blooms, July - Sept
Zebra mussel (Zebra mussel (Dreissena polymorphaDreissena polymorpha))Quagga mussel (Quagga mussel (Dreissena bugensisDreissena bugensis))Introduced by ballast water from eastern EuropeIntroduced by ballast water from eastern Europe
Impacts of zebra mussels on Impacts of zebra mussels on MicrocystisMicrocystis
promote growth of toxic promote growth of toxic Microcystis Microcystis strains 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 Map microcystin concentrations and MicrocystisMicrocystis cell numbers in Saginaw Bay cell numbers in Saginaw Bay and western Lake Erie and western Lake Erie
Identify environmental factors promoting Identify environmental factors promoting microcystin productionmicrocystin production
Develop rapid methods for detection of toxic Develop rapid methods for detection of toxic MicrocystisMicrocystis
Accumulation in fishAccumulation in fish
Current projects
Microcystis sp.
What makes a cyanobacterial bloom toxic?
Shift in community compositionShift in community composition
Mostly non-toxic Mostly toxic strains
Not producing toxin
LightNutrients
TemperatureTrace metals
Producing toxin
Stimulation of toxin production by environmental Stimulation of toxin production by environmental factorsfactors
What makes a cyanobacterial bloom toxic?
Shift in community compositionShift in community composition
Not producing toxin Producing toxin
Stimulation of toxin production by environmental Stimulation of toxin production by environmental factorsfactors
Mostly non-toxic Mostly toxic strains
10-1000 fold change in toxicity
2-10 fold change in toxicity
(Zurawell et al(Zurawell et al
2005)2005)
Intracellular total microcystin by HPLC
Saginaw Bay
August 2004
western Lake EriePut-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 strainsDifferentiate morphologically identical strains toxic and non-toxic strainstoxic and non-toxic strains
Identify geographic origin of strains and Identify geographic origin of strains and genetic diversity of populationsgenetic diversity of populations
Rapid detection Rapid detection
Advantages of molecular techniques
All toxin-producing strains of All toxin-producing strains of Microcystis Microcystis contain contain genes for microcystin production: genes for microcystin production: mcyA-JmcyA-J
Presence of Presence of mcyB mcyB = strain able to produce toxin= strain able to produce toxin
Absence of Absence of mcyB mcyB = non-toxic= non-toxic
Identifying toxic strains of Microcystis
Pearson et al 2004
Multiplex PCR for toxic Microcystis
M Saginaw Bay western Lake Erie
mcyBITS
M = molecular weight marker
Number of colonies Basin # mcyB total (ITS) Saginaw 36 40 Erie 4 16
% microcystin producers 90% 25%
Distribution of toxic Microcystis
100 20
km
Stations withmcyB
Distribution of C. raciborskii in the US
L. Y
ale
L. E
ust
is
Silv
er L
.
L. G
riff
in
L. H
arri
s
Lit
tle
L. H
arri
s
Tro
ut
L.
L. D
ora
L. B
eau
clai
r
L. O
la
L. C
arlt
on
L. M
on
roe
L. J
esu
p
+ + + + + + + + + +
Cylindrospermopsis specific nifH primers
Quantitative PCR for enumerating Quantitative PCR for enumerating toxic toxic Microcystis Microcystis coloniescolonies
ApplicationsApplications
measure temporal variation in proportion of toxic strains measure temporal variation in proportion of toxic strains biweekly sampling at 3 locations in western Lake Eriebiweekly sampling at 3 locations in western Lake Erie
identifying conditions under which cells are actively identifying conditions under which cells are actively producing toxin (expressing producing toxin (expressing mcyBmcyB))
zebra mussel grazingzebra mussel grazing changes in nutrients and light changes in nutrients and light
Tie into circulation models to predict distribution of toxic Tie into circulation models to predict distribution of toxic Microcystis Microcystis strains and forecast water qualitystrains and forecast water quality
western Lake Erie
Maumee Bay
Goal Develop predictive capabilities for presence of Develop predictive capabilities for presence of toxic cyanobacterial blooms in Great Lakes toxic cyanobacterial blooms in Great Lakes recreational and drinking water suppliesrecreational 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 PerchLake Erie, summer 2006 ng toxin (g dry mass)-1
Jun Jul Aug Oct
Tox
ins
(ng
g-1)
Monthly averages
22 24 22 90
200
400
600
800
1000
22 24 22 9
Liver
Jun Jul Aug Oct
Tox
ins
(ng
g-1)
Monthly averages
0
1
2
22 24 22 9
Muscle
Phylogenetic tree
based on mcyB
Designations according to Mikalsen et al 2003
Erie D7
Erie F2
Erie F2B
Erie D2
Microcystis botrys
Microcystis aeruginosa)
Saginaw C3E
Saginaw C3E
Saginaw A4W
Erie D2
Microcystis aeruginosa
Microcystis viridis
Erie F2B
Microcystis aeruginosa
Saginaw A4W
Erie F4
Erie E2
Microcystis aeruginosa
Microcystis botrys
Erie D2
Microcystis aeruginosa
Microcystis sp.
Saginaw C5
Saginaw C3E
Saginaw B1
Saginaw B1
Saginaw A4W
Saginaw B1
Saginaw C5
Erie D7
Erie F2B
99
98
56
93
79
69
91
87
54
100
100
70
64
100
100
0.1 substitutions per site
mcyB1(C)cluster
mcyB1(B)cluster
MC-RR
MC-LR
Purpose of cyanotoxins? Secondary metabolites Secondary metabolites
no role in primary metabolism, growth or reproduction, no role in primary metabolism, growth or reproduction, but have somehow evolved to benefit the organismbut have somehow evolved to benefit the organism
includes alkaloids, polyketides and nonribosomal includes alkaloids, polyketides and nonribosomal peptidespeptides
Anti-grazer, antibacterial, antifungalAnti-grazer, antibacterial, antifungal
Chemoattractant for microbial communityChemoattractant for microbial community
Unlikely that production of such a major cellular Unlikely that production of such a major cellular constituent would be retained through evolution if constituent would be retained through evolution if not of biological significancenot of biological significance
HAB GROWTH
NUTRIENTS
GRAZING
WIND SPEED
WATER TEMPERATURE
LIGHTMICROCYSTINPRODUCTION
Environmental factors influencing growth and toxin production in Microcystis
Microcystin concentrations above the WHO drinking water standards are common in Saginaw Microcystin concentrations above the WHO drinking water standards are common in Saginaw Bay and western Lake ErieBay and western Lake Erie Highest close to lake edges where increased human exposureHighest close to lake edges where increased human exposure
Multiple strains of Multiple strains of Microcystis Microcystis are present and toxicity may be related to genetic composition of are present and toxicity may be related to genetic composition of communitycommunity Designed assays for detecting toxic strainsDesigned assays for detecting toxic strains
Currently working to identify environmental conditions that regulate microcystin productionCurrently working to identify environmental conditions that regulate microcystin production
Summary
HPLCHPLC (high performance liquid chromatography)(high performance liquid chromatography)
Distinguishes between variantsDistinguishes between variants Based on retention timesBased on retention times Dependent on availability of standardsDependent on availability of standards
Lab-basedLab-based Detection limit: ~0.1 Detection limit: ~0.1 µµg/Lg/L
Measuring microcystins
Peak #1 - RR2 - LA3 - YR6 - LR
Harada et al 1999