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