Chemical Ecology of
Toxic Algae
Bernd Krock, Urban Tillmann, Uwe John, Sára Beszteri,
Chishimba M. Kantu, Allan D. Cembella
Toxic Algal Blooms
Distribution of Paralytic Shellfish Poisoning events
2005
1970
Lytic Effect of Alexandriumshown withOxyrrhis marina.
Black arrows: Alexandrium
Red arrows: Remainders of Oxyrrhis
Alexandrium tamarense Oxyrrhis marina
Photos: U. Tillmann
Lytic Effect of Alexandrium
Lytic Effect of A. ostenfeldii
Allelochemical potency is not related to spirolide production
Oxyrrhismarina% intactcells
10 100 1000 10000
A. ostenfeldiicell concentration (ml-1)
10 100 1000 10000
0 20 40 60 80 100 120
A
K-0287
10 100 1000 10000
0 20 40 60 80 100 120
B
BAHME136
0 20 40 60 80 100 120
C
AOSH2
A. ostenfeldii strain
0 2 4 6 8 10
AOSH2 BAHME136 K-0287
spirolides (pgcell-1 ) Not detectable Not detectable
Chemical Interaction Ecological Function Organism
Alexandrium tamarense Alexandrium minutum Alexandrium ostenfeldii
PSP-Toxins
N
N N
H H N
NH2
OH OH O
H2N R1
R2
R3
R4
?
Alexandrium tamarense Prymnesium parvum
Defense against Predators, Elimination of Competitors
?
O O
N
O
O O OH
HO
2 3
31
13
Spirolides
Towards Inter-disciplinary Science
Biological, Chemical &
Geological Oceanography
Natural Products &
Bioanalytical Chemistry
Biodiagnostics
& Ecotoxicology
Marine Biology,
Chemistry & Ecology Molecular Ecology
Marine Chemical
Ecology
Genomics, Proteomics &
Metabolomics
Strategies to answer these questions:
Can toxic strains be detectected genetically?
=> Genomic characterization (microsatellites, AFLP, rDNA sequence analysis)
What effects do toxins have?
=> Toxicological assays
What are the allelochemicals?
=> Chemical experiments & bioassays What toxins are present?
=> Bioanalytics (LC-FD, LC-MS/MS)
Which genes are responsable for growth and toxicity?
=> Gene expession analysis (EST, Data bases, microarrays)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Alexan drium os
tenfe ldii
Chryso chromuli
na polylepis
Fragil ariaops
iscylind rus
Phaeod
actylumtricornutu m
Laminar iadigitata
Arab idops
istha liana
Popu lus sp.
Coleopte
Proportion of homologousprotein Sequencesamongdifferent species ra
known unknown Algae Higher plants and animals
Genomic Characterization
John et al. 2004
10
Pseudomonas fluorescens
Desulfovibrio desulfuricans
Cryptosporidium parvum Pfiesteria shumwayae Alexandrium ostenfeldii
Microcystis aeruginosa
Anabaena sp.
Nostoc sp.
PKS EST Analysis
Genomic Characterization
16 1
4 1
18
4 3 7 7
5 11 7
2 4
6 2 3 3
Metabolism
Cellular processes
Stress, defence and toxicity
Information storing and processing
General function (prediction only) Cell structure
5 psu
0 20 40 60 80 100 120
1000 10000 100000 1000000
Prymnesium/ml
Rhodomonas viability %
control (26 psu)
0 20 40 60 80 100 120
1000 10000 100000 1000000
Rhodomonas viability%
Gene Expression Analysis
=> ESTs, Microarrays
Early exponential growth phase Late exponential growth phase
Stationary phase
Toxicity of Prymnesium parvum is high at low salinity
Bioanalytics
10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 14,5 15,0
Time, min 0,0
2,0e5 4,0e5 6,0e5 8,0e5 1,0e6 1,2e6 1,4e6 1,6e6 1,8e6 2,0e6 2,2e6 2,4e6 2,6e6 2,8e6 3,0e6 3,2e6 3,4e6 3,6e6 3,8e6 4,0e6 4,2e6 4,4e6
12,38
12,00
12,85
O O
N
O
O O
HO
2 3
32
13 HO
17
20
20-Me Spirolide G
O O
N
O
O O OH
HO
2 3
31
13
Spirolide C
604.5 > 356.3 640.5 > 164.1 650.5 > 164.1 692.5 > 150.1 692.5 > 164.1 694.5 > 164.1 706.5 > 164.1 708.5 > 164.1 720.5 > 164.1
Alexandrium ostenfeldii strain AOSH2
?
?
? ?
? ?
?
0 20 40 60 80 100 120
Control After Evaporation
undiluted 1:10 dilution
Rhodomonasmortality
Chemical Exeriments – Evaporation
Alexandrium tamarense supernatant – Lytic Effect on Rhodomonas
Chemical Exeriments – Lyophilization
0 20 40 60 80 100 120
0,0 0,1 1,0 10,0 100,0
% sample in bioassay
Rhodomonas(% of control)
original supernatant supernatant lyophilized and resuspended
EC50 original: 0.31 % EC50 „instant“: 0.44 %
Alexandrium tamarense supernatant – Lytic Effect on Rhodomonas
0 20 40 60 80 100 120
0,1 1,0 10,0 100,0
0 20 40 60 80 100 120
0,1 1,0 10,0 100,0
15°C; light (150 µE m-2 s-1)
15°C; dark
t = 0 t = 1d
t = 4d t = 7d
t = 12d t = 20d
t = 49d
Chemical Exeriments – Stability
% sample in bioassay
% sample in bioassay
Rhodomonas(% of control) Rhodomonas(% of control)
Alexandrium tamarense supernatant – Lytic Effect on Rhodomonas
0 2000 4000 6000 8000 10000
0,1 1,0 10,0 100,0
% sample in bioassay Rhodomonas(ml-1 )
A. tamarense culture 8.0 µm filter
5.0 µm filter 3.0 µm filter 1.2 µm filter 0.4 µm filter 0.2 µm filter 0.1 µm filter GF/C filter
Chemical Exeriments – Filterability
Alexandrium tamarense supernatant – Lytic Effect on Rhodomonas
Toxicological assays
Viability curve of N2a exposed to Ethanol
0.25 0.50 0.75 1.00 1.25 1.50 0
10 20 30 40 50 60 70 80 90 100
EC50= 6.5 % Ethanol
Log [% Ethanol]
Viability % control
Alexandrium ostenfeldii extract – Toxic Effect on Neoblastoma cells
Control A. ostenfeldii cell extracts
Toxicological assays
Control DesMe C A. ostenfeldii
Intermediate Cytotoxicity
A. ostenfeldii High
Cytotoxicity
Alexandrium ostenfeldii extract – Expression of stress and toxicity related genes (GEArray Q Series Mouse Stress & Toxicity Pathway Finder)
Thank You for
Your Attention!
AWI
Allan D. Cembella Uwe John
Urban Tillmann Bernd Krock
Tilman Alpermann Sascha Klöpper Ines Jung
Sára Beszteri Nina Jaeckisch Ines Marschallek Chishimba M. Kantu Chibo Chikwililwa Annegret Müller Wolfgang Drebing
GKSS
Andreas Prange Jürgen Gandraß Sandra Schäfer Beritt Schwalger
