Revealing the
Allelochemical Potential of Alexandrium ostenfeldii,
a Marine Dinoflagellate
Bernd Krock, Urban Tillmann, Uwe John, Nina Jaekisch, Allan D. Cembella
VP
Ocurrence:
Atlantic (Canada, USA, Iceland, Faroe Islands, Spain)
North Sea (Scottland, Norway, Denmark)
Mediterrean Sea (Italy, Egypt)
Pacific (USA, Russia, New Zealand)
Alexandrium ostenfeldii
Marine gonyaulacoid mixotroph dinoflagellate in temperate waters
1904: A. ostenfeldii first described as Goniodoma ostenfeldii by Paulsen in Iceland in 1904
A. Ostenfeldii – Short History
Nova Scotia
Shelburne
Ship Harbour Mahone
July 1990, 1991: Unusual mouse-deaths - lipophilic mussel extracts from Ship Harbour, Nova Scotia, Canada: “fast acting toxin” (FAT) symptoms and coincident consumer complaints of mild illness after shellfish consumption
O O
N
O
O O OH
HO 2
3
31
13
1995: Structural elucidation of spirolides
VP 1996: Identification of A. ostenfeldii as spirolide producing organism
PHOTOPERIOD
IRRADIANCE
TEMPERATURE
NUTRIENTS
TURBULENCE
ALLEOCHEMICALS SALINITY
EXTRINISIC FACTORS
PKS
GROWTH ALTERNATE LIFE HISTORIES
MITOSIS
H3N1 CH2 COO
O
acetate
C13 O
glycine
H3C1
O O
N
O
O O OH
HO 2
3
31
13
Regulation of Spirolide Biosynthesis
Toxin production is related to
extrinsic factors Toxin composition is maintained, presumably genetically determined
Effect of grazing pressure and competition with other microalgae needs further
investigation
carbon skeleton is produced by polyketide synthases (PKS) out of acetate units
Stable Isotope Feeding of A. ostenfeldii
13-desmethyl Spirolide C is a polyketide derived compound
O O
N
O H
O O
O
O H Origin
undetermined
N
Intact C2N unit from glycine Origin acetate
Genetic Analysis
Generation of a normalized cDNA library of A.
ostenfeldii.
10
Pseudomonas fluorescens
Desulfovibrio desulfuricans
Cryptosporidium parvum Pfiesteria shumwayae Alexandrium ostenfeldii
Microcystis aeruginosa
Anabaena sp.
Nostoc sp.
PKS EST Analysis
Secuencing of approx. 5000 clones.
Successful attribution of 15% of the clones to gene functions of almost all expected functional categories.
Identification of eight genes related to stress, defence and toxicity (putative PKS genes).
O O
N
O
O O OH
HO
2 3
31
13 R
Spirolide A: R = H, Δ2,3 B: R = H
C: R = Me, Δ2,3 D: R = Me
Spirolide G: R = H 20-Me Spirolide G: R = Me
O O
N
O
O O
HO
2 3
32
13
HO
17
20
R
13-DesMeSpirolide C: R = Me 13,19-DidesMe Spirolide C: R = H
O O
N
O
O O OH
HO
2 3
31
13
R
Spirolide Variability
1 0, 5 1 1, 0 11, 5 12, 0 1 2, 5 1 3, 0 13, 5 14, 0 14 , 5 1 5, 0 T i m e, m i n
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 1 1, 51
1 0, 5 1 1, 0 11, 5 12, 0 12 , 5 1 3, 0 13, 5 14, 0 14 , 5 1 5, 0
T i m e, m i n 0, 0
1, 0e5 2, 0e5 3, 0e5 4, 0e5 5, 0e5 6, 0e5 7, 0e5 8, 0e5 9, 0e5 1, 0e6 1, 1e6 1, 2e6 1, 3e6 1, 4e6 1, 5e6 1, 6e6 1, 7e6 1, 8e6 1, 9e6 2, 0e6 2, 1e6
11, 95
10, 5 11, 0 11, 5 1 2, 0 1 2, 5 1 3, 0 13 , 5 14, 0 14, 5 15, 0
T i m e, m i n 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 OH
HO
2 3
31
13
13-desMe Spirolide C
O O
N
O
O O OH
HO
2 3
31
13
Spirolide C
Spirolide Variability
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 2
3
31
13 HO
19
13,19-didesMe Spirolide C
CCMP 1773, Denmark
AOSH1, Canada
AOSH2, Canada
Lytic Effect of Alexandriumshown withOxyrrhis marina.
Black arrows: Alexandrium
Red arrows: Remainders of Oxyrrhis
Alexandrium ostenfeldii Oxyrrhis marina
Lytic Effect of A. ostenfeldii
Heterotrophs
Scale bar = 10 µm Oxyrrhis marina
Amphidinium crassum
Rimostrombidium caudatum
Gyrodinium spirale
Autotrophs
Rhodomonas baltica.
Dunaliella salina
Thalassiosira weisflogii
Scripsiella trochoidea
Microscopical observations of Alexandrium lytic effects on different target species
Lytic Effect of A. ostenfeldii
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
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
Lytic Activity of Extracellular Compounds – 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 (1 µm)
Lytic Activity – Filterability
Alexandrium tamarense supernatant – Lytic Effect on Rhodomonas
Secondary Metabolite Ecological Function
?
Defense against Predators Elimination of Competitors
?
O O
N
O
O O OH
HO
2 3
31
13
Spirolides, marine toxins
