toxin production in alexandrium tamarense investigating ... · toxin production in alexandrium...
TRANSCRIPT
![Page 1: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/1.jpg)
1
University of Bremen and
Max Planck International Research School for Marine
Microbiology
Toxin production in Alexandrium tamarense:
Investigating clonal variation and extra-cellular
bacterial effects
Chibola Chikwililwa
Master Thesis
First reviewer: Allan Cembella
Second reviewer: Uwe John
![Page 2: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/2.jpg)
2
Index
1 . Summary 4
2. Introduction 5 3. Thesis object ives 9
4 . Materials and methods 10
4 .1 Sampling s i te 11
4 .2 Strains and cul ture condi t ions 11
4 .2 .1 Axenic and xenic cul tures 11
4 .2 .2 Sub-clone cul tures 13
4 .3 Sampling procedure 14
4.3.1 Axenic and xenic cul ture 14
4.3.2 Sub-clone cul tures 14
4.4 Cel l and bacter ial counts 15
4.4.1Cel l counts 15
4.4.2 Bacterial counts 15
4.5 PSP extract ion and determinat ion 16
4.6Allelochemical t es ts 21
4 .7Amplif ied Fragment Length Polymorphism 22
4.7.1 DNA extract ion 22
4.7.2 DNA Restr ict ion 23
4.7.3 DNA Ligation 23
4.7.4 Preamplification 23
4.7.5 Amplification 24
4.7.6 Capillary electrophoresis 24
4.7.7 AFLP Analysis 23
5. Results 26
5.1 Axenicity of cultures 26
5.2Growth of Axenic and Xenic cultures 26
5. 3 Production of PSP toxins by axenic and xenic cultures 30
5.3.1 Toxin profiles 30
5.4 Toxin content verses cell growth in axenic and xenic cultures 34
5.5 Total toxin content 38
5.6 Allelochemical tests 39
5.7 Sub clones 39
5.7.1Toxin profiles 39
5.7.2 Variations in total toxicity 42
5.8 AFLP 45
![Page 3: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/3.jpg)
3
6. Discussion 49
7. Acknowledgments 59
8. References 60
![Page 4: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/4.jpg)
4
Su mma r y :
My thesis investigated the role of extra-cellular bacteria on the PSP toxin producing A
tamarense. Results showed that bacteria have no effect on the dinoflagellate growth rate.
The toxin profile remained relatively constant through out the experiment. Three
samples exhibited low production of GTX1/4. This could be further investigated to
determine if these cultures lose the ability to produce this STX derivative.
Bacteria have no effect allelochemical produced by A. tamarense. This effect was seen in
both lytic and non-lytic strains. The toxin profile remained constant for sub-clonal
cultures, signifying that PSP toxin production is in actual fact a genetically determined
characteristic. Variations in toxin content were observed, but they where not large
enough to claim that PSP toxigenesis was non chromosomally linked.
There is still
![Page 5: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/5.jpg)
5
1 ) In t r odu ct ion :
M a r i n e e c os ys t em s h os t a l ga e t h a t p l ay a c r i t i c a l r o l e a s t h e so u r c e
o f p r i m ar y p r o du c t io n . T h e y n o t on l y h a v e a b e ne f i c i a l ro l e , t h e y
c a n a l s o b e d e t r im en t a l . On e o f t h e w ays o f do in g t h i s i s b y c a u s in g
H a r m fu l Al ga l Bl oo ms (H A Bs) . H A Bs a r e a wid e sp r e ad m a r i n e
p h en om e na t h a t c on s i s t o f up t o m i l l i o ns o f c e l l s pe r l i t r e a nd c aus e
n um e ro us p r ob le ms to h um an h e a l th , f i sh e r i e s r es ou r c es a n d m ar in e
e c o s ys t e m s . Th e y o c c u r i n c o as t a l w a t e r s wo r l dw id e ( H a l l g r a e f f
1 9 93 ) a nd ex h ib i t va r yi n g e f f e c t s on th e a re a wh e r e t h e y a r e fo un d .
T h e i r e f f e c t r a n ge s f r om s t r i pp in g t h e w a t e r co lu mn o f oxyg e n ( d u e
t o t h e h i gh b iom a ss c a us i n g t h e d ea th o f mi c r o f lo r a a nd m a c ro f a un a )
t o p ro du c i n g p o t en t t ox i ns t h a t c au s e ga s t r o i n t es t i n a l an d
n e u ro l o g i c a l i l l n es s i n hu ma ns (H a l l gr a e f f 1 99 5 ) .
P a r a l yt i c sh e l l f i s h p o i so n i n g (P SP) i s o n e su c h n eu ro lo g ic a l i l l n es s .
I t i s s o c a l l e d du e to t h e s ym p t oms exh ib i t ed b y h i gh e r an im al s ,
p a r t i cu l a r l y m a mma l s an d b i r ds t h a t i n ge s t c on t am in a t e d sh e l l f i s h
( i . e . c on t ami n a t ed th r ou gh t h e a c cum ula t i on o f t he c au s a t iv e a l ga l
s p e c i es b y s u sp e ns io n f e e d in g in t o t h e t i s su e s o f s h e l l f i sh ) . T h es e
t ox in s wo r k b y b i nd in g to r e c e p to r s o f mu sc l e o r n e r ve ce l l s a nd
d i s r up t t he f l ow o f s od iu m i on s . Th i s i n t e r ru p t i on o f s i gna l
t r an smi s s io n i s w ha t ca us e s p a r a l ys i s . Du e to i t s s e r io us h um an
h e a l th an d e c on omic imp a c t i t h as b e en o f g r e a t im po r t a nc e t o s t ud y
t h e c au s a t i v e mi c roo r ga n i sm s .
T h e c a us a t iv e t ox i ns w er e fo un d t o s yn t h es i s ed b y a g r o up o f
m ic r o a l ga l sp e c i es , n am el y: G y mn o d ium c a t en a t um , P yr od in iu m
b a ha m en se an d th e m em b e rs o f t h e genu s Al e x an dr i u m (Ce m b el l a
1 9 98 )
![Page 6: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/6.jpg)
6
T h es e n e u r o t ox in s , i n c l ud in g s ax i tox in an d 20 o th e r c h em ic a l l y
r e l a t e d de r iv a t iv es d i f f e r i n ch em i c a l s t ru c t u re (G a l l a ch er & S mi t h
1 9 99 ) a nd v a r y i n t ox i c i t y .
T h e ge n us A l ex an dr iu m c on s i s t s o f 27 sp e c i e s , o f w h i c h 9 a r e kn ow n
t o p ro du c e PSP tox i ns i n va r yi n g a mou n t s . A l th ou gh i t i s a r gu ab l y
t h e b es t ch a r a c t e r i z e d h a rm f u l a l ga l gen us t o d a t e (C em b el l a 1 99 8 ,
S c ho l in 1 99 8) l i t t l e i s k no wn ab ou t t h e e nz ym e s a nd ge n es i n vo lv ed
i n t h e s yn t h e s i s o f PSP t ox ins (P l uml ey 1 9 9 7 ) .
S om e s c i en t i s t s h av e s u gge s t e d e ub a c t e r i a l i nv o l ve m en t i n t h i s
p r o c es s e i t h e r d i r ec t l y ( i . e . a s au t on om ou s PS T p ro du c e rs ) o r
i nd i r e c t l y ( i . e . b y a f f e c t in g th e g ro wt h r a t e , nu t r i en t u p t ak e , e t c . ) .
T h i s c on c ep t i s s t i l l s om e wh at co n t ro ve r s i a l . T h e on l y n on -
d in o f l a ge l l a t e sp p th a t a r e ge n e r a l l y a c c e p t e d to b e a b l e t o p r od u c e
PSP t ox ins au to nom ou s l y a r e s e v e ra l s p e c i es o f c ya n o b ac t e r i a ,
n a m el y; A p ha n i z o me n on f l o s - aq ua e ( J ak im a nd G en t i l 19 68 ; Mo ho od
a n d Ca r mi ch a e l 1 98 6 ) , A na ba e na c i r c i na l i s (H um p a ge e t a l . 1 99 4 ;
N e gr i e t a l . 1 99 7) a n d Ly ng b ya w o l l e i ( Ca rm i ch a e l e t a l . 1 9 97 ;
O n od e r a e t a l . 19 97 ) .
T h e no t i on o f b a c t e r i a e i t h e r p ro du c ing o r h a v i n g a n e f f ec t o n PS P
t ox i gen e s i s w as f i r s t h yp o t h es i z e d b y S i lv a ( 19 62 , 19 82 ) . I t s t em e d
f r om t h e f a c t t h a t sh e o bs e r ve d b a c t e r i a - l i k e s t ru c t u r es wi t h i n A.
t a mar e ns e ce l l s . Th i s w a s f o l l ow e d up b y N e l in d a (1 98 5) w ho
d e t ec t ed PSP tox i ns i n a n on tox i c d i no f l a ge l l a t e cu l tu r e a f t e r i t w as
t r an s f e r r e d i n t o m ed iu m f rom t ox ic cu l tu r es . Sh e a t t r i bu t e d t h i s t o
t h e ex t r a c e l l u l a r ba c t e r i a p r es en t i n t h e m e d iu m. Fu r th e r s t ud i e s
s ho w e d t h a t t h e r e w a s n o t a s i gn i f i c an t am ou n t o f t ox i ns t r a ns f e r r ed
b y e x t r a - ce l l u l a r ba c t e r i a . Th es e f i n d in gs d id n o t r e f u t e S i lv a ’ s
p os tu l a t i o n du e t o t h e f ac t t h a t h e r ob se r v a t io ns w e r e b ase d o n
i n t r a c e l l u l a r b ac t e r i a . Ko d am a ( 19 88 ; 1 9 90 ) f o cus e d on in t ra c e l lu l a r
![Page 7: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/7.jpg)
7
b a c t e r i a an d w as ab l e t o i so l a t e a b a c t e r iu m sp e c i e s c a l l ed
M o ra x e l la f r om a t ox i c s t r a i n o f A. t am a r ens e . I t s ho w ed th e ab i l i t y
t o a u to no mo us l y p r o d uc e PSP tox i ns . S e ve r a l p a p er s f o l l o w e d t h i s
b y c l a imi n g p r oo f o f t he ab i l i t y o f b ac t e r i a t o p r od u ce PSP t ox ins
( K o da m a e t a l . 19 88 , 19 90 ; O ga t a e t a l . 1 9 90 ; Do uc e t t e & T r i ck ,
1 9 95 ; Le v a s se u r e t a l . 19 96 ; S h imiz u e t a l . 1 99 6 ; Ko pp e t a l . 19 97 ;
G a l l a ch e r e t a l . 19 96 , 1 99 7) . M o re r e ce n t p ap e rs r e f u t i n g th e se
c l a i ms f o l l o w ed b y r e po r t i n g t h e i n co r r e c t i d e n t i f i c a t i o n o f b a c t e r i a l
m e t a bo l i t e s a s PSP tox i ns b y H P LC a na l ys i s ( N e gr i e t a l . 1 9 97 ,
O n od e r a e t a l . 19 97 , S a to e t a l . 1 99 8 , P e r e i r a e t a l . 2 00 0 a nd Ba ke r
e t a l . 2 00 3 ) . Th i s h a s l e a d to t h e co n t i nu a l co n t ro v e rs y s u r r ou nd i n g
a u to som a l o r a s s i s t e d b a c t e r i a l PSP t ox i ge n es i s . W i th i n t h i s wh o l e
d e b a t e , a n ew ap p ro a c h w as u s ed to de t e rmi n e th e e f f e c t o f
ex t r a c e l lu l a r b a c t e r i a o n d in o f l a ge l l a t e c u l t u r e t ox i c i t y; na m el y t h e
r e mo v a l o f t h e ex t ra c e l l u l a r mi c r o f l o ra w e r e us e d . T h e me t ho ds
e m plo ye d t o ob t a in ax en ic cu l tu r e s w er e ; s t e r i l e wa sh in g ( S i n gh e t
a l . 19 82 ; Kim e t a l . 1 99 3) an t ib io t i c s (D a n tz e r e t a l . 19 97 , Ho ld e t
a l . 2 00 1 ) an d l ys o z ym e / SDS t r e a tm e n t (D ou c e t t e an d Pow e l l 1 99 8 ) .
T h i s h as l e ad to an o n go in g d eb a t e o n th e ac tu a l e f f e c t s o f b a c t e r i a
o n PST t ox i gen e s i s w i th p a p er s p ro f es s in g a nd r e f u t i n g th i s a s pe c t .
A n o t he r u ns o lv e d as p e c t o f P SP t ox i gen e s i s i s t h e ge n e t i c c on t r o l o f
t ox in p ro du c t io n . Fe w s tu d i e s ha v e be e n co nd u c t ed t o de t e rmi n e th e
ge n e s i nv o lv e d i n t h i s . I t i s n o t c l e a r w h e t h e r ch r om os om al ,
m i to c ho nd r i a l , c h l o r o p l as t D NA o r n u c l e i c a c id s f ro m b a c t e r i a ,
p l asm id s o r v i rus e s a r e i n vo lv e d . On e o f t h e f i r s t s t ud i e s t o t a ck l e
t h i s w as c on du c t ed b y O ga t a e t a l . ( 198 7 ) . Th e y l o ok e d a t t he
t ox i c i t y o f n um e rou s su b - c lo n es o r i g in a t in g f r om on e c lon a l c u l t u r e
a n d f ou nd v a r i a t i o ns . Th e y a t t r i bu t ed t h i s t o t h e f a c t t h a t t ox i n
![Page 8: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/8.jpg)
8
p r od u c t i on i s no t he r e d i t a r y, t hu s l e a ds t o t h e v a r i a t i on ob s e rv e d .
K o d am a ( 19 90 ) ob se r v e d t h e s am e d i f fe r e n c es an d su gge s t e d t h a t
e n do nu c le a r b a c t e r i a w h er e as s o c i a t e d i n t ox in p r od u c t i on .
S oo n a f t e r , S ak o ( 19 9 5) an d Is h i d a (1 99 8 ) sh ow e d th a t t ox i ge n es i s
o f PSP tox i ns i s ch r o mo som al l y b a s e d , t hu s a h e r ed i t a r y c h a r a c t e r .
T h i s w as d on e b y c o n du c t i n g m at in g ex p e r im e n t s o n d i f f e r e n t
s t r a in s o f A. t a m are n s e a nd A . c a t en e l l a w i th d i f f e r in g PSP p r o f i l e s
a n d d e t e rmi n i n g wh e th e r t h e PSP p ro f i l e m e as u r ed w a s a
c o mbi n a t i on o f b o th pa r e n t s . T h e tox i n p ro f i l e wa s us e d a s a
p h en o t yp i c ch a r a c t e r i s t i c s i nc e t h e t ox in p r o f i l e i s t h ou gh t t o b e
r e a d i l y s t a b l e i n A le x a nd r i um s p ec i es ( C em b el l a . , e t a l 19 8 7 ; O ga t a
e t a l . , 1 98 7 ; Kim e t a l 19 93 ) . T he s e s t ud i e s sh o we d th a t t h e
p r o ge n y e x h ib i t ed a c om bin a t io n o f bo th p a r en t a l t ox in s a n d mo s t
s t r a in s sh ow e d a Me n d e l i a n p a t t e rn o f i n h er i t an c e . Th i s l e a d t o t h e
c o n c l us i on t ha t t h e ge n e s c od i n g fo r PS T p ro du c i n g enz ym e s wh e r e
o n n uc l ea r DN A. N o n e th e l e s s , no t a l l F1 p r o ge n y o n t h es e m at in g
ex p e r im e n t s ex h ib i t e d t h e M en d e l i a n p a t t e rn o f i n h er i t anc e . I t w a s
p r op os e d t h a t t h i s w a s d ue t o r e co mbin a t io n b e t w e en ge ne s .
O n e mo r e asp e c t o f PSP p ro du c t io n i s t h e r e l e va n c e o f t he s e t ox i ns
t o t h e o r ga n i s m. Mo s t s c i e n t i s t s v i ew t h es e t ox i ns a s s e co n da r y
m et a bo l i t e s (P l umle y 1 9 9 7 ) . Th i s t e rm wa s c o i ne d b y V i n i n g in
1 9 90 a nd d e f i n ed as “ com p ou nds t h a t d o n o t f u l f i l a ro l e i n
i n t e rm ed i a r y m e t abo l i s m ” L i t t l e i s kn o wn as t o t h e i mpo r t a n c e o f
t ox in p ro du c t io n wi th r e sp e c t t o t h e ca u s a t i v e o r ga n i sm . I t h a s b e en
s u gge s t e d th a t PSP tox i ns m a y p l a y a n in t r i n s i c r o l e i n DN A
m et a bo l i s m (M i ck e l so n & Y en t s ch 1 979 ; A nd e rs on & Ch en g 1 99 8)
o r a id i n n i t r o ge n s t o ra ge ( An d e rs on e t a l 19 90 ) o r a c t a s
p h e rom o ne s (W ya t t a nd J e nk ins on 1 997 ) t o p r om ot e m at in g o r a s
a n t i -p r ed a t io n / a l l e lo c h emi c a l co mp ou nd s (W hi t e & M a ra nd a 1 97 8 ;
H a n e y e t a l 19 95 ) . S i n c e t h e e f f e c t i v en e ss o f a n a l l e l oc he mi c a l i s
![Page 9: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/9.jpg)
9
i t s a b i l i t y t o b e r e l e a s e d i n t o t h e e nv i r o nm en t , PSP tox i ns d o no t f i t
i n t o t h i s de f in i t i o n ( i . e . PSP t ox ins a re k ep t i n t h e c e l l an d o n l y
r e l e as e d du r i n g s e ne s c e nc e ) . R e c en t s t ud i e s a r e i n d i c a t i ng t h es e
a l l e lo c he mi c a l s a l so c au s e an e f f e c t on s p e c i f i c mi c ro o r ga n i sm s .
A l l e l o ch e mi c a l a r e d e f i n ed as ch em ic a l s t h a t ca us e i nh i b i t i o n o r
s t im u l a t io n i n a no th e r o r ga n i sm du r ing t h e i r i n t e r a c t i on ( M ol i s ch
1 9 37 ) . A rz u l e t a l ( 1 99 8 ) t e s t ed t h e a l l e l o ch e mi c a l e f f e c t s o f t h r e e
t ox i c Al e xa nd r iu m s p e c i es an d f ou nd th a t t h e c e l l - f r e e f i l t r a t e s o f
t h es e c u l t u r es h ad a n a l l e lo ch e mi c a l e f f e c t on Ch a e t oc e ro s gr a c i l l e ,
G y m no d i n iu m m i k im o to i a nd S c r ip ps i e l l a t r o ch i od e a . Th e y
s u gge s t e d th a t t h i s e f f e c t w as d u e t o co mp ou nd s o t he r t han PSP
t ox in s . S ev e r a l p ape r s f ou nd t h a t t h i s w a s t ru e i n m or e r ec e n t c as e
s tu d i es . ( O ga ta & K o d am a 19 86 ; Lu s h & Ha l l e g r a e f f 199 6 ; T i l l ma nn
& J oh n 20 02 ) .
H o w ev e r n o th in g i s k no wn ab ou t t h e f a c t o r s a f f e c t i n g p ro d uc t io n o f
t h es e a l l e l o ch emi c a l b y A . t am ar e ns e . O n e o f t h e po ss i b i l i t i e s
c o u l d b e t h a t ex t r a - c e l l u l a r b ac t e r i a a r e i nv o l v ed in t h i s p r o c es s .
T h i s qu e s t i on w i l l b e ad d re s s e d in m y th e s i s .
![Page 10: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/10.jpg)
10
3 . M y t h e s i s p ro j e c t d e a l s wi th t h r e e as p e c t s r e l a t ed t o t ox in
p r od u c t i on i n A. t am a r ens e , n am el y:
1 ) D e t e rmi n in g th e e f f e c t s o f ex t r a - c e l l u l a r b a c t e r i a on t wo A .
t a mar e ns e s t a ins by o b s e r v in g th e g row t h r a t e , t ox in p ro f i l e
a n d ov e r a l l t ox in co n t e n t wi th in ax en ic a nd x e n i c c u l t u r es .
2 ) D e t e rmi n in g th e p os s i b l e ge n e t i c b i as o f t ox i gen e s i s b y
s tu d yi n g t h e ph e no t yp e ( t ox i n p r o f i l e ) o f su b -c lo n es .
D e t e rmi n in g o f t h i s v a r i a t i on c an b e s ee n a t a ge n e t i c l ev e l
u s i n g A FLP .
3 ) D e t e rmi n in g th e a l l e l o ch em ic a l e f fe c t s o f t h e f o r e m e n t i on e d
ax e n i c , x e n i c a nd cu l t u r e s us i n g a b i oa s s a y w i th t h e
C r yp t op h yt e R h od om o na s ba l t i ca t o de t e rmi n e th e b a c t e r i a l
i n f lu e n c e on th e u nk n ow n a l l e lo c h emic a l .
![Page 11: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/11.jpg)
11
4 ) M a te r ia l s and me t h od s :
4 . 1 Sa mpl e s i t e :
T h e Al e xa nd r iu m ta m ar e ns e s t r a in s us e d i n t he s e ex p er im e n t s w e r e
c o l l e c t ed f rom t wo s t a t i o ns o f f t h e c oa s t o f S co t l an d d eno t ed b y S 2
( 5 6° N , b e t w e en D un d e e an d Ed i nb u r gh ) a nd S6 ( 57 ° 4 0 ’ N , n o r th o f
A b e rd e e n ) . T h es e c u l t u r es w e r e i so l a t ed f r om n a t u ra l p l a nk to n
s a mpl e s a s s in g l e ce l l s .
4 . 2 S t ra ins and Cul tu r e c ond i t ion s :
4 . 2 . 1 Ax en ic a nd xe n i c cu l tu r es
T w o s t r a i ns o f A. t a m ar e ns e w he r e u se d fo r t h e b a c t e r i a l
ex p e r im e n t s : S 2 - P4- D 1 a nd S 2-P 4 - F2 .
K me d i um ( K el l e r e t a l 19 87 ) m ad e wi th N o r th S e a w a t e r a d j us t e d t o
p H 8 , wi th t h e ad d i t i o n o f s to c k s o l u t io ns ( s e en i n T ab le 1 ) . T h es e
c u l tu r e s wh e r e t h e n t r a ns f e r r ed to K me d ium co n t a i n i n g an
a n t ib io t i c c o ck t a i l ( i n T ab l e 2 ) . Th es e c u l t u r es w h e re m ain t a in e d a t
1 5 °C , w i t h a r t i f i c i a l l i gh t a t a ph o t on f l ux d e ns i t y o f 10 0µ Em- 2
s- 1
a n d a l i gh t :d a r k c yc l e o f 14 :1 0 . Th e x e n i c c u l tu r es w h e re
m ai n t a i n ed i n t h e sa m e f a sh i on wi t h t h e o mis s i on o f a n t ib io t i c
m e d iu m. To r ed uce th e a mo un t o f ex t r a c e l l u l a r ba c t e r i a w i t h i n t h e
c u l tu r e s , gauz e f i l t e r in g ( us in g a 20 um m es h ) wi th s t e r i l e - f i l t e r ed
s e a w at e r w as co ndu c t ed d u r in g t h e s ec o nd w ee k . T h i s en t a i l e d
ge n t l y p o u r i n g th e c u l tu r e t h r ou gh s t e r i l e ga uz e , a nd r i n s i n g t h r e e
t im es w i th s t e r i l e - f i l t e re d s e aw a te r . Th e s e c u l t u r e s w e r e t h en
t r an s f e r r e d i n t o K m e d iu m a nd m ai n t a i n ed as p r ev i ou s l y d e s c r ib e d .
A f t e r t h e s ec on d we e k , c u l t u re s w he r e t e s t e d f o r b a c t e r i a l
![Page 12: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/12.jpg)
12
c o n t am in a t io n ( as d e s c r ib e d b e l l ow ) an d u s ed as i n o cu l a f o r g r o wth
b o t t l e s co n t a i n in g 5 L o f K m ed ium ( to en su r e e no u gh s am pl i n g
v o l um e ) . Th e i no cu l a wh e r e us e d to a l l o w f o r a f i n a l c onc e n t r a t i o n
o f ap p rox im at e l y 2 0 0 c e l l sml- 1
. E a ch t r e a tm e n t w as co ndu c t ed in
t r i p l i c a t e
T a b l e 1 : L i s t o f r ea ge n t s u s ed i n m a k i n g K m e d i um.
A mo un t add ed /L S to ck s o lu t i on
1 ml N a N O 3 ( 75 .0 g / L H 2 O )
1 ml N H 4 C l ( 2 . 68 g / L H 2 O )
1 ml N a H 2 P O 4 . H 2 O (5 g /L H 2 O )
0 . 1ml H 2 S eO 3 ( 1 . 29 m g/ L)
1 ml T r i s Bas e (p H 7 . 2 ) (1 2 1 . 1 g/ L H 2 O)
1 ml K T r ac e m et a l s o l u t io n
1 ml F / 2 Vi t ami n so l u t i on
![Page 13: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/13.jpg)
13
T abl e 2 : A n t i b io t i c c o ck t a i l u s e d in ax e n i c m ed ium .
A n t ib i o t i c S to ck s o lu t i on A ddi t i on /L me d ium
A m pi c i l l i n
1 5m g/m l 3 . 33m l
G e n t a m yc i n e
1 m g/ml 3 . 33m l
S t r e p to m yc i n
2 5m g/m l 1 ml
C h l o r am ph e n i c o l
3 4m g/m l 3 0 u l
C i p ro f l ox a c i n
1 m g/ml 1 u l
4 . 2 . 2 Sub - c l on e cu l tu r es
Si n g l e c e l l s o f A. t a m ar e ns e f ro m th e s t r a in s : S2 -P4 - D1 , S 2- P4 - F2 ,
S 2 - P6 - G1 2 an d S6 -P 1- G 7 w e re p i ck e d f r om ex i s t i n g cu l t u r e s u s in g a
f i ne t i p pe d p l a s t i c p ip e t t e a t t ac h ed t o a r ub b er p ip e . Cu l t u re s w e r e
d i l u t ed an d p l a c ed i n a P e t r i d i sh . C e l l s t h a t w er e s us p en d ed wi t h in
t h e m e d i um o r mot i l e w e r e p i c k ed . T he y w e r e p i c k ed b y a p p l yi n g a
s l i gh t s u c t i on , wh ic h a l l o we d th e ce l l s t o b e su ck e d in t o t h e p i p e t t e
t i p a nd t r an s f e r r e d t o a w e l l o f a 9 6 we l l p l a t e c on ta in i n g 2 00 u l o f
d i l u t ed (1 : 10 ) K me d ium ( Ke l l e r e t a l . , 1 98 7) . Th e p l a t es w h er e t he n
c h e c k ed to co n f i rm th a t o n l y o n e ce l l p e r w e l l w as t r an s fe r r e d . T h e
p l a t es fo r e a c h s t r a i n w e r e i n cu b a t ed an d m ain t a in e d a t 15 °C ,
1 0 0µ E .m- 2
. s- 1
p ho to f l ux d e ns i t y a n d a l i gh t :d a rk c yc l e o f 14 :1 0 .
O n c e th e c lo n es w er e f o r me d th e y w e r e t r a ns f e r re d to a g l a s s c u l t u r e
![Page 14: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/14.jpg)
14
v i a l c on t a in in g 10 m l o f u nd i lu t ed K m e d iu m. O nc e t he c l on es ha d
r e a c h ed s ub s t an t i a l c on c e n t r a t i on s t h ey w e r e t r an s fe r r ed i n t o 20 0 m l
f r e sh K m e d i um wi t h p en i c i l l i n ( 0 . 1 g /L) i n 25 0ml cu l tu r in g f l a sk s
a n d nu mb e r ed 1 to 20 wi t h du p l i c a t e s am pl es f rom t he cu l tu r e w h e r e
t h e s in g l e c e l l s whe r e p i ck e d ( i . e . p a re n t c l on e ) . T h e y w e r e
m ai n t a i n ed a t t h e s a m e g ro wt h c on d i t i o ns m e n t i on e d p r e v i ou s l y.
4 . 3 S a mpl in g p r o ce du r e :
4 . 3 . 1 Ax en ic a nd xe n i c cu l tu r es
S am pl es fo r c e l l c ou n t s a nd PSP m e asu r em e n t s w e re t a ken ev e r y
s e c on d d a y t i l l t h e c u l tu r e s r e a ch ed t he l a g -p h as e o f t h e i r g r o wt h
c u r v e ( i . e . 2 0 da ys i n t o t a l ) . Fu r th e r s a m pl es we r e t ak e n d u r i n g
t h re e s t a ge s o f t h e ex p e r im e n t f o r b a c t e r i a l d e t e rm in a t io n us in g
A c r id i n e o r an ge . Sa m pl es fo r d e t e rmin in g th e a l l e l o ch em ic a l e f f ec t
o f t he cu l tu r e s w e re t ak en o n d a ys 8 an d 1 0 .
4 . 3 . 2 Sub - c l on e cu l tu r es
C ul t u re s w e r e h a r ve s t e d w h en ce l l num b er r e ac h ed ap p rox im at e l y
3 0 00 c e l l s /m l . 1ml w a s us e d f o r c e l l co u n t s , 40 ml f o r PSP
d e t e r min a t io n , 2 0ml fo r a l l e l oc h emi c a l t e s t s a nd 10 0ml fo r DN A
ex t r a c t io n f o r A FLP an a l ys i s .
![Page 15: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/15.jpg)
15
4 . 4 C e l l and b ac t er i a l c oun ts :
4 . 4 . 1C el l c oun ts
T h e Ut e rm oh l -m et ho d w a s us ed fo r t h e ax en ic , x en i c cu l tu r e s a nd
s ub - c l on e s am pl e s t o d e t e rmi n e c e l l co n c en t r a t i o n . Th e se r e su l t s
w e r e u s ed to d e t e rm in e th e g ro wt h r a t e o f e a ch s t r a in i n t h e ax e n i c
a n d x en ic cu l tu r e s a n d to ev a l u a t e P ST co n c en t r a t i o ns pe r c e l l fo r
b o t h ex p e r im e n t s . C ou n t i n g wa s do n e b y m ix in g 1ml o f s a m pl e wi th
2 0 u l Lu go l ’ s so lu t io n ( t o o b t a i n a f i n a l Lu go l ’ s s o l u t i on o f 2 % ) .
1 0 0u l o f t h i s s am ple w as t h en ad de d to a c ou n t i n g ch am be r
c o n t a i n i n g 2 00 u l o f K m ed ium . Th e to t a l n um b er o f c e l l s w a s
c o un t ed in t h e ch am b er wi t h a n i nv e r t e d mi c r os co p e ( Ze i s s Ax io v e r t
4 0 C ) an d th e n umbe r s w e r e c on v e r t ed t o c e l l s p e r ml . The g r o wt h
r a t e w as c a l cu l a t ed b y:
G r o wt h r a t e = ln ( N 1 / N 0 )
T 1 - t 0
W h e r e N r e f e r s t o t h e c e l l co n ce n t r a t i o n a t t im e t .
4 . 4 . 2 B a ct e r i a l coun ts :
St a in i n g wi t h A c r id in e o r an ge ( Ho bb ie e t a l , 19 77 ) w a s u s ed t o
d e t e r min e t h e am oun t o f b ac t e r i a i n t he Ax en i c an d x e n i c c u l t u r es .
T h i s w as d on e b y f i r s t a dd i n g 20 µ l Fo r ma ld e h yd e t o 1ml o f s am pl e .
T h es e s am pl es w ere s t o r e d in t h e f r i dge t i l l f u r th e r an a lys i s . In
o r d e r t o b e gi n s t a in in g , a c e l l u l os e n i t r a t e f i l t e r ( po r e s i z e 0 . 45 µ m ,
M i l l i p o r e ) w a s m oun t ed o n to t h e f i l t e r i n g u n i t fo l l o w ed by a b l a ck
N u c l e op o r e f i l t e r (p o r e s i z e 0 . 2 µ m) .
1 ml o f t h e f i x e d s am pl e w a s ad d ed t o t h e f i l t e r in g un i t w i th 1 00 µ l
o f Ac r id i n e O r an ge ( 1m g/ml ) an d i n cub a t ed f o r 3 m in u t es . Th en t h e
![Page 16: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/16.jpg)
16
s a mpl e wa s f i l t e r ed o f f , t h e n u c l eo po re f i l t e r w as re mo v ed wi t h
p in c e r s , mo un t e d on to a s l i d e wi t h c i t i f l uo r a nd c ov e r e d w i th
c o v e r - s l i p s . T h e y w h e r e t h en s to r ed in t h e f r i d ge t i l l a na l ys i s . T he
b l an k w as p r ep a r e d in t he s a me m a nn er wi th f i l t e r ed M i l l i - Q w at e r
a s t he s a mpl e .
Ba c t e r i a l co un t s o f e a c h sa mp le w er e d o ne as fo l l o ws : 20 g r id s on
e a c h f i l t e r w h er e co u n t ed u nd e r a n e p i f l uo r e sc e n c e m i c ro s co p e wi th
1 0 0X o i l imm e rs ion ob j e c t iv e .
4 . 5 PS P ex t ra c t i on and d e te r mi na t i on :
T hi s me th od wa s ap p l i e d t o t he ax e n ic , x e n i c a nd s ub - c lo n e
c u l tu r e s . ST X a nd i t s d e r iv a t iv es ( F i gu r e 1 ) w er e d e t e rm ine d u s i n g
k n ow n s t an d a rds . T h e s am pl es w h e r e p r e p ar e d a s fo l l o w s :
V o lum e s r an g i n g f ro m 30 ml to 9 00m l ( i . e . t o o b t a i n a pp rox i ma t e l y
3 0 00 c e l l s /m l ) o f t h e s am pl e cu l tu r es w e r e c en t r i f u ge d a t 1 30 00
r p m, 40C f o r 15 min u t e s t o ob t a in a pe l l e t f ro m t h e c u l t u r e . T h e
p e l l e t w a s t h en f r oz en an d s to r e d a t -2 5 o
C p r io r t o ex t ra c t i on .
Fo r PSP t ox in ex t ra c t i on , 1ml o f a ce t i c a c i d ( 0 . 03 N ) w as a d d ed t o
t h e c e l l pe l l e t . T h i s w as t h e n hom o gen iz ed u s in g l ys i n g m at r ix D
b e a ds (T he r mo sa v an t ) i n a h om o gen iz e r ( Fas t P r e p FP 120 ,
T h e rm os av a n t ) f o r 4 0 s e co nd s fo l l ow ed b y c e n t r i fu ga t i on
( E pp e nd o r f 5 41 5R ) f o r 15 mi nu te s a t 1 3 00 0 rp m. 50 0 µ l o f t h e
s up e r n a t an t w as t he n p a s s e d t h ro u gh a 0 . 5ml s p i n f i l t e r ( M i l l i po r e )
a n d c e n t r i fu g i n g f o r 30 s e c a t 30 00 r pm . T h e f i l t r a t e w as co l l e c t ed
i n 2ml c r im p g l as s v i a l s (A g i l e n t T e c hn o l o g ie s ) a nd c a ppe d . A
p o r t i on o f t he f i l t r a t e w a s h yd r o l yz e d to d e t e rm in e th e N-
s u l f oc a r ba mo yl c on t e n t ( T ab le 3 ) . T h i s w as do n e b y a d d in g 7 5µ l
![Page 17: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/17.jpg)
17
H Cl (1M ) t o 30 0µ l o f t he ex t r a c t e d s am pl e f o l l o we d b y 9 0 °C
i n cu b a t i on f o r 15 m in . 1 50µ l o f s od iu m a c e t a t e (1 M) wa s t he n
a d d ed a f t e r t h e s a mp l e c oo l e d to r oo m t em p e r a tu r e . T h e N -
s u l f oc a r ba mo yl c on t e n t w a s de t e r min ed i nd i r e c t l y b y c a l c u l a t i n g t he
c o n c en t r a t i o n d i f f e r e n c e b e t w e en th e h yd r o l yz e d a nd n on-
h yd r o l yz e d s am pl e . A l l s am pl e s w er e k e p t a t - 25 °C p r io r t o
a n a l ys i s .
T h e HP LC m et ho d b a s ed o n io n - p a i r ch r om at o gr ap h y w i th
o c t an es u l fo n i c ac id , pos t -c o l um n de r i va t i z a t i o n f o l l ow e d b y
f l uo r es c en c e d e t e c t i on (H umm e r t e t a l . , 1 99 7) wa s us e d to an a l yz e
t h e c on c en t r a t i o ns o f PSP d e r i v a t i v es . Th e m eth od u s ed t w o
a q u eo us e lu e n t s (Ta b l e 4A ) w i th d i f f e r e n t g r a d i en t s o ve r t im e
( T a b l e 4 B) . Th e ox id i z in g r e a ge n t f o r t h e p os t co l um n
d e r i v a t i z a t i o n c on s i s t ed o f a 10m M p er i od i c a c id , 55 0m M N H 3
s o l u t i on an d 0 . 75 M n i t r i c a c i d . F lu o re s c e n t t ox in d e r i v a t iv es w er e
d e t ec t ed a t ex c i t a t i o n a nd emi s s io n w av e l e n gt hs o f 3 33 nm an d
3 9 5nm r esp e c t iv e l y .
S t a nd a r ds f o r s ax i tox i n (S T X) , n eo s ax i tox in (N E O ) , gon ya u tox in s
( G T X 1 - 4 ) an d D ec a r b omo yl t ox i ns (d c ST X, d c G TX 2 an d d c GT X
3 ) p u r ch as e d b y t h e N a t io n a l Re s e a r c h C ou n c i l C an a d a , Ha l i f ax ,
N o v a S c o t i a , C a n ada , w er e u s ed . T h ese w e r e us e d t o c a l i b r a t e t h e
s a mpl e PSP tox i n co n c en t r a t i o ns .
![Page 18: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/18.jpg)
18
Fi g ur e 1 : S T X an d i t s d e r iv a t iv e s d ep ic t i n g t h e fo u r ma jo r g r o up s
o f PSP tox i ns . (d e pe n d i n g o n t h e g r o up s ub s t i t u t e d on R 4 p os i t i o n )
n a m el y, C a r b amo yl , N -su l fo c a rb am o yl a nd D ec a r ba mo yl t ox i ns .
S ax i t ox in a nd Ne oS TX a r e t h e m os t po t en t o f t he s e t ox ins , w i th N -
s u l f oc a r ba mo yl b e in g th e l e as t t ox i c an d D e c a rb am o yl t ox in s h av i n g
i n t e rm ed i a t e t ox in c o n t e n t .
![Page 19: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/19.jpg)
19
T abl e 3 : T ab le o f t h e N -s u l fo c a rb am oyl c o mp ou n ds a nd th e
h yd r o l yz e d d e r iv a t iv e s t h e y a r e h yd r o lyz e d t o i n d i r e c t l y d e t e r min e
t h e i r c on c en t ra t i on .
N - s u l f o c ar ba my l
H yd ro ly se d
c o mp ound f o r me d
B1 � S TX
B2 � N E O
C 1 � G T X 2
C 2 � G T X 3
C 3 � G T X 1
C 4 � G T X 4
![Page 20: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/20.jpg)
20
T abl e 4 A : Elu e n t s u s ed i n l i q u id ch ro m ato gr a p h y
El u ent
A 6 mM O c t an es u l fo n ic a c i d
6 mM T et r a su l f on i c a c id
4 0m M
A mm on ium ph os ph a t e
0 . 75 % T e t r ah yd r o fu r a n e
B 1 3m M O ct a ne su l f on i c a c i d
5 0m M P ho sp ho r i c a c i d
a d ju s t ed t o ph 6 .9 w i t h
N H 3 .
1 5 % A c e t on i t r i l e
1 . 5% T e t r a su l f on ic a c id
T abl e 4B : E lu e n t g r a d i en t s u se d f o r c h r om at o gr ap h y. In j e c t i on :
5 µ l , f l ow 1 .0 ml min- 1
.
Ti me ( mi n ) Gr a d i en t
0 1 0 0% A
1 5 1 0 0% A
1 6 1 0 0% B
3 5 1 0 0% B
3 6 1 0 0% A
4 5 1 0 0% A
C on c en t ra t i ons o f G T X 1a nd GT X4 , GT X 2 a nd G T X3 , C 1 a nd C 2 a nd
C 3 a nd C4 we r e r ep o r t ed as t he s um o f t h e t wo in d i v id u a l s du e to
t h e po t e n t i a l o f ep im e r i z a t io n du r i n g th e ex t r a c t io n p ro c es s .
![Page 21: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/21.jpg)
21
4 . 6 Al l e l o ch e mi ca l t e s t s :
T h e a l l e l o ch emi c a l e f f e c t s o f A l ex an dr iu m ta ma r en s e cu l t u re s w e r e
q u an t i f i ed wi t h a b i o as sa y u s in g t h e c r yp t o p h yt e R ho do mo n as
b a l t i ca . A f t e r ex p os in g R. b a l t i ca t o A. t a mar e ns e , t h e t a rge t c e l l s
i n c r e a s ed s l i gh t l y i n s i z e du e to s w e l l i n g , fo rm e d b l i s t e r s a nd
f i na l l y l ys e d . Co un t in g t h e nu mb e r o f i n t a c t R . ba l t i c a c e l l s ( wi th
n o rm al c e l l s ha p e s t i l l v i s i b l e ) a f t e r a c e r t a i n i n cu b a t i on p e r i od
a l l o w ed f o r t h e qu an t i f i c a t i o n o f t h e l y t i c e f f e c t .
T h e b i o as sa y w a s pe r f o rm ed as fo l l o ws : t h e c e l l d en s i t y o f an
ex po n en t i a l l y g r o w i n g cu l tu r e o f R. ba l t i c a ( cu l t u r e d a t 1 5 °C ,
u n de r a r t i f i c i a l l i gh t a t a ph o t on f l ux d e ns i t y o f 1 00 µ E m- 2
s- 1
o n a
1 6 : 8 h l i gh t -d a r k cyc l e ) w a s e s t i ma t ed b y m i c ro s co p e c e l l co un t s . A
d i l u t i on to a f i n a l c o n c en t r a t i o n o f abo u t 4 x 1 05 m l
- 1 c e l l s w as
p r e p ar e d . In t r i p l i ca t e , 3 . 9 m l o f a p r ed e f i n ed co n c en t r a t i o n o f A.
t a mar e ns e ce l l s w as p ip e t t ed i n to 6m l g l a s s s c in t i l l a t i o n v i a l s
( W h e a t on S c i n t i l l a t i on Vi a l s ) . F r esh K - m ed i um wa s us ed as
c o n t ro l . V i a l s w er e t h e n sp i k ed wi t h 0 . 1 ml o f t h e R . ba l t i c a s t o c k
c u l tu r e ( f i n a l co n ce n t r a t i o n : 1 0 x 103 m l
- 1) . Af t e r ad d i n g R . ba l t i ca ,
0 . 5 m l o f e a ch c on t r o l v i a l w a s f i x ed w i t h 2 % Lu go l ´ s so l u t i on i n
o r d e r t o d e t e rmi n e t h e a c t u a l i n i t i a l R . b a l t i c a c on c en t ra t i o n .
S am pl es w er e t he n i n cu b a t ed a t 15 ° C a t l o w l i gh t ( ab ou t 5 µ m ol m- 2
s- 1
) on a 16 : 8 h l i gh t -d a rk c yc l e . A f t e r 24 h , s amp l es w e re f i x ed
w i t h 2 % Lu go l ´ s so lu t io n a nd c e l l con c e n t r a t i on s o f bo th A.
t a mar e ns e an d R. ba l t i c a w e r e de t e r min e d . Al l co un t s o f w e r e
p e r f o r me d w i th an i nv e r t e d m i c r os c ope ( Ze i s s Ax io v e r t 40 C ) . Th e
v o l um e f o r c e l l co un t s w a s 0 . 5 ml . Fo r A . t a ma re ns e , wh o l e
c h a mb e rs w e r e c oun t ed , ex c e p t f o r t h e h i gh l y c o n c e n t r a t ed cu l tu r e
o f S2 -P 4 - F2 , w he r e r e p re s en t a t i v e s ub - a r e a s ( > 40 0 c e l l s p e r
s a mpl e ) w e r e c ou n te d . A s ub a r e a o f a t l e as t 80 0 c e l l s i n t h e c on t r o l
w a s c ou n t e d fo r R . b a l t i ca . In o rd e r t o qu an t i f y l yt i c e f fe c t s , o n l y
![Page 22: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/22.jpg)
22
i n t a c t ce l l s o f t he t a r ge t s p e c i es w e r e s c o r ed . F i n a l c e l l co u n t s o f R .
b a l t i ca w e r e c on v er t ed t o bo th c on c e n t r a t i o n ( c e l l s m l- 1
) a n d
p e r c en t mo r t a l i t y ( t r e a tm e n t c om pa r e d t o c on t r o l ) .
In o r d e r t o qu a n t i t a t i v e l y c o mp a r e l yt i c po t en c y o f a n t ib io t i c -
t r e a t e d an d un t re a t e d cu l tu r es , do s e - re s po ns e cu rv e s c ove r in g a wid e
r a n ge o f d i f f e r en t A . t a m ar en s e c on c en t ra t i ons w er e p lo t t e d . Be fo r e
s t a r t i n g th e ex pe r im e n t , cu r r en t a l ga l c e l l c on c e n t r a t i ons o f t he A.
t a mar e ns e cu l tu r es ( 3 r ep l i c a t es a n t i b io t i c t r e a t ed , 3 r ep l i c a t es
u n t r e a t e d ) we r e d e t e r mi n ed b y a l ga l co u n t s an d a pp ro p r i a t e
d i l u t i on s w e re p r e pa r e d to ga in a s e r i e s o f s e ve n f i n a l ce l l
c o n c en t r a t i o ns r a n gi n g f r om 5 0 to 2 000 c e l l s ml- 1
. E a ch d i lu t i o n w as
s e t up in t r i p l i c a t e . In t h i s ex p e r im e n t , s i x v i a l s c o n t a in in g K -
m e d iu m s e r v ed as co n t r o l s . Th e R ho dom o na s b io a s s a y w a s t h en
p e r f o r me d a s d es c r i b ed ab ov e .
4 . 7 A mpl i f i ed Fr ag me n t L eng th Po ly mo r ph i s m:
4 . 7 . 1 DN A ex t ra c t io n
D N A w a s ex t r ac t ed f r om 10 0m l o f ex po n en t i a l l y g r o w i n g c u l tu r e s .
T h e c u l t u r es w h er e f i l t e r ed o n t o 3 µ m p or e s i z e p o l yc a r b o n a t e
f i l t e r s ( Mi l l i p o r e ) a n d w as h ed wi t h s t e r i l e - f i l t e r e d s ea w at e r t o
r e mo v e ex t r a -c e l lu l a r b ac t e r i a . Th e f i l t e r s c on t a in i n g t h e s am pl es
w e r e p l a c e d in 2m l r e a c t io n v i a l s (E pp e nd o rp h ) an d imme d i a t e l y
f r oz en i n l i q u i d n i t r o ge n an d s to r e d a t - 80oC u n t i l ex t r ac t i on . T h e
D N A ex t r a c t io n w as d on e fo l lo wi n g t he i n s t ru c t io ns i n t he DN e a s y
P l a n t Mi n i k i t ( Q IA G E N ) .
Be f o r e D N A ex t r a c t io n , t h e c e l l s w er e d i s ru p t ed u s in g a
t i s s u e l ys e r ( Q IA G E N ) . A 0 . 5 mm s t a in l es s s t e a l b e ad w as ad d ed t o
t h e Ep p en do r f t u be c on t a in in g t h e f roz en f i l t e r s . Th e s am pl es w er e
p u t i n l i q u id n i t ro ge n fo r 3 0 s e c an d p l a c ed i n t o t h e t i s sue l ys e r .
![Page 23: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/23.jpg)
23
C e l l s w e r e b ro k en b y v i go r ou s l y s h a k i n g a t a n i n t e ns i t y o f 3 0 Hz fo r
o n e min u t e . T h i s w a s r e p e a t ed th r e e t im es , wi t h r e f r e ez i n g a f t e r
e v e r y c yc l e . Th e DN A p r e p ar a t i o n w as co nd u c t ed as p e r p r o t oc o l
w i t h a mi no r a d j us tm e n t . T he DN A wa s e lu t ed in a sm al l e r vo l um e
t h an s u gge s t e d t o ob t a in h i gh er co n c en t ra t ed D N A. T h e D N A w a s
s to r ed a t - 80 t i l l f u r t he r an a l ys i s .
4 . 7 . 2 DN A R e s tr i c t i on
T h e D N A w as d i lu t e d a c co rd in g l y t o ob t a in 2 50 n g /5 0µ L. T he DN A
w a s d i ge s t e d us in g a M as t e r c yc l e r ( E pp o nd or ph ) wi t h t h e f o l l o wi n g
c yc l e s : 16oC fo r 9 h o ur s , 2 0
oC f o r 6 ho u rs an d 65
oC f o r 10 m in ( i . e .
t o i n a c t i v a t e t h e enz ym e s ) . Th e enz ym e s us ed fo r t h e d ige s t i on
w h e r e 10 U E coR I a n d 10 U Ms e I ( N e w En g l a nd Bi o l ab s ) . S amp le s
w h e r e do n e in d up l i c a t e .
4 . 7 . 3 DN A Li ga t i on
1 7µ l o f t he l i ga t e d s a mpl e wa s a dd e d to a mix tu r e o f w a t e r ( 2 . 5µ l ) ,
1 0m M A TP( 1 . 7µ l ) , 1 0x T4 l i ga s e bu f f e r ( 0 . 8µ l ) , 1 0 pm ol µ l- 1
E c oR I a d a p te r (1µ l ) , 5 0 pm ol µ l- 1
M s eI a d a p t e r ( 1µ l ) a n d T 4 D NA
l i gas e (1µ l ) . T h e l i ga t i on w a s c a r r i ed o u t u s in g a M as t e rc yc l e r
g r a d i en t a t 1 6oC f o r 9h , 20
o C fo r 6 h ou r s a nd 6 5
oC fo r 1 0 mi nu t e s .
T h e l i ga t ed mix t u re w as t h en d i lu t e d 1 :5 wi t h 0 . 1 x TE bu f f e r a nd
s to r ed a t – 25oC .
4 . 7 . 4 Pr e a mpl i f i c a t i on
1 0µ l o f t he d i l u t ed l i ga t e d D NA wa s ad d ed t o a r e a c t io n m ix tu r e o f
w a t e r (1 2 . 5µ l ) , 10x T aq DN A p o l ym e r a s e b u f f e r ( 3µ l ) , 10 pmo l /µ l
E c oR I+ A p r im e r ( 0 . 5µ l ) , 10 p mol /µ l M s e I+ C p r im e r ( 0 .5 µ l ) , 1 mM
d N TPs (3µ l ) an d Ta q DN A p l o ym e r a s e . T h e PCR r e a c t i on w as
c a r r i ed o u t i n a m as t e r c yc l e r g r a d i e n t ( E pp e nd o r f , ) w i th 2 2 c yc l e s
![Page 24: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/24.jpg)
24
o f : 9 4oC fo r 1 mi nu t e (d e n a t u r a t i o n ) , 5 6
oC f o r 30 se c on ds
( a n n ea l i n g ) a nd 7 0oC fo r 1 min u t e ( ex t e n s i on ) . Fo l lo w ed b y 7 0
oC
f o r 5 mi nu t e s . Th e p ro du c t w as d i lu t ed 1 :1 0 wi th 0 .1x TE bu f f e r
a n d s t o r e d a t -2 5oC.
4 . 7 . 5 A mpl i f i c a t ion
5 µ l o f t h e p r e amp l i f i ed m ix tu r e w as ad d ed t o a mix tu r e o f : w a t e r
( 9 .9µ l ) , PCR - Buf f e r ( 2µ l ) , 1 mM d NTPs (2µ l ) , P r im e rs Mse I ( 0 . 6µ l ) ,
P r i me r E c oR I ( 0 . 1µ l ) a nd Ta q ( 0 . 4µ l )
T w o p r im e r c om bina t i on s w h er e u s ed : M s e I+ C C , E co R I+ A A ;
E c oR I+ A G a n d Ms eI+ C T . Th e a mpl i f i c a t i on wa s c a r r i ed o u t i n a
M a s t e r c yc l e r g r a d i e n t ( Ep pe nd o r f ) wi t h 12 c yc l e s o f : 94oC fo r 1 min
( d e n a tu r a t i o n ) , 65oC fo r 30 s ( an n e a l i ng ) a n d 70
oC fo r 1 m in
( ex t e ns io n ) an d 22 c yc l e s o f 94oC fo r 3 0s , 65
oC fo r 3 0s e co n ds a nd
7 0oC f o r 1 m in . T he p ro du c t w as s t o r ed a t - 2 5
oC.
4.7.6 Capillary electrophoresis
1 µ l o f t h e a mpl i f i ed p r od u c t w a s m ix ed wi t h 15 µ l o f H iD i
f o rm a mid e (A pp l i ed Bi os ) an d 0 . 5 µ l G e n e -S c an 5 00 m a rk e r (A BI) .
T h e mix tu r e w a s den a tu r ed a t 9 00C fo r 3 mi nu t e s t o ob t a in s in g l e
s t r a nd e d D NA . The s amp l es w e r e a n a l yz e d u s in g c a p i l l a r y
e l e c t r op ho r es i s .
4 . 7 . 7 A FL P An al ys i s :
G e n eM a pp e r ( A pp l i e d Bi os ys t e ms ) w as us e d to an a l ys e t he d a t a w as
a n a l ys e d us in g BIO N U ME R IC S so f t w ar e . Th i s p ro gr a m ca l c u l a t ed
t h e d i s t an c e m at r ix b y t h e f o r mul a fo r t h e “s i mpl e m at c h
c o e f f i c i e n t ” (K osm a n & Le o n a r d 20 05 ) Th e d e nd o gr ams t h r es h -
h o l d o f 40 0 . Th e co mp a r i so n w as d e t e r mi n ed b y l o ok i n g a t a l l e l e
![Page 25: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/25.jpg)
25
s imi l a r i t i e s b e tw e en s ub c l on es . Th e a l l e l e s i z e s t a r t ed a t 7 5b p a nd
w a s c om p ar e d f o r sm al l es t a l l e l e s .
![Page 26: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/26.jpg)
26
5 R esu l t s :
5 . 1 Ax en ic i ty o f cu l tu r e s :
Ba c t e r i a l co n t am ina t i on w as d e t e rmi ne d u s i n g A c r i d i n e o r a n ge
d u r i n g 4 s am pl i n g p e r i od s t h r ou gh ou t t h e wh o l e ex p e r i m en t . Th e s e
v a lu es a r e sum ma r i s e d in T a b l e 6 . T h e ax en ic cu l tu r e s bec a m e
c o n t am in a t e d wi th b a c t e r i a b e tw e e n da ys 1 5 an d 20 .
5 . 2 Grow th o f Ax en i c an d X en i c cu l tu r es :
T h e g r o wt h o f t h e x e n i c a nd ax e n i c cu l t u r e s w as m on i to r e d b y c e l l
c o un t s o v er t he 20 d a y p e r io d .
St r a i n S2 -P4 - D1 ( F i gu r e2 ) s ho w ed a s l i gh t d i f f e r e nc e i n t h e g r ow th
c u r v es b e t w e en th e x en i c an d ax en ic cu l t u r e s . Th e ax en ic c u l t u r es
r e a c h ed a h i gh e r c e l l co n c en t r a t i o n wi t h t h e i r h i gh e s t c e l l
c o n c en t r a t i o n a t d ay 2 0 w h i ch co r r es po n de d to a c e l l co nc e n t r a t i o n
o f 6 x 1 04 c e l l sm l
- 1. T he x e n i c c u l tu re r e a ch e d i t s h i ghes t
c o n c en t r a t i o n on da y 1 0 w h i ch co r r esp o nd ed t o a c e l l c on c e n t r a t i on
o f 4 .2 x 1 04 c e l l sml
- 1.
S t r a i n S2 -P4 - F2 ( F i gu r e 3 ) sh ow e d a s i mi l a r t r e nd , wi th t h e ax e n i c
c u l tu r e r e a c h in g h igh e r c e l l co n c en t r a t i o n t h an t he x e n i c c u l tu r e .
T h e h i gh e s t c on c e n t r a t i o n r e a ch ed b y t h e ax e n i c cu l tu r e w a s
a t t a in e d on D a y 1 6 . Th i s c o r r e sp on d ed t o a ce l l c on c e n t ra t i on o f
6 . 2 x 103c e l l sml
- 1. T h i s t he n l e ad to a sh a r p d ec r e as e i n c e l l
c o n c en t r a t i o n . Th e x en i c cu l t u r e s r e ac h e d a m ax imu m c e l l
c o n c en t r a t i o n o f 5 .4 x 103 c e l l sm l
- 1 on da y 1 8 wh e r e i t su r p as se d th e
c e l l co n c en t r a t i o n o f t he ax e n i c c u l t u r e s t o r ea c h a m ax im um o f 5 . 8
x 103 c e l l sm l
- 1.
![Page 27: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/27.jpg)
27
0.00E+00
1.00E+03
2.00E+03
3.00E+03
4.00E+03
5.00E+03
6.00E+03
7.00E+03
0 2 4 6 8 10 12 14 16 18 20 22
Time (days)
Ce
lls
ml -1
axenic
xenic
Fi g ur e 2 : G ro wt h o f t h e c u l t u r e s t r a in S 2- P4 - D1 o ve r 2 0 d a ys .
0.00E+00
2.00E+03
4.00E+03
6.00E+03
8.00E+03
0 2 4 6 8 10 12 14 16 18 20 22
Time (days)
ce
lls
/m
l
axenic
xenic
Fi g ur e 3 : G r ow th o f t he cu l tu r e s t r a in S 2- P4 - F2 ov e r t he 20 d a y
ex p e r im e n t i n ax en i c an d x e n i c cu l tu re s .
T h e g r o wt h r a t e v a r i ed s l i gh t l y b e t w ee n t h e t w o S 2 -P4 - D1 cu l tu r e s
( F i gu r e 4 ) . Th e axe n i c cu l t u r e s h ad a g r o w th r a t e o f 0 . 42 , wh i l e t h e
![Page 28: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/28.jpg)
28
x e n i c c u l tu r es h a d o n e o f 0 . 41 . T h e ax e n i c c u l tu r es h a d a 2 d a y l a g
p h as e an d w en t i n to ex po n en t i a l g ro wt h ( sum ma r i s ed i n t a b l e 6 ) .
T h e x en i c cu l t u r e s f o l l o w e d ro u gh l y t h e s am e p a t t e rn , w i t h l o w e r
c e l l co n c en t r a t i o ns . T he l a g ph as e l a s t e d t i l l t h e fo u r th da y, a n d
t h en th e cu l tu r es we n t i n t o ex p on e n t i a l g r ow th . T h e s t a t i o n a r y
p h as e w a s s im i l a r fo r b o t h c u l t u r e s a nd l a s t ed fo r t h e r em ai nd e r o f
t h e ex p e r im e n t .
T h e g r o wt h r a t e s o f b o th ax e n i c a nd x en i c c u l t u r e s o f S 2 -P 4- F2
( F i gu r e 5 ) w e r e v i r t u a l l y t h e s am e , n am el y 0 . 31 an d 0 . 33
r e s p ec t iv e l y. T h e d e f i n ed g r ow th p h as e s a r e su mm a r i s e d in T a b l e 6 .
Bo t h cu l tu r e s r e a che d t h e s t a t i on a r y p h a s e a t Da y 1 4 an d r e m ai n ed
t h e r e wi t h no s i gn i f i c an t c e l l co n c en t ra t i on ch a n ge s t i l l t h e
r e m ai nd e r o f t h e ex p e r i m en t .
2.00E+00
2.50E+00
3.00E+00
3.50E+00
4.00E+00
4.50E+00
0 2 4 6 8 10 12 14 16 18 20 22
Time (Days)
log
cell
s/m
l-1
axenic
xenic
Fi g ur e 4 : G r ow th c u r v e o f cu l tu r e o f s t r a in S2 -P 4 -D 1 d ep i c t i n g t he
l a g , ex po ne n t i a l a nd s t a t i on a r y p h a s e s .
![Page 29: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/29.jpg)
29
2.00E+00
2.50E+00
3.00E+00
3.50E+00
4.00E+00
4.50E+00
0 2 4 6 8 10 12 14 16 18 20 22
Time (Days)
log c
ellsm
l -1
axenic
xenic
Fi g ur e 5 : G ro wt h cu r v e o f S 2- P4 - F2 sh o wi n g th e l a g , ex p o ne n t i a l
a n d s t a t i o na r y p h a se s d e f i n ed in t a b l e 6 .
T abl e 5 : Ba c t e r i a l c ou n t s fo r ax e n i c a n d x en ic cu l tu r e s u s i n g
A c r id i n e o r an ge o n f ou r s am pl i n g d a ys .
B a ct e r i a l coun t s ml- 1
S 2 - P4 - D 1 D a y 0 D a y 8 D a y1 4 D a y 2 0
Ax e n i c 0 0 0 5 . 8 x 1 02
X e n i c 9 . 4 x 105 9 . 2 x 10
5 8 . 4 x 10
5 7 . 1 x 10
5
S 2- P4 - F2
Ax e n i c 0 0 0 6 . 2 x 102
X e n i c 9 . 1 x 105 8 . 8 x 10
5 8 . 6 x 10
5 7 . 3 x 10
5
![Page 30: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/30.jpg)
30
T abl e 6 : Th e g r owt h ph a s es a s d e f in ed . Th e g ro wt h r a t e w a s
c a l cu l a t ed u s i n g the d e f in e d ex p on e n t i a l p h as e s .
S t ra in L ag
ph as e ( Da ys )
E xpon en t ia l
ph as e ( D ay s )
S ta t ion a ry
ph as e ( D ay s )
Ax e n i c S 2 -P4 - D1 0 - 2 2 - 8 8 - 20
X e n i c S2 -P 4 -D 1 0 - 4 4 - 10 1 0 -2 0
Ax e n i c S 2 -P4 - F2 0 - 4 4 - 10 1 0 -2 0
X e n i c S2 -P 4 - F2 0 - 6 6 - 12 1 2 -2 0
5 . 3 Pr odu ct ion o f PS P t ox i ns by a xen i c an d xe n i c cu l tu r es :
5 . 3 . 1 T ox in p r of i l e s
In o r d e r t o d e t e r min e t h e PSP t ox in c on t en t , s am pl es w e re t ak en
e v e r y s e c on d d a y f o r a p e r io d o f 20 da ys .
T h e ax en ic an d x en i c cu l tu r es o f s t r a in S 2- P4 - D1 ( F i gu r e 6 A )
p r od u c ed th e fo l l ow i n g PSP tox in s : S TX , N e oST X, GTX 1 /4 , dc
S TX , C1 / 2 a nd C 3 /4 . ST X w as t h e mo s t ab un d an t , a c cou n t in g fo r
r o u gh l y 4 8 – 5 0% of t h e mo la r p e r c en t i n t he ax e n i c c u l t u r e s , wh i l e
i t a c co un te d f o r s l i gh t l y m o r e i n t h e x e n i c cu l t u r e s (5 3 -54 mo l% ) .
T h e s e co nd l a r ges t f r a c t i o n w a s f rom N e oS T X wh ic h a c co u n t ed fo r
3 1 % in t h e x e n i c an d 2 9% i n ax e n i c cu l t u r e s . Th e C1 /2 t ox in s
a c c o un t e d f o r 1 0 – 1 3 % i n bo t h c u l t u re s . G TX 2/ 3 , d c GT X 2/ 3 , d c
S TX an d C3 / 4 w e r e t h e l e as t ab un d an t a n d on l y a c c o u n t e d f o r 1 ,
0 . 5 , 0 .0 6 an d 0 . 04 % r es p e c t i v e l y.
T h e ax en ic an d x en i c cu l tu r es o f s t r a in S 2- P4 - F2 ’ s ( Fi gur e s 7 A &
B ) h ad a s im i l a r t ox in p r o f i l e t o t h a t o f S2 -P 4 -D 1 . ST X a c c o un t e d
f o r r ou gh l y 3 3 - 39 % in b o th t r e a tm e n t s . N eoS T X f o l l owe d wi t h a n
a b un d an c e o f 32 - 35% in b o th cu l tu r es . C 1 / 2 w as t h e t h i rd m os t
![Page 31: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/31.jpg)
31
a b un d an t t ox in , a c co u n t i n g f o r 16 - 19 % o f t h e t o t a l mo l a r
c o n c en t r a t i o n / pe r ce n t . G TX 1/ 4 , G T X2 /3 , d cG T X2 /3 , dcS TX w er e
t h e l e a s t a bu nd a n t w i t h a v er a ge v a lu es o f 9 , 2 , 1 an d 0 . 05
r e s p ec t iv e l y.
T h es e p r o f i l e s r e ma i n ed c on s t a n t t i l l t h e c u l t u r e s h ad r ea c h e d t h e
s t a t i on a r y p h a s e . T h e 20t h
d a y o f s am pl i n g s ho w ed a s l i gh t
v a r i a t i o n in b o t h t he ax en i c an d x en i c t r i p l i c a t e s . Th e v a r i a t i o n w as
i n t h e f o rm o f a m ar k e d r ed u c t i on i n GT X 1/ 4 p r od u c t i on (F i gu r e
6 B) . Th es e l ev e l s w h e r e so l ow t ha t t h e y c o u ld n o t b e qu a n t i f i e d .
S 2 - P4 - F2 h a s s aw va r i a t i on i n t h e x en ic t r i p l i c a t e . I t s how e d a h i gh
d e c r e as e i n S TX p ro d uc t io n ( F i gu re 6 ) . T he m ol a r p e r c e n t s a nd
t o t a l t ox i c i t y d i f f e re d o v e r t h e c ou rs e o f t he ex p e r im e n t . S TX an d
N e oS T X s ho w e d t he g r e a t es t v a r i a t i o n w h er e b y t h e 2 0t h
d a y t h e
m ol a r r a t i o o f S T X h ad d e c r e as e d f rom 5 0 to 2 0% i n bo th t h e x en i c
a n d ax en i c cu l tu r es o f S 2 -P4 - D1 . Th e s am e w a s obs e r v ed f o r S 2 -
P 4- F2 w hi ch s a w the ST X mol a r r a t i o d e c r e as e f r om 3 5 to 16 %.
![Page 32: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/32.jpg)
32
A)
0%
20%
40%
60%
80%
100%
D+1 D+2 D+3 D-1 D-2 D-3
% composition
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
B)
0%
20%
40%
60%
80%
100%
D+1 D+2 D+3 D-1 D-2 D-3
% composition
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
Fi g ur e 6 A and B : PST p r o f i l e o f S 2 -P 4- D 1 sh ow in g t h e m ol a r
p e r c en t c omp os i t i on o f e ac h PST a t a ) t = 0 ( s am pl i n g t im e z e r o )
a n d b ) T =2 0 ( en d o f t h e ex p e r i m en t )
![Page 33: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/33.jpg)
33
A)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
F+1 F+2 F+3 F-1 F-2 F-3
% composition
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
B)
0%
20%
40%
60%
80%
100%
F+1 F+2 F+3 F-1 F-2 F-3
% composition
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
Fi g ur e s 7 A and B : P ST p ro f i l e o f S2 -P 4 - F2 i n m ol a r pe r c e n t a t a )
t = 0 ( s a mpl in g t ime z e ro ) an d b ) T= 20 ( en d o f t h e ex p e r i me n t )
![Page 34: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/34.jpg)
34
5 . 4 T ox in c ont en t v e r s es c e l l g row th in a x en i c and x en ic cu l t u re s :
T ot a l t ox in co n t en t w a s p lo t t ed a ga i ns t t he g r o wth cu r ve t o
d e t e r min e t h e am oun t o f t ox in p r od uc t i on a t e a c h g r o wth p h as e .
T h e ax en ic cu l tu r e s o f S 2 -P4 - D1 ( F i gur e 8 a ) s ho w ed a d ec r e a s e i n
t ox i c i t y d u r i n g th e l a g ph a se ( f r om 9 0 f m ol / c e l l t o 60 fmol / c e l l ) .
T h e PST co n ce n t r a t i on f l u c tu a t e d du r in g th e ex po ne n t i a l p h as e ,
r e a c h i n g a h i gh o f 7 0 f mol / ce l l . T h e s t a t i on a r y p h a s e s ho w e d a
s h a rp d e c r ea s e i n t ox i n co n t en t ( f rom 7 0 – 49 f mol / ce l l ) f o l l o w e d
b y a n in c r e as e t o 60 fm ol / c e l l , w h i ch w a s t h e n f o l l ow e d b y a
p e rm a n en t de c r e as e f o r t h e r em ai nd e r o f t he ex p e r im e n t t o 25
f m ol / c e l l . T h e va r i a t i on s i n t ox i n c on te n t t h a t w h e re s t a t i s t i c a l l y
s i gn i f i c an t w as d u r i n g t he l a g / ex po n en t i a l p h as e a nd t h e s t a t i on a r y
p h as e . S2 -P 4 -D 1 x e n i c cu l t u r e h a d a h i gh er t ox in c on t en t t ha n th e
ax e n i c c u l t u r e . The l a g p ha s e s aw a s l i gh t i n c r e as e i n c e l l t ox in
c o n t e n t ( i . e . f r om 10 0 -1 10 fmo l / c e l l ) . T he ex po n en t i a l ph a s e
ge n e r a l l y s a w a n inc r e a s e i n t ox i c i t y . T h i s c o r r e sp on d ed t o t h e
h i gh es t t ox in c on t en t r e a ch e d b y t h e cu l t u r e o f 2 10 f mol / c e l l a t d a y
8 . Th e s t a t i o n ar y p h as e s a w a co n t i nu o us d e c re a s e i n t ox in co n t en t .
T h e r e w as a s t a t i s t i c a l l y s i gn i f i c a n t d i f f e r e n ce b e tw e e n th e t h r e e
g r o w th p h as es .
T h e ax en ic cu l tu r e s o f S 2 -P4 - F2 ( F i gu r e 8 B) sh ow e d a n in c r e as e i n
t ox i c i t y f r om t h e l ag t o ex p on e n t i a l p ha s e t h a t m imi c k ed th e g r ow th
c u r v e . I t i n c r e as e d s t e ad i l y t o 15 0 fmo l / c e l l t i l l t h e mi d
ex po n en t i a l p ha s e w h e r e i t d ro pp e d o f f s l i gh t l y t o 13 0 fmo l / c e l l . I t
i n c r e a s ed to 1 50 fm ol / c e l l a t t h e be g in n i n g o f t h e s t a t i on a r y p h a se
a n d s t e ad i l y d e c r e a s e d th e r e a f t e r t i l l i t r e a ch ed a v a l ue o f 5 0
f m ol / c e l l . Th e tox in co n t e n t o f t h e x en i c c u l t u r e s ho w ed l e s s
v a r i a t i o n . I t i n c r e as e d e v en l y f r o m the l a g p ha s e t o t h e ex p on en t i a l
p h as e t o i t s m ax imu m v a lu e o f 4 00 fmo l / c e l l . As t h e cu l tu r e mo v ed
![Page 35: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/35.jpg)
35
i n t o i t s s t a t i o n ar y p h as e , t h e t ox i c i t y d r op p ed o f f s t e ad i ly t i l l i t
r e a c h ed t h e va lu e o f 70 fm ol / c e l l .
0.00E+00
1.00E+00
2.00E+00
3.00E+00
4.00E+00
0 2 4 6 8 10121416182022
Time (Days)
log c
ellsm
l-1
0
50
100
150
200
250
300
Toxin
conte
nt
(fm
olc
ell-1
)
log cells/ml
Toxincontent
A )
0
50
100
150
200
250
300
0 2 4 6 8 10121416182022
Time (Days)
To
xin
co
nte
nt
(fm
olc
ell-1
)
0.00E+00
1.00E+00
2.00E+00
3.00E+00
4.00E+00
log
ce
lls
ml-1
toxincontentlog cellsml-1
B)
Fi g ur e 8 A & B : Ch a n ge s i n t o t a l P SP tox i n co n c en t r a t i on an d c e l l
c o n c en t r a t i o n a s a f u n c t i on o f c u l tu r e a ge o f A ) ax en i c and B)x e n i c
S 2- P4 - D1 .
![Page 36: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/36.jpg)
36
0
100
200
300
400
500
0 2 4 6 8 10121416182022
Time (days)
To
xin
co
nte
nt
(fm
ol/
ce
ll)
0.E+005.E-011.E+002.E+00
2.E+003.E+003.E+004.E+004.E+00
log
ce
llm
l-1
Toxin content
Log cell/ml
A )
0
100
200
300
400
500
0 2 4 6 810121416182022
Time (days)
To
xin
co
nte
nt
(fm
ol/
ce
ll)
0.E+00
5.E-01
1.E+00
2.E+00
2.E+00
3.E+00
3.E+00
4.E+00
4.E+00
log
ce
lls
ml-1
toxin content
log cells/ml
B )
Fi g ur e 9 A & B: Ch a n ge s i n t o t a l P ST a nd c e l l co n ce n t r a t i o n as a
f u n c t i on o f c u l tu r e a ge o f A ) ax en i c and B) x en i c S 2 -P4 - F2 .
![Page 37: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/37.jpg)
37
C om pa r i n g t h e t w o s t r a in s i n t ox in p r od u c t i on a t t h e d i f f e r e n t
g r o w th p h as es yi e l d e d s im i l a r r e s u l t s ( F i gu r es 1 0 & 11 ) . T he x e n i c
c u l tu r e s sh ow e d a m a rk e d in c r e as e i n t ox i c i t y d u r i n g th e l a g a nd
ex po n en t i a l p ha s es . T he s t a t i on a r y p h a s e s a w th e t o t a l t ox i n
p r od u c t i on o f t h e ax e n i c c u l tu r es s u r pa s s t h a t o f t h e x e n ic c u l t u r es
i n bo th s t r a i ns . Th es e d i f f e r en c es wh e re on l y s t a t i s t i c a l l y
s i gn i f i c an t i n t h e ex p on en t i a l p h as e o f t h e x e n i c c u l t u re s o f b o t h
s t r a in s .
0
100
200
300
400
500
600
lag exponential stationary
Growth Phase
To
xin
co
nte
nt
(fm
ol/
cell
)
axenic
xenic
F i g ur e 1 0 : T ot a l t ox i n c on t e n t w i th r e s p e c t t o g ro wt h ph a s e o f
s t r a in S2 -P 4 -D 1 wi t h a s t a nd a rd d e v i a t i o n o f 6 .4 x 1 02(n= 3 ) .
0
200
400
600
800
1000
1200
lag exponential stationary
Growth phase
Toxin
conte
nt
(fm
ol/cell)
axenic
xenic
F i g ur e 1 1 : T ot a l t ox i n c on t e n t a s wi th r es p ec t t o g r o wt h p ha s e o f
s t r a in S2 -P 4 - F2 w i th a s t a nd a rd d e v i a t i on o f 1 . 2 x 102 ( n= 3 ) .
![Page 38: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/38.jpg)
38
T o ta l to x in c on t en t :
T h e to t a l t ox i c i t y f o r t he ax e n i c a nd xe n i c cu l t u r e s o f s t r a i n S 2 -P4 -
D 1 ( f i gu r e 1 2 ) wh er e 6 . 4 x 102 a nd 1 .3 x 10
3 fm o l / c e l l r e s p e c t i v e l y.
T h e x en i c cu l t u r e s p r od u c ed t wi c e as m u ch PSP tox i n as t h e ax e n i c
c u l tu r e s . Th e ax en i c an d x e n i c cu l tu re s o f S2 -P 4- F2 ( F igu r e 11 )
w h e r e mo r e t ox i c t h a n S2 -P4 - D1 ( F i gu r e 1 0 ) , w i th a t o t a l t ox i c i t y o f
1 . 2x 1 03 an d 1 . 6x 10
3 fmo l / c e l l r e sp e c t iv e l y. Th e x e n i c cu l t u r e s
w h e r e 1 . 3 t im e s m or e t ox i c t h an t h e axe n i c cu l t u r e s . T h i s v a r i a t i o n
i n t ox in co n t en t w as s t a t i s t i c a l l y s i gn i f i c an t
0
500
1000
1500
2000
axenic xenic
Strain
To
tal
toxin
co
nte
nt
(fm
ol/
cell
)
S2-P4-D1
S2-P4-F2
F i gu r e 1 2 : To t a l t ox i c i t y o f b o th s t r a i ns o v er t h e 20 d a y p e r io d .
![Page 39: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/39.jpg)
39
A l l e c l o ch e mi ca l t e s t s :
St r a i n S2 -P4 - D1 w as s ho w n t o h av e l yt i c p o t en t i a l . C e l l
c o n c en t r a t i o ns o v er 4 x 1 03
s a w no s u rv iv in g Rh od om on as c e l l s .
T h i s w as s e e n i n bo th cu l tu r es .
0
10000
20000
30000
40000
50000
0 2000 4000 6000 8000
A. tamarense concentration (ml-1)
Su
rviv
ing
Rh
od
om
on
as
(cell
sm
l-1)
axenic
xenic
Fi g ur e 1 3 : D os e - re s po ns e cu rv e d e p ic t i n g R ho dom on a s s u rv iv a l
w i t h r es p e c t t o ch an ge s i n A. t am ar e ns e c e l l co nc e n t r a t i on s .
5 . 7 Sub c l on es
5 . 7 . 1To x in p rof i l e s :
T h e tox i n p r o f i l e o f 4 s t r a i ns o f A. t am a r ens e wa s ob s e rv e d . A l l
s t r a in s h ad d i f f e re n t t ox in p ro f i l e s .
A l l 2 0 su b -c lo n es o f S 2 -P6 - G1 2 ( F i gu re 14 ) h a d s imi l a r t ox i n
p r o f i l e s t o t h a t o f t h e p a r en t c l on e s (PC 1 a nd PC2 ) . T h i s c on s i s t ed
o f B1 ( GT X 5 ) , C1 /2 , G T X1/ 4 , Ne oS TX , ST X , C 3 / 4 ( no t p r e s e n t i n
a l l s a mpl e s ) an d GT X 2/ 3 . Th e mo l a r r a t i o s w h e r e as fo l l o w s : B1
( G T X 5 ) : 32 - 54 %, C 1 / 2 : 20 – 3 0 % , GT X 1/ 4 : 3 -9 % , N eoST X : 8 - 20 ,
S TX : 4 -9 %, C 3 /4 : 1 - 8 % an d GT X 2/3 : 1 -7 % .
![Page 40: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/40.jpg)
40
S 2- P1 - G7 ( F i gu r e ) s ho w e d t h e s am e t r e n d o f t h e s am e tox i n p ro f i l e
b e tw e e n P C’ s a nd th e 2 0 su b c lo ne s . T h e tox i n p r o f i l e s c o ns i s t e d o f
t h e fo l l ow in g ( wi t h t h e i r av e r a ge mo l a r r a t i o i n b r a ck e t s ) : GT X
5 ( 17 - 40 % ) , C1 /2 (30 - 49 ) , GT X 1/4 (5 - 12 ) , G TX 2 /3 ( 4 -1 0 ) , N eoS T X
( 2 - 11 ) a nd S TX (4 -7 ) . Lo w v a lu e s o f d c ST X a nd d c GT X2 /3 wh e r e
a l so o bs e rv e s an d ac c o un te d f o r a mo la r p e r c e n t r a n gi n g f r om 0 .0 1
t o 0 . 1 .
S e ve n o f t h e tw e n ty S 2 - P4 - D1 ( F i gu r e ) su b c l on e s sh ow ed
d i f f e r en c e s i n t ox in p r o f i l e s co mp a r ed t o P C1 an d 2 . T he tox in
p r o f i l e s co ns i s t e d o f t he fo l l o wi n g (wi th t h e i r av e r a ge mo l a r r a t i o i n
b r a c ke t s ) : GT X 2 / 3 ( 3 3 -4 0) , N e oST X (2 5 -3 5 ) , ST X ( 20 - 28) , GT X1 /4
( 2 - 6 ) , w i th l ow l eve l s o f d cS T X a nd dc G TX 2 /3
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2
% composition
subclone S2-P6-G12
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
Fi g ur e 1 4 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins
p r e s en t i n a l l s ub co n es o f S2 -P 6- G 12 .
![Page 41: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/41.jpg)
41
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2
% composition
subclone S6-P1-G7
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
Fi g ur e 1 5 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins
p r e s en t i n a l l s ub co n es o f S1 -P 6- G 7 .
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2
% composition
subclone S2-P4-D1
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
F i g ur e 1 6 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins
p r e s en t i n a l l s ub co n es o f S2 -P 4- D 1 .
![Page 42: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/42.jpg)
42
0%
20%
40%
60%
80%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2
% composition
subclone S2-P4-F2
C3/4
C1/2
B2
B1
dcGTX2/3
dcSTX
GTX 2/3
GTX 1/4
NEO
STX
F i g ur e 1 7 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins
p r e s en t i n a l l s ub co n es o f S2 -P 4- F2 .
5 . 7 . 2 Va r i a t i ons in t o ta l to x i c i ty :
T h e su b c lo ne s h ad v a r yi n g t ox i c i t y ( f i gu r e 1 8 ) wi t h t h e h i gh es t an d
l o we s t v a l u es o f 115 an d 34 fm ol / c e l l r e s p ec t iv e l y. T h i s a c c o un t s
f o r a 3 fo ld d i f f e r en c e i n t ox i c i t y . 10 o f t h e 2 2 s ub c l one s h a d
t ox i c i t y l e s s t h a n th a t o f t h e a ve r a ge tox i c i t y o f a l l t h e su b c lo n es
( F i gu r e 21 ) .
![Page 43: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/43.jpg)
43
0
20
40
60
80
100
120
140
160
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20PC
1
PC
2
Sub clone
Tota
l to
xin
conte
nt (f
mol/cell)
Fi g ur e 1 8 : T o t a l t ox i c i t y o f s ub c lo ne s S2 -P 6 -G 12 . P a re n t c l on e s
d e no t ed a s PC1 an d PC2 wi t h a s t a nd a rd de v i a t i o n o f 2 5 . 25 (n = 20 )
-60
-40
-20
0
20
40
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20PC
1
PC
2
Sub clones
To
tal
toxin
co
nte
nt
(fm
ol/
cell
)
Fi g ur e 1 9 : V a r i a t i o n in t ox in c on te n t o f S 2 -P6 - G1 2 a s a f un c t io n o f
t h e d i f fe r e n ce b e twe e n t he av e r a ge PSP tox i n c on t e n t o f t h e p a r en t
c l on es an d th e 2 0 su b c lo n es .
![Page 44: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/44.jpg)
44
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 1011121314151617181920PC
1
PC
2
Sub clone
To
tal to
xin
co
nte
nt
(fm
ol/cell)
Fi g ur e 2 0 : To t a l t ox i n c on t e n t o f 20 s ub c l on es o f S2 -P1 - G 7 .
P a r en t c l on es d e no te d as PC 1 an d PC2 , w i th a s t an d ar d d ev i a t i on o f
1 0 .4 8 .
-20
-10
0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 1011 1213 1415 1617 1819 20PC
1
PC
2
Sub-clone
To
tal
toxin
co
nte
nt
(fm
ol/
cell
)
Fi g ur e 2 1 : V a r i a t i o n in t ox in c on te n t o f S 2 -P1 - G7 as a fu n c t i on o f
t h e d i f fe r e n ce b e twe e n t he av e r a ge PSP tox i n c on t e n t o f t h e p a r en t
c l on es an d th e 2 0 su b c lo n es .
![Page 45: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/45.jpg)
45
Fi g ur e 2 2 : To t a l PS T c on c en t r a t i o n o f su b c l on e s S 2 -P4 -D 1 .
P a r en t c l on es d e no te d as PC 1 an d PC2 . W i th a S T DE V o f 3 8 .1 6
-100
-50
0
50
100
150
1 2 3 4 5 6 7 8 9 10111213141516 17181920PC1PC2
Subclones
To
tal to
xin
co
nte
nt
(fm
ol/cell)
Fi g ur e 2 3 : V a r i a t i o n in t ox i c i t y o f S2 - P4 - D1 as a fu n c t i on o f t h e
d i f f e r en c e b e t w e en th e t o t a l t ox i c i t y o f t h e i n d i v id u a l s and av e r a ge
t ox i c i t y o f t h e g r oup .
5 . 6 A FL P:
T w o p r im e r c om bina t i on s w e r e us e d . T h e r e su l t s o f t h e d i f f e re n t
p r im e r co mb in a t io ns a r e s um ma r i s ed in Ta b l e 6 . T h e p r im e r
c o mbi n a t i on o f E _A G _M _C T w as u s ed du e to t h e h i gh er v a l u es
o b t a i n ed wh en co mp a r i n g t h e s imi l a r i t y o f t h e s ub - c l on e s
T h e d en do gr a m o f S 2- P4 - D1 ( F i gu r e ) s ho w e d t h e mos t v a r i a t i o n i n
s ub c l on e g ro up in g . I t ha d a l ow s imi l a r i t y o f 37 %. S ub - c l on e 5
w a s t h e m os t o bv iou s ou t l i e r . T he r em ai n i n g s ub - c l on es h ad a
s imi l a r i t y v a l u e o f 8 2 %. Th e s e wh e r e f u r t h e r d iv id e d i n to t h re e
![Page 46: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/46.jpg)
46
d i s t i n c t g r ou ps w i th a s im i l a r i t y o v e r 9 0 %. T h e S 2 -P4 - F2 s ub -
c l on es s ho w e d l e s s v a r i a t i on an d h ad a h i gh e r bo o t s t r ap v a lu e o f
8 6 % wi th o n e ou t l i e r su b - c lo n e . Th e r e m ai n in g su b - c lo ne s wh e r e
d iv id ed i n t o t w o d i s t i n c t g r ou ps wi t h a bo o t s t r ap v a l u e o f 91 % .
S 6 - P1 - G7 s ho w ed a h i gh e r l ev e l o f s im i l a r i t y w i t h a b oo t s t r ap v a l u e
o f 8 3 % . T h e y w h e r e f u r th e r d i v id e d in to 2 d i s t i n c t g r oup s wi th
b o o t s t r a p v a lu e s o f 8 9 an d 97 % r es p ec t i v e l y. S2 -P 6 -G 12 ha d a
b o o t s t r a p v a lu e o f 8 9 %. Th e s ub c lo ne s wh e r e d iv id e d i n t o 2
d i f f e r en t g r o up s wi t h bo o t s t r a p v a l u es o f 97 % . D u e to t im e
c o ns t r a in t s t h i s d a t a c ou ld n o t b e a n a lys e d f u r t h e r t o d e t e r mi n e
w h e t h e r t h e d i f f e r en c e s s ee n w h er e s t a t i s t i c a l l y s i gn i f i c an t .
![Page 47: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/47.jpg)
47
T ab l e 6 : C omp a r i so n o f p r im e r c om bin a t io ns u s ed i n A FLP .
Primer combination
E_AA_CC_
S2-P4-D1 TH75 TH100 TH350 TH400
av similarity 89.23867 89.23867 88.30536 88.22725 av sim duplicates 92.96455 92.85091 88.52773 91.05638
S2-P4-F2 TH75 TH100 TH350 TH400
av similarity 88.35896 88.35896 83.53075 84.48922 av sim duplicates 97.73091 97.73045 93.75227 95.79773
S6-P1-G7 TH75 TH100 TH350 TH400
av similarity 88.51943 88.51943 90.0107 91.46266 av sim duplicates 93.07682 93.41773 91.025 94.32182
S2-P6-G12 TH75 TH100 TH350 TH400
av similarity 88.46586 88.46586 91.8222 90.10232 av sim duplicates 93.305 92.6908 95.68909 93.07318
Primer combination
E_AG_CT_
S2-P4-D1 TH75 TH100 TH350 TH400
av similarity 88.61405 86.22132 79.88836 83.64942 av sim duplicates 92.50909 92.96364 90.0876 91.5389
S2-P4-F2 TH75 TH100 TH350 TH400
av similarity 92.52902 91.90526 93.18651 94.24694 av sim duplicates 96.59864 96.25818 95.00409 95.34591
S6-P1-G7 TH75 TH100 TH350 TH400
av similarity 90.93088 91.36789 96.7437 96.71742 av sim duplicates 92.62136 94.44091 97.04818 97.05
S2-P6-G12 TH75 TH100 TH350 TH400
av similarity 93.82936 91.8222 94.43128 95.84905
av sim 96.03182 95.91727 95.345 96.36773
![Page 48: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/48.jpg)
48
duplicates
S im pl e m atch in g (> 50 % ME AN )
A G _C T _ D u p T H _4 00
100
95
90
85
8075
70
6560
55
5045
40
D 1_ 03 _P la teA
D 1_ 04 _P la teA
D 1_ 11 _P la teA
D 1_ 12 _P la teA
D 1_ PC 1_ Pla teA
D 1_ 08 _P la teA
D 1_ 06 _P la teA
D 1_ 19 _P la teA
D 1_ 20 _P la teA
D 1_ 02 _P la teA
D 1_ PC _2 _P la te A
D 1_ 01 _P la teA
D 1_ 09 _P la teA
D 1_ 13 _P la teA
D 1_ 14 _P la teA
D 1_ 15 _P la teA
D 1_ 17 _P la teA
D 1_ 18 _P la teA
D 1_ 07 _P la teA
D 1_ 10 _P la teA
D 1_ 16 _P la teA
D 1_ 05 _P la teA
F i g ur e 2 4 : Dendogram depicting similarity between sub-clones of strain S2-P4-D1
S im pl e m atch in g (> 50 % ME AN )
A G _C T _ D u p T H _4 00
100
999897
96
95
94
93
92
91
90
89
88
8786
F 2_ 01 _P la te A
F 2_ 19 _P la te A
F 2_ 03 _P la te A
F 2_ 02 _P la te A
F 2_ 17 _P la te A
F 2_ 18 _P la te A
F 2_ 06 _P la te A
F 2_ PC _ 1 _P la te A
F 2_ PC _ 2 _P la te A
F 2_ 04 _P la te A
F 2_ 07 _P la te A
F 2_ 09 _P la te A
F 2_ 10 _P la te A
F 2_ 11 _P la te A
F 2_ 12 _P la te A
F 2_ 13 _P la te A
F 2_ 14 _P la te A
F 2_ 15 _P la te A
F 2_ 20 _P la te A
F 2_ 05 _P la te A
F 2_ 16 _P la te A
F 2_ 08 _P la te A
![Page 49: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/49.jpg)
49
Figure 25: Dendogram depicting similarity between sub-clones of strain S2-P4-F2
S im ple m atch in g (> 50 % ME AN)
A G _C T _ D u p T H _4 00
100
99
98
97
96
95
94
93
929190
89
88
87
86
85
84
83
G 7 _0 8_ P late A
G 7 _0 9_ P late A
G 7 _0 4_ P late A
G 7 _0 5_ P late A
G 7 _0 6_ P late A
G 7 _1 6_ P late A
G 7 _0 2_ P late A
G 7 _0 3_ P late A
G 7 _0 7_ P late A
G 7 _1 1_ P late A
G 7 _1 2_ P late A
G 7 _1 3_ P late A
G 7 _1 4_ P late A
G 7 _1 5_ P late A
G 7 _1 7_ P late A
G 7 _1 8_ P late A
G 7 _1 9_ P late A
G 7 _2 0_ P late A
G 7 _P C_ 1_ Plate A
G 7 _P C_ 2_ Plate A
G 7 _0 1_ P late A
G 7 _1 0_ P late A
Figure 26: Dendogram depicting similarity between sub-clones of strain S6-P1-G7.
S im pl e m atch in g (> 50 % ME AN )
A G _C T _ D u p T H _4 00
100
99
98
97
96
959493
92
91
90
89
G 1 2_ 13 _ Pla teA
G 1 2_ 15 _ Pla teA
G 1 2_ 14 _ Pla teA
G 1 2_ 20 _ Pla teA
G 1 2_ 02 _ Pla teA
G 1 2_ 16 _ Pla teA
G 1 2_ 01 _ Pla teA
G 1 2_ 03 _ Pla teA
G 1 2_ 04 _ Pla teA
G 1 2_ 05 _ Pla teA
G 1 2_ 06 _ Pla teA
G 1 2_ 07 _ Pla teA
G 1 2_ 08 _ Pla teA
G 1 2_ 09 _ Pla teA
G 1 2_ 10 _ Pla teA
G 1 2_ 11 _ Pla teA
G 1 2_ 12 _ Pla teA
G 1 2_ 17 _ Pla teA
G 1 2_ 18 _ Pla teA
G 1 2_ 19 _ Pla teA
G 1 2_ P C _1 _P la teA
G 1 2_ P C _2 _P la teA
F i g ur e 2 7 : Dendogram depicting similarity between sub-clones of strain S 2 -P6 -
G 1 2 .
![Page 50: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/50.jpg)
50
6 . D i s cus s i on :
T h e ex t e n t o f b ac t e r i a ’ s i n v o lv em e n t i n PSP t ox in p ro du c t io n i s s t i l l
c o n t i nu in g . I t h a s b e e n su gge s t ed t ha t b a c t e r i a c om pl em en t t h e
d in o f l a ge l l a t e P SP p r od u c er s b y a f f e c t i n g t he g r o wth r a t e o r t h e
a c t ua l PS P t ox in p ro d uc t io n . In o r d e r t o a s s es t h i s e f f e c t ,
ex p e r im e n t s w h e r e c o nd u c t ed t o d e t e rm in e w h i ch p a r am et e r i s
a f f e c t e d b y b a c t e r i a .
Gr ow th ra t e :
T h e g r o wt h r a t e s o f b o th s t r a i ns o f A . t a mar e ns e w h e re ve r y s i mi l a r .
T h e s im i l a r i t i e s i n g r o w th r a t e a r e c ons i s t e n t w i th Ho l d e t a l ( 20 01 )
f i nd i n gs on A . t a ma r en s e . T h e i r ex p e r i me n t s u s ed A . l u s t i t a n i cu m
a n d A. t a mar e ns e an d c om p ar e d th e g r o w th r a t e an d tox in p ro du c t io n
b e tw e e n ax e n i c an d x en i c cu l t u r e s . A nt ib i o t i c s wh e r e us ed t o ob t a in
ax e n i c c u l t u r es . Th e ax e n i c a nd x e n ic A. t a mar e ns e cu l tu r e s
s ho w e d eq u a l g ro wt h r a t e s . Th e a u t h en t i c i t y o f t h e ax en ic c u l t u r es
w a s t h o ro u gh l y i n ve s t i ga t e d ( so l id and l i q u id m e d i a ,
e p i f l u o re s c en c e mic r o s co p y w i th S yb r g r e e n 1 a n d PC R us in g
u n i v e r s a l e ub a c t e r i a l p r i me r s t a r ge t i ng 1 6S r RN A , ) .
E v en t ho u gh th e x en i c c u l t u r e s o f bo th s t r a in s t oo k 4 d ays t o r e a c h
t h e i r ex p on e n t i a l g r o wt h ph a s e ; t h i s d i d no t s e e m t o a f f ec t t h e
o v e r a l l g ro wt h r a t e . T he d i f f e r en c e i n c e l l c on c e n t r a t i ons w as
p r ob a b l y d u e to t h e d i f f e re n c es i n i n i t i a l i n o cu l a . W h e n th e ax e n i c
c u l tu r e s wh e r e t r a ns f e r r ed in to n e w me d ium , t h e x e n i c c u l tu r es
w h e r e a l so t r an s f e r r e d . T h i s i d en t i c a l t r e a tm en t r ed u c ed t h e c ha n c e
o f h a v i n g c u l t u r es t h a t w h er e n o t i n t h e s am e g r ow th p ha s e . T h e
ex t en de d l a g p h as e o bs e r v ed in t he x e n i c cu l tu r es s u gge s t s t h a t t h e
b a c t e r i a l m i c r o f l o ra i n t h e m e d iu m m igh t a f f e c t n u t r i e n t u p t a k e
i n i t i a l l y d u e to t he f a c t t h a t t h e o th e r f a c t o r s t h a t co u ld a f f e c t
![Page 51: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/51.jpg)
51
g r o w th wh e r e c ons t a n t ( i . e . l i gh t i n t e ns i t y , t e mp e r a t u r e , s a l i n i t y ,
p H ) . Th ou gh th e b ac t e r i a a f f e c t e d t h e l a g ph a se i n i t i a l l y , t h e y s e e m
t o h av e n o e f fe c t on t h e g r o wt h r a t e o f c u l t u re s a f t e r . Th i s
d i f f e r en c e co u ld a l s o h av e b e e n du e to t h e l o w er c e l l con c e n t r a t i on s
u s ed i n i t i a l l y i n t he no n - ax en i c c u l t u re s .
T o ta l to x in c on t en t :
O n e o f t h e f i r s t s t ud i es t h a t co mp a r ed ax e n i c a nd x e n i c cu l t u r e s
w i t h r es p e c t t o g r ow t h an d tox i c i t y w e r e r ep o r t ed b y D a n tz e r &
Le v i n ( 19 97 ) . T hey u s e d A . t a ma r en se c u l t u r es a nd fo und t h a t t h e r e
w e r e n o d i f f e r e n c es i n t ox i c i t y b e t w ee n t h e ax e n i c a nd xe n i c
c u l tu r e s . Th e y t h en co n c l ud ed t h a t b ac t e r i a h ad n o e f f e c t o n
t ox i c i t y . Th i s i s co n t r ad i c to r y t o m y r e s u l t s b ec a us e , ove r a l l t h e
x e n i c c u l tu r es o f bo th s t r a i ns wh e r e m o re t ox i c t h an th e ax en ic
c u l tu r e s . X e n i c S2 -P 4- D 1 w as t wi c e a s t ox i c , wh i l e x en i c S 2- P4 - F2
w a s 1 .3 t im e s a s t ox i c . Th i s i s co ns i s t e n t wi th f i n d i n gs b y D o u c e t t e
& Po w el l (1 99 8 ) a nd Ho ld ( 20 01 ) .
T h e un i fo rmi t y o f t ox i n c on t e n t s e en by D a n tz e r & Le v i n ( 1 99 7 )
c o u l d h av e b e en d ue to t h e m et ho ds u se d t o d e t e rmi n e ba c t e r i a l
c o n t am in a t io n . T h ey g r e w th e i r cu l tu re s i n p en i c i l l i n - r i ch m e d iu m
a n d t e s t e d t h e ax en i c i t y o f t h e cu l tu r es b y u s in g m a r i n e aga r . T h i s
m et ho d w a s no t v e ry e f f e c t iv e d u e t o t h e f ac t t h a t 9 5 % o f ma r in e
b a c t e r i a a r e un cu l tu r a b l e (S c hu t e t a l 1 9 93 ) . S o l a ck o f b a c t e r i a l
g r o w th co u l d s im ply h a v e b e en d ue t o t h e un su i t ab i l i t y o f t h e m ed i a
u s ed . D ou c e t t e an d Po w el l (1 99 8) u s ed l ys o z ym e / SD S t o r e mo v e
b a c t e r i a f rom t h e i r A . l u s t i t a n i cu m c u l t u re s . T he y f o u nd t h a t t h e
ax e n i c c u l t u r es w he r e l e s s t ox i c t h a n t h e x en i c o n es a nd t h a t t ox i n
p r od u c t i on co u ld be r e s to r ed b y t h e ad d i t i o n o f b ac t e r i a b e l i ev e d to
b e PS T p ro du c e rs . T he y c o n c lu de d tha t t h e i n t e r ac t io n be t w e en A .
l u s t i t an i c um an d b ac t e r i a a c c ou n t ed fo r 50 % o f t h e ce l l u l a r t ox in s
![Page 52: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/52.jpg)
52
p r od u c ed . T h i s i s c o ns i s t e n t w i th t he s t r a i n S 2 -P4 - D1 wh i ch
s ho w e d a tw o f o l d i n c r e a s e i n t h e t o t a l t ox in p ro du c t io n . So th i s
c o u l d po i n t t o t h e i d e a o f b a c t e r i a e nha n c in g t ox in p ro duc t i on .
H o w ev e r t h e ax e n ic n a t u r e o f D a n tz e r & Le v i ns (1 99 7 ) cu l t u r e s i s
q u es t i o n ab le d u e to t h e m et ho ds u se d fo r b a c t e r i a l co n t ami n a t i on .
T h e y u t i l i s ed ep i f l u o r es c en c e mi c ro s co p y a n d l i q u i d me d i um to
d e t e r min e t h e ex t en t o f b ac t e r i a l c on ta mi n a t i on . Th e y d i d no t
r e p o r t t h e f r eq u en cy o f t h e i r m on i to r in g o f b a c t e r i a l g row t h .
A n o t he r f a c to r w as t h a t t h e ob se r v a t i on s d i d no t t a k e i n t o a c c ou n t
t h e e f f ec t l ys o z ym e /S DS t r e a tm e n t mi gh t h av e h ad o n A .
l u s t i t an i c um ’ s g r ow t h r a t e .
T h e h i gh e s t t ox in p r o du c t io n o c cu r r ed d u r i n g th e ex po n en t i a l ph a se
f o r a l l t h e cu l tu r es . Th i s i s c on s i s t en t w i th r esu l t s b y Bo ye r e t a l
( 1 98 7 ) a nd A nd e rso n e t a l (1 99 0) . SX T an d N eoS T X in b o th x e n i c
a n d ax en i c cu l tu r es i n c r e as e d du r i n g th e ex po n en t i a l ph as e an d
s ho w e d a c l ea r d e c r e a s e du r i n g t h e s t a t i on a r y p h as e .
W h e n c omp a r i n g t he t o t a l t ox in p r od uc t i on a t t h e d i f f e r en t g r o wt h
p h as e s , m a rk e d ch an ge s a re o bs e r ve d . T he d i f f e r en c e s i n t h e
ex po n en t i a l g ro wt h p ha s e o f t w o da ys i n t h e ax en ic an d x e n i c
c u l tu r e s co u l d h av e l e ad to t h e d i f f e r en c e i n t o t a l t ox in co n t e n t .
T h e x en i c cu l t u r e s s p en t l on ge r i n t h e ex po n en t i a l g ro wt h p ha s e
w h e r e t ox in p r od u c t io n i s a t i t s h i gh e s t a l l ow in g th em t o p r od u c e
m o re t ox ins . Th i s o bs e r v a t i on w a s c on s i s t en t wi t h bo th s t r a in s . T h e
ax e n i c c u l t u r es i n S 2- P4 - F2 s ho w e d a d i f f e re n c e i n t h e s t a t i o n ar y
p h as e w h e r e m o r e t ox i ns w h e r e p r od u ce d t h an in t h e x e n ic c u l t u r es .
T h i s w as p r ob a b l y d u e to t h e f ac t t h a t t h e y h a d a l on ge r s t a t i on a r y
p h as e . E v en th o u gh t ox in p ro du c t io n d e c r e as e s i n t h i s g r o w th
p h as e , i t do e s so s t e a d i l y ( i . e . no s h a rp de c r e as e s ) . Th i s e n a b l ed
t h e ax e n i c cu l tu r es t o p r od u ce m o re t ox in s bu t t h i s d i f f e re n c e w a s
n o t s t a t i s t i c a l l y s i gn i f i c an t .
![Page 53: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/53.jpg)
53
Bo ye r e t a l (1 98 7) c o nd u c t ed n u t r i e n t l im i t a t i o n ex p e r i men t s w i t h
n i t ro ge n an d ph os ph o ru s on A . t a ma r en s e . Th e y f o u nd tha t t h e
t ox i c i t y d e c r e as e d i n n i t r o ge n l imi t e d c u l tu r e s , w h i l e an i n c r e as e
w a s f ou nd i n ph os ph o ru s l im i t ed c u l tu r e s . T h e i n c re a s e i n PS T
p r od u c t i v i t y w a s du e t o t h e f a c t t ha t t h e c e l l s wh e r e un ab l e t o
d iv id e , c au s in g c e l l s t o ge t b i gge r . PS T p ro du c t i o n d id n o t s to p .
S o t h e P ST co nc e n t r a t i on i nc r e as e s p e r c e l l , b u t t h e ce l l
c o n c en t r a t i o n r em ai ns co ns t a n t l e ad i ng t o a n ob s e r v ed PS T i n c r ea s e .
S o i t c ou l d b e su gge s t e d th a t t h e b ac t e r i a mi gh t a f f e c t t h e n u t r i en t
u p t a k e o f t h e d in o f l a ge l l a t e , n am el y p h os ph o ru s s in c e t he x en ic
c u l tu r e s wh e r e mo re t ox i c . Th i s i s u n l ik e l y s in c e K m e d iu m i s n o t
n i t ro ge n o r p ho sp ho r us l i m i t e d an d the c e l l s i z e b e t w ee n th e ax en i c
a n d x en ic cu l tu r e s d id n o t v a r y e x t e ns i v e l y.
T h e d i f f e re n c es i n t ox i c i t y m i gh t no t h a v e b e en d ue t o t he b a c t e r i a ,
b u t d u e t o t h e e f f e c t o f t h e an t ib io t i c s u s ed . S t ud ie s on th e e f f e c t s
o f s t r e p t om yc i n a nd ch lo r am ph e n i co l ( w h i c h wh e r e b o th u s ed i n t h e
a n t ib io t i c c o ck t a i l ) sh o w ed h a rm fu l e f f e c t s o n so me a l ga l c e l l s
( M a r t i n s e t a l 20 04) . S t re p t om yc i n cau s ed l o s s o f p i gm en t a t i on an d
c h a n ge s i n c h l o r op la s t m o rp ho l o g y i n C hl or e l l a . In b a c t e r i a i t
i nh ib i t s p ro t e in s yn t h es i s i n g r am n e ga t iv e b a c t e r i a ( M ck an e an d
K a n d e l , 1 99 6 ) . W hi l e ch lo r am ph e n i co l c aus e d in h ib i t i on o f g r ow th
i n Eu gl e na a s w e l l a s a f f e c t i n g c h lo r op h yl l f o rm at i on . Th e r e i s n o
d o cu m en t a t io n o f t h e e f f e c t s o f a n t i b io t i c s o n d i no f l a ge l l a t es
( M a r t i n s e t a l 20 04) . T h e po ss i b l e e f f e c t s o n d i no f l a ge l l a t e c ou ld
b e d i f f e r en t i n t h e s e ns e o f a f f e c t i n g tox i n p ro du c t io n wh i c h wo u l d
l e a d t o a d e c r e as e i n t o t a l t ox i c i t y . O r i t c ou ld a f f e c t t h e c e l l w a l l
b y p e r f o r a t i n g i t , ca u s i n g l e ak a ge o f so m e PS P t ox in s . S i n c e t h e
PS T m ea su r e d i s f ro m in t a c t c e l l s , t he p oss ib l e PSP tox i ns i n t h e
m e d iu m o f t h e ax en i c cu l tu r es w ou ld no t b e d e t e c t ed . O r i t co u ld
a t t a c k th e o r ga n e l l e s o f en dos ym b i o t i c o r i g in ( “ p ro k ar yo t i c
![Page 54: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/54.jpg)
54
c o mp on e n t s ” T h es e a r e b e l i e v ed t o h av e b e e n e n gu l f ed du r in g t h e
e v o l u t i on o f t h e euk a r yo t i c c e l l . ) , n am el y t h e mi to ch ond r i a an d
c h lo ro p l a s t . Th e s e v es t i g i a l o r ga ne l l e s co u l d b e a f fe c t e d b y t h e
a n t ib io t i c s t h a t t a rge t t h e p ro k ar yo t i c c e l l s o f b a c t e r i a ca u s i n g an
e f f e c t .
A n d er so n e t a l (1 99 0 ) c on du c t e d ex p er im e n t s wh e r e t h e y lo ok e d a t
t h e e f f ec t o f N a nd P l imi t a t i ons as we l l a s d e t e rm in in g th e r a t e o f
t ox in p ro du c t io n . T h e y f o u n d th a t c e l l s p ro du c ed t ox ins a t r a t es
e q u a l t o t ho se n e e de d t o r ep l ac e l o s s es t o d au gh t e r c e l l s d u r i n g ce l l
d iv i s i on ( i . e . t h e “g r o w th d i lu t i o n ” e f f e c t ) . I d o no t b e l i e v e t ha t
t h i s ha s a n e f f ec t on t h e s t r a in s us e d du e t o t h e f a c t t ha t i f t h i s l o s s
w a s su bs t an t i a l , t he g r e a t es t l o s s wo u l d b e du r i n g t he exp o ne n t i a l
p h as e s i n c e t h a t i s w h e n t h e mo s t d augh t e r c e l l s a r e p r odu c e d , s o
PSP t ox in c on t en t w o u l d de c r e as e . Bu t t h e h i gh e s t a mou n t o f PSP
t ox in p ro du c e d i s i n t h e ex p on en t i a l ph a s e . Th e y s u gge s t e d t h a t
t ox in p ro du c t io n wa s com pl ex a nd c oup l ed to c e l l g r ow th a t c e r t a i n
c o nd i t i on s a nd u n re l a t e d i n o t h e r co nd i t i on s .
T h i s c ou l d b e du e to t h e f a c t t h a t t h e b a c t e r i a co u l d p l a y a ro l e i n
r e mi n er a l i s a t i o n o f n u t r i en t s . T he y c o u l d a l so p r od u c e m et a bo l i t e s
t h a t a c t a s co - f a c to r s an d s t im u l a t e PSP tox i n p ro du c t i o n i n t h a t
m a nn e r .
T h e PSP tox in p r o f i l e r em ai n ed c on s t an t t h ro u gh ou t t h e 2 0 d a y
ex p e r im e n t a l t h ou gh on e o f t h e t r i p l i ca t e s am pl es o f e a c h s t r a i n
s ho w e d t h e l o s s o f a PSP tox in . T h i s w a s s e en i n on e t r i p l i c a t e o f
t h e s a mp le s : aux e n i c an d x e n i c S2 -P 4- D 1 a nd x e n i c S2 -P4 - F2 .
S i n c e t h i s d i f f e r e nc e w as s e e n i n bo th a ux e n i c a nd x e n i c c u l tu r e s ,
t h i s e f f e c t c an no t be a t t r i b u t e d t o b a c t e r i a l i n f lu en c e .
![Page 55: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/55.jpg)
55
E n do nu c l e a r b a c t e r i a co u ld s t i l l h a v e a n e f f e c t o n tox i n p r od u c t i on .
T h i s c a nn o t b e d e t e r mi n ed wi t h t h e met ho d us e d s in c e w e w h e r e
f o c us i n g o n ex t r a - ce l l u l a r b a c t e r i a .
M y d a t a su gge s t s t h a t t h e e f f e c t o f b ac t e r i a i n PSP p r od uc t i on i s
s i gn i f i c an t . Th e y p r ob a b l y i n f lu e n c e PSP t ox in p ro du c t io n b y
p r od u c i n g c om po und s t h a t s t im u l a t e PSP t ox in p ro du c t ion .
A l l e l o ch e mi ca l t e s t s on a x en i c and xe n i c cu l tu r es
N u me r ou s s tu d i es ha v e b e e n co nd u c t e d in v es t i ga t in g t h e
a l l e lo c he mi c a l e f f ec t o f A . t a ma re ns e o n v a r i ous p h yt o p la n k t on
s p e c i es (O ga t a & Ko d om a 19 86 , Lu s h & H a l l e g r a e f f 19 96 , T i l l m an n
& J oh n 20 02 ) a nd ha v e s ho wn t h a t t h es e l yt i c c om po un ds a r e n o t
r e l a t e d t o P SP t ox in s . T h i s l e ad t o sugge s t io ns o f o t h e r f a c to r s
i n f lu e n c in g th e o bse r v e d a l l e lo p a th i c e f f e c t s . Co l e (1 982 )
s u gge s t e d th a t b a c t e r i a m a y m e d ia t e t h i s b y a f f e c t i n g th e
m et a bo l i s m o f t h e c a u s a t i v e s p e c i es , o r a l t e rn a t iv e l y t h ey c o u l d
r e l e as e a l l e l o ch em ic a l o f t h e i r o wn . Th e r es u l t s s ho w th a t t h i s i s
n o t t ru e i n t h e c as e o f s t r a i n S 2- P4 - D1 . T he s e r es u l t s a r e c on s i s t en t
w i t h t h os e r e po r t e d b y T i l l m an n & J ohn (2 00 2 ) a nd Su i kka n e n e t a l
( 2 00 4 ) . S 2 -P4 - D1 s ho w e d no d i f f e r enc e i n l yt i c e f f e c t on
R h od om on us c e l l s b e tw e e n t h e ax en i c a nd n on - ax en i c cu l t u re s . T h i s
s u gge s t s t h a t b a c t e r i a h av e n o e f f e c t on t h e a l l e lo c h emi c a l s
r e l e as e d . Th e m ed iu m i s t h ou gh t t o h av e b e e n ax e n i c d u r in g th e
s a mpl in g t im e b y. T h i s me a ns t ha t t h e a l l e l o ch em ic a l s a re p ro du c ed
b y t h e d i no f l a ge l l a t e . T h e p lo t o f dose c u rv e sh o w ed th a t t h i s e f f e c t
i s d ep e nd an t on th e c e l l c on c e n t r a t i ons . Th e no n - l yt i c S2 -P 4 - F2
s t r a in s ho w ed n o d i f f e r e n ce i n l yt i c e f f e c t b e t w e en ax en ic a nd n on -
ax e n i c c u l t u r es . Th i s g iv es fu r t h e r p ro o f t o t h e f a c t t h a t b a c t e r i a d o
n o t p r od u ce t h es e a l l e lo c h emi c a l s .
![Page 56: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/56.jpg)
56
Sub c l on es
T ox in p r of i l e :
T h e tox i n p r o f i l e o f f ou r s t r a i ns o f A. t a mar e ns e w h e re an a l ys e d to
s e e i f t h e r e i s a n y v a r i a t i o n in t he t ox i ns p r od uc e d . S ome a u t ho rs
h a v e r ep o r t ed t h a t t h es e p ro f i l e s r em ai n c on s t an t ( Bo ye r e t a l 19 86 )
w h i l e o th e r s ha v e re p o r t ed v a r i a t i o ns ( Bo cz a r e t a l . 19 88 , An d er so n
e t a l 19 90 ) . Bo cz ar e t a l ( 19 86 ) s u gge s t e d th a t t h e p r o f i l e co u ld b e
m od i f i ed s i gn i f i ca n t l y w i th en v i ro nm en t a l c h an ge s . W hi l e Sa ko e t
a l ( 19 9 2) an d Ki m e t a l ( 19 93 ) f ou nd th a t t h e i r t ox i n p r o f i l e s d i d
n o t ch a n ge s i gn i f i ca n t l y u n d e r v a r i ou s g r o wt h c on d i t i o ns . Th e y a l s o
c o nd u c t ed m at in g ex p e r im e n t s an d f oun d th a t t ox in p ro f i l e s wh e r e
i nh e r i t e d i n a 1 :1 M e nd e l i an r a t i o . Th i s a l l ow e d th em to co n c l ud e
t h a t t ox i n co mp os i t i on i s de t e r min e d on a ge n e t i c b as i s . I s h i d a e t a l
( 1 99 8 ) c on du c t e d m at in g ex p e r im e n t s a s w e l l w i t h bo th A .
t a mar e ns e an d A. ca n t e n e l l a . Th e y t oo k s t r a i ns w i th d i f fe r e n t t ox in
p r o f i l e s an d m at ed t h em. T he y a n a l yz e d t h e t ox in co n t en t t o s e e i f
i t wo u l d be a mix tu r e o f bo t h p a re n t s t r a in s a nd fo un d t h a t m os t o f
t h e i r s t r a in s ex h ib i t e d a t ox i n p r o f i l e o f t he co mbi n ed p a r e n t
s t r a in s . T h e y s u gge s t ed th a t
I d e t e r mi n ed th e t ox in p r o f i l e s o f s i n g l e ce l l s p i ck e d f rom fo ur
P a r en t s t r a in s . Th i s wo u ld s u gge s t t ha t t h e y w h e r e ge ne t i c a l l y
i d en t i c a l du e t o t h e i r o r i g in . Tw o s t r a i ns n am e l y, S 2 -P6 -G 1 2 a nd
S 6- P1 - G7 s ho w ed so m e v ar i a t i on s i n t ox i n p ro f i l e . On c lo s e r
i n s p ec t i on i t w a s fo u nd th a t t h es e v a r i a t i o ns w h e re d u e to t h e f a c t
t h a t so m e o f t h e t ox in s wh e r e p ro du c ed i n su c h l o w am oun t s t ha t
t h e y w h e r e un qu an t i f i a b l e ( i . e . d cS TX ) . T he i n t ro du c t io n o f C 3 /4 i n
8 o f t h e 40 su b c lon e s w h i ch w a s no t p r e s en t i n t h e p a r en t c l on e s
c o u l d b e ex p l a i n ed a s an e r r o r du r i n g h yd r o l ys i s . S t r a i ns S 2- P4 - D1
a n d S2 -P4 - F2 sh o we d m or e d i f f e r en c es i n t h e i r t ox i n p r o f i l e s .
W h e r e mo r e t h a t h a l f o f t h e s ub c lo n es h ad a d i f f e r in g t ox i n p ro f i l e
![Page 57: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/57.jpg)
57
t o t h a t o f t h e p a r en t c lo n es . S ev e n o f S 2- P4 - D1 s s ub c l on e s
d i f f e r ed t o t h a t o f t h e p a r en t c l on e . Th e s e d i f fe r e n ce s wh e r e d ue t o
t h e i n t r od uc t i on o f b o t h C3 / 4 a nd B2 . B2 w as n o t o bs e rv ed i n t h e
o r i g i n a l c h ro ma to gr a m. T he r e w a s no B2 s t a nd a r d s o i t s
c o n c en t r a t i o n w as m e as u r ed l i k e t h e o t h e r s u l f oc a r ba mo yl t ox ins b y
d e t e r min in g th e d i f f e r e n c e b e t we e n the no n h yd r o l ys e d an d
h yd r o l ys e d p e a ks o f N eo ST X. Th e h a r s h c on d i t i o ns o f h yd r o l ys i s
( a d d i t i o n o f HCl a nd t h e i n cu b a t i on a t 9 0oC ) su c h as an ex t en d ed
a m ou n t o f h e a t o r t h e v a r i a t i o ns i n pH mo s t l i ke l y f u r t h e r c on v e r t ed
C 3 / 4 i n t o N eoS T X. Th i s c ou l d h av e be e n d on e b y c l e a v in g th e
s u l f a t e g ro up p r e sen t on p os i t i on s R 2 a n d R3 r e sp e c t iv e ly . A s a
r e s u l t t h e d i f f e r en ce s e en w ou ld b e a t t r i bu t e d to t h e p r es en c e o f B2 .
I b e l i e v e t h a t t h e va r i a t i on s e e n i n t h es e s t r a i ns c an b e a cc o un t ed
f o r b y m e th od o lo g ic a l e r r o r s , s o no v a r i a t i o n i n t ox in p r o f i l e s i s
a c t ua l l y o bs e r v ed . T h i s d a t a s up po r t s t h e n o t i on o f t ox i n p ro du c t io n
b e in g a gen e t i c a l l y d e t e rmi n ed s t a b l e c h a r a c t e r i s t i c .
T h e ex t e n t o f t h i s h yd r o l ys i n g e r r o r ca n b e d e t e rm in e d by
c o nd u c t i n g ex p er ime n t s w i th v a r yi n g ac i d c on c en t r a t i o ns a n d t im e a t
9 0oC to es t a b l i sh t h e o p t i m al c on d i t i on s f o r t h i s m e th od . Th e
s a mpl e s c ou l d a l so b e ru n on c e mo r e t o s e e o f t h e r e i s a ny
d i f f e r en c e i n ch r om at o gr am p e a ks . Th e mi n i ma l p ro du c t i on o f s om e
PSP t ox ins i n t h e su b c lo n es c ou ld a l so be i nv e s t i ga t ed to s e e o f t h i s
w i l l ch a n ge o v er t he t im e p e r i od . A re c e n t p a p e r (M a r t in s e t a l
2 0 04 ) r e po r t e d t h e l o s s o f t ox in p ro duc t i on i n a o n ce t ox i c s t r a i n o f
A . l u s t an i c u m . Th ey a t t r i b u t ed t h i s t o e i t h e r ge n e t i c m ut a t io ns o r
e f f e c t s f r om th e p ro lo n ge d t r e a t m en t o f t he cu l tu r e s b y a n t i b io t i c s .
I t w o u ld b e i n t e r e s t i n g t o s e e o f t h es e c u l t u r es w i l l f o l l ow th e s am e
t r en d b y n o t p r od uc i n g s om e P SP t ox in s o r b y l o os in g the i r PSP
t ox in p ro du c t io n c om pl e t e l y.
![Page 58: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/58.jpg)
58
V a ri a t i ons in t ox in con t en t :
T h e to t a l t ox in co n t e n t w a s de t e r min ed fo r t h e fo u r s t r a in s . T o t a l
t ox i c i t y w a s d e t e rm in ed an d c on t r a s t e d b e tw e e n t h e p a re n t c l on e s
a n d th e s ub - c lo n es .
O ga t a e t a l (1 98 7 ) c o nd u c t ed a s im i l a r ex p e r i m en t , u s in g
m on o c lo n a l cu l tu r es o b t a i n ed f r om th e s am e a r e a du r in g th e s am e
s e a so n . Th e se c l one s an d su b c l on e s w h e r e c u l t u re d un de r t h e s am e
c o nd i t i on s a nd th e i r g r o wt h r a t e a nd PSP t ox in p ro du c i n g
c a p a b i l i t i e s wh e r e d e t e r min e d a nd co n t r as t ed . T h e y f o u nd
s imi l a r i t i e s i n g r ow t h r a t es an d t ox in p ro f i l e s . In a dd i t i o n , g r e a t
d i f f e r en c e s i n t ox i c i t y b e t w e en t he v a r i ou s su b c l on e s whe r e
o bs e r v ed . T h i s m ax im um d i f f e r e n c e i n t ox i c i t y w a s 2 0 fo ld b e t w e en
s om e s am pl es an d 1 0 0 fo ld b e t w e en c l on e s . Th e y a t t r i bu t ed th i s
d i f f e r en c e t o t h e f ac t t h a t t ox in p r od uc t i on w as n o t a h e re d i t a r y
c h a r a c t e r i s t i c . M y c u l tu r e s d id n o t exh ib i t su c h a h i gh de gr e e o f
v a r i a t i o n . T he h i gh e s t PSP tox i n va r i a t i on s e e n w as 9 fo l d . Th i s
w a s o n l y s e e n i n 5 o f t he 22 su b c lo n es o bs e r ve d . Th i s i s n o w h er e
n e a r w h a t O ga t a e t a l ( 19 8 7) r ep or t e d , b u t t he s ou r c e o f t h i s
v a r i a t i o n i s s t i l l s om et h i n g t o de l i be r a t e d u e t o t h e f a c t t h a t t h es e
p r e sum e d ge n e t i ca l l y s im i l a r s t r a in s wh e r e c u l tu r ed u nd e r i d e n t i c a l
c o nd i t i on s , s o i t f o l l o ws re a so n to ex pe c t mo d es t v a r i a t i on s b e t w e en
s ub c l on es .
T h es e v a r i a t i o ns i n t o t a l c e l l t ox in con t en t co u l d s t em f ro m th e f a c t
t h a t d i no f l a ge l l a t e D N A i s n o t eq ua l l y sh a r e d /d iv i d ed b e tw e e n
d a u gh t e r c e l l s d u r in g a s ex u a l r e p r od uc t i on . Ev en t ho u gh th e y
p r ob a b l y h a v e mul t i p l e c op i es o f t h e s a m e gen e , so me m ec h a n i s m
c a u s es t h e p r od uc t io n o f P SP t ox in s t o b e no n un i fo rm .
![Page 59: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/59.jpg)
59
T h i s l e a ds t o qu es t i on s a s t o w ha t c on t r o l s t he PS T p ro duc t i on .
S om e p ro du c e PST i n su c h l o w l e v e l s t h a t t h e y c o u l d no t b e
q u an t i f i ed . W h a t ca u s es t h e r ed uc t io n in t he p r od u c t i on o f s p e c i f i c
t ox in s? I t w ou ld b e i n t e r e s t i n g to an a lys e t h e t ox i n p r o f i l e o f t h e
s a mpl e s t h a t s ho w ed t h e l o w p ro du c t i on i n so me PSP t ox in s t o s e e i f
t h es e t ox i ns a r e p ro d uc e d in g r e a t e r qu a n t i t i e s , o r i f t he y a r e l o s t
e n t i r e l y .
D u e to t i m e c ons t ra i n t s , t h e A FLP d a t a c ou ld n o t b e su f f i c i e n t l y
a n a l ys e d to d e t e rm in e wh e t h er t h e r e wh e r e a c tu a l ge n e t i c
d i f f e r en c e s wi t h i n s ub - c l on e s . Th e p re l i mi na r y d a t a su gge s t s t h a t
t h e r e i s co n gr u en c e w i th - i n su b c lo ne s o f t h e s am e s t r a i n .
![Page 60: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/60.jpg)
60
A c know l ed g me n ts :
I w o u l d l i k e t o t h an k th e fo l l o wi n g f o r a l l t h e i r mu c h n ee d e d h e lp
a n d su pp o r t :
P ro f . D r . A l l an C em b el l a
D r Uw e J oh n
D r U rb an T i l l m an n
D r Be rn d K ro c k
T i lm a n A pp e rm an
A n n e- g r e t Mü l l e r
In e s J u n g
![Page 61: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/61.jpg)
61
R ef e r en c es :
A nd e rs on , D . M . , J . J . Su l l i van , and B . R e gu e ra . 19 89 .
P a r a l yt i c sh e l l f i s h p o i so n i n g in No r t hw e s t Sp a in : t h e t ox i c i t y o f t h e
d in o f l a ge l l a t e G y mn o d in iu m c a t en a t um . T ox i co n 27 :6 65– 6 74 .
A nd e rs on , D . M . , D . M. Kul l i s , J . J . Su l l i van , S . Ha l l , a nd
C . L e e . 1 99 0 . D yn a mi c s a nd p h ys i o log y o f s ax i tox i n p r od u c t i on b y
t h e d in o f l a ge l l a t es A l e xa nd r iu m s pp . M a r . B io l . 10 4 :5 11– 5 24 .
B o cz a r , B . A . , M . K . Be i t l e r , J . L i s to n , J . J . Su l l i v an , a nd
R . A . C at t o l i c o . 19 8 8 . P a r a l yt i c s h e l l f i sh t ox i ns i n P ro to g on ya u l a x
t a mar e ns i s a nd P ro t og on y au l a x c a t en e l la i n ax e n i c c u l t u re . P l a n t
P h ys i o l . 8 8: 12 85 –12 9 0 .
Carmichael W.W., Evans W.R., Yin Q.Q., Bell P .and E. Moczydlowski,
Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei
(Farlow ex Gomont) comb. nov. Appl. Environ. Microbiol. 63 (1997), pp. 3104–3110
C e mb e l l a , A . D . , J . J . Su l l i v an , G . L . Bo y er , F . J . R .
T ay lo r , a nd R. J . A nd e rs en . 19 87 . Va r i a t i on o n p a r a l yt i c sh e l l f i s h
t ox in c om po s i t i o n w i t h i n t h e Pr o t og on y au la x t a m ar en s i s / c a t en e l la
s p e c i es com pl ex : r e d t i d e d i no f l a ge l l a t e s . Bio ch e m. S ys t . Ec o l .
1 5 :1 71 –1 86 .
C e mb e l l a , A . D . , J . C . Th e r r i au l t , and P . B e land . 19 88 .
T ox i c i t y o f c u l t u r ed i so l a t es a nd n a tu r a l po p u l a t i on s o f
P r o to go n ya u l ax t am a r ens i s f r om th e S t . La w r e n c e e s t u a ry . J .
S h e l l f i sh R es . 7: 611 – 62 1 .
C o le , J . J . , 19 82 . In t e r a c t io ns b e t w e en ba c t e r i a an d a l gae i n
a q u a t i c e cos ys t e ms . A nn . R e v . E c o l og . S ys t . 1 3 , 2 91 - 314 .
![Page 62: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/62.jpg)
62
D an t z e r , W . R . , and R . E . L ev i n . 1 997 . Bac t e r i a l i n f l u enc e
o n th e p r od uc t io n o f p a ra l yt i c sh e l l f i sh t ox in s b y d i no f l age l l a t e d
a l ga e . J . Ap p l . M i c r o b i o l . 8 3 :4 64 - 46 9 .
D ou c e t t e , G. J . , and C . G . T r i ck . 199 5 . C h ar a c t e r i s a t i on o f
b a c t e r i a a s so c i a t e d w i th d i f f e r en t i so l a t es o f A l ex an dr ium
t a mar e ns e , p . 3 3– 38 . I n P . La s s u s ,
Ga l l a ch e r , S . , and E . S mi th . 1 99 9 . Ba c t e r i a a nd p a r a l yt i c
s h e l l f i sh t ox ins . P ro t i s t 15 0 :2 45 -2 55
G . A r zu l , E . E ra rd -L e D enn , P . Ge n t i en , and C .
M a r c a i l l ou -L e Bau t ( e d . ) , H a rm fu l ma r in e a l ga l b l oom s . La v o i s i e r ,
P a r i s , F r a n c e .
H al l e g ra ef f , G. M. 1 99 5 . H a rm f u l a l ga l b l oo ms : a g l ob a l
o v e rv i e w , p . 1 -2 2 . I n In G . M . H a l l e g r a e f f , D . M. A nd e rso n , an d A .
D . C em b el l a ( e d . ) , M a nu a l on h a rm fu l m a r i n e m i c r o a l ga e . IO C
m a nu a l s a nd gu id e s n o . 3 3 . In t e r gov e rn m en t a l O c e an o gr ap h i c
C omm iss io n o f UNE SC O, P ar i s , F r an ce .
D ou c e t t e . 1 99 5 . P r o k a r yo t e s i n as so c i a t i o n wi th a t ox i c
A l e xa nd r iu m lu s i t an i cu m i n cu l tu r e , p . 4 5– 51 . In P . La s s us , G . A rz u l ,
E . E r ad - Le D e n n , P . G e n t i e n , a nd C . Ma r c a i l l ou - Le Ba u t ( e d . ) ,
H a r m fu l m a r i ne a l ga l b l oo ms . La v o i s i e r , P a r i s , F r a nc e .
Fr a n ca , S . , L . P in to , P . Al v i to , I . Sous a , a nd V.
V a s con c e l os . 19 96 . S tu d i e s on p ro k a r yo t es a s s o c i a t e d wi th PSP
p r od u c i n g d i no f l a ge l l a t es . In
Fi s t ar o l , G .O . , L eg r and , C . , Gr an e ´ l i , E . , 20 03 .
A l l e l op a th i c e f f e c t o f P r ym n e s ium p a rv um o n a n a t u r a l p l a n k t on
c o mmu ni t y. M a r . Ec o l . P ro g . S e r . 25 5 , 1 15 – 12 5 .
![Page 63: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/63.jpg)
63
Fi s t ar o l , G .O . , L eg r and , C . , S e land er , E . , Hu mme r t , C . ,
S to l te , W. , Gr an e ´ l i , E . , 20 04 M a r . Ec o l . P ro g . S e r . A l l e l op a t h y i n
A l ex a nd r i um s pp . : e f f e c t on a n a tu r a l p l an k to n c omm un i ty a n d on
a l ga l mo no c u l t u r e s . Aq u a t . M i c r ob . Ec o l .
H obb i e , J .E . . Da l ey R. J and J asp e r S . , U s e o f n u c l ep o r e
f i l t e r s f o r co un t in g b a c t e r i a b y f l u o r esc e n c e mi c ro s co p y. A p pl .
E n v i ro n . Mi cr ob i o l . 33 ( 19 77 ) , pp . 122 5 –1 22 8
H o ld , G . L . , E . A . S mi th , T . H . B i rkb e rk , and S . Ga l lac h e r .
2 0 01 . C om pa r i so n o f p a r a l yt i c s he l l f i s h t ox i n (PS T) p r od u c t i on b y
t h e d in o f l a ge l l a t es A l e xa nd r iu m lu s i t an i cu m Al ex a nd r i um
l us i t an i c um N EPCC 2 53 an d Al e xa ndr iu m ta ma r en s eA lex a nd r i um
t am a r e ns e N EPCC 4 0 7 i n t h e p r es e nc e a nd ab s en c e o f b ac t e r i a .
FE MS Mi c r ob i o l . Ec o l . 3 6 :2 23 -2 34 .
I sh id a , Y . , C . H . Ki m, Y . S ako , N . H ir oo ka , and A . Uc h id a .
1 9 93 . PSP t ox in i s c h r omo so m e de p end e n t i n Al e xa nd r ium sp p . , p .
8 8 1– 88 7 . I n T. J . Sm a yd a a nd Y. Sh imiz u ( ed . ) , T ox ic
p h yt o p l an k t on b lo om s i n t h e s e a . E l s ev i e r , A ms t e rd a m, Th e
N e th e r l a nd s .
Ki m, C . H . , Y . Sako , and Y . I sh ida . 19 9 3 . V a r i a t i o n o f t ox i n
p r od u c t i on an d c om po s i t i o n i n ax en ic c u l t u r es o f Al e xan dr iu m
c a t en e l la a nd A. t am a r ens e .
![Page 64: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/64.jpg)
64
Ko da ma , M. , T . Og a ta , and S . Sa t o . 1 9 88 . Ba c t e r i a l
p r od u c t i on o f s ax i tox i n . A gr i c . B io l . C h em. 5 2 :1 07 5– 107 7 .
Ko da ma , M. , T . Og a ta , S . Sak a mo to , S . Sa t o , T . H onda ,
a nd T . M iw a tan i . 1 9 90 . P ro du c t io n o f p a ra l yt i c sh e l l f i sh t ox in s b y
a b a c t e r iu m M ora xe l l a sp . i s o l a t ed f ro m Pr o t og on y au l a x t a m ar en s i s .
T ox i co n 28 : 70 7– 714 .
Kuw a e , T . , and Y H os okaw a . 19 99 . D e t e rmi n a t i on o f
a b a nd a n ce an d b i ovo lu me o f b a c t e r i a i n s e d im e n t s b y d u a l s t a t i n i g
w i t h 4 ’6 -d i ami no -2 - p h en yl i nd o l e a nd a c r id in e o r an ge : r e l a t i o ns h ip
t o d i sp e r s i on t r e a tm e n t a nd s e d im e n t c h a r a c t e r i s t i c s . A pp l . E nv i r on .
M i cr ob io l . 6 5 : 34 07 - 34 12 .
O ga t a , T . , M . Koda ma , K . Ko ma ru , S . S aka mo t o , S . S a t o ,
a nd U. S i mi du . 199 0 . P ro du c t io n o f pa r a l yt i c sh e l l f i s h t ox i ns b y
b a c t e r i a i so l a t ed f ro m tox i c d i no f l a ge l l a t e s , p . 31 1– 31 5 . In E.
G r a n e l i , B . Su nd s t ro m, L . Ed l e r , a nd D . M .A nd e r so n ( ed . ) , Tox i c
m a r i n e ph yt o p l a nk to n . E l s e v i e r , A ms te r d am , Th e Ne th e r l a n ds .
O shi ma , Y . , S . I . B l a ckbu rn , and G. M . Hal l eg r aef f . 19 9 3 .
C om pa r a t iv e s t ud y o n p a r a l yt i c sh e l l f i s h t ox i n p r o f i l e s o f t h e
d in o f l a ge l l a t e G y mn o d in iu m c a t en a t um f ro m t h r e e d i f f e re n t
c o un t r i e s . Ma r . B io l . 11 6 :4 71 –4 76 .
S ako , Y . , C . H . Kim, an d Y. I sh id a . 1 9 92 . M e d e l i a n
i nh e r i t a n c e o f pa r a l yt i c s h e l l f i sh p o i so n i n g t ox in i n t h e m a r i n e
![Page 65: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/65.jpg)
65
d in o f l a ge l l a t e Al e xa n dr i um ca t en e l l a . B io s c i . B io t e c hn o l . B i o ch e m.
5 6 :6 92 –6 94 .
S chu t , F . , E . J . d e V r i es , J . C . Go t t s ch a l , B . R . Rob e r tso n ,
W . Ha rd e r , R . A . Pr i ns , and D . K . Bu t ton . 19 93 . I s o l a t i on o f
t yp i c a l m a r i n e b a c t e r i a b y d i lu t i on c u l t u re : g r o wt h , m a i n t e n a n c e ,
a n d c h ar a c t e r i s t i c s o f i so l a t e s un d e r l ab o r a to r y c o n d i t i o ns . Ap p l .
E n v i ro n . Mi c r ob i o l . 59 :2 15 0 -2 16 0 .
Si lv a , E . S . 19 82 . R e l a t i o ns h i p be tw ee n d in o f l a ge l l a t es a n d
i n t r a c e l l u l a r b ac t e r i a . Ma r . A l . P h a rma c o l . S c i . 2 : 26 9– 288 .
Si lv a , E . S . 19 90 . In t ra c e l lu l a r b a c t e r i a : t h e o r i g i n o f
d in o f l a ge l l a t e t ox i c i t y . J . Pa th o l . Tox i c o l . On o l . 19 :1 24 –1 2 8 .
Si ngh , H . T . , Y . Os h i ma , and T . Y asu mo t o . 1 98 2 . G r o wt h
a n d tox i c i t y o f Pr o t og on y au l a x t a ma re n s i s i n ax en i c cu l tu r e . Bu l l .
J pn . So c . S c i . F i s h . 4 8: 1 34 1– 13 43 .
Su ikkan en , S . , F i s t a r o l . G . and Grane l i E . 20 04 .
A l l e op a t h i c e f f e c t s o f t h e Ba l t i c c ya n o b a c t e r i a No du l ar ia
s pu mi g en a , A ph an i z o m en on F l os - aq uae an d A na ba en a
![Page 66: Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial](https://reader031.vdocument.in/reader031/viewer/2022020109/5b9ba39009d3f2aa588d796a/html5/thumbnails/66.jpg)
66
L e mm e rm an n i i on a l ga l mo no c u l t u re s . J . Ex pe r . M ar in e b i o l o g y a n d
E c o l o g y. 30 8 : 8 5 -1 0 1 .
Ti l l man n , U . & J ohn , U . 20 02 . Tox i c e f f e c t s o f A l ex an dr iu m
s pp o n h e t e r t ro ph i c d i no f l a ge l l a t e s : a n a l l e l o ch em ic a l de fe n c e
m e c ha n i sm in d ep e nd e n t o f PSP tox in s . M a r E c o l . P r o g . Se r . , 23 0 : 47 -
5 8
U r ib e , P and Ro mi l io . T . 2 00 3 . E f fe t c s o f Ass o c i a t e d
b a c t e r i a o n th e g row t h an d tox i c i t y o f Al e x an dr i u m c a t ene l l a . Ap p
e n v i ro