FEMS Microbiology Ecology 12 (1993) 127-130 127

© 1993 Federation of European Microbiological Societies 0168-6496/93/$06.00

Published by Elsevier

FEMSEC 00460

Isolation and verification of anatoxin-a producing

clones of Anabaena flos-aquae (Lyngb.) de Breb.
from a eutrophic lake
N. Kangatharalingam a n d J o h n C. P r i s c u

Biology Department, Montana State University, Bozeman, MT, USA

(Received 30 December 1992; revision received 4 March 1993; accepted 8 March 1993)

Abstract: We present a technique to isolate and confirm anatoxin-a producing clones (single trichome-isolates) of Anabaena
flos-aquae (Lyngb.) de Breb. from blooms of this eyanobacterium. A single trichome is isolated from a field sample and grown in
ASM medium. Single trichomes are then isolated from this culture and grown in ASM medium to produce single clone cultures.
Mouse bioassay, and thin-layer chromatography (TLC) using purified anatoxin-a as reference is then used to confirm the anatoxin-a
producing clones. Using this methodology, Anabaena flos-aquae samples collected during July 1991 from Hebgen Lake, Montana,
were found to contain only 8.7% anatoxin-a producing clones. This minor proportion of anatoxin-a producing clones apparently
accounts for the anatoxin-a produced by the entire population of A. flos-aquae. Our technique is simple and reproducible. A
selected clone of A. flos-aquae that produces anatoxin-a and one that does not produce anatoxin-a were deposited in the UTEX
culture collection, University of Texas at Austin.

Key words: Anatoxin-a; Toxic cyanobacteria; Cyanobacterial toxin; Cyanobacterial clone; Cyanobacterial strain; Intra-species
variants of cyanobacteria; Anabaena

Introduction ( M o o r e 1977; J u d a y et al., 1981; C a r m i c h a e l ,

C y a n o b a c t e r i a t h a t p r o d u c e toxins a r e f o u n d
in v a r i o u s p a r t s o f t h e w o r l d i n c l u d i n g T h e U n i t e d
S t a t e s a n d C a n a d a ( C a r m i c h a e l et al., 1985). M o s t
c y a n o b a c t e r i a l p o i s o n i n g s in N o r t h A m e r i c a a r e
a s s o c i a t e d with b l o o m s o f A n a b a e n a flos-aquae
Correspondence to: N. Kangatharalingham, Department of
Microbiology & Immunology, Sherman Fairchild Science
Bldg., Stanford University, Stanford, CA 94305. Tel.: (415)
723-5210 or (415) 988-0733
1981). A n a b a e n a flos-aquae p r o d u c e s t h e p o t e n t
nicotinic c h o l i n e r g i c alkaloid, a n a t o x i n - a , a bi-
cyclic s e c o n d a r y a m i n e ( 2 - a c e t y l - 9 - a z o b i c y c l o
[4.2.1]non-2-ene) ( D e v l i n et al., 1977; M o o r e ,
1977; Stevens a n d K r i e g e r , 1988). H o w e v e r , all
strains o f A. flos-aquae d o n o t p r o d u c e a n a t o x i n - a
( C a r m i c h a e l a n d G o r h a m , 1978; Stevens a n d
K r i e g e r , 1988; M o o r e , 1977). H e n c e it is i m p o r -
t a n t to f o r m u l a t e t e c h n i q u e s t h a t a r e simple, effi-
cient a n d r e l i a b l e to isolate a n d c h a r a c t e r i z e a n a -
toxin-a p r o d u c i n g clones o f A. flos-aquae so t h a t
s o u n d w a t e r quality j u d g e m e n t s c a n b e m a d e .
128
Because A. flos-aquae produces at least four
different kinds of anatoxin (anatoxin a, b, c and
d) anatoxin-a being the most potent (Carmichael
and Gorham, 1978), it is important that the tech-
nique should be able to accurately confirm the
presence of anatoxin-a.
Materials and Methods
Unialgal populations of A. flos-aquae (Lyngb.)
de Breb. collected during July 1991 from four
random locations in the Greyling Arm of Hebgen
Lake, Montana were combined and maintained
for up to 7 days at a temperature of 25 + I°C and
a photon flux density of 150 /zmol quanta m -2
s-1 supplied by cool white fluorescent lamps un-
der a 12 h L / D cycle.
Five random aliquots of 5 ml each were re-
moved from the stock culture while stirring, and
combined for use in the isolation procedure. The
25 ml aliquot thus obtained was gently passed
twice through 22-gauge hypodermic needle fitted
on a 30 ml syringe to disperse aggregates of the
cyanobacterium. The dispersed cyanobacterial
sample was then treated with 0.1 ml Tween-80 to
prevent the trichomes from reaggregating. The
dispersed aliquot was then diluted with ASM
medium (Allen, 1968) with 10 /zM NaNO 3 to
obtain approximately 15-25 trichomes m1-1.
While stirring, 0.15-0.20 ml aliquots of the
cyanobacterial suspension were transferred to 9
cm diameter plastic petri dishes and viewed un-
der a dissecting microscope at a magnification of
25-30 × . A 23-gauge hypodermic needle fitted
on a 1 ml syringe was then used to remove a
single trichome from the 0.15-0.20 ml aliquot
under the dissecting microscope. The syringe con-
tent was transferred to a new petri dish to view
under the dissecting microscope to ensure that a
single trichome only was removed. After adding a
few more drops of ASM medium to the culture
sample containing the single trichome, it was
transferred using a fresh 1 ml syringe to a test
tube containing 5 - 6 ml ASM medium with 20
/zM NaNO 3. This isolate was incubated under
the same conditions as the stock culture with
gentle agitation every 2 days for up to 10 days.
After 10 days, samples from each culture tube
were viewed under the dissecting microscope.
Those cultures that contained growing A. flos-
aquae were subjected one more time to the same
procedure of dispersion, dilution and single tri-
chome isolation in order to be sure the final
culture samples were derrived from a single tri-
chome. The A. flos-aquae clones thus obtained
were allowed to grow for about 2 weeks in the
test tubes before transferring to ASM medium of
the same composition in 250 ml conical flasks.
We produced a total of 23 clone-cultures (C-1 to
C-23) of A. flos-aquae.
Each one of the 23 single trichome clones were
tested for anatoxin-a production using (1) mouse
bioassay, and (2) thin-layer chromatography
(TLC) combined with mouse bioassay. 75 /zl
packed cell volume obtained by centrifuging acid-
ified (using 0.2 mM HCI to pH 3.0) culture was
disrupted thoroughly in 1 ml ice-cold ASM
medium at p H 8.2 using an ultrasonic probe-dis-
ruptor (Braun, at a power setting of 5 on a 5 0 / 5 0
duty cycle for 3-5 min). The 0.5-0.6 ml extract
(pH adjusted to 7.0 using 0.1 N N a O H ) obtained
after centrifugation was intra-peritoneally (IP) in-
jected into a male B a l b / c B y mouse with an aver-
age body weight of 26 g. A 0.55 ml portion of
0.9% (w/v) saline was injected into a control
mouse. The injected mice were closely observed
for at least 30 min.
For TLC, 125 /zl packed cell volume of each
clone of A. flos-aquae was extracted in a 2 : 1 : 2
( v / v / v ) mixture of water/absolute ethanol/acet-
one according to the sonication procedure de-
scribed for the mouse bioassay. The supernatant
was concentrated to about half its original volume
using a N2-jet from a hypodermic needle. The
concentrated supernatant was then used to
spotdry polyester TLC plates (Sigma Chemical
Co.) coated with 2 5 0 / z m thick layer of silica gel
with a mean particle size of 2-25/~m, and mean
pore diameter of 60 ,&. A control spot (0.02 /zg)
containing pure anatoxin-a ((+)-isomer obtained
from Biometric Systems Inc., Eden Prairie, MN)
was used as a control on every TLC plate. The
extract from each clone was sufficient for about
5 - 6 spots. The silica gel had a fluorescent indica-
tor at a wave length of 254 nm. The plates were

run at 4 °C for over 2 h using a solvent mixture
containing 2 : 1 : 2 ( v / v / v ) w a t e r / a b s o l u t e
ethanol/acetone in a closed TLC jar pre-equi-
librated with the solvent mixture. The plates were
dried using a blow-dryer, viewed under a broad
spectrum UV source and the UV sensitive spots
along the elution path of each spot including that
corresponding to the authentic anatoxin-a spot
was demarcated with a pencil. The spots having
the same Rf value (relative to the solvent front,
i.e., ratio of distance of the spot to the distance of
the final solvent front from the original sample
spot) in each clone were accumulated (excluding
the control elution path having the authentic
anatoxin-a) after carefully scraping individual
spots. The silica gel samples from spots accumu-
lated were then separately suspended in 1.5 ml of
0.3% (w/v) saline, vortexed to ensure dissolution
of the soluble compounds from the samples, and
centrifuged to eliminate silica particles. The 0.6
+ 0.05 ml supernatant obtained, was tested by
mouse bioassay as described above. Both experi-
ments were repeated to yield two replicate obser-
vations.
Results
Out of 23 clones of A. flos-aquae isolated, 2
clones (C-2 and C-18) killed test mice within 5
min of injection. Death was preceeded by acute
convulsions, gasps and tremors, all of which are
characteristic symptoms of anatoxin-a poisoning
(Devlin et al., 1977; Moore, 1977; Stevens and
Krieger, 1988). Three clones (C-5, C-8 and C-13)
produced laziness and slight ataxia in the injected
mice but they recovered after about 30 min. All
other clones had no noticable influence on the
test mice. These results were consistant in both
experiments.
Anabaena flos-aquae clones C-2 and C-18
showed prominent UV sensitive spots (Rf value
of 0.30-0.35) corresponding to the reference ana-
toxin-a spot on the TLC plates. Of these, clone
C-2 consistantly showed the most intense spot.
The rest of the clones of A. flos-aquae showed no
visible UV sensitive spots corresponding to the
129
authentic anatoxin-a. In the mouse bioassays
when spots were eluted from the TLC plates and
injected into test mice the spot with an Rf value
0.30-0.35 from clone C-2 killed the mouse in 5-6
min whereas that from C-18 killed the mouse
after about 10 min. Symptoms were characteristic
of anatoxin-a poisoning. None of the other clones
or other spots from clones C-2 and C-18 pro-
duced significant toxic symptoms. These results
were consistant in both experiments.
The most toxic clone C-2, and clone C-3 that
produced no symptoms or UV sensitive spot cor-
responding to the authentic anatoxin-a in any of
our experiments were selected for our further
studies on anatoxin-a. These two selected clones
were deposited in the UTEX culture collection
(Department of Botany, University of Texas at
Austin) under reference numbers LB 2557 (C-2)
and LB 2558 (C-3).
Discussion
Our studies show that less than 9% of the A.
flos-aquae clones from our study area produce
anatoxin-a; only 8.7% (2 out of 23 clones) were
able to produce anatoxin-a. This minor propor-
tion of anatoxin-a producing clones of A. flos-
aquae is apparently enough to kill cattle and
wildlife in Hebgen Lake, Montana, a phe-
nomenon which has been documented on numer-
ous occasions since the mid 1970's (Juday et al.,
1981; Priscu, unpublished data). The symptoms of
laziness and slight ataxia caused by the clones
C-5, C-8, and C-13 may have been caused by
other kinds of toxic substances that may be preva-
lent in those clones.
The technique we used to isolate the anatoxin-a
producing clones of A. flos-aquae is simple and
reliable due to the stringent steps used to isolate
and culture a single trichome from which the
entire clone-culture is produced. Even though we
lost about 8-9% of the isolated single trichomes
during prolonged culturing, our culture technique
to induce a single trichome to produce a popula-
tion was very successful. The 2-step isolation of a
single trichome used in our technique adds to the
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reliability o f p u r i t y a n d g e n e t i c h o m o g e n e i t y o f
t h e c l o n e s we isolated. Toxic strains o f A . flos-
aquae i s o l a t e d by o t h e r s (e.g., G o r h a m et al.,
1964), a p p a r e n t l y d i d not o r i g i n a t e f r o m a single
t r i c h o m e . S o m e c o l o n i e s t h e y p r o d u c e d m a y have
originated from a micro-aggregate of trichomes.
H e n c e , t h e d e g r e e o f g e n e t i c purity o f t h o s e
strains is q u e s t i o n a b l e . F u r t h e r , t h e c o n f i r m a t i o n
o f a n a t o x i n - a p r o d u c t i o n by t h o s e strains was not
p e r f o r m e d using r e f e r e n c e a n a t o x i n - a . E v e n
t h o u g h this is n o t the first t i m e a t e c h n i q u e to
isolate c l o n e s of A. flos-aquae is r e p o r t e d , o u r
t e c h n i q u e has several a d v a n t a g e s a n d i m p o r t a n t
merits. O n e of t h e i m p o r t a n t a d v a n t a g e s o f o u r
isolation t e c h n i q u e is t h a t it will allow q u a n t i t a -
tive d a t a on a n a t o x i n - a p r o d u c t i o n by a g e n e t i -
cally u n i f o r m culture. B e c a u s e it is difficult to
s e p a r a t e v i a b l e single cells f r o m t r i c h o m e s , a sin-
gle t r i c h o m e m a y b e the a l t e r n a t i v e to p r o d u c e
clones. B a s e d on t h e fact t h a t t h e o r e t i c a l l y s h o r t
t r i c h o m e s f r o m fresh c u l t u r e s w o u l d very r a r e l y
have cells t h a t a r e g e n e t i c a l l y d i f f e r e n t , t h e single
t r i c h o m e clones we have p r o d u c e d s h o u l d b e
genetically homogeneous.
O u r T L C t e c h n i q u e even t h o u g h c o m b i n e d
with m o u s e b i o a s s a y was a significant s t e p in the
i d e n t i f i c a t i o n o f a n a t o x i n - a in t h e i s o l a t e d c l o n e s
o f A. flos-aquae. I m p o r t a n t l y , by c o n v e r t i n g t h e
a n a t o x i n - a into its h y d r o c h l o r i d e f o r m by acidifi-
c a t i o n in t h e initial e x t r a c t i o n p r o c e s s we p r o -
t e c t e d t h e s e c o n d a r y a m i n e N o f this toxin f r o m
o x i d a t i o n (see also Stevens a n d K r i e g e r , 1988).
T h e a u t h e n t i c a n a t o x i n - a s a m p l e we u s e d in o u r
T L C was a h y d r o c h l o r i d e f o r m too, a n d a ( + ) -
i s o m e r b e c a u s e it is in this f o r m a n a t o x i n - a is
p r o d u c e d n a t u r a l l y by A. flos-aquae (Spivak et al.,
1982).
In s u m m a r y , we have d e v e l o p e d a s i m p l e tech-
n i q u e for isolating a n d verifying a n a t o x i n - a p r o -
d u c i n g c l o n e s o f A. flos-aquae. U s i n g this m e t h o d
we s h o w e d t h a t less t h a n 9 % o f t h e t r i c h o m e s o f
A. flos-aquae f r o m a e u t r o p h i c l a k e c o n t a i n e d
anatoxin-a. Selected anatoxin-a producing and
n o n - a n a t o x i n - a p r o d u c i n g clones i s o l a t e d a n d
i d e n t i f i e d by this p r o c e d u r e have b e e n d e p o s i t e d
with t h e U T E X c u l t u r e c o l l e c t i o n (LB 2557-toxic,
L B 2558-non toxic) a n d a r e r e a d i l y available to
scientists.
Acknowledgments
T h e a u t h o r s a r e g r a t e f u l to T. G a l l i a n d N.
S h e r i f f for l a b o r a t o r y assistance, W. F r o s t a n d M.
S c h m a u t z for p r o v i d i n g mice, facilities a n d h e l p
for m o u s e bioassays. This r e s e a r c h was s u p p o r t e d
by a g r a n t f r o m T h e S o a p a n d D e t e r g e n t A s s o c i a -
tion.
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