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Allelopathy of Baltic Sea cyanobacteria:
no evidence for the role of nodularin
SANNA SUIKKANEN1*, JONNA ENGSTRÖM-ÖST1, JOUNI JOKELA2, KAARINA SIVONEN2 AND MARKKU VIITASALO1
1
2
FINNISH INSTITUTE OF MARINE RESEARCH, PO BOX 2, FI-00561 HELSINKI, FINLAND AND DEPARTMENT OF APPLIED CHEMISTRY AND MICROBIOLOGY,
DIVISION OF MICROBIOLOGY, PO BOX 56, FI-00014 UNIVERSITY OF HELSINKI, HELSINKI, FINLAND
*CORRESPONDING AUTHOR: [email protected]
Received December 2, 2005; accepted in principle January 18, 2006; accepted for publication February 13, 2006; published online February 15, 2006
Communicating editor: K.J. Flynn
Extracts of Aphanizomenon flos-aquae and Nodularia spumigena, the two most common cyanobacteria forming recurrent blooms in the Baltic Sea, decrease the abundance of some phytoplankton
species via the release of allelopathic substances. We investigated how cell-free filtrates of the two
cyanobacteria, as well as purified hepatotoxin nodularin, produced by N. spumigena affected cell
numbers, chlorophyll a content and 14CO2 uptake of the cryptophyte Rhodomonas sp. Both
cyanobacterial filtrates significantly retarded the growth of Rhodomonas sp., A. flos-aquae filtrate
up to 46%, whereas purified nodularin showed no significant effect on any of the growth parameters
of the cryptophyte. These results suggest that the allelopathic effect of N. spumigena is most probably
due to metabolite(s) other than nodularin, possibly acting via the damage of the target cells.
INTRODUCTION
The term allelopathy refers to inhibitory and stimulatory
effects of plants and microorganisms on other plant species or microorganisms through the release of organic
compounds (Rice, 1984). Such interactions occur in all
aquatic habitats and can be caused by members from all
groups of aquatic primary producers (Gross, 2003). The
two nitrogen-fixing, brackish-water cyanobacteria,
Aphanizomenon flos-aquae and Nodularia spumigena, are the
most common species that form recurrent cyanobacterial
blooms in the Baltic Sea. Recently, both species were
found to decrease the abundance of the cryptophyte
Rhodomonas sp. and the diatom Thalassiosira weissflogii
grown in monocultures (Suikkanen et al., 2004). Also
when applied to a natural Baltic Sea plankton community, cyanobacterial cell-free filtrates decreased the
amounts of cryptophytes, whereas they increased the
abundance of other cyanobacteria, the chlorophyte
Oocystis sp., the dinoflagellate Amphidinium sp. and nanoflagellates (Suikkanen et al., 2005). However, the
mechanisms of the observed allelopathic actions, underlying the decrease in cell numbers, or the chemical compound(s) causing them, are still unknown.
Most of the cyanobacterial inhibitory allelochemicals
are directed against oxygenic photosynthetic processes of
other cyanobacteria or algae; more specifically, they
inhibit the electron transport in the vicinity of photosystem II (Smith and Doan, 1999). Thus, through the inhibition of photosynthesis, the allelochemicals decrease the
production and cell numbers of the target species. The
effects are probably first expressed at the level of the
photosynthetic pigments and primary production capacity and, finally, in cell numbers.
To date, some cyanobacterial allelochemicals have
been isolated and characterized (Smith and Doan, 1999
and references therein), and, e.g., the antialgal compounds produced by the freshwater Microcystis aeruginosa
and Anabaena spiroides have been identified as peptides
(Ishida and Murakami, 2000; Kaya et al., 2002). The
cyanobacterial peptide hepatotoxin, microcystin, has
been found to cause allelopathic effects both on higher
plants (MacKintosh et al., 1990; Pflugmacher, 2002) and
phytoplankton (Kearns and Hunter, 2000, 2001; Singh
et al., 2001). In a comparison between the allelopathic
effects of exponential and stationary N. spumigena cultures
on cell numbers of a diatom and a cryptophyte species,
allelopathic activity was only expressed by the exponential culture, with a lower nodularin concentration
(Suikkanen et al., 2004). Thus it was concluded that
nodularin was unlikely the cause of the observed
doi:10.1093/plankt/fbi139, available online at www.plankt.oxfordjournals.org
Ó The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
JOURNAL OF PLANKTON RESEARCH
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allelopathic activity. Nodularin, however, being structurally closely related to microcystin, may also be capable
of acting as an allelochemical, and therefore its potential
allelopathic effects warrant more specific attention.
The aims of the present study were (i) to investigate and
compare the allelopathic effects of the selected cyanobacteria on different growth parameters, i.e. cell numbers,
chlorophyll a (Chl a) concentration and 14CO2 uptake, of
the target species and (ii) to test if purified nodularin had an
effect on the target species, comparable to that of N. spumigena filtrate with a corresponding nodularin concentration.
We hypothesized that both cyanobacteria would inhibit
photosynthetic processes of Rhodomonas sp. (demonstrated
by inhibition of 14CO2 uptake and Chl a concentration),
that the effects on Chl a and 14CO2 uptake would not
necessarily be reflected in cell numbers and that purified
nodularin would not have as strong allelopathic effects as
the crude N. spumigena filtrate.
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The cyanobacterial strains, N. spumigena AV1 and A. flosaquae Tr183, were isolated at the Division of Microbiology,
University of Helsinki, Finland. The N. spumigena strain
produces nodularin, and the A. flos-aquae strain is nontoxic (Lehtimäki et al., 1997; Lyra et al., 2001). Based on
the enzyme-linked immunosorbent assay (ELISA; procedure described below), conducted shortly before the experiments, the N. spumigena culture filtrate contained 27.7 ng
nodularin equivalents (mg Chl a)–1, whereas the A. flos-aquae
filtrate was non-toxic. As target algae we used the cryptophyte Rhodomonas sp. (cf. salina) KAC 30, obtained from the
Kalmar Algal Collection, University of Kalmar, Sweden.
All strains were originally isolated from the Baltic Sea,
where Rhodomonas spp. typically occur during summer
(Hill, 1992), approximately simultaneously with the cyanobacterial summer maximum.
All strains were grown as batch cultures in f/10 medium
(Guillard, 1975) at 188C, in 30 mmol photons m–2 s–1, in a
16:8 h light:dark regime, and with continuous air supply.
The culture medium was prepared from filtered seawater
(Whatman GF/F glass microfibre filter) with a salinity of
5.1, and with pH adjusted to 8.2. The culture biomass was
monitored by Chl a measurements to confirm exponential
growth of the strains in the beginning of the experiment.
N. spumigena blooms in the Baltic Sea and in strains
isolated from the same source, including the AV1 strain
that was used in the present experiment (Sivonen and
Jones, 1999). For nodularin extraction, 1.6 g of freezedried cyanobacterial bloom sample from sampling station SR5 (Kononen et al., 1993) was loaded into a 11-mL
stainless steel extraction cell and extracted with Dionex
ASE 200 Accelerated Solvent Extractor (Dionex Corp.,
Sunny Vale, CA, USA) by using one extraction cycle
with methanol and the following method settings: preheat time 2 min, heat-up time 5 min, static extraction
time 15 min and pressure 10.3 MPa, extraction temperature 408C, flush% 60 and purge time 20 s with nitrogen
gas.
The methanol extract (40 mL) was prepurified with an
SPE cartridge (10 g/60 mL Mega Bond Elut C18,
Varian, Palo Alto, CA, USA). The methanol from the
cartridge was evaporated with a Heto Maxi dry plus
vacuum centrifuge (Heto-Holten A/S, Allerød,
Denmark). The residue was dissolved into 1 mL 30%
(v/v) acetonitrile in water and filtered over a 0.2 mm
Gelman Acrodisk filter (13 mm).
Nodularin was purified from the extract with an
Agilent 1100 high-performance liquid chromatography
(HPLC), equipped with a diode array detector and a
Phenomenex Luna C18(2) column (150 4.6 mm,
5 mm particle size) at 408C. The extract was injected
into the column in 100 mL portions, and isocratically
eluted (1 mL min–1) with 24% (v/v) acetonitrile and 76%
of 0.1% trifluoracetic acid (TFA) in water (pH adjusted to
7.1 with 24% ammonium hydroxide solution).
Nodularin was detected at 238 nm, and the effluent
was collected from 19.6 to 24.5 min. The fractions were
pooled, diluted with an equal volume of water and
desalted with an SPE cartridge (0.2 g/6 mL Oasis
HLB, Waters, Milford, MA, USA). After washing with
10 mL of water, purified nodularin was eluted from the
cartridge with 4 mL of acetonitrile. The solution contained 395 mg of nodularin, calculated as microcystin-LR
equivalents at 238 nm. The purity of nodularin in the
solution was 99% (238 nm), when analysed with the
HPLC method employed in the purification. For storage,
the solvent was removed by evaporation using a Heto
Maxi dry plus vacuum centrifuge.
For the experiments, dry toxin was dissolved in 99.8%
methanol and subsequently diluted to 20% with MilliQ
water. The final methanol concentration did not exceed
5 ppm (v/v) in the experimental units.
Preparation of purified nodularin
Experimental design
Nodularin-R (hereafter nodularin; Rinehart et al., 1988)
is the dominant nodularin variant both in natural
We investigated the effects of (i) cell-free filtrates of N.
spumigena and A. flos-aquae and that of (ii) purified
METHOD
Culture conditions
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nodularin, on the cell numbers, Chl a content and 14CO2
uptake of Rhodomonas sp.
To test the effects of cyanobacterial cell-free filtrates,
we gently filtered aliquots of the cyanobacterial cultures
through Whatman GF/F filters, with a pressure difference lower than –2 kPa. Freshly prepared filtrates were
added to triplicate 1-L erlenmeyer flasks containing the
target algae (final volume 400 mL). Based on the Chl a
contents of the original cultures, the ratio of cyanobacteria to target species in the flasks was adjusted to 2:1
(256.1 mg Chl a L–1 cyanobacteria and 128.0 mg Chl a L–1
Rhodomonas). This ratio represents an underestimate of
those in cyanobacterial bloom conditions, where the
amounts of cyanobacteria, in relation to other phytoplankton species, may be much higher (Kononen, 1992).
Concentrations of up to 400 mg Chl a L–1 have been
reported during blooms of N. spumigena (Potter et al.,
1983). Triplicate controls were made by adding a volume
of f/10 medium, equal to the volume of cyanobacterial
filtrate added to the treatment flasks. Prior to the experiment, the concentrations of nitrate (NO
3 -N) and phosphate (PO3
-P)
were
measured
in
the
cyanobacterial
4
cultures (Koroleff, 1979). In the filtrates used in the experiment, the nitrate and phosphate concentrations were
adjusted to the same level as in the f/10 growth medium.
Therefore both the treatments and the controls contained
the same amount of nutrients. The experiment lasted 6
days, and samples for cell counts, Chl a and 14CO2 uptake
were taken and pH was measured (from one bottle per
treatment) at least every second day. The volume removed
during every sampling occasion (50 mL) was replaced with
an equal volume of fresh cyanobacterial filtrate or control
medium.
In another experiment, we investigated the effects of
purified nodularin on Rhodomonas sp. We used a nodularin concentration of 10 mg L–1, which corresponded to
the nodularin concentration in the experiment using cellfree filtrate of N. spumigena (7 mg L–1). In the field, the
concentration of free nodularin in the water may vary
from 0.01 to 18.7 mg L–1 (Kononen et al., 1993). The
initial Chl a concentration of the target algae was
125 mg L–1, corresponding to ca. 161 106
Rhodomonas sp. cells L–1. The total volume in the 1-L
experimental flasks was 400 mL. At the beginning of
the experiment, 85 mL 570 mM nodularin solution (corresponding to 10 mg nodularin L–1) was added to triplicate experimental units, and 85 mL distilled water, with a
methanol concentration corresponding to that of the
nodularin solution (2 mL MeOH mL–1), was added to
triplicate control units. Every second day of the 6-day
experiment, we took samples for determination of Chl a,
14
CO2 uptake and cell numbers, measured pH and
replaced the sampling volume (50 mL) with an equal
amount of f/10 medium, containing 11 mL of either
570 mM nodularin or the control solution. Nodularin
uptake of the target algal cells or adherence to their
surface was monitored by taking samples for ELISA on
the first and the last experimental day.
Analyses
For Chl a analysis, 5 ml samples of the cultures were
filtered over GF/F filters (Whatman, Middlesex, UK),
which were sonicated for 10 min (Branson GWB 2200)
and extracted in 96% ethanol (Jespersen and
Christoffersen, 1987). The Chl a fluorescence was measured with a Shimadzu spectrofluorophotometer RF
5000 (Japan).
Cell numbers of Rhodomonas sp. were determined from
live samples with an electronic particle analyser (Elzone
282 PC, Particle Data, Elmhurst, IL, USA) directly after
sampling. The particle analyser was calibrated with a
standard solution of latex particles of two different sizes.
Apparent net production (14CO2 uptake) was measured according to Niemi et al. (1983). From each experimental unit, duplicate 10 mL light samples and a dark
sample were incubated with 0.5 mCi (18.5 kBq) of
NaH14CO3 (14C Agency, Denmark) for 4 h in experimental conditions. Thereafter, 4 mL of water were transferred to a glass scintillation vial and acidified with 100 mL
of 1 N HCl. The open vials were allowed to stand in a fume
hood for 24 h, in order to release inorganic 14CO2, after
which 7 mL of OptiPhase ‘HiSafe’ 3 scintillation cocktail
(Wallac) was added. Radioactivity was assayed with a
Wallac WinSpectral 1414 liquid scintillation counter,
using the external standard channel ratio method for
standardization.
The hepatotoxin concentration in the cyanobacterial
filtrates was analysed from 10-mL samples taken from
GF/F filtered N. spumigena and A. flos-aquae cultures. The
transfer of purified nodularin from the medium to the
target algal cells, and/or its adsorption on the cell surfaces, was quantified by taking duplicate 20-mL samples
from both the nodularin treatment and the control and
filtration through Whatman GF/C filters. All samples
were stored in –208C until analysis. Before the measurement, the filters were freeze-dried (GWB Edwards Super
Modulyo, West Sussex, UK), dissolved in 5 mL 100%
methanol, sonicated (MSE Soniprep 150 Ultrasonic
Disintegrator, Sanyo Gallenkamp, UK) and extracted
for 12 h. Subsequently the samples were filtered through
GF/F filters and dried with N2 gas. Then 40 mL 50%
methanol (v/v) and 280 mL MilliQ water were added to
the samples, starting with the methanol. During the
following 4 days, 70 mL MilliQ was added each day,
until a final concentration of 6.3% methanol was
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reached. On the fifth day, the samples were analysed by
means of ELISA (Chu et al., 1990), using a microcystin
plate kit (EnviroLogix, Portland, ME, USA), according
to the kit instructions. The nodularin concentration was
determined from absorbance at 450 nm, measured with
an EMS Reader MF (Labsystems, Vantaa, Finland)
photometer. The nodularin concentration values
obtained for the controls, without nodularin additions,
were probably false positive results of the ELISA. They
were thus considered as a background level and subtracted from the concentrations obtained for the respective nodularin treatments of the target species.
Statistical analyses
Repeated measures analysis of variance (ANOVA) (Zar,
1999) was used to test for differences in the growth
parameters between the target algae cultures treated
with cyanobacterial filtrates or purified nodularin and
the control, over the entire experimental period. A post
hoc test (Tukey’s HSD) was used to show which treatments significantly differed from the control and from
each other. The data were tested for normality and
homogeneity of variances. All tests are two-tailed, with
a significance level of P = 0.05, and data are reported as
means ± SD. The statistical analyses were performed
using the software SPSS 10.0.7 for Windows.
RESULTS
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cyanobacterial filtrates had decreased the cellular Chl a
content by 34% (A. flos-aquae) and 12% (N. spumigena).
There was also a significant difference in the cellular
14
CO2 uptake between the filtrate treatments and the
control (Table I, Fig. 1C), although the effects of the
two cyanobacteria did not differ. At first, the A. flosaquae filtrate increased the 14CO2 uptake of Rhodomonas
sp., but from day 4 onwards, the 14CO2 uptake was lower
in both filtrate treatments, compared to the control. By
day 6, the 14CO2 uptake was 46 or 43% lower in the
cultures treated with A. flos-aquae and N. spumigena filtrates, respectively.
The average pH was 8.3 ± 0.2 (mean ± SD, n = 4)
both in the control Rhodomonas sp. cultures and in those
treated with N. spumigena filtrate, and 8.1 ± 0.2 in the
cultures treated with A. flos-aquae filtrate.
Effects of purified nodularin
The addition of purified nodularin did not significantly
affect any of the growth parameters of Rhodomonas sp.
(Table I, Fig. 1D–F).
According to the ELISA, the cells of Rhodomonas sp.
contained more nodularin (73.5 10–6 pg nodularin
equivalents cell–1; mean, n = 2) shortly after the first
nodularin addition at the beginning of the experiment,
than at the last experimental day (2.2 10–6 pg nodularin equivalents cell–1), after three subsequent nodularin
additions.
The average pH of both the control and nodularintreated target cultures was 9.4 ± 0.6 (mean ± SD, n = 4).
Effects of cyanobacterial filtrates
The growth and production of Rhodomonas sp. were
retarded by both cyanobacterial filtrates (Fig. 1A–C).
Repeated measures ANOVA revealed significant differences in the cell numbers, as well as cellular Chl a content
and CO2 uptake of Rhodomonas sp. between the cyanobacterial filtrate treatments and the control (Table I). Both
cyanobacterial filtrates significantly decreased the cell
number of Rhodomonas sp., compared to the control (Fig. 1A).
This inhibition was already observed after the first experimental day, but the effects of the two cyanobacteria only
started to diverge after 3 days, after which A. flos-aquae
extract inhibited the cell number development significantly
more than that from N. spumigena. By day 6, A. flos-aquae and
N. spumigena filtrates had decreased the cell numbers of
Rhodomonas sp. by 29 and 14%, respectively.
A similar pattern was observed for the cellular Chl a
content of Rhodomonas sp. (Table I, Fig. 1B), but in this
case, the effects of the cyanobacterial filtrates only differed significantly from the control after two experimental days. By the end of the experiment, the
DISCUSSION
The results indicate that nodularin is not the cause of
allelopathic effects of N. spumigena. Nodularin did not
significantly affect Rhodomonas sp. at an environmentally
relevant concentration, corresponding to the nodularin
concentration in N. spumigena culture filtrate, which significantly decreased the growth and production of
Rhodomonas sp. Moreover, the non-toxic A. flos-aquae filtrate caused even stronger inhibition of Rhodomonas sp.
growth than the toxic filtrate of N. spumigena. The
observed allelopathic effects of both cyanobacterial filtrates were thus most likely caused by other biologically
active substances than the known toxins. This conclusion
agrees with other studies that have not found any correlation between toxicity and antibiotic or allelopathic
effects in cyanobacteria (Campbell et al., 1994; Lahti
et al., 1995; Østensvik et al., 1998; Suikkanen et al.,
2004) or other phytoplankton species (Arzul et al., 1999;
Fistarol et al., 2004; Sugg and VanDolah, 1999; Tillmann
546
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Cells (×103 mL–1)
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A
350
Aph filtrate
Nod filtrate
Control
300
280
260
250
240
200
220
1.4
Chl a (pg cell–1)
Nodularin
Control
300
150
200
CO2 uptake (pg C cell–1 h–1)
D
B
1.6
1.2
1.4
1.0
1.2
0.8
1.0
0.6
0.8
0.4
0.6
0.8
C
1.4
E
F
1.2
0.6
1.0
0.4
0.8
0.2
0.6
0.4
0.0
0.2
0
1
2
3
4
5
6
0
Time (days)
1
2
3
4
5
6
Time (days)
Fig. 1. Cell numbers (A, D), chlorophyll a (B, E) and 14CO2 uptake (C, F) of Rhodomonas sp. treated with (A–C) cell-free filtrates of Aphanizomenon flos-aquae
(Aph filtrate) and Nodularia spumigena (Nod filtrate) as well as the control medium (Control) and (D–F) treated with purified nodularin (Nodularin) and the
control medium (Control; n = 3, mean ± SD).
and John, 2002) and studies where stronger effects on
aquatic organisms have been detected by crude cyanobacterial extracts or a co-exposure of hepatotoxins and
endotoxins, e.g. lipopolysaccharides, than by equivalent
concentrations of purified cyanotoxins (Best et al., 2002;
Bury et al., 1996; Pietsch et al., 2001). Nodularin may
have some effect on phytoplankton under field conditions
with much smaller phytoplankton concentrations than
those used in the present study, but metabolites other
than nodularin are likely to be mainly responsible for the
allelopathic effects of N. spumigena. These metabolites
appear to be extracellular, because the effects are caused
by the cell-free culture medium.
Suggestions for the ecological role of cyanobacterial toxins have ranged from cell–cell signalling to defence against
grazers and competitors, but a consensus is still lacking
(Kaebernik and Neilan, 2001). The mechanism of action
is similar in nodularin and closely related microcystins: they
inhibit protein phosphatases in eukaryotic cells (Yoshizawa
et al., 1990). In addition to various animal poisonings,
microcystins have also been observed to inhibit microalgal
growth (Kearns and Hunter, 2000, 2001; Singh et al., 2001).
On one hand, it would be logical to assume that nodularin
has similar effects on the aquatic primary producers, especially as it is capable of direct action on cell membranes
(Spassova et al., 1995), although in the present study, no
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Table I: Repeated measures analysis of variance (ANOVA) and Tukey’s HSD post hoc test were applied
to reveal differences in the three growth parameters between the target algae cultures in the control and
treatments with cyanobacterial filtrates or purified nodularin
Parameter
Filtrate experiment
Nodularin experiment
Repeated measures
Tukey’s HSD post hoc test
Repeated measures
ANOVA
ANOVA
F(2,6)
P-value
n
P-value (Aph-Nod)
P-value (Aph-ctrl)
P-value (Nod-ctrl)
F(1,4)
P-value
n
Cell number L–1
57.2
0.000
3
0.009
0.000
0.002
0.11
0.76
3
Chl a cell–1
55.5
0.000
3
0.002
0.000
0.01
1.3
0.32
3
14
21.3
0.002
3
0.43
0.007
0.002
5.1
0.09
3
CO2 uptake cell–1
such effects were found. On the other hand, the allelopathic
effects of microcystins and nodularin have been studied
using different target algae. The effects of microcystins
have been tested on chlorophytes and cyanobacteria
(Kearns and Hunter, 2000, 2001; Singh et al., 2001),
whereas effects of nodularin have only been studied using
cryptophytes. Although it is possible that nodularin has
negative effects on target organisms other than cryptophytes, cell-free extracts of N. spumigena, the principal nodularin producer, added to a whole phytoplankton
community were only found to negatively affect cryptophytes and diatoms (Suikkanen et al., 2005).
Cryptophytes generally seem to be sensitive to chemicals released by other phytoplankton groups, including
cyanobacteria (Granéli and Johansson, 2003; Infante and
Abella, 1985; Rengefors and Legrand, 2001). In the
present study, cell-free filtrates of both cyanobacteria,
but especially A. flos-aquae, significantly inhibited the
growth and production of Rhodomonas sp. In another
monoculture experiment, A. flos-aquae was also observed
to inhibit Rhodomonas sp. more strongly than N. spumigena
(Suikkanen et al., 2004). In an experiment employing a
natural phytoplankton community, only N. spumigena
inhibited cryptophytes (Suikkanen et al., 2005), but the
cryptophytes present in the community were other species than Rhodomonas sp.
Cyanobacteria produce bioactive substances affecting
a range of biochemical processes, but most of their allelochemicals characterized to date are directed against
photosynthesis (Gross et al., 1991; Smith and Doan,
1999; Sukenik et al., 2002). Light-dependent processes
of prokaryotic cyanobacteria and eukaryotic algae
would be the logical targets for competing organisms
capable of producing bioactive metabolites. In the present study, however, the pattern of inhibition by the
cyanobacterial extracts proceeded from the effects on
cell numbers of Rhodomonas sp. after the first day, to
inhibition of 14CO2 uptake and Chl a concentration
only after two or three days. Also there was first an
increase in primary production simultaneously with the
decrease in cell numbers. Therefore the results do not
support the hypothesis that the growth inhibition caused
by the filtrates would be associated directly with the
inhibition of photosynthetic processes. The observed
decrease in cell numbers may have been caused by, e.g.
breakage of the cell membranes, as has been demonstrated in several studies of phytoplankton allelopathy
(reviewed by Legrand et al., 2003).
At least part of the purified nodularin added to the
target algal cultures was incorporated into the algal cells
by either transport into the cells or adsorption on the cell
surface. To our knowledge, nodularin has not previously
been reported to attach or be transported into phytoplankton cells. The decrease of the toxin concentration in
the cells of Rhodomonas sp. over the course of the experiment may have been due to either a dilution effect
during cell division, or to the utilization of nodularin by
Rhodomonas sp., as, e.g., a nutrient source. Also, detoxifying enzyme systems have been found in many plant
species, including marine macroalgae (Pflugmacher and
Steinberg, 1997). It is unlikely that the observed decrease
in the cellular nodularin concentration was due to bacterial or physical degradation of the toxin, because nodularin was added to the cultures every second day.
To conclude, our results support the hypothesis that the
hepatotoxin, nodularin is not the main allelopathic compound produced by N. spumigena, although nodularin is
incorporated, at least to some extent, into phytoplankton
cells. The growth inhibition of Rhodomonas sp. caused by N.
spumigena and A. flos-aquae filtrates may be associated with
the action on target cell surfaces, causing damage to the
cell membranes rather than affecting photosynthesis after
entry to the target cells. In the field, allelopathy is probably
one of the competitive strategies of cyanobacteria,
mediated via specific chemicals that decrease cell numbers
of certain phytoplankton species.
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ACKNOWLEDGEMENTS
Kaebernik, M. and Neilan, B. A. (2001) Ecological and molecular investigations of cyanotoxin production. FEMS Microbiol. Ecol., 35, 1–9.
Prof E. Granéli kindly provided the Rhodomonas sp. strain
KAC 30. We are also grateful to A.-M. Åström and
U. Sjölund for nutrient analyses and to M. Wahlsten for
help in nodularin purification. R. Autio and M.
Karjalainen are acknowledged for assistance with 14CO2
uptake and nodularin analyses, respectively. This study was
financed by the Maj and Tor Nessling Foundation and the
Walter and Andrée de Nottbeck Foundation.
Kaya, K., Mahakhant, A., Keovara, L. et al. (2002) Spiroidesin, a novel
lipopeptide from the cyanobacterium Anabaena spiroides that inhibits
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