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PRIMARY RESEARCH PAPER
The effects of Microcystis aeruginosa (cyanobacterium)on Cryptomonas ovata (Cryptophyta) in laboratory cultures:why these organisms do not coexist in steady-stateassemblages?
Viktoria B-Beres • Istvan Grigorszky •
Gabor Vasas • Gabor Borics • Gabor Varbıro •
Sandor A. Nagy • Gyorgy Borbely • Istvan Bacsi
Received: 7 May 2011 / Revised: 4 February 2012 / Accepted: 26 February 2012 / Published online: 8 March 2012
� Springer Science+Business Media B.V. 2012
Abstract The inhibitory effects of cyanobacterial
compounds as possible explanation of the lack of
stable Cyanobacteria–Cryptophyta coexistence in
steady-state phytoplankton assemblages were studied.
The possible interactions between two phytoplankton
species, a toxic Microcystis aeruginosa (cyanobacte-
ria) and a non-toxic Cryptomonas ovata (Cryptophyta)
were investigated (i) in mixed cultures (containing
C. ovata and M. aeruginosa cells); (ii) in M. aerugin-
osa crude extract-treated C. ovata cultures and (iii) in
purified microcystin-LR (MC-LR) treated C. ovata
cultures. The results of experiments proved that the
presence of living M. aeruginosa cells have more
inhibitory effects on C. ovata cultures than the crude
extract of the M. aeruginosa cells; or the presence of
the purified MC-LR. These results suggest that MCs
does not play as important role in cyanobacteria–
Cryptophyta interaction as it was presumed; hence
more complex effects (allelopathy among them) can
be significant in shallow lakes ecosystems.
Keywords Microcystis–Cryptomonas interaction �Mixed culture � Crude extract � Purified toxin
Introduction
Cyanobacteria are one of the most prominent compo-
nents of shallow lakes’ phytoplankton. The notorious
bloom formers like Planktothrix, Limnothrix, Micro-
cystis spp., and the nostocalean Anabaena, Aphani-
zomenon spp., and Cylindrospermopsis raciborskii
frequently constitute steady-state assemblages (Nasel-
li-Flores et al., 2003) both in large lakes (Padisak,
1997; Reskone & Torokne, 2000) and in small ponds
(Borics et al., 2000). There are many reasons for the
competitive success of cyanobacteria. Shade tolerance,
capability of vertical migration, resistance to grazing,
N2-fixation, production of allelopathic/toxic substances
are those characteristics that make cyanobacteria
successful organisms and dominating elements of the
Handling editor: Judit Padisak
V. B-Beres (&)
Environmental Protection, Nature Conservation and
Water Authority, Trans-Tiszanian Region, Hatvan u. 16,
Debrecen 4025, Hungary
e-mail: beres.viktoria@gmail.com
V. B-Beres � I. Grigorszky � S. A. Nagy � I. Bacsi
Department of Hydrobiology, University of Debrecen,
Egyetem Ter 1, Debrecen 4010, Hungary
G. Vasas � G. Borbely
Department of Botany, University of Debrecen, Egyetem
Ter 1, Debrecen 4010, Hungary
G. Vasas
CETOX Analytical and Toxicological, Researching and
Consulting Ltd, Debrecen 4032, Hungary
G. Borics � G. Varbıro
Department of Tisza River Research, Balaton
Limnological Research Institute, Hungarian Academy of
Sciences, Klebelsberg Kuno u. 3, Tihany 8237, Hungary
123
Hydrobiologia (2012) 691:97–107
DOI 10.1007/s10750-012-1061-9
phytoplankton in late successional state (Padisak et al.,
2003). Although cyanobacteria-dominated assem-
blages are common in eutrophic lakes, stable coexis-
tence of cyanobacteria with dinoflagellates can be
observed frequently in stratified, mesotrophic, and
shallow lakes. The Lo and LM codons proposed by
Reynolds et al. (2002) refer to this type as a functional
association. Co-occurrence of heterocytic cyanobacte-
ria with dinoflagellates were reported in Lake Balaton
(Hajnal & Padisak, 2008) and in oxbow lakes (Krasznai
et al., 2010). The common features of these groups are
the grazing resistance and capability of active
locomotion.
In contrast to cyanobacteria, which are a poor
quality food for zooplanktic grazers (Ahlgren et al.,
1990; George et al., 1990; Rohlack et al., 2001;
Stutzman, 2006) cryptomonads are preferred food
organisms (Ahlgren et al., 1990; George et al., 1990;
Stutzman, 2006) for both planktonic (Daphnia spp.,
Chydorus spp., Eubosmina spp.—Ahlgren et al., 1990;
Chen & Folt, 1993) and freshwater invertebrate
(Dreissena polymorphya—Vanderploeg et al., 1996)
grazers as well. In the lack of large sized zooplanktic
grazers, which is one of the most important features
of eutrophic lakes in summer, the large sized cryp-
tomonads are also characteristic members of the
phytoplankton (Krasznai et al., 2010). Although
cyanobacteria–cryptomonads coexistence would also
be expected, it has never been reported from this type
of waters. Next to the abiotic factors like temperature,
light, turbulence, or differences in available nutrients
one of the possible explanations is the production of
allelopathic compounds.
Allelopathy is the phenomenon when a species has
inhibitory or stimulatory effects on other species
through the production and release of chemical
compounds into the environment (Rice, 1984). Cya-
nobacteria can produce different secondary metabo-
lites, which might act as allelochemicals, antibiotics,
hormones, or toxins. The effects of cyanotoxins on
humans and animals or on higher plants are well
known (Chorus & Bartram, 1999); especially in the
case of microcystins (MacKintosh et al., 1990; Sivo-
nen et al., 1990; Carmichael, 1992; Runnegar et al.,
1995) but the allelopathic aspects of cyanotoxins are
much less clear. Nevertheless, it is also not clear
whether microcystins themselves or other released
compounds of cyanobacteria are responsible for
allelopathy (Leao et al., 2009a). The number of
studies using exudates (molecules, which are released
from intact cyanobacterial cells) or crude extracts
(total intracellular content of disrupted cells) instead
of purified toxins increased in recent years (Suikkanen
et al., 2006; Oberhaus et al., 2008; Leao et al., 2009b;
El-Sheekh et al., 2010). These approaches imitate field
conditions therefore the findings can be extrapolated
to the real life situation.
The possible interactions between Cryptomonas
ovata (non-toxic eukaryotic alga) and Microcystis
aeruginosa (toxic cyanobacterium) were studied in
laboratory experiments. Purified microcystin, crude
extracts, and living Microcystis cells were applied
during the investigations. We hypothesized that
microcystins act as allelochemicals to C. ovata,
therefore growth rates of C. ovata (Cryptophyta) are
affected by the presence of the toxins. We also
hypothesized that M. aeruginosa and its other com-
pounds have also an inhibitory effect on C. ovata.
Materials and methods
Strains, culturing conditions, and experimental
setup
Cryptomonas ovata (CCAP 979/61, United Kingdom)
and M. aeruginosa (BGSD 243, Hungary) strains were
used in this study. All experiments were carried out in
batch cultures. Cells were grown in sterile air
bubbled Jaworski’s medium in a final volume of
400 ml under continuous irradiation (20 lmol m-2
s-1), at 22–23�C. The exposure time was 14 days
(except for toxicity tests; see below); the cultures were
sampled each day. 1 9 10 ll samples were taken and
counted to determine the cell numbers of the cultures.
The abundances (cell numbers) of the samples were
determined by using a Zeiss Jenaval light microscope
at 250-fold magnification.
Mixed cultures (containing both C. ovata
and M. aeruginosa cells)
The inoculated cell numbers of C. ovata were
0.1 9 106 cells ml-1 in every case. Earlier observa-
tions showed that this cell density is optimal for
inoculation under our culturing practice. Lower cell
densities results too long lag phase and do not allow
reaching possible maximal biomass; while higher
98 Hydrobiologia (2012) 691:97–107
123
inoculating cell densities leads to early stationary
phase. M. aeruginosa cell numbers in mixed cultures
were 0.1 9 106 cells ml-1 (1:1 ratio), 0.2 9 106 cells
ml-1 (1:2 ratio), 0.4 9 106 cells ml-1 (1:4 ratio),
2.5 9 106 cells ml-1 (1:25 ratio), and 5 9 106 cells
ml-1 (1:50 ratio). The inoculated cell numbers of
C. ovata control cultures (C. ovata cultures without
M. aeruginosa cells) were 0.1 9 106 cells ml-1. The
inoculated cell numbers of M. aeruginosa control
cultures (M. aeruginosa cultures without C. ovata
cells) were the same, as the inoculated cell numbers of
M. aeruginosa in mixed cultures. In the case of
M. aeruginosa control cultures, shaded cultures were
also used where the inoculated cell numbers were
0.1 9 106 cells ml-1, to avoid photoinhibition.
Treatment of C. ovata cultures with M. aeruginosa
crude extracts
The inoculated cell numbers of C. ovata were set to
approximately 0.1 9 106 cells ml-1, both in treated
and in control cultures. C. ovata cultures were treated
with M. aeruginosa extracts equivalent to the
M. aeruginosa cell numbers in mixed cultures. Final
concentrations of MCs in the crude extract-treated
C. ovata cultures were 0.0213, 0.0425, 0.0851, 0.5317,
and 1.0633 lg ml-1. The composition of MCs in the
crude extract according to MECK analyses were MC-
LR (50.6%), MC-YR (39%), MC-YA (10%), and
traces of MC-RR.
Treatment of C. ovata with purified toxin
of M. aeruginosa
The inoculated cell numbers of C. ovata were
0.1 9 106 cells ml-1, in treated and control cultures
as well. MC-LR was used for the purified toxin
treatments; concentration of MC-LR in the toxin
treated C. ovata cultures were 0.0213, 0.0425, 0.0851,
0.5317, and 1.0633 lg ml-1. These concentrations of
the purified toxin were equal to the total toxin contents
of M. aeruginosa crude extracts.
Toxicity tests
To determine the toxic effects of MC-LR after 72 h
exposure, C. ovata cultures were treated with 2.5, 5,
10, 15, 20, 25, and 50 lg ml-1 toxin. Tests were
carried out on 12 wells plates (in a final volume of
4 ml Jaworski’s medium); the inoculated cell numbers
of C. ovata were 0.1 9 106 cells ml-1; the plates were
incubated for 72 h with continuous irradiation
(20 lmol m-2 s-1) at 22–23�C. Cultures were sam-
pled at 0th and 72nd hours. All experiments were done
in triplicate.
Crude extract preparation, MC-LR purification,
and measurement
300 ml of M. aeruginosa culture with known cell
number was centrifuged (20 min, acceleration
6,0009g; type: Beckman Avanti J-25) to prepare
M. aeruginosa extract. The pellet was stored at -20�C
until the start of the experiments. Cells were disrupted
by freezing and thawing thrice following 2 min of
sonication (Transsonic T470/H sonicator). The toxin
content of the crude extract stock was measured by
capillary electrophoresis (see details below); the MC
content of M. aeruginosa cells was calculated on the
basis of this data; the appropriate amounts of crude
extracts and purified toxin needed for the treatments
were possible to calculate knowing the intracellular
MC content.
MC-LR purification was carried out according to
Harada et al. (1988), later modified by Vasas et al.
(2006). Microcystis aeruginosa cells were harvested
by centrifugation, the pellet was extracted with 90%
methanol. The methanolic extract was evaporated to
dryness in a rotary evaporator at 40�C and then
resuspended in 10 mM Tris–HCl (pH 7.5). The
purification step was carried out on DEAE-cellulose
column (2 9 25 cm; DE-52, Whatman) equilibrated
with 10 mM Tris–HCl, pH 7.5. The column was
washed with the same buffer and eluted with a gradient
between 0 and 0.2 M NaCl in 10 mM Tris–HCl, pH
7.5. The collected fractions were measured spectro-
photometrically (Shimadzu UV-1601 UV/VIS Spec-
trophotometer) at 239 nm. The fractions were tested by
Blue-Green Sinapis Test (BGST; Vasas et al., 2004).
The toxic fractions were combined and further
purified by semi-preparative HPLC method (Shima-
dzu LC-10AD VP HPLC instrument; SUPELCO
SupelcosilTM
LC-18 25 cm 9 10 mm, 5 lm column;
diode-array UV–VIS detector). The distinctive peaks
of the chromatogram were tested with the BGST. After
rechromatography of the fractions, the purified MC-
LR was free of contaminants and was used for the
experiments.
Hydrobiologia (2012) 691:97–107 99
123
The cyanotoxin content of the culturing media in
the mixed cultures was measured by micellar electro-
kinetic chromatography (MEKC) applying the param-
eters described by Vasas et al. (2006) with minor
modifications. Briefly: Prince CEC-770 instrument;
polyimide coated fused silica capillary (Supelco,
60 cm 9 50 lm id., effective length: 52 cm); hydro-
dynamic injection 100 mbar s-1; applied voltage:
25 kV; 25 mM sodium-tetraborate—100 mM SDS
buffer, pH: 9.3; detection by diode-array detector at
238 nm). Dax 3D 8.1 software was used for the
evaluation of the electropherograms. The limit of
detection (LOD) values at 239 nm were determined
and the calculations were based on a signal-to-noise
ratio of 3. LOD values were 3–4 lg ml-1 for MCs;
lower toxin contents were calculated from the results
of concentrated samples. For sample concentration,
3 ml culture was centrifuged on the 0th, 3rd, 7th, 10th,
and 14th day of the experiment; the supernatant was
removed and lyophilized (Christ Alpha 1-2 LD plus
lyophilizer). The residue was resuspended in 50 ll
methanol (this meant a 60-fold concentration), and the
MC content of this solution was measured by MECK
described above. According to the LOD values, the
lowest actual detectable concentration of MCs was
0.05–0.067 lg ml-1 in the culturing medium.
Statistical analyses
One-way ANCOVA was used to determine the
significances among the tendency-differences of
growth curves of control and treated cultures (Zar,
1996; Hammer et al., 2001).
Results
Mixed cultures
Changes of C. ovata cell numbers in mixed cultures
Cell numbers of C. ovata decreased in mixed cultures in
all cases (Fig. 1). There were differences only in the
time when C. ovata cell numbers decreased under
100 cells ml-1 (the detection limit of our count tech-
nique); in contrast to C. ovata control cultures, where
cell number increased during the 14 days of incubation.
In cultures with 1:1 ratio, C. ovata cells could be
found even on the last (14th) day of the experiment but
the cell number was only 0.0247 9 106 cells ml-1
(Fig. 1a). Growth of the cultures showed gradually
decreasing tendency from the 2nd day (Fig. 1a).
Cryptomonas ovata cells disappeared from cultures
with 1:2 ratios to the 14th day. Cell numbers were
continuously decreasing from the 2nd day of the
experiment (Fig. 1b).
Cryptomonas ovata cell numbers in mixed cultures
with 1:4 ratios were oscillating during the first 7 days
and showed gradually decreasing tendency from the
8th day (Fig. 1c). C. ovata cells disappeared from the
cultures by the 14th day.
Cell number of C. ovata in mixed cultures with 1:25
and 1:50 ratios decreased under 100 cells ml-1 by the
1st day, i.e., the cells in this culture disappeared within
1 day (Fig. 1d–e).
Statistical analysis (one-way ANCOVA) showed
that there are significant differences (P \ 0.01) in
growth characteristics among the control and treated
cultures.
Changes of M. aeruginosa cell numbers
in mixed cultures
Microcystis aeruginosa cells in mixed cultures were
able to proliferate in all cases (Fig. 1). There were
differences only among the tendencies of growth
curves of the cultures compared to control cultures.
Microcystis aeruginosa cells in mixed cultures
with 1:1 ratios started to proliferate in contrast to
cells of non-shaded control cultures of M. aerugin-
osa with an inoculated cell number of 0.1 9 106
cells ml-1 (Fig. 1a). This means that M. aeruginosa
cells need shading at this low cell density to avoid
photodestruction (Fig. 1a, gray squares). The pres-
ence of C. ovata cells in the same density provides
this shading for Microcystis cells (Fig. 1a, black
squares).
Supposedly due to the shading effect of C. ovata
cells the cell numbers of M. aeruginosa in mixed
cultures with 1:2 and 1:4 ratios were higher than in
control cultures on the first 3 days; from the 4th–5th
day M. aeruginosa control cultures grew faster
(Fig. 1b–c). Growth of M. aeruginosa differed signif-
icantly from the control only in mixed cultures with
1:2 ratio, but not in cultures with 1:4 ratio (P \ 0.01).
However, C. ovata seemed to have some impact on
Microcystis growth at these intermediate cell number
ratios.
100 Hydrobiologia (2012) 691:97–107
123
There were no significant differences among the
growth tendencies of control cultures and mixed
cultures when 1:25 and 1:50 ratios were used
(Fig. 1d–e).
Extracellular, dissolved MC content of the cultur-
ing media was under the detection limit
(0.05–0.067 lg ml-1) in all of the M. aeruginosa
control and mixed cultures.
Fig. 1 Changes of cell numbers in control (containing C. ovataor M. aeruginosa cells only) and in mixed (containing C. ovataand M. aeruginosa cells as well) cultures. C. ovata cell numbers
were 0.1 9 106 ml-1 in control and mixed cultures in all cases
(a–e). Initial M. aeruginosa cell numbers were in control and
mixed cultures: a 0.1 9 106 ml-1 (1:1 ratio); b 0.2 9 106 ml-1
(1:2 ratio); c 0.4 9 106 ml-1 (1:4 ratio); d 2.5 9 106 ml-1 (1:25
ratio); e 5 9 106 ml-1 (1:50 ratio). Significant differences
among C. ovata growth curves indicated with aC–eC; significant
differences among M. aeruginosa growth curves indicated with
aM–fM
Hydrobiologia (2012) 691:97–107 101
123
Cultures treated with crude extract
of M. aeruginosa
Microcystis aeruginosa crude extract had no effect on
growth of C. ovata cultures in the case of treatments
with crude extracts equal to 1:1 and 1:2 cell number
ratios (Fig. 2, open squares and open triangles).
Cell numbers of C. ovata cultures treated with
crude extracts equal to 1:4 cell number ratios were
significantly higher than those of control cultures
(P \ 0.01) (Fig. 2, open circles). The same phenom-
enon was observed in the case of treatment with crude
extract equal to 1:25 cell number ratio (Fig. 2,
crosses).
The cell number of C. ovata culture treated with
crude extract equal to 1:50 cell number ratio was lower
than that of the control culture and the other treated
cultures (Fig. 2, black diamonds).
Cultures treated with purified toxin
of M. aeruginosa
Cell numbers of C. ovata cultures treated with MC-LR
were lower than cell numbers of control cultures in
each case (Fig. 3). Statistical analysis of the growth
curves showed significant differences among the
growth curves of control and treated cultures in all
of the applied toxin concentrations (which were equal
to the toxin content of the crude extracts used in the
previous experiments), at least in the first 4–6 days
(Fig. 3). The results suggest that MC-LR has a slight
inhibitory effect on the growth of C. ovata in the first
few days of incubation at the applied concentrations
(0.02–1.1 lg ml-1). From the 4th–6th day, C. ovata
cultures seemed to ‘‘recover’’ and grow almost the
same rate as the control cultures.
Short time toxicity tests
Changes in growth of C. ovata cultures on plates were
detected after 72 h exposition. As results show
(Fig. 4), at 2.5 lg ml-1 toxin concentration the cell
number of the treated culture decreased by more than
50% compared to those of the control cultures by the
end of the incubation time. The differences among the
cell numbers of treated and control cultures on the 3rd
day increased with the increasing toxin concentrations
(Fig. 4). Cell number of C. ovata decreased with
99.52% to the 3rd day applying 50 lg ml-1 toxin
concentration (Fig. 4). On the basis of these results it
can be established that MC-LR at concentration
2.5 lg ml-1 caused *50% effect, whereas almost
complete reduction of C. ovata cell numbers was
caused by 50 lg ml-1.
Fig. 2 Changes of cell
numbers of M. aeruginosacrude extract-treated
C. ovata cultures and in
control cultures. Amount of
the crude extract is given in
equivalent Microcystis cell
numbers: 0.1 9 106 ml-1
(1:1 ratio); 0.2 9 106 ml-1
(1:2 ratio); 0.4 9 106 ml-1
(1:4 ratio); 2.5 9 106 ml-1
(1:25 ratio); 5 9 106 ml-1
(1:50 ratio). Significant
differences among growth
curves indicated with a–c
102 Hydrobiologia (2012) 691:97–107
123
Discussion
Mixed cultures
Cell number of C. ovata in mixed cultures showed
decreasing tendency in all cases. Positive correlation
was observed between the decrease of growth rates of
C. ovata, and the abundance of M. aeruginosa.
Cell numbers of M. aeruginosa in mixed cultures
with 1:2 and 1:4 ratios were lower than those of the
control cultures from the 4th–5th day, but there were
no differences between the tendencies of growth
curves of control cultures and mixed cultures when
1:25 and 1:50 ratios were used. This means, that
C. ovata also had effect on M. aeruginosa in the
intermediate cell density circumstances. The way of
this interaction is not clear. It is known that crypto-
monads can produce extracellular compounds (Lan-
celot, 1984; Myklestad, 1995; Biddanda & Benner,
1997) but these are mainly polysaccharides so far
Fig. 3 Changes of cell
numbers of purified toxin
(MC-LR)-treated C. ovatacultures and in control
cultures. MC-LR
concentrations were
equivalent to the total toxin
content of crude extracts:
0.0213; 0.0425; 0.0851;
0.5317; 1.0633 lg ml-1.
Significant differences
among growth curves
indicated with a–c
Fig. 4 Cell numbers of C.ovata in toxicity test at the
0th and 72nd hours. Inserted
values show the extent of
inhibition related to control
Hydrobiologia (2012) 691:97–107 103
123
known to have allelopathic impact exclusively on
bacteria. Lam and Silvester (1979) investigated the
interactions between M. aeruginosa, Chlorella sp.,
and Anabaena oscillatorioides in mixed cultures. In
cultures containing Microcystis and Chlorella or
Anabaena and Chlorella cells, the growth of Chlorella
was inhibited. The authors established that a bilateral
antagonism could be observed between Anabaena and
Chlorella cells, while a certain kind of inhibition
occurred between Microcystis and Chlorella cells. Our
results suggest that at high Microcystis/Cryptomonas
ratio this kind of bilateral antagonism might exist.
The capillary-electrophoretic measurements
showed that extracellular MC content of the culturing
media was under the LOD in all of the mixed cultures;
namely there were observable active MC release
neither in the control M. aeruginosa cultures, nor in
the mixed cultures. So the question is why the growth
of C. ovata was inhibited by living M. aeruginosa
cells, and why C. ovata cells disappeared from mixed
cultures where the inoculated cell numbers of
M. aeruginosa were much higher. The growth of
M. aeruginosa in control cultures with ratio 1:25 and
1:50, and in mixed cultures with ratio 1:25 and 1:50
was not significantly different (Fig. 1). Since CE
measurements proved that there is no detectable MC
release by M. aeruginosa cells, one possible explana-
tion of the strong inhibition of C. ovata in mixed
cultures could be that M. aeruginosa release com-
pounds other than MCs, which inhibits the prolifera-
tion of C. ovata. Growth rates of M. aeruginosa in
control cultures were not higher than growth rates of
C. ovata in control cultures; still, more efficient
nutrient uptake by M. aeruginosa could be an other
possible explanation of the observed phenomenon. It
is also known that cyanobacteria able to change the pH
of their environment, hence hinder the proliferation of
other species (Møgelhøj et al., 2006). The proof of
these assumptions needs further experiments.
Cultures treated with crude extract
of M. aeruginosa
The maximum release of microcystin from cells
occurs during the decomposition period of Microcystis
cells when a bloom collapses in the aquatic environ-
ment (Park et al., 1998). The use of crude extracts of
cyanobacterial cells could be a good representation of
this natural situation.
Our results proved that the crude extract could
significantly influence the growth rate of C. ovata.
The relationship was highly variable. Inhibitory,
neutral, and facilitative interactions were also
observed depending on the amount of the crude
extract. Exudates of living cyanobacterial cells or
filtrates of cyanobacterial culturing media can inhibit
the growth; the polysaccharides production and
release; GSH and ROS values of certain microalgal
species (Babica et al., 2006; Mohamed, 2008; Ober-
haus et al., 2008; Leao et al., 2009b; El-Sheekh et al.,
2010). At high concentration of crude extract (equiv-
alent to 1:50 ratio) we experienced this inhibition. MC
content of this amount of crude extract was equivalent
to the 1.0633 lg ml-1 purified toxin treated C. ovata
cultures; however, the crude extract seemed to be
more toxic at the first 7–8 days at this high concen-
tration. It could be explained by the presence of the
other MC variant (MC-YR); the presence of other
inhibitory compounds of M. aeruginosa (Suikkanen
et al., 2006; Palıkova et al., 2007), or the occurrence of
protein bound cyanotoxin. MCs could be present
bound to phycobilin (ab) monomers (Juttner & Luthi,
2008) which are not detectable by our MECK method.
The present but undetected toxin could explain the
stronger toxicity of the crude extract. Furthermore,
different MCs could have different toxic properties
(McElhiney et al., 2001) and there could be synergistic
interactions among MC variants (Mohamed, 2008).
The facilitative impact that we experienced at low
crude extract concentration can be attributed to the
nutritional value of the extract. It seems that this
beneficial effect of the extract compensate for its lower
inhibitory effect. At medium concentration of the
extract these effects were neutralized.
Cultures treated with purified toxin
of M. aeruginosa
The used MC-LR concentrations were equal to total MC
content of the cells applied in mixed cultures. These
concentrations resulted only slight decrease of the cell
number of C. ovata. The inhibition occurred mainly in
the first few days of the incubation, but from the 4th–6th
days, the C. ovata cultures seem to ‘‘recover’’ and grow
almost at the same rate as the control cultures. These
findings are in accordance with that of other studies.
Kearns and Hunter (2001) investigated the effects of
MC-LR on Chlamydomonas reinhardtii cultures, and
104 Hydrobiologia (2012) 691:97–107
123
they found that at low toxin concentration the mobility
of the cells in MC-LR-treated cultures was equal to
those of the control cultures at the end of the
experiments. Babica et al. (2007) studied the effects of
microcystins on growth of five representatives of
Chlorophyta, and they found that 0.001–0.01 lg ml-1
toxin concentration (general environmentally relevant
concentrations) did not cause significant growth alter-
ations. It is also true for the microcystins analogous
nodularin (Suikkanen et al., 2006). Sedmak & Kosi
(1997) reported heterogeneous effects of 1–5 9 10-7 M
(0.1038–0.5192 lg ml-1) MC-RR under low light
conditions on different algal species. While the cell
number of Monoraphidium contortum and Scenedesmus
quadricauda increased during exposure, growth of
Cryptomonas erosa was inhibited under the same
conditions. The authors concluded that the strong
toxicity manifested only among specific light circum-
stances. Toxic effects could be enhanced by the
microenvironment; the same toxin concentration could
be more toxic under certain conditions depending on the
sensitivity of algal species.
Compared to control, *50% decrease of C. ovata
cell numbers was achieved by MC-LR at concentration
2.5 lg ml-1 in short term (72 h) toxicity tests, however
the same reduction of cell concentration after 3 days of
exposure was observed in the cultures treated only with
0.0213 lg ml-1 MC-LR in 14-day experiment. Inter-
estingly, the extent of cell number reduction did not
change with MC-LR concentration increasing up to
1.0633 lg ml-1 in 14-day experiment and the growth of
C. ovata population recovered after the 3rd day for the
remaining exposure time. In the short-term experiment,
MC-LR at concentration 2.5 lg ml-1 decreased the cell
number of C. ovata to 50% of control after 72 h,
whereas 20 lg ml-1 reduced the cell number to 50% of
cell density inoculated at the start of the experiment. So
to discuss toxic effect to other organisms the concen-
tration and/or the frequency; furthermore, the number of
individuals suffering the toxic effects is really impor-
tant. The undetectable MC release of M. aeruginosa
suggests that cyanotoxins have only a secondary
importance role in the strong inhibitory effects of intact
M. aeruginosa cells.
Ecological considerations
From the results of purified toxin treated C. ovata
cultures and toxicity tests, we can conclude that
C. ovata is not highly sensitive to MC-LR. Neverthe-
less it has to be noted that in some cases extremely
high toxin concentrations: 19.5 lg ml-1, Japan (Nag-
ata et al., 1997); 25 lg ml-1, Germany (Fastner et al.,
1999); 8.43–20 lg ml-1, Australia (Kemp & John,
2006); 18.04 lg ml-1, Hungary (Mathe et al., 2007);
can occur in natural situation after the collapse of
massive cyanobacterial blooms.
The results of crude extract-treated C. ovata culture
in 1:50 ratio suggest that there could be allelopathic
effects (synergistic interactions among MC variants
(Mohamed, 2008); occurrence of protein bound
cyanotoxin which are not detectable by our MECK
method (Juttner & Luthi, 2008); presence of other
inhibitory compounds than MCs (Suikkanen et al.,
2006; Palıkova et al., 2007), but based on the results of
crude extract and purified toxin treated cultures, we
can say that the decrease of C. ovata cell number in
mixed culture was not exclusively due to the dissolved
MC content of the medium.
In conclusion, we can say that the physical con-
straints are of primary importance in the shaping the
phytoplankton composition (Padisak et al., 2010; Zoh-
ary et al., 2010), but inhibition caused by cyanobacterial
compounds can play an important role in species
selection and could be one reason of the lack of stable
cyanobacteria–Cryptophyta coexistence in steady-state
assemblages. Supposedly the phenomenon is more
complicated: it could be a combination of the presence
of MCs and other biologically active compounds;
shading effects or possible pH changes induced by
cyanobacteria (Møgelhøj et al., 2006) and competition
for resources. Exact explanation requires further
investigations.
Acknowledgments The work has been supported by the
Hungarian National Research Foundation Grants (OTKA)
SAB81459 and K-81370; Universitas Foundation 2010.
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