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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