Allelopathic interactions between microcystin-producing
and non-microcystin-producing cyanobacteria and green
microalgae: implications for microcystins production
Maria do Carmo Bittencourt-Oliveira&Mathias Ahii Chia&Helton Soriano Bezerra de Oliveira&Micheline Kézia Cordeiro Araújo& Renato José Reis Molica&Carlos Tadeu Santos Dias
Received: 10 March 2014 / Revised and accepted: 24 April 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Most mixed culture studies on the allelopathic in-teractions between toxic and nontoxic cyanobacteria with phytoplankton species rarely investigate the role of microcystins (MC) production and regulation in the course of the studies. This study investigated the interactions between intact cells of toxic (Microcystis aeruginosa (Kützing) Kützing) and nontoxic (Microcystis panniformis Komárek e t a l . ) c y a n o b a c t e r i a w i t h t h o s e o f g r e e n a l g a e (Monoraphidium convolutum (Corda) Komárková-Legnerová and Scenedesmus acuminatus (Largerheim) Chodat) as well as the effects of their respective crude extracts (5 and 10 μg.L−1) on their growth under controlled conditions. M. aeruginosa and M. panniformis were able to significantly (p < 0.05) inhibit the growth of the green algae with M. convolutum being the most affected. The green alga S. acuminatus in return was able to inhibit the growth of the both cyanobacteria. In response to the presence of a compet-ing species in the growth medium, M. aeruginosa significant-ly increased its MC production per cell with the progression of the experiment, having the highest concentration at the end of
the experiment. On the other hand, the extracts of the cyanobacteria had no significant inhibitory effect on the green algal strains investigated, while those of the green algae also had significant inhibitory effect on the growth of M. aeruginosa. In conclusion, both cyanobacterial and green algal strains investigated were negatively affected by the presence of competing species. M. aeruginosa responded to the presence of green algae by increasing its MC production. The green algal strains significantly inhibited the growth of M. aeruginosa.
Keywords Allelopathy . Species competition .
Phytoplankton . Mixed algal cultivation . Cyanotoxins . Monoraphidium . Scenedesmus . Microcystis
Introduction
In recent decades, algal blooms have become widespread in many water bodies around the world. These have also been characterized by the increased occurrence of toxic cyanobacteria blooms, in addition to those of diatoms, dino-flagellates, and green algae (Zhang et al.2013), which have serious social and economic implications due to the degrada-tion of water resources, and as a result, generated a lot of interest into investigations on the environmental factors and mechanisms that promote these blooms as well as how cyanotoxins confer a competitive advantage to the producing species (Jonsson et al.2009).
Different water bodies around the world are characterized by dominant species of phytoplankton that alternate between cyanobacteria and other microalgae (diatoms, green algae). The dominance of different algal species changes from season to season during bloom formation in most water bodies world-wide (Chen et al. 2003; Frossard et al. 2014). In order to
M. C. Bittencourt-Oliveira
:
M. A. Chia:
H. S. B. de Oliveira:
M. K. Cordeiro AraújoDepartment of Biological Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, Piracicaba, SP 13418-900, Brazil
M. C. Bittencourt-Oliveira (*)
:
H. S. B. de Oliveira:
M. K. Cordeiro Araújo:
R. J. R. MolicaGraduate Program in Botany, Rural and Federal University of Pernambuco, Rua D. Manoel de Medeiros, S/N, Dois Irmãos, Recife, PE 52171-030, Brazil
e-mail: mbitt@usp.br C. T. S. Dias
Department of Exact Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, Piracicaba, SP 13418-900, Brazil
understand the succession mechanisms responsible for cyanobacteria and other phytoplankton species, experiments involving mixed cultures have been encouraged to be carried out by researchers. This has led to investigations on the interactions of cyanotoxin-producing and nonproducing-cyanobacteria with microalgae strains as a means of contrib-uting to the understanding of how successional processes operate in nature. A comprehensive understanding of the competition between algae and cyanobacteria can be crucial to control strategies for bloom formation and outbreaks in aquatic ecosystems (Zhang et al.2013).
Most studies have considered the effect of chemical and physical factors on the competition between cyanobacteria and other microalgae (Takeya et al. 2004; Shen and Song
2007; Li and Li2012), and shown that nutrient concentration of the medium does not always determine the phytoplankton succession in mixed cultures (Kuwata and Miyazaki2000; Zhang et al.2013). According to You et al. (2007), Dunker et al. (2013), and Zhang et al. (2013), the competition between species and the ecological success of a competitor can be described by resource exploitation and interference models. Whereas most studies have focused on the indirect interaction by exploitation that is based on the competition for limited resources (Zhang et al.2013), in hypertrophic environments, interference, for example, the production of microcystins (MCs) and other allelochemicals is a direct form of interspecific inter-action that is very important (Leflaive and ten-Hage L,2007; Bar-Yosef et al.2010; B-Beres et al.2012; Magrann et al.2012). The microcystins belong to a family of cyclic heptapeptide known to inhibit protein phosphatases. They cause liver fail-ure in wild and domestic animals and humans and have heterogenous effects on the physiology of plants (Jochimsen et al.1998; Papadimitriou et al.2013). Unfortunately, most of the mixed culture experiments rarely monitor the production of microcystins during the experiments (B-Beres et al.2012; Zhang et al.2013), in addition to the fact that most of the published results are conflicting. Due to the varied responses of different microalgae species to MCs (Sedmak et al.2008, Li and Li2012; Bittencourt-Oliveira et al.2013; Campos et al.
2013), it becomes imperative to investigate the behavior of different phytoplankton lineages in the presence of toxic and nontoxic cyanobacteria and microalgae. In addition, it is not clear whether microcystins or other released compounds by cyanobacteria or other microalgae are responsible for allelop-athy (Leao et al.2009; B-Beres et al. 2012). This makes it difficult for generalizations or conclusions to be made on the relationship between dominant phytoplankton species in-volved in standing water species succession.
Investigations on allelo pathic effects b etween cyanobacteria and green algae should not only be based on the mutual interactions in mixed culture experiments but also determine the possible role of bioactive substances like MCs in order to elucidate their allelopathic mechanisms by
monitoring changes in their concentrations (Yang et al.
2014). Studies of the action of MC on microalgae are mostly restricted to lysates of toxic cyanobacterial extracts or purified MC, and often with much higher concentrations than those commonly found in natural environments (Nagata et al.1997; Kemp and John2006; Máthé et al.2007; B-Beres et al.2012). Conclusions on the allelopathy of MC resulting from studies that used non-environmentally relevant concentrations can be misleading and not applicable in real life situations (Leao et al.
2009). Therefore, our study aimed to (1) evaluate the possible allelopathic interaction between intact cells of microcystin-producing and -nonmicrocystin-producing cyanobacteria with the green microalgae Scenedesmus acuminatus and Monoraphidium convolutum under controlled conditions and (2) the effect of crude extracts of cyanobacteria and green algae on the growth of the green algal and cyanobacterial strains, respectively, using environmentally relevant concentrations.
Materials and methods
The microcystin-producing (MC+) Microcystis aeruginosa BCCUSP232 (Bittencourt-Oliveira et al. 2011) and non-microcystins-producing (MC−) Microcystis panniformis BCCUSP200 (Bittencourt-Oliveira2003) cyanobacterial strains from the Brazilian Cyanobacteria Collection of University of São Paulo (BCCUSP). The two strains of green microalgae used in this study were Monoraphidium convolutum (CMEA/UFF0201) obtained from Elizabeth Aidar Collection of Microalgae (CMEA/UFF) and Scenedesmus acuminatus (UFSCar036) from the Federal University of São Carlos (UFSCar). For all exper-iments, the cyanobacteria and green algae were maintained in environmentally controlled growth chambers set to 24±1 °C, 14 h:10 h (light/dark) photoperiod, and 40 μmol photons m−2s−1light intensity. Light intensity was measured using a LI-COR model 250 light meter equipped with a spherical underwater sensor. The cyanobacteria and green microalgae were grown in BG-11 (Rippka et al.1979) at pH 7.4 according to the modifications of Bittencourt-Oliveira et al. (2011) that involved the substitution of iron for ferric ammonium citrate FeCl3·H2O chloride. The cultures were stirred manually, and
their positions changed on a daily basis. Mixed culture experiments
All experiments were carried out in 1,000-mL Erlenmeyer flasks containing 600 mL final culture volume. In the mixed cultures, 300 mL of the culture of each strain with an equal number of cells (ratio, 1:1) was used. Briefly, intact cells of M. aeruginosa (BCCUSP232) were cocultured with those of M. convolutum CMEA/UFF0201 in one experimental setup having an initial cell density of 7.0×105cells.mL−1, and with
S. acuminatus UFSCar036 having an initial density of 1.0× 105cells.mL−1in the second mixed culture experiment. The same procedure was repeated for intact cells of M. panniformis (BCCUSP200) in the mixed culture with both green microalgal strains. For the controls, each strain (i.e., cyanobacteria and green algae) was grown separately in 600 mL, using the same cell densities as those used in the respective experiments. The choice of different cell densities to combine with the Microcystis spp. was because Scenedesmus quadricauda had a much larger biovolume (491.39μm3cell−1) than M. convolutum (25.92μm3cell−1). The experiments were monitored daily for 10 days by mea-suring cell densities of the strains, with the first count occur-ring 24 h after the mixture of the strains.
To evaluate the influence of green microalgae on the p r o d u c t i o n o f m i c r o c y s t i n s b y M . a e r u g i n o s a BCCUSP232, 1 mL culture aliquots were collected after homogenization on days 1, 7, and 10 of the experiment. Immediately after collection, the aliquots were frozen in liquid nitrogen and stored at −80 ° C in a freezer until the time of toxin analysis. Although microcystin analy-sis was not done in the M. panniformis BCCUSP200 experiments, aliquots of the same volume were also taken at the same rates to maintain the same volume and growth conditions for all cultures.
Cyanobacterial and green algal extracts
Crude extracts of the MC+ and MC− strains from M. aeruginosa (BCCUSP232) and M. panniformis (BCCUSP200) were obtained by growing each strain in 20 L of BG-11 medium under the growth conditions shown above. At exponential growth phase, the resulting cul-tures were harvested by low speed centrifugation and the resultant pellet flash frozen in liquid nitrogen, ly-ophilized and stored at −80 °C in a freezer until the time of use. The lyophilized biomass was resuspended in a total volume of 3 mL of deionized water and ultrasonicated (Microson Ultrasonic Cell Disruptor, USA) at 15 W and 22.5 kHz to disrupt the cells and release the intracellular toxins. From the crude extracts obtained from the MC+ strain, 5 and 10 μg.L−1 MC were obtained and used in the treatments. The volumes of the crude extracts applied to the treatments were 0.5 and 1.0 mL corresponding to 5 and 10 μg.L−1 MC, respectively. Crude extract concentration equivalents of the non-MC producing strain was obtained and used in the treatments. The concentration MC in the extracts was determined using enzyme-linked immuno sorbent assay (ELISA) as described below in the microcystins analysis section.
Crude extracts of the green algal strains (M. convolutum CMEA/UFF0201 and S. acuminatus UFSCAr036) were
o b t ai n e d in th e s a m e m a n n e r a s t h o s e o f t h e cyanobacterial strains. Equivalents of the extract concen-trations (5 and 10 μg.L−1) used in the MC+-producing strains were applied to the two cyanobacterial strains to see if there may be any influence on the growth of the cyanobacterial strains. The extract exposure experiments lasted for 13 days.
Cell density and growth rate
Cell density and growth rates were obtained by counting the number of cells using a Fuchs-Rosenthal chamber with the aid of a binocular microscope, following the method of Guillard (1973). The specific growth rate (μ)
was obtained according to Fogg and Thake (1987). For all the experiments and their controls, 2 mL aliquots of the cultures were removed and immediately preserved with 10 % Lugol solution on a daily basis. As a means of increasing the reliability of results, a minimum of 400 cells were quantified to keep the estimated error within ±10 % (Lund et al. 1958). Regardless of the number of cells counted, a minimum of three chambers were counted. The cell density was expressed in cells per milliliter.
Microcystins analysis
The culture aliquots previously removed from mixed cultures and the control (n=3) were homogenized by ultrasonication (Microson Ultrasonic Cell Disruptor, USA) for 3 min (15 W and 22.5 kHz) to totally disrupt the cells. The disruption was confirmed by microscopic observations.
Total microcystin concentrations (intra and extracellu-lar) were determined by ELISA technique using the BEACON microcystins plate kit (Beacon Analytical Systems Inc., USA), following the manufacturer’s in-structions. Analyses were performed using a microplate reader (ASYS Hitech, model A-5301, Austria) set to 450 nm. The detection range of the assays was 0.10 to 2.0 parts per billion (PPB) (ng mL−1). Analyses were done in triplicates. The intracellular and extracellular concentration was defined as the total microcystins (intra- and extracellular) per cell quota.
Data treatment
The cell density data were tested for normality and homogeneity of variance. Where the normality and ho-mogeneity of variance test were positive, the cell den-sity data were subjected to repeated measure general linear model (GML) analysis of variance (ANOVA). Where significant differences were observed, the
Fig. 1 Growth curves (cells.mL−1) for the different green microalgal and cyanobacterial strains cultured in mono and mixed culture conditions. a M. convolutum CMEA/UFF0201 control and mixed culture experiment with M. aeruginosa BCCUSP232 (MC+). b M. convolutum control and mixed culture with M. panniformis BCCUSP200 (MC−). c S. acuminatus UFSCar036 control and mixed culture with M. aeruginosa BCCUSP232 (MC+). d S. acuminatus UFSCar036 control and mixed culture with M. panniformis BCCUSP200 (MC−). e M. aeruginosa BCCUSP232
(MC+) control and mixed culture with M. convolutum CMEA/UFF0201. f M. aeruginosaBCCUSP232 (MC+) control and mixed culture with S. acuminatus UFSCar036. g M. panniformis BCCUSP200 (MC−) con-trol and mixed culture with M. convolutum CMEA/UFF0201. h M. panniformis BCCUSP200 (MC−) control and mixed culture with S. acuminatus UFSCar036. Asterisks mean that there was no statistical difference between the mixed cultures and controls of the respective treatment combintations. Error bars represent standard deviation for n=3
Tukey’s HSD post hoc test was performed to separate the significantly different means. All analyses were done at 5 % significance level. All statistical analyses were done using the SAS version 9.2 software for Windows.
Results
The effect of mixed cultures of the microcystin-producing (MC+) and -nonproducing (MC−) strains of cyanobacteria with green algae can be seen in Fig.1. Over 50 % growth reduction of the green microalga M. convolutum was recorded when cocultured with both MC+ and MC− Microcystis (Fig. 1a, b). Similarly, the growth of S. acuminatus was significantly inhibited (p < 0.05) in the presence of both MC+ and MC− Microcystis (Fig.1c, d).
The cell density of M. aeruginosa (MC+) was affected by the presence of either of the green microalgae species (Fig.1e, f). The mixed culture with S. acuminatus had a more significant effect on the cell density of M. aeruginosa than did M. convolutum. The cell density results of M. panniformis (MC−) showed a similar variation as observed for M. aeruginosa (MC+) in the presence of either of the green microalgae with S. acuminatus having the most significant inhibitory effect on it (Fig.1g, h).
Specific growth rates of all the strains used in the mixed culture experiments are shown in Fig.2a–d. Both green algae and cyanobacteria strains were significantly affected by the presence of other competing species in the culture, where the controls always had a significantly higher growth rate than those of the strains in the presence of a competitor.
The green microalgae strains were not significantly affected by the crude extracts of the MC+ and MC− Microcystis (Fig.3a, b, p>0.05). Unlike what happened in the case of the green algae exposed to cyanobacteria extracts, the exposure of M. aeruginosa to 5 and 10μg L−1extracts of M. convolutum and S. acuminatus caused a significant reduction in its growth (Fig.4a, b).
Microcystin production per cell by M. aeruginosa (MC+) changed with the progression of the experiments (Fig.5a, b). In the presence of both green algae species, the maximum production of microcystins was ca. 30 fg.cell−1at the end of the experiment. However, the production per cell in the con-trols for both mixed experiments showed that the production of microcystins per cell decreased from the first day of the experiment, having the lowest concentration generally at the end of the experiment.
The effect of different extract concentrations of M. convolutum and S. acuminatus was somewhat different as toxin production did not increase with the progression of the experiment (Fig. 5c, d). The lowest MC production by
Fig. 2 Specific growth rates (μ) of cyanobacterial and green microalgal controls (filled bar) and mixed cultures (empty bar). a M. convolutum CMEA/UFF0201 with M. aeruginosa BCCUSP232 (MC+). b M. convolutum CMEA/UFF0201 with M. panniformis BCCUSP200 (MC−). c S. acuminatus UFSCar036 with M. aeruginosa BCCUSP232
(MC+). d S. acuminatus UFSCar036 with M. panniformis BCCUSP200 (MC−). MC+ means microcystins-producing strain and MC− non-microcystins-producing strain. Error bars represent standard deviation for n=3
M. aeruginosa was recorded when it was exposed to the 10μg.L−1extract concentrations.
Discussion
Our study showed that when grown in mixed cultures, signif-icant differences are observed in terms of the cell densities of the competing strains in the culture medium and microcystin production by M. aeruginosa. This study started with a 1:1 ratio (i.e., each strain in the mixed culture having equal cell density) to eliminate the bias caused by such a factor. Past studies have shown that initial cell density determines which species will be dominant, especially giving advantage to the species with higher cell density at the start of the experiment (Li and Li2012).
Microcystins are very important cyanotoxins that have different negative effects on aquatic organisms including pho-toautotrophs (Wiegand and Pflugmacher2005; Babica et al.
2006; Bártova et al.2011). The toxin was produced in much higher concentrations when M. aeruginosa (MC+) was cul-tured under mixed culture conditions compared to the control. This implied that the increased production of MC was due to competitive pressure in the mixed culture experiment. The synthesis of this toxin requires high energy input that means a high cost for the cell. However, studies have shown that the gains of producing the toxin under competitive conditions far outweigh the cost of production to the cell (Briand et al.2008,
2012; Li and Li 2012). MCs are capable of suppressing growth of microalgae when produced in sufficient amounts by acting as inhibitors of photosynthetic activity (Sukenik et al. 2002; Hu et al. 2004). According to Kaplan et al. (2012), cells exposed to MCs suffer oxidative stress due to the diversion of photosynthetic electrons to oxygen, as an electron acceptor (Mehler reaction), thereby resulting in the production of reactive oxygen species and induction of a programmed cell death cascade. Furthermore, the production of MC can be seen as a defense mechanism by M. aeruginosa when faced with competitors for common resources in their environment. In support of our assumption, we observed that S. acuminatus was able to inhibit up to 50 % of growth of M. aeruginosa when grown together, a situation that was dif-ferent when the cyanobacteria was grown with M. convolutum (results not shown). Hence, we observed that the production of MC was significantly higher when M. aeruginosa was cocultured with S. acuminatus than M. convolutum.
It is impossible to attribute changes in the cell den-sity of the microalgae to the production of MC only, as similar results were observed for the non-MC-producing M. panniformis that significantly inhibited the growth of the green algae. This is further supported by the fact that the use of MC-containing extracts and non-MC-containing extracts of Microcystis did not significantly affect the growth and biomass production (cell density) of the green algae except for some slight but statistical-ly insignificant differences observed from day 9 to the end of the experiment. From the experimental design and conditions used in this study, the effect of nutrient limitation was removed due to the use of BG-11 medi-um which is a very nutrient-rich growth medimedi-um. A situation similar to Dunker et al. (2013) where they were able to show that interspecific interference can be implicated when these other factors are controlled. Fur-thermore, the identical initial cell density ratio (1:1) used in the mixed experiment further supports the fact that nutrient competition and limitation may not have been a problem among the strains. However, it is im-portant to note that the requirements for irradiance are clearly different among different phytoplankton species
Fig. 3 Cell density (cells.mL−1) of different green microalgae during the course of the experiment (13 days) as a function of different crude extract concentrations. a M. convolutum with crude extracts of M. aeruginosa (MC+) and M. panniformis BCCUSP200 (MC−). b S. acuminatus with crude extracts of M. aeruginosa (MC+) and M. panniformis BCCUSP200 (MC−). control. M. convolutum and S. acuminatus with M. aeruginosa (MC+) crude extract of 10μg.L−1. M. convolutum and S. acuminatus with M. aeruginosa (MC+) crude extract of 5μg.L−1. M. convolutum and S. acuminatus with M. panniformis (MC−) crude extract of 10 μg.L−1. M. convolutum and S. acuminatus with M. panniformis (MC−) crude extract of 5μg.L−1. Arrow crude extract addition. Single asterisk means that the control is statistically different (p<0.05) from the 5μg.L−1and double asterisks from the 10μg.L−1treatments
Fig. 4 Cell density (cells.mL−1) of M. aeruginosa (MC+) during the course of the experiment (14 days) as a function of different green microalgae crude extract concentrations. a M. aeruginosa with crude extracts of M. convolutum. b M. aeruginosa with crude extracts of S. acuminatus. Control ( ). M. aeruginosa with crude extract of
10 μg.L−1 ( ). M. aeruginosa with crude extracts of 5μg.L−1. ( ). Arrow crude extract addition. Single asterisk means that the control is statistically different (p<0.05) from the 5μg.L−1and double asterisks from the 10μg.L−1treatments
and as the culture density increased toward the end of the experiments, irradiance may have also been a limit-ing factor. And the ability of the species to adapt to differing irradiances with changing cell density may have determined not only the outcome of interspecific competition among the species but also the uptake and utilization of nutrients (Xu et al. 2010; Briand et al.
2012). An important observation was that the inhibitory effect was higher each time the MC-producing strain was used, implying that the toxin tended to increase the allelopathic effect of the cyanobacteria. In addition, the proportional reduction in cell density of M. aeruginosa per time compared to the control was much lower than that of M. panniformis when grown with similar green microalgae strains. As both cyanobacteria were able to reduce the cell density of both green microalgal species, inferences can be made that secondary metabolites in addition to MCs may have been involved in the control of their growth. Unlike what has been reported by Zhang et al. (2013), Harel et al. (2013) and Dunker et al. (2013), where Scenedesmus obliquus benefited from the competition with M. aeruginosa, the situation was
somewhat different in our study as neither phytoplankton groups were gainers because they all showed significant re-duction in biomass prore-duction in the presence of competing strains.
The higher growth reduction of the cyanobacteria experienced in the presence of S. acuminatus could mean this green microalga also produced secondary metabolites that were capable of reducing their growth. S. acuminatus was less sensitive to the Microcystis strains due to the lower proportional reduction per time in its cell densities compared to the cyanobacteria. This is supported by the studies of Harel et al. (2013) and Zhang et al. (2013) that demonstrated that Scenedesmus sp. was capable of producing secondary metabolites like dibutyl phthalate and beta-sitosterol that had inhibitory effects on the growth of different strains of Microcystis. The authors showed that spent cell-free media from Scenedesmus sp. (Scenedesmus huji) caused severe cell lysis in various Microcystis strains. This may explain why natural Microcystis blooms are terminated by rapid cell lysis, which have been said to be a result of biotic
Fig. 5 Cell density variation (cells.mL−1) and total microcystin produc-tion per cell quota (fg.cell−1) of M. aeruginosa BCCUSP232 cultured with M. convolutum CMEA/UFFO201 (a) and S. acuminatus UFSCAR036 (b) on days 1, 7, and 10 of the experiment, and exposed to crude extracts of M. convolutum (c) and S. acuminatus (d) at 5 and 10μg.L−1concentrations. Unfilled bars represent MC production in the control and dark ash filled bars MC production by M. aeruginosa under
mixed culture conditions and 10μg.L−1in the extract treatment and light ash filled bars represent 5μg.L−1treatment. Unfilled circles represent MC production in the control and dark filled circles MC production by M. aeruginosa under mixed culture conditions and 10μg.L−1extract treatment and light ash filled circle represents MC production at 5μg.L−1. Error bars represent standard deviation for n=3
and abiotic factors (Rashidan and Bird2001; Ross et al.
2006; Sevilla et al. 2008; Sedmak et al. 2008, Harada et al. 2009). Since the abiotic factors in this study were controlled, it is possible to link the reduction in cell density in this study to biotic factors.
Extracts from the cyanobacteria did not have significant negative effect on the growth of the green algae. This is opposite with what was observed when both cyanobacterial and green algal strains were grown together in mixed culture, where significant growth inhibition was observed. It is possi-ble that the lack of inhibitory effect observed by exposing the studied strains to different extracts may be because they were from monocultured conditions and not from mixed cultured conditions. In allelopathic investigations between different cyanobacterial strains, Mello et al. (2012) showed that only the exudates from the mixed culture conditions inhibited the growth of the M. aeruginosa. Campos et al. (2013), Pinheiro et al. (2013), and Bittencourt-Oliveira et al. (2013) also showed that crude extracts from toxinproducing and -nonproducing cyanobacterial strains had varied effects on different phytoplankton species with some having stimulatory, inhibitory, and no effect. Our results with green algal extracts agree with the findings of Harel et al. (2013) and Zhang et al. (2013) that members of the chlorophyta group are capable of producing chemical substances with the ability to inhibit the growth of other species. The inhibitory effect of the green algal extract became smaller with the progression of the experiment probably due to the dilution effect of the crude extracts with increasing cell density of the M. aeruginosa.
We c o n c l u d e t h a t M . a e r u g i n o s a ( M C + ) a n d M. panniformis (MC−) were able to inhibit the growth of the green algae with M. convolutum being the most affected. The green alga S. acuminatus in return was able to inhibit the growth of both cyanobacteria with M. panniformis being the most affected in the mixed culture with the green alga. In response to the presence of a competing species in the growth medium, M. aeruginosa significantly increased its MC pro-duction per cell with the progression of the experiment, hav-ing the highest concentration at the end of the experiment. However, the reverse was the case for the control treatments that generally had reduced concentrations of the toxin as the experiments progressed. The extracts of both cyanobacteria had no significant inhibitory effect on the strains investigated, while those of the green algae also had significant inhibitory effect on the growth of M. aeruginosa.
Acknowledgments This study was supported by grants from São Paulo Research Foundation (FAPESP—2011/50840-0 and 2013/11306-3 to M.A. Chia) and Brazilian National Research Council (CNPq—301739/ 2011-0).
References
Babica P, Bláha L, Marsalek B (2006) Exploring the natural role of microcystins—a review of effects on photoautotrophic organisms. J Phycol 42:9–20
Bártova K, Hilscherova K, Babica P, Marsálek B, Bláha L (2011) Effects of microcystin and complex cyanobacterial samples on the growth and oxidative stress parameters in green alga Pseudokirchneriella subcapitata and comparison with the model oxidative stressor-herbicide paraquat. Environ Toxicol 26:641–648
Bar-Yosef Y, Sukenik A, Hadas O, Viner-Mozzini Y, Kaplan A (2010) Enslavement in the water body by toxic Aphanizomenon ovalisporum, inducing alkaline phosphatase in phytoplanktons. Curr Biol 20:1557–1561
B-Beres V, Grigorszky I, Vasas G, Borics G, Varbiro G, Nagy SA, Borbely G, Bacsi I (2012) The effects of Microcystis aeruginosa (cyanobacterium) on Cryptomonas ovata (Cryptophyta) in labora-tory cultures: why these organisms do not coexist in steady-state assemblages? Hydrobiologia 691:97–107
Bittencourt-Oliveira MC (2003) Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molec-ular marker. Harmful Algae 2:51–60
Bittencourt-Oliveira MC, Oliveira MC, Pinto E (2011) Diversity of microcystin-producing genotypes in Brazilian strains of Microcystis (Cyanobacteria). Braz J Biol 71:209–216
Bittencourt-Oliveira MC, Camargo-Santos D, Moura AN, Francisco IB, Dias CTS, Molica RJR, Cordeiro-Araújo MK (2013) Effect of toxic and non-toxic crude extracts on dif-ferent Microcystis species (Cyanobacteria). Afr J Microbiol Res 7:2596–2600
Briand E, Gugger M, François JCC, Bernard J, Humbert F, Quiblier C (2008) Temporal variations in the dynamics of potentially microcystin-producing strains in a bloom-forming Planktothrix agardhii (cyanobacterium) population. Appl Environ Microbiol 74:3839–3848
Briand E, Bormans M, Quiblier C, Salençon MJ, Humbert JF (2012) Evidence of the cost of the production of microcystins by Microcystis aeruginosa under differing light and nitrate environ-mental conditions. PLoS One 7(1):e29981
Campos L, Araújo P, Pinheiro C, Azevedo J, Osório H, Vasconcelos V (2013) Effects on growth, antioxidant enzyme activity and levels of extracellular proteins in the green alga Chlorella vulgaris exposed to crude cyanobacterial extracts and pure microcystin and cylindrospermopsin. Ecotoxicol Environ Saf 94:45–53
Chen YW, Qin BQ, Teubner K, Dokulil MT (2003) Long-term dynamics of phytoplankton assemblages: Microcystis-domination in Lake Taihu, a large shallow lake in China. J Plankton Res 25:445–453
Dunker S, Jakob T, Wilhelm C (2013) Contrasting effects of the cyanobacterium Microcystis aeruginosa on the growth and physiology of two green algae, Oocystis marsonii and Scenedesmus obliquus, revealed by flow cytometry. Freshw Biol 58:1573–1587
Fogg GE, Thake B (1987) Algae Cultures and Phytoplankton Ecology. 3rd edn London: The University of Wisconsins Press Ltd p 269 Frossard V, Versanne-Janodet S, Aleya L (2014) Factors supporting
harmful macroalgal blooms in flowing waters: a 2-year study in the lower Ain River, France. Harmful Algae 33:19–28
Guillard RRL (1973) Division rates. In: Stein J (ed) Handbook of phy-cological methods: culture methods and growth measurements. Cambridge University Press, Cambridge, pp 289–311
Harada KI, Ozaki K, Tsuzuki S, Kato H, Hasegawa M, Kuroda E, Arii S, Tsuji K (2009) Blue color formation of cyanobacteria with b-cyclocitral. J Chem Ecol 35:1295–1301
Harel M, Weiss G, Lieman-Hurwitz J, Gun J, Lev O, Lebendiker M, Temper V, Block C, Sukenik A, Zohary T, Braun S, Carmeli S, Kaplan A (2013) Interactions between Scenedesmus and Microcystis may be used to clarify the role of secondary metabolites. Environ Microbiol Rep 5:97–104
Hu ZQ, Liu YD, Li DH (2004) Physiological and biochemical analyses of microcystin-RR toxicity to the cyanobacterium Synechococcus elongatus. Environ Toxicol 19:571–577
Jochimsen EM, Carmichael WW, An JS, Cardo DM, Cookson ST, Holmes CE, Antunes MB, de Melo Filho DA, Lyra TM, Barreto VS, Azevedo SM, Jarvis WR (1998) Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. New Engl J Med 338:873–878
Jonsson PR, Pavia H, Toth G (2009) Formation of harmful algal blooms cannot be explained by allelopathic interactions. Proc Natl Acad Sci 106:11177–11182
Kaplan A, Harel M, Kaplan-Levy RN, Hadas O, Sukenik A, Dittmann E (2012) The languages spoken in the water body (or the biological role of cyanobacterial toxins). Front Microbiol 3:138
Kemp A, John J (2006) Microcystins associated with Microcystis dom-inated blooms in the southwest wetlands, Western Australia. Environ Toxicol 21:125–130
Kuwata A, Miyazaki T (2000) Effects of ammonium supply rates on competition between Microcystis novacekii (Cyanobacteria) and Scenedesmus quadricauda (Chlorophyta): simulation study. Ecol Modell 135:81–87
Leao PN, Vasconcelos MTSD, Vasconcelos VM (2009) Allelopathic activity of cyanobacteria on green microalgae at low cell densities. Eur J Phycol 44:347–355
Leflaive J, Ten-Hage L (2007) Algal and cyanobacterial secondary me-tabolites in freshwaters: a comparison of allelopathic compounds and toxins. Freshw Biol 52:199–214
Li Y, Li D (2012) Competition between toxic Microcystis aeruginosa and nontoxic Microcystis wesenbergii with Anabaena PCC7120. J Appl Phycol 24:69–78
Lund JWG, Kipling C, Lecren ED (1958) The invert microscope method of estimating algae numbers and statistical basis of estimations by counting. Hydrobiologia 11:143–170
Magrann T, Dunbar SG, Boskovic DS, Hayes WK (2012) Impacts of Microcystis on algal biodiversity and use of new technology to remove Microcystis and dissolved nutrients. Lakes Reserv Res Manag 17:231–239
Máthé C, M-Hamvas M, Vasas G, Surányi G, Bácsi I, Beyer D, Tóth S, Tímár M, Borbély G (2007) Microcystin-LR, a cyanobacterial toxin, induces growth inhibition and histological alterations in common reed (Phragmites australis) plants regenerated from embryogenic calli. New Phytol 176:824–835
Mello MM, Soares MCS, Roland F, Lurling M (2012) Growth inhibition and colony formation in the cyanobacterium Microcystis aeruginosa induced by the cyanobacterium Cylindrospermopsis raciborskii. J Plankton Res 34:987–994
Papadimitriou T, Katsiapi M, Kormas KA, Moustaka-Gouni M, Kagalou I (2013) Artificially-born“killer” lake: phytoplankton based water quality and microcystin affected fish in a reconstructed lake. Sci Total Environ 452–453:116–124
Pinheiro C, Azevedo J, Campos A, Loureiro S, Vasconcelos V (2013) Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton species. Hydrobiologia 705:27–42
Rashidan KK, Bird DF (2001) Role of predatory bacteria in the termina-tion of a cyanobacterial bloom. Microb Ecol 41:97–105
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61
Ross C, Santiago-Vazquez L, Paul V (2006) Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa. Aquat Toxicol 78:66–73
Sedmak B, Carmeli S, Elersek T (2008)“Non-toxic” cyclic peptides induce lysis of cyanobacteria—an effective cell population density control mechanism in cyanobacterial blooms. Microb Ecol 56:201–209 Sevilla E, Martin-Luna B, Vela L, Bes MT, Fillat MF, Peleato ML (2008)
Iron availability affects mcyD expression and microcystin-LR syn-thesis in Microcystis aeruginosa PCC 7806. Environ Microbiol 10: 2476–2483
Shen H, Song L (2007) Comparative studies on physiological responses to phosphorus in two phenotypes of bloom-forming Microcystis. Hydrobiologia 592:475–486
Sukenik A, Eshkol R, Livne A, Hadas O, Rom M, Tchernov D, Vardi A, Kaplan A (2002) Inhibition of growth and photosynthesis of the dinoflagellate Peridinium gatunense by Microcystis sp. (cyanobacteria): a novel allelopathic mechanism. Limnol Oceanogr 47:1656–1663
Takeya K, Kuwata A, Yoshida M, Miyazaki T (2004) Effect of dilution rate on competitive interactions between the cyanobacterium Microcystis novacekii and the green alga Scenedesmus quadricauda in mixed chemostat cultures. J Plankton Res 26:29–35
Wiegand C, Pflugmacher S (2005) Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review. Toxicol Appl Pharm 203:201–218
Xu N, Duan S, Li A, Zhang C, Cai Z, Hu Z (2010) Effects of temperature, salinity and irradiance on the growth of the harmful dinoflagellate Prorocentrum donghaienense Lu. Harmful Algae 9:13–17 Yang J, Deng X, Xian Q, Qian X, Li A (2014) Allelopathic effect of
Microcystis aeruginosa on Microcystis wesenbergii: microcystin-LR as a potential allelochemical. Hydrobiologia 727:65–73 You XH, Wang ZL, Shi XY et al (2007) Advances in the studies of
phytoplankton interspecific competition. Trans Oceanol Limn 4: 161–166
Zhang P, Zhai C, Wang X, Liu C, Jiang J, Xue Y (2013) Growth competition between Microcystis aeruginosa and Quadrigula chodatii under controlled conditions. J Appl Phycol 25:555– 565
