J Appl Phycol DOI 10.1007/s10811-014-0326-2

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- the experiment. On the other hand, the extracts of the teractions between toxic and nontoxic cyanobacteria with cyanobacteria had no significant inhibitory effect on the green phytoplankton species rarely investigate the role of algal strains investigated, while those of the green algae also microcystins (MC) production and regulation in the course had significant inhibitory effect on the growth of of the studies. This study investigated the interactions between M. aeruginosa. In conclusion, both cyanobacterial and green intact cells of toxic (Microcystis aeruginosa (Kützing) algal strains investigated were negatively affected by the Kützing) and nontoxic (Microcystis panniformis Komárek presence of competing species. M. aeruginosa responded to et al.) cyanobacteria with those of green algae the presence of green algae by increasing its MC production. (Monoraphidium convolutum (Corda) Komárková-Legnerová The green algal strains significantly inhibited the growth of and Scenedesmus acuminatus (Largerheim) Chodat) as well M. aeruginosa. as the effects of their respective crude extracts (5 and 10 μg.L−1) on their growth under controlled conditions. Keywords Allelopathy . Species competition . M. aeruginosa and M. panniformis were able to significantly Phytoplankton . Mixed algal cultivation . Cyanotoxins . (p<0.05) inhibit the growth of the green algae with Monoraphidium . Scenedesmus . Microcystis 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- Introduction ing species in the growth medium, M. aeruginosa significant- ly increased its MC production per cell with the progression of In recent decades, algal blooms have become widespread in the experiment, having the highest concentration at the end of many water bodies around the world. These have also been characterized by the increased occurrence of toxic M. C. Bittencourt-Oliveira : M. A. Chia : H. S. B. de Oliveira : cyanobacteria blooms, in addition to those of diatoms, dino- M. K. Cordeiro Araújo flagellates, and green algae (Zhang et al. 2013), which have Department of Biological Sciences, Luiz de Queiroz College of serious social and economic implications due to the degrada- Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, tion of water resources, and as a result, generated a lot of Piracicaba, SP 13418-900, Brazil interest into investigations on the environmental factors and * : : M. C. Bittencourt-Oliveira: ( ) H. S. B. de Oliveira mechanisms that promote these blooms as well as how M. K. Cordeiro Araújo R. J. R. Molica cyanotoxins confer a competitive advantage to the producing Graduate Program in Botany, Rural and Federal University of species (Jonsson et al. 2009). Pernambuco, Rua D. Manoel de Medeiros, S/N, Dois Irmãos, Recife, PE 52171-030, Brazil Different water bodies around the world are characterized e-mail: [email protected] by dominant species of phytoplankton that alternate between cyanobacteria and other microalgae (diatoms, green algae). C. T. S. Dias The dominance of different algal species changes from season Department of Exact Sciences, Luiz de Queiroz College of Agriculture, University of São Paulo, Av. Pádua Dias, 11, São Dimas, to season during bloom formation in most water bodies world- Piracicaba, SP 13418-900, Brazil wide (Chen et al. 2003;Frossardetal.2014). In order to J Appl Phycol understand the succession mechanisms responsible for monitoring changes in their concentrations (Yang et al. cyanobacteria and other phytoplankton species, experiments 2014). Studies of the action of MC on microalgae are mostly involving mixed cultures have been encouraged to be carried restricted to lysates of toxic cyanobacterial extracts or purified out by researchers. This has led to investigations on the MC, and often with much higher concentrations than those interactions of cyanotoxin-producing and nonproducing- commonly found in natural environments (Nagata et al.1997; cyanobacteria with microalgae strains as a means of contrib- Kemp and John 2006; Máthé et al. 2007; B-Beres et al. 2012). uting to the understanding of how successional processes Conclusions on the allelopathy of MC resulting from studies operate in nature. A comprehensive understanding of the that used non-environmentally relevant concentrations can be competition between algae and cyanobacteria can be crucial misleading and not applicable in real life situations (Leao et al. to control strategies for bloom formation and outbreaks in 2009). Therefore, our study aimed to (1) evaluate the possible aquatic ecosystems (Zhang et al. 2013). allelopathic interaction between intact cells of microcystin- Most studies have considered the effect of chemical and producing and -nonproducing cyanobacteria with the green physical factors on the competition between cyanobacteria microalgae Scenedesmus acuminatus and Monoraphidium and other microalgae (Takeya et al. 2004; Shen and Song convolutum under controlled conditions and (2) the effect of 2007;LiandLi2012), and shown that nutrient concentration crude extracts of cyanobacteria and green algae on the growth of the medium does not always determine the phytoplankton of the green algal and cyanobacterial strains, respectively, succession in mixed cultures (Kuwata and Miyazaki 2000; using environmentally relevant concentrations. 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. Materials and methods Whereas most studies have focused on the indirect interaction by exploitation that is based on the competition for limited The microcystin-producing (MC+) Microcystis aeruginosa resources (Zhang et al. 2013), in hypertrophic environments, BCCUSP232 (Bittencourt-Oliveira et al. 2011) and non- interference, for example, the production of microcystins (MCs) microcystins-producing (MC−) Microcystis panniformis and other allelochemicals is a direct form of interspecific inter- BCCUSP200 (Bittencourt-Oliveira 2003) cyanobacterial strains action that is very important (Leflaive and ten-Hage L, 2007; from the Brazilian Cyanobacteria Collection of University of São Bar-Yosef et al. 2010; B-Beres et al. 2012; Magrann et al. 2012). Paulo (BCCUSP). The two strains of green microalgae used in The microcystins belong to a family of cyclic heptapeptide this study were Monoraphidium convolutum (CMEA/UFF0201) known to inhibit protein phosphatases. They cause liver fail- obtained from Elizabeth Aidar Collection of Microalgae ure in wild and domestic animals and humans and have (CMEA/UFF) and Scenedesmus acuminatus (UFSCar036) from heterogenous effects on the physiology of plants (Jochimsen the Federal University of São Carlos (UFSCar). For all exper- et al. 1998; Papadimitriou et al. 2013). Unfortunately, most of iments, the cyanobacteria and green algae were maintained in the mixed culture experiments rarely monitor the production environmentally controlled growth chambers set to 24±1 °C, of microcystins during the experiments (B-Beres et al. 2012; 14 h:10 h (light/dark) photoperiod, and 40 μmol photons − − Zhang et al. 2013), in addition to the fact that most of the m 2 s 1 light intensity. Light intensity was measured using a published results are conflicting. Due to the varied responses LI-COR model 250 light meter equipped with a spherical of different microalgae species to MCs (Sedmak et al. 2008,Li underwater sensor. The cyanobacteria and green microalgae and Li 2012; Bittencourt-Oliveira et al. 2013;Camposetal. were grown in BG-11 (Rippka et al. 1979) at pH 7.4 according 2013), it becomes imperative to investigate the behavior of to the modifications of Bittencourt-Oliveira et al. (2011)that different phytoplankton lineages in the presence of toxic and involved the substitution of iron for ferric ammonium citrate nontoxic cyanobacteria and microalgae. In addition, it is not FeCl3·H2O chloride. The cultures were stirred manually, and clear whether microcystins or other released compounds by their positions changed on a daily basis. cyanobacteria or other microalgae are responsible for allelop- athy (Leao et al. 2009; B-Beres et al. 2012). This makes it Mixed culture experiments difficult for generalizations or conclusions to be made on the relationship between dominant phytoplankton species in- All experiments were carried out in 1,000-mL Erlenmeyer volved in standing water species succession. flasks containing 600 mL final culture volume. In the mixed Investigations on allelopathic effects between cultures, 300 mL of the culture of each strain with an equal cyanobacteria and green algae should not only be based on number of cells (ratio, 1:1) was used. Briefly, intact cells of the mutual interactions in mixed culture experiments but also M. aeruginosa (BCCUSP232) were cocultured with those of determine the possible role of bioactive substances like MCs M. convolutum CMEA/UFF0201 in one experimental setup in order to elucidate their allelopathic mechanisms by having an initial cell density of 7.0×105 cells.mL−1, and with J Appl Phycol

S. acuminatus UFSCar036 having an initial density of 1.0× obtained in the same manner as those of the 105 cells.mL−1 in the second mixed culture experiment. The cyanobacterial strains. Equivalents of the extract concen- same procedure was repeated for intact cells of M. panniformis trations (5 and 10 μg.L−1) used in the MC+-producing (BCCUSP200) in the mixed culture with both green strains were applied to the two cyanobacterial strains to microalgal strains. For the controls, each strain (i.e., see if there may be any influence on the growth of the cyanobacteria and green algae) was grown separately in cyanobacterial strains. The extract exposure experiments 600 mL, using the same cell densities as those used in the lasted for 13 days. respective experiments. The choice of different cell densities to combine with the Microcystis spp. was because Cell density and growth rate Scenedesmus quadricauda had a much larger biovolume μ 3 −1 μ 3 −1 (491.39 m cell ) than M. convolutum (25.92 m cell ). Cell density and growth rates were obtained by counting The experiments were monitored daily for 10 days by mea- the number of cells using a Fuchs-Rosenthal chamber suring cell densities of the strains, with the first count occur- with the aid of a binocular microscope, following the ring 24 h after the mixture of the strains. method of Guillard (1973).Thespecificgrowthrate(μ) To evaluate the influence of green microalgae on the wasobtainedaccordingtoFoggandThake(1987). For production of microcystins by M. aeruginosa all the experiments and their controls, 2 mL aliquots of BCCUSP232, 1 mL culture aliquots were collected after the cultures were removed and immediately preserved homogenization on days 1, 7, and 10 of the experiment. with 10 % Lugol solution on a daily basis. As a means Immediately after collection, the aliquots were frozen in of increasing the reliability of results, a minimum of − liquid nitrogen and stored at 80 ° C in a freezer until 400 cells were quantified to keep the estimated error the time of toxin analysis. Although microcystin analy- within ±10 % (Lund et al. 1958). Regardless of the sis was not done in the M. panniformis BCCUSP200 number of cells counted, a minimum of three chambers experiments, aliquots of the same volume were also were counted. The cell density was expressed in cells taken at the same rates to maintain the same volume per milliliter. and growth conditions for all cultures.

Cyanobacterial and green algal extracts Microcystins analysis

Crude extracts of the MC+ and MC− strains from The culture aliquots previously removed from mixed cultures M. aeruginosa (BCCUSP232) and M. panniformis and the control (n=3) were homogenized by ultrasonication (BCCUSP200) were obtained by growing each strain in (Microson Ultrasonic Cell Disruptor, USA) for 3 min (15 W 20 L of BG-11 medium under the growth conditions shown and 22.5 kHz) to totally disrupt the cells. The disruption was above. At exponential growth phase, the resulting cul- confirmed by microscopic observations. tures were harvested by low speed centrifugation and Total microcystin concentrations (intra and extracellu- the resultant pellet flash frozen in liquid nitrogen, ly- lar) were determined by ELISA technique using the ophilized and stored at −80 °C in a freezer until the BEACON microcystins plate kit (Beacon Analytical ’ time of use. The lyophilized biomass was resuspended Systems Inc., USA), following the manufacturer sin- in a total volume of 3 mL of deionized water and structions. Analyses were performed using a microplate ultrasonicated (Microson Ultrasonic Cell Disruptor, reader (ASYS Hitech, model A-5301, Austria) set to 450 nm. The detection range of the assays was 0.10 USA) at 15 W and 22.5 kHz to disrupt the cells and −1 release the intracellular toxins. From the crude extracts to 2.0 parts per billion (PPB) (ng mL ). Analyses were obtained from the MC+ strain, 5 and 10 μg.L−1 MC done in triplicates. The intracellular and extracellular were obtained and used in the treatments. The volumes concentration was defined as the total microcystins of the crude extracts applied to the treatments were 0.5 (intra- and extracellular) per cell quota. and 1.0 mL corresponding to 5 and 10 μg.L−1 MC, respectively. Crude extract concentration equivalents of Data treatment the non-MC producing strain was obtained and used in the treatments. The concentration MC in the extracts The cell density data were tested for normality and was determined using enzyme-linked immuno sorbent homogeneity of variance. Where the normality and ho- assay (ELISA) as described below in the microcystins mogeneity of variance test were positive, the cell den- analysis section. sity data were subjected to repeated measure general Crude extracts of the green algal strains (M. convolutum linear model (GML) analysis of variance (ANOVA). CMEA/UFF0201 and S. acuminatus UFSCAr036) were Where significant differences were observed, the J Appl Phycol

Fig. 1 Growth curves (cells.mL−1) for the different green microalgal and (MC+) control and mixed culture with M. convolutum CMEA/UFF0201. cyanobacterial strains cultured in mono and mixed culture conditions. a f M. aeruginosa BCCUSP232 (MC+) control and mixed culture with M. convolutum CMEA/UFF0201 control and mixed culture experiment S. acuminatus UFSCar036. g M. panniformis BCCUSP200 (MC−)con- with M. aeruginosa BCCUSP232 (MC+). b M. convolutum control and trol and mixed culture with M. convolutum CMEA/UFF0201. h mixed culture with M. panniformis BCCUSP200 (MC−). c S. acuminatus M. panniformis BCCUSP200 (MC−) control and mixed culture with UFSCar036 control and mixed culture with M. aeruginosa BCCUSP232 S. acuminatus UFSCar036. Asterisks mean that there was no statistical (MC+). d S. acuminatus UFSCar036 control and mixed culture with difference between the mixed cultures and controls of the respective M. panniformis BCCUSP200 (MC−). e M. aeruginosa BCCUSP232 treatment combintations. Error bars represent standard deviation for n=3 J Appl Phycol

Tukey’s HSD post hoc test was performed to separate Specific growth rates of all the strains used in the mixed the significantly different means. All analyses were done culture experiments are shown in Fig. 2a–d. Both green algae at 5 % significance level. All statistical analyses were and cyanobacteria strains were significantly affected by the done using the SAS version 9.2 software for Windows. 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. Results The green microalgae strains were not significantly affected by the crude extracts of the MC+ and MC− Microcystis (Fig. 3a, b, The effect of mixed cultures of the microcystin-producing p>0.05). Unlike what happened in the case of the green algae (MC+) and -nonproducing (MC−) strains of cyanobacteria exposed to cyanobacteria extracts, the exposure of M. aeruginosa with green algae can be seen in Fig. 1. Over 50 % growth to 5 and 10 μgL−1 extracts of M. convolutum and S. acuminatus reduction of the green microalga M. convolutum was recorded caused a significant reduction in its growth (Fig. 4a, b). when cocultured with both MC+ and MC− Microcystis Microcystin production per cell by M. aeruginosa (MC+) (Fig. 1a, b). Similarly, the growth of S. acuminatus was changed with the progression of the experiments (Fig. 5a, b). significantly inhibited (p<0.05) in the presence of both In the presence of both green algae species, the maximum MC+ and MC− Microcystis (Fig. 1c, d). production of microcystins was ca. 30 fg.cell−1 at the end of The cell density of M. aeruginosa (MC+) was affected by the experiment. However, the production per cell in the con- the presence of either of the green microalgae species trols for both mixed experiments showed that the production (Fig. 1e, f). The mixed culture with S. acuminatus had a more of microcystins per cell decreased from the first day of the significant effect on the cell density of M. aeruginosa than did experiment, having the lowest concentration generally at the M. convolutum. The cell density results of M. panniformis end of the experiment. (MC−) showed a similar variation as observed for The effect of different extract concentrations of M. aeruginosa (MC+) in the presence of either of the green M. convolutum and S. acuminatus was somewhat different microalgae with S. acuminatus having the most significant as toxin production did not increase with the progression of inhibitory effect on it (Fig. 1g, h). the experiment (Fig. 5c, d). The lowest MC production by

Fig. 2 Specific growth rates (μ) of cyanobacterial and green microalgal (MC+). d S. acuminatus UFSCar036 with M. panniformis BCCUSP200 controls (filled bar) and mixed cultures (empty bar). a M. convolutum (MC−). MC+ means microcystins-producing strain and MC− non- CMEA/UFF0201 with M. aeruginosa BCCUSP232 (MC+). b microcystins-producing strain. Error bars represent standard deviation M. convolutum CMEA/UFF0201 with M. panniformis BCCUSP200 for n=3 (MC−). c S. acuminatus UFSCar036 with M. aeruginosa BCCUSP232 J Appl Phycol

Microcystins are very important cyanotoxins that have different negative effects on aquatic organisms including pho- toautotrophs (Wiegand and Pflugmacher 2005; 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;LiandLi2012). MCs are capable of suppressing growth of microalgae when produced in sufficient amounts by acting as inhibitors of photosynthetic activity (Sukenik et al. 2002;Huetal.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 Fig. 3 Cell density (cells.mL−1) of different green microalgae during the S. acuminatus was able to inhibit up to 50 % of growth of course of the experiment (13 days) as a function of different crude extract M. aeruginosa when grown together, a situation that was dif- concentrations. a M. convolutum with crude extracts of M. aeruginosa (MC+) ferent when the cyanobacteria was grown with M. convolutum and M. panniformis BCCUSP200 (MC−). b S. acuminatus with crude extracts (results not shown). Hence, we observed that the production of − of M. aeruginosa (MC+) and M. panniformis BCCUSP200 (MC ). MC was significantly higher when M. aeruginosa was control. M. convolutum and S. acuminatus with M. aeruginosa (MC+) − cocultured with S. acuminatus than M. convolutum. crude extract of 10 μg.L 1. M. convolutum and S. acuminatus with M. aeruginosa (MC+) crude extract of 5 μg.L−1. M. convolutum and It is impossible to attribute changes in the cell den- S. acuminatus with M. panniformis (MC−) crude extract of 10 μg.L−1. sity of the microalgae to the production of MC only, as M. convolutum and S. acuminatus with M. panniformis (MC−) crude extract of similar results were observed for the non-MC-producing μ −1 5 g.L . Arrow crude extract addition. Single asterisk means that the control is M. panniformis that significantly inhibited the growth of statistically different (p<0.05) from the 5 μg.L−1 and double asterisks from the 10 μg.L−1 treatments 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 M. aeruginosa was recorded when it was exposed to the affect the growth and biomass production (cell density) 10 μg.L−1 extract concentrations. 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 Discussion and conditions used in this study, the effect of nutrient limitation was removed due to the use of BG-11 medi- Our study showed that when grown in mixed cultures, signif- um which is a very nutrient-rich growth medium. A icant differences are observed in terms of the cell densities of situation similar to Dunker et al. (2013)wherethey the competing strains in the culture medium and microcystin were able to show that interspecific interference can be production by M. aeruginosa. This study started with a 1:1 implicated when these other factors are controlled. Fur- ratio (i.e., each strain in the mixed culture having equal cell thermore, the identical initial cell density ratio (1:1) density) to eliminate the bias caused by such a factor. Past used in the mixed experiment further supports the fact studies have shown that initial cell density determines which that nutrient competition and limitation may not have species will be dominant, especially giving advantage to the been a problem among the strains. However, it is im- species with higher cell density at the start of the experiment portant to note that the requirements for irradiance are (Li and Li 2012). clearly different among different phytoplankton species J Appl Phycol

Fig. 4 Cell density (cells.mL−1)ofM. aeruginosa (MC+) during the 10 μg.L−1 ( ). M. aeruginosa with crude extracts of 5 μg.L−1. course of the experiment (14 days) as a function of different green ( ). Arrow crude extract addition. Single asterisk means that the microalgae crude extract concentrations. a M. aeruginosa with crude control is statistically different (p<0.05) from the 5 μg.L−1 and double extracts of M. convolutum. b M. aeruginosa with crude extracts of asterisks from the 10 μg.L−1 treatments S. acuminatus.Control( ). M. aeruginosa with crude extract of J Appl Phycol

Fig. 5 Cell density variation (cells.mL−1) and total microcystin produc- mixed culture conditions and 10 μg.L−1 in the extract treatment and light tion per cell quota (fg.cell−1)ofM. aeruginosa BCCUSP232 cultured ash filled bars represent 5 μg.L−1 treatment. Unfilled circles represent with M. convolutum CMEA/UFFO201 (a)andS. acuminatus MC production in the control and dark filled circles MC production by UFSCAR036 (b) on days 1, 7, and 10 of the experiment, and exposed M. aeruginosa under mixed culture conditions and 10 μg.L−1 extract to crude extracts of M. convolutum (c)andS. acuminatus (d)at5and treatment and light ash filled circle represents MC production at 5 μg.L−1. 10 μg.L−1 concentrations. Unfilled bars represent MC production in the Error bars represent standard deviation for n=3 control and dark ash filled bars MC production by M. aeruginosa under

and as the culture density increased toward the end of somewhat different in our study as neither phytoplankton the experiments, irradiance may have also been a limit- groups were gainers because they all showed significant re- ing factor. And the ability of the species to adapt to duction in biomass production in the presence of competing differing irradiances with changing cell density may strains. have determined not only the outcome of interspecific The higher growth reduction of the cyanobacteria competition among the species but also the uptake and experienced in the presence of S. acuminatus could utilization of nutrients (Xu et al. 2010;Briandetal. mean this green microalga also produced secondary 2012). An important observation was that the inhibitory metabolites that were capable of reducing their growth. effect was higher each time the MC-producing strain S. acuminatus waslesssensitivetotheMicrocystis was used, implying that the toxin tended to increase strains due to the lower proportional reduction per time the allelopathic effect of the cyanobacteria. In addition, in its cell densities compared to the cyanobacteria. This the proportional reduction in cell density of M. aeruginosa per is supported by the studies of Harel et al. (2013)and time compared to the control was much lower than that of Zhang et al. (2013) that demonstrated that Scenedesmus M. panniformis when grown with similar green microalgae sp. was capable of producing secondary metabolites like strains. As both cyanobacteria were able to reduce the cell dibutyl phthalate and beta-sitosterol that had inhibitory density of both green microalgal species, inferences can be effects on the growth of different strains of Microcystis. made that secondary metabolites in addition to MCs may have The authors showed that spent cell-free media from been involved in the control of their growth. Unlike what has Scenedesmus sp. (Scenedesmus huji) caused severe cell been reported by Zhang et al. (2013), Harel et al. (2013)and lysis in various Microcystis strains. This may explain Dunker et al. (2013), where Scenedesmus obliquus benefited why natural Microcystis bloomsareterminatedbyrapid from the competition with M. aeruginosa, the situation was cell lysis, which have been said to be a result of biotic J Appl Phycol and abiotic factors (Rashidan and Bird 2001;Rossetal. References 2006; Sevilla et al. 2008;Sedmaketal.2008, Harada et al. 2009). 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