Competitiveness of alga Microcystis aeruginosa co-cultivated with cyanobacterium Raphidiopsis raciborskii conf irms its dominating position*

Zengling MA , Xiaoqiao ZHANG , , Renhui LI , Min WANG , Wenli QIN ,He ZHANG , Gang LI , Henguo YU , Chuanjun DAI , Min ZHAO ,**

1 Zhejiang Provincial Key Laboratory for Subtropical Water Environment and Marine Biological Resources Protection, Wenzhou University, Wenzhou 325035, China

2 National and Local Joint Engineering Research Center of Ecological Treatment Technology for Urban Water Pollution, Wenzhou University, Wenzhou 325035, China

3 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

Abstract Microcystis aeruginosa has always been regarded as the main culprit of cyanobacterial blooms in freshwater. However, in recent years, Raphidiopsis raciborskii has gradually replaced M. aeruginosa as the culprit of cyanobacterial blooms in some tropical and subtropical shallow lakes. To reveal which one plays a more dominant role, interactions between cylindrospermospin (CYN)-producing R. raciborskii and microcystins (MCs)-producing or non-MCs-producing M. aeruginosa strains were studied using bialgal cultures at diff erent initial ratios of biomasses of the two species at 25 °C. During the co-cultivation,the M. aeruginosa strains inhibited the growth and heterocyst formation of R. raciborskii f ilaments, and thus occupied a dominant position during the co-cultivation regardless of the initial biomass ratios in the cultures. In addition, the MCs-producing M. aeruginosa strain contributed to a higher portion of the total biomass and exerted a stronger inhibitory eff ect on R. raciborskii compared with the non-MCs-producing strain. However, the growth of both MCs-producing and non-MCs-producing M. aeruginosa strains was stimulated by R. raciborskii in the co-cultures compared with M. aeruginosa monoculture, indicating that M. aeruginosa could outcompete R. raciborskii if given enough time, enabling it to develop into the dominant species even in very low initial concentration. To our best knowledge, this is the f irst report on the loss of heterocyst formation by a species of cyanobacteria that resulted from interactions between two diff erent species of cyanobacteria. These f indings indicate that it is diffi cult for R. raciborskii to replace the dominant position of M. aeruginosa under the experimental environmental condition, and the allelopathic eff ects of M. aeruginosa on R. raciborskii could signif icantly contribute to the success of M. aeruginosa.

Keyword: competition; growth; heterocyst; Microcystis aeruginosa; morphology; Raphidiopsis raciborskii

1 INTRODUCTION

Cyanobacterial harmful algal blooms (CyanoHABs)are almost a ubiquitous phenomenon in stagnant water worldwide (Huisman et al., 2018; Paerl,2018). These algal blooms have presented a great environmental challenge because of their severe ecological impacts and associated health threats not only to humans but also to the many aquatic animals that inhabit the water (Olokotum et al., 2020;Amorim and Moura, 2021; Plaas and Paerl, 2021).Microcystisaeruginosa,Raphidiopsisraciborskii,Dolichospermumflos-aquae,Aphanizomenonflosaquae, andPlanktothrixsp. are the most successful CyanoHAB-forming species in many freshwater ecosystems (Padisák and Reynolds, 1998; Nixdorf et al., 2003; Hoeger et al., 2004; Paerl et al., 2011).Among them,M.aeruginosais the most important and the blooms it causes are the most frequent and widely distributed algal blooms as well as being the largest scale worldwide (Harke et al., 2016). In addition, during the proliferation ofM.aeruginosa,the odor compounds and microcystins (MCs) that it produces can lead to serious environmental disaster(Dokulil and Teubner, 2000; Dittmann and Wiegand,2006; Zhang et al., 2010; Kim et al., 2020).

In recent years,R.raciborskiihas become the second most concerning species in terms of the causation of CyanoHABs.Raphidiopsisraciborskiihas a particularly broad growth temperature range(Briand et al., 2004; Chonudomkul et al., 2004; Soares et al., 2013), well suited to diff erent light habitats(Pierangelini et al., 2014a, 2015b; Kovács et al.,2016) and inorganic carbon centration (Pierangelini et al., 2014b, 2015a). In addition, it is more competitive when phosphorus and nitrogen availability is low and/or variable availability (Amaral et al., 2014;Burford et al., 2014, 2018; Willis et al., 2015).Therefore,R.raciborskiialso causes algal blooms globally, and there seem to be signs ofR.raciborskiireplacingM.aeruginosaas the main culprit of algal blooms in some tropical and subtropical shallow lakes(Chislock et al., 2014; Burford et al., 2016; Lei et al.,2020; Xiao et al., 2020). Therefore,R.raciborskiiandM.aeruginosaare of particular concern and these two species of cyanobacteria have received widespread attention (Nixdorf et al., 2003; Tomioka et al., 2011).

Currently,R.raciborskiiis well regarded as an invasive cyanobacterial species in natural waters(Hamilton et al., 2005; Stüken et al., 2006; Moreira et al., 2015), and has gradually become the dominant species in some tropical and subtropical lakes whereM.aeruginosaused to be the dominant species (Saker and Griffi ths, 2001; Moisander et al., 2012). According to the results of f ield investigation,R.raciborskiiandM.aeruginosausually coexist in the same bodies of natural water as dominant species (Rzymski et al.,2014; Jia et al., 2020). However, to our knowledge,there has been a lack of studies focusing on the interactions between these two species using a bialgal cultivation system in the lab, and only non-MCsproducing strains ofM.aeruginosawere used in the previous studies that investigated the competition for dominance betweenR.raciborskiiandM.aeruginosa(Rzymski et al., 2014; Bai et al., 2020; Jia et al., 2020).In addition, it has been reported that the competition betweenR.raciborskiiandM.aeruginosacan be aff ected by environmental factors such as temperature,light, and nutrients (Marinho et al., 2013; Soares et al., 2013; da Silva Brito et al., 2018). Furthermore,the biological invasion is a dynamic process from low-density cell introduction to high-density cell replacement of the dominant species, and the biomass ofR.raciborskiiand that ofM.aeruginosaare almost unequal in every natural body of water that has been tested (Mallon et al., 2015; Sukenik et al.,2015). Therefore, the competition for dominance betweenR.raciborskiiandM.aeruginosaas initiated by diff erent biomass ratios are more suitable for simulating their competition in natural waters.

Compared with the non-MCs-producingM.aeruginosastrain, the MCs-producing strain exerts a higher inhibitory eff ect on other co-existing microalgae because of its MCs-producing ability, and therefore, it usually acquires a greater advantage in such competition for dominance (Li and Li, 2012; Lei et al.,2015; Ma et al., 2015). MCs are well-known toxicants that are primarily responsible for the inhibitory eff ects ofM.aeruginosastrains, which can induce oxidative stress and eventually inhibit the photosynthesis and growth of their competitors (Campos et al., 2013;Yang et al., 2014; Kaur et al., 2019). Besides MCs,other compounds released byM.aeruginosastrains are found to be inhibitory to its competitors, and together with MCs, they can exert a synergistic eff ect(Campos et al., 2013; Yang et al., 2014). A few studies have demonstrated thatR.raciborskiistrains with the ability to synthesize cylindrospermopsin (CYN),and can rely on its contribution to enhance their competitiveness and extend their population size (Bar-Yosef et al., 2010; Sukenik et al., 2012). In addition, the CYN pool size ofR.raciborskiiis constitutive and not aff ected by light and CO2conditions, thus toxicity of blooms formed byR.raciborskiiis determined by the absolute abundance ofR.raciborskiicells within the water column and the relative abundance of toxic and nontoxic strains (Pierangelini et al., 2015b), although diff erent CYN-producing strains possess diff erent constituent CYN cell quotas (Willis et al., 2015).As a hepatotoxic alkaloid, the main targets of CYN included green algae (Bar-Yosef et al., 2010; Campos et al., 2013, Pinheiro et al., 2013) and cyanobacterial species (Rzymski and Poniedzialek, 2014; Rzymski et al., 2014). For example, it was reported that 10- and 50-μg/L CYN (both of which are environmentallyrelevant concentrations) can induce growth inhibition and cell necrosis ofM.aeruginosa, respectively(Rzymski et al., 2014). Both MCs and CYN can exert similar eff ects on the cyanobacteria with respect to the process of their specif ic competition for dominance,but the eff ects of direct cell-to-cell contact between CYN-producingR.raciborskiiand MCs-producingM.aeruginosaremain unclarif ied.

The consequences of competition between two diff erent species depend on the niche diff erence and relative f itness diff erence between the two species,as well as the spatial and resource constraints(MacDougall et al., 2009; Narwani et al., 2013).These consequences can be the replacement of one of the species by the other, the failure of one of the species to maintain a steady population growth, or the coexistence of both species in a more balanced stage. Coexistence is def ined as the ability of each of the two species to invade an established population of the other species from rarity (Chesson, 2000). BothR.raciborskiiandM.aeruginosaare CyanoHABsforming species with similar environmental adaptation strategies, such as high growth rate, high genetic diversity, and the possession of gas vesicles to attain buoyancy, indicating that they may occupy the same niche (Gugger et al., 2005; Duan et al.,2009). However, as two distinct species, they also have their own biological characteristics besides the obvious diff erences in morphology. For example,R.raciborskiican f ix nitrogen with its heterocysts to alleviate nitrogen def iciency (Plominsky et al.,2013; Yang et al., 2018b), whereasM.aeruginosahas no heterocyst but can form colonies to gain greater buoyancy and withstand higher tolerance to a stressful environment (Mantzouki et al., 2016; Xiao et al.,2018). These biological similarities and diff erences determine their diff erent competitive potentials for dominance in the same water body.

Based on the above-mentioned f ield results and the biological similarities and diff erences, it is reasonable to speculate thatR.raciborskiican coexist withM.aeruginosa, but the consequences of such competition would depend on whether theM.aeruginosastrain is capable or incapable of producing MCs. To verify the hypothesis, a CYNproducing strain ofR.raciborskiiwas co-cultivated with either an MCs-producing or non-MCs-producing strain ofM.aeruginosaat diff erent initial biomass ratios, and the contribution of biomass from each species to the total algal biomass in the culture and the morphological changes of the two species were determined to assess their competitive potentials as well as to predict the success ofRaphidiopsisandMicrocystisblooms in natural waters.

2 MATERIAL AND METHOD

2.1 Strain and culture condition

Pure cultures of CYN-producingR.raciborskiiFACHB-3438, MCs-producingM.aeruginosaFACHB-905, and non-MCs-producingM.aeruginosaFACHB-526 were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences located in Wuhan City of China. These species were grown in 250-mL Erlenmeyer f lasks, each containing 150 mL of sterilized CT liquid medium (Watanabe and Ichimura, 1977). The cultures were incubated at 25±0.5 °C and exposed to 40 μmol photons/(m2·s) in a 12-h∶12-h light-dark cycle, as it somewhat replicates the environmental conditions of night and day.

2.2 Co-cultivation experiments

To explore the competitive potentials ofR.raciborskiiand the twoM.aeruginosastrains under conditions of unequal biomass densities, diff erent initial biomass ratios ofR.raciborskiito eitherM.aeruginosastrain were used, and these were as follows: (1) 100%R.raciborskii(100R); (2) 95%R.raciborskiiand 5%M.aeruginosa(95R∶5M); (3) 75%R.raciborskiiand 25%M.aeruginosa(75R∶25M); (4) 50%R.raciborskiiand 50%M.aeruginosa(50R∶50M); (5) 25%R.raciborskiiand 75%M.aeruginosa(25R∶75M); (6)5%R.raciborskiiand 95%M.aeruginosa(5R∶95M);and (7) 100%M.aeruginosa(100M).

The cultures containing the 95R∶5M and 5R∶95M biomass ratios were regarded as the invasive groups,based on the 95∶5 resident-to-invader ratios (Narwani et al., 2013; Gallego et al., 2019).M.aeruginosawas the invader andR.raciborskiiwas the defender in the culture with 95R∶5M ratio, while the roles were reversed in the culture with the 5R∶95M ratio. To obtain the above-mentioned co-cultures, all the strains at the logarithmic growth phase were harvested by centrifugation at 10 000×gand resuspended in fresh medium to acquire the designated biomass ratios.Both monocultures and co-cultures were prepared to have an equal initial total biomass density of 0.016 mg/mL, which correspond to 2×108, 7×108,and 6×108cells/mL, respectively, in the monoculture of CYN-producingR.raciborskii, MCs-producing and non-MCs-producingM.aeruginosa. To get the biomass densities of the three microalgae used in this study, the cell concentrations were determined by cell counting with an inverted microscope (ECLIPSE Ts2, Nikon, Tokyo, Japan) and their morphologies were recorded using a digital camera system (Nikon DS-Ri2, Tokyo, Japan). The biovolume calculation was performed according the method of Hillebrand et al. (1999) and their biomasses were estimated by assuming an average density for phytoplankton was 1 g/mL. All the cultures were prepared in 250-mL flasks, each containing 150-mL cell suspensions.Triplicate samples were prepared for each treatment and all the cultures were grown under the same temperature and light intensity as stated above.During the cultivation period, all flasks were shaken three times per day and their positions in the incubator were randomly adjusted after shaking to eliminate the possible uneven irradiance levels caused by the arrangement of their positions in the incubator. The experiment lasted for 24 days, and samples of the cultures were taken every three days.

2.3 Determination of specif ic growth rate

The sampled cell suspensions were f ixed with 1%Lugol’s iodine solution and the numbers of cells in the samples were then counted under a microscope(ECLIPSE Ts2, Nikon, Tokyo, Japan). The biovolume of each species was calculated by multiplying its abundance with its measured cell size (Hillebrand et al., 1999). The specif ic growth rate (μ) of the species was calculated using the following formula (Orr and Jones, 1998):

whereC1andC2represent the biomass concentrations(mg/mL) of CYN-producingR.raciborskiiplus MCsproducing or non-MCs-producingM.aeruginosaat timet1andt2.

2.4 Morphological examination

To explore the f inal diff erences in morphologies ofR.raciborskiif ilaments in the co-cultivation, the photographs of the f ilaments were recorded with a digital microscope camera system (Nikon DS-Ri2),and the widths of 100 f ilaments were randomly measured with the Nikon software NIS Element Ar 3.0 (Nikon, Tokyo, Japan) at the end of cultivation.The width of the vegetative cell was equal to that of the f ilament. Furthermore, the ratios of the number of f ilaments containing heterocysts to the total number of f ilaments in diff erent treatments were determined as we accidentally discovered that the heterocysts in the f ilaments were disappearing during the co-cultivation process. In addition, at the end of cultivation, only a small quantity ofR.raciborskiif ilaments was observed when the initial biomass ofR.raciborskiirelative toM.aeruginosawas less than 50%. Therefore, the ratios ofR.raciborskiif ilaments containing the heterocysts to the total f ilaments were only determined for cocultures where the initial biomass ofR.raciborskiiwas more than 50%. About 1 000 f ilaments in each culture were examined for this purpose.

2.5 Determination of total dissolved nitrogen (TN)and phosphorus (TP) in the cultures

To address the changes of the extracellular microenvironment during the cultivation, the concentrations of total dissolved nitrogen (TN) and total dissolved phosphorus (TP) were measured every three days. After sampling, the cell suspension was immediately f iltered through a 0.45- μ m cellulose acetate f ilm, and the f iltrate was used for determination of the concentrations of TN and TP by potassium persulfate oxidation and Mo-Sb antispectrophotometry.For each culture, the determination of TN and TP was carried out for three replicates.

2.6 Statistical analysis

Data were presented as mean ± standard deviation for each set of measurements. Shapiro-Wilk and Levene tests were used to assess the normality and homogeneity of variance. Diff erences between groups were analyzed byt-test or one-way ANOVA followed by Tukey’s multiple comparison test, and statistical signif icances were considered at theP＜0.05 level.Statistical analysis was performed with SPSS V.16.0(SPSS Inc., USA), and all f igures were generated using Origin 9.0 (OriginLab, USA).

3 RESULT

3.1 Eff ects of biomass ratios and incubation time on the growth of R. raciborskii and M. aeruginosa in the co-cultures

Fig.1 Changes in algal biomass for cultures with diff erent initial R. raciborskii to M. aeruginosa biomass ratios

In the monocultures (Control, 100R), the biomass densities ofR.raciborskiiincreased with prolonged incubation (Fig.1a-b). However, whenR.raciborskiiwas co-cultivated withM.aeruginosa,its density signif icantly decreased at all tested initialR.raciborskiitoM.aeruginosabiomass ratios. In addition, the inhibition ofR.raciborskiigrowth was stronger when it was co-cultivated with the MCsproducingM.aeruginosastrain compared with the non-MCs-producingM.aeruginosastrain. At the end of the cultivation, the densities ofR.raciborskiiwere 0.25, 0.23, 0.09, 0.07, and 0.01 mg/mL in the co-cultures where the initial biomass ratios ofR.raciborskiito the MCs-producingM.aeruginosastrain were 95R∶5M, 75R∶25M, 50R∶50M,25R∶75M, and 5R∶95M, respectively (Fig.1a). The corresponding densities ofR.raciborskiiin the cases of the co-cultures containing the non-MCs-producingM.aeruginosastrain at the same initial biomass ratios were 0.73, 0.31, 0.16, 0.10, and 0.02 mg/mL (Fig.1b).

ForM.aeruginosa, the biomass density increased with prolonged cultivation, and when higher initial biomass relative toR.raciborskiiwas used at the time of inoculation, higher biomass was also achieved at the end of the co-cultivation (Fig.1c-d). Furthermore,at the sameR.raciborskiitoM.aeruginosabiomass ratios, the MCs-producingM.aeruginosastrain grew better than the non-MCs-producing one (Fig.1c-d).At the end of the cultivation, the densities of the MCsproducingM.aeruginosastrain co-cultivated withR.raciborskiiat initial biomass ratios of 95R∶5M,75R∶25M, 50R∶50M, 25R∶75M, and 5R∶95M increased to 1.68, 1.75, 2.00, 2.20 and 2.38 mg/mL,respectively, from an initial density of 0.016 mg/mL(Fig.1c). For the sameR.raciborskiitoM.aeruginosabiomass ratios, the corresponding densities of the non-MCs-producingM.aeruginosastrain reached 0.56,0.96, 1.75, 1.84, and 1.88 mg/mL at the end of the co-cultivation (Fig.1d). Thus, regardless of the initial biomass ratios ofR.raciborskiitoM.aeruginosa(both MCs-producing and non-MCs-producing strains), the biomass ofM.aeruginosarelative to that ofR.raciborskiiexhibited an increasing trend at the end of the co-cultivation. This suggested the ability ofM.aeruginosato proliferate out-competedR.raciborskiiin the co-culture system.

Fig.2 Specific growth rates of R. raciborskii and M. aeruginosa co-cultivated at diff erent initial biomass ratios

The average specif ic growth rate ofR.raciborskiiin the co-cultures was suppressed by both strains ofM.aeruginosa(Fig.2a-b). The inhibition ofR.raciborskiigrowth exerted by the MCs-producingM.aeruginosastrain was stronger than that exerted by the non-MCs-producing strain at all initial biomass ratios tested (Fig.2a). At the end of the cultivation,the average specific growth rate ofR.raciborskiiin the monoculture was 0.22/d, but the specif ic growth rates ofR.raciborskiiin the co-cultures with initialR.raciborskiito MCs-producingM.aeruginosabiomass ratios of 95R∶5M, 75R∶25M, 50R∶50M,25R∶75M and 5R∶95M decreased to 0.12, 0.12, 0.10,0.12, and 0.10/d, respectively (Fig.2a). Similarly, a decrease in the specif ic growth rate ofR.raciborskiiwas also evident when it was co-cultured with the non-MCs-producingM.aeruginosastrain, reaching 0.16,0.13, 0.12, 0.13, and 0.13/d, respectively when the biomass ratios were 95R∶5M, 75R∶25M, 50R∶50M,25R∶75M, and 5R∶95M (Fig.2a). However, the average specif ic growth rates of the MCs-producing and non-MCs-producingM.aeruginosastrains in the cocultures were signif icantly (P＜0.01) higher than those in the monocultures (Fig.2b). The specif ic growth rates ofM.aeruginosastrain at initial biomass ratios of 95R∶5M, 75R∶25M, 50R∶50M, 25R∶75M and 5R∶95M were 0.32, 0.25, 0.23, 0.22, and 0.21/d, respectively,in the case of the MCs-producing strain, and were 0.27, 0.23, 0.22, 0.21, and 0.20/d, respectively, for the non-MCs-producing strain (Fig.2b).

3.2 Contribution of each algal biomass to the total biomass during co-cultivation

Fig.3 Contribution of R. raciborskii and the two M.aeruginosa strains to the total biomass in the cocultures at diff erent initial biomass ratios and incubation times

Fig.4 Morphological characteristics of the f ilaments of R. raciborskii co-cultivated with M. aeruginosa

To visually show the dominating competitiveness of the microalgae during the co-cultivation, the contribution of each algal biomass to the total biomass during the entire co-cultivation period was determined. Overall, the contribution ofR.raciborskiito the total biomass was positively correlated with its initial biomass in the culture, but such contribution decreased with prolonged cultivation time, with the decrease occurring at a faster rate when the co-cultivatedM.aeruginosastrain was the MCsproducing strain (Fig.3a-e). Correspondingly, the contribution ofM.aeruginosato the total biomass also increased with prolonged cultivation time and the contribution of the MCs-producing strain exceeded that of the non-MCs-producing strain. At the end of the cultivation, the biomass contribution of eitherM.aeruginosastrain was signif icantly higher(P＜ 0.01) than that ofR.raciborskii, except in the culture where the initial biomass ofR.raciborskiiwas 95% of the total initial biomass and thatR.raciborskiiwas co-cultured with the non-MCs-producing strain ofM.aeruginosa(Fig.3a). For the co-cultures with initial biomass ratios of 95R∶5M, 75R∶25M,50R∶50M, 25R∶75M, and 5R∶95M, the biomass contribution ofR.raciborskiidecreased by 84.94%,88.36%, 95.49%, 96.90%, and 99.97%, respectively,when MCs-producingM.aeruginosawas the cocultured strain, or by 43.39%, 75.76%, 91.85%,95.09%, and 99.91%, respectively, when non-MCsproducingM.aeruginosawas the co-cultured strain(Fig.3a-e). The results suggested that loss in biomass contribution byR.raciborskiiwas less severe when it was co-cultured with the non-MCs-producingM.aeruginosastrain, and in cultures with higher initialR.raciboskiibiomass than those with higher initialM.aeruginosabiomass.

3.3 Morphological changes of R. raciborskii cocultured with M. aeruginosa

The morphological changes inR.raciborskiiandM.aeruginosathat have been co-cultivated for 24 days are shown in Fig.4. Changes in the morphological parameters ofM.aeruginosawere ignored in the present study because no visible morphological changes were found. For the monoculture ofR.raciborskii,the f ilaments contained heterocysts (Fig.4a & g),but an obvious loss of heterocysts in the f ilaments occurred when the strain was co-cultured with the MCs-producing strain ofM.aeruginosa(Fig.4b-f),and the percent of heterocyst-containing f ilaments also decreased with increased initial biomass ratios of MCs-producingM.aeruginosatoR.raciborskii(Fig.5a). The loss of heterocyst inR.raciborskiif ilaments also occurred when the strain was cocultured with the non-MCs-producingM.aeruginosa strain (Fig.4h-l), but the loss occurred at a lower rate (Fig.5b). For the mono-culturedR.raciborskii,the heterocyst-containing f ilaments accounted for 97.36% of the total number of f ilaments. However,whenR.raciborskiiwas co-cultivated with the MCsproducingM.aeruginosastrain at initial biomass ratios of 95R∶5M, 75R∶25M, and 50R∶50M, the percentage of heterocyst-containing f ilaments decreased to 37.13%, 25.75% and 18.30%, respectively (Fig.5a).As forR.raciborskiico-cultivated with the non-MCsproducingM.aeruginosa, this percentage decreased to 40.17%, 26.40%, and 19.77%, respectively (Fig.5b).

Fig.5 Percentage of heterocyst-containing R. raciborskii f ilaments co-cultivated with M. aeruginosa

In addition to the changes in the heterocysts of the f ilaments, the average length and width of the f ilaments ofR.raciborskiico-cultivated with the MCs-producingM.aeruginosastrain also decreased signif icantly (P＜0.05) when the initial biomass ratio was 95R∶5M (Fig.6a-b). In contrast, no signif icant diff erence in the average length and width of the f ilaments were detected whenR.raciborskiiwas co-cultivated with the non-MCs-producingM.aeruginosastrain regardless of the initial biomass ratios of the cultures and incubation time.

3.4 Changes in TN and TP during the cultivation period

Fig.6 Changes in f ilament length (a) and width (b) of R. raciborskii co-cultivated with M. aeruginosa at diff erent initial biomass ratios for 24 days

Changes in TN and TP in the media of bothR.raciborskiimonoculture andR.raciborskii-M.aeruginosaco-culture showed a gradually decreasing trend with prolonged incubation (Fig.7),but the rate of decrease for TN was slower in the case of the monoculture (Fig.7a-b). In addition, the rates of decrease for TN were similar in the co-cultures containing either the MCs-producing or non-MCsproducingM.aeruginosastrain with no signif icant diff erence between the two cultures regardless of incubation time. As for TP, its concentration in both the monoculture and co-culture decreased sharply from the point of inoculation to Day 9, followed by a more gradual decrease that also f luctuated slightly among the diff erent set of cultures (Fig.7c-d).Unlike the change in TN, the TP concentration in bothR.raciborskiimonoculture andR.raciborskii-M.aeruginosaco-culture displayed almost identical trends, with no signif icant diff erence between the two cultures regardless of incubation time and the initial biomass ratios in the case of the co-culture (Fig.7c-d).

4 DISCUSSION

Fig.7 Changes in total soluble nitrogen (TN) and total soluble phosphorus (TP) concentrations in R. raciborskii- M. aeruginosa co-cultures at diff erent initial biomass ratios and incubation times

Co-cultivation is considered to be a good method for studying the allelopathic interaction between two diff erent phytoplankton species because direct evidence for allelopathic interactions under natural aquatic conditions is diffi cult to provide owing to the interferences from other processes (Sukenik et al., 2002; Legrand et al., 2003; Ma et al., 2015;Dunker et al., 2017; Chia et al., 2018). Through cocultivation, we have shown in the present study that the growth rates ofR.raciborskiicould be inhibited by both MCs-producing and non-MCs-producingM.aeruginosastrains (Figs.1a-b & 2a). In addition,the inhibition ofR.raciborskiigrowth exerted by the MCs-producingM.aeruginosastrain was greater than that exerted by the non-MCs-producing one (Figs.1a,b, & 2a). This is in accordance with the reported allelopathic eff ects ofM.aeruginosaon several other species of green algae and dinof lagellates (Sukenik et al., 2002; Bittencourt-Oliveira et al., 2015; Ma et al., 2015; Yang et al., 2018a; Omidi et al., 2019).MCs-producingM.aeruginosastrains can suppress the growth and photosynthesis of their competitors by producing MCs as well as other odor substances(Pietsch et al., 2001; Singh et al., 2001; Pf lugmacher,2002; Hu et al., 2004; Babica et al., 2007; Wang et al., 2017), which give them an advantage in such competition (Ma et al., 2015; Wang et al., 2017).

MCs are allelochemicals that can induce suppression of the growth of cyanobacteria and eukaryotic algae (Sukenik et al., 2002; Qian et al.,2009; Tan et al., 2019). The mechanism of growth suppression is rather diverse, and it includes reducing carbon dioxide f ixation and nitrogen fixation activities(Singh et al., 2001), obstructing electron transport by specif ically binding to relevant sites in photosystem II (PSII) (Keating, 1999), hindering adenosine triphosphate (ATP) synthesis (Zhu et al., 2010),abolishing intracellular carbonic anhydrase activity(Sukenik et al., 2002), and causing oxidative damage(Qian et al., 2009). Besides the negative allelopathy, a recent study has found thatM.aeruginosacan inhibit the growth ofChlorellavulgarisvia the release of linoleic acid (Song et al., 2017).

However, forM.aeruginosastrains, co-cultivation withR.raciborskiistimulated their growth rates and the stimulating eff ects decreased with increased proportions of initial biomass ofR.raciborskiiin the co-cultures (Figs.1c-d & 2b). In addition, under such co-cultivation, the toxicM.aeruginosastrain achieved a higher growth rate than the non-toxic one (Fig.2b).This is also supported by other previous experimental studies. For example, environmentally relevant concentrations of CYN were found to have no eff ect on the growth of the cyanobacteriaNannochloropsissp. andChlamydomonasreinhardti(Pinheiro et al., 2013), whereas CYN was found to stimulate the growth of the green algaC.vulgarisdespite its relatively high concentration, up to 179 μg/L (Campos et al., 2013). Taken together, these results may suggest thatM.aeruginosa, similar to other microalgae, can cope with CYN-induced oxidative stress through increasing the activity of antioxidant enzymes(Campos et al., 2013). Furthermore, the biomass contribution of the MCs-producing or non-MCsproducingM.aeruginosastrain to the total biomass increased regardless of the initialR.raciborskiitoM.aeruginosabiomass ratios, and a ratio of 5R∶95M resulted in the complete elimination ofR.raciborskiifrom the co-culture on the 9thday and 21thday for the MCs-producing and non-MCs-producingM.aeruginosastrains, respectively (Fig.3e).According to the trend of biomass contribution by each algal strain to the total biomass during the entire co-cultivation period (Fig.3a-e), it could be inferred that with further incubation time, both the MCsproducing and non-MCs-producingM.aeruginosastrains could eventually excludeR.raciborskiicompletely regardless of the initial biomass ratios, but the non-MCs-producing strain would need a longer incubation to completely replaceR.raciborskii.

Raphidiopsisspecies have been reported to exert stronger inhibitory eff ects than pure CYN on the growth of green algae and cyanobacteria (Campos et al., 2013; Rzymski et al., 2014).RaphidiopsisraciborskiiFACHB-3438 is a CYN-producing species(Jiang et al., 2014). However, the higher growth rates of both MCs-producing and non-MCs-producingM.aeruginosastrains observed when they were cocultivated withR.raciborskiiFACHB-3438 (Fig.2b)suggested that CYN could stimulate the growth ofM.aeruginosa. Furthermore, it could also mean that the MCs-producing species are more likely to gain a competitive advantage over their competitors than the CYN-producing species.

Under natural conditions, most of theR.raciborskiif ilaments contain heterocysts needed for nitrogen f ixation, a function that gives this alga an advantage in the competition with non-nitrogen-f ixing algae,especially in N-def icient environments (Moisander et al., 2012; Yang et al., 2017). In the present study, the formation of heterocysts inR.raciborskiif ilaments was inhibited byM.aeruginosa(Figs.4-5), and the inhibitory eff ect increased with an increased proportion ofM.aeruginosabiomass in the coculture. In addition, co-cultivation with the MCsproducingM.aeruginosastrain led to a greater loss of heterocysts than co-cultivation with the non-MCs-producing one (Fig.5a-b). To our knowledge,this is the f irst report to mention the disappearance of heterocysts in cyanobacterial f ilaments caused by allelopathy although the physiological mechanism is unclear. A previous study has reported thatM.aeruginosacan exert an inhibitory eff ect on the diff erentiation of heterocysts in the f ilamentous cyanobacteriumTrichormusvariabilisthrough an unknown mechanism (Bártová et al., 2011).In addition, co-culture withM.aeruginosacan also result in the growth reduction ofPlanktothrix(Briand et al., 2019). In either case, the molecular mechanism responsible for the eff ect has not been clarif ied. However, the disappearance of heterocysts in theR.raciborskiif ilaments and the morphological changes observed for the f ilaments could be an adaptation feature resulting from the pressure brought by the presence ofM.aeruginosain the culture. This could increase the biotic stress forR.raciborskii,although the direct eff ects on the competition for dominance betweenR.raciborskiiandM.aeruginosaremain unclear. Therefore, a further study involving the use of multiple environmental factors and multiple technical methods should be carried out to reveal the precise eff ects of the extracellular microenvironment on the morphological changes ofR.raciborskiif ilaments and the underlying molecular mechanisms.

The competition for dominance between the two algae is aff ected by many factors, such as temperature(Ma et al., 2015; Yang et al., 2018a), nutrient levels(Marinho et al., 2013; Chia et al., 2018), algae species even strains of the competitors (Sukenik et al., 2002;Ma et al., 2015; Wang et al., 2017). For example,in the co-cultivation ofScendesmusobliquuswithM.aeruginosa,S.obliquusis more competitive at 15 °C, the two species are equally competitive at 20-30 °C, butM.aeruginosais more competitive at 35 °C (Yang et al., 2018a). Furthermore, studies have shown thatR.raciborskiiis able to utilize a range of nitrogen sources such as ammonium, nitrate, and urea with a clear preference for ammonium (Burford et al., 2006; Stucken et al., 2014), thus appears to gain competitive advantage under low and f luctuating dissolved nitrogen concentrations, in line with the term“facultative diazotroph” coined by Moisander et al.(2012). In addition,R.raciborskiihas high dissolved inorganic phosphorus uptake rates and storage capacity relative to a range of other cyanobacterial species (Isvánovics et al., 2000; Prentice et al., 2015).Consistent with this,R.raciborskiican dominate in a range of phosphorus levels (Chislock et al., 2014). In this study, the changes in the concentrations of TN and TP in the co-cultures displayed a similar trend without any signif icant diff erences among the diff erent sets of cultures regardless of the initial biomass ratios and incubation time (Fig.7a-d). This may be due to the demand for nitrogen and phosphorus by the two algae being completely satisf ied throughout the incubation period. Therefore, the diff erent eff ects that the MCsproducing and non-MCs-producingM.aeruginosastrains had onR.raciborskiias shown in the present study, were unlikely to be caused by variations in nutrients in co-cultivations.

5 CONCLUSION

In the co-cultivation ofR.raciborskiiwithM.aeruginosaat diff erent biomass ratios, the growth ofR.raciborskiiwas inhibited by both MCsproducing and non-MCs-producingM.aeruginosastrains, with the former exerting a stronger inhibition than the latter. On the other hand, co-cultivation ofM.aeruginosawithR.raciborskiistimulated the growth of theM.aeruginosa. Therefore, in the cocultivation,R.raciborskiicould be completely eliminated byM.aeruginosaeven whenR.raciborskiiwas present in dominating proportion at the start of the cultivation (R∶M=95∶5 in biomass ratio), and during the cultivation, the MCs-producingM.aeruginosastrain took less time to replaceR.raciborskiithan the non-MCs-producing strain. Furthermore, the co-cultivation ofR.raciborskiiwithM.aeruginosaled to the disappearance of heterocysts containing in f ilaments ofR.raciborskii. Compared with the non-MCs-producingM.aeruginosastrain, the MCsproducing strain exerted a more potent inhibitory eff ect onR.raciborskii, thereby, enabling it to acquire stronger competitiveness for dominance These results indicated that it is diffi cult forR.raciborskiito replace the dominant position ofM.aeruginosain the same natural water if based purely on the allelopathy between the two species. This study has provided important evidence pertaining to the competition for dominance betweenR.raciborskiiandM.aeruginosa, and essentially ruled out the possibility thatR.raciborskiicould replace the dominant position ofM.aeruginosain natural waters.

6 DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGMENT

The authors highly appreciate Dr Alan K. Chang(Wenzhou University) for his eff ort in the revision of the manuscript’s language, and the anonymous reviewers for their very useful and insightful comments.