# Freshwater Salinization Impacts the Interspecific Competition between Microcystis and Scenedesmus

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Microalgae and Salinity Adaptation

^{−2}s

^{−1}. Then algae were inoculated in fresh culture media every 3 days to maintain an exponential growth phase prior to the experiment. Two algae were cultured in 1 L Erlenmeyer flasks containing 500 mL of BG-11 medium. The salinity of the algal culture was raised every two days at an interval of one until it reached six. The measured salinities of the three target salinities (0, 3, 6) were 0.99, 3.97 and 6.98, respectively. No difference was found between different mono- or cocultures.

#### 2.2. Experimental Protocol

^{−1}NaNO

_{3}and 0.039 mg L

^{−1}KH

_{2}PO

_{4}. Three salinities were set to simulate freshwater salinization: zero, three, and six, according to the predictive results of salinity levels and trends across and within the seven regional river basins [30]. Algal were cultured semi-continuously in different salt conditions (0, 3, 6) to remain in exponential growth prior to the experiments, and then exponential-phase cells were inoculated into Erlenmeyer flasks at different initial algal densities.

^{5}cells mL

^{−1}and S. obliquus was 1.0 × 10

^{5}cells mL

^{−1}. Three cocultures with a uniform initial total biovolume were set with the following biovolume ratios, and initial cell densities were set as below: (1) 75% Ma + 25% So: M. aeruginosa was 3.75 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.25 × 10

^{5}cells mL

^{−1}; (2) 50% Ma + 50% So: M. aeruginosa was 2.5 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.5 × 10

^{5}cells mL

^{−1}; (3) 25% Ma + 75% So: M. aeruginosa was 1 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.75 × 10

^{5}cells mL

^{−1}. Both monoculture and coculture groups were incubated in climate-controlled chambers in 250 mL Erlenmeyer flasks at three different salinities (0, 3, and 6). The experiments were performed in triplicate. All the cultures were grown axenically in a climate-controlled chamber at a constant temperature of 20 °C, illuminated with a 14:10 h light:dark cycle at 60 μmol photons m

^{−2}s

^{−1}and shaken manually three times daily.

#### 2.3. Data Analyses

^{−1}) was assumed to be exponential and determined as the slope of ln cell density vs time. One-way ANOVA was used to compare the differences in growth rates at different salinities of the two algal monocultures. In the cocultures, the algal cell density versus time was fitted by the Gaussian distribution ${N}_{t}={N}_{max}\times {e}^{(-0.5\times {(\frac{t-{t}_{max}}{SD})}^{2})}$, where N

_{0}, N

_{t}and N

_{max}represent the algal abundance at time zero and t, and the maximal number of algal abundances, t and t

_{max}represent the cultural time and the time when reached N

_{max}, and SD was the width of the Gaussian distribution. In cocultures, the natural log of the ratio of the abundances of the two algae, $Y\left(t\right)=ln(\frac{{N}_{Microcystis}}{{N}_{Scnedesmus}})$, were calculated and regressed against time. The slope of the linear regression of Y(t) versus t is adopted as the competitive displacement rate. To reflect the changes of Y(t) with time, they were fitted by the Gaussian distribution: $Y\left(t\right)={Y}_{max}\times {e}^{(-0.5\times {(\frac{t-{{t}^{\prime}}_{max}}{{SD}^{\prime}})}^{2})}+{Y}_{0}$, where Y

_{0}, Y

_{max}represent the initial Y(t), Y(t) at time t, and the maximal value of Y(t), t and t′

_{max}represent the cultural time and the time when reached Y

_{max}, and SD′ was the width of the Gaussian distribution.

_{i}is the number of particles in different morphs.

#### 2.4. Chlorophyll Fluorescence Measurements

_{v}/F

_{m}$=\frac{{F}_{m}-{F}_{0}}{{F}_{m}}$, where F

_{m}and F

_{0}are the maximal and minimal chlorophyll fluorescence yields, respectively, of dark-adapted (15 min) algal suspensions. Three-way ANOVA tests were run to analyze the effects of salinity, initial algal composition, and incubation time on F

_{v}/F

_{m}of both algae, and Holm-Sidak tests were adopted for all multiple pairwise comparisons.

## 3. Results

#### 3.1. Response of Algae Growth and Competition to Salinity Stress

_{(2,6)}= 71.37, p < 0.0001) and S. obliquus (F

_{(2,6)}= 7.715, p = 0.0219). The algal population growth rate of M. aeruginosa significantly decreased with increasing salinity (Figure 1a), and the growth rate of S. obliquus was significantly lower under salinity stress but had no significant difference between the two salinity groups (3, 6) (Figure 1b).

_{max}) was slightly advanced under higher salinity conditions (Table 1). The results of two-way ANOVA tests showed a significant effect of salinity on N

_{max}and t

_{max}of S. obliquus, and t

_{max}of M. aeruginosa. The initial composition of two algal species had no effect on neither N

_{max}nor t

_{max}of the two algae. Furthermore, there was no significant interaction between salinity and initial composition (Table 2). In the treatments with an initial cell density ratio of 75% Ma + 25% So, M. aeruginosa occupied dominance in the early several days. The competitive advantage of M. aeruginosa was maintained for 6.546, 5.208, and 3.989 days, respectively, in zero, three, and six (Figure 2a,d,g). When the initial density proportion of M. aeruginosa decreased to 50%, the competitive advantage could only maintain for 2.950, 3.547 and 3.289 days, respectively, in zero, three, and six (Figure 2b,e,h). However, in treatments with an initial ratio of 25% Ma + 75% So, S. obliquus occupied dominance from second day (Figure 2c,f,i).

#### 3.2. Morphological Variations of S. obliquus

#### 3.3. Photosynthetic Performance of Both Algae in Cocultures

_{v}/F

_{m}) was significantly affected by the increase in salinity, the presence of competitors, and the incubation time for both M. aeruginosa and S. obliquus (Figure 5, Table 3). Additionally, there were statistically significant interactions on F

_{v}/F

_{m}of both algae between time, salinity, and initial proportion of algae. For M. aeruginosa, F

_{v}/F

_{m}differed significantly between the treatments of zero and three (p = 0.016), also zero and six (p = 0.006), but not significantly between the two high salinity treatments (p = 0.635). F

_{v}/F

_{m}increased with time when cultured at zero (Figure 5a), when at three and six, F

_{v}/F

_{m}increased more rapidly than at zero (Figure 5b,c). Interestingly, the treatment of 75% Ma + 25% So had the lowest F

_{v}/F

_{m}value in zero and six (Figure 5a,c). For S. obliquus (Figure 5d–f), similar to M. aeruginosa, F

_{v}/F

_{m}differed significantly between the treatments of zero and three (p < 0.001), also zero and six (p < 0.001), but not significantly between the two high salinity treatments (p = 0.230). The initial algal proportion affected F

_{v}/F

_{m}differently with M. aeruginosa: monoculture (100% So) differed significantly with all three cocultures, whereas F

_{v}/F

_{m}of the cocultures had no significant difference. Especially in the treatment of six, F

_{v}/F

_{m}was higher in monoculture than in cocultures (Figure 5f).

## 4. Discussion

^{+}extrusion, accumulation and synthesis of certain solutes, and metabolic modifications like synthesizing compatible solutes, such as sugars, amino acids, and fats, which act as osmoprotectants. The differences in interspecies sensitivity to salinity were likely due to the inherent morphological and physiological aspects of each species. S. obliquus can form colonies through the attachment of daughter cells during cell division and can produce a mucilage envelope, which serves as a protective mechanism against environmental stressors, such as salinity and heavy metals toxicity [32,33]. Zhu et al. [34] reported that the presence of M. aeruginosa affected the formation of morphological defence against grazers in S. obliquus. This suggested that competition can influence the ability of organisms to defend themselves against stressors. Nevertheless, the formation of cell colonies can relieve the stress caused by elevated salinity levels, although this mechanism of defence may have an impact on algal growth, such as reducing the surface-to-volume ratio and intensifying nutrient competition among cells. Small-sized unicells have a relatively high surface-to-volume ratio, which facilitates nutrient uptake and utilization of light [35]. M. aeruginosa has been found to have weak osmotic regulating abilities because of its limited ability to synthesize compatible solutes. Even decreases in microcystin production in M. aeruginosa under high osmotic conditions have been reported in former studies [36,37], but contrary change has been found in Planktothrix agardhii [38]. Further, the relatively thin cell wall of M. aeruginosa makes it susceptible to osmotic stress. In natural waters, M. aeruginosa can often form large colonies with a mucilage sheath, which could act as a hydrated matrix and maintain a more stable osmotic environment within the colonies [39]. In our study, M. aeruginosa maintained unicellular or bicellular morphs; no mucilage sheath was synthesized during the experiment, which might cause less tolerance to high salinity.

_{v}/F

_{m}, especially in the treatment of six. The influence of competition on photosynthetic efficiency has also been documented, Sun et al. [47] found that ultraviolet-B (UV-B) radiation stress changed the competitive outcome of algae and that this change was related to changes in photosynthetic efficiency. Light limitation from the shading effect of the coexisting algae might be responsible for the reduction of F

_{v}/F

_{m}[48]. Additionally, allelopathic substances secreted by competitors could also affect photosynthesis. Hernández-Zamora et al. [49] reported the diminishment of chlorophyll-a, b of M. aeruginosa and two green algae in combined cultures. The allelopathic activity of cyanobacteria on other microalgae can vary, Zak et al. [50] found that while Anabaena variabilis had a strong inhibitory effect on C. vulgaris, Nodularia spumigena mostly stimulated its growth. Further, Different species of Scenedesmus have been found to restrict the growth of cyanobacteria through various means of exposure, including the use of conditioned water and crude extracts [51,52].

## 5. Conclusions

## Author Contributions

## Funding

## Data Availability Statement

## Conflicts of Interest

## References

- Kaushal, S.S.; Likens, G.E.; Pace, M.L.; Utz, R.M.; Haq, S.; Gorman, J.; Grese, M. Freshwater salinization syndrome on a continental scale. Proc. Natl. Acad. Sci. USA
**2018**, 115, E574–E583. [Google Scholar] [CrossRef][Green Version] - Castillo, A.M.; Sharpe, D.M.; Ghalambor, C.K.; De León, L.F. Exploring the effects of salinization on trophic diversity in freshwater ecosystems: A quantitative review. Hydrobiologia
**2018**, 807, 1–17. [Google Scholar] [CrossRef] - Metz, B.; Davidson, O.; Bosch, P.; Dave, R.; Meyer, L. Climate Change 2007: Mitigation of Climate Change; Intergovernmental Panel on Climate Change: Geneva, Switzerland, 2007. [Google Scholar]
- Metz, B.; Davidson, O.R.; Bosch, P.R.; Dave, R.; Meyer, L.A. (Eds.) Contribution of Working Group III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change; Cambridge University Press: Cambridge, UK, 2007; ISBN 9780521880114. [Google Scholar]
- Vidal, N.; Yu, J.; Gutierrez, M.F.; de Mello, F.T.; Tavşanoğlu, Ü.N.; Çakiroglu, A.I.; He, H.; Meerhoff, M.; Brucet, S.; Liu, Z.W.; et al. Salinity shapes food webs of lakes in semiarid climate zones: A stable isotope approach. Inland Waters
**2021**, 11, 476–491. [Google Scholar] [CrossRef] - El Hamidi, L.; Larabi, A.; Faouzi, M. Numerical modeling of saltwater intrusion in the rmel-oulad ogbane coastal aquifer (Larache, Morocco) in the climate change and sea-level rise context (2040). Water
**2021**, 13, 2167. [Google Scholar] [CrossRef] - El Shinawi, A.; Kuriqi, A.; Zelenakova, M.; Vranayova, Z.; Abd-Elaty, I. Land subsidence and environmental threats in coastal aquifers under sea level rise and over-pumping stress. J. Hydrol.
**2022**, 608, 127607. [Google Scholar] [CrossRef] - Cunillera-Montcusí, D.; Beklioğlu, M.; Cañedo-Argüelles, M.; Jeppesen, E.; Ptacnik, R.; Amorim, C.A.; Arnott, S.E.; Berger, S.A.; Brucet, S.; Dugan, H.A.; et al. Freshwater salinisation: A research agenda for a saltier world. Trends Ecol. Evol.
**2022**, 37, 440–453. [Google Scholar] [CrossRef] - Talebi, A.F.; Tabatabaei, M.; Mohtashami, S.K.; Tohidfar, M.; Moradi, F. Comparative salt stress study on intracellular ion concentration in marine and salt-adapted freshwater strains of microalgae. Not. Sci. Biol.
**2013**, 5, 309–315. [Google Scholar] [CrossRef][Green Version] - Demetriou, G.; Neonaki, C.; Navakoudis, E.; Kotzabasis, K. Salt stress impact on the molecular structure and function of the photosynthetic apparatus—The protective role of polyamines. BBA-Bioenerg.
**2007**, 1767, 272–280. [Google Scholar] [CrossRef][Green Version] - Christensen, I.; Pedersen, L.K.; Søndergaard, M.; Lauridsen, T.L.; Tserenpil, S.; Richardson, K.; Amorim, C.A.; Pacheco, J.P.; Jeppesen, E. Impact of zooplankton grazing on phytoplankton in north temperate coastal lakes: Changes along gradients in salinity and nutrients. Hydrobiologia
**2022**. [Google Scholar] [CrossRef] - He, H.; Jeppesen, E.; Bruhn, D.; Yde, M.; Hansen, K.J.; Spanggaard, L.; Madsen, N.; Liu, W.; Søndergaard, M.; Lauridsen, T.L. Decadal changes in zooplankton biomass, composition and body mass in four temperate shallow brackish lakes subjected to various degrees of eutrophication. Inland Waters
**2020**, 10, 186–196. [Google Scholar] [CrossRef] - Jensen, E.; Brucet, S.; Meerhoff, M.; Nathansen, L.; Jeppesen, E. Community structure and diel migration of zooplankton in brackish lakes: Role of salinity and predators. Hydrobiologia
**2010**, 646, 215–229. [Google Scholar] [CrossRef] - Szöcs, E.; Coring, E.; Bäthe, J.; Schäfer, R.B. Effects of anthropogenic salinization on biological traits and community composition of stream macroinvertebrates. Sci. Total Environ.
**2014**, 468, 943–949. [Google Scholar] [CrossRef] [PubMed] - Li, T.; Liu, G.; Yuan, H.; Chen, J.; Lin, X.; Li, H.; Yu, L.; Wang, C.; Li, L.; Zhuang, Y.; et al. Eukaryotic plankton community assembly and influencing factors between continental shelf and slope sites in the northern South China Sea. Environ. Res.
**2023**, 216, 114584. [Google Scholar] [CrossRef] [PubMed] - Omidi, A.; Pflugmacher, S.; Kaplan, A.; Kim, Y.J.; Esterhuizen, M. Reviewing interspecies interactions as a driving force affecting the community structure in lakes via cyanotoxins. Microorganisms
**2021**, 9, 1583. [Google Scholar] [CrossRef] [PubMed] - Borics, G.; Abonyi, A.; Salmaso, N.; Ptacnik, R. Freshwater phytoplankton diversity: Models, drivers and implications for ecosystem properties. Hydrobiologia
**2021**, 848, 53–75. [Google Scholar] [CrossRef] - Hutchinson, G. The paradox of the plankton. Am. Nat.
**1961**, 95, 137–145. [Google Scholar] [CrossRef][Green Version] - Roy, S.; Chattopadhyay, J. Towards a resolution of ‘the paradox of the plankton’: A brief overview of the proposed mechanisms. Ecol. Complex.
**2007**, 4, 26–33. [Google Scholar] [CrossRef] - Suttle, C.A.; Chan, A.M.; Cottrell, M.T. Infection of phytoplankton by viruses and reduction of primary productivity. Nature
**1990**, 347, 467–469. [Google Scholar] [CrossRef] - Pal, M.; Yesankar, P.J.; Dwivedi, A.; Qureshi, A. Biotic control of harmful algal blooms (HABs): A brief review. J. Environ. Manag.
**2020**, 268, 110687. [Google Scholar] [CrossRef] - Dawson, R.M. The toxicology of microcystins. Toxicon
**1998**, 36, 953–962. [Google Scholar] [CrossRef] - Tonk, L.; Bosch, K.; Visser, P.M.; Huisman, J. Salt tolerance of the harmful cyanobacterium Microcystis aeruginosa. Aquat. Microb. Ecol.
**2007**, 46, 117–123. [Google Scholar] [CrossRef] - Preece, E.P.; Hardy, F.J.; Moore, B.C.; Bryan, M. A review of microcystin detections in Estuarine and Marine waters: Environmental implications and human health risk. Harmful Algae
**2017**, 61, 31–45. [Google Scholar] [CrossRef][Green Version] - Tanabe, Y.; Hodoki, Y.; Sano, T.; Tada, K.; Watanabe, M.M. Adaptation of the freshwater bloom-forming cyanobacterium Microcystis aeruginosa to brackish water is driven by recent horizontal transfer of sucrose genes. Front. Microbiol.
**2018**, 9, 1150. [Google Scholar] [CrossRef][Green Version] - Takeya, K.; Kuwata, A.; Yoshida, M.; Miyazaki, T. 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.
**2004**, 26, 29–35. [Google Scholar] [CrossRef] - Ji, X.; Verspagen, J.M.; Stomp, M.; Huisman, J. Competition between cyanobacteria and green algae at low versus elevated CO
_{2}: Who will win, and why? J. Exp. Bot.**2017**, 68, 3815–3828. [Google Scholar] [CrossRef][Green Version] - Beardall, J.; Raven, J.A. Cyanobacteria vs green algae: Which group has the edge? Environ. Dev. Sustain.
**2017**, 68, 3697–3699. [Google Scholar] [CrossRef][Green Version] - Ray, J.G.; Santhakumaran, P.; Kookal, S. Phytoplankton communities of eutrophic freshwater bodies (Kerala, India) in relation to the physicochemical water quality parameters. Environ. Dev. Sustain.
**2021**, 23, 259–290. [Google Scholar] [CrossRef] - Thorslund, J.; Bierkens, M.F.P.; Oude Essink, G.H.P.; Sutanudjaja, E.H.; van Vliet, M.T.H. Common irrigation drivers of freshwater salinisation in river basins worldwide. Nat. Commun.
**2021**, 12, 4232. [Google Scholar] [CrossRef] - Brown, A.F.M.; Dortch, Q.; Van Dolah, F.M.; Leighfield, T.A.; Morrison, W.; Thessen, A.E.; Steidinger, K.; Richardson, B.; Moncreiff, C.A.; Pennock, J.R. Effect of salinity on the distribution, growth, and toxicity of Karenia spp. Harmful Algae
**2006**, 5, 199–212. [Google Scholar] [CrossRef] - Zhu, X.; Wang, Y.; Hou, X.; Kong, Q.; Sun, Y.; Wang, J.; Huang, Y.; Yang, Z. High temperature promotes the inhibition effect of Zn
^{2+}on inducible defense of Scenedesmus obliquus. Chemosphere**2019**, 216, 203–212. [Google Scholar] [CrossRef] - Jia, X.; Pan, Y.; Zhu, X. Salinization and heavy metal cadmium impair growth but have contrasting effects on defensive colony formation of Scenedesmus obliquus. Sci. Total Environ.
**2023**, 862, 160693. [Google Scholar] [CrossRef] [PubMed] - Zhu, X.; Wang, J.; Lu, Y.; Chen, Q.; Yang, Z. Grazer-induced morphological defense in Scenedesmus obliquus is affected by competition against Microcystis aeruginosa. Sci. Rep.
**2015**, 5, 12743. [Google Scholar] [CrossRef] [PubMed][Green Version] - Huang, Y.; Cui, G.; Li, B.; Zhu, X.; Yang, Z. Elevated atmospheric CO
_{2}enhances grazer-induced morphological defense in the freshwater green alga Scenedesmus obliquus. Limnol. Oceanogr.**2018**, 63, 1004–1014. [Google Scholar] [CrossRef] - Martin-Luna, B.; Sevilla, E.; Bes, M.T.; Fillat, M.F.; Peleato, M.L. Variation in the synthesis of microcystin in response to osmotic stress in Microcystin aeruginosa PCC 7806. Limnetica
**2015**, 34, 205–2014. [Google Scholar] - Black, K.; Yilmaz, M.; Phlips, E.J. Growth and toxin production by Microcystis aeruginosa pcc 7806 (kutzing) lemmerman at elevated salt concentrations. J. Environ. Prot.
**2011**, 2, 669–674. [Google Scholar] [CrossRef][Green Version] - Vergalli, J.; Fayolle, S.; Combes, A.; Franquet, E.; Comte, K. Persistence of microcystin production by Planktothrix agardhii (Cyanobacteria) exposed to different salinities. Phycologia
**2020**, 59, 24–34. [Google Scholar] [CrossRef] - Wang, W.; Sheng, Y.; Jiang, M. Physiological and metabolic responses of Microcystis aeruginosa to a salinity gradient. Environ. Sci. Pollut. Res.
**2022**, 29, 13226–13237. [Google Scholar] [CrossRef] - Helmus, M.R.; Mercado-Silva, N.; Vander Zanden, M.J. Subsidies to predators, apparent competition and the phylogenetic structure of prey communities. Oecologia
**2013**, 173, 997–1007. [Google Scholar] [CrossRef] - Song, H.; Lavoie, M.; Fan, X.; Tan, H.; Liu, G.; Xu, P.; Fu, Z.; Paerl, H.; Qian, H. Allelopathic interactions of linoleic acid and nitric oxide increase the competitive ability of Microcystis aeruginosa. ISME J.
**2017**, 11, 1865–1876. [Google Scholar] [CrossRef][Green Version] - Qian, H.; Xu, J.; Lu, T.; Zhang, Q.; Qu, Q.; Yang, Z.; Pan, X. Responses of unicellular alga Chlorella pyrenoidosa to allelochemical linoleic acid. Sci. Total Environ.
**2018**, 625, 1415–1422. [Google Scholar] [CrossRef] - Yang, J.; Tang, H.; Zhang, X.; Zhu, X.; Huang, Y.; Yang, Z. High temperature and pH favor Microcystis aeruginosa to outcompete Scenedesmus obliquus. Environ. Sci. Pollut. Res.
**2018**, 25, 4794–4802. [Google Scholar] [CrossRef] - Wei, Y.; Liu, J.; Dai, M.; Chen, X. Competition between Microcystis aeruginosa and Scenedesmus obliquus under different temperature and light regimes. J. Environ. Manag.
**2021**, 286, 111809. [Google Scholar] - von Alvensleben, N.; Magnusson, M.; Heimann, K. Salinity tolerance of four freshwater microalgal species and the effects of salinity and nutrient limitation on biochemical profiles. J. Appl. Phycol.
**2016**, 28, 861–876. [Google Scholar] [CrossRef] - Yamamoto, M.; Chiba, T.; Tuji, A. Salinity responses of benthic diatoms inhabiting tidal flats. Diatom Res.
**2017**, 32, 243–250. [Google Scholar] [CrossRef] - Sun, Y.; Chen, Y.; Wei, J.; Zhang, X.; Zhang, L.; Yang, Z.; Huang, Y. Ultraviolet-B radiation stress alters the competitive outcome of algae: Based on analyzing population dynamics and photosynthesis. Chemosphere
**2021**, 272, 129645. [Google Scholar] [CrossRef] - Zhang, M.; Kong, F.; Xing, P.; Tan, X. Effects of Interspecific Interactions between Microcystis aeruginosa and Chlorella pyrenoidosa on Their Growth and Physiology. Int. Rev. Hydrobiol.
**2007**, 92, 281–290. [Google Scholar] [CrossRef] - Hernández-Zamora, M.; Santiago-Martínez, E.; Martínez-Jerónimo, F. Toxigenic Microcystis aeruginosa (Cyanobacteria) affects the population growth of two common green microalgae: Evidence of other allelopathic metabolites different to cyanotoxins. J. Phycol.
**2021**, 57, 1530–1541. [Google Scholar] [CrossRef] - Zak, A.; Musiewicz, K.; Kosakowska, A. Allelopathic activity of the Baltic cyanobacteria against microalgae. Estuar. Coast. Shelf Sci.
**2012**, 112, 4–10. [Google Scholar] [CrossRef] - Sychrová, E.; Štěpánková, T.; Nováková, K.; Bláha, L.; Giesy, J.P.; Hilscherová, K. Estrogenic activity in extracts and exudates of cyanobacteria and green algae. Environ. Int.
**2012**, 39, 134–140. [Google Scholar] [CrossRef] - Li, B.; Yin, Y.; Kang, L.; Feng, L.; Liu, Y.; Du, Z.; Tian, Y.; Zhang, L. A review: Application of allelochemicals in water ecological restoration—Algal inhibition. Chemosphere
**2021**, 267, 128869. [Google Scholar] [CrossRef]

**Figure 1.**Growth rates of M. aeruginosa (

**a**) and S. obliquus (

**b**) in monocultures at three salinities (0, 3, 6). The values were presented as mean ± standard error. Lowercase letters in italic style represented significant differences caused by different salinities).

**Figure 2.**The increase of cell density of M. aeruginosa (blue square) and S. obliquus (green circle) over the 17-day experiment in three different salinities (0, 3, 6).

**Figure 3.**Displacement rates for M. aeruginosa against S. obliquus with different initial algal compositions at different salinities ((

**a**,

**d**,

**g**): 75% Ma/25% So at 0, 3, 6; (

**b**,

**e**,

**h**): 50% Ma/50% So at 0, 3, 6; (

**c**,

**f**,

**i**): 25% Ma/75% So at 0, 3, 6).

**Figure 4.**Proportions of unicells and 2-, 4- and 8-celled colonies in S. obliquus populations of monocultures and cocultures with different initial algal compositions. The “rest” group represents 3-, 5-, 6-, and 7-celled colonies.

**Figure 5.**The maximal efficiency of PSII photochemistry (F

_{v}/F

_{m}) of M. aeruginosa and S. obliquus in monocultures and cocultures with different initial algal compositions at three salinities (0: (

**a**,

**d**), 3: (

**b**,

**e**), 6: (

**c**,

**f**)) on different days.

**Table 1.**Gaussian distribution formulae of the algal cell density versus time of both M. aeruginosa and S. obliquus in cocultures, and the formulae of competitive displacement rate against time of S. obliquus by M. aeruginosa.

Salinity | Algae | 25% So + 75% Ma | 50% So + 50% Ma | 75% So + 25% Ma |
---|---|---|---|---|

0 | Ma | ${N}_{t}=2.250\times {e}^{\left(-0.5\times {\left(\frac{t-12.59}{6.985}\right)}^{2}\right)}-0.248$ R ^{2} = 0.6799 | ${N}_{t}=1.351\times {e}^{\left(-0.5\times {\left(\frac{t-11.98}{6.004}\right)}^{2}\right)}-0.049$ R ^{2} = 0.6004 | ${N}_{t}=0.742\times {e}^{\left(-0.5\times {\left(\frac{t-14.14}{7.824}\right)}^{2}\right)}-0.113$ R ^{2} = 0.5069 |

So | ${N}_{t}=6.040\times {e}^{\left(-0.5\times {\left(\frac{t-14.27}{4.584}\right)}^{2}\right)}-0.143$ R ^{2} = 0.9180 | ${N}_{t}=6.413\times {e}^{\left(-0.5\times {\left(\frac{t-15.57}{6.286}\right)}^{2}\right)}-0.484$ R ^{2} = 0.9543 | ${N}_{t}=7.084\times {e}^{\left(-0.5\times {\left(\frac{t-15.28}{6.418}\right)}^{2}\right)}-0.640$ R ^{2} = 0.9140 | |

Y(t) | $Y\left(t\right)=10.46\times {e}^{\left(-0.5\times {\left(\frac{t+12.86}{9.568}\right)}^{2}\right)}-1.318$ R ^{2} = 0.9398 | $Y\left(t\right)=381.8\times {e}^{\left(-0.5\times {\left(\frac{t+54.23}{17.78}\right)}^{2}\right)}-1.793$ R ^{2} = 0.8693 | $Y\left(t\right)=17.73\times {e}^{\left(-0.5\times {\left(\frac{t+18.73}{9.850}\right)}^{2}\right)}-2.472$ R ^{2} = 0.8013 | |

3 | Ma | ${N}_{t}=1.380\times {e}^{\left(-0.5\times {\left(\frac{t-8.251}{5.333}\right)}^{2}\right)}-0.049$ R ^{2} = 0.6753 | ${N}_{t}=1.987\times {e}^{\left(-0.5\times {\left(\frac{t-7.999}{9.493}\right)}^{2}\right)}-1.132$ R ^{2} = 0.5851 | ${N}_{t}=0.399\times {e}^{\left(-0.5\times {\left(\frac{t-3.851}{0.148}\right)}^{2}\right)}+0.311$ R ^{2} = 0.1538 |

So | ${N}_{t}=6.213\times {e}^{\left(-0.5\times {\left(\frac{t-13.98}{5.072}\right)}^{2}\right)}-0.268$ R ^{2} = 0.9041 | ${N}_{t}=5.941\times {e}^{\left(-0.5\times {\left(\frac{t-13.62}{5.241}\right)}^{2}\right)}-0.274$ R ^{2} = 0.9003 | ${N}_{t}=8.253\times {e}^{\left(-0.5\times {\left(\frac{t-16.14}{9.598}\right)}^{2}\right)}-2.397$ R ^{2} = 0.9016 | |

Y(t) | $Y\left(t\right)=5.808\times {e}^{\left(-0.5\times {\left(\frac{t+3.185}{7.274}\right)}^{2}\right)}-2.547$ R ^{2} = 0.8627 | $Y\left(t\right)=4.695\times {e}^{\left(-0.5\times {\left(\frac{t+0.911}{5.128}\right)}^{2}\right)}-2.523$ R ^{2} = 0.8746 | $Y\left(t\right)=3.562\times {e}^{\left(-0.5\times {\left(\frac{t-1.157}{2.961}\right)}^{2}\right)}-2.832$ R ^{2} = 0.8574 | |

6 | Ma | ${N}_{t}=1.143\times {e}^{\left(-0.5\times {\left(\frac{t-9.356}{6.643}\right)}^{2}\right)}-0.061$ R ^{2} = 0.6174 | ${N}_{t}=0.812\times {e}^{\left(-0.5\times {\left(\frac{t-10.36}{7.320}\right)}^{2}\right)}-0.032$ R ^{2} = 0.5690 | ${N}_{t}=0.425\times {e}^{\left(-0.5\times {\left(\frac{t-10.48}{5.329}\right)}^{2}\right)}+0.049$ R ^{2} = 0.4568 |

So | ${N}_{t}=4.374\times {e}^{\left(-0.5\times {\left(\frac{t-11.82}{5.121}\right)}^{2}\right)}-0.482$ R ^{2} = 0.8361 | ${N}_{t}=4.351\times {e}^{\left(-0.5\times {\left(\frac{t-11.84}{4.567}\right)}^{2}\right)}-0.246$ R ^{2} = 0.9269 | ${N}_{t}=5.095\times {e}^{\left(-0.5\times {\left(\frac{t-12.42}{5.045}\right)}^{2}\right)}-0.320$ R ^{2} = 0.7749 | |

Y(t) | $Y\left(t\right)=6.225\times {e}^{\left(-0.5\times {\left(\frac{t+5.596}{5.858}\right)}^{2}\right)}-1.369$ R ^{2} = 0.9349 | $Y\left(t\right)=3.296\times {e}^{\left(-0.5\times {\left(\frac{t-0.417}{3.326}\right)}^{2}\right)}-1.656$ R ^{2} = 0.8829 | $Y\left(t\right)=3.199\times {e}^{\left(-0.5\times {\left(\frac{t-1.432}{1.915}\right)}^{2}\right)}-2.365$ R ^{2} = 0.7747 |

**Table 2.**Summary of two-way ANOVAs for the effects of salinity and initial algal composition on the maximal values of algal abundance (N

_{max}) and the time to N

_{max}(t

_{max}) of M. aeruginosa and S. obliquus.

Algae | Factors | N_{max} | t_{max} | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|

SS | DF | MS | F | p Value | SS | DF | MS | F | p Value | ||

Microcystis | Salinity | 2.014 | 2 | 1.007 | 0.1493 | 0.8624 | 173.7 | 2 | 86.85 | 19.14 | <0.0001 |

Composition | 5.766 | 2 | 2.883 | 0.4273 | 0.6587 | 2.169 | 2 | 1.085 | 0.2391 | 0.7898 | |

Interaction | 2.319 | 4 | 0.5799 | 0.08594 | 0.9857 | 44.16 | 4 | 11.04 | 2.434 | 0.085 | |

Scenedesmus | Salinity | 25.62 | 2 | 12.81 | 5.596 | 0.0129 | 47.52 | 2 | 23.76 | 10.43 | 0.001 |

Composition | 9.455 | 2 | 4.727 | 2.065 | 0.1558 | 7.698 | 2 | 3.849 | 1.689 | 0.2126 | |

Interaction | 2.877 | 4 | 0.7193 | 0.3142 | 0.8647 | 6.962 | 4 | 1.74 | 0.7640 | 0.5623 |

**Table 3.**Summary of three-way ANOVAs for the effects of salinity, proportion, and culture time on the maximal efficiency of PSII photochemistry (F

_{v}/F

_{m}) of M. aeruginosa and S. obliquus.

Algae | Source of Variation | SS | DF | MS | F | p Value |
---|---|---|---|---|---|---|

Microcystis | Time | 0.437 | 7 | 0.0625 | 47.108 | <0.001 |

Salinity | 0.0153 | 2 | 0.00765 | 5.773 | 0.004 | |

Proportion of Ma | 0.0531 | 3 | 0.0177 | 13.341 | <0.001 | |

Time × Salinity | 0.185 | 14 | 0.0132 | 9.973 | <0.001 | |

Time × Proportion of Ma | 0.0773 | 21 | 0.00368 | 2.778 | <0.001 | |

Salinity × Proportion of Ma | 0.0242 | 6 | 0.00403 | 3.043 | 0.007 | |

Time × Salinity × Proportion of Ma | 0.0847 | 42 | 0.00202 | 1.522 | 0.031 | |

Scenedesmus | Time | 0.152 | 7 | 0.0217 | 62.987 | <0.001 |

Salinity | 0.00898 | 2 | 0.00449 | 13.009 | <0.001 | |

Proportion of So | 0.00543 | 3 | 0.00181 | 5.243 | 0.002 | |

Time × Salinity | 0.0285 | 14 | 0.00203 | 5.889 | <0.001 | |

Time × Proportion of So | 0.0233 | 21 | 0.00111 | 3.216 | <0.001 | |

Salinity × Proportion of So | 0.0115 | 6 | 0.00191 | 5.539 | <0.001 | |

Time × Salinity × Proportion of So | 0.0421 | 42 | 0.001 | 2.906 | <0.001 |

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## Share and Cite

**MDPI and ACS Style**

Gao, T.; Li, Y.; Xue, W.; Pan, Y.; Zhu, X.
Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*. *Water* **2023**, *15*, 1331.
https://doi.org/10.3390/w15071331

**AMA Style**

Gao T, Li Y, Xue W, Pan Y, Zhu X.
Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*. *Water*. 2023; 15(7):1331.
https://doi.org/10.3390/w15071331

**Chicago/Turabian Style**

Gao, Tianheng, Yinkang Li, Wenlei Xue, Yueqiang Pan, and Xuexia Zhu.
2023. "Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*" *Water* 15, no. 7: 1331.
https://doi.org/10.3390/w15071331