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Effects of the submerged macrophyte Ceratophyllum demersum

(Ceratophyllaceae) and the cladoceran Moina micrura

(Cladocera: Moinidae) on microalgal interactions

Vitor Ricardo de Souza1; https://orcid.org/0000-0002-7291-3605

Cihelio Alves Amorim1,2; https://orcid.org/0000-0002-7171-7450

Ariadne do Nascimento Moura1*; https://orcid.org/0000-0001-5093-2840

1. Departamento de Biologia, Universidade Federal Rural de Pernambuco, Dom Manoel de Medeiros, s/n, Dois Irmãos,

52171-030, Recife, Pernambuco, Brazil; vitorricardo.biologia@gmail.com, ariadne_moura@hotmail.com

2. DepartmentofBiologicalSciences,MiddleEastTechnicalUniversity,ÜniversitelerMahallesi,DumlupınarBulvarı,

60800, Ankara, Turkey; alvescihelio@gmail.com (*Correspondence)

Received 29-VI-2020. Corrected 10-XI-2021. Accepted 25-XI-2021.

ABSTRACT

Introduction: Cyanobacterial blooms in tropical water bodies are increasingly common, because of eutrophica-

tion and rising temperatures. Consequently, many freshwater systems are affected, by reducing water quality,

biodiversity, and ecosystem services. With the increased frequency of harmful algal blooms, the development of

biological tools to improve water quality is an urgent issue.

Objective: To evaluate the effects of a submerged macrophyte and a cladoceran on the microcystin-producing

cyanobacteria Microcystis aeruginosa (NPLJ-4) and the chlorophyte Raphidocelis subcapitata (BMIUFRPE-02)

in mixed cultures.

Methods: Two parallel experiments were carried out for ten days to evaluate the effects of the submerged mac-

rophyte Ceratophyllum demersum and the cladoceran Moina micrura on microalgal interactions. Microalgal

strains were cultivated in the ASM1 culture medium, under controlled laboratory conditions. The first experi-

ment presented four treatments: M (C. demersum), Z (M. micrura), MZ (C. demersum and M. micrura), and

C (control). Meanwhile, the second experiment consisted of five treatments, in which the microalgae were

cultivated together at different Microcystis:Raphidocelis ratios: 1:0, 3:1, 1:1, 1:3, and 0:1. Biomass and growth

rates of the strains were evaluated every two days, which were statistically treated with three-way or two-way

repeated-measures ANOVA.

Results: In the first experiment, M. aeruginosa was significantly inhibited in M and MZ treatments from the

second day, and Z from the fourth, while R. subcapitata showed no reduction in its biomass in any treatment.

On the other hand, R. subcapitata was stimulated from the eighth and tenth days in M treatment and only on the

eighth day in Z treatment. In the second experiment, M. aeruginosa was significantly inhibited when cultivated

with R. subcapitata in low ratios (Microcystis:Raphidocelis ratio of 1:3) throughout the experiment, while the

chlorophyte was stimulated in that treatment.

Conclusions: The coexistence of a cyanobacterium with a green alga did not alter the main negative response

of M. aeruginosa to the submerged macrophyte and zooplankton but stimulated the green alga. Accordingly, the

introduction of submerged macrophytes and cladocerans already adapted to eutrophic conditions, both isolated

and combined, proved to be a good method to control cyanobacterial blooms without negatively affecting other

coexisting phytoplankton species.

Key words: microalgal blooms; allelopathy; biomanipulation; competition; grazing.

de Souza, V. R., Alves Amorim, C., & do Nascimento Moura, A.

(2021). Effects of a submerged macrophyte, Ceratophyllum

demersum (Ceratophyllaceae) and a cladoceran, Moina

micrura (Cladocera: Moinidae) on microalgal interactions.

Revista de Biología Tropical, 69(4), 1276-1288. https://doi.

org/10.15517/rbt.v69i4.42589

https://doi.org/10.15517/rbt.v69i4.42589

AQUATIC ECOLOGY

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The occurrence of cyanobacterial blooms

in freshwater environments, in response to

anthropogenic eutrophication and climate

change (Moura et al., 2018; O’Neil et al.,

2012), has seriously damaged aquatic biota

(Paerl & Otten, 2013), leading to the death of

fish, mollusks, and crustaceans (Ibelings &

Chorus, 2007; Zurawell et al., 2005). For the

most part, blooms affect aquatic ecosystems

through the production of toxic metabolites

(e.g., Microcystins, Li et al., 2021) by certain

cyanobacteria species (Cirés & Ballot, 2016;

Harke et al., 2016). Moreover, algal blooms can

represent a serious threat to freshwater biodi-

versity, reducing water quality and ecosystem

functioning (Amorim & Moura, 2021).

Recurrent toxic blooms of these organ-

isms seriously compromise the quality of water

and make it unsuitable for human consump-

tion, generating economic (Carmichael &

Boyer, 2016) and health problems (Chen et

al., 2009; Zurawell et al., 2005) and, in more

serious cases, can be fatal (Azevedo et al.,

2002). Among the main bloom-forming genera,

Microcystis stands out with wide geographical

distribution and several microcystin-producing

morphospecies (Harke et al., 2016; O’Neil et

al., 2012; Wiegand & Pflugmacher, 2005). To

control algal blooms, laboratory and in situ

studies using aquatic plants (e.g., Amorim &

Moura, 2020; Amorim et al., 2019a;) and zoo-

plankton organisms (e.g., Amorim & Moura,

2020; Amorim et al., 2019b; Diniz et al., 2019;

Severiano et al., 2018; Severiano et al., 2021)

have been carried out in tropical regions, spe-

cifically in the Northeastern region of Brazil. In

that region, the biomanipulation of fish to con-

trol eutrophication and phytoplankton blooms

can show negative (Menezes et al., 2010),

positive or no effects (e.g., Dantas et al., 2019;

Okun et al., 2008).

Submerged macrophytes can produce

allelochemicals that are important tools for

controlling cyanobacteria (Amorim & Moura,

2020; Zhu et al., 2010). The secondary metabo-

lites released by plants into the water column

can inhibit the activity of photosystem II and

rupture the cell membrane, which kills the

cyanobacteria (Mohamed, 2017). Macrophyte

allelopathy is a biological alternative that can

minimize impacts caused by harmful cyano-

bacteria and improve water quality (Chen et al.,

2012; Hilt & Gross, 2008; Vanderstukken et al.,

2014). However, unlike cyanobacteria, green

algae are resistant to the inhibitory effect of

allelochemicals from submerged macrophytes

(Amorim et al., 2019a; Dong et al., 2014; Zhu

et al., 2010). Nevertheless, it is believed that

when in coexistence with a green alga, cya-

nobacteria can be stimulated by macrophyte

allelochemicals instead of being inhibited

(Chang et al., 2012).

Another way to reduce cyanobacterial

blooms is through zooplankton since the her-

bivory pressure exerted by these organisms

negatively affects the biomass of phytoplank-

ton species (Amorim et al., 2019b; Diniz et

al., 2019; Severiano et al., 2018). However,

that method does not always negatively affect

cyanobacteria, because they can produce large

filaments and colonies, along with the pro-

duction of toxic metabolites, which reduce

the grazing of zooplankton on cyanobacteria

(Ger et al., 2016). Cyanotoxins can negatively

affect the quality of life for zooplankton, lead-

ing to death, retarding growth, and changing

the ingestion rate (Bownik, 2016; Santos et

al., 2021). Moreover, cyanotoxins can affect

more seriously larger zooplankton than small

organisms, however, the former ones can graze

more efficiently on phytoplankton (Guo & Xie,

2006). With that, some zooplankton species

can select palatable phytoplankton organisms,

which can, in turn, increase cyanobacterial

biomass (Leitão et al., 2018; Severiano et al.,

2021).

Therefore, the present study aims to (1)

verify the effect of the submerged macrophyte

Ceratophyllum demersum L. and the cladoc-

eran Moina micrura Kurz, 1874 on the inter-

action between the cyanobacteria Microcystis

aeruginosa (Kützing) Kützing and the chloro-

phyte Raphidocelis subcapitata (Korshikov)

Nygaard, Komárek, Kristiansen & Skulberg;

and (2) understand the relationships between

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cyanobacteria and chlorophytes under different

dominance scenarios for both groups.

MATERIALS AND METHODS

Phytoplankton organisms and culture

conditions: For this study, two non-axenic

strains of microalgae were selected: the cya-

nobacterium M. aeruginosa, and the chloro-

phyte R. subcapitata. The M. aeruginosa strain

(NPLJ-4) (Cyanobacteria), which has been

proven to produce four variants of microcys-

tins (Amorim et al., 2017), was provided by

the Laboratory of Ecophysiology and Toxicol-

ogy of Cyanobacteria (LETC) at the Federal

University of Rio de Janeiro (Rio de Janeiro,

Brazil). The R. subcapitata strain (BMIU-

FRPE-02) (Chlorophyceae) was obtained from

the Microalgae Culture Collection at the Fed-

eral Rural University of Pernambuco - BMIU-

FRPE, Recife (Pernambuco, Brazil).

Cyanobacteria and chlorophyte were grown

in 1 000 ml Erlenmeyer flasks filled with 800

ml of ASM1 culture medium (Gorham et al.,

1964), in a climatic chamber with controlled

conditions of temperature (25 °C ± 1.5), light

intensity (40 µmol m-2 s-1), pH (7.5), and 12

h photoperiod. All cultures were homogenized

three times a day to avoid agglomeration and

sedimentation of cells. Microalgae were culti-

vated until biomass of 50 mg L-1 was obtained.

Collection and maintenance of sub-

merged macrophyte Ceratophyllum demer-

sum: The submerged macrophyte C. demersum

was collected from the Carpina reservoir,

Lagoa do Carro municipality (Pernambuco,

Brazil). During the sampling, 20 cm apical

branches from young plants were selected

and transported to the laboratory, where they

were washed with distilled water jets and a

soft brush to remove epiphytic microalgae,

small invertebrate animals, and adhered sedi-

ments. The macrophytes were cultivated in 8 l

aquaria (20 cm²) that were filled with filtered

tap water and maintained under the same con-

ditions described for microalgae, however, the

aquaria were constantly aerated with aquarium

aerators. Until the experiments were carried

out, the water was renewed twice a week to

prevent the proliferation of insects, small mol-

lusks, and microorganisms.

Obtaining and culture of the cladocer-

an: The cladoceran M. micrura was collected

from the Mundaú reservoir, Garanhuns munici-

pality (Pernambuco, Brazil). This reservoir

presents intense cyanobacteria blooms formed

mainly by Microcystis spp. and Raphidiopsis

raciborskii (Woloszynska) Aguilera, Berren-

dero Gómez, Kastovsky, Echenique & Salerno

(Moura et al., 2015; Amorim et al., 2020). This

cladoceran was collected by filtering 100 l of

water with a 68 µm mesh plankton net and

identified under an optical microscope using

specialized bibliography. The individuals were

selected and cultivated separately in 30 ml test

tubes, filled with 20 ml of reconstituted water

(70 % mineral water + humic acid and 30 %

water from the environment that was filtered

through a 20 μm mesh net), for subsequent

selection of genetically identical clones from

the same mother.

After at least 20 individuals grew in each

tube, one clonal lineage was selected and the

cladocerans were transferred to 250 ml Erlen-

meyer flasks filled with 200 ml of reconstituted

water and constant aeration. Thirty days before

the experiments, the cladocerans were cultivat-

ed in 1 000 ml Erlenmeyer flasks at 27 °C, 40

µmol m-2 s-1 light intensity, constant aeration,

and 12 h photoperiod; and were fed with the

chlorophyte R. subcapitata every day.

Experimental design: The experiments

were carried out in an aseptic room with the

same conditions described for the cultiva-

tion of the microalgae strains. Twenty-four

1 000 ml Erlenmeyer flasks were filled with

500 ml of ASM1 nutrient medium containing

cyanobacteria and chlorophyte inoculum. Two

parallel experiments were carried out for ten

days to observe the allelopathic effects of C.

demersum and the grazing pressure of the cla-

doceran M. micrura, both isolated and together,

on the interaction between M. aeruginosa and

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R. subcapitata; as well as to observe possible

allelopathic interactions between cyanobacteria

and green algae at different dominance ratios.

The experimental design consisted of eight

treatments with three replicates that were divid-

ed between the two parallel experiments (Table

1). For both experiments, all microalgal treat-

ments were grown in coexistence or isolated

with initial biomass of approximately 35 mg

l-1. In the first experiment, the proportion of

microalgae biomass was 1:1 in all treatments.

In the second experiment, the interaction treat-

ments consisted of different concentrations

of Microcystis and Raphidocelis, while the

controls consisted of the isolated cultures of

the cyanobacterium and the chlorophyte. Thus,

there was a gradient in the Microcystis and

Raphidocelis ratios of M:R = 1:0; 3:1; 1:1; 1:3;

and 0:1 (Table 1).

To determine the exact ratio between

Microcystis and Raphidocelis cultures for the

experiments, the biomasses of the stock cul-

tures were analyzed. The proportions were

achieved using the ASMI medium dilution

method. Three days before the experiments

began, young and apical branches of C. demer-

sum were selected, washed several times with

distilled water, cut to obtain an 8 gFW l-1 bio-

mass (g of fresh weight), then grown in ASM1

medium for acclimatization. Similarly, three

days before the experiment, visibly healthy

(actively swimming) cladocerans of the same

age were selected (120 ind l-1) and transferred

to 250 ml Erlenmeyer flasks containing ASM1

medium for acclimatization.

At the beginning of the experiment, one

macrophyte branch was added to each experi-

mental unity in the M and MZ treatments.

Similarly, 50 M. micrura individuals were

transferred to each experimental unit in the

Z and MZ treatments. During the transfer of

cladocerans to microalgae cultures, about 5 ml

of ASM1 medium was also transferred, thus,

the same amount of medium from the cla-

doceran cultures was added to all treatments.

Aliquots of 1 ml were collected every two

days for 10 days (0, 2, 4, 6, 8, and 10) from

all experimental units to determine cell density

and biovolume. Microalgal density was deter-

mined by counting cells in a Fuchs-Rosenthal

chamber (hemocytometer) under an optical

microscope. Also, the length and width of the

cells were measured to obtain the biovolume

as proposed by Hillebrand et al. (1999), which

was multiplied by density for conversion into

biomass. In the second experiment, growth

rates were determined for each species in all

treatments, following the formula described by

Wood et al. (2005).

Statistical analyses: A three-way repeat-

ed-measures ANOVA was used to compare the

biomass of microalgae in the first experiment,

based on the factors Macrophyte, Zooplankton,

TABLE 1

Descriptions of the treatments used in the allelopathy and grazing experiment (Exp. 1) and

microalgae interaction experiment (Exp. 2) with different proportions of microalgae

Experiment / Treatment Microcystis

Percentage (%)

Raphidocelis

Percentage (%) Ceratophyllum demersum Moina micrura

Exp.1 / M 50 50 X -

Exp.1 / Z 50 50 - X

Exp.1 / MZ 50 50 X X

Exp.2 / 1:0 100 0 - -

Exp.2 / 3:1 75 25 - -

Exp.1 and 2 / C and 1:1 50 50 - -

Exp.2 / 1:3 25 75 - -

Exp.2 / 0:1 0 100 - -

X: presence; -: absence. The 1:1 treatment of the second experiment was also used as a control for the first since they were

developed in parallel.

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Time, and their interactions. Likewise, a two-

way repeated-measures ANOVA was used to

compare the growth rates of the cultures in

the second experiment, based on the factors

Ratios, Time, and their interactions. Before the

analysis of variances, the data were tested for

normality with the Kolmogorov-Smirnov test

and homoscedasticity with Bartlett’s test. For

statistical analyses, the R program was used,

with a significance level of P < 0.05 (R Core

Team, 2021).

RESULTS

Experiment 1: The cyanobacterium and

green alga strains responded differently to the

treatments and time (Table 2). Ceratophyllum

demersum significantly reduced the biomass

of M. aeruginosa from the second day of the

experiment in treatment M (P < 0.05) (Fig. 1A).

However, R. subcapitata was not significantly

affected in treatment M when compared to the

control (P > 0.05) until the sixth day. After that,

R. subcapitata showed significantly higher

biomass in the treatment M, compared to the

control (P < 0.05) (Fig. 1B).

With the addition of cladocerans, there was

a significant reduction in the biomass of M.

aeruginosa (P < 0.05) from the fourth day (Fig.

1A). Regarding the green alga, M. micrura

stimulated the growth of R. subcapitata on the

eighth day, with significant differences when

compared to the control (P < 0.05) and showing

no significant grazing by cladocerans (Fig. 1B).

The coexistence of aquatic macrophytes

with the cladoceran in the MZ treatment sig-

nificantly reduced the biomass of M. aerugi-

nosa from the second day of the experiment

compared to the control (P < 0.05) but did not

show significant differences to the M treat-

ment (P > 0.05) (Fig. 1A). For green alga, the

combined treatment of the plant and micro-

crustacean (MZ) did not alter the biomass of R.

subcapitata (P > 0.05) (Fig. 1B). Raphidocelis

subcapitata also did not show any significant

differences between the treatments M, Z, and

MZ (P > 0.05). During the experiment, R. sub-

capitata tended to form small to large colonies

TABLE 2

Results of the three-way repeated-measures ANOVA comparing the effects of Ceratophyllum demersum

(macrophyte), Moina micrura (zooplankton), and time on the biomass of Microcystis aeruginosa

and Raphidocelis subcapitata in the first experiment

Factors df F p

Microcystis aeruginosa

Macrophyte 1 6681.14 < 0.001

Zooplankton 1 1129.62 < 0.001

Time 5 120.38 < 0.001

Macrophyte:Zooplankton 1 726.46 < 0.001

Macrophyte:Time 5 206.70 < 0.001

Zooplankton:Time 5 182.68 < 0.001

Macrophyte:Zooplankton:Time 5 194.49 < 0.001

Raphidocelis subcapitata

Macrophyte 1 25.65 0.037

Zooplankton 1 23.12 0.041

Time 5 174.30 < 0.001

Macrophyte:Zooplankton 1 73.75 0.013

Macrophyte:Time 5 8.27 0.003

Zooplankton:Time 5 13.26 < 0.001

Macrophyte:Zooplankton:Time 5 20.39 < 0.001

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in all treatments with the macrophyte and the

cladoceran (data not shown).

Experiment 2: The mixed cultures, with

different ratios of M. aeruginosa and R. sub-

capitata, showed distinct responses to the

treatments and time (Table 3). In the controls,

M. aeruginosa (M:R 1:0) maintained a constant

growth (Fig. 2A), while R. subcapitata (M:R

0:1) decreased its growth during the experi-

ment (Fig. 2B). For the treatment with 75

% Microcystis and 25 % Raphidocelis (M:R

3:1), both cyanobacterium and green alga did

not differ from the controls throughout the

experiment (P > 0.05) (Fig. 2A, Fig. 2B). The

opposite effect was observed for the treatment

with 25 % Microcystis and 75 % Raphidocelis

(M:R 1:3), when the green alga strain sig-

nificantly inhibited the growth of Microcystis

from the second day of the experiment (P <

0.05) (Fig. 2A). Also, Raphidocelis presented

higher growth rates in treatment M:R 1:3 than

in M:R 1:1 on the fourth and eighth days. In

equal proportions of microalgal biomass, i.e.

50 % of both strains (M:R 1:1), neither strain

differed significantly from the controls (P >

0.05) (Fig. 2).

Regarding the ratio between the biomass of

M. aeruginosa and R. subcapitata in the treat-

ments with the dominance of M. aeruginosa

Fig. 1. Isolated effects of Ceratophyllum demersum (M) and Moina micrura (Z), besides the combined addition of C.

demersum and M. micrura (MZ), and control (C) on A. Microcystis aeruginosa and B. Raphidocelis subcapitata in mixed

cultures for ten days. The different letters indicate significant differences between treatments for each day (P < 0.05). Lines

and shaded areas represent the mean and 95 % confidence interval, respectively.

TABLE 3

Results of the two-way repeated-measures ANOVA comparing the effects of different ratios

between Microcystis and Raphidocelis and time on the growth rate of Microcystis aeruginosa

and Raphidocelis subcapitata in the second experiment

Factors df F p

Microcystis aeruginosa - Growth rate

Ratios 3 1049.02 < 0.001

Time 4 16.27 < 0.001

Ratios:Time 12 22.26 < 0.001

Raphidocelis subcapitata - Growth rate

Ratios 3 5.74 0.034

Time 4 152.95 < 0.001

Ratios:Time 12 2.75 0.017

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(M:R 3:1) or in equal proportions (M:R 1:1),

there was an increase in the relative participa-

tion of cyanobacteria compared to green algae

from the fourth day until the end of the experi-

ment. On the other hand, under the dominance

of R. subcapitata (M:R 1:3), M. aeruginosa

showed a reduction in relative participation

from the second day (Fig. 3).

DISCUSSION

Competition for nutrients (Zhang et

al., 2013), light (Marinho et al., 2013), and

allelopathy (Bittencourt-Oliveira et al., 2015;

Harel et al., 2013) are variables that strongly

regulate the development of phytoplankton

species. The availability of nitrogen and phos-

phorus contributes to the growth and helps

to maintain the metabolism of microalgae,

increasing the biomass of species that better

absorb inorganic compounds (Carey et al.,

2012; Markou et al., 2014). Nevertheless, in

our study, we excluded the effect of nutrient

competition by using ASM1 culture medium

in optimum quantities for the development of

both strains.

The presence of morphological structures

that facilitate fluctuation, as well as the pres-

ence of accessory pigments that protect cells

from excessive light, are adaptive advantages

that cyanobacteria species present (Carey et

al., 2012). Microcystis aeruginosa is a strong

competitor for light, as verified by Marinho et

al. (2013). However, we discarded this type of

competition through the uniform and random

light supply between treatments, as well as

by manually agitating the experimental units

three times a day to reduce cell sedimentation.

Fig. 2. Growth rate of A. Microcystis aeruginosa and B.

Raphidocelis subcapitata in treatments with different M:R

(Microcystis:Raphidocelis) ratios: 1:0, 3:1, 1:1, 1:3, and

0:1 during ten days of the experiment. The different letters

indicate significant differences between treatments for each

day (P < 0.05). Bars and error bars represent the mean and

standard deviation, respectively.

Fig. 3. The ratio between the biomass of M. aeruginosa

and R. subcapitata in treatments with 75 % Microcystis

and 25 % Raphidocelis (3:1), 50 % Microcystis and 50

% Raphidocelis (1:1), and 25 % Microcystis and 75 %

Raphidocelis (1:3) during 10 days of the experiment. Lines

and shaded areas represent the mean and 95 % confidence

interval, respectively.

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Finally, by excluding other possible forms of

competition, we suggest that allelopathy was

the main mechanism of action among photo-

synthesizing organisms, especially the macro-

phyte Ceratophyllum demersum.

In treatments with C. demersum, cyano-

bacterial biomass was reduced too much after

the second day of coexistence. Previously,

Amorim et al. (2019a) reported that the bio-

mass of M. aeruginosa was inhibited by the

presence of C. demersum in unialgal cultures.

Therefore, the macrophyte effect could be

attributed to allelopathy herein, as also veri-

fied by Nakai et al. (1999), Dong et al. (2014),

and Amorim et al. (2019a). Furthermore, in a

field experiment, Amorim and Moura (2020)

showed a significant reduction in cyanobacte-

rial blooms composed mainly of Microcystis

spp. (reduction of 85 % in the total biomass and

99 % in the biomass of filamentous morphot-

ypes) by C. demersum in a tropical reservoir in

Northeast Brazil.

Studies highlight that M. aeruginosa is

sensitive to chemical compounds released by

several macrophyte species (Nakai et al., 1999;

Zhu et al., 2010). In this case, Ceratophyllum

demersum, a free-living submerged macro-

phyte, can produce secondary metabolites (Hilt

& Gross, 2008) which inhibit competitors’ pho-

tosystem II, compromising the photosynthetic

activities of the target cell (Körner & Nicklisch,

2002). Sulfur or lipophilic labile sulfur com-

pounds are the major allelopathic substances

released by C. demersum (Wium-Andersen

et al., 1983). Also, some volatile compounds

from C. demersum, including fatty compounds,

terpenoids, phenolic compounds, and phthal-

ates, can show a strong inhibitory effect on M.

aeruginosa (Xian et al., 2006). Further com-

pounds present in C. demersum tissues, such

as hexanoic acid, phthalic acid, octanedioic

acid, butenoic acid, azelaic acid, palmitic acid,

alpha-linolenic acid, and pentanedioic acid,

can also inhibit the growth and induce colony

formation in green algae species (Dong et al.,

2019). Therefore, allelopathy can act as one of

the main adaptative strategies of submerged

macrophytes, especially C. demersum, in their

competition with phytoplankton (Gross et al.,

2003), as well as to maintain the clear state of

shallow lakes (Hilt & Gross, 2008). So, this

macrophyte can support the eutrophic and cya-

nobacterial blooms conditions, as it has potent

antioxidant and biotransformation mechanisms

to alleviate the effects of cyanotoxins on its

physiology, besides removing cyanotoxins

from the water (Pflugmacher, 2004).

In our study, M. aeruginosa was signifi-

cantly inhibited in coexistence with an allelo-

chemical-producing macrophyte and green

algae, differing from the results observed by

Chang et al. (2012), who recorded the stimulus

of M. aeruginosa in coexistence with a sub-

merged macrophyte and the chlorophyte Des-

modesmus armatus (R. Chodat) E. Hegewald.

Furthermore, R. subcapitata was not inhibited

in coexistence with C. demersum (M and MZ

treatments), corroborating the results of Amor-

im et al. (2019a), who found that biomass from

R. subcapitata was not significantly affected by

C. demersum throughout the experiment. This

response could be attributed to the low sensitiv-

ity of chlorophytes to the inhibitory metabolites

released by macrophytes in comparison to

cyanobacteria, as verified by Zhu et al. (2010).

Moreover, Körner and Nicklisch (2002) sug-

gest that phytoplankton species can physiologi-

cally adapt to allelochemicals. One important

evolutionary adaptation of green algae to coex-

ist with allelopathically active macrophytes is

colony formation (Dong et al., 2018), as also

observed in our study for R. subcapitata.

In tests with the addition of cladocerans,

we observed that the cyanobacterial biomass

was significantly reduced from the fourth day

and the green alga was stimulated on the eighth

day, differing statistically from the control.

This result differs from previous findings in

the literature, where zooplankton are assumed

to select the palatable food source in co-cul-

tures, and thus stimulate cyanobacterial growth

(Leitão et al., 2018; Severiano et al., 2021).

However, in experimental studies, Guo and Xie

(2006) found that populations of M. micrura

pre-exposed to toxic strains M. aeruginosa

may become more resistant to cyanobacteria

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metabolites compared to other large cladocer-

ans, enabling the predation of cyanobacteria.

Herein, the cladoceran M. micrura proved

to be resistant to toxins from the Microcystis

strain, which may have favored its predation on

cyanobacteria, considering that the cladoceran

was isolated from a reservoir with a history of

Microcystis blooms (Moura et al., 2015; Amor-

im et al., 2020). Similarly, Santos et al. (2021)

showed that cladocerans isolated from lakes

with cyanobacterial blooms are less affected by

cyanotoxins through the diet or the absorption

of dissolved toxins.

Although being considered a palatable

food source for zooplankton, R. subcapitata

was stimulated in treatment Z, instead of being

grazed. This can be attributed to the colony

formation in the presence of predators or com-

petitors. Both the presence of macrophytes and

zooplankton grazers can induce colony forma-

tion in green algae species, with a strong effect

in the presence of zooplankton cues, which can

act as an important anti-grazer defense (Zhu et

al., 2021). The stimulus of Raphidocelis in cla-

doceran treatments can also be attributed to a

reduction in Microcystis biomass. Furthermore,

zooplankton contributes to nutrient cycling in

the water column, favoring the growth of phy-

toplankton species (Attayde & Hansson, 1999),

which may justify the significant stimulus of R.

subcapitata in Z treatment.

As for the interaction between microalgae,

different results between Microcystis and Raph-

idocelis were observed. Raphidocelis subcapi-

tata inhibited M. aeruginosa in the treatment

with low concentrations of cyanobacteria (M:R

ratio 1:3). Li and Li (2012), in co-cultivation

experiments with M. aeruginosa and Anabaena,

found that the species with higher proportions

at the beginning of cultivation (1:9 and 9:1)

remained dominant throughout the experiment.

In our study, although M. aeruginosa did not

inhibit R. subcapitata growth when dominant

(M:R 3:1), the ratio between Microcystis and

Raphidocelis increased throughout the experi-

ment, showing that M. aeruginosa remained

dominant, corroborating with Li and Li (2012).

Another factor that may be strictly related to

the inhibition of M. aeruginosa is allelopathy.

Some chlorophyte species can release chemical

compounds that inhibit cyanobacteria, as veri-

fied by Harel et al. (2013), who demonstrated

that Scenedesmus sp. inhibited the growth

of Microcystis sp. by producing secondary

metabolites that disrupted the cell membranes

of cyanobacteria.

Bittencourt-Oliveira et al. (2015) showed

that the density of M. aeruginosa decreased in

mixed cultures with 1:1 ratios of cyanobacteria

and chlorophytes Monoraphidium convolutum

(Corda) Komárková-Legnerová and Scenedes-

mus acuminatus (Lagerheim) Chodat, with

more significant effects when coexisting with

the latter species. However, in our experi-

ment, the growth rates of M. aeruginosa and

R. subcapitata did not differ statistically from

controls in the treatment with proportions of

M:R 1:1. Li and Li (2012) found that M. aeru-

ginosa and Anabaena, in 1:1 ratios, maintained

similar growth for 15 days, reinforcing that the

dominance of a given strain is related to the

values of inoculated biomass on the first day.

This result also reinforces the main effects of

the submerged macrophyte and the cladoceran

in the first experiment, proving that the micro-

algae species do not affect each other under the

same proportion of biomass.

The submerged macrophyte C. demersum

considerably inhibited M. aeruginosa in coex-

istence with the green alga, controlling the

cyanobacteria biomass, differing from previ-

ous research (e.g., Chang et al., 2012). How-

ever, this macrophyte stimulated R. subcapitata

growth since the chlorophytes present physi-

ological mechanisms that protect them against

the allelopathic effects of C. demersum. The

cladoceran M. micrura also reduced the bio-

mass of M. aeruginosa and favored the growth

of R. subcapitata, but in a less remarkable way

than the macrophyte C. demersum, also differ-

ing from previous findings where zooplankton

can graze on palatable food and stimulate

cyanobacterial growth (Leitão et al., 2018). As

we used a cladoceran isolated from a hyper-

eutrophic reservoir, already adapted to cyano-

bacterial blooms, it could efficiently graze on

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cyanobacteria. In small ratios, M. aeruginosa

was severely inhibited by R. subcapitata in

comparison to the competitor. However, in

equal and dominant proportions, no cyano-

bacteria inhibition was observed. Unlike M.

aeruginosa, R. subcapitata was not inhibited

in low proportions but maintained constant

growth when compared to the control. On the

other hand, in cultures with lower proportions

of M. aeruginosa, chlorophyte was stimulated.

These results reinforce the applicability of

submerged macrophytes and cladocerans, both

isolated and combined, to control cyanobacte-

rial blooms. The coexistence with other micro-

algal species does not reduce the allelopathic

effect of the submerged macrophytes or grazing

efficiency of the cladoceran on cyanobacteria

but can increase the inhibitory effect when the

proportion of cyanobacteria is low. However, it

is important to use organisms already adapted

to the cyanobacterial blooms and eutrophic con-

ditions. Besides that, the reduction of external

sources of nutrients and fish biomanipulation

should be considered to allow the development

of macrophytes and zooplankton.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is

no conflict of interest of any kind; and that we

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowled-

gements section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

This work was supported by the Bra-

zilian National Council of Technological

and Scientific Development (CNPq) Brazil

(grant ID 305829/2019-0), and Fundação de

Amparo à Ciência e Tecnologia do Estado

de Pernambuco (FACEPE), Brazil (grant ID

BIC-1020-2.03/18).

RESUMEN

Efectos de un macrófito sumergido

Ceratophyllum demersum (Ceratophyllaceae)

y un cladócero Moina micrura (Cladocera: Moinidae)

sobre las interacciones de microalgas

Introducción: Las proliferaciones de cianobacterias en

los cuerpos de agua tropicales son cada vez más comunes,

debido a la eutrofización y al aumento de las temperaturas.

En consecuencia, muchos sistemas de agua dulce se ven

afectados por la reducción de la calidad del agua, la biodi-

versidad y los servicios de los ecosistemas. Con el aumento

de la frecuencia de la proliferación de algas nocivas, el

desarrollo de herramientas biológicas para mejorar la cali-

dad del agua es urgente.

Objetivo: Evaluar los efectos de una macrófita sumer-

gida y un cladócero sobre la cianobacteria productora de

microcistina llamada Microcystis aeruginosa (NPLJ-4) y

la clorofita Raphidocelis subcapitata (BMIUFRPE-02) en

cultivos mixtos.

Métodos: Se realizaron dos experimentos paralelos duran-

te diez días para evaluar los efectos de la macrófita

sumergida Ceratophyllum demersum y el cladócero Moina

micrura sobre las interacciones microalgales. Se cultivaron

cepas de microalgas en el medio de cultivo ASM1, en con-

diciones controladas de laboratorio. El primer experimento

presentó cuatro tratamientos: M (C. demersum), Z (M.

micrura), MZ (C. demersum y M. micrura) y C (control).

El segundo experimento consistió en cinco tratamientos, en

el que las microalgas se cultivaron juntas en diferentes pro-

porciones de Microcystis:Raphidocelis: 1:0, 3:1, 1:1, 1:3 y

0:1. La biomasa y las tasas de crecimiento de las cepas se

evaluaron cada dos días, y se trataron estadísticamente con

ANOVA de medidas repetidas de dos o tres factores.

Resultados: En el primer experimento, M. aeruginosa se

inhibió significativamente en los tratamientos M y MZ a

partir del segundo día, y en Z a partir del cuarto, mientras

que R. subcapitata no mostró reducción de su biomasa en

ningún tratamiento. Por otro lado, R. subcapitata fue esti-

mulada a partir del octavo y décimo día en el tratamiento M

y solo en el octavo día en el tratamiento Z. En el segundo

experimento, M. aeruginosa se inhibió significativamente

cuando se cultivó con R. subcapitata en proporciones bajas

(proporción de Microcystis:Raphidocelis de 1:3) durante

todo el experimento, mientras que la clorófita se estimuló

en ese tratamiento.

Conclusiones: La coexistencia de una cianobacteria con

un alga verde no alteró la principal respuesta negativa de

M. aeruginosa a la macrófita sumergida y al zooplanc-

ton, sino que estimuló al alga verde. En consecuencia, la

introducción de macrófitos y cladóceros sumergidos ya

adaptados a las condiciones eutróficas, tanto aislados como

combinados, resultó ser un buen método para controlar las

proliferaciones de cianobacterias sin afectar negativamente

a otras especies de fitoplancton coexistentes.

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Palabras clave: proliferaciones de microalgas; alelopatía;

biomanipulación; competencia; pastoreo.

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