# New insights on the species-specific allelopathic interactions between macrophytes and marine HAB dinoflagellates

Hela Ben Gharbia, Ons Kéfi-Daly Yahia, Philippe Cecchi, Estelle Masseret, Zouher Amzil, Fabienne Hervé, Georges Rovillon, Habiba Nouri, Charaf M'rabet, Douglas Couet, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Hela Ben Gharbia, Ons Kéfi-Daly Yahia, Philippe Cecchi, Estelle Masseret, Zouher Amzil, et al. New insights on the species-specific allelopathic interactions between macrophytes and marine HAB dinoflagellates. PLoS ONE, 2017, 12 (11), pp.e0187963. 10.1371/journal.pone.0187963. [https://doi.org/10.1371/journal.pone.0187963](https://doi.org/10.1371/journal.pone.0187963).

HAL Id: hal-01929204

https://hal.umontpellier.fr/hal-01929204

Submitted on 26 May 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH ARTICLE

# New insights on the species-specific allelopathic interactions between macrophytes and marine HAB dinoflagellates

Hela Ben Gharbia¹*, Ons Kéfi-Daly Yahia¹, Philippe Cecchi²*, Estelle Masseret², Zouher Amzil³, Fabienne Herve³, Georges Rovillon³, Habiba Nouri⁴, Charaf M'Rabet¹, Douglas Couet⁴, Habiba Zmerli Triki¹, Mohamed Laabir²

¹ Research Group on Oceanography and Plankton Ecology, Tunisian National Institute of Agronomy (INAT), IRESA-Carthage University. U.R.13ES36 Marine Biology (University of Tunis El Manar), Tunis, Tunisia,

² Center for Marine Biodiversity, Exploitation and Conservation (MARBEC): IRD, IFREMER, CNRS, Montpellier University, Montpellier, France, 3 IFREMER-Phycotoxins Laboratory, Nantes, France, 4 Institut de Recherche pour le Développement (IRD), Tunis, Tunisia

Current address: CRO (Centre de Recherches Océanologiques), Abidjan, Ivory Coast.

* hela.bg@hotmail.fr

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

## OPEN ACCESS

**Citation:** Ben Gharbia H, Kéfi-Daly Yahia O, Cecchi P, Masseret E, Amzil Z, Herve F, et al. (2017) New insights on the species-specific allelopathic interactions between macrophytes and marine HAB dinoflagellates. PLoS ONE 12(11): e0187963.
https://doi.org/10.1371/journal.pone.0187963

**Editor:** Hans G. Dam, University of Connecticut, UNITED STATES

**Received:** May 29, 2017

**Accepted:** October 30, 2017

**Published:** November 17, 2017

**Copyright:** © 2017 Ben Gharbia et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Funding:** This work was supported by the LAGUNOTOX project funded by Fondation TOTAL. Thanks to LMI COSYS-Med (Laboratoire Mixte International Contaminants et Ecosystèmes Marins Sud Méditerranéens) and to IRD (Institut de Recherche pour le Développement) for funding Hela Ben Gharbia's Ph.D. The funders had no role in study design, data collection and analysis,

## Abstract

Macrophytes are known to release allelochemicals that have the ability to inhibit the proliferation of their competitors. Here, we investigated the effects of the fresh leaves of two magnoliophytes (Zostera noltei and Cymodocea nodosa) and thalli of the macroalgae Ulva rigida on three HAB-forming benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima, and Coella monotis). The effects of *C. nodosa* and *U. rigida* were also tested against the neurotoxic planktonic dinoflagellate *Alexandrium pacificum* Litaker sp. nov (former *Alexandrium catenella*). Co-culture experiments were conducted under controlled laboratory conditions and potential allelopathic effects of the macrophytes on the growth, photosynthesis and toxin production of the targeted dinoflagellates were evaluated. Results showed that *U. rigida* had the strongest algicidal effect and that the planktonic *A. pacificum* was the most vulnerable species. Benthic dinoflagellates seemed more tolerant to potential allelochemicals produced by macrophytes. Depending on the dinoflagellate/macrophyte pairs and the weight of leaves/thalli tested, the studied physiological processes were moderately to heavily altered. Our results suggest that the allelopathic activity of the macrophytes could influence the development of HAB species.

## Introduction

Allelopathy is a prevalent natural phenomenon in terrestrial and aquatic ecosystems. It is now widely accepted that plants, macrophytes and various microorganisms can produce and release chemicals into the surrounding environment [1–3]. Allelopathy has been extensively studied in terrestrial habitats and harmful effects of plants on other plants or crops are quite well known [4]. The involved allelopathic compounds (allelochemicals) have been explored as natural substitutes of pesticides for pest control [5,6]. In aquatic ecosystems, allelopathy has been more investigated in freshwater environments than in marine habitats [7,8].
decision to publish, or preparation of the
manuscript.

**Competing interests:** We, the authors, declare that no competing interests exist concerning TOTAL foundation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Harmful algal blooms (HAB) occur frequently in both freshwater and marine areas, and represent a significant threat to water-supply reservoirs, fisheries, aquaculture, public health, and tourism [9–11]. In order to control and mitigate the proliferation and dispersion of HAB species, different strategies have been adopted [12]. However, the most well known methods (such as the dispersion of flocculant clays [13] or the use of copper sulfate [14]) are still expensive, time-consuming, and may have dangerous environmental consequences.

It has been demonstrated, *in situ*, that microalgae are less abundant in the presence of macrophytes [15,16], which suggests that seaweeds and seagrasses might produce and release allelochemicals acting as natural biological mitigation agents. It has been reported that different macrophytes were able to reduce the proliferation of red tide species with relatively low detrimental effects on the surrounding environment [17–19]. The inhibitory properties of macrophytes are mainly attributed to the action of the released bioactive molecules [2,7,20]. These allelochemicals are mainly secondary metabolites [21] that might be released either actively by exudation from intact living tissue or passively by leaching, leaf wounds or decaying shoots [8,21]. The most well known molecules are phenolic acids, flavonoids, tannins, terpenoids, alkaloids, and various polyunsaturated fatty acids (PUFAs) [7,17].

Knowing the structural diversity of allelochemicals, it is evident that allelopathy must involve more than one mechanism of action. It has been shown that allelochemicals are likely to disturb a variety of physiological processes of the target organisms, such as mitosis, cell division, membrane permeability, ion and water uptake, cell structure and morphology, respiration, photosynthesis, enzyme activity, signal transduction, and protein and nucleic acid synthesis [20,22–25].

Studies on the allelopathic interactions between macrophytes and HAB-forming benthic dinoflagellates are rare [26], since most of the research focuses on the potential effects of allelochemicals on planktonic species. Investigations on the allelopathy exerted by macrophytes on marine benthic dinoflagellates will be of great interest for mitigation purposes. In fact, many of these organisms are emergent HAB species involved in the production of potent toxins, that may threaten both ecosystem functioning and human health [27,28].

Here, we investigated the nature of the allelopathic interactions between three widely distributed macrophytes (*Zostera noltei*, *Cymodocea nodosa*, and *Ulva rigida*) and three HAB-forming marine benthic dinoflagellates (*Ostreopsis cf. ovata*, *Prorocentrum lima*, and *Coolia monotis*). The potential allelopathic effects of the magnoliophyte *C. nodosa* and the green macroalgae *U. rigida* were also tested on the neurotoxic planktonic dinoflagellate *Alexandrium pacificum*, whose sensitivity to *Zostera* spp. allelochemicals has already been demonstrated [29].

The studied benthic dinoflagellates (*O. cf. ovata*, *P. lima*, and *C. monotis*) constitute a significant part of epibenthic assemblages in marine ecosystems worldwide. *O. cf. ovata* can produce palytoxin, ovatoxins, and mascarenotoxins [30–34] and is responsible for recurrent toxic blooms in the Mediterranean Sea (up to 1.8 x 10⁶ cells.L⁻¹) with notable effects on socio-economic activities and public health [35,36]. *P. lima*, a cosmopolitan toxic dinoflagellate, is also known to produce several toxins, such as okadaic acid, dinophysistoxins, prorocentrolide, and prorocentin [37–39], that can cause diarrhetic shellfish poisoning episodes. Recently, a bloom of *P. lima* was recorded in Cartagena Bay (Cartagena de Indias, Colombian Caribbean) with cell abundances reaching 2.1 to 4.5 x 10⁶ cells.L⁻¹[40]. *C. monotis* is a bloom forming species able to reach high cell densities (5 x 10⁵ cells.L⁻¹ reported in the North Lake of Tunis-Tunisia [41]), but its toxic properties are not confirmed [34]. The planktonic dinoflagellate *A. pacificum* produces potent neurotoxins responsible for paralytic shellfish poisoning syndrome, and is known to induce extensive blooms in marine waters worldwide (up to 1.4 x 10⁷ cells.L⁻¹ reported in Thau lagoon, French Mediterranean coast [42]) with disastrous effects on fisheries and aquaculture [43].
The three tested macrophytes are cosmopolitan species. *Z. noltei* occurs in European, African, and Atlantic coasts [44]. It has been suggested that the chemical content of *Zostera* species might negatively affect growth and/or photosynthesis of microalgae [29,45–47]. *Cymodocea nodosa* is one of the most important magnoliophytes in the Mediterranean Sea, although data on allelo-chemicals released by this macrophyte and information about their potential effects on surrounding organisms are very limited [48–50]. *Ulva rigida* is a common green subtidal marine seaweed distributed worldwide. *Ulva* species are “green tide” forming macroalgae, which are known to efficiently weaken HABs due to their negative allelopathic properties [51–54].

The aim of our study was to examine the potential allelopathic interactions induced by macrophytes (*Z. noltei*, *C. nodosa*, and *U. rigida*) on HAB-forming dinoflagellate species, including benthic (*O. cf. ovata*, *P. lima*, and *C. monotis*) and planktonic (*A. pacificum*) microorganisms. Co-culture experiments of each microalgae with fresh macrophyte leaves/thalli were performed through controlled laboratory experiments in microcosms. The allelopathic effects of the tested macrophytes on various physiological processes of the dinoflagellate species including growth, photosynthesis, and toxin production were investigated.

# Material and methods

## Dinoflagellate cultures

Non-axenic monoclonal cultures of the three thermophilic benthic dinoflagellates *Ostreopsis cf. ovata* (OOBZT14), *Prorocentrum lima*, (PLBZT14) and *Coolia monotis* (CMBZT14) were grown in enriched natural seawater culture medium (ESNW medium; NO₃⁻ and PO₄³⁻ concentrations: 549 µmol.L⁻¹ and 22.4 µmol.L⁻¹, respectively [55]) at stable conditions (salinity: 36; temperature: 25°C; irradiance: 100 µmol photons.m⁻².s⁻¹ in a 12:12 light:dark cycle). The planktonic dinoflagellate *Alexandrium pacificum* (ABZ1) (former *A. catenella*, [56]) was cultured under the same conditions but at a temperature of 20°C, which corresponds to its optimal growth [57]. OOBZT14, PLBZT14, and CMBZT14 strains were isolated from the Bizerte Bay [34] while the ABZ1 strain was obtained from the culture collection of the Center for Marine Biodiversity, Exploitation and Conservation (Montpellier University, France) and was originally isolated from the Bizerte lagoon [58]. Both sites are located in Northern Tunisia, Southern Mediterranean Sea.

## Macrophyte collection site

Fresh leaves/thalli of the three macrophytes *Zostera noltei*, *Cymodocea nodosa* and *Ulva rigida* were collected between April and October 2015 in the Bizerte lagoon (Fig 1). This lagoon is dominated by dense *C. nodosa* beds. It is also characterized by *U. rigida* mats in its eastern part and by the presence of patchy *Z. noltei* meadows in its western part. The collection of macrophyte samples for scientific purposes didn't require a specific authorization. Macrophytes were carefully gathered to keep belowground parts intact. All samples were placed in plastic boxes containing *in situ* seawater to prevent evaporation, and then immediately transported to the laboratory. Plant material was initially washed with freshwater to remove sand and salt, then carefully cleaned with filtered seawater before being briefly rinsed with distilled water to remove potential attached organisms.

## Dinoflagellate-macrophyte co-incubations

Four different weights of each macrophyte were tested on each dinoflagellate species. For experiments with the two magnoliophytes (*Z. noltei* and *C. nodosa*), the dinoflagellates were cultured with 0.1 g, 0.3 g, 0.75 g, and 1.5 g fresh weight (FW) of leaves. For experiments with the macroalgae (*U. rigida*), 0.08 g, 0.16 g, 0.5 g and 1.0 g FW of thalli were tested. Cleaned fresh leaves/thalli
<figure><img src="image_5.png" /><figcaption>**Fig 1. Macrophyte collection sites (North of Tunisia, Southern Mediterranean Sea).** Circle: Menzel Jemil station; Triangle: Menzel Bourguiba station.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0187963.g001

were blotted dry, weighed, and placed in 250 mL culture flasks filled with culture medium. Each flask was inoculated with dinoflagellates in order to obtain an initial concentration of ca. 800–1000 cell.mL⁻¹ in a final volume of 200 mL. The obtained concentrations of leaves/thalli were comparable to those observed *in situ* [15,59–61]. All strains were cultured to the exponential phase before inoculation, and all experiments were conducted in triplicate over a time course of 10 days. For each experiment, controls (10-day incubations of the four dinoflagellates in ESNW without macrophytes) were also performed in triplicate. One gram FW of each macrophyte was dried in a drying oven in order to determine the equivalent dry weights (DW) of leaves/thalli. Allelopathic effects of *Z. noltei*, *C. nodosa*, and *U. rigida* fresh leaves/thalli were tested on the three benthic strains, while only *C. nodosa* and *U. rigida* were tested on the planktonic *A. pacificum*.

At the beginning (Day 0) and the end (Day 10) of each experiment, aliquots (15 mL) were taken from each flask. pH and dissolved oxygen were measured with a multiparameter HACH (HQ40d multi) sensor. Samples were then filtered (Whatman GF/F, diameter 47 mm, porosity 0.7 µm) and stored at -20°C for nutrient analysis. Concentrations of the main nutrients (NO₃⁻ and PO₄³⁻) were analyzed with an automated channel Technicon autoanalyzer (Seal Analytical continuous flow AutoAnalyzer AA3) using conventional colorimetric methods [62]. These measurements were performed in order to ensure that no deleterious effects, potentially associated with an eventual nutrient limitation or drastic variations of pH and/or oxygen level occurred during the time course of the incubation experiments.

## Effect of fresh leaves/thalli on dinoflagellate growth

Dinoflagellate cell densities were monitored at Days 0, 1, 3, 6, 8, and 10 by direct microscopic counts of cells. Maximum growth rates (µmax; expressed in day⁻¹) were calculated according to Guillard [63] from the slope of a linear regression over the entire exponential phase of growth
by the least squares fit of a straight line to the data after logarithmic transformation: $\mu_{max} = [\text{Ln}(N_t)-\text{Ln}(N_0)/(T_t-T_0)]$ where $N_0$ and $N_t$ are the cell densities (cells.mL$^{-1}$) at the beginning ($T_0$) and the end ($T_t$) of the exponential phase, respectively. The EC$_{50}$ (effective concentrations inducing a 50% reduction of dinoflagellates growth when compared to the control) were determined using curves that link the observed growth rates to the tested macrophyte weights (FW).

## Effect of fresh leaves/thalli on dinoflagellate photosynthetic activity

The efficiency of the photosynthetic apparatus of the four dinoflagellate species was assessed with a portable pulse amplitude modulated fluorometer AquaPen-C AP-C 100 device (Photon Systems Instruments, Czech Republic), measuring chlorophyll fluorescence parameters and by using the FluorPen 1.0.4.2 software to access the data. The OJIP protocol (Chlorophyll Fluorescence Induction Kinetics, [64]) was applied after a 30-min dark-adaptation period before measurements. Photosynthetic activity was monitored at Days 1, 3, 6, and 10 and the ratio Fv/Fm, corresponding to the maximum quantum yield of Photosystem II (PSII), was used to characterize the physiological status of the microalgae, as it is classically done [65].

## Effect of fresh leaves/thalli on dinoflagellate morphology

Qualitative observations of the dinoflagellate cell morphology were performed microscopically (at 400x magnification) at the end of each experiment (Day 10). Up to 30 cells of each culture (controls and treatments) were photographed and analyzed using an inverted microscope (Zeiss Axiovert 25) connected to a camera (Canon G3). For some experiments, cells were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and nuclear DNA was observed (at 630x magnification) with a Zeiss microscope (Zeiss Axioimager Z1 upright microscope).

## Effect of fresh leaves/thalli on dinoflagellate toxin production

For experiments with *O. cf. ovata*, *P. lima*, and *A. pacificum*, toxin profiles and contents were analyzed in order to assess the effect of the tested macrophytes on the toxin production of each dinoflagellate. A defined volume of the cultures was harvested at Day 10. Cells were centrifuged at 3500 x g for 10 min at 4°C and the supernatant was carefully removed. The pellets were stored at -20°C until toxin analysis. For *O. cf. ovata* and *P. lima*, the methods used to analyze the toxins were those described in Ben Gharbia et al. [34]. Toxin analyzes were performed as described by Laabir et al. [42] for *A. pacificum*.

## Dinoflagellate behavior

During the time course of the experiments (Days 1, 3, 6 and 10), the adhesion of the dinoflagellate species to the macrophyte leaves/thalli (vicinity and, attachment) was monitored and observed using an inverted microscope (Zeiss Axiovert 25).

## Statistical analyzes

Data were analyzed using one-way analysis of variance (ANOVA) in order to check for the existence of significant differences between control and treatments (different macrophyte weights). Two-way ANOVA (in considering both macrophyte weights and dinoflagellate species) was performed in order to clarify the relative sensitivity of the different dinoflagellates to each macrophyte, when exposed to the same gradient of thalli/leaf weights. In both cases, Tukey post-hoc tests were performed to segregate groups of similar responses within the
different series of results (growth rate, photosynthetic activity and toxin production). The sig-
nificance level was set at p < 0.05. Statistical analyzes were performed using the software Sig-
maStat (v3.5, Systat Software Inc.).

**Results**

Experimental conditions

For all experiments, no significant differences (p > 0.05) were observed between the pH and dissolved oxygen values of the controls and the different treatments. Nutrient analyzes showed a decrease in NO₃⁻ and PO₄³⁻ concentrations throughout the duration of the incubation experiments (see S1 Appendix). Nutrient concentrations at the end of our experiments remained at saturated levels for dinoflagellate requirements. Mean initial and final concentrations ranged between 418.5–484.4 (Day 0) and 192.4–376.6 µmol.L⁻¹ (Day 10) for NO₃⁻ and between 16.8–18.0 (Day 0) and 9.1–10.1 µmol.L⁻¹ (Day 10) for PO₄³⁻. The minimum values for NO₃⁻ and PO₄³⁻ were recorded in the presence of *U. rigida* thalli and were equal to 177.8 µmol.L⁻¹ and 8.0 µmol.L⁻¹, respectively.

Effects of fresh macrophyte leaves/thalli on dinoflagellate growth

Zostera noltei fresh leaves reduced the growth of the three benthic dinoflagellates. Compared to the controls (macrophyte free), cell density reduction at the end of the experiment (Day 10), was about 20–47% for *P. lima* and 21–24% for *O. cf. ovata* (across all treatments), but none of these apparent inhibitions was statistically significant (one-way ANOVA, F = 2.572, p = 0.103 and F = 0.581, p = 0.683, respectively; Fig 2A). In contrast, a pronounced inhibition was observed for *C. monotis* (one-way ANOVA, F = 5.813, p = 0.011), which was up to 55% for the highest weight tested. This treatment was statistically different from all the others, which were aggregated by the Tukey post-hoc procedure (Fig 2A). The effect of *Z. noltei* on growth rates was statistically significant only for *P. lima* (one-way ANOVA, F = 4.254; p = 0.029) with a decline ranging between 26% and 43%. Nevertheless, this decrease was independent from the weight of the tested macrophyte (Fig 3A). Growth rate reduction varied between 11% and 24% for *O. cf. ovata* and between 4% and 18% for *C. monotis*, but in both cases, no statistically significant differences were recorded (one-way ANOVA, F = 1.588, p = 0.252 and F = 2.912, p = 0.078, respectively; Fig 3A). Two-way ANOVA, performed on growth rates, confirmed that the effects of the dinoflagellate species and those of the different treatments were statistically significant (F = 8.201, p = 0.001 and F = 4.099, p = 0.009, for the effects of the dinoflagellate species and of the different treatments, respectively). Post-hoc tests failed to separate *C. monotis* and *O. cf. ovata*, but have discriminated *P. lima*, which suggests a higher sensitivity of this species to *Z. noltei* when compared to the two other benthic dinoflagellates.

In the presence of *C. nodosa* leaves, *O. cf. ovata*, *P. lima* and *C. monotis* cell densities were not statistically different from those of the controls at the end of the experiments (one-way ANOVA, F = 0.280, p = 0.884; F = 1.670, p = 0.233 and F = 0.728, p = 0.593 respectively; Fig 2B).

The growth rates of the three benthic dinoflagellates were affected differently. A slight inhi-
bition, ranging between 1–12% for *O. cf. ovata* (one-way ANOVA: F = 0.262, p = 0.896) and
between 3–11% for *C. monotis* (one-way ANOVA: F = 1.457, p = 0.286), was recorded (Fig
3B). A statistically significant negative effect was observed only on *P. lima* growth rates (one-
way ANOVA: F = 4.138, p = 0.031), and two different clusters of treatments have been segre-
gated by the Tukey post-hoc test with a decrease of about 30–40% for the two most concen-
trated treatments. In contrast, the planktonic *A. pacificum* was dramatically inhibited by the
presence of *C. nodosa* leaves, which induced a strong decrease in cell densities (one-way
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Fig 2.** Normalized final cell densities (% of the control) of the tested dinoflagellates, exposed to different weights of fresh leaves/thalli of *Zostera noltei* (a), *Cymodocea nodosa* (b) and *Ulva rigida* (c) at the end of the experiments (Day 10). Error bars correspond to the standard deviation (N = 3 replicates). The inscription 'ns' above bars indicates a statistically non-significant one-way ANOVA. 'p-values' associated with significant one-way ANOVA are provided; in such cases and for each dinoflagellate species, values that did not differ at the 0.05 level (Tukey post-hoc test) are assigned the same letter. (*C.m.*: *Coolia monotis*; *O.cf.*: *Ostreopsis cf. ovata*; *P.l.*: *Prorocentrum lima*; *A.p.*: *Alexandrium pacificum*).

https://doi.org/10.1371/journal.pone.0187963.g002
<figure><img src="image_9.png" /><figcaption>**Fig 3.** Normalized maximum growth rates (% of the control) of dinoflagellate cells growing with fresh leaves/thalli of *Zostera noltei* (a), *Cymodocea nodosa* (b) and *Ulva rigida* (c). Error bars correspond to the standard deviation (N = 3 replicates). The inscription ‘ns’ above bars indicates a statistically non-significant one-way ANOVA. ‘p-values’ associated with significant one-way ANOVA are provided; in such cases and for each dinoflagellate species, values that did not differ at the 0.05 level (Tukey post-hoc test) are assigned the same letter. (C.m: *Coolia monotis*; O.cf.o: *Ostreopsis cf. ovata*; P.l: *Prorocentrum lima*; A.p: *Alexandrium pacificum*).</figcaption></figure>

https://doi.org/10.1371/journal.pone.0187963.g003

ANOVA: F = 59.932, p < 0.001), up to 93% for the highest weight tested (Fig 2B). Highly significant growth-rate inhibitions (ranging between 35% and 58%) were observed (one-way ANOVA: F = 51.925, p < 0.001). The effect levels depended on the macrophyte weight as highlighted by the Tukey post-hoc test, which distinguished three groups of treatments (Fig 3B). The EC₅₀ value for *A. pacificum* cultured with fresh *C. nodosa* leaves was 3.4 g.L⁻¹ FW
(equivalent to 0.72 g.L⁻¹ DW). Two-way ANOVA, performed on growth rates, confirmed that the effects of the dinoflagellate species and those of the different treatments were statistically significant (F = 13.732, p < 0.001 and F = 21.900, p < 0.001, for both factors, respectively). Post-hoc tests highlighted the highest sensitivity of the planktonic dinoflagellate *A. pacificum*, which was systematically segregated regardless of the concentration tested. *P. lima* and *A. pacificum* were grouped in a cluster significantly different from *C. monotis* and *O. cf. ovata* for the two highest concentrations, indicating again a more pronounced sensitivity of *P. lima* when compared to the two other benthic species.

The macroalgae *U. rigida* induced the most important and significant decrease in cell abundances of the three benthic species after 10 days of co-cultures (p < 0.001, for the three dinoflagellates; Fig 2C). Compared to the controls, growth rates decreased between 42% and 62% for *O. cf. ovata* (one-way ANOVA: F = 6.002, p = 0.010), between 38% and 51% for *P. lima* (one-way ANOVA: F = 3.949, p = 0.036), and between 35% and 47% for *C. monotis* (one-way ANOVA: F = 7.055, p = 0.006) (Fig 3C). The EC₅₀ values for *U. rigida* were 1 g.L⁻¹ (equivalent to 0.153 g.L⁻¹ DW) for *O. cf. ovata* and 2.35 g.L⁻¹ (equivalent to 0.36 g.L⁻¹ DW) for *P. lima*. Inhibition did not exceed 47% for *C. monotis* cells exposed to *U. rigida*, and the calculation of the EC₅₀ value was thus not possible. The growth of *A. pacificum* was highly affected by *U. rigida* (one-way ANOVA: F = 529.376, p < 0.001): all of the *A. pacificum* cells exposed to the three highest weights tested died at Day 6, and the EC₅₀ value was lower than 0.4 g.L⁻¹ (equivalent to 0.06 g.L⁻¹ DW) (Fig 3C). In all cases, Tukey post-hoc tests clearly separated the controls from the different treatments that were similar. Two-way ANOVA, performed on growth rates, confirmed that the effects of the dinoflagellate species and those of the different treatments were statistically significant (F = 41.918, p < 0.001 and F = 37.847, p < 0.001, for both factors, respectively). Tukey post-hoc tests clearly separated *A. pacificum* from the three benthic dinoflagellates, whose responses were not statistically different from each other. When a two-way ANOVA was performed without considering *A. pacificum*, no more additional effects of the dinoflagellate species were identified (F = 2.784, p = 0.078), which confirmed their similar responses when exposed to *U. rigida* thalli.

## Effect of fresh leaves/thalli on dinoflagellate photosynthetic activity

For all experiments, Fv/Fm values of controls increased systematically during the time course of the incubations, and were associated with healthy cultures (median values: 0.56, 0.60, 0.62, and 0.62 for Days 1, 3, 6, and 10, respectively, and in considering all the control replicates of the 11 co-incubation experiments).

Results did not reveal any effects of fresh leaves from the two magnoliophytes *Z. noltei* and *C. nodosa* on the photosynthetic activity of the three benthic dinoflagellates. The maximum quantum yields of PSII (Fv/Fm ratio) remained consistently elevated, between 0.6 and 0.7, even for the highest macrophyte weights tested. In contrast, *C. nodosa* leaves induced a statistically significant decrease of the maximum quantum yield of PSII of *A. pacificum* cultures (one-way ANOVA performed on Day 3: F = 6.909, p = 0.006 and F = 6.139, p = 0.009 on Day 10), ranging between 13% and 44% at the end of the experiment (Day 10) (Fig 4A). This emphasizes again the higher sensitivity of the planktonic dinoflagellate in its physiological responses.

A dramatic effect was observed when *A. pacificum* was co-incubated with *U. rigida*. This macroalgae induced a strong inhibition of the photosynthetic efficiency after 3 days of exposure to the thalli (one-way ANOVA performed on Day 3: F = 7.302, p = 0.005) for all treatments (Fig 4B). At Day 6, the reduction of Fv/Fm values for the lowest weight tested reached 86% when compared to the control (one-way ANOVA performed on Day 6:
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

**Fig 4.** Fv/Fm ratio (maximum quantum yield of Photosystem II) of *Alexandrium pacificum* cells in co-culture with fresh leaves/thalli of *Cymodocea nodosa* (a) and *Ulva rigida* (b); and of *Ostreopsis cf. ovata* (c) and *Prorocentrum lima* (d) cells growing with *Ulva rigida* thalli. Error bars correspond to the standard deviation (N = 3 replicates). The inscription 'ns' above bars indicates a statistically non-significant one-way ANOVA. 'p-values' associated with significant one-way ANOVA are provided; in such cases and for each day, values that did not differ at the 0.05 level (Tukey post-hoc test) are assigned the same letter.

https://doi.org/10.1371/journal.pone.0187963.g004

F = 21.382, p < 0.001), and all cells were dead for the three other treatments. No measurements were performed at Day 10, because, except for the controls, all *Alexandrium* cells died. The effect of *U. rigida* on the photosynthetic activity of the three benthic species was clearly less important than that observed for *A. pacificum*. A statistically significant decrease of the Fv/Fm ratio was observed for *O. cf. ovata* at Day 6 (one-way ANOVA: F = 4.213, p = 0.030) for the two highest weights tested (0.5 g and 1.0 g FW). This decrease was not statistically different between the two treatments (Tukey post-hoc test). The inhibition seemed more pronounced at the end of the experiment (one-way ANOVA performed on Day 10: F = 6.731, p = 0.007) for the three highest treatments (0.16 g, 0.5 g and 1.0 g FW) with Fv/Fm decreases ranging between 13 and 15% (Fig 4C). The Tukey post-hoc test failed to separate these three treatments that have been clustered into a single group. Except for the treatment 0.16 g FW of *U. rigida* thalli, which was not significantly effective on Day 6 (clustered in the same group 'a' as the control) but was active on Day 10 (clustered in group 'b'), the overlapping of error bars for the two more concentrated treatments (on Day 6 and Day 10) indicated that the observed inhibitions at the two dates were not statistically different. For *P. lima*, a statistically significant reduction of Fv/Fm (4%-12%) was recorded only on Day 10 (one-way ANOVA: F = 16.720, p < 0.001) (Fig 4D). No significant effect was observed for *C. monotis* cultures, with a marginal decrease of the Fv/Fm ratio on Day 10 that did not exceed 6% for the highest treatment when compared to the control (one-way ANOVA: F = 0.667, p = 0.629).
## Effect of the macrophytes on dinoflagellate cell morphology

Cell morphology observations at the end of the co-culture experiments did not reveal any major morphological damage on the cells of the three benthic dinoflagellates in the presence of *Z. noltei* and *C. nodosa*. In contrast, after 10 days of *A. pacificum* co-culture with *C. nodosa* (0.3, 0.75 and 1.5 g FW) deformed cells, membrane lysis and an important degradation of the intracellular contents were observed at a large scale (Fig 5A–5E).

In the presence of *U. rigida*, empty thecae or lysed and deformed *A. pacificum* cells were observed for all treatments at the end of the experiment (Fig 5F–5J). *O. cf. ovata* cells co-cultured with *U. rigida* thalli exhibited structural damage and aberrant forms (lysed or small-rounded cells) (Fig 5K–5O) while *P. lima* and *C. monotis* cells were not impacted.

Dinoflagellate vegetative cells exposed to *U. rigida* thalli were also observed under fluorescent light after staining the nucleus with DAPI, and we noticed scattered and irregular DNA for *A. pacificum* and *O. cf. ovata* (Fig 6A and 6B). No effects were found on *P. lima* and *C. monotis* cells, which were characterized by a regularly shaped nucleus and condensed chromosomes despite the treatment.

## Effect of fresh leaves/thalli on dinoflagellate toxin production

Toxin contents measured in dinoflagellate cells at the end of the different co-culture experiments revealed contrasting patterns. The exposure of *O. cf. ovata* cells to *Z. noltei* leaves seemed to induce a stimulation of ovatoxin production (OVTX-a and OVTX-b). However, results were not statistically significant due to the important within-treatments variability (Fig 7A; one-way ANOVA performed at Day 10: F = 2.124, p = 0.140 for OVTX-a, and F = 1.874, p = 0.247 for OVTX-b). For *P. lima*, the observed concentrations of okadaic acid (OA) and of dinophysistoxin-1 (DTX-1) after ten days of co-incubation with *Z. noltei* leaves were not

<figure><img src="image_14.png" /><figcaption>**Fig 5.** Light microscope observations of morphological damages of vegetative cells of the targeted dinoflagellate species. Photographs of *Alexandrium pacificum* cells cultured with *Cymodocea nodosa* (a-e) and *Ulva rigida* (f-j); and of *Ostreopsis* cf. *ovata* cultured with *Ulva rigida* (k-o). a,f,k = control cells; b-e, g-j, l-o = cells under increasing macrophyte weights. Scale bars, 10 µm.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0187963.g005
<figure><img src="image_15.png" /><figcaption>**Fig 6.** Light (a₁,a₁',b₁,b₁'), epifluorescence (a₂,a₂',b₂,b₂') and superposed light-epifluorescence (a₃,a₃',b₃,b₃') microscope photographs of dinoflagellate vegetative cells cultured with *Ulva rigida* thalli. **A:** *Alexandrium pacificum* cells (a₁-a₂-a₃ = control, a₁'-a₂'-a₃' = cell exposed to 0.16g (FW) of *Ulva rigida* after 3 days of co-culture). **B:** *Ostreopsis* cf. *ovata* cells (b₁-b₂-b₃ = control; b₁'-b₂'-b₃' = cell exposed to 1g FW of *Ulva rigida* after 10 days of co-culture). Scale bars, 10 µm.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0187963.g006

different from those measured in the controls (Fig 7B; one-way ANOVA: F = 0.481, p = 0.750 for OA, and F = 1.165, p = 0.363 for DTX-1).

The toxin content of *O. cf. ovata* cells exposed to *C. nodosa* leaves (Fig 7C) was not statistically different from that of the controls after ten days of co-incubations despite the treatment (one-way ANOVA performed at Day 10: F = 0.421, p = 0.791 for OVTX-a, and F = 0.259, p = 0.897 for OVTX-b). This was also the case for *P. lima* (Fig 7D; one-way ANOVA performed at Day 10: F = 0.439, p = 0.779 for OA, and F = 0.889, p = 0.493 for DTX-1). For *A. pacificum* (Fig 7E), the apparent increase of the cellular toxin contents was once again not statistically significant (one-way ANOVA performed at Day 10: F = 5.669, p = 0.096; F = 0.747, p = 0.479; F = 15.213, p = 0.060; and F = 1.566, p = 0.314 for GTX4, GTX3, C1 and C2 respectively). Some measurements were below the limit of detection (GTX1) or below the limit of quantification (Neo-STX), and it was not possible in such cases to perform one-way ANOVA.

In the presence of *U. rigida*, ovatoxin-a production by the *O. cf. ovata* strain was significantly enhanced (Fig 7F; one-way ANOVA performed at Day 10: F = 17.280, p < 0.001). Tukey post-hoc test clearly separated the controls from the different treatments. Several ovatoxin-b analyzes were below the limits of quantification (Fig 7F), and it was not possible to formally assess the potential impact of the macrophyte thalli. The stimulating effect of *U. rigida* on the toxin content of *P. lima* cells was also statistically confirmed (Fig 7G; one-way ANOVA performed at Day 10: F = 5.657, p = 0.005 for OA, and F = 4.969, p = 0.009 for DTX-1). Tukey post-hoc test clearly separated the control from the third treatment (0.5g), while all the other
<figure><img src="image_16.png" /><figcaption>**Fig 7.** Cellular toxin contents (pg.cell⁻¹) at the end of the experiments (after 10 days) of *Ostreopsis cf. ovata* (O. cf. ovata) and *Prorocentrum lima* (*P. lima*) in presence of the leaves/thalli of *Cymodocea nodosa* (*C. nodosa*), *Zostera noltei* (*Z. noltei*) and *Ulva rigida* (*U. rigida*), and of *Alexandrium pacificum* (*A. pacificum*) in presence of *C. nodosa* leaves. OVTX-a: Ovatoxin-a; OVTX-b: Ovatoxin-b; OA: Okadaic Acid; DTX-1: Dinophysistoxin-1; Neo-STX, GTX1, GTX3 and GTX4: Carbamoyl toxins; C1 and C2: N-sulfocarbamoyl toxins. ‘< LoD’ and ‘< LoQ’ indicate ‘< Limit of Detection’ and ‘< Limit of Quantification’, respectively. Error bars correspond to the standard deviation (N = 3 replicates, except for control (O. cf. *ovata* and *P. lima*) for which the controls of the three experiments have been pooled, N varying between 3 and 9 depending on the considered toxin). When only one among the three triplicates of each treatment was above LoD or LoQ, standard deviation was not calculable, and there is thus no error bar in such cases.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0187963.g007

treatments corresponded to intermediary responses. Quantification of *A. pacificum* toxin content has not been performed: all cells died before the end of the experiment.

## Dinoflagellate behavior

Observations of dinoflagellate behavior in co-cultures revealed that cells of the three benthic strains covered the bottom of the flasks or were suspended in water and embedded in mucus rather than attached to the macrophyte leaves/thalli. For the planktonic *A. pacificum*, we did not observe any cell attachment to the macrophytes. In the presence of *Z. noltei* and *C. nodosa*, O. cf. *ovata* and *C. monotis* cells were observed on the leaf edges but not on the surface. They formed aggregates around the ends/extremities of the leaf and used it as a support to form mucus that encompassed the cells. *P. lima* cells colonized both the edges and the entire surface of *Z. noltei* and *C. nodosa* leaves. The same pattern was observed for *P. lima* co-cultured with *U. rigida*, even if the adhesion of the cells to the thalli was less important in comparison to the two other magnoliophytes. For O. cf. *ovata*, cells adhered mainly to the edges of *U. rigida* thalli, but were also in contact with the whole surface. We noticed that O. cf. *ovata* cell attachment was less important when the weights of *Ulva* thalli increased. As for *C. monotis*, cells did not cling a lot to the thalli, and were observed only on the edges.
Thus, direct contact between the dinoflagellate cells and the entire surface of the leaves/thalli (not only the edges) was observed solely for *P. lima* co-cultured with the three tested macrophytes and for *O. cf. ovata* co-cultured with *U. rigida* (See S2 Appendix).

## Discussion

In aquatic ecosystems, primary producers (microalgae and macroalgae) are known to compete for light and nutrients [66]. Macrophytes can make an environment unsuitable for microalgal growth by reducing light and $O_2$ levels, increasing pH,uptaking nutrients, and releasing various allelopathic substances. In our study, co-cultures were conducted under stable environmental conditions. Normal pH and oxygen levels were recorded during the incubation period, and the residual concentrations of $NO_3^-$ and $PO_4^{3-}$ measured at the end of our experiments for both controls and treatments were above the limiting levels [67–69]. Thus, our results suggested that the observed inhibitory effects were mainly due to potential algalidal allelopathic compounds that could be released by the tested macrophytes, and that a shortage in nutrients or unsuitable pH or $O_2$ levels could not be incriminated.

## Effect on growth and cell morphology

To our knowledge, studies characterizing the allelopathic effect of macrophytes on benthic dinoflagellates are rather rare. Table 1 summarizes the current knowledge of the allelopathic effects associated with *Ulva* spp. and *Zostera* spp. on the growth of HAB-forming dinoflagellate species investigated in various marine ecosystems.

The present study highlighted contrasting effects of the three tested macrophytes on the growth of the four targeted dinoflagellate species. *U. rigida* exerted the highest algalidal effect, and the planktonic *A. pacificum* was the most sensitive dinoflagellate. Our results are in agreement with the observations of Accoroni et al. [26] who reported a significant allelopathic inhibitory effect of *U. rigida* thalli on *O. cf. ovata* (OoAPn0807/E). Alamsjah et al. [53] tested the effect of three algalidal compounds extracted from *Ulva* thalli on several planktonic HAB species, including *A. catenella* (NIES-677), and found a reduction of the growth of this dinoflagellate. Other authors showed that *Ulva* species could suppress the growth of different harmful dinoflagellates (Table 1). Here, for *U. rigida*, the $EC_{50}$ values were 1 $g.L^{-1}$ FW for *O. cf. ovata*, 2.35 $g.L^{-1}$ FW for *P. lima*, and less than 0.4 $g.L^{-1}$ FW for *A. pacificum*. Close $EC_{50}$ values were reported for the effects of *Ulva pertusa* (1.8 $g.L^{-1}$ FW) and *Ulva linza* (2.3 $g.L^{-1}$ FW) species on the growth of *Prorocentrum micans* [70]. In contrast, it has been shown that *Ulva pertusa* may inhibit *Alexandrium tamarense* with an $EC_{50}$ ranging between 2 and 2.5 $g.L^{-1}$ FW [51], which is much higher than that found for the *U. rigida*/*A. pacificum* pair investigated in our experiments. This suggests that the allelopathic effect is highly species-specific.

Our results demonstrated that the growth of *A. pacificum* was highly affected in comparison to the three benthic species, which highlights an increased sensitivity of the physiological processes of this planktonic dinoflagellate to potential allelochemicals produced by the macrophytes. Benthic strains seem more resistant to substances that could be released by macrophytes; this could be explained by their permanent vicinity to the leaves/thalli, since they grow attached to the plant material. Hilt [71] supported our finding and highlighted a lower sensitivity of epiphyte species to allelochemicals. This author has reported in particular that epiphytic algae and cyanobacteria would be less vulnerable than planktonic species to the allelopathic effect of *Myriophyllum spicatum* and she has suggested that these organisms might have developed resistance against allelopathic substances released by macrophytes by a co-evolutionary process [7,72].
<table><caption>Table 1. Reported allelopathic effects of *Ulva* spp. and *Zostera* spp. on harmful algal blooms dinoflagellate species in various marine ecosystems.</caption><thead><tr><th>Macrophyte species and origin</th><th>Target dinoflagellate species and strains</th><th>Effects</th><th>Tested concentrations</th><th>Time course experiments</th><th>References</th></tr></thead><tbody><tr><td colspan="6"><strong>Ulva spp.</strong></td></tr><tr><td rowspan="3">Ulva rigida (Conero Riviera, Italy)</td><td rowspan="3">Ostreopsis cf. ovata (OoAPn0807/E)</td><td>FT: GI (CR = 94%)</td><td>FT:1g.500mL<sup>-1</sup> FW</td><td>20 Days</td><td rowspan="3">[26]</td></tr><tr><td>FTF: GS</td><td>FTF: 24 g.L<sup>-1</sup> FW</td><td>23 Days</td></tr><tr><td>DP: GI (CR = 17–37%), vegetative cells replaced by cysts</td><td>DP: 0.4–0.8–1.6 g.L<sup>-1</sup> DW</td><td>18 Days</td></tr><tr><td rowspan="4">Ulva lactuca (Old Fort Pond, Long Island, NY, USA)</td><td>Prorocentrum minimum (CCMP696)</td><td>FT: (CR = 24%<sub>72h-725mg.L-1 DW</sub>), DPAE: GI<sub>180-360mg.L-1 DW</sub></td><td>FT ≈ 45-180-400-725 mg. L<sup>-1</sup> DW</td><td>5 Days</td><td rowspan="4">[54]</td></tr><tr><td>Karlodinium veneficum (FR-6)</td><td>FT: (CR = 38%<sub>72h-725mg.L-1 DW</sub>), DPAE: GI<sub>360mg.L-1DW</sub> + cells lysed after 5 days</td><td>DPAE ≈ 36-180-360-1800 mg. L<sup>-1</sup> DW</td><td>5 Days</td></tr><tr><td>Karenia brevis (CCMP2228)</td><td>FT: (CR = 50%<sub>72h-725mg. L-1DW</sub>), DPAE: GI<sub>1800mg.L-1DW</sub> + cells lysed after 5 days</td><td></td><td></td></tr><tr><td>Cochlodinium polykrikoides (CP1)</td><td>FT: (CR = 29%<sub>120h-400mg.L-1DW</sub>), DPAE: **</td><td></td><td></td></tr><tr><td rowspan="11">Ulva fasciata, Ulva pertusa, Ulva arasakii, Ulva conglobota (Nagasaki, Japan)</td><td>Alexandrium catenella (NIES-677)</td><td>PM: GI (HDTA-ALA:CR&lt;30%, ODTA: 30&lt;CR&lt;69%<sub>25µg.mL-1</sub> / CR&lt;30%<sub>5µg.mL-1</sub>)</td><td>PM (HDTA-ALA-ODTA): 5–25 µg.mL<sup>-1</sup></td><td>4 Hours</td><td rowspan="11">[53]</td></tr><tr><td>Cochlodinium polykrikoides (ND-14)</td><td>PM: GI (HDTA-ALA-ODTA:CR&lt;30%)</td><td></td><td></td></tr><tr><td>Karenia mikimotoi (NIES-680)</td><td>PM: GI (HDTA-ALA-ODTA:CR&gt;70%)</td><td></td><td></td></tr><tr><td>Heterocapsa circularisquama (ND-12)</td><td>PM: GI (HDTA-ALA-ODTA:30&lt;CR&lt;69%)</td><td></td><td></td></tr><tr><td>Heterocapsa triquetra (NIES-7)</td><td>PM: GI (HDTA-ALA: 30&lt;CR&lt;69%<sub>25µg.mL-1</sub>/CR&lt;30%<sub>5µg.mL-1</sub>, ODTA: CR&gt;70%<sub>25µg.mL-1</sub>/30&lt;CR&lt;69%<sub>5µg.mL-1</sub>)</td><td></td><td></td></tr><tr><td>Scrippsia sweeneyae (NIES-684)</td><td>PM: GI (HDTA-ALA: 30&lt;CR&lt;69%<sub>25µg.mL-1</sub>/CR&lt;30%<sub>5µg.mL-1</sub>, ODTA: CR&gt;70%<sub>25µg.mL-1</sub>/30&lt;CR&lt;69%<sub>5µg.mL-1</sub>)</td><td></td><td></td></tr><tr><td>Prorocentrum minimum (ND-34)</td><td>PM: GI (HDTA-ALA-ODTA: 30&lt;CR&lt;69%<sub>25µg.mL-1</sub> / CR&lt;30%<sub>5µg.mL-1</sub>)</td><td></td><td></td></tr><tr><td>Prorocentrum sigmoides (NIES-683)</td><td>PM: GI (HDTA-ALA-ODTA: 30&lt;CR&lt;69%<sub>25µg.mL-1</sub> / CR&lt;30%<sub>5µg.mL-1</sub>)</td><td></td><td></td></tr><tr><td>Scrippsia trochoidea (NIES-369)</td><td>PM: GI (HDTA-ALA-ODTA:CR&gt;70%<sub>25µg.mL-1</sub> / 30&lt;CR&lt;69%<sub>5µg.mL-1</sub>)</td><td></td><td></td></tr><tr><td rowspan="3">Ulva lactuca (Nanao island, South China Sea)</td><td rowspan="3">Alexandrium tamarense (**)</td><td>FT: GI (CR = 48%)</td><td>FT: 0.8 g.L<sup>-1</sup>FW</td><td>12 Days</td><td rowspan="3">[112]</td></tr><tr><td>FTF: GI first 2 days (recovered in the following days)</td><td>FTF: 80 g.L<sup>-1</sup>FW</td><td>**</td></tr><tr><td>DP: GI (EC<sub>50</sub> = 0.19 g.L<sup>-1</sup> DW)</td><td>DP ≈ 0.5-1-2 g.L<sup>-1</sup>DW</td><td>3 Days</td></tr><tr><td rowspan="4">Ulva fasciata (**)</td><td>Alexandrium tamarense (**)</td><td>PM: GI (α-linolenic acid: LC<sub>50</sub> = 66.06; linoleic acid: LC<sub>50</sub> = 98.40 µg.mL<sup>-1</sup>)</td><td>PM: α-linolenic acid and linoleic acid</td><td rowspan="4">24 Hours</td><td rowspan="4">[113]</td></tr><tr><td>Alexandrium taylori (**)</td><td>PM: GI (α-linolenic acid: LC<sub>50</sub> = 35.30; linoleic acid: LC<sub>50</sub> = 72.47µg.mL<sup>-1</sup>)</td><td>≈10<sup>-2</sup>–10<sup>-1</sup>–10<sup>0</sup>–10<sup>1</sup>–10<sup>2</sup>–10<sup>3</sup> µg.mL<sup>-1</sup></td></tr><tr><td>Gymnodinium impudicum (**)</td><td>PM: GI (α-linolenic acid and linoleic acid: LC<sub>50</sub> &gt; 1,000 µg.mL<sup>-1</sup>)</td><td></td></tr><tr><td>Heterocapsa circularisquama (**)</td><td>PM: GI (α-linolenic acid and linoleic acid: LC<sub>50</sub> &gt; 1,000 µg.mL<sup>-1</sup>)</td><td></td></tr><tr><td rowspan="5">Ulva linza (Taiping Angle of Qingdao, China)</td><td rowspan="5">Prorocentrum donghaiense (**)</td><td>FT: GI (EC<sub>50</sub> = 0.4 g.L<sup>-1</sup> DW)</td><td>FT: 0.625–1.25–2.5-5-10 g.L<sup>-1</sup>FW</td><td>10 Days</td><td rowspan="5">[114]</td></tr><tr><td>FTF: GI (CR≈94%)</td><td>FTF: 80 g.L<sup>-1</sup>FW</td><td>≈10 Days</td></tr><tr><td>DP: GI (EC<sub>50</sub> = 0.1 g.L<sup>-1</sup> DW)</td><td>DP ≈ 0.15–0.3–0.6–1.2–2.4 g.L<sup>-1</sup>DW</td><td>10 Days</td></tr><tr><td>AE: GI (EC<sub>50</sub> = 1.5 ppt)</td><td>AE ≈ 0.1–0.2–0.4–0.8–1.6 ppt</td><td>5 Days</td></tr><tr><td>ME: GI (EC<sub>50</sub> = 0.02 ppt)</td><td>ME≈ 0.025–0.05–0.1–0.2–0.4 ppt</td><td>5 Days</td></tr></tbody></table>

(Continued)
<table><caption>**Table 1. (Continued)**</caption><thead><tr><th>Macrophyte species and origin</th><th>Target dinoflagellate species and strains</th><th>Effects</th><th>Tested concentrations</th><th>Time course experiments</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Ulva pertusa (Huiquan Bay, China)</td><td>Prorocentrum donghaiense (**)</td><td>FT: GI (LT<sub>50</sub> = 37.9 h), FTF: GI (≈5%)</td><td>FT: 3.4 g.L<sup>-1</sup>FW</td><td>≈216 Hours</td><td rowspan="4">[115]</td></tr><tr><td>Alexandrium tamarense (**)</td><td>FT: GI (LT<sub>50</sub> = 59.8 h), FTF: GI (≈27%)</td><td>FTF: 3.4 g.L<sup>-1</sup>FW</td><td>≈216 Hours</td></tr><tr><td>Scrippsiella trochoide (**)</td><td>FT: GI (LT<sub>50</sub> = 63.6 h), FTF: GI (≈7%)</td><td></td><td></td></tr><tr><td>Amphidinium carterae (**)</td><td>FT: GI (≈34%), FTF: GS (≈78%)</td><td></td><td></td></tr><tr><td rowspan="5">Ulva pertusa<sup>a</sup>, Ulva linza<sup>b</sup> (Coast of Qingdao, China)</td><td>Prorocentrum micans (**)</td><td>FT: GI (EC<sub>50</sub> = 1.8<sup>a</sup> - 2.3<sup>b</sup> g.L<sup>-1</sup>FW)</td><td>FT ≈ 0.625-1.25-2.5-5-10 g.L<sup>-1</sup>FW</td><td>10 Days</td><td rowspan="5">[70]</td></tr><tr><td></td><td>FTF: No significant inhibitory effects</td><td>FTF: 40 g.L<sup>-1</sup>FW</td><td>10 Days</td></tr><tr><td></td><td>DP: GI (EC<sub>50</sub> = 0.7<sup>a</sup>—0.8<sup>b</sup> g.L<sup>-1</sup>DW)</td><td>DP ≈ 0.15-0.3-0.6-1.2-2.4 g.L<sup>-1</sup>DW</td><td>10 Days</td></tr><tr><td></td><td>AE: GI (EC<sub>50</sub> = 0.7<sup>a</sup>—1.0<sup>b</sup> ppt)</td><td>AE: 0.1 to 1.6 ppt</td><td>6 Days</td></tr><tr><td></td><td>ME: GI (EC<sub>50</sub> = 0.015<sup>a</sup>—0.017<sup>b</sup> ppt)</td><td>ME: 0.025 to 0.4 ppt</td><td>6 Days</td></tr><tr><td>Ulva conglobota<sup>a</sup>, Ulva fasciata<sup>b</sup>, Ulva pertusa<sup>c</sup> (Nagasaki Beach, Japan)</td><td>Gymnodinium mikimotoi (NIES-680)</td><td>ME: GI (Mortality = 10.0%<sup>a</sup>—71.3%<sup>b</sup>—10.1%<sup>c</sup>)</td><td>ME: ** (10µL of extract. mL<sup>-1</sup>)</td><td>4 Hours</td><td>[52]</td></tr><tr><td rowspan="2">Ulva pertusa (**)</td><td rowspan="2">Alexandrium tamarense (**)</td><td>FT: GI (≈70%)</td><td>FT: 12.5 g.L<sup>-1</sup>FW</td><td>≈11 Days</td><td rowspan="2">[116]</td></tr><tr><td>FTF: No significant inhibitory effects</td><td>FTF: 80 g.L<sup>-1</sup>FW</td><td>≈7 Days</td></tr><tr><td rowspan="3">Ulva pertusa: Non-sexual<sup>a</sup> (**) Sexual<sup>b</sup> strain (Coast of Qingdao, China)</td><td rowspan="3">Alexandrium tamarense (**)</td><td>FT: GI (EC<sub>50</sub> = 2<sup>a</sup>—2.5<sup>b</sup> g.L<sup>-1</sup>FW)</td><td>FT: 0.625-1.25-2.5-5-10 g.L<sup>-1</sup>FW</td><td>10 Days</td><td rowspan="3">[51]</td></tr><tr><td>FTF: Slight, not significant GS</td><td>FTF: 100 g.L<sup>-1</sup>FW</td><td>10 Days</td></tr><tr><td>DP: GI (EC<sub>50</sub> = 0.6<sup>a</sup>—0.8<sup>b</sup> g.L<sup>-1</sup>DW)</td><td>DP: 0.15-0.3-0.6-1.2-2.4 g.L<sup>-1</sup>DW</td><td>10 Days</td></tr><tr><td colspan="6"><strong>Zostera spp.</strong></td></tr><tr><td rowspan="3">Zostera noltii<sup>a</sup>, Zostera marina<sup>b</sup> (Thau lagoon and Arcachon bay, France)</td><td rowspan="3">Alexandrium catenella (ACT03)</td><td>AE: GI (EC<sub>50</sub> = 0.76<sup>a</sup>—0.82<sup>b</sup> g.L<sup>-1</sup>DW)</td><td>AE ≈ 0.13 to 3.34g of extract.L<sup>-1</sup></td><td>72 Hours</td><td rowspan="3">[29]</td></tr><tr><td>ME: GI (EC<sub>50</sub> = 0.12 to 0.42<sup>a</sup>—0.09 to 0.29<sup>b</sup> g.L<sup>-1</sup>DW)</td><td>ME ≈ 0.065 to 2g of extract.L<sup>-1</sup></td><td>72 Hours</td></tr><tr><td>AE+ME: Loss of motility / Loss of thecae / Retracted intracellular contents / Degradation in intracellular organelles / Some cells stopped their division / Scattered and irregular DNA.</td><td></td><td></td></tr><tr><td rowspan="2">Zostera marina (Roberts Bank, Canada)</td><td>Gonyaulax polyedra (**)</td><td>ME: GI (CR = 100%: No viable cells after 30 Days). Reduced swimming speed / Loss of motility / Loss of thecae / Extruded protoplasts / Cells disintegration.</td><td>ME(black leaves): 1.1 mg.mL<sup>-1</sup>DW</td><td>30 Days</td><td rowspan="2">[45]</td></tr><tr><td>Protogonyaulax tamarenensis (**)</td><td></td><td></td><td></td></tr></tbody></table>

FT = Fresh Tissues, FTF = Fresh Tissue Filtrate (initial dose addition), DP = Dry Powder, DPAE = Dry Powder Aqueous Extracts, PM = Pure Molecules, AE = Aqueous Extracts, ME = Methanol Extracts.

GI = Growth Inhibition, CR = Cell Density Reduction, GS = Growth Stimulation, FW = Fresh Weight, DW = Dry Weight, ≈ = from graphs/tables
** = No Data.

EC<sub>50</sub> = Effective Concentration inducing 50% reduction of dinoflagellate growth, LC<sub>50</sub> = 50% Lethal Concentration, LT<sub>50</sub> = Time at which 50% of the dinoflagellate cells are dead.

HDTA-ALA-ODTA: Hexadeca-4,7,10,13-tetraenoic acid (HDTA), α-linolenic acid (ALA), Octadeca-6,9,12,15-tetraenoic acid (ODTA).
(Macrophyte and Dinoflagellate species are named as cited in the references).

https://doi.org/10.1371/journal.pone.0187963.t001

Z. noltei induced a moderate growth inhibition with statistically significant effects exerted only on *P. lima* whereas *O. cf. ovata* and *C. monotis* were not significantly affected. The potential effects of Z. noltei fresh leaves were not tested on *A. pacificum* in our study. However, Laabir et al. [29] have shown that Z. noltei and Z. marina crude extracts induced a strong inhibitory effect on *A. catenella* cells (ACT03 strain) even at very low concentrations (0.09 g
of extract.L⁻¹). De Wit et al. [73] have observed a delay in phytoplankton growth in the presence of *Z. soltei* in situ and hypothesized a direct interference related to the excretion or leaching of allelopathic substances by this macrophyte. Harisson and Chan [45], Harisson [47], and Harisson and Durance [46] have also suggested that chemicals released by *Zostera* leaves might reduce the growth of epiphytic microorganisms and decrease carbon uptake rates in diatoms.

In our study, *C. nodosa* induced the weakest inhibition on dinoflagellate growth in comparison with the other tested macrophytes. *C. nodosa* was not significantly efficient against *O. cf. ovata* and *C. monotis* (*p* > 0.05), moderately efficient against *P. lima* (*p* = 0.031) and significantly active against *A. pacificum* (*p* < 0.001). To our knowledge, there are no data in the literature about the allelopathic activity of *C. nodosa* leaves on dinoflagellates. However, some studies have examined the biological activity of this species. Kontiza et al. [48] have reported an antiproliferative effect of two biphenyl compounds isolated from *C. nodosa* on two lung cancer cell lines. Kontiza et al. [49] have also demonstrated an antibacterial activity of metabolites isolated from *C. nodosa* against multidrug-resistant and methicillin-resistant strains of *Staphylococcus aureus* as well as rapidly growing mycobacteria.

Our results showed that severe structural anomalies were induced by *C. nodosa* on *A. pacificum* cells. Similar effects were caused by *U. rigida* thalli that altered the cellular morphology of *A. pacificum* and *O. cf. ovata*. Previous studies have reported important degradations in intracellular contents, membrane disruption, and cell shrinkage of microalgal cells exposed to the allelopathic compounds of macrophytes [29,45,74]. Potential allelochemicals also seem to have genotoxic properties, as a DNA damage was observed for *O. cf. ovata* and *A. pacificum* cells co-cultured with *U. rigida*. DNA fragmentation and chromatin dispersion have been previously observed for *O. cf. ovata* cells when exposed to aldehydes from diatoms [75] and for *A. catenella* cells when exposed to *Zostera* extracts [29]. In our study, alterations in cell structures are in agreement with the observed high mortality rates associated with *U. rigida* thalli.

## Effect on photosynthesis

At the end of the experiment (Day 10), the photosynthetic efficiency of the three benthic species was not altered by *Z. soltei* and *C. nodosa* fresh leaves. Only *U. rigida* thalli induced a moderate decrease in Fv/Fm values of *O. cf. ovata* and *P. lima*. In contrast, the photosynthetic activity of the planktonic *A. pacificum* was strongly reduced by *C. nodosa* (up to 40% after 10 days in some cases) and by *U. rigida* with an important inhibition of the Fv/Fm ratio (up to 86% at Day 6 at the lowest weight tested).

Inhibition of the photosynthetic process by allelochemicals is a well known phenomenon in aquatic ecosystems, with the Photosystem II (PSII) being the main target [2,76]. Analysis of chlorophyll a fluorescence transient is a useful tool to assess several biophysical parameters related to the efficiency of PSII (fluxes of photons, excitons, electrons, and further metabolic events; [77]). Ye et al. [77] have found that the main photosynthetic inhibition targets by the macroalgae *Gracilaria lemaneiformis* on the dinoflagellate *Scrippsiella trochoidea* were a decrease in the quantity and size of antenna chlorophyll, in the number of active reaction centers, and in the photochemical efficiency of PSII, in addition to the blocking of the electron transport chain and the damage to the oxygen-evolving complex. Ye and Zhang [78] have also shown that dried thalli of *Gracilaria tenuistipitata* inhibited the photosynthesis of *P. micans*. They have attributed this effect to the decrease or the alteration of relevant parameters such as Fv/Fm, density of reaction centers, and electron transport per PSII cross-sections (RC/CS₀ and ET₀/CS₀ when using OJIP terminology).
In freshwater ecosystems, different studies focused on the allelopathic effects of the macrophyte *Myriophyllum spicatum* on the photosynthetic activity of various microorganisms. Zhu et al. [25] have reported that allelochemicals isolated from this macrophyte were key agents in inhibiting the PSII and the whole chain activities of *Microcystis aeruginosa*. Purified tellima-grandin II and lipophilic extracts from *M. spicatum* were also found to inhibit the PSII of the cyanobacterium *Anabaena* sp. by the interruption of the photosynthetic electron transport between the primary and the secondary quinone electron acceptors (Q<sub>A</sub> and Q<sub>B</sub>). These compounds were found to inhibit electron transport between Q<sub>A</sub> and Q<sub>B</sub> due to interference with non-heme iron [76]. It has been also reported that the cyclic sulfur compounds dithiolane and trithiane from *Chara globularis* can affect carbon uptake of diatoms and other phytoplankton species [79]. Nevertheless, the impairment of the photosynthetic activity of the targeted organisms by allelochemicals remains unclear, and the detailed mechanisms of action need further investigation.

## Effect on toxin production

To our knowledge, no data are available in the literature concerning the allelopathic effect of macrophytes on the toxin production of dinoflagellates. Our results revealed contrasting patterns. The observed increase in toxin contents of *O. cf. ovata* cells exposed to *Z. noltei* and *A. pacificum* cells exposed to *C. nodosa* leaves was not statistically confirmed due to the important within-treatment variability. Only, *U. rigida* thalli induced a significant stimulation of the toxin production of the two benthic dinoflagellates *O. cf. ovata* and *P. lima*. The induced effects were not dose-dependent for *O. cf. ovata*, as it seemed to be the case for *P. lima*. We can hypothesize that microalgae exposed to stressful conditions first enhance their toxin production, then, when the cell metabolism is altered and/or structural damages appear, the microorganisms may reduce or lose their capacity to produce toxins. Factors affecting the toxin production remain poorly known and results are often contradictory [80]. An enhancement of toxicity levels in P-limited dinoflagellate cultures and low toxin contents in N-limited cultures have been reported [80–82]. But, Vanucci et al. [83] found a significant increase in okadaic acid amounts of *P. lima* cells under both N and P limitations, while Vanucci et al. [84] observed a decrease in toxin content of *O. cf. ovata* cells under N- and P-limited conditions. Further research is needed to clarify how allelochemicals from macrophytes could influence dinoflagellate toxin production since an increase in the toxin contents could have important implications when using macrophytes as bloom mitigation agents.

## Dinoflagellate behavior and natural association with macrophytes

In our study, the magnoliophytes *Z. noltei* and *C. nodosa* induced a lower inhibitory effect compared to *U. rigida*. Previous *in situ* studies have reported that *Cymodocea* spp. and *Zostera* spp. represent typical host species and are usually colonized by epiphytes. Turki and El Abed [85], Turki [86], and Aligizaki et al. [87] have indeed reported a high abundance of *P. lima* cells on *C. nodosa* leaves. Foden et al. [88] have identically observed high densities of *P. lima* associated with *Zostera* beds. However, our results showed that among all the benthic dinoflagellates, *P. lima* was the most sensitive to the bioactivity of the tested magnoliophytes, with systematic significant responses whatever the macrophyte considered, whereas *O. cf. ovata* and *C. monotis* appeared sensitive only to the presence of *U. rigida* thalli. In addition to different physiological adaptations acquired along co-evolutionary processes, it can be hypothesized that the highest vulnerability of *P. lima* might be due to its distinct behavior in culture. *P. lima* cells were motionless and attached to the entire surface of the leaves, thus enhancing the cellular exposure to the allelochemicals. In contrast, *O. cf. ovata* and *C. monotis* were suspended in
the water column and attached only to the edges of the magnoliophyte leaves. Direct contact between *P. lima* cells and macrophyte leaves may therefore promote the inhibitory effect. Our results suggest that dinoflagellate adhesion patterns should be also taken into account in order to better explain the observed allelopathic effects. Concerning *Ulva* thalli, field surveys showed contradictory observations. Low epiphytic dinoflagellate abundances have been reported [89,90], while moderate to high cell densities were also observed on the macroalgae depending on the species and the marine habitats studied [91–93]. Otherwise, it has been suggested that macrophyte morphotypes can drive host preference trends. Parsons and Preskitt [91] have thus found that *P. lima* and *C. monotis* preferred microfilamentous macroalgae, while *O. ovata* cells were more abundant on microblades thalli.

Nevertheless, it has been suggested [26,91] that epiphytic regulation and host preferences seem to depend mostly on the specific requirements of dinoflagellates and on the inhibitory efficiency of the allelochemicals released by the macrophytes.

## Chemicals potentially responsible for the observed allelopathic effects

Table 2 summarizes the known chemicals identified, produced, and released by *U. rigida*, *Z. noltei*, and *C. nodosa* species and their reported potential biological activity (See also S3 Appendix). From the data gathered in Tables 1 and 2 and in the S3 Appendix, we can hypothesize that the inhibitory effect caused by *Z. noltei* and *C. nodosa* on dinoflagellate species was mainly related to the production of polyphenols, whereas for *U. rigida*, polyunsaturated fatty acids (PUFAs) seem to be the most incriminated inhibitory compounds. However, the identification of these potential allelochemicals in our own macrophyte species and the evaluation of their inhibitory activity using biological tests are required.

Phenolics play a key role in the defense strategy of plants against pathogens and herbivores [94]. They are also known to induce inhibitory effects on phytoplankton growth [20]. Several biological activities, including antioxidant and antimicrobial properties, have been attributed to polyphenols [95–97]. Laabir et al. [29] have reported that *Z. noltei* and *Z. marina* crude extracts, which inhibited the growth of *A. catenella*, contained significant amounts of phenolics (zosteric acid, rosmarinic acid, and flavonoids). Achamlale et al. [98,99] and Grignon-Dubois et al. [100] have found substantial concentrations of phenolic acids (rosmarinic, zosteric, and caffeic acids) in *Z. noltei* detrital leaves and in crude extracts. *Z. noltei* is also characterized by the presence of flavonoids [101]. Like other polyphenols, these compounds have the capacity to act as antioxidants and can also affect growth and important metabolic functions of harmful microalgal species, including cyanobacteria and dinoflagellates [74].

Less is known about allelochemicals produced by *C. nodosa* and evaluation of their bioactivity needs more clarification. Grignon-Dubois and Rezzonico [50] have screened detrital and fresh specimens and identified chicoric acid as the major polyphenol of *C. nodosa*. Kontiza et al. [48] and Kontiza et al. [102] have isolated two diarylheptanoids (cymodienol and cymodien) and four 3-keto steroids from *C. nodosa*, respectively. They reported moderate to strong cytotoxic activity of these compounds. Four other metabolites (deoxycymodienol, isocymodiene, nodosol, and briarane diterpene) have been isolated from the organic extract of this seagrass and exhibited weak to strong antibacterial activity [49]. Among the extracted compounds, cymodienol and nodosol were the most active substances.

For *Ulva* species, there is an increasing evidence that these macroalgae have a strong inhibitory allelopathic activity [70,103]. It has been reported that PUFAs produced by *Ulva* spp. have potent algicidal activity and can act as allelochemicals [52–53]. These compounds were highly active against several red tide phytoplankton species even at low concentrations [52–53]. Alamsjah et al. [52] have reported that PUFAs were released into the seawater and gradually
<table><caption>Table 2. Phytochemicals associated with *Ulva rigida*, *Zostera noltei* and *Cymodocea nodosa* species with their reported biological activity.</caption><thead><tr><th>Macrophyte species and origin</th><th>Detected and identified compounds</th><th>Reported biological activities</th><th>Reference</th></tr></thead><tbody><tr><td colspan="4"><strong>Ulva rigida</strong></td></tr><tr><td>Ulva rigida (Ras-Djebel, Tunisia)</td><td><strong>Polyphenols:</strong> Phloroglucinol / Feruloyl-hexose / Fucodiphloroethol / Vanillic acid / Fucophloroethols derivatives / Quinin acid / Dieckol / Fucophloroethol / Syringic acid / Phloroeckol / Dihydroxybenzoic acid / Phenylethanol / Dioxinodehydroeckol / Eckol / Diphroroethohydroxycarmalol.</td><td>Radical-scavenging activity.<br/>Not toxic to HeLa cells culture.</td><td>[105]</td></tr><tr><td>Ulva rigida (Ria Formosa, Portugal)</td><td><strong>Fatty acids:</strong> Linoleic / α-linolenic / Stearidonic / γ-linolenic / Arachidonic / Eicosapentaenoic / Oleic / Palmitoleic.<br/><strong>Polyunsaturated aldehydes</strong> (detected upon tissue damage): 2,4-Heptadienal / 2,4-decadienal / 2,4,7-decatrienal.</td><td>Not Tested</td><td>[117]</td></tr><tr><td>Ulva rigida (Sidi Mansour, Sfax, Tunisia)</td><td><strong>Fatty acids:</strong> Palmitic / Oleic / Linolenic / Eicosenoic / Linoleic / Palmitoleic / Stearic / Myristic / Arachidic.</td><td>Antibacterial, antimicrobial and antioxidant activities. Acetylcholinesterase inhibitory capacity.</td><td>[104]</td></tr><tr><td>Ulva rigida (Black Sea)</td><td><strong>Sterols:</strong> Fucosterol (= main sterol component)</td><td>Not Tested</td><td>[118]</td></tr><tr><td colspan="4"><strong>Zostera noltei</strong></td></tr><tr><td>Zostera noltei (Algarve, Southern Portugal)</td><td><strong>Phenolic acid:</strong> Rosmarinic acid.<br/><strong>Fatty acids:</strong> Palmitic / Linoleic / α-linolenic / Myristic / Margaric / Stearic / Arachidic / Behenic / Lignoceric / Palmitoleic / Oleic / Hexadecatrienoic / Arachidonic / Eicosapentaenoic / Docosahexaenoic.</td><td>Radical scavenging activity. Capacity to chelate copper and iron ions. Toxicity against HepG2, S17 and neuroblastoma cell lines.</td><td>[119]</td></tr><tr><td>Zostera noltii (Thau lagoon and Arcachon bay, France)</td><td><strong>Phenolics:</strong> Zosteric acid / Rosmarinic acid / Flavonoids.</td><td>Allicidal activity against the neuro-toxic dinoflagellate <em>Alexandrium catenella</em>.</td><td>[29]</td></tr><tr><td>Zostera noltii (Bays of Arcachon, France; Cadiz, Spain)</td><td><strong>Phenolics:</strong> Zosteric acid / Caffeic acid / Luteolin 7-sulfate / Apigenin 7-glucoside / Apigenin 7-sulfate / Diosmetin 7-sulfate / Luteolin / Apigenin / Diosmetin.</td><td>Not Tested</td><td>[101]</td></tr><tr><td>Zostera noltii (Bays of Cadiz, Sa Nitja and Alfacs, Spain; Arcachon lagoon, France)</td><td><strong>Phenolics:</strong> Rosmarinic acid / Zosteric acid / Caffeic acid.</td><td>Not Tested</td><td>[100]</td></tr><tr><td>Zostera noltii (Arcachon lagoon, France)</td><td><strong>Phenolics:</strong> Rosmarinic acid / traces of Caffeic acid.</td><td>Not Tested</td><td>[99]</td></tr><tr><td>Zostera noltii (Arcachon lagoon, France)</td><td><strong>Phenolic acid:</strong> Zosteric acid.</td><td>Not Tested</td><td>[98]</td></tr><tr><td>Zostera noltii (Spain)</td><td><strong>Phenolics:</strong> p-Coumaric / p-Hydroxybenzoic acids.</td><td>Not Tested</td><td>[120]</td></tr><tr><td>Zostera nana (Bucknall; Isle of Wight, U.K)</td><td><strong>Two Flavone sulfates:</strong> Luteolin 7-sulphates / Diosmetin.</td><td>Not Tested</td><td>[121]</td></tr><tr><td colspan="4"><strong>Cymodocea nodosa</strong></td></tr><tr><td>Cymodocea nodosa (Chebba coast, Tunisia)</td><td>Sulfated polysaccharide</td><td>Anti-hypertensive properties.</td><td>[122]</td></tr><tr><td>Cymodocea nodosa (Gran Canaria, Canary Islands; Cadiz and Alfacs bays, Spain; Zeytineli, Turkey; Sahline Sebkha beach-Monastir, Tunisia)</td><td><strong>Phenolic acids:</strong> Chicoric acid / Caftaric acid.</td><td>Not Tested</td><td>[50]</td></tr><tr><td>Cymodocea nodosa (Porto Germeno, Greece)</td><td>Deoxycymodienol / Isocymodiene / Meroterpenoid (nodosol) /Brominated briarane diterpene / Cymodienol</td><td>Antibacterial activity.</td><td>[49]</td></tr><tr><td>Cymodocea nodosa (Ag. Cosmas Gulf, Greece)</td><td><strong>Four 3-keto steroids:</strong> (20R)-22E-24-ethylcholesta-4,22-dien-3-one / (20R)-24-ethylcholest-4-en-3-one / (20R)-22E-6β-hydroxy-24-ethylcholesta-4,22-dien-3-one / 6β-hydroxy-(20R)-24-ethylcholest-4-en-3-one.</td><td>No data</td><td>[102]</td></tr><tr><td>Cymodocea nodosa (Ag. Cosmas Gulf, Greece)</td><td><strong>Diarylheptanoids:</strong> Cymodienol / Cymodiene.</td><td>Cytotoxic activity against two lung cancer cell lines (NSCL-N6 and A549).</td><td>[48]</td></tr><tr><td>Cymodocea nodosa (Bay of Naples, Italy)</td><td><strong>Sterols:</strong> Most abundant compounds: Sitosterol / Cholesterol / Stigmasterol.</td><td>Not Tested</td><td>[123]</td></tr></tbody></table>

(Continued)
<table><caption>**Table 2. (Continued)**</caption><thead><tr><td>Macrophyte species and origin</td><td>Detected and identified compounds</td><td>Reported biological activities</td><td>Reference</td></tr></thead><tbody><tr><td>Cymodocea nodosa (Bat-Yam)</td><td>Sulfated phenolic acids</td><td>Not Tested</td><td>[124]</td></tr><tr><td>Cymodocea nodosa (Ganzirri, Sicily; Marsaxlokk, Malta)</td><td>1-chiro-inositol / myo-inositol / muco-inositol.</td><td>Not Tested</td><td>[125]</td></tr></tbody></table>

(Macrophyte species are named as cited in the references).

https://doi.org/10.1371/journal.pone.0187963.t002

decomposed with time. Antibacterial and antimicrobial activities were reported for *U. rigida*, and the fatty acid composition of this macroalgae was investigated [104]. Otherwise, *U. rigida* has shown biological activities that are related not only to fatty acids but also to the presence of polyphenols [105]. Reports dealing with the phytochemistry of *U. rigida* and the identification of its polyphenols remain scarce. Mezghani et al. [105] have reported that *U. rigida* extracts contained various polyphenols and they also noted the presence of phlorotannins such as phloroglucinol, a compound usually reported to occur in brown marine algae [106]. All of these molecules are well known for their potent antioxidant and radical scavenging activities, which confirms the inhibitory effects observed in our study.

## Conclusion

Our results highlight the potential of the macrophytes *Z. noltei*, *C. nodosa*, and *U. rigida* to reduce the proliferation of the HAB-forming benthic marine dinoflagellates *O. cf. ovata*, *P. lima*, and *C. monotis* but with contrasted efficiencies. We demonstrated that the planktonic *A. pacificum* can be strongly affected by the presence of *C. nodosa* and *U. rigida* fresh leaves/thalli. Our findings suggest that benthic dinoflagellates seem more resistant than planktonic species to potential allelochemicals released by the macrophytes. The variable sensitivity of target phytoplankton species to the same macrophyte provides some insights for a better understanding of the complex species-specific allelopathic interactions occuring in marine ecosystems. In freshwater ecosystems, an important feature associated with the allelopathical relationship between macrophytes and microalgae is related to their species-specific nature [107,108]. Some macrophytes are more potent than others and may exert stronger effects on phytoplankton [109,110], whereas differential sensitivities of phytoplankton taxa have been described [107,111]. Allelopathy might play a crucial role in determining species compositions and dominance patterns by regulating the diversity and structure of phytoplankton communities. Depending on the macrophyte species, inhibitory effects were observed on growth and photosynthesis. Cell toxin production also seemed to respond to the stress induced by the presence of the macrophytes.

Future investigations have to isolate and identify the allelopathic substances that are effectively exudated in the seawater by the macrophytes investigated in our study. Their modes of action and the physiological processes that are impacted, have also to be thoroughly explored for a potential use of macrophytes in bloom control and mitigation. A better understanding of the co-evolutionary relationships between epibenthic dinoflagellates and macrophytes would be of great interest, as these processes could explain the resistance or tolerance of the targeted species to allelochemicals released by the macrophytes with which they have co-evolved.

## Supporting information

S1 Appendix. NO₃⁻ and PO₄³⁻ concentrations (µmol.L⁻¹) in all experiments according to the macrophyte species.

(DOCX)
**S2 Appendix. Dinoflagellate colonization patterns.**

(PDF)

**S3 Appendix. Phytochemicals associated with *Ulva*, *Zostera* and *Cymodocea* species with their reported biological activity.**

(DOCX)

**Acknowledgments**

We thank Abdessalem Shili from the Tunisian National Institute of Agronomy for his help for macrophyte identification and Vicky Diakou from the Technical Platform of Imagery MRI-UM-Biocampus DS Biologie Santé, Montpellier for her help in epifluorescence microscopy observations. We also thank the four anonymous reviewers for their help in improving our manuscript.

**Author Contributions**

**Conceptualization:** Ons Kéfi-Daly Yahia, Mohamed Laabir.

**Data curation:** Philippe Cecchi, Habiba Nouri.

**Formal analysis:** Hela Ben Gharbia, Philippe Cecchi, Zouher Amzil, Mohamed Laabir.

**Funding acquisition:** Mohamed Laabir.

**Investigation:** Hela Ben Gharbia, Fabienne Herve, Georges Rovillon, Charaf M'Rabet, Douglas Couet, Habiba Zmerli Triki, Mohamed Laabir.

**Methodology:** Philippe Cecchi, Estelle Masseret, Zouher Amzil, Mohamed Laabir.

**Project administration:** Mohamed Laabir.

**Resources:** Ons Kéfi-Daly Yahia, Mohamed Laabir.

**Software:** Philippe Cecchi, Habiba Nouri.

**Supervision:** Ons Kéfi-Daly Yahia, Philippe Cecchi, Estelle Masseret, Mohamed Laabir.

**Validation:** Ons Kéfi-Daly Yahia, Philippe Cecchi, Zouher Amzil, Mohamed Laabir.

**Visualization:** Hela Ben Gharbia, Philippe Cecchi, Estelle Masseret, Habiba Nouri, Mohamed Laabir.

**Writing – original draft:** Hela Ben Gharbia, Mohamed Laabir.

**Writing – review & editing:** Hela Ben Gharbia, Philippe Cecchi, Mohamed Laabir.

**References**

1. Rice EL. Allelopathy. New York, USA: Academic Press Inc.; 1984.

2. Gross EM. Allelopathy of Aquatic Autotrophs. CRC Crit Rev Plant Sci. 2003; 22: 313–339.

3. Legrand C, Rengefors K, Fistarol GO, Granéli E. Allelopathy in phytoplankton–biochemical, ecological and evolutionary aspects. Phycologia. 2003; 42 (4): 406–419.

4. Reigosa MJ, Pedrol N, Gonzalez L, editors. Allelopathy: a physiological process with ecological implications. Springer, Dordrecht, Netherlands; 2006. 637 pp.

5. Dilday RH, Frans RE, Semiday N, Smith RJ, Oliver LR. Weed control with crop allelopathy. Arkansas Farm Res. 1992; 4: 14–15.

6. Yang RZ, Tang CS. Plants used for pest control in China: a literature review. Eco Bot. 1988; 42: 376–406.
7. Macias FA, Galindo JLG, Garcia-Diaz MD, Galindo JCG. Allelopathic agents from aquatic ecosystems: potential biopesticides models. Phytochem Rev. 2008; 7: 155-178.

8. Erhard D. Allelopathy in aquatic environments. In: Reigosa MJ, Pedrol N, Gonzalez L, editors. Allelopathy: a physiological process with ecological implications. Springer, Dordrecht, Netherlands; 2006. pp. 433-450.

9. Hallegraeff GM. Harmful Algal Blooms: A Global Overview. In: Hallegraeff GM, Anderson DM, Cembella AD, editors. Manual on Harmful Marine Microalgae. UNESCO Publishing; 2003. pp. 25-46.

10. Lewitus AJ, Horner RA, Caron DA, Garcia-Mendoza E, Hickey BM, Hunter M, et al. Harmful algal blooms along the North American west coast region: History, trends, causes, and impacts. Harmful Algae. 2012; 19: 133-159.

11. Cecchi P, Garrido M, Collos Y, Pasqualini V. Water flux management and phytoplankton communities in a Mediterranean coastal lagoon. Part II: mixotrophy of dinoflagellates as an adaptive strategy?. Mar Pollut Bull. 2016; 108: 120-133. https://doi.org/10.1016/j.marpolbul.2016.04.041 PMID: 27126183

12. Kim HG. Mitigation and Controls of HABs. In: Granéli E, Turner JT, editors. Ecology of Harmful Algae. Springer-Verlag, Berlin, Heidelberg, Ecological Studies; 2006. pp. 327-338.

13. Park TG, Lim WA, Park YT, Lee CK, Jeong HJ. Economic impact, management and mitigation of red tides in Korea. Harmful Algae. 2013; 30S1: S131-S143.

14. Rounsefell GA, Evans JE. Large-scale experimental test of copper sulfate as a control for the Florida red tide. US Fish Wildlife Serv Spec Sci Rep. 1958; 270 pp.

15. Sfriso A, Pavoni B. Macroalgae and phytoplankton competition in the central Venice lagoon. Environ Technol. 1994; 15: 1-14.

16. Thomas S, Cecchi P, Corbin D, Lemoalle J. The different primary producers in a small African tropical reservoir during a drought: temporal changes and interactions. Freshwater Biol. 2000; 45: 43-56.

17. Hu H, Hong Y. Algal-bloom control by allelopathy of aquatic macrophytes-a review. Front Environ Sci Eng China. 2008; 2 (4): 421-438.

18. Tang YZ, Kang Y, Berry D, Gobler CJ. The ability of the red macroalga, *Porphyra purpurea* (Rhodophyceae) to inhibit the proliferation of seven common harmful microalgae. J Appl Phycol. 2015; 27: 531-544.

19. Jeong JH, Jin HJ, Sohn CH, Suh KH, Hong YK. Algicidal activity of the seaweed *Corallina pilulifera* against red tide microalgae. J Appl Phycol. 2000; 12: 37-43.

20. Gross EM, Meyer H, Schilling G. Release and ecological impact of algicidal hydrolysable polyphenols in *Myriophyllum spicatum*. Phytochemistry. 1996; 41: 133-138.

21. Gross EM. Allelopathy in benthic and littoral areas: case studies on allelochemicals from benthic cyanobacteria and submerged macrophytes. In: Inderjit Dakshini KMM, Foy CL, editors. Principles and Practices in Plant Ecology: Allelochemical Interactions. CRC Press, Boca Raton, FL; 1999. pp. 179-199.

22. Dziga D, Suda M, Bialczyk J, Czaja-Prokop U, Lechowski Z. The alteration of *Microcystis aeruginosa* biomass and dissolved microcystin-LR concentration following exposure to plant-producing phenols. Environ Toxicol. 2007; 22: 341-346. https://doi.org/10.1002/tox.20276 PMID: 17607725

23. Wu C, Chang X, Dong H, Li D, Liu J. Allelopathic inhibitory effect of *Myriophyllum aquaticum* (Vell.) Verdc. on *Microcystis aeruginosa* and its physiological mechanism. Acta Ecol Sin. 2008; 28 (6): 2595-2603.

24. Rojo C, Segura M, Rodrigo MA. The allelopathic capacity of submerged macrophytes shapes the microalgal assemblages from a recently restored coastal wetland. Ecol Eng. 2013; 58: 149-155.

25. Zhu J, Liu B, Wang J, Gao Y, Wu Z. Study on the mechanism of allelopathic influence on cyanobacteria and chlorophytes by submerged macrophyte (*Myriophyllum spicatum*) and its secretion. Aquatic Toxicol. 2010; 98: 196-203.

26. Accoroni S, Percopo I, Cerino F, Romagnoli T, Pichierri S, Perrone C, et al. Allelopathic interactions between the HAB dinoflagellate *Ostreopsis cf. ovata* and macroalgae. Harmful Algae. 2015; 49: 147-155.

27. Abadie E, Muguet A, Berteaux T, Chomérat N, Hess P, D'OrbCastel ER, et al. Growth Responses of the Neurotoxic Dinoflagellate *Vulcanodinium rugosum* to Varying Temperature and Salinity. Toxins. 2016; 8: 136.

28. Rhodes LL, Smith KF, Murray S, Harwood DT, Trnski T, Munday R. The Epiphytic Genus *Gambierdiscus* (Dinophyceae) in the Kermadec Islands and Zealandia Regions of the Southwestern Pacific and the Associated Risk of Ciguatera Fish Poisoning. Mar Drugs. 2017; 15: 219.
29. Laabir M, Grignon-Dubois M, Masseret E, Rezzonico B, Soteras G, Rouquette M, et al. Algicidal effects of *Zostera marina* L. and *Zostera noltii* Hornem. extracts on the neuro-toxic bloom-forming dinoflagellate *Alexandrium catenella*. Aquat Bot. 2013; 111: 16-25.

30. Rossi R, Castellano V, Scalco E, Serpe L, Zingone A, Soprano V. New palytoxin-like molecules in Mediterranean *Ostreopsis* cf. *ovata* (dinoflagellates) and in *Palythoa tuberculosa* detected by liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Toxicon. 2010; 56: 1381-1387. https://doi.org/10.1016/j.toxicon.2010.08.003 PMID: 20797402

31. Ciminiello P, Dell'Aversano C, Dello Iacovo E, Fattorusso E, Forino M, Tartaglione L, et al. Unique toxin profile of a Mediterranean *Ostreopsis* cf. *ovata* strain: HR LC-MS⁹ characterization of ovatoxin-f, a new palytoxin congener. Chem Res Toxicol. 2012; 25: 1243-1252. https://doi.org/10.1021/tx300085e PMID: 22502872

32. Amzil Z, Sibat M, Chomerat N, Grossel H, Marco-Miralles F, Lemee R, et al. Ovatoxin-a and Palytoxin Accumulation in Seafood in Relation to *Ostreopsis* cf. *ovata* Blooms on the French Mediterranean Coast. Mar Drugs. 2012; 10 (2): 477-496. https://doi.org/10.3390/ md10020477 PMID: 22412814

33. Brissard C, Herrenknecht C, Sechet V, Herve F, Pisapia F, Harcouet J, et al. Complex Toxin Profile of French Mediterranean *Ostreopsis* cf. *ovata* Strains, Seafood Accumulation and Ovatoxins Purpurification. Mar Drugs. 2014; 12 (5): 2851-2876. https://doi.org/10.3390/ md12052851 PMID: 24828292

34. Ben Gharbia H, Kéfi-Daly Yahia O, Amzil Z, Chomérat N, Abadie E, Masseret E, et al. Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (*Ostreopsis* cf. *ovata*, *Prorocentrum* *lima* and *Coolia monotis*) Developing in the Southern Mediterranean Basin. Toxins. 2016; 8: 297.

35. Mangialajo L, Ganzin N, Accoroni S, Asnaghi V, Blanfuné A, Cabrini M, et al. Trends in *Ostreopsis* proliferation along the Northern Mediterranean coasts. Toxicon. 2011; 57: 408-420. https://doi.org/10.1016/j.toxicon.2010.11.019 PMID: 21145339

36. Ciminiello P, Dell'Aversano C, Fattorusso E, Forino M, Magno GS, Tartaglione L, et al. The Genoa 2005 Outbreak. Determination of Putative Palytoxin in Mediterranean *Ostreopsis ovata* by a New Liquid Chromatography Tandem Mass Spectrometry Method. Anal chem. 2006; 78 (17): 6153-6159. https://doi.org/10.1021/ac606250j PMID: 16944897

37. Murakami Y, Oshima Y, Yasumoto T. Identification of okadaic acid as a toxic component of a marine dinoflagellate *Prorocentrum lima*. Nippon Suisan Gakkai Shi. 1982; 48: 69-72.

38. Torigoe K, Murata M, Yasumoto T. Prorocentrolide, a toxic nitrogenous macrocycle from a marine dinoflagellate, *Prorocentrum lima*. J Am Chem Soc. 1988; 110: 7876-7877.

39. Nascimento SM, Purdie DA, Morris S. Morphology, toxin composition and pigment content of *Prorocentrum lima* strains isolated from a coastal lagoon in Southern UK. Toxicon. 2005; 45: 633-649. https://doi.org/10.1016/j.toxicon.2004.12.023 PMID: 15777960

40. Salon-Barros J, Arregoces L. *Prorocentrum lima* and *Prorocentrum balticum* blooms in Cartagena de Indias, Colombian Caribbean. Harmful Algae News. 2016; 53: pp. 9.

41. Armi Z, Turki S, Trabelsi E, Ben Maiz N. First recorded proliferation of *Coolia monotis* (Meunier, 1919) in the North Lake of Tunis (Tunisia) correlation with environmental factors. Environ Monit Assess. 2010; 164: 423-433. https://doi.org/10.1007/s10661-009-0903-z PMID: 19404758

42. Laabir M, Collos Y, Masseret E, Grzebyk D, Abadie E, Savart V, et al. Influence of Environmental Factors on the Paralytic Shellfish Toxin Content and Profile of *Alexandrium catenella* (Dinophyceae) Isolated from the Mediterranean Sea. Mar Drugs. 2013; 11: 1583-1601. https://doi.org/10.3390/ md11051583 PMID: 23676417

43. Anderson DM, Alpermann TJ, Cembella AD, Collos Y, Masseret E, Montresor M. The globally distributed genus *Alexandrium*: Multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae. 2012; 14: 10-35. https://doi.org/10.1016/j.hal.2011.10.012 PMID: 22308102

44. Green EP, Short FT. World atlas of seagrasses. University of California Press, Berkeley, CA; 2003. pp. 5-26.

45. Harrison PG, Chan AT. Inhibition of the growth of micro-algae and bacteria by extracts of eelgrass (*Zostera marina*) leaves. Mar Biol. 1980; 61: 21-26.

46. Harrison PG, Durance CD. Reductions in photosynthetic carbon uptake in epiphytic diatoms by water-soluble extracts of leaves of *Zostera marina*. Mar Biol. 1985; 90: 117-120.

47. Harrison PG. Control of microbial growth and of amphipod grazing by water-soluble compounds from leaves of *Zostera marina*. Mar Biol. 1982; 67: 225-230.

48. Kontiza I, Vagias C, Jakupovic J, Moreau D, Roussakis C, Roussis V. Cymodienol and cymodiene: new cytotoxic diarylheptanoids from the sea grass *Cymodocea nodosa*. Tetrahedron Lett. 2005; 46: 2845-2847.

49. Kontiza I, Stavri M, Zloh M, Vagias C, Gibbons S, Roussis V. New metabolites with antibacterial activity from the marine angiosperm *Cymodocea nodosa*. Tetrahedron. 2008; 64: 1696-1702.
50. Grignon-Dubois M, Rezzonico B. The economic potential of beach-cast seagrass–Cymodocea nodosa: a promising renewable source of chicoric acid. Botanica Marina. 2013; 56 (4): 303–311.

51. Jin Q, Dong S. Comparative studies on the allelopathic effects of two different strains of *Ulva pertusa* on *Heterosigma akashiwo* and *Alexandrium tamarense*. J Exp Mar Bio Ecol. 2003; 293: 41–55.

52. Alamsjah MA, Hirao S, Ishibashi F, Fujita Y. Isolation and structure determination of algicidal compounds from *Ulva fasciata*. Biosci Biotechnol Biochem. 2005; 69: 2186–2192. https://doi.org/10.1271/bbb.69.2186 PMID: 16306701

53. Alamsjah MA, Hirao S, Ishibashi F, Oda T, Fujita Y. Algicidal activity of polyunsaturated fatty acids derived from *Ulva fasciata* and *U. pertusa* (Ulvaceae, Chlorophyta) on phytoplankton. J Appl Phycol. 2008; 20: 713–720.

54. Tang YZ, Gobler CJ. The green macroalga, *Ulva lactuca*, inhibits the growth of seven common harmful algal bloom species via allelopathy. Harmful Algae. 2011; 10: 480–488.

55. Harrison PJ, Waters RE, Taylor FJR. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J Phycol. 1980; 16: 28–35.

56. John U, Litaker RW, Montresor M, Murray S, Brosnahan ML, Anderson DM. Formal Revision of the *Alexandrium tamarense* Species Complex (Dinophyceae) Taxonomy: The Introduction of Five Species with Emphasis on Molecular-based (rDNA) Classification. Protist. 2014; 165 (6): 779–804. https://doi.org/10.1016/j.protis.2014.10.001 PMID: 25460230

57. Laabir M, Jauzein C, Genovesi B, Masseret E, Grzebyk D, Cecchi P, et al. Influence of temperature, salinity and irradiance on the growth and cell yield of the harmful red tide dinoflagellate *Alexandrium catenella* colonising Mediterranean waters. J Plank Res. 2011; 33: 1550–1563.

58. Fertouna-Bellakhala M, Dhib A, Fathalli A, Bellakhal M, Chomérat N, Masseret E, et al. *Alexandrium pacificum* Litaker sp. nov (group IV): resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia, Southern Mediterranean Sea). Harmful Algae. 2015; 48: 69–82.

59. Plus M, Chapelle A, Lazure P, Auby I, Levavasseur G, Verlaque M, et al. Modelling of oxygen and nitrogen cycling as a function of macrophyte community in the Thau lagoon. Cont Shelf Res. 2003; 23: 1877–1898.

60. Plus M, Chapelle A, Ménesguen A, Deslous-Paoli JM, Auby I. Modelling seasonal dynamics of bio-masses and nitrogen contents in a seagrass meadow (Zostera noltii Hornem.): application to the Thau lagoon (French Mediterranean coast). Ecol Model. 2003; 161 (3): 211–236.

61. Sghaier YR, Zakhama-Sraieb R, Charfi-Cheikhrouha F. Seasonal variation of *Cymodocea nodosa* in the Ghar El Melh Lagoon (Tunisia), with reference to insolation, temperature and salinity effects. Bull Inst Natn Scien Tech Mer de Salammbô. 2012; 39: 117–125.

62. Tréguer P, LeCorre P. Manuel d'analyse des sels nutritifs dans l'eau de mer. Utilisation de l'autoanalysteur II Technicon. Université de Bretagne Occidentale, Laboratoire de Chimie marine, Brest, France; 1975. 110 pp.

63. Guillard RLR. Division rates. In: Stein JR, editor. Handbook of Phycological Methods: Cultures Methods and Growth Measurements. Cambridge University Press, Cambridge, UK; 1973. pp. 290–311.

64. Strasser RJ, Srivastava A, Tsimilli-Michael M. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus M, Pathre U, Mohanty P, editors. Probing Photosynthesis: Mechanism, Regulation and Adaptation. Taylor and Francis, UK, Chapter 25; 2000. pp. 445–483.

65. Garrido M, Cecchi P, Vaquer A, Pasqualini V. Effects of samples conservation on photosynthetic efficiency assessment of phytoplankton using PAM fluorometry. Deep-Sea Res Pt I. 2013; 71: 38–48.

66. Fong P, Donohoe RM, Zedler JB. Competition with macroalgae and benthic cyanobacterial mats limits phytoplankton abundance in experimental microcosms. Mar Ecol Prog Ser. 1993; 100 (1–2): 97–102.

67. Smayda T. Harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnol Oceanogr. 1997; 42 (5, part 2): 1137–1153.

68. Guillard RRL, Ryther JH. Studies on marine planktonic diatoms I. *Cystotella nana* (Hustedt) and *Detomula confervaceae* (Cleve). Can J Microbiol. 1962; 8: 229–239. PMID: 13902807

69. Provasoli L. Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A, editors. Cultures and Collections of Algae. Proc. US and Japan Conference, Hakone. Japanese Society of Plant Physiology, Hakone; 1968. pp. 63–75.

70. Jin Q, Dong SL, Wang CY. Allelopathic growth inhibition of *Prorocentrum micans* (Dinophyta) by *Ulva pertusa* and *Ulva linza* (Chlorophyta) in laboratory cultures. Eur J Phycol. 2005; 40: 31–37.

71. Hilt S. Allelopathic inhibition of epiphytes by submerged macrophytes. Aquat Bot. 2006; 85: 252–256.

72. Reigosa J, Sánchez-Moreiras A, González L. Ecophysiological Approach in Allelopathy. CRC Crit Rev Plant Sci. 1999; 18 (5): 577–608.
73. De Wit R, Troussellier M, Courties C, Buffan-Dubau E, Lemaire E. Short-term interactions between phytoplankton and intertidal seagrass vegetation in a coastal lagoon (Bassin d'Arcachon, SW France). Hydrobiologia. 2012; 699 (1): 55-68.

74. Huang H, Xiao X, Ghadouani A, Wu J, Nie Z, Peng C, et al. Effects of Natural Flavonoids on Photosynthetic Activity and Cell Integrity in *Microcystis aeruginosa*. Toxins. 2015; 7: 66–80. https://doi.org/10.3390/toxins7010066 PMID: 25584428

75. Pichierri S, Pezzolesi L, Vanucci S, Totti C, Pistocchi R. Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate *Ostreopsis cf. ovata*. Aquat Toxicol. 2016; 179: 125–133. https://doi.org/10.1016/j.aquatox.2016.08.018 PMID: 27606904

76. Leu E, Krieger-Liszkay A, Goussias C, Gross EM. Polyphenolic Allelochemicals from the Aquatic Angiosperm *Myriophyllum spicatum* Inhibit Photosystem II. Plant Physiol. 2002; 130(4): 2011–2018. https://doi.org/10.1104/pp.011593 PMID: 12481084

77. Ye C, Liao H, Yang Y. Allelopathic inhibition of photosynthesis in the red tide-causing marine alga, *Scrippsiella trochoidea* (Pyrrophyta), by the dried macroalga, *Gracilaria lemaneiformis* (Rhodophyta). *J Sea Res.* 2014; 90: 10–15.

78. Ye C, Zhang M. Allelopathic effect of macroalga *Gracilaria tenuistipitata* (Rhodophyta) on the photosynthetic apparatus of red-tide causing microalga *Prorocentrum micans*. *IERI Procedia*. 2013; 5: 209–215.

79. Wium-Andersen S, Anthoni U, Christophersen C. Allelopathic effects on phytoplankton by substances isolated from aquatic macrophytes (Charales). Oikos. 1982; 39: 187-190.

80. Anderson DM, Kulis DM, Sullivan JJ, Hall S, Lee C. Dynamics and physiology of saxitoxin production by the dinoflagellate *Alexandrium* spp. Mar Biol. 1990; 104: 511–524.

81. Boyer GL, Sullivan JJ, Andersen RJ, Harrison PJ, Taylor FJR. Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate *Protogonyaulax tamarensis*. Mar Biol. 1987; 96: 123–128.

82. Béchemin C, Grzebyk D, Hachame F, Humrert C, Maestrini SY. Effect of different nitrogen/phosphorus nutrient ratios on the toxin content in *Alexandrium minutum*. Aquat Microb Ecol. 1999; 20: 157–165.

83. Vanucci S, Guerrini F, Milandri A, Pistocchi R. Effects of different levels of N- and P-deficiency on cell yield, okadaic acid, DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate *Prorocentrum lima*. Harmful Algae. 2010; 9: 590–599.

84. Vanucci S, Pezzolesi L, Pistocchi R, Ciminiello P, Dell'Aversano C, Dello Iacovo E, et al. Nitrogen and phosphorus limitation effects on cell growth, biovolume, and toxin production in *Ostreopsis cf. ovata*. Harmful Algae. 2012; 15: 78–90.

85. Turki S, El Abed A. On the presence of potentially toxic algae in the lagoons of Tunisia. Harmful Algae News. 2001; 22: pp.10.

86. Turki S. Distribution of toxic dinoflagellates along the leaves of seagrass *Posidonia oceanica* and *Cymodocea nodosa* from the Gulf of Tunis. Cah Biol Mar. 2005; 46: 29–34.

87. Aligizaki K, Nikolaidis G, Katikou P, Baxevanis AD, Abatzopoulos TJ. Potentially toxic epiphytic *Prorocentrum* (Dinophyceae) species in Greek coastal waters. Harmful Algae. 2009; 8: 299–311.

88. Foden J, Purdie DA, Morris S, Nascimento S. Epiphytic abundance and toxicity of *Prorocentrum lima* populations in the Fleet Lagoon, UK. Harmful Algae. 2005; 4: 1063–1074.

89. Aligizaki K, Nikolaidis G. The presence of the potentially toxic genera *Ostreopsis* and *Coolia* (Dinophyceae) in the North Aegean Sea, Greece. Harmful Algae. 2006; 5: 717–730.

90. Cohu S. Ecologie du Dinoflagellé benthique toxique Ostreopsis cf. ovata Fukuyo en Méditerranée Nord-Occidentale. Thesis, The University of Nice-Sophia Antipolis. 2012. Available from: http://www.theses.fr/2012NICE4107.

91. Parsons ML, Preskitt LB. A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawai'i. Harmful Algae. 2007; 6: 658–669.

92. Okolodkov YB, Campos-Bautista G, Gárate-Lizárraga I, González-González JAG, Hoppenrath M, Arenas V. Seasonal changes of benthic and epiphytic dinoflagellates in the Veracruz reef zone, Gulf of Mexico. Aquat Microb Ecol. 2007; 47: 223–237.

93. Kim HS, Yih W, Kim JH, Myung G, Jeong HJ. Abundance of Epiphytic Dinoflagellates from Coastal Waters off Jeju Island, Korea During Autumn 2009. Ocean Sci J. 2011; 46(3): 205–209.

94. Smolders AJP, Vergeer LHT, Van der Velde G, Roelofs JGM. Phenolic contents of submerged, emergent and floating leaves of aquatic and semiaquatic macrophyte species: why do they differ? Oikos. 2000; 91: 307–310.
95. Nakai S, Inoue Y, Hosomi M, Murakami A. *Myriophyllum spicatum*-released allelopathic polyphenols inhibiting growth of blue-green algae *Microcystis aeruginosa*. Water Res. 2000; 34: 3026–3032.

96. Haslam E. Plant Polyphenols. Vegetable Tannins Revisited. Cambridge: Cambridge University Press; 1989.

97. Gross EM, Sütfeld R. Polyphenols with algicidal activity in the submerged macrophyte *Myriophyllum spicatum* L. Acta Hortic. 1994; 381: 710–716.

98. Achamlale S, Rezzonico B, Grignon-Dubois M. Evaluation of *Zostera detritus* as a potential new source of zosteric acid. J Appl Phycol. 2009; 21: 347–352.

99. Achamlale S, Rezzonico B, Grignon-Dubois M. Rosmarinic acid from beach waste: Isolation and HPLC quantification in *Zostera detritus* from Arcachon lagoon. Food Chem. 2009; 113: 878–883.

100. Grignon-Dubois M, Rezzonico B, Alcoverro T. Regional scale patterns in seagrass defences: Phenolic acid content in *Zostera noltii*. Estuar Coast Shelf Sci. 2012; 114: 18–22.

101. Grignon-Dubois M, Rezzonico B. First Phytochemical Evidence of Chemotypes for the Seagrass *Zostera noltii*. Plants. 2012; 1: 27–38. https://doi.org/10.3390/plants1010027 PMID: 27137638

102. Kontiza I, Abatis D, Malakate K, Vagias C, Roussis V. 3-Keto steroids from the marine organisms *Den-drophyllia cornigera* and *Cymodocea nodosa*. Steroids. 2006; 71: 177–181. https://doi.org/10.1016/j.steroids.2005.09.004 PMID: 16280145

103. Nelson TA, Lee DJ, Smith BC. Are "green tides" harmful algal blooms? Toxic properties of water-soluble extracts from two bloom-forming macroalgae, *Ulva fenestrata* and *Ulvaria obscura* (Ulvophyceae). J Phycol. 2003; 39: 874–879.

104. Trigui M, Gasmi L, Zouari I, Tounsi S. Seasonal variation in phenolic composition antibacterial and antioxidant activities of *Ulva rigida* (Chlorophyta) and assessment of antiacetylcholinesterase potentiel. J App Phycol. 2013; 25: 319–328.

105. Mezghani S, Csupor D, Bourguiba I, Hohmann J, Amri M, Bouaziz M. Characterization of Phenolic Compounds of *Ulva rigida* (Chlorophyceae) and Its Antioxidant Activity. European J Med Plants. 2016; 12 (1): 1–9.

106. Singh IP, Bharate SB. Phloroglucinol compounds of natural origin. Nat Prod Rep. 2006; 23: 558–591. https://doi.org/10.1039/b600518g PMID: 16874390

107. Mulderij G, Van Donk E, Roelofs JGM. Differential sensitivity of green algae to allelopathic substances from *Chara*. Hydrobiologia. 2003; 491: 261–271.

108. Pakdel FM, Sim L, Beardall J, Davis J. Allelopathic inhibition of microalgae by the freshwater stonewort, *Chara australis*, and a submerged angiosperm, *Potamogeton crispus*. Aquat Bot. 2013; 110: 24–30.

109. Vanderstukken M, Mazzeo N, Van Colen W. Biological control of phytoplankton by the subtropical submerged macrophytes *Egeria densa* and *Potamogeton illinoensis*: a mesocosm study. Freshwater Biol. 2011; 56: 1837–1849.

110. Pelechata A, Pelechaty M. The in situ influence of *Ceratophyllum demersum* on a phytoplankton assemblage. Oceanol Hydrobiol Stud. 2010; 39: 95–101.

111. Eigemann F, Vanormelingen P, Hilt S. Sensitivity of the Green Alga *Pediastrum duplex* Meyen to Allelochemicals Is Strain-Specific and Not Related to Co-Occurrence with Allelopathic Macrophytes. PLoS ONE. 2013; 8 (10): e78463. https://doi.org/10.1371/journal.pone.0078463 PMID: 24167626

112. Nan CR, Zhang HZ, Lin SZ, Zhao GQ, Liu XY. Allelopathic effects of *Ulva lactuca* on selected species of harmful bloom-forming microalgae in laboratory cultures. Aquat Bot. 2008; 89: 9–15.

113. Alamsjah MA, Ishibe K, Kim D, Yamaguchi K, Ishibashi F, Fujita Y, et al. Selective Toxic Effects of Polyunsaturated Fatty Acids Derived from *Ulva fasciata* on Red Tide Phytoplankter Species. Biosci Biotechnol Biochem. 2007; 71 (1): 265–268. https://doi.org/10.1271/bbb.60475 PMID: 17213644

114. Wang RJ, Xiao H, Wang Y, Zhou WL, Tang XX. Effects of three macroalgae, *Ulva linza* (Chlorophyta), *Corallina pilulifera* (Rhodophyta) and *Sargassum thunbergii* (Phaeophyta) on the growth of the red tide microalga *Prorocentrum donghaiense* under laboratory conditions. J Sea Res. 2007; 58: 189–197.

115. Wang Y, Yu ZM, Song XX, Tang XX, Zhang SD. Effects of macroalgae *Ulva pertusa* (Chlorophyta) and *Gracilaria lemaneiformis* (Rhodophyta) on growth of four species of bloom-forming dinoflagellates. Aquat Bot. 2007; 86: 139–147.

116. Nan CR, Zhang HZ, Zhao GQ. Allelopathic interactions between the macroalga *Ulva pertusa* and eight microalgal species. J Sea Res. 2004; 52: 259–268.

117. Alsufyani T, Engelen AH, Diekmann OE, Kuegler S, Wichard T. Prevalence and mechanism of polyunsaturated aldehydes production in the green tide forming macroalgal genus *Ulva* (Ulvales, Chlorophyta). Chem Phys Lipids. 2014; 183: 100–109. https://doi.org/10.1016/j.chemphyslip.2014.05.008 PMID: 24915501
118. Popov SS, Marekov NL, Konaklieva MI, Panayotova MI, Dimitrova-Konaklieva S. Sterols from some Black Sea Ulvaceae. Phytochemistry. 1985; 24: 1987-1990.

119. Custódio L, Laukaityte S, Engelen AH, Rodrigues MJ, Pereira H, Vizetto-Duarte C, et al. A comparative evaluation of biological activities and bioactive compounds of the seagrasses *Zostera marina* and *Zostera noltei* from southern Portugal. Nat Prod Res. 2016; 30 (6): 724-728. https://doi.org/10.1080/14786419.2015.1040791 PMID: 26189828

120. Zapata O, McMillan C. Phenolic acids in seagrasses. Aquat Bot. 1979; 7: 307-317.

121. Harborne JB, Williams CA. Occurrence of sulphated flavones and caffeic acid esters in members of Fluviales. Biochem Syst Ecol. 1976; 4: 37-41.

122. Ben Abdallah Kolsi R, Fakhfakh J, Krichen F, Jribi I, Chiarore A, Patti FP, et al. Structural characterization and functional properties of antihypertensive *Cymodocea nodosa* sulfated polysaccharide. Carbohydr Polym. 2016; 151: 511-522. https://doi.org/10.1016/j.carbpol.2016.05.098 PMID: 27474595

123. Sica D, Piccialli V, Masullo A. Configuration at C-24 of sterols from the marine phanerogames *Posidonia oceanica* and *Cymodocea nodosa*. Phytochemistry. 1984; 23: 2609-2611.

124. McMillan C, Zapata O, Escobar L. Sulphated phenolic compounds in seagrasses. Aquat Bot. 1980; 8: 267-278.

125. Drew EA. Carbohydrate and Inositol metabolism in the seagrass, *Cymodocea nodosa*. New Phytol. 1978; 81: 249-264.