ORIGINAL RESEARCH

published: 21 August 2020

doi: 10.3389/fmars.2020.00683

Exploring the Role of Macroalgal

Surface Metabolites on the
Settlement of the Benthic
Dinoflagellate Ostreopsis cf. ovata
Eva Ternon 1,2,3* † , Benoît Paix 4† , Olivier P. Thomas 5 , Jean-François Briand 4 and
Gérald Culioli 4*
1
CNRS, OCA, IRD, Université Côte d’Azur, Géoazur, Valbonne, France, 2 Laboratoire d’Océanographie de Villefranche,
CNRS UMR 7093, Sorbonne Universités, Villefranche-sur-Mer, France, 3 Center for Marine Biotechnology and Biomedicine,
Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, United States, 4 Laboratoire MAPIEM,
Université de Toulon, Toulon, France, 5 Marine Biodiscovery, School of Chemistry and Ryan Institute, National University
of Ireland Galway, Galway, Ireland
Edited by:
Elisa Berdalet,
Institute of Marine Sciences (CSIC), Macroalgae constitute one of the preferred substrates of the benthic dinoflagellate
Spain Ostreopsis cf. ovata. Across the Mediterranean Sea, this toxic microalga has been
Reviewed by: shown to thrive on the surface of various species of macroalgae, including Chlorophyta,
Stefano Accoroni,
Marche Polytechnic University, Italy Rhodophyta, and Phaeophyceae. Interestingly, some Dictyotaceae are characterized by
Olga Carnicer, a low abundance of cells of O. cf. ovata on their surface. Based on the antifouling
Dalhousie University, Canada
properties of some specialized metabolites produced by seaweeds, macroalgal
Leo Lai Chan,
City University of Hong Kong, metabolites have been proposed to contribute to the settlement and development of O.
Hong Kong cf. ovata. To address this question, the composition of the surface of four Dictyotaceae,
*Correspondence: Dictyota dichotoma, Dictyota spiralis, Taonia atomaria, and Padina pavonica was
Eva Ternon
eva.ternon@imev-mer.fr investigated through an integrative approach combining the analysis of their eukaryotic
Gérald Culioli diversity (18S rRNA gene metabarcoding), their surface metabolome (untargeted LC-
culioli@univ-tln.fr
MS-based metabolomics) as well as the bioactivity of their surface extracts on O. cf.
† These authors have contributed
equally to this work
ovata. Altogether, the data suggest an influence of the macroalgal surface chemistry
on the growth of the dinoflagellate, with D. dichotoma being the most bioactive. Some
Specialty section: metabolites are proposed to be involved in the observed bioactivity. Other biotic factors
This article was submitted to
Marine Ecosystem Ecology,
are also likely to be entailed in the control of the O. cf. ovata population and they may
a section of the journal even prevail on the influence of the macroalgal surface chemistry.
Frontiers in Marine Science
Keywords: Ostreopsis cf. ovata, Dictyotaceae, surface colonization, metabolomics, metabarcoding, eukaryotes,
Received: 13 March 2020 biofilms
Accepted: 28 July 2020
Published: 21 August 2020
Citation: INTRODUCTION
Ternon E, Paix B, Thomas OP,
Briand J-F and Culioli G (2020) Recurrent outbreaks of the toxic dinoflagellate Ostreopsis cf. ovata have been reported over the
Exploring the Role of Macroalgal
past 15 years in Mediterranean coastal waters (Ciminiello et al., 2006; Mangialajo et al., 2011).
Surface Metabolites on
the Settlement of the Benthic
Despite recent research efforts (reviewed in Pistocchi et al., 2011; Carnicer et al., 2015), the abiotic
Dinoflagellate Ostreopsis cf. ovata. factors fostering the proliferation of this species remain unclear (Cohu et al., 2011; Meroni et al.,
Front. Mar. Sci. 7:683. 2018) although high temperature, low hydrodynamic conditions seem to constitute a pre-requisite
doi: 10.3389/fmars.2020.00683 (Totti et al., 2010; Pezzolesi et al., 2012; Meroni et al., 2018). O. cf. ovata is a benthic species

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Ternon et al. Role of Macroalgal Surface Metabolites

that colonizes a variety of substrates, including macrophytes and settlement of O. cf. ovata remains largely to be assessed.
(Totti et al., 2010; Mangialajo et al., 2011). The surface of Results of such studies will provide valuable information on
these seaweeds can constitute an ideal microenvironment the pattern of colonization of this toxic dinoflagellate along the
for the growth of highly diverse prokaryotic and eukaryotic NW Mediterranean shoreline that leads to ecological (Gorbi
communities, including benthic microalgae. While the et al., 2013; Giussani et al., 2016; Pavaux et al., 2019) and health
interactions between bacteria and macroalgae have been (Ciminiello et al., 2006; Casabianca et al., 2013) concerns.
investigated to some extent during the past decades (Egan et al., The epiphytic community colonizing the surface of
2013; Hollants et al., 2013), less information has been provided macroalgae is not only expected to contribute to the surface
about the drivers guiding the settlement of microalgae on the metabolome but also to directly interact with O. cf. ovata, adding
surface of macroalgae. another level of complexity to its settlement preferences. Among
Ostreopsis cf. ovata has been shown to thrive on the surface co-occurring species on surfaces colonized by O. cf. ovata,
of various species of macroalgae across the Mediterranean Roseobacter bacteria (Vanucci et al., 2016; Guidi et al., 2018),
Sea, including Chlorophyta (Battocchi et al., 2010; Accoroni diatoms of the genera Licmophora, Navicula, and Amphora, and
et al., 2015; Hosny and Labib, 2019), Rhodophyta (Vila et al., the dinoflagellates Prorocentrum lima, Amphidinium carterae,
2001; Battocchi et al., 2010; Cohu et al., 2013; Blanfuné and Coolia monotis (Accoroni et al., 2015; Marro et al., 2019)
et al., 2015; Hosny and Labib, 2019; Gémin et al., 2020) and have been reported to be the most abundant. Recent evidences
Phaeophyceae (Vila et al., 2001; Battocchi et al., 2010; Cohu of chemically mediated interactions between microalgae and
et al., 2013; Blanfuné et al., 2015; Hosny and Labib, 2019; their predators (Ianora et al., 2004; Selander et al., 2015),
Gémin et al., 2020), without any clear species preference. competitors (Tillmann and Hansen, 2009; Poulson-Ellestad
Remarkably, some Phaeophyceae of the family Dictyotaceae et al., 2014; Ternon et al., 2018), and the bacterial community
are usually characterized by a low abundance of O. cf. ovata (Wichard and Beemelmanns, 2018 and references therein)
as for instance Taonia atomaria, Dictyota fasciola, or Padina suggest that biotic interactions within members of the epiphytic
pavonica (Blanfuné et al., 2015; Hosny and Labib, 2019; Gémin community may also influence the settlement of O. cf. ovata on
et al., 2020). The antifouling properties of some specialized macroalgal surfaces.
metabolites produced by Dictyotaceae have been proposed to This study aims to investigate the influence of the surface
drive the settlement and development of O. cf. ovata (Cohu et al., metabolome of four brown algae of the Dictyotaceae family
2013; Blanfuné et al., 2015; Gémin et al., 2020). Approaches in (T. atomaria, P. pavonica, D. dichotoma, and Dictyota spiralis)
chemical ecology have revealed that metabolites produced by on the settlement of the toxic dinoflagellate O. cf. ovata in
macroalgae can be detrimental or beneficial to the settlement the NW Mediterranean. An integrative approach was used to
of bacteria and microalgae (Goecke et al., 2010; Hellio et al., investigate both the chemical and eukaryotic compositions at
2002; Wichard and Beemelmanns, 2018). In particular, several the surface of each algal species combining untargeted LC-
metabolites produced by species of the family Dictyotaceae MS-based metabolomics and high throughput sequencing (18S
have been shown to inhibit the growth of bacteria (e.g., dictyol rRNA Illumina MiSeq sequencing), respectively. Applications of
C and fucoxanthin; Salvador Soler et al., 2007; Viano et al., dedicated experimental protocols allowed the selective extraction
2009), microalgae (e.g., dictyolactone and sanadaol; Kim et al., of the surface metabolites of each algal species (Othmani et al.,
2006), bryozoan (e.g., dictyol E, pachydictyol A, dictyodial; 2016a) and the activity of these extracts were further tested on
Schmitt et al., 1998) and various other benthic species (e.g., monocultures of the toxic dinoflagellate.
diterpenoids, gleenol, and geranylgeranylglycerol; Bianco et al.,
2009; Othmani et al., 2016b).
To the best of our knowledge, only one study has investigated MATERIALS AND METHODS
the effect of the metabolites from brown, red and green
macroalgae (Dictyota dichotoma, Rhodymenia pseudopalmata, Field sampling was performed at 9 a.m. on the 7th of July 2017
and Ulva rigida, respectively) on the growth of the dinoflagellate during an O. cf. ovata bloom at the Rochambeau site in the bay
O. cf. ovata (Accoroni et al., 2015). The exposure to dried algal of Villefranche-sur-Mer (France, Ligurian Sea; 43◦ 410 34.8300 N,
cells, fresh thalli and dissolved metabolites of D. dichotoma 7◦ 180 31.6600 E) known for the recurrence of such events (Cohu
caused an intense stress to O. cf. ovata cells yielding to et al., 2013; Jauzein et al., 2018; Gémin et al., 2020). The seawater
growth inhibition as well as formation of double-walled cysts. temperature and salinity were 23.9◦ C and 39, respectively, at the
Conversely, this inhibiting activity of D. dichotoma metabolites time of the collection. Thirteen specimens of each macroalgal
on the toxic dinoflagellate does not mirror the frequent species (D. dichotoma, D. spiralis, P. pavonica, and T. atomaria)
development of O. cf. ovata on D. dichotoma observed in previous were collected at 0.5 m depth by snorkeling. The collection of the
studies performed in the NW Mediterranean Sea (Cohu et al., macroalgae followed the protocol recommended by Totti et al.
2013; Blanfuné et al., 2015; Gémin et al., 2020). Following (2010) and consisted in holding specimens of each species with
common practices in the chemical ecology of macroalgae zip bags avoiding any agitation of the surrounding seawater and
(Puglisi et al., 2014), the authors used whole-cell macroalgal resuspension of benthic cells, followed by a careful removing
extracts which are not representative of real ecological situations of their holdfast from the rocky substrate using tweezers. All
occurring at the surface of macroalgae. Therefore, the influence zip bags containing macroalgae specimens and surrounding
of the surface metabolome of brown algae on the growth seawater were immediately closed underwater and processed

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Ternon et al. Role of Macroalgal Surface Metabolites

in the lab the same morning using: (i) four specimens of preserved at −80◦ C. After DNA extraction, the V7 region
each species to measure the abundance of O. cf. ovata and of 18S rRNA gene was amplified using 960F and NSR1438R
diatoms at the algal surface, (ii) six specimens of each species primers following Debroas et al. (2017). Amplicons were
to extract the surface and the whole macroalgal metabolomes, sent to GeT Platform (Toulouse, France) for MiSeq Illumina
and (iii) three specimens of each species for the analysis of sequencing (2 × 250 bp).
epiphytic eukaryotic communities through 18S rRNA gene high-
throughput sequencing. After sampling for macroalgae, a volume 18S rRNA Gene Metabarcoding Data
of 1 L of seawater was also collected from the water column at the
sampling site for 18S rRNA gene high-throughput sequencing.
Processing and Analysis
18S rRNA gene reads were processed using the FROGS workflow
under Galaxy environment (Escudié et al., 2017). Sequences were
Microalgal Cell Abundance at the
quality filtered by removing those for which primers sequences
Surface of the Macroalgae were not present. The primer search accepts 10% of differences.
Four specimens of each macroalga were transferred from their Primers sequences were then removed in the remaining sequence
zip bag to 15 mL Falcon tubes filled with 4 mL of filtered seawater using “cutadapt.” Clustering step was performed using SWARM
(0.2 µm) and 150 µL of acidic Lugol’s iodine solution. The tubes with a clustering aggregation distance set to 3 (Mahé et al., 2014).
were vigorously shaken and then vortexed during 5 s to remove Chimeric sequences were removed de novo using VSEARCH
epiphytes. Supernatants were kept in the Falcon tubes while the (Rognes et al., 2016). Rare OTUs representing less than 0.005% of
macroalgal thalli were removed with tweezers and air-dried on all sequences were removed. OTUs were affiliated with the Silva
a filter paper. No pre-filtration of the supernatant was needed 132 18S rRNA. The final matrix was obtained by performing a
(Totti et al., 2010) as sediments were not abundant. Pictures rarefaction to the minimum library size (8975 reads) using the
of the macroalgae were taken to further estimate their surface “phyloseq” R package (McMurdie and Holmes, 2013). Eukaryotic
area using the Mesurim software and to allow the expression
R
β-diversity was analyzed with a non-metric multidimensional
of the cell abundance in cell cm−2 . Epiphytic cell abundances in scaling (NMDS) using Jaccard distances with the “phyloseq”
the rinsed water were determined in a 1 mL volume Sedgwick package (McMurdie and Holmes, 2013). PERMANOVA test was
Rafter counting chamber under an inverted phase contrast performed using the distance matrix constructed with the Jaccard
microscope (Zeiss Axiovert 40C) at a magnification of ×40. index, using the “vegan” R package (Oksanen et al., 2013).
Identification of diatoms and dinoflagellates was performed
using data from the literature (Bertalot et al., 2000; Horiguchi,
2014, respectively). Bioassays
A bioassay was designed to test the activity of the SEs in 48-well
Chemical Extraction and Analysis of plates for which the well surface is ∼1 cm2 . The low amounts
of SEs available (0.2–5 mg) allowed only one bioassay. The
Macroalgal Samples activity of compounds of the SEs being unknown, the highest
The surface metabolome of the four seaweed species was concentrations prepared from the SEs were tested to ensure a
investigated on six replicates by a dipping method previously biological response: 2.5, 3.5, 1, and 2 mg cm−2 for T. atomaria,
developed for T. atomaria (Othmani et al., 2016a) that caused P. pavonica, D. dichotoma, and D. spiralis, respectively. These
no disruption of its membranes. P. pavonica is expected to concentrations are higher than those naturally encountered at the
have similar membrane strength as its surface is calcified surface of the macroalgae by a factor 5, 4, 1, and 3, respectively
(Benita et al., 2018). The integrity of the cell walls of the (Table 1). All SEs were re-suspended in dimethyl sulfoxide
two softer Dictyota species was confirmed after soaking using (DMSO) to reach concentrations of 50, 70, 20, and 40 mg mL−1 ,
an optical microscope. Briefly, surface extracts (SEs) were respectively for each alga. A volume of 50 µL of each SE was
obtained by soaking each algal thallus during 5s in an 8 mL added to the wells containing a thin layer of fresh unsolidified
glass vial filled with 5 mL of methanol (MeOH). The total agar and further dried at room temperature (0.3% of DMSO final
extracts (TEs) were obtained by soaking each thallus in 7 mL volume). A same volume of DMSO was similarly added to six
of MeOH during 18 h. Both resulting extracts (SEs and more wells as a control.
TEs) were concentrated under reduced pressure and stored at A monoclonal strain of O. cf. ovata obtained from the MCCV
−20◦ C until analyses and bioassays. The approximate extracted (Mediterranean Culture Collection of Villefranche, MCCV054)
surface area of each thallus was measured therefore leading was grown in L1 medium (Guillard and Ryther, 1962) prepared
to an estimation of the amount of extracts per cm2 of with autoclaved aged and filtered seawater (from the Bay of
macroalga (Table 1). Villefranche), adjusted to a salinity of 38 and maintained at 24◦ C
under a 14:10 light/dark cycle with a light intensity of 250 µmol
DNA Extraction and Amplification of the m−2 s−1 . After four days of growth (early exponential phase),
Epiphytic Communities 2 mL of the microalgal culture were sampled and fixed with
The epiphytic microbiota was sampled using a sterile scalpel, acidic Lugol’s iodine solution (4% v/v) and the cell abundance
introduced into plastic Eppendorf tubes (2 mL) and flash frozen was determined using a Sedgwick rafter counting chamber and
in liquid nitrogen. DNA extraction was carried out using the an inverted phase contrast microscope (Zeiss Axiovert 40C) at
PowerBiofilm DNA isolation Kit (Qiagen) and samples were a magnification of ×40. Appropriate volumes of culture and L1

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Ternon et al. Role of Macroalgal Surface Metabolites

TABLE 1 | Mean concentration (mg cm−2 ) of the SEs obtained from each macroalgal species given with the mean surface area measured for each species (n = 4).

Species Natural concentration Tested concentration Macroalgal surface

Mean SD Concentration Enrichment factor Mean SD

mg cm−2 mg cm−2 cm2

Taonia atomaria 0.5 0.1 2.5 5 7.4 1.8

Padina pavonica 0.9 0.3 3.5 4 7.3 2.3
Dictyota dichotoma 0.8 0.2 1.0 1 2.5 1.8
Dictyota spiralis 0.7 0.1 2.0 3 6.0 2.3

The concentration of the SEs tested in the bioassays is also given together with anestimation of the enrichment factors compared to natural concentrations. The
tested
enrichment factor was calculated as follow: Enrichment factor = Concentration natural .

medium were added to each well previously filled with SEs or analyzed on a reversed phase column (150 × 2.1 mm, 1.7 µm,
DMSO (blank) to obtain a concentration of 100 cell mL−1 of O. Kinetex Phenyl-Hexyl equipped with a SecurityGuard cartridge;
cf. ovata cells with a final volume of 1.4 mL. All well plates were Phenomenex, Le Pecq, France) with a column temperature of
incubated 24 h in the same conditions as for the cultures. 40◦ C and a flow rate of 0.5 mL min−1 . Mobile phases were (A)
To monitor the bioactivity of the SEs on O. cf. ovata, both water and (B) acetonitrile (Chromasolv; Sigma-Aldrich-Merck,
the physiological state and the abundance of the cells were Darmstadt, Germany) containing each 0.1% (v/v) of formic acid
measured after 24 h of incubation. All well plates were placed (Ultra grade; Fluka, Fischer Scientific, Illkirch, France). The
in the dark for 15 min and the well content are successively elution gradient started at 5% B and kept for 2 min, then to 100%
transferred to a 2 mL glass cuvette immediately moved to B (linear ramp) in 8 min and kept for 4 min; then back to 5%
a MC-PAM (Multi-Color Pulse-Amplitude-Modulated, Heinz B (linear ramp) over 0.01 min and maintained 1.99 min, for a
Walz Gmbh, Effeltrich, Germany) equipped with a blue LED total run time of 16 min. Mass spectrometry (MS) analyses were
(440 nm) as a source for the actinic light and a white LED performed in the full scan positive mode (ESI+). The capillary
used for the saturating pulses. The maximum quantum yield voltage of the MS spectrometer was set at 4500 V and the
(Fv /Fm ) of the photosystem II (PSII) was used as a proxy of the nebulizing parameters were set as follows: nebulizing gas (N2 )
microalga physiological state. It was calculated as (Fm −F0 )/Fm , pressure at 0.4 bar, drying gas (N2 ) flow at 4 L min−1 , and drying
where F0 is the fluorescence of a dark-adapted sample and temperature at 180◦ C. Mass spectra were recorded from m/z 50
Fm is measured after application of a saturation pulse of light to 1200. Tandem mass spectrometry analyses were performed
(intensity 431 µE m−2 s−1 , 300 ms duration). Curve-fitting with a collision induced dissociation (CID) and collision energy
software provided with the instrument (PAMwin V3.20W) was of 25 eV. A solution of formate/acetate forming clusters was
used to obtain Fv /Fm . All curve fits and fluorescence transients automatically injected before each sample for internal mass
were manually inspected in real time. Each sample was placed calibration, and the mass spectrometer was calibrated with the
back in its well after the PAM measurement and immediately same solution before each sequence of samples. Raw UHPLC-
fixed with acidic Lugol’s iodine solution (4% v/v) for cell counting HRMS data were analyzed using DataAnalysis (version 4.3;
under microscope as described earlier. The growth rate of O. cf. Bruker Daltonics), converted into netCDF files and processed for
ovata was calculated for each well as follows: peak finding, integration and alignment using the open source
XCMS package (version 1.46.0; Smith et al., 2006) in the R 3.2.3
ln(N2/N1)
µ= environment. The resulting variables list was filtered using three
t2 − t1 successive steps (signal/noise ratio, coefficient of variation and
where N1 and N2 are the cell abundances at times t1 (beginning coefficient of autocorrelation) with an in-house script running
of the experiment) and t2 (sampling after 24 h). on R. These variables were log10 -transformed, mean-centerd and
normalized using the sum of the chromatographic peak areas
UHPLC-HRMS Analysis of Macroalgal as described in Paix et al. (2020), and analyzed by Principal
Extracts Component Analysis (PCA) and partial least-square discriminant
analysis (PLS-DA) using the MetaboAnalyst 3.5 online resource1 .
Before injection, samples were solubilized in 1 mL of LC-
The global annotation was assessed by comparison of MS/MS
MS grade MeOH. Eleven quality control samples (QCs) and
spectra with those of an in-house database (purified compounds
four analytical blanks were also prepared as described in Paix
and commercial standards), and public databases (MarinLit,
et al. (2020), and all samples were randomly injected to avoid
MetLin, MoNA, or Lipidmaps). Subsequently, the annotation
systematic errors. Acquisition was performed by UHPLC-HRMS
procedure of analogs of known compounds was facilitated by
using a Dionex Ultimate 3000 rapid Separation chromatographic
molecular networking using the GNPS platform2 .
system (Thermo Fisher Scientific, Waltham, MA, United States)
coupled to a QToF Impact II mass spectrometer (Bruker
Daltonics, Bremen, Germany) equipped with an electrospray 1
http://www.metaboanalyst.ca
source. A volume of 5 µL of each sample was injected and 2
https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp

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Ternon et al. Role of Macroalgal Surface Metabolites

RESULTS determined by cell counting was highly consistent with the

results obtained by 18S rDNA gene sequencing. The major
Assessment of the Natural Abundance of OTU identified belonged to the genus Ostreopsis, while other
dinoflagellate taxa, without specific affiliation at the genus level,
Benthic Microalgae Using Microscopy
were also detected with very few sequences. Dinoflagellates
All four macroalgal species had their surface colonized by
represented only 1% of the total eukaryotic diversity on
diatoms, including species of the genera Licmophora, Amphora.,
T. atomaria, P. pavonica, and D. spiralis while it reached 6%
Navicula, Cylindrotheca, Striatella, and Gyrosigma, as well as
on D. dichotoma. The Harpacticoids, Amphiascoides, and
the dinoflagellate O. cf. ovata (Figure 1). O. cf. ovata showed
Paramphiascella were the main Arthropoda families and the
a lower abundance than diatoms, representing 14–23% of the
species Amonardia coreana, Parastenhelia sp., Amphiascoides
total number of counted cells, with densities ranging from 1.5
atopus, and Paramphiascella fulvofasciata were annotated. Several
to 7.9 × 103 cell cm−2 . Significant different concentrations
Annelida species of the Phyllodocida family were detected,
of O. cf. ovata cells were observed between macroalgal hosts
including Platynereis dumerilii and Syllis pigmentata; they
(ANOVA: p = 0.0175). Post hoc analysis revealed a significantly
were mainly distributed on D. dichotoma and P. pavonica. The
higher concentration of cells on D. dichotoma compared to
major platyhelminthe species was Convoluta convoluta which
D. spiralis and P. pavonica, while no significant differences
was reported on the two Dictyota spp. The Arthropoda were
were detected between T. atomaria, P. pavonica, and D. spiralis,
particularly represented on D. spiralis (67%) and T. atomaria
and neither between T. atomaria and D. dichotoma – due to
(32.5%) while P. pavonica showed the highest Annelida
one outlier sample.
community (39.5%). The Florideophycideae also largely
contributed to the eukaryotic diversity on T. atomaria (22.4%).
Diversity of Epiphytic Eukaryotes These same taxa were shown to colonize D. dichotoma, none
Through 18S rRNA Gene HTS of them being predominant: Dinoflagellata (6%), Arthropoda
When compared to the number of OTUs, the Chao1 index (18%), Ochrophyta (10%), Annelida (23%), Florideophycideae
reflects the richness that could be reached if a higher number (12%), Mollusca (8%), and Xenacoelomorpha (16%).
of sequences has been obtained. Here, Chao1 indicated that
the highest richness of eukaryotic communities was observed
in seawater as well as on the surface of T. atomaria and Chemical Analysis of Macroalgal
P. pavonica (Supplementary Figure S1). Considering the Extracts
β-diversity, seawater communities were clearly dissimilar The chemical diversity of the four macroalgal extracts was
from those found on the macroalgal surfaces (Figure 2 and assessed by untargeted LC-MS-based metabolomics and led
Supplementary Figure S2). The dinobiontes seemed to to 375 features after filtering. The PCA score plot of the
constitute the major taxa in seawater, with Amphidinium being surface and total (SEs and TEs) macroalgal extracts (Figure 3)
the most represented genus (54%) followed by Alexandrium showed a clear separation between the two Dictyota spp.
(9%), while Ostreopsis only represented 3%. Among epiphytes, and the two other species (T. atomaria and P. pavonica),
a clear clustering at the algal species level could be noticed, mainly on the first component (48% of the total variance)
with the two Dictyota species communities on the one highlighting a distinct chemical composition between these
hand, and T. atomaria and P. pavonica communities on species. Furthermore, the second component (18.5% of the total
the other hand (Supplementary Figure S2). Using the variance) highlighted differences between SEs and TEs within
Jaccard index with a NMDS analysis, the β-diversity of the each of these two algal groups. A list of the features responsible
eukaryotic communities revealed clear differences between the for the differences between SEs and TEs for each species is
epiphytic and the planktonic communities, but also between provided in Supplementary Table S1. The vast majority of
macroalgal hosts (Figure 2 and Supplementary Figure S2, these metabolites being minor ions on the chromatograms, very
PERMANOVA: p = 0.001). few could be annotated. Most metabolites specific to the SEs
Even though they serve as support of common taxa, were small/polar compounds as suggested by their retention
like Dinoflagellata, Arthropoda, Ochrophyta, Annelida, time (RT < 1 min) whereas TEs were more enriched in less
Florideophycideae, and Mollusca, the four macroalgae species polar metabolites (RT > 8 min). Dimethylsulfoniopropionate
exhibited a contrasted composition of their epiphytic eukaryotic (DMSP), Lyso-diacylglyceryl-3-O-carboxyhydroxymethylcholine
communities. Among the major detected taxa, two belonged to (C16:0) and a compound, putatively annotated as acetyl-L-
microalgae (Ochrophyta and Dinoflagellata), one to copepods cysteine, were found to be enriched in the SEs of T. atomaria,
(Arthropoda), two to red algae (Florideophycideae) and two to P. pavonica, and D. dichotoma, respectively.
upper benthic organisms (Annelida and Mollusca). The major The score plot of the PLS-DA only built with SEs (Figure 4)
species of diatoms present on the macroalgae belonged to the offered a more focused view of the distribution of these extracts.
genera Berkeleya, Navicula, Haslea, Nitzschia, Actinocyclus, None of the SEs overlapped with 95% confidence, suggesting
Amphora, Cylindrotheca, Cocconeis, Psammoneis, Hyalosira, significant differences in their chemical content, even for the
and Frustulia and their relative abundance was the highest two Dictyota spp. The distribution of the samples on both the
on P. pavonica (19%) and the lowest on D. spiralis (2%). PCA and the PLS-DA score plots was highly driven by the large
The abundance of Dinoflagellata in the epiphytic community number of metabolites produced by D. dichotoma and D. spiralis.

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Ternon et al. Role of Macroalgal Surface Metabolites

4x10⁴ a

3x10⁴
)
-2
Cell density (cell.cm

Ostreopsis ovata
Amphora sp.
Cylindrotheca sp.
2x10⁴
Gyrosigma sp.
Licmophora sp.
Navicula sp.
Striatella sp.
ab b b
1x10⁴

Taonia Padina Dictyota Dictyota

atomaria pavonica dichotoma spiralis

FIGURE 1 | Cell density (cell cm−2 ) of major diatoms and O. cf. ovata at the surface of the four macroalgae. Statistical significance was evaluated only for O. cf.
ovata abundances using an ANOVA test (*p = 0.0175) followed by a post hoc Tukey’s test (results indicated by a,b indexes).

The first 50 Variable Importance of Projection (VIPs) responsible were putatively identified in cluster B, and dictyotadimer A
for the separation of the samples on the PLS-DA score plot (VIP (VIP n◦ 13) in cluster I. Finally, even if not appearing in the
score >1.1) are listed in Table 2. molecular network, dictyol B (VIP n◦ 29) was also putatively
The Search of matches of MS data from compounds of annotated. Several other VIPs were not identified but were
our in-house database, the MarinLit database and molecular characterized as diterpenes or diterpene derivatives using their
network built from MS/MS spectra analysis (Figure 5) allowed MS and MS/MS data.
the annotation of several of the VIPs characteristic of each Both the lipid geranylgeranylglycerol and the sulfur
macroalga (Table 2). compound DMSP (VIPs n◦ 20 and 27, respectively) were
Several chemical markers enriched in D. dichotoma and identified by the mean of chemical standards as the major
D. spiralis SEs were detected in cluster B and I of the markers found in T. atomaria SEs. Five other compounds (VIPS
molecular network, allowing their putative annotation as n◦ 31, 36, 37, 40, and 42), putatively identified as diacylglyceryl-
diterpene derivatives. More precisely, the diterpenes dictyotalide 3-O-carboxyhydroxymethylcholines [DGCCs (C22:6), (C44:12),
A (or an isomer) (VIP n◦ 4), ent-erogorgiaene (VIP n◦ 10), (C36:6); Lyso-DGCCs (C16:0), and (C38:6), respectively), were
dictyotetraene (VIP n◦ 14), dictyol A (VIP n◦ 15), isopachydictyol also detected in T. atomaria and gathered in cluster D. Finally,
A (VIP n◦ 19), 17,18-18,19-bisepoxyxenic-19-methoxy-6,9,13- the VIP n◦ 46 was putatively annotated as the sesquiterpene
triene (VIP n◦ 25) and tricyclodictyofuran C (VIP n◦ 6 and 41) trans-calamenene using an in-house standard.

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Ternon et al. Role of Macroalgal Surface Metabolites

Dictyota dichotoma

Dictyota spiralis

Padina pavonica

Taonia atomaria

Seawater

0 25 50 75 100
Relative percentage of sequences (%)

Annelida ; Polychaeta Florideophycidae ; Corallinophycidae

Taxonomic affiliation

Apicomplexa ; Conoidasida Florideophycidae ; Rhodymeniophycidae

Arthropoda ; Maxillopoda Mollusca ; Bivalvia
Arthropoda ; Ostracoda Mollusca ; Gastropoda
Chlorophyta ; Chlorodendrophyceae Nematoda ; Chromadorea
Chlorophyta ; Ulvophyceae Ochrophyta ; Diatomea
Ciliophora ; Intramacronucleata Platyhelminthes ; Rhabditophora
Cnidaria ; Hydrozoa Xenacoelomorpha ; Acoela
Dinoflagellata ; Dinophyceae Other

FIGURE 2 | Eukaryotic communities colonizing the surface of the four macroalgae and present in the surrounding seawater. Replicate samples of a same species
were averaged (n = 3) and relative colonization to all sequences is given (%). Taxonomic affiliation corresponds to the Phylum and Class level. “Other” combines
Classes that represent less than 2% of the total diversity.

Padina pavonica was the algal species that showed the least (1 mg cm−2 ), the inhibition of O. cf. ovata growth induced by
chemical richness on its surface with only two unidentified D. dichotoma SEs was similar to those of P. pavonica while being
markers (VIPs n◦ 34 and 49) being found in its corresponding SEs. significantly lower than the one of T. atomaria. Although similar
More compounds not listed as VIPs were identified from other concentrations were used for T. atomaria and D. spiralis SEs
clusters of the molecular network: diacylglycerylhydroxymethyl- (2.5 and 2 mg cm−2 , respectively), the inhibition on O. cf. ovata
N,N,N-trimethyl-β-alanines (DGTAs) and phosphatidylcholines growth induced by T. atomaria SEs was significantly higher.
(PCs) present in cluster A, other PCs in cluster E, carotenoids All but P. pavonica macroalgal SEs induced a significant
in cluster F, sesquiterpenoids in cluster G, and pheophytin a stress on O. cf. ovata physiology as conveyed by the significant
derivatives in cluster N. alteration of the efficiency of the PSII shown by the Fv/Fm
ratio (p < 0.001; Figure 6B). Despite being tested at the lowest
concentration, the SEs of D. dichotoma caused among the most
Bioactivity of Surface Extracts on O. cf. intense inhibition of the PSII efficiency. Such an inhibition was
ovata comparable to those observed for D. spiralis and T. atomaria
To ensure a measurable effect on O. cf. ovata cells, the maximal SEs. Whether the growth rate of O. cf. ovata was not affected
concentration that could be obtained from the SEs was tested for by D. spiralis, the PSII efficiency was significantly altered to a
their bioactivity. Therefore, tested concentrations varied from 1 similar extent than with D. dichotoma. Mirroring the effects on
to 3.5 mg cm−2 according to the species and comparison between O. cf. ovata growth rate, T. atomaria SEs tested at 2.5 mg cm−2
bioactivities should be treated cautiously. Three macroalgal SEs also caused an intense stress on the PSII of the dinoflagellate. The
out of four induced a significant growth reduction of O. cf. related reduction in the Fv/Fm ratio measured was significantly
ovata (p < 0.001; Figure 6A), with D. spiralis SE found to be higher than for D. spiralis and P. pavonica, although tested at a
not statistically active. Despite tested at the lowest concentration similar and lower concentration, respectively.

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Ternon et al. Role of Macroalgal Surface Metabolites

that the development of this bloom followed a classic trend. In

terms of intensity, the bloom peaked at 9.2 ± 7.5 105 cells g−1
20
FW (Gémin et al., 2020) corresponding to a moderate bloom for
this site (Guidi-Guilvard et al., 2012; Cohu et al., 2013) or other
Mediterranean sampling sites (Totti et al., 2010). Consistently
10
with our results but also with other observations from the NW
Mediterranean Sea (Cohu et al., 2013; Blanfuné et al., 2015),
PC 2 (18.5 %)

0
Gémin et al. (2020) found the highest cell concentration of O. cf.
ovata on Dictyota spp. and the lowest on P. pavonica.

−10
Potential Control of the Abundance of O. cf. ovata by
the Epiphytic Community
A preferred colonization by O. cf. ovata on branched, three-
−20 dimensional thalli with a high surface/volume ratio has been
previously suggested by several authors (Vila et al., 2001; Totti
et al., 2010; Gémin et al., 2020) but such an hypothesis is not
−20 0 20 40 fully supported by the low abundance of O. cf. ovata measured
PC 1 (48.1 %) on the highly branched T. atomaria or by settlement differences
Extract type Seaweed
Taonia atomaria
observed between morphologically similar Dictyota species (this
Surface extract
Total extract
Padina pavonica study and Blanfuné et al., 2015). The 18S rRNA gene sequencing
Dictyota dichotoma
revealed a high diversity of eukaryotes colonizing the macroalgae,
Dictyota spiralis
suggesting that various biotic interactions may take place within
the epiphytic community having the potential to regulate the
FIGURE 3 | PCA score plot obtained from LC-(+)-ESI-MS metabolomics
analyses of both the total extracts (TEs) and surface extracts (SEs) of the four
colonization of O. cf. ovata.
macroalgae. Inter-specific competition for nutrients, light and space may
occur between the dinoflagellate O. cf. ovata and the various
species of diatoms that co-occurred within the epiphytes of
the macroalgae. Species of the genera Licmophora, Navicula,
and Amphora were the most abundant diatoms co-occurring
with O. cf. ovata, in line with previous findings (Accoroni
et al., 2016), while Coscinodiscus spp. usually detected in the
Catalan Sea (Vila et al., 2001; Carnicer et al., 2015) was
missing. Other dinoflagellate species were barely detectable
conversely to previous studies that reported the co-occurrence
of P. lima and C. monotis in various sampling sites across
the Mediterranean Sea, including the bay of Villefranche (Vila
et al., 2001; Aligizaki and Nikolaidis, 2006; Cohu et al., 2011;
Blanfuné et al., 2015; Accoroni et al., 2016). In recent co-
culture experiments, weak deleterious allelopathic effects induced
by Licmophora paradoxa toward O. cf. ovata were highlighted
(Ternon et al., 2018). A chemical control of the colonization
of O. cf. ovata by co-occurring benthic microalgae is therefore
possible although it may be counterbalanced by the existence of
an additional weak inhibiting allelopathic effects applied by O.
cf. ovata on co-occurring microalgae (Monti and Cecchin, 2012;
García-Portela et al., 2016).
Predation by benthic copepods is another factor that may
modulate the colonization pattern of benthic microalgae. Benthic
FIGURE 4 | PLS-DA score plot obtained from LC-(+)-ESI-MS metabolomics harpacticoids, such as Sphaeroma serratum, Tigriopus fulvus,
analyses of the surface extracts (SEs) of the four macroalgae.
Acartia clausi, and Sarsamphiascus cf. propinquus, have been
recently shown to feed on O. cf. ovata (Prato et al., 2011;
Faimali et al., 2012; Furlan et al., 2013; Pavaux et al., 2019) with
DISCUSSION similar ingestion rates as for diatoms (Boisnoir et al., 2020).
The resistance of benthic copepods to O. cf. ovata’s toxicity was
The simultaneous monitoring of both the abundance and toxin found to be highly variable among species, with LC50 ranging
content of benthic O. cf. ovata cells undertaken by Gémin et al. from 10 to 20,000 cells mL−1 (Prato et al., 2011; Faimali et al.,
(2020) at the same sampling site in the bay of Villefranche showed 2012; Pavaux et al., 2019). Ingestion of O. cf. ovata leads to

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Ternon et al.
TABLE 2 | Putative annotation of the first 50 chemical markers (VIP score >1.1) driving the distribution of the samples on the PLS-DA score plot obtained from LC-(+)-ESI-MS metabolomics analyses of the SEs of the
four macroalgae.

Color
VIP Cluster m/z Annotation Formula err msigma D. dichotoma D. spiralis P. pavonica T. atomaria code

1 – 472.236753 Unknown C26 H34 NO7 −9.6 9.5 −4

2 – 390.263585 Diterpene derivative (methoxylated and acetylated)? C23 H36 NO4 −2.8 1.1 −2
3 SL 461.300916 Unknown C26 H41 N2 O5 1.0 13.0 0
4 B 319.226939 Dictyotalide A or isomer C20 H31 O3 −1.0 12.9 2
5 J 447.285584 Unknown C25 H39 N2 O5 −0.6 6.7 4
6 – 317.247489 Tricyclodictyofuran C or isomer C21 H33 O2 −1.2 12.4
7 – 422.254062 Diterpene derivative (methoxylated and acetylated)? C23 H36 NO6 −1.6 4.6
8 I 378.264217 Diterpene derivative (acetylated) C22 H36 NO4 −3.8 9.3
9 – 364.284964 Diterpene derivative (acetylated) C22 H38 NO3 −2.7 17.0
10 B 271.242119 ent-erogorgiaene C20 H31 −1.0 4.4
11 – 342.242938 4-methylaminoacarone C22 H32 NO2 −2.7 11.9
12 – 573.466831 Carotenoid (pirardixanthin derivative?) C40 H61 O2 −0.3 28.1
9

13 I 637.448128 Dictyotadimer A C40 H61 O6 −2.7 14.0

14 B 269.226492 Dictyotetraene C20 H29 −0.3 66.9
15 B 285.221393 Dictyol A* C20 H29 O −1.8 12.4
16 – 413.280297 Apo-carotenoid C30 H37 O 8.5 4.4
17 B 283.20567 Unknown C20 H27 O −1.0 7.9
18 – 743.450601 Unknown C46 H63 O8 −1.0 38.4
19 B 289.252582 Isopachydictyol A or dollabellatrienone C20 H33 O 0.5 6.7
20 – 382.331504 Geranylgeranylglycerol* C23 H44 NO3 −1.7 6.1
21 – 373.235224 Unknown C21 H34 NaO4 −0.3 2.6
22 – 410.290343 Diterpene derivative (methoxylated and acetylated)? C23 H40 NO5 −3.4 4.7
23 (B) 333.242627 Diterpene derivative (methoxylated) C21 H33 O3 −1.8 7.7
24 SL 446.290738 Pharmacin B C26 H40 NO5 −1.0 7.2

Role of Macroalgal Surface Metabolites

25 B 333.242496 Diterpene derivative (methoxylated) C21 H33 O3 −0.8 4.4
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26 – 406.258778 Diterpene derivative (methoxylated and acetylated)? C23 H36 NO5 −2.9 5.0
27 – 135.047248 DMSP* C5 H11 O2 S 0.2 1.0

(Continued)
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Ternon et al.
TABLE 2 | Continued

VIP Cluster m/z Annotation Formula err msigma D. dichotoma D. spiralis P. pavonica T. atomaria

28 – 489.343493 Unknown C26 H49 O8 −3.5 13.7

29 — 327.229615 Dictyol B C20 H32 NaO2 −0.5 14.2
30 – 654.47347 Carotenoid C40 H64 NO6 −5.5 18.9
31 D 562.374243 DGCC (C22:6) C32 H52 NO7 −2.3 1.4
32 – 356.25884 Unknown C23 H34 NO2 −2.7 16.0
33 – 426.231272 Unknown C22 H36 NO5 S −2.6 15.7
34 SL 653.297738 Unknown C29 H49 O16 2.3 10.3
35 E 741.614801 Unknown C46 H81 N2 O5 −2.5 9.7
36 D 872.605027 DGCC (C44:12) C54 H82 NO8 −3.1 2.1
37 D 772.573549 DGCC (C36:6) C46 H78 NO8 −2.7 2.1
38 – 317.247669 Tricyclodictyofuran C or isomer C21 H33 O2 −3.0 3.5
10

39 SL 318.242795 Unknown C20 H32 NO2 −1.6 18.5

40 D 490.374196 Lyso-DGCC (C16:0) C26 H52 NO7 −0.7 14.3
41 B 287.237035 Tricyclodictyofuran B C20 H31 O −0.8 27.2
42 D 800.604167 DGCC (C38:6) C48 H82 NO8 −2.1 6.6
43 B 343.226662 Diterpene derivative (acetylated) C22 H31 O3 −2.2 9.6
44 SL 260.112865 Unknown C11 H18 NO6 −0.6 3.2
45 – 453.27291 Apo-carotenoid C30 H38 NaO2 4.0 10.3
46 – 201.16362 Trans-calamenene* C15 H21 −2.8 25.8
47 SL 376.787236 FA (C24:5) C24 H42 NO2 3.3 9.9
48 SL 445.292694 Amphidinolide C25 H42 NaO5 −0.1 5.3
49 – 704.546719 Unknown C42 H74 NO7 −2.5 7.7
50 – 319.226828 Dictyotalide A or isomer C20 H31 O3 −0.3 27.0

Role of Macroalgal Surface Metabolites

Identification of the compounds marked by * was confirmed with a chemical standard. Clusters “SL” corresponds to a self-loop (a metabolite only connected to itself). The color code represents the mean concentration
August 2020 | Volume 7 | Article 683

values obtained for each macroalga. DGCC, diacylglyceryl-3-O-carboxyhydroxymethylcholines; FA, fatty acid.
Ternon et al. Role of Macroalgal Surface Metabolites

Cluster A: PCs and DGTAs Cluster B: Diterpenes

Cluster C
Diterpene
(ent-erogorgiaene Diterpene
?) C20H31 derivative
(acetylated)?
C22H31O3

Diterpene
(Dictyotetraene?) Dictyol A
C20H29 [M-H2O+H]+
C20H31O2
Diterpene ?
C20H27O
Dictyol A [M+H]+
C20H29O
Diterpene:
Dictyotalide A ?
Diterpene (Isopachydictyol A or C20H31O3
Dollabellatrienone ?) C20H33O

Diterpene Diterpene derivative

(Tricyclodictyofuran (17,18-18,19-Bisepoxyxenic- 19-
B ?) C20H31O methoxy-6,9,13-triene ?) C21H33O3

Cluster D: DGCCs Cluster E: Cluster F Cluster G

DGCC (C46:2)
DGCC (C36:6)
PCs Carotenoid?
C41H59O
PC (C36:5) Acetylated
Acetylated

DGCC (C44:12)
PC (C40:8)
Carotenoid?
C40H57O
sesquiterpene?
C17H32NO4
sesquiterpene?
C17H30NO4 Cluster H
Lyso -DGCC (C18:0) Lyso -PC (C16:0)
DGCC (C38:6)
Carotenoid?
C40H55O Sesquiterpene?
PC (C40:7)
Lyso -DGCC (C16:0) PC (C38:6) C15H28NO4
Sesquiterpene?
DGCC (C22:6)
C15H30NO3
beta-carotene

C40H55O2
Lyso -DGCC (C14:0)

Acetylated sesquiterpenes ?
Carotenoids ?
Cluster I Cluster J Cluster K Cluster L
Carotenoid or
diterpene dimer C25H39N2O5
C40H61O6

Carotenoid or
diterpene dimer
C40H59O4 Cluster M Cluster O
Acetylated
Cluster N
diterpene? C46H81N2O5 Pheophytin
C22H33O4 Acetylated derivative
diterpene?
C22H36NO4 Pheophytin Pheophytin a
derivative

Carotenoids or Pheophytin a and derivatives

diterpenes dimers ? VIP biomarker for Dictyota spiralis
VIP biomarker for Dictyota dichotoma
VIP biomarker for Taonia atomaria

FIGURE 5 | Molecular network obtained from LC-(+)-ESI-MS/MS data of the SEs of the four macroalgal species. Only clusters with three or more nodes are
represented. Nodes represent MS/MS spectra which are connected based on their spectral similarity. Colored nodes indicate biomarkers according to the four
macroalgae (orange: D. dichotoma, green: D. spiralis, red: T. atomaria, no VIPs for P. pavonica were observed). The edge width is proportional to the cosine score.
PC, phosphatidylcholines; DGTA, diacylglycerylhydroxymethyl-N,N,N-trimethyl-b-alanines; DGCC,diacylglyceryl-3-O-carboxyhydroxymethylcholines.

reduced fecal pellets production and fecundity rates indicating a weight (9.2 ± 7.5 105 cells g−1 FW; Gémin et al., 2020) likely
reprotoxic effect (Pavaux et al., 2019) that may cause a reduction exceeded the toxic level of 20,000 cells per mL (Pavaux et al.,
in nauplii abundance (Guidi-Guilvard et al., 2012) but not on 2019). Nevertheless, the amount of toxins per cell reported in situ
adults’ abundance. Our study was conducted at the peak of the was lower compared to the MCCV054 strain of O. cf. ovata
bloom, when the highest concentrations of O. cf. ovata and used in Pavaux et al. study (5–10 against 13 pg cell−1 ; Gémin
toxin content per cell were reported (Gémin et al., 2020). In situ et al., 2020; Pavaux et al., 2020, respectively). Additionally, Gémin
cell concentrations reported in cells per g of macroalga fresh et al. (2020) showed that the toxin content in O. cf. ovata varied

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Ternon et al. Role of Macroalgal Surface Metabolites

0.12 a
p < 0.001
ab
0.09 b
bc

(A) Growth rate

0.06
c

0.03

T. atomaria
0.00 P. pavonica

p < 0.001 ab a D. dichotoma

D. spiralis
0.3 b Control

bc
0.2

(B) Fv/Fm
c

0.1

0.0
T. atomaria P. pavonica D. dichotoma D. spiralis Control

FIGURE 6 | (A) Growth rate (µ) of O. cf. ovata and (B) Maximum quantum yield of PSII (Fv /Fm ) of dark-adapted samples of O. cf. ovata after exposure of 24 h to
macroalgal surface extracts (SEs) and blanks (n = 6 per condition). p-values and a, b, c indexes correspond to results of the one-way ANOVA and Tukey’s tests,
respectively.

according to the macroalgal species they colonized. The toxicity similarly abundant on D. spiralis, T. atomaria, and P. pavonica
of O. cf. ovata toward grazers may therefore vary according to (1,505; 2,251, and 1,759 cells cm−2 , respectively), not mirroring
the substrate. Therefore, it is difficult to conclude whether the their different ratio Ostreopsis:maxillopods (1:67, 1:33, and 1:17,
maxillopods are suffering from the natural concentrations of O. respectively) in term of comparison of 18S rRNA gene sequences.
cf. ovata encountered on the four macroalgae. Moreover, the Other predators, like polychaetes, were also shown to graze
resistance of the copepod species (P. fulvofasciata, A. atopus, O. cf. ovata despite an important sensitivity to the ovatoxins
A. coreana, Harpaticus spp., and Nemesis spp.) detected on the (Simonini et al., 2011). Concentrations of O. cf. ovata as low
four macroalgae to O. cf. ovata has not been experimentally as 200 cells mL−1 caused the death of 100% of a population of
tested and may differ from already tested species. It is worth Dinophylus gyrociliatus in less than 48 h (Simonini et al., 2011).
noting that the relative abundance of benthic copepods and O. However, similarly to copepods, the capacity to feed on O. cf.
cf. ovata did not show any obvious correlation. In contrast, ovata as well as the resistance to the ovatoxins may differ among
if maxillopods have the capacity to feed on O. cf. ovata, they species and according to the toxicity of O. cf. ovata cells linked
unlikely are the major factor that regulates its colonization on with the substrate they colonize (Gémin et al., 2020). The species
the macroalgae. Indeed, the toxic dinoflagellate was found to be S. pigmentata, P. dumerilii, and Neodexiospira brasiliensis, for

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Ternon et al. Role of Macroalgal Surface Metabolites

which no LC50 is available yet, were the most abundant while Heterosigma akashiwo (Kim et al., 2006), dictyol C that reduces
D. gyrociliatus was not detected on the macroalgae. the adhesion of Pseudoalteromonas spp. (Viano et al., 2009), as
The eukaryotic richness of the epiphytic macroalgal well as dictyol E, dictyol B acetate, pachydictyol A and dictyodial
communities was high, particularly on T. atomaria and that all cause significant larval mortalities of invertebrates
P. pavonica, and interactions that may result from it have not (Schmitt et al., 1998). Although none of the cited compounds
been fully investigated yet. For instance, the bacterial community were major chemical markers in any of the two Dictyota spp.,
which was not described in this study may play an important the high diterpenes content of their SEs suggests that this family
role in the regulation of epiphytic communities (Tait et al., 2005; of metabolites could be responsible for the observed bioactivity
Wheeler et al., 2006; Saha and Weinberger, 2019), including of both species. Nevertheless, considering the higher stress
O. cf. ovata. The present data set did not highlight a specific induced by D. dichotoma compared to D. spiralis on O. cf. ovata,
biotic interaction within the epiphytic community that may chemical markers more specific to D. dichotoma were sought.
favor or inhibit the settlement of O. cf. ovata on the different Two metabolites listed as VIPs n◦ 5 and 12 were significantly
macroalgal species. more enriched in D. dichotoma SEs (Supplementary Figure S3)
with the molecular formulae C25 H39 N2 O5 and C40 H61 O2
Algal Surface Chemistry Influences the Growth of O. corresponding to an unidentified compound (cluster J) and a
cf. ovata carotenoid-like compound likely to belong to the pirardixanthin
Several studies have shown deleterious effects of macroalgal family (Tsushima et al., 2001), respectively. While the activity
metabolites on microalgae using whole cells dried and grounded of these families of compounds has not been determined so
(Tang and Gobler, 2011; Accoroni et al., 2015) or organic whole- far, some carotenoids like fucoxanthin are known to possess
cell extracts (Hellio et al., 2002; Nagayama et al., 2003). To inhibiting properties against bacteria (Viano et al., 2009; Saha
our knowledge, this study is the first attempt in evaluating the et al., 2011) which could also target benthic microalgae.
bioactivity of SEs on settlement and growth of a microalgal Taonia atomaria showed a higher bioactivity than D. spiralis
species. Previous study on T. atomaria demonstrated a satisfying and P. pavonica, tested at a similar and higher concentration,
extraction of surface metabolites without disturbance of the respectively. This bioactivity is, however, likely overestimated
macroalgal membranes by the soaking protocol used in the compared to an ecological situation (enrichment factor of
present study (Othmani et al., 2016a). The differences observed 5). Both geranylgeranylglycerol and DMSP were the major
in the chemistry of the SEs and TEs for all four macroalgae biomarkers in T. atomaria SEs, in agreement with previous
species further validated the soaking protocol as an efficient findings (Othmani et al., 2016a; Paix et al., 2019), and were
method to extract surface metabolites of the three other species therefore enriched in this species compared to the three others
P. pavonica, D. spiralis, and D. dichotoma. Most chemical markers (Supplementary Figure S3). These two metabolites have shown
characteristics of the SEs were found to be polar metabolites activity against bacterial settlement (Saha et al., 2012; Othmani
(RT < 1 min), including DMSP, whereas TEs were characterized et al., 2016a,b) and may have the potential for inhibiting
by less polar compounds (RT > 8 min). the growth of O. cf. ovata. However, DMSP is a metabolite
The chemical fingerprint of the four macroalgae species largely biosynthesized by O. cf. ovata itself (personal data; Chen
draws two distinct groups: the two Dictyota spp. clustering et al., 2020), leaving geranylgeranylglycerol as the most likely
together and P. pavonica and T. atomaria gathering in the candidate. The present dataset did not allow to propose any
second group. In terms of eukaryotic β-diversity, the same two metabolite for P. pavonica that could explain the unspecific
groups emerged, suggesting that the metabolites at the surface bioactivity of its SEs. If all four macroalgae species have the
of the macroalgae are influencing the settlement of eukaryotes. ability to affect O. cf. ovata’s growth or physiology, bidirectional
Since both T. atomaria and P. pavonica showed a higher effects may also be considered since some macroalgae have been
richness of their overall epiphytic eukaryotic community, their shown to rely on microbial metabolites for cell division and
surface metabolites could be considered less bioactive toward differentiation (Wichard and Beemelmanns, 2018). The bioactive
these communities, although higher antibacterial activities have metabolites produced by the benthic dinoflagellate (Pavaux et al.,
been previously observed for Taonia and Dictyota spp. among 2020) may be detrimental or beneficial to the macroalgal host.
Phaeophyceae species (Salvador Soler et al., 2007). When looking The bioactivity of the SEs combined to the natural settlement
specifically at O. cf. ovata, the surface chemistry of the four of O. cf. ovata suggests that the colonization preferences of
macroalgae has the potential to influence the settlement of O. cf. ovata are not driven by the sole bioactivity of the
the toxic dinoflagellate by altering both its physiology and surface chemistry of macroalgae. Indeed, the highest abundance
its growth. Nevertheless, the use of different concentrations of O. cf. ovata’s cells were found on D. dichotoma that
(1–3.5 mg cm−2 ) in the bioassay does not allow to order simultaneously presented the highest bioactivity at ecological
the bioactivity of the four macroalgae, however D. dichotoma concentrations (1 mg cm−2 ). The toxicity of D. dichotoma
clearly induced a strong inhibition of both the growth and surface chemistry is likely faded by other unknown factors
the PSII efficiency of O. cf. ovata at ecological concentrations that may include reduced grazing and interspecific competition
(1 mg cm−2 ) and can therefore be considered as the most as shown by the lower ratio dinoflagellates:maxillopods and
bioactive species. Many diterpenes isolated from Dictyota spp. dinoflagellates:diatoms observed at the surface of D. dichotoma
have been found to exhibit antifouling properties as for instance compared to other macroalgae. Diterpenes of Dictyota spp.
the algicidal dictyolactone toward the red-tide microalgae have been shown to have deterrent effects on a wide range of

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Ternon et al. Role of Macroalgal Surface Metabolites

herbivores (Hay and Steinberg, 1992; Pereira et al., 2000) and DATA AVAILABILITY STATEMENT
could also target grazers like copepods since they have been
shown to be sensitive to various allelochemicals (Ianora et al., The datasets presented in this study can be found in online
2004). The lower bioactivity of D. spiralis SEs compared to those repositories. The names of the repository/repositories and
of D. dichotoma (tested at 2 mg cm−2 ) could not explain the accession number(s) can be found below: NCBI Sequence Read
low abundance of O. cf. ovata on its surface, suggesting the Archive (SRA) (accession: PRJNA612893).
existence of other inhibiting factors controlling the colonization
of the dinoflagellate on this macroalga as for instance the large
relative abundance of copepods compared to that found at the
surface of D. dichotoma.
AUTHOR CONTRIBUTIONS
The outcome of this experiment considered neither a spatial ET, BP, J-FB, and GC designed the experiments. ET and BP
nor temporal variability, and rather describes an interaction conducted the experiments and treated the data. All the authors
between macroalgae and O. cf. ovata at high temperature participated in the analysis of the data, contributed to the
(∼24◦ C) and salinity (∼39) conditions commonly found in the manuscript and approved the submitted version.
NW Mediterranean coastal waters in summer (Vila et al., 2001). If
O. cf. ovata mostly blooms in summer across the Mediterranean
Sea (Ciminiello et al., 2006; Cohu et al., 2013), late blooms are
also reported in several areas (Aligizaki and Nikolaidis, 2006; FUNDING
Accoroni et al., 2012). The macroalgae colonized by O. cf.
ovata were shown to differ throughout the year (Aligizaki and This work benefited from the support of the project OCEAN-
Nikolaidis, 2006; Battocchi et al., 2010; Hosny and Labib, 2019) 15 (ANR-15-CE35-0002-01) of the French National Research
likely in link to the macroalgal seasonality that modifies both Agency (ANR) and from the project CMAPO (2017) of the
their distribution and chemotypes (Paix et al., 2019) as well as French GdR Mediatec. This work was also funded by the French
their related bioactivity (Salvador Soler et al., 2007). In addition, “Sud Provence-Alpes-Côte d’Azur (Sud PACA)” regional council
the growth of O. cf. ovata was shown to be salinity sensitive (Ph.D. grant of BP). The open access publication was supported
(Pistocchi et al., 2011) and is optimal at salinity 36. Salinity also by the MSCA project CHEMICROS (H2020-MSCA-IF-841051).
determines the distribution of seaweeds species (Martins et al.,
1999) by altering various physiological aspects, including the
production of metabolites (Polo et al., 2015). Therefore, at lower ACKNOWLEDGMENTS
salinities areas (e.g., Northern Adriatic) a modified macroalgal
bioactivity may lead to a different colonization pattern by O. cf. The authors are grateful to Anaïs Lebrun for her help
ovata (Accoroni et al., 2016). with bioassays and to Dr. Stéphane Greff (Aix-Marseille
University, IMBE) for his help during the acquisition of
LC-MS profiles. LC-MS experiments were conducted on the
CONCLUSION regional platform MALLABAR funded by the Institute of
The present dataset confirms that some Phaeophyceae of the Ecology and Environment (INEE) of the French National Centre
family Dictyotaceae like T. atomaria, Dictyota spp. or P. pavonica for Scientific Research (CNRS) and the French Sud PACA
may be characterized by a lower abundance of O. cf. ovata regional council.
cells. Whether the surface chemistry of the macroalgae have
the potential to handicap the growth of O. cf. ovata, it is not
enough to explain the settlement preference on D. dichotoma SUPPLEMENTARY MATERIAL
that was found to be the most bioactive species. Complex
interlacing factors involving several members of the epiphytic The Supplementary Material for this article can be found
community are likely to modulate the growth of the toxic benthic online at: https://www.frontiersin.org/articles/10.3389/fmars.
dinoflagellate O. cf. ovata on macroalgae. 2020.00683/full#supplementary-material

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Frontiers in Marine Science | www.frontiersin.org 16 August 2020 | Volume 7 | Article 683