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# Prokaryotic Diversity and Dynamics during Dinoflagellate Bloom Decays in Coastal Tunisian Waters

Rim Lajnef, Marianne Quéméneur, Moufida Abdennadher, Lamia Dammak

Walha, Asma Hamza, Malika Belhassen, Amel Bellaaj Zouari

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Rim Lajnef, Marianne Quéméneur, Moufida Abdennadher, Lamia Dammak Walha, Asma Hamza, et al.. Prokaryotic Diversity and Dynamics during Dinoflagellate Bloom Decays in Coastal Tunisian Waters. Diversity, 2023, 15 (2), pp.273. 10.3390/d15020273. hal-04114131

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Article

# Prokaryotic Diversity and Dynamics during Dinoflagellate Bloom Decays in Coastal Tunisian Waters

Rim Lajnef <fnref n="1" />, Marianne Quéméneur <fnref n="2" />,* , Moufida Abdennadher <fnref n="1" />, Lamia Dammak Walha <fnref n="1" />, Asma Hamza <fnref n="1" />, Malika Belhassen <fnref n="1" /> and Amel Bellaaj Zouari <fnref n="1" />,*

**Abstract:** (1) Background: Harmful algal blooms (HABs) can negatively impact marine ecosystems, but few studies have evaluated the microbial diversity associated with HABs and its potential role in the fates of these proliferations. (2) Methods: Marine prokaryotic diversity was investigated using high-throughput sequencing of the 16S rRNA gene during the bloom declines of two dinoflagellates detected in the summer of 2019 along the northern and southern Tunisian coasts (South Mediterranean Sea). The species *Gymnodinium impudicum* (Carthage, Tunis Gulf) and *Alexandrium minutum* (Sfax, Gabes Gulf) were identified using microscopy and molecular methods and were related to physicochemical factors and prokaryotic compositions. (3) Results: The abundance of *G. impudicum* decreased over time with decreasing phosphate concentrations. During the *G. impudicum* bloom decay, prokaryotes were predominated by the archaeal MGII group (*Thalassarchaeaceae*, *Pelagibacterales* (SAR11), *Rhodobacterales*, and *Flavobacteriales*. At Sfax, the abundance of *A. minutum* declined with decreasing phosphate concentrations and increasing pH. At the *A. minutum* peak, prokaryotic communities were largely dominated by anoxygenic phototrophic sulfur-oxidizing *Chromatiaceae* (*Gammaproteobacteria*) before decreasing at the end of the survey. Both the ubiquitous archaeal MGII group and *Pelagibacterales* were found in low proportions during the *A. minutum* decay. Contrary to the photosynthetic *Cyanobacteria*, the photo-autotrophic and -heterotrophic *Rhodobacterales* and *Flavobacteriales* contents remained stable during the dinoflagellate bloom decays. (4) Conclusions: These results indicated changes in prokaryotic community diversity during dinoflagellate bloom decays, suggesting different bacterial adaptations to environmental conditions, with stable core populations that were potentially able to degrade HABs.

**Keywords:** bacteria; archaea; microbial diversity; HABs; dinoflagellates; Tunisia; Mediterranean Sea; marine ecosystems; *Alexandrium*; *Gymnodinium*

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**Citation:** Lajnef, R.; Quéméneur, M.; Abdennadher, M.; Dammak Walha, L.; Hamza, A.; Belhassen, M.; Bellaaj Zouari, A. Prokaryotic Diversity and Dynamics during Dinoflagellate Bloom Decays in Coastal Tunisian Waters. *Diversity* **2023**, *15*, 273. https://doi.org/10.3390/d15020273

Academic Editors: Ana C. Sampaio and Bert W. Hoeksema

Received: 22 December 2022

Revised: 9 February 2023

Accepted: 10 February 2023

Published: 14 February 2023

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**Copyright:** © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).

## 1. Introduction

Phytoplankton are a major component of marine ecosystems and form the basis of the ocean's food chains. They drive, with the terrestrial biosphere, the chemical composition of the global atmosphere and thereby the climate [1]. They contribute to the production of more than half of terrestrial oxygen [2] and approximately 50% of global primary production. However, under certain conditions, some phytoplankton taxa can grow out of control and form harmful algal blooms (HABs), which can negatively impact aquatic ecosystems and associated economic activities (e.g., aquaculture, fishing activities, and seaside tourism) as well as human health [3,4]. Most harmful microalgae species (planktonic and benthic) belong to the dinoflagellate group, such as species of the genera *Alexandrium* and *Gymnodinium* [5,6], which proliferate recurrently in various marine ecosystems worldwide by forming large blooms in coastal waters [5–7]. Their proliferation can cause contamination and mortality of the seafood chain (including mollusks and fish)

<footnote n="1">¹ Institut National des Sciences et Technologies de la Mer, 28 Rue 2 Mars 1934, Salammbô 2025, Tunisia</footnote>

<footnote n="2">² Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO UM 110, Marseille, France</footnote>

<footnote>* Correspondence: marianne.quemeneur@ird.fr (M.Q.); amel.zouari@instm.rnrt.tn (A.B.Z.)</footnote>
through the production of different kinds of toxins or the creation of anoxic areas by de-
pleting the dissolved oxygen consumed by bacteria during the decomposition of organic
matter from microalgae [8].

In recent decades, the increase in the frequency, intensity, and geographical distri-
bution of HABs worldwide has intensified scientific investigations of the ecological and
physiological factors that trigger the initiation and the decline of these events and influence
their magnitude [9]. HAB dynamics include three critical phases (i.e., the triggering pro-
cess, maintenance, and decline) and are generally influenced by several synergic abiotic
factors, such as hydrodynamic processes, environmental conditions (e.g., temperature,
salinity, and vertical mixing turbidity due to wind) [10,11], and nutrient availability [12].
In addition, some biotic factors, such as the compositions of microbial communities, in
particular that of bacteria, have been increasingly mentioned as parameters influencing
HAB dynamics because of their key roles in biogeochemical cycles and their production
of algae-stimulating or -inhibiting elements [13,14]. Bacteria can stimulate HAB growth
via nutrient regeneration or vitamin production [15,16] and can induce bloom decay via
algicidal activities [17,18]. Recently, it has also been shown that HABs affect bacterial
community abundance and diversity [19,20], depending on the type of microalgal species,
their physiological state, and the environmental conditions [20,21]. However, the mon-
itoring of archaeal community dynamics during HABs is still largely undervalued [20].
A global consideration of the prokaryotic diversity and the interactions between these
microorganisms via an evaluation of their dynamics could improve our understanding
of the triggering and resilience mechanisms during HABs and could help in predicting
such events.

In Tunisia, the majority of HAB studies have focused on the spatiotemporal distribu-
tion of HAB species in relation to abiotic factors, considered as environmental enhancers,
such as nutrients and physical parameters (salinity, temperature, and tide amplitude),
meteorological constraints (evaporation, air temperature, insolation, rainfall, atmospheric
pressure, and humidity), and nutrients [22–28]. Despite their major roles in the function-
ing of marine ecosystems, no previous data have been collected related to biotic factors,
especially the prokaryotic community composition during HABs occurring along Tunisian
coasts or other southern Mediterranean coasts.

In this context, the general objective of this study was to analyze for the first time the di-
versity and dynamics of the marine prokaryotic communities associated with HAB decays
in two South Mediterranean coastal ecosystems. Two dinoflagellate bloom events caused
by *G. impudicum* and *A. minutum* were, respectively, monitored using microscopy during
the summer of 2019 at northern (Tunis Gulf) and southern (Gabès Gulf) Tunisian sites that
differed in their functioning and their degree of anthropization/exploitation. Morphology
and sequence analyses of ribosomal regions (LSU and ITS) of the *G. impudicum* Gy.imp1 and
*A. minutum* Am.6 clonal strains were also investigated. Then, the bloom-forming species
were related to environmental variables (i.e., temperature, pH, salinity, and nutrients), and
the variability in prokaryotic community composition was investigated using 16S rRNA
gene high-throughput sequencing (Illumina MiSeq) to better understand the role of prokary-
otes in the decline stages of HABs and potentially propose bioremediation solutions.

**2. Materials and Methods**

## 2.1. *Study Sites*

This study was carried out in two Tunisian coastal sites (southeastern Mediterranean Sea) that were impacted by dinoflagellate blooms during summer 2019 (Figure 1). Since the 1980s, the toxic armored dinoflagellate *A. minutum* has formed many blooms in eutrophic and semi-enclosed ecosystems along the northern (the Gulf of Tunis) [22] and southern (the Gulf of Gabès) Tunisian coasts [23], whereas HAB events associated with *G. impudicum* were observed only in the Bay of Tunis [24–26]. *Alexandrium* blooms have often coincided with mass fish kills [27] and contamination with paralytic shellfish poisoning (PSP) [28].
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Locations of the sampling sites on the coast of the South Mediterranean Sea. The site of Salammbô is located at Carthage (CAR) in the Gulf of Tunis (northeastern Tunisia), and the site of Sidi Mansour (SM) is located at Sfax on the coast of the Gulf of Gabès (southeastern Tunisia) (A). Photographs of brownish (B) and red (C) seawater discoloration caused by the bloom species *G. impudicum* and *A. minutum* at Carthage and Sidi Mansour, respectively.</figcaption></figure>

The Salammbô site (CAR; 36°83′84″ N, 10°32′03″ E) was in Carthage, one of Tunisia's most important tourist and historic cities, on the Gulf of Tunis (northeastern Tunisia). The sampling site was a bathing area that was impacted by a bloom of *G. impudicum*, a non-toxic chain-forming species, occurring in early July and causing a brown water coloration (Figure 1B). The site of Sidi Mansour (SM; 34°47′55″ N, 10°51′57″ E) was situated on the northern coast of Sfax, Tunisia's second-largest city, in the Gulf of Gabès (south of Tunisia). This gulf is known to display the highest tides in the Mediterranean Sea (up to 2.3 m) [29]. The SM site has been impacted by fishing, trade, harbor activities, and a former phosphogypsum deposit in the Taparura area [30,31]. During the period between the end of June and the beginning of July, this SM site was affected by the red-tide dinoflagellate *A. minutum*, a paralytic shellfish poisoning (PSP)-toxin-producing species (Figure 1C).

## 2.2. *Sampling Procedures*

During dinoflagellate blooms, daily sampling was carried out for 4 and 7 days at the Carthage and Sidi Mansour sites, respectively. Seawater samples were collected at a depth of 1 m with a Van Dorn bottle. For microscopic examination, one liter of seawater was fixed with Lugol's iodine solution (4% final concentration). To determine nutrient concentrations (i.e., nitrate ($NO_3^−$), nitrite ($NO_2^−$), ammonium ($NH_4^+$), phosphate ($PO_4^{3−}$), and silicate ($SiO_4^{4−}$), 250 mL of seawater was frozen at −20 °C in a plastic bottle that was previously
washed with acid and rinsed with distilled water. Subsamples (250 mL) were filtered in duplicate through a 47 mm diameter membrane filter with a pore size of 0.2 µm, then immediately stored at -80 °C until DNA extraction.

## 2.3. Dinoflagellate Enumeration, Cultures, and Microscopy Analyses

Cell enumeration was performed using a 10 mL sedimentation chamber under an inverted microscope (Nikon Eclipse TS100) according to the Utermöhl method [32] and was expressed as cells L⁻¹ [33]. *A. minutum* and *G. impudicum* cells were isolated from the collected seawater samples using the micropipette technique [34] under an inverted light microscope (Olympus CK40, Tokyo, Japan). Isolated cells were first inoculated into 96-well culture plates (Sigma-Aldrich, St. Louis, MO, USA) in L1 medium [35] and then transferred to a Nunclon culture flask once established (Sigma-Aldrich, St. Louis, MO, USA). Clonal cultures of the *G. impudicum* (Gy.imp1) and *A. minutum* (Am.6) strains were grown at a salinity of 40 in L1 medium at 22 °C on a 12:12 h light/dark cycle with 100 µmol photons m⁻²s⁻¹. Light microscope (LM) observations were performed on living or Lugol-fixed cultured cells using a Carl Zeiss Microscopy GmbH (Jena, Germany) microscope at magnifications of 100, 200, and 400. Images were captured with a Carl Zeiss Axiocam 105 color digital camera and capture software (ZEN core v2.7 acquisition and analysis; Carl Zeiss, Jena, Germany). Cell length (L) and width (W) were measured using images of living cells at magnifications of 200× or 400×. The identification of *A. minutum* was based on the caliper plate tabulation [36] following the calcofluor method [37] and using fluorescent brightener 28 (Sigma-Aldrich Co., St. Louis, MO, USA). The cells were examined under 400× magnification in an upright Carl Zeiss Axioimager Z2 Apotome microscope.

## 2.4. Environmental Parameters and Nutrient Analyses

The temperature, salinity, and pH were measured in situ with a multi-parameter probe (Multi 340i/SET). Nutrient analyses were performed with a BRAN and LUEBBE type III AutoAnalyser using standard methods [38]. Dissolved inorganic nitrogen (DIN) is the sum of the NO₂⁻, NO₃⁻, and NH₄⁺ values.

## 2.5. Molecular Analyses

### 2.5.1. DNA Extraction

A DNeasy PowerWater kit (Qiagen, Hilden, Germany) was used to extract DNA from dinoflagellate clonal cultures and planktonic biomass recovered on a 0.22 µm filter, following the manufacturer's instructions with minor modifications. The vortexing time was increased to 10 min, and the DNA elution was performed in a volume of 100 µL. The quality and concentration of DNA extracts were determined using a NanoDrop2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNA samples were stored at -20 °C until the PCR analyses.

### 2.5.2. PCR and Sequencing of 16S rRNA Gene Fragments

Bacterial and archaeal 16S rRNA gene V4 variable regions were amplified via PCR using the Pro341F/Pro805R prokaryotic universal primer set [39] with a barcode on the forward primer as previously described by Dowd et al. [40] and were sequenced using the MiSeq Illumina (paired-end: 2 × 300 bp) platform of the Molecular Research Laboratory (Shallowater, TX, USA). In brief, sequences were joined and depleted of barcodes. Then, sequences < 150 bp and sequences with ambiguous base calls removed. Sequences were denoised. Then, operational taxonomic units (OTUs) were generated, and chimeras were removed. OTUs were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against the NCBI non-redundant (NR) reference database. The 16S rRNA gene sequences of dominant OTUs were deposited in the GenBank database under the accession numbers ON195826-ON195869.
### 2.5.3. PCR and Sequencing of ITS Regions and 28S rRNA Gene

The internal transcribed spacer (ITS1-5.8S-ITS2) regions and the D1-D3 region of the large subunit (LSU) of the ribosomal DNA (28S rDNA) were amplified using PCR with the primer sets ITS1F/ITS1R [41,42] and D1R/D3ca [43], respectively.

Amplification reactions were conducted in volumes of 50 µL each using Go Taq G2 Green Master Mix, 2x (Promega, Madison, WI, USA), 3 µL of DMSO, and 20 ng of genomic DNA as a template under the following program: a denaturing cycle of 3 min at 95 °C; 35 cycles of denaturing at 95 °C for 30 s, annealing for 45 s at 51 °C for the IT1F/ITS1R primers and at 58 °C for the D1/D3ca primers, and extension at 72 °C for 60 s; and a 5 min elongation step at 72 °C was ultimately carried out. The primers ITS1F/ITS1R and D1R/D3ca were used at final concentrations of 0.2 and 0.35 µM, respectively. The purification and sequencing of amplification products were carried out as described previously in the study by Abdennadher et al. [44]. Sequencing reactions were performed with the same primer pairs used for PCR amplification. The nucleotide sequences of ITS-5.8S rDNA and LSU rDNA D1-D3 were submitted to GenBank under the following accession numbers: A. minutum Am.6, MZ66321 and MZ489652, respectively, and G. impudicum Gy.imp1, MZ366320 and MZ489666, respectively.

## 2.6. Statistical Analyses

All statistical analyses (Spearman correlations and principal component analyses (PCA)) were performed using XLSTAT 2020.5.1 (Microsoft Excel add-in program; Addinsoft, Paris, France).

# 3. Results

## 3.1. Identification of HABs and Cell Abundances

During the HAB monitoring in summer 2019, red and brown tides were observed in Sfax and Carthage, respectively (Figure 1). The dinoflagellate abundance was higher at the Carthage site (impacted by *G. impudicum*) than at the Sfax site (impacted by *A. minutum*) over the sampling period (Table 1). The *G. impudicum* and *A. minutum* cell densities ranged from 2.59 × 10⁵ to 33.85 × 10⁵ cells L⁻¹ and from 0.47 × 10⁴ to 30 × 10⁴ cells L⁻¹, respectively.

<table><caption>**Table 1.** Environmental variables obtained at the Sfax (Sidi Mansour (SM)) and Carthage (CAR) sites during the blooms of *Alexandrium minutum* and *Gymnodinium impiducum*, respectively.</caption><thead><tr><th rowspan="2">Stations</th><th colspan="8">Sfax (Gulf of Gabès)</th><th colspan="4">Carthage (Gulf of Tunis)</th></tr><tr><th>SM01</th><th>SM02</th><th>SM1<sup>2</sup></th><th>SM2<sup>2</sup></th><th>SM3<sup>2</sup></th><th>SM4<sup>2</sup></th><th>SM5</th><th>CAR1<sup>3</sup></th><th>CAR2<sup>3</sup></th><th>CAR3<sup>3</sup></th><th>CAR4<sup>3</sup></th></tr></thead><tbody><tr><td>Sampling Date (dd/mm)</td><td>29/06</td><td>30/06</td><td>01/07</td><td>02/07</td><td>04/07</td><td>08/07</td><td>09/07</td><td>05/07</td><td>06/07</td><td>07/07</td><td>08/07</td></tr><tr><td>Temperature (°C)</td><td>32</td><td>33</td><td>31.2</td><td>33</td><td>31</td><td>34.4</td><td>33</td><td>25.7</td><td>25</td><td>25.5</td><td>26.1</td></tr><tr><td>Air Temp Max (°C)</td><td>36</td><td>33</td><td>31</td><td>32</td><td>34</td><td>39</td><td>37</td><td>37</td><td>39</td><td>38</td><td>40</td></tr><tr><td>Air Temp Min (°C)</td><td>23.5</td><td>23.5</td><td>24</td><td>23</td><td>24.5</td><td>24.5</td><td>25</td><td>24</td><td>22</td><td>25</td><td>26</td></tr><tr><td>Sea-Level Pressure (hPa)</td><td>1013</td><td>1015</td><td>1016</td><td>1015</td><td>1015</td><td>1013</td><td>1013</td><td>1015.5</td><td>1015.52</td><td>1013.5</td><td>1014.5</td></tr><tr><td>Wind Max (km/h)</td><td>8</td><td>11</td><td>21</td><td>18</td><td>11</td><td>9</td><td>11</td><td>24</td><td>15</td><td>13</td><td>26</td></tr><tr><td>Wind Direction (°)</td><td>130</td><td>137</td><td>135</td><td>135</td><td>135</td><td>135</td><td>135</td><td>23</td><td>23</td><td>1</td><td>180</td></tr><tr><td>Day Length (h)</td><td>14:29</td><td>14:28</td><td>14:28</td><td>14:26</td><td>14:25</td><td>14:23</td><td>14:22</td><td>14:36</td><td>14:35</td><td>14:34</td><td>14:34</td></tr><tr><td>Sun Hours</td><td>8</td><td>8</td><td>8</td><td>8</td><td>7</td><td>7</td><td>7</td><td>13</td><td>13</td><td>11</td><td>10</td></tr><tr><td>pH</td><td>7.59</td><td>7.90</td><td>7.95</td><td>8.10</td><td>8.00</td><td>8.23</td><td>8.40</td><td>7.90</td><td>7.95</td><td>8.20</td><td>8.00</td></tr></tbody></table>
<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th>Stations</th><th colspan="5">Sfax (Gulf of Gabès)</th><th colspan="4">Carthage (Gulf of Tunis)</th></tr></thead><tbody><tr><td>Salinity (psu)</td><td>39.1</td><td>39.1</td><td>38.8</td><td>39.0</td><td>38.6</td><td>39.1</td><td>38.8</td><td>37.9</td><td>37.8</td><td>38.2</td><td>37.9</td></tr><tr><td>NO<sub>2</sub><sup>-</sup> (µM)</td><td>0.369</td><td>0.912</td><td>0.782</td><td>0.956</td><td>0.391</td><td>0.999</td><td>0.304</td><td>9.281</td><td>14.193</td><td>15.84</td><td>7.651</td></tr><tr><td>NO<sub>3</sub><sup>-</sup> (µM)</td><td>3.21</td><td>11.27</td><td>8.05</td><td>5.02</td><td>3.58</td><td>15.60</td><td>1.95</td><td>92.19</td><td>114.90</td><td>120.40</td><td>66.04</td></tr><tr><td>NH<sub>4</sub><sup>+</sup> (µM)</td><td>96.79</td><td>1.72</td><td>4.93</td><td>6.04</td><td>0.89</td><td>5.65</td><td>44.57</td><td>328.08</td><td>1283.26</td><td>826.51</td><td>257.39</td></tr><tr><td>PO<sub>4</sub><sup>3-</sup> (µM)</td><td>1.095</td><td>0.705</td><td>2.074</td><td>1.653</td><td>0.031</td><td>0.063</td><td>0.031</td><td>11.118</td><td>10.465</td><td>3.274</td><td>1.958</td></tr><tr><td>Silicates (µM)</td><td>0.721</td><td>0.434</td><td>2.306</td><td>0.757</td><td>2.45</td><td>0.508</td><td>0.577</td><td>4.923</td><td>5.302</td><td>4.292</td><td>4.04</td></tr><tr><td>DIN (µM)<sup>1</sup></td><td>100.37</td><td>13.90</td><td>13.76</td><td>12.01</td><td>4.86</td><td>22.25</td><td>46.83</td><td>429.54</td><td>1412.34</td><td>962.75</td><td>331.09</td></tr><tr><td>Microphytoplankton (10<sup>3</sup> cells/L)<sup>2</sup></td><td>300</td><td>50</td><td>72.2</td><td>80</td><td>65</td><td>10.2</td><td>4.7</td><td>3385</td><td>600</td><td>756</td><td>259.2</td></tr></tbody></table>

Morphologically, *A. minutum* cells isolated from the SM site were single, oval to elliptical in a ventral view, and small (Figure 2). Their lengths averaged 23.49 ± 1.72 µm (19.80–27.44 µm, *n* = 20) and their widths averaged 17.53 ± 1.41 µm (14.3–19.8 µm, *n* = 20). The main distinctive characteristics described by Halim [45] and Balech [36] are shown, including the presence of a ventral pore at the 1' right-anterior, the direct connection of the 1'-Po plates, a narrow 6" plate, and a posterior sulcal plate quadrangular.

<figure><img src="image_6.png" /><figcaption>**Figure 2.** *A. minutum* strain Am.6. (A,B) Light micrographs (LMs) of living cultured cells. (A) LM of dorsal view of cell. (B) LM of ventral view of cell. (C,D) Fluorescence microscopy of CalcoFlour-white-stained cells. (C) Ventral view showing the ventral pore (Pv) on the 1' plate, the narrow 6" plate, and the sulcal anterior plate (S.a.). (D) Lateral view showing the connection between the Po and the 1' plate.</figcaption></figure>

*G. impudicum* cells isolated from the CAR site were 23.31 ± 3.27 µm long (18.12–32.07 µm, *n* = 20) and 18.54 ± 2.05 µm wide (15.19–22.57 µm, *n* = 20). They often formed chains of four

<footnote n="1">¹ DIN means dissolved inorganic nitrogen (i.e., NH₄⁺ plus NO₂⁻ plus NO₃⁻); ² Microphytoplankton corresponds to the dinoflagellates studied at Sfax (*Alexandrium minutum*) and at Carthage (*Gymnodinium impudicum*); ³ Samples collected for diversity analyses of procaryotic communities.</footnote>
cells, although longer and shorter chains, as well as solitary cells, were observed (Figure 3). The size of the cells generally grew towards the posterior end of the chain (Figure 3C,D). Numerous banded chloroplasts were located in the cells' peripheries (Figure 3A,D). The intermediate cells in the chain had flattened epicones and hypocones, whereas the apical cells had dome-shaped epicones and flattened hypocones (Figure 3C,D).

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Live cells of *G. impudicum* strain Gy.imp1. (A) Single cell showing chloroplasts. (B) Ventral view of a pair of cells showing cingular displacement. (C) Dorsal view of a chain of three cells in which the posterior cell is much larger than the anterior one. (D) Dorsal view of a chain of four cells in which the epicones are clearly larger than the hypocones. The size of the cells increases towards the posterior end of the chain.</figcaption></figure>

Sequencing of both the LSU and ITS regions confirmed the morphological identification of *A. minutum* and *G. impudicum* clonal cultures. The ITS and LSU rDNA D1-D3 nucleotide sequence lengths for Am.6 were 566 and 881 pb, respectively. The sequence lengths were 563 and 949 pb for G.imp1, respectively.

## 3.2. *Environmental Variables and Correlations with HAB Cell Abundances*

The physicochemical variables measured at Sfax and Carthage are summarized in the Table 1. During the sampling period, a significant increase in the seawater pH values from 7.59 to 8.40 was observed at Sfax, while at Carthage a lower variation in pH values was detected (ranging between 7.9 and 8.2). The temperature and salinity values for CAR samples ranged from 25.0 to 26.1 °C and 37.9 to 38.2 psu (practical salinity unit), respectively, and were lower than for the SM samples, which ranged from 31 to 34.4 °C and 38.6 to 39.1 psu, respectively. The concentrations of dissolved inorganic nitrogen (DIN, i.e., NH₄⁺ plus NO₂⁻ plus NO₃⁻) and phosphate (PO₄³⁻) in Carthage, ranged from 0.33 to 1.42 mM and from 1.95 to 11.11 × 10⁻³ mM, respectively, while in Sfax they varied from 4.8 × 10⁻³ to 0.1 mM and from 0.031 to 2.074 µM.

At Sfax, a negative correlation was observed between *A. minutum* and the minimum air temperature (r<sub>s</sub> = -0.76; *p* < 0.05), while the abundance of *A. minutum* was positively correlated with the length of the day (r<sub>s</sub> = 0.79; *p* < 0.05; Table S1). The abundance of *A. minutum* tended to decline over time with decreasing PO₄³⁻ concentrations and increasing pH. The peak bloom concentration of *A. minutum* was also observed at the maximum concentration of DIN (and more specifically at the highest NH₄⁺ contents) (Table 1).
At Carthage, no relationship between phytoplankton densities and DIN concentrations was observed during the bloom of *G. impudicum*. However, we noticed that the $PO_4^{3-}$ concentrations decreased during the HAB decline stage (between the bloom's peak stage and the end of sampling, corresponding to the end of the bloom) at both Carthage and Sfax.

### 3.3. Prokaryotic Community Composition during Dinoflagellate Bloom Declines

Molecular surveys based on a 16S rRNA gene analysis provided evidence of diverse prokaryotic communities in coastal waters during dinoflagellate bloom monitoring. Overall, seventeen different phyla were identified across the seawater samples: Acidobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Euryarchaeota, Fibrobacteres, Firmicutes, Fusobacteria, Nitrosperae, Planctomycetes, Proteobacteria, Spirochaetes, Tenericutes, Thaumarchaeota, and Verrucomicrobia (Figure 4). Proteobacteria were predominant in all samples ($64.8 \pm 9.8\%$, 54.0–83.3%), followed by Bacteroidetes ($12.6 \pm 2.7\%$, 10.2–16.8%), Euryarchaeota ($7.5 \pm 8.6\%$, 0.1–8.2%), Actinobacteria ($6 \pm 4.5\%$, 1.5–14.0%), Cyanobacteria ($5.6 \pm 4.8\%$, 0.7–15.6%), and Verrucomicrobia ($1 \pm 0.8\%$).

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Compositions of prokaryotic communities at the phylum level in the waters of the Sfax site (Sidi Mansour (SM)) and the Carthage site (CAR) during dinoflagellate blooms.</figcaption></figure>

Differences in microbial community composition were observed at different taxonomic ranks between the SM and CAR sampling sites. At the phylum level, the contents of Proteobacteria (>50% on average) and Bacteroidetes (>10%) were similar, but Cyanobacteria and Actinobacteria were more abundant at Sfax than at Carthage (Figure 4). In addition, Euryarchaeota were abundant at CAR (>10–20%) but rare at SM (<1%). Marked differences were observed at lower taxonomic levels, i.e., the order and family levels (Figure 5). The archaeal MGII group (>10%), Pelagibacterales (>10%), Rhodobacterales (>10%), and Flavobacterales (>5%) clearly dominated at Carthage, while both archaeal MGII and Pelagibacterales were found in lower proportion at SM, with a dominance of Rhodobacterales (Rhodobacteraceae) and Chromatiales (Chromatiaceae).
<figure><img src="image_9.png" /><figcaption>**Figure 5.** Compositions of prokaryotic communities at the family level in the waters of the Sfax site (Sidi Mansour (SM)) and the Carthage site (CAR) during dinoflagellate blooms.</figcaption></figure>

Pronounced variation in the prokaryotic community structure was also observed at Sfax over the sampling period (Figure 5). At the beginning of the monitoring, Rhodobacteraceae were the dominant bacteria (SM1), but they decreased with the increase in Chromatiaceae at SM2 (47%), the second monitored day, before decreasing progressively to represent only 2–3% of the community at the end of the survey (Figure 5). On the contrary, the prokaryotic diversity at these taxonomic levels was more stable during the decline of the *G. impudicum* bloom at Carthage, but a decrease in *Ca. Thalassarcheaceae* (MGII) with an increase in *Pelagibacteriaceae* (SAR11) was observed on the second sampling day (CAR2) (Figure 5).

3.4. *Dynamics of Abundant Prokaryotic Species during Dinoflagellate Bloom Declines*

Three and four OTUs dominated the prokaryotic communities (>5% of total reads) during the progressive declines of *G. impudicum* and *A. minutum*, respectively (Figure 6).

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Relative abundances of the dominant prokaryotic OTUs (>5% of total sequences in at least one sample) associated with the abundance of *A. minutum* at the Sfax site (A) and *G. impudicum* at the Carthage site (B) during the decline phases of their respective blooms occurring in the summer of 2019. The BLAST affiliations of the dominant prokaryotic OTUs can be found in Table S2.</figcaption></figure>
At Carthage, the dominant prokaryotes associated with *G. impudicum* decay were mostly represented by *Candidatus* Pelagibacter (OTU #1, SAR11 subclade Ia, >99% identity) that displayed variations during the monitoring period with an increase directly correlated to the dinoflagellate decrease between CAR1 and CAR2, followed by decline. On the contrary, we observed that *Ca*. Thalassoarchaea (OTU #7, Euryarchaea MGIIb, ~90% identity) increased at the end of the monitoring between CAR2 and CAR4.

At Sfax, the prokaryotic communities associated with *A. minutum* decay were dominated by two main OTUs affiliated with *Rhodovulum adriaticum* (OTU #5, >99% identity) and the *Halochromatium/Thiohalocapsa* genera (OTU #2, ~96% identity) (Figure 6 and Table S2). *Rhodovulum* OTU #5 dramatically decreased between SM1 and SM2, while Chromatiaceae OTU #2 increased during the same period before decreasing at the end of the monitoring, corresponding to the disappearance of the red color of the waters.

A PCA was performed to identify the factors that affected the prokaryotic communities during the dinoflagellate decays (Figure 7). The first two principal components explained 87.9% and 84.2% of the variability in the data from Sfax and Carthage, respectively. A Spearman's rank correlation analysis showed positive correlations between the dominant prokaryotes, *A. minutum*, and $PO_4^{3-}$ at Sfax, while no relationship was observed between *G. impudicum* and the dominant prokaryotic OTUs at Carthage, where they were influenced by the DIN concentrations.

<figure><img src="image_11.png" /><figcaption>**Figure 7.** Principal component analysis (PCA) biplots showing the variation among the samples based on the relative abundances of microorganisms and environmental variables. Black squares represent samples from Sfax (A) and Carthage (B). Arrows indicate the direction of the maximum increase and strength (through the length) of each variable to the overall distribution.</figcaption></figure>

# 4. Discussion

## 4.1. HAB Identification and Densities

HABs are usually characterized by the proliferation and occasional dominance of a particular species of toxic or otherwise harmful alga, and in many instances these proliferations discolor the water. Sea surface discolorations vary according to the pigments within the causative species of algae [46,47]. During the summer of 2019, two dinoflagellate species, *A. minutum* and *G. impudicum*, respectively, caused spectacular water discoloration in Sfax (Gabès Gulf) and Carthage (Tunis Gulf).

The *A. minutum* isolated from Sfax largely conformed to the emended descriptions by Halim [45] and Balech [36], with the presence of a ventral pore (Vp.), which has also been observed in several strains of *A. minutum* collected from the Mediterranean Sea [22,48]. The morphological features of the *G. impudicum* strain from Carthage were consistent with previous descriptions, particularly in the production of cell chains [49,50].

The highest abundance of *A. minutum* ($3 \times 10^5$ cells L$^{-1}$) observed at Sfax was comparable to other records of *A. minutum* found in the northwestern Mediterranean Sea ($10^5$ cells L$^{-1}$) and the North Atlantic coasts ($4.6 \times 10^5$ cells L$^{-1}$) [51] and $3.3 \times 10^5$ cells
L⁻¹ [52]. However, relatively higher cell densities (>10⁶ cells L⁻¹) have been reported during *A. minutum* blooms on both the Mediterranean and Atlantic coasts [45,53–61]. The highest concentration of *G. impudicum* (33.9 × 10⁵ cells L⁻¹) observed in this study was higher than that (2.0–6.3 × 10⁵ cells L⁻¹) recorded on the Catalan coast [49] but lower than the highest abundance (71.2 × 10⁵ cells L⁻¹) of *G. impudicum* observed in Alexandria (Egypt, southeastern Mediterranean coast) [62].

## 4.2. Relationships between HAB Species and Environmental Factors

At Sfax, *A. minutum* bloomed at high sea surface temperatures (31–34.4 °C) and salinity (38.6–39.1 psu). However, several studies reported lower temperatures for *A. minutum* cell proliferations, from 12 °C to 24 °C in Mediterranean coastal locations [63] and less than 15° on the Atlantic coasts [52,56]. The salinity tolerance of *A. minutum* is also broad, ranging from 11 [51] to 46 [22] in natural samples. Our observations support the euryhaline and eurythermal character of *A. minutum* species, which has also been demonstrated in culture studies [64,65]. The abundance of *A. minutum* decreased as pH increased, with *A. minutum* peaks at pH 7.6 and the lowest densities at pH 8.4. This finding is consistent with that of Hwang et al. [66] showing higher growth rates of *A. minutum* at pH 7.5 than at pH 8.5. Flores-Moya et al. [67] also found an optimal growth pH of 7.5, with two *A. minutum* strains growing 1.5–1.6 times faster at pH 7.5 than at pH 8. The major nitrogen form, NH₄⁺, may have an important role in shaping *A. minutum* blooms on Sfax's northern shore. Accordingly, Maguer et al. [68] demonstrated in experimental research that *A. minutum* preferentially takes up this N form. Field investigations, on the other hand, revealed that nitrate plays a significant role in the development of *A. minutum* blooms [69,70]. The decline in *A. minutum* abundance with decreasing PO₄³⁻ concentrations was consistent with previous field studies showing decreases in cell numbers with decreasing PO₄³⁻ intake [22,69]. Le Bec et al. [70] reported that over 14 years in the Rance macrotidal estuary (Brittany, France), a gradual decrease in phosphorus caused the bloom phenomenon to disappear.

Based on two-year observations, Daly Yahia-Kéfi et al. [26] reported that *G. impudicum* was only observed in the Bay of Tunis during the summer. Here, a *G. impudicum* bloom also occurred in the gulf of Tunis on Salammbô beach (Carthage) during the summer of 2019 (July), with temperatures ranging from 25.0 to 26.1 °C and salinities ranging from 37.9 to 38.2 psu. Previous blooms of this species were also recorded during summer at similar temperatures (22–28 °C) and salinity (36–38 psu) on the northern Mediterranean coasts [58,71] and at much higher temperatures (30.4–32.9 °C) and lower salinity (28.6–29.3 psu) on the southeastern Mediterranean coast [71]. In this study, a decline in *G. impudicum* cell numbers was also observed with decreasing PO₄³⁻ concentrations. Friligos and Gotsis-Skretas [72] reported that *G. impudicum* blooms in the Pagassitikos Gulf (Greece) were linked to pollution and eutrophication. Furthermore, Daly Yahia-Kéfi et al. [26] found that *G. impudicum* blooms appeared in the Bay of Tunis under high PO₄³⁻ levels and nitrogen-limited conditions. Experiments confirmed these field observations; *G. impudicum* uses a wide range of dissolved organic phosphorus compounds in addition to dissolved inorganic phosphorus (DIP). It has been also shown that cultures containing nucleotides (AMP, ADP, and ATP) or phosphomonoesters as well as DIP promote cell growth [73].

Compared to the Sfax site, the Carthage site was very rich in nutrients and had more than 10 h of sunlight with decreasing wind speeds, which could explain why the cell density of microphytoplankton in Carthage was approximately 10 times higher at the bloom peak. The low wind speed and high light intensity may have contributed to the blooms of *G. impudicum* and *A. minutum* in Carthage and Sfax, respectively. These factors tend to affect phytoplankton community structure, dominant species, and biomass. Climatic changes over the past 20 years include a decrease in wind speed [74] and an increase in sun radiation [75]. In fact, light conditions (such as solar radiation and water column transparency) were found to be the most important determinants of phytoplankton community biovolume and structure, which were then followed by the nutrient concentrations and
wind speed [76]. The physical mechanism that best predicts the date of the spring bloom is mixed-layer shoaling, which is characterized by a decrease in wind-driven mixing [77–79]. Accordingly, research by Merlivat et al. [80] in the northwestern Mediterranean Sea (BOUS-SOLE site) demonstrated that decreases in mixing and mixed-layer depths caused the start of phytoplankton growth due to wind speed easing after storms. Merlivat et al. [80] demonstrated in the northwest Mediterranean Sea (BOUSSOLE location) that decreases in mixing and mixed-layer depths caused phytoplankton development.

### 4.3. Taxonomic Composition of the Prokaryotic Community during HAB Decays

In this study, we evaluated the diversity of prokaryotes (bacteria and archaea) associated with *G. impudicum* and *A. minutum* HABs. Several studies have shown that bacteria could play a fundamental role in maintaining [81] or, on the contrary, in the decline of algal blooms [82]. This impact can also be reciprocal, as algal blooms are also known to cause significant changes in planktonic bacterial communities [83–85].

Our monitoring showed that *Proteobacteria* and *Bacteroidetes* were the two major dominant phyla associated with the decline stages of *G. impudicum* and *A. minutum* blooms. Accordingly, Zhou et al. [20] showed that both phyla dominated the prokaryotic community during all stages of the dinoflagellate *Alexandrium catanella*'s spring bloom (northeastern USA, Atlantic Ocean). These authors observed a high proportion of *Bacteroidetes* during the bloom's peak (65.3%), with substantial contents during the post-bloom stages of *A. catanella* (21.5–53.1% versus <20% in our study). As observed at Sfax, they also showed that *Alphaproteobacteria* (*Rhodobacterales*) were predominant during the *Alexandrium* peak and decline stages, suggesting that both *Rhodobacterales* and *Flavobacteriales* (*Bacteroidetes*) may be important regulators of HAB dynamics [86]. Indeed, both photo-autotrophic and -heterotrophic *Rhodobacteraceae* and *Flavobacteriaceae* form a core stable population in Carthage and Sfax waters during dinoflagellate decays and could play important roles in HAB degradation. In addition, Zhou et al. [20] mentioned a predominance of heterotrophic *Gammaproteobacteria*, mainly during the pre-bloom stage of *A. catanella*, while *Chromatiaceae* (*Gammaproteobacteria*) dominated during the decline of *A. minutum* at Sfax. Together or successively, these bacterial populations belonging to the classes *Alphaproteobacteria*, *Flavobacteriia*, and *Gammaproteobacteria* contribute to the remineralization of the released organic matter during phytoplankton blooms in coastal marine environments [20,86].

In addition to this typical heterotrophic bacterioplankton, the prokaryotic communities associated with *G. impudicum* at Carthage were dominated by *Pelagibacterales* (SAR11) and *Thalassoarchaea* (MGIIb), showing a photoheterotrophic lifestyle. Both cosmopolitan SAR11 and MGII were previously found to be the dominant prokaryotic groups inhabiting coastal Tunisian waters [87]. Here, the relative abundances of the dominant SAR11 and MGII OTUs #1 and #7 displayed opposite variations during the *G. impudicum* decline. According to the work of Pernthaler et al. [88], MGII has been shown to increase in abundance in response to phytoplankton blooms and can account for up to ~30% of the total microbial community after a bloom terminates. Several studies have shown that MGII correlates with specific genera of phytoplankton [89] during and after blooms [90]. Generally, the relative abundance of the two dominant clades of MGII, called MGIIa and MGIIb (the latter is called *Thalassoarchaea*) [90], responds to different environmental conditions, such as temperature and nutrients [91]. *Thalassoarchaea* (MGIIb) populations dominated winter seawater samples, as previously observed in Tunisian seawater [87], whereas MGIIa dominated the prokaryotic community at the beginning of summer. The relative abundance of the class *Thalassoarchaea* in the summer waters at Carthage is thus surprising and could have played an important role in the decline of *G. impudicum*.

### 4.4. Dominance of Gammaproteobacterial Chromatiaceae during A. minutum Decays

At Sfax, the bloom of *A. minutum* (followed by an increase in the *Chromatiaceae* content during its decline) dramatically modified the typical prokaryotic communities of Tunisian coastal waters analyzed in a previous study [87]. The prokaryotic communities associated
with *A. minutum* decay were dominated by two main OTUs affiliated with alphaproteobacterial *Rhodovulum* (OTU #5) and gammaproteobacterial *Halochromatium/Thiohalocapsa genera* (OTU #2). Here, our results contradicted those of previous studies that used older technologies (plate isolation, DGGE, and FISH) and concluded that the *Rosebacter* genus was the dominant bacterial group associated with *Alexandrium* [92,93]. The high relative abundance of anoxygenic phototrophic sulfur-oxidizing *Chromatiaceae* (also called purple sulfur bacteria (PSB)) may explain the *Alexandrium* decay. In our study, the increase in the PSB proportion (reaching almost half of the prokaryotes at SM2) could be explained by the depletion of dissolved oxygen caused by the dinoflagellate bloom and the development of sulfate-reducing bacteria (SRB) producing sulfide at the Sfax coast sediment surface.

Indeed, the family *Chromatiaceae*, including the genera *Halochromatium* and *Thiohalocapsa*, and SRB are generally abundantly detected in the sediment of the Sfax coast, which is impacted by the release of phosphogypsum, a sulfate-rich coproduct of phosphate-transformation industries [94,95]. Thus, purple sulfur gammaproteobacterial *Halochromatium/Thiohalocapsa* may have also played an important role in intensifying the red color of the Sfax waters during the monitoring period. As observed in our study, Hiraishi et al. [96] showed that red and pink blooms in coastal Japanese environments contained PSB belonging to genera *Thiolamprovum/Thiodictyon* (*Chromatiaceae*) as the major populations, accompanied by smaller but significant densities of purple non-sulfur bacteria, with members of *Rhodovulum* predominating. In addition, red-pink blooms of phototrophic PSB belonging to the family *Chromatiaceae* have also previously been observed during warm summers in shallow brackish coastal lagoons of the Mediterranean coast [97,98], including Tunisian sites [99].

Overall, our study showed a change in the diversity of marine prokaryotes during the decline stages of dinoflagellate bloom events caused by *G. impudicum* and *A. minutum* along the northern (Carthage, Gulf of Tunis) and southern (Sfax, Gulf of Gabes) Tunisian coasts during the summer of 2019. Pronounced differences in the prokaryotic community structure between the waters of the northern and southern Tunisian coasts were previously highlighted during winter observations [87]. In this previous study, *Gammaproteobacteria* were prevalent in the waters of the Gulf of Gabès (near Sfax) but were represented and dominated by different genera, i.e., *Pseudoalteromonas* and *Alteromonas*, which increased with increasing salinity, density, and nutrients ($NH_4^+$ and/or $PO_4^{3-}$). The detection of different gammaproteobacterial genera, i.e., *Halochromatium* and *Rhodovulum*, in the present study suggests potential reciprocal interactions between HABs and these bacterial species as well as environmental factors. Temperature and salinity were key environmental factors associated with changes in bacterial and archaeal community structures, respectively, whereas inorganic nitrogen and $PO_4^{3-}$ were associated with eukaryotic variation [20]. According to these authors, temperature was the major abiotic force shaping the microbial community structure in Salt Pond, which was consistent with earlier studies that documented a pronounced impact of temperature on plankton composition [100,101].

**5. Conclusions**

In this study, the use of high-throughput sequencing of 16S rRNA genes provided a detailed overview of the prokaryotic communities associated with HABs caused by *G. impudicum* and *A. minutum* in the South Mediterranean Sea. To our knowledge, this is the first study evaluating the diversity of the prokaryotic community and its dynamics during HABs occurring along the Tunisian coast and more generally along the southern Mediterranean coast. This study revealed the great influence of dinoflagellate blooms on prokaryotic community structure, with stimulation (e.g., Gammaproteobacteria) or inhibition of specific prokaryotic groups (e.g., Cyanobacteria, SAR11, and MGII), depending on the dinoflagellate species and environmental variables. Dinoflagellate bloom declines stimulated different heterotrophic bacterial taxa, suggesting different abilities to utilize organic matter from dinoflagellates or adaptation to the oxygen depletion caused by dinoflagellate growth.
This study, performed in 2019 and targeting the peaks and declines of dinoflagellate blooms, is currently being continued for several years and includes the pre-bloom phase and more frequent sampling to assess the microbial and abiotic triggers of HABs. Long-term, high-frequency, and in situ monitoring will be necessary to predict and better understand which processes control bloom onset timing.

**Supplementary Materials:** The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d15020273/s1, Table S1: Spearman's rank correlation coefficients ($r_s$) between the dinoflagellate concentrations and the environmental variables obtained at the Sfax and Carthage sites; Table S2: Blast analysis of the dominant prokaryotic OTUs (>5% of total sequences in at least one sample) obtained from the Sfax (Sidi Mansour (SM)) and Carthage (CAR) sites during dinoflagellate blooms.

**Author Contributions:** Conceptualization, M.Q., M.B. and A.B.Z.; methodology, R.L., M.Q., M.A., L.D.W., A.H. and A.B.Z.; formal analysis, R.L., M.Q. and A.B.Z.; investigation, R.L. and M.A.; writing—original draft preparation, R.L., M.Q. and A.B.Z.; supervision, M.Q. and A.B.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Higher Education and Scientific Research (Tunisia) and the French National Research Institute for Sustainable Development (IRD).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database under the accession numbers ON195826-ON195869. The ITS-5.8S rDNA and LSU rDNA D1-D3 sequences were deposited in the GenBank database under the following accession numbers: *A. minutum* Am.6, MZ66321 and MZ489652, respectively, and *G. impudicum* Gy.imp1, MZ366320 and MZ489666, respectively.

**Acknowledgments:** This work was conducted at the National Institute of Marine Sciences and Technologies (INSTM) in the framework of the Tunisian project LittoHABs (HABs in the Tunisian coastline; LR16INSTM04) and under the MOBIDOC scheme, funded by The Ministry of Higher Education and Scientific Research through the PromESsE project and managed by the ANPR. This work was partially supported by the French National Research Institute for Sustainable Development (IRD, the French-Tunisian International Joint Laboratory "LMI COSYS-Med").

**Conflicts of Interest:** The authors declare no conflict of interest.

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