# Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate Alexandrium pacificum grown in metal-contaminated conditions

Cherif Chetouhi, Estelle Masseret, Cecilia Teodora Satta, Thierry Balliau, Mohamed Laabir, N. Jean

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Cherif Chetouhi, Estelle Masseret, Cecilia Teodora Satta, Thierry Balliau, Mohamed Laabir, et al. Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate Alexandrium pacificum grown in metal-contaminated conditions. Science of the Total Environment, 2020, 715, pp.136834. 10.1016/j.scitotenv.2020.136834. hal-02959936

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# Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate *Alexandrium pacificum* grown in metal-contaminated conditions

Cherif Chetouhi<sup>a</sup>, Estelle Masseret<sup>b</sup>, Cecilia Teodora Satta<sup>c</sup>, Thierry Balliau<sup>d</sup>, Mohamed Laabir<sup>b</sup>, Natacha Jean<sup>a,*</sup>

## HIGHLIGHTS

* Growth inhibited under metal stress for *A. pacificum* SG C10-3, not for TAR C5-4F.

* SG C10-3 shows increased cell sizes under metal stress, not TAR C5-4F.

* Metals down-regulate proteins, decrease the number of proteins in the proteomes.

* Protein down-regulation affects photosynthesis/energy metabolism in both strains.

* Proteomic modifications promote cell adaptation in metal-contaminated ecosystems.

## GRAPHICAL ABSTRACT

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

## ABSTRACT

Over the past decades, the occurrence, distribution and intensity of harmful algal blooms involving the dinoflagellate *Alexandrium pacificum* have increased in marine coastal areas disturbed by anthropogenic inputs. This invasive species produces saxitoxin, which causes the paralytic shellfish poisoning syndrome in humans upon consumption of contaminated seafood. Blooms of *A. pacificum* have been reported in metal-contaminated coastal ecosystems, suggesting some ability of these microorganisms to adapt to and/or resist in metal stress conditions. This study seeks to characterize the modifications in membrane proteomes (by 2-D electrophoresis coupled to LC-MS/MS), cell growth and morphometry (measured with an inverted microscope), in response to metal stress (addition of Zn²⁺, Pb²⁺, Cu²⁺ and Cd²⁺), in two Mediterranean *A. pacificum* strains: SG C10-3 and TAR C5-4F, respectively isolated from the Santa Giusta Lagoon (Sardinia, Italy) and from the Tarragona seaport (Spain), both metal-contaminated ecosystems. In the SG C10-3 cultures grown in a metal cocktail, cell growth was significantly delayed, and cell size increased (22% of 37.5 µm cells after 25 days of growth). Conversely, no substantial change was observed for cell growth or cell size in the TAR C5-4F cultures grown in a metal cocktail (*P* > 0.10), thus indicating intraspecific variability in the responses of *A. pacificum* strains to metal contamination. Regardless of the

Keywords:
*Alexandrium pacificum*
Harmful algal bloom
Proteomics
Membrane proteome
Trace metals

<footnote><sup>a</sup> Mediterranean Institute of Oceanography, Equipe Microbiologie Environnementale et Biotechnologie, UM 110 CNRS/IRD Aix-Marseille Université, Université de Toulon, CS 60584, 83 041 Toulon Cedex 9, France</footnote>

<footnote><sup>b</sup> Marbec, University of Montpellier, IRD, Ifremer, CNRS, 34 095 Montpellier Cedex 5, France</footnote>

<footnote><sup>c</sup> University of Sassari, via Piandanna 4, Agenzia Regionale per la Ricerca in Agricoltura, Loc. Bonassai, Olmedo, 07 100 Sassari, Italy</footnote>

<footnote><sup>d</sup> PAPSO-GQE-Le Moulon, INRA, Université Paris-Sud, CNRS, AgroParisTech, Université Paris-Saclay, 91 190 Gif-sur-Yvette, France</footnote>

<footnote>* Corresponding author at: Mediterranean Institute of Oceanography, Equipe Microbiologie Environnementale et Biotechnologie, UM 110 CNRS/IRD Aix-Marseille Université, Université de Toulon, CS 60584, 83 041 Toulon Cedex 9, France.
E-mail address: jean@univ-tln.fr (N. Jean).</footnote>
conditions tested, the total number of proteins constituting the membrane proteome was significantly higher for TAR C5-4F than for SG C10-3, which may help TAR C5-4F to thrive better in contaminated conditions. For both strains, the total number of proteins constituting the membrane proteomes was significantly lower in response to metal stress (29% decrease in the SG C10-3 proteome: 82 ± 12 proteins for controls, and 58 ± 12 in metal-contaminated cultures; 17% decrease in the TAR C5-4F proteome: 101 ± 8 proteins for controls, and 84 ± 5 in metal-contaminated cultures). Moreover, regardless of the strain, proteins with significantly modified expression in response to stress were mainly down-regulated (representing 45% of the proteome for SG C10-3 and 38% for TAR C5-4F), clearly showing the harmful effects of the metals. Protein down-regulation may affect cell transport (actin and phospholipid scramblase in SG C10-3), photosynthesis (RUBISCO in SG C10-3, light-harvesting protein in TAR C5-4F, and high-CO₂-inducing periplasmic protein in both strains), and finally energy metabolism (ATP synthase in both strains). However, other modifications in protein expression may confer to these *A. pacificum* strains a capacity for adaptation and/or resistance to metal stress conditions, for example by (i) limiting the metal entry through the plasma membrane of the SG C10-3 cells (via the down-regulation of scramblase) and/or (ii) reducing the oxidative stress generated by metals in SG C10-3 and TAR C5-4F cells (due to down-regulation of ATP-synthase).

# 1. Introduction

Among the dinoflagellates involved in harmful algal blooms (HABs), the genus *Alexandrium* causes HABs that induce the paralytic shellfish poisoning (PSP), due to its production of saxitoxin and derivates (Anderson et al., 2012). Saxitoxins cause human neural system syndromes with approximately 2000 cases of toxicity per year worldwide (Quod and Turquet, 1996), lead to mass mortalities of fish, shellfish, marine mammals, and birds (Hallegraeff, 1993), and trigger severe economic impacts on aquafarming (Hoagland et al., 2002; Park et al., 2013).

The species *Alexandrium pacificum* Litaker (ex *A. catenella* (Whedon and Kofoid), Balech) is widely distributed throughout the world, observed along Mediterranean, Pacific, and Antarctic coasts (Vila et al., 2001; Masseret et al., 2009; John et al., 2014). In the Mediterranean area, proliferations of *A. pacificum* can occur in marine areas contaminated with trace metals, such as the Tyrrhenian coasts (Italy), the Balearic and Catalan coasts, the Thau Lagoon (France), the Bizerte Lagoon (Tunisia) (Penna et al., 2005; Bravo et al., 2008; Laabir et al., 2013; Fertouna-Bellakhal et al., 2015), and *Alexandrium* spp. have been observed in Toulon Bay (western Mediterranean, France), where the water column and sediments are severely metal-contaminated (Jean et al., 2005; Tessier et al., 2011; Jean et al., 2012). Occurrences of *A. pacificum* in such areas suggest that it can adapt and/or is resistant to metal contamination. To date, much effort has been devoted to understanding *A. pacificum* physiology, via the study of toxin biosynthesis (Wang et al., 2006; Zhang et al., 2017), mechanisms underlying cell growth (Hadjadji et al., 2014), and the impact of diverse nutrient and environmental conditions on cells (Uribe and Espejo, 2003; Yoo and Shin, 2004; Jauzein et al., 2008; Laabir et al., 2011; Herzi et al., 2013). However, few studies have investigated the possible ability of this dinoflagellate to adapt and/or resist in disturbed environments, such as metal-contaminated marine coastal ecosystems. Thus, studies characterizing the possible tolerance and/or resistance of *A. pacificum* to metal stress are needed to better understand its potential capacity to develop in certain metal-polluted marine environments.

"Omics" approaches provide insight into the responses and the adaptation mechanisms developed by organisms exposed to environmental stresses (Jean et al., 2012). Proteomic approaches have revealed an intraspecific variability between a toxicity-lost mutant and the wild-type of *A. pacificum*, through differentially expressed proteins involved in its toxicity (Zhang et al., 2015). The proteome involved in the various toxin biosynthesis stages by *A. pacificum* has been characterized (Wang et al., 2013). An ecotoxicoproteomic study on a Mediterranean strain (ACT03) of *A. pacificum* showed that single-stress by lead or zinc induced the down-regulation of many proteins constituting its soluble proteome, compensated by the up-regulation of some proteins like ATP-synthase able to contribute to an adaptive proteomic response in

metal-contaminated ecosystems (Jean et al., 2017). Using an *in silico* approach, the secretome of *A. pacificum* has been predicted, showing that 47% of the secreted proteins are enzymes (peptidases) active in the extracellular medium during stress responses, which could help this dinoflagellate to develop in various environmental conditions, such as in metal-contaminated areas (Chetouhi et al., 2019).

The plasma membrane, the first barrier against biotic or abiotic stresses in all organisms, mediates the exchange of molecular information between the intracellular and the extracellular environment. Biomolecules located in this membrane play important roles in stress signal transduction. Some studies have reported that the plasma membrane is involved in a variety of mechanisms for metal detoxification and tolerance, for example, by (i) reducing metal uptake, (ii) stimulating the efflux pumping of metals that have entered the cytosol, (iii) repairing metal stress-damaged proteins, (iv) chelating metals and/or (v) compartmenting metals away from metabolic processes by transport into the vacuole (Hall, 2002). Consequently, the study of the effects of metal stress on the proteins constituting the *A. pacificum* membrane proteome will help understand how metals affect plasma membrane proteins, by degrading them and/or down- or up-regulating their expression, and as a result, by inducing a modified protein expression that can lead to cell-resistance mechanisms and adaptation in *A. pacificum*.

Investigations about changes in the proteome during the cell cycle of *Prorocentrum triestinum* showed strong relationships between its protein expression and its cell cycle (Chan et al., 2004). The growth cycle controls the DNA cycle which is mediated by proteins necessary for the DNA replication (Alberghina and Sturani, 1981). Besides, there is a relationship between cell division and cell size during cell growth, some studies reporting that delays in cell division, due to increased interdivision time, lead to higher cell sizes (Vadia and Levin, 2015).

Multiple effects of the metal stress are expected simultaneously on the proteomes, the cell growth and the cell sizes of *A. pacificum*. As a result, the present study combines ecotoxicoproteomic and physiological approaches to compare the responses to a multiple-metal stress in two different strains of *A. pacificum*, coming from two metal-contaminated Mediterranean areas: the SG C10-3 strain isolated from the Santa Giusta Lagoon (Luglié et al., 2002), and the TAR C5-4F strain from the Tarragona seaport (Bravo et al., 2008). The main objectives of this study were to (i) evaluate the extent physiological variations in *A. pacificum* cells by following their density and morphometry during growth in the presence of metals (ii) determine the changes in the plasma membrane proteome induced by metal stress. Based on these results, we built a model to depict how certain strains of *A. pacificum* react and could improve their resistance to metals in disturbed marine ecosystems.
## 2. Material and methods

### 2.1. *Alexandrium pacificum* cultures, growth kinetics and morphometry

#### 2.1.1. Strains

The two studied strains of *Alexandrium pacificum* were obtained from germination of single cysts isolated from sediments collected in respectively the Santa Giusta Lagoon (Sardinia, Italy) for the SG C10-3 strain and in the Tarragona seaport (Spain) for the TAR C5-4F strain

(Fig. 1). In the Santa Giusta Lagoon, contamination by diverse trace metals has been reported in the surface sediments of the lagoon (for example: Zn²⁺ = 47.6 µg g⁻¹, Pb²⁺ = 9.9 µg g⁻¹, Cu²⁺ = 9.6 µg g⁻¹, and Cd²⁺ = 3.8 µg g⁻¹) (Luglié et al., 2002). Similarly, the shallow sediments of the Tarragona seaport, an area of intense shipping traffic that receives discharges from major rivers, are contaminated by various trace metals (for example: Zn²⁺ = 45.0 µg g⁻¹, Pb²⁺ = 19.9 µg g⁻¹, Cu²⁺ = 12.1 µg g⁻¹, and Cd²⁺ = 0.6 µg g⁻¹) (Bravo et al., 2008; Pinedo et al., 2014).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Location of the sampling stations. (A) Sampling station for the *Alexandrium pacificum* SG C10-3 strain (B) Sampling station for the *A. pacificum* TAR C5-4F strain.</figcaption></figure>
### 2.1.2. Cultures

The obtained monoclonal cultures were maintained in f/2 culture medium (i.e. containing: NaNO₃, NaH₂PO₄, Na₂EDTA, FeCl₃, ZnSO₄, CoSO₄, MnSO₄, Na₂MoO₄, Na₂SeO₃, NiCl₂, thiamine HCl, biotin, cyanocobalamin) (Guillard and Ryther, 1962), at 20 °C in sterile 250 mL flasks (75 cm² Greiner, Dominique Dutscher SAS), under light intensity of 135 µmol photons m⁻² s⁻¹, with a photoperiod of 12 h:12 h (light:dark) (Herzi et al., 2013, 2014). Natural seawater, filtered through a GF/F grade glass fiber filter (Ø = 47 mm, Whatman) and then sterilized, was used for the culture medium.

To study the variations in cell growth, morphometry, and membrane proteomes in *A. pacificum* cultures exposed to metal stress, the f/2 medium was supplemented with a cocktail of sterile stock solutions of metals, prepared with ZnSO₄·7H₂O, Pb(CH₃COO)₂·3H₂O, CuSO₄·2H₂O or 3CdSO₄·8H₂O salts (chosen for their solubility) dissolved in ultrapure water, to reach a total concentration of 1.5 µM for each metal (Zn²⁺, Pb²⁺, Cu²⁺ and Cd²⁺), giving a total metal concentration (Mₐ⁺) of 6 µM in the obtained metal cocktail culture medium. A metal-free culture medium was used as the control (Fig. 2A). Trace metals used in this study have been chosen since they contamine in general marine metal-contaminated coastal ecosystems: Zn²⁺ and Cu²⁺ are essential for cells as oligo-elements but toxic at high levels, whereas Pb²⁺ and Cd²⁺ are always toxic for the cells (Tessier et al., 2011; Jean et al., 2012). Based on the MINEQL-program (Garnier et al., 2004) and on the known composition of the f/2 medium, the estimated corresponding free metal concentrations (Mₐ⁺) bioavailable for toxicity towards cells were then calculated (Herzi et al., 2013, 2014): Znₐ⁺ = 0.67 × 10⁻⁷ M, Pbₐ⁺ = 2.56 × 10⁻⁹ M, Cuₐ⁺ = 4.96 × 10⁻¹⁰ M and Cdₐ⁺ = 0.70 × 10⁻⁸ M. These used concentrations have been chosen similar in units to those directly measured in marine metal-contaminated coastal ecosystems (Tessier et al., 2011; Jean et al., 2012). Three independent biological replicates of the SG C10-3 and TAR C5-4F *A. pacificum* cultures were achieved in control and cocktail conditions, in order to measure their respective cell sizes and growth kinetics.

### 2.1.3. Morphometric measurements

The effects of trace metals used in cocktail on morphometry were evaluated by measuring cell sizes (in diameter) in three independent biological replicates of the SG C10-3 and TAR C5-4F *A. pacificum* cultures. These measurements were carried out with an inverted microscope at a magnification of 100×, during the 30 days of their growth (Fig. 2B). At each point of growth (5 day intervals), the sizes of 50 cells chosen randomly were thus measured for both strains in each condition (control and cocktail).

### 2.1.4. Growth kinetics measurements

The effects of trace metals on the growth kinetics were evaluated by counting all the cells present in 30 µL subsample of 1 mL Lugol-fixed culture sample during the growth (every 5 days), with an optical microscope at a magnification of 40×, so as to obtain the density in number of cells per liter of culture (cells L⁻¹) (Fig. 2B). These measurements were performed on three independent biological replicates of the SG C10-3 and TAR C5-4F *A. pacificum* cultures.

### 2.1.5. Statistical analysis

Student's t-tests were used to compare the means in cell densities and in cell sizes measured in the three independent biological replicates of the SG C10-3 and TAR C5-4F *A. pacificum* cultures, in control and cocktail conditions (Shapiro-Wilk's test showed that the data followed a normal distribution: *P* > 0.05).

## 2.2. Proteomics analysis and protein identification using mass spectrometry

Proteomics analysis and protein identification were carried out on three independent biological replicates of the SG C10-3 and TAR C5-4F *A. pacificum* cultures, obtained in control or cocktail conditions, at the end of exponential phase-early stationary phase of their growth (after 15 days of growth). Cell density was then calculated to ensure that each culture sample studied contained 8 × 10⁶ cells.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic representation of the experimental procedure. (A) Cells were grown with or without metals for 30 days (B) Cells were counted, and their sizes measured, every 5 days during growth (C) After extraction, membrane proteins were separated by 2-D electrophoresis. Proteins (spots) of interest were picked to be identified by LC-MS/MS. Protein names/ functions were determined with Uniprot/NCBI database queries.</figcaption></figure>
### 2.2.1. Preparation of protein extracts

The culture samples were centrifuged for 15 min at 1500g, at 15 °C. The obtained cell pellet was washed twice with sterile filtered natural seawater for 10 min at 15,000g (Wang et al., 2008). The pellet was then re-suspended in an extraction solution containing 2 mL of 40 mM Tris at pH 8.7, 2.4 µL benzonase nuclease (Sigma-Aldrich) and 10 µL of protease inhibitor cocktail (Sigma-Aldrich) (Fig. 2C). The obtained suspension was sonicated in ice-water bath, using a microtip Vibra Cell 734 24 (Bioblock Scientific) for 3 min (10 s on and 10 s off cycles) at 50 W and 25 kHz. Then, the solution was centrifuged for 30 min at 15,000g. From the pellet containing membrane proteins, 50 mg were used for the membrane protein extraction using the Ready Prep™-Protein Extraction Kit Membrane I (Bio-Rad). This kit is based on the separation of membrane proteins with the X-114 detergent (Bordier, 1981; Santoni et al., 2000). The extracted sample was then cleaned using the Kit Ready Prep™ 2D Cleanup (Bio-Rad), which facilitated the preparation of low conductivity samples, suitable for isoelectric focusing (IEF) and SDS-PAGE, during 2-D gel electrophoresis.

### 2.2.2. Protein determination

Protein determination of the extracts was achieved using the Reagent Compatible Detergent Compatible Protein Assay (RC DC Protein Assay, Bio-Rad) based on the Lowry method (Lowry et al., 1951), with bovine serum albumin (BSA) as standard.

### 2.2.3. 2-D electrophoresis

2-D electrophoresis was performed using 350 µL of extract containing 200 µg of membrane proteins loaded under a pre-prepared immobilized pH gradient (IPG) strip (17 cm length, linear gradient, pH 3-10, Bio-Rad) (Linares et al., 2016) (Fig. 2C). Rehydration, and then, isoelectric focusing (IEF) of the extract within the IPG strip, was performed in a PROTEAN IEF Cell (Bio-Rad) horizontal electrophoresis system at 20 °C. The program used was: 18 h at 50 V (active rehydration), 2 h at 100 V, 2 h at 250 V, 2 h at 500 V, 2 h at 1000 V, 2 h at 4000 V and 5 h at 10,000 V, to reach 60,000 Vh for each loaded IPG strip. At the end of IEF, the equilibration of each IPG strip was performed for 10 min at room temperature, in equilibration buffer 1 (6 M urea, 2% w/v SDS, 0.375 M Tris pH 8.8, 20% v/v glycerol and 2% w/v DTT) and buffer 2 (6 M urea, 2% w/v SDS, 0.375 M Tris pH 8.8, 20% v/v glycerol and 2.5% w/v iodoacetamide). The proteins separated by IEF underwent SDS-PAGE (Laemmli, 1970) in 18-cm 12% polyacrylamide gels on the top of which the strip was positioned and sealed with 0.7% agarose. A volume of 20 µL of molecular weight marker solution (10–250 kDa range, Precision Plus Protein Standards Dual Color, Bio-Rad) was loaded at the left top of the gel. The gels were run at 4 °C in a Protean II XL (Bio-Rad), with a constant current of 20 mA per gel for 1 h, and then, with a constant current of 30 mA per gel until the dye reached the bottom of the gel. At the end of SDS-PAGE, gels were washed three times for 5 min in ultrapure water, stained with the Imperial Protein Stain (Thermo Fisher Scientific) under orbital shaking for 1.5 h. The gels were then destained in ultrapure water until visualization of separated protein spots. Each gel presented is representative of three different biological gel replicates. The protein spots on the gels were analyzed using PD Quest 2-D Analysis Software 8.0.1 (Bio-Rad). Abundance of a given protein spot was obtained after normalization, based on the ratio (in %) of its individual abundance to the abundance of all the standard marker bands.

### 2.2.4. Tracking of proteins of interest

Proteins of interest showed significantly modified expression in response to metal stress, either by up-regulation (or appearance) of protein spots on the contaminated 2-D gels, relatively to those of controls, or by down-regulation (or disappearance) of protein spots on the control 2-D gels, relatively to those obtained in metal stress conditions (Jean et al., 2017). As a result, presence/absence of these proteins corresponded to spots respectively appearing/disappearing on at least

two of the three gel replicates obtained in metal stress conditions, in comparison with controls. To be considered as up-regulated, a protein showed a ratio ≥ 2 of the normalized abundance of its spot on the contaminated 2-D gels to the normalized abundance of its spot on the control 2-D gels (Bae et al., 2003). Similarly, a down-regulated protein had a ratio ≥ 2 of its normalized abundance of its spot on the control 2-D gels to the normalized abundance of this spot on the contaminated 2-D gels. Then, a Student's t-test, comparing the normalized abundances obtained for these protein candidates, in control and in metal-contaminated conditions, was applied to determine if the differences in abundances observed between each condition were significant (highly significant: $P^{***} \le 0.01$; significant: $0.01 < P^{**} \le 0.05$; nearly significant: $0.05 < P^{*} \le 0.10$), in which case the corresponding proteins were considered as proteins of interest (up- or down-regulated).

### 2.2.5. Protein identification using liquid chromatography-tandem mass spectrometry

To identify the proteins of interest by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 2C), the corresponding protein spots were excised from gels. The digestion of proteins was performed according to a standard trypsin protocol (Aloui et al., 2018). Ultimate 3000 RSLC nano system (Thermo Scientific) was used for HPLC. 4 µL of solution contained peptide sample was loaded at 30 µL min⁻¹ on a precolumn cartridge (stationary phase: C18 PepMap, 5 µm; column: 300 µm inner diameter, 5 mm; Thermo Scientific) and desalted with 0.08% trifluoroacetic acid and 2% acetonitrile (ACN) in water. Then, 3 min later, the precolumn cartridge was connected to the separating PepMap C18 column (stationary phase: C18 PepMap, 3 µm; column: 75 µm inner diameter, 150 mm; Thermo Scientific). The buffer A was prepared with 0.1% HCOOH and 3% ACN in water, and buffer B with 0.1% HCOOH and 80% ACN in water. The peptides were separated with a linear gradient from 4 to 39% B for 14 min at µL min⁻¹. Including the regeneration step at 99% B and the equilibration step at 4% A, one run took 22 min. LTQ Orbitrap Discovery (Thermo Electron) was used to analyze the eluted peptides by nanoelectrospray interface. Liquid junction and a non-coated capillary probe (10 µm inner diameter; New Objective) were used for ionization (1.4 kV ionization potential). Then, peptide ions were analyzed using Xcalibur 2.5.5 SP1 with the following data-dependent acquisition steps: (1) full MS scan (mass-to-charge ratio (m/z) 300–1400, centroid mode in orbitrap) and (2) MS/MS (qz = 0.22, activation time = 50 ms, and collision energy = 35%; centroid mode in linear trap). Step 2 was repeated for the three major ions detected in step 1. Dynamic exclusion was set to 30 s. Database searches were achieved using X!Tandem (Craig and Beavis, 2004). For protein identification, the *A. pacificum* database of Zhang et al. (2014) (11,437 entries) and a common contaminant database (keratins, trypsin, etc.: 55 entries) were used. Protein identification was parsed/validated using the X!TandemPipeline tool, when at least two peptides (at 0.05 E-value), originating from a single protein (at 0.001 E-value), were significant (Langella et al., 2017). All the proteins constituting the membrane proteomes could not be identified, when: (i) corresponding spots were too small (thus impossible to excise in the gels), (ii) proteins could not be sequenced for other technical reasons, (iii) corresponding protein sequences remained unknown (or hypothetical) in the proteomic databases.

## 3. Results

### 3.1. Effects of metal stress on cell growth/morphometry of *Alexandrium pacificum* strains

#### 3.1.1. Effects on cell growth

For the SG C10-3 strain, the initial cell density was systematically $0.77 \times 10^6$ cells L⁻¹ in the metal-contaminated and control cultures (Fig. 3). During the exponential phase (days 5, 10, and 15), cell growth slowed down in the metal-contaminated cultures, with cell densities
becoming significantly lower than in controls ($0.01 < P \le 0.10$) (Table 1 and Supp data 1). Under metal stress, cell densities were 2.0 fold lower on day 5, 1.5 fold lower on day 10 and 1.3 fold lower on day 15. After the exponential phase (on days 20 and 25), cell growth ultimately reached that of controls, with no significant difference ($P > 0.10$) in cell densities between metal-contaminated and control cultures.

For the TAR C5-4F strain, the initial cell density was systematically $0.65 \times 10^6$ cells L$^{-1}$ in the metal-contaminated and control cultures (Fig. 3). Cell growth of TAR C5-4F in metal-contaminated conditions showed no significant difference from that of controls ($P > 0.10$), whatever the day of growth (Table 1 and Supp data 1).

### 3.1.2. Effects on the morphometry

Regardless of the *A. pacificum* strain, cell size (in diameter) ranged between 22.5 and 37.5 µm (Fig. 4A). Boxplots of the morphometric data obtained for the SG C10-3 strain during its cell growth, show that the median cell sizes in metal-contaminated cultures were generally higher than those measured for controls (13% higher on day 5, 20% on day 15, and 18% on day 30) (Fig. 4B), with significantly higher cell sizes ($P < 0.10$) for cells under metal stress (Table 1 and Supp data 2). The proportion of cells at the different sizes (22.5, 25.0, 27.5, 30.0, 32.5, 35.0 and 37.5 µm) revealed that, in metal-contaminated conditions, there was a non-negligible proportion of larger cells (37.5 µm) in the SG C10-3 cultures, from day 5 until the end of the cell growth (10% of 37.5 µm cells on day 20; 22% of 37.5 µm cells on day 25; 16% of 37.5 µm cells on day 30) (Fig. 4C). By contrast, in the SG C10-3 control cultures, such large cells were observed only at the end of cell growth, and moreover, in much lower proportions (2% of 37.5 µm cells on day 25; 4% of 37.5 µm cells on day 30).

The boxplots of the morphometric data obtained for the TAR C5-4F strain show that median cell sizes were often lower in cultures exposed to metal stress (8% lower on day 15, and 17% on day 25) (Fig. 4B), with significantly lower cell sizes ($P < 0.10$) on day 15 and day 25 in contaminated conditions (Table 1 and Supp data 2). Low proportions of larger cells (37.5 µm) were only observed at the end of the growth in metal-contaminated TAR C5-4F cultures (2% of 37.5 µm cells on day 20; 4% of 37.5 µm cells on day 25; 18% of 37.5 µm cells on day 30), whereas such large cells were observed in TAR C5-4F control cultures on day 30 (8% of 37.5 µm cells) (Fig. 4C). However, on days 20 and 25,

proportions of larger cells (37.5 µm) were five times lower in the metal-contaminated TAR C5-4F cultures than in metal-contaminated SG C10-3 cultures, suggesting a greater impact of metals on the SG C10-3 strain.

## 3.2. Effects of metal stress on the membrane proteomes of the *A. pacificum* strains

### 3.2.1. Effects of metal stress on the protein expression profiles

The protein expression profiles (PEPs) obtained for the SG C10-3 and the TAR C5-4F strains showed protein spots distributed in the range 22–131 kDa/3.8–6.8 pI and 23–123 kDa/4.0–6.0 pI, respectively (Figs. 5A and B, 6A and B).

Protein spot detection led to a total number of 82 ± 12 and 58 ± 12 spots on the membrane proteome of the SG C10-3 strain respectively grown in control and in metal-contaminated conditions. The total number of membrane proteins expressed by SG C10-3 in the metal cocktail was nearly significantly lower than in control conditions (Student's *t*-test: $P = 0.0702$, $0.05 < P^* \le 0.10$) (Fig. 5A and B).

For the TAR C5-4F strain, protein spot detection led to a total number of 101 ± 8 and 84 ± 5 spots on the membrane proteome of the TAR C5-4F strain respectively grown in control and in metal-contaminated conditions. The total number of membrane proteins expressed by TAR C5-4F in the metal cocktail was thus significantly lower than in control conditions (Student's *t*-test: $P = 0.0325$, $0.01 < P^{**} \le 0.05$) (Fig. 6A and B).

Finally, we found that the total number of spots on the membrane proteomes, obtained in metal-contaminated or in control conditions, was significantly higher for the TAR C5-4F strain than for the SG C10-3 strain (metal-contaminated conditions: $P = 0.0229$, $0.01 < P^{**} \le 0.05$; control: $P = 0.0921$, $0.05 < P^* \le 0.10$) (Figs. 5A and B, 6A and B).

### 3.2.2. Effects of metal stress on the membrane proteomes

LC-MS/MS identification and functional annotations for the membrane proteins expressed by the two *A. pacificum* strains are shown in Tables 2 and 3, respectively (Supp data 3 and 4).

In comparison with controls, exposure to the multiple metals led to the following proportions of proteins of interest in the membrane proteomes of the two *A. pacificum* strains: 45% proteins of interest for SG C10-3 and 38% for TAR C5-4F. Among these proteins principally down-regulated, (i) 34 disappeared from the membrane proteome of

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Temporal change in cell densities (cells L$^{-1}$) during growth of *Alexandrium pacificum* SG C10-3 and TAR C5-4F strains, in metal-contaminated and control conditions.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

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

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Effects of metal stress on the cell size of the *Alexandrium pacificum* strains (A) Panel of the cell sizes measured between 22.5 µm (min) and 37.5 µm (max) (B) Boxplots of the temporal change in cell size distribution around the median: cell sizes for the SG C10-3 strain and the TAR C5-4F strain (C: control; CK: cocktail). (C) Temporal change in the proportions of cells in the different measured cell sizes (C: control; CK: cocktail).</figcaption></figure>
<caption>**Table 1**</caption>

P values (Student's test) for the comparison of cell densities and cell sizes during growth of the *Alexandrium pacificum* SG C10-3 and TAR C5-4F strains, in control and in metal-contaminated conditions.

<table><thead><tr><th rowspan="2">Days of growth</th><th colspan="2">Alexandrium pacificum SG C10-3 (Control vs cocktail)</th><th colspan="2">Alexandrium pacificum TAR C5-4F (Control vs cocktail)</th></tr><tr><th>Cell density</th><th>Cell size</th><th>Cell density</th><th>Cell size</th></tr></thead><tbody><tr><td>5</td><td>3.8E-02**</td><td>3.2E-05***</td><td>7.4E-01</td><td>4.8E-01</td></tr><tr><td>10</td><td>7.7E-02*</td><td>2.9E-02**</td><td>9.6E-01</td><td>5.8E-01</td></tr><tr><td>15</td><td>5.7E-02*</td><td>1.9E-07**</td><td>6.3E-01</td><td>8.9E-02*</td></tr><tr><td>20</td><td>8.0E-01</td><td>6.6E-02*</td><td>1.6E-01</td><td>8.9E-04***</td></tr><tr><td>25</td><td>4.9E-01</td><td>6.7E-02*</td><td>8.6E-01</td><td>5.8E-03***</td></tr><tr><td>30</td><td>4.4E-02**</td><td>3.2E-03***</td><td>1.4E-01</td><td>7.5E-01</td></tr></tbody></table>

each *A. pacificum* strain, (ii) one was down-regulated (spot 1: −6.7) for SG C103 (Fig. 5C), and two proteins were down-regulated for TAR C5-4F (spot 1: −11.5; spot 2: −16.7) (Fig. 6C), (iii) whereas only one protein appeared for SG C10-3 (spot 23) (Fig. 5C). Among the proteins disappearing from the SG C10-3 membrane proteome, five proteins were identified: actin (spot 3) and phospholipid scramblase (spot 5) (involved in cell transport), RUBISCO (spot 10) and CO₂ inducible periplasmic protein (spot 20) (key proteins involved in cell photosynthesis), and mitochondrial ATP synthase F1 beta subunit (spot 15) (involved in energy metabolism) (Fig. 5C). Among the proteins disappearing from the TAR C5-4F membrane proteome, six proteins were identified: high-CO₂-inducible periplasmic protein (spots 22, 30 and 33) and light-harvesting protein (spots 26 and 35) (involved in photosynthesis), and ATP synthase CF1 beta chain (spot 34) (involved in energy metabolism) (Fig. 6C).

# 4. Discussion

## 4.1. Effects of metal stress on cell growth and morphometry of the *Alexandrium pacificum* strains

The characterization of cell growth and morphometry under metal stress highlighted differences between the SG C10-3 and TAR C5-4F strains, suggesting intraspecific variability in response to contamination. Indeed, the SG C10-3 strain showed higher susceptibility in cell growth and morphometry to metals than the TAR C5-4F strain, for which no significant effect on these parameters was observed in the tested conditions. For SG C10-3, the metal cocktail induced increased cell sizes and delayed cell growth, but not for TAR C5-4F. Similarly, chromium stress inhibits cell growth in the green microalga *Micrasterias denticulate* (Volland et al., 2014) and cadmium stress reduces growth in maize (Figlioli et al., 2019), and in rhizosphere bacteria (Kowalkowski et al., 2019).

Some studies mention that delays in cell division can lead to increased cell sizes (Vadia and Levin, 2015). Indeed, delays in cell division results from increased interdivision time during which the cell sizes can increase too. Though not verified in the present study, our results suggest that the delayed SG C10-3 growth also could result from increased interdivision time in response to metal stress, potentially contributing to the observed increase in SG C10-3 cell size.

Intraspecific variability observed in the cell growth and morphometry can also be explained by various genomic/proteomic regulations of the strains exposed to metal stress. The TAR C5-4F strain has probably developed a best adaptive response leading to nearly normal growth when exposed to metal contamination, in comparison to the SG C10-3 strain which showed disturbed growth and morphometry in metal-contaminated conditions.

## 4.2. Effects of metal stress on the membrane proteomes expressed by the *A. pacificum* strains

### 4.2.1. Comparison of the membrane proteomes expressed by the *A. pacificum* strains

Analysis of the membrane proteomes revealed distinct/specific PEPs for the *A. pacificum* strains, regardless of the experimental conditions tested (metal-contaminated or control). A proteomic approach applied on two distinct *Trypanosoma cruzi* strains also revealed significant differences in their expression profiles (181 proteins were considered common, 119 were found solely in a first strain, and 114 proteins were present exclusively in the second strain) (Kikuchi et al., 2010). In our study, the membrane proteomes showed significant differences in their constitutive total number of proteins, which was significantly higher for TAR C5-4F than for SG C10-3, across both tested conditions (metal-contaminated or control). Similarly, intraspecific variability of three strains of *Vibrio tapetis* depicted different numbers of protein spots in 2D-gels despite loading the same quantities of proteins (there were 729 ± 13, 681 ± 2 and 556 ± 6 spots for the three respective strains, with similarity of 79% found between two of them, whereas the third showed similarities lower than 70%) (Balboa et al., 2011). For *A. pacificum*, this means that TAR C5-4F expresses a greater panel of membrane proteins, which may be used by the cells to better survive in the presence of metals, without be affected in its growth.

The membrane proteomes were made of proteins that differed between the two strains, with only three identified proteins expressed in common (ATP synthase, high-CO₂-inducible periplasmic protein, and light-harvesting protein). In both strains, part of the total membrane proteome remained unchanged in expression under metal stress (55% of the membrane proteome is unchanged for SG C10-3, and 62% for TAR C5-4F). In SG C10-3, the unchanged proteins were identified as HSP 90 known for its chaperone activity (Jean et al., 2017), peridinin chlorophyll a binding protein involved in photosynthesis (Jean et al., 2017), and mitochondrial ATP synthase F1 beta subunit (Fig. 5C), whereas in TAR C5-4F, these unchanged proteins were ferredoxin-NADP(+) reductase involved in the photophosphorylation step of photosynthesis (Jean et al., 2017), chloroplast cytochrome f known for its role in respiratory and photosynthetic electron transport (Hurt and Hauska, 1981) and ATP synthase CF1 beta chain involved in energy metabolism (Jean et al., 2017) (Fig. 6C). As a result, involved in photosynthesis, chaperone activity or energy metabolism, these unchanged proteins can be candidates for maintaining each strain in its respective metal-contaminated ecosystem.

### 4.2.2. Effects of metal stress on the *A. pacificum* membrane proteomes

Under metal stress, the proteins of interest were principally down-regulated, with total numbers of spots constituting the membrane proteomes significantly lower than in controls, for both studied *A. pacificum* strains. The proteins of interest (changing in expression in response to metal stress) represented 45% of the membrane proteome expressed by SG C10-3, compared with 38% of the membrane proteome expressed by TAR C5-4F. The higher susceptibility in terms of growth of the SG C10-3 strain exposed to metal stress, relatively to TAR C5-4F, may result from its higher percentage of down-regulated membrane proteins in these metal-contaminated conditions. It exists a strong relationship between the cell cycle and the protein expression, as shown in investigations about changes in the proteome during the cell cycle of *Prorocentrum triestinum* (Chan et al., 2004). Significant fluctuations in the relative abundance of proteins were observed during the cell cycle of this dinoflagellate: preblooming proteins PB1, PB2, and PB3 were prominently found in the vegetative stages but blooming-related proteins BP1 and BP2 were expressed at high abundance and greatly enhanced in the blooming stages. Indeed, the growth cycle controls the DNA cycle which is mediated through the attainment of a level of protein necessary for DNA replication (Alberghina and Sturani, 1981). In our study, the higher down-regulation (or degradation) of some

<footnote>** Highly significant: *P* ≤ 0.01.</footnote>

<footnote>** Significant: 0.01 < *P* ≤ 0.05.</footnote>

<footnote>* Nearly significant: 0.05 < *P* ≤ 0.10.</footnote>
<figure><img src="image_9.png" /></figure>

C

<table><thead><tr><th>Spot n°</th><th>Protein name</th><th>Fold change</th></tr></thead><tbody><tr><td>1</td><td>Unidentified protein</td><td>-6.7"</td></tr><tr><td>3</td><td>Actin</td><td>D</td></tr><tr><td>5</td><td>Phospholipid scramblase</td><td>D</td></tr><tr><td>10</td><td>Ribulose bisphosphate carboxylase</td><td>D</td></tr><tr><td>11</td><td>Unidentified protein</td><td>D</td></tr><tr><td>15</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>D</td></tr><tr><td>20</td><td>High-CO<sub>2</sub>-inducible periplasmic protein</td><td>D</td></tr><tr><td>23</td><td>Unidentified protein</td><td>A</td></tr><tr><td>24</td><td>Heat shock protein 90</td><td>-</td></tr><tr><td>25</td><td>Unidentified protein</td><td>-</td></tr><tr><td>27</td><td>Unidentified protein</td><td>-</td></tr><tr><td>31</td><td>Unidentified protein</td><td>-</td></tr><tr><td>32</td><td>Unidentified protein</td><td>-</td></tr><tr><td>33</td><td>Peridinin chlorophyll a binding protein</td><td>-</td></tr><tr><td>34</td><td>Unidentified protein</td><td>-</td></tr><tr><td>35</td><td>Unidentified protein</td><td>-</td></tr><tr><td>36</td><td>Hypothetical protein</td><td>-</td></tr><tr><td>37</td><td>Hypothetical protein</td><td>-</td></tr><tr><td>38</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>-</td></tr><tr><td>39</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>-</td></tr><tr><td>40</td><td>High-CO<sub>2</sub>-inducible periplasmic protein</td><td>D</td></tr><tr><td>41</td><td>Ribulose bisphosphate carboxylase</td><td>-</td></tr><tr><td>45</td><td>Unidentified protein</td><td>-</td></tr><tr><td>46</td><td>Unidentified protein</td><td>-</td></tr><tr><td>48</td><td>Ribulose bisphosphate carboxylase</td><td>-</td></tr><tr><td>49</td><td>Light-harvesting protein</td><td>-</td></tr></tbody></table>

**Fig. 5.** Representative 2-D maps comparing the membrane proteomes of *Alexandrium pacificum* SG C10-3 obtained in control and metal-contaminated conditions. (A) Protein expression profile of the *Alexandrium pacificum* SG C10-3 strain grown under control conditions (B) Protein expression profile of the *Alexandrium pacificum* SG C10-3 strain grown in the metal cocktail. Proteins are identified by their spot numbers (C) Names of the proteins are shown with their fold differences in expression; in black (A): appeared proteins; in grey: down-regulated or (D) disappeared proteins; in white (-): proteins unchanged in expression. *: protein whose expression was nearly significantly different under metal-contaminated conditions (0.05 < *P*° ≤ 0.10).

proteins, subsequent to metal exposure, could further impact the growth of SG C10-3 strain, relatively to TAR C5-4F.

As detailed below, several biological functions in the cells of the studied strains may be affected by the changes in their protein expression under polymetallic contamination. These disturbed functions include cell transport (changes in expression of actin and phospholipid scramblase), photosynthesis (changes in expression of high-CO₂-inducing periplasmic protein, RUBISCO, and light-harvesting protein), and energy metabolism (changes in expression of ATP synthase).

### 4.2.2.1. Effects of metal contamination on cell transport proteins.
Cytoskeletal actin disappeared specifically from the proteome of the *A. pacificum*

SG C10-3 strain. Similarly, in the alga *Spirogyra decimina*, actin gradually disappeared with increasing time and cadmium concentration, which indicated disruption of the actin cytoskeleton (Pribyl et al., 2005).

Actin is a family of multi-functional proteins that form microfilariae in the cytoskeleton of all eukaryotic cells (Hall, 1998). Mg²⁺ and Ca²⁺ are involved in the polymerization of actin (Maruyama et al., 1981) and some metals may act similarly to Ca²⁺ (cadmium and calcium have similar ionic radii). Actin participates in many important cellular processes, including cell motility, vesicle and organelle movement, cell signaling, establishment and maintenance of cell junctions/shape, cytokinesis, and cell division (Soyer-Gobillard et al., 1996). As a result, in the present study, the down-regulation (or degradation) of actin in
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Representative 2-D maps comparing the membrane proteomes of *Alexandrium pacificum* TAR C5-4F obtained in control and metal-contaminated conditions. (A) Protein expression profile of the *Alexandrium pacificum* TAR C5-4F strain grown under control conditions (B) Protein expression profile of the *Alexandrium pacificum* TAR C5-4F strain grown in metal-contaminated conditions. Proteins are identified by their spot numbers (C) Names of the proteins are shown with their fold differences in expression; in grey: down-regulated or (D) disappeared proteins; in white (-): proteins unchanged in expression. *: protein whose expression was nearly significantly different under metal-contaminated conditions (0.05 < P² ≤ 0.10).</figcaption></figure>

SG C10-3 may help explain the delay in cell growth and the subsequent increase in cell size observed in metal-contaminated conditions (not occurring in the TAR C5-4F strain).

The phospholipid scramblase, down-regulated in the SG C10-3 cells exposed to metal stress, belongs to a family of transmembrane proteins responsible for the Ca²⁺ mediated-bidirectional phospholipid translocation in plasma membranes (Sivagnanam et al., 2017). Divalent metals may mimic calcium (Fox et al., 1998), especially during scramblase-mediated Ca²⁺ transport into the cells. Consequently, the down-regulation of scramblase in SG C10-3 may reduce metal toxicity in cells by limiting metal entry through the plasma membrane, which may help this strain adapt and/or be resistant to metal stress. Many reports have shown the multifunctional roles of phospholipid scramblase in key cellular processes, including protein/DNA interactions, transcriptional regulation and apoptosis. Trace metals such as cadmium,

chromium, lead, and mercury are known to induce apoptosis, via proteins like phospholipid scramblase (Pulido and Parrish, 2003). In our study, down-regulation of phospholipid scramblase in SG C10-3 cells may lead to less apoptosis at the end of its growth, as observed for this strain during the stationary phase.

4.2.2.2. Effects of metal contamination on photosynthetic proteins. Decreases in the abundance of many photosynthetic proteins (high-CO₂-inducing periplasmic protein, RUBISCO, and light-harvesting protein) were recorded, indicating that photosynthesis is a metabolic pathway sensitive to metal contamination in *A. pacificum* cells. This echoes many studies reporting the harmful effects of metals on photosynthesis, either by chlorophyll degradation, or by destruction of chloroplast ultrastructure and down-regulation of many proteins involved in the Calvin
<caption>**Table 2**</caption>

Liquid chromatography-tandem mass spectrometry identification of the proteins constituting the membrane proteome of the *Alexandrium pacificum* SG C10-3 strain.

<table><thead><tr><th rowspan="2">Spot no</th><th rowspan="2">Peptide number</th><th colspan="2">MW (kDa)/pl</th><th rowspan="2">Protein name</th><th rowspan="2">Species Accession number</th><th rowspan="2">Putative function</th></tr><tr><th>Obs</th><th>Theo</th></tr></thead><tbody><tr><td>3</td><td>2</td><td>41.7/3.9</td><td>41.7/5.3</td><td>Actin</td><td>Alexandrium fundyense ABO47874.1</td><td>Vesicular cellular import (20.09.18.09)</td></tr><tr><td>5</td><td>2</td><td>28.7/4.8</td><td>29.9/5.3</td><td>Phospholipid scramblase</td><td>Perkinsus marinus XP_002768242.1</td><td>Membrane lipid metabolism (01.06.02)</td></tr><tr><td>10</td><td>4</td><td>25.7/5.2</td><td>30.2/8.5</td><td>Ribulose-1,5-bisphosphate carboxylase</td><td>Symbiodinium microadriaticum OLP97657.1</td><td>Photosynthesis (02.30)</td></tr><tr><td>15</td><td>5</td><td>26.4/5.5</td><td>54.8/5.1</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>Karlodinium veneficum ADV91188.1</td><td>Transported compounds (20.01)</td></tr><tr><td>20</td><td>8</td><td>40.1/5.7</td><td>42.9/4.9</td><td>High-CO<sub>2</sub>-inducible periplasmic protein</td><td>Heterocapsa triquetra AAW79380.1</td><td>Calcium binding (16.17.01)</td></tr><tr><td>24</td><td>6</td><td>29.3/5.1</td><td>81.6/5.0</td><td>HSP 90</td><td>Karlodinium veneficum ABI14419.1</td><td>Chaperone (14.01)</td></tr><tr><td>33</td><td>2</td><td>36.8/3.8</td><td>38.0/9.1</td><td>Peridinin-chlorophyll α-binding protein</td><td>Symbiodinium sp. AFH88375.1</td><td>Light absorption (02.45.03)</td></tr><tr><td>36</td><td>8</td><td>44.5/5.0</td><td>57.0/4.4</td><td>Hypothetical protein</td><td>Aureococcus anophagefferens XP_009037435.1</td><td>Unclassified protein (99)</td></tr><tr><td>37</td><td>15</td><td>47.1/5.2</td><td>57.0/4.4</td><td>Hypothetical protein</td><td>Aureococcus anophagefferens XP_009037435.1</td><td>Unclassified protein (99)</td></tr><tr><td>38</td><td>12</td><td>49.9/5.5</td><td>54.8/5.1</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>Karlodinium veneficum ADV91188.1</td><td>Transported compounds (20.01)</td></tr><tr><td>39</td><td>14</td><td>50.1/5.7</td><td>54.8/5.1</td><td>Mitochondrial ATP synthase F1 beta subunit</td><td>Karlodinium veneficum ADV91188.1</td><td>Transported compounds (20.01)</td></tr><tr><td>40</td><td>13</td><td>40.3/5.6</td><td>42.9/4.9</td><td>High-CO<sub>2</sub>-inducible periplasmic protein</td><td>Heterocapsa triquetra AAW79380.1</td><td>Calcium binding (16.17.01)</td></tr><tr><td>41</td><td>3</td><td>44.8/5.7</td><td>30.2/8.5</td><td>Ribulose-1,5-bisphosphate carboxylase</td><td>Symbiodinium microadriaticum OLP97657.1</td><td>Photosynthesis (02.30)</td></tr><tr><td>48</td><td>2</td><td>99.5/5.4</td><td>30.2/8.5</td><td>Ribulose-1,5-bisphosphate carboxylase</td><td>Symbiodinium microadriaticum OLP97657.1</td><td>Photosynthesis (02.30)</td></tr><tr><td>49</td><td>2</td><td>108.2/5.1</td><td>45.9/8.3</td><td>Light-harvesting protein</td><td>Symbiodinium sp. CBI83418.1</td><td>Transmembrane signal transduction (30.05)</td></tr></tbody></table>

cycle (Gillet et al., 2006; Führs et al., 2008; Kieffer et al., 2008; Ahsan et al., 2009).

Regarding the high-CO₂-inducing periplasmic protein, this protein disappeared from the membrane proteomes of both studied strains in contaminated conditions. Among the molecular mechanisms developed by photosynthetic organisms to acclimate and adapt in response to high CO₂ concentrations needed by photosynthesis, expression of multiple high-CO₂-inducible periplasmic proteins has been shown, for example in the extracellular proteome of the unicellular green alga *Chlamydomonas reinhardtii* exposed to 3% CO₂ in air for three days (Hanawa et al., 2007; Baba et al., 2011). The high-CO₂-inducible periplasmic H43/Fea1 homologous genes are expressed by the chlorophytes *Scenedesmus obliquus*, *Chlorococcum littorale*, *Volvox carteri*, and the dinoflagellate *Heterocapsa triquetra* (Allen et al., 2007). This protein, involved in the iron-assimilation pathway, is linked to changes in CO₂ concentration (Sasaki et al., 1998; Allen et al., 2007). For example, H43/Fea1 is the most abundant extracellular soluble protein (26%) expressed by the high-CO₂ (3%)-grown cells of *C. reinhardtii* (Baba et al., 2011). A homology search of DNA sequences showed that H43/Fea1 expression is induced in *C. reinhardtii* cells by iron deficiency (< 1 µM) and by excessive levels of cadmium (> 25 µM), which show contrasted responses of this green alga in response to metal exposure (Rubinelli et al., 2002; Allen et al., 2007).

Thanks to RUBISCO, photosynthetic microorganisms use CO₂ efficiently during the Calvin cycle of photosynthesis. However, RUBISCO, which catalyzes carboxylation of the substrate ribulose-1,5-bisphosphate (RuBP) during the second step of photosynthesis (Reumann and Weber, 2006), disappeared in the membrane proteome of the metal-contaminated SG C10-3 cultures. Some studies have also shown that trace metals decrease RUBISCO abundance in *A. pacificum* ACT03, *Zea mays*, and the microalga *Pseudokirchneriella*

subcapitata (Vannini et al., 2009; Jean et al., 2017; Figlioli et al., 2019). The known high RUBISCO sensitivity to metal-induced oxidative conditions, which leads to methionine sulfoxide residues then targeted for ubiquitination, may increase its proteolytic degradation (Ge et al., 2009). Similarly, significant decreases in RUBISCO activity have been observed in the aquatic plant *Salvinia natans* contaminated by metals (Dhir et al., 2011). The main consequence of this decrease may be lower CO₂ assimilation by the cells (Dhir et al., 2011), and lower efficiency of RuBP recycling (which is a substrate of the Calvin cycle) (Kieffer et al., 2008).

In our study, a light-harvesting polyprotein precursor and a light-harvesting protein were under-expressed by the TAR C5-4F strain in metal-contaminated conditions. Light-harvesting complex of a photosystem (functional unit in photosynthesis) is made of subunit proteins and photosynthetic pigments. The constituent transmembrane proteins, occurring in the reaction center of photosystem II, bind to chlorophyll a and beta-carotene, and pass the excitation energy on to the reaction center (Barber, 2002; Standfuss et al., 2005). These light-harvesting protein complexes are used by photosynthetic organisms to collect more of the incoming light than would be captured by the photosynthetic reaction center alone. In the alga *Euglena gracilis*, the polyprotein precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is inhibited in the presence of Zn²⁺ and Cu²⁺ (Enomoto et al., 1997). Similarly, in the diatom *Thalassiosira pseudonana*, the transcription of three *LI818*-like genes (*LhcX1*, *LhcX5*, and *LhcX6*) is down-regulated under iron or copper deprivation, whereas the protein encoded by *LhcX1* is up-regulated under iron limitation (Zhu et al., 2010).

### 4.2.2.3. Effects of metal contamination on energy metabolism proteins.
In response to the tested metal stress conditions, ATP synthase F1 beta subunit and the membrane mitochondrial ATP synthase CF1 beta

<caption>**Table 3**</caption>

Liquid chromatography-tandem mass spectrometry identification of the proteins constituting the membrane proteome of the *Alexandrium pacificum* TAR C5-4F strain.

<table><thead><tr><th rowspan="2">Spot no</th><th rowspan="2">Peptide number</th><th colspan="2">MW (kDa)/pl</th><th rowspan="2">Protein name</th><th rowspan="2">Species Accession number</th><th rowspan="2">Putative function</th></tr><tr><th>Obs</th><th>Theo</th></tr></thead><tbody><tr><td>11</td><td>3</td><td>42.8/4.3</td><td>33.4/6.0</td><td>Unknown protein</td><td></td><td></td></tr><tr><td>22</td><td>9</td><td>41.5/4.3</td><td>42.9/4.9</td><td>High-CO₂-inducible periplasmic protein</td><td>Heterocapsa triquetra AAW79380.1</td><td>Calcium binding (16.17.01)</td></tr><tr><td>26</td><td>2</td><td>29.1/5.7</td><td>45.9/8.3</td><td>Light-harvesting protein</td><td>Symbiodinium sp. CBI83418.1</td><td>Transmembrane signal transduction (30.05)</td></tr><tr><td>30</td><td>9</td><td>41.7/5.2</td><td>42.9/4.9</td><td>High-CO₂-inducible periplasmic protein</td><td>Heterocapsa triquetra AAW79380.1</td><td>Calcium binding (16.17.01)</td></tr><tr><td>33</td><td>8</td><td>42.2/5.6</td><td>42.9/4.9</td><td>High-CO₂-inducible periplasmic protein</td><td>Heterocapsa triquetra AAW79380.1</td><td>Calcium binding (16.17.01)</td></tr><tr><td>34</td><td>6</td><td>24.5/5.8</td><td>53.2/5.1</td><td>ATP synthase CF1 beta chain</td><td>Nageia nagi YP_008965117.1</td><td>Transported compounds (20.01)</td></tr><tr><td>35</td><td>7</td><td>19.6/5.9</td><td>87.9/5.9</td><td>Light-harvesting polyprotein precursor</td><td>Amphidinium carterae CAA08771.1</td><td>Photosynthesis (02.30)</td></tr><tr><td>40</td><td>2</td><td>34.7/4.1</td><td>45.0/7.0</td><td>Chloroplast ferredoxin NADP(+) reductase</td><td>Heterocapsa triquetra AAW79380.1</td><td>Electron transport (20.01.15)</td></tr><tr><td>43</td><td>6</td><td>34.7/4.4</td><td>45.0/7.0</td><td>Chloroplast ferredoxin NADP(+) reductase</td><td>Heterocapsa triquetra AAW79380.1</td><td>Electron transport (20.01.15)</td></tr><tr><td>44</td><td>3</td><td>31.2/4.6</td><td>37.4/7.1</td><td>Chloroplast cytochrome f</td><td>Heterocapsa triquetra AAW79342.1</td><td>Electron transport (20.01.15)</td></tr><tr><td>47</td><td>2</td><td>45.4/4.4</td><td>53.2/5.1</td><td>ATP synthase CF1 beta chain</td><td>Nageia nagi YP_008965117.1</td><td>Transported compounds (20.01)</td></tr></tbody></table>
chain disappeared from the membrane proteomes of the SG C10-3 and TAR C5-4F strains, respectively. These subunits belong to a large ATP-synthase complex located in the mitochondrial membranes, which converts ADP and orthophosphate during oxidative phosphorylation of cell respiration, into ATP-energy rich molecules (Kühlbrandt, 2015). Few studies report similar results, except regarding the effects of trace metals on the ATP-synthase activity that decreased in the plasma membrane fraction of wheat and sunflower roots that had been exposed to cadmium treatments (Fodor et al., 1995). Consequently, reduced ATP-synthase activity and subsequent ATP synthesis may correspond to a decreased aerobic pathway/mitochondrial respiratory chain, leading to less O₂ consumption and less associated reactive oxygen species (ROS) production, which may counteract the quantities of ROS generated by the metal stress, and thus, helping limit the oxidative stress.

## 5. Conclusion

This study explored modifications in the membrane proteomes, cell growth, and morphometry in two distinct Mediterranean strains of *A. pacificum*, in response to exposure to a metal cocktail. Cell growth and morphometry were different between strains in these conditions. Susceptibility of SG C10-3 to metals was high during its growth, with an increase in cell size, which was not observed for TAR C5-4F. This intraspecific variability may be related to different biomolecular regulations in these strains coming from metal-contaminated ecosystems. The total number of proteins constituting the membrane proteome of TAR C5-4F was higher than that of SG C10-3, which may help this strain to better adapt and/or be resistant to metal-contaminated conditions. However, metal stress led to mainly down-regulated proteins of interest, showing the harmful effects of metals on the membrane proteomes from both strains. These proteomic modifications may affect metabolic functions in SG C10-3 and TAR C5-4F cells, such as cell transport, photosynthesis, and energy metabolism. However, other modifications observed in their membrane proteomes may promote adaptation and/or resistance to metal stress, contributing, more generally, to an increase in the occurrence, distribution, and intensity of the *A. pacificum* HAB, particularly in marine metal-contaminated ecosystems.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2020.136834.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

This work was supported by the french research network GdR "PHYCOTOX: Des micro-algues aux risques pour l'Homme et l'écosystème". We would like to thank D's Antonella Luglié and Esther Garcés for providing us the *A. pacificum* strains used in this study, and D's Yong Zhang and Da-Zhi Wang (State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University, Xiamen, P.R. China) for the *A. pacificum* protein database sequences used for protein identification.

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