Ali Kadar
email: ali.kadar@etu.univ-amu.fr

Ludovic Peyre

Georges De Sousa

Henri Wortham

Pierre Doumenq

Roger Rahmani

Georges De Souza

An accurate and robust LC-MS/MS method for the quantification of chlorfenvinphos, ethion and linuron in liver samples

Keywords: Pesticide mixtures, Liver samples, LC-MS/MS, Method validation

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Introduction

For many years, pesticides have been used on a broad scale for pest control in agriculture. Despite their outstanding positive influence on farm productivity, these active ingredients are harmful for the environment. Owing to their physicochemical properties and their wide use, many of the pesticide residues end-up in water resources and in agricultural products. Consequently, the entire food chain is exposed to such toxic molecules, which may ultimately reach human beings through bioaccumulation or directly by the consumption of contaminated water or foodstuffs [START_REF] Cao | Relationship between serum concentrations of polychlorinated biphenyls and organochlorine pesticides and dietary habits of pregnant women in Shanghai[END_REF][START_REF] Damalas | Pesticide Exposure, Safety Issues, and Risk Assessment Indicators[END_REF][START_REF] Ding | Revisiting pesticide exposure and children's health: focus on China[END_REF]. Most of studies aiming to estimate dietary exposure of the general population highlighted that the consumers were simultaneously exposed to different residues (Iñigo-Nuñez et al., 2010, Claeys et al., 2011, Chen et al., 2011, Nougadère et al., 2012, Bakırcı et al., 2014[START_REF] Betsy | Assessment of dietary intakes of nineteen pesticide residues among five socioeconomic sections of Hyderabad-a total diet study approach[END_REF], Lozowicka, 2015[START_REF] Szpyrka | Evaluation of pesticide residues in fruits and vegetables from the region of south-eastern Poland[END_REF][START_REF] Lemos | Risk assessment of exposure to pesticides through dietary intake of vegetables typical of the Mediterranean diet in the Basque Country[END_REF]. In France, Crepet et al. (2013) established that the general population was mainly exposed to 7 different pesticide mixtures consisting of 2 to 6 compounds. Among them, a mixture including chlorfenvinphos, linuron and ethion was significantly correlated to basic food items such as carrots and potatoes. After the consumption of these potentially contaminated vegetables, and once these xenobiotics have passed into the body, the blood flow delivers them to the liver for degradation and subsequent elimination. Thus, to evaluate the importance of the liver contamination, a sensitive and reliable analytical method is required. Unfortunately, to the best of our knowledge, no analytical procedure determining simultaneously chlorfenvinphos, linuron and ethion in a liver matrix has been published so far.

To date, literature survey reveals that very few articles have been reported on the analysis of linuron, chlorfenvinphos or ethion in biological samples. [START_REF] Nguyen | Quantification of atrazine, phenylurea, and sulfonylurea herbicide metabolites in urine by high-performance liquid chromatography-tandem mass spectrometry[END_REF] proposed a methodology based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the quantification of linuron in urine sample. [START_REF] Cazorla-Reyes | Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry[END_REF] also developed a method using LC-MS/MS for the determination of this polar herbicide in the same matrix. By contrast, the same authors quantified the non-polar compounds chlorfenvinphos and ethion thanks to gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). [START_REF] Pitarch | Rapid multiresidue determination of organochlorine and organophosphorus compounds in human serum by solid-phase extraction and gas chromatography coupled to tandem mass spectrometry[END_REF] and [START_REF] Raposo | Determination of eight selected organophosphorus insecticides in postmortem blood samples using solid-phase extraction and gas chromatography/mass spectrometry[END_REF] also used GC-MS/MS for the determination of ethion in blood samples. Even if gas chromatography is adequate [START_REF] Deme | LC-MS/MS determination of organophosphorus pesticide residues in coconut water[END_REF][START_REF] Sinha | A liquid chromatography mass spectrometry-based method to measure organophosphorous insecticide, herbicide and nonorganophosphorous pesticide in grape and apple samples[END_REF] for the separation of organophosphorus, it is a less suitable option for phenyl urea herbicides, since these are thermolabiles [START_REF] Liska | Comparison of gas and liquid chromatography for analysing polar pesticides in water samples[END_REF]. As a result, an analytical protocol which uses a LC separation followed by MS/MS detection would be suitable to estimate the pesticides liver contamination.

Thus, the aim of this work was to develop, optimize and fully validate a simple, sensitive and reproducible analytical method for quantitative determination of linuron, chlorfenvinphos and ethion in human liver samples (hepatocytes, microsomes…).

Experimental

Chemicals, materials and biological samples

Trichloroacetic acid, ammonium sulfate salts of research grade purity and, anhydrous dimethylsulfoxide were supplied by Sigma Aldrich (Saint-Quentin Fallavier, France). LC-MS grade methanol and acetonitrile were obtained from Carlo Erba (Val de Reuil, France). Chlorfenvinphos, chlorfenvinphos-d10 (internal standard; IS), ethion and linuron certified standards of purity higher than 99.5% were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Standard stock solutions were prepared by dissolving the pure compounds in acetonitrile and further diluted as required in acetonitrile, for calibration standards and sample treatment. Sample extracts were centrifuged using a Thermo IEC Micromax TM RF benchtop centrifuge acquired from Thermo Fisher Scientific (Illkirch, France). Oasis™ HLB (10 mg/1 mL), Strata X ® (10 mg/1 mL) and Sola TM (10 mg/1 mL) solid phase extraction (SPE) cartridges were provided by Waters (Guyancourt, France), Phenomenex (Le Pecq, France) and Thermo Fisher Scientific (Courtaboeuf, France), respectively. A 12 ports SPE manifold (J.T. Baker ® ) connected to a KNF Neuberger LABOPORT ® filtration pump (VWR, Paris, France) was used for conditioning, sample loading, drying of the cartridges and elution of the targeted compounds.

All experiments on human tissue were carried out according to the ethical standards of the responsible committee on human experimentation and the Helsinki Declaration. Liver tissue can be mechanically decomposed into cellular (hepatocytes) or subcellular (S9, cytosolic and microsomal) fractions. Here we chose to carry out the study with thermally inactivated hepatocytes (100 °C for 3 min) previously isolated as described by [START_REF] Berry | High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study[END_REF].

Sample treatment

400 µL of thermally inactivated liver cells at a total protein concentration of 0.5 mg/mL in 100 mM phosphate potassium buffer (pH 7.4) were pipetted into 1.8 mL Eppendorf ® tubes. The samples were spiked with the required amounts of chlorfenvinphos, ethion, linuron and ITSD before being briefly vortex-mixed. Then, 400 µL of glacial acetonitrile was added to the tubes. Centrifugation performed at 16000g for 5 min allowed the denatured proteins to precipitate. The supernatant was purified according to the optimized following SPE protocol. The samples were diluted in 6 mL borosilicate glass tubes by addition of purified water in order to obtain a ratio of acetonitrile-water (25:75, v/v) in the mixture. The samples were then loaded onto Thermo Sola TM extraction cartridges, which had been pre-cleaned using 1mL of methanol, followed by 1 mL of acetonitrile and finally conditioned using 1 mL of acetonitrile-water (25:75, v/v). Compounds of interest were trapped on the cartridges while interferents were successively eluted with 1 mL of acetonitrile-water (25:75, v/v) and 1 mL of purified water. After 5 min SPE manifold vacuum drying (-10 PSI) of the cartridges, compounds of interest were eluted under vacuum with 2×0.2 mL of pure acetonitrile. Finally, the eluates were diluted 1:1 in purified water prior to analysis.

LC-MS/MS analysis

Compounds were separated and quantified using a Surveyor HPLC analytical system purchased from Thermo Fisher Scientific (Courtaboeuf, France). It consisted in a quaternary low pressure mixing pump equipped with an integrated degasser, a 20 µL injection loop, a temperature controlled autosampler set at 10 °C and a column oven kept at 25 °C. A Hypersil end-capped Gold PFP reversed phase column (100 mm×2.1 mm, 3 µm) purchased from Thermo Fisher Scientific (Gif-sur-Yvette, France) equipped the Surveyor module. LC separation was achieved at a flow rate of 280 µL.min -1 using a mobile phase composed of 10 mM ammonium formate in methanol (solvent A) and 10 mM ammonium formate in water (solvent B). The gradient program was run as follows: maintain 35% A from 0 to 7 min, linear increase to 100% A from 7 to 9 min, hold 100% A from 9 to 12 min, return to the initial conditions from 12 to 17 min and stabilization during 3 min before the next injection.

The detection and quantification were performed using a TSQ Quantum triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. To prevent a contamination of the ESI source, from 0 to 6 min, the column effluent was systematically diverted to the waste by means of a motorized Divert/Inject valve.

The mass spectrometer was operated in multiple reaction monitoring (MRM) positive acquisition mode. The ion capillary temperature was heated to 350 °C and the ESI needle voltage was set at 4000 V. The sheath and auxiliary gas (N 2 ) pressures were respectively tuned at 40 and 30 (arbitrary units). A 4 V source collision induced dissociation (CID) offset and a 1.5 mTorr collision gas (Ar) pressure were applied at the collision cell. For each compound, sensitive quantitative determination was performed using an addition of the MRM transitions displayed in Table 1.

All MS parameters were optimized by direct infusion and the source parameters were subsequently adjusted by flow injection. Data analysis was accomplished using Xcalibur™ 2.1 software.

Analytical method validation

Validation of the analytical method was carried out in accordance with the general guidelines for bioanalytical methods established by the FDA (US Food and drug Administration, 2013). Validation criteria including lower limit of quantification (LLOQ) sensitivity, linearity, selectivity, accuracy, precision, recovery and stability were investigated.

Limit of quantification

The LLOQ is defined as the lowest concentration that can be measured with acceptable precision and accuracy. For its assessment, four serial dilutions of sample containing 0.400 µM mL of each analyte were made by mixing equal volumes of spiked microsomal sample with blank microsomal sample (six replicates). The peak areas of these fortified extracts should be at least five times higher than the background of blank samples (i.e. signal-to-noise ratio, S/N=5/1) to be considered as a proven LLOQ.

The precision and mean accuracy of back-calculated LLOQ replicate concentrations must be of <20% and ±20%, respectively.

Selectivity and matrix effects

The evaluation of the selectivity was conducted after the pretreatment and instrumental analysis of ten different blank human microsomes samples. Selectivity was assessed to ensure the absence of any potential endogenous interference co-eluting with analytes, including the chlorfenvinphos-d10 (IS).

Chromatographic signals of pesticides were discriminated on the basis of their specific retention times and MRM responses.

In addition, to assess matrix effects, ten different blank matrices were extracted, further spiked with the standard solution at the LLOQ level and compared with aqueous standards of the same concentration level. According to the guidance the difference of response should not exceed ±5%.

Linearity

For each compound, the calibration curve range varied from the validated LLOQ (0.005 µM) to 2.0 µM. Calibration curve standard samples were prepared in replicates (n=6) in a mixture of purified water and acetonitrile (50:50, v/v), and then analyzed. Data was reprocessed and validity of the linearity was checked through ANOVA statistical analyses (Microsoft Excel). The goodness of fit (GoF) and lack of fit (LoF) were determined and correlated with the corresponding Fisher theoretical table value. The fitting of the calibration curves was obtained with a 1/x weighted least squares linear regression.

Recovery

Recovery rates of pesticides from thermally inactivated human hepatocytes samples were assessed at three concentration levels: low (0.05 µM), medium (5.0 µM) and high (50.0 µM). Three replicates were prepared for each level and extracted. After their analysis, the peak areas from these samples were compared to those from post-extracted blank inactivated hepatocytes samples fortified with the targeted compounds at the same concentration and analyzed. The ratio of mean peak areas of preextracted samples to mean post-extracted spiked samples enabled to calculate individual percentage recovery.

Precision and accuracy

Thanks to freshly prepared calibration curves, imprecision (intra-and inter-day) and accuracy were back calculated for the mixture respectively at four concentration levels: low (0.05 µM), medium 1

(2.0 µM), medium 2 (5.0 µM) and high (50.0 µM).

For intra-day imprecision and accuracy, five replicate samples per concentration were prepared and consecutively analyzed on the same day. For inter-day imprecision, the samples' preparation and analysis were carried out in duplicate at the same spiking levels, and repeated on six different days.

Imprecision was expressed as the relative standard deviation (RSD%) and accuracy was calculated as the mean percentage deviation (A%) from the spiked value. The acceptance criteria for intra-and inter-day imprecision were ≤15% and, for accuracy, were between 85 and 115% of the nominal concentrations.

Stability of pesticides in the matrix extract

Stability tests were conducted in triplicate with processed samples that had been previously spiked at a concentration level of 5 µM. Different storage conditions were tested: 72 h in the autosampler tray at +12 °C, after 15 h of three cycles of freezing (-20 °C) and thawing (room temperature) for either 1 month, 3 months or 9 months at -20 °C. The results were calculated using freshly prepared calibration curves. The imprecision and accuracy calculated for samples' stability should be below 15% and between 85 and 115% of their nominal levels, respectively.

Results and discussion

Method development

Mass spectrometry optimization

Direct infusion of individual compound and IS solutions at a concentration of 1 mg.L -1 in watermethanol (50:50, v/v), 10 mM ammonium acetate in water-methanol (50:50, v/v) or 10 mM ammonium formate in water-methanol (50:50, v/v) was carried out to select the best solvent mixture phase, precursor and product ions. The individual mass spectra of each molecule obtained in positive ion mode showed the presence of both an abundant pseudo-molecular ion [M+H] + and a reproducible stable sodium adduct [M+Na] + signal. For all the compounds, the best [M+H] + /[M+Na] + signal ratio was obtained with the standard solution containing 10 mM ammonium formate. Once the precursor ion was chosen, the optimum tube lens voltage was automatically optimized. Then, analytes were fragmented by applying the collision energy giving the highest abundance for each product ion. The optimized source parameters, MRM transitions and settings were then included in the mass spectrometry acquisition method.

Chromatographic conditions

After optimization of the mass spectrometry parameters, different liquid chromatography columns were tested. The first evaluations were achieved on two core-shell LC columns from Phenomenex (Le Pecq, France): a Kinetex ® C 18 and a Kinetex ® PFP (100 mm × 2.1 mm, 3 µm). Both columns enabled a satisfactory separation of linuron, chlorfenvinphos, ethion and IS. Less peak tailing was nevertheless obtained with the Kinetex PFP, therefore contributing to a significant improvement on sensitivity, especially for the chlorinated molecules such as chlorfenvinphos and linuron. This column was initially selected, but the drawback of using this specific stationary phase appeared later during the study. In fact, a high back pressure was observed only after a short time use. As a consequence, a fully porous Thermo Hypersil TM Gold PFP column was used instead. A new elution gradient was therefore optimized for this column and enabled to achieve optimum resolution and compound detection, as already depicted in "Experimental -LC-MS/MS analysis". Finally, the analytes' signals were appropriately separated and chromatograms displayed good peak shapes as presented on Fig. 1. The carry-over in the chromatographic system was measured by injecting three blank solvents after the highest calibration standard.

Sample purification

A suitable optimization of the extraction step was then needed to achieve a satisfactory LLOQ and selectivity for the detection of the pesticides mixture in such small volumes of human liver extracts.

For this purpose, after the protein precipitation step described above, an additional solid phase extraction step was preferred against a liquid-liquid extraction to remove potential additional endogenous interferents such as phospholipids or inorganic salts contained in the cell seeding medium (Yaroshenko and Kartsova, 2014) and polyethylene glycol leached from plastic containers [START_REF] Weaver | Identification and reduction of ion suppression effects on pharmacokinetic parameters by polyethylene glycol 400[END_REF].

SPE sorbent types such as polymeric and silica based reversed-phase sorbents seemed appropriate for the simultaneous extraction of organophosphorus and neutral phenylurea pesticides from biological matrices [START_REF] Cazorla-Reyes | Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry[END_REF]. The few studies dedicated to their purification from human body fluids indicated that both polymeric reversed-phase cartridges [START_REF] Nguyen | Quantification of atrazine, phenylurea, and sulfonylurea herbicide metabolites in urine by high-performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Raposo | Determination of eight selected organophosphorus insecticides in postmortem blood samples using solid-phase extraction and gas chromatography/mass spectrometry[END_REF] and silica based reversed-phase sorbent cartridges [START_REF] Pitarch | Rapid multiresidue determination of organochlorine and organophosphorus compounds in human serum by solid-phase extraction and gas chromatography coupled to tandem mass spectrometry[END_REF] could be used. In this study, the aim was to minimize elution volumes in order to simplify sample treatment by avoiding the concentration step after the elution of the analytes. As a result, the polymeric reversed-phase (PRP) sorbents were chosen for their higher loading capacities which allowed the use of lower sorbent amounts, associated with reduced elution volumes. In this work, three different commercial PRP cartridges were compared (Oasis TM HLB, Strata X ® and Sola TM ) with the goal to choose the one which could give the maximum recoveries with the minimum elution volume. Aiming at obtaining comparable results, a common SPE methodology was applied for all the cartridges. First, after being successively rinsed using 1 mL of methanol and 1 mL acetonitrile, the cartridges were conditioned with 1 mL of purified water. Then, a blank liver cells sample containing all the test compounds at 20 µM was loaded on the cartridge. Then, a washing step consisting of 1 mL of purified water, followed by a 5 min drying step and an elution of the target compounds with 2×0.2 mL of pure acetonitrile under vacuum (-5 PSI), were applied. Acetonitrile was chosen for its LC-MS compatibility and its higher elution strength in comparison with methanol. Indeed, the latter did not allow for satisfactory elution of ethion even if a larger volume was used (up to 2 mL). After dilution (50:50, v/v) in purified water, the extract was transferred to an injection vial for analysis. The performance of the SPE cartridges tested are displayed in Table 2. The best results, in terms of relative recoveries and RSD values, were achieved using Sola TM cartridges with values ranging from 97 to 100%.

As incubation of both hepatocytes and microsomes samples was stopped by adding cold-acetonitrile (50/50, v/v), further optimization was performed to determine the maximum acceptable amount of acetonitrile in the liver extracts to be purified by SPE. To this goal, as described above, blank samples were prepared and loaded onto Sola TM cartridges. Then, an elution/retention profile of the analytes was established with the successive addition of mixtures of water and acetonitrile, as shown in Table 3.

The experiments were performed in duplicate. All the analytes were retained with acetonitrile proportion below 30%. Linuron, as the most polar compound, was the first one to be desorbed. As a consequence, we considered that the liver extracts from kinetic experiments should be diluted with the appropriate amount of water (25:75, v/v) prior to the solid phase extraction.

Performance of the analytical method

The chromatograms of liver cells samples were visually checked and compared with chromatogram obtained from standard references in neat solvents. As they showed no disturbing peaks, the selectivity was approved. Besides, the matrix effect assessed at the LLOQ level did not exceed 0.6% and was considered negligible for all the pesticides. Additionally, no cross-contamination was observed when three blank solvent samples were injected consecutively to the highest calibration standard.

The results of the linearity, the intra-and inter-day precision and accuracy, as well as the stability of pesticides in the matrix extracts are summarized in Table 4.

The results from the Goodness of Fit and Lack of Fit of the Fisher significance tests (GoF-LoF) indicated that the linear regression model was validated in the defined range of concentrations for all compounds. In addition, the determination coefficient was systematically verified and always gave satisfactory values (r 2 >0.998).

Recovery data collected on three replicates of three wide range covering levels ranged from 92.9% to 99.5%, with a maximum RSD of 2.3%, demonstrating the efficiency of the SPE purification process.

Moreover, intra-and inter-day imprecision and accuracy were all within the established ranges of acceptance. Finally, stability data revealed that whichever test used, no significant loss was noticed, indicating that all the analytes were stable within the studied working conditions.

All the evaluated performance parameters were in accordance with FDA recommendations, making this method reliable and rugged for future in vitro liver metabolism studies.

Conclusions

To our knowledge, this is the first reported analytical method for determination of linuron, chlorfenvinphos and ethion in liver samples. This LC-MS/MS method developed with one stable isotope-labeled internal standard exhibited very satisfactory performance in terms of selectivity, limit of quantification, linearity, recovery, precision and accuracy, in compliance with current FDA requirements. A user-friendly sample treatment process providing excellent recoveries and high sample cleanliness was obtained after appropriate optimization of conditions. Indeed, protein precipitation, solid phase extraction sorbent type, volume and solvent elution strength were optimized. This method is optimal for conducting metabolism studies through the accurate monitoring of the parent compound loss in in-vitro human liver samples. Furthermore, it would probably be also convenient for the determination of the above-mentioned pesticides mixture in human liver biopsies or mammalian liver samples.

Table 1

 1 Ions monitored under the MRM mode by LC-MS/MS a and their relative intensities (%). The compounds were quantified with both product 1 and product 2 ions. Ionized in the positive mode with a 4 V CID offset.

	Compound	precursor ion b	product ion 1	product ion 2	collision energy
		(m/z)	(m/z)	(m/z)	(V)
	Linuron	249	182(100)	161(23)	20-25
	chlorfenvinphos	359	155(100)	127(45)	18-22
	chlorfenvinphos-d10 (IS)	369	165(100)	133(66)	16-25
	Ethion	385	143(100)	97(88)	35-45

a b

Table 2

 2 Percentage recoveries and associated RSD (in brackets) of the target analytes testing different SPE cartridges (n=3).

		Oasis TM HLB	Strata X ®	Thermo Sola TM
	Linuron	92 (4)	87 (11)	100 (2)
	chlorfenvinphos	90 (8)	83 (12)	97 (2)
	Ethion	85 (9)	43 (3)	98 (5)

Table 3

 3 Elution of the pesticides mixture from Sola TM sorbent cartridge (n=2).

	elution mixture		Elution rate from (%)
	acetonitrile/water
	(v:v, 1 mL)	linuron	chlorfenvinphos	Ethion
	0/1	0	0	0
	0.05/0.95	0	0	0
	0.10/0.90	0	0	0
	0.15/0.85	0	0	0
	0.20/0.80	0	0	0
	0.25/0.75	0	0	0
	0.30/0.70	1	0	0
	0.35/0.65	14	3	0
	0.40/0.60	56	29	0
	1/0	100	100	100

Table Click here to download Table: Tables-MS1_170224.docx

 Click

Table 4

 4 Results of the analytical method validation: linearity (n=6), recovery (n=3), intra-day accuracy (n=5), inter-day accuracy (n=2, 6 days), stability (n=3). LC-MS/MS chromatograms obtained from fortified inactivated hepatocytes extract (LLOQ)

	Parameter	Linuron		chlorfenvinphos	Ethion		Limits
	Linearity	GoF-LoF
	LoF	0.164		1.019		0.055		<4.51
	GoF	4840		21868		3084		>>5.39
	Recovery	R%	RSD%
	Low	99.2	1.0	97.0	2.0	92.9	1.9
	Medium	98.7	2.2	95.1	1.8	94.2	1.6	n.a
	High	99.2	1.3	97.6	1.7	95.0	1.2
	Accuracy
	Intra-day	Ar%	RSD%
	Low	100.0	1.3	96.6	4.9	93.8	2.6	±20, ≤20 %
	Medium 1	98.4	3.5	99.6	4.2	93.6	0.8	±15, ≤15 %
	Medium 2	97.6	6.2	94.6	7.1	93.9	3.7
	High	100.0	0.8	99.0	1.6	93.3	4.2
	Inter-day	Br%	RSD%
	Low	99.4	4.1	98.2	6.9	92.8	4.8	±20%, ≤20 %
	Medium 1	99.8	6.7	100	6.6	91.6	2.9	±15%, ≤15 %
	Medium 2	98.9	8.1	97.2	8.2	90.9	5.9
	High	99.5	2.6	100.0	3.1	93.1	6.3
	Stability
	Freeze-thaw	SFt%
	-20/20 °C-15 h	101.1	5.3	99.9	5.7	100.0	4.2	±15 %, ≤15 %
	Long term	SLt%
	1 month	100.7	6.1	99.0	5.5	97.8	3.0	±15 %, ≤15 %
	3 months	102.1	5.7	101.1	5.8	104.2	4.1
	9 months	99.8	6.0	99.5	5.1	102.2	3.7
	Autosampler	SA%
	72 h	99.9	5.8	101.2	4.9	100.2	4.6	±15 %, ≤15 %

GoF-LoF : Goodness of Fit -Lack of Fit Fig. 1.

Bioanalytical Methods Using Chromatography-Mass Spectrometry. J.Anal. Chem. 69,[311][312][313][314][315][316][317]

Acknowledgements

This work was funded by the Agence Nationale de la Recherche (under reference ANR-2008-CESA-016-01), by the Office National de l'Eau et des Milieux Aquatiques (ONEMA) and by the Agence Nationale de SÉcurité Sanitaire de l'alimentation, de l'environnement et du travail (ANSES) in the frame of the MEPIMEX Project. The authors also gratefully acknowledge the supply of the LC-MS/MS system at Laboratoire de l'environnement de Nice as a collaboration agreement with Patricia Pacini.

ABSTRACT

A generic method for the determination of linuron, chlorfenvinphos and ethion in liver samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. In vitro sample treatment was performed by using solid phase extraction (SPE) after protein precipitation. The lowest elution solvent volumes providing the highest recoveries were obtained with Sola TM polymeric reversed-phase cartridges. Gradient elution using 10 mM ammonium formate in methanol (A) and 10 mM ammonium formate in water (B) was used for chromatographic separation of analytes on a

Hypersil TM end-capped Gold PFP column (100 mm×2.1 mm, 3 µm). All analytes were quantified without interference, in positive mode using multiple reaction monitoring (MRM) with chlorfenvinphos-d10 as internal standard. The whole procedure was successfully validated according to the Food and Drugs Administration (FDA) guidelines for bioanalytical methods. The calibration curves for all compounds were linear over the concentration range of 0.005 -2 µM, with coefficients of determination higher than 0.998. A Lower limit of quantification of 0.005 µM was achieved for all analytes. Compounds extraction recovery rates ranged from 92.9 to 99.5% with a maximum relative standard deviation (RSD) of 2.3%. Intra--and inter--day accuracies were within 90.9% and 100%, and imprecision varied from 0.8 to 8.2%. Stability tests proved all analytes were stable in liver extracts during instrumental analysis (+12°C in autosampler tray for 72 h) at the end of three successive freeze-thaw cycles and at -20°C for up to 9 months. This accurate and robust analytical method is therefore suitable for metabolism studies of pesticide mixtures.

Abstract Click here to download Abstract: Abstract-MS1_170224.docx


A V Ereskovsky
email: alexander.ereskovsky@imbe.fr

A Geronimo

T Pérez

Asexual and puzzling sexual reproduction of the Mediterranean sponge Haliclona fulva (Demospongiae): life cycle and cytological structures

Keywords: asexual reproduction, budding, Haplosclerida, oviparity, reproductive cycle

Despite the common assumption that most Haplosclerida are viviparous sponges, this study of the reproductive cycle of Haliclona fulva demonstrates that this species is actually oviparous and gonochoric. Intriguingly, not a single male was recorded in 15 months of sampling. Oogenesis is synchronous, starting in late April and terminating in September. Asexual reproduction is represented by cyclic budding, which occurs from late November to early March. During the season of asexual reproduction, the reproductive effort represents from 0.21% to 1.49% of the parental tissue, with the highest values being recorded in winter. During the season of sexual reproduction, the female reproductive effort ranges 0.05-1.15%, with the highest effort appearing in early summer. However, no significant correlation between reproductive efforts and seawater temperature fluctuations could be detected. We describe the ultrastructural morphogenesis of the buds for the first time in this species. This process is asynchronous, with buds of variable size being attached to the maternal apical surface via a short stalk. Young buds lack any particular anatomical organization, whereas bud maturity is characterized by the development of mesohyl and by the appearance of an increasing number and volume of lacunae in the central part of each bud. At this stage, buds harbor numerous small choanocyte chambers scattered throughout the inner region, and all cell types known from the mesohyl of parental sponges: microgranular cells, granular cells, archaeocytes, endopinacocytes and exopinacocytes, central cells, and sclerocytes.

 Pérez-Porro et al. 2012;Di Camillo et al. 2012;[START_REF] Mercurio | Sexual reproduction in Sarcotragus spinosulus from two different shallow environments[END_REF][START_REF] Zarrouk | Sexual Reproduction of Hippospongia communis (Lamarck, 1814) (Dictyoceratida, Demospongiae): comparison of two populations living under contrasted environmental conditions[END_REF][START_REF] Reverter | Secondary Metabolome Variability and Inducible Chemical Defenses in the Mediterranean Sponge Aplysina cavernicola[END_REF]. However, there is less knowledge about the sponges from order Haplosclerida, which are relatively common and abundant in the Mediterranean Sea. Up to now, we have data on reproductive cycles for only four species of Haplosclerida [START_REF] Scalera Liaci | Raffronto tra il comportamento sussuale di alcune Ceractinomorpha[END_REF][START_REF] Maldonado | Gametogenesis, embryogenesis, and larval features of the oviparous sponge Petrosia ficiformis (Haplosclerida, Demospongiae)[END_REF].

In this work, we investigate Haliclona fulva (TOPSENT 1893), a common shallow-water Mediterranean demosponge of the order Haplosclerida, family Chalinidae. We chose H. fulva for this study because this species regularly uses budding in its life cycle, unlike other haplosclerids, which have never been described to possess this mode of asexual reproduction. Moreover, as we show here, it is the only oviparous species in the family Chalinidae. We evaluate the incidence of sexual and asexual reproduction in the life cycle of Haliclona fulva and describe budding in H. fulva thoroughly, detailing the cellular composition of buds at different stages of development and comparing them with parental tissue.

Methods

Biological model

Haliclona (Halichoclona) fulva is a common Mediterranean sponge species, individuals of which form thick crusts (5-15 mm thickness) that vary in color from orange to red and which inhabit shaded benthic communities, such as semi-dark submarine caves or coralligenous formations, at depths of 5-50 m (Fig. 1A).

Sampling site

Sponges were collected by SCUBA diving at depths of between 14 and 16 m at a site called Grotte à Corail, located at Maire Island (Marseilles Bay). This sampling site was equipped with a permanent temperature recorder (HOBO Tidbit Data Logger). From September-March 2007 to December 2008, between four and nine individuals were collected monthly (during June and August 2008, the sponges were sampled twice), which represents a total of 103 individuals studied (Table 1). Only one month (October 2007) could not be sampled due to bad weather conditions. In order to pinpoint the period of budding, we also examined a great number of underwater photographs taken by scientific divers at different sites in the Marseille region from 1999 to 2009.

Morphological and ultrastructural analysis

To characterize the life cycle and assess the reproductive effort, samples were preserved in Bouin's fixative. Tissue fragments were then dehydrated through an ethanol series and embedded in paraffin. Serial sections of 6 μm thickness were mounted on glass slides and stained with Masson-Goldner's trichrome hematoxylin, and then observed under a WILD M20 light microscope.

For semithin sections and for transmission electron microscopy (TEM) investigations, sponges were fixed in a solution composed of one volume of 25% glutaraldehyde, four volumes of 0.2 M cacodylate buffer, and five volumes of filtered seawater for 2 h before being post-fixed in 2% OsO 4 in seawater at room temperature for 2 h. After fixation, samples were washed in 0.2 M cacodylate buffer and distilled water successively, and finally dehydrated through a graded ethanol series. Specimens were embedded in Araldite resin. Semithin sections (1 µm in thickness) were cut on a Reichert Jung ultramicrotome equipped with a "Micro Star" 45° diamond knife before being stained with toluidine blue, and observed under a WILD M20 microscope. Digital photos were taken with a Leica DMLB microscope using the Evolution LC color photo capture system. Ultrathin sections (60-80 nm) were cut with a Leica UCT ultramicrotome equipped with a Drukkert 45° diamond knife. Ultrathin sections, contrasted with uranyl acetate, were observed under a Zeiss-1000 transmission electron microscope (TEM).

Data analysis

Calculations of sexual reproductive effort (sRE) were carried out on serial histological sections. For each specimen, digital photographs of 16 histological sections were analyzed. Four photographs per serial section were taken. To avoid the overlapping of reproductive products that would lead to over-estimation, photographs of tissue were taken at least 200 µm from each other. The four photographs provided a total surveyed area of 1 mm 2 per sponge. We determined the number of sexually active sponges over time, counted the number of reproductive elements, and calculated the area of each reproductive element within the tissue sample, using ImageJ Software (http://rsb.info.nih.gov/ij/index.html). Reproductive elements were related to the overall surface of the section, and reproductive effort could thus be expressed as a percentage of reproductive tissue (mean ± SD).

In order to calculate the number of asexual reproductive efforts (aRE), the number of buds per individual was counted and bud area was estimated. These data allowed us to obtain the total surface of buds relative to the sponge surface, with aRE also expressed as a percentage of reproductive tissue.

The nonparametric Kruskal-Wallis test was used to determine the seasonal variability of each reproductive effort. To assess the putative influence of seawater temperature, we then applied a Spearman correlation test. Statistics and graphs were performed using RStudio (R Development Core Team, 2012 ).

Results

Reproductive cycle

No occurrence of gametogenesis was found between November 2007 and April 2008, nor from November to December 2008. In 2008, we observed oogenesis starting in late April and ending in September (Table 1, Fig. 2). No hermaphroditic individuals were observed; thus, Haliclona fulva appears to be gonochoric and oviparous, given the absence of observable embryo development. Through examination of our sponge collection and in situ photographs, we were able to observe the beginning of the budding period of H. fulva in early November and the end in April (Table 1, Fig. 2), which indicates an alternation between sexual and asexual processes.

Sexual reproduction

No male reproductive elements were recorded during this study of 103 individuals collected from this subpopulation. Of the 44 individuals observed during this period, 7 individuals lacked reproductive elements, but not a single male was recorded.

Oocytes were observed throughout the mesohyl, with the exception of the most superficial layer of the sponge tissue below the exopinacoderm. Oogenesis was synchronous within a mother sponge and in the entire population. Young oocytes were of small size, 14-24 µm in diameter. At this previtellogenic stage, oocytes (35-47 µm in diameter) had an irregular shape, a consequence of their active movement in the maternal mesohyl and phagocytosis of amoebocytes (Fig. 3A,B). During vitellogenesis, oocytes increased significantly in size to reach 50-77 µm in diameter. When the yolk started to accumulate, the oocytes amassed near exhalant canals (Fig. 3C,D). At this stage, they were surrounded by a follicle. At the end of oogenesis, the eggs penetrated into the exhalant canals to be released into the seawater column through the aquiferous system.

Reproductive effort

The calculation of monthly mean sexual reproductive effort (sRE) was possible for females only and revealed significant variation over time (Kruskal-Wallis test: χ 2 =24.40, K=24.28, df=8, p=0.002), with a minimum of 0.05% in April 2007 and a maximum of 1.15% in late August 2008 (Fig. 4A). Furthermore, the observed interannual variation is supported by a significant difference between sRE in September 2007 (2.70%) and September 2008 (0.44%) (Mann-Whitney-Wilcoxon: U=3, p=0.009).

The calculation of asexual reproductive effort (aRE) revealed a minimum of 0.21% for March 2008 and a maximum of 1.49% for January 2008 (Fig. 4B). We used the Kruskal-Wallis test to compare median aRE values for January, February and March, which showed that the apparent differences were not significant (χ 2 =3.84, K=3.84, df=2, p=0.147). A Spearman correlation statistic was used to quantify the association between reproductive effort and temperature fluctuations. No significant correlation could be detected between temperature fluctuations and either sRE or aRE (Spearman rank correlation: r=0.130,p=0.843;and r=0,p=1,respectively).

Morphological observations of budding

Buds were mostly concentrated on the central part of the sponge surface (Fig. 1B). They were attached to the maternal apical surface by a short stalk (Fig. 5A), and were of different sizes and shapes due to their asynchronous development. Buds were oval or spherical, their mean surface was 0.5 mm 2 and their diameter ranged 0.5-3 mm (Fig. 5A,B). Their density also differed among specimens.

The beginning of budding was marked by the formation of small, irregular protuberances that emerged from the sponge surface. The buds then grow gradually as their apical region swelled (Fig. 5A,B). Bud skeletons consisted of oxea arranged uniformly at the periphery of the bud and in the mesohyl, but at this early stage of development the bud surface remained smooth. At the histological level, early buds did not possess any particular anatomical organization: there was no developed ectosome or choanosome (Fig. 5C). They consisted of a mass of compact cells, with a higher density of cells in the central part than at the periphery (Fig. 5C). Cells with inclusions (microgranular, granular cells), as well as cells with irregular shapes and long, thin cytoplasmic extensions (lophocytes, archaeocytes), were uniformly distributed throughout the bud tissue.

Just before their detachment, the external morphology and anatomy of mature buds changed. At this stage, buds clearly protruded from the sponge surface. They were oval in shape and had a pleated surface. Their central part slackened due to a significant increase in the number and volume of internal lacunae. Meanwhile, the mesohyl developed with a synthesis of collagen bundles and spicules (Fig. 5D). In contrast with the early stage, many small choanocyte chambers, composed of a few choanocytes and a central cell, were scattered through the inner region of the buds (Fig. 5D).

Cytological composition of the buds

Many choanocytes could be observed in aggregates in the central part of young buds, but at this stage of development, they did not yet form choanocyte chambers (Figs. 5C,6A). These cells were spherical or oval and measured about 3.2 µm in width and 4.3 µm in height. Their nucleus was oval (~1.5 µm in diameter), lacked a nucleolus, but featured prominent heterochromatin (Fig. 6A). In mature buds, choanocyte chambers measured 15-18.5 µm in diameter (Figs. 5D,6B).

The central cells were large amoeboid-like cells that were present inside of each choanocyte chamber (Fig. 6B,C). The central cells were irregular and branched in shape and had numerous cytoplasmic projections. Their nucleus was large (2.3-2.9 µm in diameter), spherical, and without a nucleolus. The cytoplasm of these cells was perforated by one large canal (1.83.5 µm in diameter) and other, smaller, canals (~0.5 µm in diameter) into which entered the flagellum of each choanocytes in the chamber (Fig. 6C).

The buds and the stalk were covered with an exopinacoderm composed of exopinacocytes, which were flat cells (~17.8 µm wide and 5.1 µm high) that harbored an oval anucleolated nucleus (5.02.6 µm in diameter) (Figs. 5C,7A).

Microgranular cells with small inclusions had an amoeboid, slightly irregular shape (~8.54.9 µm), with a nucleus of 1.8 µm in diameter (Fig. 7B). These cells had vacuoles measuring 1-1.5 µm in diameter, with small, electron-dense inclusions. Microgranular cells were more numerous at the periphery of the buds (where they represented ~30% of total cells) than in the central part (where they represented only 8%) (Fig. 5C,D).

Granular cells had an oval or amoeboid shape (~9.5 µm in diameter) and harbored an anucleolated nucleus and large, homogenous electron-dense inclusions which were spherical in shape (1.2-2.7 µm in diameter) (Figs. 5C,7C). These cells were less numerous than the previous cell type.

Archaeocytes were amoeboid or spheroid cells with no particular inclusions (~8.2 µm wide and 4.6 µm high). These cells were also abundant and harbored a large nucleus (2.5 µm in diameter) and a prominent nucleolus (Fig. 7D). Another amoeboid cell was the lophocyte. They were the biggest cells found in the buds (~18 µm wide and 5 µm high), with a large oval nucleus (4.52.5 µm) that may have had a nucleolus (Fig. 7E). The cytoplasm of lophocytes contained different sorts of inclusions such as granules and phagosomes, and these cells presented a developed, rough endoplasmic reticulum.

Sclerocytes were oval or amoeboid cells (~7.0 µm in diameter) with a large vacuole (Fig. 7F). Endopinacocytes were flat cells (~19.6 µm wide and 3.0 µm high) with an oval nucleus (4.22.0 µm) and no nucleolus, and the cytoplasm of these cells included numerous vacuoles (Fig. 7G). These cells formed the canals of the aquiferous system at the late stage of bud development. Collagen fibrils were quite abundant in the central part of the bud where they were sometimes arranged in fine bundles; they were, however, rare at the periphery. Finally, many extracellular symbiotic bacteria were found in the mesohyl (Figs. 6A,B; 7B,F).

Discussion

It is considered that asexual reproduction together with sexual reproduction accompanied multicellular animals throughout their entire evolution. Asexual reproduction in one form or another has repeatedly appeared and disappeared in different groups of Metazoa (Ivanova-Kazas 1977;Adiyodi & Adiyodi 1993). In some animals, such as flatworms, annelids, and holothurians [START_REF] Christensen | Asexual propagation and reproductive strategies in aquatic Oligochaeta[END_REF][START_REF] Franke | Reproduction of the Syllidae (Annelida: Polychaeta)[END_REF][START_REF] Reuter | Flatworm asexual multiplication implicates stem cells and regeneration[END_REF][START_REF] Dolmatov | Asexual Reproduction in Holothurians[END_REF], asexual reproduction is a sporadic process. In other organisms with mixed life history strategies, such as most cnidarians, tunicates, bryozoans and some sponges, asexual reproduction is an obligatory stage of their life cycle [START_REF] Om | Asexual reproduction of animals[END_REF]Adiyodi & Adiyodi 1993;[START_REF] Brusca | Unvertebrates[END_REF]). In the latter case, sexual reproduction can be an adaptive advantage in unstable or unpredictable environments, while asexual reproduction is the competitively superior tactic for colonization of the parental environment (Williams 1975;[START_REF] Smith | Optimization Theory in Evolution[END_REF].

According to the available limited data, budding might promote the maintenance and growth of marine sponge populations [START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF][START_REF] Cardone | The budding process in Tethya citrina Sar. & Melone (Porifera, Demospongiae) and the incidence of post-buds in sponge population maintenance[END_REF][START_REF] Singh | Field and laboratory investigations of budding in the tetillid sponge Cinachyrella cavernosa[END_REF]. Indeed, buds have been shown to contribute significantly to the dispersal and recruitment of new sponges [START_REF] Wulf | Effects of a hurricane on survival and orientation of large erect coral reef sponges[END_REF], in particular under conditions of environmental stress. This strategy thus may improve the survival of individual sponge genotypes, and enhance the growth of a sponge population, as is the case for clonal organisms [START_REF] Jackson | Life cycles and evolution of clonal (modular) animals[END_REF].

Reproductive pattern

Viviparity (brooding) is more widespread and perhaps an ancestral reproductive mode in Porifera [START_REF] Riesgo | Inferring the ancestral sexuality and reproductive condition in sponges (Porifera)[END_REF]. However, oviparity is the general rule in the Demospongiae orders Tetractinellida, Polymastiida, Suberitida, Clionaida, Tethyida, Chondrosida, Verongida, Agelasida, Biemnida, and Axinellida [START_REF] Ereskovsky | The comparative embryology of sponges[END_REF]. Viviparous representatives are found in some these orders, however; these representatives include, for example, the viviparous genera Alectona and Thoosa (order Suberitida), genus Stylocordyla (order Suberitida), and genus Halisarca (order Chondrosida) [START_REF] Vacelet | Planktonic armoured propagules of the excavating sponge Alectona (Porifera: Demospongiae) are larvae: evidence from Alectona wallichii and A. mesatlantica sp[END_REF][START_REF] Sarà | Viviparous development in the Antarctic sponge Stylocordyla borealis Loven, 1868[END_REF][START_REF] Bautista-Guerrero | Reproductive cycle of the coralexcavating sponge Thoosa mismalolli (Clionaidae) from Mexican Pacific coral reefs[END_REF][START_REF] Ereskovsky | New data on embryonic development of Halisarca dujardini Johnston, 1842 (Demospongiae: Halisarcida)[END_REF]. It is largely accepted today that these two reproductive traits do not have any phylogenetic value and cannot be used for taxonomical revision of Porifera [START_REF] Hoppe | Reproductive patterns in three species of large coral reef sponges[END_REF][START_REF] Van Soest | Demosponge higher taxa classification re-examined[END_REF][START_REF] Ereskovsky | The comparative embryology of sponges[END_REF][START_REF] Riesgo | Inferring the ancestral sexuality and reproductive condition in sponges (Porifera)[END_REF].

The studies by Fromont (1994a, b) and [START_REF] Fromont | Reproductive biology of three sponges species of the genus Xestospongia (Porifera: Demospongiae: Petrosiida) from the Great Barrier Reef[END_REF] led to the general acceptance that all Haplosclerida families are characterized by viviparity, with the only exception being the oviparity in some Petrosiidae (Table 2). Although a good number of representatives of Chalinidae, especially in the Haliclona genus, have been shown to be viviparous, the present work demonstrates surprisingly that the Mediterranean Haliclona fulva is oviparous.

Another feature of the life cycle of H. fulva that is unusual in Haplosclerida is that this species includes budding as an obligatory phase of asexual reproduction, alternating with a sexual one. This case once more supports the observations that budding in Demospongiae is correlated with oviparity [START_REF] Fell | Porifera. In: Asexual propagation and reproductive strategies[END_REF][START_REF] Ereskovsky | The comparative embryology of sponges[END_REF]). Up to now there are only two exceptions to this rule: the viviparous demosponges Mycale (Aegogropila) contarenii (LIEBERKÜHN 1859) (Poecilosclerida) (Corrierro et al. 1998) and Radiospongilla cerebellata (BOWERBANK 1863) (Spongillida) [START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF].

While different modes of reproduction often occur within the same genus in various marine metazoans, for instance in Echinodermata and Bivalvia [START_REF] Strathmann | The evolution and loss of feeding larvae stages of marine invertebrates. Study of larval settlement in the sea[END_REF][START_REF] Kasyanov | Reproductive Strategy of Marine Bivalves and Echinoderms[END_REF][START_REF] Byrne | Reproduction and Larval Morphology of Broadcasting and Viviparous Species in the Cryptasterina Species Complex[END_REF], in sponges this phenomenon has previously been observed only in two genera of Petrosiidae (Haplosclerida). In the genus Xestospongia, X. bergquistia FROMONT 1991, X. testudinaria (WILSON 1925), and X. muta (SCHMIDT 1870) are oviparous, releasing sperm and eggs, while X. bocatorensis DIAZ, THACKER, RÜTZLER & PIANTONI, 2007 is viviparous, releasing brooded larvae [START_REF] Fromont | Reproductive biology of three sponges species of the genus Xestospongia (Porifera: Demospongiae: Petrosiida) from the Great Barrier Reef[END_REF][START_REF] Becerro | Spawning of the giant barrel sponge Xestospongia muta in Belize[END_REF][START_REF] Collin | Phototactic responses of larvae from the marine sponges Neopetrosia proxima and Xestospongia bocatorensis (Haplosclerida: Petrosiidae)[END_REF]). In the genus Neopetrosia, N. exigua (KIRKPATRICK 1900) is oviparous, while N. proxima (DUCHASSAING & MICHELOTTI 1864) is viviparous [START_REF] Fromont | Reproductive biology of three sponges species of the genus Xestospongia (Porifera: Demospongiae: Petrosiida) from the Great Barrier Reef[END_REF][START_REF] Collin | Phototactic responses of larvae from the marine sponges Neopetrosia proxima and Xestospongia bocatorensis (Haplosclerida: Petrosiidae)[END_REF]).

Another explanation for the phenomenon of oviparity in Haliclona fulva and different reproductive patterns in Xestospongia and Neopetrosia follows from the systematics of the order Haplosclerida. The phylogenetic relationships among haplosclerids are not clear yet, and suggestions of the polyphyletic nature of the various taxa within this order have appeared in various publications [START_REF] Hill | Reconstruction of Family-Level Phylogenetic Relationships within Demospongiae (Porifera) Using Nuclear Encoded Housekeeping Genes[END_REF][START_REF] Mccormack | Major discrepancy between phylogenetic hypotheses based on molecular and morphological criteria within the Order Haplosclerida (Phylum Porifera: Class Demospongiae)[END_REF][START_REF] Raleigh | Mitochondrial cytochrome oxidase 1 phylogeny supports alternative taxonomic scheme for marine Haplosclerida[END_REF]Redmond et al. 2007;[START_REF] Redmond | Phylogenetic relationships of the marine Haplosclerida (Phylum Porifera) employing ribosomal (28S rRNA) and mitochondrial (cox1, nad1) gene sequence data[END_REF][START_REF] Redmond | Phylogeny and Systematics of Demospongiae in Light of New Small Subunit Ribosomal DNA (18S) Sequences[END_REF]. It is important that the biggest genus Haliclona is distributed across the order Haplosclerida. [START_REF] Redmond | Phylogeny and Systematics of Demospongiae in Light of New Small Subunit Ribosomal DNA (18S) Sequences[END_REF] showed that members of this genus are positioned within Clade C with Niphatidae and Petrosiidae. The latter family, as mentioned above, includes both oviparous and viviparous species, particularly the viviparous Neopetrosia proxima [START_REF] Collin | Phototactic responses of larvae from the marine sponges Neopetrosia proxima and Xestospongia bocatorensis (Haplosclerida: Petrosiidae)[END_REF]) in Clade C. Species of Haliclona are also positioned within Clade B and has high support for a sister relationship with the viviparous Xestospongia bocatorensis [START_REF] Collin | Phototactic responses of larvae from the marine sponges Neopetrosia proxima and Xestospongia bocatorensis (Haplosclerida: Petrosiidae)[END_REF]). It should be noted that this Clade B also includes the oviparous X. muta. According to the results of [START_REF] Redmond | Phylogenetic relationships of the marine Haplosclerida (Phylum Porifera) employing ribosomal (28S rRNA) and mitochondrial (cox1, nad1) gene sequence data[END_REF], H. fulva is located in the cluster including the oviparous Petrosia ficiformis (POIRET 1789) and three other Petrosia species, as well as H. mucosa (GRIESSINGER 1971) and Cribrochalina vascuum (LAMARCK 1814). Therefore, it is possible that H. fulva might be misplaced in the genus Haliclona, and relationships with other oviparous clades might be the key to understanding reproductive behavior in H. fulva.

The general pattern of H. fulva oogenesis is similar to that reported for other oviparous Haplosclerida: no degeneration of mesohyl is observed during oogenesis and the majority of the eggs that develop to maturity retain an ovoid shape [START_REF] Fromont | Aspects of the reproductive biology of Xestospongia testudinaria (Great Barrier Reef)[END_REF][START_REF] Lepore | The ultrastructure of the mature oocyte and the nurse cells of the Ceractinomorpha Petrosia ficiformis[END_REF][START_REF] Maldonado | Gametogenesis, embryogenesis, and larval features of the oviparous sponge Petrosia ficiformis (Haplosclerida, Demospongiae)[END_REF]. During vitellogenesis, the oocytes have an amoeboid shape and participate in the phagocytosis of somatic cells, rich of phagosomes, like other investigated Haplosclerida [START_REF] Ereskovsky | The comparative embryology of sponges[END_REF].

It is always a challenge to describe the reproductive cycle of oviparous sponges, but it is even more difficult when the males are hidden. Despite the large number of specimens of H. fulva investigated, no spermatocysts were observed. A very low ratio of male : female individuals has been reported for many marine sponges [START_REF] Hogg | Approaches to the systematics of the Demospongiae[END_REF][START_REF] Scalera Liaci | Osservazioni sui cicli sessuali di alcune Keratosa (Porifera) e loro interesse negli studi filogenetici[END_REF][START_REF] Ayling | Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae[END_REF][START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF][START_REF] Mercurio | A 3-year investigation of sexual reproduction in Geodia cydonium (Jameson 1811) (Porifera, Demospongiae) from a semi-enclosed Mediterranean bay[END_REF][START_REF] Ereskovsky | Pluriannual study of the reproduction of two Mediterranean Oscarella species (Porifera, Homoscleromorpha): cycle, sex-ratio, reproductive effort and phenology[END_REF]. For example, after two years of fortnightly monitoring, [START_REF] Ayling | Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae[END_REF] found males of Aaptos aaptos (SCHMIDT 1864) in only one year of the study. No males were found in Mediterranean oviparous sponges Tethya citrina SARA &MELONE 1965 andT. auranrium (PALLAS 1766) during monthly collection over an 18-month period [START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF]. In a striking parallel to our study of H. fulva, the same author was unable to detect a single male among populations of Raspailia topsenti DENDY 1924 and Polymiastia sp. over a 2-year monitoring period [START_REF] Ayling | Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae[END_REF]. [START_REF] Giesel | Sex ratio, rate of evolution and environmental heterogeneity[END_REF] proposed that deviations in sex ratio may internally regulate the size of a population by affecting its reproductive potential. In brooding corals, for example, female biased sex ratios may be an evolutionary adaptation caused by the physical limitations of the incubation chamber inside of polyps [START_REF] Szmant | Reproductive ecology of Caribbean reef corals[END_REF]. [START_REF] Whalan | Sexual reproduction of the brooding sponge Rhopaloeides odorabile[END_REF] discussed some possible problems that may lead to biased sex ratios in sessile or slow moving marine invertebrates: proximity to mates [START_REF] Sewell | Fertilisation success in a natural spawning of the dendrochirote sea cucumber Cucumaria miniata[END_REF][START_REF] Babcock | Fertilisation biology of the abalone Haliotis laevigata: laboratory and Weld studies[END_REF], sperm limitation [START_REF] Brazeau | Reproductive success in the Caribbean octocoral Briareum absestinum[END_REF], and dilution of gametes [START_REF] Oliver | Aspects of fertilisation ecology of broadcast spawning corals: sperm dilution effects and in situ measurements of fertilisation[END_REF]. Other possible factors that could influence the sex ratio are temperature, salinity, and the quantity and quality of food available [START_REF] Simonini | Effects of temperature on two Mediterranean populations of Dinophilus gyrociliatus (Polychaeta: Dinophilidae). I. Effects on life history and sex ratio[END_REF].

In this study of H. fulva, we remain perplexed as to where the males are. One explanation for the absence of males could be that the species has a very short period of spermatogenesis. In demosponges the period of spermatogenesis often is shorter compared to oogenesis [START_REF] Ayling | Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae[END_REF][START_REF] Diaz | Variations, differentiations et functions des categories cellulaires de la demosponge d'eaux saumatres, Suberites massa, Nardo, au cours[END_REF][START_REF] Ereskovsky | Reproduction cycles and strategies of cold-water sponges Halisarca dujardini (Demospongiae, Dendroceratida), Myxilla incrustans and Iophon piceus (Demospongiae, Poecilosclerida) from the White Sea[END_REF][START_REF] Riesgo | Differences in reproductive timing among sponges sharing habitat and thermal regime[END_REF][START_REF] Mercurio | A 3-year investigation of sexual reproduction in Geodia cydonium (Jameson 1811) (Porifera, Demospongiae) from a semi-enclosed Mediterranean bay[END_REF][START_REF] Whalan | Sexual reproduction of the brooding sponge Rhopaloeides odorabile[END_REF][START_REF] Ereskovsky | Pluriannual study of the reproduction of two Mediterranean Oscarella species (Porifera, Homoscleromorpha): cycle, sex-ratio, reproductive effort and phenology[END_REF][START_REF] Stephens | Reproductive cycle and larval characteristics of the sponge Haliclona indistincta (Porifera: Demospongiae)[END_REF]). This trend is more pronounced in oviparous sponges, particularly in oviparous Haplosclerida. For example, in Xestospongia bergquistia and X. testudinaria from the Great Barrier Reef, the period of oogenesis lasts more than 5 months, while spermatogenesis lasts less than 5 days [START_REF] Fromont | Reproductive biology of three sponges species of the genus Xestospongia (Porifera: Demospongiae: Petrosiida) from the Great Barrier Reef[END_REF]. In Mediterranean Petrosia ficiformis, oogenesis duration is 7-8 months, and the duration of spermatogenesis is only 2-2.3 weeks [START_REF] Scalera Liaci | Raffronto tra il comportamento sussuale di alcune Ceractinomorpha[END_REF][START_REF] Maldonado | Gametogenesis, embryogenesis, and larval features of the oviparous sponge Petrosia ficiformis (Haplosclerida, Demospongiae)[END_REF]. [START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF] proposed that a very short period of spermatogenesis accounted for the absence of males during monthly collections of Tethya citrina, T. auranrium, and Mycale contarenii over 18 months of monitoring. Thus, it's possible to assume that the nine individuals of Haliclona fulva that lacked any reproductive elements (of the 37 individuals observed during the period of oogenesis) could be males with a short spermatogenesis period.

Reproductive cycle and effort

Numerous external factors could be responsible for induction of sexual or asexual reproduction in individuals within a population of heterogonic invertebrate species. Among the more important are the physicochemical quality of water, hydrodynamic, food availability, population density, habitat stability, and seasonal variation in temperature (for review see: Adiyodi & Adiyodi 1993). Nevertheless, it seems that water temperature and food availability play the principal roles in this process in the case of various marine invertebrates, such as sea anemones [START_REF] Bucklin | Growth and asexual reproduction of the sea anemone Metridium: comparative laboratory studies of three species[END_REF], scyphozoans [START_REF] Lucas | Jellyfish life histories: role of polyps in forming and maintaining scyphomedusa populations[END_REF][START_REF] Purcell | Temperature effects on asexual reproduction rates of scyphozoan species from the northwest Mediterranean Sea[END_REF], echinoderms [START_REF] Lawrence | Stress and deviant reproduction in Echinoderms[END_REF], and bryozoans [START_REF]Asexual propagation in the marine bryozoan Cupuladria exfragminis[END_REF]. In sponges, the increase in the frequency of specimens with buds and in the number of buds per sponge happens concomitantly with rapid decreases in water temperature as, for example, in the Mediterranean species Tethya citrina T. aurantium, Mycale contarenii [START_REF] Connes | Etude histologique, cytologique et expérimentale de la régénération et de la reproduction asexuée chez Tethya lyncurium Lamarck (= Tethya aurantium (Pallas) (Demosponges)[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF][START_REF] Cardone | The budding process in Tethya citrina Sar. & Melone (Porifera, Demospongiae) and the incidence of post-buds in sponge population maintenance[END_REF] and in White Sea species Polymastia arctica (MEREJKOWSKY 1878) [START_REF] Plotkin | Ecological aspects of asexual reproduction of the White Sea Sponge Polymastia mammillaris (Demospongiae, Tetractinomorpha) in Kandalaksha Bay[END_REF]. In H. fulva, budding takes place from late November to early March, which is the coldest season. This is also the case for the two Mediterranean Tethya citrina and T. aurantium [START_REF] Connes | Etude histologique, cytologique et expérimentale de la régénération et de la reproduction asexuée chez Tethya lyncurium Lamarck (= Tethya aurantium (Pallas) (Demosponges)[END_REF][START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF][START_REF] Gaino | Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)[END_REF] and Mycale contarenii [START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF]). However we were unable to demonstrate a significant correlation between the natural variations of seawater temperature and asexual reproductive effort.

Reproductive effort is an integrative indicator of resource allocation to reproductive compartments, and it can vary greatly depending on the reproductive strategy. Haliclona fulva allocates energy to the differentiation of gametes or buds at very different times, and as in other marine sponges, there is a clear alternation of sexual and asexual phases of reproduction [START_REF] Fell | Postlarval reproduction and reproductive strategy in Haliclona loosanoffi and Halichondria sp[END_REF][START_REF] Corriero | Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF]. Both reproductive efforts are of roughly the same intensity, with oogenesis representing 0.7% on average over the period, and budding representing 0.9% H. fulva demonstrates low annual variability of both reproductive processes, whereas marine organisms can exhibit highly variable sexual or asexual reproductive efforts which are sometimes related to the natural variations of the sea water temperature (Adiyodi & Adiyodi 1993;[START_REF] Olive | Annual breeding cycles in marine invertebrates and environmental temperature: probing the proximate and ultimate causes of reproductive synchrony[END_REF][START_REF] Llodra | Fecundity and Life-history Strategies in Marine Invertebrates[END_REF]. Overall, biotic and abiotic factors that regulate the proportion of asexual and sexual reproduction in the life cycle of heterogonic species are poorly understood. Unfortunately, in the case of H. fulva we were unable to fill this knowledge gap.

Although our sampling strategy has proven its efficiency in a number of previous studies of sponge life cycles (see for instance [START_REF] Pérez | Oscarella balibaloi, a new sponge species (Homoscleromorpha: Plakinidae) from the Western Mediterranean Sea: cytological description, reproductive cycle and ecology[END_REF]Ivanisevic et al. 2011a, b;[START_REF] Ereskovsky | Pluriannual study of the reproduction of two Mediterranean Oscarella species (Porifera, Homoscleromorpha): cycle, sex-ratio, reproductive effort and phenology[END_REF][START_REF] Zarrouk | Sexual Reproduction of Hippospongia communis (Lamarck, 1814) (Dictyoceratida, Demospongiae): comparison of two populations living under contrasted environmental conditions[END_REF]Reveter et al. 2016), we are here confronted with a limitation with this strategy for H. fulva.

Bud formation, structure and cell composition

The development of buds differs among sponge species. For example, the formation of the aquiferous system can occur either during bud development [START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF][START_REF] Ereskovsky | Asexual reproduction of homoscleromorph sponges (Porifera; Homoscleromorpha)[END_REF][START_REF] Gaino | Choanocyte chambers in unreleased buds of Tethya seychellensis (Wright, 1881) (Porifera, Demospongiae)[END_REF]this work), or after detachment from the parental sponge [START_REF] Ayling | Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae[END_REF][START_REF] Battershill | The influence of storms on asexual reproduction, recruitment, and survivorship of sponges[END_REF][START_REF] Gaino | Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)[END_REF].

Haliclona fulva presents three stages in its bud formation: an ectosomal protuberance caused by cell migration; growth of the apical part of the bud with formation of the stalk connected to the parental sponge; and thin-stalked buds protruding noticeably from the sponge surface, followed by detachment of the bud. These stages are very similar to what was described in Tethya aurantium [START_REF] Connes | Etude histologique, cytologique et expérimentale de la régénération et de la reproduction asexuée chez Tethya lyncurium Lamarck (= Tethya aurantium (Pallas) (Demosponges)[END_REF][START_REF] Gaino | Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)[END_REF], Radiospongilla cerebellata [START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF], T. seychellensis (WRIGHT 1881) [START_REF] Gaino | Choanocyte chambers in unreleased buds of Tethya seychellensis (Wright, 1881) (Porifera, Demospongiae)[END_REF], and in Cinachyrella cavernosa (LAMARCK 1815) [START_REF] Singh | Field and laboratory investigations of budding in the tetillid sponge Cinachyrella cavernosa[END_REF].

Cells in buds are amoeboid-like in shape and show cytoplasmic extensions. They tend to align either in parallel rows or along the spicule bundles. These features suggest that these cells may be able to migrate from the parental sponge to the newly formed buds. The migration of specialized and polypotent cells, and their subsequent differentiation into definitive cells, is a typical feature of the budding process (Ereskovsky 2003[START_REF] Ereskovsky | The comparative embryology of sponges[END_REF][START_REF] Gaino | Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)[END_REF]. In this work we have shown that unreleased buds of H. fulva possess choanocyte chambers. The occurrence of choanocyte chambers is very uncommon in demosponge buds. Until now, these structures have been observed in unreleased buds of only four demosponge species: in Mycale contarenii [START_REF] Devos | Le bourgeonnement externe de l'éponge Mycale contarenii[END_REF][START_REF] Corriero | Reproductive strategies of Mycale contarenii (Porifera: Demospongiae)[END_REF], in the freshwater sponge Radiospongilla cerebellata [START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF], in Tethya seychellensis [START_REF] Gaino | Choanocyte chambers in unreleased buds of Tethya seychellensis (Wright, 1881) (Porifera, Demospongiae)[END_REF], and in T. wilhelma SARA, SARA, NICKEL & BRÜMMER 2001 [START_REF] Hammel | Sponge budding is a spatiotemporal morphological patterning process: insights from synchrotron radiation-based xray microtomography into the asexual reproduction of Tethya wilhelma[END_REF], as well as in homoscleromorph sponges of the genus Oscarella [START_REF] Ereskovsky | Asexual reproduction of homoscleromorph sponges (Porifera; Homoscleromorpha)[END_REF].

Cell composition in demosponge buds has been poorly studied. Optic microscopy has provided only two good descriptions of bud cell composition in Mycale contarenii and Axinella damicornis (ESPER 1794) [START_REF] Devos | Le bourgeonnement externe de l'éponge Mycale contarenii[END_REF][START_REF] Boury-Esnault | Un phénomène de bourgeonnement externe chez l'éponge Axinella damicornis (Exper.)[END_REF], and electron microscopy has illustrated this condition in Tethya aurantium, T. citrina, T. seyshellensis, Radiospongilla cerebellata, and Cinachyrella australiensis (CARTER 1886) [START_REF] Connes | Structure et développement des bourgeons chez l'éponge siliceuse Tethya lyncurium Lamarck[END_REF][START_REF] Connes | Etude histologique, cytologique et expérimentale de la régénération et de la reproduction asexuée chez Tethya lyncurium Lamarck (= Tethya aurantium (Pallas) (Demosponges)[END_REF][START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF][START_REF] Chen | Budding cycle and bud morphology of the globeshaped sponge Cinachyra australiensis[END_REF][START_REF] Gaino | Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae)[END_REF][START_REF] Gaino | Choanocyte chambers in unreleased buds of Tethya seychellensis (Wright, 1881) (Porifera, Demospongiae)[END_REF]. According to these descriptions, it seems that cells with inclusions represent the main cellular components of buds, and that archaeocytes are the second most abundant cell type observed. In A. damicornis, M. contarenii, and R. cerebellata, all cell types of the parental sponge are present in equal proportions in the buds at their later developmental stages [START_REF] Devos | Le bourgeonnement externe de l'éponge Mycale contarenii[END_REF][START_REF] Boury-Esnault | Un phénomène de bourgeonnement externe chez l'éponge Axinella damicornis (Exper.)[END_REF][START_REF] Saller | Formation and construction of asexual buds of the fresh-water sponge Radiospongilla cerebellata (Porifera, Spongillidae)[END_REF]. In this respect, the buds of H. fulva do not differ from these sponges, as they are composed of microgranular cells, granular cells, archaeocytes, endopinacocytes (and exopinacocytes), choanocytes, central cells, and sclerocytes. On average, all these cells are similar in size to the cells of the parental sponge. Further research is needed to determine whether cell inclusions represent stored material useful in sustaining morphogenetic processes [START_REF] Connes | Structure et développement des bourgeons chez l'éponge siliceuse Tethya lyncurium Lamarck[END_REF], and whether they might thus be pivotal for the acquisition of complete functionality. Endopinacocyte. Scale: A-D,F,G=5 µm; E=10 µm. af, axial filament of a spicule; ar, archaeocytes; ch, choanocyte; en, endopinacocyte; ex, exopinacocytes; g, granule; lo, lophocyte; mgc, microgranular cells; n, nucleus; nu, nucleolus; sb, symbiotic bacteria; sc, sclerocytes; ss, spongin of spicule; v, vacuole. Table 1

Figure legends

Fig. 1 .

 1 Fig. 1. Specimen of Haliclona fulva, in situ, (A) without buds, in June 2008, and (B) with buds, in February 2008. Scale bars=30 mm. b, buds; o, oscula.

Fig. 2 .

 2 Fig. 2. Diagram of the reproductive cycle of Haliclona fulva.

Fig. 3 .

 3 Fig. 3. Oogenesis in Haliclona fulva, histological section (Masson-Goldner's trichrome hematoxylin staining). A. The mesohyl with oocytes at previtellogenic stage. B. Previtellogenic oocyte phagocyting the cells (arrowhead). C. The mesohyl with the eggs. D. Egg of H. fulva. Scale: A=50 µm; B,D=20 µm; C=100 µm. cc, choanocyte chambers; eg, egg; exc, exhalant canal; gc, granular cell; n, nucleus; oo, oocytes; s, spicules.

Fig. 4 .

 4 Fig. 4. Reproductive efforts of Haliclona fulva. A. Boxplot distribution of reproductive efforts during oogenesis and corresponding water temperatures during the study. B. Boxplot distribution of reproductive efforts during budding.

Fig. 5 .

 5 Fig. 5. Buds of Haliclona fulva. A. Buds in vivo at different stages of development. B. Buds in vivo at last stages of development. C. Semithin section of early bud. D. Semithin section of the bud at last stage. Inset: TEM micrograph of collagen bundles in the mesohyl of a bud. Scale: A=5 mm, B=1.5 mm, C=100 µm, D=150 µm, Inset=4 µm. b, buds; cb, collagen bundles; cc, choanocyte chambers; ch, choanocytes; ep, exopinacocytes; gc, granular cells; l, lacuna; mgc, microgranular cells; o, osculum; sb, symbiotic bacteria; ss, spongin of spicules.

Fig. 6 .

 6 Fig. 6. TEM images of the choanocytes in buds from individuals of Haliclona fulva. A. The aggregate of separated choanocytes in early bud. B. Choanocyte chamber with central cell in the bud at last stage. C. Central cell inside of a choanocyte chamber. Scale: A,B=5 µm; C=2 µm. c, canal in a central cell cytoplasm; ce, central cell; ch, choanocyte; f, flagella of choanocytes; mv, microvilli; n, nucleus; sb, symbiotic bacteria.

Fig. 7 .

 7 Fig. 7.TEM images of the cells in buds from individuals of Haliclona fulva. A. Exopinacocytes. B. Microgranular cells. C. Granular cell. D. Archaeocytes. E. Lophocyte. F. Sclerocytes. G.Endopinacocyte. Scale: A-D,F,G=5 µm; E=10 µm. af, axial filament of a spicule; ar, archaeocytes; ch, choanocyte; en, endopinacocyte; ex, exopinacocytes; g, granule; lo, lophocyte; mgc, microgranular cells; n, nucleus; nu, nucleolus; sb, symbiotic bacteria; sc, sclerocytes; ss, spongin of spicule; v, vacuole.

Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Fig. 7

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Table legend Table 1 .

 legend1 The number of individuals of Haliclona fulva collected during the sampling period for histological investigation.

Acknowledgments. This work has been supported by grant n° 1.38.209.2014 from Saint-Petersburg State University, and the Russian Science Foundation #17-14-01089 (for the microscopy). The authors gratefully thank Joël Courageot and Alexandre Altié of Service Commun de Microscopie Électronique et Photographie Faculté de Médecine La Timone, Aix-Marseille Université and Daria Tokina for technical support.