Bastien Dalzon
email: bastien.dalzon@gmail.com

Joanna Bons
email: joanna.bons@etu.unistra.fr

Hélène Diemer
email: hdiemer@unistra.fr

Véronique Collin-Faure
email: veronique.collin@cea.fr

Caroline Marie-Desvergne
email: caroline.desvergne@cea.fr

Muriel Dubosson
email: muriel.dubosson@cea.fr

Sarah Cianferani
email: sarah.cianferani@unistra.fr

Christine Carapito
email: ccarapito@unistra.fr

Thierry Rabilloud
email: thierry.rabilloud@cea.fr

A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes

Keywords: anticancer copper complex, hydroxyquinoline copper complex, leukemic cells, proteomics, two-dimensional electrophoresis, proteasome, glutathione, actin cytoskeleton, antioxidant defense

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.

Introduction

Metal-containing drugs, i.e., metal-organic complexes, also known as metallodrugs, have been used to combat cancer for quite a long time. The oldest metal-based drugs in this field are clearly platinum complexes [START_REF] Rosenberg | Platinum coordination complexes in cancer chemotherapy[END_REF], which act mainly by inducing severe DNA damage [START_REF] Ho | Platinum-based anticancer agents: Innovative design strategies and biological perspectives[END_REF][START_REF] Dilruba | Platinum-based drugs: Past, present and future[END_REF]. Because of the side effects of these drugs and of the emergence of resistances, other metal complexes have been investigated as anticancer agents. Various metals such as gold [START_REF] Milacic | A novel anticancer gold(III) dithiocarbamate compound inhibits the activity of a purified 20S proteasome and 26S proteasome in human breast cancer cell cultures and xenografts[END_REF][START_REF] Arsenijevic | Cytotoxicity of gold(III) complexes on A549 human lung carcinoma epithelial cell line[END_REF], copper [START_REF] Daniel | Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells[END_REF][START_REF] Chen | New uses for old copper-binding drugs: Converting the pro-angiogenic copper to a specific cancer cell death inducer[END_REF], zinc [START_REF] Brar | Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease[END_REF] or ruthenium [START_REF] Sava | Na[trans-RuCl4(DMSO)Im], a metal complex of ruthenium with antimetastatic properties[END_REF][START_REF] Hartinger | From bench to bedside-Preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A)[END_REF][START_REF] Gianferrara | Synthetic strategies towards ruthenium-porphyrin conjugates for anticancer activity[END_REF] have been investigated (reviewed in [START_REF] Schmitt | New applications of old metal-binding drugs in the treatment of human cancer[END_REF]). Various mechanisms of actions have been proposed for these non-platinum metallodrugs. For ruthenium complexes, their binding to proteins have been extensively studied, they suggest that different ruthenium metallodrugs bind to (and may inhibit) different proteins [START_REF] Merlino | Interactions between proteins and Ru compounds of medicinal interest: A structural perspective[END_REF].

Taking gold complexes as another example, the suggested mechanisms range from DNA lesions [START_REF] Ronconi | Gold(III) dithiocarbamate derivatives for the treatment of cancer: Solution chemistry, DNA binding, and hemolytic properties[END_REF] to mitochondrial stress [START_REF] Chiara | Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3alpha/beta and Bax, leading to permeability transition pore opening and tumor cell death[END_REF] and to proteasome inhibition [START_REF] Milacic | A novel anticancer gold(III) dithiocarbamate compound inhibits the activity of a purified 20S proteasome and 26S proteasome in human breast cancer cell cultures and xenografts[END_REF]. The facts that proteasome inhibition is a well-established avenue in anticancer treatments (reviewed in [START_REF] Manasanch | Proteasome inhibitors in cancer therapy[END_REF]), and that metals are known inhibitors of the proteasome [START_REF] Kim | Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis[END_REF][START_REF] Xiao | Molecular study on copper-mediated tumor proteasome inhibition and cell death[END_REF][START_REF] Verani | Metal complexes as inhibitors of the 26S proteasome in tumor cells[END_REF], have paved the way to thorough investigations of this effect in metallodrugs [START_REF] Milacic | A novel anticancer gold(III) dithiocarbamate compound inhibits the activity of a purified 20S proteasome and 26S proteasome in human breast cancer cell cultures and xenografts[END_REF][START_REF] Daniel | Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells[END_REF][START_REF] Kim | Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis[END_REF][START_REF] Daniel | Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells[END_REF][START_REF] Chen | Clioquinol, a therapeutic agent for Alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts[END_REF][START_REF] Daniel | Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer[END_REF][START_REF] Milacic | Novel 8-hydroxylquinoline analogs induce copper-dependent proteasome inhibition and cell death in human breast cancer cells[END_REF][START_REF] Zhai | Tumor cellular proteasome inhibition and growth suppression by 8-hydroxyquinoline and clioquinol requires their capabilities to bind copper and transport copper into cells[END_REF][START_REF] Schimmer | Clioquinol-A Novel Copper-Dependent and Independent Proteasome Inhibitor[END_REF][START_REF] Oliveri | Amino-and chloro-8-hydroxyquinolines and their copper complexes as proteasome inhibitors and antiproliferative agents[END_REF][START_REF] Zhang | Novel copper complexes as potential proteasome inhibitors for cancer treatment[END_REF]. Nevertheless, other mechanisms have been put forward to explain the anticancer activities of the metallodrugs, including direct induction of apoptosis [START_REF] Chiara | Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3alpha/beta and Bax, leading to permeability transition pore opening and tumor cell death[END_REF], topoisomerase inhibition [START_REF] Ikotun | Pyrophosphate-bridged complexes with picomolar toxicity[END_REF] or redox imbalance [START_REF] Chen | Synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and MAPK pathway activation[END_REF][START_REF] Jungwirth | Anticancer Activity of Metal Complexes: Involvement of Redox Processes[END_REF].

One of the attractive features of the metallodrugs proposed to date is that they often rely on organic moieties that are established drugs by themselves in other medical fields, so that their toxicology is already well known. As an example, disulfiram, which has been proposed against cancer as a copper [START_REF] Allensworth | Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer[END_REF] or zinc [START_REF] Brar | Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease[END_REF] complex is also well known for treatment against alcoholism [START_REF] Wright | Disulfiram treatment of alcoholism[END_REF]. Clioquinol, a copper complex which has also been proposed against cancer [START_REF] Daniel | Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells[END_REF][START_REF] Chen | Clioquinol, a therapeutic agent for Alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts[END_REF][START_REF] Schimmer | Clioquinol-A Novel Copper-Dependent and Independent Proteasome Inhibitor[END_REF], was first used alone as an antiparasitic drug [START_REF] Gholz | Prophylaxis and Therapy of Amebiasis and Shigellosis with Iodochlorhydroxyquin[END_REF][START_REF] Rohde | Hydroxyquinolines inhibit ribonucleic acid-dependent deoxyribonucleic acid polymerase and inactivate Rous sarcoma virus and herpes simplex virus[END_REF].

Besides these simple metal-drugs complexes, other research avenues in metallodrugs have focused on more elaborate chemistries, in which a metal binding moiety is covalently grafted onto drug-related organic molecules. The use of ferrocene as the metal component, grafted onto various prodrugs, has been proposed in several approaches [START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF][START_REF] Patra | The medicinal chemistry of ferrocene and its derivatives[END_REF][START_REF] Kowalski | Insight into the Biological Activity of Organometallic Acetylsalicylic Acid (Aspirin) Derivatives[END_REF]. In addition to iron, gold and silver metal carbene complexes have been proposed as metallodrugs [START_REF] Liu | Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs[END_REF].

Knowing as much as possible about the actions of these metallodrugs on cancer cells is not just an academic exercise; it may impact their future use as drugs in clinics, which is still in progress [START_REF] Wehbe | Development of a copper-clioquinol formulation suitable for intravenous use[END_REF]. For example, antioxidant capacity has been proposed as a mechanism for cancer cells to resist to proteasome inhibitors [START_REF] Weniger | Treatment-induced oxidative stress and cellular antioxidant capacity determine response to bortezomib in mantle cell lymphoma[END_REF]. Thus, if a metallodrug is able to inhibit proteasome and antioxidant defenses at the same time, it may overcome classical cell resistances. Furthermore, it has been demonstrated on ferrocene-based metallodrugs that different organic moieties coupled to the same metal-containing moiety lead to different biological consequences [START_REF] Zanellato | Antiproliferative effect of ferrocifen drug candidates on malignant pleural mesothelioma cell lines[END_REF].

When multiple mechanisms of action of a chemical on cells are suspected, omics approaches are very relevant and interesting, as they do not need pre-conceived hypotheses other than the homeostatic principles [START_REF] Rabilloud | Proteomics in mechanistic toxicology: History, concepts, achievements, caveats, and potential[END_REF]. It is therefore not surprising that such approaches have been used to increase our knowledge on the mechanisms of actions of anticancer metallodrugs, as exemplified by work carried out on ruthenium [START_REF] Guidi | The molecular mechanisms of antimetastatic ruthenium compounds explored through DIGE proteomics[END_REF] and gold complexes [START_REF] Gamberi | Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipy(c)[END_REF], leading to the discovery of new modes of action for these chemicals.

We therefore decided to use a proteomic approach to further investigate the effects of copper complexes on leukemic cells, as they are attractive therapeutical targets [START_REF] Mao | Clioquinol inhibits the proteasome and displays preclinical activity in leukemia and myeloma[END_REF]. As a cellular model, we have used the RAW 264.7 cell line, which is a myeloid leukemic mouse cell line. As the copper complex, we have used a complex of copper with 8-hydroxyquinoline. This organic compound shows an interesting pharmacology [START_REF] Prachayasittikul | 8-Hydroxyquinolines: A review of their metal chelating properties and medicinal applications[END_REF], including a noticeable anticancer activity on cells [START_REF] Daniel | Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells[END_REF][START_REF] Zhai | Tumor cellular proteasome inhibition and growth suppression by 8-hydroxyquinoline and clioquinol requires their capabilities to bind copper and transport copper into cells[END_REF], and is the parent compound for several metallo-prodrugs currently under investigation [START_REF] Milacic | Novel 8-hydroxylquinoline analogs induce copper-dependent proteasome inhibition and cell death in human breast cancer cells[END_REF][START_REF] Oliveri | Amino-and chloro-8-hydroxyquinolines and their copper complexes as proteasome inhibitors and antiproliferative agents[END_REF][START_REF] Chen | Synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and MAPK pathway activation[END_REF][START_REF] Tardito | Copper-Dependent Cytotoxicity of 8-Hydroxyquinoline Derivatives Correlates with Their Hydrophobicity and Does Not Require Caspase Activation[END_REF] including the classical drug clioquinol [START_REF] Daniel | Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells[END_REF][START_REF] Chen | Clioquinol, a therapeutic agent for Alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts[END_REF][START_REF] Wehbe | Development of a copper-clioquinol formulation suitable for intravenous use[END_REF]. Furthermore, copper is a very attractive metal to be investigated, as it appears to be more toxic on cancer cells than on normal cells, provided that its concentration can be kept high enough in cells [START_REF] Wang | Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation[END_REF][START_REF] Barresi | ATOX1 gene silencing increases susceptibility to anticancer therapy based on copper ionophores or chelating drugs[END_REF].

Materials and Methods

Unless specified otherwise, the chemicals used in this work were purchased from Sigma-Millipore and were at least 99% pure. As a copper source for the copper complexes, we used a titrated copper sulfate solution (4% catalog number C2284).

Cell Culture

The mouse monocyte/macrophage cell line RAW264.7 was purchased from the European Cell Culture Collection (Salisbury, UK). The cells were cultured in RPMI 1640 medium + 10% fetal bovine serum. Cells were seeded every two days at 200,000 cells/mL and harvested at 1,000,000 cells per ml. For treatment with chemicals, the following scheme was used: cells were first seeded at 500,000 cells/mL in T175 flasks (50 mL per flask). They were exposed to 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex (2:1) on the following day and harvested after a further 24 h in culture. Cell viability was measured by the neutral red uptake assay [START_REF] Repetto | Neutral red uptake assay for the estimation of cell viability/cytotoxicity[END_REF]. All experiments were carried out at least in triplicate on independent cultures.

Ion Quantification by ICP-MS

To measure ion uptake in cells, the cells were harvested after exposure to 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex by scraping in buffer A (Hepes 50 mM pH 7.5, sorbitol 200 mM, magnesium acetate 2 mM), and collected by centrifugation (500 g, 5 min). After a further rinse in buffer A, the cells were lysed by incubation in 10 cell pellet volume of lysis buffer (Hepes 50 mM pH 7.5, magnesium acetate 2 mM, tetradecyldimethylammonio propane sulfonate (SB 3-14) 0.15% (w/v)) for 20 min on ice, and the protein concentration in the extracts, determined by a modified dye-binding assay [START_REF] Rabilloud | Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers[END_REF].

The extracts were mineralized by the addition of three volumes of suprapure 65% HNO 3 per volume of extract and incubation on a rotating wheel at room temperature for 18 h.

Mineralized samples were then diluted in ultrapure grade HNO 3 (1% (v/v)) and analyzed on a Nexion 300X ICP-MS (Perkin Elmer, Waltham, MA, USA) equipped with a concentric nebulizer and operated in standard mode. External calibration was performed using certified ionic Cu and Zn ICP-MS standards and Yttrium was used as an internal standard.

The technique was validated based on linearity, repeatability, reproducibility, accuracy, inter-sample contamination criterions, and the analytical limit of detection (LoD) determined as the sum of the mean and three standard deviations of 20 ultrapure water samples. For this experiment, the LoD was 0.0017 µg/L for Cu and 0.022 µg/L for Zn.

Proteomics

The 2D gel based proteomic experiments were essentially carried out as previously described [START_REF] Dalzon | Differential proteomics highlights macrophage-specific responses to amorphous silica nanoparticles[END_REF], on independent biological triplicates. However, detailed material and methods are provided for the sake of consistency.

Sample Preparation

The cells were collected by scraping, and then washed three times in PBS. The cells were then washed once in TSE buffer (Tris-HCl 10 mM pH 7.5, sucrose 0.25 M, EDTA 1 mM), and the volume of the cell pellet was estimated. The pellet was resuspended in its own volume of TSE buffer. Then 4 volumes (respective to the cell suspension just prepared) of concentrated lysis buffer (urea 8.75 M, thiourea 2.5 M, CHAPS 5% (w/v), TCEP-HCl 6.25 mM, spermine base 12.5 mM) were added and the solution was let to extract at room temperature for 1 h. The nucleic acids were then pelleted by ultracentrifugation (270,000 g at room temperature for 1 h), and the protein concentration in the supernatant was determined by a modified dye-binding assay [START_REF] Guidi | The molecular mechanisms of antimetastatic ruthenium compounds explored through DIGE proteomics[END_REF]. Carrier ampholytes (Pharmalytes pH 3-10) were added to a final concentration of 0.4% (w/v), and the samples were kept frozen at -20 • C until use.

Isoelectric Focusing

Home-made 160 mm long 4-8 linear pH gradient gels [START_REF] Gianazza | Formulations for immobilized pH gradients including pH extremes[END_REF] were cast according to published procedures [START_REF] Rabilloud | Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension[END_REF]. Four mm-wide strips were cut, and rehydrated overnight with the sample, diluted in a final volume of 0.6 mL of rehydration solution (urea 7 M, thiourea 2 M, CHAPS 4%, carrier ampholytes 0.4% (Pharmalytes 3-10) and dithiodiethanol 100 mM [START_REF] Luche | About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis[END_REF].

The strips were then placed in a Multiphor plate (GE Healthcare), and IEF was carried out with the following electrical parameters: 100V for 1 h, then 300 V for 3 h, then 1000 V for 1 h, then 3400 V up to 60-70 kVh. After IEF, the gels were equilibrated for 20 min in Tris 125mM, HCl 100mM, SDS 2.5%, glycerol 30% and urea 6 M [START_REF] Gorg | Elimination of Point Streaking on Silver Stained Two-Dimensional Gels by Addition of Iodoacetamide to the Equilibration Buffer[END_REF]. They were then transferred on top of the SDS gels and sealed in place with 1% agarose dissolved in Tris 125 mM, HCl 100 mM, SDS 0.4% and 0.005% (w/v) bromophenol blue.

SDS Electrophoresis and Protein Detection

Ten percent gels (160 × 200 × 1.5 mm) were used for protein separation. The Tris taurine buffer system [START_REF] Tastet | A versatile electrophoresis system for the analysis of high-and low-molecular-weight proteins[END_REF] was used and operated at a ionic strength of 0.1 and a pH of 7.9. The final gel composition is thus Tris 180 mM, HCl 100 mM, acrylamide 10% (w/v), bisacrylamide 0.27%. The upper electrode buffer is Tris 50 mM, Taurine 200 mM, SDS 0.1%. The lower electrode buffer is Tris 50 mM, glycine 200 mM, SDS 0.1%. The gels were run at 25V for 1hour, then 12.5 W per gel until the dye front has reached the bottom of the gel. Detection was carried out by a tetrathionate silver staining [START_REF] Sinha | A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis[END_REF].

Image Analysis

The gels were scanned after silver staining on a flatbed scanner (Epson perfection V750), using a 16 bits grayscale image acquisition. The gel images were then analyzed using the Delta 2D software (v 4.7). Spots that were never expressed above 100 ppm of the total spots were first filtered out. Then, significantly-varying spots were selected on the basis of their Student T-test p-value between the treated and the control groups. Spots showing a p-value lower than 0.05 were selected.

The false discovery rate was controlled by a combination of approaches, including the classical Benjamini-Hochberg FDR [START_REF] Yekutieli | Resampling-based false discovery rate controlling multiple test procedures for correlated test statistics[END_REF] or the more recent Sequential Goodness of Fit approaches [START_REF] Diz | Multiple hypothesis testing in proteomics: A strategy for experimental work[END_REF][START_REF] Carvajal-Rodriguez | Assessing significance in high-throughput experiments by sequential goodness of fit and q-value estimation[END_REF]. Global analysis of the proteomic results was carried out by the PAST statistical software [START_REF] Hammer | Paleontological statistics software package for education and data analysis[END_REF].

Mass Spectrometry

The spots selected for identification were excised from silver-stained gels and destained with ferricyanide/thiosulfate on the same day as silver staining in order to improve the efficiency of the identification process [START_REF] Gharahdaghi | Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity[END_REF]. In gel digestion was performed with an automated protein digestion system, MassPrep Station (Waters, Milford, MA, USA). The gel plugs were washed twice with 50 µL of ammonium hydrogen carbonate (NH 4 HCO 3 ) 25 mM and 50 µL of acetonitrile. The cysteine residues were reduced by 50 µL of 10 mM dithiothreitol at 57 • C and alkylated by 50 µL of iodoacetamide 55 mM. After dehydration with acetonitrile, the proteins were cleaved in gel with 10 µL of 6.5 ng/µL of modified porcine trypsin (Promega, Madison, WI, USA) in NH 4 HCO 3 25 mM. The digestion was performed overnight at room temperature. The generated peptides were extracted with 30 µL of 60% acetonitrile in 0.1% formic acid. Acetonitrile was evaporated under vacuum before nanoLC-MS/MS analysis.

NanoLC-MS/MS analysis was performed using a nanoACQUITY Ultra-Performance-LC (Waters Corporation, Milford, MA, USA) coupled to the Synapt TM High Definition Mass Spectrometer TM (Waters Corporation, Milford, MA, USA), or to the TripleTOF 5600 (Sciex,Framingham, MS, USA).

The nanoLC system was composed of ACQUITY UPLC ® CSH130 C18 column (250 mm × 75 µm with a 1.7 µm particle size, Waters Corporation, Milford, MA, USA) and a Symmetry C18 precolumn (20 mm × 180 µm with a 5 µm particle size, Waters Corporation, Milford, MA, USA). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). 4 µL of sample were loaded into the enrichment column during 3 min at 5 µL/min with 99% of solvent A and 1% of solvent B. Elution of the peptides was performed at a flow rate of 300 nL/min with a 8-35% linear gradient of solvent B in 9 min.

The Synapt TM High Definition Mass Spectrometer TM (Waters Corporation, Milford, MA, USA) was equipped with a Z-spray ion source and a lock mass system. The system was fully controlled by MassLynx 4.1 SCN639 (Waters Corporation, Milford, MA, USA). The capillary voltage was set at 2.8 kV and the cone voltage at 35 V. Mass calibration of the TOF was achieved using fragment ions from Glu-fibrino-peptide B on the [50;2000] m/z range. Online correction of this calibration was performed with Glu-fibrino-peptide B as the lock-mass. The ion [M + 2H] 2+ at m/z 785.8426 was used to calibrate MS data and the fragment ion [M + H] + at m/z 684.3469 was used to calibrate MS/MS data during the analysis.

For tandem MS experiments, the system was operated with automatic switching between MS (0.5 s/scan on m/z range [150;1700]) and MS/MS modes (0.5 s/scan on m/z range [50;2000]). The two most abundant peptides (intensity threshold 20 counts/s), preferably doubly and triply charged ions, were selected on each MS spectrum for further isolation and CID fragmentation using collision energy profile. Fragmentation was performed using argon as the collision gas.

Mass data collected during analysis were processed and converted into pkl files using ProteinLynx Global Server 2.3 (Waters Corporation, Milford, MA, USA). Normal background subtraction type was used for both MS and MS/MS with 5% threshold and polynomial correction of order 5. Smoothing was performed on MS/MS spectra (Savitsky-Golay, 2 iterations, window of 3 channels). Deisotoping was applied for MS (medium deisotoping) and for MS/MS (fast deisotoping).

The TripleTOF 5600 was operated in positive mode, with the following settings: ionspray voltage floating (ISVF) 2300 V, curtain gas (CUR) 10, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 2, declustering potential (DP) 80 V. Information-dependent acquisition (IDA) mode was used with Top 10 MS/MS scans. The MS scan had an accumulation time of 250 ms on m/z [400;1250] range and the MS/MS scans 100 ms on m/z [150;1800] range in high sensitivity mode. Switching criteria were set to ions with charge state of 2-4 and an abundance threshold of more than 500 counts, exclusion time was set at 4 s. IDA rolling collision energy script was used for automatically adapting the CE. Mass calibration of the analyser was achieved using peptides from digested BSA. The complete system was fully controlled by AnalystTF 1.7 (Sciex) Raw data collected were processed and converted with MSDataConverter in mgf peak list format.

For protein identification, MS/MS data were interpreted using a local Mascot server with MASCOT 2.5.1 algorithm (Matrix Science, London, UK) against UniProtKB/SwissProt (version 2018_11, 558,898 sequences), without taxonomical restrictions. Spectra were searched with a mass tolerance of 15 ppm for MS and 0.05 Da for MS/MS data, allowing a maximum of one trypsin missed cleavage. Carbamidomethylation of cysteine residues and oxidation of methionine residues were specified as variable modifications. Protein identifications were validated with at least two peptides with Mascot ion score above 30.

Classical contaminants from human skin (keratins, filaggrin, desmoglein, involucrin) were removed from identifications. To cope with multiple identification issues from single 2D gel spots, univocal identifications were reported when the fist candidate was identified by at least twice more unique peptides than the next candidate or represented at least twice more spectra than the next candidate, and corresponded to the correct species (Mus musculus). This allowed removal of contaminating serum proteins (Bos taurus) from the identifications. In order to decrease the severity of the multiple identification problem, a graded identification strategy was used. A first identification of the spots of interest was attempted on analytical gels (loaded with 150µg protein, silver stained) when unsuccessful, more heavily loaded gels were used (250 µg protein, silver stained). If still unsuccessful, micropreparative gels (1 mg protein, colloidal Coomassie Blue-stained) were used. This strategy limited the problems associated with increased diffusion and streaking of proteins on 2D gels at high protein loads [START_REF] Zhan | How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?[END_REF].

Phagocytosis and Particle Internalization Assay

The phagocytic activity was measured using fluorescent latex beads (1 µm diameter, green labelled, catalog number L4655 from Sigma, St Louis, MO, USA), as described previously [START_REF] Triboulet | Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses[END_REF]. After a 2h30 treatment with the fluorescent beads in culture medium at 37 • C, the cells were harvested in PBS). Phagocytic activity was measured by flow cytometry on a FacsCalibur instrument (Beckton Dickinson, Franklin Lakes, NJ, USA).

F-Actin Staining

The experiments were performed essentially as previously described [START_REF] Aude-Garcia | A combined proteomic and targeted analysis unravels new toxic mechanisms for zinc oxide nanoparticles in macrophages[END_REF]. The cells were cultured on coverslips placed in 6-well plates, and exposed to 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex as described above. At the end of the exposure time, cells were washed twice for 5 min at 4 • C in PBS, fixed in 4% paraformaldehyde for 30 min at room temperature. After two washes (5 min/4 • C in PBS), they were permeabilized in 0.1% Triton X100 for 5 min at room temperature. After two more washes in PBS, Phalloidin-Atto 550 (Sigma (500 nM) was added to the cells and let for 20 min at room temperature in the dark. Coverslips-attached cells were washed, placed on microscope slides (Thermo Scientific, Waltham, MS, USA) using a Vectashield mounting medium containing DAPI (Eurobio, Paris, France) and imaged using a Zeiss LSM 800 confocal microscope (Zeiss, Jena, Germany). The images were processed using the ImageJ software.

Mitochondrial Transmembrane Potential Measurement

The mitochondrial transmembrane potential was assessed by Rhodamine 123 uptake [START_REF] Dalzon | Differential proteomics highlights macrophage-specific responses to amorphous silica nanoparticles[END_REF], using a low Rhodamine concentration (80 nM) to avoid intramitochondrial fluorescence quenching that would result in a poor estimation of the mitochondrial potential [START_REF] Perry | Mitochondrial membrane potential probes and the proton gradient: A practical usage guide[END_REF]. The samples were analyzed by flow cytometry on a FacsCalibur instrument (Beckton Dickinson). Both the proportion of rhodamine positive cells and the mean fluorescence of this population were recorded in the analysis.

Glutathione Assay

Classical glutathione assays are difficult to apply to macrophage-like cells, as the oxidizing activities present in their phagolysosomes oxidize the cytosolic reduced glutathione when the cells are homogenized. To alleviate this problem, we used a spectrophotometric assay utilizing the cellular glutathione S-transferases and the chlorodinitrobenzene substrate [START_REF] Warholm | Glutathione transferases from human liver[END_REF]. After exposure to 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex in 6 well plates, cells were treated for 30 min with chlorodinitrobenzene (25 µM, final concentration in the culture medium), resulting in the conjugation of glutathione to the substrate and consumption of free reduced glutathione [START_REF] Arrick | Glutathione depletion sensitizes tumor cells to oxidative cytolysis[END_REF][START_REF] Bruggeman | Differential effects of raising and lowering intracellular glutathione levels on the cytotoxicity of allyl isothiocyanate, tert-butylhydroperoxide and chlorodinitrobenzene[END_REF]. The culture medium was removed and the cell layer was rinsed twice in Hepes NaOH buffer (10 mM pH 7.5) containing 2 mM magnesium acetate and 0.25 M sucrose. The cells were then lysed on a rocking table for 15 min in 800 µl of Hepes NaOH buffer (10 mM pH 7.5) containing 2 mM magnesium acetate and 1 mg/mL tetradecyldimethylammonio propane sulfonate (SB 3-14). The suspension was collected and centrifuged at 15,000 g for 15 min to pellet particulate material. The concentration of glutathione was determined by measuring the absorbance at 340 nm, using an extinction coefficient of 9600 M -1 •cm -1 [START_REF] Warholm | Glutathione transferases from human liver[END_REF]. Results were normalized to the protein concentration of the cell extracts, determined by a modified dye-binding assay [START_REF] Rabilloud | Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers[END_REF]. The final results were expressed in nmoles glutathione/mg protein.

NO Production

The cells were grown to confluence in a 6 well plate and pre-treated with 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex as described above. For the final 18 h of culture, half of the wells were treated with 100 ng/mL LPS (from salmonella, purchased from Sigma), and arginine monohydrochloride was added to all the wells (5 mM final concentration) to secure a high concentration of substrate for the nitric oxide synthase. After 18 h of incubation, the cell culture medium was recovered, centrifuged at 10,000 g for 10 min to remove cells and debris, and the nitrite concentration in the supernatants was read at 540 nm after addition of an equal volume of Griess reagent and incubation at room temperature for 30 min.

Results

We first investigated the working dose for the hydroxyquinoline-copper complex. To this purpose, cells were treated for 24 h with various doses of the copper complex, and with the corresponding doses of hydroxyquinoline. The results, shown on Figure 1, indicated a very strong decrease of viability at a complex concentration corresponding to 6 µM hydroxyquinoline and 3 µM copper. This led us to carry out all experiments with 4 µM hydroxyquinoline ± 2 µM copper. The cells were treated for 24 h with either 8-hydroxyquinoline alone (black curve, square symbols) or with the 8-hydroxyquinoline-copper 2:1 complex (grey curve, diamond symbols). The concentrations on the abscissae are expressed in μM 8-hydroxyquinoline.

As hydroxyquinoline and its derivatives have been shown to behave as ionophores for copper and zinc [START_REF] Ding | Anticancer activity of the antibiotic clioquinol[END_REF][START_REF] Yu | Clioquinol targets zinc to lysosomes in human cancer cells[END_REF][START_REF] Ding | Metal Ionophores-An Emerging Class of Anticancer Drugs[END_REF], we analyzed the intracellular concentration of these two metals after treatment with either hydroxyquinoline alone or with the hydroxyquinoline-copper complex. The results, shown in Table 1, indicated that the intracellular zinc concentration was constant in all conditions, and that treatment with hydroxyquinoline alone did not induce an influx of copper, while treatment with the copper complex resulted into a massive increase in intracellular copper concentration.

Global Analysis of the Proteomic Results

We then performed a proteomic screen using two-dimensional electrophoresis. The raw gel images are provided in Supplementary Figures 123. Upon computerized analysis, 2444 spots were detected across the gel series, with a median CV of 21% (control), 20% (copper complex) and 16% (hydroxyquinoline alone). As a first test, we performed a global analysis of the results. To this purpose, we selected all spots showing a p-value < 0.25 by a Student T-test analysis in at least one of the two comparisons: copper complex vs. control or hydroxyquinoline vs. control. This allowed to decrease the noise provided by proteins with random variability, while selecting more than 50% of the spots (1426 out of 2444). This spot list was then analyzed by hierarchical clustering, and the results are shown on Figure 2. They indicated first that the within-group variability was much lower than the inter-group variability, which validated the overall quality of the proteomic analysis. It also indicated that the copper complex-treated group was separated from the other two groups, showing that the copper complex had a much stronger effect on the proteome than hydroxyquinoline alone. The cells were treated for 24 h with either 8-hydroxyquinoline alone (black curve, square symbols) or with the 8-hydroxyquinoline-copper 2:1 complex (grey curve, diamond symbols). The concentrations on the abscissae are expressed in µM 8-hydroxyquinoline.

As hydroxyquinoline and its derivatives have been shown to behave as ionophores for copper and zinc [START_REF] Ding | Anticancer activity of the antibiotic clioquinol[END_REF][START_REF] Yu | Clioquinol targets zinc to lysosomes in human cancer cells[END_REF][START_REF] Ding | Metal Ionophores-An Emerging Class of Anticancer Drugs[END_REF], we analyzed the intracellular concentration of these two metals after treatment with either hydroxyquinoline alone or with the hydroxyquinoline-copper complex. The results, shown in Table 1, indicated that the intracellular zinc concentration was constant in all conditions, and that treatment with hydroxyquinoline alone did not induce an influx of copper, while treatment with the copper complex resulted into a massive increase in intracellular copper concentration.

Global Analysis of the Proteomic Results

We then performed a proteomic screen using two-dimensional electrophoresis. The raw gel images are provided in Supplementary Figures S1-S3. Upon computerized analysis, 2444 spots were detected across the gel series, with a median CV of 21% (control), 20% (copper complex) and 16% (hydroxyquinoline alone). As a first test, we performed a global analysis of the results. To this purpose, we selected all spots showing a p-value < 0.25 by a Student T-test analysis in at least one of the two comparisons: copper complex vs. control or hydroxyquinoline vs. control. This allowed to decrease the noise provided by proteins with random variability, while selecting more than 50% of the spots (1426 out of 2444). This spot list was then analyzed by hierarchical clustering, and the results are shown on Figure 2. They indicated first that the within-group variability was much lower than the inter-group variability, which validated the overall quality of the proteomic analysis. It also indicated that the copper complex-treated group was separated from the other two groups, showing that the copper complex had a much stronger effect on the proteome than hydroxyquinoline alone.

Figure 2.

Global analysis of the proteomic experiment by hierarchical clustering. All the spots that showed a p-value <0.25 in either the hydroxyquinoline vs. control or the copper complex vs. control comparisons in the proteomic experiments were used for the calculation of the clustering tree. The PAST software package was used for the calculations, using a Euclidean distance and the Ward's algorithm. This tree indicates the similarity between the various experimental groups (the higher the distance of the branching point between groups, the more dissimilar they are). Ctrl: unexposed cells. OHQ: cells exposed to 4 μM 8-hydroxyquinoline. QCu: cells exposed to 4 μM 8-hydroxyquinoline +2 μM copper sulfate. 3.2. Detailed Analysis of the Proteomic Results.

We then selected the spots modulated by at least one of the treatments, on the strict basis of the statistical tests, without any fold change threshold. This allowed to take into account coordinated but low amplitude changes that can be highly significant, as shown before [START_REF] Herrmann | Total variance should drive data handling strategies in third generation proteomic studies[END_REF]. The proteins were then identified by mass spectrometry. The results are shown on Figures 345, the raw images, quantitative data and identification data being supplied in Supplementary Figures 123, Supplementary Tables 1 and2, respectively. Global analysis of the proteomic experiment by hierarchical clustering. All the spots that showed a p-value <0.25 in either the hydroxyquinoline vs. control or the copper complex vs. control comparisons in the proteomic experiments were used for the calculation of the clustering tree. The PAST software package was used for the calculations, using a Euclidean distance and the Ward's algorithm. This tree indicates the similarity between the various experimental groups (the higher the distance of the branching point between groups, the more dissimilar they are). Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. QCu: cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate. 3.2. Detailed Analysis of the Proteomic Results.

We then selected the spots modulated by at least one of the treatments, on the strict basis of the statistical tests, without any fold change threshold. This allowed to take into account coordinated but low amplitude changes that can be highly significant, as shown before [START_REF] Herrmann | Total variance should drive data handling strategies in third generation proteomic studies[END_REF]. The proteins were then identified by mass spectrometry. The results are shown on Figures 345, the raw images, quantitative data and identification data being supplied in Supplementary Figures S1-S3, Supplementary Tables S1 andS2, respectively. The same conditions as described in Figure 3 were applied to this figure The proteins involved in mitochondria, central metabolism, protein production, as well as unclassified proteins are shown in this figure

In order to take into account the multiple testing issue, we submitted the modulated protein lists to statistical tests as the Benjamini-Hochberg FDR test [START_REF] Yekutieli | Resampling-based false discovery rate controlling multiple test procedures for correlated test statistics[END_REF] and the sequential goodness of fit tests [START_REF] Diz | Multiple hypothesis testing in proteomics: A strategy for experimental work[END_REF][START_REF] Carvajal-Rodriguez | Assessing significance in high-throughput experiments by sequential goodness of fit and q-value estimation[END_REF]. The results, shown in Supplementary Tables 3 and4, indicated that most of the proteins The same conditions as described in Figure 3 were applied to this figure Only the proteins involved in the RNA and nucleotide metabolism, lipid metabolism, redox control, and signaling are shown in this figure

The same conditions as described in Figure 3 were applied to this figure The proteins involved in mitochondria, central metabolism, protein production, as well as unclassified proteins are shown in this figure

In order to take into account the multiple testing issue, we submitted the modulated protein lists to statistical tests as the Benjamini-Hochberg FDR test [START_REF] Yekutieli | Resampling-based false discovery rate controlling multiple test procedures for correlated test statistics[END_REF] and the sequential goodness of fit tests [START_REF] Diz | Multiple hypothesis testing in proteomics: A strategy for experimental work[END_REF][START_REF] Carvajal-Rodriguez | Assessing significance in high-throughput experiments by sequential goodness of fit and q-value estimation[END_REF]. The results, shown in Supplementary Tables S3 andS4, indicated that most of the proteins selected on the basis of the T-test also had a low FDR value with all the tested algorithms, validating their selection for further functional analysis.

As a first step, we analyzed the lists of modulated proteins by the DAVID pathway analysis tool. The results, shown in Supplementary Tables S5 andS6, indicated strongly modulated pathways, such as the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, carbon metabolism or oxido-reduction. As could be expected from the global analysis, more pathways were modulated upon treatment with the copper complex than with hydroxyquinoline alone.

Targeted Validation Experiments

Even though the statistical tests indicated a strong robustness of the proteomic analysis, we decided to validate the proteomic results by targeted analyses. Based on the pathway analysis, we first measured the free glutathione concentration in cells, and obtained the following values:

Control cells: 16.68 ± 1.52 nmoles/mg protein (range 15.15-18.02) Cells treated with hydroquinoline alone: 16.22 ± 3.50 nmoles/mg protein (range 11.32-18.18) Cells treated with the copper complex: 2.59 ± 3.37 nmoles/mg protein (range 0.89-7.66) This very strong decrease upon treatment with the copper complex was statistically significant (p < 0.05 in Mann Whitney U test, p < 0.01 in Student T-test).

As the adhesion-cytoskeleton and the lysosome pathways were also highlighted in the pathway analysis we also investigated the phagocytic activity, as it is dependent on both the integrity of the lysosomal pathway and on the actin cytoskeleton. We also studied the actin cytoskeleton by confocal microscopy. The results, shown on Figure 6, indicated once again no effect of hydroquinoline alone, but a strong effect of the copper complex, with the disappearance of pseudopodia and a strong decrease in the phagocytic activity, both in the proportion of phagocytic cells and in the number of beads internalized per cell. On the basis of the pathway analysis, we also investigated the mitochondrial activity by testing the mitochondrial transmembrane potential. The results, shown on Figure 7A, indicated a slight decrease in the proportion of cells with a high mitochondrial potential. However, no change in the value of this potential was observed upon treatment with hydroxyquinoline alone or with added copper.

Lastly, we investigated the production of nitric oxide, which is one of the many differentiated functions of monocytes/macrophages. The results, shown on Figure 7B, indicated an increase in the basal production of nitric oxide, both by hydroxyquinoline alone and-even more strongly-with added copper. However, when lipopolysaccharide was also added to stimulate the cells and mimic a On the basis of the pathway analysis, we also investigated the mitochondrial activity by testing the mitochondrial transmembrane potential. The results, shown on Figure 7A, indicated a slight decrease in the proportion of cells with a high mitochondrial potential. However, no change in the value of this potential was observed upon treatment with hydroxyquinoline alone or with added copper.

Lastly, we investigated the production of nitric oxide, which is one of the many differentiated functions of monocytes/macrophages. The results, shown on Figure 7B, indicated an increase in the basal production of nitric oxide, both by hydroxyquinoline alone and-even more strongly-with added copper. However, when lipopolysaccharide was also added to stimulate the cells and mimic a bacterial infection, the treatment with hydroxyquinoline alone showed no effect while the treatment with the copper complex induce a slight decrease in nitric oxide production.

bacterial infection, the treatment with hydroxyquinoline alone showed no effect while the treatment with the copper complex induce a slight decrease in nitric oxide production.

Discussion

With the increase in sensitivity of mass spectrometers, multi-identification of several proteins in a single two-dimensional gel spot has become increasingly frequent; this has been perceived as a problem by some authors [START_REF] Campostrini | Spot overlapping in two-dimensional maps: A serious problem ignored for much too long[END_REF] but not others [START_REF] Hunsucker | Is protein overlap in two-dimensional gels a serious practical problem?[END_REF]. It has been demonstrated that in most cases, the different proteins contained in a single spot are present in very different amounts, and that the variations detected via image analysis of 2D gels impact the most abundant protein in the spot [START_REF] Colignon | Evaluation of three-dimensional gel electrophoresis to improve quantitative profiling of complex proteomes[END_REF]. As expected, the severity of the multi-identification issue increases with protein loads [START_REF] Rabilloud | When 2D is not enough, go for an extra dimension[END_REF], both

Discussion

With the increase in sensitivity of mass spectrometers, multi-identification of several proteins in a single two-dimensional gel spot has become increasingly frequent; this has been perceived as a problem by some authors [START_REF] Campostrini | Spot overlapping in two-dimensional maps: A serious problem ignored for much too long[END_REF] but not others [START_REF] Hunsucker | Is protein overlap in two-dimensional gels a serious practical problem?[END_REF]. It has been demonstrated that in most cases, the different proteins contained in a single spot are present in very different amounts, and that the variations detected via image analysis of 2D gels impact the most abundant protein in the spot [START_REF] Colignon | Evaluation of three-dimensional gel electrophoresis to improve quantitative profiling of complex proteomes[END_REF]. As expected, the severity of the multi-identification issue increases with protein loads [START_REF] Rabilloud | When 2D is not enough, go for an extra dimension[END_REF], both because of the increased diffusional spreading of proteins at high loads and because of the streaking induced by gel overloading. However, even in the harshest possible conditions, i.e., the coupling of a highly sensitive mass spectrometry with sub-milligram protein loads [START_REF] Zhan | How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?[END_REF], the list of proteins identified in a single spot lengthens, but the first candidate usually accounts for more than 70% of the total mass spectrometry signal.

On the basis of these data, we decided to report only "winner takes all" identifications, but only in cases where this situation is most likely to occur, i.e., when the first candidate accounts for more than twice the number of unique peptides or spectra than the second candidate. A post-hoc validation of this strategy in the case of this study is represented by the fact that the proteins found as modulated belong to well identified pathways and are not randomly distributed.

A good example of this is represented by the numerous proteins identified in the proteasome/ubiquitin pathway, known to be a target of the copper complexes [START_REF] Daniel | Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells[END_REF][START_REF] Daniel | Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells[END_REF][START_REF] Daniel | Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer[END_REF][START_REF] Oliveri | Amino-and chloro-8-hydroxyquinolines and their copper complexes as proteasome inhibitors and antiproliferative agents[END_REF]. With the exception of the immunoproteasome subunit psb8, all modulated proteasome subunits detected in the proteomic screen are decreased upon treatment with the copper complex, suggesting a destabilization of the proteasome itself. This effect has been shown to be copper-dependent [START_REF] Zhai | Tumor cellular proteasome inhibition and growth suppression by 8-hydroxyquinoline and clioquinol requires their capabilities to bind copper and transport copper into cells[END_REF], and coupling the data on the abundances of the proteasome subunits to our data on ion transport mediated by hydroxyquinoline in the absence of added copper further corroborated this finding.

Indeed, beside the effects of the copper complexes, hydroxyquinoline derivatives by themselves have been shown to impact important biochemical processes such as the histone deacetylase pathway [START_REF] Cao | The Antiparasitic Clioquinol Induces Apoptosis in Leukemia and Myeloma Cells by Inhibiting Histone Deacetylase Activity[END_REF], the mTOR [START_REF] Cao | Clioquinol induces pro-death autophagy in leukemia and myeloma cells by disrupting the mTOR signaling pathway[END_REF] or autophagy [START_REF] He | Combination treatment with fasudil and clioquinol produces synergistic anti-tumor effects in U87 glioblastoma cells by activating apoptosis and autophagy[END_REF] pathways, or even the proteasome activity [START_REF] Mao | Clioquinol inhibits the proteasome and displays preclinical activity in leukemia and myeloma[END_REF]. Our proteomic screen did not detect important effects of hydroxyquinoline alone. However, the effects reported to date have been observed only with higher doses (>10 µM) than the one used here (4 µM).

In addition to proteasome inhibition, other effects such as redox effects have been reported for copper complexes [START_REF] Chen | Synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and MAPK pathway activation[END_REF][START_REF] Jungwirth | Anticancer Activity of Metal Complexes: Involvement of Redox Processes[END_REF]. Here again, the proteomic screen corroborated this hypothesis, with a decrease in the abundances of two abundant cytosolic peroxiredoxins (prx2 and prx6) and in a thioredoxin-like protein (txnl1) upon treatment with the copper complexes.

These two examples suggest that a proteomic screen could be a powerful tool for the detection of other targets of the copper complexes, in a strategy similar to the one used for gold complexes [START_REF] Gamberi | Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipy(c)[END_REF]. Indeed, proteomics suggested that the level of reduced glutathione could be impacted by the copper complexes, which was confirmed by targeted experiments. This result is in line with what we previously showed for copper nanoparticles [START_REF] Triboulet | Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses[END_REF].

Another pathway that we found modulated by the copper treatment was the actin cytoskeleton, and here again our targeted experiments on cell shape and on phagocytosis confirmed the impact of the copper complexes on this pathway. This finding may be of interest in anticancer strategies, as the actin cytoskeleton is key to cell shape and motility, i.e., parameters that are important in the metastasis phenomenon.

Regarding the mitochondria, whose proteins are well represented in the proteomic screen, we tested the mitochondrial potential. While a small decrease in the proportion of cells with a high mitochondrial potential was noted after treatment with the copper complex, corresponding to the slight increase in mortality induced by this treatment, the value of the mitochondrial potential did not change appreciably upon treatment by 8-hydroxyquinoline or to the 8-hydroxyquinoline-copper complex. This may mean that the changes observed in the mitochondrial proteins are sufficient to maintain the mitochondrial potential to a normal value.

Lastly, we investigated one differentiated function of monocytes/macrophages, namely the production of nitric oxide, either alone or after stimulation with lipopolysaccharide. Interestingly, both 8-hydroxyquinoline and its copper complex were able to induce by themselves a significant increase in the production of nitric oxide, a fact that may be related to the reported release of TNF alpha by clioquinol, a hydroxyquinoline derivative [START_REF] Du | Clioquinol promotes cancer cell toxicity through tumor necrosis factor alpha release from macrophages[END_REF]. Regarding the nitric oxide release after stimulation with lipopolysaccharide, we observed a decrease upon treatment with the copper complex, in line with what we observed with copper oxide nanoparticles [START_REF] Triboulet | Comparative proteomic analysis of the molecular responses of mouse macrophages to titanium dioxide and copper oxide nanoparticles unravels some toxic mechanisms for copper oxide nanoparticles in macrophages[END_REF].

Conclusions

In conclusion, the main output of the proteomic study of the effects of the 8-hydroxyquinolinecopper complex on leukemic cells is the demonstration that beyond its well-documented effects on the proteasome, it also has multi-target effects on cells, including the oxidative stress response and the cytoskeleton. On the negative side, such a pleiotropic effect may increase the toxicity of the drug toward normal, non-cancerous cells. On the positive side, a pleiotropic effect strongly decreases the probability of emergence of resistant cells, and thus, may be highly beneficial as a cancer treatment. In line with this observation, metallodrugs, including copper complexes, are among the rare drugs which are able to target cancer stem cells [START_REF] Laws | The Next Generation of Anticancer Metallopharmaceuticals: Cancer Stem Cell-Active Inorganics[END_REF], thereby representing a very important actor in the emergence of resistance and, therefore, in cancer relapses. Moreover, copper appears to have some selectivity against cancer cells [START_REF] Wang | Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation[END_REF][START_REF] Barresi | ATOX1 gene silencing increases susceptibility to anticancer therapy based on copper ionophores or chelating drugs[END_REF]. Thus, the multipotency of metal complexes clearly deserves further research.

Figure 1 .

 1 Figure 1. Dose-response viability curve.

Figure 1 .

 1 Figure 1. Dose-response viability curve.

Figure 2 .

 2 Figure 2. Global analysis of the proteomic experiment by hierarchical clustering. All the spots that showed a p-value <0.25 in either the hydroxyquinoline vs. control or the copper complex vs. control comparisons in the proteomic experiments were used for the calculation of the clustering tree. The PAST software package was used for the calculations, using a Euclidean distance and the Ward's algorithm. This tree indicates the similarity between the various experimental groups (the higher the distance of the branching point between groups, the more dissimilar they are). Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. QCu: cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate. 3.2. Detailed Analysis of the Proteomic Results.

Figure 3 .

 3 Figure 3. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline + 2μM copper sulfate; (C): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline.

Figure 3 .

 3 Figure 3. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline + 2µM copper sulfate; (C): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline. Total cell extracts of RAW274.7 cells were separated by two-dimensional gel electrophoresis. The first dimensions covered a 4-8 pH range and the second dimension a 15-200 kDa range. Total cellular proteins (150 µg) were loaded on the first dimension gel. Only the proteins involved in the proteasome/ubiquitin pathway, cytoskeleton, homeostasis and vesicles/lysosomes are shown in this figure.

Figure 4 .

 4 Figure 4. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline + 2 μM copper sulfate; (C): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline

Figure 4 .

 4 Figure 4. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline + 2 µM copper sulfate; (C): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline.

Figure 5 .

 5 Figure 5. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline +2 μM copper sulfate; (C): gel obtained from cells exposed to 4 μM 8-hydroxyquinoline

Figure 5 .

 5 Figure 5. Proteomic analysis of total cell extracts by 2D electrophoresis. (A): gel obtained from unexposed cells; (B): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate; (C): gel obtained from cells exposed to 4 µM 8-hydroxyquinoline.

Figure 6 .

 6 Figure 6. Actin cytoskeleton and phagocytosis. In the top panels, the actin cytoskeleton was visualized with fluorescent phalloidin and confocal microscopy. Only one confocal plane inside the cells is shown (going through the cell nucleus). Ctrl: unexposed cells. OHQ: cells exposed to 4 μM 8-hydroxyquinoline. Qcu: cells exposed to 4 μM 8-hydroxyquinoline +2 μM copper sulfate. In the bottom panel, the phagocytic capacity was assessed by fluorescent latex beads internalization. grey bars: proportion of positive cells in the viable cell population. black bars: mean cellular fluorescence. Ctrl: unexposed cells. OHQ: cells exposed to 4 μM 8-hydroxyquinoline. QCu: cells exposed to 4 μM 8-hydroxyquinoline +2 μM copper sulfate. Symbols indicate the statistical significance (Student Ttest): **: p < 0.01.

Figure 6 .

 6 Figure 6. Actin cytoskeleton and phagocytosis. In the top panels, the actin cytoskeleton was visualized with fluorescent phalloidin and confocal microscopy. Only one confocal plane inside the cells is shown (going through the cell nucleus). Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. Qcu: cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate. In the bottom panel, the phagocytic capacity was assessed by fluorescent latex beads internalization. grey bars: proportion of positive cells in the viable cell population. black bars: mean cellular fluorescence. Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. QCu: cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate. Symbols indicate the statistical significance (Student T-test): **: p < 0.01.

Figure 7 .

 7 Figure 7. Analysis of the mitochondrial transmembrane potential and of the nitric oxide production. panel (A), the mitochondrial potential was analyzed by the rhodamine 123 accumulation method. Grey bars: proportion of positive cells in the viable cell population. Black bars: mean cellular fluorescence; Ctrl: unexposed cells. OHQ: cells exposed to 4 μM 8-hydroxyquinoline. QCu: cells exposed to 4 μM 8-hydroxyquinoline +2 μM copper sulfate. Symbols indicate the statistical significance (Student T-test): **: p < 0.01. In panel (B), the production of nitric oxide was analyzed. Black bars: production after treatment for 24 h with 4μM 8-hydroxyquinoline ± 2 μM copper sulfate. Grey bars: production after treatment for 24 h with 4 μM 8-hydroxyquinoline ±2 μM copper sulfate and stimulation with lipopolysaccharide for the last 18 h of culture. Ctrl: unexposed cells. OHQ: cells exposed to 4 μM 8-hydroxyquinoline. QCu: cells exposed to 4 μM 8-hydroxyquinoline + 2 μM copper sulfate. Symbols indicate the statistical significance (Student T-test): † p < 0.1; **: p < 0.01; ***: p < 0.001

Figure 7 .

 7 Figure 7. Analysis of the mitochondrial transmembrane potential and of the nitric oxide production. panel (A), the mitochondrial potential was analyzed by the rhodamine 123 accumulation method. Grey bars: proportion of positive cells in the viable cell population. Black bars: mean cellular fluorescence; Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. QCu: cells exposed to 4 µM 8-hydroxyquinoline +2 µM copper sulfate. Symbols indicate the statistical significance (Student T-test): **: p < 0.01. In panel (B), the production of nitric oxide was analyzed. Black bars: production after treatment for 24 h with 4µM 8-hydroxyquinoline ± 2 µM copper sulfate. Grey bars: production after treatment for 24 h with 4 µM 8-hydroxyquinoline ±2 µM copper sulfate and stimulation with lipopolysaccharide for the last 18 h of culture. Ctrl: unexposed cells. OHQ: cells exposed to 4 µM 8-hydroxyquinoline. QCu: cells exposed to 4 µM 8-hydroxyquinoline + 2 µM copper sulfate. Symbols indicate the statistical significance (Student T-test): † p < 0.1; **: p < 0.01; ***: p < 0.001.

Table 1 .

 1 Ion levels in cells.

	condition control	OHQ	Qcu
	Zn	263 ± 81 257 ± 23	280 ± 79
	Cu	16 ± 17	13 ± 10 1076 ± 258

The ion concentrations are expressed in ng ion/mg cellular protein. OHQ: treatment with 4 μM 8-hydroxyquinoline. Qcu: treatment with 4 μM 8-hydroxyquinoline +2 μM copper sulfate.

Table 1 .

 1 Ion levels in cells.

	Condition	Control	OHQ	Qcu
	Zn	263 ± 81	257 ± 23	280 ± 79
	Cu	16 ± 17	13 ± 10	1076 ± 258

The ion concentrations are expressed in ng ion/mg cellular protein. OHQ: treatment with 4 µM 8-hydroxyquinoline. Qcu: treatment with 4 µM 8-hydroxyquinoline +2 µM copper sulfate.

Proteomes 2019, 7, 26; doi:10.3390/proteomes7020026 www.mdpi.com/journal/proteomes

Proteomes 2019, 7, x FOR PEER REVIEW

Funding: This work was funded by the CNRS, The University of Grenoble, the University of Strasbourg Unistra, the Région Alsace. This work used the platforms of theFrench Proteomic Infrastructure (ProFI) project (grant ANR-10-INBS-08-03).

Supplementary Materials:

The following are available online at http://www.mdpi.com/2227-7382/7/2/26/s1, Figure S1:Raw gel images for the control cells, Figure S2: Raw gel images for the cells treated with the copper complex, Figure S3: Raw gel images for the cells treated with hydroxyquinoline alone, Table S1: Quantitative data from computerized gel analysis, Table S2: Protein identification data by mass spectrometry, Table S3: Expected false discovery rates for the proteins modulated by the hydroxyquinoline-copper complex, Table S4: Expected false discovery rates for the proteins modulated by hydroxyquinoline alone, Table S5 Modulated pathways highlighted by the DAVID annotation tool. Cells treated with hydroxyquinoline copper complex, Table S6

Conflicts of Interest:

The authors declare no conflict of interest


Keith & Marquet

Kalfatak

Valentin De Mazancourt
email: valentin.demazancourt@laposte.net

Gerard Marquet

Werner Klotz
email: wklotz@aon.at

Philippe Keith

Magalie Castelin

C Gueryi Marquet

Keith & Kalfatak

Valentin De Mazancourt

When molecules and morphology work together: lines of evidence for the validity of Caridina buehleri Roux (Crustacea : Decapoda : Atyidae) and for C. gueryi

Keywords: Freshwater shrimp, Indo-Pacific, Integrative taxonomy, 16S

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

One of the aims of the Muséum national d'Histoire naturelle (MNHN) in Paris is to carry out faunistic inventories of rivers in tropical islands in order to establish a better protection of these fragile ecosystems and, in this context, to clarify taxonomy of poorly known organisms.

The freshwater shrimp Caridina H. Milne-Edwards, 1837, comprising 298 species (WoRMS database as of Oct. 2016) and mostly present in the Indo-Pacific region, is the most diversified genus of the family Atyidae [START_REF] De Grave | Dead Shrimp Blues: A Global Assessment of Extinction Risk in Freshwater Shrimps(Crustacea: Decapoda: Caridea)[END_REF] and an important ecological component in the tropical streams [START_REF] Covich | The role of benthic invertebrate species in freshwater ecosystems[END_REF][START_REF] Pringle | Effects of omnivorous shrimp in a montane tropical stream: sediment removal, disturbance of sessile invertebrates and enhancement of understory algal biomass[END_REF]. Their high diversity combined with the lack of informative morphological characters has led to a confused taxonomy [START_REF] Richard | African Caridina (Crustacea: Decapoda: Caridea: Atyidae): redescriptions of C. africana Kingsley, 1882, C. togoensis Hilgendorf[END_REF]. Indeed, until recently, their taxonomy was mainly based on morphological characters. Some have been proven highly variable within a single species (e.g. rostrum shape and indentation or coloration) and so taxonomically noninformative, making it difficult to identify boundaries among species [START_REF] Von Rintelen | Radiation of endemic species flocks in ancient lakes: systematic revision of the freshwater shrimp Caridina H. Milne Edwards, 1837 (Crustacea: Decapoda: Atyidae) from the ancient lakes of Sulawesi, Indonesia, with the description of eight new species[END_REF]. There is thus a need for an integrative and standardized approach to improve the systematic of the group, focusing on informative morphological features and using molecular characters [START_REF] Page | The taxonomic feedback loop: symbiosis of morphology and molecules[END_REF][START_REF] Page | Neither molecular nor morphological data have all the answers; with an example from Macrobrachium (Decapoda: Palaemonidae) from Australia[END_REF]. To illustrate this problem, we here focused on two species that are morphologically very close: Caridina buehleri Roux, 1934 and Caridina gueryi Marquet, Keith & Kalfatak, 2009. Roux (1934) described Caridina buehleri from a single non-ovigerous female from New Ireland (Papua New-Guinea) as possessing a distinctively long stylocerite and a long and pointed rostrum. In 2007, this species has been reported from Luwuk Peninsula, Central Sulawesi, Indonesia and re-described with more morphological characters but without examining the type specimen (Klotz et al. 2007). Caridina buehleri was later reported from Upolu Island (Western Samoa) (Keith et al. 2013) and collected by some of the authors (P. Keith and G. Marquet) in Kolobangara Island (Solomon Islands) in 2015.

Caridina gueryi, was described after examination of specimens collected from Vanuatu (Santo and Malekula islands) by the Vanuatu Environment Unit (VEU) and the MNHN (Keith et al. 2010) but they were not compared to the holotype of C. buehleri, despite showing strong similarities between the two species.

As we collected more and more specimens from different localities, we started to raise doubts regarding the validity of C. gueryi, as the specimens identified as such fitted well with the description of C. buehleri. Consequently, we here examined type specimens of these two species and combined morphological data with a 16S mtDNA analysis in order to clarify their respective status.

MATERIALS AND METHODS

Abbreviations for

Molecular analyses

DNA extraction, amplification and sequencing. DNA was extracted from abdominal tissues using the semi-automatic Ependorf ep-Motion 5075 robot. Fragments of the mitochondrial 16S rRNA (~ 520 bp) were amplified using the primers 16Sa-L (CGCCTGTTTATCAAAAACAT) and 16Sb-H2 (CTCCGGTTTGAACTCAGATCA) [START_REF] Palumbi | Nucleic acids II: the polymerase chain reaction[END_REF]. DNA amplification was performed in 25µl PCR reactions, containing approximately 3 ng of template DNA, 2.5 mM MgCL2, 0.26 mM of each nucleotide, 0.3 µM of each primer, 5% DMSO, 1 ng of BSA and 1.5 units of QBIOTAQ polymerase (MPBiomedicals). Amplification products were generated by an initial denaturation step of 4 min at 94°C followed by 30 cycles of denaturation at 94°C for 30s, annealing at 51°C for 60 s and extension at 72°C for 60 s. PCR products were sequenced using same primers and in both directions to insure the accuracy of base calls. Chromatograms were edited using BioEdit Sequence Alignment Editor Version 7.0.4. 1 (Hall, 1999). All sequences were deposited in GenBank (GenBank Accession N°s: XXXXXXXXXX, Table 1). Sequences for the genera Caridina and Paratya published by Page et al., (T.J. Page, von Rintelen, & Hughes, 2007) were included in our analysis, Paratya sequences being used as outgroup.

(Table 1 here) Sequence and phylogenetic analyses.

DNA sequences were aligned using MEGA6 software [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF] with Muscle algorithm [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF].

Since non-coding markers can be difficult to align, two datasets were produced; a first one was obtained by analyzing our alignment through GBLOCKS [START_REF] Talavera | Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[END_REF] in order to remove poorly aligned portions of sequences, and a second by leaving the sequences as they were after the alignment.

Using Bayesian information criterion in PartitionFinder [START_REF] Lanfear | PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses[END_REF] we retained the GTR + G + I model for both datasets. Best-scoring ML trees were estimated for each dataset using RAxML HPC2 v.8.2.4 [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] on Teragrid v.7.2.7, implemented in the Cyber Infrastructure for phylogenetic Research (CIPRES) portal v.3.1. [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF] (https://www.phylo.org/). One hundred independent searches, each starting from distinct random trees, were conducted. Robustness of the nodes was assessed using non-parametric bootstrapping [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF] with 1,000 bootstrap replicates. Bayesian trees were calculated using MrBayes software [START_REF] Huelsenbeck | MrBayes: a program for the Bayesian inference of phylogeny[END_REF] with the previously determined model, running for 5,000,000 generations, a sampling frequency of 1,000 and a burn in of 25%. Support for nodes was determined using posterior probabilities (PP) calculated by MrBayes. We considered a group to be 'moderately supported' if it had a bootstrap support value (B) between 75 and 89% and posterior probability (PP) between 0.95 and 0.97, and 'highly supported' when B ≥ 90% and PP ≥ 0.98.

Morphological comparisons.

The rostrum, the general cephalothorax, the pereiopods 1, 2, 3 and 5 and the abdomen were observed using a stereoscopic microscope. The proportions of the various joints of the appendages were measured using microphotographs and the AnalySIS Works software (Olympus).

The examinations were made on the type specimens of C. buehleri and C. gueryi, and on a total of 12 specimens grouped by locality (6 from Sulawesi, 2 from Kolobangara and 4 from Malekula). The ranges were then compared side by side and a global range of all the specimens was given. Drawings were made using the "Digital Inking" method (Coleman 2003;Coleman 2006) by tracing vectorial paths on high resolution photographs using Adobe Illustrator (CS6) and a WACOM MPTZ-1230 graphic tablet. SEM micrographs were obtained from ethanol preserved specimens, some treated with 10% bleach for a night in order to remove the setae.

RESULTS

Molecular results.

A 16S gene fragment was successfully sequenced for seven specimens (Table 1). Sequences included 490 bp, containing 10 variable sites. After alignment with 87 atyid sequences ( 83Caridina and 4 Paratya as outgroup) from Page et al. (T.J. [START_REF] Page | An island in the stream: Australia's place in the cosmopolitan world of Indo-West Pacific freshwater shrimp (Decapoda: Atyidae: Caridina)[END_REF], the 16S datasets of 94 sequences of 513 bp included 193 variable sites, of which 174 were phylogenetically informative. After removal of ambiguous blocks, 374 bp of sequence remained to be used in phylogenetic analyses (73% of the original 513 positions), of which 131 bp were variable and 112 bp phylogenetically informative.

No supported conflict was found between full-length and reduced datasets, however, both the BI and ML trees produced using the full-length dataset (Figure 1) were overall more supported than the trees produced using the reduced dataset (Appendix 1). Therefore only the results obtained for the full-length dataset are presented here.

In each of the BI and ML analysis, the consensus tree showed C. buehleri and C. gueryi as a monophyletic and highly supported group (PP=0.99; B=97%), which was well differentiated from all other Caridina analyzed (p-distance to the closest relative: 12.3%), and appeared as belonging to the C. weberi species complex. Within this monophyletic group, sequences from C. buehleri and C. gueryi were clustered in 2 well supported genetic groups; the first one (PP=0.94; B=100%) including 2 specimens identified as C. buehleri from Indonesia, and the second one (PP=0.99; B=100%) including 3 specimens identified as C. gueryi from Vanuatu, 1 specimen identified as C. buehleri from Indonesia and 1 specimen identified as C. buehleri too, from Solomon Islands. Clade 1 is 2% (p-distance) distant from Clade 2.

(Figure 1 First pereiopod stout, chela about 1.9-2.7 times as long as wide, dactylus 1.3-2.9 times as long as wide, 0.4-0.8 length of palm; tips of both fingers with prominent claws, carpus 1.2-1.7 times as long as wide.

Second pereiopod more slender and longer than P1; chela 2.2-3.2 times as long as wide, dactylus 3.0-4.8 times as long as wide, 1.0-1.6 times length of palm, tips of fingers rounded, carpus slender 4.4-7.0 times as long as wide.

Third pereiopod slender, dactylus 2.7-4.0 times as long as wide, terminal spiniform setae included) with 5-8 spiniform setae on flexor margin in addition to the terminal one; propodus 8.0-14.0 times as long as wide, 3.0-5.3 times as long as dactylus.

Fifth pereiopod with dactylus 3.3-5.6 as long as wide with 26-36 spiniform setae on flexor margin; propodus 9.4-20.5 times as long as wide, 3.8-6.2 as long as dactylus.

Endopod of first pleopod with well developed appendix interna in male specimens; Appendix masculina on second pleopod reaching to about 0.6 times length of endopod; appendix interna reaching about 2/3 of appendix masculina. Telson ending in broadly convex margin bearing a median projection; distal margin with 5-10 long plumose setae flanked by one pair of stout spiniform setae. Uropodal diaeresis with 16-20 short cuspidate teeth. Preanal carina high, without a spine. Eggs size: 0.36-0.49 x 0.23-0.32 mm. Distribution. C. buehleri is known from Indonesia (Sulawesi), Papua New Guinea (New Ireland), Vanuatu (Santo, Malekula), Western Samoa (Upolu) and Solomon islands (Kolobangara) (Figure 3).

(Figure 3 here) Habitat. All specimens were collected in a typical brackish water environment (brackish water pools or lower part of rivers, near the estuary). The species is rarely found over the superior limit of the tide-influenced zone, and was not found in the true freshwater zone.

Live colour pattern.

This species shows a rather specific pattern of seven white stripes (one on each somite and one on the cephalothorax), upon a varying colour that tends to match the environment, ranging from bright yellow to black, with reddish dots (Figure 4).

(Figure 4 here)

Morphological comparison

A total of 24 variables were obtained for each group and were compared in Table 2. It appears that all of the variations of the measures are globally overlapping, and no significant differences were observed.

(Table 2 here) DISCUSSION

Caridina gueryi, a junior synonym of C. buehleri.

Genetic results showed that specimens attributed to both of the two species clustered in the same clade, and that the two groups were too close (2% p-distance) to support the hypothesis of separate species, and thus suggesting that one of the two species names has to be synonymized. After morphological examination of both the type specimens and the populations from the known distribution of the two species (Sulawesi, Vanuatu and Solomon Islands), we found that no morphological feature could split our specimens in two clear groups. Through the integrative taxonomy approach applied here, we regard both populations as belonging to C. buehleri, since no morphological or ecological difference was observed between them. Therefore, our results suggested that C. gueryi Marquet, Keith & Kalfatak, 2009 should be treated as a junior synonym of C. buehleri Roux, 1934.

Diagnostic morphological characters of C. buehleri

From our morphological observations we found 4 very specific characters that are diagnostic of this species and allow to separate it from all other Caridina species: 1) a long styliform rostrum, reaching the end of scaphocerite, distodorsal margin of the rostrum unarmed without apical teeth, with 8-16 teeth on basal part of dorsal margin, 1-4 of them situated on carapace behind orbital margin and 3-6 teeth on ventral margin; 2) the stylocerite is long, reaching at least about half of the second segment of antennular peduncle; 3) the first pereiopod is stout, with a very short dactylus and both fingers of chela with prominent distal claws; 4) a high pre-anal carina with an indistinct tooth. According to [START_REF] Richard | Decapoda: Caridea: Atyoidea: Atyidae) and the synonymy by Johnson[END_REF], the number of teeth and their placement on the rostrum (character 1) remain an important identification character, in contrast to its length, which has been shown to be greatly plastic in atyid shrimps [START_REF] Jugovic | The atyid shrimp (Crystacea: Decapoda: Atyidae) rostrum: phylogeny versus adaptation, taxonomy versus trophic ecology[END_REF]. Indeed, we found that the disposition of the teeth on the rostrum of C. buehleri is quite characteristic (without an apical dorsal tooth and few ventral teeth) and cannot be confused with any other species of the genus. The long stylocerite reaching to the second segment of the antennular peduncle (character 2) is also found in C. serratirostris De Man, 1892, C. celebensis De Man, 1892, C. lobocensis Cai, Choy & Ng, 2009 and in all members of the Caridina serrata species group [START_REF] Cai | A revision of the Caridina serrata species group, with descriptions of five new species (Crustacea: Decapoda: Caridea: Atyidae)[END_REF][START_REF] Klotz | To "bee" or not to be-on some ornamental shrimp from Guangdong Province, Southern China and Hong Kong SAR, with descriptions of three new species[END_REF].

However, these species can be easily differentiated from C. buehleri by the rostrum denticulations and the proportions of the carpus of the first pereiopod for C. serratirostris and C. celebensis, the spines of the telson, the shape of the male first pleopod and the size of the eggs for species of the C. serrata group (Klotz et al. 2007) and the setae of the fifth pereiopod for C. lobocensis. The chelae of the first pereiopods exhibit a distinctive claw on both fingers (character 3). This character was not reported by Roux (1934) in the original description of C. buehleri nor in C. gueryi. It might thus be more widely distributed in this genus than it appears, as it is quite difficult to see, hidden in the setae. According to Klotz et al. (2007), C. buehleri shows a high pre-anal carina with an indistinct tooth (character 4). [START_REF] Marquet | Caridina gueryi, a new species of freshwater shrimp (Decapoda, Atyidae) from Santo island, Vanuatu[END_REF] have reported a spine on this structure in C. gueryi. After re-examination of the holotype of C. gueryi, it appears that there is actually no tooth on the pre-anal carina and that the carina is similar to the one that C. buehleri possesses.

Perspectives on the taxonomy of Caridina: C. buehleri, part of the C. weberi complex?

Our single-gene phylogenetic analysis (Figure 1) corroborated the statement of Roux (1934), identifying C. buehleri as closely related to C. weberi De Man, 1892 and C. multidentata Stimpson, 1860, and more generally to species traditionally included in the C. typus group (sensu [START_REF] Bouvier | Recherches sur la Morphologie, les variations et la Distribution systématique des Crevettes d'eau douce de la famille des Atyidés[END_REF]. This whole group includes species with rather stout bodies, short antennular peduncle, short sixth abdominal segment, a high pre-anal carina and a high number of spinules on the uropodial diaeresis.

In the present work (Figure 1), this group was genetically supported by BI (PP = 0.99), thus suggesting the existence of a C. weberi species complex in the same way that there exist species complexes for the groups C. nilotica (Karge & Klotz, 2008), C. serrata [START_REF] Cai | A revision of the Caridina serrata species group, with descriptions of five new species (Crustacea: Decapoda: Caridea: Atyidae)[END_REF]; [START_REF] Klotz | To "bee" or not to be-on some ornamental shrimp from Guangdong Province, Southern China and Hong Kong SAR, with descriptions of three new species[END_REF], and C. gracilirostris [START_REF] Cai | A revision of the Caridina gracilirostris De Man, 1892, species group, with descriptions of two new taxa (Decapoda; Caridea; Atyidae)[END_REF]. Indeed our sequences clustered with C. cf. weberi from Moorea in both datasets, but also with C. weberi longicarpus from New Caledonia, C. multidentata from Taiwan, C. multidentata from Madagascar and Caridina cf. weberi papuana from Solomon Islands, in the BI analyses.

Caridina sumatrensis from Indonesia and Caridina aff. weberi from Papua New Guinea are not supported in this group, but due to the low resolution of the node that bring them together with the "C. weberi complex", some further analyses are needed. Caridina weberi from Micronesia and Caridina aff. weberi from Vanuatu are far from this group in the tree and are most likely a misidentification. Multi-locus phylogenetic approach combined with morphological and ecological data is highly needed to clarify the taxonomy of Caridina.

New insights on the biology of C. buehleri

Caridina buehleri exhibits a stout first pereiopod with both fingers showing a prominent claw (Figure 5). These features are also found in other species of Caridina such as C. typus (Klotz, pers. com.). Clawed fingers might prove advantageous in feeding on discrete particles, as shown in Limnocaridina species [START_REF] Fryer | Evolution in ancient lakes: radiation of Tanganyikan atyid prawns and speciation of pelagic cichlid fishes in Lake Malawi[END_REF] or small invertebrates, like oligochaetes or juvenile insect or larvae, while species without clawed finger tips are primarily feeding on detritus or single-celled periphyton (Klotz et al. 2007). This morphological feature could be linked to a predatory behaviour by scrapping fixed invertebrates that evolved as an adaptation to the strong competition in the part of the rivers where C. buehleri lives.

(Figure 5 here) It is interesting to note that C. buehleri is a species inhabiting brackish water environment, and that ovigerous females carry small and numerous eggs, indicative of a prolonged larval development. A wide distribution range could therefore be expected [START_REF] Von Rintelen | News from a small island -first record of a freshwater shrimp (Decapoda, Atyidae, Caridina) from Peleng, Banggai Islands, Indonesia[END_REF] rather than a scattered distribution as known until today. Further fieldworks are necessary to collect more specimens and get a better picture of the extent of the distribution range of C. buehleri, whose identification should be facilitated by this study.

AKNOWLEDGEMENTS

Legends to figures

  Abbreviations forMuseums. Muséum national d'Histoire naturelle, Paris: MNHN; Naturhistorisches Museum Basel, Basel: NMB; Museum für Naturkunde, Berlin: ZMB.

(

  Figure 2 here)Caridina gueryi IU-MNHN-2015-23 Marquet, Keith & Kalfatak, 2009 Vanuatu, Malakula Island, Brenwe river, 16°07.849'S 167°17.342'E; coll. Kalfatak, Keith & Lord, 20-11-2008, det. Marquet : 4 ovigerous females (CL 5-6 mm).Diagnosis. Carapace smooth, glabrous, with sharp antennal spine placed at lower orbital angle. Pterygostomial margin blunt. Rostrum long, 0.5-1.4 of CL, styliform, reaching to the end of scaphocerite, curved upwards, distal margin unarmed without apical teeth, armed with 8 to 16 teeth on basal part of dorsal margin, 1-4 of them situated on carapace behind orbital margin, ventral margin with 2-7 teeth. Stylocerite sharp, long, reaching to about half of second segment of peduncle.

Figure 1 :

 1 Figure 1: Caridina buehleri, holotype female (cl 7 mm): a. cephalothorax; b. first pereiopod;c. tips of fingers of first pereiopod; d. second pereiopod; e. fifth pereiopod; f. dactylus of fifth pereiopod. Caridina gueryi holotype female (cl 6.4 mm) g. third pereiopod; h. dactylus of third pereiopod; i. preanal carina.

Figure 2 :

 2 Figure 2: Collecting sites of Caridina buehleri in Pacific Ocean.

Figure 3 :

 3 Figure 3: A, C, D = Caridina buehleri from Kolobangara, Solomon Islands (Photo P. Keith) respectively specimen CA1519, and 2 not sequenced specimens and; B = Caridina gueryi from Santo, Vanuatu (Photo P. Keith), not sequenced; E = Caridina buehleri from Sulawesi, Indonesia (Photo H. Evers), not sequenced.

Figure 4 :

 4 Figure 4: Molecular phylogeny obtained from the full dataset showing the position of Caridina buehleri (16S). Numbers above branches are the Bayesian posterior probabilities; numbers under branches are the bootstrap from the Maximum Likelihood analysis. Dark grey shading indicates the genetic "C. weberi species complex" and light grey shading indicates taxa morphologically close to the "C. weberi species complex". Locality codes: AUS = Australia; FRP = French Polynesia; HKG = Hong Kong; HOW = Lord Howe Island; IND = Indonesia; MAD = Madagascar; MAL = Malaysia; MIC = Micronesia; NC = New Caledonia;

Table 1 : Accession number Specimen ID Collection ID Locality Date CollectorsTable 2 :

 12

				Unarmed distal
				portion of dorsal
		Rostrum times	Dorsal teeth (Ventral	margin / armed
		length of cl	teeth)	portion	Teeth placed on carapace
	C. buehleri
	Holotype cl 7 mm	1.3	15 (5)	0.5	4
	C. gueryi
	Holotype cl 6.4 mm	0.8	12 (6)	0.7	3
	Allotype cl 3.2 mm	0.9	16 (5)	0.3	3
	Sulawesi specimens N = 6	0.7 -1.4	13 -15 (4 -7)	0.33	3 -4
	(Klotz et al., 2007)
	Kolobangara specimens N =	0.5	12 -14 (2)	0.4	3 -4
	2
	Malekula specimens N = 4	0.7	14 -16 (3 -5)	0.2 -0.5	3 -4
	Global range	0.5 -1.4	12 -16 (2 -7)	0.2 -0.7	3 -4
		P1	P1	P1	P1
		Dactylus times	Chela times	Carpus times	Dactylus times
		as long as wide	as long as wide	as long as wide	length of palm
	C.buehleri
	Holotype cl 7 mm	2.1	2.2	1.6	0.7
	C. gueryi
	Holotype cl 6.4 mm	2.9	2.1	1.5	0.7
	Allotype cl 3.2 mm	2.5	2.3	1.7	0.7
	Sulawesi specimens N = 6	1.3 -2.3	1.9 -2.3	1.2 -1.7	0.4 -0.7
	(Klotz et al., 2007)
	Kolobangara specimens N =	2.4 -2.8	2.2 -2.4	1.4 -1.7	0.5 -0.7
	2
	Malekula specimens N = 4	2.3 -2.7	2.2 -2.4	1.5 -1.7	0.6 -0.8
	Global range	1.3 -2.9	1.9 -2.4	1.2 -1.7	0.4 -0.8

P2 Dactylus times as long as wide P2 Chela times as long as wide P2 Carpus times as long as wide P2 Dactylus times length of palm

	C.buehleri
	Holotype cl 7mm	4.0	2.7	4.4	1.4
	C. gueryi
	Holotype cl 6.4 mm
	Allotype cl 3.2 mm

We thank the following persons and institutions for help and assistance during the various trips and expeditions to Vanuatu: the Environment Unit of Vanuatu, especially J. White, R.

Nari, and Ernest Banni, for their interest and concerns for the conservation of flora and fauna native to Vanuatu, Mr Alsen Obed and Mr Glen Alo, Fisheries Office in Santo; Mr Kenery Alvea, Environment Officer, Sanma Province, Ms. Rollenas Tavue, Vatthe Office, and all people of Vanuatu along the rivers prospected for their kindness. The SANTO 2006 Expedition was organized by Muséum national d'Histoire naturelle, Paris, Pro Natura International (PNI), and Institut de Recherche pour le Développement (IRD). It operated under a permit granted to Philippe Bouchet (MNHN) by the Environment Unit of the Government of Vanuatu. During the expedition, the scientists operated under the "Forests, Mountains, Rivers" theme coordinated by Bruno Corbara, Philippe Keith and Jérôme Munzinger. We would like to acknowledge the customary landowners, villages and tribes of Hunda-Kena, Jack Harbour, Poitete, Lodumoe and Vanga who welcomed us on Kolobangara Island and for allowing the expedition team to enter their customary lands. We would also like to thank KIBCA and ESSI for the meetings in the villages prior to the expeditions and for the organisation of the trip. Thanks to Rex Loka and Piokera Holland, our very efficient partners on the Kolobangara field work. Thanks also to the MNHN and the BOREA team. The authors also wish to thank Dr Urs Wuest and Edi Stöckli (NMB) for providing holotype of C. buehleri, Dr. Oliver Coleman (ZMB) for providing specimens of C. buehleri collected by Thomas von Rintelen, Paula Martin-Lefèvre and Laure Corbari (MNHN) for providing holotype and paratypes of C. gueryi and Géraldine Toutirais of the Plateau Technique de Microscopie Electronique et de Microanalyses of the MNHN for providing the SEM micrographs.

Finally, thanks to Tim Page and an anonymous reviewer for their precious comments and suggestions on the manuscript.