Karen Burga

Anna Maria Becker

Cédric Mondy

Hugues Clivot

Audrey Cordi

Anne- Sophie Foltete

Eric Gismondi

Nelly Jacquet

Stéphane Jomini

Huong Thi

Thuy Ngo

Nubia Quiroz

Guillermo Restrepo

Benjamin Schmidt

Didier Techer

Rosy Toda

Hela Toumi

Philippe Wagner

O Santini

Vasseur P 1&2

Frank, H 1&2

Article 2: Copper effects on Na + /K + -ATPase and H + -ATPase in the freshwater bivalve Anodonta anatina

Keywords: calcium homeostasis, copper, Na + /K + -ATPase, H + -ATPase, Anodonta anatina Anodonta cygnea metal homeostasis, freshwater bivalve, phytochelatins, Anodonta cygnea 8 Article 4: Phytochelatins, a group copper, freshwater bivalve, phytochelatins, Anodonta cygnea, metal tolerance

Effects of copper on the activities of the cell plasma membrane H + -ATPase and Na + /K + -ATPase of the freshwater mussel Anodonta anatina were assessed after 4, 7, and 15 days of exposure to Cu 2+ at the environmentally relevant concentration of 0.35 µmol L -1 . The H + -ATPase was measured in the mantle, and the Na + /K + -ATPase in the gills, the digestive gland and the mantle. The Na + /K + -ATPase activities showed significant inhibition upon 4 days in the gills (72 %) and in the digestive gland (80 %) relative to control mussels: in the mantle, no inhibition of the enzyme activities was noted. Incipient recovery of the Na + /K + -ATPase activity was registered after 7 and 15 days in the gills and the digestive gland yet, in the gills not returning to basal level, within 15 days. H + -ATPase activity remained unaffected by Cu 2+ at the test concentration.

 [START_REF] Kang | Metallothionein redox cycle and function[END_REF], MT: metallothionein, ROS: reactive oxygen species, GSH: glutathione, GSSG: glutathione disulfide 61 Fig. 8: Fig. 8: PC synthesis [START_REF] Vatamaniuk | Worms take the 'phyto' out of 'phytochelatins[END_REF] 62 Peak "a" is an unidentified compound originating from the derivatization reaction with reagent. Standard concentration was 10 µmol L -1 for cysteine (Cys), glutathione (GSH), -glutamylcysteine ( -GluCys), 5 µmol L -1 for the internal standard N-acetylcysteine (NAC), and 2 µmol L -1 for phytochelatins 2-5 (PC 2-5 ). 114 Peak "a" is an unidentified compound originating from the derivatization reaction with reagent. Standard concentration was 10 µmol L -1 for cysteine (Cys), glutathione (GSH), -glutamylcysteine ( -GluCys), 5 µmol L 1 for the internal standard N-acetylcysteine (NAC), and 2 µmol L -1 for phytochelatins 2-5 (PC 2-5 ). 128 List of tables Background Table 1: Functions of the main cuproproteins (Tapiero et al., 2003) 40

Extended summary

Extended summary

Table 1: Basal enzymatic activities of plasma membrane ATPase(s) (µmol P i /mg protein/min) and cytoplasmic carbonic anhydrase (U/mg protein) in Anodonta anatina. 72 Table 2: Basal levels of phytochelatines 2-4 (µg PC/g tissue wet weight), -GluCys (µg -GluCys/g tissue wet weight), and metallothionein (mg MT/g protein) in Anodonta cygnea. 76

Article 1 Table 1: PMCA activities (µmol P i -1 L -1 mg -1 protein min -1 ) in different tissues of freshwater and marine organisms.

88 Table 2: Carbonic anhydrase activities in freshwater and marine organisms evaluated by measurement of pH decrease caused by enzymatic hydration of CO 2 . 89

Article 2 Table 1: Plasma membrane Na + /K + -ATPase and H + -ATPase activities (µmol/L P i /mg protein/min) in different tissues of freshwater and marine organisms. 103

Article 3 Table 1: Average retention time in minutes (n = 20) ± SD, limit of detection (LOD), and limit of quantification (LOQ) in pmol per 20 µL injected for cysteine-rich metal-binding peptides standards. Standard curves were run with 7 concentrations: 1 to 10 µmol L -1 for Cys, GSH, -GluCys, and 0.2 to 2 µmol L -1 for PC 2-5 . 112 Table 2: PC content [µg PC g -1 tissue wet weight] in digestive gland and gills of Anodonta cygnea, means (n = 6) ± SD. 117

Article 4 Table 1: -GluCys and MT content in the digestive gland and the gills of A. cygnea exposed to 0.35 µmol L -1 Cu 2+ for 0 h, 12 h, 48 h, 4 d, 7 d, and 21 d. Means of results (n = 6) ± SD. *= significant difference between exposed group and its respective control (Kruskal-Wallis and Mann-Whitney two sided test (n = 6, = 0.05)) 131

Summary

Copper (Cu) is one of the metals contaminating European fresh water ecosystems. Filter feeding bivalves have high bioaccumulation potential for transition metals as Cu. While copper is an essential micronutrient for living organisms, it causes serious metabolic and physiological impairments when in excess.

The objectives of this thesis are to get knowledge on toxic effects and detoxification mechanisms of copper in Anodonta cygnea and Anodonta anatina, two mussel species widely distributed in continental waters. Because Ca plays a fundamental role in shell formation and in numerous biological processes, Cu 2+ effects on cellular plasma membrane calcium transport were studied first. In the second step, the investigations focused on Cu 2+ detoxification mechanism involving cysteine (Cys) rich compounds known to play a major role in homeostasis of essential trace metals and in cellular metal detoxification.

Under our experimental conditions, copper inhibition of Ca 2+ -ATPase activity was observed in the gills and the kidneys, and inhibition of Na + /K + -ATPase in the gills and the digestive gland (DG) upon 4 d of exposure. At day 7 of exposure to environmental Cu 2+ concentrations total recoveries was observed in the kidneys and the gills for Ca 2+ -ATPase activity, and in the DG for Na + /K + -ATPase, but not at high doses. Ca and Na transport inhibition may entail disturbance of osmo-regulation and lead to continuous under-supply of Ca. Recoveries of Na + /K + -ATPase and Ca 2+ -ATPase enzymes function suggest that metaldetoxification is induced.

Phytochelatins (PC) are Cys-rich oligopeptides synthesised by phytochelatin synthase from glutathione in plants and fungi. Phytochelatin synthase genes have recently been identified in invertebrates; this allows us to hypothesize a role of PC in metal detoxification in animals.

In the second part of this work, PC and their precursors as well as metallothionein were analyzed in the gills and in the DG of Anodonta cygnea exposed to Cu 2+ . Our results showed for the first time the presence of PC 2-4 in invertebrates. PC were detected in control mussels not exposed to metal, suggesting a role in essential metal homeostasis. Compared to control, PC 2 induction was observed during the first 12 h of Cu 2+ exposure. Those results confirm the role of PC as a first line detoxification mechanism in A. cygnea.

Key words: calcium homeostasis, copper, Anodonta freshwater bivalve, phytochelatins Zusammenfassung Kupfer ist eines der Übergangs-Metalle, die in Süsswasser-Ökosystemen Europas am weitesten verbreitet sind und in Konzentrationen auftreten, die ökotoxikologische Bedeutung haben können. Die filtrierenden, zweischaligen Muscheln haben die Fähigkeit, solche Übergangs-und Spuren-Metalle wie Kupfer durch Bioakkumulation anzureichern, da Kupfer auch ein wesentliches essentielles Metall für alle lebenden Organismen ist; bei übermässiger Belastung mit diesem Spuren-Metall werden jedoch schwere metabolische und physiologische Störungen hervorgerufen.

Diese Doktorarbeit zielt darauf ab, die Kenntnisse über toxische Wirkungen sowie Entgiftungsmechanismen von Kupfer in Anodonta cygnea und Anodonta anatina zu erweitern, zwei Arten von zweischaligen Süsswassermuscheln, die in Gewässern Europas weit verbreitet sind. Da Calcium eine fundamentale Rolle für die Zusammensetzung der Muschel-Schalen sowie bei zahlreichen biologischen Prozessen spielt, wurden zuerst die Wirkungen von Cu 2+ auf den zellulären Transport von Calcium auf der Ebene der Plasmamembran studiert. Dann wurde der Schwerpunkt der Studie auf die Entgiftungsmechanismen des Kupfers gelegt, wobei cysteinreiche Peptide und Proteine im Vordergrund stehen. Cystein ist bekannt für seine Rolle als funktionelles Element für die Homöostase der essenziellen Spurenmetalle sowie für die zelluläre Metallentgiftung.

Unter den gewählten experimentellen Bedingungen wurde nach vier Tagen Cu 2+ -Exposition bei 0,35 µmol L -1 die Hemmung der Ca 2+ -ATPase in den Kiemen und Nieren und die Hemmung der Na + /K + -ATPase in den Kiemen und der Mitteldarmdrüse beobachtet. Nach sieben Tagen Exposition wurde die Erholung der Enzymtätigkeit in den Nieren und in den Kiemen für die Ca 2+ -ATPase beobachtet, in der Mitteldarmdrüse für die Na + /K + -ATPase. Die Erholung der Enzymtätigkeiten weist auf die Induktion der Metallentgiftungs-Kapazität hin. Bei doppelt so hoher Cu-Konzentration dauerte die Hemmung aller genannten Enzyme über den gesamten Expositionszeitraum von 15 Tagen an. Die Hemmung des Transports von Calcium und Natrium kann Störungen in der Osmoregulation verursachen und zu einem Defizit an Calcium führen.

Phytochelatine (PC) sind cysteinreiche Polypeptide, die in Pflanzen und Hefen durch PC Synthase aus Glutathion synthetisiert werden. Gene, die zu funktionellen PC Synthasen führen können, wurden in Wirbellosen identifiziert, was uns annehmen ließ, dass PC auch bei Tieren eine Rolle bei der Metallentgiftung spielen könnte. Daher wurden im zweiten Teil der Arbeit PC und deren Vorläufer sowie die Metallothioneine in den Kiemen und in der Mitteldarmdrüse von kupferexponierten Anodonta cygnea analysiert. Die Ergebnisse zeigen zum ersten Mal die Präsenz von PC 2-4 in Wirbellosen. Die PC wurden auch in Kontrollmuscheln gefunden, was auf ihre Rolle bei der Homöostase essenzieller Metalle hinweist. Eine Induktion von PC 2 wurde in den ersten 12 Stunden der Cu 2+ -Exposition beobachtet, eine Bestätigung seiner Rolle als erster Metallentgiftungsmechanismus in A. cygnea.

Schlüsselwörter: Calcium Homöostase, ionisches Kupfer, Zweischalige Süsswasser-Muscheln Anodonta, Phytochelatine Résumé Le cuivre est l'un des métaux contaminants des écosystèmes d'eau douce le plus représenté en Europe. Les bivalves filtreurs ont une grande capacité de bioaccumulation des métaux de transitions tel que le cuivre. Le cuivre est un oligo-élément essentiel pour les organismes vivants, mais en excès il provoque de graves perturbations métaboliques et physiologiques.

L'objectif de cette thèse est d'acquérir des connaissances sur les effets toxiques et les mécanismes de détoxification du cuivre chez Anodonta cygnea et Anodonta anatina, deux espèces de bivalves dulcicoles largement distribuées dans les eaux continentales. Parce que le calcium joue un rôle fondamental dans la composition de la coquille et pour de nombreux processus biologiques, les effets du Cu 2+ ont été étudiés d'abord sur le transport cellulaire du calcium au niveau de la membrane plasmique. Dans un deuxième temps, l'étude a été axée sur les mécanismes de détoxification du Cu 2+ impliquant des composés riches en cystéine (Cys), connus pour jouer un rôle majeur dans l'homéostasie des métaux traces essentiels et dans la détoxification des métaux dans les cellules.

Dans nos conditions expérimentales (0,35 et 0,64 µmol L -1 ), l'inhibition de la Ca 2+ -ATPase par le Cu 2+ a été observée dans les branchies et les reins, et l'inhibition de la Na + /K + -ATPase dans les branchies et la glande digestive, après 4 jours d'exposition. Au delà de 7 jours d'exposition à la concentration de 0,35 µmol L -1 Cu 2+ , une récupération totale de l'activité enzymatique a été observée dans les reins et les branchies pour Ca 2+ -ATPase, et dans la glande digestive pour la Na + /K + -ATPase. A dose élevée (0,64 µmol L -1 ), l'inhibition persiste. L'inhibition du transport du calcium et du sodium peut entraîner des perturbations de l'osmorégulation et conduire à des carences en calcium. La récupération de l'activité enzymatique de la Ca 2+ -ATPase et de la Na + /K + -ATPase suggère une induction de fonctions de détoxification des métaux.

Les phytochélatines (PC) sont des oligopeptides riches en Cys synthétisés par la phytochélatine synthase à partir du glutathion, chez les plantes et les champignons. Des gènes codant pour des phytochélatine synthases fonctionnelles ont étés identifiés chez des invertébrés tel que le ver de fumier. Ceci nous a incités à rechercher la présence de PC dans les bivalves. Nos résultats ont montré pour la première fois, la présence de PC 2-4 chez les invertébrés. Les PC ont été détectés dans des moules témoins non exposées aux métaux, ceci suggère une fonction dans l'homéostasie des métaux essentiels. Nous avons donc étudiés leur rôle éventuel dans la détoxification des métaux chez ces organismes et les animaux en général. Jusqu'ici les phytochélatines étaient considérées jouer un rôle uniquement chez les végétaux.

Dans la seconde partie de ce travail, les PC et leurs précurseurs, ont été recherchés dans les branchies et la glande digestive d'Anodonta cygnea exposé au Cu 2+ . Une induction de PC 2 a été observée dés les 12 premières heures d'exposition au Cu 2+ , comparé aux bivalves témoins. Ces résultats confirment le rôle du PC en tant que mécanisme de première ligne de détoxification des métaux chez A. cygnea. Les métallothioneines ont été analysées en parallèle, mais aucune induction n'a été prouvée en présence de cuivre. (Tapiero et al., 2003). Les propriétés qui rendent ce métal essentiel Les bivalves sont en contact étroit avec leur environnement, ils sont largement utilisés pour la surveillance de la pollution dans les écosystèmes aquatiques. Parmi ces animaux, les bivalves Unionidae sont largement utilisés comme organismes indicateurs de la bioaccumulation et des effets toxiques des polluants métalliques et organiques (Winter, 1996;Falfushynska et al., 2009). Pour ces raisons, nous avons choisi comme modèles biologiques pour l'étudier de la toxicité du cuivre, Anodonta cygnea et Anodonta anatina qui appartiennent aux Unionidae. (Coimbra et al., 1993). Dans notre étude des inhibitions de l'activité des Ca L'identification de gènes en mesure de donner des PCS fonctionnelles chez les invertébrés (Clemens et al., 2001;Vatamanuik et al., 2001;Brulle et al., 2008), nous avons fait des recherches de PC chez les organismes animaux. Des gènes homologues de phytochélatine synthase ont été trouvés répandus chez les invertébrés, (Clemens et Peršoh, 2009)

Matériel et méthodes

Acclimatation des bivalves

L'entretien de moules a été décrit en détail dans notre premier article précédent (Santini et al., 2011a)

Exposition au cuivre

Les effets du cuivre ont étés évalués chez A. anatina sur les activités enzymatiques de Ca 2+ -ATPase, Na + /K + -ATPase, H + -ATPase de la membrane plasmique et AC cytosolique. Les activités des Ca 2+ -ATPase, Na + /K + -ATPase, et H + -ATPase ont été déterminées dans chacun des organes à partir du culot C75600 remis en suspension (fig. 1). La quantification du phosphate inorganique libéré lors de la dégradation de l'ATP a été réalisée avec la méthode de Chifflet et al. (1988) par dosage spectrophotométrique du complexe molybdate d'ammonium à 850 nm. L'activité de CA a été déterminée avec la méthode de Vitale et al. (1999) par la mesure de la chute de pH en présence des extraits de tissus et de CO 2 .

Analyse des composés riches en thiol

Résultats

Ca 2+ -ATPase de la membrane plasmique et anhydrase carbonique L'activité moyenne des Ca 2+ -ATPase (± écart type) chez les animaux témoins était de 0,087 ± 0,023 µmol P i /mg de protéine/min dans les reins, 0,45 ± 0,13 dans les branchies, et 0,095 ± 0,03 dans la glande digestive.

Le cuivre a inhibé les activités PMCA dans le rein de façon significative après 4 jours d'exposition à toutes les concentrations testées, soit de 0,26 à 1,15 µmol L -1 Cu 2+ . A 0,35 µmol L -1 Cu 2+ , une récupération de l'activité PMCA a été observée au delà de 7 jours d'exposition. A forte concentration (0,64 µmol L -1 ), une inhibition de 20 % de l'activité PMCA a été observée sur les 15 jours d'exposition sans aucune récupération. Dans les branchies un profil similaire à celui des reins, mais avec des variations non significatives est observé.

Aucun effet significatif de l'éxposition à 0,35 µmol L -1 de Cu 2+ a été noté sur l'anhydrase carbonique, à l'exception d'une légère inhibition, mais non significative, après 15 jours d'exposition.

Na + /K + -ATPase et H + -ATPase

La moyenne Na + /K + -ATPase (± écart type) chez les animaux témoins en septembre était de 0,098 ± 0,006 µmol P i /mg de protéine/min dans les branchies, 0,045 ± 0,007 dans la glande digestive, et 0,042 ± 0,009 dans le manteau. L'activité de la H + -ATPase était de 0,002 ± 0,0012 dans le manteau.

Une inhibition significative de l'activité de la Na + /K + -ATPase par rapport aux témoins a été observée à 4 jours d'exposition à 0,35 µmol L -1 de Cu 2+ dans les branchies (72 % d'inhibition) et la glande digestive (80 % d'inhibition). Une reprise de l'activité est observée entre le quatrième et quinzième jour d'exposition à 0,35 µmol L -1 de Cu 2+ , en fin de test une inhibition (54 %) persiste.

Dans le manteau, l'activité de la Na + /K + -ATPase baisse de la même façon (26 % d'inhibition à 4 jours) mais la différence n'est pas significative par rapport aux témoins.

Aucun effet significatif du cuivre sur la H + -ATPase n'a été noté dans le manteau de moules après 15 jours d'exposition. D'une façon générale et dans tous les tissus, les activités des deux ATPases ont été plus élevées en juillet et septembre par rapport aux valeurs mesurées en janvier, mars et avril.

Métallothionéine, phytochélatines et ses précurseurs

Le temps d'élution moyenne des standards de phytochélatines ont été: PC 2 à 13,09 min, PC 3 à 16,62 min, PC 4 à 18,59 min, et PC 5 à 19,75 min. Des composés marqués au mBBr avec des temps de rétention correspondant aux standards de PC 2 , PC 3 , PC 4 ont été détectés dans les extraits de glande digestive et de branchies de moules témoins. La PC 5 était au dessous de la limite de détection. La PC 2 présentait la concentration la plus forte avec 2,17 ± 0,59 et 0,88 ± 0,15 mg PC 2 / g de poids frais dans les tissus de la glande digestive et des branchies, respectivement. Dans ces deux organes, l'ordre de grandeur des concentrations en PC 2-4 était classé de la façon suivante : PC 2 > PC 3 > PC 4 . Les proportions de PC 2 et de PC 3 sont deux ou trois fois plus élevées dans la glande digestive que dans les branchies, tandis que le niveau de PC 4 est à peu près équivalent dans ces tissus.

Le niveau de PC 2 a nettement et significativement augmenté dans les branchies des moules exposées à 0,35 µmol L -1 de Cu 2+ . Comparé aux bivalves témoins respectifs, une induction de 50 % de PC 2 a été observée dès les 12 premières heures jusqu'à 4 jours

Discussion

Nous avons étudié les effets du cuivre sur les enzymes impliquées dans le transport du calcium et dans les processus de ionorégulation, et les mécanismes de détoxification par phytochélatines et métallothionéines chez les bivalves Anodonta.

Le calcium joue un rôle fondamental dans de nombreux processus biologiques (production d'énergie, métabolisme cellulaire, contraction musculaire, reproduction) et a d'importantes fonctions mécaniques (coquille, squelette) chez les organismes vivants (Mooren et Kinne, 1998). Contrairement aux mollusques des écosystèmes marins qui sont généralement hypoosmotiques et pour lesquels l'absorption du calcium est facilitée par de plus fortes concentrations ambiantes de calcium, les bivalves d'eau douce sont hyperosmotiques et nécessitent une régulation stricte de leur métabolisme calcique.

La présente étude nous a permis de déterminer des niveaux de base des activités enzymatiques de Ca 2+ -ATPase, Na + /K + -ATPase et H + -ATPase de la membrane plasmique et de l'anhydrase carbonique cytosolique dans les organes impliqués dans l'homéostasie du calcium. Il y a un manque de données physiologiques chez les bivalves d'eau douce par rapport aux modèles marins tels que Mytilus edulis ou Mytilus galloprovincialis. Ces données sont nécessaires à la compréhension des mécanismes de transport du calcium puisque les bivalves d'eau douce sont soumis à une osmo-iono-régulation différente des bivalves marins.

Dans notre étude, l'activité de la Ca 2+ -ATPase de la membrane plasmique (PMCA) des branchies d'A. anatina était quatre fois supérieure à celle trouvée chez Mytilus edulis (Burlando et al., 2004). Ceci reflète l'importance du transport actif du calcium chez Anodonta anatina. Les concentrations de calcium en eau douce sont inférieures à celles trouvées dans l'eau de mer, où l'absorption du calcium est plus facile pour Mytilus edulis.

L'activité de la PMCA est également élevée dans les reins et la glande digestive, favorisant l'absorption du calcium provenant des aliments et la réabsorption du calcium dans l'ultrafiltrat rénal. Ces données physiologiques nous ont permis de comprendre dans quelle mesure la PMCA est importante pour l'homéostasie du calcium chez les bivalves d'eau douce par rapport aux organismes marins. En outre, chez les unionidés, le calcium joue un rôle direct dans la reproduction, puisque les glochidies sont incubées dans le marsupium (branchies), il est donc déterminant pour la croissance des populations de moules. La Na + /K + -ATPase maintient le gradient de sodium cellulaire transmembranaire nécessaire pour la diffusion facilitée du calcium par le Na + /Ca 2+ antiporteur. L'augmentation significative de l'activité de la Na + /K + -ATPase a été mesurée dans les branchies et la glande digestive en juillet et septembre par rapport au reste de l'année. Ces deux mois correspondent à la période de biominéralisation chez Anodonta sp. (Moura et al., 2000).

Les reins ont un rôle essentiel dans la filtration et la réabsorption des ions, de l'eau et des molécules organiques de l'ultrafiltrat. Comme les bivalves d'eau douce sont hyperosmotiques, la pression osmotique résultant du gradient de concentration entre les compartiments internes et l'environnement conduit à l'absorption d'eau par osmose et à une perte par diffusion ionique. L'absorption de l'eau est compensée par la production d'urine et la perte ionique est limitée par la réabsorption ionique. Chez les bivalves d'eau douce, la production journalière d'urine est élevée. Les reins jouent un rôle essentiel dans l'homéostasie du Ca en limitant les pertes ioniques dans l'urine, par réabsorption active des ions Ca 2+ dans le filtrat (Turquier, 1994). Une inhibition de la réabsorption de calcium peut donc considérablement perturber l'homéostasie calcique.

Notre étude a montré une inhibition de l'activité enzymatique de la PMCA dans les branchies et les reins chez les moules exposées à 0,35 µmol L -1 de Cu 2+ . Une reprise totale de l'activité de la PMCA a été observée à partir de 7 j d'exposition à 0,35 µmol L -1 de Cu 2+ , mais l'inhibition a persisté à concentration plus élevée (0,64 µmol L -1 ). En raison de problèmes analytiques inhérents à la faible masse des reins, ce tissu a été peu étudié chez les bivalves d'eau douce jusqu'alors. Cet organe joue pourtant un rôle important dans les processus de détoxification (Viarengo et Nott, 1993) ; de plus le rein est essentiel pour la iono-régulation.

Nos résultats ont montré la grande sensibilité de cet organe au Cu 2+ à des concentrations réalistes au plan environnemental (0,35 µmol L -1 = 22,3 µg L -1 ).

Dans le présent travail, une inhibition de la Na + /K + -ATPase a été observée dans les branchies et dans la glande digestive. Une telle perturbation de la iono-régulation pourrait conduire à une carence en calcium ; elle peut également affecter les voies de signalisation cellulaires calciques, en plus de perturber la biominéralisation et la formation de la coquille des moules et des glochidies. Une reprise des fonctions enzymatiques (Ca 2+ -ATPase et Na + /K + -ATPase) suggère qu'un mécanisme de détoxification des métaux est induit. Par conséquent, dans la deuxième partie de cette étude, nous nous sommes concentrés sur les mécanismes de détoxification par les composés riches en Cys, chélateurs de métaux par leur groupement thiol.

Les bivalves dulcicoles unionidés sont largement reconnus pour leur capacité à accumuler dans leurs tissus, une grande variété de contaminants de l'environnement, y compris les métaux (Bonneris et al., 2005). Cette tolérance aux métaux est permise par des stratégies biochimiques impliquant leur séquestration. La séquestration intracellulaire des métaux se fait selon une séquence d'événements en cascade impliquant différents ligands avec une force croissante de liaison vis-à-vis des éléments métalliques. A concentration élevée, les métaux peuvent inhiber ces mécanismes de détoxication. Dans nos résultats, le retour au niveau basal des Ca 2+ -ATPase et Na + /K + -ATPase observé au-delà de 7 jours d'exposition à 0,35 µmol L -1 de Cu 2+ indique des capacités d'adaptation des bivalves, via des systèmes de détoxification efficace à faible concentration de cuivre. A une concentration plus élevée de Cu 2+ (0,64 µmol L -1 ), aucune récupération de l'activité enzymatique n'a été notée. Il est intéressant de remarquer que la récupération a été observée uniquement à faible concentration (0,35 µmol L -1 ) environnementalement pertinente. Ceci souligne l'importance d'utiliser des concentrations environnementales dans la recherche écotoxicologique; l'extrapolation de résultats observés à des doses élevées pour des situations environnementales peut être critiquée en raison des différents mécanismes de toxicité développés à faibles et fortes doses.

L'étude de phytochélatines chez Anodonta cygnea, nous a amené à développer et optimiser un protocole analytique par HPLC pour la quantification des PC dans les tissus animaux, basée sur la méthode de Minocha et al. (2008). Le présent travail a confirmé la présence de phytochélatines chez les invertébrés. A notre connaissance, c'est la première fois que les PC ont été trouvés chez les animaux et chez les invertébrés.

Chez les végétaux, les PC sont rapidement induites dans les cellules et les tissus exposés aux métaux de transition. Les PC jouent un rôle important dans la détoxification des métaux. Les PC pourraient également être impliquées dans l'homéostasie des métaux essentiels (Hirata et al., 2005). Dans notre étude, la PC 2 , PC 3 et PC 4 , ont été détectés dans les branchies et la glande digestive d'A. cygnea chez les témoins non exposés. Dans les deux organes, la PC 2 a été trouvée en concentration la plus forte suivie de la PC 3 elle-même en concentration supérieure à celle de la PC 4 . Les concentrations de PC 2 et PC 3 étaient environ deux à trois fois plus élevée dans la glande digestive que dans les branchies. Le niveau de base des PC 2-4 en l'absence de cuivre et d'autres métaux d'exposition suggère leur rôle dans l'homéostasie des métaux essentiels.

A 12 h d'exposition au cuivre, une induction significative de PC 2 a été observée par rapport aux témoins correspondants dans les branchies et à un moindre niveau dans la glande digestive. Ces résultats confirment le rôle des PC comme chélateur de métaux impliqués en première ligne dans les mécanismes de détoxification chez A.cygnea. Au delà de 7 jours et jusqu'à 21 j, dans les branchies des moules exposées au cuivre, la PC 2 diminue pour revenir au niveau basal, identique à celui des témoins. Cette diminution suggère que la détoxification du cuivre a été prise en charge par d'autres mécanismes, sur le long terme. Chez les Unionidae, les métallothionéines et les granules (concretions intracellulaire insolubles le plus souvent minérale, mais également organiques) sont connues pour jouer ce rôle. Bonneris et al. (2005) ont montré que les concentrations de cadmium, de zinc et de cuivre dans la fraction de granules des branchies étaient significativement corrélées avec les concentrations de ces métaux sur l'environnement. Les granules sont connues pour être des sites privilégiés pour le stockage de cuivre dans les branchies des unionidés. Environ 65 % du cuivre total dans les branchies a été trouvé séquestré dans les granules chez Anodonta grandis grandis, où les concrétions de calcium représentent 51 % du poids sec des branchies. Des valeurs similaires ont été trouvées chez Anodonta cygnea (Bonneris et al., 2005).

Aucune variation significative du niveau de MT n'à été observée lors de l'exposition des bivalves au cuivre durant les 21 jours de la présente étude. L'isoforme de MT (<10 kDa) dans l'extrait de moules élué, après séparation dans nos conditions HPLC, n'a pas été induit par le cuivre. Une détoxification par d'autres isoformes de MT non détectées par notre méthode HPLC chez A. cygnea, ne peut être exclue. En effet un polymorphisme important des MT est connu chez les invertébrés (Amiard et al., 2006).

Les métallothionéines, les granules et les systèmes antioxydants ont été décrits comme étant impliqués dans les mécanismes de détoxication des bivalves d'eau douce. Cossu-Leguille et al. (1997) ont montré le rôle majeur joué chez les Unionidae par les antioxydants et en particulier par le glutathion réduit (GSH) pour la détoxification des métaux. Dans les branchies de Unio tumidus une diminution de 45 % du GSH a été observée chez les moules exposées en site contaminés par des métaux, par rapport aux témoins. Cette diminution du niveau de GSH chez les Unionidae exposés au cuivre a été confirmée par Doyotte et al. (1997) dans des conditions contrôlées de laboratoire, indiquant la séquestration des métaux directement par le groupe SH du GSH ou de son utilisation comme substrat par les enzymes antioxydantes. En effet, une implication en parallèle des enzymes antioxydantes a été décrite. L'activité de ces enzymes augmente lors d'exposition à faibles concentrations en métaux (Vasseur et Leguille, 2004). Le GSH joue également un rôle dans la synthèse des PC. Ce double rôle dans la détoxification directe des métaux, et comme précurseur de synthèse des PC pourrait expliquer cette diminution.

Les populations d'Unionidae représentent la plus grande partie de la biomasse totale dans de nombreux systèmes aquatiques. Ils prennent une part active dans la purification de l'eau, dans la sédimentation, la modification des communautés de phytoplancton et des invertébrés détritivores (Aldridge, 2000). Par conséquent la disparition des Unionidae pourrait produire des perturbations structurelles et fonctionnelles dans les écosystèmes aquatiques. Les effets potentiellement négatifs de la compétition entre bivalves, autochtones et invasifs est une question qui fait débat. Les bivalves d'eau douce invasifs Corbicula fluminea, Dreissena polymorpha n'appartenant pas aux Unionidae colonisent les hydrosystèmes dulcicoles avec des effets préjudiciables pour les autres invertébrés. Ces espèces invasives ne jouent pas le même rôle fonctionnel dans les écosystèmes que les unionidés. Leur présence est l'une des hypothèses expliquant la baisse des populations d'unionidés.

La combinaison de différents facteurs explique la disparition progressive des Unionoidea au profit des espèces invasives. L'appauvrissement en salmonidés qui sont de potentiels poissons hôtes pour les larves d'unionidés est présenté comme une autre hypothèse possible à celle d'une compétition par des éspeces invasives. Les populations de salmonidés sont touchées par la pollution de l'eau, pouvant indirectement renforcer les maladies naturelles tels que le parasitisme connu pour son impact négatif sur les populations de salmonidés (Voutilainen et al., 2009). Les autres principales raisons de ce déclin sont la dégradation physique des ruisseaux par la modification des lits de rivières et canaux, ainsi que la dégradation de la qualité de l'eau. En effet les interactions entre polluants, sont une cause de perturbation même à faible concentration (Vighi et al., 2003). Afin de protéger les populations autochtones d'Unionidae, il est important de déterminer comment et dans quelle mesure ces facteurs sont impliqués dans leur disparition. Les résultats acquis au cours de ce travail de thèse contribuent à la compréhension des effets de la pollution par les métaux sur l'homéostasie du calcium chez les bivalves d'eau douce. La pollution aquatique par les métaux est susceptible d'être l'une des raisons du déclin généralisé des populations d'Unionoidea dans les rivières européennes (Frank et Gerstmann, 2007). Nos résultats sur les effets du cuivre sur A. anatina ont confirmé cette hypothèse.

Conclusion

Les communautés du macrobentos sont d'excellents indicateurs de qualité de l'eau (Ippolito et al., 2010). Les objectifs du notre travail ont été l'étude des effets toxiques du cuivre et des mécanismes de détoxification chez les espèces de bivalves dulsicoles du genre Anodonta appartenant aux Unionidae. Tout d'abord, l'étude du Cu 2+ comme un inhibiteur potentiel des protéines enzymatiques jouant un rôle dans le transport du calcium et les processus biominéralisation a été réalisée chez Anodonta anatina. Deuxièmement, l'étude s'est orientée sur les mécanismes de détoxification du Cu 2+ par les composés riches en Cys chélateurs de métaux chez Anodonta cygnea.

Dans une première partie de la présente étude, les effets de l'exposition au Cu 2+ sur les activités enzymatiques de la Ca 2+ -ATPase, la Na + /K + -ATPase, et la H + -ATPase de la membrane plasmique, et de l'AC cytosolique ont été évalués chez le bivalve dulcicole Anodonta anatina.

Dès 4 jours, une inhibition de l'activité enzymatique de la Ca 2+ -ATPase des branchies et des reins, et de la Na + /K + -ATPase des branchies et la glande digestive, a été observée chez Anodonta anatina exposée à 0,35 µmol L -1 de Cu 2+ . Une récupération totale de l'activité de la Ca 2+ -ATPase a été observée à 7 jours d'exposition à 0,35 µmol L -1 de Cu 2+ , indiquant l'induction de mécanismes de détoxication des métaux. Chez les bivalves exposés à 0,64 µmol L -1 de cuivre aucune récupération de l'activité enzymatique n'a été observée sur 15 jours.

Il serait intéressant d'étudier les conséquences à long terme de la perturbation des fonctions osmorégulatrices des reins chez A. anatina exposés à 0,64 µmol L -1 de Cu 2+ .

Dans un deuxième temps nous avons recherché la présence de phytochélatines chez les bivalves. Au cours de notre recherche nous avons identifié les phytochélatines PC 2 , PC 3 et PC 4 . C'est la première fois que la présence de PC est mise en évidence chez des organismes animaux et chez A. cygnea en particulier. Chez Anodonta cygnea exposés à 0,35 µmol L -1 de Cu 2+ , la PC 2 a été induite par rapport aux témoins à 12 h dans la glande digestive et les branchies, l'induction a persisté après 7 jours dans les branchies. Ces résultats suggèrent un rôle des PC dans l'homéostasie des métaux essentiels, et confirme le rôle des PC en tant que mécanisme de première ligne pour la détoxification des métaux chez A.cygnea.

Le cuivre a été signalé comme un faible inducteur des polypeptides riches en thiols chélateur de métaux (Zenk, 1996). L'exposition à un métal non essentiel et puissant inducteur de PC comme le cadmium pourrait être intéressante pour l'étude de l'induction des PC 3-4 chez A. cygnea. Par ailleurs la comparaison des effets de l'exposition à un métal essentiel comme le cuivre et un métal non essentiel tel que le cadmium pourrait nous permettre de déterminer le rôle joué par les PC dans l'homéostasie des métaux essentiels et les mécanismes de détoxification.

Nos résultats d'analyses de métallothionéine (MT) par HPLC n'ont montré aucune variation de son niveau d'expression après exposition au cuivre. Ceci indique que l'isoforme MT quantifié avec notre méthode n'est pas induit par ce métal. Des analyses complémentaires des MT chez A. cygnea exposés au cuivre dans les mêmes conditions, avec une méthode de spectrométrie qui permet la quantification de la totalité des isoformes de MT seraient intéressantes afin de déterminer si d'autres isoformes de MT sont induites chez cette espèce.

De plus en parallèle, il serait nécessaire d'optimiser notre protocole d'analyse HPLC de manière à séparer et mesurer les isoformes de MT induites par le cuivre chez A. cygnea.

Le cuivre est un inducteur d'espèces réactives de l'oxygène (ERO) à travers la réaction de Fenton. Le rôle antioxydant joué par les PC (Hirata et al., 2005) serait intéressant à étudier pour une meilleure compréhension du mécanisme de détoxification du cuivre par les PC chez A. cygnea.

La pollution chimique est l'une cause de dégradation de l'environnement aquatique responsable du déclin des populations de bivalves dulcicoles. La toxicité dépend non seulement de la biodisponibilité des polluants et de leur toxicité intrinsèque, mais également de l'efficacité des systèmes de détoxification dans l'élimination des espèces chimiques réactives.

Cette thèse est une contribution significative à l'étude des systèmes de détoxification des métaux chez les bivalves dulsicoles A. cygnea et A. anatina.

L'originalité de notre travail est la mise en évidence, pour la première fois, de phytochélatines chez des organismes animaux. Nous avons démontré leur rôle dans la protection vis-à-vis de la toxicité du Cu 2+ , à des concentrations réalistes et pertinentes au plan environmental.

Introduction 1 General introduction

Human activity is associated with the development of industry and agriculture which have become indispensable. These sectors are responsible for the production and release of many pollutants. The chemical and physical properties and the different types of transport determine pollutant diffusion in all compartments of ecosystems. The aquatic environment is a sink for most many pollutants including metals. Copper belongs to the metals most commonly used because of its physical (particularly its electrical and thermal conductivity) and chemical properties. As pure metal, as alloy, or in the ionic state it is employed in many industrial and agricultural sectors. Therefore, copper is a metal frequently detected in the aquatic continental environment where it is present in the water column and accumulates in sediments [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. The chemical properties of copper (including as catalyst) make it essential to many biological processes involving such vital functions as respiration or photosynthesis (Tapiero et al., 2003). The properties that make this metal an essential element are at the same time the reasons for its toxicity when in excess. Copper is bioaccumulative and can become a threat to biocenoses. Mechanisms regulating the copper concentration and detoxification are essential to all living organisms.

Molluscs represent an important group of macroinvertebrates in aquatic ecosystems.

Among this group, bivalves are particularly interesting. Through their important filtering activity to satisfy respiratory and nutrition, bivalves have the capacity to accumulate a variety of environmental contaminants. In freshwater ecosystems they play an important role in matter transfer from the water column to the sediment. Faeces and pseudofaeces of bivalves make the plankton fraction available to detrivores and can change the sediment quality by pollutant sedimentation and concentration. In close contact with their environment, they are widely used for pollution monitoring in aquatic ecosystems. Among these animals, freshwater mussels of the Unionidae family are employed for controlling the bioaccumulation and toxic effects of metallic and organic pollutants (Winter, 1996;Falfushynska et al., 2009). For these reasons we chose Anodonta cygnea and Anodonta anatina which belongs to the Unionidae as biological models to study copper toxicity. These invertebrates are autochtonous species in European hydrosystems, but regression of Unionidae populations, like many other freshwater bivalves, has been observed in the last decades, the main reason for such ecotoxicological studies as this one.

The objective of this thesis was to gain knowledge on the mechanisms of disturbance of calcium metabolism in Anodonta anatina by copper. Calcium is a critical element in the functioning of eukaryotic organisms. It controls multiple processes of reproduction, life, and death (Ermak and Davies, 2001). Absorption, biomineralization, and maintenance of intracellular calcium concentrations are effected and controlled by its passage through cell membranes. This takes place by simple diffusion and also by means of transport proteins. In bivalves, calcium is also very important for exo-skeleton synthesis by biomineralization. In addition to calcium, for biomineralization carbonate ions are required, produced by carbonic anhydrase (CA) catalysis. The effects of Cu 2+ on calcium transport have been tested in Anodonta anatina by assessment of the enzymatic activities of Ca 2+ -ATPase, Na + /K + -ATPase, H + -ATPase of the plasma membrane, and of the cytosolic CA. These enzymes are involved in calcium absorption and in the biomineralization processes. Organs studied were the gills, the digestive gland, the kidney, and the mantle which play an important role in the absorption of calcium and the synthesis of the shell (Coimbra et al., 1993). Enzymatic inhibition in A.

anatina exposed to Cu 2+ at concentrations ranging from 0.26 to 1.15 µmol L -1 was observed.

At low concentration of 0.35 µmol L -1 , a total recovery following the inhibition indicated the induction of detoxication mechanisms.

The second part of this study focused specifically on metal binding Cys-rich compounds. These compounds are oligopeptides with high Cys content such as phytochelatins, or proteins such as metallothioneins. They play a major role in animals and plants as metal chelator, in essential metal homeostasis, and in non-essential metal detoxication. Phytochelatins (PC) are thiol rich oligopeptides with ( -Glu-Cys) n -Gly as general structure synthesised by phytochelatin synthase from glutathione. PC binds metal ions and forms metal complexes that reduce the intracellular free metal ion concentration in cells of plants, fungi, and microalgae. Since the identification of homologous genes able to give a functional phytochelatin synthase in invertebrates (Clemens et al., 2001;Vatamaniuk et al., 2001;Brulle et al., 2008), PC are strongly suspected to play a wider role in trace metal detoxification in animals. In invertebrates including bivalves, PC synthase genes were found to be widespread (Clemens and Peršoh, 2009). Therefore invertebrates such as Unionidae, known to bioaccumulate metals, are likely to express PC. Induction of PC and their precursors and of MT were assessed in the gills and the digestive gland of Anodonta cygnea under Cu 2+ exposure.

This thesis manuscript is divided into eight chapters. After this general introduction, the second chapter contains a literature review on freshwater bivalves and data on copper, on its chemical and biological properties, its distribution in aquatic ecosystems, bioaccumulation, toxic effects, and detoxification mechanisms. An extended summary is presented in the third chapter followed by a conclusion (chapter 4). A published article on the inhibition of Ca 2+ -ATPase and CA activities by Cu 2+ is presented in the fifth chapter, and the sixth chapter is on the inhibition of Na + /K + -ATPase and the H + -ATPase activities; both dealing with the

Origin and use

Copper (symbol Cu, atomic number 29, atomic mass 63.546, two stable isotopes) is a transition metal belonging to the group 11 IB (silver, gold) of the periodic table of elements.

Copper is a ductile metal with good electrical and thermal conductivities. It occurs naturally in oceans, lakes, rivers, and soils. The average natural levels of copper in the Earth's crust vary from 40 to 70 mg kg -1 . Values in ores range from 80 to 150 mg kg -1 (IFEN, 2011).

Copper is found mainly as the sulfides CuS and Cu 2 S in tetrahedrite (Cu 12 Sb 4 S 13 ) and enargite (Cu 3 AsS 4 ) and as the oxide Cu 2 O (cuprite). The most important ore is chalcopyrite (Cu 2 S, Fe 2 S 3 ). It is also found in malachite (CuCO 3 (OH) 2 ), azurite Cu 3 (CO 3 ) 2 (OH) 2 , chalcocite (Cu 2 S), and bornite (Cu 5 FeS 4 ). Since 1980, an increase in global mining production has been observed due to the growing demand for copper in the fields of renewable energy, Copper is commonly used because of its physical properties, particularly its electrical and thermal conductivity and its resistance to corrosion. It is mainly used as metal in electronics (42 %), construction (28 %), and in vehicles (12 %). Seventy percent of pure copper are used for electrical wire laminates, and pipes. In alloys, the main families are the brasses (copper-zinc), the bronzes (copper-tin), copper-aluminium, and copper-nickel blends.

Many other alloys are also in use for special purposes, such as copper-nickel-zinc, coppersilicon, copper-lead, copper-silver, copper-gold, copper-zinc-aluminium-magnesium, as well as copper alloyed with cadmium, tellurium, chromium, or beryllium [START_REF] Blazy | Cuivre: ressources, procédés et produits[END_REF].

Salts of copper at different levels of oxidation are also used for their chemical and biochemical properties.

Copper is encountered in many fields of technical application:

-Electricity, used pure or alloyed in wires of electrical equipment (coils, generators, transformers, connectors) [START_REF] Blazy | Cuivre: ressources, procédés et produits[END_REF].

-Electronics and communication: pure or alloyed it plays an important role in communications technology, computers, and mobile phones [START_REF] Blazy | Cuivre: ressources, procédés et produits[END_REF], in printed circuit boards, in solar cells of solar panels, and in the semiconductor industry.

-Building: copper and brass are used for pipes and plumbing fixtures, in heating and guttering, as copper sheets for cover.

-Transportation: alloys of copper and nickel are used in boat hulls and in anti-fooling paint to reduce hull fouling by algae. It is also in motors, radiators, connectors, and brakes; an automobile contains about 22.5 kilograms of copper. Trains can contain between 1 and 4 tons [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF].

-In various industrial equipments, copper and copper alloys are used in the manufacture of turbine blades, gears and bearings, heat exchangers, tanks and pressure equipment, marine equipment, pressed steel, and smelters. Some commercial preparations are particularly polluting for the aquatic compartment since they contain copper in ionic form directly soluble in water. Salts of copper are used in many preparations and in industrial quantities:

-As, copper acetate, cupric chloride, and copper sulphate for colouring of textiles, glass, ceramics, paints, and varnishes, and for tanning (OECD, 1995). Copper acetate, cuprous and cupric oxide, cuprous and cupric chloride are used as catalysts in petrochemical organic synthesis and for rubber production. They are also used in solder pastes, in electroplating, as polishing agent for optical glass, and for refining of copper, silver, and gold.

-Pyrotechnics, oxides and cupric copper to colour firework, brass in ammunition.

-General consumer products, pool algicides, decorative objects made in copper and its alloys (buttons, zippers, jewelry), coins.

-In the agriculture, the farming of cattle, pigs, and poultry is a large sector, using copper as a dietary supplement [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. Copper acetate, copper sulphate, copper hydroxide, cuprous oxide, and tetracupric oxychloride are used in fungicides, herbicides, insecticides, molluscicides, and as antiseptics. Copper treatment is practiced in vinyards and on fruit trees with average doses from 1000 to 2500 g Cu / ha / year and from 3750 to 5000 g Cu / ha / year respectively (ADEME-SOGREAH, 2007).

-In wood preservation, copper sulphate, cupric oxide, and cupric chloride are used (INERIS, 2005). Copper is also used as a substitute for other substances such as lead, arsenic, and tributyltin. This enumeration illustrates the widespread use of copper.

Sources of emissions and environmental levels

Natural inputs in the order of decreasing importance are volcanic eruptions, plant decomposition, forest fires, and marine aerosols [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. Water and soil surrounding sites of agricultural and industrial activities are most heavily exposed to copper and, to a lesser extent, the air contaminated by road and rail traffic. In the European Union, some countries represent a large part of copper emission such as France with 43% in water, 15% in soil, and 13% in air. According to IFEN (2011), anthropogenic inputs of copper originating from industrial activities are mostly into waters and soils, while urban and agricultural activities as well as road traffic emit mostly into the air. In 2007, the emissions of copper in-to the environment in Europe were estimated as 371,000 kg year -1 to water, 146,000 kg year -1 to air, and 139,000 kg year -1 to soil [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF].

Air emissions are mainly non-industrial. The road transport sector is a major issue representing 51 % of emissions in France in 2004. These are mainly caused by wear of brake pads containing copper. The railway transport sector represents 32 % of total air emissions of copper which is caused by wear of overhead lines (ADEME-SOGREAH, 2007). Emissions of copper-related road transport are increasing with the development of this sector. Industrial discharges of copper are mainly from the synthesis of organic chemicals, in the production of nonferrous metals from ore, ferrous metal smelting, and the production of iron and steel. In the atmosphere, copper metal oxidizes slowly to Cu 2 O which covers the metal with a protective layer against corrosion. Copper is released into the atmosphere as particulate oxide, sulphate, or carbonate as particulate matter.

Releases to the aquatic environment are mainly due to corrosion of equipment made from copper or brass, and urban waste is another important source of release. One of the most significant non-industrial discharges of copper is the treatment of urban wastewater. This sector represents 28 % (15617 kg year -1 ) of the total French emissions of copper and its derivatives to the aquatic environment [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. Copper is one of the compounds always detected in the input and output of secondary sewage treatment plants (but not always in sewage treatment plants with tertiary treatment). Copper is one of the highest concentrated compounds in sewage treatment plant inflow with concentrations generally greater than 10 µg L -1 . At the outlet it is usually found at concentrations between 1 and 10 µg L -1 . In Europe in 2007, the main emitters of industrial origin are the United Kingdom, France, Germany, and Romania. They represent respectively 35 % (129000 kg), 15 % (55700 kg), 11 % (39800 kg) and 7 % (24500 kg) of the European industrial emissions to water [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. In the European Union, the most significant sectors are thermal power plants and other combustion plants. In France, another important sector is the production of nonferrous metals from ore from concentrates and from secondary materials. Speciation and behaviour of copper in the environment will directly influence its bioavailability. In aquatic environments, the fate of copper is influenced by many processes and factors such as chelation by organic ligands (especially on NH 2 and SH groups, to a lesser extent on the OH group). Adsorption phenomena can also occur on metal oxides, clays, or particulate organic matter, bioaccumulation, presence of competing cations (Ca 2+ , Fe 2+ , Mg 2+ )

or of anions (OH, S 2-, PO 4 3- , CO 3 2-
) plays also a role in copper behaviour [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF].

The great portion of copper released into the water is in the particulate form and tends to settle, to precipitate, or adsorb to organic matter, hydrous iron, manganese oxides, or clay particles (ATSDR, 1990;INERIS, 2005). In hard water (carbonate concentration up to 1 mg L -1 ), the largest fraction of copper is precipitated as insoluble compounds. Cuprous oxide Cu 2 O is insoluble in water. Except in the presence of a stabilizing ligand such as sulfides, cyanide, or fluoride the oxidation state Cu(I) is easily oxidized to Cu(II) as CuSO 4 , Cu(OH) 2 and CuCl 2 more soluble in water. The Cu 2+ ion forms many stable complexes with inorganic ligands such as chloride or ammonium, or organic ligands. When copper enters the aquatic environment, the chemical equilibrium of oxidation states and of soluble and insoluble species is usually reached within 24 hours (INERIS, 2005). In aquatic environments, copper is mainly absorbed on particles, and suspended solids are often heavily loaded. In Europe, according to the forum of the European geological surveys (FOREGS, 2010), copper concentrations up to 3 µg L -1 are found in continental water. In regions with intensive human activities (agricultural, urban), copper concentrations of 40 µg L -1 and even 100 µg L -1 can be found seasonally [START_REF] Neal | A summary of river water quality data collected within the Land-Ocean Interaction Study: core data for eastern UK rivers draining to the North Sea[END_REF]Falfushynska et al., 2009). Trace metals as copper are mainly transported to the marine environment by rivers through estuaries. The magnitude of metal input to the marine environment depends on the levels in the river waters and on the physico-chemical processes that take place in the estuaries [START_REF] Waeles | Distribution and chemical speciation of dissolved cadmium and copper in the Loire estuary and North Biscay continental shelf, France[END_REF]. In sea water, copper is found in concentrations ranging from 0.1 to 4 µg L -1 [START_REF] Waeles | Seasonal variations of dissolved and particulate copper species in estuarine waters[END_REF][START_REF] Levet | Agence de l'eau Seine-Normandie[END_REF].

Depositions of copper into soils are from different sources. In respect to industrial activities in Europe, in 2007 the major industrial emitters to soil are the United Kingdom, France, and Germany with respectively 74 tons, 60 tons, and 5 tons. The most significant activities are landfilling or recycling of non-hazardous waste (45 %), abattoirs (21 %) and sewage sludge from urban waste water treatment plants (12 %) (E -PRTR, 2010). The main agricultural sources identified (ADEME-SOGREAH, 2007) for the release of copper are animal wastes (faeces and manure), sewage sludge from water treatment plants, compost, mineral fertilizers, lime and magnesium soil amendment. Animal waste represents an important input into soils (53 %). This reflects the fact that the feed of cattle, pig and poultry production is supplemented with copper for promotion of growth and prevention of diseases.

As copper is poorly absorbed, it is added in great quantities to the feed (sometimes added at levels 30 times in excess of the needs of the animal), and the surplus is then found in the droppings. The spreading of animal manure is important for agricultural soils [START_REF] Jondreville | Le cuivre dans l'alimentation du porc : oligoélément essentiel, facteur de croissance et risque potentiel pour l'Homme et l'environnement[END_REF]. Thirty four % of the copper amount found in agricultural soils comes from phytosanitary treatment (ADEME-SOGREAH, 2007). Furthermore, mineral fertilizers and lime and magnesium soil amendments contribute to copper inputs to agricultural soils. The burden of copper to soil by urban activities is mainly through spreading and composting of sludge from wastewater treatment plants. For example, the average concentration of copper in sewage sludge of the Rhone Mediterranean basin was about 350 mg kg -1 in 2004 (Agence de l'eau Rhône Méditerranée Corse, 2004). In soils, copper is in the oxidation states I or II in the form of sulfides, sulphates, carbonates, and oxides. The behavior of copper in soils depends on many factors such as soil pH, redox potential, cation exchange capacity, type and distribution of organic matter, presence of oxides, rate of decomposition of the organic matter, proportions of clay, silt, and sand, climate, and type of vegetation [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF].

Acidification for instance causes a decrease of the metal bound to solids and thus release from solid matter [START_REF] Hlavackova | Evaluation du comportement du cuivre et du zinc dans une matrice de type sol à l'aide de différente méthodologie[END_REF]. Copper binds preferentially to organic matter representing between 25 to 50 % of copper, to iron oxides, manganese oxides, carbonates, and to clays. This characteristic makes the majority of copper strongly adsorbed in the upper few centimeters of soil, especially on organic matter. Copper does not migrate deeply, except under special circumstances of drainage or in highly acidic environment [START_REF] Ineris | Données technico-économiques sur les substances chimiques en France : cuivre, composés et alliages[END_REF]. In the European Union, in 2010 the most polluted solid matters are the sediments of alluvial plains with 25 mg kg -1 , the sediments of rivers with 22 mg kg -1 , soil with 17 mg kg -1 . The maximum concentration determined in a river sediment was found to be 877 mg kg -1 , more than 40 times the average (FOREGS, 2010).

In France, concentrations of copper in the surface layers of soil can reach 46 mg kg -1 .

The levels of copper in surface and deep layers of the most polluted soils are found mainly in the South of France and the West of Bretagne.

Copper metabolism and its physiological role in animals (generalities)

Copper is an essential trace element in microorganisms, plants, and animals. It plays a basic role being in the active centre of enzymes involved in connective tissue formation with lysyl oxidase, in respiration with cytochrome C oxidase (López de Romana, 2011), in photosynthesis with plastocyanin [START_REF] Grotz | Molecular aspects of Cu, Fe and Zn homeostasis in plants[END_REF], and in controlling the level of oxygen radicals with Cu / Zn-superoxide dismutase (Table 1). It allows also the transport of oxygen in the haemolymph of many invertebrates. Copper is used in several cell compartments, and the intracellular distribution of copper is regulated in response to metabolic demands and changes in the cell environment (Tapiero et al., 2003). The same properties that make transition metal ions indispensable for life at low exposure level are also the ones that are responsible for toxicity when present in excess. Copper metabolism must be tightly regulated, ensuring a sufficient supply without toxic accumulation. Copper homeostasis involves a balance between absorption, distribution, use, storage, and detoxification.

Table 1: Functions of the main cuproproteins (Tapiero et al., 2003)

Metalloenzymes Functions

Superoxide

Photosynthesis

In the mammalian intestine where low pH and the presence of ligands promote its solubility, copper passes by the enterocytes. Copper is absorbed by saturable active transport through membrane transporters. In the cytosol of enterocytes, copper binds to metallothioneins which play a role in copper sequestration and transport. Once absorbed, copper is transported to the liver, predominantly by albumin, but also by transcuprein and free amino acids including histidine. In the liver, depending on the status of the animal, copper is bound to metallothioneins to be stored, incorporated into ceruloplasmin [START_REF] Cousins | Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin[END_REF], and then transported to other organs, or secreted into the intestine via the bile. In monogastric animals, copper homeostasis is largely maintained by increased excretion avoiding excessive accumulation in the liver (fig. 1). Biliary excretion is the major route, losses via urine, skin, or via cellular desquamation in the gut are minor [START_REF] Arredondo | Iron and copper metabolism[END_REF].

In aquatic organisms, especially bivalves, copper is ingested and internalized after solubilization in the gastrointestinal tract and absorbed by the intestinal tract (associated with the particulate phase or in solution). Copper in the dissolved phase can also be directly absorbed by the tissues (gills, mantle, etc.) in contact with the surrounding water (Viarengo and Nott, 1993).

Fig. 1: Copper metabolism in mammals [START_REF] Jondreville | Le cuivre dans l'alimentation du porc : oligoélément essentiel, facteur de croissance et risque potentiel pour l'Homme et l'environnement[END_REF].

The entry of copper into the cell occurs mainly by mechanisms that depend on copper transporting membrane channels. In this process, Cu 2+ ion is first reduced to Cu + by reductases associated with the membrane to facilitate its entry. Once inside the cytoplasm, it is likely that reduced glutathione (GSH) and metallothioneins (MT) bind the copper, serving as intracellular stores. Copper appears to be interchanged between MT forming a stable complex and its bound state with GSH. As copper bound to GSH turns over more rapidly than when bound to MT, copper becomes available for other uses and for its transport. The delivery of copper ions to their specific pathways into the cell is mediated by metallochaperones that protect the metal from intracellular scavengers and delivers it directly to the respective target proteins and cellular compartments [START_REF] Kozlowski | Copper, iron, and zinc ions homeostasis and their role in neurodegenerative disorders (metal uptake, transport, distribution and regulation)[END_REF]. The copper chaperone for Cu/Zn superoxide dismutase guides copper to superoxide dismutase (Cu/Zn SOD) which participates in the defence against oxidant stress within the cytoplasm.

Cytochrome c oxidase copper chaperone is another protein that channels copper to cytochrome c oxidase in the inner mitochondrial membrane which plays a critical role in the electron transport chain for cellular respiration. Antioxidant protein 1 presents copper to the ATPase of type P, ATP7A of ATP7B [START_REF] Lutsenko | Human copper homeostasis: a network of interconnected pathways[END_REF]. These three transporters play an essential role in copper homeostasis. They perform distinct functions depending on their cellular localization: when at the Golgi apparatus they enable the management of copper by ceruloplasmin and incorporation of copper into enzymes which require it as cofactor. When localized in vesicular compartments, they allow the removal of copper from the cell and thus participate in copper homeostasis [START_REF] Arredondo | Iron and copper metabolism[END_REF][START_REF] Hejl | Du gène à la maladie : les anomalies des transporteurs du cuivre From gene to disease: Copper-transporting P ATPases alteration[END_REF].

Toxicology of copper (general)

In living organisms, cupric ion (Cu 2+ ) is fairly soluble whereas cuprous (Cu + ) solubility is in the sub-micromolar range. Cu is present mainly as Cu 2+ since in the presence of oxygen or other electron acceptors, Cu + is readily oxidized. Strong reductants such as ascorbate or reduced glutathione can reduce Cu 2+ back to Cu + [START_REF] Arredondo | Iron and copper metabolism[END_REF].

As in the case of iron through the Haber-Weiss and Fenton reactions, free copper ions can catalyse the production of hydroxyl radicals (HO • ). Copper toxicity results also from nonspecific binding which can inactivate important regulatory enzymes by displacing other essential metal ions from catalytic sites, by binding to catalytic Cys groups or by allosterically altering the functional conformation of proteins [START_REF] Mason | Metal detoxification in aquatic organisms[END_REF].

Thus, the mechanisms of toxicity are associated both with oxidative stress and direct interactions with cellular compounds.

Free copper ions have high affinity to sulfur-, nitrogen-, and oxygen-containing functional groups in biological molecules which can inactivate and damage them.

Cytotoxicity observed in copper poisoning results from inhibition of the pyruvate oxidase system by competing for the protein's sulfhydryl groups. Glucose-6-phospho-dehydrogenase and GR are also inhibited (competitive inhibition) proportionally to the concentration of intracellular copper [START_REF] Barceloux | Copper[END_REF]. The same applies to some transporters as the ATPase(s) which are also inhibited by copper, causing disruption of homeostasis of the respective transported entities. Toxic effects of copper can also result from its affinity to DNA [START_REF] Agarwal | Effects of copper on mammalian cell components[END_REF][START_REF] Bremner | Manifestations of copper excess[END_REF][START_REF] Sagripanti | Interaction of copper with DNA and antagonism by other metals[END_REF]. Another mechanism of toxicity of excessive concentrations of copper is the modification of the zinc finger structures of transcriptional factors which cannot any longer bind to DNA [START_REF] Pena | A delicate balance: homeostatic control of copper uptake and distribution[END_REF]. Copper in excess can also promote apoptosis [START_REF] Kozlowski | Copper, iron, and zinc ions homeostasis and their role in neurodegenerative disorders (metal uptake, transport, distribution and regulation)[END_REF] wile copper deficiency may be the cause of many diseases due to cuproprotein and copper dependent reaction inhibition.

In mammals, copper homeostasis is primordial. Wilson's and Menkes diseases are caused by genetic mutations in copper transporter proteins. The former results from accumulation of copper in several organs and tissues. There are different varieties, the most common being liver disease and anaemia [START_REF] Hejl | Du gène à la maladie : les anomalies des transporteurs du cuivre From gene to disease: Copper-transporting P ATPases alteration[END_REF]. The accumulation of copper arises from a defect in the P-type Cu-protein ATP7B (called Wilson protein), a specific transporter of copper. The gene which encodes this protein is located on autosome number 13 in humans. It also allows the incorporation of copper in cuproproteins and excretion of copper into the bile. Accumulation of copper leads to liver cirrhosis and neurodegeneration.

Menkes disease is a neurodegenerative disease. Copper, after ingestion, accumulates in the intestine and absorption by other organs and tissues (blood, liver, brain) is defective.

Menkes syndrome is caused by a mutation in the ATP7A gene located on chromosome X which encodes a protein ATP7A Cu-type P. This membrane protein is the first specific transporter of copper found in eukaryotes.

Copper (Cu), besides cadmium, is one of the major metals causing environmental problems in fresh water ecosystems. Since it is highly toxic to fish, it is also used as piscicide [START_REF] Manzl | Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes[END_REF] For Anodonta cygnea common names are: swan mussel (English), anodonte des cygnes (French), Große Teichmuschel (German); for Anodonta anatina: duck mussel (English), mulette des cannards (French), Gemeine Teichmuschel (German).

Description:

In both species, the shell is nacreous, and there are no teeth on the ligament.

Anodonta anatina (fig. 2A): Shell slightly elongated, triangular because of the presence of a large rear wing or crest, the top and bottom form an angle of greater or lesser extent, the upper edge rises in an almost straight line towards the back to the highest point of the crest, then descends in a more concave line towards the posterior end; the anterior is broadly rounded, rather short, the posterior is longer, ending posteriorly by an obtuse rostrum;

the ligament is rather long and prominent; the top is a little bulging, covered with fine wrinkles, a little curved, obliquely cutting the growth lines; the shell is rather thick, solid, shiny, greenish gray, or brownish. (Length: 14 cm, height: 10.5 cm, thickness: 6 cm) [START_REF] Vrignaud | Clef de détermination des Naïades d'Auvergne[END_REF].

Anodonta cygnea (fig. 2B): Shell oval, more or less elongated with the top and bottom edges roughly parallel or convex, the upper edge is more straight than the lower, the posterior area being much longer; the ligament is rather long, and prominent; the top is a little bulging, covered with fine wrinkles parallel to growth lines; the shell is thin, not strong, fairly shiny, greenish-yellow. (Length: 20 cm, height: 10 cm, thickness: 6 cm) [START_REF] Vrignaud | Clef de détermination des Naïades d'Auvergne[END_REF].

A B

Fig. 2: A : Anodonta anatina, B: Anodonta cygnea (www.biopix.eu)

Geographic distribution

Anodonta cygnea and Anodonta anatina are autochthonous bivalves, widely distributed under the polar circle in European continental hydrosystems. The two mussels have a similar distribution, but wider for Anodonta cygnea. The geographical distribution (fig.

3) of Anodonta anatina extends from the British Isles to the eastern limit of Europe and from Sweden to Spain. Anodonta cygnea is present from the British Isles to Siberia and from Sweden to North Africa [START_REF] Başçınar | A Preliminary study on reproduction and larval development of swan mussel [Anodonta cygnea (Linnaeus, 1758)] (Bivalvia: Unionidae), in Lake Çıldır (Kars, Turkey)[END_REF]; www.discoverlife.org).

Life Cycle

Of the unionids, the individual animals are usually gonochoric. No age limit is known for gametogenesis. The spermatozoids are released into the water through the exhalant siphon.

They are filtered by mussels located downstream. After fertilization, the eggs are incubated in the marsupium which is a modification of the gills of the mussel where the larvae or glochidia will hatch (fig. 4). They are produced in large quantities, from 50000 to 2 million [START_REF] Başçınar | A Preliminary study on reproduction and larval development of swan mussel [Anodonta cygnea (Linnaeus, 1758)] (Bivalvia: Unionidae), in Lake Çıldır (Kars, Turkey)[END_REF], some of them attaching themselves with hooks at the end of their valves on the gills of a fish host, and then live as encysted parasites. After a few weeks, the cyst bursts and releases a small bivalve similar in its anatomy to an adult mussel. This juvenile will grow buried in the substratum before becoming adult and gradually rising to the surface. The expulsion of glochidia begins at the end of winter and may continue until September [START_REF] Mouthon | Les mollusques dulcicoles -Données biologiques et écologiques -Clés de détermination des principaux genres de bivalves et de gastéropodes de France[END_REF]. The Unionidae produce only one generation per year. It is estimated that mussels of the genus Anodonta can live very long, about fifteen to twenty years [START_REF] Taskinen | Age-, size-and sex-specific infection of Anodonta piscinalis (Bivalvia, Unionidae) with Rhipidocotyle fennica (Digenea, Bucephalidae) and its influence on host reproduction[END_REF].

Diet

According to the conventional nomenclature, among the freshwater molluscs one distinguishes vegetarians, detritivores, and more rarely omnivores; there is no true carnivor.

Most bivalves have a mixed diet with detritivore and vegetarian dominance throughout the year, as for the Unionidae. Except during their larval stage when the glochidium parasites a host fish and feeds on his plasma [START_REF] Uthaiwan | Culture of glochidia of the freshwater pearl mussel Hyriopsis myersiana (Lea, 1856) in artificial media[END_REF], Anodonta mussels are filter feeders, although their diet is not precisely known. They feed on seston (phytoplankton, filamentous algae, detritus, protists, epipelic) suspended in water. Water enters the mantle cavity through the inhalant siphon, passes through the gills and is exhaled through the exhalant siphon. Food particles in the water are intercepted by rows of cilia or cirri which extend between the gill filaments. The particles are either transferred and incorporated into mucus strings or carried in a concentrated suspension as they are transported to the labial palps. Particles selected for ingestion are transferred to the mouth, while rejected particles are bound up into a mucus ball and expelled from the inhalant siphon or pedal gap (gap between the valves) as pseudofaeces.

Anatomy and ecology

Anatomy

The mantle cavity is divided by the gills into a ventral inhalant chamber and a dorsal exhalant chamber. Water passes through the inhalant siphon to enter the branchial chamber, flows across the gills and then into the exhalant suprabranchial chamber. As the water crosses the gills, food particles are filtered from it and oxygen is removed. From the suprabranchial chamber the water flows out of the exhalant siphon. The gills consist of a sheet of coalescent filaments folded into a "W" shape and attached to the dorsal wall of the mantle. This sheet divides the mantle cavity into the ventral chamber and the dorsal suprabranchial chamber. To get from the ventral chamber to the dorsal, water must pass through pores in the gills. The

Ecology

Anodonta cygnea and Anodonta anatina are species that frequent ponds, oxbow lakes, slow rivers, and canals with weak stream which often have high trophic level. They prefer bottom with fine granulometry (silt, sand, gravel), often with accumulations of organic matter.

These species are very tolerant to average temperature of the water as evidenced by their presence in Spain to Sweden. They are localised in bed areas, out of strong stream undergoing some erosion, in general in a depth range between 0.2 and 2.5 m. These are burrowing organisms, living vertically partially embedded in the substrate. These bivalves are generally sedentary organisms. In Spain, a displacement of about ten meters by year was found for mussels A. cygnea and A. anatina marked by Aldridge (2000). However, flooding can cause passive dispersal from upstream to downstream. The most mobile stage is the larval stage, which is capable of moving long distances through the host fish.

Threats

Over the past 50 years, a general decline of freshwater mussel has-been observed. It seems that some factors are combined to cause the gradual disappearance of these species:

among the potential causes, human activities leading to chemical pollution and habitat exchange have been suggested (Aldridge, 2000;[START_REF] Kádár | Avoidance responses to aluminium in the freshwater bivalve Anodonta cygnea[END_REF]. Given the lack of ecological and ecotoxicological knowledge for these bivalves (including the tolerance to -The virtual disappearance or severe depletion of potential host-fish.

-The physical degradation of streams and reworks of the beds of rivers and canals (Aldridge, 2000), rectification, dams and channel dredging, but also the impact of intensive agriculture, quality and quantity of sediments and their transit.

-Degradation of water quality, including eutrophication, as well as pollutants from human activity.

-The fragmentation of populations, one of the main causes of biodiversity loss.

-Introduced species, the potential effects of competition in bivalves such as Corbicula fluminea, Sinanodonta woodiana as well as Dreissena polymorpha are poorly documented.

Zebra mussels appear to have a negative impact by binding to the valves of certain freshwater mussels, hampering their opening. S. woodiana also seems to have a negative impact on native unionid populations [START_REF] Adam | L'Anodonte chinoise Sinanodonta woodiana (Lea, 1834) (Mollusca, Bivalvia, Unionidae) : une espèce introduite qui colonise le bassin Rhône-Méditerranée[END_REF].

Ecotoxicological interest

Molluscs are common, highly visible, ecologically and commercially important on a global scale as food and as non-food resources [START_REF] Rittschof | Molluscs as multidisciplinary models in environment toxicology[END_REF]. In some aquatic ecosystems (lakes, slow streams) molluscs can represent up to 80% of the total biomass of the benthic macroinvertebrates, so their impact can become major. Populations of bivalves filter large amounts of water (Unionidae 300ml/individual/h) and take an active part in sedimentation and water purification. Faeces and pseudofaeces concentrate sometimes a large fraction of planktonic microorganisms not used, making them accessible to detritivorous invertebrates such as oligochaetes, and many diptera. But they also change the quality of sediment by concentration and excretion of many substances (metals, pesticides, radionuclides). Because of their sedentary lifestyle, their filtration capacity, and their wide distribution, molluscs and bivalves are excellent sentinels for monitoring the fraction of bioavailable pollutants in their environment [START_REF] Hayer | Accumulation of extractable organic halogens (EOX) by the freshwater mussel Anodonta cygnea L., exposed to chlorine bleached pulp and paper mill effluents[END_REF]. In close contact with water-suspended particles and sediment, they are widely used for controlling the bioaccumulation and toxic effects of metallic and organic pollutants in aquatic ecosystems [START_REF] Viarengo | Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program[END_REF][START_REF] Rittschof | Molluscs as multidisciplinary models in environment toxicology[END_REF]. Several Unionidae mussels, especially from the Anodonta genus have since been used as biomonitor organisms in toxicity assessment of numerous compounds released in continental water. The anatomy and physiology of these animals has been studied for a long time, allowing the study of toxic effects of compounds but also the mechanisms of detoxification. Anodonta cygnea and Anodonta anatina are biological models widely used in ecotoxicology (Falfushynska et al., 2009). They are present in large quantities, sedentary, easy to collect and to acclimatize in aquaria.

2.3 Effects of copper exposure and detoxification mechanisms

Copper exposure effects

Copper bioaccumulation

Trace elements are known to be highly accumulated by aquatic molluscs. Bivalves are in close contact with sediments which constitute a major environmental sink for metals, with an important filtering activity to satisfy respiration and nutrition, and tolerance mechanisms that involve metal sequestration rather than metal exclusion or elimination. They provide accurate and integrated information about the environmental impact and bioavailability of chemicals. They are therefore extensively applied in marine environments using mussels and oysters, but are also implemented in freshwater systems using other bivalve species such as Anodonta sp., Dreissena polymorpha, Elliptio complanata, and Asiatic clams. Among freshwater organisms, unionid molluscs are widely recognised for their capacity to accumulate a variety of environmental contaminants including metals in their tissues (Winter, 1996;[START_REF] Bilos | Trace metals in suspended particles, sediments and Asiatic clams (Corbicula fluminea) of the Rio de la Plata Estuary, Argentina[END_REF][START_REF] Kádár | Avoidance responses to aluminium in the freshwater bivalve Anodonta cygnea[END_REF]Falfushynska et al., 2009). The widespread recent decline in the species diversity and population density of freshwater mussels (Lydeard et al., 2004) may be partly related to chronic, low-level exposure to toxic metals (Frank and Gerstmann, 2007). Freshwater mussels are exposed to metals that are dissolved in water, associated with suspended particles, and deposited in bottom sediments. Thus, freshwater mussels can bioaccumulate certain metals to concentrations that greatly exceed those dissolved in water. In adult mussels, the most common site of metal uptake is the gills, followed by the digestive gland, the mantle, and the kidneys [START_REF] Pagliarani | Mussel microsomal Na + /Mg 2+ -ATPase Sensitivity to Waterborne Mercury, Zinc and Ammonia[END_REF]Bonneris et al., 2005). Bioaccumulation of metals varies strongly according the water chemistry conditions, pH and water hardness being important parameters. Aqueous concentrations of calcium probably enhance the bioavailability and toxicity of metal cations, because the permeability of membranes is inversely related to aqueous calcium concentration; calcium ions apparently compete with other metal cations for binding sites on the gill surface, decreasing the direct uptake of other cationic metals. The pH influences both the chemistry of metals and macromolecules of surface structures. A modification of membrane permeability causes an alteration in metal diffusion. Additionally, changes in membrane potential modify the transport of polar metal species (Winter, 1996). In bivalves, the biological barriers are the gill epithelium, the wall of the digestive tract, and the shell (which is often reported as a site of bioaccumulation). Metals or metalloids in solution are more easily absorbed by the surfaces in direct contact with the external environment, while those associated with the particulate phase are rather ingested and internalized after solubilization in the digestive tract, or transferred by endocytosis to then undergo lysosomal digestion (Wang and [START_REF] Rainbow | Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates[END_REF].

Once past the first barrier, the mechanisms of transfer of metals or metalloids into the cell involve intracellular diffusion (passive or facilitated), active transport, and endocytosis (phagocytosis and pinocytosis). The oxygen concentration of the water or the density of microalgae are parameters that will directly influence the ventilatory activity of the bivalve and so its exposure to metals in solution or particulate [START_REF] Tran | Mechanism of oxygen consumption maintenance under varying levels of oxygenation in the freshwater clam Corbicula fluminea[END_REF][START_REF] Tran | Copper detection in the Asiatic clam Corbicula fluminea: optimum valve closure response[END_REF].

Copper and metal bioaccumulation can change between individuals of the same species with the difference in sizes, or between different species mussels due to different physiology (breathing, eating) (Winter, 1996;[START_REF] Bilos | Trace metals in suspended particles, sediments and Asiatic clams (Corbicula fluminea) of the Rio de la Plata Estuary, Argentina[END_REF]Gundacker, 2000;[START_REF] Hédouin | Allometric relationships in the bioconcentration of heavy metals by the edible tropical clam Gafrarium tumidum[END_REF]Falfushynska et al., 2009). In bivalves, the determination of trace metal concentrations in whole individuals presents little interest, since determination of bioconcentration factors in various tissues suggested that the principal accumulating organs are: the gills, the digestive gland, the kidney, and the mantle, also the shell acting as a storage matrix (Viarengo and Nott, 1993;[START_REF] Roméo | Metal distribution in different tissues and in subcellular fractions of the Mediterranean clam Ruditapes decussatus treated with cadmium, copper, or zinc[END_REF][START_REF] Das | Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.)[END_REF]Bonneris et al., 2005). The intracellular sequestration of metals is based on a sequence of cellular events involving a cascade of different ligands with increasing metal binding strengths. Anodonta sp. and other molluscs accumulate metals to high levels in their tissues (Falfushynska et al., 2009). Metal tolerance in such accumulator organisms involves sequestration of metals in non-toxic forms.

Among the best studied intracellular sites involved in the sequestration of essential and nonessential metals in aquatic invertebrates are lysosomes, granules, and soluble Cys-rich ligands as metal-binding peptide and proteins. Unionids also lay down calcium microspherule concretions, particularly in the connective tissue of the gills, in the mantle, and in the digestive gland [START_REF] Pynnönen | Occurrence of calcium concretions in various tissues of freshwater mussels, and their capacity of cadmium sequestration[END_REF]Moura et al., 2000;[START_REF] Lopes-Lima | Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea[END_REF].

Dissimilar mechanisms for copper and metal uptake, storage, mobilisation, and excretion performed by different cell types in different organs explain the pattern of metal accumulation and tissue distribution [START_REF] Soto | The contribution of metal/shell-weight index in target-tissues to metal body burden in sentinel marine molluscs. 1. Littorina littorea[END_REF].

Oxidative stress

Reactive oxygen species comprise a variety of unstable oxygen derivatives. Some [START_REF] Viarengo | Heavy metal effects on lipid peroxidation in the tissues of mytilus galloprovincialis lam[END_REF]Géret et al., 2002a;[START_REF] Sheehan | Oxidative stress and bivalves: a proteomic approach[END_REF]). An excess of ROS causes a risk of oxidative stress to the cell.

Free radicals are naturally produced and may also have a physiological role. The major role of ROS is an activity of regulation of molecules containing sulfhydryl groups. This type of regulation can in particular affect molecules implied in the mechanisms of signal transduction like protein kinase C [START_REF] Lander | An essential role for free radicals and derived species in signal transduction[END_REF][START_REF] Dalton | Regulation of gene expression by reactive oxygen[END_REF]. Significant sources of free oxygenated radicals are redox cycles and oxidations catalysed by cytochrome P450 monooxygenases implicated in detoxification. Like many exogenous compounds that can stimulate the production of ROS, copper is known as hydroxyl radical inducer [START_REF] Company | Antioxidant biochemical responses to long-term copper exposure in Bathymodiolus azoricus from Menez-Gwen hydrothermal vent[END_REF]. Metal deficiency can also lead to oxidative stress. Indeed, copper belong to the molecular composition of antioxydent enzyme such as Cu / Zn-SOD. Thus copper deficiency disrupts in this case the dismutation of the superoxide anion.

ROS can potentially react with every cellular component. They influence in particular the thiol groups of proteins, leading to the formation of intra-or inter-molecular disulphide bridges. The most widely studied action of ROS is lipid peroxidation, mainly carried out by HO • . After rearrangement and addition of oxygen, ROO • and RO • radicals are generated.

Oxidation of phospholipids modifies the membranes with loss in permeability and membrane potential, leading to inactivation of membrane receptors and enzymes. In a general way, during this reaction, various compounds are produced such malondialdehyde (MDA) and 4hydroxynonenal, both able to bind to proteins and to form DNA adducts [START_REF] Blair | Lipid hydroperoxide-mediated DNA damage[END_REF]. Thus, lipid peroxidation may trigger endogenous DNA damage as nuclear DNA and mitochondrial DNA are the targets of ROS. Four main classes of damage can be noted: single and double strand breaks, modified bases, DNA-DNA and DNA-protein bridges, and abasic sites.

Proteins are essential structural and functional cellular components which may undergo oxidative modification, thus inducing their aggregation or digestion by proteases [START_REF] Davies | The oxidative environment and protein damage[END_REF]. Oxidation of amino acids, particularly of sulphur-containing and aromatic amino acids, result in structural modifications of proteins. HO • is the main initiator of the oxidation of polypeptide chains producing free radicals. In the absence of oxygen, two radicals can react together to form intra-or interchain cross-links. In the presence of oxygen, an addition reaction may take place to yield a peroxyl radical. A series of reactions follows, leading to the formation of alkoxyl radicals, precursors to the fragmentation of polypeptide chains. The oxidation of glucose can be performed in the presence of metal ions, leading to the release of aldehydes and hydrogen peroxide. This leads to glycation of proteins by attachment of aldehydes, often entailing the cleavage of protein chains [START_REF] Wolff | Autoxidative glycosylation": free radicals and glycation theory[END_REF]. Glycation of proteins promotes their oxidizability. The structural modifications induce functional changes, in particular of cellular metabolism. The oxidation by ROS can disturb ionic transport, enzymatic activities, and calcium homeostasis. Antioxidant defense systems can directly inhibit the production of ROS, limit their propagation, or destroy them. These antioxidant systems may act by reducing ROS species or by trapping them to form stable compounds.

Two categories of antioxidant systems are generally defined, i. In the antioxydent system glutathione peroxidases (GPxs) / glutathione reductase (GR), GPxs are also able to reduce H 2 O 2 and other peroxides. Their enzymatic activities of GPxs are coupled with the oxidation of glutathione (GSH). Oxidized glutathione (GSSG) will be reduced by GR using NADPH. These enzymes are localised in the cytoplasm and the mitochondrial matrix [START_REF] Lushchak | Environmentally induced oxidative stress in aquatic animals[END_REF].

Antioxidant molecules or free radical scavengers such as Cys thiol rich compounds, principally GSH and the metallothioneins, will be discussed later. Their antioxidant capacity is conferred by the thiol group of the Cys residue. Vitamin E is also known for its powerful anti-radical activity operating in lipid membranes. Other scavenger molecules include vitamin C, carotenoids, and uric acid.

Calcium transport and perturbation of bio-mineralization

Calcium transport perturbation

Normally, intracellular Ca homeostasis is maintained by a balance between extrusion and compartmentation systems (Mooren and Kinne, 1998). Alteration of these processes during cell injury can result in inhibition of Ca 2+ extrusion or intracellular compartmentation mechanisms, as well as in enhanced Ca 2+ influx and release of Ca 2+ from intracellular stores such as the endoplasmic reticulum and mitochondria [START_REF] Marchi | Mercury-and copper-induced lysosomal membrane destabilisation depends on [Ca 2+ ] i dependent phospholipase A2 activation[END_REF]. This can lead to uncontrolled rises in cytosolic Ca 2+ concentration. The biological functions of Ca 2+ are versatile; they control multiple processes of birth, life and death. Location, duration, and amplitude of calcium signals form a complex code that can control vital physiological processes. Any disturbance in these signals would change the "Ca 2+ code" and modify multiple life processes which are usually associated with loss of cell viability (Ermak and Davies, 2001). Filter feeding bivalves have high bioaccumulation potential for trace metals which at higher concentration can cause serious metabolic, physiological, and structural impairments. Continuous decline of freshwater mussels during the past decades, could be attributed to calcium homeostasis perturbation by trace metals (Frank and Gerstmann, 2007).

Slight deregulations of Ca 2+ homeostasis like those deriving from low-dose trace metal contamination can affect the cellular ability to maintain and modulate Ca 2+ signaling. Copper is known to increase Ca 2+ intracellular concentration by release of the Calcium stock of the endoplasmic reticulum, leading to lysosomal membrane destabilisation in the mussels [START_REF] Marchi | Mercury-and copper-induced lysosomal membrane destabilisation depends on [Ca 2+ ] i dependent phospholipase A2 activation[END_REF] and apoptosis or cytotoxicity and necrosis (Ermak and Davies, 2001). Cell Ca 2+ extrusion systems allowing physiological calcium concentration maintenance appear to be essential for cell viability.

The absorption and release of calcium in the environment, biomineralization, and the maintenance of intracellular calcium concentration in involved its passage through cell membranes. Ca 2+ passively enters the apical membrane down to its concentration gradient through carrier-mediated facilitated diffusion Na + /Ca 2+ antiporters and via simple diffusion through Ca 2+ channels. Intracellular Ca 2+ gradients are maintained by membrane Ca 2+ -ATPases across the plasma membrane (PMCA) and the membranes of intracellular stores. In Mytilus edulis copper is known to inhibit PMCA. Thiol groups of PMCA proteins are directly oxidized or they bind copper because of high affinity to the metal. Hydroxyl radicals OH • are induced by copper which indirectly causes PMCA protein impairment [START_REF] Viarengo | Possible role of Ca 2+ in heavy metal cytotoxicity[END_REF]Viarengo et al., 1996;Burlando et al., 2004). Besides the Ca 2+ -ATPases, another important system for the maintenance of calcium homeostasis is the plasma membrane Na + /Ca 2+ antiporter, the activity of which is based upon the transmembrane Na + electrochemical gradient. Inhibition of the Na + /K + -ATPase modifies the Na + gradient, and therefore it can also affect the activity of the Na + /Ca 2+ antiporter. Na + /K + -ATPase inhibition was observed in Mytilus edulis and Perna viridis after Ag, Cr, and copper exposure (Viarengo et al., 1996;[START_REF] Vijayavel | Sublethal effect of silver and chromium in the green mussel Perna viridis with reference to alterations in oxygen uptake, filtration rate and membrane bound ATPase system as biomarkers[END_REF].

Due to their physiological function for respiratorion, nutrient absorption and excretion, the gills, the digestive gland, and the kidneys are preferential sites of metal uptake and bioaccumulation in bivalves. These organs play important roles in iono-regulation, in particular for calcium homeostasis in freshwater bivalves (Coimbra et al., 1993). Effects on the Ca 2+ transport systems in these tissues leads to perturbation of calcium homeostasis in the whole organism.

Perturbation of biomineralization

Biomineralization is a complex process; in bivalves principally it enables the formation, growth, and repair of the shell. In addition to the shell, mineral concretions are produced which also play a role in detoxification mechanisms (Viarengo and Nott, 1993). In freshwater molluscs such as Anodonta, several internal tissues, bathed by haemolymph, also produce calcified structures, namely microspherules [START_REF] Lopes-Lima | Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea[END_REF].

Microspherules are usually present between both epithelia of the mantle under CaCO 3 and / or Ca 3 (PO 4 ) 2 deposits. These calcium transitory reserves are devoted to shell growth or glochidia larvae. The mantle of lamellibranchs is a leaflet that covers the internal surface of the shell and surrounds the body of the animal. It consists of two epithelia: internal in contact with the external medium, and external, the outer mantle epithelium (OME) facing the shell. The mantle is the tissue responsible for shell synthesis (Coimbra et al., 1993). Shell growth and maintenance of its mineral content are thus dynamic equilibrium involving a continuous exchange of Ca 2+ between the shell and the OME through the extrapalleal fluid dividing the two. The dynamic calcium exchange may result in a net accumulation of Ca 2+ in the shell, or its re-absorption, depending on the developmental stage and the metabolic state of the animal.

The biomineralization mechanism depends on pH changes or its tendency [START_REF] Lopes-Lima | Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea[END_REF]. H + -ATPases in cells of the OME regulate the deposition of calcium or its reuptake into the haemolymph by pH control. H + -ATPases probably play a role equivalent to those of the two bone cell lines, the osteoblasts and the osteoclasts [START_REF] Machado | Ultrastructural and cytochemical studies in the mantle of Anodonta cygnea[END_REF]. H + -ATPases of the OME induce calcium mobilisation from the shell mainly by pumping protons into the extrapalleal fluid. H + -ATPase inhibition under bis(tributyltin)-oxide exposure was observed (Machado et al., 1989). CA is a ubiquitous enzyme; in the mantle of bivalves it controls bicarbonate balance between haemolymph and extrapalleal fluid for CaCO 3 formation. CA is a bioindicator of biomineralization already studied in ecotoxicology of metals (Vitale et al., 1999[START_REF] Rousseau | Biomineralisation markers during a phase of active growth in Pinctada margaritifera[END_REF]. So, if some alteration of the ion transport mechanisms across the mantle occurred in bivalves, it may depend on a direct inhibition of biomineralization. In freshwater bivalves, trace metals and in particular copper, seem to interfere with the shell calcification conditions by direct action on the proton pump and CA. Perturbation of biomineralization could cause subsequent shell thickening (Machado et al., 1989) or thinning.

Detoxification mechanisms

Cysteine thiol rich compounds

Glutathione

The tripeptide glutathione (GSH, -glutamyl-cysteinylglycine) is a non-protein thiol.

The GSH/glutathione disulfide (GSSG) system is the most abundant redox system in eukaryotic cells, playing a fundamental role in cell homeostasis and metal detoxification, and being involved in signalling processes associated with programmed cell death termed apoptosis [START_REF] Canesi | Heavy metals and glutathione metabolism in mussel tissues[END_REF][START_REF] Camera | A nalytical methods to investigate glutathione and related compounds in biological and pathological processes[END_REF]. It is present mainly in its reduced form, GSH, and represents the most significant thiol in eukaryotic cells (0.2 to 10 mM). GSH biosynthesis requires two enzymes (fig. 6): first, glutamate-cysteine-ligase ( -ECS) catalyzing the fusion of glutamic acid (Glu) and Cys to -glutamyl-cysteine ( -GluCys), which in turn is converted into GSH by addition of glycine (Gly) by glutathione synthase (GS). -ECS is inhibited in feedback by GSH [START_REF] Monostori | Determination of glutathione and glutathione disulfide in biological samples: An in-depth review[END_REF]. GSH exerts many functions in the cell.

It intervenes in processes of reduction such as the synthesis and degradation of proteins, and the formation of deoxyribonucleotides. GSH plays a role as co-enzyme of various reactions, and it is also used for being combined with either endogenous (oestrogens, prostaglandins, and leucotrienes) or exogenous compounds (drugs and xenobiotics), thus taking part in their metabolism. GSH is thus regarded as a central component in antioxidant defense [START_REF] Cossu | Antioxidant biomarkers in freshwater bivalves, Unio tumidus, in response to different contamination profiles of aquatic sediments[END_REF][START_REF] Manduzio | The point about oxidative stress in molluscs[END_REF]. The biologically active site of GSH is represented by the thiol group of the Cys residue. The high nucleophilicity of the thiol function facilitates the role of GSH as a free radical scavenger both under physiological conditions and in xenobiotic toxicity. GSH also helps in the regeneration of other antioxidants such as vitamin E, ascorbic acid, and metallothionein [START_REF] Knapen | Glutathione and glutathione-related enzymes in reproduction: A review[END_REF]. GSH is a cofactor for glutathione peroxidase in the decomposition of hydrogen peroxide or organic peroxides (fig. 6), for glyoxalase 1 in the detoxification of methylglyoxal and other oxo-aldehydes, and for maleylacetoacetate isomerase in the conversion of maleylacetoacetate and maleylpyruvate to the corresponding fumaryl derivatives [START_REF] Monostori | Determination of glutathione and glutathione disulfide in biological samples: An in-depth review[END_REF]. GS-conjugates of endogenous compounds are involved in metabolism, transport, and storage in cell. In addition, conjugation of xenobiotics to GSH initiates a detoxification pathway that generally leads to excretion or compartmentation of the biotransformed compound. Although some adducts can be formed directly, glutathione-S-transferases (GST) mediated reactions generally predominate. GST belong to cellular mechanisms of detoxification and elimination of molecules (Wünschmann et al., 2009). In animals, GS-conjugates are hydrolyzed and degraded by c-glutamyltransferases, followed by carboxypeptidation of the glycine residue. GR reduces GSSG to GSH which, with synthesis of new GSH, maintains the cellular GSH stock (fig. 6). [START_REF] Vašák | Advances in metallothionein structure and functions[END_REF]. The designation MT reflects the extremely high thiolate sulfur and metal content, both of the order of 10% (w/w). MT have been identified in a wide range of organisms, from bacteria to mammals, in many fish and aquatic invertebrates, mainly molluscs. Classically these extremely heterogeneous polypeptides were grouped into three classes of MT [START_REF] Fowler | Nomenclature of metallothionein[END_REF]Viarengo and Nott, 1993) Metallothioneins are playing a role in the homeostatic control of essential metals (Cu, Zn) as they can act as essential metal stores ready to fulfil enzymatic and other metabolic demands (Amiard et al., 2006). Their involvement in metal metabolism is based on their capacity to complex metals, effectively buffering free-metal ion concentrations in the intracellular environment. Additionally, the biosynthesis of these metalloproteins may be induced by exposure to essential and non-essential metals (Bonneris et al., 2005). Vital roles for this pleiotropic protein result in its involvement in homeostasis of essential trace metals, zinc and copper, or sequestration of the environmental non essential metals. Moreover, MT could protect cells from oxidative stress (Géret et al., 2002b). In the presence of ROS, zinc can be removed [START_REF] Vašák | Advances in metallothionein structure and functions[END_REF]. The release of zinc is accompanied by the formation of the MTdisulfide (or thionin, oxidized form of the protein) which in turn can be reduced by the ratio GSH / GSSG to restore the ability of the protein to bind zinc. This redox cycle of MT (fig. 7) plays a crucial role in maintaining physiological homeostasis of metals, detoxification of toxic metals and protection against oxidative stress [START_REF] Kang | Metallothionein redox cycle and function[END_REF]. MT plays an essential role in other metabolic processes, since MT expression is rapidly induced by a variety of factors such as cold, heat, hormones, cytokines [START_REF] Monserrat | Pollution biomarkers in estuarine animals: Critical review and new perspectives[END_REF], in some molluscs important seasonal variations in MT levels also correlate with the gametogenesis process. MT, like other proteins, are degraded in lysosomes [START_REF] Ng | Metallothionein turnover, cytosolic distribution and the uptake of Cd by the green mussel Perna viridis[END_REF]. ) (Hirata et al., 2005). These results suggest that PC play important roles not only in the chelation of trace metals but also as antioxidant defence. PC are synthesized from glutathione, homo-glutathione, hydroxymethyl-glutathione or -glutamylcysteine, catalysed by a transpeptidase, named phytochelatin synthase (fig. 8), which is a constitutive enzyme requiring post-translational activation by trensition metals [START_REF] Grill | Phytochelatins, the heavy-metalbinding peptides of plants, are synthesized from glutathione by a specic -glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase)[END_REF]Clemens, 2006).

Phytochelatin synthase has been shown to be activated by a broad range of metals and

MT

Reductant

GSH Oxidant GSSG

MT-disulfide MT-thiols

Metal (Zn 2+ ,…)

Oxidant GSSG, ROS,…

Reductant + Metal

Synthesis Degradation metalloids, in particular Cd, Ag, Pb, Cu, Hg, Zn, Sn, Au, and As, both in vivo and in vitro.

After the completion of the full genome sequence of the nematode Caenorhabditis elegans two publications independently described a functional PC synthase enable to synthesize PC in this model invertebrate (Clemens et al., 2001;Vatamaniuk et al., 2001). In addition sequences similar to PC synthase gene have been identified from the aquatic midge, Chironomus, and a species of earthworm (Brulle et al., 2008;[START_REF] Cobbett | A family of phytochelatin synthase genes from plant, fungal and animal species[END_REF]. There is, yet, no evidence that these animal genes encode PC-synthase activity, however it seems likely that they to encode PC synthase. It became clear that PC could play a wider role in trace metal detoxification than has previously been thought. Organisms with an aquatic or soil habitat are more likely to express PC (Cobbett, 2000). Clemens et al. (2001) hypothesized that PC may be ubiquitously involved in the tolerance and homeostasis of metals in all eukaryotic organisms. Clemens and Peršoh (2009) suggest how PC synthase genes are far more widespread than anticipated, homologous sequences are found through the entire animal kingdom including in bivalves.

Metal detoxification mechanisms in bivalves

Bivalves and particularly freshwater mussel unionids are widely recognised for their capacity to accumulate a variety of environmental contaminants, including metals, in their

tissues and yet survive in these polluted environments (Winter, 1996;Cossu et al., 1997;[START_REF] Das | Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.)[END_REF]Bonneris et al., 2005). Such tolerance depends on the ability of these animals to regulate the essential metal concentration and detoxify nonessential metal. In molluscs three physiological and biochemical ways allow metal regulations: by binding to specific, soluble, Cys-rich ligand; by compartmentalization within organelle; and by formation of insoluble non-toxic precipitates (Viarengo and Nott, 1993;[START_REF] Rainbow | Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates[END_REF]. Metal sub-cellular partitioning depending of organs and metal nature. For example in mussels, copper bound to granules are dominant in the gills, in the digestive gland copper is principally bound to soluble Cys-rich compounds (Bonneris et al., 2005). For a long period of exposure, metals might be displaced from soluble metal-binding ligands to granules [START_REF] Roméo | Metal distribution in different tissues and in subcellular fractions of the Mediterranean clam Ruditapes decussatus treated with cadmium, copper, or zinc[END_REF].

Insolubles storage, compartmentalization and elimination of metals

In mollusc an important storage or detoxification system is carried out by sequestration of metals in intracellular cytosolic and compartmentalized precipitated structures named granules [START_REF] Howard | The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa)[END_REF]Viarengo and Nott, 1993). Granules are observed in different tissues of bivalves, mainly in the digestive gland, gills, and kidneys which are implicated in metal homeostasis and detoxification (Wang and [START_REF] Rainbow | Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates[END_REF].

Lysosome is mainly involved in the catabolism of both endogenous and exogenous molecules. Ni and appear to be linked with metal detoxification. These insoluble granules are often present in cells from which they can be eliminated by exocytosis [START_REF] Howard | The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa)[END_REF]Viarengo and Nott, 1993). Granules alone or with the lysosome are finally eliminated, principally by exocytosis in urine, haemolymph, and faeces.

In addition, a specificity of unionids is calcium extracellular concretions named microspherules. They are found in extrapalial fluid and haemolymph particularly in the gills, and in the mantle. The calcium of these microspherules is usually bound to phosphate and carbonate, either as orthophosphate Ca 3 (PO 4 ) 2 , and carbonate calcium CaCO 3 (Moura et al., 2000;[START_REF] Lopes-Lima | Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea[END_REF]. These microspherules appear to act as a calcium reservoir, serving as a source of calcium for embryonic shell development, but can also play a role in the detoxification of metals [START_REF] Pynnönen | Effects of episodic low pH exposure on the valve movements of the freshwater bivalve Anodonta cygnea L[END_REF]Bonneris et al., 2005).

An other site of metal storage in mollusc is the shell which may act as safe storage matrix for toxic contaminants resistant to soft tissue detoxification mechanisms [START_REF] Das | Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.)[END_REF]. In shell of bivalves and unionids, substantial bioaccumulation of copper and other metals was shown (Gundacker, 2000).

Cysteine rich metal binding compounds detoxification mechanisms

A strategy for cells to detoxify non-essential metal ions and essential metal ions excess is the synthesis of high-affinity binding sites to suppress blockage of physiologically important functional groups (Clemens, 2006). Metal ions have high reactivity with thiol, amino or hydroxyl groups making molecules carrying these functional groups candidates for metal detoxification processes (Viarengo and Nott, 1993). Therefore, the best-known and presumably the first line to Cu, Zn and non-essential metal chelation are the Cys-containing peptides glutathione and phytochelatins and small Cys-rich proteins metallothioneins.

Metal cation with a high affinity for SH residues displace Zn 2+ from a metallothionein physiological pool always present in the cells. An excess of trace metal cations, including Zn 2+ released from pre-existing metallothioneins, induces in the nucleus the synthesis of the mRNA coding for metallothioneins, consequently increasing MT on the cytosolic compartment. These MT chelate the trace metal cations, thus reducing their cytotoxic effects (Viarengo and Nott, 1993). The metal-thiolate clusters within the MT molecules allow rapid exchanges of metallic ions between clusters and with other MT molecules [START_REF] Monserrat | Pollution biomarkers in estuarine animals: Critical review and new perspectives[END_REF]. MT are usually not saturated by a single metal but contain several atoms of Cu, Zn, Cd, or Hg and Ag when present (Amiard et al., 2006). In Zn-thioneins the seven metal atoms of Zn or Cd are distributed between two clusters: cluster of 3 Zn and cluster of 4 Zn [START_REF] Maret | Fluorescent probes for the structure and function of metallothionein[END_REF]. Functionally, the two clusters show different affinities for metal cations. At pH 7 or below, cluster is the first to be saturated and cluster the first to release the metal. Data ion is different for MT (Géret et al., 2002b). Indeed, the stability constant for copper is 100 times higher than for cadmium and 1000 times higher than for zinc. Due to the high affinity of Cu(I) for the SH residues, the complex is stable and the metal is not easily released. It is important to note that the Cu-thionein has distinct chemical characteristics, including the capacity to produce oxidized insoluble polymers. In the digestive gland of bivalve, the metal is detoxified into the lysosomes trapped in the form of oxidized insoluble Cu-thioneins, which is subsequently eliminated by exocytosis of residual bodies. Nevertheless, literature data indicate that MT synthesis is not always induced in freshwater mussel, studies carried out in laboratory in A. cygnea by [START_REF] Tallandini | Regulation and subcellular distribution of copper in the freshwater molluscs Anodonta cygnea (L.) and Unio elongatulus (Pf.)[END_REF] and in Dreissena polymorpha by [START_REF] Lecoeur | Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha[END_REF] showed no induction of MT after Cu 2+ exposure. MT in response to a metal exposure might be reflected in increased turnover (synthesis and breakdown) of the protein, but not necessarily in changes in MT concentration [START_REF] Mouneyrac | Partitioning of accumulated trace metals in the talitrid amphipod crustacean Orchestia gammarellus: a cautionary tale on the use of metallothionein-like proteins as biomarkers[END_REF].

The Cys-containing peptides PC and GSH have been shown to form complexes with various metals, through their thiolate sulfur atom function. In aquatic organisms, glutathione is believed to play a fundamental role in detoxifying metals. The soluble tripeptide GSH complexes and detoxifies trace metal cations soon after their entrance in the cells, thus representing a first line of defence against trace metal cytotoxicity [START_REF] Canesi | Heavy metals and glutathione metabolism in mussel tissues[END_REF]. In particular, reduction of Cu(II) by GSH produces a stable Cu(I)-SG complex which is also a physiological donor of Cu(I) to copper apoproteins in both mammalian and marine invertebrate cells. Methylmercury has also a high affinity for SH groups of GSH, methylmercury-SG complexes have been identified in different animals tissues. Glutathione metal complexes are transported across the plasma membranes and, therefore represent a carrier for the elimination of the metal from the cells (Viarengo and Nott, 1993). Conjugation of glutathione to metals prevents them from interacting deleteriously with other cellular components. Enhanced cadmium toxicity after glutathione depletion has been observed in both in vitro and in vivo mammalian studies. There is also evidence that glutathione depletion enhances metal toxicity in aquatic organisms. Glutathione depletion by inhibitor of glutathione synthesis (buthionine sulfoximine) enhances copper toxicity in the oysters Crassostrea virginica [START_REF] Conners | Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)[END_REF].

Biochemical and genetic studies have confirmed that GSH is the substrate for PC biosynthesis. PC have been assumed to function in the cellular homeostasis of essential transition metal nutrients, particularly Cu and Zn [START_REF] Schat | The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and nonhyperaccumulator metallophytes[END_REF]. PC are high-affinity chelators of metals, and play major roles in the detoxification. Unequivocal evidence was shown for the involvement of PC synthesis in metal detoxification [START_REF] Courbot | Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus[END_REF]Morelli and Scano, 2004;[START_REF] Thangavel | Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg.) cell suspension cultures under cadmium and zinc stress[END_REF]. Induction of PC is triggered by exposure to various physiological and non-physiological metal ions. Some of these: Cd, Ag, Pb, Cu, Hg, Zn, Sn, Au, and As form complexes with PC in vivo in algae, plants, and fungi (Clemens, 2006). They are suspected to play a role in animals as well as in plant (Cobbett, 2000;[START_REF] Vatamaniuk | Worms take the 'phyto' out of 'phytochelatins[END_REF]Clemens and Peršoh, 2009). In the structural model of a PC-Cd complex, for example, the Cd coordinately binds one, two, three or, at maximum capacity, four sulfur atoms from either single or multiple PC molecules, resulting in amorphous complexes. In vivo the size of the PC chain molecules, and the pH stability are essential and determined the metal binding capacity per molecule of PC (Hirata et al., 2005). [START_REF] Konishi | Enhancing the tolerance of zebrafish (Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase[END_REF], introduced into the early embryos of zebrafish, mRNA coding for PC synthase from Arabidopsis thaliana. As a result, the heterogeneous expression of PC synthase and the synthesis of PC from GSH in embryos could be detected. The developing embryos expressing PC synthase and became more tolerant to Cd exposure.

Extended summary 3 Extended summary

The present work was undertaken in order to investigate some potential causes involved in the Europe-wide observed phenomenon of the decline of various freshwater bivalve species. As stated in the IUCN Red List of Threatened Species, 44 % of all freshwater molluscs are under threat (http://www.iucnredlist.org/news/european-red-list-press-release).

One of the potential causes is the increasing use and corresponding local or regional release of industrially and technologically important metals, besides many other potential factors involved in such a complex eco-toxicologically sequence of events. Clearly, such limited toxicological-pathobiochemical study as presented in the few investigations described here is far too limited to come to a distinct and exclusive delineation of one single, individual explanation for the widespread decline of freshwater molluscs including the bivalves, one class of this important, threatened phylum. On the other hand, without such detailed and necessarily limited studies of concentrating on the potential patho-biochemical and toxicological mechanisms of a single likely pollutant, the delineation of the multitude of facets of such ecotoxicological effects will remain blurred. Therefore, the overall conclusions derived from the investigations presented in more detail in the following is not meant as proof of the only relevance of the increasingly used, technologically important metal copper, but as a demonstration of its potential relevance when considering various possibilities which can have an impact on the stability of bivalve populations, including the most spectacular and most strongly threatened, the European pearl mussel.

Optimization of analytical protocols

Bivalve maintenance and copper exposure Particular attention was paid to mussel maintenance in order to carry out the tests under stable, homogenous, and reproducible conditions (detailed in article 1). The bottom of the tank was covered with a layer of glass beads of 10 mm of diameter, so the mussels could find conditions for burying. This was important for giving the animals a substrate for close-tonature behaviour, at the same avoiding the use of natural river sediment as substrate; thus, unpredictable or unaccountable metal accumulations by adsorption onto sediment particles or interferences of microorganisms by metal species conversion could be avoided without excessively disturbing the behaviour of the animals, as indicated by their regular ventilation activity. Moreover, the large beads facilitated a deep daily cleaning of the tank by suction.

The water, artificially reconstituted from deionised water, was renewed daily in order to ensure equal conditions during the whole experimental course for all replicates and avoid any uncontrolled metal accumulation or cross-contamination. Under these conditions, the copper concentrations could be maintained within close limits of the target concentration during the whole exposure period, as verified by graphite furnace atomic absorption spectrometry (GFASS) or inductively coupled plasma mass spectrometry (ICPMS).

Enzymatic analyses

The enzymatic analyses were carried out as described in article 1 and 2. The activities of Ca 2+ -ATPase and Na + /K + -ATPase were determined in the suspension of the microsomal pellet (as shown in fig. 1) obtained by homogenization of the mantle, digestive gland, gills, and kidney, and by centrifugation at 75600 g, the activity of H + -ATPase was determined in the supernatant of the first centrifugation step at 2000 g as shown in (fig. 1).

Inorganic phosphate released by the ATPases was quantified by spectrophotometry of the ammonium molybdate complex according to Chifflet et al. (1988). The CA activity was evaluated in the supernatants (S 2000, fig. 1) by measuring the pH decrease according to Vitale et al. (1999). The study of phytochelatins in Anodonta cygnea lead us to develop and optimise an HPLC analytical protocol for phytochelatin (PC) quantification in animal tissues, based on the method for plants developed by Minocha et al. (2008). A protein removal step by acidification and centrifugation was added (fig. 2) and the mobile phase gradient profile was modified as described in articles 3 and 4. PC were quantified in the cytosolic fractions of deproteinized tissue homogenates and metallothionein (MT) in the non-deproteinized cytosolic fractions, after thiol reduction with tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) and 1,4-dithiothreitol (DTT), respectively. Monobromobimane (mBBr) was used as a fluorescent tag. Initially non-fluorescent, the dye mBBr became fluorescent after its binding to thiol groups under dehalogenation. It is selective for thiol groups and allows their quantification with high sensitivity by fluorimetry. Each sample was spiked with 0.6 µmol L -1 of each standard PC 2-5 to certify PC identity. The quantification limits per 20 µL sample injected into the HPLC column were 1.5 pmol for PC 2-3 , 2.7 pmol for PC 4 , 5 pmol for PC 5 .

Effects of copper on calcium transport in Anodonta anatina

Physiological data

A good physiological knowledge of the biomarkers is important for their ecotoxicological interpretation.

The present study (article 1 and article 2) allowed us to determine basal levels of the enzymatic activities of Ca 2+ -ATPase, Na + /K + -ATPase, and H + -ATPase of the plasma membrane. These enzymes were studied in gills, digestive gland, kidneys, and mantle, organs playing an important role in calcium input. The Ca 2+ -ATPase controls the cellular calcium concentration by active transport. The Na + /K + -ATPase and the H + -ATPase are maintaining sodium and proton gradients necessary for calcium Na + /Ca 2+ and H + /Ca 2+ transporters. H + -ATPase and cytoplasmic CA were also studied for their implication in the biomineralization process of the shell and in the maturation of glochidia. There is a lack of physiological data in freshwater bivalves compared to marine models such as Mytilus edulis or M. galloprovincialis. These data are useful in understanding calcium transport mechanisms since freshwater bivalves are subject to osmo-and iono-regulations different from marine mussels. Enzymatic activity found in the gills of A. anatina (table 1) were consistent to those reported in the gills of A. cygnea for Na + /K + -ATPase and Ca 2+ -ATPase 0.11 µmol P i /mg protein/min, (Lagerspetz and Senius, 1979) and 0.58 µmol P i /mg protein/min [START_REF] Pivovarova | Effect of cadmium on the ATPase activity in gills of Anodonta cygnea at different assay temperatures[END_REF] respectively, and in the gills of A. anatina for CA 2.1 U/mg protein [START_REF] Ngo | Sub-chronic effects of environment-like cadmium levels on the bivalve Anodonta anatina (Linnaeus 1758): III. Effects on carbonic anhydrase activity in relation to calcium metabolism[END_REF]. The plasma membrane Ca 2+ -ATPase (PMCA) activity in the gills (article 1) was fourfold higher than in the marine bivalve Mytilus edulis (Burlando et al., 2004). This reflects how important calcium active transport is to the freshwater mussel Anodonta anatina.

Calcium concentrations in seawaters are higher than in freshwaters which explains that calcium absorption is easier for Mytilus edulis. PMCA activity is also high in the kidneys and the digestive gland, favouring calcium absorption from food and calcium reuptake from renal filtrate. These physiologic data allowed us to understand how PMCA is crucial for calcium homeostasis in freshwater bivalves compared to marine organisms. Moreover in Unionidae, calcium plays a direct role in reproduction since glochidia are incubated in the marsupium (gills) and is therefore important for population growth of mussels. Na + /K + -ATPase maintained the sodium transmembrane cellular gradient necessary for calcium facilitateddiffusion by Na + /Ca 2+ antiporters. In Anodonta anatina, Na + /K + -ATPase activity varied in a consistent manner as function of the seasons (article 2). The enzymatic activities of Na + /K + -ATPase and H + -ATPase were found to be maximum in summer, the season of shell and glochidia calcification in Anodonta mussels (Taskinen, 1998;Moura et al., 2000). This seasonal variation is an important aspect which is imperative to consider for data interpretation in ecotoxicological investigations.

signalling pathways in addition to biomineralization and shell formation in mussels and in glochidia.

The kidneys have an essential role in filtration and reabsorption of ions, water, and organic molecules from the ultrafiltrate. As freshwater organisms are hyperosmotic, the osmotic pressure resulting from the gradient of concentrations between internal and surrounding compartments leads to water uptake by osmosis and ionic loss by diffusion. The water uptake is compensated by urine production and ionic loss is limited by active ion reabsorption. In freshwater bivalves, the daily output of urine is high. The kidneys play an essential role in calcium homeostasis by limiting ionic loss in urine through active Ca 2+ reuptake from the filtrate (Turquier, 1994). An inhibition of calcium reabsorption may therefore dramatically disturb calcium homeostasis. Because of analytic problems inherent to the small biomass of kidneys, this tissue has been poorly studied in freshwater bivalves. This organ plays a role in detoxification (Viarengo and Nott, 1993) and is essential to ionoregulation. Our results (article 1) showed the high sensitivity of Ca 2+ -ATPase to Cu 2+ in this organ.

The Unionidae freshwater bivalves are widely recognized for their capacity to accumulate a variety of environmental contaminants in their tissues including metals (Bonneris et al., 2005). This marked metal tolerance is effected by biochemical strategies that involve metal sequestration. The intracellular sequestration of metals is based on a sequence of cellular events involving a cascade of different ligands with increasing metal binding strengths. High concentration of metal could also inhibit detoxification mechanisms. In our results (article 1 and 2), recovery of Ca 2+ -ATPase and Na + /K + -ATPase activity within 7 days at the low Cu 2+ concentration (0.35 µmol L -1 ) indicated adaptive ability. This suggested the mobilisation of detoxification systems efficient at low Cu 2+ concentrations. At higher Cu 2+ concentration (0.64 µmol L -1 ), no recovery was noted. It is interesting to note that recovery was observed only at 0.35 µmol L -1 , a concentration environmentally more relevant than the higher concentration studied (article 1). Therefore, it is important to use environmental concentrations in ecotoxicological research; extrapolation of the results observed at high doses to environmental situations may be critical and not pertinent due to different mechanisms of adaptation and toxicity at high and low doses.

Metal detoxification mechanism in Anodonta cygnea

Aquatic molluscs have a number of properties that make them one of the most popular sentinels for monitoring water quality. The molluscs take up and accumulate high levels of trace metals, although the body concentrations show wide variability across metals and invertebrate taxa. The study of the capacity of bivalves for assessment of water quality is primarily linked to their tendency to accumulate trace metals even at high concentrations (Doyotte et al., 1997). Tolerance depends on the ability to regulate the metal concentrations in the cells and to accumulate excess metals in non-toxic forms (Viarengo and Nott, 1993).

In our study, the recovery of Ca 2+ -ATPase (article 1) and Na + /K + -ATPase (article 2) activity observed within 7 days of exposure indicate an induction of detoxification mechanisms. Copper belongs to the transition metals most of which are known to show various degrees of affinity for thiol group. This property makes chelating ligands carrying thiol functions the first mechanism of metal detoxification. In articles 3 and 4, the study was focused on the mechanisms of metal detoxification by thiol rich compounds. In most animals, tolerance to trace metals depends on the induction of MT, a family of thiol-rich proteins of low molecular weight. These metallo-proteins are known to play an important role in the homeostatic control of essential metals such as Cu and Zn but also in the detoxification of excessive amounts of essential and non-essential trace metals. In the tissues of metal-exposed mussels is a rapid increase of metallothioneins, the soluble proteins involved in transition metal detoxification which is synergetic with lysosome compartmentalization (Amiard et al., 2006).

Phytochelatins (PC) are thiol rich oligopeptides which have been characterized in a wide range of plant species (Grill et al., 1985). PC plays a major role in the detoxification of trace metals in plants by chelating metals with high affinity. In 2001, two publications independently described a functional phytochelatin synthase in the invertebrate nematode

Caenorhabditis elegans (Clemens et al., 2001;Vatamaniuk et al., 2001). Brulle et al. (2008) provided the first evidence of a phytochelatin synthase from the earthworm Eisenia fetida implicated in dose-dependent cadmium detoxification. Neither study furnished evidence of the existence of the peptide PC in the invertebrates and animals. Recent cloning of the gene encoding phytochelatin synthase in E. fetida (Brulle et al., 2008;[START_REF] Bernard | Metallic trace element body burden sand gene expression analysis of biomarker candidates in Eisenia fetida, using an ''exposure/depuration'' experimental scheme with field soils[END_REF] suggested the existence of this detoxification pathway in this species. A superficial view of the limited selection of species in which such sequences have been identified might suggest that invertebrates with an aquatic or soil habitat are more likely to express PC (Cobbett, 2000). The objectives of our study were to determine the ability of A. cygnea to synthesize PC (article 3), and to study the possible role played by PC in copper detoxification in this freshwater bivalve (article 4).

In plants, PC are rapidly induced in cells and tissues exposed to a range of transition metals and play an important role in detoxification. The results obtained in the present work (articles 3 and 4) showed the presence of phytochelatins in invertebrates; to our knowledge this is the first time. Basal levels were determined for PC 2-4 , -GluCys, and MT in the gills and the digestive gland (table 2). Besides the interest of these data for freshwater bivalve studies, the detection of PC 2-4 in the absence of excessive copper and other metals suggests their role in essential metal homeostasis. In the gills and the digestive gland, PC 2 was found in higher concentrations, followed by PC 3 which again was higher in concentration than PC 4 .

PC 2 and PC 3 were found to be two or three times higher in the digestive gland than in the gills.

Table 2: Basal levels of phytochelatines 2-4 (µg PC/g tissue wet weight), -GluCys (µg -GluCys/g tissue wet weight), and metallothionein (mg MT/g protein) in Anodonta cygnea. G: gills; DG: digestive gland; PC: phytochelatin; MT: metallothionein

In the European continental hydrosystem affected by agricultural and / or urban activities, levels of up to 0.6 µmol L -1 of Cu 2+ are seasonally found [START_REF] Neal | A summary of river water quality data collected within the Land-Ocean Interaction Study: core data for eastern UK rivers draining to the North Sea[END_REF]Falfushynska et al., 2009). In our work (article 1 and 2), inhibition followed by recovery of Ca 2+ -ATPase and Na + /K + -ATPase activities were observed in mussels exposed to 0.35 µmol L -1 Cu 2+ , an environmentally relevant concentration. Therefore, in our studies on the detoxification mechanisms by metal binding Cys-rich compounds (articles 3 and 4), the mussels were exposed to the same Cu 2+ concentration.

A statistical significant PC 2 induction in the gills of A. cygnea exposed to of 0.35 µmol L -1 Cu 2+ was observed (article 4), i.e. 50 % from 12 h to 4 d exposure, and 30 % upon 7 d. In the digestive gland, significant PC 2 induction was observed only at 12 h of Cu 2+ exposure.

Relative to the respective control, -GluCys increased significantly in the gills within 48 h and 7 d exposure and within 48 h and 4 d in the digestive gland.

The induction of PC 2 in A. cygnea exposed to Cu 2+ suggests its key role as metal chelator in a first line of detoxification, together with other compounds such as GSH. Higher sensitivity of PC 2 induction in the gills could be explained by the water route of Cu-exposure.

The gills may play a more important role than the digestive gland in the uptake of copper dissolved in the test media. However, beyond 7 d of exposure, PC 2 declined to control values within 21 d of exposure. This decrease suggests that long-term Cu 2+ detoxification was shifting to other mechanisms.

In Unionidae, MT and insoluble granules are known to play a role in metal detoxification in the long term. Bonneris et al. (2005) showed that cadmium, zinc, and copper concentrations in the gill granule fraction were significantly correlated with environmental concentrations of these metals. The granules are known to be a preferential site for copper storage in the gills of Unionidae. Around 65 % of the total copper in the gills was found to be bound to granules in Anodonta grandis grandis, were the calcium concretion represented 51 % of the gills dry weight. Similar values were found in Anodonta cygnea (Bonneris et al., 2005).

No increase of MT was observed in the present study upon Cu 2+ exposure for 21 d.

The MT isoform (< 10 kDa) in the mussel extract identified by HPLC separation in our study was not induced by Cu 2+ . Detoxification in A. cygnea by other MT isoforms not detected by the HPLC method cannot be excluded. Indeed, MT polymorphism is known to be important in invertebrates (Géret et al., 2002b;Amiard et al., 2006). A good example of such polymorphism and functional divergence is found for MT in the snail Helix pomatia. These snails can tolerate exceptionally high concentrations of cadmium. Additionally, they accumulate relatively high amounts of copper, needed for the biosynthesis of the oxygen carrier hemocyanin. The specific metal accumulation is paralleled by the presence MT forms which bind specifically one type of metal (Cd or Cu), both containing 18 conserved Cys residues but differing in other amino acids [START_REF] Vašák | Advances in metallothionein structure and functions[END_REF].

Metallothioneins, granules, and antioxidant systems have been described to be involved in detoxification mechanisms in freshwater bivalves. Cossu et al. (1997) have shown that in Unionidae antioxidants and especially GSH play major roles in metal detoxification. In the gills of Unio tumidus, a decrease by 45 % of GSH was found in mussels exposed at a metal-contaminated site. A decrease in GSH level in Unionidae exposed to copper was confirmed by Doyotte et al. (1997) under controlled laboratory conditions, indicating either metal blockage by SH groups or the use of GSH as substrate of antioxidant enzymes. Indeed, a parallel involvement of antioxidant enzymes had been described with increased activity at low metal concentration (Vasseur and Leguille, 2004). GSH also plays a role in PC synthesis.

This double role in direct metal detoxification and as a PC precursor could explain this decrease (fig. 1).

Decline of the Unionidae populations

Over the past 50 years, a world-wide decline of autochthonous freshwater molluscs has been observed (Lydeard et al., 2004). Among the different species, the Unionoida taxon seems to be particularly endangered. Species such as the pearl mussels Margaritifera margaritifera and Margaritifera auricularia are declining dramatically in European rivers.

Other species belonging to the Unionidae family are included in the red list of threatened species established by the World Conservation Union and is the case for Anodonta anatina and Anodonta cygnea.

The Unionidae populations represented the largest part of the total biomass in many aquatic systems. They take an active part in sedimentation and water purification, modifying the phytoplankton community, and are detritivorous invertebrates (Aldridge, 2000;[START_REF] Vaughn | Community and foodweb ecology of freshwater mussels[END_REF]. Therefore, the disappearance of Unionidae may result in structural and functional perturbations of aquatic ecosystems. The competition with invasive bivalves is also a matter of debate. Invasive freshwater bivalves as Corbicula fluminea or Dreissena polymorpha not belonging to the Unionidae colonise freshwater hydrosystems with detrimental effects to other invertebrates. These invasive species do not play the same functional role as Unionidae in ecosystems. Their presence is hypothesized to contribute to the decline of the Unionidae.

A combination of various factors may explain the gradual disappearance of Unionoidea in their competition with invasive species. Depletion of salmonids as host-fish has also been mentioned as a possible cause. Salmonid fish populations are affected by water pollution; moreover, pollutants may indirectly promote natural disease factors such as parasitism which is known to endanger salmonids (Voutilainen et al., 2009), and bivalves; trematode parasitism of Anodonta population entails complete infertility [START_REF] Taskinen | Age-, size-and sex-specific infection of Anodonta piscinalis (Bivalvia, Unionidae) with Rhipidocotyle fennica (Digenea, Bucephalidae) and its influence on host reproduction[END_REF]. Other major reasons for the decline are the physical degradation of streams and the reworking of river beds and canals as well as the degradation of water quality. Indeed, interactions between pollutants may disturb these biocoenoses even at low concentrations (Vighi et al., 2003). In order to protect the autochthonous Unionidae it is important to determine how and to which extent those factors contribute to their disappearance.

The present work is meant as a contribution to understand the role of metal pollution in affecting calcium homeostasis in fresh water mussels, likely to be one of the reasons for the widespread decline of mussel populations in European rivers (Frank and Gerstmann 2007).

Conclusion 4 Conclusion

A macrobentos community is an excellent indicator of water quality (Ippolito et al., 2010). The objectives of the present work were to study some toxic effects of ionic copper and its detoxification mechanisms in the freshwater mussel Anodonta species belonging to the Unionidae. First, Cu 2+ as a potential inhibitor of proteins playing a role in calcium transport and biomineralization was assessed with A. anatina. Secondly, mechanisms of copper detoxification by MT, PC, and -GluCys were studied in A. cygnea.

In the present study, the effects of Cu 2+ exposure on the activities of Ca 2+ -ATPase, Na + /K + -ATPase, and H + -ATPase of the plasma membrane, and of cytosolic CA have been evaluated in the freshwater bivalve A. anatina.

Upon 4 d exposure, inhibition of Ca 2+ -ATPase activity in the gills and the kidneys, and of the Na + /K + -ATPase activity in the gills and the digestive gland was observed in A. anatina exposed to 0.35 µmol L -1 Cu 2+ . A total recovery of Ca 2+ -ATPase was observed upon 7 d of exposure, indicating that detoxication mechanisms are activated, except for the higher Cu 2+ concentration of 0.64 µmol L -1 .

Ca 2+ -ATPase inhibition was particularly sensitive in the kidney, the organ playing an important role in calcium reuptake and iono-regultion. It would be interesting to study the long-term consequences on disturbances of osmoregulatory functions in the kidney of A.

anatina exposed to 0.64 µmol L -1 Cu 2+ .

The present work is the first to identify the phytochelatins, i.e. PC 2 , PC 3 , and PC 4 , in freshwater mussels. In A. cygnea exposed to 0.35 µmol L -1 of Cu 2+ , PC 2 was induced in the digestive gland and the gills within 12 h. The induction in the gills persisted for 7 days. These results suggest a role of PC in essential metal homeostasis and their involvement in first-line detoxification.

Our HPLC results showed no variation of MT levels after Cu 2+ exposure, at least the MT isoform quantified with our method. Additional MT experiments with A. cygnea exposed to Cu 2+ at the same conditions and employing a spectrometric method which allows the quantification of the total MT isoforms would be interesting, in order to compare our results with those found in A. anatina by Nugroho and Frank (2012). In parallel the HPLC method should be optimized to allow the detection of other MT isoforms induced by copper.

Copper has been reported as a weak inducer of metal binding thiol peptides (Zenk, 1996). Exposure to a non-essential metal being a strong inducer of PC, such as cadmium, could be interesting. Comparison of effects of an essential metal like copper and a non essential metal like cadmium could allow to determine the role played by PC in homeostasis of essential metal and detoxfication mechanisms.

Copper is an inducer of reactive oxygen species (ROS) through Fenton type reactions.

The activity of PC in scavenging ROS in A. cygnea exposed to copper would be interesting to study.

Chemical pollution is one of the environmental factors that may impact bivalve populations. Toxicity depends not only on the bioavailability of pollutants and their intrinsic toxicity, but also on the efficiency of detoxifying systems in eliminating reactive chemical species.

Articles

INTRODUCTION

Na + /K + -ATPase and H + -ATPase are important enzymes mainly involved in osmoregulation and acid-base balance. H + -ATPase plays as proton pump also an essential role in shell synthesis by pH control, a decisive factor in the biomineralization process (Machado et al., 1989). The plasma membrane Na + /K + -ATPase and H + -ATPase maintain the sodium and proton gradients necessary for the calcium antiporter system. Although calcium uptake is mainly achieved by active flux generated by Ca 2+ -ATPase. Facilitated diffusion through Ca 2+ /Na + or Ca 2+ /H + antiporters are other important ways of entry of calcium into the cell (Wheatly et al., 2002).

Perturbation of calcium homeostasis has been hypothesized as a possible cause in the decline of the pearl mussel populations (Frank and Gerstmann 2007). Another freshwater mussel belonging to the Unionoida order known to bioaccumulate copper is Anodonta anatina (Cossu et al., 1997;Nugroho and Frank, 2011). Inhibition of Ca 2+ -ATPase activity in the plasma membrane was shown in A. anatina exposed to 0.35 µmol L -1 of copper (Santini et al., 2011a).

Both H + -ATPase and Na + /K + -ATPase of the plasma membrane are metal-sensitive enzymes with functional SH groups. Due to its thiol affinity, copper is likely to affect the activity of these enzymes (Viarengo et al., 1991) and calcium homeostasis could thus be indirectly affected by copper; alteration of Na + and H + gradients will entail decreased efficacy of Ca 2+ /Na + or Ca 2+ /H + antiporters. Therefore, it seemed interesting to evaluate the activities of both enzymes in mussels in order to determine their sensitivity to this transition metal which nowadays is widely found in the environment at elevated levels.

Therefore, in the present study the effects on the activities of Na + /K + -ATPase and H + -ATPase have been evaluated with A. anatina mussels exposed to copper ions (Cu 2+ ) at 0.35 µmol L -1 . The H + -ATPase activity was measured in the mantle, which plays the dominant role in the generation, growth and repair of the shell. Na + /K + -ATPase activity was determined in the mantle, the gills, and the digestive gland, organs which are mainly involved in calcium uptake and homeostasis (Coimbra et al., 1993).

MATERIAL AND METHODS

Chemicals

All chemicals used for maintenance and exposure of bivalves, for sample preparation, and the biochemical assays were of analytical grade.

Ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), ouabain, phenylmethylsulfonic fluoric acid (PMSF), sodium azide, sodium dodecyl sulphate (SDS), Nethylmaleimide (NEM), and sodium orthovanadate were from Fluka (Schnelldorf, Germany).

Adenosine-5'-triphosphate (ATP), N-(2-hydroxyethyl) piperazine N'-(2-ethanesulfonic acid) (HEPES), and tris-(hydroxymethyl)-aminomethane (Tris) were from Carl Roth (Karlsruhe, Germany).

Animal maintenance and copper exposure

Mussel maintenance was described in detail in a previous article (Santini et al., 2011a). Briefly, adult mussels A. anatina with shell lengths of 6.5 -7.5 cm were kept at 17° C in a glass aquarium containing 1.5 L per animal of filtered and aerated artificial pond water ). The water was renewed every 48 h. The bottom of the tank was covered with a 5-cm layer of glass beads (about 10 mm diameter), so the mussels could find conditions for burying. They were fed daily with unicellular algae Chlorella kessleri from a culture in the exponential growth phase, added to a final algal density of 2×10 5 cells mL -1 . Animals were acclimatized for 3 weeks in September before any experiment.

For the copper (Cu 2+ ) exposure, animals were placed in a 20 L aquarium lined with dye-and pigment-free high-density polyethylene foils, with a glass beads layer and filled with 1.5 L per mussel of experimental media (pH 7.2 temperature: 17 ± 0.5° C). The bivalves were fed daily as described above and the test media were renewed every day. Concentration of 0.35 µmol L -1 Cu 2+ was chosen for exposure test. The concentration was controlled by inductively-coupled plasma mass spectrometry, with a limit of Cu detection of 0.5 µg L -1 = 8 nmol L -1 . The test was conducted with 12 mussels randomly assigned by groups of 3 to each treatment. At the beginning (time 0) a group of 3 mussels unexposed to Cu 2+ was used as control. A group of 3 mussels was taken from the aquarium after 4 d, 7 d, and 15 d of Cu 2+ exposure. Mussels used on each treatment were dissected separately for biochemical analyses, i.e. 3 replicates per treatment.

Tissue sample preparation for enzymatic analysis

Tissue samples of the gills, the mantle, and the digestive gland were prepared. The samples were suspended in 6 vol. ice-cold HEPES buffer (10 mmol L -1 HEPES, 250 mmol L -1 sucrose, 1 mmol L -1 EDTA, 1 mmol L -1 phenylmethylsulfonic fluoric acid (PMSF) as protease inhibitor, adjusted to pH 7.4 with HCl, 1 mol L -1 ) and homogenized by means of a motor-driven Teflon pestle homogeniser with 30 up-and-down strokes. The resulting homogenates were centrifuged at 2000 g (10 min, 4° C), the supernatants (S2000) were diluted with 12 mL ice-cold HEPES buffer per g tissue wet weight and centrifuged at 10000 g (20 min, 4° C). The supernatants (S10000) were ultracentrifuged at 75600 g (60 min, 4° C), and the final pellets were suspended in 6 vol. ice-cold Tris-HCl buffer (25 mmol L -1 Tris, 1 mmol L -1 PMSF, adjusted to pH 7.4 with HCl, 1 mol L -1 ) for plasma membrane Na + /K + -ATPase test. Plasma membrane H + -ATPase activity was determined in the S2000 supernatant used as mantle extract. All samples were frozen in liquid nitrogen and stored at -80° C until analyses were carried out, not later than one week after sampling.

Protein concentrations were determined according to Bradford (1976), using bovine serum albumin as standard.

Plasma membrane H + -ATPase and Na + /K + -ATPase activity assay

Plasma membrane H + -ATPase and Na + /K + -ATPase activities were determined by measurement of inorganic phosphate released (Chifflet et al., 1988) and quantified by spectrophotometry of the ammonium molybdate complex at 850 nm (Spectrophotometer Unicon, Kontron 930).

The H + -ATPase reaction medium contained (final concentrations) 6 mmol L -1 MgSO 4 , 50 mmol L -1 hepes, 0.5 mmol L -1 sodium orthovanadate (P-ATPase inhibitor), 0.5 mmol L -1 sodium azide as inhibitor of mitochondrial ATPase activities, 4 mmol L -1 Na 2 ATP, adjusted to pH 7.4. The mantle extract (160 µg protein) was incubated at 1 mL final volume for 60 min in a shaking water bath at 25° C, with or without addition of NEM as H + -ATPase inhibitor [START_REF] Lin | H + -ATPase activity in crude homogenates of fish gill tissue: inhibitor sensitivity and environmental and hormonal regulation[END_REF]. The reaction was stopped by addition of 400 µL sample to 400 µL of a 12 % solution of sodium dodecyl sulphate (SDS). Blanks were prepared in the same way except that the tissue samples were added after the SDS-dilution step. H + -ATPase activity, expressed in micromoles of P i released per mg protein and min, was determined as the difference between the ATPase activity in the presence of 10 mmol L -1 NEM and the ATPase activity without NEM.

The Na + /K + -ATPase reaction medium contained (final concentrations) 100 mmol L -1 NaCl, 0.5 mmol L -1 EGTA, 5 mmol L -1 MgCl 2 , 25 mmol L -1 Tris, 0.5 mmol L -1 sodium azide as inhibitor of mitochondrial ATPase activities, 4 mmol L -1 Na 2 ATP, adjusted to pH 7.4.

Samples of the membrane fractions (suspensions of the 75600 g pellet) of the gills, the digestive gland, and the mantle (20, 70, 60 µg protein, respectively) were incubated at 1 mL final volume for 20 min in a shaking water bath at 37° C, with or without addition of K + and ouabain as Na + /K + -ATPase inhibitor (Lionetto et al., 1998). The reaction was stopped by addition of 400 µL sample to 400 µL of a 12 % solution of sodium dodecyl sulphate (SDS).

Blanks were prepared in the same way except that the tissue samples were added after the SDS-dilution step. Na + /K + -ATPase activity, expressed in micromoles of P i released per mg protein per min, was determined as the difference between the ATPase activity in the presence of 20 mmol L -1 KCl, and the ATPase activity without KCl and in the presence of ouabain (1 mmol L -1 ).

Statistical analyses

Data distributions were not normal, so statistical analysis for comparison of the enzymatic activities between treated and control mussels, was done using the non-parametric Mann-Whitney test (Statistica, StatSoft France 2001, data analysis software, version 6, Maison-Alfort, France). All data are reported as mean (n = 3) ± standard deviation (S.D.).

Differences were considered as significant when p < 0.05. No significant effect of copper on H + -ATPase activity was noted (fig. 2) in the mantle of mussels upon 15 days of exposure. The H + -ATPase activities exhibited a continuously declining trend, although statistically not significant due to high variability between mussels. These basal activities (expressed in micromoles of P i released per mg protein per min) varied in consistent manner in function of seasons. The activities were the highest in July and September compared to the values measured in January, March and April (fig. 3). In July, the Na + /K + -ATPase activity expressed as µmol P i mg/protein/min was 0.107 ± 0.027 in the gills, 0.047 ± 0.050 in the digestive gland, and 0.053 ± 0.025 in the mantle (fig. 3). The H + -ATPase of the mantle did not significantly differed over the study period (from July to April) with 0.0025 ± 0.001 µmol P i /mg protein/min in July (fig. 4). Minimal values of Na + /K + -ATPase and H + -ATPase activities were observed in April in every tissue with a strong decrease by half and ten of the July values, depending on organ.

RESULTS

Fig

DISCUSSION

In the general decline of freshwater bivalve observed (Lydeard et al., 2004), the Unionoida taxon is particularly endangered. Alteration of ionic transport systems as Na + /K + -ATPase and H + -ATPase by copper is poorly investigated in the freshwater mussel Anodonta anatina belonging to this taxon. Copper, a metal extensively used in industry, building sector, public energy supply and transportation systems, and agriculture was studied in the present work as a potential factor implicated in this complex phenomenon of decline.

The magnitude of mean basal activities of Na + /K + -ATPase in the different organs studies were gills > mantle > digestive gland. The high activity measured in the gills reflects the specialization and implication of this organ in ionoregulation. The gills are known to play a major role in active uptake of mineral ions from surrounding water (Turquier, 1994). The mantle assumes the ionic balance between haemolymph and the extrapallial fluid necessary for shell growth or calcium body reabsorption (Coimbra et al., 1993).

The basal activities of Na + /K + -ATPase determined in the gills of control A. anatina mussels were in the same order of magnitude as found by Lagerspetz and Senius (1979) in A.

cygnea, and in the freshwater fish Bidyanus bidyanus (Alam and Frankel, 2006) (Table 1).

The hyperosmotic status of A. anatina and A. cygnea is expressed by the high basal activities of the enzyme. In comparison, the marine mussel Mytilus galloprovincialis showed a Na + /K + -ATPase activity threefold lower than A. anatina (Viarengo et al., 1996).

There is little information in the scientific literature on the H + -ATPase activity in the mantle of mussels. Investigations on the enzyme activity expressed as µmol P i mg/protein/min (table 1) were carried out mostly in the gills of crustacean 0.023 and fish 0.003 to 0.041. In the present work the mean H + -ATPase activity 0.0025 ± 0.001 found in the mantle of A.

anatina was lower.

In this study, seasonal fluctuations of Na + /K + -ATPase basal activity were observed with clear and significantly elevated levels in July and September. It is known that the freshwater mussel shows seasonal changes in calcification (Taskinen, 1998). Our results correspond to the cycle of calcification found in Anodonta cygnea a close species. The growth of clam shells and the level of glycosaminoglycans know to be important for biomineralisation, increased both in summer with a maximum in July and August (Taskinen, 1998;Moura et al., 2000). These fluctuations, parallel to the calcification cycle, suggest an indirect implication of Na + /K + -ATPase in the calcium transport by the Ca 2+ /Na + antiporter.

H + -ATPase activities showed not statistically significant comparable seasonal fluctuations.

Na + /K + -ATPase of the gills and the digestive gland were strongly affected by exposure to 0.35 µmol L -1 of Cu 2+ upon 4 days of exposure, to a lesser extent in the mantle. Therefore, even though our results do not provide direct evidence of the Ca 2+ /Na + antiporter inhibition, a decrease of calcium ionic transport can be assumed. This is consistent with the results observed by Viarengo et al. (1996) in M. galloprovincialis exposed 4 d to 0.6 µmol L -1 of Cu 2+ . In A. anatina, inhibition of Na + /K + -ATPase modified the cellular Na + gradient, which could result in a reduced activity of Ca 2+ /Na + antiporter and disturbance of calcium homeostasis. A similar profile of inhibition of the H + -ATPase activity was observed in the mantle, but inter-individual variations were too high to be statistically significant. Some recovery of the Na + /K + -ATPase was observed in the digestive gland of A.

anatina within 7 days of Cu 2+ exposure. Viarengo et al. (1996) also showed a return to basal level of the Na + /K + -ATPase activity in M. galloprovincialis exposed during 7 d to 0.6 µmol L - 1 of Cu 2+ . This may indicate the occurrence of detoxification systems mobilized in the first times of exposure to cope with stressors through metal sequestration or binding to metallothioneins.

Despite a trend indicating partial recovery beyond 4 days the Na + /K + -ATPase activity in the gills of A. anatina was still significantly inhibited upon 15 days of experiment. An inhibition of Ca 2+ -ATPase activity was also observed in the gills and the kidneys of A.

anatina exposed to Cu 2+ in same conditions as in the present study (Santini et al., 2011a).

These whole results indicate an inhibition of ionic transport by Cu 2+ , which may perturb calcium homeostasis and ionoregulation more generally. In the long term, this may lead to biomineralization perturbation (shell slimmer, glochidia viability decrease), and physiologic impairments.

INTRODUCTION

Several transition metals (Cr, Mn, Fe, Co Cu, Zn, Mo) have chemical properties that make them essential for biological systems but toxic in excess. Essential and non-essential metals pose the problem of being toxic in the micromolar concentration range. For homeostasis and detoxification of such metals, bacteria, plants, and animals employ a strategy by synthesizing cysteine-rich peptides and proteins with high-affinity metal binding sites in the form of thiol groups (Clemens, 2006), viz. glutathione, phytochelatins, and metallothioneins.

Until recently, in animal studies on trace metal detoxification, only reduced glutathione (GSH) and the family of metallothioneins (MT) were taken into consideration.

The ubiquitous tripeptide GSH ( -GluCysGly) is present in most eucharyotic cells at concentrations of about 0.2 to 10 mmol L -1 . Metallothioneins are cysteine-rich, ubiquitous cytosolic proteins of low molecular weights (~ 4 -14 kDa). In addition to GSH and MT, in plants, some fungi, and yeasts, phytochelatins (PC n ) represent a third type of thiol-bearing entity (Grill et al., 1985;Clemens, 2006). Ni, Cu, Zn, Ag, Hg, and Pb, and the metalloid arsenic (Clemens, 2006). Homologous genes enabling to give a functional PCS were identified in the nematode Caenorhabditis elegans (Clemens et al., 2001;Vatamaniuk et al., 2001) and more recently in the oligochaete Eisenia fetida (Brulle et al., 2008). These findings suggest that PC n may play a role in metal homeostasis also in animals.

Bivalves are known to bio-accumulate persistent organic pollutants and metals, which is one of the reasons suspected of the freshwater bivalve general decline (Franck and Gerstmann, 2007). According to Clemens and Peršoh (2009), bivalves also present a homologous gene of PCS. Anodonta cygnea is a freshwater bivalve belonging to the Unionidae, a filter-feeding and burrowing species living at the water/sediment interface. As bivalves are in close contact with the aquatic environment, they are known to bioaccumulate transition metals (Gundacker, 2000;Nugroho and Frank, 2011). The aim of the present study was to determine whether PC n are eventually synthesised by A. cygnea in the gills and the digestive gland. Indeed the presence of PC n in animals has been established for the first time.

MATERIALS AND METHODS

Chemicals

Phytochelatins standards (PC 2 , PC 3 , PC 4 , and PC 5 ) [PC n , ( -Glu-Cys) n -Gly, where n = 2-5], and monobromobimane (mBBr) were from Anaspec (San Jose, CA, USA). Acetonitrile pooled in order to obtain sufficient mass for analysis. The samples were immediately analysed after sampling or frozen in liquid N 2 and stored at -80°C for no more than 4 weeks until analysis. To avoid enzymatic degradation or PC n oxidation, tissues were homogenised in acid buffer (6.3 mmol L -1 DTPA, 0.1% TFA) with a manual Potter-Elvehjem homogenizer with glass pestle. The homogenization was performed with 500 mg tissues in 1 mL buffer. The homogenate was centrifuged at 3500 g for 10 min (model 1-15PK, Sigma-Aldrich, St.

Quentin Fallavier, France). The resulting supernatant (500 µL) was deproteinized by addition of 125 µL 5 mol L -1 perchloric acid, and centrifuged again at 13000 g for 30 min. The supernatant (500 µL) was neutralised with 100 µL 5 mol L -1 NaOH, and used for PC n analyses.

Reduction and derivatization:

Just after extraction, 99 µL of tissue extract was mixed with 244 µL of HEPPS buffer (200 mmol L -1 HEPPS, 6.3 mmol L -1 DTPA, pH 8.2), 10 µL TCEP solution (20 mmol L -1 TCEP in HEPPS buffer, pH 8.2) as disulfide reductant, and 4 µL of a solution of 0.5 mmol L -1 NAC as internal standard. Reduction was conducted in a water-bath at 45°C for 10 min. The sample injection volume was 20 µL. Fluorescence of mBBr-labeled compounds was monitored at an excitation wave length of 382 nm and 470 nm of emission. Derivatized PC n were separated in a reversed-phase column (Phenomenex-Synergi-Hydro RP C18), 100 mm × 4.6 mm, 4 µm particle size, protected by a C18 guard column, 4 mm × 3 mm, 5 µm (Phenomenex Security guard cartridge). The temperature of the column oven was 40°C. Peak areas were integrated using proper software (UniPoint system, version 1.90, Gilson, Villier-le-Bel, France). The bimane derivatives were separated using a gradient of mobile phase A (99.9 vol-% ACN, 0.1 vol-% TFA) and B (89.9 vol-% water, 10 vol-% ACN, 0.1 vol-% TFA). The gradient profile was a linear gradient of mobile phase A from 0 to 10.6% run for 11.2 min at 1 mL min -1 . Further, the linear gradient of solvent A was raised from 10.6 to 28.6% in 13.6 min.

Before injecting a new sample, the column was rinsed with 100% of solvent A for 5.5 min at a flow rate of 2.5 mL min -1 . The column was equilibrated with 100% of solvent B for a total of 10 min at 1 mL min -1 . Total run time for each sample was 40.3 min including column rinsing and re-equilibration.

Purified standards at seven increasing concentrations ranging from 0.2 to 2 µmol L -1 for PC 2-5 , and 1 to 10 µmol L -1 for Cys, GSH, and -GluCys, were used to plot calibration curve. In samples thiol compound concentration were determined using equations of calibration curve. PC content was expressed in µg of PC per g of tissue wet weight. Each sample was spiked with 0.6 µmol L -1 of each standard PC 2-5 to certify PC identity.

Statistical analyses

Homogeneity of variances and normality of data were not verified (Bartlett and Shapiro-Wilk tests), so statistical analysis was done using the non parametric Kruskal-Wallis and Mann-Whitney two sided tests (R Development Core Team, 2010. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.Rproject.org). All data means were reported with standard deviations (SD). Differences were considered as significant when p < 0.05.

RESULTS AND DISCUSSION

Table 1: Average retention time in minutes (n = 20) ± SD, limit of detection (LOD), and limit of quantification (LOQ) in pmol per 20 µL injected for cysteine-rich metal-binding peptides standards. Standard curves were run with 7 concentrations: 1 to 10 µmol L -1 for Cys, GSH, -GluCys, and 0.2 to 2 µmol L -1 for PC 2-5 . -Not determined for the NAC used as an internal standard r 2 : Pearson correlation coefficients of standard curves

In this study, with precolumn mBBr derivatization and reversed-phase HPLC analyses, an excellent linearity was obtained for the calibration curves as shown by the Pearson coefficients (table 1). The quantification limits per 20 µL sample injected into the HPLC column were 1.5 pmol for PC 2-3 , GSH, -GluCys, 2.7 pmol for PC 4 , 5 pmol for PC 5 , and 1 pmol for Cys. In the samples a part of mBBr underwent reductive dehalogenation with TCEP to give tetramethylbimane (Me 4 B) (Graham et al., 2003). i.e. 2.2 ± 0.6 and 0.9 ± 0.2 µg g -1 wet weights respectively. In both organs, the PC n levels decreased in the order PC 2 > PC 3 > PC 4 . The concentrations of PC 2 and PC 3 were two to three times higher in the digestive gland than in the gills, whereas the PC 4 levels were nearly equivalent in both organs. Wilcoxon, Mann-Whitney test showed no significant difference in PC 2 content (fig. 4) and PC 3-4 (data not shown) between fresh and frozen tissues. PC are important for detoxification of the non-essential metal Cd in plants and fungi. were renewed every day, and the bivalves were fed daily as previously. For sampling, the 12 mussels used on each treatment were dissected. The gills and digestive glands of two mussels were pooled, which gives 6 replicates per treatment for analysis.

Determination of PC and other cysteine-rich metal-binding peptides

PC were determined according to Minocha and al. (2008) with a slight modification in the protein removal step and the HPLC mobile phase gradient profile (Santini et al., 2011b).

Cysteine-rich peptides were determined in the cytosolic fractions of the deproteinized tissue homogenates after reduction.

Extraction:

All dissection, extraction, and centrifugation steps were carried out at 4°C. The gills and digestive gland were carefully dissected, the digestive content was removed, and the tissues were washed in Tris buffer (Tris 25 mmol L -1 , NaCl 50 mmol L -1 , pH 8.0). The organs from two mussels were pooled in order to obtain sufficient mass for analysis. The samples were immediately analysed after sampling, or frozen in liquid N 2 and stored at -80°C until analysis. For extraction to avoid enzymatic degradation or PC oxidation, the tissues were homogenised in acid buffer (6,3 mM DTPA, 0,1% TFA) with a manual Potter-Elvehjem homogenizer with glass pestle. The homogenization was performed with 500 mg of tissues in 1 mL of buffer. The homogenate was centrifuged at 3500 g for 10 min (model 1-15PK, Sigma-Aldrich, St. Quentin Fallavier, France). The resulting supernatant (500 µL) was deproteinized by addition of 125 µL perchloric acid 5 mol L -1 , and centrifuged again at 13000 g for 30 min. The supernatant, 500 µL, was neutralised with 100 µL aqueous NaOH 5 mol L - 1 , and used as tissues extract for PC analyses. (UniPoint system version 1.90, Gilson, Villier-le-Bel, France). The bimane derivatives were separated using a gradient of mobile phase A (99.9 vol-% ACN, 0.1 vol-% TFA) and B (89.9 vol-% water, 10 vol-% ACN, 0.1 vol-% TFA). The gradient profile was a linear gradient of mobile phase A from 0 to 10.6 vol-% was run for 11.2 min at 1 mL min -1 . Further, the linear gradient of solvent A was raised from10.6 to 28.6 vol-% in 13.6 min at 1 mL min -1 . Before injecting a new sample, the column was rinsed with 100 % of solvent A for 5.5 min at a flow rate of 2.5 mL min -1 . The column was equilibrated with 100 % of solvent B for a total of 10 min at 1 mL min -1 . Total run time for each sample was 40.3 min, including column rinsing and re-equilibration.

Purified standards at seven increasing concentrations ranging from 0.2 to 2 µmol L -1 for PC 2-5 , and 1 to 10 µmol L -1 for Cys, GSH, and -GluCys, were used to plot calibration curve. In samples thiol compound concentration were determinate using equations of calibration curve. PC content was expressed in µg of PC per g of tissue wet weight. Each sample was spiked with 0.6 µmol L -1 of each standard PC 2-5 to certify PC identity. The quantification limits per 20 µL sample injected into the HPLC column were 1.5 pmol for PC 2-

Determination of metallothionein

The MT content was determined by the method of Romero-Ruiz et al. (2008). All steps were carried out at 4°C. Organs from two mussels were pooled in order to obtain sufficient mass for analysis. The samples were immediately frozen in liquid N 2 and stored at -80°C until analysis. The gills and digestive gland were homogenised in buffer (0.1 mol L -1

Tris, 1 mmol L -1 DTT, 50 mmol L -1 PMSF, 6 mmol L -1 leupeptin, pH 9.5) at a ratio of 5 mL g -1 with a manual Potter-Elvehjem homogenizer with glass pestle. The homogenate was centrifuged at 3500 g for 10 min. The resulting supernatant was centrifuged again at 25000 g for 30 min, and the supernatant was used as extract for MT analyses.

Tissue extract, 125 µL was mixed with 30 µl of Tris buffer, 0.23 mol L -1 , pH 9.5, 10 µL, DTT 0.3 M, added to 5 µL EDTA, 0.1 mol L -1 , pH 7, and 63 µL of 12 % SDS aqueous solution. Reduction and denaturation was conducted in a water-bath at 70°C for 20 min. For derivatization, 16.7 µL of mBBr solution in ACN, 0.18 mol L -1 , was added and the sample was incubated for 15 min at room temperature away from light.

The derivatized samples were chromatographically analyzed on VWR reversed-phase column (LiChrocart LiChrosphere RP C18, Strasbourg, France) 250 mm × 4 mm, 5 µm particle size, fitted to a C-18 guard column, 4 mm × 3 mm, 5 µm (Phenomenex Securityguard cartridge). The fluorescence detector parameters were the same than for PC analyses.

MT was separated using a gradient of mobile phase A (99.9 vol-% ACN, 0.1 vol-% TFA) and B (99.9 vol-% water, 0.1 vol-% TFA). After injection, the mobile phase A was kept for 10 min at 30 %, and changed by 1 min linear rise to 70 % of phase A. These conditions were maintained for 10 min, before the initial conditions (30 % phase A) were re-established by 1min linear decrease. Before injecting a new sample, the column was rinsed and equilibrated at the initial conditions for 8 min. T flow was kept at 1 mL min -1 throughout analysis. The injection volume was 20 µL.

A plot of peak area versus purified standard content was established using seven increasing concentrations from 0.4 to 1 µmol L -1 rabbit liver MT-I. The equation obtained from this calibration line was used for MT quantification in the samples. The MT content was expressed in mg per g of protein. Protein was determined according to Bradford (1976) using bovine serum albumin as standard.

Statistical analyses

As homogeneity of variances and normality of data were not verified (Bartlett and Shapiro-Wilk tests), statistical analysis was done using the non parametric Kruskal-Wallis and Mann-Whitney two sided tests (R Development Core Team (2010). R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.Rproject.org). All data were reported as means ± standard deviations (SD). Differences were considered as significant when p < 0.05.

RESULTS

A broad peak of tetramethylbimane resulting from the reaction between mBBr and TCEP eluted approximately at 10.5 min (fig. 1 A). At 17.96 min an unidentified compound (peak "a") from reagents eluted. In standards profile (fig. 1 B), the phytochelatin mean elution times, were: PC 2 at 13.09 min, PC 3 at 16.62 min, PC 4 at 18.59 min, and PC 5 at 19.75 min. (Amiard et al., 2006). In the present work, MT identification and quantification was carried out with purified rabbit liver MT-I standard. After HPLC separation, the MT in the mussel extract eluted at the same time as the rabbit liver standard, i.e. have a molecular weight and cysteine content similar to MT-I of mammals (Alhama et al., 2006). This MT isoform from A. cygnea does not appear to be induced by copper. Cu 2+ induction of MT in A. anatina was shown using a spectrophotometric method which allowed total MT isoform quantification (Nugroho and Frank, 2012).

Since the discovery of the PC synthase gene, able to give a functional PC synthase in invertebrates, the question of the implication of PC in metal detoxification and homeostasis in animals has arisen. The present study shows a clear PC 2 induction in freshwater mussels under Cu 2+ exposure, indicating a key role of this cysteine rich polypeptide as metal chelator.

PC 2 increased in the gills from 12 h to 7 d, so it may serve as first line defense, with other compounds such as GSH. The PC 2 decline to control values upon 21 d suggests that at later time copper is taken in charge by other detoxication mechanisms such as MT and insoluble granules (Viarengo and Nott, 1993). Sequestration of 65 % of the intracellular copper in granules was shown in the gills of Unionidae bivalves (Bonneris et al., 2005).

In the digestive gland, PC 2 increased significantly only up to 12 h of Cu 2+ exposure.

The water media route of Cu-exposure could be the reason of higher sensitivity of PC 2 induction in the gills, which may play a more important role than digestive gland, in the uptake of copper dissolved in the surrounding water. Moreover, PC induction depends on the metal species, Cu having been reported as a weak inducer compared to Cd (Zenk, 1996).

Further studies with Cd would be interesting, in order to determin whether PC 2-4 may be efficient in detoxification of such a non-essential metal. Additional investigations are necessary to assess the relevance of PC as potential biomarker for metal exposure of molluscs.

Annexe 2: Algae culture medium Summary: Copper (Cu) is one of the metals contaminating European fresh water ecosystems. Filter feeding bivalves have high bioaccumulation potential for transition metals as Cu. While Cu is an essential micronutrient for living organisms, it causes serious metabolic and physiological impairments when in excess. The objectives of this thesis are to get knowledge on toxic effects and detoxification mechanisms of copper in Anodonta cygnea and Anodonta anatina, two mussel species widely distributed in continental waters. Because Calcium (Ca) plays a fundamental role in shell formation and in numerous biological processes, Cu 2+ effects on cellular plasma membrane Ca transport were studied first. In the second step, the investigations focused on Cu 2+ detoxification mechanisms involving Cys-rich compounds known to play a major role in homeostasis of essential trace metals and in cellular metal detoxification. Under our experimental conditions, Cu 2+ inhibition of Ca 2+ -ATPase activity was observed in the gills and the kidneys, and inhibition of Na + /K + -ATPase in the gills and the digestive gland upon 4 d of exposure. At day 7 of exposure to 0,35 µmol L -1 Cu 2+ : total recovery was observed in the kidneys and the gills for Ca 2+ -ATPase activity, and in the digestive gland for Na + /K + -ATPase, but not at high doses. Ca and Na transport inhibition may entail disturbance of osmoregulation and lead to continuous under-supply of Ca. Recoveries of Na + /K + -ATPase and Ca 2+ -ATPase enzymes function suggest that metal-detoxification is induced. Phytochelatins (PC) are Cysrich oligopeptides synthesised by phytochelatin synthase from glutathione in plants and fungi. Phytochelatin synthase genes have recently been identified in invertebrates; this allows us to hypothesize a role of PC in metal detoxification in animals. In the second part of this work, PC and their precursors as well as metallothionein were analyzed in the gills and in the digestive gland of A. cygnea exposed to Cu 2+ . Our results showed for the first time the presence of PC 2-4 in invertebrates. PC were detected in control mussels not exposed to metal, suggesting a role in essential metal homeostasis. Compared to control, PC 2 induction was observed during the first 12 h of Cu 2+ exposure. Those results confirm the role of PC as a first line detoxification mechanism in A. cygnea. Key words: calcium homeostasis, copper, Anodonta freshwater bivalve, phytochelatins Résumé : Le cuivre (Cu) est l'un des métaux contaminants les écosystèmes dulcicoles Européens. Les bivalves filtreurs ont une grande capacité de bioaccumulation des métaux de transitions tel que le Cu. Le Cu est un oligo-élément essentiel pour les organismes vivants, mais en excès il provoque de graves perturbations métaboliques et physiologiques. L'objectif de cette thèse est d'acquérir des connaissances sur les effets toxiques et les mécanismes de détoxification du cuivre chez Anodonta cygnea et Anodonta anatina, deux espèces de bivalves largement distribuées dans les eaux continentales. Parce que le Calcium (Ca) joue un rôle fondamental dans la composition de la coquille et pour de nombreux processus biologiques, les effets du Cu 2+ ont été étudiés d'abord sur le transport cellulaire du Ca au niveau de la membrane plasmique. Dans un deuxième temps, l'étude a été axée sur les mécanismes de détoxification du Cu 2+ impliquant des composés riches en Cys, connus pour jouer un rôle majeur dans l'homéostasie des métaux traces essentiels et dans la détoxification des métaux dans les cellules. Dans nos conditions expérimentales, l'inhibition de la Ca 2+ -ATPase par le Cu 2+ a été observée dans les branchies et les reins, et l'inhibition de la Na + /K + -ATPase dans les branchies et la glande digestive, après 4 jours d'exposition à concentrations environnementales. Au delà de 7 jours d'exposition à 0,35 µmol L -1 Cu, une récupération totale de l'activité enzymatique a été observée dans les reins et les branchies pour Ca 2+ -ATPase, et dans la glande digestive pour la Na + /K + -ATPase. A dose élevée l'inhibition persiste. L'inhibition du transport du Ca et du Na peut entraîner des perturbations de l'osmorégulation et conduire à des carences en Ca. La récupération de l'activité enzymatique de la Ca 2+ -ATPase et de la Na + /K + -ATPase suggère une induction de fonctions de détoxification des métaux. Les phytochélatines (PC) sont des oligopeptides riches en Cys synthétisés par la phytochélatine synthase à partir du glutathion, chez les plantes et les champignons. Des gènes codant pour des phytochélatine synthases fonctionnelles ont étés identifiés chez des invertébrés. Dans la seconde partie de ce travail, les PC et leurs précurseurs, ainsi que les métallothioneines ont été étudiés dans les branchies et la glande digestive d'A. cygnea exposé au Cu 2+ . Nos résultats ont montré pour la première fois, la présence de PC 2-4 chez les invertébrés. Les PC ont été détectés dans des moules témoins non exposées aux métaux, ceci suggère une fonction dans l'homéostasie des métaux essentiels. Une induction de PC 2 a été observée dés les 12 premières heures d'exposition au Cu 2+ , comparé aux bivalves témoins. Ces résultats confirment le rôle du PC en tant que mécanisme de première ligne de détoxification des métaux chez A. cygnea.

Mots clés : homéostasie du calcium, cuivre, bivalves dulcicoles Anodonta, phytochélatines

  Copper metabolism in mammals[START_REF] Jondreville | Le cuivre dans l'alimentation du porc : oligoélément essentiel, facteur de croissance et risque potentiel pour l'Homme et l'environnement[END_REF]. 41 Fig.2: A: Anodonta anatina, B: Anodonta cygnea (www.biopix.eu) 45 Fig. 3: Geographical distribution of Anodonta anatina, and distribution of Anodonta cygnea 46 Fig. 4: Biological cycle of Unionidae (http://biodidac.bio.uottawa.ca) 47 Fig. 5: Longitudinal-section of an Unionidae (Mouthon, 1982), p.a.m.: posterior adductor muscle, a.a.m.: anterior adductor muscle 49 Fig. 6: GSH metabolism (Mendoza-Cózatl and Moreno-Sánchez, 2006), -ECS: glutamatecysteine-ligase, GS: glutathione synthase, GR: glutathione reductase, GPx: glutathione peroxidase, GST: glutathione transferase, Xe: xenobiotic, GSH: glutathione, GSSG: glutathione disulfide 59 Fig. 7: Redox cycle of MT

Fig. 1 :Fig. 1 :Fig. 1 :

 111 Protocol of tissue fraction preparation for enzymes analysis of Ca 2+ -ATPase, Na + /K + -ATPase, H + -ATPase, and carbonic anhydrase (CA), in the mantle (M), digestive gland (DG), gills (G), and kidneys (K) of the fresh water mussel A. anatina. 69 Fig. 2: Protocol of tissue fraction preparation for thiol rich compound analysis, i.e. of metallothionein (MT), phytochelatins (PC) and precursors in the digestive gland (DG), and gills (G) of the fresh water mussel A. anatina. 70 Fig. 3: Phytochelatin and precursor biosynthesis in plants (Mendoza-Cózatl and Moreno-Sánchez, 2006), PCS: phytochelatin synthase, -ECS: glutamate-cysteine-ligase, GS: glutathione synthase, GST: glutathione transferase, Xe: xenobiotic, GSH: glutathione, GSSG: glutathione disulfide, HMWC: high molecular weight complexes. 78 Article 1 Schematic presentation of the Ca flow through the different compartments of a freshwater bivalve, and inhibition of PMCA by Cu in kidney and gills. MCE: mantle cavity epithelium, DG: digestive gland, PMCA: plasma membrane Ca 2+ -ATPase. 83 Fig. 2: PMCA activity in kidneys (K), gills (G), and digestive gland (DG) of A. anatina upon 0 (control), 4, 7, 15 days of exposure to 0.35 µmol L -1 (a) and 0.64 µmol L -1 Cu (b). Means of results, (n = 3) ± SE, are presented as ratios of enzymatic activity at the respective Cu concentration and time vs. control (mentioned on the graph). Notes: For K, the error bars reflect the variability of the analytical determination of the pooled tissue sample, for G and DG the biological variation between animals. * = significantly lower than control, = significantly higher than control (U test p < 0Na + /K + -ATPase activity determination in September in gills (G), the digestive gland (DG), and mantle (M) of A. anatina upon 0 (control), 4, 7, 15 days of exposure to 0.35 µmol L -1 Cu 2+ . Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. *= significantly lower than control (Mann-Whitney test two sided test = 0.05). 98 Fig. 2: H + -ATPase activity determination in September in mantle (M) of A. anatina upon 0 (control), 4, 7, 15 days of exposure to 0.35 µmol L -1 Cu 2+ . Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. *= significantly lower than control (Mann-Whitney test two sided test = 0.05). 99 Fig. 3: Na + /K + -ATPase basal activity in gills (G), the digestive gland (DG), and mantle (M) of A. anatina in July, September, January, March, and April 2007 / 2008. Means of results, n = 3 ± SD, are presented as enzymatic activities in µmol P i /mg protein/min. # = significantly higher than in January, March, and April (Mann-Whitney test two sided test = 0.05). 100 Fig. 4: H + -ATPase basal activity in mantle (M) of A. anatina in July, September, January, March, and April 2007 / 2008. Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. # = significantly higher than in January, March, and April (Mann-Whitney test two sided test = 0.05). 101 Article 3 Chromatograms of (A) reagents blank with homogenisation buffer, and (B) mix of the eight cysteine-rich peptide standards. The broad peak is tetramethylbimane (Me 4 B).

Fig. 2 :Fig. 1 :

 21 Chromatograms of digestive gland samples obtained with the same extract: alone (A), and spiked with 0.6 µmol L -1 of each standard PC 2-5 (B). 115 Fig. 3: Chromatograms of gills samples made with the same extract: alone (A), and spiked with 0.6 µmol L -1 of each standard PC 2-5 (B). 116 Fig. 4: PC 2 content in the digestive gland (DG) and the gills (G) of Anodonta cygnea, means (n = 6) ± SD. No significant difference was found between fresh (white bars) and frozen (grey bars) tissues (Wilcoxon, Mann-Whitney test). 117 Article 4 Chromatograms of (A) reagent blank with homogenisation buffer, and (B) mix of the eight cysteine-rich peptide standards. The broad peak is tetramethylbimane (Me 4 B).

Fig. 2 :

 2 Chromatograms of gills samples: mussels exposed for 4 days to 0.35 µmol L -1 Cu 2+ (A), mussel controls 4 days (B), mussel controls 4 days spiked with 0.6 µmol L -1 of each standard PC 2-5 (C), a.u.: area unit. 129 Fig. 3: PC 2 content in the digestive gland (DG) and the gills (G) of A. cygnea exposed to 0.35 µmol L -1 of Cu 2+ for 0 h, 12 h, 48 h, 4 d, 7 d, and 21 d. Means of results (n = 6) ± SD. *= significant difference between exposed group and its respective control (Kruskal-Wallis and Mann-Whitney two sided test (n = 6, = 0.05)) 130

  Mots clés: homéostasie du calcium, cuivre, bivalves dulcicoles Anodonta,

(

  respiration avec le cytochrome C oxydase, formation des tissus connectifs avec la lysyl oxydase) sont aussi à l'origine de sa toxicité lorsqu'il est en excès. Le cuivre est bioaccumulable et peut devenir une menace pour la biocénose. Des mécanismes de régulation de la concentration et de la détoxication du cuivre sont essentiels pour les organismes vivants. Les mollusques représentent une forte proportion de macroinvertébrés dans les écosystèmes aquatiques. Parmi ce groupe, les bivalves sont particulièrement intéressants. Du fait de leur importante activité de filtration nécéssaire pour satisfaire leur respiration et leur nutrition, les bivalves ont la capacité d'accumuler de nombreux contaminants. Dans l'écosystème, ils jouent un rôle important dans le transfert de matière de la colonne d'eau vers les sédiments. Les excréments et pseudofèces de bivalves rendent le phytoplancton disponible aux détritivores, et peuvent modifier la qualité des sédiments par concentration des polluants.

  2+ -ATPase et Na + /K + -ATPase ont été observées chez A. anatina exposés au Cu 2+ . Une inhibition suivie d'une reprise totale de l'activité enzymatique, observée à la concentration d'exposition de 0,35 µmol L -1 de Cu 2+ mais non à forte concentration (0.64 µmol L -1 ) indique un mécanisme de détoxication. Ces résultats suggérent l'induction de mécanismes de détoxication métallique. La deuxième étape de cette étude a porté spécifiquement sur les composés riches en Cys chélateurs de métaux. Nous avons posé l'hypothèse que des phytochélatines pouvaient être présentes chez les bivalves et jouer un rôle dans la détoxification du cuivre. Les composés riches en Cys sont des polypeptides ou des protéines telles que les phytochélatines ou les métallothionéines, avec une teneur en Cys élevée. Ils jouent un rôle majeur comme chélateur de métaux, dans l'homéostasie des métaux essentiels et pour la détoxication des métaux nonessentiels. Les phytochélatines (PC) sont des polypeptides riches en thiol de formule générale : ( -Glu-Cys) n -Gly, synthétisés par la phytochélatine synthase (PCS) à partir de glutathion. Les PC complexent les ions métalliques réduisant la concentration intracellulaire en ions métalliques libres chez les plantes, les champignons et les microalgues.

  . Brièvement A. cygnea et A. anatina adultes (7,5 ± 0,5 cm de long) ont été placées en aquariums à 20 ± 0,5 ° C. L'eau d'étang artificiel 1,5 L / moule, pH 7,25 ± 0,10 (en mmol L -1 : 0.40 Ca 2+ , 0.20 Mg 2+ , 0.70 Na + , 0.05 K + , 1.35 Cl -, 0. Les bivalves ont été nourris quotidiennement avec une culture de Chlorella kessleri en phase exponentielle de croissance, ajoutée à une densité finale d'algues de 2 × 10 5 cellules / ml. Les animaux ont été acclimatés à ces conditions pendant deux semaines avant toute expérimentation.

Fig. 2 :

 2 Fig. 2 : Protocole d'analyse des composés riches en thiols GD : glande digestive, B : branchies.

  d'exposition au cuivre et 30 % d'augmentation à 7 jours d'exposition. Au delà de 7 jours, la PC 2 revient au même niveau que celui mesuré dans les témoins. Dans la glande digestive, aucune variation significative de la PC 2 n'a été mise en évidence, excepté à 12 h d'exposition au Cu 2+ . Les concentrations de -GluCys sont significativement supérieures dans les branchies des moules exposées au cuivre durant 48 heures et 7 jours. La concentration de -GluCys augmente également dans la glande digestive des bivalves à 48 h et 4 j d'exposition au cuivre. Aucune variation significative du niveau de MT n'a été observée dans les branchies et la glande digestive des moules sur les 21 jours d'exposition au cuivre.

  disruption of calcium transport and biomineralization mechanisms by copper. Detoxification mechanisms by metal binding thiol compounds are presented in the seventh and

  construction, and transport (LME, 2011). Primary production of copper increased from 9.6 million in 1980 to 16.9 million in 2006 (INERIS, 2010). In 2007, according to INERIS (2010), 35 % of the world copper consumption came from recycled copper. In Europe, in 2007 the recycling rate of copper was 41 %, and in 2006 copper consumption was about 4.7 million tons of copper (21 % of global demand). The demand for copper in Europe was estimated in 2007 at 3.85 million tons (European Copper Institute, 2009).

Fig. 3 :

 3 Fig. 3: Geographical distribution (enclosed by the red line) of Anodonta anatina, and distribution of Anodonta cygnea in European continental hydrosystems[START_REF] Başçınar | A Preliminary study on reproduction and larval development of swan mussel [Anodonta cygnea (Linnaeus, 1758)] (Bivalvia: Unionidae), in Lake Çıldır (Kars, Turkey)[END_REF] www.discoverlife.org)

Fig. 4 :

 4 Fig. 4: Biological cycle of Unionidae (http://biodidac.bio.uottawa.ca)

  Fig. 5: Longitudinal-section of an Unionidae (Mouthon, 1982), p.a.m.: posterior adductor muscle, a.a.m.: anterior adductor muscle

  , but especially of larvae and juveniles), little is available to define the main threats and causes of extinction:

  ROS are free radicals, presenting unpaired electrons (hydroxyl radical HO • ), others are nonradical species such as hydrogen peroxide H 2 O 2 . To reach a better level of stability, these radicals will capture electrons from reductor molecules, ROS reduction causing oxidations in chains. All the bio-molecules of the cell (nucleic acids, lipids, proteins, polysaccharides) are potential reductor substrates of ROS. The level of ROS instability characterized their diffusion capacity. A low-reactive form tends to act far from its site of production as it has a significant diffusion radius. On the contrary, a very reactive species acts very quickly and its diffusion is limited. ROS include the superoxide anion radical (2 ), hydroperoxy (ROO • ), and alkoxy (RO • ) radicals, nitric oxide (NO • ), and hydroxyl radical (HO • ). Molecular oxygen O 2 can also be regarded as a radical species since it has two single electrons. The superoxide anion is produced during various reactions with transition metals, and enzymes are implied in its formation. H 2 O 2 reacts weakly, diffuses freely, and has a long lifetime. The ROO • and RO • radicals arise from the peroxidation of lipids. These radicals allow the gradual propagation of lipid peroxidation. Free radicals are produced physiologically during normal cell metabolism. They can also be formed in response to a wide range of exogenous agents including radiation, metal ions, solvents, particulate matter, nitrogen oxides, and ozone. NO • plays at the same time a role in the destruction and the production of radicals. It is not very reactive with cellular components and reacts with radicals generating less reactive species. Combined with the superoxide anion radical, it may be involved in the formation of peroxynitrite, a highly toxic species. Due to its high reactivity, HO • is quite non-specific in its targets for oxidation, whereas ROS with lower rate constants react more specifically. ROS are at the origin of lipid peroxidation. Copper ions are involved in redox reactions which result in the production of ROS. In the Fenton reaction, cuprous ions react with H 2 O 2 giving rise to the extremely reactive HO •[START_REF] Labieniec | Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H 2 O 2 or Cu 2+ ions[END_REF]. In the case of organic hydroperoxides (ROOH), a homologous reaction is thought to occur leading to the formation of ROO • and of the more reactive RO • . Copper ions may participate both in the initiation and the propagation steps of lipid peroxidation, thus stimulating the in vivo degradation of membrane lipids

  e. antioxidant enzymes, and molecules without enzymatic activity. Antioxidant enzymes are superoxide dismutases (SODs) and catalase (CAT): SODs catalyze the dismutation of the superoxide anion radical under formation of molecular oxygen and H 2 O 2 , the latter detoxified by CAT. SOD and CAT are localised in peroxysomes, and also in mitochondria and cytosol. Isoenzymes of SOD are found in various compartments of the cell.

Fig. 6 :

 6 Fig. 6: GSH metabolism (Mendoza-Cózatl and Moreno-Sánchez, 2006), -ECS: glutamatecysteine-ligase, GS: glutathione synthase, GR: glutathione reductase, GPx: glutathione peroxidase, GST: glutathione transferase, Xe: xenobiotic, GSH: glutathione, GSSG: glutathione disulfide

  : Class I, including the MT which show biochemical homology and close elution time by chromatography with horse MT, Class II, including the rest of the MT with no homology with horse MT, and Class III, which includes phytochelatins, Cys-rich enzymatically synthesised peptides. A second classification was performed[START_REF] Binz | Metallothionein: molecular evolution and classification[END_REF]; http://www.bioc.uzh.ch/mtpage/classif.html) and takes into account taxonomic parameters and the patterns of distribution of Cys residues along the MT sequence. Cysteine (Cys) residues are distributed in typical motifs consisting of Cys-Cys, Cys-X-Cys or Cys-X-X-Cys sequences (X denoting amino-acid residues other than Cys). It results in a classification of 15 families for proteinaceous MT. Mollusc MT belong to family 2, divided in two subfamilies.

Fig. 7 :

 7 Fig. 7: Redox cycle of MT[START_REF] Kang | Metallothionein redox cycle and function[END_REF], MT: metallothionein, ROS: reactive oxygen species, GSH: glutathione, GSSG: glutathione disulfide

Fig. 8 :

 8 Fig.8: PC synthesis[START_REF] Vatamaniuk | Worms take the 'phyto' out of 'phytochelatins[END_REF]

for

  Zn/Cu-thionein indicate that copper is present in the form of Cu(I). Unlike to Zn, Cu is arranged differently in the MT clusters. Cu(I) atoms are binding by one bivalent connection which allows 10 copper atoms to bind per MT protein. The relative affinities of each metal

Fig. 1 :Fig. 2 :

 12 Fig.1: Protocol of tissue fraction preparation for enzymes analysis of Ca 2+ -ATPase, Na + /K + -ATPase, H + -ATPase, and carbonic anhydrase (CA), in the mantle (M), digestive gland (DG), gills (G), and kidneys (K) of the fresh water mussel A. anatina.

Fig. 3 :

 3 Fig. 3: Phytochelatin and precursor biosynthesis in plants (Mendoza-Cózatl and Moreno-Sánchez, 2006), PCS: phytochelatin synthase, -ECS: glutamate-cysteine-ligase, GS: glutathione synthase, GST: glutathione transferase, Xe: xenobiotic, GSH: glutathione, GSSG: glutathione disulfide, HMWC: high molecular weight complexes.

(

  in mmol L -1 : 0.40 Ca 2+ , 0.20 Mg 2+ , 0.70 Na + , 0.05 K + , 1.35 Cl -, 0

  Fig. 1: Na + /K + -ATPase activity determination in September in the gills (G), the digestive gland (DG), and the mantle (M) of A. anatina upon 0 (control), 4, 7, 15 days of exposure to 0.35 µmol L -1 Cu 2+ . Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. *= significantly lower than control (Mann-Whitney test two sided test = 0.05).

Fig. 2 :

 2 Fig. 2: H + -ATPase activity determination in September in mantle (M) of A. anatina upon 0 (control), 4, 7, 15 days of exposure to 0.35 µmol L -1 Cu 2+ . The Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. *= significantly lower than control (Mann-Whitney test two sided test = 0.05).

Fig. 3 :

 3 Fig. 3: Na + /K + -ATPase basal activity in gills (G), the digestive gland (DG), and mantle (M) of A. anatina in July, September, January, March, and April 2007 / 2008. Means of results, n = 3 ± SD, are presented as enzymatic activities in µmol P i /mg protein/min. # = significantly higher than in January, March, and April (Mann-Whitney test two sided test = 0.05).

  Fig. 4: H + -ATPase basal activity in mantle of A. anatina in July, September, January, March, and April 2007 / 2008. Means of results, n = 3 ± SD, are presented as enzymatic activity in µmol P i /mg protein/min. # = significantly higher than in January, March, and April (Mann-Whitney test two sided test = 0.05).

  PC n are synthesised by phytochelatin synthase (PCS) which catalyzes the transpeptidation of the -Glu-Cys moiety of GSH onto a second GSH moiety or with PC n ( -Glu-Cys) n -Gly (n=2-11) to form PC n+1 . PC n are rapidly induced in cells and tissues when exposed to a range of transition metal ions, including the cations Cd,

  Derivatization was carried out by addition of 4 µL of a solution of 50 mmol L -1 mBBr acetonitrile and incubation in a water-bath at 45°C for 30 min in the dark. The reaction was stopped by addition of 40 µL of an aqueous solution of MSA 1 mol L -1 .The high-performance liquid chromatographic analysis was performed with an HPLC instrument (Gilson, Roissy, France) equipped with a dual solvent pump (model 322), autosampler (model 234), a 100 µL injection loop, and fluorescence detector (model 122).

Fig. 1 :Fig. 2 :Fig. 3 :

 123 Fig. 1: Chromatograms of (A) reagents blank with homogenisation buffer, and (B) mix of the eight cysteine-rich peptide standards. The broad peak is tetramethylbimane (Me 4 B). Peak "a" is an unidentified compound originating from the derivatization reaction with reagent.Standard concentration was 10 µmol L -1 for cysteine (Cys), glutathione (GSH),glutamylcysteine ( -GluCys), 5 µmol L -1 for the internal standard N-acetyl-cysteine (NAC), and 2 µmol L -1 for phytochelatins 2-5 (PC 2-5 ).

Fig. 4 :

 4 Fig. 4: PC 2 content in the digestive gland (DG) and the gills (G) of Anodonta cygnea, means (n = 6) ± SD. No significant difference was found between fresh (white bars) and frozen (grey bars) tissues (Wilcoxon, Mann-Whitney test).

  Reduction and derivatization:Just after extraction, 244 µL HEPPS buffer (HEPPS 200 mmol L -1 , DTPA 6.3 mmol L -1 , pH 8.2) was mixed with 10 µL TCEP (TCEP 20 mmol L -1 in HEPPS buffer, pH 8.2) used as disulfide reductant, 4 µL of NAC 0.5 mmol L -1 as an internal standard, and 99 µL of tissue extract. Disulfide reduction was conducted in a water-bath at 45°C for 10 min. Derivatization was carried out by addition of 4 µL of mBBr (50 mmol L -1 in acetonitrile), and incubated in a water-bath at 45°C for 30 min in the dark. The reaction was stopped by addition of 40 µL of an aqueous solution of MSA 1 M.Analyses were performed using HPLC instrument (Gilson, Roissy, France) equipped with a dual solvent pump (model 322), an autosampler (model 234), a 100 µL injection loop, and a fluorescence detector (model 122). The fluorescence of mBBr-labeled molecules was monitored with an excitation wave length of 382 nm and emission at 470 nm. The injection volume was 20 µL. The derivatized PC were separated on a reversed-phase column (Phenomenex-Synergi-Hydro RP C18), 100 mm × 4.6 mm, 4 µm particle size, fitted with a C-18 guard column (Phenomenex Securityguard cartridge) 4 mm × 3 mm, 5 µm. The temperature of the column oven was 40°C. Peak areas were integrated using proper software

Fig. 1 :Fig. 2 :

 12 Fig. 1: Chromatograms of (A) reagent blank with homogenisation buffer, and (B) mix of the eight cysteine-rich peptide standards. The broad peak is tetramethylbimane (Me 4 B). Peak "a" is an unidentified compound originating from the derivatization reaction with reagent.Standard concentration was 10 µmol L -1 for cysteine (Cys), glutathione (GSH),glutamylcysteine ( -GluCys), 5 µmol L 1 for the internal standard N-acetyl-cysteine (NAC), and 2 µmol L -1 for phytochelatins 2-5 (PC 2-5 ).

Fig. 3

 3 Fig. 3 PC 2 content in the digestive gland (DG) and the gills (G) of A. cygnea exposed to 0.35 µmol L -1 Cu 2+ for 0 h, 12 h, 48 h, 4 d, 7 d, and 21 d. Means of results (n = 6) ± SD. *= significant difference between exposed group and its respective control (Kruskal-Wallis and Mann-Whitney two sided test (n = 6, = 0.05))

  Le milieu aquatique est le réservoir final pour la plupart des polluants, dont les métaux. Le cuivre appartient aux métaux les plus couramment utilisés du fait de ses propriétés physiques et chimiques (particulièrement pour ses qualités de conductivité électrique et thermique).Comme métal pur, en alliage, ou à l'état ionique, il est utilisé dans un grand nombre de

	Introduction
	L'activité humaine est associée au développement de l'industrie et l'agriculture, qui
	sont devenus indispensables. Ces secteurs sont responsables de la production et de la diffusion
	de nombreux polluants. Les propriétés chimiques et physiques et les différents types de
	transport déterminent la diffusion des polluants dans tous les compartiments des écosystèmes.
	PC :	phytochélatine(s)
	PCS :	phytochélatine synthase
	P i :	phosphate inorganique
	PMCA :	Ca 2+ -ATPase de la membrane plasmique
	R :	reins
	S :	surnageant
	SH :	groupement thiol
	TCEP :	tris-(2-carboxyethyl)-phosphine hydrochloride

secteurs industriels et agricoles. De ce fait, le cuivre est un métal fréquemment détecté dans les milieux aquatiques continentaux où il est présent dans la colonne d'eau et s'accumule dans les sédiments (INERIS, 2010). Les propriétés chimiques du cuivre en font un élément surtout utilisé en tant que catalyseur biologique des réactions enzymatiques, et un élément essentiel à de nombreux processus biologiques impliquant des fonctions vitales comme la respiration ou la photosynthèse

  Ces deux invertébrés sont autochtones des systèmes hydrologiques européens. Au cours des dernières décennies, une régression des populations d'Unionidae a été observée en Europe. L'objectif de cette thèse a été d'acquérir des connaissances sur les mécanismes de perturbation par le cuivre du métabolisme du calcium chez Anodonta anatina. Le calcium est un élément essentiel dans le fonctionnement des organismes eucaryotes. Il contrôle plusieurs processus vitaux(Ermak et Davies, 2001). L'absorption, le maintien de la concentration intracellulaire de calcium dans l'organisme, et les processus de biominéralisation sont rendus possible par le contrôle de son passage à travers les membranes cellulaires. Ce passage se fait par simple diffusion, mais aussi par des protéines de transport. Le phénomène de biominéralisation nécessite en plus du calcium, des ions carbonate produits en partie par l'anhydrase carbonique (AC). Nous avons étudié les effets du Cu 2+ sur le transport du calcium

chez Anodonta anatina par l'évaluation des activités enzymatiques de Ca 2+ -ATPase, Na + /K + -ATPase et H + -ATPase de la membrane plasmique et de l'activité enzymatique cytosolique de AC, enzymes impliquées dans l'absorption du calcium et dans les processus de biominéralisation. Les organes étudiés ont été les branchies, la glande digestive, le rein, et le manteau, qui jouent un rôle important dans l'absorption du calcium et la synthèse de la coquille

  . Les invertébrés appartenant aux Unionidae, connus pour leur facilité à bioaccumuler les métaux, pourraient être susceptibles de synthétiser des PC, c'est l'hypothèse que nous émettons dans la deuxiéme partie de la thèse. Leur présence est connue chez les végétaux, non chez les organismes animaux. L'induction de PC, de leurs précurseurs ont été évalués dans les branchies et la glande digestive chez Anodonta cygnea exposée au cuivre.

  Comme l'homogénéité des variances et la normalité des données ne se sont pas révélées vérifiées (tests de Bartlett et de Shapiro-Wilk), l'analyse statistique a été réalisée par tests non paramétriques de Kruskal-Wallis et de Mann-Whitney. Les différences ont été considérées comme significatives lorsque p <0,05.

	Analyses statistiques
	Analyses

Des expériences préliminaires ont été effectuées avec trois bivalves exposés à chacune des concentrations de Cu 2+ : 0,26, 0,54 et 1,15 µmol L -1 pendant quatre jours, pour trouver les concentrations les plus pertinentes. Pour l'étude des effets du cuivre sur ces activités enzymatiques l'exposition a été effectuée à 0,35 et 0,64 µmol L -1 de Cu 2+ sur une durée de 15 jours. Trois moules ont été échantillonnées et disséquées pour chaque traitement : jour 0 (témoin), 4 j, 7 j et 15 j. Les organes de chaque individu ont été préparés et étudiés séparément, ce qui donne 3 réplicats par traitement. Les effets du cuivre sur les mécanismes de détoxification des métaux ont étés évalués chez A. cygnea sur les paramétres suivants : PC et leurs précurseurs, et MT. Les bivalves ont été exposés à 0,35 µmol L -1 de Cu 2+ durant 0 h, 12 h, 48 h, 4 j, 7 j et 21 j, les témoins ont étés maintenus en parallèle, sur la même durée, dans l'eau artificielle. Les milieux tests ont été renouvelés chaque jour, et les bivalves ont été nourris quotidiennement, comme décrit précédemment. Douze moules ont étés échantillonnées par traitement avant d'être disséquées. Les branchies et la glande digestive de deux moules ont été regroupées, donnant 6 réplicats par traitement pour l'analyse. enzymatiques Fig. 1 : Protocole d'analyse enzymatique M : manteau, GD : glande digestive, B : branchies, R : reins, S : surnageant, C : culot.

  Ca 3 (PO 4 ) 2 , Mg 3 (PO 4 ) 2 ) and pyrophosphate (Ca 2 P 2 O 7 , Mg 2 P 2 O 7 ) can contain Mn, Zn, Cu, Fe, Co, Cd and

This organelle accumulate high concentrations of trace metals in non-toxic granules forms and thus represent an important detoxification way. The ferritin-rich and copper-sulphur granules are related to Fe and Cu metabolism in the respiratory pigment and also in copper detoxification. Lipofuscins are mainly lipid peroxidation end-products which are accumulated in the lysosomes as insoluble lipoprotein granules. Metals such as Cu, Cd, and Zn are trapped by the lipofuscin and sterically prevented from moving in or out of the granule. In mollusc intracellular granules composed to calcium / magnesium orthophosphate (

Table 1 :

 1 Basal enzymatic activities of plasma membrane ATPase(s) (µmol P i /mg protein/min) and cytoplasmic CA (U/mg protein) in Anodonta anatina.

		G	DG	M -	K	Reference
	Ca 2+ -ATPase Na + /K + -ATPase H + -ATPase	0.45 0.098 -	0.095 0.015 --	0.042 0.002	0.087 ---	Article 1 Article 2 Article 2
	CA	2.4		1.76		Article 1

G: gills; DG: digestive gland; M: mantle; K: kidneys, CA: carbonic anhydrase; ─: not determined

Table 1 :

 1 Plasma membrane Na + /K + -ATPase and H + -ATPase activities (µmol/L P i /mg protein/min) in different tissues of freshwater and marine organisms.

	Species	Na + /K + -ATPase	H + -ATPase	Tissues	References
	Freshwater bivalve
	Anodonta anatina	0.098		G	The present study
		0.015		DG
		0.042	0.002	M
	Anodonta cygnea	0.109		G	Lagerspetz and Senius, 1979
	Asellus aquaticus	0.018		G	Bouskill et al. , 2006
	Dreissena polymorpha	0.006		G
	Marine bivalve
	Mytilus galloprovincialis	0.033		G	Viarengo et al. , 1996
	Freshwater crustacean
	Dilocarcinus pagei		0.023	G	Firmino et al. , 2011
	Brackishwater crustacean
	Acartia tonsa	0.014		total	Pedroso et al. , 2007
	Freshwater fish
	Oncorhynchus mykiss		0.025	G	Lin and Randall, 1993
	Trichogaster microlepis	0.015	0.003	G	Huang et al. , 2010
	Bidyanus bidyanus	0.124	0.041	G	Alam and Frankel, 2006
	Macquaria ambigua	0.052	0.032	G
	Perca flavescens	0.064		G	Packer and Garvin, 1998

Plasma membrane ATPase activity µmol L -1 Pi/mg protein/min

G: gills, DG: digestive gland, M: mantle.

Component name Retention time (min) LOD pmol / 20 µl LOQ pmol / 20 µl r 2

	Cys	3.14 ± 0.08	0.21	1.09	0.999
	GSH	5.99 ± 0.12	0.24	1.40	0.999
	-GluCys	6.43 ± 0.13	0.37	1.46	0.999
	NAC	9.16 ± 0.23	-	-	-
	PC 2	13.09 ± 0.33	0.59	1.39	0.999
	PC 3	16.62 ± 0.28	0.79	1.52	0.998
	PC 4	18.59 ± 0.25	1.93	2.70	0.991
	PC 5	19.75 ± 0.18	4.71	5.09	0.961

Table 2 :

 2 PC content [µg PC/g tissue wet weight] in the digestive gland and the gills of Anodonta cygnea, means (n = 6) ± SD.Table2shows the PC n content in the gills and the digestive gland in µg per g tissue wet weight. The concentrations in the digestive gland and the gills were the highest for PC 2 ,

		PC 2	PC 3	PC 4	PC 5
	Digestive gland	2.17 ± 0.59	1.10 ± 0.12	0.47 ± 0.52	< LOD
	Gills	0.88 ± 0.15	0.72 ± 0.57	0.40 ± 0.44	< LOD
	< LOD: below limit of detection

  Clemens and Peršoh (2009) andGonzalez-Mendoza et al. (2007) support the hypothesis that PC have dual functions: metal detoxification and essential metal homeostasis (Zn, Cu). In our work, before analyses the mussels were kept at least for 2 weeks in artificial pond water (see animal maintenance) without exposure to non-essential metals like Cd or the like, but PC 2-4 could be detected anyway. The presence of PC 2-4 without the animals being exposed to Cd or other transition metals suggests that PC could have functions comparable to other cysteinerich compounds, i.e. for homeostasis of essential trace metals or as reducing agent. The evidence of homologous genes for functional PCS in animals suggests that PC n play a wider role in heavy-metal detoxification than previously thought. The results obtained in this study highlight, for the first time, the ability of the freshwater bivalve Anodonta cygnea to synthesize PC 2-4 .6 tons, worldwide about 17 × 10 6 tons (INERIS, present work 0.35 µmol L -1 Cu 2+ (controlled by graphite furnace atomic absorption spectrometry GFAAS, limit of detection for Cu = 0.5 µg L -1 = 8 nmol L -1 ) was chosen as concentration for exposure test. The experiments were performed in aquaria lined with dyeand pigment free high-density polyethylene foil, filled with 1.5 L of experimental medium (pH 7.25) per mussel. The mussels were kept at 20° C in a thermo-regulated room with a photoperiod of 16 h light and 8 h darkness. The animals were divided in groups of 12 mussels.

	These groups were exposed to 0.35 µmol L -1 Cu 2+ for 0 h, 12 h, 48 h, 4 d, 7 d, and 21 d, or
	kept for the same duration in artificial pond water as corresponding time controls. Test media
	INTRODUCTION
	Copper is extensively used for various technical applications, especially conducting
	metal for electro-technical equipment, electrical power lines, as catalyst in the chemical

industry, and in the building industry for water pipes and roofings; a small amount goes into the use as fungicide by which way it is directly emitted into the environment. Annual copper consumption in Europe alone is 3.5 × 10

, 2.7 pmol for PC

,

pmol for PC 5 .

Acknowledgements I am very grateful to Professor Konrad Dettner, Professor Britta Planer-Friedrich, and Professor Jouni Taskinen, to participate in the jury of defense of this Ph.D, and to accept reviewing this Ph.D. thesis. I thank Professor Pascale Bauda, Professor Laure Giambérini, and Professor Jean François Ferard, the present and past directors of the laboratory, for welcoming me into the Laboratory Interactions Ecotoxicology Biodiversity Ecosystems (LIEBE). I wish to express my sincere gratitude to my two Supervisors, Professor Hartmut Frank and Professor Paule Vasseur who welcomed me in their teams. I am indebted to my supervisors for their guidance, for all of their valuable advices and remarks throughout this Ph.D. thesis. I thank Doctor Naima Chahbane, Lecturer-HDR Carole Cossu-Leguille, and Doctor Silke Gerstmann for their interest in this research and their help. My warm thanks to Mrs Agnes Bednorz for her kindness and her help. I am indebted to Lecturer Sylvie Cotelle for allowing me to use the HPLC equipment. I acknowledge Mr Philippe Rousselle for copper determinations by GFAAS and FAAS. Many thanks to Mrs Catherine Drui, Mrs Maryline Goergen and Mrs Irmgard Lauterbach, for their help in administrative issues. I am pleased to acknowledge all the colleagues for their kindness and their cheerfulness: ACKNOWLEDGEMENTS This research was supported by the Universities of Bayreuth (Germany) and Lorraine (France) and the CPER (Contrat de Projet Etat Région) in Lorraine. The authors thank Dr. Silke Gerstmann for helpful discussion. Financial support of the Oberfranken-Stiftung and by Dr. Robert Klupp of the fisheries department of the Regional Government of Upper Franconia is appreciated. ACKNOWLEDGEMENTS This research was supported by the Universities of Lorraine (France) and Bayreuth (Germany), the PRST Region Lorraine, and the French Ministry of Research. ACKNOWLEDGEMENTS This research was supported by the Universities of Lorraine (France) and Bayreuth (Germany), PRST Region Lorraine in France, and the French Ministry of Research.

List of abbreviations a.a.m.:

anterior adductor muscle

ACN: acetonitrile

Copper and enzymatic perturbation

The enzymatic activity of the plasma membrane Ca 2+ -ATPase was significantly inhibited in the kidneys of A. anatina upon 4 days of exposure at all concentrations of Cu 2+ tested in the rage of 0.26 to 1.15 µmol L -1 (article 1). In the kidneys and the gills, a significant inhibition of Ca 2+ -ATPase activity was observed upon 4 d of exposure at 0.35 µmol L -1 followed by a recovery at 7 d of exposure. Significant Ca 2+ -ATPase activity inhibition with no recovery was observed upon 15 d in the kidneys at the higher concentration of 0.64 µmol L -1 of Cu 2+ . No significant effect was noted on CA activity in gills and digestive gland of A.

anatina exposed to Cu 2+ (article 1).

Compared to controls mussels, the Na + /K + -ATPase activity was significantly inhibited in the gills (72 % inhibition) and the digestive gland (80 % inhibition) of A. anatina upon 4

days of exposure at 0.35 µmol L -1 of Cu 2+ (article 2). The Na + /K + -ATPase activity was inhibited by 26 % in the mantle of mussels exposed 4 d to the same conditions, but not significantly relative to controls.

No recovery was observed upon the 15 days of exposure in the gills which still showed 54 % of inhibition at the end of test, and a partial recovery was observed at day 7 in the digestive gland. In the mantle, the H + -ATPase activity declined continuously but the decline was not statistically significant due to high variability between mussels (article 2).

Calcium plays a fundamental role in numerous biological processes (energy production, cellular metabolism, muscle contraction, reproduction) and has important mechanical functions (shell, skeleton) in many organisms (Mooren & Kinne, 1998). Contrary to molluscs from marine ecosystem which are generally hyposmotic and for which calcium uptake is easy, freshwater bivalves are hyperosmotic and require tight regulation of their calcium metabolism.

Despite some recovery beyond 4 days in the digestive gland, the Na + /K + -ATPase activity in the gills of A. anatina was still significantly inhibited upon 15 days of experiment;

an inhibition of Ca 2+ -ATPase activity was also observed in the gills and the kidneys under the same conditions (article 1 and 2). Inhibition of these enzymes was consistent with the results obtained with Mytillus galloprovincialis exposed to copper by Viarengo et al. (1996) and Burlendo et al. (2004). Inhibition of Na + /K + -ATPase modified the cellular Na + gradient which could result in a reduced activity of Ca 2+ /Na + antiporter; this, in addition to direct inhibition of Ca 2+ -ATPase, may lead to a disturbance of calcium homeostasis. Such a disturbance of ionoregulations could lead to continuous under-supply of calcium which may also affect Ca 2+ VWR), Water was purified (18.2 M by a Milli-Q system (Millipore, France).

Animal maintenance

Mussel maintenance is described in detail in a previous article (Santini et al., 2010a).

Adult mussel A. cygnea with shell lengths of 7.5 ± 0.5 cm were provided by a commercial supplier (Amazon fish, Pfaffenhoffen, France). The mussels were kept in 10 L aquaria under a photoperiod of 16 h illumination and 8 h darkness. The bottom of the tank was covered with a layer of glass beads (10 mm diameter) so mussels could find conditions for burying. The , 0.20 HCO 3 -mmol L -1 , was renewed every day. Bivalves were fed daily with unicellular algae Chlorella kessleri from a culture in the exponential growth phase, which were added to a final algal density of 2×10 5 cells mL -1 . Air was bubbled continuously to ensure aeration and water column homogeneity. Animals were acclimatized to these conditions for 2 weeks before any experiment.

Determination of PC n and other cysteine-rich metal-binding peptides

PC n s were determined according Minocha et al. (2008) with a slight modification in the protein removal step and in the gradient profile of the mobile phase in HPLC. Cysteinerich peptides levels were studied in the cytosolic fractions of deproteinized tissue homogenates after reduction.

Extraction:

All dissection, extraction, and centrifugation steps were carried out at 4°C. The gills and the digestive gland were carefully dissected, digestive content was removed, and washed in Tris buffer (Tris 25 mmol L -1 , NaCl 50 mmol L -1 , pH 8.0). Organs from two mussels were 2010). Another possible source is fly ash from coal combustion, containing up to 20 g Cu per ton coal. Copper is easily found in aquatic ecosystems [START_REF] Waeles | Distribution and chemical speciation of dissolved cadmium and copper in the Loire estuary and North Biscay continental shelf, France[END_REF] since they are the ultimate way for numerous contaminants.

Copper is an essential trace element for the function of many cellular enzymes and proteins. However, copper become toxic when excessive intracellular accumulation occurs (Viarengo and Nott, 1993). Copper toxicity results both from non-specific metal binding to proteins, from is involvement in Fenton reactions leading to formation of reactive oxygen species (ROS) and oxidative stress. Through their important filtering activity they satisfy their respiratory and nutrition needs, by which bivalves have the capacity to accumulate a variety of environmental contaminants. The freshwater bivalve Anodonta cygnea belongs to Unionidae family, and is a species well distributed in continental waters. Unionidae are widely recognised for metal bioaccumulation including copper (Cossu et al., 1997). The level up to which mussels can tolerate transition metals depends on their ability to regulate the metal cation concentration in cells.

The proteins of the metallothionein (MT) group, the polypeptides glutathione (GSH)

and phytochelatins (PC) are protective compounds rich in the amino acid cysteine containing a thiol group (SH). Cu, as other transitions metals have high affinity for SH groups, making cysteine rich peptides the principal biological reagents for transition metal sequestration (Viarengo andNott, 1993, Clemens, 2006). PC bind transition metals with high thiol complexation constants, reducing the intracellular concentration of free ions of such metals in plants, fungi and microalgae. The general structure of PC is (γ -Glu-Cys) n -Gly (n = 2 to 11), synthesized by the constitutive enzyme phytochelatin synthase, which is activated by the presence of metal ions with GSH as substrate. (Grill et al., 1985). A PC synthase homologous sequence has been found in the genomes of the invertebrates Caenorhabditis elegans (Clemens et al., 2001;Vatamaniuk et al., 2001), Eisenia fetida (Brulle et al., 2008) and

Chironomus (Cobbett, 2000) genomes and more generally throughout the invertebrates (Clemens and Peršoh, 2009). In our previous article (Santini et al., 2011b)

Animal maintenance and copper exposure

Mussel maintenance was described in detail in our previous article (Santini et al., 2011a). Briefly adult individuals of A. cygnea with shell lengths of 7.5±0.5 cm were kept in aquaria at a temperature of 20 ± 0.5° C. The bottom of the tank was covered with a layer of glass beads so the mussels could find conditions for burying. Artificial pond water 1.5 L/mussel, pH 7.25 ± 0.10 (in mmol L -1 : 0.40 Ca 2+ , 0.20 Mg 2+ , 0.70 Na + , 0.05 K + , 1.35 Cl -, 0.20 SO 4 2-, 0.20 HCO 3 -) was renewed every day. The bivalves were fed daily with Chlorella kessleri from a culture in the exponential growth phase and added to a final algal density of 2×10 5 cells mL -1 . The animals were acclimatized to these conditions for two weeks before any experiment.

Previous results (Santini et al., 2011a) showed inhibition of enzymes involved in osmoregulationin A. cygnea exposed to 0.35 µmol L -1 of Cu 2+ . A total recovery of Ca 2+ -ATPase upon 7 d followed, indicating the induction of detoxication mechanisms. In the organs varied as follows: PC 2 > PC 3 > PC 4 , no significant induction of PC 3-4 was observed.

The quantities of PC 2-4 found in digestive gland were higher than in gills (data not shown).

In the gills of mussels exposed to Cu 2+ upon 48 h and 7 d -GluCys levels (table 1) are significantly superior to the respective controls. -GluCys increase as well, in the digestive gland of bivalves at 48 h and 4 d of exposure.

Table 1: No induction of MT was observed upon Cu 2+ exposure for 21 d, a result similarly to the one found in A. cygnea (Amiard et al., 2006). MT polymorphism appears to be particularly important in invertebrates compared to mammals. Different isoforms of MT play Controlled by inductively coupled plasma mass spectrometry (ICPMS) (detection limit: Cu = 0.5 µg L -1 = 0.008 nmol L -1 ), means (n = 3) ± standard deviations (SD) Controlled by graphite furnace atomic absorption spectrometry (GFAAS) (detection limit Cu = 0.5 µg L -1 = 8 nmol L -1 ), means (n = 3) ± standard deviations (SD)

Annexe 4: Pictures of mussel maintenance and exposure

Anodonta cygnea in acclimatization aquarium

Anodonta cygnea copper exposure in aquaria lined with polyethylene foil, in a thermoregulated room