Scientia Marina, Vol 78, No 1 (2014)

Effects of water-borne copper on the survival, antioxidant status, metallothionein-I mRNA expression and physiological responses of the Chinese mitten crab, Eriocheir sinensis (Decapoda: Brachyura) larvae

Shengming Sun
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences - School of Life Science, Dalian Ocean University, China

Xianping Ge
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, China

Jian Zhu
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, China

Hongtuo Fu
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, China

Zhiqiang Jiang
School of Life Science, Dalian Ocean University, China


The lethal concentration of water-borne copper in Chinese mitten crab Eriocheir sinensis larvae was tested by exposing the animals to 0, 0.1, 0.2, 0.3, 0.5 and 0.8 mg Cu L-1 at 20°C for 96 h. The 96-h median lethal concentration (LC50) and its corresponding 95% confident interval was estimated on zoea 1 larvae and megalopa larvae, respectively. Acute dissolved copper toxicity was higher for zoea 1 larvae (0.16 mg L-1) than for megalopa larvae (0.21 mg L-1). The antioxidant status, metallothionein-I mRNA expression and physiological response of the crab to copper toxicity was further investigated by exposing the megalopa larvae to 0, 0.08 and 0.16 mg Cu L-1 for 96 h. The superoxide dismutase activity, catalase activity, glutathione S-transferase (GST) activity and lipid peroxidation content of megalopa larvae increased concomitantly with the exposure time and copper concentration. MT-I mRNA expression levels were positively correlated with both the concentration and duration of copper exposure. The oxygen consumption and respiratory quotient of megalopa larvae in response to 0.16 mg L-1 copper were significantly higher than those in the control group after 96 h of exposure (P < 0.05). The results of this study highlight the potential effects of copper as a common stressor in E. sinensis larvae. MT-I and GST appear to be suitable biomarkers of environmental copper exposure stress in E. sinensis larvae


Eriocheir sinensis; copper toxicity; physiological; larvae; antioxidant enzyme; metallothionein

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Aebi H. 1984. Catalase in vitro. In: Packer, L. (ed.), Methods in Enzymology, vol. 105. Academic Press Inc., San Diego, pp. 121-126.

Agrahari KC. 2009. Heavy metals in aquatic ecosystem: some environmental implications. Everyman's Science, Vol. XLIV No. 2. pp. 88-93.

Amin O.A., Comoglio L.I. 2010. Effects of copper on the physiological responses of the commercial crab Lithodes santolla (Decapoda: Anomura) larvae. Sci. Mar. 74: 25-31.

Amin O., Comoglio L., Rodríguez E. 2003. Toxicity of Cadmium, Lead, and Zinc to larval stages of Lithodes santolla (Decapoda, Anomura). Bull. Environ. Contam. Toxicol. 71: 527-534.

Bambang Y., Thuet P., Charmantier-Daures M., Trilles J.P., Charmantier G. 1995. Effect of copper on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus Bate (Crustacea, Decapoda). Aquat. Toxicol. 33: 125-139.

Barata C., Varo I., Navarro J.C., Arun S., Porte C. 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comp. Biochem. Physiol. C 140: 175-186.

Beuge J.A., Aust S.D. 1972. Microsomal Lipoperoxidation. Methods. Enzymol. 52: 302-307.

Brouwer M., Schlenk D., Ringwood A.H., Brouwer-Hoexum T.M. 1992. Metal-specific induction of metallothionein isoforms in the blue crab, Callinectes sapidus. Arch. Biochem. Biophys. 294: 461-468.

Brouwer M., Enghild J., Hoexum-Brouwer T.M., Thogersen I., Truncali A. 1995. Primary structure and tissue-specific expression of blue crab (Callinectes sapidus) metallothionein isoforms. Biochem. J. 311: 617-622.

Brouwer M., Syring R., Brouwer T.H. 2002. Role of a copper-specific metallothionein of the blue crab, Callinectes sapidus, in copper metabolism associated with degradation and synthesis of hemocyanin. J. Inorg. Biochem. 88: 228-239.

Chen J.R. 1998. Experimental Direction Book for Aquatic Chemistry. China agricultural Press, Beijing, pp. 120-126.

Chourpagar A.R., Kulkarni G.K. 2011. Heavy metal toxicity to a freshwater crab, Barytelphusa cunicularis (Westwood) from Aurangabad region. Recent. Res. Sci. Technol. 3: 1-5.

Correia A.D., Livingstone D.R., Costa M.H. 2002. Effects of water-born copper on metallothionein and lipid peroxidation in the marine amphipod Gammarus lacusta. Mar. Environ. Res. 54: 357-360.

Coyle P., Philcox J.C., Carey L.C., Rofe A.M. 2002. Metallothionein: the multipurpose protein. Cell. Mol. Life. Sci. 59: 627-647.

D'Adamo R., Di Stasio M., Fabbrocini A., Petitto F., Roselli L., Volpe M.G. 2008. Migratory crustaceans as biomonitors of metal pollution in their nursery areas. The Lesina lagoon (SE Italy) as a case study. Environ. Monit. Assess. 143: 15-24.

De Boeck G.D., Ngo T.T.H., Campenhout K.V., Blust R. 2003. Differential metallothionein induction patterns in three freshwater fish during sublethal copper exposure. Aquat. Toxicol. 65: 413-424.

Dissanayake A., Galloway T.S., Jones M.B. 2008. Physiological responses of juvenile and adult shore crabs Carcinus maenas (Crustacea: Decapoda) to pyrene exposure. Mar. Environ. Res. 66: 445-450.

El-Bahr S.M. 2013. Biochemistry of free radicals and oxidative stress. Sci. Int. 1: 111-117.

Fang Y., Yang S., Wu G. 2002. Free radicals, antioxidants, and nutrition. Nutrition. 18: 872-879.

Ferrer L.D., Andrade J.S., Contardi E.T., Asteasuaian R.O., Marcovecchio J.E. 2003. Copper and zinc concentrations in Bahía Blanca Estuary (Argentina), and their acute lethal effects on larvae of the crab Chasmagnathus granulata. Chem. Speciat. Bioavailab. 15: 7-14.

Finney D.J. 1971. Probit analysis. 3rd edition. Cambridge University Press, Cambridge.

Habig W., Jakobi W.B. 1981. Glutathione S-transferase (rat and human). Methods of Biochemical alterations in rats subjected to dermal application of dodine. Ind. Comput. Physiol. 9: 27-31.

Haq F., Mahoney M., Koropatnick J. 2003. Signaling events for metallothionein induction. Mutat. Res. 533: 211-226.

Hasspieler B.M., Behar J.V., Di Giulio R.T. 1994. Glutathione-dependent defense in channel catfish (Ictalurus punctatus) and brown bullhead (Ameriurus nebulosus). Ecotoxicol. Environ. Safety. 28: 82-90.

Hou Y.X., Wang Y, Li H.Y., Li X.X., Hu X.J. 2011. Accumulation and distribution of heavy metals in Phragmites australis in the wetland of Liaohe river estuary. Adv. Mater. Res. 994: 356-360.

Hotard S, Zou E. 2008. Activity of glutathione S-transferase in the hepatopancreas is not influenced by the molting cycle in the fiddler crab, Uca pugilator. Bull. Environ. Contam. Toxicol. 81: 242–244.

Jing T.Y., Zhao X.Y. 1995. The improved pyrogallol method by using terminating agent for superoxide dismutase measurement. Prog. Inorg. Biochem. Biophys. 1: 13-15.

Leung K.M.Y., Furness R.W. 1999. Induction of metallothionein in dogwhelk Nucella lapillus during and after exposure to cadmium. Ecotoxicol. Environ. Safe. 43:156-164.

Li Q.S., Li Y.T. 2003. Practical manual for water environmental monitoring. China Waterpower Press, Beijing, China, 433.

Li N., Zhao Y.L., Yan J. 2008. Effects of water-borne copper on digestive and metabolic enzymes of the giant freshwater prawn Macrobrachium rosenbergii. Arch. Environ. Contam. Toxicol. 55: 86-93.

Li E.H., Chen L.Q., Zeng C., Chen X.M., Yu N., Lai Q.M., Qin J.G. 2007. Growth, body composition, respiration and ambient ammonia nitrogen tolerance of the juvenile white shrimp, Litopenaeus vannamei, at different salinities. Aquaculture. 265: 385-390.

Liang X.Q., Yan S.L., Zheng D.C., Guo D.D. 1974. Larval development of Eriocheir sinensis H. Milne-Edwards. Acta. Zoologica. Sinica. 20: 61-75. (in Chinese with English abstract).

Lin S., Hsieh I.J., Huang K.M., Wang C.H. 2002. Influence of the Yangtze River and grain size on the spatial variations of heavy metals and organic carbon in the East China Sea continental shelf sediments. Chem. Geol. 182: 377-394.

Livak K.J., Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.

López Greco L.S., Sánchez M.V., Nicoloso G.L., Medesani D.A., Rodríguez E.M. 2001. Toxicity of cadmium and copper on larval and juvenile stages of the estuarine crab Chasmagnathus granulata (Brachyura, Grapsidae). Arch. Environ. Contam. Toxicol. 41: 333-338.

Lowry O.H., Rosenbrough N.J., Farr A.L., Randall R.J. 1951. Protein measurement with a Folin reagent. J. Biol. Chem. 193: 265-275.

Marklund S., Marklund G. 1974. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47: 469-474.

Moksnes P.O., Lindahl U., Haux C. 1995. Metallothionein as a bioindicator of heavy-metal exposure in the tropical shrimp Penaeus vanname, a study of dose-dependent indication. Mar. Environ. 39: 143-146.

Martins Cde M., Barcarolli I.F., de Menezes E.J., Giacomin M.M., Wood C.M., Bianchini A. 2011. Acute toxicity, accumulation and tissue distribution of copper in the blue crab Callinectes sapidus acclimated to different salinities: In vivo and in vitro studies. Aquat. Toxicol. 17: 88-99.

Ministry of Agriculture, State Environmental Protection Administration. 2004. Report on state of the fishery eco-environment in China.

Ministry of Agriculture, State Environmental Protection Administration, Beijing, China, pp. 3-7.

Munshi A.B., Quan S.Y., Li, S.J. 1996. Acute toxicity of cop-per, cadmium and copper-cadmium mixture to the larvae of the shrimp Penaeus monodon. Pak. J. Sci. Res. 39: 68-71.

Olafson R.W., Kearns A., Sim R.G. 1979. Heavy metal induction of metallothionein synthesis in the hepatopancreas of the crab Scylla serrata. Comp. Biochem. Physiol. B 62: 417-424.

Rainbow P.S., 1992. The significance of accumulated heavy metal concentrations in marine organisms. In: Proceedings of bioaccumulation workshop, Water Board and Australian Marine Sciences Association Inc., Sydney.

Ren F., Jiang H., Sun J., He L., Li W.W., Wang Y, Wang Q. 2011.Cloning, characterization, expression, and copper sensitivity of the metallothionein-1 gene in the Chinese mitten crab, Eriocheir sinensis. Mol. Biol. Rep. 38: 2383-2393.

Sharonov B.P., Churilova I.V. 1992. Inactivation and oxidative modification of Cu, Zn superoxide dismutase by stimulated neutrophils: the appearance of new catalytically active structures. Biochem. Biophys. Res. Commun. 189: 1129-1135.

Stohs S.J., Bagchi D. 1995. Oxidative mechanisms in the toxicity of metal ions. Free. Radic. Biol. Med. 18: 321-336.

Sunda W.G., Hanson A.K. 1987. Measurement of free cupric ion concentration in seawater by a ligand competition technique involving copper sorption onto C18 SEP-PAK cartridge. Limnol. Oceanog. 32: 537-551.

Syring R.A., Brouwer T.H., Brouwer M. 2000. Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus. Comp. Biochem. Physiol. C 125: 325-332.

van der Oost R., Beyer J., Vermeulen N.P.E. 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13: 57-149.

Vosloo A., Van Aardt W.J., Mienie L.J. 2002. Sublethal effects of copper on the freshwater crab Potamonautes warreni. Comp. Biochem. Physiol. A 133: 695-702.

Yang Z.B., Zhao Y.L., Zhou Z.L., Zhou X., Yang J. 2005. Effects of copper in water on distribution of copper and digestive enzymes activities in Eriocheir sinensis. J. Fish. China. 29: 496-501. (in Chinese with English abstract).

Yang Z.B., Zhao Y.L., Zhou Z.L., Yang J. 2006a. Effects of CuSO4 on molting, growth, and survival of Eriocheir sinensis. Acta. Hydrobiol. Sin. 30: 563-569. (in Chinese with English abstract).

Yang Z.B., Zhao Y.L., Zhou Z.L., Li N., Yang J. 2006b. Effects of water-borne copper on activities of metabolism enzymes in the crab Eriocheir sinensis. Oceanol. Limnol. Sin. 37: 118-124. (in Chinese with English abstract).

Yang Z.B., Zhao Y.L., Li N., Yang J. 2007. Effect of water-borne copper on the microstructures of gill and hepatopancreas in Eriocheir sinensis and its induction of metallothionein synthesis. Arch. Environ. Contam. Toxicol. 52: 222-228.

Yeh S.T., Liu C.H., Chen J.C. 2004. Effect of copper sulphate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei. Fish. Shellfish. Immunol. 17: 437-446.

Zapata V., López Greco L., Rodríguez E. 2001. Effect of copper on hatching and development of larvae of the estuarine crab Chasmagnathus granulata (Decapoda, Brachyura). Environ. Toxicol. Chem. 20: 1579-1583.<1579:EOCOHA>2.0.CO;2

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