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A novel TCTP gene from the crustacean Eriocheir sinensis: possible role involving metallic [Cu.sup.2+] stress.


The translationally controlled tumor protein (TCTP) was initially described as an abundant growth-related (Yenofsky et al., 1983) and highly conserved 19-kDa protein (Thaw et al.,7 2001). This protein is widely distributed in mammals (Chitpatima et al, 1988; Thiele et al., 1998), plants (Pay et al., 1992), invertebrates (Sturzenbaum et al, 1998; Bhisutthibhan et al., 1998; Gachet et al, 1999), and yeast (Ras-mussen, 1994). In earlier studies, the synthesis and expression of TCTP was considered to be distributed mainly in tumor cells and controlled at the translational level (Bom-mer et al., 2002). However, subsequent intensive studies have shown that TCTP orthologs are found in most normal and tumor mammalian tissues, as well as in common cell lines, with the exception of kidney cells (Sanchez et aL, 1997; Thiele et aL, 2000). In fact, TCTPs are calciumbinding (Kim et at, 2000), heat-stable (Mak et aL 2001), and anti-apoptotic proteins (Rho et al., 2011). TCTP are also involved in multiple biological processes, such as cell growth (Kang et at, 2001), cell cycle progression (Cans et al, 2003), malignant transformation (Tuynder et al, 2002), and enhancement of the anti-apoptotic activity (Liu et al, 2005; Rho et al. 2011); and they are often designated as stress-related proteins (Bhisutthibhan et aL, 1998; Bonnet et al, 2000; Gnanasekar et aL, 2009). These combined functions indicate that TCTPs may be regulated in response to a wide range of extracellular signals and cellular conditions (Bommer and Thiel, 2004), such as proapoptotic and cytotoxic signals (Oikawa et aL, 2002; Sinha et aL, 2000), starvation (Bonnet et aL, 2000), heat shock (Gnanasekar et aL, 2009), calcium stress (Xu et aL, 1999), and heavy metals (Sturzenbaum et aL, 1998). In fact, studies of many different species have indicated that copper stress may result in the up-regulation of TCTP. In the earthworm Lumbricus rubellus. among 1536 SSH-derived cDNA clones sequenced, TCTP was identified as highly expressed during metallic exposure, and subsequent research also buttresses these results (Sturzenbaum et aL, 1998; Spurgeon et aL, 2004; Brulle et aL, 2008). In Cannabis sativa, TCTP was also found to be one of the major up-regulated proteins induced by copper stress (Bona et aL, 2007). Copper exposure affected the yeast mitochondrial proteome, and higher Levels of TCP protein were expressed at high copper concentrations (Barcia ET all, 2011). Further, in Calu-6 and Cos-7 cells, the rate of induction of TCTP was highest under the influence of copper stress compared to treatments with 4[beta]-phorbol 12-myristate 13-acetate, forskolin, dioxin, cobalt, and nickel: the increase was 4.5- to 5.0-fold at the mRNA level and 3.4- to 4.0-fold at the protein level (Schmidt et al, 2007).

However, knowledge about the effects of these processes remains limited in the mitten crab Richer saneness (De-caporal), despite it being a commercially important freshwater aquaculture species. In China, the aquaculture of this crab is a rapidly developing industry, with a peak yield of 40 million tons in 2005 (Yang and Zhang, 2005). However, the quality of the water used for the aquaculture of these crabs is threatened by high concentrations of copper pollution as a result of discharge from urban sewerage, mines, industrial processes, and agriculture. Copper is in fact an essential micronutrient for all living organisms for a variety of physiological and biochemical processes, including respiration (in the respiratory pigment hemocyanin) and oxygen transport (Engel and Brouwer, 1987; Rainer and Brouwer, 1993). However, it is potentially toxic to aquatic organisms when present in excess in water (Paganini et al., 2008). Hence, a better understanding of the molecular mechanism of tolerance for metal stress has become a research priority (Phillips and Hickey, 2010). In the present study, we analyzed the nucleotide sequence of TCTP from hepatopancreatic tissue of the mitten crab, and evaluated the expression of TCTP mRNA after treatment with CuS[O.sub.4] We apply the results of this work to consider the role of TCTP in the anti-stress mechanisms of animals.

Materials and Methods

Preparation of the samples

Healthy 1-year-old crabs (mean weight of 90 [+ or -] 12 g, sexually mature) were collected from the Tongchuan Road Aquatic product market in Shanghai, China. Crabs were placed in an ice bath for 3-5 min until lightly anesthetized prior to sacrifice. Tissues from different organs (hepatopancreas, hemocytes, brain, eye stalk, gills, thoracic ganglia, muscle, stomach, heart, ovaries, and testis) were harvested, snap-frozen in liquid nitrogen, and stored at -80 [degrees] C until nucleic acid analysis. For cloning and expression analysis, tissues from six crabs were pooled and ground with a mortar and pestle prior to extraction.

Total RNA isolation and cDNA synthesis

Total RNA was extracted using an RNA Extraction kit (Axygen, USA), according to the manufacturer's protocol. RNA concentration and quality were estimated by using spectrophotometry at an absorbance of 260 nm (Eppendorf, Germany) and agarose gel electrophoresis (Bio-Rad, USA), respectively. Total RNA (2[micro]g) that was isolated from the hepatopancreas was reverse-transcribed using the SMART cDNA kit (Clonetech, USA) for cDNA cloning. Total RNA (2[micro]g) was reverse-transcribed using the PrimeScript RT-PCR kit (TaKaRa, Japan) for semi-quantitative RT-PCR (RT-PCR) analysis, or the PrimeScript Real-time PCR kit (TaKaRa, Japan) for real-time quantitative RT-PCR (qRT-PCR) analysis.

Cloning and sequencing of Es-TCTP

A partial sequence of Eriocheir sinensis TCTP (Es-TCTP) was obtained from the clone that had been isolated from the Chinese mitten crab hepatopancreas cDNA library, which had been constructed by our laboratory (Jiang et aL, 2009). RACE technology was used to obtain the total coding sequence. The SMARTer RACE eDNA Amplification kit (Clotech, USA) was selected according to the protocol for 3' and 5' RACE reactions. The gene-specific primers (GSP) were GSP-F and GSP-R. All primers used in this study are presented in Table 1. The PCR program was carried out at 94 [degrees]C for 3 min, followed by 94 [degrees]C for 30 s, with a subsequent 45-s touch-down from 68 [degrees]C to 57.5[degrees] C in 0.7 [degrees]C steps, followed by 20 cycles at 57.5[degrees] C and 1 min at 72 [degrees]C. A final extension step was performed at 72 [degrees]C for 3 min. The amplified coda fragments were cloned into the PMD18-T vector (TaKaRa, China), following the manufacturer's instructions. Recombinant bacteria were identified by blue/white screening and confirmed by the PCR analysis. Plasmids containing the inserted Es-TCTP fragment were used as a template for DNA sequencing.
Table 1
Sequences of primers

Primer             Sequence                         Code

TCTP cDNA cloning
sqRT-PCR primers
TCTP 5 'primer     5'-TTGTAGTCGCCAAGCACCAC-3'       QR1
TCTP 3' primer     5'-AGGGCACAGAATCCAGCAGT-3'       QF1
qRT-PCR primers
[beta]-actin F     5'-GCATCCACGAGACCACTTACA-3'      [beta]-F
[beta]-actin R     5'-CTCCTGCTTGCTGATCCACATC-3'     [beta]-R

Sequence analysis

The homology of nucleotide and protein sequences was confirmed from the BLAST algorithm at the National Center for Biotechnology Information (NCBI) web site ( The deduced amino acid sequence was analyzed using the Expert Protein Analysis system (Gasteiger et aL, 2003). The Signal P 3.0 program was utilized to predict the presence and location of signal peptide and to predict the cleavage sites in amino acid sequences.

Alignment of multiple sequences and phyla genetic analysis

The full-length multiple alignment of the Es-TCTP sequence was compared to the TCTP of other species. Amino acid sequences from various crustacean species were retrieved from the NCBI GenBank and analyzed using ClustalW, ver. 2 (Larkin et aL, 2007). The Multiple Alignment Program and Multiple Alignment Show Program (Sto-hard, 2000) were used for multiple sequence alignment. A neighbor-joining phylogenetic tree was constructed using the MEGA software version 4.0 package (Tamura et aL, 2007). The reliability of the branching was tested using bootstrap resampling, with 1000 pseudo replicates.

Real-time polymerase chain reaction analysis

Tissue-dependent mRNA expression analysis of transcripts was conducted via semi-quantitative RT-PCR. The first-strand cDNA was synthesized following the methods described in the current study. The gene-specific primer pairs QF1 and QR1 were designed based on the cloned Es-TCTP cDNA to produce a 271-bp amplicon. The primers - actin F and [pounds sterling]-actin R [beta]-F, [beta]-R) were designed based on a cloned E. sinensis [beta]-actin cDNA fragment to produce a 276-bp amplicon. All RT-PCR reactions were completed in triplicate, using independently extracted RNA. RT-PCR was performed in a final volume of 25[micro]l of solution, containing 2.5[micro] of 10X PCR buffer, 1[micro]l of [10[micro]mol1.sup.-1]1 dNTP mix, 0.5 /xl of [10[micro]mol.sup.-1] primer, 18.75[micro]l of sterile deionized water, 0.25[micro]l of Ex-Taq HS DNA polymerase (TaKaRa, Japan), and 2 [micro]of first-strand cDNA that was used as template. PCR conditions were as follows: 94 [degrees]C for 3 min, 30 cycles of 94 [degrees]C for 30 s, 56 [degrees]C for 30 s, and 72 [degrees]C for 2 min. RT-PCR products were separated according to size on a 1.2% agarose gel stained with ethidium bromide, and visualized under ultraviolet light. Images were captured using a Gel Doc 2000 system (Tannon, China).

SYBR quantitative real-time polymerase chain reaction analysis

Real-time qRT-PCR was performed in a C1000 thermal cycler (BioRad CFX 96 Real-Time system), according to the manufacturer's instructions. Gene-specific primers QF2 and QR2 were designed based on the cloned Es-TCTP cDNA to produce a 185-bp amplicon. Samples were run in triplicate and normalized to the control gene /3-actin. PCR conditions were as follows: 95[degrees]C for 30 s, 40 cycles of 95[degrees]C for 5 s, and 55[degrees]C for 30 s. The final volume of each qRT-PCR reaction was 25.0 ^tl of solution, containing 12.5 jLtl of 2X SYBR Premix ExTaq (TaKaRa, Japan), 0.5 pi diluted cDNA template, 11.0 p of PCR-grade water, and 0.5 /xl of each 10 /xmol l_l primer. The two pairs of primers, /3-F and j3-R, were used to amplify /3-actin fragments, serving as a positive control. Samples were run in triplicate, and Es-TCTP expression levels were calculated by the 2-AACt comparative CT method (Livak and Schmittgen, 2001). Data are represented as triplicate mean [+ or -] S.E. (stan-dard deviation), and are presented as the n-fold difference relative to /3-actin. The resultant data were analyzed using the CFX Manager software (ver. 1.0).

[Cu .sup.2+ ]stress experiment and tissue isolation

Crabs were subjected to a total of 72 h of copper sulfate (CuS04) at median lethal concentrations (LC50), through acute copper toxicity tests following the method of Pinho (Pinho et al.r 2007).

Based on the LC50 of [Cu.sup.2+] on E. sinensis, one test concentration of 5 mg/1 of CuS04 was selected for the immersion experiment. Live healthy crabs (n = 128, 80 [+ or -] 5 g wet weight) were used in the test, and were acclimated for 1 week at 20-25 [degrees]C in filtered, aerated fresh water prior to the immersion experiment. Crabs maintained under normal conditions (i.e., without CuS04 stress) served as controls. Each treatment had three replicates, and each replicate was placed in one plastic aquarium (50 X 35 X 30 cm) with 42 1 of filtered water that was aerated continuously using an air stone; a constant temperature was maintained (26 [+ or -] 2 [degrees]C). Crabs (4 from the test and 4 from the control) were randomly selected at 0, 2, 4, 8, 12, 24, 48, and 72 h after the onset of the experiment. Hepatopancreas and gills were collected and preserved at -80 [degrees]C for subsequent RNA extraction experiments.

Statistical analysis

Statistical analysis was performed using SPSS software (ver. 11.0). Data represent the mean [+ or -] standard error (S.E.). Statistical significance was determined by one-way ANOVA (Snedecor and Cochran, 1971) and post hoc Duncan multiple range tests. Significance was set at P Less than 0.05.


Cloning and characterization of Es-TCTP cDNA

Full-length cDNA of Es-TCTP from Eriocheir sinensis was 727 bp in length and contained an open reading frame (ORF) of 507 bp, beginning with a methionine codon at position 86 and ending with a TGA termination codon at nucleotide position 592. The 3'-untranslated region (UTR) has 121 by with a poly (A) tail of 23 bp. The 5'-UTR has a CG-rich domain (nearly 70%), which is highly indicative of secondary structure (Thayanithy et al., 2005; Bangrak et ah, 2004). The complete sequence was deposited in GenBank under the accession number HM231279. The encoded 168 amino acid polypeptide had a calculated molecular mass of 18.9 kDa and a predicted isoelectric point of 4.27.

Motifs analysis and amino acid sequence alignment of Es-TCTP

Sequence analysis isolated two similar TCTP protein signatures at the 45-55 aa (XGXNPSXEXXD) and 123-145 aa (FXXXXFFXGEXXXXDG ...) motif in Es-TCTP. Potential phosphorylation sites were identified from Es-TCTP through the prosite scan at Pole Bioinformatique Lyonnais (Perriere et aL, 2003). Es-TCTP contained (a) a potential Asn-glycosylation site (N-glycosylation site) at 34-37 aa (NTTV); (b) four CK2-phospho-sites (casein kinase II phosphorylation site) at 9-12 aa (SGDE), 36-39 aa (TVTE), 50-53 aa (SAEE), and 64-67 aa (SGID); (c) two N-myristoylation sites at 46-51 aa (GANPSA) and 57-62 aa (GTESSS); and (d) three protein kinase C phosphorylation sites at 17-19 aa (TYK), 95-97 aa (SLK), and 103-105 aa (TPK). A similar conservative "ion response element (IRE)-like element"(Fig. 1 in the first rectangle) was presented in the 5'-UTR of Es-TCTP. The deduced amino acid sequence and Es-TCTP motifs are shown in Figure 1.


We aligned the TCTP sequences from 10 species, each representing one kingdom. Twenty-six of the approximately 170 residues comprised a total of nearly 15% absolutely conserved amino acids. Blast analysis revealed that Es-TCTP showed high identities with other registered TCTPs. The Es-TCTP shared 92%, 89%, 80%, 80%, and 79% identity with Penaeus monodon, Scylla paramamosain, Fenneropenaeus chinensis, Litopenaeus vannamei, and Marsupenaeus japonicas, respectively; and shared 50%-78% identity with the TCTPs of the other examined species. Es-TCTP Blast analysis is shown in Figure 2, and the accession numbers of the TCTPs of the other species are presented in Figure 3.


Phylogenetic analysis f Es-TCTP

The phylogeny of global TCTP sequences is shown in Figure 3. The protein sequences of the TCTP of other species were obtained from the NCBI database. As a result, a neighbor-joining phylogenetic tree was produced that contained two distinct branches, vertebrates and invertebrates, supporting traditional taxonomic relationships (Kinch and Grishin, 2002). Polygenetic analysis also revealed that Es-TCTP constitutes a well-defined subgroup within the TCTPs of other crustaceans, and E. sinensis was found to be a sister species of Scylla serrata, with high bootstrap values and posterior probabilities support.


Organic distribution of Es-TCTP transcripts in normal Chinese mitten crabs

From the RT-PCR analysis, Es-TCTP mRNAs were de-tected in all target tissues at different levels of expression, and were predominantly detected in the tissues of hepatopancreas, hemocytes, brain, thoracic ganglia, muscle, stomach, heart, ovaries, and testis (Fig. 4). Expression was highest in the hepatopancreas. The levels in the hemocytes, stomach, and gonads were all similar to one another. The lowest levels were found in the gills. As determined by real-time qRT-PCR, Es-TCTP expression was widely detectable, with highest level in the hepatopancreas when compared with other tissues.


Temporal expression of Es-TCTP in hepatopancreas and gills under [Cu.sup.2+] stress

The temporal expression of Es-TCTP in hepatopancreas and gills as a result of [Cu.sup.2+] stress was measured by using a qRT-PCR method with[beta]-actin as internal control. The method was comparable to that used for the normal crabs. For both Es-TCTP and [beta]-actin genes, there was only one peak at the corresponding melting temperature in the disso-ciation curve analysis, indicating that the PCR was specif-ically amplified. The effect of [Cu.sup.2+] stress on Es-TCTP expression is presented in Figure 5. Es-TCTP expression in hepatopancreas showed no significant difference during the first 4 h (P > 0.05) with respect to the control. The difference was up-regulated immediately following 8 (1.8-fold), 12 (2.5-fold), 24 (4.3-fold), and 48 h (4.2-fold). Up-regula-tion peaked at 4.3-fold above that of the control after 24 h and was maintained even after 72 h (2.6-fold) in the immersion group. However Es-TCTP expression in gills was significantly greater than in the control at 4 and 8 h after induction of [Cu.sup.2+] stress, and decreased in the later phase. Control reactions without [Cu.sup.2+] stress yielded no significant increase in expression levels.



TCTP, the translationally controlled tumor protein, is involved in various biologically relevant processes (Bangrak et al., 2004; Thayanithy, 2005; Meyvis et alt 2009), with the expression of TCTP mRNA possibly being subject to rapid induction by growth signals (Chen et al, 2007), cytokines (Nielsen et al, 1998), and metal pollution or stress (Bona et al, 2007; Schmidt et al, 2007; Hinojosa et al, 2008; Bancia et ai, 2011). Although TCTP is considered to play an important role in the anti-stress program of many organisms, knowledge of its gene expression has remained limited, particularly in invertebrates. The present study is therefore the first to identify the full-length sequence of TCTP in the Chinese mitten crab Eriocheir sinensis and to assess the mRNA expression profile of TCTP after [acuteCU.sup.2+]metal ion stress.

TCTP sequences usually contain two conserved domains, TCTPl and TCTP2 (Gachet et al., 1999; Bommer and Thiele, 2004). In our study, the folding of the TCTPl domain displayed significant similarity to that of the Mss4 and Dss4 proteins (mammalian suppressor of Sec4), which are two small transporter proteins that have been reported to bind to the nucleotide-free form of Rab proteins (Thaw et aL, 2001). As a result, TCTP is now grouped into one protein family with Mss4/Dss4 (Bommer and Thiele, 2004). TCTP2 comprises the flexible loop and the helical domain that are both specific to TCTP. The middle of the loop contains a highly conserved area with the TCTP signature 1, which contains binding regions for tubulin (Gachet et alf 1999) and [Ca.sup.2+ ](Kim et aly 2000). Our analysis of the Es-TCTP amino acids showed that the crab also has both TCTPl and TCTP2 domains. This result indicates that Es-TCTP should be included as a new member of the TCTP family. Iron response-like elements in the 5'-UTR that were found in the Es-TCTP cDNA sequence showed high similarity with the ferritin of the crayfish Pacifastacus leniusculus (Huang et al., 1996), the giant tiger prawn Penaeus monodon (Bangrak et al., 2004), and the fleshy prawn Fenneropenaeus chinensis (Zhang et al., 2004). The element ferritin has the characteristic of combining metal ions, such as copper. We therefore suggest that Es-TCTP is a stress-related protein and may have a role in interactions with [Cu.sup.2+].

In humans and rabbits, it has been demonstrated that the TCTP expression profile is high in mitotically active tissues but low in postmitotic tissue, such as the brain (Thiele et al, 2000; Andree et al., 2006). In our study, Es-TCTP transcripts were detected in all examined tissues, which may be related to the specific function of each organ. For example, expression was high in the brain, stomach, muscles, and gonads, and was especially high in the hepatopancreas and hemocytes. The hepatopancreas is the main toxin-expelling and anti-stress organ, and the major function of the hemocytes is defense and transportation (Rusaini and Owens, 2010). Infiltration of the hemocytes may also contribute toward explaining the wide expression of Es-TCTP in all other tissues (Rusaini and Owens, 2010; Martins et al., 2011). In addition, the distinct difference that was recorded in our study in the expression of Es-TCTP in the brain compared to previous studies may be due to species differences.

CCTCTP levels are regulated in response to various stress conditions. In fact, an increase in TCTP has been previously reported to be associated with increased chemoresistance (Sinha et ai, 2000; Rusaini and Owens, 2010). Research has shown that the over-expression of mammalian TCTP results in microtubule stabilization and the alteration of cell morphology (Cachet et aL, 1999). When combining this infor-mation with the recent characterization of TCTP as an anti-stress protein, it may be interpreted that TCTP generally exerts a cytoprotective function (Li et al., 2001; Schmidt et al., 2007). In the CuS04 immersion experiment, Es-TCTP expression in hepatopancreas was found to have no significant difference from that in the control during the first 4 h. However Es-TCTP expression in gills was significantly different during the first 4 h and 8 h, while there was no obvious reactivity in the control. These results may imply that hepatopancreas and gills participate in metal stress response. Expression in gills was more rapid than in hepatopancreas owing to direct water exposure. Es-TCTP expression in hepatopancreas increased sharply in subsequent hours, resulting in a total of five expression peaks in response to the Cu stress (Fig. 5). Heavy metal ions may bioaccumulate in the organism, and as their levels increase, the synthesis of TCTP increases correspondingly (Andree etal., 2006; Bona et al.f 2007; Schmidt et aL, 2007; Hinojosa et aL, 2008; Bancia et aL, 2011). Not coincidently, the hepatopancreas is the most important tissue for heavy metal accumulation as well as the central location of TCTP thesis. These findings also support the results published for other species. In a study of the red worm Eisenia fetida, TCTP transcripts of coelomocytes were up-regulated (to about 2-fold) after Cu/Cd exposure treatment (Brulle et al., 2008). TCTP levels were highly expressed (to 335-fold) in the earthworm (Lumbricus rubellus) after the South Caradon Copper Mine treatment (Stiirzenbaum et aL, 1998). In the study of Cannabis sativa, TCTP was detected in its roots as one of five major proteins up-regulated after copper stress (Bona et al., 2007). In the study of TCTP expression in human Calu-6 cells and in monkey Cos-7 cells under the influence of the heavy metals nickel, cobalt, and copper, the highest induction rates, 4.5-5.0-fold at the mRNA level and 3.5-4.0-fold at the protein level, were observed with copper (Schmidt et aL, 2007). The TCTP-inducing capability of copper was known from experiments with earthworms inhabiting copper-contaminated soils (Stiirzenbaum et al., 1998). In Calu-6 and Cos-7 cells, copper turned out to be a potent inducing agent, comparable to the situation in earthworms, suggesting that the action of copper involves both a transcriptional and a post-transcriptional effect (Schmidt et aL, 2007). These results combined with our study suggest that copper may also be a potent inducing regulator of TCTP at both a transcriptional and a post-transcriptional level. However, because of the lack of cell line research in decapods, it remains unclear as to whether this response is linked to a direct mechanistic function of TCTP to copper or is a by-product caused by stress or inflammation.

Of interest, the up-regulated expression level of Es-TCTP after [Cu.sup.2+] stress took longer than in the other species, which showed peaks at about 2-h post challenge (Li et aL, 2010). This delayed response in the crab may be related to different levels of copper tolerance between species. Alternatively, copper is an essential trace element that is required by organisms for metabolic function, particularly crustaceans (Nasreddine et aL, 2010). For example, at least 12 major proteins require copper as an integral part of their structure, including the respiratory enzyme cytochrome oxidase (Martins et al., 2011). In addition^ most crustaceans possess many metabolic enzymes that contain large amounts of copper as their main oxygen-carrying blood protein, particularly hemocyanin (Jaenicke and Decker, 2004). Alternatively, [Cu.sup.2+] may have negatively regulated the transcription of TCTP at the beginning of the challenge (Yoon et aL, 2006). It is also possible that [Cu.sup.2+] stress may have prompted the programmed cell death pathway, leading to the down-regulation of survival genes, including TCTP (Andree et aL, 2006). The data presented in this report suggest that TCTP expression is anti-stress responsive, and that TCTP may possess anti-metal stress functionality in E. sinensis.

In the current study, we cloned a novel gene, Es-TCTP, related to metallic stress and characterized its molecular structure and expression pattern. Our results showing that Es-TCTP has the characteristic of anti-metal stress explain how Chinese mitten crabs maintain health in waters that may contain toxins. In addition, our findings may provide new insights about invertebrate tolerance mechanisms. In the future, the molecular mechanisms linking TCTP expression to heavy metal stress require determination, particuiarly if this gene is to be expioited as a molecular biomarker in ecotoxicological studies. Further research is also required to explain the function of TCTP in response to stress from [Cu.sup.2+ ] and other heavy metals. Overall, this study provides the first step toward understanding heavy metal tolerance in marine invertebrates, which will contribute toward improving the quality of aquaculture stocks and the selection of the water in which they are reared.


This research was supported by the National Natural Science Foundation of China (Grant Nos. 30671607, 30972241), program of Shanghai Education Commission (No. 08SG24).

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QUN WANG (1) *, DI-AN FANG (1, WEL-WEL LI, JUAN WANG, AND HUI JIANG School of Life science, East China Normal univerity, shanghai 200062, China

Received 28 June 2011; accepted 25 October 2011.

* To whom correspondence should be addressed. E-mail:

(1.) These two authors contributed equally to the work.
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Author:Wang, Qun; Fang, Di-an; Wei-Wei, Li; Wang, Juan; Jiang, Hui
Publication:The Biological Bulletin
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Geographic Code:9CHIN
Date:Dec 1, 2011
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