Differential HSP70 expression in Mytilus coruscus under various stressors

Gene 543 (2014) 166–173

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Short Communication

Differential HSP70 expression in Mytilus coruscus under various stressors Hui-hui Liu a,b,⁎, Jian-yu He a, Chang-feng Chi a, Jianzhong Shao b a b

National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316004, PR China College of Life Sciences, Zhejiang University, Hangzhou 310012, PR China

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 19 March 2014 Accepted 2 April 2014 Available online 3 April 2014 Keywords: Mytilus coruscus Heat shock protein 70 (HSP70) Vibrio infection Thermal stress Environmental stress

a b s t r a c t Heat shock proteins (HSPs) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. The full-length HSP70 cDNA of Mytilus coruscus (designated as McHSP70, GenBank accession no. KF322135) was obtained, which was 2319 bp, including an ORF of 1965 bp to encode a polypeptide of 655 amino acid with predicted pI/MW5.29/71.38 kDa, a 81 bp-5′UTR and a 270 bp-3′-UTR. The BLAST analysis and phylogenetic relationship strongly suggested that this amino acid sequence was a member of HSP70 family and highly homologous with Mytilus galloprovincialis (95%). Multiple sequence alignment revealed that McHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-Binding site motif and ‘EEVD’ motif. The mRNA of McHSP70 in hemolymph was constitutively expressed in all treatments including Vibrio challenge, thermal stress, metals (copper and cadmium) and 180 CST fuel exposure based on SYBR Green quantitative RT-PCR analysis. The temporal expression of HSP70 mRNA in hemolymph was significantly affected by Vibrio alginolyticus and Vibrio harveyi challenge. The maximum level appeared at 24 h post-injection with 5.44-fold in V. alginolyticus and dropped back to the original level at 72 h post-injection. In V. harveyi infection the transcripts of McHSP70 was significantly induced to the highest at 2 h after post-injection with 13.52-fold, then decreased until 36 h appearing another expression peak with 10.29-fold, and returned gradually to physiological level at the end of this experiment. In heat shock experiment the maximum expression appeared at 12 h with 15-fold higher than that of blank mussels. Time-dependent mRNA expression pattern of McHSP70 was found in exposure to copper, cadmium and 180 CST fuel, but the highest expression and time were different in various treatments (copper of 9.41-fold at day 15, cadmium of 10.82-fold at day 10, and 180 CST fuel of 5.94-fold at day 25), and then dropped progressively. All these results indicated that HSP70 in marine bivalve had a significant role in mediating the environmental stress and immune response. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Heat shock proteins (HSPs) are a family of proteins that have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins (Joly et al., 2010; Mjahed et al., 2012). Under external environmental stress such as heat shock, heavy metals, pathogenic infections or almost any sudden changes inducing protein damage in the cellular environment, HSPs play crucial roles in protecting organisms from the damage and restoring the damaged proteins to their functional three-dimensional structure (Gao et al., 2007; Song et al., 2006; Welch, 1992). Heat shock protein 70 (HSP70) is an important member

Abbreviations: Degenerate primers: S, C/G; M, A/C; Y, C/T; W, A/T; R, A/G; K, G/T; bp, base pair(s); cDNA, DNA complementary to RNA; dNTP, deoxyribonucleoside triphosphate; kb, kilobase(s) or 1000 bp; kDa, kilodalton(s); nt, nucleotide(s); ORF, open reading frame; oligo, oligodeoxyribonucleotide; UTR, unstranslated region(s). ⁎ Corresponding author at: National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316004, PR China.

http://dx.doi.org/10.1016/j.gene.2014.04.008 0378-1119/© 2014 Elsevier B.V. All rights reserved.

of the heat shock protein superfamily and appears in almost all species except for some archaea (Krenek et al., 2013; Tavaria et al., 1996). As a kind of highly conserved protein HSP70 is associated with intracellular chaperone and extracellular immunoregulatory functions to protect cells against environmental stress, and shows tissue-/time-/dosedependent changes (Snyder et al., 2001). Therefore, HSP70 protein is known to be involved in immune response and promote its use in environmental monitoring (Steinert and Pickwell, 1993). In recent years, marine pollution has become more and more serious than before, and marine organisms have been suffering from increasing environmental stress (Silvia and Elena, 2005). For the sensitive reaction to marine or aquatic environment, mollusks, especially marine bivalves, have been regarded as ideal indicators in the assessment of environmental pollution because they are ubiquitous, sedentary, filter-feeders inhabiting in the coastal and estuarine areas (Goldberg et al., 1983; Pascal et al., 2004). Bivalves, particularly Mytilus sp. representing the sentinel species, have been frequently used for the assessment of potential biological exposure to anthropogenic contaminants in benthic environments.

H. Liu et al. / Gene 543 (2014) 166–173

Mytilus coruscus is an important economic shellfish among aquatic invertebrates, widely distributed in the eastern coast of East China Sea, especially Zhoushan (China) coast (Liao et al., 2013; Maria et al., 2009). In view of few related reports of this mussel and its acute response to marine pollution, M. coruscus was regarded as our experimental animals, and HSP70 was the candidate gene for its biomarker for pollutants in aquatic invertebrates. The full-length HSP70 cDNA sequence was cloned from M. coruscus, and the mRNA expression profiles were analyzed emphatically in hemolymph after thermal stress, Vibrio pathogen infection and marine contaminants (copper, cadmium and 180 CST, Critical Solution Temperature) stimulation based on SYBR Green quantitative RT-PCR analysis. All of the results will contribute to better understanding of HSP70 diversity in mollusca and the biochemical resistance mechanisms used by marine consumers to cope with their allelochemically defended prey. 2. Material and methods 2.1. Experimental animals Adult mussels (M. coruscus, 5–8 cm in length) were obtained from Zhoushan (Zhejiang Province, PR China), and immediately transferred to tanks in the laboratory. The mussels were maintained at 25 °C with salinity of 27–28‰ for a week before the beginning of the experiment. Seawater was changed daily. Animals were fed with microalgae during the acclimation and experimental period. No mortality was observed in either the experimental or control groups. Hemolymph was collected from the adductor muscles using a syringe. Total RNA was isolated from the hemolymph using Trizol reagent (TaKaRa) and treated with DNAse-1 (Sigma, USA) to remove genomic DNA. The concentration was quantified for the ratio of A260/A280 by measuring the absorbance at 260 and 280 nm with a UV–spectrophotometer (Bio-Rad, USA). The cDNA synthesis was carried out with M-MLV RTase cDNA Synthesis Kit (TaKaRa). 2.2. cDNA of M. coruscus HSP70 identification and full-length amplification The primers (HSP-F and HSP-R, as shown in Table 1) were designed by the software of Primer 5.0 according to the homologous HSP70 sequences of M. galloprovincialis (AY861684), M. edulis (AF172607), Chlamys farreri (AY206871), Argopecten purpuratus (FJ839890), Pteria penguin (EF011060) and Pinctada fucata (EF011061). The reaction system was performed in a 20 μL volume, including 10 × PCR Buffer 2 μL, dNTPs 0.4 μL, HSP-F 0.8 μL, HSP-R 0.8 μL, template cDNA 0.6 μL, and 0.4 μL of Taq DNA polymerase (TaKaRa). The PCR amplification was conducted on the Thermal Cycler (Bio-Rad, USA), and amplification conditions were: 4 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 53 °C, and 45 s at 72 °C, with a final extension of 10 min at 72 °C. The PCR products were subjected to 1.5% agarose gel electrophoresis, purified by NucleoTrap Gel Extraction Kit (TIANGEN), and sequenced at Shanghai Invitrogen Biological Technology Company (China). Table 1 PCR primer sequences for HSP70 cloning of Mytilus coruscus. Degenerate primers as follows, S: C/G; M: A/C; Y: C/T; W: A/T; R: A/G; K: G/T. Primer For HSP70 cDNA clone HSP-F HSP-R For 5′ and 3′ RACE 5P-R 3P-F For qRT-PCR McHSP70-F McHSP70-R β-actin-F β-actin-R

Sequences 5′-GATYTKGGWACHACATAYTCCTG-3′ 5′-ACTCCYTCMARYTCYTTCTGTT-3′ 5′–CACCACCCAAGTCAAAGATGAGA-3′ 5′–CAGCTGGAGGTGTGATGACAGCTC-3′ 5′-GAAATTGTCTTAGTAGGTGGATC-3′ 5′-CAGGCTGGTTGTCGGAGTAAGT-3′ 5′-ATGAAACCACCTACAACAGT-3′ 5′-TAGACCCACCAATCCAGACG-3′

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Full-length cDNA sequence of HSP70 was performed with the other primers (5P, 3P as shown in Table 1) and the primers in the Smart RACE cDNA amplification kit (Clontech, USA). Both 5′-RACE and 3′-RACE were carried out according to the manufacturer's instructions. The PCR products were cloned into the MD18-T simple vector (TaKaRa) and sequenced from both directions. The full-length cDNA of clone was obtained by overlapping the forward and reverse strand sequences. The resulting sequences were verified by the amplification of the whole full length and further subjected to cluster analysis. 2.3. Sequence analysis The cDNA sequences (sequencing was repeated for three times) were spliced by the software of DNAstar v7.0. The BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast) was used to analyze the cDNA sequence. The conserved domains were predicted using SMART (http:// smart.embl-heidelberg.de/smart/set_mode.cgi) online tool. The theoretical MW and pI were determined by Expasy-ProtParam online tool (www.expasy.org/tools/protparam.html) (Gasteiger et al., 2005). Multiple sequence alignments were performed with ClustalW (http:// www.genome.jp/tools/clustalw/) (Thompson et al., 1994) and software of Genedoc. The phylogenetic tree was constructed by Boostrapped Neighbor-Joining rule method from a distance matrix with the software of MEGA 4.0 software (Kumar et al., 2004). 2.4. The temporal expression of HSP70 mRNA in hemolymph after Vibrio harveyi and Vibrio alginolyticus challenge For the aquatic pathogenic bacterial challenge experiment, 100 mussels were collected and kept in aerated tanks. The live V. harveyi and V. alginolyticus that were isolated from marine shellfish before, were injected into the adductor respectively of 100 μL diluted with PBS (pH 7.4, final concentration of O.D. 600 = 0.4). Forty-five individuals of the challenged mussels by different Vibrio were randomly collected at 2, 4, 6, 8, 12, 24, 36, 48 and 72 h post-challenge. The mussels that were injected with the same amount of PBS, were used as the blank group. Hemolymph (about 0.35 ml for each individual) from the blank and the challenged group were collected from the adductor muscles with a syringe. The hemolymph from five mussels of the same time were pooled together as one sample and immediately centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes for total RNA extracting. Two micrograms of total RNA from each group (n = 5) was reverse transcribed in a final volume of 40 μL with a PrimeScript™ RT reagent kit (Perfect Real Time) (TaKaRa) following the manufacturer's instructions. Real-time PCR was performed in a reaction mixture of 20 μL, containing McHSP70-F 0.8 μL, McHSP70-R 0.8 μL, 2 × SYBR® Premix Ex Taq™ II (TaKaRa) 10 μL, cDNA sample 0.8 μL, ROX II 0.4 μL, ddH2O 7.2 μL. The standard cycling conditions were: 95 °C for 1 min (initial polymerase activation), followed by 40 cycles of 30 s at 95 °C and 45 s at 65 °C. The PCR specificity was checked with dissociation curve analysis from 55 to 95 °C, and the β-actin of M. coruscus was used as the internal standard (primers as shown in Table 1). The 2−ΔΔCT method was used to analyze the mRNA expression level (Livak and Schmittgen, 2001). All data were given in terms of relative mRNA expressed as mean ± S.E. (N = 5). Statistical analysis of differences was done using SPSS 13.0 by one-way analysis of variance (ANOVA) followed by an unpaired, twotailed t-test. Differences were considered significantly at P b 0.05. 2.5. The temporal expression of HSP70 mRNA in hemolymph after thermal stressed and contaminated treatments The mussels were divided into 4 groups with 50 animals each. The mussels in the first group were stimulated with heat shock (35 °C for 1 h). Five normal mussels were reared in an individual tank as the control. The other two groups were exposed to 10 L seawater with heavy metal press, Cu2+ (final concentration of 20 μg/L) from CuSO4·5H2O,

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and Cd2+ (final concentration of 200 μg/L) from CdCl2·5H2O. The last group was cultured in 10 L seawater with 180 CST fuel (final concentration of 40 μg/L). The control groups were reared as described above. All mussels were kept in static tanks at 25 °C. Fresh seawater was changed daily and resupplied with the corresponding concentration of pollutants. Sampling was performed in the four groups at different time intervals. The first group was at 3, 6, 9, 12, 15, 18 and 24 h after thermal stress, and the other three groups were on 3, 5, 10, 15, 20, 25 and 30 d after the stimulation. The methods of hemolymph collection, total RNA extraction, cDNA synthesis and real-time PCR analysis were performed as described above.

3. Results 3.1. cDNA sequence analysis and characterization of McHSP70 full-length Total RNA from hemolymph of M. coruscus was isolated and the ratio of A260/A280 was 1.93. The complete cDNA sequence of McHSP70 (GenBank accession no. KF322135) was 2319 bp, containing an ORF of 1965 bp with a coding capacity of 655 amino acid residues. The

5′-UTR was 81 bp, and the 3′-UTR was 270 bp with a polyadenylation signal (AATAAA) appearing at position 167 nt downstream of stop codon (TAA) (Fig. 1). The BLASTn program demonstrated that the obtained HSP70 sequence shared high homologies with HSP70 from bivalve mollusca, such as M. galloprovincialis (95%), Perna viridis (83%) and Meretrix meretrix (80%). It was concluded that the sequence cloned from M. coruscus belonged to HSP70 family. The theoretical MW of McHSP70 was 71.38 kDa and its estimated pI was 5.29.

3.2. The multiple sequence alignment between M. coruscus and other species The deduced amino acid sequence of McHSP70 was aligned with the known HSP70 of other species (as shown in Fig. 2). The amino acid sequences in most marine mollusca were highly homologous, especially between M. galloprovincialis and M. coruscus in N terminal and C terminal. In addition, multiple sequence alignment of McHSP70 with other known HSP70 revealed that they were highly conserved, especially in the regions of HSP70 family signatures, including HSP70 family signatures 1, 2 and 3, the bipartite nuclear targeting sequence and ATP/ GTP-Binding site motif A (P-loop) (Fig. 2). The last four amino acids

Fig. 1. The full-length cDNA and deduced amino acid sequence of HSP70 in M. coruscus. The stop codon (TAA) is indicated with an asterisk (*); the polyadenylation signal (AATAAA) is marked with thick line; the cDNA sequences have been submitted to GenBank with accession no. KF322135.

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Fig. 2. Multiple alignment of the deduced amino acid sequences of HSP70 in M. coruscus with other species. Comparison of the amino acid sequence deduced from M. coruscus with some similar protein sequences of HSP70 or the like. The GenBank accession no. for the sequences are as follows: Mytilus coruscus (KF322135), Chlamys farreri (AY206871), Argopecten purpuratus (FJ839890), Pinctada fucata (EF011061), Pteria penguin (EF011060), Mizuhopecten yessoensis (AY485262), Argopecten irradians (AY485261), and Mytilus galloprovincialis (AY861684). In the figure, the red box indicates HSP70 family signatures, included HSP70 family signatures 1, 2 and 3; the blue box indicates ATP/GTP-Binding site motif; the purple box indicates Bipartite Nuclear Targeting Sequence and the last four amino acids that form a motif ‘EEVD’ are marked the green box.

To reveal the molecular phylogenetic position of McHSP70, an unrooted phylogenetic tree was constructed with Neighbor-Joining method from a distance matrix (Fig. 3). The phylogenetic tree was composed of five major groups: bivalve, shrimp, fish, chicken, and mammalians. M. galloprovincialis and M. coruscus were firstly gathered into the same branch, and further grouped with HSP70 from P. viridis and other mollusca. The relationship displayed in the phylogenic tree was generally in agreement with the traditional taxonomy.

RT-qPCR with β-actin as internal control. The relative expression of McHSP70 was up-regulated after being infected by V. alginolyticus in the first 6 h, and reached 2.91-fold compared with the blank at 6 h after challenge (Fig. 4A). The maximum expression was 5.44-fold at 24 h post-injection. As time progressed, the expression of McHSP70 transcript was down-regulated and dropped back to the original level at 72 h post-injection. Not surprisingly, the transcripts showed a time dependent expression pattern in V. harveyi infection similar to V. alginolyticus, but the time and expression quantity were different. McHSP70 expression in hemolymph was significantly induced to the highest level at 2 h after post-injection with 13.52-fold higher than that of the blank, then decreased until 36 h appearing another expression peak with 10.29-fold, and returned to physiological level at the end of this experiment (Fig. 4B). An unpaired two-tailed t-test with the blank and challenged groups showed a significant difference in McHSP70 mRNA expression at the highest level (P b 0.05).

3.4. Temporal expression of HSP70 in hemolymph after Vibrio challenge

3.5. The expression profile of McHSP70 in different treatment groups

The temporal expression of HSP70 mRNA in hemolymph was significantly affected by V. alginolyticus and V. harveyi challenge using

The temporal expression of the McHSP70 mRNA showed similar trend after all environmental stimulation (as shown in Fig. 5). The

formed a motif ‘EEVD’ at the C-terminus, which is highly conserved throughout all species. The presence of five consecutive repeats of the tetrapeptide motif GGMP in McHSP70 C-terminal region was another notable feature in this protein. 3.3. Phylogenetic relationship of HSP70 between M. coruscus and other species

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Fig. 3. Phylogenetic tree depicting the relationship of M. coruscus HSP70 with other species. All protein sequences are obtained from GenBank of the NCBI, GenBank accession number is in parentheses, and phylogenetic tree is constructed using Neighbor-Joining rule method by the software of MEGA v4.0.

clear time-dependent expression profile of McHSP70 was observed after heat shock, and the high expression of HSP70 mRNA was explicit during the period of heat shock stress from 3 to 24 h. At 3 h poststressed initially, the expression of McHSP70 was up-regulated significantly, then increased to the highest peak approximately 15.46-fold compared with the blank at 12 h, and decreased to original 2-fold level at 24 h after heat shock. The time-dependent expression pattern was also observed in fuel exposure. The McHSP70 mRNA expression increased as time went on, and reached a peak approximately 5.53-fold higher than the blank in 180 CST fuel exposure. The maximum peak appear at 25 d with

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Relative expression of HSP70 in M.coruscus

Relative expression of HSP70 in M.coruscus

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5.94-fold, and then dropped until 30 d. In the groups treated by heavy metals, the expression of McHSP70 mRNA kept the same tendency as that in fuel exposure, but the expression values in metal groups were higher than that in 180 CST fuel exposure. McHSP70 mRNA sustainedly increased until 15 d in Cu2+ exposure (approximately 9.41-fold higher than the blank). In Cd2 + treatment the highest expression level appeared at 9 d (approximately 10.82-fold higher than the blank group, P b 0.05), and then reduced approximately 7-fold higher than the blank group at 15 d. The results indicated that McHSP70 took part in the cellular immune process and played important roles in protecting organisms from various environmental stresses.

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Fig. 4. Temporal expression of the McHSP70 transcripts in hemocytes after Vibrio alginolyticus and Vibrio harveyi challenge as measured by Quantitative real-time RT-PCR. Vertical bars represent the mean ± S.E. (N = 5). Significant difference compared to the control expression level at the different sampling times is marked by an asterisk “*” at P b 0.05 and “**” at P b 0.01.

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Fig. 5. Expression analysis of HSP70 in hemocytes from M. coruscus. Statistical analysis of differences is done by oneway analysis of variance (ANOVA) using SPSS 13.0 software in thermal stressed (as shown in subpanel A), Cd2+ pressed (as shown in subpanel B), Cu2+ pressed (as shown in subpanel C) and 180 CST fuel contaminated groups (as shown in subpanel D). Vertical bars are mean ± SD of three technical replicates, and the asterisks above the bars represent statistically significant differences from the control samples, “*” at P b 0.05 levels and “**” at P b 0.01.

4. Discussion HSP70 is responsible for the folding or assembly of native proteins and extracellular immunoregulatory functions (Reina et al., 2012). The protective mechanism of HSP70 is the induction of stress response proteins as molecular chaperones involved in heat, heavy metals, desiccation, diseases and parasites (Tukaj and Tukaj, 2010). Up to now HSP70 gene had been studied in several marine bivalve species, such as scallop C. farreri (Wu et al., 2003), bay scallop A. irradians (Reina et al., 2012), and M. galloprovincialis (Anestis et al., 2007), and the characterization of HSP70 in M. coruscus (McHSP70) would provide further insights into the functional research of this protein family. In the present study the full-length cDNA sequence of McHSP70 was obtained from hemolymph of M. coruscus, which contained an ORF of 1965 bp coding 655 amino acid residues, and the predicted theoretical molecular weight was 71.38 kDa. Generally, the molecular weight (MW) of HSP70 family was ranged from 68 to 73 kDa (Song et al., 2006). Searching for sequence similarities revealed that the deduced amino acid sequence of McHSP70 shared high identity and similarity with other known HSP70s, especially with M. galloprovincialis and the HSP70s from bivalve species. In the phylogenetic tree M. coruscus and M. galloprovincialis were firstly gathered into the same branch, further grouped with HSP70 from P. viridis and other mollusca. The relationship

was generally in agreement with the traditional taxonomy, consistent with the occurrence of HSP70 gene duplication events during evolution. The conserved sequences and characteristic motifs of HSP70 family also appeared in the McHSP70 including the signatures 1, 2 and 3, the bipartite nuclear targeting sequence and ATP/GTP-Binding site motif A (P-loop). At the C-terminus, a notable feature in HSP70 is the presence of the consecutive repeats of the tetrapeptide motif GGMP, and the last four amino acids form a cytosolic HSP70-specific motif ‘EEVD’ (Freeman et al., 1995). The function of the GGMP repeats at the C-terminus has not been revealed, and it might form a structural entity together with the helical subdomain and the EEVD motif to mediate chaperone cofactor binding (Demand et al., 1998). The molecular chaperones in HSP70 are the large and diverse family of allosteric twodomain proteins: the NH2-terminal ATP-binding domain (ABD) and the COOH-terminal peptide-binding domain (PBD) (Smock et al., 2010; Steinert and Pickwell, 1993). The conserved interdomain linker sequence motif ‘389VLLL392’ in the sector stimulates ATPase activity when present on truncated nucleotide-binding domain constructs (Freeman et al., 1995). Therefore, the sequence alignment, structure comparison and phylogenetic analysis revealed that McHSP70 was a member of the HSP70 family. Living cells are continually challenged by external environmental conditions which cause acute and chronic stress (Santoro, 2000). The

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HSP70 family, one of the most abundant of various HSPs, has been confirmed maintaining cellular homeostasis and playing important roles in protecting organism from pathogenic stress (Encomio and Chu, 2004). In the present study, the McHSP70 expression was thus investigated after challenge by Vibrio in the hemolymph, the tissue in which immune cells are likely to manage the toxic effect of ROS to kill phagocytosed pathogens in defense mechanisms. McHSP70 transcription was upregulated after Vibrio challenge, and a clearly time-dependent expression pattern was observed, which in accordance with the previous studies in other molluscs (Wang et al., 2009). The findings suggested that McHSP70 was involved in mussel's response to pathogenic infection, and the up-regulated mRNA expression of McHSP70 following infection response indicated that the HSP70 gene was inducible and might play an important role in the immune response. HSP70 genes are typically intron-less (Encomio and Chu, 2004), the absence of introns allows HSP70 genes to be rapidly induced to avoid post-transcriptional editing of the related mRNA and to be effectively transcribed when splicing processes may be inhibited by stressful conditions. In the heat shock experiment, the transcription of McHSP70 was significantly enhanced by acute heat shock, in agreement with its classification as an inducible gene product. The rapid induction of the heat shock response by means of the inducible McHSP70 gene product represented another component of the molecular adaptation of this mussel species to transitional environments, providing an effective tool to cope with rapidly changing environmental conditions. This result was corroborated by the occurrence of analogous mechanisms in other mussel species (Encomio and Chu, 2004) and aquatic vertebrates (Wang et al., 2009) exposed to fluctuating environments. As intertidal organisms, mussels live in rapidly fluctuating habitats where protection from environmental stressors including changes in temperature, salinity, and anthropogenic contaminants, is essential for survival. At present marine waters and sediments receive anthropogenic inputs, and contain a multitude of chemical contaminants that are potentially toxic to aquatic organisms. Cu2 + and Cd2 + were the major toxic heavy metals, CST fuel was the major toxic organic hydro carbons as major components of crude oil in the marine environment (Deane and Woo, 2005), and these environmental pollutants could induce a series of HSPs to protect various living organisms (Andrew et al., 2003; Ovelgonne et al., 1995). HSP70 expression is frequently used as a component of physiological mechanisms through which bivalve mussels cope with environmental challenges. Time-dependent mRNA expression of McHSP70 was found in copper (the highest expression of 9.41-fold at 15 d), cadmium (the highest expression of 10.82-fold at 10 d) and 180 CST fuel (the highest expression of 5.94-fold at 25 d). McHSP70 mRNA levels in hemolymph kept the same tendency in all contaminants stressed, but the highest expression appeared at various times, and the rate of descent was different. The differential expression of the three groups was mainly due to a specific mechanism of shortand long-term cell protection (Franzellitti and Fabbri, 2005). Gao found that other HSPs, such as HSP90 in C. farreri, were more sensitive to the concentration variation of Cd2+ (Gao et al., 2007). HSP70, as a molecular chaperon may be related to long-term cell protection due to its expression manner under heavy metal stress. The changes in HSP70 expression of mRNA observed in the mussel exposed to heavy metals and organic hydro carbons indicated that McHSP70 could play an important role in the detoxification of these contaminants. Therefore, the results provided us an opportunity to speculate that HSP70 gene in hemolymph of M. coruscus might be a potential biomarker of metals and fuels. In summary, this was the first report of full-length cloning, characterization and inducible expression of HSP70 in hemolymph of M. coruscus after heat shock, Vibrio infection and contaminant exposure. The analysis of phylogenetic and structural features might contribute to the understanding of adaptation and evolutionary processes of HSPs in invertebrate. The upregulated mRNA expression of HSP70 in the M. coruscus following heat shock, infection response and contaminants

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