Characterization of calmodulin in the clam Anodonta woodiana: differential expressions in response to environmental Ca²⁺ and Cd²⁺

Introduction

An array of test organisms, including amphipods, polychaetes, mollusks, crustaceans and fish have been recommend for use in sediment toxicity tests [1]. While this array of test organisms covers a diverse group of fauna, many of them may not be indigenous to all geographic locations [2]. As a group of sedimentary organism, the bivalve filter feeds on suspended particulates and phytoplankton [2]. Their habitat is vulnerable to pollution from urban and industrial development. In addition, adults do not migrate, repopulation of over-fished hard-clam beds depends on the transport of larvae from other areas and several years are required for growth, maturation and reproduction [3]. Using water concentrations associated with response of organism as an indicator of sensitivity, bivalves appear more sensitive to environmental pollution than do other invertebrates [4]. Freshwater clams, Anodonta woodiana are widely distributed in the world and functions as a main criteria required for a bio-indicator organism. Earlier studies have revealed the ability of A. woodiana to accumulate trace elements and pesticides, as well as its potential to detect genotoxicity [5]. In addition, these mussels have important ecosystem functions such as particle filtration and processing, nutrient release and sediment mixing [6]. Unfortunate, metal pollution has resulted in a tremendous threat on their population that can profoundly affect food chain and aquatic ecosystem. Cadmium (Cd), a non-essential metal element required for many of living organisms, can easily enter the environment as a pollutant from many anthropogenic activities [7]. Now, Cd is one of seven most common released heavy metals for the environment (Cd, Cr, Cu, Hg, Ni, Pb and Zn) and its existence causes a serious threat on many invertebrates in aquatic environment, such as crustacean, gastropod mollusks and bivalve [8].

Great evidences have demonstrated intracellular Ca²⁺ mediates a large number of cellular responses with the high affinity and specificity required for a regulatory second messenger [9]. Calmodulin (CaM), a Ca²⁺-binding protein, is a ubiquitous, highly conserved, eukaryotic protein that binds and regulates a number of diverse target proteins involved in programmed cell death, autophagy, muscle contraction, inflammation and the immune response [9]. When bound to Ca²⁺, CaM is able to further bind to CaM-binding proteins (CaMBPs) and directly regulate activities of target proteins [9]. Through the action of these CaMBPs, CaM is involved into a great variety of cellular processes, such as protein translation, protein phosphorylation, cell cycle and cyclic nucleotide metabolism [10]. Notably, several lines of in vitro evidence indicate Cd²⁺, which has a similar ionic radius to Ca²⁺, can also bind CaM influencing these downstream effector proteins [11]. A recent in vitro study using osteoblasts derived from rat fetal, demonstrates that Cd²⁺ treatment significantly increases intracellular Ca²⁺, leads to CaM activation and ultimately apoptotic death of cells [12]. Other studies specifically implicate the CAMKII pathway as being activated by Cd²⁺ exposure resulting in apoptosis in cultured mesangial and neuronal cells [13].

Studies on Cd²⁺-induced toxicological effects have been underdone in the vertebrate, but the mechanisms underlying of observed responses are needed to further elucidate in the invertebrate. Take into consideration of great interest to investigate the effect of metals on A. woodiana, in the current study, one complete cDNA sequence and two premature termination codon mutations of CaM have been cloned and respectively named AwCaM1, AwCaM2 and AwCaM3. In addition, spatio-temporal expressions of AwCaM1 derived from Ca²⁺ and Cd²⁺ exposure were determined by quantitative real-time PCR. The present study should be helpful to elucidate the regulatory network of AwCaM1 derived from Ca²⁺ and Cd²⁺ treatment. Meanwhile, it also contribute to government paid attention to heavy metals diffusion and take measures to mitigate extensive negative impacts on freshwater organisms and conserve aquatic organism biodiversity.

Materials and methods

Results

Characterization of AwCaM1 cDNA

The complete cDNA sequence of AwCaM1 was obtained and deposited in GenBank under accession number KY996397, and comprised of 692 bp, including a 5′ untranslated region (UTR) of 21 nucleotides, a 516 bp open reading frame (ORF) which was encoded 172 amino acids, and a long 3′-UTR of 155 nucleotides containing a stop codon (TAG), a putative polyadenylation consensus signal (AATAAA), and a poly (A) tail (Figure 1). The calculated molecular mass of deduced mature AwCaM1 was 19.22 kDa and had a theoretical isoelectric point of 4.07. AwCaM1 contained four putative Ca²⁺-binding EF-hand motifs which were respectively located at 34–63 aa, 71–99 aa, 108–136 aa and 144–172 aa (Figure 1). Comparing the variance of amino acid sequences, the conserved amino acid residues were highly fund in AwCaM1 and CaM of others. Two crucial phosphorylation sites of the human CaM (Thr⁷⁹ and Ser⁸¹) were respectively identified in AwCaM1 (102 aa and 104 aa) (Figure 2). Notably, alignment result showed only three residues of Arg⁷⁵ and Ile⁸⁶ and Tyr¹⁰⁰ in human were respectively substituted by Lys, Leu and Phe in AwCaM1 (Figure 2).

Figure 1:

Nucleotide and deduced amino acid sequences of AwCaM1.

The start and stop codons are indicated with bold. Putative polyadenylation signal “AATAAA” is showed with wavy line. The four Ca²⁺-binding domains are marked with box.

Figure 2:

Multiple alignment of AwCaM1 with other CaMs.

Two crucial phosphorylation sites of Thr and Ser are indicated with shadow. The four Ca²⁺-binding domains are marked with box.

Furthermore, predicted secondary structure of AwCaM1 protein contained seven alpha-helices and eight β-sheets (Figure 3A) that was very similar to CaM secondary structure of other species, especially to that of human. 3D structure of AwCaM1 showed a high-degree similarity with CaMs (Figure 3B).

Figure 3:

Predicted secondary and 3D structures of AwCaM1 deduced amino acids.

(A) The secondary structure of AwCaM1. (B) The 3D structure of AwCaM1.

Evolutionary relationship of AwCaM1

BLAST analysis revealed that amino acid sequence of AwCaM1 was close with other CaMs. The overall deduced amino acid sequence of AwCaM1 exhibited 96% to freshwater bivalve Pinctada fucata, 94% to insect Daphnia magna, 96% to Drosophila melanogaster and 95% with Homo sapiens. A molecular phylogenetic tree was generated to further analyze the evolutionary relationships between AwCaM1 and other CaMs sequences (Figure 4). In evidenced sequences of mollusk, AwCaM1 shared the higher relationship of evolution with clam of freshwater. Interestingly, the freshwater calms and seawater ones were not grouped in one cluster although they were belonged to same class (Figure 4). In addition, species were not orderly arranged according to evolutional relationship, such as spider Cupiennius salei, holothuroidea Apostichopus japonicas, fish Branchiostoma belcheri tsingtauense located in one cluster as well as chordata Ciona intestinalis, camel Camelus dromedaries, Homo sapiens, bird Cariama cristata distributed in one clade (Figure 4). Notably, few lower bootstrap values were observed between two classes (Figure 4).

Figure 4:

Phylogenetic relation of AwCaM1 from other organisms.

AwCaM1 is marked with underline. Crassostrea gigas (accession number XP_011436578.1), Ciona intestinalis (accession number NP_001027633.1), Cupiennius salei (accession number CFW94154.1), Drosophila melanogaster (accession number NP_523710.1), Papilio xuthus (accession number KPI95926.1), Caenorhabditis elegans (accession number NP_503386.1), Procambarus clarkii (accession number ACI15835.1), Camelus dromedarius (accession number XP_010996006.1), Cariama cristata (accession number XP_009702107.1), Pinctada fucata (accession number JAS03530.1), Litopenaeus vannamei (accession number AEK21539.1), Hyriopsis cumingii (accession number ADT61781.1), Apostichopus japonicus (accession number AAY41437.1), Bombyx mori (accession number XP_012546645.1), Homo sapiens (accession number NP_001734.1), Branchiostoma belcheri tsingtauense (accession number ABM53481.1).

Analysis of two premature termination codon mutations of AwCaM1

Two premature termination codon mutations of CaM from A. woodiana were isolated and named as AwCaM2 (GenBank accession number KY996398) and AwCaM3 (GenBank accession number KY996399), their full cDNA lengths were 610 bp and 546 bp, respectively. The AwCaM2 and AwCaM3 share similar sequence composition with AwCaM1 from 1st to 535th bp (Figure 5). In contrasted with cDNA sequence of AwCaM1, AATAA signal sequence was missed in 3′ UTR of AwCaM2, stop-code and AATAA signal sequences lacked in the AwCaM3 (Figure 5). Therefore, AwCaM2 and AwCaM3 were the individuals of premature termination codon mutations of CaM transcription.

Figure 5:

Alignment of nucleotide sequences of AwCaM1, AwCaM2 and AwCaM3.

Stop codons (TGA) are indicated with green box. Putative polyadenylation signal “AATAAA” is showed with red underline.

Tissue distribution of AwCaM1

Quantitative real-time PCR was employed to investigate the distribution of AwCaM1 in different tissues. The constitutive expression levels of AwCaM1 were examined in different tissues including foot, mantle, adductor muscle, heart, hepatopancreas, hemocytes and gill, and showed different expression profiles (Figure 6). Higher expressions of AwCaM1 were observed in mantle and gill, a moderate level in hepatopancreas, foot and heart, but a lower level in adductor muscle and hemocytes (Figure 6).

Figure 6:

Real-time PCR analysis of AwCaM1 transcript from different tissues.

n=3 Replicates.

Ca²⁺ and Cd²⁺ effect on the siphoning behavior

No statistical difference of the siphoning behavior was observed between the control group and lower concentration Ca²⁺ treated groups (0.01, 0.02, 0.04 and 0.08 mg/L) (Figure 7). However, the filtration rate at 0.16 mg/L Ca²⁺ (24.84±3.04 mL/animal/h) was significantly decreased compared with that of the control group (38.38±3.12 mL/animal/h) (p=0.0039) (Figure 7). In addition, administration different concentrations of Cd²⁺ could result in a decrease of filtration rate (Figure 7). Compared with that of control group, filtration rate of individual clam reduced more than 31.37% (p=0.0106), 56.04% (p=0.0006), 64.35% (p=0.0003) and 87.38% (p=0.0001) in the 2, 4, 8 and 16 mg/L of Cd²⁺ treated group, respectively.

Figure 7:

Filtration rate (mL/animal/h) of Anodonta woodiana exposed to Ca²⁺ and Cd²⁺ treated water.

Average (n=5) filtration rate (mL/animal/h) of clams in Ca²⁺ and Cd²⁺ treated water containing a 1 mg/L neutral red solution (0–50 mg/L) following a 72-h exposure period to the concentrations indicated. The experiments were performed in triplicate and repeated three times with similar results. The bars display the mean±SE. A statistically significant difference between treatment group and control (p<0.05) is indicated by differences in the letters above the bars. ^ap<0.05, ^bp<0.01 vs. control group at the treated group.

Temporal expressions of AwCaM1 in hepatopancreas exposured to Ca²⁺ and Cd²⁺

Temporal expressions of AwCaM1 mRNA in hepatopancreas were significantly affected by Ca²⁺ and Cd²⁺ challenge using RT-qPCR with β-actin as internal control (Figure 8). In the Ca²⁺ treated groups (0.01, 0.02, 0.04 and 0.08 mg/L), profiles of AwCaM1 expression showed a time and dose dependent pattern with respect of that of control group during experiment observed (Figure 8A). Meanwhile, in the Ca²⁺ treated groups (0.08 and 0.16 mg/L), AwCaM1 expression increased more than 56.15% (p=0.0003) from 6 h to 72 h (Figure 8A). In the Cd²⁺ treated groups (1, 2 and 4 mg/L), up-regulation of AwCaM1 expression showed a timed and dose dependent patter compared with that of control group (Figure 8B). In the Cd²⁺ treated groups (8 and 16 mg/L), AwCaM1 mRNA levels increased more 65.04% (p=0.0001) from 6 h to 72 h (Figure 8B).

Figure 8:

Temporal expressions of AwCaM1 in hepatopancreas after Ca²⁺ and Cd²⁺ challenge as measured by quantitative real-time PCR.

(A) Effect of Ca²⁺ on AwCaM1 expression in hepatopancreas. (B) Effect of Cd²⁺ on AwCaM1 expression in hepatopancreas. Bars represented means±SE; n=3/each group/each time point. ^ap<0.05, ^bp<0.01 vs. control group at the same time.

Temporal expressions of AwCaM1 in gill exposured to Ca²⁺ and Cd²⁺

In gill, treatment of different concentrations of Ca²⁺ caused a significant induction of AwCaM1 expression. Up-regulation of AwCaM1 expressions characterized with a timed and dose dependent patter was observed in the Ca²⁺ treated groups (0.01, 0.02, 0.04 and 0.08 mg/L) from 6 h to 72 h (Figure 9A). Among of all the Ca²⁺ treated groups, AwCaM1 expression increased more than 79.41% (p=0.0001) compared with that of control group (Figure 9A). Like mode of Ca²⁺ treatment, administration of Cd²⁺ could result in a significant up-regulation of AwCaM1 expression. In all the Cd²⁺ treated groups, AwCaM1 expression increased more than 88.23% (p=0.0001) in contrasted with that of control group (Figure 9B).

Figure 9:

Temporal expressions of AwCaM1 in gill after Ca²⁺ and Cd²⁺ challenge as measured by quantitative real-time PCR.

(A) Effect of Ca²⁺ on AwCaM1 expression in gill. (B) Effect of Cd²⁺ on AwCaM1 expression in gill. Bars represented means±SE; n=3/each group/each time point. ^ap<0.05, ^bp<0.01 vs. control group at the same time.

Temporal expressions of AwCaM1 in mantle exposured to Ca²⁺ and Cd²⁺

In the mantle, up-regulation of AwCaM1 expression with a time and dose dependent pattern was observed in the Ca²⁺ treated groups (0.01, 0.02, 0.04 and 0.08 mg/L) during experiment observed (Figure 10A). With Ca²⁺ treatment at a concentration of 0.16 mg/L, AwCaM1 expression increased more than 1.69 times (p=0.0001) from 6 h to 72 h compared with that of control group (Figure 10A). In the Cd²⁺ treated groups (1, 2 and 4 mg/L), up-regulation of AwCaM1 expression showed a timed and dose dependent patter. In the Cd²⁺ treated groups (8 and 16 mg/L), AwCaM1 mRNA levels increased more 1.65 times (p=0.0001) from 6 h to 72 h in contrasted with that of control group (Figure 10B).

Figure 10:

Temporal expressions of AwCaM1 in mantle after Ca²⁺ and Cd²⁺ challenge as measured by quantitative real-time PCR.

(A) Effect of Ca²⁺ on AwCaM1 expression in mantle. (B) Effect of Cd²⁺ on AwCaM1 expression in mantle. Bars represented means±SE; n=3/each group/each time point. ^ap<0.05, ^b p<0.01 vs. control group at the same time.

Discussion

CaM is a pivotal calcium metabolism regulator and involved into the shell formation process of the pearl oyster. In current study, one complete cDNA sequence of CaM gene was isolated from freshwater bivalve A. woodiana and named AwCaM1, and its expression was also examined in different tissues and different times.

The deduced primary amino acid sequence of AwCaM1 showed a high identity with the published CaMs of a range of invertebrate and vertebrate species. With respect of difference of amino acid composition, only three amino acid residues were observed between AwCaM1 and human CaM. Meanwhile, the CaM genes from different vertebrate species encode the same CaM molecule with high identical amino acid sequences. In current study, AwCaM1 was also identified with that from crustacean D. magna, and insect D. melanogaster, which indicates that the CaM proteins are highly conserved during invertebrate evolution. Notably, residue substitution, an interesting phenomenon, was detected in the present study. Compared to the marine pearl oyster P. fucata CaM, there is a single-amino acid substitution at position 147 in which Met is occupied by the Thr in freshwater bivalve Hyriopsis cumingii and Ser in A. woodiana, respectively. The Met at this position in P. fucata is considered to be a characteristic residue of the oyster [18], but it is not found in these freshwater clams. What reasons of substitution between oyster and freshwater clams are needed be further elucidate. In addition, compared with the deduced protein sequences, an important phosphorylation site Tyr100 in the Ca²⁺-binding ligand position of the third EF-hand motif of human Cam was substituted by Phe100 in AwCaM1. It has demonstrated that phosphorylation of Tyr100 in human works as important role of increase binding affinity for its target proteins [19]. The substitution of phosphorylation sites of 100 aa is likely associated with the regulation of AwCaM1 at the posttranscriptional level. On another hand, as a key binding site of EF-hand motif, event of substitution should result in a conformational change of this protein that further effect its affinity for most of its target proteins and its ability to activate target proteins. To shed light of these phenomena is a very interesting work in the future.

CaM, a multifunctional Ca²⁺ binding protein, is involved in the regulation of numerous important physiological functions, including neural activity, gene expression, enzyme regulation and muscle contraction. CaM is highly conserved across different species, and comprised of four EF hands that generally form two structurally similar domains connected by a flexible central linker. Although CaMs of these species are highly conserved and share similar structure, phylogenetic result showed that few lower bootstrap values were observed in different clusters in these CaMs located. An obvious distance of phylogenetic relationship was also detected between freshwater calms and seawater ones in spite they all belong to one class. Meanwhile, different species derived from vertebrate and invertebrate were grouped into similar cluster. Based on mentioned above, it is suggested CaM is not a candidate for reflecting the phylogenetic relationship, but an important player for sustaining normal physiological function of animals.

In all the tissues, strong expression of AwCaM1 was observed in mantle, suggested that is associated with the shell formation. The original hypotheses indicate that CaM expression should be greater in epithelial tissues (such as the gill and mantle) and lesser in non-epithelial tissues (such as muscle) [20]. In mollusks, the mantle, especially the outer epithelium of the mantle, plays a key role in shell formation [21]. In current study, the AwCaM1 mRNA expression level was obvious higher in the mantle compared to the adductor muscle. Similar results are also reported from other animals. In addition, the higher expression level of AwCaM1 mRNA was observed in the gill that supports previous conclusion in which gill is considered as a key organ of calcium uptake and accumulation in the bivalve. It has demonstrated that the gill is a primary organ for calcium uptake from the water in bivalves. Particularly in the scallop, the gill is thought to play a regulatory role in the membrane Ca²⁺-ATPase system or to act as a “calcium sink” [18].

In the current study, AwCaM2 and AwCaM3, two premature termination codon mutations of CaMs, were also isolated from A. woodiana, suggesting there is a complex mechanism involved into controlling AwCaM1 transcription. As to phenomena, studies of loss-of-function mutations pave an avenue to elucidate mechanism, it has gain great attention. Loss-of-function mutations are generally believed to be deleterious and have been discussed in the context of human medicine [22]. Now, genome wide surveys in human and fly populations reveal an unexpected prevalence of loss-of-function mutations with hundreds or thousands of genes harboring deletions and/or premature termination codon mutations [23]. These genes are narrowly transcribed and encode high numbers of paralogs. But, gene expression in eukaryotes is a compartmentalized process consisting of several different, yet connected steps. In the process, importance of gene expression for living cells and organisms is exemplified by the existence of diverse molecular mechanisms that detect errors and thereby ensure the accuracy of gene expression. A well-known quality control process, referred to as nonsense-mediated mRNA decay or alternatively mRNA surveillance, limits the expression of mRNAs with premature termination codons and other aberrant termination events. So, in order to tolerate existence of these genes, a sophisticated mechanism should be involved in the regulated process of transcription [22, 23].

Filtration rates of Cd²⁺ treated groups were significant reduced compared with that of control group, suggesting that treatment of Cd²⁺ could result in an obvious injury on the health clams. Combination of siphoning behavior and valve movement has been utilized as indicators for continuous biomonitoring of water supplies and effluents [24]. Bivalve siphons play important roles in nutritional physiology, defense, and reproduction and are therefore a signal of general healthy or stress. Previous studies have demonstrated that bivalve siphoning can be influenced by metals or chlorpyrifos [17, 25]. Valves of the clams are always closed during exposure to 3.13 mg/L chlorpyrifos treatments [26]. In addition, a reduction of siphoning activity as a response to chemical stress has also been observed that is associated with ammonia accumulation in the tissue, a reduction in oxygen exchange, and reduced feeding which could all have significant implications for survival, growth and reproduction of bivalves [25, 27]. Herein, we found that Cd²⁺ exposure decreased the filtration rates of clams, although no significant valve closure was observed. It is therefore reasonable to conclude that the decreased siphoning caused by Cd²⁺ exposure reflects an overall negative health impact and an indicator of chemical stress.

In the present study, treatment of different Ca²⁺ concentrations could result in a significant up-regulation of AwCaM1 expression in the hepatopancreas, gill and mantle, suggested that stimulating AwCaM1 transcription might help A. woodiana to maintain cytosolic Ca²⁺ homeostasis and gain compensatory Ca²⁺ absorption as well as adapt to environmental stress. CaM plays a pivotal role in cellular Ca²⁺ homeostasis by activating Ca²⁺-ATPase, which is the major transporter in Ca²⁺ fluxes [15, 28]. Up-regulation of AwCaM1 is contributed to increase Ca²⁺ absorption of cells. On another hand, in these tissues, expression of AwCaM1 was down-regulated at 72 h compared with that of 48 h in the Ca²⁺ treated group at a concentration of 0.16 mg/L. Significant up-regulation of AwCaM1 expression is also associated with coping environmental stress derived from Ca²⁺ treatment, especially in the higher concentration. Expression of CaMs of animals could be induced by environmental stressors, such as hypo-osmotic stress and pathogenic organism [21, 29]. Evidence has indicated that CaM is considered as one of the most important molecular biomarkers in teleost exposed to the chronic stressors [30]. Previous studies demonstrated that the cold adaptation in Dissostichus mawsoni is highly related to CaM expression [31]. Given that CaM is an important subunit for several ion channels, the significant up-regulation of AwCaM1 expression likely function as dual role in the different concentrations of Ca²⁺ treatment.

In the hepatopancreas, gill and mantle, up-regulations of AwCaM1 with a time and dose dependent pattern were occurred in the Ca²⁺ treated groups at concentrations from 0.01 to 0.08 mg/L. In addition, filtration rates of Ca²⁺ treated groups showed a gradually up-regulation trend with respect of increase of treated concentrations between 0.01 and 0.04 mg/L. Combination of characterizations of AwCaM1 expression and filtration rate, we postulated, under 0.04 mg/L, administration of Ca²⁺ is contribute to increase absorption of Ca²⁺ and further maintain cytosolic Ca²⁺ homeostasis as well as boost shell formation of calms.

In the current study, treatment of Cd²⁺ at different concentrations could result in a significant up-regulation of AwCaM1 expression in the hepatopancreas, gills and mantile, suggested stimulation of AwCaM1 transcription is associated with Cd²⁺ toxicity. Due to their filtration activity, bivalves are exposed to numerous heavy metals and thus are subject to a considerable amount of oxidative damage [32]. It has found Cd²⁺ can induced oxidative DNA lesions in bivalves of freshwater and sea water. In the Mytilus edulis, low concentrations of Cd²⁺ enhance the genotoxicity of H₂O₂ and Cd²⁺ inhibits the DNA repair of 8-oxodG [33]. Further study shows that Cd²⁺ induced an increase of the DNA strand break levels and a low level of 8-hydroxy guanine DNA glycosidase-sensitive sites [34]. Undoubtedly, existence of Cd²⁺ can cause tremendous threat on cell survival. Notably, what is mechanism of Cd²⁺ entering into cells? Studies have indicated treatment of Cd²⁺ can cause break of Ca²⁺ homeostasis, and result in apoptosis of a variety of cells [35]. In the process, some Ca²⁺ related proteins are significantly up-regulated after Cd²⁺ exposure, such as neuronal calcium sensor, sarcoplasmic calcium-binding protein and CaM [35]. In addition, it was assumed that Ca²⁺ allow to be displaced by Cd²⁺ in some Ca²⁺-binding proteins with respect of the biophysicochemical similarities of Cd²⁺ and Ca²⁺ that disrupt Ca-mediated signaling pathways. Notably, research indicates Cd²⁺ exposure can lead to a transient rise in intracellular Ca²⁺ ions in several cell types, especially in osteoblasts [12]. Osteoblasts can uptake Cd²⁺ ions via membrane-bound transporters, such as transient receptor potential channels [36]. Consequently, it is possible that a combination of increased intracellular Cd²⁺ and Ca²⁺ ions can bind CaM leading to activation or inhibition of Ca²⁺/calmodulin dependent kinase (CAMK) pathways. Other studies specifically implicate the CAMKII pathway as being activated by Cd²⁺ exposure that result in apoptosis in cultured mesangial and neuronal cells [37]. However, the roles of the other two pathways, CaM-dependent PDE and CAMKK, in Cd²⁺ toxicity are under-investigated. Taken together, these studies provide evidence in support of the current research. Therefore, up-regulation of AwCaM1 is related with accumulation of Cd²⁺ that should lead to a risk of cell damage.

Notably, the AwCaM1 expressions of hepatopancreas, gill and mantle showed a dose- and time-dependent manner in lower concentration of Cd²⁺ treated groups. However, AwCaM1 expression showed a biphasic profile in the 8 and 16 mg/L of Cd²⁺ treated groups characterized by up-regulation matter in the early stage and down-regulation trend in the latter stage. This pattern of AwCaM1 is associated with persistent accumulation of reactive oxygen species (ROS) derived from Cd²⁺ injury. With elongation of Cd²⁺ treated time, ROS should increasingly be produced and accumulated in the cells. Levels of abruptly increased ROS is exceed the eliminating ability of cells that cause redox imbalance possibly via a shift between oxidants and antioxidants in favor of oxidants, and result in chronic inflammation, apoptosis of immune cells, etc. [38]. In the condition, if animals cannot produce new cells to compensate for death ones, number of living cells should be gradually decreased. Following, expressions of AwCaM1 are also decline alone treated time. Thus, a biphasic phenomenon was detected in higher concentration groups of Cd²⁺.

In conclusion, one complete cDNA sequence and two premature termination codon mutations of CaMs (AwCaM1, AwCaM2 and AwCaM3) were cloned and characterized from the freshwater mussel A. woodiana. With respect of complex process from binding CaM to activation, effect of Ca²⁺ and Cd²⁺ on the freshwater bivalve A. woodiana is an sophisticated event. Use of AwCaM1 expression is contributed to provide insight into the important landscape of change at the molecular level derived from Ca²⁺ and Cd²⁺ treatment. This work is helpful to elucidate the potential mechanism of Cd²⁺ toxicity and provide a key regulator of Cd²⁺ toxicity in freshwater clam A. woodiana. The continuous functional investigation of proteins of network into Ca²⁺ involved could eventually solve the key targets of Cd²⁺ toxicity. Meanwhile, government should concern heavy metals diffusion and take measures to mitigate extensive negative impacts on freshwater organisms and conserve aquatic organism biodiversity. Notably, the present work is only the laboratory results. So, great efforts are required to explore the application of A. woodiana in the environmental engineering and environmental monitoring.