Aquatic Toxicology 148 (2014) 55–64

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Molecular cloning of manganese superoxide dismutase gene in the cladoceran Daphnia magna: Effects of microcystin, nitrite, and cadmium on gene expression profiles Kai Lyu a , Xuexia Zhu a , Rui Chen a , Yafen Chen b , Zhou Yang a,∗ a Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China b State Key Laboratory for Lake Science and Environment, Nanjing Institute of Geography and Limnology, the Chinese Academy of Sciences, Nanjing 210008, China

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Article history: Received 23 October 2013 Received in revised form 26 December 2013 Accepted 29 December 2013 Keywords: Cladoceran Antioxidation Oxidative stress Enzyme activity Biomarker

a b s t r a c t Superoxide dismutases (SODs) are metalloenzymes that represent one important line of defense against oxidative stress produced by reactive oxygen species in aerobic organisms. Generally, waterborne pollutants caused by irregular anthropogenic activities often result in oxidative damage in aquatic organisms. The aim of this study was to molecularly characterize the manganese superoxide dismutase gene (DmMnSOD) in the waterflea, Daphnia magna, and evaluate the mRNA expression patterns quantified by real-time PCR after exposure to three common waterborne pollutants (microcystin-LR, nitrite, and cadmium). The results showed that the full-length Dm-MnSOD sequence consists of 954 bp nucleotides, encoding 215 amino acids, showing well-conserved domains that are required for metal binding and several common characteristics, such as two MnSOD domains. The deduced amino acid sequence of Dm-MnSOD shared over 70% similarity with homologues from Bythograea thermydron, Dromia personata, Cancer pagurus, and Scylla paramamosain. Dm-MnSOD gene expression was up-regulated in response to exposure to the three chemicals tested. The overall results indicated that Dm-MnSOD gene is an inducible gene and potential biomarker indicating these pollutants in the environment. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Reactive oxygen species (ROS) generally occur as unwanted side products of aerobic metabolism for production of energy in cells (Sharma, 2013). Normally, living organisms maintain steady-state ROS level through a balance between production and elimination. Besides performing an important role in signal transduction (Ray et al., 2012), the production of ROS is one of the earliest cellular responses in living organisms under stress (Lushchak, 2011). Excessive ROS may lead to disturbance of the redox status causing oxidative stress. Consequently, oxidative damage changes cell function leading to cell apoptosis, increased sensitivity to pathogens, and reduced reproduction potentials (Lushchak, 2011). In order to balance the harmful and positive effects of ROS, aerobic organisms have developed various antioxidant defense mechanisms (Becker et al., 2011; Valavanidis et al., 2006). Superoxide dismutases (SODs; EC 1.15.1.1) are considered to be the prime antioxidant enzymes by catalyzing the dismutation reaction of ROS into H2 O2 and O2 , which is subsequently transformed into

∗ Corresponding author. Tel.: +86 25 85891671; fax: +86 25 85891526. E-mail address: [email protected] (Z. Yang). 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.12.031

H2 O by catalase (den Hartog et al., 2003; Kim et al., 2010b; Mruk et al., 2002). Importantly, the SODs were also revealed to have an outstanding function in immune response induced by bacteria (Umasuthan et al., 2012), virus infection (Zhang et al., 2007), toxic chemical exposure (Kim et al., 2011; Lyu et al., 2013b) and thermal stress (Choi et al., 2006). Eukaryotes possess two major kinds of SOD, CuZnSOD, which is present mostly in the cytosol and nucleus, and MnSOD, which is present in mitochondria (Wang et al., 2010). Great importance of the SOD gene which is very highly conserved has been well confirmed in model species such as Danio rerio, Drosophila melanogaster and Caenorhabditis elegans (Duttaroy et al., 1994; Hunter et al., 1997; Malek et al., 2004). Even though some drawbacks exist, such as the need to recognize that changes in expression of mRNA do not necessarily translate in changes of expression of the gene product, it is currently recognized that gene expression holds great potential for ecotoxicity testing and environmental risk assessment, especially as it is frequently presumed to be more sensitive than traditional ecotoxicity testing where effects are assessed on life-cycle end points (Marinkovic´ et al., 2012). Although SOD has been extensively employed as an ecotoxicity biomarker in various aquatic species based on its enzyme activity (Arzate-Cárdenas and Martínez-Jerónimo, 2011; Semedo et al., 2012; Vega and Pizarro, 2000), the molecular characterization

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of the SOD gene across other model taxa remains unexplored. Again, gene expression has advantages over traditional enzymatic assays, as not only the large amount of samples required for enzymatic assays (Arzate-Cárdenas and Martínez-Jerónimo, 2011; Tang et al., 2011), but also RNA preservation in the field (i.e. using specific buffers) can be easier than for proteins (Kasahara et al., 2006). Daphnia, a genus of common lake-dwelling crustaceans, plays a pivotal role in aquatic food webs and shows an outstanding performance in aquatic ecotoxicity assessment, which has been the focus of study by limnologists for well over a century (reviewed by Sarma and Nandini, 2006). Hence, characterization of SOD genes in important ecotoxicity model species, such as Daphnia magna, will promote future studies to understand oxidative stress responses in aquatic invertebrates caused by exposure to various waterborne pollutants. More importantly, it will also heighten the potential use of this species in sensitive, gene-expression-based ecotoxicity monitoring. In this study, we tested three environmentally common toxicants, microcystin-LR (MC-LR), nitrite, and cadmium (Cd), which have different toxic modes in inducing oxidative stress. Cyanobacterial blooms occur worldwide in eutrophic lakes and reservoirs and are likely to increase in prevalence and magnitude in the future, especially with climate change (Paerl and Huisman, 2008). Some cyanobacterial taxa produce noxious secondary metabolites, such as MC-LR, a commonly studied class of cyclic heptapeptide hepatotoxins (White et al., 2011; Wilson et al., 2006), which can induce oxidative stress and inhibit protein phosphatases (Amado and Monserrat, 2010; Mikhailov et al., 2003; Ortiz-Rodríguez and Wiegand, 2010). Recent studies have revealed that microcystin levels can be over 15 ␮g L−1 in localized regions of eutrophic lakes: e.g. this has been observed in Lake Taihu (Zhang et al., 2010; Zhang et al., 2008). Furthermore, in a few cases, levels can reach above 1800 ␮g L−1 , immediately after the collapse of a highly toxic bloom (Jones and Orr, 1994; Svrcek and Smith, 2004). Nitrite is a natural component of the nitrogen cycle in ecosystems, and its presence at high concentrations in the environment is a potential problem for animal and ecosystem health. The concentration of nitrite is low in unpolluted waters (typically well below 50 ␮g L−1 ), but it can reach as high as 46 mg L−1 or more due to eutrophication (Jensen, 2003; Kamstra et al., 1996). High concentrations of nitrate may cause oxidative stress on aquatic animals by causing lipid peroxidation and protein denaturation (Jensen, 2003). Additionally, irregular anthropogenic activities may further promote heavy metal pollution, which can cause oxidative damage in aquatic organisms. Cadmium (Cd), one of the most toxic heavy metals, is especially pronounced in some freshwater systems (Bandara et al., 2008; Satarug et al., 2003). Cd concentrations in the river Scheldt located in Belgium have been observed as high as 20 ␮g L−1 (Bervoets and Blust, 2003). Worse, Cd in localized regions of the Guadiamar river basin can reach 286 ␮g L−1 (Alonso et al., 2004). Cd has been shown to negatively impact growth and cellular energy allocation in Daphnia, and recent work suggests that Cd may affect molecular pathways, including inducing oxidative response (Connon et al., 2008). In the present study, we used a partial sequence of the wellconserved Mn-SOD gene present across other taxa to design a universal primer pair. Subsequently, the full-length MnSOD cDNA from D. magna (Dm-MnSOD) was cloned and sequenced. We then characterized the molecular structural levels of Dm-MnSOD. Finally, we elucidated the mRNA expression patterns with total SOD activity under the exposure to three oxidative stressors (MC-LR, nitrite, and cadmium) commonly found in aquatic environment. This is the first report of gene information about MnSOD and its role after exposure to various waterborne pollutants in branchiopod crustaceans.

2. Materials and methods 2.1. Animal maintenance D. magna strain was provided by the State Key Laboratory of Pollution Control and Resource Reuse, Nanjing University (Nanjing, China); this strain has been maintained successfully under laboratory conditions for over 10 years in 3 L beakers filled with M4 medium (Elendt and Bias, 1990) and fed with Scenedesmus obliquus (algal density: 5 × 105 cells mL−1 ) at 25 ◦ C under fluorescent light at 40 ␮mol photons m−2 s−1 with a light-dark period of 12:12 h (Yang et al., 2012). Media was gently aerated with filtered air for 24 h before use and renewed totally twice weekly.

2.2. Exposure to stress and sampling To investigate the expressions of MnSOD in D. magna at different ages, organisms aged 1, 4, 6, and 14-days were selected that corresponded with newly-born neonate (1-day), juveniles (4days), maturing adults (6-days), and reproductive adults (14-days). To investigate the expressions of MnSOD under 48-h exposure to MC-LR, nitrite and Cd, D. magna juvenile organisms were placed randomly in 500-mL beakers. MC-LR, nitrite and Cd test solutions were prepared by dissolving purified MC-LR (Express, Beijing, China), sodium nitrite (NaNO2 ), and CdCl2 ·2.5H2 O, respectively. The concentrations of test solutions in the beakers were set as: 0, 10, 50, and 100 ␮g L−1 for MC-LR; 0, 10, 20, 30 mg L−1 for nitrite; and 0, 2, 10 and 50 ␮g L−1 , for Cd. All treatment concentrations were set up based on environmental relevance reported by previous studies (Jones and Orr, 1994; Kim et al., 2010b; Xiang et al., 2010; Yang et al., 2011). The bioassays were performed in quadruplicate for each treatment and 30 animals were placed into each 500 ml beaker replicate of each treatment. All experiments were conducted under the same conditions used for D. magna maintenance.

2.3. RNA isolation and cloning of MnSOD cDNA We collected the experimental D. magna individuals and homogenized them using a pestle prior to extract total RNA using the Trizol technique following manufacturer instructions (Takara, Japan). The total RNA concentrations were determined by measuring the absorbance at OD260 . RNA integrity was checked by electrophoresis. Total RNA was reverse-transcribed to cDNA with oligo-dT primers and a cDNA Synthesis Kit (Takara, Japan), according to manufacturer instructions. Next, we used Mn0623-F1 and Mn0623-R1 (Table 1 in Supplementary Data, SD) as the PCR primers to amplify SOD coding sequences. The primers set was designed against the conserved regions of the corresponding genes in Eriocheir sinensis (GenBank accession number: JX101466.1), Cherax quadricarinatus (JQ763321.1), and Procambarus clarkia (KC333178.1). PCR was carried out using 1.5 mM MgCl2 , 0.2 mM dNTP, 0.2 mM of each of primers, 1 U Taq DNA polymerase (Takara, Japan) and 5 ng of cDNA. The amplification program consisted of initial denaturing for 5 min at 94 ◦ C followed by 35 cycles of 94 ◦ C for 30 s, 45 ◦ C for 40 s, 72 ◦ C for 40 s and a final elongation step at 72 ◦ C for 5 min. PCR amplicons were size separated and visualized on an ethidium bromide stained 1% agarose gel. Amplicons of expected sizes were purified with an Agarose Gel DNA Purification Kit (Takara, Japan), and then sub-cloned into the pMD-19 T cloning vector (Takara, Japan). Positive clones containing inserts of an expected size were sequenced using M13 primers, and sequenced at BGI (Shanghai), China.

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2.4. Rapid amplification of cDNA ends (RACE) The D. magna MnSOD partial cDNA sequence was extended using 5 and 3 RACE (SMARTTM cDNA kit, Life Technologies). The 3 RACE PCR reaction was carried out in a total volume of 50 ␮L containing 2.5 ␮L (800 ng ␮L−1 ) of the first-strand cDNA reaction as a template, 5 ␮L of 10 × Advantage 2 PCR buffer, 1 ␮L of 10 mM dNTPs, 5 ␮L (10 mM) gene-specific primer (3 -Mn-RA-1 and 3 -Mn-RA-2; Table 1 in SD), 1 mL of Universal Primer A Mix (UPM; Clonetech, USA), 34.5 ␮L of sterile deionized water, and 1 U 50 × Advantage 2 polymerase mix (Clonetech, USA). For the 5 RACE, UPM was used as forward primers in PCR reactions in conjunction with the reverse gene-specific primers (5 -Mn-RA-1, 5 -Mn-RA2 and 5 -Mn-RA-3; Table 1 in SD). All RACE PCR was performed based on manufacturer instructions. After linking into the vector, the samples were sequenced at BGI (Shanghai), China. 2.5. Sequence analysis of MnSOD The cDNA sequence and deduced amino acid sequence of MnSOD were analysed using the BLAST algorithm (http://www. ncbi.nlm.nih.gov/blast). Translation and protein analyses were performed using ExPASy tools (http://us.expasy.org/ tools/). The ClustalW Multiple Alignment program (http://www.ebi.ac.uk/clustalw/) was used to create the multiple sequence alignment. The predicted molecular weight was calculated using online tool (http://expasy.org/cgi-bin/pi tool). To predict in silico the tertiary structure of mature Dm-MnSOD, a 2.4 Å crystal structure of Homo MnSOD dimer from PDB database (ID, 2adqB) was selected as template by Swiss-Model (http://swissmodel.expasy.org/). The monomer structure of Dm-MnSOD was constructed by the I-TASSER server through multiple-threading alignments with potential templates. The structural models were visualized with DeepView and RasMol programs. An unrooted phylogenetic tree was constructed from different species (Table 2 in SD), based on genetic distance (Poisson model) of amino sequences by the neighbor-joining (NJ) algorithm embedded in MEGA 5.0 program (http://www.megasoftware.net/). The reliability of the branching was tested by bootstrap resampling (1000 pseudoreplicates). 2.6. Quantitative PCR Total RNA of exposed individuals was isolated as mentioned above, and 5 ␮g of total RNA was used to synthesize the first strand cDNA. For quantification of the Dm-MnSOD mRNA expression, a pair of gene specific primers (Mn-Q-F and Mn-Q-R; Table 1 in SD) was used, and the primers ␤-actin-F and ␤-actin-R (Table 1 in SD) were used to amplify ␤-actin as an internal control. The amplification efficiencies of two primers are between 95% and 105%, examined by using the Ct slope method. The thermal conditions were: one cycle of 95 ◦ C for 10 s, followed by 40 cycles of 95 ◦ C for 5 s, 54 ◦ C for 10 s and 72 ◦ C for 20 s. Dissociation curve analysis of amplification products was performed at the end of reaction to confirm that a specific single PCR product was amplified and detected. The differential gene expression was calculated by the 2-CT method (Livak and Schmittgen, 2001). Previously, the housekeeping gene (internal control) ␤-actin was measured with the same cDNA sample. Validation process followed by Kim et al. (2010b) confirmed that the efficiencies of the target and endogenous control (␤-actin) amplifications were approximately equal.

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microcentrifuge tube and immediately frozen at −70 ◦ C until further biochemical analysis. Individuals were homogenized on ice in 500 ␮L extraction buffer (50 mM phosphate buffer, pH 7.4) containing 100 mM KCl and 1 mM EDTA. Samples were kept on ice during the entire procedure. Homogenates were centrifuged at 13,000g for 15 min and the supernatants were used for measurement of TSEA. Protein concentrations were estimated using the Diagnostic Reagent Kit (Coomassie protein assay dye); TSEA was determined using the Diagnostic Reagent Kits purchased from Nanjing Jiancheng Bioengineering Institute (China). 2.8. Statistical analyses All biochemical data were expressed as mean ±1 standard error (SE). Significant differences were evaluated by one-way analysis of variance (ANOVA) followed by the Duncan multiple range test (˛ = 0.05). All tests were run with the SigmaPlot 11.0 software package. 3. Results and discussion 3.1. cDNA and amino acid sequences of Dm-MnSOD The full-length cDNA of MnSOD from D. magna (Genbank accession no. KF005233) and its deduced amino acid (AA) sequence are shown in Fig. 1. The Dm-MnSOD cDNA comprised 954 bp with a 648 bp CDS (including a stop codon, TAA), flanked by a 159 bp 5 -untranslated region (UTR) and a 147 bp 3 -UTR containing a canonical polyadenylation signal sequence (843 AATAAA848 ) 82 bp upstream of poly (a) tail. The open reading frame (ORF) of DmMnSOD encoded a putative polypeptide of 215 residues with a calculated molecular mass of 24,565.25 Da and a theoretical pI of 7.99. Profile of the Dm-MnSOD deduced protein illustrated the following dominating features in its primary structure: I. a mitochondrial targeting sequence (MTS) of 18 residues at N-terminus (Fig. 2); II. three potential N-glycosylation sites (57 NQTE60 , 91 NHSI94 and 146 NKTT149 ) (Fig. 1); III. two MnSOD domains (MSD) or signatures (84 FNGGGHLNHSIFW96 and 176 DVWEHAYY183 ) (Fig. 2); and IV. potential metal-binding sites (Fig. 2) for Mn2+ (His44 , His92 , Asp176 and His180 ) which mediate its catalytic activity. Prediction analysis of the identified protein revealed the presence of a 26 amino acid long mitochondrial targeting sequence (MTS) at N-terminal suggesting its localization to the mitochondria, whereas Cu/ZnSOD of D. magna was characterized and had a 21-AA signal peptide, indicating the Cu/ZnSOD was transported into extracellular (Lyu et al., 2013b). Generally, MnSOD is synthesized as a precursor protein in the cytosol and its energy-dependent translocation into mitochondrial matrix is mediated by MTS (Umasuthan et al., 2012). Then, the premature MnSOD is proteolytically cleaved within mitochondria to form a mature protein (Karnati et al., 2013; Wang et al., 2013). Despite that MnSODs are the chief antioxidant enzymes in mitochondrial matrix of aerobic eukaryotes, evidence of cytosolic MnSOD isoforms without MTS (cytosolic forms) have been reported in several species, which suggested their significance as antioxidant enzymes in the entire cellular compartment (Brouwer et al., 2003; Cheng et al., 2006; Gómez-Anduro et al., 2006). In addition, the presence of N-glycosylation sites suggested that similar to its orthologs, Dm-MnSOD also might be a glycoprotein (Cheng et al., 2006). 3.2. Dm-MnSOD protein homology and alignments

2.7. Measurement of total SOD enzymatic activity (TSEA) To determine the responses in TSEA of D. magna, 15 individuals were sampled from each replicate beaker, pooled in a

Multiple sequence alignment showed that the deduced AA sequence of Dm-MnSOD shares high similarity with other MnSOD AA sequences from Bythograea thermydron, Dromia personata,

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Fig. 1. The nucleotide sequence and deduced amino acid sequence of Dm-MnSOD. Nucleotides are numbered from the first base at 5 end. Amino acids are numbered from the initiating methionine start codon. The MTS is shown in blue highlights. The MnSOD domains (84 FNGGGHLNHSIFW96 and 176 DVWEHAYY183 ) are shown in single linear underlining. The potential metal-binding sites for Mn2+ (His44 , His92 , Asp176 and His180 ) are boxed. The secondary structure of Dm-MnSOD consists of 10 ␣ helixes and 5 ␤ strands and is boxed in pink and green, respectively. Three potential N-glycosylation sites (57 NQTE60 , 91 NHSI94 and 146 NKTT149 ) were shown in bold letters. The asterisk (*) indicates the stop codon.

Cancer pagurus, Scylla paramamosain, Procambarus clarkia, and Cherax quadricarinatus (Fig. 3; Table 2 in SD). Specifically, DmMnSOD displays over 70% similarity with homologues from B. thermydron, D. personata, C. pagurus, S. paramamosain. There is 100% conserved regions in Dm-MnSOD (Fig. 3), including the Mn family signature motifs, invariant AAs responsible for the coordination of Mn. Furthermore, since no significant sequence conservation was found in MTS region of different lineages (Fig. 3), it has been suggested that these divergent sequences of MTS in different species

may retain the same functional attributes through specific motif(s) (Kong et al., 2003). 3.3. Secondary and tertiary structures of Dm-MnSOD The Dm-MnSOD had 10 ␣ helixes and 5 ␤ strands (Fig. 1). Based on human MnSOD protein 3D templates, which shared 65.63% of identity with Dm-MnSOD, the potential tertiary structures of DmMnSOD was constructed (Fig. 4). The MnSOD enzyme exists as

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Fig. 2. The domain architecture of Dm-MnSOD. The Dm-MnSOD is composed of a MTS of 18 residues (yellow), two Sod Fe domains (green) containing of three MnSOD domains (red) and metal-binding sites of His44 , His92 , Asp176 and His18 .

either homodimer in bacteria as well as homotetramer in human (Borgstahl et al., 1992; Parker and Blake, 1988). In this study, the homodimeric structure of Dm-MnSOD was established by automated platform in Swiss-Model using human MnSOD (PDB ID, 2adqB) template and monomeric model was custom built by ITASSER server. The homology model of Dm-MnSOD revealed its distinctiveness in structural identity, 3D folding and evolutionary conservation of the active site Mn2+ (His44 , His92 , Asp176 and His180 ) as shown in Fig. 4C. Furthermore, the metal-binding site of the MnSOD is located at the junction of ␣-helical (hairpin) domain at N-terminus and ␣/␤ domain at C-terminus (Fig. 4B and C). Geometry of the Dm-MnSOD metal-binding site showed that not only

Mn2+ binding residues of 3 His and 1 Asp, but also the second shell residues consisting of His48 , Tyr52 ,Trp140 , Gln160 and Trp178 which is essential for stabilizing the active site topology (Porta et al., 2010; Umasuthan et al., 2013), were also conserved (Fig. 4D). 3.4. Phylogenetic analysis of Dm-MnSOD The phylogenetic analysis (neighbor-joining algorithm) indicated that MnSODs can be classified into two groups corresponding to mitochondrial MnSOD (mMnSOD) and cytosolic (cMnSOD) (Fig. 5). All the mMnSODs were clustered together as one subgroup and all cMnSODs as another group (Fig. 5), consistent with the belief

Fig. 3. Multiple alignment of mMnSOD amino acid sequences from Bythograea thermydron, Dromia personata, Cancer pagurus, Scylla paramamosain, Procambarus clarkii, Cherax quadricarinatus and Daphnia magna. Identical (*) and similar (. or:) amino acid residues are indicated. Gaps (−) are introduced to maximize the alignment. MTS is boxed in blue. The potential metal-binding sites for Mn2+ (His44 , His92 , Asp176 and His180 ) are shown in a black rectangle box. The two conserved MnSOD signatures are shown with single linear underlining in orange.

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Fig. 4. Molecular modeling of the Dm-MnSOD tertiary structure. (A) Homodimeric structure of Dm-MnSOD, based on human MnSOD (PDB ID, 2adqB); ␣-helices are in red, and ␤-strands are in yellow. (B) Ribbon diagram of an individual monomer; N- and C-termini and active site residues are marked. (C) The location of the active site in a monomer. (D) Closer view of the active site; Mn ion ligand, Mn ion-binding residues, His44 , His92 , Asp176 and His180 , second shell residues, such as His48 , Tyr52 , Trp140 , Gln160 and Trp178 are marked with corresponding color of residues.

Fig. 5. Neighbor-joining phylogenetic tree of MnSODs amino acid sequences across multiple taxa, including arthropods, nematodes, mollusks, and vertebrates.

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Fig. 6. Real-time PCR analysis of Dm-MnSOD (A) mRNA expression and (B) TSEA in Daphnia magna during different developmental stages (day 1, 4, 6, and 14). All values represent the mean ± SE (n = 3). Different letters denote significant difference using Duncan multiple range test (P < 0.05).

of common origin for these two SODs with different subcellular compartmentalization (Zelko et al., 2002). Dm-MnSOD was positioned within the mitochondrial branch of the arthropoda cluster, but it was not the sister group of mMnSOD of decapod crustaceans which was sister group of Insecta. This result was in line with the paraphyly hypothesis of Crustacea related to Insecta (Cook et al., 2005; García-Machado et al., 1999; Legg et al., 2013). 3.5. Changes in MnSOD mRNA expression and TSEA of D. magna To better understand the possible age-dependent effects of Dm-MnSOD and SOD enzymatic activity across four age classes (day 1, 4, 6, and 14) of D. magna, its mRNA expression and TSEA were detected, respectively (Fig. 6). The mRNA expression levels of Dm-MnSOD and TSEA in the 6-day-old and 14-day-old were significantly lower than the 1-day-old and 4-day-old (P < 0.05), with most exhibiting higher expression levels in the 1-day-old individual (neonate). Changes in D. magna physiology during its developmental and aging periods would therefore result in age related differences in its antioxidant status as was seen for the DmMnSOD mRNA expression levels and for TSEA. ROS are continuously produced as by-products of normal oxidative metabolism, principally from mitochondrial respiration (Cadenas and Davies, 2000). Since oxygen demand is usually higher in neonate and juvenile D. magna than adults (Bohrer and Lampert, 1988; Glazier, 1991), the former are likely to require higher SOD to protect against potential increased production of ROS, as evidenced in our study. Similarly, SOD activity was highest in neonate D. schodler and decreased as animals aged to 28 days (Arzate-Cárdenas and Martínez-Jerónimo, 2011). In general, consistent response patterns of the Dm-MnSOD gene and SOD enzymatic activity occurred in different developmental D. magna, i.e. both of the transcription and translation levels reduced along with ages. To identify the potential of Dm-MnSOD gene as a biomarker of waterborne pollutants, its mRNA expression was first assessed under exposure to common waterborne chemicals, including MC-LR, nitrite and Cd, which are well-known oxidative stress inducers. Also, TSEA, which has already been used as an appropriate biomarker tool in ecotoxicological assessments (Choi et al., 1999; Valavanidis et al., 2006), was conducted to test whether changes in Dm-MnSOD gene expression were consistent with changes in TSEA. An increase of Dm-MnSOD mRNA expression and TSEA levels occurred after exposure to MC-LR at high concentrations (50 ␮g L−1 and 100 ␮g L−1 ) (Fig. 7A and B). MC-LR can induce the production of ROS, including O2− and H2 O2 (Li et al., 2003; Ortiz-Rodríguez and Wiegand, 2010). O2− is known to be converted into H2 O2 and O2 by SOD. Our results further suggested that individuals elevate the SOD transcript and enzyme levels to scavenge ROS caused by MC to maintain a balance between oxidants and antioxidants (Lyu et al., 2013b). In addition, MC-LR also stimulated SOD translation

levels, as well as other antioxidants (e.g. catalase, glutathione Stransferase) that may relieve oxidative reactions with toxins when D. magna are exposed to MC > 100 ␮g L−1 (Chen et al., 2005). Acute 48-h nitrite exposure also stimulated MnSOD mRNA expression. In nitrite-exposed D. magna, the MnSOD mRNA expression levels significantly increased at low and high concentration, while TSEA remained unchanged until exposed to >20 mg NO2 –N L−1 . The toxicity of nitrite to crustaceans has been well-studied recently (Hannas et al., 2010; Lyu et al., 2013a; Romano and Zeng, 2013; Yang et al., 2011), and elevated environmental nitrite has been illustrated to induce oxygen-transport proteins structural change, cause hypoxia in tissue, and impair the respiratory process in aquatic crustaceans (Jensen, 2003; Wang et al., 2006). It is logically suggested that O2 deficiency increase ROS generation, as a result of the operation of xanthine reductase/xanthine oxidase system. Under hypoxic conditions, the first enzyme can be theoretically converted to the second via limited proteolysis or oxidation and be transformed in efficient ROS producer. Therefore, the transcription of antioxidant enzyme was up-regulated in D. magna, similarly evidenced by Rahman and Thomas (2012). Additionally, oxidative stress can be induced by heavy metals, which are always big challenges for many aquatic animals, for example, copper inducing elevated Cu/Zn SOD gene expressions has been demonstrated in D. magna (Lyu et al., 2013b). Wang et al. (2010) reported that MnSOD expression in Phascolosoma esculenta was increasingly responsive to the treatment of Cd showing a 7-fold increase relative to the control group. In a marine crab, Charybdis japonica, Cd induced ROS generation and TSEA of the gill was significantly higher when exposed to 0.025 mg L−1 for 3 days (Pan and Zhang, 2006). Previously, increased expression of MnSOD as a result of Cd exposure was also investigated in aquatic species such as Takifugu obscurus and Hemibarbus mylodon (Cho et al., 2009; Kim et al., 2010a). In the present study, experimental exposure of D. magna in Cd significantly induced the mRNA expression of MnSOD, albeit to different degrees. Although Cd does not involve the redox cycling mechanism, there are reports that Cd could induce oxidative stress by ROS production. Shi et al. (2005) revealed that Cd could increase ROS production and result in oxidative damage in the liver of Carassius auratus in a concentration-dependent manner. Importantly, Cd is shown to induce production of superoxide anion and hydrogen peroxide in mitochondria using a cell line study (Miccadei and Floridi, 1993). It is also noted that the inhibition of electron transfer chain in mitochondria by Cd is explained as a probable mechanism of ROS production (Wang et al., 2004). It was illustrated that the ubiquitous pro-oxidant inducers (MCLR, nitrite, and Cd at environmentally relevant concentrations) elevated Dm-MnSOD gene expression and TSEA levels, and therefore our findings indicated the possibility of using MnSOD mRNA expression as a biomarker of oxidative stressors in D. magna. As

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Fig. 7. Real-time PCR of Dm-MnSOD mRNA expression and TSEA after treatment with MC-LR (A for transcript, B for TSEA), nitrite (C for transcript, D for TSEA), and Cd (E for transcript, F for TSEA). All values represent the mean ± SE (n = 3). Different letters denote significant difference using Duncan multiple range test (P < 0.05).

mentioned above, reducing Dm-MnSOD mRNA expression and TSEA occurred with respect to age, indicating that antioxidant activity in D. magna is probably affected by age (Arzate-Cárdenas and Martínez-Jerónimo, 2011; Barata et al., 2005). Age, therefore, is a key factor to be considered for the experimental design of toxicity bioassays with D. magna (Alberto et al., 2011). In addition, to accurately measure the relative expression of a target gene, the Dm-MnSOD mRNA expression data were calculated by means of an internal reference gene, D. magna ␤-actin, which is considered to be constantly maintained its expression level (Dhar et al., 2009). Despite ubiquitously employed, however, the expression of reference genes, such as ␤-actin or GADPH, could be influenced during individual developmental stages as well as experimental conditions (Heckmann et al., 2006). Therefore, such limitation should be recognized when interpreting the results. 4. Conclusion Dm-MnSOD gene was successfully cloned and characterized the full length in D. magna. Also, Dm-MnSOD gene showed wellconserved domains that are required for metal bingding and several common characteristics. Common aquatic toxicants (MCLR, nitrite, and Cd) respectively elevated Dm-MnSOD mRNA levels, suggesting that the Dm-MnSOD gene seems to react to oxidative stress inducers in aquatic environment. Thus, Dm-MnSOD has the potential to be used as a biomarker for waterborne pollutants,

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