Genome-wide identification and expression of the entire 52 glutathione S-transferase (GST) subfamily genes in the Cu2+-exposed marine copepods Tigriopus japonicus and Paracyclopina nana

Aquatic Toxicology 209 (2019) 56–69

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Genome-wide identification and expression of the entire 52 glutathione Stransferase (GST) subfamily genes in the Cu2+-exposed marine copepods Tigriopus japonicus and Paracyclopina nana

T

Jun Chul Parka, Min-Chul Leea, Deok-Seo Yoona, Jeonghoon Hana, Heum Gi Parkb, ⁎ Un-Ki Hwangc, Jae-Seong Leea, a b c

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 25457, South Korea Marine Ecological Risk Assessment Center, West Sea Fisheries Research Institute, National Institute of Fisheries Science, Incheon 46083, South Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Glutathione S-transferase Genome-wide identification Detoxification Copepod Tandem duplication

In this study, the entire glutathione S-transferases (GSTs), the major phase II detoxification enzyme, were identified in two marine copepod species Tigriopus japonicus and Paracyclopina nana. The genome-wide identification of GSTs in T. japonicus and P. nana resulted in 32 and 20 GSTs in total, respectively. Among the identified GSTs, two specific classes of GSTs, specifically sigma and delta/epsilon GSTs were the dominant form of cytosolic GSTs in T. japonicus, while delta/epsilon and mu classes were dominant cytosolic GSTs in P. nana. In addition, Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) family were found in relatively higher proportion compared to other classes. Moreover, sigma, delta/epsilon, and microsomal GSTs have shown to expand through tandem duplication. To validate the detoxification function of the identified GSTs, both copepods were exposed to copper (Cu2+) and the reactive oxygen species (ROS) level and GST activity were measured. With integration of phylogenetic analysis and xenobiotic-mediated GST mRNA expression patterns along with previous enzymatic activities, the functional divergence among species-specific GST genes was clearly observed. This study covers full identification of GST classes in two marine copepod species and their important role in marine environmental ecotoxicology.

1. Introduction The glutathione S-transferases (GSTs) are crucial group of enzymes in living organisms which detoxify both endogenous and exogenous compounds such as pharmaceuticals and environmental pollutants (Nebert and Vasiliou, 2004). In addition, it is also widely accepted as one of the predominant antioxidant enzyme family against reactive oxygen species (ROS) and oxidative stress (Enayati et al., 2005; Hayes et al., 2005) by catalyzing the conjugation of glutathione (GSH) to electrophilic compounds thioether linkages (Hayes et al., 2005; Sheehan et al., 2001; Townsend and Tew, 2003), which renders xenobiotics more soluble (Suzuki et al., 2001) for elimination in phase III detoxification. To date, GSTs are largely divided into four classes, namely cytosolic, mitochondrial, microsomal (Atkinson and Babbitt, 2009), and further classified according to the protein sequence and structure (Suzuki et al., 2001). Cytosolic GSTs include alpha (Sinning et al., 1993), beta (Rossjohn et al., 1998), delta, epsilon (Wilce et al.,

⁎

1995; Oakley et al., 2001; Sawicki et al., 2003), mu (Ji et al., 1993), nu (Schuller et al., 2005), omega (Board et al., 2000), phi (Reinemer et al., 1996), pi (Reinemer et al., 1992), sigma (Ji et al., 1995; Kanaoka et al., 1997), tau (Thom et al., 2002), theta (Rossjohn et al., 1998), and zeta (Polekhina et al., 2001). As for the mitochondrial GST, kappa was first identified in the mitochondrial matrix of rat liver (Harris et al., 1991); microsomal GST was first identified and purified from the human liver (McLellan et al., 1989). Due to largely diversified GST classes, variability of the active site, composed of highly conserved G site which binds reduced glutathione (GSH) and the highly variable H site (CheMendoza et al., 2009) have been validated by many previous studies, demonstrating their specific detoxification potential against specifictypes of xenobiotics. For example, delta and epsilon subclasses have shown resistance against pesticides such as organophosphates, organochlorines, and pyrethroids (Enayati et al., 2005), whereas those belonging to the same cytosolic class, omega, theta, and zeta were involved mainly in cellular processes such as protection against oxidative

Corresponding author. E-mail address: [email protected] (J.-S. Lee).

https://doi.org/10.1016/j.aquatox.2019.01.020 Received 31 December 2018; Received in revised form 23 January 2019; Accepted 23 January 2019 Available online 24 January 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.

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and other macromolecules (Atli et al., 2006; Craig et al., 2007; Pandey et al., 2008). Moreover, generation of oxidative stress via Cu2+ has been reported from previous studies (Ahmad et al., 2005; Bopp et al., 2008), which is critical in living aquatic organisms. To date, clarification on the information of the whole-genome identification of GSTs in the model aquatic invertebrate species T. japonicus and P. nana is required despite various isoforms of GST genes identified from previous investigations. This is the first study characterizing the whole GST gene family in the copepod T. japonicus and P. nana with in-depth detailed information on the synteny analysis, which could be applied to other genetically complex organisms without currently available whole genome sequences.

stress (McLellan and Wolf, 1999). Yet, compared to cytosolic GST subclasses, studies on mitochondrial GST kappa and MAPEG microsomal GSTs are limited that likely be shown to be associated other than detoxification response (Bresell et al., 2005; Jakobsson et al., 1999). This study highly focuses on the identification of the GST genes of the superfamily, which is the key enzymes for phase II of the detoxification process (Frova, 2006). As superfamily of multifunctional proteins with fundamental roles in the cellular detoxification of both endoand exogenous compounds via catalysis of the conjugation of the tripeptide glutathione (GSH: γ-Glu-Cys-Gly) with compounds containing an electrophilic center, thus forming more soluble, non-toxic peptide derivatives to be excreted or compartmentalized by phase III enzymes (e.g., ABC transporters) (Coleman et al., 1997). As catalytic function, GSTs serve as peroxidases, isomerases, and thiol transferases (Jensson et al., 1986; Bartling et al., 1993; Fernández-Cañón and Peñalva, 1998; Board et al., 2000). As non-catalytic function, GST also functions in binding of non-substrate ligands and modulation of signaling processes (Smith et al., 2003; Axarli et al., 2004, Loyall et al., 2000). In addition to the three large subfamilies, the unique structure of GSTs also attribute to their capacity to encompass even more subfamilies such as glutaredoxins (GRX) (Xia et al., 2001; Collison and Grant, 2003), chloride intracellular channels (CLIC) (Harrop et al., 2001; Dulhunty et al., 2001; Cromer et al., 2002), dehydroascorbate reductases (DHAR) (Dixon et al., 2002), selenocysteine glutathione peroxidases (SecGPx) (Epp et al., 1983), and eukaryotic protein elongation factors (eEF1Bγ) (Jeppesen et al., 2003), mainly due to the unique structural motif of GST, the thioredoxin fold. Up to date, abundant information on GSTs in of marine species (fish) are available, whereas limited information is available on the GST in the aquatic invertebrates species (e.g., Calanus finmarchicus [Roncalli et al., 2015], Daphnia pulex [Colbourne et al., 2011]). Insufficient reports on the aquatic invertebrates are primarily due to intricacies of GST structure and function of aquatic organisms (Blanchette et al., 2007) and ecological (e.g., habitats, dietary sources, and etc) factors that may have influenced evolutionary paths. Thus, it is important to identify one of the main xenobiotic detoxifier gene families, GST, in aquatic invertebrate organisms, since they bridge between lower and higher trophic levels. Among marine invertebrates, the benthic copepod Tigriopus japonicus, the widely distributed along the coast of Northwest Pacific rim, is an indicator species of the family Harpacticidae. Tigriopus japonicus is considered as a model organism for aquatic toxicology due to favorable traits (e.g. small size [∼1 mm], short life cycle [∼2 weeks], distinct developmental stages, high fecundity, ease of maintenance) (Raisuddin et al., 2007). The importance of GST genes as biomarkers in T. japonicus has been highlighted in studies of the detoxification mechanisms of diverse chemicals and environmental stressors (Lee et al., 2006, 2007). In addition, another pelagic marine copepod Paracyclopina nana was chosen as a comparative experimental species to T. japoncius, since P. nana also features several advantages such as small size (∼0.6 mm), short generation cycle (∼2 weeks), high fecundity, distinctive postembryonic developmental stages, and sensitive responses to environmental stressors (Dahms et al., 2016). Based on these favorable characteristics for ecotoxicological studies, along with recent construction of the genome and RNA-seq database for P. nana (Lee et al., 2015, Dahms et al., 2016), both copepods serve as suitable comparative ecotoxicological studies. Yet, previous studies on antioxidant role of GSTs in T. japonicus and P. nana were performed based on non genomewidely identified GSTs (Lee et al., 2017; Park et al., 2017; Lee et al., 2006, 2007; Lee et al., 2008), thus, genome-wide identification of GSTs in marine copepods are crucial to understand molecular detoxification mechanism and further application of important biomarkers of xenobiotics. Further verification of identified GSTs were analyzed by treating one of the most abundant transition metals in nature, the copper (Cu) (Eyckmans et al., 2011), since Fenton reactions of Cu affect anti-oxidants, leading to peroxidative damage to cell membranes, DNA,

2. Materials and methods 2.1. Culture and maintenance of copepods The copepod T. japonicus was collected from a single rock pool at Haeundae beach (35°9′29.57”N, 129°9′36.60”E) in Busan, South Korea (kindly provided by Prof. Heum Gi Park, Gangneung-Wonju National University in South Korea) in 2003, and maintained under controlled incubator conditions with a 12 h light/12 h dark cycle at a temperature of 25 °C and maintained culture medium at 30 practical salinity units (psu) with pH 8.0. The cyclopoid copepod P. nana was identified by morphometric analysis followed by molecular characterization of the animal to confirm the species identity. P. nana was maintained in filtered artificial seawater (ASW) (TetraMarine Salt Pro, TetraTM, Cincinnati, OH, USA) under confined laboratory conditions (15 psu salinity, 12:12 h [light: dark] photoperiod at 25 °C). The identity of copepod species used for this experiment was verified by morphological characteristics and sequence analysis of T. japonicus and P. nana mitochondrial cytochrome oxidase 1 (mt CO1) as the barcoding gene for animals (Jung et al., 2006; Ki et al., 2009). Both copepods were fed Tetraselmis suecica as dietary source (approximately 6 × 104 cells/mL) on a daily basis. 2.2. In silico mining of glutathione S-transferase genes To identify GST genes, genomic DNAs from the two copepods T. japonicus and P. nana were sequenced using the Illumina HiSeq 2500 platform (300 bp, 500bp, and 800 bp as paired-end libraries and 2 kb, 5 kb, and 10 kb as mate-pair libraries) at the National Instrumentation Center for Environmental Management (NICEM), Seoul National University, in Seoul, South Korea. After sequencing, pre-processing of the raw sequenced reads was conducted using Trimmomatic (http:// www.usadellab.org/cms/?page=trimmomatic). De novo assembly of pre-processed raw reads was performed using the Platanas assembler v1.2.4 (http://platanus.bio.titech.ac.jp) and HaploMerger 2 v20151124 (http://mosas.sysu.edu.cn/genome/download_softwares.php). Whole genome assembly yielded a total genome length of 196,587,744 bp (scaffold Nos. 358; N50 = 1.69 Mb), 85,709,251 bp (scaffold Nos. 203; N50 = 1.68Mb), in T. japonicus and P. nana (unpublished data), respectively. To obtain GSTs, in silico analysis of T. japonicus (GenBank GCHA00000000) and P. nana (GenBank GCJT00000000) RNA-seq information was performed. Genes were subjected to BLAST analysis in the GenBank non-redundant (NR; including all GenBank, EMBL, DDBJ, and PDB sequences except EST, STS, GSS, and HTGS) amino acid sequence database (http://blast.ncbi.nlm.nih.gov/). All acquired contigs were mapped to the genome for obtaining the complete DNA sequence using Geneious (v.10.0.7; Biomatters Ltd., Auckland, New Zealand) (Kearse et al., 2012). Annotation and nomenclature of all GST genes were completed based on amino acid sequence similarities and phylogenetic analysis under the guidance of the recommendations from the HUGO Gene Nomenclature Committee (Nebert and Vasiliou, 2004). 57

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[0.098 μM], 0.0125 [0.197 μM], and 0.0250 [0.393 μM] mg/L), respectively, for 24 h. Intracellular ROS were measured as protocols provided by Kim et al. (2016). Samples were homogenized with Teflon pestle in a buffer (0.32 M sucrose, 20 mM HEPES, 1 mM MgCl2, and 0.4 mM PMSF at pH 7.4). The homogenate was centrifuged at 10,000×g for 20 min at 4 °C and the supernatant was collected for the measurement purpose. ROS level was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR, USA), which oxidizes to fluorescent dichlorofluorescein (DCF) by the intracellular ROS. The mixture of phosphate-buffered saline (PBS), probe (H2DCFDA at a final concentration of 40 μM), and the supernatant fraction in a ratio of 170: 20:10, respectively, with the total volume of 200 μL was transferred to the Black 96-well plates (SPL Life Sciences, Seoul, South Korea). Each well was measured at an excitation wavelength of 485 nm and emission wavelength of 520 nm under a spectrophotometer (Thermo™ Varioskan Flash, Thermo Electron, Vantaa, Finland). For GST activity measurement, GST Assay kit, purchased from Sigma Aldrich (CS0410) was used. The increasing absorbance at 340 nm was measured for the conjugation of GSH with 1-chloro-2,4dinitrobenzene (CDNB), which acts as a substrate and detected the absorbance at 340 nm at 25 °C (Regoli et al., 1997). The total protein content obtained from the supernatant for ROS level and GST activity was determined by dye-binding method (Bradford, 1976) with bovine serum albumin standard (9–200 μg BSA/mL PBS).

2.3. Phylogenetic analysis of GST gene families To analyze the evolutionary relationships of GSTs of two nichedistinct copepods benthic copepod T. japonicus and the pelagic copepod P. nana, two species-specific GSTs were subjected to phylogenetic analysis and compared with the GSTs from other organisms, including Calanus finmarchicus (Roncalli et al., 2015), Caligus clemensi, Caligus rogercresseyi, Eurytemora affinis, T. californicus, Acartia pacifica, Calanus helgolandicus, Daphnia magna, Lepeophtheirus salmonis, Pseudodiaptomus poplesia, and Tortanus forcipatus. These organisms were chosen as analytical candidates due to access to identified GSTs and phylogeny based hierarchies and their beings as aquatic organisms. The translated amino acid sequences of GSTs from the two copepods were first subjected to multiple alignments with ClustalW algorithm. To establish the best-fit substitution model for phylogenetic analysis, the model with the lowest score according to the Bayesian Information Criterion (BIC) (Schwarz, 1978) and Akaike Information Criterion (AIC) (Hurvich and Tsai, 1989; Posada and Buckley, 2004) was analyzed by maximum likelihood (ML) analysis. The phylogenetic tree was constructed using MEGA software (ver.7.0) under the best-fit model (LG + G+F) (Center for evolutionary Medicine and Informatics, Tempe, AZ, USA) (Tamura et al., 2013). Full lengths protein sequences were aligned and a phylogenetic tree was obtained as described above and the reliability of tree topology was evaluated by bootstrapping test (1000 replicates). 2.4. Syntenic comparison of tandem duplicated GSTs

2.7. Modulation of mRNA expression of entire GST genes in Cu2+-exposed Tigriopus japonicus and Paracyclopina nana

For synteny analysis, gene models of two copepods (T. japonicus and P. nana) were constructed by gene prediction pipeline using BRAKER1 v1.9.0 (http://bioinf.uni-greifswald.de/augustus/downloads/) and MAKER v2.31.9 (http://www.yandell-lab.org/software/maker.html). Synteny analysis was carried out by separately by comparing the GST gene clusters in individual copepod by analyzing 10k bp in both direction of the strand from gene models. The identified homologous genes from ± 10k bp of the GST gene clusters were verified by performing BLAST analysis against NCBI NR database. A significant hit was defined as a hit with an E-value ≤10−5.

Based on previous literatures on exposure to Cu and Cd in T. japonicus (Lee et al., 2008) and P. nana (Hwang et al., 2010), both copepods were exposed to Cu2+under NOEC concentration, 800 μg/L (12.589 μM) and 25 μg/L (0.393 μM) of Cu2+ respectively. Total RNA extraction was performed prior to the quantitative real-time polymerase chain reaction (qRT-PCR). Approximately 300 copepods exposed to Cu (0.800 mg/L [12.589 μM]) for T. japonicus and (0.0250 mg/L [0.393 μM]) for P. nana for 24 h, were homogenized in five volumes of TRIZOL® reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) with a tissue homogenizer and stored at −80 °C until RNA extraction was isolated under manufacturer’s protocol. DNase I (Sigma–Aldrich Co.) was used to remove genomic DNA. Total RNA concentration was measured at 230, 260, and 280 nm (A230/260, A260/280) using a spectrophotometer (Ultrospec 2100 pro, Amersham Bioscience, Freiburg, Germany). Two μg of RNA with the reverse transcriptase (SuperScript™ II RT kit, Invitrogen, Carlsbad, CA, USA) were used to synthesize a single-stranded cDNA under the following condition; 65 °C/5 min, 42 °C/2 min, 42 °C/60 min, and 72 °C/15 min. The mRNA expression levels of GST genes (GSTd/e, -mu, -o and -DHAR, -s, -t, -z, -GDAP, -k, and -microsomal) were analyzed by qRT-PCR experiments. qRT-PCR was performed in triplicate using a CFX96™ RT-PCR (Bio-Rad, Hercules, CA, USA). Amplifications were performed in the presence of SYBR® Green (Molecular Probes Inc., Invitrogen, Waltham, MA, USA) using 1 μl cDNA and 0.2 μM gene-specific primers (Tables S1 and S2). The thermal profile for qRT-PCR was 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s operated in triplicate. Melting curve analyses were also monitored to confirm the precision and accuracy of the specific amplification. The quantification cycle (Cq) between samples was normalized by the elongation factor 1-α (EF1-α) gene for both T. japonicus and P. nana, as an endogenous control. The relative gene expression was determined using the comparative threshold cycle 2−ΔΔCT method (Livak and Schmittgen, 2001) and represented as heat-map to compare modulations in each GSTs transcriptional levels. Sequences for the primers sets used in this study are appended in Tables S1 and S2.

2.5. Acute toxicity analysis of Cu2+ in Tigriopus japonicus and Paracyclopina nana The no observed effect concentration (NOEC) and median lethal concentration (LC50) at 48 and 96 h were evaluated to assess the acute toxicity of copper in T. japonicus and P. nana. Copper sulfate pentahydrate (CuSO4⋅5H2O MW 249.69; CAS Number 7758-99-8) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and it was dissolved in ultrapure distilled water. In brief, copepods were exposed to copper under identical laboratory conditions to those used for acclimation but test organisms were not fed during the entire 96-h experiment. Mortality was recorded once every 24 h, and a copepod was considered dead when it showed no sign of movement. Half (2 mL) of coppercontaining solution was renewed daily. Finally, NOEC and LC50 values were calculated using Probit analysis (ToxRat Ver.2.09, Alsdorf, Germany, GmbH, 2005). Three biological replicates were performed in a 12-well culture plates (SPL Life Sciences, Seoul, South Korea) in 4 ml of seawater, and each well contained 10 adult copepods. 2.6. Measurement of ROS and GST activity in response to Cu2+ in Tigriopus japonicus and Paracyclopina nana To measure the oxidative stress induced by Cu2+in T. japonicus and P. nana, approximately 200 adult individuals were collected by 200 μm and 150 μm sieve, respectively, prior to the experiment to acquire sufficient amount of protein extract. To induce stress, T. japonicus and P. nana were exposed to Cu2+ under NOEC (con, 0.200 [3.147 μM], 0.400 [6.295 μM], and 0.800 [12.589 μM] mg/L) and (con, 0.00625 58

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Fig. 1. A) Phylogenetic tree analysis of GSTs obtained in silico from Tigriopus japonicus and Paracyclopina nana and other aquatic invertebrate species (Calanus finmarchicus, Eurytemora affinis, Tigriopus californicus, Acartia pacifica, Caligus clemensi, Calanus helgolandicus, Caligus rogercresseyi, Lepeophtheirus salmonis, Pseudodiaptomus poplesia, and Tortanus forcipatus), using maximum likelihood model (LG + G+F: InL -53344.07893). Each GST class belonging to Tigriopus japonicus and Paracyclopina nana is indicated by the colors shown in the legend box: Red indicates GST sigma, Purple indicates GST mu, Turquoise indicates GST omega, Light green indicates GST delta/epsilon, Green indicates GST theta, Pink indicates GST DHAR, Gray indicates GST zeta, Light purple indicates GST GDAP, and Teal indicates GST kappa, and Orange indicates microsomal GST. B) Pi-chart representation of GSTs belonging to different classes in Tigriopus japonicus and Paracyclopina nana. C) Comparison of GST classes and genes among two copepods.

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Fig. 2. Localization of gene analysis of the 52 GSTs identified in A) Tigriopus japonicus and B) Paracyclopina nana. Genes are represented by specific colored arrows, indicating the direction of the reads, in each scaffold. Each gene locus is presented in proportion to the length of the scaffold. Clustered regions are represented are indicated by a set of vertical bars. C) Synteny analysis of tandem duplicated regions of GSTs within 10 kb region forward and reverse direction in Tigriopus japonicus and D) in Paracyclopina nana.

3. Results

were identified within each GST class with similar number of exons and introns in each sub-class. Based on a conserved domain analysis, total 32 and 20 GST genes of T. japonicus and P. nana, respectively, were further sub-classified into distinct classes, namely, GST-alpha (GDAP), -delta/epsilon, -theta, -omega, -DHAR, -mu, -sigma, –zeta, -kappa, and -microsomal. Interestingly, among the 10 identified classes of GSTs, highest number of genes was identified within the sigma and microsomal classes in T. japonicus, while highest number of genes was identified within the delta/epsilon class in P. nana. Among the identified GSTs, sigma class 2-1 and 2-2 showed orthologous relationship of 3 and 4 exons, respectively, between the two

3.1. Identification and phylogenetic analysis of GST genes in copepod Tigriopus japonicus and Paracyclopina nana Based on the full genome and transcriptome assembly data of T. japonicus and P. nana, the total number of 32 and 20 putative GSTs were identified, respectively. (Fig. 1A). Of 32 and 20 GSTs identified, genes were mapped onto 25 and 13 scaffolds in T. japonicus and P. nana, respectively (Fig. 2A and B); the number of genes in each scaffold varied with no particular patterns. In two copepods, conserved gene structures 60

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copepods. GSTz also consisted of 5 exons for both species. Overall, all subclasses of GSTs exhibited their lengths of open reading frames (ORF) ranging from 417 and 753 base pairs (bp) (i.e. 139 to 251 amino acid length). In particular GSTk, belonging to mitochondrial GST family contained the comparatively longer ORF lengths, compared to GSTs belonging to cytosolic (e.g., delta/epsilon, DHAR, mu, omega, sigma, theta, and zeta) with an exception of GDAP of T. japonicus which consisted of 999 bp in length. Moreover the shortest ORF length was observed in all microsomal GSTs belonging to MAPEG family. Phylogenetic tree analysis revealed the origin of diversification of GST genes into two large branches: the microsomal, mitochondrial, while the cytosolic branch that is further divided into three large clades, namely, GST theta/delta&epsilon, sigma, mu, and omega/DHAR. Among the identified GST classes, GST sigma was further divided into three smaller clades (Fig. 1).

GST genes was demonstrated between two copepods. GSTs belonging to delta/epsilon classes contained two to six exons at the maximum (Table 1). In T japonicus, four delta/epsilon, four sigma, and five microsomal GSTs were located in tandem on scaffolds (18 and 35, 2 and 62, and 79 and 130, respectively). In comparison, two delta/epsilon, two mu, two sigma, and two microsomal GSTs were located in tandem on scaffolds (9, 5, 18, and 20, respectively) in P. nana. In addition, the total ORF lengths of GSTs belonging to different classes were varied, yet, the overall ORF lengths of each GSTs belonging to each class were highly conserved in class-specific manner. In both copepods, microsomal GSTs contained the shortest ORFs, while GDAP of T. japonicus and GSTk of two copepods was comparatively longer than other GSTs. One to one orthologous relationship of GSTs belonging cytosolic, mitochondrial families of T. japonicus and P. nana was demonstrated within GST DHAR, theta, and zeta of cytosolic, and GST kappa of mitochondrial.

3.2. Comparative homology and genomic organization of GST genes between Tigriopus japonicus and Paracyclopina nana

3.3. Syntenic analysis of tandem duplication of GST genes in Tigriopus japonicus and Paracyclopina nana

To compare the presence and number of GST belonging to different classes in T. japonicus and P. nana, the inter-linkage between GST subfamilies among the 10 identified classes, namely delta/epsilon, GDAP, DHAR, kappa, microsomal, mu, omega, sigma, theta, and zeta, was constructed (Fig. 1). Among the identified GST classes, delta/epsilon, sigma, and microsomal classes were highly duplicated in T. japonicus, whereas high duplication of delta/epsilon and microsomal GSTs were found in P. nana. (Fig. 1 and Table 1). In general, high similarity in the number of exons in each identified

Synteny analysis of the GST genes was conducted by confirming the relative locations of the genes in the scaffolds of the T. japonicus and P. nana genome (Fig. 2A and B). Among the 10 identified GST classes (i.e. GST-alpha [GDAP], -delta/epsilon, -theta, -omega, -DHAR, -mu, -sigma, –zeta, -kappa, and -microsomal), only three specific classes, namely, delta/epsilon, sigma, and microsomal GSTs, demonstrated gene duplication through tandem duplication. In general, tandemly duplicated genes were closely clustered within short distance of scaffold, ranging between 1.6 kb to 5.6 kb in lengths, in two copepods. Synteny structure analysis demonstrated similar class-specific GST genes belonging to each copepod. For example, GSTd/e4b and 4c of T. japonicus and GSTd/ e4a and 4b of P. nana were duplicated through tandem duplication within 2.4 kb and 3.3 kb of scaffold 18 and 9, respectively. In addition to GST delta/epsilon class, GST sigma class, specifically, GSTs3a and 3b of T. japonicus and P. nana were clustered within 2 and 3.9 kb of scaffold 2 and 18, respectively. (Fig. 2C and D). Moreover, another gene duplication of GSTs1a and 1b was shown in T. japonicus, specifically, clustered within 5.6 kb in scaffold 62. Microsomal GSTs belonging to MAPEG family, have also shown tandem duplication, specifically mGST3, 4a, and 4b/9a and 9b of T. japonicus and mGST5a and5b of P. nana, located within 2.7 kb for mGST3, 4a, and 4b in scaffold 130; 4.0 kb for mGST9a and 9b in scaffold 79 in T. japonicus, while mGST5a and 5b were located within 1.6 kb in scaffold 20 of P. nana.

Table 1 Genome-wide identification of GSTs in Tigriopus japonicus and Paracyclopina nana. The open reading frame length is represented by open reading frame (ORF) in bp, exon numbers are represented with Ex. Family

Cytosolic

Mitochondrial Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG)

Tigriopus japonicus

Paracyclopina nana

Gene

ORF

Ex

Gene

ORF

Ex

D/E1 D/E2 D/E3 D/E4a D/E4b D/E4c DHAR GDAP Mu1 Mu2

735 666 657 660 687 651 687 999 741 666

5 3 3 3 3 2 3 1 2 5

D/E1 D/E3a D/E3b D/E4a D/E4b

753 672 687 666 660

6 3 3 4 4

DHAR

645

2

Omega1a Omega1b Sigma1a Sigma1b Sigma1c Sigma1-2a Sigma1-2b Sigma2a Sigma2b Sigma3a Sigma3b Theta Zeta Kappa mGST1 mGST3 mGST4a mGST4b mGST8 mGST9a mGST9b mGST9c

753 771 651 654 588 663 702 657 654 618 534 663 651 975 456 429 438 438 426 426 426 414

4 4 3 3 4 4 5 3 3 4 3 4 5 5 4 3 3 3 4 1 1 1

Mu1 Mu2a Mu2b Mu2c Omega

783 639 654 654 729

1 3 3 2 5

Sigma3a Sigma3b

621 582

4 3

Theta Zeta Kappa mGST5a mGST5b mGST8 mGST9

702 645 921 438 441 426 417

5 5 3 4 4 4 2

3.4. Acute toxicity assessment of Cu2+ in Tigriopus japonicus and Paracyclopina nana The 48 h-LC50 and 96 h-LC50 values of copper (Cu2+) on the copepod T. japonicus were determined as 8.755 mg/L and 1.353 mg/L, respectively (Table 2). In contrast, the 24 h-LC50 and 48 h-LC50 values on P. nana, were 0.252 mg/L and 0.118 mg/L, respectively (Table 2). Species-specific sensitivity to copper was also observed in 48 h- and 96NOEC values, where P. nana was 5 and 32 folds more sensitive to copper compared to T. japonicus (Fig. 3A and B). Table 2 The no observed effect concentrations (NOEC), LC50, and 95% confidence intervals (CI) for T. japonicus and P. nana exposed to copper (Cu2+) for 48 h and 96 h. Species

Time (h)

NOEC (mg/L)

LC50 (95%CI; mg/L)

Tigriopus japonicus

48 96 48 96

0.800 0.800 0.150 0.025

8.755 1.353 0.252 0.118

Paracyclopina nana

a

61

n.d; not determined.

(4.404–17.406) (1.095–1.760) (0.209–0.303) (n.d.)a

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Fig. 3. Acute toxicity assessment of copper (Cu2+) in A) Tigriopus japonicus and B) Paracyclopina nana. Cu2+ concentration exposed to Tigriopus japonicus and Paracyclopina nana are: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.5, and 5.0 mg/L for 96 h and 0.005, 0.010, 0.025, 0.050, 0.100, 0.150., 0.200, and 0.400 mg/L for 96 h, respectively. The LC50 and NOEC values of 48 and 96 h are represented by closed and open circle, respectively. Effects of Cu2+ exposure in intracellular ROS level and GST activity under different concentrations (0, 0.200, 0.400, and 0.800 mg/L) to C) Tigriopus japonicus and (0.00625, 0.0125, and 0.0250 mg/L) to D) Paracyclopina nana at 24 h. Results represent the mean ± S.E. of three replicate samples as percentage of controls. Significant differences are indicated as the letters above each bar (P < 0.05).

upregulated GSTs were GSTd/e2, GSTo1a, GSTs2b, GSTs1c, and mGST4b. In P. nana, most of the GSTs have been upregulated, with significant upregulation of GSTm2b, m2c, m1, z, o, k, d/e3b, and mGST5a/b.

3.5. Copper-induced ROS level and GST activities in Tigriopus japonicus and Paracyclopina nana The intracellular ROS level and GST activity were analyzed based on the NOEC 96 h of copper exposure in two copepods. In both copepods, the intracellular ROS level was significantly increased (P < 0.05) in concentration-dependent manner in response to Cu2+ (Fig. 3C and D), with higher ROS level detected in P. nana compared to T. japonicus. Cu2+-induced oxidative stress also resulted in the significant increase (P < 0.05) in GST activity in both copepods, under maximum concentration, which corresponds the NOEC 96 h.

4. Discussion In silico analysis of GST transcripts from T. japonicus and P. nana database resulted in the total of 32 and 20 GSTs, respectively. According to previous reports on GST, GSTs are divided into three major groups cytosolic, mitochondrial, and microsomal (Jakobssen et al., 1996; Sheehan et al., 2001; Robinson et al., 2004). In consistent, GSTs identified from two copepods were divided into three families, namely cytosolic, mitochondrial, and microsomal of MAPEG. Phylogenetic analysis revealed the existence of sigma, omega, mu, DHAR, delta/epsilon, theta, and zeta in both copepods. In addition to commonly found GSTs in invertebrates, one single GST GDAP was also identified in T. japonicus. In general, insect specific GSTs belonging to cytosolic family are further classified into subclasses, namely, delta, epsilon, omega, sigma, theta, and zeta (Chelvanayagam et al., 2001; Ranson et al., 2001; 2002; Ding et al., 2003; Ranson and Hemingway,

3.6. Modulation of mRNA expression of the entire GST genes identified in Tigriopus japonicus and Paracyclopina nana under Cu2+ exposure The temporal-mRNA expression of the entirely identified GSTs of both P. nana and T. japonicus under NOEC 96 h, 0.800 mg/L and 0.025 mg/L, respectively, demonstrated various modulation in expression levels at different time exposure (3, 6, 12, and 24 h) (Fig. 4A and B). In T. japonicus, significantly downregulated GSTs belonging to cytosolic family were GSTd/e1, GSTm1, and mGST9b, while significantly 62

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Fig. 4. Temporal-transcription profiles of A) 32 Tigriopus japonicus and B) 20 Paracyclopina nana GST genes under exposure to copper (Cu2+) under NOEC (0.800 mg/L and 0.025 mg/L for T. japonicus and P. nana, respectively) at 3, 6, 12, and 24 h time intervals. Transcription profiles are represented based on topological phylogenetic tree and transcription levels are expressed as mean fold transcription relative to the control, represented in heat map. Red indicates upregulation and Blue indicates downregulation.

have suggested the conservation/shared amino acids of mGST1 between both arthropods and vertebrates (Bresell et al., 2005; Holm et al., 2006; Haas et al., 2013; Shi et al., 2012). As previously suggested, high conservation of amino acid sequence is likely associated with relatively smaller overall length of microsomal GSTs compared to other GSTs belonging to cytosolic and mitochondrial family indicating high conservation of microsomal GSTs within two copepods. The proportion of GSTs belonging to cytosolic family each class were different among two copepods, where sigma and microsomal were the most abundant form in T. japonicus compared to delta and mu/ microsomal GSTs in P. nana, indicating difference in the species-specific gene expansion mechanism. Specifically, massive gene expansion of GST delta/epsilon and sigma classes was previously shown in many invertebrate species, including beetle Tribolium castaneum (Shi et al., 2012), fruit fly Drosophila melanogaster (Ranson et al., 2002; Zdobnov et al., 2002), mosquito Anopheles gambiae (Ding et al., 2003; Ranson et al., 2002; Zdobnov et al., 2002), and silkworm Bombyx mori (Yu et al., 2008). In addition, aquatic invertebrate species, crustacean C. finmarchicus (Roncalli et al., 2015) and water flea D. pulex (Colbourne et al., 2011), also demonstrated massive expansion of GST delta/epsilon and sigma, suggesting the conservation of gene expansion mechanism

2005; Tu and Akgül, 2005; Yu et al., 2008). In contrast, however, mammalian cytosolic GSTs are classified into eight classes, which include: alpha, mu, omega, pi, sigma, theta, and zeta (Hayes and McLellan, 1999; Board et al., 2000; Sheehan et al., 2001; Pearson, 2005), suggesting phylogenetic divergence of GSTs, evidenced by nonorthologous GSTs shared among vertebrates and invertebrates (i.e., presence of specific GSTs in either vertebrates or invertebrates). In addition to cytosolic GST family, mitochondrial and microsomal GST families were identified in T. japonicus and P. nana through in silico analysis. In particular, GST kappa belonging to mitochondrial GST family, has shown one to one orthologous relationship between T. japonicus and P. nana, which is suggestive of the evolutionary conservation of GST kappa. Interestingly, despite the absence of GST kappa in insects (Morel and Aninat, 2011), GST kappa has been predicted from either genomic or transcriptomic data (Roncalli et al., 2015) from Daphina pulex (Colbourne et al., 2011), Lepeophtheirus salmonis (Accession no. AC011809), C. clemensi (Accession no. AC015728), and C. rogercresseyi (Accession no. AC010845), which can also suggest the unique mode of evolutionary conservation of GST kappa across the animal taxa, and in particular of copepods. Lastly, microsomal GSTs in T. japonicus highly outnumbered those identified in P. nana. Previous reported literatures 63

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increase in ROS levels. Antioxidant enzymatic activities including GST activity have been used as a biomarker for adverse effects of xenobiotics and ROS induction (Lemaire and Livingstone, 1993; Dourado et al., 2008). Due to high ROS inducibility of Cu2+, Cu2+ is also associated with GST activity. For example, dietary Cu in the bivalve Diplodon chilensis (Sabatini et al., 2011) and the fish Catla catla (Rajasekar et al., 2017) demonstrated significant increase of antioxidant enzymatic activities including GST, which were positively correlated with the increased GST activity in Cu2+-exposed T. japonicus and P. nana. Mixed function oxygenase and GST are detoxification enzymes with low levels of substrate specificity that are employed in the metabolism of different classes of organic xenobiotics (Domingues et al., 2010). Their activity can be enhanced in response to xenobiotics, making them a potentially suitable candidate as a stress-specific indicator of organic xenobiotic pollution (Barata et al., 2005). Taken together, elevation of intracellular ROS level and GST activity suggest modulation of response mechanism in response to Cu2+ as a result of induction of oxidative stress in T. japonicus and P. nana. Furthermore, despite the large scale difference in NOEC of 800 and 25 μg/L respective of T. japonicus and P. nana, the degree of the increase in ROS level and GST activities in response to Cu2+ was relatively identical to one another, compared to the control, suggesting the higher sensitivity of P. nana compared to T. japonicus. However, specific GST gene responsible for the induction of the total GST activity is yet to be elucidated. In this study, the application of genome-widely identified GSTs transcriptomic profiles have demonstrated significant differences among the expressional levels in response to Cu2+ in both copepods. Among 32 and 20 GSTs identified in T. japonicus and P. nana, respectively, significantly upregulated genes were TjGSTs2b, TjGSTd/e2, TjGSTo1a, and TjmGST4b of T. japonicus, and PnGSTm2b, PnGSTm2c, PnGSTm1, PnGSTz, PnGSTo, PnGSTk, PnGSTd/e3b, PnmGST5a and b of P. nana. Difference in the modulation of GSTs in response to Cu exposure is likely due to multiple factors including, the presence of multiple isoforms as a result of gene duplication (Ranson et al., 2002; Shi et al., 2012), xenobiotic/substrate specificity, and time-exposure. For example, in the silkmoth Bombyx mori, GSTz and GSTo have been significantly upregulated in response to permethrin (Yamamoto et al., 2009), whereas, GSTd/e, GSTo, GSTt, and GSTz have shown upregulation at different time intervals in response to malathion in the fruit fly Bactrocera dorsalis (Hu et al., 2014), suggesting specificity of GSTs function in metabolic activity and detoxifying potential of each GST class. While less number of GSTs responded to Cu2+ exposure stress in T. japonicus, higher number of GSTs were upregulated in P. nana. Despite the difference in the number of upregulated genes, GSTs belonging to each class have shown similar upregulatory pattern in both copepods, indicating functional homology of GSTs across copepod species. For example, GST delta/epsilon classes, in general, are highly found in insects (e.g., Aedes aegypti and Drosophila melanogaster) and confer their role in resistance to organophophorus, organochlorine and pyrethroid insecticides (Kostaropoulos et al., 2001; Enayati et al., 2005; Alias and Clark, 2010; Lumjuan et al., 2011). In summary, genome-wide identification of GSTs in T. japonicus and P. nana have demonstrated their gene expansion of delta/epsilon, sigma of cytosolic and microsomal GSTs in T. japonicus, while delta/epsilon, mu, and microsomal GSTs in P. nana. In addition, phylogenetic analysis of the identified GSTs has shown convergence and divergence of multiple GST classes in two copepod species. The detoxification potential of the identified GSTs was validated by gene expression levels in response to significant increase in oxidative stress and GST activity, induced by one of the heavy metal contaminant, copper. The inducibility of each gene in response to copper was different, suggesting the function of each GST belonging to specific classes, function in chemical-specific manner, yet further studies are required to understand chemical and substrate specificity of GSTs.

through evolution among insects, including copepods. Further synteny analysis of GSTs in two copepods concluded that majority of expanded genes (delta/epsilon, sigma, and microsomal) in copepods was mainly attributed to tandem duplication, which give a rise to genetic novelty for the development of novel traits (Ohno, 1970; Conant and Wolfe, 2008). The significance of tandem duplication is its involvement in the co-amplification, which contribute to the increase in the fitness of organism (Roth et al., 1996). Indeed, tandemly arrayed clusters of GST sigma in invertebrates including T. japonicus and the rotifer Brachionus koreanus have demonstrated clear detoxification potential in response to various xenobiotics (Lee et al., 2007, 2017; Park et al., 2017; Zhou et al., 2019), suggesting the importance of tandem duplication of expansion of GST families in gene stability (Romero and Palacios, 1997; Reams and Neidle, 2004) and consequently, detoxification potential of GSTs. To validate the identified GSTs from two model copepod species, induction of oxidative stress and the consequent antioxidant activities were assessed in response to one of the major heavy-metal contaminant, copper (Cu2+). According to previous studies, acute toxicity assessment has been widely used and demonstrated its potential role as a reliable parameter against emerging chemicals in molecular toxicity and biomarker analysis (Chinedu et al., 2013; Akhila et al., 2007). In T. japonicus and P. nana, large difference in NOEC was observed in response to Cu2+, where observed NOEC at 96 h was 0.800 mg/L and 0.025 mg/L, respectively. Species-specific differences in acute toxicity have been reported among many aquatic organisms, including crustacean Echinogammarus olivii, Sphaeroma sprratum, and Palaemon elegans in response to heavy metals (Bat et al., 1999). In addition to heavy metals, a large variation in ecotoxicity values among aquatic invertebrates in response to pharmaceutical compounds, have also been reported (Ferrari et al., 2003; Huggett et al., 2002; Isidori et al., 2007; Marques et al., 2004), indicating species-specific sensitivity to various chemicals. On the basis of previously reported literatures, species-specific sensitivity between the two copepods is inevitable, as tolerance and/or sensitivity to various xenobiotics is associated with body size and genome size of organisms (Bennett, 1976; Beaton and Hebert, 1988). Thus, the difference in the copper-induced acute toxicity in two comparative copepods T. japonicus and P. nana is possible suggested with difference in genome their genome size along with their body size, resulting in higher tolerance of T. japonicus against copper. Copper is associated with induction of oxidative stress upon dietary and waterborne exposure (Lushchak, 2010), thus, based on the acquired acute toxicity, intracellular ROS level and the total GST activities have been measured in response to copper in two copepods, under various concentration lower than NOEC 96 h. In this study, significant increase in intracellular ROS levels was observed in T. japonicus compared to the control, whereas concentration-dependent significant increase in ROS levels was demonstrated in P. nana. In addition, the total GST activity of T. japonicus and P. nana was positively correlated with the increase in the intracellular ROS level. Indeed, metals including copper, are well known inducers of oxidative stress, which were validated by increased lipid peroxidation with exposure to metals including copper (Livingstone, 2003). Furthermore, increase in malondialdehyde and lipofuscins, which are the products of increase in the lipid peroxidation, has been demonstrated in the mussels Mytilus galloprovincialis (Viarengo et al., 1990), Perna perna (Almeida et al., 2004), and oyster Crassostrea virginica (Ringwood et al., 1998). Because the cupric copper (Cu2+) ion participates in the formation of ROS, oxidation, and reduction reactions (Gaetke and Chow, 2003), Cu2+-exposed copepods T. japonicus and P. nana, in agreement, have shown significant increase in intracellular ROS level indicating overall induction of oxidative stress. Modulations of antioxidant defenses depend on the stressors, intensity, and duration of the applied stress and the susceptibility of species being exposed (Bebianno et al., 2005) (Table 3). In this study, T. japonicus and P. nana demonstrated significant increase in the total GST activity in response to Cu2+, which were positively correlated to the 64

Adult

14-day-old

Juvenile

Paracyclopina nana

Daphnia pulex

Daphnia magna

65

Copepod

Copepod Copepod

Copepod

Paracyclopina nana Paracyclopina nana

Tigriopus japonicus

Copepod Nauplii Male

Adult

both male and female

Paracyclopina nana

Tigriopus japonicus

Macrobrachium alcolmsonii Tigriopus japonicus

Signal crayfish Pacifastacus leniusculus clam Ruditapes philippinarum freshwater snail Physella acuta Hyalella azteca

marine clams Ruditapes decussatus oysters Crassostrea brasiliana

Adult

Tigriopus japonicus

Adult

Developmental stage

Species

437.476 μg/L 51.759 μg/L 6.664 (5.005–8.873) μg/L 4.052 (3.501–5.053) μg/L

water-accommodated fraction (WAF) of crude oil

BDE-47 Multi-walled carbon nanotubes

Polystyrene microbead

Methylmercury

Triclosan

LC50 48h

LC50 96h

103.8 mg/L

48 and 96 h

Chlorprifos

24 h

24 h 6, 12, 24, 48 h

24 h

24 h

12 and 24h

21 days

5, 24, 48, 72, 96h

Bisphenol A (BPA)

Endosulfan

0, 2, 7, 14 days

Pb

96 h

24 h

24 h 48 h 24 h 48 h 14 days

72 h

LC50 96h LC50 96h LC50 48h

n.d

(0.65–1.05) (0.42–0.74) (0.38–0.60) (0.24–0.50)

48 h 96 h

7 (5-9) μg/L 80.02 mg/L

LC50 48h LC50 48h

0.83 0.56 0.48 0.35

48 hr 24 h

122 (92-161) μg/L 16 (12-22) μg/L

LC50 48h LC50 24h

24h 48h 24h 48h

24 h

329 (229-475) μg/L

LC50 24h

LC50 LC50 LC50 LC50

Exposure time

Measured value

Toxicity value

Dimethoate and HgCl2

Gold Octahedra nanoparticles (Au_0.03 and Au_0.045) Phenanthrene

DEHP

nanoplastic

Cypermethrin

Chemicals

Table 3 Toxicity values and significant changes in biomarker antioxidant genes, including GST genes in various aquatic invertebrates.

Significant decrease of ROS level and GPx activity Concentration dependent increase in SOD activity Significant changes in GST activity with increase at 50 ng/L Significant downregulation of GSTs at 24 h exposure to 500 ng/L

Lee et al. (2016)

Jeong et al. (2017)

Lee et al. (2017)

Morales et al. (2018) Steevens and Benson (1999) Bhavans and Geraldine (2001) Park et al. (2017)

Aouini et al. (2018)

Gunderson et al. (2018)

Lima et al. (2018)

Fkiri et al. (2018)

Wang et al. (2018)

Liu et al. (2018)

Zhou et al. (2019)

References

(continued on next page)

- Increase in GST activity during 21 days of exposure under endosulfan - Increase in ROS, GSH, GST, GPx, and SOD activities - Increase in mRNA expression of GST-Theta3 - GPx, GR, and GST activities increased in a concentrationdependent manner up to 100 ng/L - Decrease in SOD activity - Increase in mRNA expression of GST-Sigma and SOD activities - Increase in ROS, GPx, GR, GST, and SOD activities in response to 0.05-m diameters of polystyrene microbeads - Increase in ROS, GST and GPx activities - Decrease in ROS - Decrease in GSH but increase in 6 hour exposured copepod - Increase in GST, GR, GPx and SOD activities - Increase in GSH, GST, GR and CAT activities

- Concentration and time-dependent changes in antioxidant activities (CAT, GPx, GR, GST, and SOD) - Inhibition of GST activities were observed under chronic exposure - no changes in GST activity were observed in P. acuta under BPA exposure during 48 h - Inhibition of GST activity at 96 h

- Temperature dependent changes in mRNA expression of CYPs and antioxidants (SOD, GST, and GPx) - Temperature dependent changes in mRNA expression of CYPs and antioxidants (SOD, GST, and GPx) - Lower temperature treatment induced upregulation of antioxidant genes - Decrease in GST activity

- Increased mRNA expressions of antioxidants and defensome Hsps 70 and 90) in adult species (21 days) in response to 0.1 and 1 mg of nanoplastics - Life-stage dependent modulations in GST activity and mRNA expression with significant downregulation in juveniles compared to significant upregulation of GST mRNA expression in adults under 0.06 and 0.1 mg/L - Concentration-dependent increase in SOD, CAT, and GST activities

-

- Significant decrease of GR, SOD, GPx at concentrations 0.25, 2.5, and 25 μg/L) - Significant increase of ROS level and GST activity at 0.25 and 0.025 μg/L, respectively - Significant upregulation of GST sigma at 25 μg/L within 24 h

Key points

J.C. Park et al.

Aquatic Toxicology 209 (2019) 56–69

LC50 96h

Ag

Cu Mn

H2O2

LC50 96h

As

Copepod

LC50 96h

Cd

Tigriopus japonicus

LC50 96h

Cu

Toxicity value

Tigriopus japonicus

Chemicals

β-naphthoflavone

Developmental stage

Tigriopus japonicus

Species

Table 3 (continued)

0.024 (0.022–0.028) mg/L

9.7 (5.57–16.92) mg/ L

25.2 (17.81–35.55) mg/L

3.9 (2.60–5.87) mg/L

Measured value

10, 20, 30, 60, 120, 180 min 6, 12, 24, 48 h 6, 12, 24, 48 h

96 h

96 h

96 h

96 h

12, 24, 36, 72 h

Exposure time

- Increase in mRNA expression of GST-Sigma, Omega, Mu5, Theta3 and Delta1 - Increase in ROS, GSH, GST, GPx, GR and SOD activities - Increase in mRNA expression of GST-Delta-E(1) in 0.5 mg/L - Increase in mRNA expression of GST-Omega mRNA in 1 and 2 mg/ L - Increase in mRNA expression of mGST1 in 1 mg/L - Increase in mRNA expression of GST-Sigma in all concentrations - Increase in mRNA expression of GST-Omega and mGST1 mRNA in 5 mg/L - Increase in mRNA expression of GST-Sigma in 10 and 15 mg/L - Increase in mRNA expression of GST-Delta-E(1) in 6 mg/L - Increase in mRNA expression of GST-Mu5 in 4 and 6 mg/L - Increase in mRNA expression of GST-Sigma in all concentrations - Increase in mRNA expression of GST-Delta-E(1), GST-Omega and GST-Sigma in 4 μg/L - Decrease in mRNA expression of GST-Sigma until 60 min, but increase in 180 min - Increase in mRNA expression of GST-Sigma - Increase in mRNA expression of GST-Sigma until 24 hr, but decrease in 48 h

Key points

References

J.C. Park et al.

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Acknowledgements This work was supported by a grant from the Development of Techniques for Assessment and Management of Hazardous Chemicals in the Marine Environment program of the Korean Ministry of Oceans and Fisheries to Jae-Seong Lee and also supported by a grant from the National Fisheries Research and Development Institute, Korea (R2019025). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquatox.2019.01.020. References Ahmad, I., Oliveira, M., Pacheco, M., Santos, M.A., 2005. Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without β-naphthoflavone pre-exposure. Chemosphere 61, 267–275. Akhila, J.S., Deepa, S., Alwar, M.C., 2007. Acute toxicity studies and determination of median lethal dose. Curr. Sci. 93, 917–920. Alias, Z., Clark, A.G., 2010. 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