Introduction

Salinity regulates physiological conditions of aquatic organisms. Thus, changes in salinity affect survival and growth (Hwang et al. 2012), and modulates stress-related gene expression (Kim and Kang 2015). Sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) is a membrane bound protein, which is responsible for regulation of cellular homeostasis by transporting sodium ions out of the cell and potassium ions into the cell with the consumption of ATP against a concentration gradient (Lingrel and Kuntzweiler 1994; Palmgren and Axelsen 1998; Kaplan 2002; Geering 2008). It is a heterodimeric protein composed of alpha (110-120 kDa), beta (40-60 kDa) and gamma (8-14 kDa) subunits that are encoded by different genes (Blanco and Mercer 1998). The gamma subunit is only found in vertebrates (Therine and Blostein 2000).

The alpha subunit belongs to the P-type ATPase family is a functional protein containing catalytic domains, and is conserved in eukaryotes, except for plants (Zhu 2001; Saez et al. 2009). The beta subunit composes of a cytoplasmic tail, an extracellular protein, and a single transmembrane domain. This subunit is known to be involved in stability, cell adhesion and membrane orientation of the Na⁺/K⁺- ATPase (McDonough et al. 1990; Noguchi et al. 1990; Hasler et al. 1998; Geering 2001; Genova and Fehon 2003; Paul et al. 2003; Barwe et al. 2005).

Cloning and characterization of Na⁺/K⁺-ATPase have been documented in the few aquatic organisms, such as teleost (Semple et al. 2002; Deane and Woo 2005) and crustacean (Lind et al. 2013; Sathapondecha et al. 2014). Complete mRNA sequences of Na⁺/K⁺-ATPase alpha subunit in several invertebrates have been registered in the National Center for Biotechnology Information (NCBI) (see review by Thabet et al. (2016)), but those of rotifer is still vacant. In silico analysis showed that most of invertebrates including Arthropoda, Cnidaria, Prifera, and Mollusca have one copy of the alpha subunit gene, whereas the nematodes have 2 to 4 copies (Thabet et al. 2016).

It is known that Na⁺/K⁺-ATPase is involved in hypo- and hyper-osmoregulation in mainly halophilic and euryhaline organisms (Lima et al. 1997; Lucu et al. 2000). Its activity increased in intertidal crustaceans upon exposure to hyposalinity (Lucu and Flik 1999) and in the freshwater shrimp Artemia franciscana upon exposure to hypersalinity (Holliday 1990). In European sea bass, activity of Na⁺/K⁺-ATPase increased in both freshwater and hypersaline water (50 and 60 ppt) (Jensen et al. 1998). However, in osmoconforming marine crustaceans that exhibit osmotic equilibrium with the environment, such as decapods, activity of Na⁺/K⁺- ATPase was not affected by salinity (Lucu et al. 2000). These findings suggest that status of Na⁺/K⁺-ATPase activity is a powerful biomarker of osmotic stress against environmental stressors.

The monogonont rotifer, Brachionus koreanus, is abundant in freshwater and seawater all over the world (Hagiwara et al. 1995; Gómez and Snell 1996). These animals have several advantages, such as short life cycle (approximately 1 day), high fecundity, ease of maintenance, and genetic homozygosity (Dahms et al. 2011), making them a useful model organism for aquaculture, ecotoxicology, ecophysiology, and environmental genomics. Animals belonging to the genus Brachionus have been known as osmoconformers. However, Lowe et al. (2005) recently reported that the euryhaline Brachionus plicatilis is in fact an osmoregulator, because the activity of its Na⁺/K⁺-ATPase was modulated by salinity. Our recent study showed that Bk Na⁺/K⁺-ATPase may be involved in cellular defense against heavy metal - mediated osmotic stress (Kim et al. 2016). Despite of their ecological importance as energy transfer, physiological and molecular study on osmoregulation was still limited in the rotifer.

The aims of this study were to 1) identify and molecular characterize the Na⁺/K⁺-ATPase in B. koreanus, and 2) analyze the enzyme activity of the Na⁺/K⁺-ATPase in response to hypo- and hyper-salinity condition.

Materials and methods

1.Culture and maintenance

The monogonont rotifer, Brachionus koreanus (S type) was collected at Uljin (36°58′43.01″N, 129°24′28.40″E) in South Korea and transported to the laboratory of genetics in Sangmyung University, Seoul, South Korea. They were reared and maintained in filtered artificial seawater (TetraMarine Salt Pro, Tetra™, OH, USA) adjusted to 25°C with a light/dark cycle of 12:12 h photoperiod, dissolved oxygen ≥6.3 mg L^-1, and salinity of 15 practical salinity units (psu). Culture vessel (clear square plastic aquaria; 12 L capacity) was filled with 10 L artificial seawater, and approximately 50% of the water was changed every 3 days. The green algae, Chlorella ellipsoidea (approximately 1×10⁵ cells mL^-1; AQUANET Co., Kyungnam, South Korea) was supplied daily as a food source.

2.Cloning and sequence analysis

A partial sequence of the rotifer Na⁺/K⁺-ATPase was obtained from the Rotifer Local Genome Database (supplied by the Molecular and Environmental Biology Lab of Sungkunkwan University). To confirm the sequence, the Na⁺/ K⁺-ATPase gene was amplified from B. koreanus cDNA using appropriate PCR primers (Table 1), and the sequence was searched using NCBI blastx (http://www.ncbi.nih.gov/BLAST/). To obtain the full-length sequence of Na⁺/K⁺- ATPase cDNA, 5′- and 3′-rapid amplification of cDNA ends (RACE) was performed using the CapFishing™ Full-Length cDNA Premix Kit (Seegene, South Korea) according to manufacturer’s protocol. The amplicon was ligated into pGEM®-T Easy Vector (Promega, USA) at 4°C, and the plasmid was subsequently transformed into Escherichia coli JM109. Selected plasmids were sequenced using ABI3700 DNA Analyzer (Genotech, Co., Daejeon, South Korea). NCBI Conserved Domain Database (CDD) and TMHMM Server v. 2.0 were used to search for conserved domains and transmembrane domains, respectively.

3.Phylogenetic analysis

To investigate the phylogenetic relationship of B. koreanus Na⁺/K⁺-ATPase, sequences from other species were obtained from GenBank, and subsequently aligned using ClustalX 1.83 (Tompson et al. 1997). GenBank accession No. of species used for this study are the following: Rhabdosargus sarba (AAT48993.1), Fundulus heteroclitus (AAL18002.1), Oreochromis mossambicus (Q9YH26.2), Oncorhynchus mykiss (AAQ82788.1), Artemia franciscana (X56650.1), Dugesia japonica (BAA32798.1), Drosophila melanogaster (CCJ09645.1), Anopheles gambiae (XP_003436270.1), Aedes aegypti (XP_001662218.1), Bathypolypus bairdii (AEH68839.1), Brachionus plicatilis (AAZ53208.1), and Brachionus plicatilis (AAZ53211.1). Multiple aligned sequences were analyzed by MEGA5.0 (ver. 5.1) using neighbor- joining (NJ) method. Bootstrap value was set to 1000 replications. The phylogenetic tree was generated by Kimura2- parameter model and visualized by TreeExplorer.

4.Measurement of Na⁺/K⁺-ATPase activity in different salinity

After acclimation to 15 psu for 24 h, adult B. koreanus (20,000 individuals 50 mL^-1) were transferred to hyposaline conditions (6 psu) and hypersaline conditions (32 psu) for 48 h. Activity of Na⁺/K⁺-ATPase was measured by using QuantiChromTM ATPase/GTPase Assay Kit (DATG-200, Bioassay system, USA) according to manufacturer’s instructions and methods by Brooks and Mills (2003) and Jorge et al. (2013) with a minor modification. In brief, phosphate standard solutions of 0, 15, 30, and 50 μM were prepared. After exposure, each sample was homogenized in 0.1 M Tris-HCL (pH 7.5) using a sonicator (VCX130, Vibra cell, USA), and total protein was quantified by Bradford assay (Bradford 1976). Standard and experimental samples were transferred into 96-well plates, and then buffer 1 (20 μL 2× Assay buffer (containing 40 mM Tris, 80 mM NaCl, 8 mM MgAC₂, 1 mM EDTA, pH 7.5), 10 μL enzyme, 10 μL 4 mM ATP (SIGMA, USA), 10 μL distilled water (D.W.) and 2 mM ouabain (SIGMA, USA), known as a specific inhibitor of Na⁺/K⁺-ATPase) and buffer 2 (20 μL 2×Assay buffer, 10 μL enzyme, 10 μL 4 mM ATP) with 20 mM KCl (JUNSEL, Japan) were added to the experimental samples. Absorbance was measured at 620 nm at 30 minutes after the addition of 200 μL of reagent in each well, using in microplate absorbance spectrophotometer (xMarkTM, Bio-rad, USA). Na⁺/ K⁺-ATPase activity was determined as follows: absorbance of buffer 2 - absorbance of buffer 1. Enzyme activity was expressed as a percentage of control activity.

5.Statistical analysis

Data are expressed as means±S.D. of three replicates. Comparison of relative mRNA expression was analyzed using one-way analysis of variance (one-way ANOVA) followed by Tukey’s test. PASW statistics ver. 18.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A p value below 0.05 was considered statistically significant.

Results

1.loning and sequence analysis of the B. koreanus Na⁺/K⁺-ATPase alpha subunit

The B. koreanus Na⁺/K⁺-ATPase (designated as Bk Na⁺/K⁺-ATPase) open reading frame (ORF) was 3069 bp-long, encoding a 1022-amino acid (aa) polypeptide. Polyadenylation signal (AATAAA) was observed in the 3′-untranslated region (UTR) (Suppl. Fig. 1). Sequence of the Bk Na⁺/K⁺- ATPase was deposited in GenBank with the accession number KP405838. Its theoretical pI value was 5.83, and calculated molecular weight was 113.82 kDa. Conserved domain analysis showed that the Bk Na⁺/K⁺-ATPase has five domains, such as cation ATPase N superfamily domain (40-113 aa), E1-E2 ATPase superfamily domain (138-366 aa), Hydrolase-like2 superfamily domain (427-520 aa), HAD-like superfamily domain (626-705 aa), and cation transporting ATPase domain (798-1007 aa) (Fig. 1). Prediction of transmembrane helices using PROTTER server v. 1.0 showed that the Bk Na⁺/K⁺-ATPase possesses eight membrane spanning regions, the same number as Oncorhynchus mykiss and Drosophila melanogaster (Fig. 2). We found four conserved phosphorylation motifs (TGES (219-222 aa), DKTGTLT (376-382 aa), KGAP (507-510 aa), TGDGVNDSP (714-722 aa)) (Colina et al. 2007; Morth et al. 2007) (Fig. 2). From the NCBI blastx search, the Bk Na⁺/K⁺-ATPase amino acid sequence had the highest identity with the migratory locust Locusta migratoria Na⁺/K⁺-ATPase alpha 2 (77%) and the yellow fever mosquito Aedes aegypti Na⁺/K⁺-ATPase alpha (77%). Based on these findings, we designated this putative protein as the Bk Na⁺/K⁺-ATPase alpha subunit.

2.Phylogenetic relationships

To investigate phylogenetic relationship, the amino acid sequence of the Bk Na⁺/K⁺-ATPase alpha subunit was compared with those of other species retrieved from GenBank. Full-length sequence of the Bk Na⁺/K⁺-ATPase alpha subunit showed high identity to the one from the African malaria mosquito Anopheles gambiae (75%) to goldlined seabream Rhabdosargus sarba (70%). The Bk Na⁺/K⁺-ATPase sequence showed 95% identity to partial regions of the Brachionus plicatilis Na⁺/K⁺-ATPase. In the phylogenetic tree, the Bk Na⁺/K⁺-ATPase was clustered with other Brachionus sp. and separately located from flatworm, arthropods, and chordates (Fig. 3).

3.Altered Na⁺/K⁺-ATPase activity in response to salinity

After exposure to different salinity for 48 h, Bk Na⁺/K⁺- ATPase activity was significantly elevated in both hyposaline (6 psu) and hypersaline (32 psu) up to approximately 1.6-fold (p<0.001), compared with that in the control (15 psu) (Fig. 4).

Discussion

Na⁺/K⁺-ATPase is known to play a key role in osmoregulation across the epithelial cell membrane. Identification and molecular characterization of Na⁺/K⁺-ATPase was reported in several aquatic invertebrates (see review by Tabet et al. 2016). However, physiological and molecular study on osmoregulation are still lack in the rotifer. In the present study, we cloned and molecular characterized Na⁺/K⁺- ATPase in the monogonont rotifer, B. koreanus, and subsequently investigated the effect of salinity on this enzyme.

The total length of Bk Na⁺/K⁺-ATPase ORF was similar with those of other organisms compared in this study, ranging from 3003 bp (A. gamiae XP_003436270.1) to 3090 bp (B. bairdii AEH68839.1). The Bk Na⁺/K⁺-ATPase had five conserved domains which are observed in ATPase. Like it was proposed by cited in NCBI site, the N-terminal domain (40 to 133 aa) and the C-terminal domain (798 to 1007 aa) are cation- (Na⁺/K⁺, H⁺/K⁺, Ca²⁺, and Mg²⁺) transporting ATPase domains. The E1-E2 ATPase superfamily domain, which plays a role in auto-phosphorylation, is found in 138 to 366 aa. Hydrolase-like 2 superfamily domain (427 to 520 aa) and HAD-like superfamily domain (626 to 705 aa) are common in hydrolases.

The Bk Na⁺/K⁺-ATPase had eight transmembrane spanning regions, and were same as those predicted using amino acid sequences of eight species retrieved from GenBank (Rhabdosargus sarba AAT48993; Fundulus heteroclitus AAL18002; Precursor Tilapia Q9YH26; Oncorhynchus mykiss AAQ82788; Artemia franciscana X56650; Dugesia japonica BAA32798; Drosophila melanogaster CCJ09645; Anopheles gambiae XP_003436270; Aedes aegypti XP_00 1662218; and Bathypolypus bairdii AEH68839). Among them, the Oncorhynchus mykiss and Drosophila melanogaster Na⁺/K⁺-ATPase transmembrane structures with Bk Na⁺/ K⁺-ATPase are shown in Fig. 2. Together with domain searching data, this finding suggests that this protein seems to be conserved both structurally and functionally. In addition, when amino acid sequence of the Bk Na⁺/K⁺-ATPase was compared with those of the alpha and beta subunits of human, rat, and mouse proteins, phylogenetic analysis showed that the Bk Na⁺/K⁺-ATPase was clustered with the alpha subunits, but not beta subunits (data not shown). Based on these findings, this protein of B. koreanus was designated Na⁺/K⁺-ATPase alpha subunit.

Under hyposaline and hypersaline conditions (6 and 32 psu, respectively), Bk Na⁺/K⁺-ATPase activity was significantly induced compared to the control group (15 psu) in the present study. This is similar to the findings by Lowe et al. (2005), which demonstrated that Na⁺/K⁺-ATPase activity was elevated with increasing salinity in Brachionus plicatilis. However, the patterns of modulation of Na⁺/K⁺-ATPase activity in response to salinity seem to be different depending on the species and their habitat, although the protein may be involved in osmoregulation. Indeed, in shore crab Carcinus maenas, the Na⁺/K⁺-ATPase activity was induced by decreased salinity (Lucu and Flik 1999), whereas that of the euryhaline European sea bass acclimated at 15 psu increased in freshwater and high salinity seawater (50 and 60 ppt) after 10 days (Jensen et al. 1998), consistent with our results. Lowe et al. (2005) reported that high Na⁺/K⁺-ATPase activity are correlated with high ATP consumption, and ultimately result in growth inhibition. Thus, chronic osmotic stress may have adverse effects in aquatic organisms.

In conclusion, the Bk Na⁺/K⁺-ATPase alpha subunit was evolutionarily conserved, and seemed to be involved in osmoregulation against changes in salinity. Thus, Na⁺/K⁺-ATPase alpha subunit may be useful molecular biomarker for evaluation of health status and environmental contaminants - mediated osmotic stress in the rotifer. In addition, this study is helpful for better understanding of the physiology of the rotifer.