ZOOLOGICAL SCIENCE 33: 414–425 (2016) © 2016 Zoological Society of Japan

A Stenohaline Medaka, woworae, Increases Expression of Gill Na+, K+-ATPase and Na+, K+, 2Cl– Cotransporter 1 to Tolerate Osmotic Stress

Jiun-Jang Juo1†, Chao-Kai Kang2†, Wen-Kai Yang1†, Shu-Yuan Yang1, and Tsung-Han Lee1,3*

1Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan 2Tainan Hydraulics Laboratory, National Cheng Kung University, Tainan 709, Taiwan 3Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan

The present study aimed to evaluate the osmoregulatory mechanism of Daisy’s medaka, O. woworae, as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of eury- haline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy’s medaka belongs to the celebensis group. The tolerance of Daisy’s medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy’s medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, “Experimental (Exp.) 10‰ SW”, was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy’s medaka significantly increased. The protein abun- dance and distribution of branchial Na+, K+-ATPase (NKA) and Na+, K+, 2Cl– cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA α-subunit protein- abundance was found in the 10‰ SW-acclimated . In gill cross-sections, more and larger NKA- immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

Key words: Oryzias woworae, gill, Na+, K+-ATPase, Na+, K+, 2Cl– cotransporter 1, stenohaline, osmotic stress

mechanisms is known as . In general, fresh INTRODUCTION and marine water-living fish tend to manage a net water Teleosts are aquatic vertebrates that cope with osmotic influx or efflux in order to keep constant plasma osmolality. and ionic gradients in aquatic environments with diverse In the branchial epithelium, a specialized cell type, iono- (Hwang and Lee, 2007). Species of genus Oryzias cytes, also known as mitochondria-rich cells, are the main exhibit the ability to adapt to a wide spectrum of environ- site for the active transport of ions. The driving force for mental salinity, although adaptability varies from species to active transport is created by Na+, K+-ATPase (NKA), which species. Inoue and Takei (2003) reported that O. javanicus exchanges two extracellular K+ molecules with three intrac- and O. dancena exhibited highly salinity adaptability. O. ellular Na+ via the hydrolysis of one molecule of ATP. javanicus and O. dancena survived a direct transfer from Previous studies have demonstrated that NKA is localized freshwater (FW) to seawater or to 1/2 seawater. Meanwhile, specifically to the tubular system (Hootman and Philpott, O. latipes survived subsequent direct transfer to full-seawater 1979; Dang et al., 2000). Significant increases in the abun- (35‰) with pre-acclimation to 1/2 seawater, whereas O. dance and activity of NKA, as well as the number and size marmoratus was intolerant to SW transfer or 1/2 seawater of NKA-immunoreactive cells (NKA-IR cells) were also transfer (Inoue and Takei, 2003). observed upon salinity challenge in gills of the salmonids The ability to adapt to environmental salinity by certain (Hiroi and McCormick, 2007) and medaka (Kang et al., 2008). Both euryhaline medakas, O. latipes and O. dancena, * Corresponding author. Tel. : +886-4-2285-6141; Fax : +886-4-2287-4740; exhibit clear seawater adaptation mechanisms on their bran- E-mail: [email protected] chial ionocytes. On salinity challenge, enhanced expression † JJJ, CKK, and WKY contributed equally to this paper. of NKA was found in gills of the O. latipes and O. dancena, Supplemental material for this article is available online. accompanied by enlarged NKA-IR cell size, increased NKA- doi:10.2108/zs150157 IR cell number, and elevation of NKA activity (Kang et al., Hypo-osmoregulation of Daisy’s medaka 415

2008). Upregulated protein or gene expressions of branchial Fish and experimental environments NKCC1 were found in both O. latipes (Hsu et al., 2014) and Adult Daisy’s medaka (Oryzias woworae) and brackish O. dancena (Kang et al., 2010) in response to salinity medaka (O. dancena) with an average standard length of 2.5 ± 0.4 challenge. Previous studies indicated that Cl– secretion in cm were obtained from local aquaria and reared in 10-liter aquarium ionocytes was dependent on basolateral Na+, K+, 2Cl– tanks containing air-exposed local tap water for at least four weeks under 14 hr:10 hr light: dark photoperiod at 28 ± 1°C. The water was cotransporter 1 (NKCC1) (Marshall, 2002; Hwang and Lee, partially refreshed (50%) every week. For our experiments, 5‰, 10‰, 2007; Seo et al., 2013; Chandrasekar et al., 2014). Basolateral 15‰, 20‰, and 35‰ saltwater (SW) were prepared from freshwa- – NKCC accumulates Cl intracellularly above its electrochem- ter with proper amounts of the synthetic sea salt “Instant ” – ical equilibrium so that Cl exits passively via anion channels (Aquarium Systems, Mentor, OH, USA). The freshwater (FW) was in the apical membrane. The gill ionocytes of euryhaline aerated local tap water. The fish were fed with commercial pellets teleosts exhibited salinity-dependent expression of NKCC in twice a day. The facilities and protocols for experimental the basolateral membrane upon hyperosmotic stress were approved by the Institutional Care and Use Committee (Tipsmark et al., 2002; Wu et al., 2003; Scott et al., 2004; of the National Chung Hsing University (IACUC approval no. 102– Hiroi and McCormick, 2007; Kang et al., 2010). Hiroi and 114 to THL). Fish were not fed one day before the following experi- McCormick (2007) reported that NKCC abundance was ments, and were anaesthetized with MS-222 (100 mg/l; 3-aminoben- zoic acid ethyl ester, Sigma, St Louis, MO, USA) before sampling. upregulated after transfer from FW to seawater in parallel with gill NKA activity in three salmonids, lake trout (Salvelinus Genomic DNA extraction namaycush), brook trout (Salvelinus fontinalis) and Atlantic Partial caudal fins of Daisy’s medaka were excised and stored (Salmo salar). Interestingly, the Atlantic salmon which at –80°C. For extracting genomic DNA, caudal fins of Daisy’s shows a 100% survival rate on direct seawater transfer, medaka were homogenized with PELLET PESTLE Cordless Motor exhibits a higher gill NKCC abundance than two other salmo- (Kimble Chase, Vineland, NJ) and PELLET PESTLE (Kimble nids. Accordingly, it was speculated that gill NKCC abun- Chase) at maximum speed (3000 rpm) in lysis buffer (100 mM dance is positively correlated with salinity tolerance. NaCl, 1% SDS, 100 mM EDTA, 50 mM Tris) containing Proteinase Daisy’s medaka (Oryzias woworae) is a recently K (Roche Molecular Biochemicals, Indianapolis, IN, USA) and ° described, remarkably colorful species. This species, how- heated to 57 C for 1 hr. Phenol/chloroform was added to the lysed mixture and centrifuged at 4°C, 15,000 g for 5 min. The supernatant ever, is only known to inhabit FW streams of , was removed and mixed with equal volume of isopropyl alcohol and (Parenti and Hadiaty, 2010). Takehana et al. 1/10 volume of 3M sodium acetate followed by cooling at –20°C for 5 (2005) have illustrated three phylogenetic groups in the phy- min. After subsequent centrifugation at 15,000 g for 10 min, the logenetic tree of medakas. Of these, two euryhaline medaka supernatants were discarded and the pellets were dissolved in ster- , the Japanese medaka (O. latipes) and the brackish ilized water. For constructing a phylogenetic tree, 12S rRNA and medaka (O. dancena) are respectively classified into the 16S rRNA gene sequences of the Daisy’s medaka were amplified latipes and javanicus species groups (Takehana et al., by PCR with primers designed according to Takehana et al. (2005). 2005). On the other hand, the O. marmoratus exhibiting The PCR products were separated by 1% agarose gel electropho- stenohalinity (Inoue and Takei, 2002, 2003) was classified to resis, and a single band area of the gel was excised for purification be a member of celebensis species group (Takehana et al., by 1-4-3 DNA Extraction (PROTECH, Taipei, Taiwan). The purified PCR product (12S rDNA, 430 bp; 16S rDNA, 525 bp) was ligated 2005). According to the experiments of previous studies to pOSI-T-vector and then transfected to DH5α competent cells (Inoue and Takei, 2002; Kang et al., 2010), different mortal- using pOSI-T PCR cloning kit (GeneMark, Taiwan). The transfected ity rates were observed when Daisy’s medaka were competent cells were cultivated on LB agar plates with kanamycin exposed to various environments with a salinity gradient for the selection of successfully transfected cells. When colonies from FW to SW. Previous studies by our group revealed that reached optimal size, the colonies were collected and sequenced the Japanese medaka and the brackish medaka shared a (ABI 3730 DNA sequencer, Tri-I Biotech Inc., Taipei, Taiwan). similar osmoregulatory mechanism including the elevated NKA activity, increased number of NKA-IR cells, and the Phylogenetic tree enlargement of the NKA-IR cells upon osmotic stress (Kang The gene sequences of 12S rRNA (O. celebensis, AB188691; et al., 2008). To reveal the phylogenetic group of O. woworae, O. curvinotus, AB188693; O. dancena, AB188694; O. hubbsi, AB188696; O. javanicus, AB188698; O. latipes, AB188701; O. the phylogenetic tree of medakas was reconstructed with luzonensis, AB188705; O. marmoratus, AB188706; O. matanensis, genomic DNA sequences of 12S rRNA and 16S rRNA in this AB188707; O. mekongensis, AB188709; O. minutillus, AB188710; study. The present study also evaluated the profiles of gill O. nigrimas, AB188712; O. profundicola, AB188713; Xenopoecilus NKA and NKCC1 abundances in Daisy’s medaka exposed to oophorus, AB188714, and O. sarasinorum, AB188715) and 16S FW and the tolerating saltwater. In addition, the branchial rRNA (O. celebensis, AB188718; O. curvinotus, AB188720; O. NKCC1 abundance between Daisy’s medaka and the brackish dancena, AB188721; O. hubbsi, AB188723; O. javanicus, medaka in the same environmental salinity was compared. AB188725; O. latipes, AB188728; O. luzonensis, AB188732; O. marmoratus, AB188733; O. matanensis, AB188734; O. mekongensis, MATERIALS AND METHODS AB188736; O. minutillus, AB188737; O. nigrimas, AB188739; O. profundicola, AB188740; X. oophorus, AB188741, and O. sarasino- Ethics statement rum, AB188742) of the other medakas were downloaded from the The facilities and protocols for experimental animals were GeneBank, and those of Cololabis saira (AB188689 and approved by the Institutional Animal Care and Use Committee of AB188716) and Cypselurus pinnatibarbatus japonicus (AB188690 the National Chung Hsing University (IACUC approval no. 102–114 and AB188717) were also downloaded, representing outgroup spe- to THL). cies. All gene sequences of 12S rRNA and 16S rRNA of all species described above were then aligned using ClustalW software (available at http://embnet.vital-it.ch/software/ClustalW.html). Subsequently, phy- 416 J.-J. Juo et al. logenetic analysis was conducted with MEGA 5.2 software (Tamura et (SM0671; Hanover, MD, USA). Aliquots containing 20 μg of protein al., 2011) via maximum likelihood method. Reliability of the tree was from the gill homogenates were heated at 65°C for 25 min and frac- assessed by bootstrapping (1000 replications). tionated by electrophoresis on SDS-containing 7.5% polyacrylamide gels. Separated proteins were transferred from unstained gels to Salinity tolerance tests PVDF (Millipore, Bedford, MA, USA) using a tank transfer system Daisy’s medaka were transferred from FW to FW, 5‰, 10‰, (Bio-Rad, Mini Protean 3). For minimizing non-specific binding, blots 15‰, 20‰, 35‰ SW, respectively, with 15 individuals used for were pre-incubated in 5% (v/v) nonfat milk that diluted with each experiment. The 7th day was set as the end-point of the exper- Phosphate Buffered Saline Tween-20 (PBST; 137 mM NaCl, 3 mM iment, from which the number of surviving individuals were summa- KCl, 10 mM Na2HPO4, and 0.2% (v/v) Tween 20, pH 7.4) at room rized. In addition, the experimental 10‰ (Exp. 10‰, 342–352 mOsm/ temperature for 2 hrs. The blots were then incubated at room tem- kg) SW group of adult Daisy’s medaka were transferred from FW to perature with the primary antibody α5 (NKA) diluted in PBST 5‰ SW for seven days first, and then transferred to 10‰ SW for (1:4000) or T4 (NKCC1) diluted in PBST (1:500). The blots were another seven days. Five individuals were exposed to each medium washed with PBST, and subsequently HRP-conjugated secondary for seven days (FW, 5‰, 10‰, 15‰, 20‰, 35‰ SW, and Exp. 10). antibody (1:10,000 dilution) was added. After incubation, the blots The survival rates for transfer to different salinity SW were deter- were developed using a SuperSignal West Pico Detection Kit mined in triplicate to reveal the salinity tolerance of Daisy’s medaka. (#34082, Pierce) and observed with a Universal hood with a cooling- CCD (charge-coupled device) camera (ChemiDoc XRS+, Bio-Rad) Plasma analysis and the associated software (Quantity One version 4.6.8, Bio-Rad). Blood was collected according to the method of Kang et al. Immunoreactive bands were analyzed using Image Lab software (2008). Each plasma sample was pooled from the blood of 10 indi- version 3.0 (Bio-Rad). According to previous studies (Kang et al., viduals. 70 individuals in total were used for determining the plasma 2008 and 2010), the target bands of NKA α-subunit and NKCC1 at osmolality. After centrifugation at 1,000 g and 4°C for 5 min, the 110 kDa and 130–170 kDa, respectively, were used for analyses. plasma was stored at –80°C. Plasma osmolality was determined by The bands were converted to numerical values to compare relative the vapor osmometer 5520 (Wescor, Logan, UT, USA). For deter- protein abundances of the immunoreactive bands. mining the concentration of sodium and chloride ions, each plasma sample was collected from one individual, and the sample size was NKA activity accumulated to seven in this experiment. The concentration of The method to determine gill NKA activity was modified from plasma Na+ was determined using a Hitachi Z-8000 polarized Kang et al. (2008). The assay solution (50 mM imidazole, 0.5 mM Zeeman atomic absorption spectrophotometer (Hitachi, Tokyo, ATP, 2 mM phosphoenolpyruvate (PEP), 0.32 mM NADH, 3.3 U Japan). The concentration of plasma Cl– was determined with the LDH mL–1, and 3.6 U PK mL–1, pH 7.5) was mixed with salt solution ferricyanide method (Franson, 1985) and a VERSAmax ELISA (189 mM NaCl, 10.5 mM MgCl2, 42 mM KCl, 50 mM imidazole, pH microplate reader (Molecular Devices, Sunnyvale, CA, USA). 7.5) in a 3:1 ratio. Before the assay, a standard curve was deter- mined from 0 to 30 nmol ADP per well at 340 nm at 28°C after add- Preparation of gill samples ing 200 μl assay mixture for at least 5 min in a 96-well plate. The Fish from the FW (control) and Exp. 10‰ groups were anes- slope of the standard curve should be –0.012 to –0.015 absorbance thetized with MS-222 then the gills were excised and immediately units nmol ADP–1. A 10 μl sample from one fish was loaded in a stored at –80°C before use. The gills were homogenized in SEID well, and 200 μl assay mixture was added with or without 1 mM buffer (150 mM sucrose, 10 mM EDTA, 50 mM imidazole, and 0.1% ouabain. Each sample was assayed in triplicate. The plate was read sodium deoxycholate, pH 7.5) containing proteinase inhibitor with a every 15 seconds for up to 10 min in a VERSAmax microplate Polytron PT1200E at maximal speed for 10 seconds on ice. The reader (Molecular Devices) at 340 nm, 28°C. The linear rate from 2 homogenates were then centrifuged at 5500 g at 4°C for 15 min. to 10 min for each pair of triplicate wells was determined. Protein The supernatants were used for western blotting and the determi- concentrations of the samples were determined by the Pierce BCA nation of NKA activity. protein assay (Thermo, Rockford, IL, USA). The NKA activity expressed as μmol ADP per mg protein per hr was calculated as Antibodies the difference in slope of ATP hydrolysis (NADH reduction) in the The primary antibodies used in this study included: (1,2) Na+, presence and absence of ouabain. K+-ATPase (NKA) α-subunit: a mouse monoclonal antibody (α5; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) raised Total RNA extraction and reverse transcription against the α-subunit of the avian NKA and a rabbit monoclonal Each total RNA sample from gills was extracted from four individ- antibody (EP1845Y; Abcam, Cambridge, UK) raised against the ual by using the TriPureTM Isolation Reagent (Roche Diagnostics, residues near the N-terminus of human NKA; (3) Na+, K+, 2Cl– Mannheim, Germany) following the manufacturer’s instruction. The cotransporter 1 (NKCC1): a mouse monoclonal antibody (T4; RNA pellet was dissolved in 50 μl Nuclease-free water (GE Healthcare, Developmental Studies Hybridoma Bank) raised against the car- Little Chalfont, Buckinghamshire, UK) and treated with the RNA boxy terminus of human colonic NKCC; (4) β-actin: a mouse mono- clean-up protocol from the RNAspin Mini RNA isolation kit (GE clonal antibody (ab8226; abcam, Cambridge, MA USA) raised Healthcare) following the manufacturer’s instructions to eliminate against the amino acid residues 1–100 of human beta actin. The genomic DNA contamination. RNA integrity was verified by 1% secondary antibody used for immunoblots was horseradish peroxi- agarose gel electrophoresis. Extracted RNA samples were stored at dase (HRP)-conjugated anti-mouse IgG (Pierce, Rockford, IL, –80°C after isolation. USA). The secondary antibodies used for double immunofluores- cence staining were Dylight-488-conjugated goat anti-Rabbit IgG Quantitative real-time PCR (qPCR) and Dylight-549-conjugated goat anti mouse IgG (Jackson Gill nkcc1a mRNA was quantified with MiniOpticon real-time Immunoresearch, West Baltimore Pike, PA, USA). PCR system (Bio-Rad Laboratories, Hercules, CA, USA). The nkcc1a primer sequences were as follows (5′ to 3′): forward— Immunoblotting of NKA and NKCC1 CTCTCCACTTCAGCCATCG and reverse—ACCACATACATG- The protein concentrations of the supernatants were deter- GCAACAGC. The β-actin primer sequences were as follows (5′ to mined with a Pierce BCA protein assay (Thermo). The pre-stained 3′): forward—TCGCTGACAGAATGCAGAAG and reverse—GCTG- protein molecular weight marker was purchased from Fermentas GAAGGTGGACAGAGAG. PCR reactions contained 8 μl of cDNA Hypo-osmoregulation of Daisy’s medaka 417

(200×), 2 μl of either 5 μM nkcc1a-QPCR primer mixture or 5 μM T4 diluted in blocking buffer at room temperature for 2 hrs. After β-actin primer mixture, and 10 μl of 2× FastStart Universal SYBR incubation, the cryosections were washed several times with PBS, Green Master (Roche Diagnostics, Mannheim, Germany). The exposed to the secondary antibody (Alexa-flour 488 goat anti- nkcc1a mRNA values were normalized using the expression of the mouse antibody) at room temperature for 1 hr and then washed with β-actin mRNA (KT031391) from the same DNA samples. The PBS again. After the first staining, the cryosections were incubated occurrence of secondary products and primer-dimers was inspected with the monoclonal anti-NKA antibody (EP1845Y) diluted in block- using melting curve analysis and electrophoresis to confirm that the ing buffer at room temperature for 2 hrs. The cryosections were amplification was specific. One identical cDNA sample from the washed with PBS several times, and then exposed to the secondary 10‰-acclimated fish was used as the internal control among differ- antibody (Dylight-549-conjugated goat anti-rabbit antibody) at room ent groups. For each unknown sample, the comparative Ct method temperature for 1 hr, followed by several PBS washes. The sections TM with the formula 2^[(Ctnkcc1a,n-Ctβ-actin,n)-(Ctnkcc1a,c-Ctβ-actin,c)] was were then covered by a coverslip with clearmount mounting solu- used to obtain the corresponding nkcc1a and β-actin values, where tion (Thermo Fisher Scientific, Carlsbad, CA, USA) and observed Ct corresponded to the threshold cycle number. with a fluorescence microscope (BX50, Olympus, Tokyo, Japan) or a laser scanning confocal microscope (FV1000, Olympus). Cryosectioning and double immunofluorescence staining Micrographs were taken using the cooling-CCD camera (DP72, The excised gills of Daisy’s medaka were fixed with 10% neutral Olympus). Only those cross sections of filaments about 135 ± 5 μm buffered formalin for 2 hrs. For cryosectioning, the gills were perme- in length were randomly selected to minimize the effects of cutting ated with methanol for 30 min at –20°C after phosphate buffer saline angles on the sections. Ten gill filaments in the field of the fluores- (PBS). The samples were then stored in methanol at –20°C before cent microscope were randomly chosen, and the area and counts performing the following experiments. The samples were washed of NKA-IR cells were averaged to represent the average size and with PBS and then infiltrated with O.C.T. (optimal cutting tempera- number of ionocytes on a single gill filament of a fish. ture) compound (Sakura, Tissure-Tek, Torrance, CA, USA) over- night at 4°C. Subsequently, the samples were mounted in the Statistical analysis O.C.T. compound. Gills were cut in cross-sections of 5 μm thick- Values were compared with the Student's t test, and P < 0.05 ness using a Leica CM3050 S cryostat (Leica Microsystems, was set as the significance level. Values were expressed as the Wetzlar, Germany) at –20°C. The sections were placed on 0.01% mean ± S.E.M. (the standard error of the mean). The survival rate poly-L-lysine (Sigma) coated slides and kept in slide boxes at –20°C was analyzed by one-way analysis of variance followed by before immunofluorescence staining. Dunnett’s test multiple comparison against a single control. Cryosections were rinsed with PBS, incubated in blocking buf- fer (1.25% BSA and 0.05% sodium azide in PBS) for 30 min, and RESULTS then washed with PBS and incubated with the monoclonal antibody Phylogenetic tree The 12S (KM582167) and 16S (KM582168) rRNA genes were cloned from Daisy’s medaka using prim- ers designed according to Takehana et al. (2005). To clarify the relationship between Daisy’s medaka and the other medakas, a phylogenetic tree of medakas was constructed and then classified into three groups according to Takehana et al. (2005), including the latipes species group, the celebensis species group, and the javanicus species group. Pair-wise sequence

Fig. 2. Survival rate of Daisy’s medaka transferred directly Fig. 1. (A) Photographs show the phenotype of male (left) and female (right) from fresh water (FW) to FW or salt water (5‰, 10‰, 15‰, Daisy’s medaka (Oryzias woworae). (B) Phylogenetic relationships of medaka 20‰) for 7 days (n = 15 for all groups). “Exp. 10‰” indicates fishes inferred from the datasets of mitochondrial 12S rRNA and 16S rRNA that the Daisy’s medaka were first transferred to 5‰ salt genes. Daisy’s medaka (shaded) genetically belongs to the celebensis species water (5‰) from FW for one week and then transferred to group. The phylogenetic relationships were analyzed by the maximum-likelihood 10‰ salt water (10‰) for another week. The asterisks indi- method. The numbers beside the branches indicate the bootstrap values. Inoue cate significant difference (P < 0.05) using Dunnett’s test for and Takei (2003) revealed that O. latipes, O. dancena, and O. javanicus exhib- comparison against a single control (FW) following one-way ited euryhalinity (*), whereas O. marmoratus exhibited stenohalinity (†). ANOVA. Values are means ± S.E.M. ND, not detected. 418 J.-J. Juo et al. comparisons of the concatenated 12S and 16S rRNA genes medaka survived in 20‰ saltwater (SW) and only 53.3% of of species in the genus Oryzias indicated that the sequence tested fish survived in 10‰ SW. The more the water salinity isolated from O. woworae shared a 94–97% nucleotide sim- increased, the more the survival rate decreased. After pre- ilarity with species in celebensis species group, a 89–91% acclimation to 5‰ SW for one week, the survival rate of the nucleotide similarity with species in latipes species group, medaka in 10‰ SW increased to 62.5% (Fig. 2). For the fol- and a 88–90% nucleotide similarity with species in javanicus lowing experiments, the medaka were pre-acclimated to 5‰ species group. Therefore, Daisy’s medaka should belong to SW for one week and subsequently transferred to 10‰ SW the celebensis species group (Fig. 1). before sampling (the “Experimental 10‰ group”).

Salinity tolerance of Daisy’s medaka Plasma analysis After direct transfer from fresh water (FW), no Daisy’s The plasma osmolality of the 10‰ SW medaka was 17% higher than that of fish acclimated to FW (Fig. 3A). On the other hand, 44% higher plasma Na+ concentration was found in the 10‰ SW medaka compared to the FW group (Fig. 3B). The Cl– concentration of the 10‰ SW medaka

Fig. 4. Expression of NKA α-subunit protein and NKA activity in Fig. 3. Plasma osmolality, Na+ and Cl– concentrations of the gills of the Daisy’s medaka acclimated to fresh water (FW) and 10‰ Daisy’s medaka acclimated to fresh water (FW) and 10‰ salt water salt water (10‰). (A) The representative immunoblot of NKA α- (10‰). (A) Plasma osmolality of the FW group was significantly subunit. (B) Relative intensities of the immunoreactive bands of higher than the 10‰ SW group (n = 4 for both groups). Concentra- NKA α-subunit between FW and 10‰ SW group (n = 25 for both tions of plasma sodium (B) and chloride (C) were also higher in the groups). (C) The NKA activity in FW and 10‰ SW group (n = 10 for FW group than in the 10‰ SW group (n = 7 for both groups). The both groups). β-actin was used as the loading control. The asterisk asterisk indicates a significant difference (P < 0.05) using Student’s t indicates a significant difference (P < 0.05) using Student’s t test. test. Values are means ± S.E.M.. Values are means ± S.E.M.. Hypo-osmoregulation of Daisy’s medaka 419

was 43% higher than that of the FW individuals (Fig. 3C). the afferent epithelium of the filaments (Fig. 5A and 5B). The average sizes of NKA-IR cells were significantly larger in the Salinity effects on gill Na+, K+ -ATPase (NKA) 10‰ SW group (61.4 ± 17.6 μm2) than those in the FW group Immunoblots of gill NKA in Daisy’s medaka probed with (37.2 ± 12.0 μm2) (Fig. 5C). In addition, the average numbers the monoclonal antibody (α5) indicated that the immunore- of NKA-IR cells significantly increased in 10‰ SW group active bands were at 110 kDa (Fig. 4A). Quantification of the (approximately 1.5-fold) compared to the FW group (Fig. 5D). immunoreactive bands of different groups revealed that the protein abundance of NKA was significantly increased Salinity effects on gill Na+, K+, 2Cl– cotransporter 1 (approximately 4-fold) in the 10‰ SW group compared with (NKCC1) in the FW group (Fig. 4B). The gill NKA activity of fish sig- Quantification of nkcc1a gene in gills of Daisy’s medaka nificantly increased (approximately 2-fold) after exposure to revealed that the mRNA level in the 10‰ SW group was 10‰ SW when compared with the FW group (Fig. 4C). The higher (approximately 1.5-fold) than that in the FW group micrographs of the gill cross-sections showed that the NKA (Fig. 6A). Immunoblots of gill NKCC1 in Daisy’s medaka immunoreactive (NKA-IR) cells were mainly distributed in probed with the monoclonal antibody (T4) revealed the

Fig. 6. Expression of Na+, K+, 2Cl– cotransporter1 (NKCC1) Fig. 5. Immunofluorescence staining and average size and number of in gills of the Daisy’s medaka acclimated to fresh water (FW) NKA-immunoreactive (IR) cells in the gills of Daisy’s medaka acclimated to and 10‰ salt water (10‰). (A) The mRNA levels of nkcc1a in fresh water (FW) and 10‰ salt water (10‰) (n = 5 for both groups). Repre- the gills of the FW and 10‰ SW Daisy’s medaka (n = 6 for sentative immunofluorescence staining of NKA in the gill filaments in (A) FW both groups). (B) The representative immunoblot probed with and (B) 10‰ SW groups. (C) The average size of NKA-IR cells in the FW a monoclonal antibody (T4). (C) Relative intensities of immu- and 10‰ SW group. (D) The average number of NKA-IR cells in the FW and noreactive bands of NKCC1 in the FW and 10‰ SW group (n = 10‰ SW groups. The asterisk indicates a significant difference (P < 0.05) 12 for both groups). β-actin was used as the loading control. using Student’s t test. C, cartilage; EF, efferent region of the gill filaments. The asterisk indicates a significant difference (P < 0.05) using Scale bar, 50 μm. Student's t test. Values are means ± S.E.M.. 420 J.-J. Juo et al.

immunoreactive bands ranged from 95 to 170 kDa (Fig. 6B). There were three bands at 95, 110, and 120 kDa, and a smear band at 130–170 kDa in the Fig. 6A. Quantification of the immunoreactive bands (from 130 to 170 kDa) of different groups showed that the protein abundance of NKCC1 was significantly higher in the 10‰ SW group than in the FW group (Fig. 6C). No significant differences were found at that band at 95 kDa between FW and 10‰ SW group. At least 10 gill slides in both experimental conditions were applied to make sure that all the expression patterns were constant. The confocal micro- graphs were taken from the best immuno- fluorescence stained slide. Immunostaining of the gill filaments with the antibody T4 revealed basolateral signals in NKA-IR cells in the 10‰ SW group, while the FW Fig. 7. Confocal micrographs of double immunofluorescence staining with the anti-NCC/ group showed apical signals in NKA-IR cells NKCC1 (red) (A, D) and anti-NKA α-subunit antibody (green) (B, E) on the afferent (Fig. 7). regions of the gill filaments in the Daisy’s medaka acclimated to fresh water (FW) and 10‰ salt water (10‰ SW). The merged images revealed that NKCC1 was localized in the Comparisons of gill NKCC1 expression basolateral membrane of NKA immunoreactive (NKA-IR) cells in the 10‰ SW group between O. dancena and O. woworae (arrows) (F). However, the antibody T4 detected the apical signal on NKA-IR cells in the Between the same quantity of total FW group (arrow heads) (C). C, cartilage; EF, efferent region of the gill filaments. Scale lysates in the gills of O. dancena and O. μ bar, 20 m. woworae exposed to 10‰ SW, different abundances of gill NKCC1 protein were detected and compared. Immunoblots of gill NKCC1 in both O. dancena and O. woworae probed with the antibody T4 indicated that the immunoreactive bands ranged from 95 to 170 kDa (Fig. 8A). Quantification of the immunoreactive bands showed that the basic level of NKCC1 protein of 10‰ SW-acclimated O. dancena was significantly higher than that of 10‰ SW-acclimated O. woworae (approximately 3- fold) (Fig. 8B). In addition, the ratios of gill NKCC1 protein between FW and 10‰ SW in both O. dancena and O. woworae were increased by approximately 2-fold (Fig. 6C, Supplementary Figure S1 online). DISCUSSION The phylogenetic tree based on mitochondrial ribosomal RNA (rRNA) is a valid representation of organismal geneal- ogy (Woese, 2000). Phylogenetic trees based on 12S and 16S rRNA genes have been constructed for many teleosts, e.g., Leuciscinae fishes (Bufalino and Mayden, 2010); Loricariinae catfish (Rodriguez et al., 2011); holocephalan fishes (Licht et al., 2012). Takehana (2005) reported that the phylogenetic tree of Oryzias species was separated into three monophylogenetic groups, the O. latipes species

Fig. 8. Expression of NKCC1 protein in the gills of O. woworae (OW) and O. dancena (OD) acclimated to 10‰ salt water (10‰ SW). (A) Immunoblots of O. woworae and O. dancena probed with the monoclonal antibody T4. (B) The basic level of NKCC1 protein in 10‰ SW-acclimated O. dancena and O. woworae (n = 6 for each group). β-actin was used as the loading control. The asterisk indi- cates a significant difference (P < 0.05) using Student’s t test. Val- ues are means ± S.E.M.. Hypo-osmoregulation of Daisy’s medaka 421 group, the O. javanicus species group, and the O. celebensis of ionocytes in gills (Marshall, 2002; Hirose et al., 2003; species group. Our phylogenetic tree also separated meda- Evans et al., 2005; Hwang and Lee, 2007). In branchial ion- kas into three phylogenetic groups and further confirmed ocytes, the activity of NKA exchanges 2K+ in and 3Na+ out that O. woworae belongs to the O. celebensis species of the cell with one cost of ATP and sustain a substantial group, which conformed to the phylogenetic tree construc- cytosol-negative membrane potential and a chemical gradi- tion using nucleotide sequence of tDNA-Val, 12S and 16S ent across cell membrane, thus provides a driving force for rDNA (Ngamniyom and Phowan, 2014). O. latipes and O. secondary ion-transporting systems (Marshall and Bryson, dancena were separated into different phylogenetic groups, 1998; Hirose et al., 2003; Hwang and Lee, 2007; Hwang et and they both exhibited euryhalinity. O. woworae exhibiting al., 2011). Upon salinity challenge, Daisy’s medaka stenohalinity, however, coincidently fell into the third phylo- increased gill NKA activity when encountered increasing genetic group, the O. celebensis species group. Accord- influx of Na+, similar to that of Trichogaster microlepis ingly, the osmoregulatory mechanism of O. woworae should (Huang et al., 2010). Meanwhile, the gill NKA activity was be different from the two other euryhaline medaka, the also found to increase significantly, parallel to the NKA Japanese medaka (O. latipes) and the brackish medaka (O. abundance in gills. In addition, Marshall (2002) stated that dancena). most euryhaline teleosts exhibit adaptive changes in gill Stenohaline freshwater (FW) teleosts typically can NKA activity following salinity changes. Accumulated survive in distill FW, hard FW, and salt water (SW) with evidences had supported this statement, including salmon salinities up to approximately 10‰ (Evans and Claiborne, (Tipsmark et al., 2002), tilapia (Lee et al., 2003), pufferfish 2006). The survival rates of teleosts following direct or grad- (Lin et al., 2004b), sea bream (Sparus aurata L.; Laiz- ual transfer between environments with different salinities Carrión et al., 2005) and pearl spot (Chandrasekar et al., are generally observed to determine their salinity tolerance 2014). O. latipes and O. dancena were also reported to (Hiroi and McCormick, 2007; Kang et al., 2010, 2012). exhibit adaptive changes in NKA activity upon salinity chal- Hence, the survival rates of adult Daisy’s medaka (Oryzias lenge (Kang et al., 2008). O. latipes, most inhabit in FW, woworae) were explored in this study to evaluate the salinity increased gill NKA activity along with increasing environ- tolerance of this Oryzias species. Pre-acclimation to lower mental salinity, whereas O. dancena, most inhabit in BW, environmental salinity is a helpful method for some teleosts revealed the lowest gill NKA activity in the BW group com- to increase salinity tolerance, including tilapia (Oreochromis pared to the FW and seawater groups. Although 10‰ SW mossambicus; Hwang et al., 1989), silver perch (Bidyanus was generally considered isotonic to the teleosts, pointing to bidyanus; Guo et al., 1995), and O. latipes (Inoue and a lower need to allocate energy to osmoregulation, the pres- Takei, 2003). The present study indicates that although ent study revealed that NKA expression and NKA activity Daisy’s medaka is a stenohaline teleost, pre-acclimation to elevated in gills of the Daisy’s medaka in response to 10‰ lower salinity also increases its survival rate to environments SW. The increased NKA activity indicated more energy allo- with higher salinities. Our findings suggest that Oryzias spe- cated to osmoregulation in gills of the Daisy’s medaka to cies exhibit varying salinity tolerance. supply more driving force for secondary ion-transporting The plasma osmolality of O. latipes was constant when system. On the other hand, a positive correlation between transferred from FW to (BW; 15‰), while it stress responses and NKA expression has been found in was significantly increased after direct transfer to seawater several other teleostean species (Yang et al., 2009; Tine et (35‰). The plasma osmolality of O. dancena in contrast sig- al., 2010; Tang and Lee, 2013). The present study sug- nificantly increased when transferred from FW to BW, but no gested 10‰ SW was stressful to Daisy’s medaka that further increase occurred when the fish was transferred to a induced elevation of gill NKA expression. Because NKA is higher salinity environment (Kang et al., 2008). In this study, abundant in the ionocytes of teleostean gills (Karnaky et al., the plasma osmolality and Na+ and Cl– concentrations of 1976; Hirose et al., 2003; Evans et al., 2005; Hwang and Daisy’s medaka significantly increased upon salinity chal- Lee, 2007; Tang and Lee, 2013; Chandrasekar et al., 2014), lenge. Similar patterns for these parameters have been the NKA immunoreactive (NKA-IR) cells in this study repre- found in other stenohaline species, including grass carp sented the ionocytes detected by the same antibody in the (Ctenopharyngodon idella; Maceina et al., 1980), catfish immunoblots. The cross-sections of the gill filaments (Heteropneustes fossilis; Goswami et al., 1983), channel showed that the NKA-IR cells of O. woworae that acclimated catfish (Ictalurus punctatus; Eckert et al., 2001), and to FW and 10‰ SW were distributed mainly in the afferent Caspian roach (Rutilus caspicus; Kolbadinezhad et al., epithelium and scarcely observed on the lamellar epithelium, 2012). Our findings suggest that stenohaline species typi- similar to the distribution of ionocytes in gills of O. latipes cally lose control of plasma electrolyte concentrations even and O. dancena (Kang et al., 2008). Previous studies in 10‰ SW. The results of survival rate indicate that the reported that the size of the NKA-IR cells (ionocytes) 10‰ SW was the survival limitation of the Daisy’s medaka. increased in seawater-acclimated chum salmon (Onco- The higher osmolality (350–360 mOsm/kg) of the 10‰ SW rhynchus keta; Uchida et al., 1996), guppy (Poecilia reticulata; group thus indicated the allostatic state of Daisy’s medaka. Shikano and Fujio, 1998), eel (Anguila japonica; Wong and In order to keep the homeostasis under osmotic stress, the Chan, 1999), Japanese medaka (O. latipes; Sakamoto et allostasis comprised changes of the mechanisms from al., 2001), and tilapia (Oreochromis mossanbicus; Shiraishi hyperosmoregulation to hypo-osmoregulation. et al., 1997; Lee et al., 2003). Our results revealed that the The gill is a target organ in research of fish osmo- and size of the NKA-IR cells in the 10‰ SW-exposed O. woworae ionoregulation (Hwang and Lee, 2007; Takei et al., 2014). was larger than that in the FW group, and the number of Several studies have described the ion transporter systems NKA-IR cells was also increased in 10‰ SW group. Upon 422 J.-J. Juo et al. salinity challenge, the number of NKA-IR cells was also noreactive band was found at 110 kDa by the polyclonal found to be significantly increased in the gills of the sailfin NCC antibody (Hsu et al., 2014). Two potential N-linked gly- molly (Poecilia latipinna; Yang et al., 2009), Trichogaster cosylation sites were predicted using the peptide sequence microlepis (Huang et al., 2010), sea bass (Dicentrarchus of NKCC1 in the other fish. Different degrees of glycosyla- labrax; Varsamos et al., 2002) and pufferfish (Tetraodon tion might lead to variable molecular weights of NKCC1 in nigroviridis; Tang et al., 2011). In the gills of O. woworae, the immunoblots (Tipsmark et al., 2002; Wu et al., 2003; increased number and size of NKA-IR cells accompanied Kang et al., 2010). Furthermore, the 130–170 kDa immuno- the higher expression of NKA activity and NKA protein reactive band only appeared in 10‰ SW group, but not in abundance. The expansion of the tubular reticulum and the FW group. Therefore, the present study quantified the increased cell number could provide more NKA for cellular target bands (130–170 kDa) for indicating that Daisy’s functions (Hirose et al., 2003). Therefore, in this study, the medaka increased gill NKCC1 abundance under osmotic increased number and size of the NKA-IR cells was found stress. The abundance of branchial NKCC1 protein was also to parallel the protein abundance and enzyme activities of found to increase in parallel to environmental salinity the stenohaline species O. woworae in response to a non- changes in euryhaline teleosts, including killifish (Fundulus lethal salinity stress from environments with a salinity differ- heteroclitus; Scott et al., 2004), striped bass (Morone saxatilis; ent from that in its natural habitat. Tipsmark et al., 2004), sea bass (Dicentrarchus labrax; The Na+, K+, 2Cl– cotransporter 1 (NKCC1), which is in Lorin-Nebel et al., 2006), salmon (Salmon salar; Pelis et al., the epithelial cells located on the basolateral membrane, 2001; Tipsmark et al., 2002; Hiroi and McCormick, 2007; plays the role of simultaneously transporting Na+, K+, and Nilsen et al., 2007), pufferfish (Tetraodon nigroviridis; Tang Cl– into cells and has been considered to be the secretory and Lee, 2007), tilapia (Oreochromis mossambicus; Wu et isoform (Lytle et al., ‘95; Starremans et al., 2003). According al., 2003; Tipsmark et al., 2008), goby (Oxyeleotris mamorata; to previous review articles, NKCC1 and the cystic fibrosis Chew et al., 2009), sturgeon (Acipenser medirostris; transmembrane conductance regulator (CFTR) participated Sardella and Kültz, 2009), and brackish medaka (O. in the mechanism underlying Cl– excretion and represented dancena; Kang et al., 2010). Kang et al. (2010) reported marker transporters in branchial ionocytes of seawater that the nkcc1a was prominently expressed in the gill iono- teleosts (Marshall, 2002; Hirose et al., 2003; Hwang and cytes of the brackish medaka. The colocalization of T4 sig- Lee, 2007; Evans, 2008; Seo et al., 2013; Chandrasekar et nal and NKA in the gills of the 10‰ SW-acclimated O. al., 2014). Our previous study indicated that the trend of woworae revealed that NKCC1 was located in the basolat- nkcc1a gene expression in gill ionocytes of the brackish eral membrane of ionocytes. The distribution of NKCC1 con- medaka was salinity-dependent (Kang et al., 2010). In this formed to the currently accepted model of Cl– secretion via study, the primers were designed for detecting the con- branchial ionocytes in seawater fish (Pelis et al., 2001; Wu served region of medaka nkcc1a. In Daisy’s medaka, mRNA et al., 2003; Evans et al., 2005; Hiroi and McCormick, 2007; levels of branchial nkcc1a also increased with environmental Katoh et al., 2008; Choi et al., 2011). By immunofluores- salinity. Furthermore, our unpublished data revealed that the cence staining, NKCC1 protein was found to be distributed lower abundance of Na+, Cl– cotansporter (ncc) mRNA was in the basolateral membrane of ionocytes (Marshall et al., found in the 10‰ Daisy’s medaka compared to the FW fish. 2002; Marshall et al., 2005). On the other hand, the apical These results indicated that the protein levels of NKCC1 NCC protein was detected by the same antibody (T4) on the would be hyper-salinity dependent rather than NCC in the NKA-IR cells in gills of FW-acclimated O. woworae, similar gills of the stenohaline medaka. On the other hand, the pro- to that in gills of O. dancena (Kang et al., 2010), as well as tein expression of branchial NKCC1 was detected using a the embryonic skin and gills of tilapia (Wu et al., 2003; monoclonal anti-human NKCC1 antibody (T4) in teleosts Hwang and Lee, 2007; Hiroi et al., 2008). Hiroi et al. (2008) (Wu et al., 2003; Hiroi and McCormick, 2007; Tang and Lee, identified that NCC was localized in the apical membrane 2007; Katoh et al., 2008; Kang et al., 2010, 2012). In this and NKCC1 was localized in the basolateral membrane of study, the molecular weights of the immunoreactive bands ionocytes on yolk sac membranes of the euryhaline tilapia. detected from gill lysates of O. woworae were from 95–170 Hsu et al. (2014) further confirmed that NCC was on the api- kDa, was similar to those reported in killifish (Fundulus cal membrane of NKA-IR cells of the Japanese medaka as heteroclitus; Marshall et al., 2002), salmon (Salmo salar; detected by both the antibody T4 and the anti-medaka NCC Pelis et al., 2001; Hiroi and McCormick, 2007), pufferfish antibody. Hence, the NCC protein that appeared on the api- (Tetraodon nigroviridis; Tang and Lee, 2007), tilapia cal membrane of ionocytes was suggested to be involved in (Oreochromis mossambicus; Hiroi et al., 2008), brackish Cl– uptake. The present study revealed that Daisy’s medaka medaka (O. dancena; Kang et al., 2010) and sailfin molly has mechanisms for both Cl– uptake and excretion, through (Poecilia latipinna; Yang et al., 2011). The NKCC1 and NCC shifting expression between NCC and NKCC1 protein. would be detected by the immunofluorescence staining by When the Daisy’s medaka exposed to the osmotic stress of the T4 antibody (Wu et al., 2003; Hwang and Lee, 2007; 10‰ SW, the increased NKA activities could provide more Hiroi et al., 2008; Hsu et al., 2014). However, no study has driving force for basolateral NKCC1 to transport Cl– into the identified all immunoreactive bands of T4 antibody in the gill gill ionocytes. Contrary to our primary expectation, even lysates. Based on the deduced protein sequence of though Daisy’s medaka is a stenohaline species, they still NKCC1a from the brackish medaka (Kang et al., 2010), its conserved the hypo-osmoregulatory capability upon salinity molecular weight was predicted to be 125 kDa. In contrast, challenge. 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