Journal of Experimental Marine Biology and 375 (2009) 41–50

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Journal of Experimental Marine Biology and Ecology

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Na+/K+-ATPase expression in gills of the sailfin molly, Poecilia latipinna, is altered in response to salinity challenge

Wen-Kai Yang a, Jinn-Rong Hseu b, Cheng-Hao Tang a, Ming-Ju Chung c, Su-Mei Wu c,⁎, Tsung-Han Lee a,⁎ a Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan b Mariculture Research Center, Fisheries Research Institute, Tainan 724, Taiwan c Department of Aquatic Biosciences, National Chiayi University, Chiayi 600, Taiwan article info abstract

Article history: Sailfin molly (Poecilia latipinna) is an of euryhaline mainly distributed in the lower reaches Received 23 December 2008 and river mouths over the southwestern part of Taiwan. Upon salinity challenge, the gill is the major organ Received in revised form 5 May 2009 responsible for ion-regulation, and the branchial Na+–K+-ATPase (NKA) is a primary driving force for the other ion Accepted 6 May 2009 transporters and channels. Hence we hypothesized that branchial NKA expression changed in response to salinity stress of sailfin molly so that they were able to survive in environments of different salinities. Before sampling, the Keywords: fish were acclimated to fresh water (FW), brackish water (BW, 15‰), or seawater (SW, 35‰) for at least one month. Gill The physiological (plasma osmolality), biochemical (activity and protein abundance of branchial NKA), cellular Glucose Heat shock protein (number of NKA immunoreactive cells), and stress (plasma glucose levels and protein abundance of hepatic and Na+/K+-ATPase branchial heat shock protein 90) indicators of osmoregulatory challenge in sailfinmollyweresignificantly increased Salinity in the SW-acclimated group compared to the FW- or BW-acclimated group. Elevated levels of stress indicators Teleost revealed that SW was stressful than FW to the sailfin molly. Meanwhile, the elevated biochemical indicators showed that more active NKA expression was necessary to match the demand of ion secretion of SW-acclimated sailfinmolly to survive in the environment with salinity challenge. The plasma osmolality of sailfin molly increased with environmental salinities within a tolerated range, while the muscle water content, another physiological indicator, was constant among different salinity groups. Taken together, through different ways of analyses, the sailfinmolly was proved to be an efficient osmoregulator in environments of different salinities with their branchial NKA expression changing in response to salinity challenge to maintain homeostasis of ion and water. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The gill is the major organ responsible for gas-exchange, ionoregula- tion, and acid-base balance in euryhaline (Perry et al., 2003; The teleosts that inhabit in either fresh water (FW) or seawater Evans et al., 2005). The mitochondrion-rich cells (MRCs; i.e., chloride − 1 (SW; about 1000 mOsmo∙kg ) have to maintain osmolality in a cells, CCs) in branchial epithelium are thought to be the “ionocytes” − 1 range (270–450 mOsmo∙kg ) suitable for their blood and tissue fluid responsible for ion uptake in FW and ion secretion in SW, respectively by regulating internal water and ion contents (Evans et al., 2005). In (Hirose et al., 2003; Evans et al., 2005; Hwang and Lee, 2007). The MRCs FW (i.e., the hypoosmotic environment), teleosts maintain hydro- are characterized by the presence of a rich population of mitochondria mineral balance by excreting diluted urine and ingesting external ions. and an extensive tubular system in the cytoplasm. The tubular system is In contrast, teleosts residing in SW (i.e., the hyperosmotic environ- continuous with the basolateral membrane, resulting in a large surface ment) maintain homeostasis by actively secreting ions and drinking area for the placement of ion transporting proteins such as Na+/K+- water (Evans et al., 1999; Marshall, 2002; Miller and Harley, 2002; ATPase (NKA; sodium–potassium pump or sodium pump), a key Hirose et al., 2003; Hwang and Lee, 2007). For euryhaline teleosts that enzyme for ion transport (Evans et al., 2005). survive in a variety of habitats, it is thus important to maintain a stable NKA is a ubiquitous membrane-spanning enzyme that actively internal environment through efficient ionoregulatory mechanisms. transports Na+ out of cells and K+ into animal cells. It is a P-

type ATPase consisting of an (αβ)2 protein complex. The molecular weights of the catalytic α-subunit and the smaller glycosylated β-subunit are about 100 and 55 kDa, respectively (Scheiner-Bobis, 2002). NKA is ⁎ Corresponding authors. Lee is to be contacted at the Department of Life Sciences, crucial for maintaining intracellular homeostasis by providing a driving National Chung-Hsing University, Taichung 402, Taiwan. Tel.: +886 4 2285 6141; force for many other ion-transporting systems (Marshall and Bryson, fax: +886 4 2287 4740. Wu, Department of Aquatic Biosciences, National Chiayi 1998; Hirose et al., 2003; Hwang and Lee, 2007). Most euryhaline teleosts University, Chiayi 600, Taiwan. Tel.: +886 5 275 4081; fax: +886 5 271 7847. E-mail addresses: [email protected] (S.-M. Wu), [email protected] exhibit adaptive changes in branchial NKA activity following salinity (T.-H. Lee). changes (Marshall, 2002; Evans et al., 2005; Fiol and Kültz, 2007; Hwang

0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2009.05.004 42 W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 and Lee, 2007). NKA in fish gills can be detected by immunocytochemical activity was thought to regulate physiological homeostasis when staining, and the number of Na+/K+-ATPase immunoreactive cells (NKA- individuals were exposed to salinity/osmotic stress. Hence we IR cells) can be counted. Most branchial NKA was found to localize in hypothesized that branchial NKA expression of sailfin molly changed MRCs; the number and distribution of the NKA-IR cells also changed with in response to salinity stress for homeostasis so that they were able to environmental salinities (Shikano and Fujio, 1998a,d; Sakamoto et al., survive in environments of different salinities. The goal of the present 2001b; Lin et al., 2003; Hwang and Lee, 2007). study was to profile the osmoregulatory mechanisms of the sailfin For acclimation and survival, fish respond to environmental stressors molly upon salinity challenge by indicators including physiological at many levels including increasing levels of blood glucose and heat parameters (plasma osmolality and muscle water content), biochem- shock proteins (HSPs). Blood glucose is an important source of energy ical expression (activity, α-subunit protein abundance, and immunor- for the metabolism necessary to maintain physiological homeostasis and eactive cells of branchial NKA), and stress responses (plasma glucose cope with environmental changes (Bashamohideen and Parvatheswar- concentrations and protein abundance of hepatic and branchial arao, 1972; Kelly et al., 1999). Usually the levels of blood glucose in fish HSP90). increase following exposure to stressful environments (Pottinger and Carrick, 1999) such as toxicants (Silbergeld, 1974), artificial habitat 2. Materials and methods (Arends et al., 1999; Kubokawa et al., 1999), abnormal temperature (Tanck et al., 2000; Hsieh et al., 2003), handling or capture (Rotllant and 2.1. Fish and experimental environments Tort, 1997; Frisch and Anderson, 2005; Tintos et al., 2006), and osmotic challenges (Bashamohideen and Parvatheswararao, 1972; Mancera Adult sailfin molly (P. latipinna)of3.1±0.4gbodyweightand et al., 1993; Soengas et al., 1995; Woo and Chung, 1995; Morgan and 51.2 ±27.3 mm standard length were captured from Linyuan, Kaoh- Iwama, 1998; Kelly et al., 1999; Claireaux and Audet, 2000; De Boeck siung, Taiwan (120.38° E 22.49° N) and transported to the laboratory. et al., 2000; Diouf et al., 2000; Palmisano et al., 2000; Yavuzcan-Yıldız Seawater (35‰) and brackish water (15‰; BW) were prepared from and Kırkağaç-Uzbilek, 2001; Sangiao-Alvarellos et al., 2003, 2005; local tap water by adding standardized amounts of synthetic sea salt Cataldi et al., 2005; Laiz-Carrión et al., 2005a,b; Arjona et al., 2007; Fiess “Instant Ocean” (Aquarium Systems, Mentor, OH, USA). The fish were et al., 2007; Choi et al., 2008). On the other hand, previous studies reared in BW at 25±1 °C for one week in the laboratory and then indicated that in fish, the HSPs with high molecular weights such as 60, acclimated to either fresh water (FW), BW, or SW with a daily 12 h 70, and 90 kDa interact with environmental stressors (Iwama et al.,1998, photoperiod for at least 4 weeks before sampling for the following tests 1999; Feder and Hofmann, 1999; Basu et al., 2002), e.g., abnormal and analyses. The parameters of the water were identical to our previous temperature (Dietz,1994; Sathiyaa et al., 2001; Cara et al., 2005; Buckley study (Wang et al., 2008). The water was continuously circulated et al., 2006; Rendell et al., 2006; Manchado et al., 2008) and external through fabric-floss filters and partially refreshed every three days. Fish osmolality challenges (Kültz, 1996; Smith et al., 1999; Palmisano et al., were fed a daily diet of commercial pellets ad libitum. In order to 2000; Pan et al., 2000; Deane et al., 2002; Deane and Woo, 2004; perform the following experiments, fish were fully anesthetized with Todgahm et al., 2005; De Jong et al., 2006; Choi and An, 2008). Among MS-222 (100–200 mg/L) and placed on ice before sampling. them, HSP90 constituted about 2% of total cellular protein in all organisms (Parsell and Lindquist, 1993). HSP90 was active in the 2.2. Plasma analyses enzymes, steroid receptors, and cytoskeleton of various components of cell signaling and was regarded as an indicator to Fish blood was collected from the caudal veins with heparinized estimate the level of environmental stressors (Iwama, 1998; Iwama 1 ml syringes and 27 G needles. After centrifugation at 13,000 g,4°C et al., 2004). Furthermore, HSP90 is expressed in various fish organs for 20 min, the plasma was stored at −20 °C prior to analysis. Plasma (Iwama, 1998; Iwama et al., 1999) including gill (Dietz, 1994; Kültz, osmolality was measured with the Wescor 5520 vapro osmometer 1996; Palmisano et al., 2000; Pan et al., 2000; Buckley et al., 2006; Choi (Logan, UT, USA). Plasma glucose concentrations were determined and An, 2008)andliver(Dietz, 1994; Palmisano et al., 2000; Sathiyaa et according to the manufacturer's protocol (Sigma, St. Louis, MO, USA) al., 2001; Deane et al., 2002; Deane and Woo, 2004; Rendell et al., 2006; using a Hitachi U-2001 spectrophotometer (Tokyo, Japan). Wang et al., 2007). The liver is an important organ with more diverse functionality than any other organ in the fish body, possible due to its 2.3. Muscle water content (MWC) large size, rich enzymatic content, HSP-sensitivity, and capacity for glycogen storage (Bond, 1996; Bruslé and Anadon, 1996; Iwama, 1998; The muscle from the dorsum of the body of the adult fish of FW-, Mwase et al.,1998; Sangiao-Alvarellos et al., 2003). Elevated branchial or BW-, and SW-acclimated groups was sampled and muscle water hepatic HSP90 expression (protein or mRNA level) upon salinity content was determined as the percentage of weight loss after drying challenge was reported as an indicator of osmotic stress in teleosts at 100 °C for three days. (Palmisano et al., 2000; Pan et al., 2000; Deane et al., 2002; Choi and An, 2008). 2.4. Sample preparation The sailfin molly (Poecilia latipinna) is a livebearing species with large dorsal fins on the males (Nelson, 1984; Berra, 2001; Froese and The gills and livers were dissected and rinsed in phosphate buffered −1 Pauly, 2008). They are natively distributed in low elevations from saline (PBS; concentrations in mmol·l : NaCl:137; KCl: 3; Na2HPO4: North Carolina, USA, to Veracruz, Mexico (Nordlie et al., 1992; Ptacek 10; KH2PO4: 2; pH 7.4). The tissues were then blotted dry before further and Breden, 1998; Froese and Pauly, 2008) and have been introduced analysis. The gills were fixed in 10% formalin for immunocytochemical to many countries (Froese and Pauly, 2008) including Taiwan (Shao, detection or homogenized for immunoblotting and other analyses; the 2008). Today the euryhaline sailfin molly is found in the shallow livers were homogenized for immunoblotting. The gill and liver surface water along the edges of lowland, ponds, streams, gullies, scrapings were suspended in the mixture of homogenization medium −1 swamps, salt marshes, and estuaries (Nordlie et al., 1992; Froese and (concentrations in mmol·l :imidazole-HCl:100;Na2EDTA: 5; sucrose: Pauly, 2008). In Taiwan, the sailfin molly was mainly distributed in the 200; 0.1% sodium deoxycholate; pH 7.6) and proteinase inhibitor (10 mg lower reaches (FW) and river mouths (brackish water, BW) over the antipain, 5 mg leupeptin, and 50 mg benzamidine dissolved in 5 ml southwestern part (Shao, 2008). Similar to the other euryhaline aprotinin) (v/v: 100/ 1). Homogenization was performed in 2 ml teleosts, branchial NKA activity and plasma osmolality of SW sailfin microtubes with a Polytron PT1200E (Kinematica, Lucerne, Switzer- molly were found to be elevated with increasing salinity such as land) at maximal speed for 30 s The homogenate was then hypersaline water (Gonzalez et al., 2005). Moreover, branchial NKA centrifuged at 13,000 g,4°Cfor20min.Thesupernatantswere W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 43 used for determination of protein concentrations, enzyme activity, or by a 1-h incubation with AP-conjugated secondary antibody diluted immunoblotting. Protein concentrations were identified by reagents 4000x in PBST. Blots were visualized after incubation with an NBT/BCIP from BCA Protein Assay (Pierce, Hercules, CA, USA), using bovine kit (Zymed, South San Francisco, CA, USA). serum albumin (BSA) as a standard (Pierce). The samples were For immunoblotting of HSP90, sample buffers were mixed with stored at −80 °C before use. aliquots containing 20 or 50 µg of hepatic or branchial homogenates. The homogenates were then heated at 95 °C for 5 min and fractionated by 2.5. Optimal conditions for determining Na+/K+-ATPase (NKA) activity electrophoresis on 7.5% SDS-polyacrylamide gels. The following protocol was identical to that of branchial NKA α-subunit immunoblotting except For determining the optimal concentrations of Na+,K+, and Mg2+ that the primary antibody to HSP90 was diluted 1000×. in the reaction medium of in vitro assay of molly NKA activity, 0.1 ml Immunoblots were scanned and imported as TIFF files. Immunore- filtered homogenates and 5 ml reaction medium (concentrations active bands were analyzed using MCID software version 7.0, rev. 1.0 in mmol·l− 1: imidazole-HCl buffer: 100; pH 7.0; NaCl: 0–300; KCl: (Imaging Research, Ontario, Canada). The results were converted to

0–300; MgCl2:0–30; Na2ATP: 5) were mixed. Each reaction numerical values in order to compare the relative protein abundance condition was repeated. The reactions with the samples exposed to of the immunoreactive bands. different environments of [Na+], [K+], or [Mg2+]wererunat37°C for 30 min. The reactions were then stopped by the addition of 2 ml 2.9. Immunohistochemical detection ice-cold 30% trichloroacetic acid. The reaction tube was centrifuged and the inorganic phosphate concentration in the supernatant was Gills were dissected and fixed for 24 h in 10% formalin in 0.1 M determined with a spectrophotometer at 700 nm according to the phosphate buffer (pH 7.2) at 25 °C. Samples were dehydrated through method proposed by Peterson (1978).TheenzymeactivityofNKA a graded ethanol series, embedded in paraffin, and serial sections of was defined as the difference between the inorganic phosphate 7 µm were mounted on gelatin-coated glass slides. The sections were liberated in the presence and absence of 3.75 mmol·l− 1 ouabain in stained immunohistochemically with the monoclonal antibody (α5) the reaction mixture. All experiments were repeated 2–4timeswith to NKA α-subunit followed by a commercial kit (PicTure™, Zymed). similar results. For comparisons, enzymatic activities were con- Negative control experiments in which PBS (phosphate buffered verted into relative activities (%) of the highest value obtained in saline) was used instead of the primary antibody were conducted to each experiment. Individual points of concentrations in each panel confirm the positive results of NKA immunoreactive (NKA-IR) cells. were the averages of duplicate samples. Immunostained sections were observed using a light microscope (Olympus BX50, Tokyo, Japan) and the micrographs were taken with a 2.6. Assay of NKA activity digital camera (Nikon COOLPIX 5000, Tokyo, Japan). The method of quantification for NKA-IR cells was modified from Aliquots of the suspension of branchial homogenates, prepared as our previous studies (Lin et al., 2003, 2006; Tang et al., 2008). The described above, were used for assaying protein concentrations and numbers of NKA-IR cells were counted on the sections immunostained enzyme activities. NKA activity was assayed by adding the super- as described in the previous paragraph. Preliminary observations natant to the optimal reaction medium (concentrations in mmol·l− 1: indicated that most NKA-IR cells in gills of the sailfin molly were imidazole-HCl buffer: 100; pH 7.6; NaCl: 200; KCl: 150; MgCl2:5; distributed in the interlamellar regions of the filaments. Hence, Na2ATP: 5). The reaction was run as described above. Each sample was longisections of the gills including lamellae and the cartilages of the assayed in triplicate. NKA activity was expressed as μmol inorganic filaments were chosen, and the numbers of immunoreactive cells in phosphate released per mg of protein per hour. the interlamellar regions of the filaments and the lamellae were counted. Cell numbers in the interlamellar regions within the range of 2.7. Antibodies 5 adjacent lamellae (approximately 40–60 µm) were counted and the lengths of the 4 interlamellar regions were measured to normalize cell The primary antibodies used in this study included (1) NKA: a counts to a fixed length (50 µm). Results were expressed as numbers of mouse monoclonal antibody (α5; Developmental Studies Hybridoma NKA-IR cells per 50 µm of filaments (interlamellar regions). For each Bank, Iowa City, IA, USA) raised against the α-subunit of the avian sample, 10 areas on the filaments including symmetrical interlamellar NKA; (2) heat shock protein 90 (HSP90): a rabbit polyclonal antibody regions were randomly selected. (#4874; Cell Signaling, Beverly, MA, USA) raised against the human HSP90. The secondary antibodies for immunoblotting were alkaline 2.10. Statistical analysis phosphatase (AP)-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Chemicon, Temecula, CA, USA). Values were compared using a one-way analysis of variance (ANOVA) (Tukey's pair-wise method) and Pb0.05 was set as the significant level. 2.8. Immunoblotting Values were expressed as the means±S.E.M. (the standard error of the mean) unless stated otherwise. For immunoblotting of branchial NKA, aliquots containing 25 µg of branchial homogenates were added to sample buffer and heated at 60 °C 3. Results for 15 min followed by electrophoresis on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The pre-stain protein molecular weight 3.1. Physiological parameters marker was purchased from Fermentas (SM0671; Hanover, MD, USA). Separated proteins were transferred from unstained gels to PVDF The plasma osmolality of sailfin molly increased with environmental membranes (Millipore, Bedford, MA, USA) by electroblotting using a salinities (Fig. 1A). The muscle water content (MWC), however, was tank transfer system (Mini Protean 3, Bio-Rad, Hercules, CA, USA). Blots constant in either FW-, BW-, or SW-acclimated fish (Fig. 1B). were preincubated for 2 h in PBST (phosphate buffer saline with Tween −1 + + 20) buffer (concentrations in mmol·l : NaCl:137; KCl: 3; Na2HPO4:10; 3.2. Branchial Na /K -ATPase (NKA) expression KH2PO4: 2; 0.05% (vol/vol) Tween 20, pH 7.4) containing 5% (wt/vol) nonfat dried milk to minimize non-specific binding, then incubated at When the concentration ratio of Na+:K+ was 4:3, the highest enzyme 4 °C with the primary antibody (α5) diluted in 1% BSA and 0.05% sodium activity was found at 200 and 150 mmol·l−1 of total Na+ and K+, azide in PBST (1: 2500) overnight. The blot was washed in PBST, followed respectively. Thus optimal concentrations of Na+ and K+ for the in vitro 44 W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50

3.3. Plasma glucose concentrations and HSP90 abundance in livers and gills

The plasma glucose concentrations of sailfin molly elevated with environmental salinities that were significantly higher in SW- acclimated fish than the FW fish (Fig. 7). Immunoblotting of HSP90 in livers and gills from fish of different salinity-groups (FW, BW, and SW) resulted in a single immunoreac- tive band centered at about 90 kDa (Figs. 8A and 9A). Quantification of immunoreactive bands revealed that relative protein abundance of hepatic HSP90 in SW sailfin molly was significantly higher than that of FW-acclimated fish (Fig. 8B), while the branchial HSP90 protein

Fig. 1. Salinity effects on (A) plasma osmolality (N=8) and (B) muscle water content (MWC; N=16) of P. latipinna. Dissimilar letters indicated significant differences among osmolality of fish acclimated to environments of various salinities (mean±S.E.M., one way ANOVA with Tukey's comparison, Pb0.05). No significant difference was found among MWC of the three salinity groups.

assay of NKA activity were 200 and 150 mmol·l−1, respectively (Fig. 2). The optimal concentration of Mg2+ required for the NKA activity assay was determined to be 5 mmol·l−1, although the relative enzyme activities between 5 and 10 mmol·l−1 [Mg2+] revealed a difference less than 10% (Fig. 2). The specific activity of branchial NKA of fish acclimated to SW was significantly higher than that of fish acclimated to BW and FW (Fig. 3). There was no significant difference, however, between branchial NKA activity among the BW and FW groups (Fig. 3). Immunoblotting of NKA in gills from the sailfin molly acclimated to environments of different salinities (FW, BW, and SW) resulted in a single immunoreactive band of about 100 kDa (Fig. 4A). Quantifica- tion of immunoreactive bands revealed that the branchial NKA α- subunit protein abundance of SW molly was significantly higher than that of FW- or BW-acclimated fish: about 1.7- and 2.2-fold, respectively (Fig. 4B). Salinity effects on the distribution of branchial NKA-immunoreactive (IR) cells were visualized using immunolocalization of the α-subunit on paraffinsections(Fig. 5). The branchial NKA-IR cells of SW-, BW- and FW- acclimated fish were distributed mainly in filaments (the interlamellar region) and rarely found in lamellae (Fig. 5A–C), and the NKA-IR cells were not found in either the filaments or the lamellar of the negative control from SW-acclimated fish (Fig. 5D). The number of NKA-IR cells of SW-acclimated fish was significantly greater than that of BW-acclimated group (Fig. 6). The number of NKA-IR cells of FW-acclimated fish, Fig. 2. Effects of different concentrations of (A) [Na+], (B) [K+], and (C) [Mg2+] in the fi however, was not signi cantly different from that in the BW or SW- assay of P. latipinna branchial NKA activity. The arrows indicated the optimal ionic acclimated group (Fig. 6). concentrations showing maximal relative ATPase activity. W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 45

environments of different salinities. Short-term observations also revealed that the MWC of rainbow trout (Oncorhynchus mykiss; Eddy and Bath, 1979; Leray et al., 1981), brook trout (Salvelinus fontinalis; Claireaux and Audet, 2000), Japanese rice fish (Sakamoto et al., 2001a), and sea trout (Salmo trutta; Tipsmark et al., 2002), Mozambique tilapia (Tipsmark et al., 2008a), and southern flounder (Paralichthys lethos- tigma; Tipsmark et al., 2008b) recovered gradually after transfer from FW to SW. Apparent differences in MWC, however, were found in other species such as black porgy (Kelly et al.,1999) that acclimated to various salinity environments. Notably, osmoregulatory capabilities may be correlated with a species' evolutionary history or natural habitat (Freire et al., 2008).

4.2. Biochemical and cellular expression

Fig. 3. Salinity effects on branchial NKA activity of P. latipinna. The asterisk indicated a significant difference among groups of various environments (N=8, mean±S.E.M., Elevated branchial NKA activity provided more driving force for ion one way ANOVA with Tukey's comparison, Pb0.05). transport, indicating increased demand for ion uptake or secretion in gills (Lin et al., 2003). In vitro activity assays demonstrated that branchial NKA of euryhaline teleosts such as Mozambique tilapia abundance of SW sailfin molly was significantly higher than that of the (Hwang et al., 1988; Lin and Lee, 2005), European seabass (Jensen FW or BW group (Fig. 9B). et al., 1998), milkfish (Chanos chanos), and spotted green pufferfish (Lin and Lee, 2005) were sensitive to the ionic strength of the assay medium. 4. Discussion In this study, the results showed that maximal NKA activities were found in the optimal assay conditions: 200 mmol·l−1 Na+, 150 mmol·l−1 K+, − 4.1. Physiological parameters and 5 mmol·l 1 Mg2+ (Fig. 2). The optimal Na+/K+ ratio in gills varied with different teleostean species (Hwang et al., 1988; Lin and Lee, 2005). Plasma osmolality and MWC were assayed in this study to determine Although the optimal branchial Na+/K+ ratio for sailfinmolly(4:3)was the osmoregulatory capacity of the sailfin molly. The plasma osmolality different from the ratio for Mozambique tilapia (5:3; Hwang et al., 1988), of sailfin molly increased with environmental salinity; the highest level the present study showed the optimal reaction conditions for determining was found in SW-acclimated fish (Fig. 1A). Although the experimental branchial NKA activity in sailfin molly. This methodology yields more designs were different, Nordlie et al. (1992) and Gonzalez et al. (2005) significant results with minimal individual variation (Hwang et al., 1988). also reported that plasma osmolality of sailfinmollyincreasedina In addition, the other sensitivities including Mg2+,ATP,pH,ouabain,or tolerated range with environmental salinity. Similar patterns of plasma osmolality were found in the other euryhaline teleosts including (Fundulus heteroclitus; Jacob and Taylor, 1983), European seabass (Dicentrarchus labrax; Jensen et al., 1998), black porgy (Acanthopagrus schlegelii; Kelly et al., 1999; Choi and An, 2008), Mozambique tilapia (Oreochromis mossambicus; Lin et al., 2004a; Fiess et al., 2007; Tipsmark et al., 2008a), spotted green pufferfish (Tetraodon nigroviridis; Lin et al., 2004b; Bagherie-Lachidan et al., 2008), and Japanese rice fish ( latipes; Kang et al., 2008); plasma osmolalities were higher in hyperosmotic environments. Moreover, previous studies revealed that changed patterns of plasma osmolality in euryhaline teleosts were correlated with their primary habitats and salinity tolerance (Kato et al., 2005; Bystriansky et al., 2006). When exposed to SWor hypersaline water (HSW)(i.e., the hyperosmotic environment), the osmolalities of external environments were higher than those of internal environments (i.e., the body fluids); the fish gained external ions and lost internal water (Miller and Harley, 2002). With the capacity for ionoregulation, the osmolality of euryhaline teleosts increased in the tolerated range depending on the external hyperosmotic environments. The muscle water content (MWC) of sailfin molly was found to be constant in the FW, BW, and SW groups (Fig. 1B) as well as in the HSW group (b80‰; Gonzalez et al., 2005). The sailfin molly could maintain internal water balance by regulating drinking rates (Varsamos et al., 2004; Gonzalez et al., 2005). These results indicated that the sailfin molly could maintain water homeostasis in varied salinity environ- ments, enabling this euryhaline species to survive in natural habitats ranging from FW to BW environments. Similarly, the MWC of em- peror angelfish (Pomacanthus imperator; Woo and Chung, 1995), goldlined seabream (Rhabdosargus sarba; Kelly and Woo, 1999), Fig. 4. (A) Representative immunoblot of P. latipinna gills probed with a monoclonal gilthead seabream (Sparus auratus;5–55‰ of environments; antibody (α5) to NKA α-subunit. (B) Relative abundance of immunoreactive bands of NKA α-subunit in gills of different groups. The asterisk indicated a significant difference Sangiao-Alvarellos et al., 2003; Laiz-Carrión et al., 2005a), spotted among groups of various environments (N=6, mean±S.E.M., one way ANOVA with fi green puffer sh (Bagherie-Lachidan et al., 2008), and marine medaka Tukey's comparison, Pb0.05). M, marker (kDa); FW, fresh water; BW, brackish water; (Oryzias dancena; Kang et al., 2008) was constant when acclimated to SW, seawater. 46 W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50

reaction temperature also affected NKA activity (Hwang et al., 1988; Blanco and Mercer, 1998; Lin and Lee, 2005; this study). The branchial NKA activity of SW-acclimated sailfin molly was found to be higher than that of BW- or FW-acclimated groups in this study (Fig. 3). The changing pattern of branchial NKA activity in the present study conformed to the results of a previous study of sailfin molly (Gonzalez et al., 2005) which compared the activity between SW and HSW, rather than common natural environments (i.e., FW or BW). In general, changing profiles of branchial NKA activity among euryhaline teleosts corresponded to their natural habitats (Hwang and Lee, 2007). Certain marine inhabitants exhibit higher NKA activities in FW than in SW, including species such as longjaw mudsucker (Gillichthys mirabilis; Doneen, 1981), emperor angelfish (Woo and Chung, 1995), European seabass (Jensen et al., 1998), black porgy (Kelly et al., 1999), milkfish (Lin et al., 2003, 2006), and gilthead seabream (Laiz-Carrión et al., 2005a,b). The pattern of branchial NKA activity of the sailfin molly was similar to the other euryhaline teleosts such as mummichog (Jacob and Taylor, 1983), golden perch (Macquaria ambigua; Langdon, 1987), Mozambique tilapia (Hwang et al., 1988, 1989; Uchida et al., 2000; Lee et al., 2003; Chang et al., 2007; Fiess et al., 2007; Inokuchi et al., 2008; Tipsmark et al., 2008a), rainbow trout (Madsen and Naamansen, 1989), coho salmon (Oncorhynchus kisutch; Morgan and Iwama, 1998), European eel (Anguilla anguilla; Marsigliante et al., 2000; Wilson et al., 2004, 2007), sea trout (Tipsmark et al., 2002), striped bass (Morone saxatilis; Tipsmark et al., 2004), spotted green pufferfish (Lin et al., 2004b; Bagherie-Lachidan et al., 2008), lake trout (Salvelinus namaycush), brook trout (Hiroi and McCormick, 2007), and Japanese rice fish (Kang et al., 2008). These latter species exhibit a pattern similar to that of freshwater or brackishwater fish with branchial NKA activities higher in SW rather than in FW. Previous studies reported that the higher branchial NKA activity of the euryhaline teleosts always accompanied a raised NKA α-subunit protein abundance, in species such as Mozambique tilapia (Lee et al., 2000, 2003; Lin et al., 2004a; Tang et al., 2008), sea trout (Tipsmark et al., 2002), milkfish (Lin et al., 2003), spotted green pufferfish (Lin et al., 2004b), European eel (Wilson et al., 2004, 2007), Atlantic salmon (Salmo salar; Nilsen et al., 2007), marine medaka (Kang et al., 2008), and southern flounder (Tipsmark et al., 2008b). In this study, the highest level of branchial NKA α-subunit protein abundance was found in the SW-acclimated sailfin molly (Fig. 4B), in agreement with the pattern of NKA activity. The positive correlation between NKA activity and NKA α-subunit protein abundance indicated that raised branchial NKA activity in the sailfin molly may derive from increasing branchial NKA α-subunit protein abundance in SW. Most branchial NKA-immunoreactive (IR) cells of the FW-, BW-, and SW-acclimated sailfinmollyweredistributedinfilaments (the inter- lamellar region) and rarely found in lamellae (Fig. 5). The distribution of branchial NKA-IR cells in sailfin molly was similar to that of other euryhaline teleosts such as guppy (Poecilia reticulata; Shikano and Fujio, 1998b,c), spotted green pufferfish (Lin et al., 2004b), gilthead seabream (Laiz-Carrión et al., 2005a), Japanese rice fish, marine medaka (Kang et al., 2008), and southern flounder (Tipsmark et al., 2008b). NKA-IR cells, normally abundant in filament epithelia of both FW and SW teleosts (Wilson and Laurent, 2002), were effective at absorbing ions in a hypotonic environment (as FW) as well as secreting ions in a hypertonic environment (as SW) (Marshall, 2002). Upon salinity challenge, the changes in distribution, number, or cell size of branchial NKA-IR cells were usually found in euryhaline teleosts (Hwang and Lee, 2007). In this study, the number of branchial NKA-IR cells of SW-acclimated sailfinmollywas found to be greater than that of BW-acclimated group (Fig. 6). The pattern

Fig. 5. Representative immunostaining of NKA-immunoreactive (IR) cells in longitudinal sections of the gill filamentsfrom(A)FW-,(B)BW-,and(C)SW-acclimatedP. latipinna and negative control from SW-acclimated P. latipinna (D). Arrows indicated that NKA-IR cells were observed in the interlamellar regions of filaments. Bar=10 µm. W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 47

Fig. 6. Densities of NKA-IR cells in gills of P. latipinna acclimated to FW, BW, and SW. Dissimilar letters indicated significant differences among fish of various environments (N=8, mean±S.E.M., one way ANOVA with Tukey's comparison, Pb0.05). of changing numbers of NKA-IR cells was similar to that of protein abundance and activity of NKA in gills of sailfinmollyasdescribedabove. Such changes of NKA protein abundance and NKA-IR cells were considered to contribute to alterations in NKA activity for ionoregulation (Dang et al., 2000; Lee et al., 2003; Lin et al., 2003; Laiz-Carrión et al., Fig. 8. (A) Representative immunoblot of P. latipinna livers probed with a polyclonal 2005a; Kang et al., 2008). antibody to HSP90. (B) Relative abundance of immunoreactive bands of HSP90 in livers of different groups. Dissimilar letters indicated significant differences among fish of various environments (N=10, mean±S.E.M., one way ANOVA with Tukey's comparison, Pb0.05). 4.3. Stress responses M, marker (kDa); FW, fresh water; BW, brackish water; SW, seawater.

When exposed to a stressful environment, increased expression of certain proteins suggests the presence of stress indicators in fish (Fig. 7; Laiz-Carrión et al., 2005a; Sangiao-Alvarellos et al., 2005; attempting to adjust to the situation (Iwama et al., 1999, 2004). The Arjona et al., 2007). Temporal patterns of plasma glucose concentra- levels of plasma glucose (Kelly et al.,1999) and hepatic or branchial HSP90 tions following environmental stress, however, varied with teleostean expression (Deane et al., 2002; Choi and An, 2008) of teleost, were used to species. Plasma glucose levels can rise quickly and return to normal indicate the stress caused by salinity challenge. In this study, higher levels within one day (Rotllant and Tort, 1997; Arends et al., 1999) or four of plasma glucose and hepatic and branchial HSP90 expression in SW- acclimated sailfin molly showed that the SW environment (non-natural habitat) was stressful to this species (Figs.7,8B,9B). Moreover, since the resistant process is energy-demanding, the animal must mobilize energy substrates for metabolism (Iwama, 1998). Plasma glucose could be used as an osmolyte for plasma osmolality or as an important energy source for metabolism to resist the stress (Kelly et al., 1999; Diouf et al., 2000). The increased concentrations of plasma glucose thus indicated that sailfin molly acclimating to SW (non-natural habitat) required more energy for branchial NKA-mediated maintenance of physiological homeostasis

Fig. 9. (A) Representative immunoblot of P. latipinna gills probed with a polyclonal antibody to HSP90. (B) Relative abundance of immunoreactive bands of HSP90 in the gills Fig. 7. Salinity effects on plasma glucose levels. Dissimilar letters indicated significant from different groups. The asterisk indicated a significant difference among groups in differences among fish acclimated to various environments (N=8, mean±S.E.M., one various environments (N=6, mean±S.E.M., one way ANOVA with Tukey's comparison, way ANOVA with Tukey's comparison, Pb0.05). Pb0.05). M, marker (kDa); FW, fresh water; BW, brackish water; SW, seawater. 48 W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 days (Mancera et al., 1993; Claireaux and Audet, 2000; Hsieh et al., Bashamohideen, M., Parvatheswararao, V., 1972. to osmotic stress in the fresh-water euryhaline teleost Tilapia mossambica. IV. 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