Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50 Contents lists available at ScienceDirect Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe Na+/K+-ATPase expression in gills of the euryhaline 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 introduced species of euryhaline teleost 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 teleosts (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 animal 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 hormone 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.