Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50
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Journal of Experimental Marine Biology and Ecology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
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
b
c
Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan
Mariculture Research Center, Fisheries Research Institute, Tainan 724, Taiwan
Department of Aquatic Biosciences, National Chiayi University, Chiayi 600, Taiwan
a r t i c l e
i n f o
Article history:
Received 23 December 2008
Received in revised form 5 May 2009
Accepted 6 May 2009
Keywords:
Gill
Glucose
Heat shock protein
Na+/K+-ATPase
Salinity
Teleost
a b s t r a c t
Sailfin molly (Poecilia latipinna) is an introduced species of euryhaline teleost mainly distributed in the lower reaches
and river mouths over the southwestern part of Taiwan. Upon salinity challenge, the gill is the major organ
responsible for ion-regulation, and the branchial Na+–K+-ATPase (NKA) is a primary driving force for the other ion
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
fish were acclimated to fresh water (FW), brackish water (BW, 15‰), or seawater (SW, 35‰) for at least one month.
The physiological (plasma osmolality), biochemical (activity and protein abundance of branchial NKA), cellular
(number of NKA immunoreactive cells), and stress (plasma glucose levels and protein abundance of hepatic and
branchial heat shock protein 90) indicators of osmoregulatory challenge in sailfin molly were significantly increased
in the SW-acclimated group compared to the FW- or BW-acclimated group. Elevated levels of stress indicators
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 sailfin molly
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 sailfin molly
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 teleosts that inhabit in either fresh water (FW) or seawater
(SW; about 1000 mOsmo∙kg− 1) have to maintain osmolality in a
range (270–450 mOsmo∙kg− 1) suitable for their blood and tissue fluid
by regulating internal water and ion contents (Evans et al., 2005). In
FW (i.e., the hypoosmotic environment), teleosts maintain hydromineral balance by excreting diluted urine and ingesting external ions.
In contrast, teleosts residing in SW (i.e., the hyperosmotic environment) maintain homeostasis by actively secreting ions and drinking
water (Evans et al., 1999; Marshall, 2002; Miller and Harley, 2002;
Hirose et al., 2003; Hwang and Lee, 2007). For euryhaline teleosts that
survive in a variety of habitats, it is thus important to maintain a stable
internal environment through efficient ionoregulatory mechanisms.
⁎ Corresponding authors. Lee is to be contacted at the Department of Life Sciences,
National Chung-Hsing University, Taichung 402, Taiwan. Tel.: +886 4 2285 6141;
fax: +886 4 2287 4740. Wu, Department of Aquatic Biosciences, National Chiayi
University, Chiayi 600, Taiwan. Tel.: +886 5 275 4081; fax: +886 5 271 7847.
E-mail addresses: [email protected] (S.-M. Wu), [email protected]
(T.-H. Lee).
0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2009.05.004
The gill is the major organ responsible for gas-exchange, ionoregulation, and acid-base balance in euryhaline teleosts (Perry et al., 2003;
Evans et al., 2005). The mitochondrion-rich cells (MRCs; i.e., chloride
cells, CCs) in branchial epithelium are thought to be the “ionocytes”
responsible for ion uptake in FW and ion secretion in SW, respectively
(Hirose et al., 2003; Evans et al., 2005; Hwang and Lee, 2007). The MRCs
are characterized by the presence of a rich population of mitochondria
and an extensive tubular system in the cytoplasm. The tubular system is
continuous with the basolateral membrane, resulting in a large surface
area for the placement of ion transporting proteins such as Na+/K+ATPase (NKA; sodium–potassium pump or sodium pump), a key
enzyme for ion transport (Evans et al., 2005).
NKA is a ubiquitous membrane-spanning enzyme that actively
transports Na+ out of animal cells and K+ into animal cells. It is a Ptype 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
crucial for maintaining intracellular homeostasis by providing a driving
force for many other ion-transporting systems (Marshall and Bryson,
1998; Hirose et al., 2003; Hwang and Lee, 2007). Most euryhaline teleosts
exhibit adaptive changes in branchial NKA activity following salinity
changes (Marshall, 2002; Evans et al., 2005; Fiol and Kültz, 2007; Hwang
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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
staining, and the number of Na+/K+-ATPase immunoreactive cells (NKAIR cells) can be counted. Most branchial NKA was found to localize in
MRCs; the number and distribution of the NKA-IR cells also changed with
environmental salinities (Shikano and Fujio, 1998a,d; Sakamoto et al.,
2001b; Lin et al., 2003; Hwang and Lee, 2007).
For acclimation and survival, fish respond to environmental stressors
at many levels including increasing levels of blood glucose and heat
shock proteins (HSPs). Blood glucose is an important source of energy
for the metabolism necessary to maintain physiological homeostasis and
cope with environmental changes (Bashamohideen and Parvatheswararao, 1972; Kelly et al., 1999). Usually the levels of blood glucose in fish
increase following exposure to stressful environments (Pottinger and
Carrick, 1999) such as toxicants (Silbergeld, 1974), artificial habitat
(Arends et al., 1999; Kubokawa et al., 1999), abnormal temperature
(Tanck et al., 2000; Hsieh et al., 2003), handling or capture (Rotllant and
Tort, 1997; Frisch and Anderson, 2005; Tintos et al., 2006), and osmotic
challenges (Bashamohideen and Parvatheswararao, 1972; Mancera
et al., 1993; Soengas et al., 1995; Woo and Chung, 1995; Morgan and
Iwama, 1998; Kelly et al., 1999; Claireaux and Audet, 2000; De Boeck
et al., 2000; Diouf et al., 2000; Palmisano et al., 2000; Yavuzcan-Yıldız
and Kırkağaç-Uzbilek, 2001; Sangiao-Alvarellos et al., 2003, 2005;
Cataldi et al., 2005; Laiz-Carrión et al., 2005a,b; Arjona et al., 2007; Fiess
et al., 2007; Choi et al., 2008). On the other hand, previous studies
indicated that in fish, the HSPs with high molecular weights such as 60,
70, and 90 kDa interact with environmental stressors (Iwama et al.,1998,
1999; Feder and Hofmann, 1999; Basu et al., 2002), e.g., abnormal
temperature (Dietz,1994; Sathiyaa et al., 2001; Cara et al., 2005; Buckley
et al., 2006; Rendell et al., 2006; Manchado et al., 2008) and external
osmolality challenges (Kültz, 1996; Smith et al., 1999; Palmisano et al.,
2000; Pan et al., 2000; Deane et al., 2002; Deane and Woo, 2004;
Todgahm et al., 2005; De Jong et al., 2006; Choi and An, 2008). Among
them, HSP90 constituted about 2% of total cellular protein in all
organisms (Parsell and Lindquist, 1993). HSP90 was active in the
enzymes, steroid hormone receptors, and cytoskeleton of various
components of cell signaling and was regarded as an indicator to
estimate the level of environmental stressors (Iwama, 1998; Iwama
et al., 2004). Furthermore, HSP90 is expressed in various fish organs
(Iwama, 1998; Iwama et al., 1999) including gill (Dietz, 1994; Kültz,
1996; Palmisano et al., 2000; Pan et al., 2000; Buckley et al., 2006; Choi
and An, 2008) and liver (Dietz, 1994; Palmisano et al., 2000; Sathiyaa et
al., 2001; Deane et al., 2002; Deane and Woo, 2004; Rendell et al., 2006;
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
large size, rich enzymatic content, HSP-sensitivity, and capacity for
glycogen storage (Bond, 1996; Bruslé and Anadon, 1996; Iwama, 1998;
Mwase et al.,1998; Sangiao-Alvarellos et al., 2003). Elevated branchial or
hepatic HSP90 expression (protein or mRNA level) upon salinity
challenge was reported as an indicator of osmotic stress in teleosts
(Palmisano et al., 2000; Pan et al., 2000; Deane et al., 2002; Choi and An,
2008).
The sailfin molly (Poecilia latipinna) is a livebearing species with
large dorsal fins on the males (Nelson, 1984; Berra, 2001; Froese and
Pauly, 2008). They are natively distributed in low elevations from
North Carolina, USA, to Veracruz, Mexico (Nordlie et al., 1992; Ptacek
and Breden, 1998; Froese and Pauly, 2008) and have been introduced
to many countries (Froese and Pauly, 2008) including Taiwan (Shao,
2008). Today the euryhaline sailfin molly is found in the shallow
surface water along the edges of lowland, ponds, streams, gullies,
swamps, salt marshes, and estuaries (Nordlie et al., 1992; Froese and
Pauly, 2008). In Taiwan, the sailfin molly was mainly distributed in the
lower reaches (FW) and river mouths (brackish water, BW) over the
southwestern part (Shao, 2008). Similar to the other euryhaline
teleosts, branchial NKA activity and plasma osmolality of SW sailfin
molly were found to be elevated with increasing salinity such as
hypersaline water (Gonzalez et al., 2005). Moreover, branchial NKA
activity was thought to regulate physiological homeostasis when
individuals were exposed to salinity/osmotic stress. Hence we
hypothesized that branchial NKA expression of sailfin molly changed
in response to salinity stress for homeostasis so that they were able to
survive in environments of different salinities. The goal of the present
study was to profile the osmoregulatory mechanisms of the sailfin
molly upon salinity challenge by indicators including physiological
parameters (plasma osmolality and muscle water content), biochemical expression (activity, α-subunit protein abundance, and immunoreactive cells of branchial NKA), and stress responses (plasma glucose
concentrations and protein abundance of hepatic and branchial
HSP90).
2. Materials and methods
2.1. Fish and experimental environments
Adult sailfin molly (P. latipinna) of 3.1 ± 0.4 g body weight and
51.2 ± 27.3 mm standard length were captured from Linyuan, Kaohsiung, Taiwan (120.38° E 22.49° N) and transported to the laboratory.
Seawater (35‰) and brackish water (15‰; BW) were prepared from
local tap water by adding standardized amounts of synthetic sea salt
“Instant Ocean” (Aquarium Systems, Mentor, OH, USA). The fish were
reared in BW at 25± 1 °C for one week in the laboratory and then
acclimated to either fresh water (FW), BW, or SW with a daily 12 h
photoperiod for at least 4 weeks before sampling for the following tests
and analyses. The parameters of the water were identical to our previous
study (Wang et al., 2008). The water was continuously circulated
through fabric-floss filters and partially refreshed every three days. Fish
were fed a daily diet of commercial pellets ad libitum. In order to
perform the following experiments, fish were fully anesthetized with
MS-222 (100–200 mg/L) and placed on ice before sampling.
2.2. Plasma analyses
Fish blood was collected from the caudal veins with heparinized
1 ml syringes and 27 G needles. After centrifugation at 13,000 g, 4 °C
for 20 min, the plasma was stored at − 20 °C prior to analysis. Plasma
osmolality was measured with the Wescor 5520 vapro osmometer
(Logan, UT, USA). Plasma glucose concentrations were determined
according to the manufacturer's protocol (Sigma, St. Louis, MO, USA)
using a Hitachi U-2001 spectrophotometer (Tokyo, Japan).
2.3. Muscle water content (MWC)
The muscle from the dorsum of the body of the adult fish of FW-,
BW-, and SW-acclimated groups was sampled and muscle water
content was determined as the percentage of weight loss after drying
at 100 °C for three days.
2.4. Sample preparation
The gills and livers were dissected and rinsed in phosphate buffered
saline (PBS; concentrations in mmol·l− 1: NaCl:137; KCl: 3; Na2HPO4:
10; KH2PO4: 2; pH 7.4). The tissues were then blotted dry before further
analysis. The gills were fixed in 10% formalin for immunocytochemical
detection or homogenized for immunoblotting and other analyses; the
livers were homogenized for immunoblotting. The gill and liver
scrapings were suspended in the mixture of homogenization medium
(concentrations in mmol·l− 1: imidazole-HCl: 100; Na2EDTA: 5; sucrose:
200; 0.1% sodium deoxycholate; pH 7.6) and proteinase inhibitor (10 mg
antipain, 5 mg leupeptin, and 50 mg benzamidine dissolved in 5 ml
aprotinin) (v/v: 100/ 1). Homogenization was performed in 2 ml
microtubes with a Polytron PT1200E (Kinematica, Lucerne, Switzerland) at maximal speed for 30 s The homogenate was then
centrifuged at 13,000 g, 4 °C for 20 min. The supernatants were
W.-K. Yang et al. / Journal of Experimental Marine Biology and Ecology 375 (2009) 41–50
used for determination of protein concentrations, enzyme activity, or
immunoblotting. Protein concentrations were identified by reagents
from BCA Protein Assay (Pierce, Hercules, CA, USA), using bovine
serum albumin (BSA) as a standard (Pierce). The samples were
stored at − 80 °C before use.
2.5. Optimal conditions for determining Na+/K+-ATPase (NKA) activity
For determining the optimal concentrations of Na+, K+, and Mg2+
in the reaction medium of in vitro assay of molly NKA activity, 0.1 ml
filtered homogenates and 5 ml reaction medium (concentrations
in mmol·l− 1: imidazole-HCl buffer: 100; pH 7.0; NaCl: 0–300; KCl:
0–300; MgCl 2 : 0–30; Na 2 ATP: 5) were mixed. Each reaction
condition was repeated. The reactions with the samples exposed to
different environments of [Na+], [K+], or [Mg2+] were run at 37 °C
for 30 min. The reactions were then stopped by the addition of 2 ml
ice-cold 30% trichloroacetic acid. The reaction tube was centrifuged
and the inorganic phosphate concentration in the supernatant was
determined with a spectrophotometer at 700 nm according to the
method proposed by Peterson (1978). The enzyme activity of NKA
was defined as the difference between the inorganic phosphate
liberated in the presence and absence of 3.75 mmol·l− 1 ouabain in
the reaction mixture. All experiments were repeated 2–4 times with
similar results. For comparisons, enzymatic activities were converted into relative activities (%) of the highest value obtained in
each experiment. Individual points of concentrations in each panel
were the averages of duplicate samples.
2.6. Assay of NKA activity
Aliquots of the suspension of branchial homogenates, prepared as
described above, were used for assaying protein concentrations and
enzyme activities. NKA activity was assayed by adding the supernatant to the optimal reaction medium (concentrations in mmol·l− 1:
imidazole-HCl buffer: 100; pH 7.6; NaCl: 200; KCl: 150; MgCl2: 5;
Na2ATP: 5). The reaction was run as described above. Each sample was
assayed in triplicate. NKA activity was expressed as μmol inorganic
phosphate released per mg of protein per hour.
2.7. Antibodies
The primary antibodies used in this study included (1) NKA: a
mouse monoclonal antibody (α5; Developmental Studies Hybridoma
Bank, Iowa City, IA, USA) raised against the α-subunit of the avian
NKA; (2) heat shock protein 90 (HSP90): a rabbit polyclonal antibody
(#4874; Cell Signaling, Beverly, MA, USA) raised against the human
HSP90. The secondary antibodies for immunoblotting were alkaline
phosphatase (AP)-conjugated goat anti-mouse IgG or goat anti-rabbit
IgG (Chemicon, Temecula, CA, USA).
2.8. Immunoblotting
For immunoblotting of branchial NKA, aliquots containing 25 µg of
branchial homogenates were added to sample buffer and heated at 60 °C
for 15 min followed by electrophoresis on 7.5% sodium dodecyl sulfate
(SDS)-polyacrylamide gel. The pre-stain protein molecular weight
marker was purchased from Fermentas (SM0671; Hanover, MD, USA).
Separated proteins were transferred from unstained gels to PVDF
membranes (Millipore, Bedford, MA, USA) by electroblotting using a
tank transfer system (Mini Protean 3, Bio-Rad, Hercules, CA, USA). Blots
were preincubated for 2 h in PBST (phosphate buffer saline with Tween
20) buffer (concentrations in mmol·l− 1: NaCl:137; KCl: 3; Na2HPO4: 10;
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
4 °C with the primary antibody (α5) diluted in 1% BSA and 0.05% sodium
azide in PBST (1: 2500) overnight. The blot was washed in PBST, followed
43
by a 1-h incubation with AP-conjugated secondary antibody diluted
4000x in PBST. Blots were visualized after incubation with an NBT/BCIP
kit (Zymed, South San Francisco, CA, USA).
For immunoblotting of HSP90, sample buffers were mixed with
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
electrophoresis on 7.5% SDS-polyacrylamide gels. The following protocol
was identical to that of branchial NKA α-subunit immunoblotting except
that the primary antibody to HSP90 was diluted 1000×.
Immunoblots were scanned and imported as TIFF files. Immunoreactive bands were analyzed using MCID software version 7.0, rev. 1.0
(Imaging Research, Ontario, Canada). The results were converted to
numerical values in order to compare the relative protein abundance
of the immunoreactive bands.
2.9. Immunohistochemical detection
Gills were dissected and fixed for 24 h in 10% formalin in 0.1 M
phosphate buffer (pH 7.2) at 25 °C. Samples were dehydrated through
a graded ethanol series, embedded in paraffin, and serial sections of
7 µm were mounted on gelatin-coated glass slides. The sections were
stained immunohistochemically with the monoclonal antibody (α5)
to NKA α-subunit followed by a commercial kit (PicTure™, Zymed).
Negative control experiments in which PBS (phosphate buffered
saline) was used instead of the primary antibody were conducted to
confirm the positive results of NKA immunoreactive (NKA-IR) cells.
Immunostained sections were observed using a light microscope
(Olympus BX50, Tokyo, Japan) and the micrographs were taken with a
digital camera (Nikon COOLPIX 5000, Tokyo, Japan).
The method of quantification for NKA-IR cells was modified from
our previous studies (Lin et al., 2003, 2006; Tang et al., 2008). The
numbers of NKA-IR cells were counted on the sections immunostained
as described in the previous paragraph. Preliminary observations
indicated that most NKA-IR cells in gills of the sailfin molly were
distributed in the interlamellar regions of the filaments. Hence,
longisections of the gills including lamellae and the cartilages of the
filaments were chosen, and the numbers of immunoreactive cells in
the interlamellar regions of the filaments and the lamellae were
counted. Cell numbers in the interlamellar regions within the range of
5 adjacent lamellae (approximately 40–60 µm) were counted and the
lengths of the 4 interlamellar regions were measured to normalize cell
counts to a fixed length (50 µm). Results were expressed as numbers of
NKA-IR cells per 50 µm of filaments (interlamellar regions). For each
sample, 10 areas on the filaments including symmetrical interlamellar
regions were randomly selected.
2.10. Statistical analysis
Values were compared using a one-way analysis of variance (ANOVA)
(Tukey's pair-wise method) and Pb 0.05 was set as the significant level.
Values were expressed as the means±S.E.M. (the standard error of the
mean) unless stated otherwise.
3. Results
3.1. Physiological parameters
The plasma osmolality of sailfin molly increased with environmental
salinities (Fig. 1A). The muscle water content (MWC), however, was
constant in either FW-, BW-, or SW-acclimated fish (Fig. 1B).
3.2. Branchial Na+/K+-ATPase (NKA) expression
When the concentration ratio of Na+: K+ was 4:3, the highest enzyme
activity was found at 200 and 150 mmol·l− 1 of total Na+ and K+,
respectively. Thus optimal concentrations of Na+ and K+ for the in vitro
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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 SWacclimated 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 immunoreactive 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, P b 0.05). 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). Quantification of immunoreactive bands revealed that the branchial NKA αsubunit 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
paraffin sections (Fig. 5). The branchial NKA-IR cells of SW-, BW- and FWacclimated 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,
however, was not significantly different from that in the BW or SWacclimated group (Fig. 6).
Fig. 2. Effects of different concentrations of (A) [Na+], (B) [K+], and (C) [Mg2+] in the
assay of P. latipinna branchial NKA activity. The arrows indicated the optimal ionic
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 lethostigma; 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.,
one way ANOVA with Tukey's comparison, P b 0.05).
abundance of SW sailfin molly was significantly higher than that of the
FW or BW group (Fig. 9B).
4. Discussion
4.1. Physiological parameters
Plasma osmolality and MWC were assayed in this study to determine
the osmoregulatory capacity of the sailfin molly. The plasma osmolality
of sailfin molly increased with environmental salinity; the highest level
was found in SW-acclimated fish (Fig. 1A). Although the experimental
designs were different, Nordlie et al. (1992) and Gonzalez et al. (2005)
also reported that plasma osmolality of sailfin molly increased in a
tolerated range with environmental salinity. Similar patterns of plasma
osmolality were found in the other euryhaline teleosts including
mummichog (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 (Oryzias 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 SW or 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 environments, enabling this euryhaline species to survive in natural habitats
ranging from FW to BW environments. Similarly, the MWC of emperor angelfish (Pomacanthus imperator; Woo and Chung, 1995),
goldlined seabream (Rhabdosargus sarba; Kelly and Woo, 1999),
gilthead seabream (Sparus auratus; 5–55‰ of environments;
Sangiao-Alvarellos et al., 2003; Laiz-Carrión et al., 2005a), spotted
green pufferfish (Bagherie-Lachidan et al., 2008), and marine medaka
(Oryzias dancena; Kang et al., 2008) was constant when acclimated to
Elevated branchial NKA activity provided more driving force for ion
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
(Hwang et al., 1988; Lin and Lee, 2005), European seabass (Jensen
et al., 1998), milkfish (Chanos chanos), and spotted green pufferfish (Lin
and Lee, 2005) were sensitive to the ionic strength of the assay medium.
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+,
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).
Although the optimal branchial Na+/K+ ratio for sailfin molly (4:3) was
different from the ratio for Mozambique tilapia (5:3; Hwang et al., 1988),
the present study showed the optimal reaction conditions for determining
branchial NKA activity in sailfin molly. This methodology yields more
significant results with minimal individual variation (Hwang et al., 1988).
In addition, the other sensitivities including Mg2+, ATP, pH, ouabain, or
Fig. 4. (A) Representative immunoblot of P. latipinna gills probed with a monoclonal
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
among groups of various environments (N = 6, mean ± S.E.M., one way ANOVA with
Tukey's comparison, P b 0.05). M, marker (kDa); FW, fresh water; BW, brackish water;
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 sailfin molly were distributed in filaments (the interlamellar 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 sailfin molly was
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 filaments from (A) FW-, (B) BW-, and (C) SW-acclimated P. 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, P b 0.05).
of changing numbers of NKA-IR cells was similar to that of protein
abundance and activity of NKA in gills of sailfin molly as described above.
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.,
2005a; Kang et al., 2008).
4.3. Stress responses
When exposed to a stressful environment, increased expression of
certain proteins suggests the presence of stress indicators in fish
attempting to adjust to the situation (Iwama et al., 1999, 2004). The
levels of plasma glucose (Kelly et al.,1999) and hepatic or branchial HSP90
expression (Deane et al., 2002; Choi and An, 2008) of teleost, were used to
indicate the stress caused by salinity challenge. In this study, higher levels
of plasma glucose and hepatic and branchial HSP90 expression in SWacclimated 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. 7. Salinity effects on plasma glucose levels. Dissimilar letters indicated significant
differences among fish acclimated to various environments (N = 8, mean ± S.E.M., one
way ANOVA with Tukey's comparison, P b 0.05).
Fig. 8. (A) Representative immunoblot of P. latipinna livers probed with a polyclonal
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, P b 0.05).
M, marker (kDa); FW, fresh water; BW, brackish water; SW, seawater.
(Fig. 7; Laiz-Carrión et al., 2005a; Sangiao-Alvarellos et al., 2005;
Arjona et al., 2007). Temporal patterns of plasma glucose concentrations following environmental stress, however, varied with teleostean
species. Plasma glucose levels can rise quickly and return to normal
within one day (Rotllant and Tort, 1997; Arends et al., 1999) or four
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
from different groups. The asterisk indicated a significant difference among groups in
various environments (N = 6, mean± S.E.M., one way ANOVA with Tukey's comparison,
P b 0.05). 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.,
2003; Frisch and Anderson, 2005; Sangiao-Alvarellos et al., 2005)
after shocks. Higher plasma glucose levels can be maintained over
20 days after salinity shocks (Fig. 7; Woo and Chung, 1995; Morgan
and Iwama, 1998; Kelly et al., 1999; Claireaux and Audet, 2000; De
Boeck et al., 2000; Fiess et al., 2007). Consistently higher glucose
levels after transfer illustrated a higher energy demand for acclimatation to the new environment (Soengas et al., 1995; Arjona et al.,
2007).
Increased expression of HSPs including HSP90 of fish upon transfer to
a hyperosmotic environment, suggests a role for HSPs in maintaining the
normal functions of proteins and cells (Iwama et al., 1999). Upon salinity
challenge, HSP90 expression varied differentially among species. HSP90
expression in livers (protein amount; Deane et al., 2002) and gills
(mRNA abundance; Choi and An, 2008) of black porgy was found to be
higher in hypoosmotic environments (BW or FW) than in SW. However,
no change in HSP90 mRNA expression was found in the livers and gills of
chinook salmon at one day after transfer from FW to SW (Oncorhynchus
tshawytscha; Palmisano et al., 2000). On the other hand, the branchial
HSP90 mRNA expression in the gills of Atlantic salmon was raised and
then returned to baseline levels within two days after transfer from FW
to SW (Pan et al., 2000). HSP90 is abundantly expressed in most
eukaryotes and it performs its role by forming a multiple protein
complex with ATPase activity as well as cooperating with other HSPs
(Wegele et al., 2004). Elevated hepatic and branchial HSP90 expression
in SW-acclimated sailfin molly might play important roles for reaching
homeostasis upon salinity challenge. More evidence such as plasma
cortisol levels will be necessary to illustrate the stress response of sailfin
molly upon salinity challenge in future work.
5. Conclusion
Euryhaline sailfin molly is able to survive in FW, BW, and SW.
Moreover, elevated plasma glucose concentrations, as well as branchial
and hepatic HSP90 protein abundance, revealed that SW was stressful
than FW to the sailfin molly. To maintain homeostasis, higher NKA
activity was necessary for ion secretion in gills of the sailfin molly in SW
(the stressful environment). Although the plasma osmolality of sailfin
molly increased with environmental salinities within a tolerated range,
the MWC were constant among different salinity groups. These results
illustrated that the sailfin molly was proved to be an efficient
osmoregulator with gill NKA expression altering in response to salinity
challenge to maintain ion and water homeostasis in environments of
different salinities.
Acknowledgements
The monoclonal antibody of Na+/K+-ATPase α-subunit (α5) were
purchased from the Developmental Studies Hybridoma Bank maintained
by the Department of Pharmacology and Molecular Sciences, John
Hopkins University School of Medicine, Baltimore, MD 21205, and the
Department of Biological Sciences, University of Iowa, Iowa City, IA 52242,
under Contract N01-HD-6-2915, NICHD, USA. This study was supported
by a grant from the National Science Council of Taiwan to T.H.L. (NSC 942311-B-005-009). [SS]
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