RESEARCH PAPER
Freshwater Acclimation Induces
Stress Responses and Expression
of Branchial Naþ/Kþ‐ATPase and
Proliferating Cell Nuclear Antigen
in Takifugu niphobles
CHENG‐HAO TANG1,2
3,4
AND TSUNG‐HAN LEE *
1
Institute of Marine Biotechnology, National Dong Hwa University, Pingtung, Taiwan
National Museum of Marine Biology and Aquarium, Pingtung, Taiwan
3
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
4
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
2
ABSTRACT
J. Exp. Zool.
319A:409–421,
2013
Almost the whole life cycle of the grass puffer (Takifugu niphobles) occurs in seawater (SW), but it is
also sometimes found in fresh water (FW) rivers. This study aims to evaluate the effects of FW
exposure on the stress, osmoregulatory, and physiological responses of the grass puffer. The grass
puffers were captured from a local wetland and acclimated to SW (35‰) or FW in the laboratory. In
the stress responses, plasma glucose concentrations and the abundances of hepatic and branchial
heat shock proteins were higher in the FW group than in the SW group. FW acclimation led to a
significant increase in the protein abundance and the specific activity of branchial Naþ/Kþ‐ATPase
(NKA). Immunochemical staining showed that the NKA immunoreactive (NKIR) cells of the FW and
SW puffer were distributed mainly in gill filaments. Although the number of NKIR cells was similar
in the two groups, the protein levels of proliferating cell nuclear antigen (PCNA) of nuclear fractions
were elevated in the gills of the FW puffer. The induction of gill PCNA might contribute to cell
proliferation which would maintain the amount of NKIR cells or repair DNA when exposed to FW, an
osmotically stressful environment. Hence, activation of stress responses would provide the
osmoprotection associated with FW adaptation of the grass puffer. Changes of branchial NKA
expression and activity for osmoregulatory adjustment were required for stable blood osmolality
and muscle water content. Based on our findings, the grass puffer was suggested to be a euryhaline
teleost with SW preference. J. Exp. Zool. 319A:409–421, 2013. © 2013 Wiley Periodicals, Inc.
How to cite this article: Tang C‐H, Lee T‐H. 2013. Freshwater acclimation induces stress
responses and expression of branchial Naþ/Kþ‐ATPase and proliferating cell nuclear antigen in
Takifugu niphobles. J. Exp. Zool. 319A:409–421.
The maintenance of a stable internal environment is important for
vertebrates to survive in a variety of habitats. Even slight changes in
ion concentration, osmolality, or the pH of body fluids can
significantly affect the survival of the animals. Strategies for
maintaining the homeostasis of body fluids vary, depending on the
species of animal and on their habitats (Evans, 2009). Osmotic stress
is one of the principal challenges that can fluctuate greatly in aquatic
environments. When osmotic stresses are present, protein synthesis,
stabilization, and biological function become disrupted, eventually
causing protein damage harmful to cellular function and viability
Grant sponsor: National Science Council of Taiwan; grant numbers: NSC
95‐2311‐B‐005‐040‐MY3, NSC 101‐2313‐B‐259‐002.
Conflicts of interest: None.
*Correspondence to: Tsung‐Han Lee, Department of Life Sciences,
National Chung Hsing University, Taichung 402, Taiwan.
E‐mail: [email protected]
Received 25 July 2012; Revised 18 February 2013; Accepted 17 April
2013
DOI: 10.1002/jez.1804
Published online 17 June 2013 in Wiley Online Library
(wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
410
(Deane and Woo, 2004; Choe and Strange, 2008). Stress responses
are energy‐demanding mechanisms in animals (Vijayan et al., '97;
Iwama et al., '99). Because glucose plays a major role in providing
energy for metabolism, a change in plasma glucose concentration is
one known stress indicator at the organismal level (Afonso
et al., 2003; Iwama et al., 2006). Moreover, cellular responses to
stressors lead to an evident increase in the levels of stress‐related
proteins. Under stressed conditions, proteins belonging to the heat
shock protein (HSP) family are rapidly synthesized and perform an
important cytoprotective function, helping to refold and repair
damaged proteins (Hightower, '91; Gething and Sambrook, '92).
Among them, HSP70 constitutes the central part of a ubiquitous
chaperone system in eukaryotic cells and HSP90 is one of the most
abundant proteins in unstressed cells (Lai et al., '84; Macario
et al., '99; Wegele et al., 2004). HSPs are also involved in the adaptive
mechanisms that fish use to resist extreme environments, which
contain various stressors (Hightower, '91; Beck et al., 2000; Iwama
et al., 2006). Without these protective mechanisms, cellular proteins
become misfolded and aggregate when organisms are exposed to
stresses (Wegele et al., 2004). HSPs have received the most attention
in organisms undergoing experimental stresses in the laboratory
(Feder and Hofmann, '99). Furthermore, HSPs with large molecular
weights may serve as biomarkers of non‐specific stressors in many
organisms because they are highly conserved among organisms
from different taxa (Iwama et al., '99). The crucial roles of HSPs in
salinity acclimation have been discussed in fish (Pan et al., 2000;
Deane et al., 2002; Deane and Woo, 2004).
Osmoregulatory mechanisms are crucial for salinity adaptation
of euryhaline teleosts (Evans et al., 2005, 2010; Marshall and
Grosell, 2006; Kaneko et al., 2008; Hwang et al., 2011). The gills are
the major osmoregulatory organs that directly contact the
external aquatic environment. The fundamental transporters
responsible for ion movement across gill epithelia have been
reported in several reviews (Evans et al., 2005; Kaneko et al., 2008;
Evans, 2010; Hwang et al., 2011). Among the transporters, Naþ/
Kþ‐ATPase (NKA) is thought to provide the primary driving force
for ion‐transporting systems. Therefore, modulation of gill NKA
protein expression and activity is a critical osmoregulatory
adjustment. Because most of the NKA protein expressed in gills is
localized in mitochondrion‐rich (MR) cells, MR cells are also called
NKA immunoreactive (NKIR) cells and are thought to be the
principal sites responsible for ion transport. The distribution of gill
NKIR cells is dependent on species and environmental salinity. In
addition, the densities of NKIR cells correlated with the NKA
responses in the gills of several studied teleosts (Hwang and
Lee, 2007; Kaneko et al., 2008). Moreover, proliferating cell
nuclear antigen (PCNA) is a biomarker of cell proliferation and a
protein involved in DNA repair (Cox, '97; Maga and
Hubscher, 2003). The changes in NKIR cell densities in response
to salinity changes are related to alteration of the rates of cell
proliferation and turnover (Uchida and Kaneko, '96; Katoh and
Kaneko, 2003).
J. Exp. Zool.
TANG AND LEE
Takifugu is a genus of puffer belonging to the Tetradontidae
family of teleost fish. Because the genome project for T. rubripes
has been completed (Aparicio et al., 2002), the Takifugu genus has
become a good model animal (Kato et al., 2005). The grass puffers
(Takifugu niphobles) are widely distributed around the coastal
areas of Korea, Japan, and China (Masuda et al., '84). Nearly the
entire life cycle of the T. niphobles occurs in the SW, including
maturation and reproduction (Nozaki et al., '76; Honma and
Ozawa, '80). However, the grass puffers are the peripheral FW fish
that adult grass puffers sometimes can be found in brackish river
mouths (Miyadi et al., '76; Masuda et al., '84), and FW rivers (Kuo
and Shao, '99; Kato et al., 2005). Moreover, Shen et al. (2007) and
our preliminary test have shown that grass puffers can survive in
fresh water (FW) for at least 2 weeks. To our knowledge, impact of
FW acclimation on the physiological responses of T. niphobles has
not been well studied. Therefore, the aim of this study is to
investigate how stress responses and osmoregulatory mechanisms
are related to the euryhalinity of the grass puffer.
MATERIALS AND METHODS
Experimental Animals and Environments
The grass puffers (T. niphobles), 11–13 cm in total length and 30–
40 g in weight, were captured from the local estuary (i.e., Gaomei
wetland at Taichung, Taiwan) (the salinity was approximately 22–
25‰) and raised in a tank with a seawater (SW; 35‰) circulating
system at 27 1°C with a 12/12‐hr light/dark photoperiod for at
least 2 weeks. The SW (35‰) used in this study was prepared from
local tap water with proper amounts of synthetic sea salt (Instant
Ocean, Aquarium Systems, Mentor, OH, USA). The composition of
the SW was [Naþ], 582.6 mM; [Cl], 526.8 mM; and [Ca2þ],
15.7 mM; pH 8.2. The water was continuously circulated through
fabric‐floss filters, and the salinity was checked daily. The fish
were fed a daily diet of commercial pellets but were not fed for a
period of 24 hr before the sampling. No mortality was observed
during the holding period.
Acclimation Experiment
After the holding period, the grass puffers were randomly divided
into two different groups for the acclimation experiment. The fish
were directly transferred to SW or fresh water (FW; dechlorinated
local tap water) tanks for 2 weeks. The composition of the
dechlorinated FW was [Naþ], 2.6 mM; [Cl], 0.2 mM; and [Ca2þ],
0.58 mM; pH 7.8. After 2 weeks, experimental animals were
randomly selected from tanks and anaesthetized by immersion in
MS‐222 (75 mg/L) before sampling.
Analysis of Plasma Glucose Levels, Plasma Osmolality and Muscle
Water Content (MWC)
Fish blood was collected from the heart using heparinized 1 mL
syringes and 21 G needles. After centrifugation at 1,000g at 4°C
for 10 min, the plasma osmolality and glucose levels were
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
measured immediately using a Wescor 5520 Vapro osmometer
(Logan, UT, USA) and an ACCU‐CHEK Go blood glucose meter
(Roche, Mannheim, Germany), respectively. The MWC was
determined according to Tang et al. (2009). The MWC was
measured gravimetrically after drying at 100°C for 48 hr.
Antibodies
The primary antibodies used in the present study included (1) anti‐
HSP70, a mouse monoclonal antibody (H 5147; Sigma, St. Louis,
MO, USA) generated using immunization with purified bovine
brain HSP70; (2) anti‐HSP90, a rabbit polyclonal antibody (#4874;
Cell Signaling Technology, Beverly, MA, USA) corresponding to
human HSP90; (3) anti‐Naþ/Kþ‐ATPase (NKA), a mouse monoclonal antibody (a5; Developmental Studies Hybridoma Bank,
Iowa City, IA, USA) raised against the a‐subunit of avian NKA;
and (4) anti‐PCNA, a mouse monoclonal antibody raised against
amino acid residues 112–121 of human PCNA (Ab‐1; Calbiochem,
Darmstadt, Germany). The secondary antibodies for immunoblotting were alkaline phosphatase‐conjugated goat anti‐mouse IgG
and goat anti‐rabbit IgG (Chemicon, Temecula, CA, USA).
Preparation of Gill and Liver Homogenates
Immediately after the fish were killed by spinal pithing, the gills
and livers were extracted, and the gill arches were removed and
blotted dry. The samples were immersed in liquid nitrogen and
placed into an ice‐cold homogenization buffer (250 mM sucrose,
1 mM EDTA, and 30 mM Tris, pH 7.4). To 1 mL of the ice‐cold
homogenization buffer, 40 mL of a proteinase inhibitor cocktail
(Roche) was added. Homogenization was performed in 2 mL tubes
using a Brinkmann Polytron Homogenizer (PT1200E, Kinematica,
Lucerne, Switzerland) operating at maximum speed for 10 strokes.
The homogenates were centrifuged at 4°C and 12,000g for 20 min,
and the supernatants were stored at 80°C. The protein
concentrations were determined colorimetrically using a BCA
Protein Assay Kit (Pierce, Hercules, CA, USA) with bovine serum
albumin (Pierce) as a standard.
Preparation of Nuclear and Non‐Nuclear Fractions of Gills
Nuclear and non‐nuclear fractions were prepared according to
Rendell and Currie (2005). Briefly, the gills were minced and
homogenized in TEDG buffer (50 mM Tris, 1.5 mM EDTA, 1 mM
dithiothreitol, and 30% glycerol). To 1 mL of the TEDG buffer, 40 mL
of a proteinase inhibitor cocktail (Roche) was added. The
homogenization was performed in 2 mL tubes with a Brinkmann
Polytron Homogenizer operating at maximum speed for 10 strokes.
The homogenates were centrifuged at 1,000g for 20 min, and the
supernatants were stored at 80°C. This supernatant represented the
non‐nuclear fraction. The nuclear pellet from the original 1,000g
centrifugation was washed twice in TMDS buffer (50 mM Tris,
5 mM MgCl2, 250 mM sucrose, and 1 mM dithiothreitol; pH 7.4)
and centrifuged at 750g for 10 min after each wash. The pellet was
re‐suspended in 500 mL of TEDG þ KCl (TEDG buffer with 0.6 mM
411
KCl; pH 7.4) and intermittently vortexed for 60 min. After vortexing,
the samples were centrifuged at 12,000g for 20 min. The
supernatant, representing the nuclear salt extract (nuclear fraction),
was stored at 80°C for further analysis.
Immunoblotting
The immunoblotting procedure was conducted according to Tang
et al. (2009) with little modification. Proteins from the samples
were heated with sample buffer at 90°C for 5 min for the HSPs and
the PCNA and 37°C for 30 min for the NKA a‐subunit. The pre‐
stained protein molecular weight marker was purchased from
Fermentas (SM0671; Hanover, MD, USA). All samples were
separated using electrophoresis on sodium dodecyl sulfate (SDS)‐
containing 7.5% (for HSPs and NKA a‐subunit) or 12% (for PCNA)
polyacrylamide gels (30 mg protein/lane). The separated proteins
were then transferred to a PVDF membrane (Millipore, Bedford,
MA, USA) by electroblotting. After preincubation for 3 hr in PBST
buffer containing 5% (w/v) nonfat dried milk (used to minimize
nonspecific binding), the blots were incubated for 2 hr at room
temperature with the primary antibodies diluted in 1% (w/v) BSA
and 0.05% (w/v) sodium azide in PBST. The dilution factor for the
primary antibodies is 1:4,000 for anti‐HSP70; 1:3,000 for anti‐
HSP90; 1:5,000 for anti‐NKA, and 1:2,000 for anti‐PCNA. The
blots were then washed in PBST and incubated at room
temperature for 1.5 hr with the secondary antibody (1:5,000
dilution). The blots were developed after incubation with a BCIP/
NBT Kit (Zymed, South San Francisco, CA, USA). The immunoblots were photographed and imported as TIFF files into the
commercial image‐analysis software package MCID version 7.0,
rev. 1.0 (Imaging Research Inc., Ontario, Canada). The results were
converted into numerical values to compare the relative intensities
of the immunoreactive bands.
Specific Activity of NKA
Gill NKA activity was determined as described by Hwang et al.
('89). Aliquots of the suspension of gill homogenates, prepared as
described above, were used for determination of protein and
enzyme activities. The NKA activity was assayed by adding the
supernatant to the reaction mixture (100 mM imidazole–HCl
buffer, pH 7.6, 125 mM NaCl, 75 mM KCl, 7.5 mM MgCl2, and
5 mM Na2ATP). The reaction was run at 37°C for 30 min and then
stopped by the addition of 200 mL of ice‐cold 30% trichloroacetic
acid. The inorganic phosphate concentration was measured
according to Peterson's method ('78). The enzyme activity of NKA
was defined as the difference between the amount of inorganic
phosphate liberated in the presence and absence of 1 mM ouabain in
the reaction mixture. Each sample was assayed in triplicate.
Cryosection and Immunohistochemical Detection of NKA
Immunoreactive (NKIR) Cells
Cryosections were performed to reveal different parts of the gills,
that is, the filaments and lamellae, for examining the distribution
J. Exp. Zool.
412
of NKIR cells. The procedure for immunohistochemical staining of
NKA was modified from Lin et al. (2006). The gills were excised
and fixed in a mixture of methanol and DMSO (4:1 v/v) at 20°C
for 3 hr before being embedded using O.C.T. (optimal cutting
temperature) compound (Sakura, Tissue‐Tek®, Torrance, CA,
USA). The fixed samples were washed with PBS, and the gill
arch and one side of the filaments were removed. Serial sections
(7 mm) were cut parallel to the long axis of the filament and
mounted on slides coated with poly‐L‐lysine. The cryosections
were incubated with the primary antibody to NKA a‐subunit (a5)
(1:200 dilution) followed by staining with a commercial kit
(PicTure TM, Zymed). The immuno‐stained sections were counterstained with hematoxylin. Negative control experiments, in which
PBS was used instead of the primary antibody, were conducted
(data not shown) to confirm the positive results. To quantify the
distribution of NKA immunoreactive cells (NKIR cells), longitudinal sections of the gills, including the lamellae and the cartilages
of the filaments, were chosen; the immunoreactive cells in the
filaments (F) and lamellae (L) were also counted. Lamellar areas
were defined as the parts projecting from filaments. The
interlamellar regions, including the regions between the lamellar
bases and filaments, were considered to be filament areas. Ten
areas on the filaments, including symmetrical lamellae, were
randomly selected in each sample. The immunostained cryosections were observed using a light microscope (Olympus BX50,
Tokyo, Japan), and the micrographs were recorded with a digital
camera (Nikon COOLPIX 5000, Tokyo, Japan).
Statistical Analysis
The significance of the difference between SW and FW was
assessed using an unpaired t‐test (P < 0.05). The values were
expressed as the means SEM.
RESULTS
Stress Responses at the Organismal and Cellular Levels: Plasma
Glucose Levels and Expression of Branchial and Hepatic Heat Shock
Proteins (HSPs)
The acclimation experiments revealed that the plasma glucose
levels were significantly higher in the fresh water (FW)‐acclimated
group (117.6 15.6 mg/dL) than in the seawater (SW)‐acclimated group (49.6 3.1 mg/dL). The relative protein abundance of
stress proteins (i.e., HSPs) in the fish gills and livers was examined.
Immunoblotting of the gills (Figs. 1A and 2A) and livers (Figs. 1C
and 2C) from the FW‐ and SW‐acclimated grass puffers, probed
with primary antibodies to HSP70 (Fig. 1) and HSP90 (Fig. 2),
resulted in single immunoreactive bands with molecular weights
at approximately 70 (Fig. 1A and C) and 90 kDa (Fig. 2A and C),
respectively. The protein expression levels of HSP70 (Fig. 1B and
D) and HSP90 (Fig. 2B and D) in the gills and livers of the FW‐
exposed individuals were significantly higher than those of the
SW‐acclimated fish.
J. Exp. Zool.
TANG AND LEE
Plasma Osmolality and Muscle Water Content (MWC)
The plasma osmolality was significantly lower in the FW‐
acclimated grass puffers (T. niphobles) (296.5 12.8 mOsm/kg)
than in the SW‐acclimated individuals (383.0 6.5 mOsm/kg).
There was no significant difference in the MWC of the FW‐ and
SW‐acclimated grass puffers (79.2 1.1% and 79.2 1.1%).
Branchial Naþ/Kþ‐ATPase (NKA) Responses
The relative protein abundance of branchial NKA was examined.
Immunoblotting of the gills from the FW‐ and SW‐acclimated
grass puffers resulted in a single immunoreactive band with
molecular weight of approximately 105 kDa (Fig. 3A). The relative
protein abundance of gill NKA a‐subunit was significantly
elevated in the FW‐acclimated grass puffer group compared with
the SW‐acclimated group (Fig. 3B). Moreover, the NKA specific
activities in the gills of the FW individuals were also significantly
higher than those of the SW fish (1.94‐fold; Fig. 3C).
Distribution and Quantification of NKA Immunoreactive (NKIR) Cells
No evident changes associated with salinity could be observed in
the distribution of gill NKIR cells (Fig. 4). The gill NKIR cells of the
FW‐ and SW‐acclimated fish were distributed in the filaments, yet
none were found in the lamellae (Fig. 4A and B). Additionally, no
significant difference was found in the densities of NKIR cells
between the FW‐ and SW‐acclimated grass puffers (Fig. 4C;
19.34 0.74 vs. 19.77 0.55 per 100 mm of the gill filament).
Expression of Proliferating Cell Nuclear Antigen (PCNA) in Non‐
Nuclear and Nuclear Fractions of Gills
Immunoblotting of the non‐nuclear (Fig. 5A and B) and nuclear
fractions (Fig. 5C and D) of the gills from the FW‐ and SW‐exposed
puffers showed a single immunoreactive band at 33 kDa (Fig. 5A
and C). Quantification of the immunoreactive bands revealed no
significant difference between the FW and SW puffers in the
amount of gill PCNA in the non‐nuclear fractions (Fig. 5B).
However, in the nuclear fractions, the protein expression level of
gill PCNA was significantly higher in the FW‐acclimated grass
puffer group than in the SW‐acclimated group (Fig. 5D).
DISCUSSION
Seawater (SW) is the major habitat of grass puffer (T. niphobles),
however, it can be found naturally in freshwater (FW) rivers (Kuo
and Shao, '99; Kato et al., 2005). The ecological distribution of T.
niphobles revealed that it is a peripheral FW species. Changes in
environmental salinity usually result in osmotic stress to aquatic
organisms have be illustrated in the previous studies (Kltz, 2001;
Iwama et al., 2006; Fiol and Kltz, 2007; Deane and Woo, 2009).
Furthermore, when marine teleosts (Deane et al., 2002; Deane and
Woo, 2004; Choi and An, 2008), lobsters (Spees et al., 2002),
mussels (Hamer et al., 2008), and sea cucumber (Dong et al., 2008)
were acclimated to hypotonic or low‐salinity environments,
diverse cellular stress responses were activated. It was therefore
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
413
Figure 1. Representative immunoblots (A, C) and relative protein abundance (B, D) of the grass puffer livers and gills probed with a
monoclonal antibody to HSP70. The immunoreactive bands of FW‐acclimated puffer livers (A) and gills (C) are more intense than those in the
SW‐acclimated individuals. The relative protein amounts of the immunoreactive bands of HSP70 in livers (B) and gills (D) are significantly
higher in the FW fish than in the SW fish (n ¼ 6). The expression of hepatic and branchial HSP70 is 2.6‐ and 2.1‐fold higher in the FW group
than the SW group, respectively. M, marker; FW, fresh water; SW, seawater.
hypothesized that diverse physiological mechanisms would be
activated to maintain organismal and cellular homeostasis in SW
grass puffers were acclimated to FW. To evaluate the effects of a
hypotonic environment on the stress responses of the grass puffer,
stress bioindicators were examined. Stress responses are energy‐
demanding processes and animals must regulate energy substrates
to metabolically resist the stresses. Thus, changes in plasma
glucose concentrations have commonly been used as a stress
bioindicator at the organismal level (Basu et al., 2001; Afonso
et al., 2003; Iwama et al., 2006). In the present study, plasma
glucose levels greatly increased in the FW‐exposed puffers
compared with the SW‐exposed fish (Table 1). Thus, it is presumed
that more energy is needed for compensation of the cost from
energy‐demanding processes when grass puffers were acclimated
from SW to FW. In contrast, osmotic stress (i.e., hypertonic and
hypotonic stress) at the cellular level would severely disrupt
protein synthesis and conformation, causing protein damage that
is harmful to cells (Beck et al., 2000; Deane et al., 2002; Deane and
Woo, 2004; Alfieri and Petronini, 2007). In response to hypotonic
shock, many cell types conduct regulatory volume decrease (RVD)
through changes in specific ionic fluxes (Margalit et al., '93).
Hypotonicity‐induced ionic balance disruption with subsequent
protein damage can increase the expression of heat shock proteins
(HSPs) (Deane et al., 2002). HSPs are crucial to the cellular defense
procedures (cytoprotection) that cells use to survive when
experiencing abnormal protein conditions or proteotoxicity. In
J. Exp. Zool.
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TANG AND LEE
Figure 2. Representative immunoblots (A, C) and relative protein abundance (B, D) of the grass puffer livers and gills probed with a polyclonal
antibody to HSP90. The immunoreactive bands of FW‐acclimated puffer livers (A) and gills (C) are more intense than those in the SW‐
acclimated individuals. The relative protein amounts of the immunoreactive bands of HSP70 in the livers (B) and gills (D) are significantly
higher in the FW group than in the SW group (n ¼ 6). The expression of hepatic and branchial HSP70 is 1.7‐ and 2.8‐fold higher in the FW
group than in the SW group, respectively. M, marker; FW, fresh water; SW, seawater.
our study, the protein expression of hepatic HSP70 and HSP90
were examined. This organ has sensitive physiological responses
and performs a wide variety of important functions; the liver is
usually considered the major biochemical factory of the body
(Sherwood et al., 2005). The abundances of HSP70 and HSP90
were also measured in the gills, the osmoregulatory organ directly
exposed to the external environment (Figs. 1 and 2). Compared
with those in the SW puffer, the protein levels of hepatic and
branchial HSPs were higher in the FW individuals (Figs. 1 and 2).
The induction of increased protein abundance of HSPs could
protect cells from injury. HSPs have roles as molecular
chaperones; thus, they repair the damage to proteins induced
by hypotonicity and maintain the folding, assembly, and
J. Exp. Zool.
biological function of proteins in grass puffers acclimated to
FW. The fish intestine also plays a critical role in osmoregulation
and is exposed to the external environment when fish drink in SW.
Although this study examined the protein expression of HSPs in
grass puffer gills and livers, it is difficult to fully characterizing the
effects of osmotic stress without the data of cellular stress
responses in intestine. On the other hand, the induction of HSP
expression is mediated by the binding of a transcriptional
activator, heat shock factor (HSF), to a short, highly conserved
upstream response element, the heat shock cis‐element (HSE)
(Xiao and Lis, '88). Importantly, it has been demonstrated that
HSF‐1 is activated by hypotonic stress in mammalian cells (Huang
et al., '95; Caruccio et al., '97). However, the expression and
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
Figure 3. Representative immunoblots of the grass puffer gills
probed with monoclonal antibody a5 to the NKA a‐subunit (A). The
immunoreactive bands of the FW‐acclimated fish are more intense
than those of the SW‐acclimated individuals. (B) Relative
abundance of the immunoreactive bands of the NKA a‐subunit
in the gills of different salinity groups (n ¼ 6). The expression of
the NKA a‐subunit is 4.8‐fold higher in the FW group than in the
SW group. (C) The NKA activity in the gills of grass puffers
acclimated to FW and SW (n ¼ 6); the activity is significantly
higher in the FW‐acclimated group than in the SW‐acclimated
group. M, marker.
415
Figure 4. NKA immunostaining in longitudinal sections of the gills
of (A) FW‐acclimated and (B) SW‐acclimated grass puffers. (C)
Quantification of NKA immunoreactive (NKIR) cells in the gills of
SW and FW fish (n ¼ 6) revealed that there is no significant
difference between these two groups in the number of NKIR cells in
the filaments. F, filaments; L, lamellae; arrowhead, NKIR cells.
J. Exp. Zool.
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TANG AND LEE
Figure 5. Representative immunoblots of the grass puffer gills probed with a monoclonal antibody (NA03; Calbiochem) to gill PCNA in
cytoplasmic (A) and nuclear (C) fractions. The relative intensities of the PCNA immunoreactive bands (n ¼ 4) showed that no significant
difference in the amounts of cytoplasmic PCNA was found between the FW and SW groups (B); however, a significant increase in the PCNA
protein abundance of the nuclear fraction was observed in the puffer exposed to FW (D). M, marker.
activation of teleostean HSF‐1 in response to osmotic stress are
poorly understood. Only the positive correlation was found
between the levels of gill HSP70 and HIF‐1 transcripts in
euryhaline silver sea bream acclimated to different salinities
(Deane and Woo, 2004). Therefore, future work will investigate
whether HSF‐1 of grass puffers can be activated by hypotonic
shock to trigger the induction of HSP expression. Combining
previous findings with our data reveals that activation of cellular
stress responses is crucial for the species with SW preference faced
with a low‐salinity challenge.
Table 1. Comparison of SW‐ and FW‐acclimated grass puffers, Takifugu niphobles.
Environments
Plasma glucose levels (mg/dL)
Plasma osmolality (mOsm/kg)
Muscle water content (%)
Values are expressed as the mean SEM, n ¼ 6 for all groups.
Significantly different between SW and FW groups values at P < 0.05.
J. Exp. Zool.
SW
FW
49.6 3.1
383.0 6.5
79.2 1.1
117.6 15.6
296.5 12.8
79.2 1.1
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
In general, marine teleosts exhibit limited capability for FW
adaptation. However, the euryhaline marine or estuarine teleosts
have been shown to be capable of tolerating low‐salinity
environments. This finding suggests that those teleosts might
have excellent hyper‐osmoregulatory ability, allowing them to
maintain blood osmolality within certain physiological ranges
(reviewed by Kaneko et al., 2008). In the long‐term (i.e., at least 2
weeks) acclimation experiment, unlike the euryhaline milkfish
(Chanos chanos) (Lin et al., 2003) and the Senegalese sole (Solea
senegalensis) (Arjona et al., 2007), salinity‐induced changes in
plasma osmolality were found in the grass puffer (Table 1), tilapia
(Oreochromis mossambicus) (Uchida et al., 2000), spotted green
pufferfish (Tetraodon nigroviridis) (Lin et al., 2004), gilthead sea
bream (Sparus aurata) (Sangiao‐Alvarellos et al., 2004), and
medaka (Oryzias latipes and O. dancena) (Kang et al., 2008).
Although the plasma osmolality of the FW group was lower than
that of the SW group, the osmolality of the FW‐ and SW‐
acclimated grass puffers were well within the ranges reported for
FW and SW teleosts (Withers, '92; Kaneko et al., 2008).
Additionally, the plasma osmolality of the grass puffer reported
by Kato et al. (2005) was similar in SW individuals (392.00 5.60
vs. 383.00 6.52 mOsm/kg) compared with the present study but
different for the FW group (244.00 9.50 vs. 296.50 12.80
mOsm/kg). The lower osmolality was consistent with the higher
mortality in the FW‐acclimated grass puffers (Kato et al., 2005).
Although the grass puffers used in Kato et al. (2005) were adult
fish, their body lengths (approximately 6–7 cm) were smaller than
the lengths of the fish used in this study (approximately 11–
13 cm). Our data showing that the captured grass puffers could
survive in FW in the laboratory reflected the fact that adult grass
puffers have sometimes appeared in FW rivers (Kuo and Shao, '99),
also mentioned in Kato et al. (2005). Moreover, muscle water
content (MWC) could be an indicator of the osmoregulatory
capacity and degree of euryhalinity in fishes (Freire et al., 2008).
The MWC in FW and SW grass puffer was similar (Table 1). As
described by numerous previous studies, the MWC in euryhaline
teleosts can be maintained within a narrow range when
acclimated to environments of different salinities reflected their
homeostasis. These species included two medaka (O. latipes and O.
dancena) (Kang et al., 2008), European sea bass (Dicentrarchus
labrax) (Giffard‐Mena et al., 2008), brown trout (Salmo trutta)
(Tipsmark et al., 2002), Mozambique tilapia (O. mossambicus), and
milkfish (C. chanos) (Tang et al., 2009). According to the
physiological parameters described above, the grass puffer should
have excellent osmoregulatory ability when subject to a salinity
challenge.
The significant role played by branchial NKA in ion transport
has been confirmed in various species (reviewed in Evans
et al., 2005; Hwang and Lee, 2007; Hwang et al., 2011). Moreover,
salinity‐dependent alterations of the protein abundances and
specific activity of gill NKA were indeed found in the grass puffer
(Fig. 3), Atlantic salmon, Salmo salar (D'Cotta et al., 2000),
417
Mozambique tilapia (Lee et al., 2000, 2003), milkfish (Lin
et al., 2003), pufferfish (Lin et al., 2004), and two medaka species
(Kang et al., 2008). In the grass puffer, gill NKA protein abundance
and activity was higher in FW individuals than in SW fish (Fig. 3).
Many euryhaline brackish water (BW) and SW species that have
been studied also displayed higher gill NKA activity during FW
acclimation, including the European sea bass (Lasserre, '71),
pupfish, Cyprinodon salinus (Stuenkel and Hillyard, '80), mullet,
Chelon labrosus (Lasserre, '71) and Mugil cephalis (Ciccotti
et al., '95), long‐jawed mudsucker, Gillichthys mirabilis (Doneen, '81), flounder, Platichthys flesus (Stagg and Shuttleworth, '82), black seabream, Mylio macrocephalus (Kelly
et al., '99), milkfish (Lin et al., 2003), and rabbitfish, Siganus
rivulatus (Saoud et al., 2007). Furthermore, in the same genus
species, the tiger puffer (Takifugu rubripes) is a well known model
species since its genome was completely sequenced. The tiger
puffers spawn in the entrance of bays and their fry of grow in
shallow and river mouths of bays for 1 year, and then go to the
ocean (Fujita, '62). In the laboratory study, Kato et al. (2005)
suggested that T. rubripes could not survive in FW. When the tiger
puffers were acclimated to a low‐salinity environment (i.e., 1%
SW), up‐regulated gill NKA activity was not found in this
stenohaline teleost (Lee et al., 2005). The results may explain the
limited salinity tolerance of T. rubripes. These findings also
illustrated the vital role of gill NKA responses for osmoregulation
in euryhaline teleosts upon hypotonic acclimation (Hwang and
Lee, 2007; Kaneko et al., 2008). In addition, salinity‐dependent
expression of two branchial NKA a‐subunit isoforms, a1a and
a1b, was reported in several species (Richards et al., 2003;
Bystriansky et al., 2006, 2007; McCormick et al., 2009; Tipsmark
et al., 2011). The future study should identify and analyze the
expression of NKA a1a and a1b in grass puffer to clarify the
involvement of switch of two isoforms in modulation of gill NKA
activity.
According to current working model for the NaCl secretion in
gill mitochondrion‐rich (MR) cells, the apically expressed cystic
fibrosis transmembrane conductance regulator (CFTR) performs
an important role in Cl secretion in SW teleosts (Hwang and
Lee, 2007; Evans, 2010; Hwang et al., 2011). The euryhalinity of the
grass puffer was examined using investigations of Cl related
transporters in gills (Shen et al., 2007). CFTR was detected in the
apical membrane of gill NKIR cells (MR cells) of the SW puffer
using immunofluorescence staining, whereas the immunoreactive
signal from CFTR completely disappeared in the FW puffer (Shen
et al., 2007), which is a similar result to that from several well‐
studied euryhaline teleosts (reviewed in Hirose et al., 2003; Evans
et al., 2005; Evans, 2010; Hwang et al., 2011). This evidence
indicated that the grass puffer has the ability to switch the
direction of ion transport when acclimated to FW (Shen et al.,
2007).
Three groups of euryhaline teleosts were identified based on the
differences between distributions of NKA‐immunoreactive (NKIR)
J. Exp. Zool.
418
cells (i.e., mitochondrion‐rich (MR) cells) in the gills: (1) NKIR cells
occurred only in the filament epithelia of both FW‐ and SW‐
acclimated fish gills; (2) NKIR cells were exhibited abundantly in
the lamellar epithelia, in addition to the filaments, of FW‐
acclimated individuals; and (3) NKIR cells were observed in both
the gill filaments and lamellae of FW and SW individuals
(reviewed in Hwang and Lee, 2007). The grass puffer was
categorized into the first group because NKIR cells were exhibited
only in the filaments of the FW and SW groups (Fig. 4A and B). The
effect of environmental salinity on the density of branchial NKIR
cells seems to vary among different euryhaline teleosts.
Interestingly, similarly to O. mossambicus (Lee et al., 2003) and
T. nigroviridis (Tang and Lee, 2011), no significant change in the
numbers of NKIR cells was found between FW and SW grass
puffers (Fig. 4C). Meanwhile, the expression of gill NKA protein
was elevated in FW‐acclimated grass puffers (Fig. 3B). These
findings suggested that changes in protein amounts of gill NKA of
grass puffer corresponding to different environmental salinities
occurred through modulating the protein amounts of NKA per cell
rather than altering the density of NKIR cells, as reported in tilapia
and killifish (Uchida et al., 2000; Katoh et al., 2001).
Changes in NKIR cell densities in response to a salinity
challenge are related to variations in the rates of cell proliferation
and turnover (Uchida and Kaneko, '96; Katoh and Kaneko, 2003).
Although the number of gill NKIR cells of grass puffer was not
affected by ambient salinity, the protein expression of proliferating cell nuclear antigen (PCNA), a marker of cell proliferation,
evidently increased in the gill nuclear fraction of FW puffers
(Fig. 5). Because PCNA is involved in DNA synthesis, cell
proliferation, and the DNA repair system (Cox, '97; Maga and
Hübscher, 2003), the induction of gill PCNA is assumed to
contribute to (1) cell proliferation for maintaining NKIR cell
numbers or (2) DNA repair in puffer exposed to FW, an osmotically
stressful condition.
In conclusion, the present study investigated multiple aspects of
the physiological strategies of the grass puffers in response to FW
acclimation. Different regulatory mechanisms were crucial for the
FW acclimation of the grass puffer, including induction of stress
responses at the organismal (plasma glucose levels) and cellular
levels (HSP expression) for conduction of energy‐demanded
regulation and cytoprotection, (2) elevation of gill NKA expression
for adjustment of osmoregulatory status, and (3) induction of gill
PCNA expression might contribute to DNA repair and gill cell
proliferation. Parallel increases in NKA activity and stress protein
expression were found in the grass puffer. According to our
findings, we suggested the grass puffer is a euryhaline species with
SW preference. Thus, the gill NKA activity may elevate in response
to hypotonicity because the FW environment is a challenge for the
grass puffer. The positive correlation between gill NKA activity
and the expression of heat shock proteins in other marine teleosts
when subjected to a salinity challenge will be intriguing to
investigate in future studies.
J. Exp. Zool.
TANG AND LEE
ACKNOWLEDGMENTS
The monoclonal antibody against the Naþ/Kþ‐ATPase a‐subunit
(a5) was purchased from the Developmental Studies Hybridoma
Bank, which is maintained by the Department of Pharmacology
and Molecular Sciences, John Hopkins University School of
Medicine, Baltimore, MD, and the Department of Biological
Sciences, University of Iowa, Iowa City, IA, under Contract N01‐
HD‐6‐2915, NICHD, USA. This study was supported partly by a
grant to T.H.L. (NSC 95‐2313‐B‐005‐040‐MY3) and partly by a
grant to C.H.T. (NSC 101‐2313‐B‐259‐002) from the National
Science Council of Taiwan.
LITERATURE CITED
Afonso LOB, Basu N, Nakano K, Devlin RH, Iwama GK. 2003. Sex‐
related differences in the organismal and cellular stress response in
juvenile salmon exposed to treated bleached kraft mill effluent. Fish
Physiol Biochem 29:173–179.
Alfieri RR, Petronini PG. 2007. Hyperosmotic stress response:
comparison with other cellular stresses. Pflugers Arch 454:173–185.
Aparicio S, Chapman J, Stupka E, et al. 2002. Whole‐genome shotgun
assembly and analysis of the genome of Fugu rubripes. Science
297:1301–1310.
Arjona FJ, Vargas‐Chacoff L, Ruiz‐Jarabo I, Martin del Rio MP, Mancera
JM. 2007. Osmoregulatory response of Senegalese sole (Solea
senegalensis) to changes in environmental salinity. Comp Biochem
Physiol 148A:413–421.
Basu N, Kennedy CJ, Hodson PV, Iwama GK. 2001. Altered stress
responses in rainbow trout following a dietary administration of
cortisol and b‐napthoflavone. Fish Physiol Biochem 25:131–140.
Beck FX, Grunbein R, Lugmayr K, Neuhofer W. 2000. Heat shock
proteins and the cellular response to osmotic stress. Cell Physiol
Biochem 10:303–306.
Bystriansky JS, Richards JG, Schulte PM, Ballantyne JS. 2006.
Reciprocal expression of gill Naþ/Kþ‐ATPase a‐subunit isoforms
a1a and a1b during seawater acclimation of three salmonid
fishes that vary in their salinity tolerance. J Exp Biol 209:1848–
1858.
Bystriansky JS, Frick NT, Richards JG, Schulte PM, Ballantyne JS.
2007. Wild arctic char (Salvelinus alpinus) upregulate gill Naþ,
Kþ‐ATPase during freshwater migration. Physiol Biochem Zool
80:270–282.
Caruccio L, Bae S, Liu AY, Chen KY. 1997. The heat‐shock transcription
factor HSF1 is rapidly activated by either hyper‐ or hypo‐osmotic
stress in mammalian cells. Biochem J 327:341–347.
Choe KP, Strange K. 2008. Genome‐wide RNAi screen and in vivo
protein aggregation reporters identify degradation of damaged
proteins as an essential hypertonic stress response. Am J Physiol Cell
Physiol 295:C1488–C1498.
Choi CY, An KW. 2008. Cloning and expression of Naþ/Kþ‐ATPase and
osmotic stress transcription factor 1 mRNA in black porgy,
Acanthopagrus schlegeli during osmotic stress. Comp Biochem
Physiol B 149:91–100.
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
Ciccotti E, Marino G, Pucci P, Cataldi E, Cataudella S. 1995.
Acclimation trial of Mugil cephalus juveniles to freshwater:
morphological and biochemical aspects. Env Biol Fish 43:163–170.
Cox LS. 1997. Who binds wins: competition for PCNA rings out cell‐
cycle changes. Trends Cell Biol 7:493–498.
D'Cotta H, Valotaire C, le Gac F, Prunet P. 2000. Synthesis of gill
Naþ–Kþ‐ATPase in Atlantic salmon smolts: differences in a‐mRNA
and a‐protein levels. Am J Physiol 278:R101–R110.
Deane EE, Kelly SP, Luk JC, Woo NY. 2002. Chronic salinity adaptation
modulates hepatic heat shock protein and insulin‐like growth factor
I expression in black sea bream. Mar Biotechnol (NY) 4:193–
205.
Deane EE, Woo NY. 2004. Differential gene expression associated with
euryhalinity in sea bream (Sparus sarba). Am J Physiol 287:R1054–
R1063.
Deane EE, Woo NY. 2009. Modulation of fish growth hormone levels by
salinity, temperature, pollutants and aquaculture related stress:
a review. Rev Fish Biol Fisheries 19:97–120.
Doneen BA. 1981. Effects of adaptation to sea water, 170% sea water
and to fresh water on activities and subcellular distribution of
branchial Naþ–Kþ‐ATPase, low‐ and high affinity Caþþ‐ATPase,
and ouabain‐insensitive ATPase in Gillichthys mirabilis. J Comp
Physiol B 145:51–61.
Dong Y, Dong S, Meng X. 2008. Effects of thermal and osmotic
stress on growth, osmoregulation and Hsp70 in sea cucumber
(Apostichopus japonicus Selenka). Aquaculture 276:196–186.
Evans DH. 2009. Osmotic and ionic regulation. Cells and animals. Boca
Raton, FL: CRC Press.
Evans DH. 2010. A brief history of the study of fish osmoregulation: the
central role of the Mt. Desert Island Biological Laboratory. Front
Physiol 18:1–13.
Evans DH, Piermarini PM, Choe KP. 2005. The multifunctional fish gill:
dominant site of gas exchange, osmoregulation, acid–base
regulation, and excretion of nitrogenous waste. Physiol Rev
85:97–177.
Feder ME, Hofmann GE. 1999. Heat‐shock proteins, molecular
chaperones, and the stress response: evolutionary and ecological
physiology. Annu Rev Physiol 61:243–282.
Fiol D, Kültz D. 2007. Osmotic stress sensing and signaling in fishes.
FEBS J 274:5790–5798.
Freire CA, Amado EM, Souza LR, et al. 2008. Muscle water control in
crustaceans and fishes as a function of habitat, osmoregulatory
capacity, and degree of euryhalinity. Comp Biochem Physiol A
149:435–446.
Fujita S. 1962. Studies on life history and aquaculture of Japanese
puffer fishes. Rep Nagasaki Pref Inst Fish 2:1–121.
Gething MJ, Sambrook J. 1992. Protein folding in the cell. Nature
355:33–45.
Giffard‐Mena I, Lorin‐Nebel C, Charmantier G, Castille R, Boulo V.
2008. Adaptation of the sea‐bass (Dicentrarchus labrax) to fresh
water: role of aquaporins and Naþ/Kþ‐ATPases. Comp Biochem
Physiol A 150:332–338.
419
Hamer B, Jaksic Z, Pavicic‐Hamer D, et al. 2008. Effect of hypoosmotic
stress by low salinity acclimation of Mediterranean mussels Mytilus
galloprovincialis on biological parameters used for pollution
assessment. Aquat Toxicol 89:137–151.
Hightower LE. 1991. Heat shock, stress proteins, chaperones, and
proteotoxicity. Cell 66:191–197.
Hirose S, Kaneko T, Naito N, Takei Y. 2003. Molecular biology of major
components of chloride cells. Comp Biochem Physiol B 136:593–
620.
Honma Y, Ozawa T, Chiba A. 1980. Maturation and spawning behavior
of the puffer, Fugu niphobles, occurring on the coast of Sado Island
in the Sea of Japan. Jpn J Ichthyol 27:129–138.
Huang LE, Caruccio L, Liu AY, Chen KY. 1995. Rapid activation of the
heat shock transcription factor, HSF1, by hypo‐osmotic stress in
mammalian cells. Biochem J 307:347–352.
Hwang PP, Lee TH. 2007. New insights into fish ion regulation and
mitochondrion‐rich cells. Comp Biochem Physiol A 148:479–
497.
Hwang PP, Lee TH, Lin LY. 2011. Ion regulation in fish gills: recent
progress in the cellular and molecular mechanisms. Am J Physiol
301:R28–R47.
Hwang PP, Sun CM, Wu SM. 1989. Changes of plasma osmolality,
chloride concentration and gill Na‐K‐ATPase activity in tilapia
Oreochromis mossambicus during seawater acclimation. Mar Biol
100:625–627.
Iwama GK, Afonso LOB, Vijayan MM. 2006. Stress in fishes. In: Evans
DH, Claiborne JB, editors. The physiology of fishes. Boca Raton, FL:
CRC Press. p 319–335.
Iwama GK, Vijayan MM, Forsyth RB. 1999. Heat shock proteins and
physiological stress in fish. Am Zool 39:901–909.
Kaneko T, Watanabe S, Lee KM. 2008. Functional morphology of
mitochondrion‐rich cells in euryhaline and stenohaline teleosts.
Aqua‐BioSci Monogr 1:1–62.
Kang CK, Tsai SC, Lee TH, Hwang PP. 2008. Differential expression of
branchial Naþ/Kþ‐ATPase of two medaka species, Oryzias latipes
and Oryzias dancena, with different salinity tolerances acclimated
to fresh water, brackish water and seawater. Comp Biochem Physiol
A 151:566–575.
Kato A, Doi H, Nakada T, Sakai H, Hirose S. 2005. Takifugu obscurus is a
euryhaline fugu species very close to Takifugu rubripes and suitable
for studying osmoregulation. BMC Physiol 5:18.
Katoh F, Hasegawa S, Kita J, Takagi Y, Kaneko T. 2001. Distinct seawater
and freshwater types of chloride cells in killifish, Fundulus
heteroclitus. Can J Zool 79:822–829.
Katoh F, Kaneko T. 2003. Short‐term transformation and long‐term
replacement of branchial chloride cells in killifish transferred from
seawater to freshwater, revealed by morphofunctional observations
and a newly established ‘time‐differential double fluorescent
staining’ technique. J Exp Biol 206:4113–4123.
Kelly SP, Chow INK, Woo NYS. 1999. Haloplasticity of black seabream
(Mylio macrocephalus): hypersaline to freshwater acclimation. J Exp
Zool 283:226–241.
J. Exp. Zool.
420
Kuo SR, Shao K. 1999. Species composition of fish in the coastal zones
of the Tsengwen estuary, with descriptions of five new records from
Taiwan. Zool Stud 38:391–404.
Kültz D. 2001. Cellular osmoregulation: beyond ion transport and cell
volume. Zool 104:198–208.
Lai BT, Chin NW, Stanek AE, Keh W, Lanks KW. 1984. Quantitation
and intracellular localization of the 85K heat shock protein by
using monoclonal and polyclonal antibodies. Mol Cell Biol 4:2802–
2810.
Lasserre P. 1971. Increase of (Naþ þ Kþ)‐dependent ATPase activity in
gills and kidneys of two euryhaline marine teleosts, Crenimugil
labrosus (Risso, 1826) and Dicentrarchus labrax (Linnaeus, 1758),
during adaptation to fresh water. Life Sci II 10:113–119.
Lee KM, Kaneko T, Aida K. 2005. Low‐salinity tolerance of juvenile fugu
Takifugu rubripes. Fish Sci 71:1324–1331.
Lee TH, Feng SH, Lin CH, et al. 2003. Ambient salinity modulates the
expression of sodium pumps in branchial mitochondria‐rich cells
of Mozambique tilapia, Oreochromis mossambicus. Zool Sci 20:
29–36.
Lee TH, Hwang PP, Shieh YE, Lin CH. 2000. The relationship between
“deep‐hole” mitochondria‐rich cells and salinity adaptation in the
euryhaline teleost, Oreochromis mossambicus. Fish Physiol Biochem
23:133–140.
Lin CH, Tsai RS, Lee TH. 2004. Expression and distribution of Na,
K‐ATPase in gill and kidney of the spotted green pufferfish,
Tetraodon nigroviridis, in response to salinity challenge. Comp
Biochem Physiol A 138:287–295.
Lin YM, Chen CN, Lee TH. 2003. The expression of gill Na, K‐ATPase in
milkfish, Chanos chanos, acclimated to seawater, brackish water
and fresh water. Comp Biochem Physiol A 135:489–497.
Lin YM, Chen CN, Yoshinaga T, et al. 2006. Short‐term effects of
hyposmotic shock on Naþ/Kþ‐ATPase expression in gills of the
euryhaline milkfish, Chanos chanos. Comp Biochem Physiol A
143:406–415.
Macario AJ, Lange M, Ahring BK, Conway de Macario E. 1999. Stress
genes and proteins in the archaea. Microbiol Mol Biol Rev 63:923–
967 table of contents.
Maga G, Hübscher U. 2003. Proliferating cell nuclear antigen (PCNA): a
dancer with many partners. J Cell Sci 116:3051–3060.
Margalit A, Sofer Y, Grossman S, et al. 1993. Hepoxilin A3 is the
endogenous lipid mediator opposing hypotonic swelling of intact
human platelets. Proc Natl Acad Sci U S A 90:2589–2592.
Marshall MS, Grosell M. 2006. Ion transport, osmoregulation, and
acid–base balance. In: Evans DH, Claiborne JB, editors. The
physiology of fishes. Boca Raton, FL: CRC Press. p 179–214.
Masuda H, Amaoka K, Araga C, Uyeno T, Yoshino T. 1984. The fishes of
the Japanese archipelago. Tokyo: Tokai University Press.
McCormick SD, Regish AM, Christensen AK. 2009. Distinct freshwater
and seawater isoforms of Naþ/Kþ‐ATPase in gill chloride cells of
Atlantic salmon. J Exp Biol 212:3994–4001.
Miyadi D, Kawanabe H, Mizuno N. 1976. Colored illustrations of the
freshwater fishes of Japan. Osaka: Hoikusha.
J. Exp. Zool.
TANG AND LEE
Nozaki M, Tsutsumi T, Kobayashi H, et al. 1976. Spawning habit of
the puffer, Fugu niphobles (Jordan et Snyder). Zool Mag 85:156–
168.
Pan F, Zarate JM, Tremblay GC, Bradley TM. 2000. Cloning and
characterization of salmon hsp90 cDNA: upregulation by thermal
and hyperosmotic stress. J Exp Zool 287:199–212.
Peterson GL. 1978. A simplified method for analysis of inorganic
phosphate in the presence of interfering substances. Anal Biochem
84:164–172.
Rendell JL, Currie S. 2005. Intracellular localization of hsp90 is
influenced by developmental stage and environmental estrogens in
rainbow trout Oncorhynchus mykiss. Physiol Biochem Zool 78:937–
946.
Richards JG, Semple JW, Bystriansky JS, Schulte PM. 2003. Naþ/
Kþ‐ATPase a‐isoform switching in gills of rainbow trout
(Oncorhynchus mykiss) during salinity transfer. J Exp Biol 206:
4475–4486.
Sangiao‐Alvarellos S, Laiz‐Carrión R, Guzmán JM, et al. 2004.
Acclimation of S. aurata to various salinities alters energy
metabolism of osmoregulatory and nonosmoregulatory organs.
Am J Physiol 285:R897–R907.
Saoud IP, Kreydiyyeh S, Chalfoun A, Fakih M. 2007. Influence of salinity
on survival, growth, plasma osmolality and gill Naþ–Kþ‐ATPase
activity in the rabbitfish Siganus rivulatus. J Exp Mar Biol Ecol
348:183–190.
Shen ID, Chiu YH, Lee TH, Hwang PP. 2007. Localization of chloride
transporters in gill epithelia of the grass pufferfish, Takifugu
niphobles. J Fisher Society of Taiwan 34:87–100.
Sherwood L, Klandorf H, Yancey P. 2005. Digestive systems. Animal
physiology from genes to organisms. Belmont: Thomon. p 612–
669.
Spees JL, Chang SA, Snyder MJ, Chang ES. 2002. Osmotic induction
of stress‐responsive gene expression in the lobster Homarus
americanus. Biol Bull 203:331–337.
Stagg RM, Shuttleworth TJ. 1982. Naþ, Kþ ATPase, quabain binding
and quabain‐sensitive oxygen consumption in gills from Platichthys
flesus adapted to seawater and freshwater. J Comp Physiol B
147:93–99.
Stuenkel EL, Hillyard SD. 1980. Effects of temperature and salinity on
gill Naþ–Kþ ATPase activity in the pupfish, Cyprinodon salinus.
Comp Biochem Physiol A 67:179–182.
Tang CH, Lee TH. 2011. Morphological and ion‐transporting plasticity
of branchial mitochondrion‐rich cells in the euryhaline spotted
green pufferfish, Tetraodon nigroviridis. Zool Stud 50:31–42.
Tang CH, Tzeng CS, Hwang LY, Lee TH. 2009. Constant muscle water
contents and renal HSP90 expression reflect the osmotic
homeostasis in euryhaline teleosts acclimatized to different
environmental salinities. Zool Stud 48:435–441.
Tipsmark CK, Madsen SS, Seidelin M, et al. 2002. Dynamics of Naþ, Kþ,
2Cl cotransporter and Naþ, Kþ‐ATPase expression in the branchial
epithelium of brown trout (Salmo trutta) and Atlantic salmon
(Salmo salar). J Exp Zool 293:106–118.
FRESHWATER ACCLIMATION AND GRASS PUFFER STRESS
Tipsmark CK, Breves JP, Seale AP, et al. 2011. Switching of Naþ,
Kþ‐ATPase isoforms by salinity and prolactin in the gill of a cichlid
fish. J Endocrinol 209:237–244.
Uchida K, Kaneko T. 1996. Enhanced chloride cell turnover in the gills
of chum salmon fry in seawater. Zool Sci 13:655–660.
Uchida K, Kaneko T, Miyazaki H, Hasegawa S, Hirano T. 2000. Excellent
salinity tolerance of mozambique tilapia (Oreochromis mossambicus): elevated chloride cell activity in the branchial and opercular
epithelia of the fish adapted to concentrated seawater. Zool Sci
17:149–160.
421
Vijayan MM, Pereira C, Grau EG, Iwama GK. 1997. Metabolic responses
associated with confinement stress in tilapia: the role of cortisol.
Comp Biochem Physiol C: Pharmacol Toxicol Endocrinol 116:89–95.
Wegele H, Muller L, Buchner J. 2004. Hsp70 and Hsp90—a relay team
for protein folding. Rev Physiol Biochem Pharmacol 151:1–44.
Withers PC. 1992. Comparative animal physiology. Orlando, FL:
Saunders College Publishing.
Xiao H, Lis JT. 1988. Germline transformation used to define key
features of heat‐shock response elements. Science 239:1139–
1142.
J. Exp. Zool.