The inner opercular membrane of the
euryhaline teleost: a useful surrogate model
for comparisons of different characteristics
of ionocytes between seawater- and
freshwater-acclimated medaka
Chao-Kai Kang, Shu-Yuan Yang, ShangTao Lin & Tsung-Han Lee
Histochemistry and Cell Biology
ISSN 0948-6143
Volume 143
Number 1
Histochem Cell Biol (2015) 143:69-81
DOI 10.1007/s00418-014-1266-2
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Histochem Cell Biol (2015) 143:69–81
DOI 10.1007/s00418-014-1266-2
ORIGINAL PAPER
The inner opercular membrane of the euryhaline teleost: a useful
surrogate model for comparisons of different characteristics
of ionocytes between seawater‑ and freshwater‑acclimated
medaka
Chao‑Kai Kang · Shu‑Yuan Yang · Shang‑Tao Lin ·
Tsung‑Han Lee Accepted: 14 August 2014 / Published online: 28 August 2014
© Springer-Verlag Berlin Heidelberg 2014
Abstract The inner opercular membranes of the brackish medaka, Oryzias dancena, have numerous ionocytes,
similar to the gill epithelia. By histological observation,
this study demonstrated that it is possible to investigate
the cellular morphology and function of ionocytes in the
opercular membrane. The mitochondria-rich ionocytes in
the opercular membranes were traced using rhodamine
123 and a cytochrome c oxidase IV antibody in vital and
fixed situations, respectively. To validate different morphologies of seawater (SW)-type and freshwater (FW)type ionocytes of the opercular membrane of euryhaline
brackish medaka, a method of dual observation including
immunofluorescence staining and subsequent scanning
electron microscopy was used. The apical morphologies
of SW- and FW-type ionocytes were hole and flat opening, respectively. In addition, the microvilli were found on
the apical surface of the FW-type ionocytes. The SW-type
ionocytes exhibited basolateral Na+, K+, 2Cl− cotransporter and the apical cystic fibrosis transmembrane conductance regulator. In contrast, in the apical region of
FW-type ionocytes, Na+, Cl− cotransporter and villin
1-like protein were expressed. In addition, histochemical
staining of AgCl precipitation counterstained with a Na+,
Electronic supplementary material The online version of this
article (doi:10.1007/s00418-014-1266-2) contains supplementary
material, which is available to authorized users.
C.-K. Kang · S.-Y. Yang · S.-T. Lin · T.-H. Lee (*) Department of Life Sciences, National Chung-Hsing University,
Taichung 402, Taiwan
e-mail: [email protected]
T.-H. Lee Department of Biological Science and Technology, China
Medical University, Taichung 404, Taiwan
K+-ATPase α-subunit antibody on the opercular membrane illustrated the role of Cl− secretion in the SW-type
ionocytes of the brackish medaka. A combination of different observations in this study indicated that the opercular membrane could be a useful surrogate model for histological and functional studies on the epithelial ionocytes
of fish gills.
Keywords Opercular membrane · Ionocyte ·
Mitochondrion · Microvilli · Chloride secretion · Medaka
Introduction
Teleosts, as osmoregulators, maintain homeostasis with
osmoregulatory organs for adaptation to aquatic environments. Being a type of epithelial cell, the ionocytes
play an important role in ionoregulation in gill filaments
of teleosts. Typical ionocytes were found in the gills as
well as the inner epithelium of the operculum of killifish
(Fundulus heteroclitus, Karnaky and Kinter 1977; Marshall et al. 2000) and tilapia (Oreochromis mossambicus,
Uchida et al. 2000; Shieh et al. 2003). The piece of epithelium lining the inside of the operculum is called the
opercular membrane. The opercular membrane consists
of a single epithelium with its apical side exposed to the
aqueous environment of the gill chamber, and the basal
side connecting to an underlying loose connective tissue
layer. Histologically, the opercular epithelium is similar to
the gill epithelium, which is composed of four major cell
types, i.e., mucous cells, pavement cells, non-differentiated cells, and ionocytes (Degnan et al. 1977). The opercular membrane, resembling a flattened gill epithelium,
was easier for observation without the respiratory leaflets
and complicated vasculature. Hence, it could be applied
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in electrophysiological and chemical physiological studies. Meanwhile, the opercular membrane revealed much
potential for investigation of the cellular characteristics
of ionocytes compared with the gill epithelium of teleosts
(Karnaky and Kinter 1977; Karnaky et al. 1977; Foskett
and Scheffey 1982; Uchida et al. 2000; Mazon et al. 2007;
Marshall et al. 2008).
The ionocytes are called mitochondrion-rich cells due
to the observation of numerous mitochondria in the cytoplasm by transmission electron microscopy (TEM; Evans
et al. 2005). Previous studies used fluorescence chemicals, such as 2-(4-dimethylaminostyryl)-1-ethylpyridinium
iodide (DASPEI) or rhodamine 123, to label the ionocytes
in gills of teleosts for observations under a fluorescence or
confocal laser scanning microscope (Kaneko et al. 2008).
Gill ionocytes of freshwater (FW) and seawater (SW)
medaka were classified into flat type and hole type, respectively, by the morphological phenotypes of apical surfaces
(Kang et al. 2013; Kang and Lee 2014). The microvilli
mainly developed on the apical surface of the flat-type
ionocytes rather than in the hole-type cells for ion absorption (Kang and Lee 2014). Remodeling morphologies of
the apical surfaces were observed in ionocytes of numerous teleosts when exposed to the environment with different salinities (Hossler et al. 1979; King and Hossler 1991;
Lee et al. 1996; Perry 1997; Katoh and Kaneko 2003).
Our previous study demonstrated that salinity-dependent
expression of villin 1-like (VILL) protein was exhibited in the cell cortex of gill ionocytes and was involved
in the formation of microvilli (Kang and Lee 2014). The
immunostained VILL protein would be a potential marker
for morphological identification of fish ionocytes with
microvilli rather than observation with scanning electron
microscopy (SEM).
According to the current ionoregulatory model of gill
ionocytes, basolateral Na+, K+-ATPase (NKA) provides
the gradient force driving secondary ion transporters to
absorb ions in FW and secrete ions in SW. In addition, the
major internal anion, Cl−, was controlled at a stable level
by the osmoregulatory system of the teleosts (Wood and
Marshall 1994; Evans et al. 1999; Marshall 2002; Hirose
et al. 2003; Hwang and Lee 2007; Evans 2008; Hwang
et al. 2011). Recent studies reported that the gill ionocytes
mediated the apical Na+, Cl− cotransporter (NCC) and
basolateral chloride channel CLC-3 for Cl− absorption
in the FW fish (Kaneko et al. 2008; Hwang et al. 2011;
Evans 2011). Meanwhile, in the SW teleosts, a basolateral Na+, K+, 2Cl− cotransporter (NKCC) and an apical
chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR), were shown to be involved
in the Cl− secretory model of ionocytes (Marshall 2002;
Hirose et al. 2003; Hwang and Lee 2007; Evans 2008;
Hwang et al. 2011). Scott et al. (2005, 2008) found that
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Histochem Cell Biol (2015) 143:69–81
correlated profiles of NKAα1a, NKCC1, and CFTR genes
were found in both the opercular membrane and gills of
killifish in response to hyperosmotic or hypoosmotic
challenges.
Karnaky et al. (1977) first isolated the inner opercular
epithelium of SW killifish to detect the short-circuit current, transepithelial potential difference, and 36Cl− fluxes.
Recently, a scanning ion-selective electrode technique
(SIET) was used to determine ion fluxes on the apical
region of the ionocytes for functional studies in living fish
embryos (Lin et al. 2006; Horng et al. 2007; Shen et al.
2011). Ionoregulatory functions of branchial ionocytes in
adult medaka, however, could not be explored with SIET
due to the very complex structures of the gills. In addition
to these electrophysiological techniques, previous studies used the silver precipitation staining method to verify Cl− secretion in ionocytes of the SW fish (Wong and
Chan 1999; Kaneko and Shiraishi 2001; Tse et al. 2006;
Tang and Lee 2011). In the present study, we modified
the silver staining process to examine the Cl− secreting
functions of ionocytes in the opercular membrane of adult
medaka.
The brackish medaka (Oryzias dancena) is a small
euryhaline teleost that inhabits river mouths and estuaries of eastern India, Bangladesh, and Myanmer (Roberts
1998). Inoue and Takei (2002, 2003) indicated that the
brackish medaka exhibited better salinity tolerance than
the Japanese medaka (O. latipes). Therefore, this species was a good experimental animal for studies on the
mechanisms of fish ionoregulation. Our previous studies
illustrated correlations between environmental salinities
and expressions of NKA, NKCC1a, and CFTR in the gills
of brackish medaka. In addition, typical characteristics
of SW-type ionocytes with apical CFTR and basolateral
NKCC1a were reported (Kang et al. 2008, 2010, 2012,
2013). A functional assay of Cl− secretion in gill ionocytes
of brackish medaka, however, needs to be performed to
clarify the mechanisms.
Being complex in structure, gill filaments exhibiting
target ionocytes are difficult to investigate after various
histological procedures. Therefore, this study performed
numerous histological observations on the inner opercular
membrane of the SW- and FW-acclimated brackish medaka
to study SW- and FW-type ionocytes from morphology to
function. Meanwhile, the present study tested a hypothesis
that critical characteristics of opercular ionocytes were similar to those of the gill ionocytes in the small euryhaline
teleost. In SW and FW types of ionocytes of the medaka
opercula, mitochondria were recognized, apical morphologies were validated, cell size and density were determined,
distributions of VILL, NKCC1a, and CFTR proteins were
detected, and the location of apical Cl− permeability was
explored.
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Materials and methods
Fish and experimental condition
Adult brackish medaka (O. dancena, 2.50 ± 0.30 cm in
length) was acclimated to aerated local fresh tap water
(FW: [Na+] 0.22 ± 0.01 mM; [K+] 0.04 ± 0.01 mM;
[Ca2+] 0.68 ± 0.01 mM; [Mg2+] 0.28 ± 0.01 mM; [Cl−]
0.14 ± 0.01 mM) and artificial seawater (SW; 35 ‰) prepared from RealOcean™ Synthetic Sea Salt (TAAM, Camarillo, CA, USA) for at least 3 weeks. The water temperature was maintained at 28 ± 1 °C. The photoperiod cycle
was 14 h light: 10 h dark every day. The fish were fed a
daily diet of commercial pellets. Water was continuously
circulated through fabric-floss filters and partially refreshed
every 2 weeks. The facilities and protocols for experimental animals were approved by Institutional Animal Care and
Use Committee of the National Chung Hsing University
(IACUC approval no. 102-114 to THL). For the following
experiments, fish were not fed and were anaesthetized with
MS-222 (100–200 mg/L) before sampling.
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(Kang and Lee 2014); (5) anti-Na+, K+, Cl− cotransporter
(NKCC): A mouse monoclonal antibody (T4; Developmental Studies Hybridoma Bank) raised against the c-terminus
of human NKCC; (6) anti-cystic fibrosis transmembrane
conductance regulator (CFTR): A mouse monoclonal antibody (R&D Systems, Boston, MA, USA) directed against
104 amino acids at the C-terminus of the human CFTR.
For double immunofluorescence staining, the secondary
antibodies were Dylight 549-conjugated AffiniPure goat
anti-mouse IgG, Dylight 488-conjugated AffiniPure goat
anti-mouse IgG, Dylight 549-conjugated AffiniPure goat
anti-rabbit IgG, and Dylight 488-conjugated AffiniPure
goat anti-rabbit IgG (Jackson, West Grove, PA, USA).
Preparation of whole‑mount sample
The opercula of medaka were fixed in the neutral formalin
(pH 7.2) in 4 °C for 3 h. After washing in phosphate buffer
saline (PBS), the samples were permeated with methanol
for 30 min at −20 °C. The samples were then stored in
the methanol at −20 °C before performing the following
experiments.
Histological examination of the paraffin sections
Double immunofluorescence staining
The heads of brackish medaka were excised and fixed in
Bouin’s solution at room temperature for 48 h. The fixed
heads were dehydrated through a graded ethanol series
(50, 70, 80, 95, and 100 % ethanol) and cleared in xylene
for 3 h. Then, the heads were infiltrated and embedded in
paraffin (Merck, Darmstadt, Germany). Cross sections of
the heads with a thickness of 2 μm were mounted on glass
slides and stained with Gill’s hematoxylin and eosin (H&E)
reagent (Merck), followed by mounting with cover slips.
The sections were observed under an optical microscope
(BX50; Olympus, Tokyo, Japan), and micrographs were
obtained using a cooled CCD camera (DP72; Olympus)
with CellSens, standard version 1.4 software (Olympus).
Antibodies
Primary antibodies used in this study included: (1) antiNa+, K+-ATPase (NKA) α-subunit: A mouse monoclonal
antibody (α5; Developmental Studies Hybridoma Bank,
Iowa City, IA, USA) raised against the α-subunit of avian
NKA; (2) anti-NKA α-subunit: a rabbit monoclonal antibody (EP1845Y, Abcam, Cambridge, UK), a synthetic
peptide corresponding to residues near the N terminus
of human NKA; (3) anti-cytochrome c oxidase IV (COX
IV): a rabbit monoclonal antibody (3E11, Cell Signaling,
Ozyme, Saint Quentin en Yvelines, France), a synthetic
peptide corresponding to residues surrounding Lys29 of
human COX IV; (4) the rabbit polyclonal antibody against
the C-terminus of medaka villin 1-like (VILL) protein
Whole-mount samples were rehydrated by rinsing with
PBS three times. Subsequently, the samples were washed
with PBS three times and incubated in 5 % bovine serum
albumin (Sigma, St. Louis, MO, USA) at room temperature
for 30 min. Samples were then incubated with the rabbit
polyclonal antibody at room temperature for 2 h. Subsequently, samples were washed three times with PBS, incubated with secondary antibody (Dylight-488 or 549 goat
anti-rabbit IgG) at room temperature for 1 h, and washed
several times with PBS. After the first staining, samples
were incubated with the mouse monoclonal antibody for
2 h at room temperature. Samples were then washed several times with PBS and incubated with secondary antibody
(Dylight-549 or 488 goat anti-mouse IgG) at room temperature for 1 h followed by several PBS washes. Samples
were then mounted in 50 % glycerol with PBS and examined using a fluorescent microscope (BX50, Olympus) with
Olympus DP72 CCD camera or a laser scanning confocal
microscope (FV1000, Olympus).
Fluorescent staining of mitochondrion
The SW- and FW-acclimated medaka (n = 6) were exposed
to 50 mL SW and FW, respectively, containing the mitochondria-specific fluorescent dye, rhodamine 123 (25 μM,
Invitrogen, Carlsbad, CA, USA) for 4 h in darkness. After
the duration of fluorescent exposure, the right opercula
were excised and mounted in a chamber slide with PBS
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after fish were euthanized. The first micrograph of the inner
opercular membrane was taken with the Olympus DP72
CCD camera (Olympus) connected to the fluorescence
microscope (BX50, Olympus). Subsequently, the operculum was fixed with the neutral formalin in 4 °C for 3 h for
double immunofluorescence staining as described previously. Since the green fluorescent signals of rhodamine 123
vanished after sample fixation, it is easier to detect different
types of ionocytes. The COX IV and α5 antibodies were
used in conjunction with the secondary antibodies with
Dylight-549 and Dylight-488, respectively. The two micrographs were compared to identify the target region of the
same operculum.
Histochem Cell Biol (2015) 143:69–81
et al. 2005), immunostaining of NKA appears all across
the cytoplasm making the entire cell visible for cell area
quantification. Micrographs of the opercular membrane
were taken by the cooled CCD camera (DP72; Olympus),
and CellSens, standard version 1.4 software (Olympus),
were applied for subsequent measurement. The number
of ionocytes in three rectangle areas (100 μm × 100 μm)
chosen in the pair of opercular membrane from each fish
was counted and averaged to represent one fish sample. In
these three areas of each fish for measurements, cell sizes
of NKA-IR cells were analyzed to determine the average
size of ionocytes.
Silver staining and NKA counterstaining
Dual observations of whole‑mount immunofluorescence
staining and scanning electron microscopy (SEM)
The method was modified from Choi et al. (2011). In the
inner opercular membranes of the SW- and FW-acclimated
medaka, dual observations including whole-mount immunofluorescence staining and SEM were used to investigate
morphologies of the apical surfaces of SW- and FW-type
ionocytes. The right opercula of 6 individuals were excised
and fixed with neutral formalin in 4 °C overnight. The opercular ionocytes were identified with the method of wholemount immunofluorescence staining as described above
with the NKA α-subunit antibody (α5). The directional
identification could refer easily from the special shapes of
opercula. After observing and photographing the images
using the fluorescence microscope with the Olympus DP72
CCD camera (Olympus), each sample was rinsed for 15 min
with three changes of 0.1 M phosphate buffer (PB) and then
post-fixed with 1 % (w/v) osmium tetroxide in 0.1 M PB for
1.5 h. After post-fixation, the opercula were rinsed in PB
and dehydrated in ascending concentrations of ethanol, from
30 % to absolute. Samples were then critical point dried
using liquid CO2 in a Hitachi HCP-2 (Tokyo, Japan) critical
point drier, mounted on aluminum stubs with silver paint,
and sputter coated for 3 min with a gold–palladium complex
in a Pt Coater (JFC-1600, JEOL, Tokyo, Japan). The coated
specimens were observed using a TM-3000 SEM (Hitachi)
with COMPO model and 15 kV. Relative localization of
identical ionocytes in the inner opercular membrane was
identified by comparing the immunofluorescence staining
images with the corresponding SEM images.
Determination of the cell size and number of ionocytes
The cell size and number of ionocytes were examined by
immunostaining with NKA in whole-mount preparations of
opercula from FW- and SW-acclimated medaka. Because
the ionocytes possess an intricate tubular system formed by
extensive invaginations of the basolateral membrane (Evans
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The method of silver staining was modified from Dickson
et al. (1991). The opercula were excised and fixed immediately in 10 % formalin in deionized water (v/v) at room
temperature and shook for 15 min. After fixation, the opercula were immersed overnight in 1 % AgNO3 solution with
shaking in the dark at room temperature. The opercula
were then washed several times with deionized water and
immersed in Kodak developer (#190 0984, Eastman Kodak,
Rochester, New York, USA) for 5 min. Subsequently, samples were immersed in 10 % formalin at room temperature
for 15 min to stop color development followed by several
PBS washes. After the silver staining, samples were incubated with the PBS-diluted primary monoclonal antibody
to NKA α-subunit (α5; 1:200) for 2 h at room temperature.
After several washes with PBS, samples were incubated
with the secondary antibody (Dylight 549 goat anti-mouse
IgG) at room temperature for 1 h to show the localization
of NKA-immunoreactive cells.
Statistical analysis
Values were compared using unpaired Student’s t test.
The significance level was set as p < 0.05. Values were
expressed as the mean ± SEM (the standard error of the
mean) unless stated otherwise.
Results
One pair of opercula
One pair of opercula was located in the outer region of the
gill chamber in the head of brackish medaka. The medaka
use the motion of the opercula to pump out the external
water from the mouth through gill slits to the gill opening for respiratory ventilation (Fig. 1). Close to the first
gill slit, the inner opercula membrane covers the opercular bone. Thus, this operculum can be easily and quickly
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Fig. 1 A pair of opercula (Op) covered the gill chamber of the brackish medaka. a The image shows that the operculum conducted respiratory ventilation with external water. The water was pumped from the
mouth through gill slits to the gill opening (the blue flow direction).
b The red arrow and box indicate the direction and site of the following micrographs of cross sections, respectively. The green dashed
line illustrates the sampling area of the operculum for the following
whole-mount experiments. AP anterior position, VS ventral side
excised from the head (Fig. 1b). The single membrane is
a stratified epithelium that contacts the aqueous media of
the gill chamber (Fig. 2a). After a serial process of experimentation, no obvious change in structure was found in the
inner opercular membrane (Fig. 3). Crossing the opercular membrane, a linear area (the asterisk in Fig. 3) without distribution of ionocytes was observed. There were two
patterns of Na+, K+-ATPase-immunoreactive (NKA-IR)
cells/ionocytes distributed in the inner opercular membrane. Above the line area, the distribution of opercular
ionocytes was similar to the pattern of ionocyte distribution
in gill filaments (Fig. 3b). This finger-like pattern was also
observed and identified as a gill-filament-like (GFL) region
using SEM (Fig. 3c). On the other hand, in the flat region
below the line area, the opercular ionocytes were randomly
distributed. Close to the gill opening (the lower curved
region of the opercular membrane in Fig. 3), the structure
of the opercular membrane was wrinkled (the position of
arrow). The central area of the intact opercular membrane
with a flat structure (the white box) was used for the following observations in the micrographs of vital tissue,
immunofluorescent staining results, and SEM (Fig. 3).
Fig.  2 a The hematoxylin- and eosin-stained section of the medaka
head shows that the pair of opercula (Op) covered the four gill slits
(from GI to GIV) to form a gill concave. The box reveals that the
inner opercular membrane was a layer of epidermal tissue. Two types
of ionocytes (eosinophilic cells) were observed in the inner opercular membranes of the brackish medaka acclimated to SW (b) and FW
(c). The apical surface morphology of the structures of ionocytes of
the SW and FW medaka were the hole type (arrows) and microvillus
flat type (arrowheads), respectively. AP anterior position, Ex external
region, FW freshwater, In inner region; SW seawater; VS ventral side
when the fish were exposed to the FW with rhodamine 123
(Fig. S1). After fixation, the fluorescent signals of rhodamine 123 disappeared. In addition, the present study used
a mitochondrion marker, i.e., a cytochrome c oxidase IV
(COX IV) antibody, to trace the mitochondria-rich cells
in the rhodamine 123-treated membranes of SW and FW
medaka (Fig. 4c, d). Immunoblots of the COX IV results
probed with the commercial antibody indicated a single
immunoreactive band of 15 kDa in the medaka opercula
(Fig. S2). Therefore, the NKA-IR cells/ionocytes exhibited
rhodamine 123-positive signals and immunoreactive signals
of the COX IV proteins (Fig. 4). The results indicated that
the easily sampled inner opercular membrane was optimal
for operating and investigating the ionocytes in either vital
or fixed situations.
The mitochondria‑rich opercular ionocytes
The green fluorescent chemical, rhodamine 123, was used
to label mitochondria of the living ionocytes in the vital
opercular membranes of the FW and SW medaka (Fig. 4a,
b). The ionocytes were also labeled on the gill filaments
Different morphologies of the SW‑ and FW‑type ionocytes
in the opercula
The ionocytes in the central area of the opercula of FWand SW-acclimated brackish medaka were analyzed
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Histochem Cell Biol (2015) 143:69–81
Fig. 4 Triple fluorescent staining with rhodamine 123 (a, b) and
the anti-COX IV antibody (c, d) to characterize that the NKA-IR
cells/ionocytes (e, f) were rich in mitochondria in the opercular membrane of the brackish medaka acclimated to SW (a, c, e) and FW (b,
d, f). The positive signals of three target molecules were localized to
identical cells (indicated by the same numbers) in both SW and FW
individuals. FW freshwater, SW seawater
Fig. 3 Different images of the inner opercular membrane from the
brackish medaka. a Anatomical structure of the inner operculum was
observed with the optical microscope. b The ionocytes distributed
on the opercular membranes were immunostained with an anti-NKA
α-subunit antibody. Ionocytes labeled in red are located on the inner
opercular membrane of the operculum. c Scanning electron microscopic (SEM) observation of the previously immunostained operculum. The flat region with numerous NKA-IR cells/ionocytes for
investigation is indicated by the white box in the picture of the opercular membrane. The asterisk indicates the line area without NKA-IR
cells/ionocytes in the opercular membrane. The water flow direction
is indicated by the arrow in the lower wrinkled part of the operculum
close to the gill opening. AP anterior position, GFL gill filament-like
structure, VS ventral side
and compared (Fig. 6). The average size of the NKA-IR
cells/ionocytes from the SW-acclimated medaka was significantly bigger (approximately 1.6-fold, p < 0.05) than
that of the FW group (Fig. 5a). However, the density of
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NKA-IR cells for the FW-acclimated medaka was threefold
higher (p < 0.05) than that of the SW fish (Fig. 5b). The
eosinophilic ionocytes were observed in the inner opercular membrane based on the higher magnified hematoxylin–
eosin-stained micrographs. Different apical structures of
ionocytes were compared in the opercular membranes of
the SW- and FW-acclimated medaka. The orifices (hole
type) were found in the apical regions of the SW-type ionocytes (Fig. 2b). Meanwhile, the FW-type (flat-type) ionocytes exhibited convex surfaces with microvilli (Fig. 2c).
In addition, identical cellular regions and an apical opening were identified by dual observations of immunostained
confocal micrographs and corresponding SEM micrographs (Fig. 6). Through the see-through pavement cells,
the underneath ionocytes and their apical surfaces (hole in
SW and flat in FW) exhibited in the intersectional points
of adjacent pavement cells were easily identified in the
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Histochem Cell Biol (2015) 143:69–81
Fig. 5 Average size (a) and density (b) of NKA-IR cells in the inner
opercular membranes of the brackish medaka acclimated to SW and
FW were quantified (n = 10 for each group). NKA-IR cells that were
higher in density and larger in size were found in the membranes of
FW and SW fish, respectively. The asterisk indicates a significant difference (p < 0.05) using Student’s t test. Values are mean ± SEM. FW
freshwater, SW seawater
SEM micrographs (Fig. 6c, d). The larger apical openings of ionocytes were found in the FW medaka (flat type;
Fig. 6f) compared with the SW group (hole type; Fig. 6e).
Using whole-mount double immunofluorescence staining,
Fig. 7 shows that the villin 1-like (VILL) protein of the
inner opercular membrane was mainly observed in the FW
medaka. The merged images (Fig. 7e, f) revealed that VILL
protein was localized in the NKA-IR cells in the opercula
of the FW fish rather than the SW group, corresponding to
the microvilli structures observed on the surfaces of FWtype NKA-IR cells/ionocytes (Figs. 2c, 6f). The magnified
merged z-axis images (Fig. 7g, h) further revealed that the
VILL signals were localized to the apical region of NKAIR cells in the opercular membrane of FW-acclimated
medaka.
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Fig. 6 Dual observations of whole-mount immunofluorescence staining (a, b) and SEM (c–f) on the same area of the opercular membranes in the brackish medaka acclimated to SW (a, c, e) and FW
(b, d, f). The opercular ionocytes were identified with an anti-NKA
α-subunit (a, b) antibody to illustrate the distributed pattern. Using
a specific protocol, the cellular area of ionocytes beneath the seethrough pavement cells could be identified using TM-3000 SEM
(c, d). The same numbers in the confocal micrograph and scanning
electron micrograph of SW (a, c) and FW (b, d) fish indicated identical ionocytes observed by different facilities. The white square (c,
d) shows observations at a higher magnification to show apical morphologies of ionocytes (e, f), typical hole type (e), and microvillus
flat type (f) in SW and FW, respectively. PVC pavement cell
Ionoregulatory mechanisms of the opercular ionocytes
The opercular ionocytes of SW- and FW-acclimated
medaka were immunostained with antibodies against
NKCC1a/NCC and CFTR. The merged image of the opercular membrane revealed that NKCC1a protein was present
in the basolateral membrane of NKA-IR cells/ionocytes
in the SW-acclimated medaka (Fig. 8a). In the FW fish,
NCC immunoreactivity was revealed in the apical regions
of the NKA-IR cells/ionocytes (Figs. 8b, S3). Meanwhile,
the merged image showed that CFTR was present in the
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Histochem Cell Biol (2015) 143:69–81
Fig. 7 Confocal micrographs of
whole-mount double immunofluorescence staining with
anti-VILL protein (green, a, b)
and anti-NKA α-subunit (red;
c, d) antibodies in the inner
opercular membranes of the
brackish medaka acclimated
to SW (a, c, e, g) and FW (b,
d, f, h). The merged images
revealed that VILL protein
was localized to the opercular
ionocytes (NKA-IR cells) in the
FW fish (f) rather than in the
SW group (e). The merged 3D
image at the Z-axis section of
the FW fish (h) revealed that the
VILL signals were at the apical
regions of the NKA-IR cells.
FW freshwater; SW seawater
apical region of NKA-IR cells/ionocytes of SW-acclimated
medaka (Fig. 8c), but not of FW fish (Fig. 8d). Notably,
more significant black signals of AgCl precipitation were
found in the opercular membrane of the SW medaka rather
than in the FW group (Fig. 9). In SW medaka, AgCl precipitation in dot and line was observed in both the opercular membrane (Fig. 9a) and gill filament (Fig. S4). Counterstaining with NKA α-subunit antibody further showed
that the line signal bordered the pavement cells while dot
signals were localized to the apical regions of NKA-IR
cells/ionocytes beneath the pavement cells of the opercula
in the SW individuals (Fig. 9e).
Discussion
In the ancestors of teleosts, the jaw, gill, and operculum were derived from pairs of pharyngeal slits (Graham
2003; Soukup et al. 2013). Compared with Agnatha and
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Chondrichthyes, the teleosts had a pair of opercula covered with a hard bone to protect the gills in the head. In
addition, the motion of opercula was controlled to conduct
respiratory ventilation. When external water entered the
mouth opening, the opercula closed the gill opening. Subsequently, the mouth closed and the opercula opened the
gill opening to lead the water through gill slits, and then,
the water went out the gill chamber (Evans et al. 2005).
Therefore, the above-described functions of the opercula
were shown in this study when the brackish medaka was
raised in a SW or FW tank.
There were four pairs of gill slits in the gill chamber
of the brackish medaka. A pair of gill filaments existed in
each gill slit. The gill ionocytes were localized in the afferent regions of the gill filaments (Kang et al. 2008; 2012).
Because gill filaments in the gill chamber were arranged in
parallel, the ionocytes were easily sheltered by the other gill
filaments during observation. In addition, the gill filaments
were fragile and easily destroyed or lost in the washing
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Histochem Cell Biol (2015) 143:69–81
77
Fig. 8 The ionocytes (NKA-IR
cells, green) showed two typical
transporter mediated Cl− secretion in the inner opercular membranes of the brackish medaka
acclimated to SW (a, c) and FW
(b, d). Confocal micrographs of
whole-mount double immunofluorescence staining with the
anti-NKCC1a/NCC antibody
(red; a, b) revealed that the
NKCC1a signals of SW-type
ionocytes were colocalized to
NKA at the basolateral membrane, while the NCC signals
of FW-type ionocytes were
found at the apical membranes
of NKA-IR cells (ionocytes).
The micrographs of anti-CFTR
antibody (red; c, d) showed that
CFTR protein was localized in
the apical regions of SW-type
ionocytes rather than the FW
ionocytes. FW freshwater; SW
seawater
procedure of whole-mount immunostaining and SEM. Our
observations indicated the shape of the gill filament was
slightly deformed after sample preparation for SEM observation. Hence, it was difficult to use the whole-mount gill
filaments of the brackish medaka with various labeling
treatments for investigating the target ionocytes. Compared with the gill filament, the operculum was tougher and
easier to use in different protocols for multiple observations. Several studies on the killifish and tilapia have used
the opercular ionocytes to demonstrate the ionoregulatory
model of gill ionocytes (Karnaky and Kinter 1977; Karnaky et al. 1977; Foskett and Scheffey 1982; Uchida et al.
2000; Mazon et al. 2007; Marshall et al. 2008). According
to Choi et al. (2011), the opercula of the brackish medaka
were observed in three conditions including vital tissue,
immunofluorescent staining, and SEM. Therefore, the present study could trace the same region of the inner opercular membrane based on the stable shape of the operculum
after various treatments. In the adult medaka, the identified
site of the inner opercular membrane was a good platform
to observe the physiological situation of ionocytes instead
of the afferent region of gill filaments.
Our previous studies showed that the ionocytes were
mitochondrion-rich cells in the medaka gill (Kang et al.
2008, 2010). The present study further clarified this characteristic by two markers of mitochondria. Katoh and Kaneko
(2003) reported that rhodamine 123 was optimal for staining vital mitochondria in the living ionocytes of fish. Rhodamine 123, with a positive charge, preferentially accumulated within the inner mitochondrial space due to the
mitochondrial inner membrane potential (negative charge
inside) (Johnson et al. 1980). This study demonstrated that
the mitochondrial-specific fluorescent dye was able to label
the ionocytes rich in mitochondria in the opercular membrane as well as gill filaments of the medaka. After fixation,
however, the fluorescent signals were absent in the rhodamine 123-positive cells. However, the present study indicated that the mitochondria of opercular ionocytes exhibit
the COX IV protein in their inner membrane. In the fish
ionocytes, this commercial antibody was applied for the
first time to examine this target protein. The COX IV protein is a common marker for identifying mitochondria in
mammalian cells (Aschrafi et al. 2008; Chacko et al. 2010).
In the mitochondrial electron transfer chain, the COX
IV protein catalyzes the terminal step in the reduction of
molecular oxygen to water coupled to the translocation of
protons across the mitochondrial inner membrane to drive
ATP synthesis and plays a major role in the regulation of
oxidative phosphorylation (Li et al. 2007). The NKA-IR
cells/ionocytes were the COX IV-IR cells in the fixed tissue
of the brackish medaka. Based on the results of multiple
staining, the present study demonstrated that the SW- and
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78
Histochem Cell Biol (2015) 143:69–81
Fig. 9 Silver precipitates of
Cl− accumulation (a, b) on the
ionocytes (NKA-IR cells; c, d)
of the inner opercular membranes of SW-acclimated (a, c,
e) and FW-acclimated (b, d, f)
brackish medaka. The merged
images (e, f) revealed that
more AgCl precipitates (black
signals) were localized to the
apical regions of the SW-type
ionocytes compared with those
in the FW-type ionocytes. FW
freshwater, SW seawater
FW-type ionocytes of the medaka were rich in mitochondria. This study showed that staining with these mitochondrial markers, in addition to TEM observation, is optimal
for recognizing the ionocytes of euryhaline teleosts. Characteristics of the opercular membrane can provide multiple
observations with double or triple staining to reveal the
localization of ionocytes as well as the distribution of target
proteins.
Similar to gill filaments, the inner opercular membrane
of the brackish medaka exhibited numerous ionocytes. The
ionocytes were also found in the opercular membranes of
the killifish and tilapia (Karnaky and Kinter 1977; Katoh
et al. 2001; Uchida et al. 2000; Shieh et al. 2003). This
study was the first to investigate the patterns of ionocyte
distribution in the opercular membrane of the euryhaline
medaka. In the region close to the eye, the gill-filamentlike pattern of opercular ionocytes was found. In contrast,
the pattern of ionocytes in the flat region of the opercular
13
membrane close to the gill opening, similar to that of the
yolk sac of the Japanese medaka and zebrafish embryos,
was a “salt-and-pepper pattern” (Hsiao et al. 2007;
Thermes et al. 2010). Hsiao et al. (2007) described the distributed pattern of ionocytes as a salt-and-pepper pattern.
In the middle area between the regions with different ionocytes patterns, no ionocytes were observed. Therefore, the
opercular membrane was similar to a gill slit exhibiting one
pair of gill filaments with ionocytes and one gill arch without ionocytes. This finding could be considered as possible
evidence for a similar origination of the gill and opercular
membrane. Based on observation of the opercular membrane, the flat area with the salt-and-pepper distribution of
ionocytes was a good platform to perform the subsequent
experiments.
Degnan et al. (1977) illustrated that the pavement cells
formed a continuous external layer except where the
mucous and ionocytes contacting the external environment.
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Histochem Cell Biol (2015) 143:69–81
In this study, the COMPO model using a high accelerating
voltage (15 kV) and Hitachi TM-3000 SEM to observe the
methanol-treated opercula revealed the transparent effect
of the outermost pavement cells. Therefore, the apical
morphologies and cell size of ionocytes could be observed
simultaneously. Previous studies have indicated that there
are morphological similarities between opercular ionocytes and gill ionocytes of the killifish and tilapia (Katoh
et al. 2001; Marshall et al. 2002; Uchida et al. 2000; Shieh
et al. 2003). The apical openings of opercular ionocytes
exposed to the gill chamber were close to the first gill slit.
The apical structure of SW-type ionocytes was a hole. In
contrast, the convex structure with microvilli was the dominant morphology of the FW-type ionocytes. Our recent
study reported that VILL was involved in the formation of
microvilli in the FW-type ionocytes (Kang and Lee 2014).
According to the results of the opercular membranes with
VILL immunofluorescence staining, the apical regions of
the FW-type ionocytes exhibited more VILL expression
than the apical regions of SW cells. The hypoosmoticdependent expression of VILL regulated the remodeling
of actin filaments to participate in the formation of ionocyte microvilli. In addition, the opercular membrane of SW
medaka exhibited larger ionocytes with lower density for
ion secretion, while numerous ionocytes with bigger openings for ion absorption were found in the opercular membrane of the FW medaka. These results revealed that the
cellular morphologies of SW- and FW-type ionocytes of
the opercular membrane conformed to typical morphologies of the gill ionocytes in the euryhaline medaka (Kang
et al. 2012; Kang and Lee 2014). Therefore, the operculum
was a useful surrogate model for studying the remodeling
of branchial epithelial cell morphologies.
The SW- and FW-type ionocytes in the teleostean gills
have been demonstrated to function in ion secretion and
absorption, respectively (Wood and Marshall 1994; Evans
et al. 1999; Marshall 2002; Hirose et al. 2003; Hwang and
Lee 2007; Evans 2011; Hwang et al. 2011). Moreover, the
opercular ionocytes of the killifish were reported to exhibit
NKCC, NCC, and the CFTR corresponding to SW or FW
gill ionocytes (Marshall et al. 2002). According to Kang
et al. (2010, 2012), gill ionocytes of the brackish medaka
were classified into SW type and FW type by immunostaining with the anti-human CFTR antibody or the antibody
T4. In the opercular membranes of the brackish medaka,
the SW-type ionocytes exhibited apical CFTR and basolateral NKCC1a expression. In contrast, the apical NCC protein was found in the FW-type ionocytes. The same profiles
of these transporter genes were found in the gills and opercular membranes of the killifish (Scott et al. 2005, 2008).
Therefore, these results suggested that the ionoregulatory
mechanisms of medaka ionocytes in the opercular membrane corresponded to those in the gill.
79
The present study provided reliable evidence to verify
the ionoregulatory models in euryhaline medaka using
the opercular membrane. Opercular membranes of killifish and tilapia were used as the detection systems for
an electrophysiological technique, i.e., the Ussing Chamber, to explore the ionoregulatory mechanisms of ionocytes (Karnaky et al. 1977; Foskett and Scheffey 1982).
Recent studies used the SIET to measure ion fluxes close
to the apical openings of embryonic ionocytes of the
zebrafish, tilapia, and medaka (Lin et al. 2006; Horng
et al. 2007; Shen et al. 2011). In addition, silver precipitation for detecting Cl− secretion of ionocytes was performed in the gills of the pufferfish (Tang and Lee 2011),
the yolk sac of tilapia embryos (Kaneko et al. 2008),
and the body epidermis of eel larvae (Lee et al. 2013).
This histochemical staining method of silver precipitation was easy to perform in laboratories compared with
the other electrophysiological systems. Therefore, the
present study modified this method by counterstaining
with immunostaining using NKA to validate different
levels of apical Cl− accumulation in the opercular ionocytes of the SW and FW medaka. Horng et al. (2009)
reported that the Cl− flux of the FW-type ionocytes was
less than that of the SW-type cells in tilapia embryos
with SIET detection. Weak signals of silver precipitation
were also reported in the ionocytes of the FW-exposed
tilapia embryos (Kaneko et al. 2008). Compared with the
SW-type ionocytes, the FW-type cells would uptake the
Cl− in low amounts, which could not be determined by
the staining method in the opercula membranes of the
brackish medaka. In contrast, strong signals of silver
precipitates indicated that the SW-type ionocytes of the
euryhaline medaka conducted Cl− secretion through apical openings to the external media. The data of the surrogate tissue illustrated that the ionoregulatory functions of
opercular ionocytes conformed to typical models for ion
transport in the gills of the teleosts.
Taken together, the typical characteristics of opercular ionocytes were correlated with gill ionocytes in the
euryhaline teleost acclimated to environments with different salinities. This study was the first to develop methods for multiple observations of the intact opercula rather
than the gill filaments. With these observations, the apical
morphologies, cytoplasmic mitochondria, and ionoregulatory functions of the ionocytes were illustrated in the
brackish medaka. Our studies demonstrated that the gills
of the brackish medaka could be used for analysis of target protein or gene expressions in epithelial cells (Kang
et al. 2008, 2010, 2012; Kang and Lee 2014), and simultaneously, the opercula could be a surrogate platform for
whole-mount dual or triple observations of ionocytes to
evaluate cellular responses to environmental challenges.
Our future work will use multiple observations of the
13
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80
opercular membrane to study the mechanisms of differentiation and transformation between SW- and FW-type ionocytes of brackish medaka.
Acknowledgments The monoclonal antibodies, T4 and α5, were
purchased from the Developmental Studies Hybridoma Bank (DSHB)
maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of Medicine, Baltimore, MD
2120521205, 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 Ministry of Science and Technology (MOST), Taiwan, to T.H.L. (NSC
102-2321-B-005-011-MY3). K.C.K. was supported by a MOST postdoctoral fellowship (NSC102-2811-B-005-024).
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