Evolutionary Ecology Research, 2019, 20: 349–363

Gill Na+/K+-ATPase in the threespine stickleback(Gasterosteus aculeatus): changes in transcript levels
and sites of expression during acclimation to seawater
Makoto Kusakabe1,2, Seiichi Mori3 and Jun Kitano4
1

Department of Biological Sciences, Faculty of Science, Shizuoka University, Surugaku,
Shizuoka, Japan, 2Department of Marine Bioscience, Atmosphere and Ocean Research Institute,
University of Tokyo, Kashiwa, Chiba, Japan, 3Biological Laboratory, Gifu-keizai University,
Ogaki, Gifu, Japan and 4Ecological Genetics Laboratory, National Institute of Genetics,
Mishima, Shizuoka, Japan

ABSTRACT++

Background: Na /K -ATPase (NKA) in the gills is a key contributor to osmoregulation infishes.
Questions: Does gill NKA contribute to the regulation of salinity tolerance in stickleback?
Do gill NKA expression patterns during acclimation to seawater differ among stickleback
populations that differ in salinity tolerance?
Organisms: Freshwater threespine stickleback (Gasterosteus aculeatus) from populations in
Ono and Gifu, Japan.
Methods: We gradually acclimated the stickleback to 100% seawater. During acclimation, wemonitored changes in plasma Na+ concentrations using atomic absorption spectrophotometry
and gill NKA transcript levels using qPCR. We determined the sites of gill NKA expression byimmunohistochemistry.
Results: Threespine stickleback from Ono were able to acclimate to 100% seawater. In contrast, stickleback from the Gifu population were able to survive in 50% seawater but not 100%
seawater. Both ecotypes maintained a plasma Na+ concentration of approximately 150 m
during the acclimation process. Patterns of change in NKA transcript and protein expression
were similar between the two populations, although they differed in tolerance to salinity. In
both populations, gill NKA-positive ionocytes were distributed on the primary filaments andsecondary lamellae in fresh water, but during acclimation to seawater the NKA-positive ionocytes on the secondary lamellae disappeared. The cell sizes and densities of NKA-positive
ionocytes were higher in fish from Ono than in fish from Gifu, which may explain the differencein salinity tolerance between these two stickleback populations.
Keywords: Gasterosteus aculeatus, gills, Na+/K+-ATPase, plasma Na+, salinity, stickleback.

Correspondence: M. Kusakabe, Department of Biological Sciences, Faculty of Science, Shizuoka University,
Surugaku, Shizuoka, Japan. email: kusakabe.makoto@shizuoka.ac.jp
Consult the copyright statement on the inside front cover for non-commercial copying policies.
© 2019 Makoto Kusakabe

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INTRODUCTION
Selection against maladaptive migrants is an important mechanism in determining species
distributions (Mayr, 1963; Nosil, 2012). In aquatic organisms, environmental salinity has a major
influence on survival, since salt concentrations in the environment directly influence body
fluid osmotic pressure as well as fitness (Schmidt-Nielsen, 1997).
To maintain homeostasis under high osmotic pressure (seawater), marine teleosts actively
excrete excess sodium, chloride, and other ions. Conversely, freshwater teleosts, under low
osmotic pressure (fresh water), actively take up these ions. Ion excretion and uptake arecontrolled by various ion transporters and channels present in gill ionocytes (Hiroi and McCormick,++2012). Among ion transporters and channels, the sodium-potassium pump Na /K -ATPase(NKA) is known to be the major driving force behind ion excretion/uptake in most animal
systems (Marshall and Bryson, 1998; Hirose et al., 2003; Lin et al., 2004; Hwang and Lee, 2007). The NKA is
localized in the plasma membrane, and pumps three sodium ions out of cells in exchange
for two potassium ions (Kaplan, 2002) using energy derived from ATP hydrolysis. Thus, inteleosts, NKA is considered a major factor in osmoregulation.
The NKA is composed of two subunits, α and β. The α-subunit is responsible for thecatalytic properties of the enzyme, while the β-subunit regulates the structural properties of
the NKA (Blanco et al., 1994). Multiple isoforms of both subunits have been identified andexhibit tissue-specific expression patterns (Lingrel et al., 1990). The characteristics of the NKA
α-subunits have been intensively studied since the functional properties of the NKA arelargely regulated by the α-subunits. The isoforms of α-subunits in teleosts are more
complicated than those of other organisms because teleosts underwent a third round ofwhole-genome duplication. Thus, the nomenclature of NKA α-subunits in teleosts has not
yet been integrated. A recent comprehensive phylogenetic analysis of NKA α-subunits inteleosts showed that they can first be classified into α1-, α2-, and α3-subunits, with α1subunits being further subdivided into α1a/b and α1c clades (Wong et al., 2016). In the current
study, we follow this nomenclature. In salmonids, the expression of α1a is high in fresh
water and that of α1b is high in seawater, but this expression pattern switches when fish
migrate between fresh water and seawater (Richards et al., 2003; Madsen et al., 2009; McCormick et al., 2009).
Consequently, the switching mechanism of multiple NKA isoforms is considered key foradaptation to diverse salinities, particularly in salmonids. Furthermore, the localization of
NKA proteins has been reported to change in salmonids depending on environmental
salinity; for example, NKA-positive ionocytes are distributed in both the primaryfilament and secondary lamellae of the gill in fresh water, but are only present in the
primary filament in seawater (Uchida et al., 1996; McCormick et al., 2009).
Stickleback inhabit diverse environments with a variety of salinities, ranging fromseawater to fresh water. Thus, the stickleback ought to be an excellent model to study thephysiological mechanisms underlying variations in salinity tolerance and habitat isolation
between populations (Heuts, 1946; Wootton, 1984; Kitano et al., 2012). Variation in salinity tolerance has
been reported among stickleback ecotypes (Kitano et al., 2012; Ishikawa et al., 2013; Kusakabe et al., 2014,2017; Divino et al., 2016), and even parapatric ecotypes in the same river may differ in salinity
tolerance. For example, a stream-resident threespine stickleback (Gasterosteus aculeatus)
population lost its ability to adapt to seawater, while an anadromous ecotype that migrates
to the same river to spawn is tolerant of seawater challenge (Ishikawa et al., 2013; Kusakabe et al., 2014,2017). However, the mechanisms involved in loss of seawater adaptability remain largely
unknown.

Gill Na+/K+-ATPase in the threespine stickleback351

The stickleback reference genome contains four genes encoding NKA α-subunits:
ENSGACG00000014324(Atp1a1)on
Chromosome
1
encodes
α1,
ENSGACG00000017683 (Atp1a2) on Chromosome 3 encodes α2, while
ENSGACG00000001959 (Atp1a3a) on Chromosome 10 and ENSGACG00000009524(Atp1a3b) on Chromosome 20 encode α3-subunits (Jones et al., 2012; Wong et al., 2016).
ENSGACG00000014324 (Atp1a1) is composed of two genes resulting from a tandem
duplication and produces at least two different transcripts, atp1a1a.4-201(ENSGACT00000018945) and atp1a1a.4-204 (ENSGACT00000018961) (Wong et al., 2016). A
previous phylogenetic analysis showed that both transcripts belong to α1a/b, whereas
no transcript corresponding to α1c has yet to be found in the stickleback genome (Wonget al., 2016). Most previous studies on NKA expression in sticklebacks examined only
atp1a1a.4-201, but showed inconsistent patterns; with increasing environmental salinity,
the expression levels increased in both marine and lacustrine populations in British
Columbia, Canada (Judd, 2012), or decreased in both marine and lacustrine populationsin the St. Lawrence River and British Columbia (McCairns and Bernatchez, 2010; Gibbons et al., 2017).
The only study to examine these two isoforms separately found that, in a Mediterranean
freshwater population, only the levels of atp1a1a.4-204 increased in seawater, while levelsof atp1a1a.4-201 were unchanged (Rind et al., 2017). Therefore, the general patterns of NKA
response to seawater/freshwater challenges in sticklebacks remain unclear.
Our long-term goal is to understand how changes in osmoregulatory ability occur among
stickleback populations inhabiting different salinity environments. As one of the first steps,in the present study we focused on variations in salinity tolerance and NKA expression
between two Japanese freshwater threespine stickleback populations collected from Ono
and Gifu. The two freshwater stickleback populations were acclimated to 100% seawater,
and changes in plasma Na+ concentrations were evaluated to examine the osmoregulatory
ability of these two populations. We further examined NKA mRNA expression levels
and protein localization in the gills to gain insights into how gill NKA contributes toosmoregulatory differences in these stickleback populations.

MATERIALS AND METHODS
Seawater acclimation experiments
We used freshwater threespine stickleback collected from Ono and Gifu, Japan, in July 2015
(Fig. 1) to test the salinity tolerance of wild-caught fish. The Ono population was collectedfrom a freshwater pond of Hongan-shozu Itoyo-no-Sato (35.976⬚N, 136.491⬚E) in Ono
City, Fukui Prefecture. The Gifu population was collected from a spring-fed pond located in
Gifu-keizai University in Ogaki City (35.366⬚N, 136.616⬚E), Gifu Prefecture. Wild-caught
stickleback were brought to the fish facility of the Atmosphere and Ocean ResearchInstitute, University of Tokyo (Chiba, Japan), and maintained at 16⬚C in 60-litre tanks witha closed recirculating system.
Fish were first acclimated in fresh water (0.3‰) for 7 days, before being transferred to
10% seawater (3.3‰) for 1 day, 30% seawater (10.2‰) for 3 days, 50% seawater (15.2‰) for
3 days, and finally 100% seawater (34.5‰) for 8 days (Fig. 2). Plasma and gills were collected
at the following sampling time points:

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Fig. 1. Sampling locations for the Ono and Gifu threespine stickleback (Gasterosteus aculeatus)
populations.

Fig. 2. Time course of the seawater acclimation experiment. Circles show the days when the plasmaand gill samples were collected. Vertical lines indicate the days when tank salinity was changed but nosamples were collected.

Gill Na+/K+-ATPase in the threespine stickleback353• immediately after the fish were collected from the wild (Day 0; N = 10 for Ono andN = 10 for Gifu);• 7 days after acclimation to fresh water in the lab (Day 7; N = 4 for Ono and N = 6 forGifu);• after acclimation to 50% seawater (Day 14; N = 3 for Ono and N = 6 for Gifu); and• after acclimation to 100% seawater (Day 22; N = 6 for Ono).
All the Gifu fish died within 10 hours of being transferred to 100% seawater (N = 10), andthus could not be sampled at Day 22.
For sampling, fish were euthanized using an overdose of MS-222 (tricaine methanesulfonate, 300 mg/L) buffered with sodium bicarbonate. Blood samples were collected fromthe caudal vasculature using heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA).
After centrifugation of the heparinized capillary tubes, plasma was obtained and stored at−80⬚C until required. Plasma Na+ concentration was measured using an atomic absorption
spectrophotometer (Z-5300 Hitachi, Tokyo, Japan) as described previously (Kusakabe et al.,2014). The left halves of gills were removed and immersed in RNAlater reagent (Thermo
Fisher, Waltham, MA) overnight at 4⬚C, and then stored at −80⬚C for RNA extraction. The
right halves of gills were removed and fixed in 4% paraformaldehyde overnight at 4⬚C.
The fixed gills were embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). All
animal experiments were approved by the Institutional Animal Care and Use Committee ofthe University of Tokyo.
Quantitative analysis of mRNA levels of the NKA 1-subunit in the stickleback gill
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The
RNA concentration was determined by measuring the absorbance at 260 nm using a
BioPhotometer D30 (Eppendorf, Hamburg, Germany), and RNA quality was assessedby A260nm/A280nm ratios, with ratios > 1.8 deemed acceptable. Total gill RNA (1 µg) was
incubated with 2 units of TURBO DNase (TURBO DNA-free, Thermo Fisher,
Waltham, MA) at 37⬚C for 2 hours to remove genomic DNA contamination. Single-strand
cDNA was synthesized from the DNase-treated RNA using a High-Capacity cDNA
archive kit as described by the manufacturer (Thermo Fisher, Waltham, MA). Real-timequantitative PCR with SYBR Green was performed to quantify the mRNA using a
LightCycler 480 (Roche, Basel, Switzerland). PCR was performed in a 10 µL reaction
volume, using 1 µL of the 10-fold diluted reverse-transcribed cDNA. The primer pairs
used for the detection of the stickleback NKA α-subunits and internal controls are listed inTable 1. The PCR conditions were as follows: 95⬚C for 5 minutes, followed by 45 cycles of95⬚C for 10 seconds, 60⬚C for 10 seconds, and 72⬚C for 10 seconds. A standard curve was
generated by a four-fold serial dilution of pooled cDNA. The transcript levels are indicatedas relative mRNA levels. The stickleback genes encoding actin beta 2 (actb2), Ribosomal
protein L13a (rpl13a), and Ubiquitin A-52 residue ribosomal protein fusion product 1(uba52) were used as the internal controls. The primer sequences for actb, rpl13a, and uba52
were obtained from a previous study of internal control genes in the stickleback (Hibbeler et al.,2008). The mean expression levels of the three genes (actb2, rpl13a, and uba52) were used asinternal controls and the expression levels of the Atp1a genes were divided by the internal
control values. The mean transcript levels of the initial controls (Day 0) were assigned avalue of 1.0.

UniGene ID

ENSGACG00000017683

ENSGACG00000001959

ENSGACG00000009524

ENSGACG00000007836
ENSGACG00000009310

ENSGACG00000008021

atp1a2

atp1a3a

atp1a3bactb2
rpl13auba52

atp1a1a.2-204 ENSGACG00000014324

atp1a1a.2-201 ENSGACG00000014324

Gene name5⬘-AGACGGGCATAGCACTTGC-3⬘

5⬘-CAGGACAAGGAAGGCATCC-3⬘

5⬘-GCGTGGCTACTCCTTCACC-3⬘
5⬘-AGGACTTCATACCGAGGAAGG-3⬘
5⬘-CACCTTGGTCAACTTGAACAGTG-3⬘ 5⬘-TCCCTCCGCCCTACGAC-3⬘

5⬘-CAACCAGATCTCCACCCACT-3⬘

5⬘-CTCCGGCACATTAGCAACAA-3

5⬘-TTCCTCGGCATGTCCTTCTT-3⬘
5⬘-AAACATGGTGCCTCAGCAAG-3⬘

5⬘-CATTATGCTGCTGCACCTGT-3⬘

5⬘-GTTTCAGGTGTGCCATCCTT-3⬘

5⬘-TATCGAGCTGTGCTGTGGAT-3⬘
5⬘-ACAATCCCTCCGGTCACATT-3⬘

5⬘-ACGATCTTTCTGCGCTCGTA-3⬘

5⬘-TCAGAGTGATGTGGGACGAC-3⬘

Na+/K+-ATPase
α1-subunit++
Na /K -ATPase
α1-subunit
Na+/K+-ATPase
α1-subunit++
Na /K -ATPase
α1-subunit
Na+/K+-ATPase
α1-subunit
Actin, beta 2
Ribosomal
protein L13a
Ubiquitin A-52
residueribosomal
protein fusion
product 1

Reverse primer

Forward primer

Description

Table 1. List of primers used for real-time quantitative RT-PCR assay

Gill Na+/K+-ATPase in the threespine stickleback355

NKA immunohistochemistry in the stickleback gill
The embedded gill samples were sectioned into 6 µm slices and mounted on MAS-coatedslides (Matsunami Glass, Osaka, Japan). Endogenous peroxidases were blocked in 0.6%
H2O2 in methanol for 15 minutes at room temperature, and non-specific binding was
blocked with 2% normal goat serum in phosphate-buffered saline at room temperaturefor 30 minutes. The primary antiserum against the NKA α-subunit of masu salmon(Oncorhynchus masou) (a gift from Dr. K. Ura, Hokkaido University) was raised in rabbits, and theantigen sequences are conserved among fish, including elasmobranchs (Ura et al., 1996; Hyodo etal., 2004; Takabe et al., 2012). The antiserum was applied at a dilution of 1:1500. Sections wereincubated with the primary antiserum for 40 hours at 4⬚C in a humidified chamber, andthen incubated with biotinylated goat anti-rabbit IgG antibody (Vector Laboratories,
Burlingame, CA) for 30 minutes and with an avidin–biotin–peroxidase complex (Vector
Laboratories, Burlingame, CA) for 45 minutes at room temperature. The antibody–antigen
complex was visualized by exposure to 0.05% 3,3⬘-diaminobenzidine and 0.01% H2O2
in 50 m Tris buffer (pH 7.3) for 5 minutes at room temperature. The sections werecounterstained with haematoxylin. The sizes and densities of ionocytes (NKA-positive
cells) were analysed using the BZ-HIC software (Keyence, Osaka, Japan). For cell size
measurement, 1735 NKA-positive cells of Ono fish and 1494 NKA-positive cells of Gifu
2fish were randomly selected from three fish of each population and area (µm ) of the
NKA-positive cells measured. For cell density measurement, 12 immunohistochemistry
images were randomly selected from three fish of each population and area ratios of the
NKA-positive cells against the entire gill area were calculated.
Statistical analysis+

Differences in plasma Na concentrations in fresh water (Days 0 and 7) were analysed byStudent’s t-test. Changes in plasma Na+ concentrations during acclimation to seawater wereassessed by two-way analysis of variance (ANOVA). Changes in mRNA levels of the gill
NKA α1-subunits were assessed by one-way ANOVA, followed by Tukey’s HSD post-hoc
test. Differences in the sizes of the NKA-positive cells were analysed using Student’s t-test.
All statistical analyses were performed using R statistical software (v.3.0.2, R Foundationfor Statistical Computing, Vienna, Austria). A P-value of 5% or less was considered statistically significant.
RESULTS
Plasma Na+ concentrations during acclimation to seawater
There was no significant difference in the plasma Na+ concentration between the Ono andGifu sticklebacks immediately after collection in the field (Day 0, t-test, P = 0.07). Afterbeing brought to the laboratory, the plasma Na+ concentrations of the Ono fish were significantly higher than those of the Gifu fish (Day 7, t-test, P < 0.001), but all the fishsurvived during acclimation to laboratory conditions (Fig. 3). There were no significant
changes in the plasma Na+ concentrations either in the Ono or Gifu fish during acclimation
to seawater (two-way ANOVA, P = 0.34). However, the Gifu fish were unable to survive in
100% seawater even after gradual acclimation to 50% seawater. There was a marginally

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Fig. 3. Plasma Na+ levels of (a) Ono and (b) Gifu threespine stickleback during acclimation toseawater. Plasma samples were taken immediately after fish were collected in the field (Day 0),
and then after 7, 14, and 22 days. The number of samples per data point is shown in parentheses.
Differences in plasma Na+ levels between sampling points were analysed using one-way ANOVA.

significant interaction between population and salinity for plasma Na+ concentrations (twoway ANOVA, P = 0.05): plasma Na+ concentration tended to increase in Ono fish, whereas
it appeared to decrease slightly in Gifu fish.
Transcript levels of the NKA 1-subunit during acclimation to seawater
The atp1a1a.4-201 transcript levels of the Ono fish decreased significantly after transfer
to 50% seawater (P < 0.05), but returned to baseline (Day 0) after acclimation to 100%
seawater (Fig. 4a). The atp1a1a.4-201 transcript levels of the Gifu fish also decreased significantly after transfer to 50% seawater (Fig. 4f, P < 0.001). There was no significant
change in atp1a1a.4-204 transcript levels either in the Ono (Fig. 4b, P = 0.07) or Gifu fish
(Fig. 4g, P = 0.36) during acclimation to seawater. The atp1a2 transcript levels of the Ono
fish increased significantly after transfer to the laboratory, and transcript levels were maintained during acclimation to seawater (Fig. 4c, P < 0.001). The atp1a2 transcript levels of
the Gifu fish increased significantly after transfer to 50% seawater (Fig. 4h, P < 0.001).
There was no significant change in atp1a3a transcript levels either in the Ono (Fig. 4d,

Gill Na+/K+-ATPase in the threespine stickleback357

Fig. 4. Gill NKA α-subunit mRNA levels (atp1a1a.4-201, atp1a1a.4-204, atp1a2, atp1a3a, andatp1a3b) of the Ono (a–e) and Gifu (f–j) threespine stickleback populations during the seawater
acclimation experiment. Gill samples were taken immediately after fish were collected in the field(Day 0), and then after 7, 14, and 22 days. The number of samples per data point is shown inparentheses. The values were corrected with internal controls and the relative expression values at
Day 0 were set to 1.0 (see Materials and methods). Differences in transcript levels between sampling
points were analysed using one-way ANOVA. Data points that do not share a letter are significantly
different by Tukey’s HSD (P < 0.05).

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P = 0.30) or Gifu fish (Fig. 4i, P = 0.13) during the seawater acclimation experiment. The
atp1a3b transcript levels of the Ono fish increased significantly after transfer to 100%
seawater (Fig. 4e, P < 0.001). The atp1a3b transcript levels of the Gifu fish increased
significantly after transfer to 50% seawater (Fig. 4j, P < 0.001). The qPCR crossing point(Cp) values, which indicate how rapidly the PCR products are detected and represent anapproximation of the overall transcript levels, showed that atp1a1a.4-201 was the mostabundant transcript, and that the levels of the other transcripts were substantially lower
in comparison (evolutionary-ecology.com/data/3177Appendix.pdf, Fig. S1). The predominance of atp1a1a.4-201 in the gills is consistent with previous gill RNA-sequencing
data for Russian populations (Appendix Fig. S2) (Artemov et al., 2017).

Fig. 5. Immunohistochemistry of the NKA in gills of the Ono (a–c) and Gifu (d, e) populations.
Arrows indicate cells positive for anti-NKA antibody. Panel (f) summarizes the structures of stickleback gills and indicates sites of gill ionocyte localization after acclimation to fresh water or seawater(grey ovals).

Gill Na+/K+-ATPase in the threespine stickleback359

Fig. 6. Sizes (a) and densities (b) of gill ionocytes of fish acclimated to 50% seawater. Values aremeans ± S.E. Sample sizes are given. The asterisk indicates a significantly higher value (*P < 0.05).

NKA immunohistochemistry in the gill
The sites of NKA-positive cells were immunohistochemically detected using an antiserum
specific for fish NKA. Under the freshwater condition, NKA-positive cells were detectedin both the gill filament and secondary lamellae in both the Ono and Gifu sticklebacks(Fig. 5a, d). After transfer to 50% seawater (Day 14), the NKA-positive cells were detected
only in the gill filament of both stickleback populations (Fig. 5b, e). The average size anddensity of NKA-positive cells of the Ono fish acclimated to 50% seawater were significantly
larger than those of the Gifu fish (Fig. 6a, b). After transfer to 100% seawater (Day 22),
NKA-positive cells could still be detected in the gill filament of the Ono fish (Fig. 5c). Since
all Gifu fish died after being transferred to 100% seawater, Gifu fish gill samples could not
be obtained. The localizations of NKA-positive cells in both freshwater and seawaterconditions are summarized in Fig. 5f.
DISCUSSION
The two Japanese freshwater stickleback populations examined differed in salinity tolerance.
The Ono population acclimated to 100% seawater, whereas the Gifu population was
unable to do so even after gradual acclimation to such salinity (10% seawater for 1 day,
30% seawater for 3 days, and 50% seawater for 3 days, rising to 100% seawater). We repeatedthe acclimation experiment with members of the same Gifu freshwater population and once
again all of them died after being transferred to 100% seawater (N = 6, data not shown).
Mean plasma Na+ concentration for both the Ono and Gifu populations during theseawater acclimation experiment was 140–160 m, a physiologically normal range for++plasma Na concentration in teleosts (Schmidt-Nielsen, 1997). The plasma Na concentration of
the Gifu freshwater stickleback remained within the normal range at 50% seawater, but they
could not survive in 100% seawater even after gradual acclimation to 50% seawater.

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Comparison of NKA α-subunit mRNA levels did not reveal substantial differences
between the two populations. Therefore, differential mRNA expression may not be theprimary reason for the difference in salinity tolerance between the two populations.
Switching between the NKA α1a- and α1b-subunits under different salinity conditions is amajor osmoregulatory mechanism in salmonids. A similar switching system of different α1
isoforms has been reported in tilapia (Tipsmark et al., 2011) and galaxiid fish (Urbina et al., 2013).
However, recent studies reported that Japanese medaka (Bollinger et al., 2016) and Japanese eel(Wong et al., 2016) did not show a switching pattern for any α1 isoform, suggesting that theswitching of the NKA α1 isoforms is not a universal osmoregulatory mechanism for euryhalinity. In the current study, the two stickleback isoforms (atp1a1a.4-201 and atp1a1a.4204) did not show a clear switching pattern. In addition, we did not observe up-regulationof atp1a1a.4-204 with the seawater challenge, which is different to that previously reported
for a Mediterranean freshwater stickleback population (Rind et al., 2017). It should be highlighted that the numbers sampled at some time points were low, particularly in the Ono
population. This might have affected the results that showed no clear switching patterns of
atp1a1a isoforms. However, previous gene expression analyses of atp1a1a.4-201 with aseawater challenge also showed inconsistent results (McCairns and Bernatchez, 2010; Judd, 2012; Gibbons
et al., 2017). Changes in the transcript levels of the stickleback NKA α-subunits may not be as
pronounced as for other euryhaline fish, such as killifish (Scott et al., 2004), salmonids (Seidelin etal., 2000; McCormick et al., 2009), and Japanese eel (Wong et al., 2016), indicating that differential NKA
mRNA levels might not explain the variations in salinity tolerance among stickleback
ecotypes.
Our immunohistochemical analysis showed clear changes in the localization sites of the
NKA protein in the gills of both populations in response to the seawater challenge. In
teleosts, NKA expression is found in ionocytes. In salmonids, ionocytes are found both ingill filaments and secondary lamellae in fresh water, whereas in seawater they are only foundin gill filaments (Uchida et al., 1996; McCormick et al., 2009). In seawater, ionocytes are typically larger
and embedded within the lamellae of filaments, whereas in fresh water they are flat andappear to be attached to the secondary lamellae (Uchida et al., 1996; McCormick, 2001; McCormick et al.,2009). We observed similar patterns in both stickleback populations. In fresh water, theionocytes (NKA-positive cells) in the Ono and Gifu fish were found both in gill filaments
and secondary lamellae. Following acclimation to 50% seawater, most ionocytes werelocated in the inter-lamellar region, and the ionocytes on the secondary lamellae could not
be detected. Cell shape also appeared to change; ionocytes in fresh water were flatter, while
those in seawater were more round. The cell measurement analysis showed that the mean
size and density of the ionocytes in the gills of Ono fish acclimated to 50% seawater wereboth significantly greater than in the Gifu fish at 50% seawater (Fig. 6). Differences in
ionocyte differentiation may be a possible mechanism underlying the differences in salinity
tolerance between these two populations.
In summary, the two Japanese freshwater stickleback populations studied showed nosignificant changes in plasma Na+ concentrations during the seawater acclimation process;however, the Gifu fish – but not the Ono fish – were unable to survive in 100% seawater. The
transcript levels of the NKA α-subunit showed similar changes in response to seawater inboth populations. The sites of NKA protein expression also showed similar patterns
with up to 50% seawater, but the sizes of immunoreactive ionocytes differed between
populations. The mechanism of ionocyte formation after mRNA synthesis could be key todetermining osmoregulatory ability. Our results indicate that analysis of mRNA expression

Gill Na+/K+-ATPase in the threespine stickleback361

levels is not sufficient on its own to investigate the physiological basis of variation in
osmoregulatory ability, and highlight the importance of immunohistochemical analysis.

ACKNOWLEDGEMENTS
This work was supported by NIG-JOINT (87B2018) to M. Kusakabe. We thank Stephen D.
McCormick, Graham Young, and Marty Wong for invaluable help and advice and Kazuhiro Ura forproviding the NKA antiserum.

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