Structures and immunolocalization of Na+, K+-ATPase, Na+/H+ exchanger 3 and vacuolar-type H+-ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas

Journal of Fish Biology (2012) 80, 2236–2252

doi:10.1111/j.1095-8649.2012.03277.x, available online at wileyonlinelibrary.com

Structures and immunolocalization of Na+, K+-ATPase,Na+/H+ exchanger 3 and vacuolar-type H+-ATPase in the

gills of blennies (Teleostei: Blenniidae) inhabiting rockyintertidal areas

M. Uchiyama*†, M. Komiyama*, H. Yoshizawa‡, N. Shimizu§,N. Konno* and K. Matsuda*

*Department of Biological Science, Graduate School of Science and Engineering, Universityof Toyama, 3190 Gofuku, Toyama 930-8555, Japan, ‡Department of Biology, Matsumoto

Dental University, Hirooka Gouhara, Shiojiri 399-0781, Japan and §Hiroshima UniversityMuseums, Hiroshima University 1-1-1 Kagamiyama, Higashi-Hiroshima 739-8524, Japan

(Received 15 April 2011, Accepted 15 February 2012)

The structure and immunolocalization of the ion transporters Na+,K+-ATPase (NKA), Na+/H+

exchanger (NHE3) and vacuolar-type H+-ATPase (VHA) were examined in the gills of teleosts ofthe family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. Thesefeatures were compared among the following species with different ecologies: the amphibious rock-skipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimaeand the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopicobservations indicated that thick gill filaments were arranged close to each other and alternately ontwo hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC)and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. Animmunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were locatedpredominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses usingserial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells(IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae,NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the baseof the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E.yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located atthe tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- andVHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. Thisstudy shows that the structure and distribution of ion transporters in the gills differ among the threeblennid species, presumably reflecting their different ecologies. © 2012 The Authors

Journal of Fish Biology © 2012 The Fisheries Society of the British Isles

Key words: gill arch intertidal fishes; ion transporters; lamella mitochondrion-rich cell; SEM; TEM.

†Author to whom correspondence should be addressed. Tel.: +81 76 445 6633; e-mail: [email protected]

2236© 2012 The Authors

Journal of Fish Biology © 2012 The Fisheries Society of the British Isles

G I L L I O N T R A N S P O RT E R S I N I N T E RT I DA L B L E N N I I DA E 2237

INTRODUCTION

Rocky shore habitats are living spaces occupied by various fishes, including severalblennid species, whose habitats show vertical zonation (Graham, 1997; Horn et al.,1999). Blennies belong to the suborder Blenniodei, which includes almost 900 rec-ognized species with a world-wide distribution, with greatest diversity in tropicaland subtropical seas (Nelson, 2006). They inhabit primarily marine shallow, inter-tidal or subtidal waters but some species have colonized other environments, suchas crevices in reefs or the lower stretches of rivers, and some species inhabit freshwater. Two blennid species that inhabit freshwater or seawater habitats, Salaria pavo(Risso 1810) and Salaria fluviatilis (Asso 1801), have the ability to osmoregulate inboth of these environments (Plaut, 1998). In the course of their evolution, blennidshave adapted to a wide variety of ecological niches. Studies of this group of fishesmay contribute significantly to the understanding of physiological adaptation and theevolutionary history of the intertidal ichthyofauna.

Tidal environments significantly influence the vertical zonation of fishes in rockyshore habitats. The subtidal zone is the most stable of the tidal environments, and thenumber of herbivorous fishes is higher in this zone than in other tidal zones. A typi-cal rocky shore can be divided into the supratidal zone and the intertidal zone. In thesupratidal environment, seaweeds are usually abundant but temperature and salinityfluctuate drastically as a result of isolation and evaporation during daytime low tides.The intertidal zone is the region that is exposed to the air at low tide and submergedat high tide. Organisms in the intertidal zone are adapted to harsh environments.Therefore, supratidal and intertidal fishes may exhibit morphological and physiolog-ical adaptations in addition to a number of behavioural adaptations. Supratidal fishesemerge from normoxic water at all phases of the tidal cycle (Bridges, 1988). In con-trast, most intertidal fishes do not voluntarily emerge but some species may be foundpassively emerged during low tides (Gibson, 1982). Although various physiologicaladaptations to an aerial habitat have been studied (Sayer, 2005), little is knownabout osmoregulatory adaptations of amphibious (supratidal) fishes in supratidal andintertidal environments.

The gills of fishes perform multiple physiological functions in addition to gasexchange, such as osmotic and ionic regulation, control of acid–base balance anddetoxification. The gill is therefore vital in maintaining systemic homeostasis in theface of changing internal (e.g. acidosis) and environmental (e.g. desiccation andsalinity) conditions (Laurent & Perry, 1991; Perry, 1998; Evans et al., 2005; Mar-shall & Grosell, 2006; Evans & Claiborne, 2009). The gill epithelium is composedof several distinct cell types, such as mitochondrion-rich cells (MRC), pavementcells (PVC), mucous cells (MC) and undifferentiated cells (Laurent, 1984; Wilson& Laurent, 2002). The detection of various ion transport proteins on the gill epithe-lium, using immunohistochemical techniques as well as molecular cloning of theseproteins, is increasing the knowledge of the roles of MRC and PVC in fishes (Mar-shall, 2002; Wilson & Laurent, 2002; Hwang & Lee, 2007; Hwang et al., 2011).Teleost MRCs are the main sites responsible for the active transport of ions in thegills. MRCs, also known as chloride cells, are characterized cytologically by elab-orate intracellular infoldings, which are continuous with the basolateral membrane(tubular system), and the presence of numerous mitochondria in the cytoplasm (Lau-rent, 1984). In seawater teleosts, NaCl secretion is essential for osmoregulation,

© 2012 The AuthorsJournal of Fish Biology © 2012 The Fisheries Society of the British Isles, Journal of Fish Biology 2012, 80, 2236–2252

2238 M . U C H I YA M A E T A L .

and the key transporters are Na+,K+-ATPase (NKA), Na+/K+/2Cl− and cysticfibrosis transmembrane conductance regulator Cl− channel (Marshall, 2002; Hiroseet al., 2003; Evans et al., 2005). Na+/H+ ion exchangers (NHE) and vacuolar-typeH+-ATPase (VHA) are important for acid–base regulation in fishes (Claiborne et al.,2002; Catches et al., 2006). Although respiratory and nitrogen excretory functionsof gills have been studied in several amphibious air-breathing fishes (Graham, 1997;Frick & Wright, 2002; Ong et al., 2007), only a few studies have investigated ionexchange (Graham, 1997; Huang et al., 2010). Respiration in many amphibious fishesis dependent on cutaneous gas exchange. The gills of mudskippers (Oxudercinae)exhibit morphological adaptations such as a reduced surface area, while there isproliferation of cutaneous blood vessels in the skin (Wilson et al., 1999; Ong et al.,2007).

Little is known regarding gill structure and function in intertidal fishes inhabitingrocky reef areas (Laurent & Perry, 1991; Perry, 1998; Evans et al., 2005; Evans &Claiborne, 2009). In this study, the structure, ultrastructure and distribution of NKA,NHE3 and VHA on the gill epithelium were investigated in the rockskipper blennyAndamia tetradactylus (Bleeker 1858) in comparison with the white-finned blennyPraealticus tanegasimae (Jordan & Starks 1906) and the yaeyama blenny Ecseniusyaeyamaensis (Aoyagi 1954). These species have different vertical distributions inrocky intertidal areas.

MATERIALS AND METHODS

A N I M A L S

Three teleost species of the family Blenniidae inhabiting the intertidal zone of the sub-tropical sea of Japan were collected using several types of nets at Kuchierabu-jima Island,Yakushima-cho, Kagoshima Prefecture. Andamia tetradactylus is a small air-breathing fish[mean ± s.e., total length (LT): 7·9 ± 0·5 cm] inhabiting the supralittoral zone of tropi-cal reefs. In previous field and laboratory observations, A. tetradactylus spent 95 and 65%of its time in air, respectively (Shimizu et al., 2006). Praealticus tanegasimae is a smallfish (total length, LT: 7·0 ± 0·1 cm) living at the mid-tide level, where it is covered bywater during neap tides, and in tide pools on rocky beaches. Ecsenius yaeyamaensis isa small fish (LT: 5·1 ± 0·2 cm) that inhabits subtidal regions (water depth 2–10 m) incoral reefs.

Immediately after capture, animals were killed by decapitation and their gills were removed.Field sampling protocols were implemented after approval of the study by the animal ethicscommittee at the University of Toyama.

S C A N N I N G E L E C T RO N M I C RO S C O P Y

The second and third gill arches were removed from three or five individuals of each ofthe three species, fixed in Bouin’s solution for 24 h at 4◦ C and stored in 70% ethanol. Thegills were dehydrated in a graded ethanol series (70, 80, 90 and three rounds of 100%) andthen dried using a critical point dryer. Dried samples were then mounted on a carbon tapeand sputter coated with 30 nm gold using a Hitachi 101 ion sputter coater (Hitachi High-Tech; www.hitachi-hitec.com). A Hitachi TM-1000 scanning electron microscope (SEM) wasused to observe and capture micrographs of the gills. Based on the micrographic data,filament and lamella sizes were measured to compare the gill structures among the threespecies.



L I G H T M I C RO S C O P Y A N D I M M U N O H I S T O C H E M I S T RYTissue samples fixed with Bouin’s solution without acetic acid were dehydrated and embed-

ded in paraffin wax. Deparaffinized sections (6 μm) were stained with haematoxylin and eosin.MCs were characterized and examined using the periodic acid-Schiff (PAS) method to detectneutral mucin (Mallatt & Paulsen, 1986). Other sections were incubated overnight at 4◦ Cwith rabbit anti-VHA antiserum [dilution 1:5000 with 1% bovine serum albumin–phosphate-buffered saline (BSA–PBS)] in 1% BSA–PBS. Serial (mirror image) sections were stainedwith rabbit anti-salmon NKA α-subunit antiserum (1:5000 dilution) or anti-tilapia type 3NHE3 antiserum (1:4000 dilution). To determine whether VHA was expressed in the samecells as NKA or NHE3, two serial (mirror image) sections were obtained with the cut surfacesfacing each other. Each section was then individually reacted with the different antiserum.After being rinsed with PBS, these sections were incubated with a second antibody, biotiny-lated swine anti-rabbit immunoglobulin G (IgG) and peroxidase-conjugated avidin, using aVectastain ABC Kit (Vector Laboratories; www.vectorlabs.com) for 2 h at room temperature.After rinsing with PBS, immunolabelling for NKA, NHE3 or VHA was visualized with 3, 3′-diaminobenzidine solution (Sigma-Aldrich; www.sigmaaldrich.com) containing 0·01% H2O2.Control sections were treated with normal rabbit serum instead of the primary antibody. Allcontrol preparations were negative for immunestaining. Sections were counterstained withhaematoxylin.

The following antibodies were used in this study. Polyclonal antisera to NKA and VHAwere raised in rabbits against synthetic peptides corresponding to the highly conserved regionsof salmon NKA α-subunit (VTGVEEGRLIFDNLKKS) and bovine VHA (AEMPADSGY-PAYLGAR) (Ura et al., 1996; Hayashi et al., 2000), respectively. These two antibodies havebeen successfully used for epithelial cells of various vertebrates (Uchida et al., 2000; Konnoet al., 2007; Kaneko et al., 2008; Uchiyama et al., 2009, 2011). A rabbit polyclonal antibodyto tilapia NHE3 (TDTKQMNNDQFPPP) was obtained from T. Kaneko of Tokyo University(Watanabe et al., 2008).

W E S T E R N B L OT T I N GThe immunoreactivity of a polyclonal antibody to tilapia NHE3 was examined. Gill arches

were quickly removed from A. tetradactylus, and epithelial tissue was then scraped fromthe gill arches using a single-edged razor blade and collected in 2 ml ice-cold extractionbuffer with Protease Inhibitor Cocktail Set III (ProteoExtract Transmembrane Protein Extrac-tion Kit, Cosmo Bio; www.cosmobio.co.jp). Protein extraction was performed according tothe manufacturer’s instructions. The tissue was homogenized using an NS-310E homoge-nizer (Physcotorn, Microtec Co.; http://nition.com/) and centrifuged at 1000 g for 5 min at4◦ C. The pellet fraction was resuspended in ice-cold extraction buffer and then subjected tohigh-speed centrifugation at 16 000 g for 15 min at 4◦ C to obtain a crude cell membranefraction. Total protein in the samples was determined by the Bradford method (Bradford,1976). The specificity of immunoreactivity was confirmed by incubating the membrane withthe preimmune serum.

For Western analysis, protein samples were separated by 7·5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose mem-branes (Hybond-C ECL, GE Healthcare; www. gelifesciences.com). After blocking non-specific-binding sites with 5% non-fat dry milk in 100 mM Tris, 500 mM NaCl and 0·1%Tween-20 (TBST), membranes were incubated overnight at 4◦ C with a primary antiserum(1:4000 dilution) against NHE3. Immunodetection was performed by enhanced chemilumines-cence using the horseradish peroxidase-conjugated goat anti-rabbit antibody. The membraneswere washed and antibody–antigen complexes were visualized using the ECL detection sys-tem (GE Healthcare) on hyperfilms.

R E L AT I V E N U M B E R O F I M M U N O R E AC T I V E C E L L SI N F I L A M E N T S A N D L A M E L L A E

Relative number of immunopositive cells was estimated by light microscopy to comparedifferences in the number of immunoreactive cells (IRC) in filaments and lamellae among


2240 M . U C H I YA M A E T A L .

the three blennid species. For each fish (n = 3–5 for each species), serial sections of sec-ond gill arches were examined. Immunoreactive and non-immunoreactive epithelial cells onfive randomly selected gill filaments and 50 lamellae in each fish were counted under lightmicroscopy, and the ratio of IRCs to non-IRCs was calculated. In this study, to avoid biasfor the counts, cells were counted individually by two observers and the data were evaluated.

T R A N S M I S S I O N E L E C T RO N M I C RO S C O P YThe gills of the three species were rapidly excised, immediately immersion-fixed in a

solution of 2·5% glutaraldehyde and 4% paraformaldehyde in 0·1 M sodium cacodylate buffer,cut into small pieces and placed in fresh fixative for 6 h. Tissue pieces were washed twicewith 0·1 M sodium cacodylate buffer and post-fixed with 1% osmium tetroxide for 1 h. Then,the tissue pieces were washed with distilled water, dehydrated in ethanol and embedded inEpon. Thin sections were stained with methanolic uranyl acetate and alkaline lead citrate andexamined using a Hitachi H-7600 transmission electron microscope (TEM). Some semithin(1 μm) sections prepared by the methods described for transmission electron microscopy werestained with toluidine blue and observed by light microscopy.

S TAT I S T I C SData are presented as mean ± s.e. Data were analysed by the one-way ANOVA with the

Bonferroni post hoc test. Statistical analyses were performed using JMP version 9 software(SAS Institute Japan; www.jmp.com). The differences were considered statistically significantat P < 0·05.

RESULTS

G I L L S T RU C T U R E B Y S C A N N I N G E L E C T RO N M I C RO S C O P Y

Observations by the naked eye and SEM showed that four gill arches were presentin A. tetradactylus, P. tanegasimae and E. yaeyamaensis, each arch bearing twohemibranches comprising up to several dozen gill filaments. Scanning electron micro-graphs revealed marked differences in morphological appearances among the threespecies. In A. tetradactylus, the thick filaments of two adjacent hemibranches werearranged close to each other and alternately in a line on the gill arch (Fig. 1). Fil-aments supported by cartilaginous gill rods were long (1101 ± 17 μm) and thick(63 ± 2 and 78 ± 4 μm on the top and base, respectively) and lamellae were highlyconvoluted and tall (147 ± 5 μm) [Fig. 2(a)]. In P. tanegasimae, filament length andlamella height were 1114 ± 38 and 99 ± 4 μm, respectively. Many short and highlyconvoluted lamellae were regularly arranged in P. tanegasimae [Fig. 2(b)]. In E.yaeyamaensis, filament length and lamella height were 732 ± 54 and 107 ± 4 μm,respectively. In comparison with the filaments and lamellae of A. tetradactylus, thin-ner filaments (53 ± 1 and 78 ± 4 μm on the top and base, respectively) and shorterand straight lamellae were observed in E. yaeyamaensis [Fig. 2(c)]. As there wasan apparent size gradient in these species, gill morphometric variables were cal-culated based on the total body length. There was a significant difference in thenumber of filaments among the three species. Length of centrally located filamentsin the gills of A. tetradactylus was slightly but not significantly shorter than thatof E. yaeyamaensis. There were significant differences in the width of filaments onthe top and base among the three species. In A. tetradactylus, the width of fila-ment on the top was thicker than that in E. yaeyamaensis. Lamella height in the



Filament

Gill arch

b

da

Lamella

c

Fig. 1. Schematic drawing of the arrangement of gill filaments in Andamia tetradactylus. Adjacent hemifila-ments are arranged close to each other and alternately on the gill arch. a, length of filament; b, width inthe basal part of filament; c, width in the top part of filament; d, lamella height.

gills of A. tetradactylus and P. tanegasimae was significantly shorter than that of E.yaeyamaensis. Morphometric data are given in Table I.

The distribution of MCs in the gill filaments was examined by the PAS method(Fig. 3). Large spherical or ovoid MCs were most common at the extreme edgesof the filament as single isolated cells [Fig. 3(a), (c), (e)]. Vase-shaped or sphericalMCs were also abundant in the filament epithelium towards the outer surface in A.tetradactylus [Fig. 3(b)] and P. tanegasimae [Fig. 3(d)], but only a few cells werepresent in the filament epithelium of E. yaeyamaensis [Fig. 3(f)].

W E S T E R N B L OT A NA LY S I S

In Western blot analysis, tilapia NHE3 antibody recognized a single protein bandwith a molecular mass of c. 95 kDa (Fig. 4). No immunoreactive band was detectedin the control.

(a) (b) (c)

Fig. 2. Scanning electron micrograph of gill filaments and lamellae of three blenny species (a) Andamiatetradactylus, (b) Praealticus tanegasimae and (c) Ecsenius yaeyamaensis. In A. tetradactylus, the gillfilaments are thick and arranged close to each other and the bent lamellae do not fuse together. Thegills of P. tanegasimae have thick filaments and short lamellae. The gills of E. yaeyamaensis have thinfilaments and straight lamellae. Scale bars, 500 μm.


2242 M . U C H I YA M A E T A L .

Tab

leI.

Mor

phom

etry

offil

amen

tsan

dla

mel

lae

ofse

cond

and

thir

dgi

llar

ches

inth

ree

spec

ies

ofB

lenn

iidae

Spec

ies

LT

(cm

)

Num

ber

offil

amen

tsin

agi

llar

ch(n

cm−1

LT)

Len

gth

ofce

ntra

llylo

cate

dfil

amen

ts*

(μm

cm−1

LT)

Wid

thof

filam

ents

:to

p*(μ

mm

m−1

LT),

base

*(μ

mcm

−1L

T)

Num

ber

ofla

mel

lain

afil

amen

t(r

ange

,n

cm−1

LT)

Lam

ella

heig

ht*

(μm

cm−1

LT)

And

amia

tetr

adac

tylu

s7·9

±0·5

(5)a

3·0±

0·1#

(9)b

137·5

±2·1

(6)c

7·7±

0·3#

(5)c

10–

12(1

0)d

18·4

±0·6

##(1

4)d

9·8±

0·5##

(5)

Pra

ealt

icus

tane

gasi

mae

7·0±

0·1(3

)a4·7

±0·1

†(5

)b15

9·2±

5·4(6

)c6·0

±0·7

†(5

)c13

–17

(10)

d14

·2±

0·6††

(20)

d

7·5±

0·6††

(5)c

Ecs

eniu

sya

eyam

aens

is5·1

±0·2

††#

(3)a

4·5±

0·2†

(3)b

146·5

±10

·0(6

)c3·8

±0·2

††##

(5)c

10–

11(1

0)d

21·6

±0·8

††##

(26)

d

10·6

±0·2

##(5

)c

Dat

aar

epr

esen

ted

asm

ean

±s.

e.D

ata

are

calc

ulat

edas

num

ber

(n)

and

leng

th(μ

m)

per

tota

lle

ngth

(LT

,cm

).a N

umbe

rof

fish

used

.bN

umbe

rof

gill

arch

esm

easu

red

inea

chfis

hsp

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umbe

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filam

ents

mea

sure

din

each

fish.

dN

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h.*S

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†,††

Sign

ifica

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#,##

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imae

,re

spec

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y.



(a)

(b)

(c)

(d)

(e)

(f)

Fig. 3. Periodic acid-Schiff (PAS) staining of gills of (a), (b) Andamia tetradactylus, (c), (d) Praealticustanegasimae and (e), (f) Ecsenius yaeyamaensis. (a), (c), (e) Efferent or leading edge and (b), (d),(f) interlamellar region of the filaments are visible in the three species. PAS-positive (dark) cellswere mainly observed in apical and interlamellar regions of the gill filaments in A. tetradactylus andP. tanegasimae. PAS-positive cells were sparsely present in these regions of the gill filaments in E.yaeyamaensis. PAS-positive cells in the gills of E. yaeyamaensis were smaller and fewer in number thanthose in the gills of A. tetradactylus and P. tanegasimae. Scale bars, 50 μm.

I M M U N O L O C A L I Z AT I O N O F N K A , V H A A N D N H E 3

Proteins homologous to NKA, VHA and NHE3 were present in the gill epithe-lium of the three species. There were marked differences in the immunohistochemicallocalization and distribution of cells immunoreactive for the three transporters in theepithelia of the gills, filaments and lamellae of the three species. The immunohisto-chemical results are summarized in Table II.

NKA immunoreactivity was observed in the ionocytes located at the base ofthe lamellae in the three species [Fig. 5(a), (d), (g)]. Significant differences in thelocation, abundance and size of gill NKA-IRCs were observed among the threespecies. Density of distribution of NKA-IRCs on the filaments and basal cells ofeach lamella were the highest in A. tetradactylus [Fig. 5(a)], high in P. tanegasimae[Fig. 5(d)] and low in E. yaeyamaensis [Fig. 5(g)]. NKA-IRCs, which were largerthan NKA-IRCs in the other cell types studied and had an apical pit, were observedon the filaments and in the basal cell population in A. tetradactylus [Fig. 5(a)]. InP. tanegasimae, large NKA-IRCs were observed in the filaments and at the baseof the lamellae [Fig. 5(d)]. In E. yaeyamaensis, NKA-IRCs were located in theinterlamellar region of the filaments [Fig. 5(g)].

In A. tetradactylus, VHA-IRCs were rarely observed on the central part of thefilament [Fig. 5(b)]. In P. tanegasimae, VHA-IRCs on the filaments and lamellaewere scattered but slightly more than those on the gill filaments of A. tetradacty-lus [Fig. 5(b), (e)]. In the gills of E. yaeyamaensis, both NKA- and VHA-IRCs


2244 M . U C H I YA M A E T A L .

120

95

50

Fig. 4. Western blotting of Andamia tetradactylus gill homogenates using polyclonal antibody raised againsta synthetic peptide of tilapia Na+/H+ exchanger 3 (NHE3). A distinct band corresponding to NHE3(96 kDa) is observed. Positions of molecular mass markers are shown on the left.

were located in the interlamellar region of the filaments [Fig. 5(g), (h)]. NKA-IRCswere observed more frequently than VHA- and NHE3-IRCs on the filaments ofA. tetradactylus [Fig. 5(a)–(c)] and P. tanegasimae [Fig. 5(d)–(f)]. NKA-IRCs wereobserved in numbers similar to those of VHA- and NHE3-IRCs in the interlamellarregion of the filaments of E. yaeyamaensis [Fig. 5(g)–(i)]. Analyses using serial

Table II. Presence of Na+,K+-ATPase, vacuolar H+-ATPase and Na+/H+ exchanger3-immunoreactive cells of filaments and lamellae in the gills of three species of Blenniidae

Species Na+,K+ -ATPase Vacuolar H+ -ATPase Na+/H+ exchanger 3

Andamia tetradactylusFilament +++ + ++Base of lamella +++ ± ++Lamella − − −Praealticus tanegasimaeFilament ++ + ++Base of lamella ++ + ++Lamella − ± ±Ecsenius yaeyamaensisFilament + ++ ++Base of lamella + + ++Lamella − ± ±Percentage of immunoreactive epithelial cells in the gills: + + +, >45% of cells; ++, 45–15% of cells;+, 14–5% of cells; ±, < 5% of cells; −, no immunoreactive cell.



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 5. Na+,K+-ATPase (NKA)-, Na+/H+ exchanger (NHE3)- and vacuolar-type H+-ATPase (VHA)-immunoreactive cells (IRC) on the gill filaments of (a)–(c) Andamia tetradactylus, (d)–(f) Praealticustanegasimae and (g)–(i) Ecsenius yaeyamaensis. (a) In the gills of A. tetradactylus, NKA-immunoreactive mitochondrion-rich cells (MRC) were localized at the base of the lamellae and onfilaments. (b) A very few VHA-immunoreactive MRCs were located sparsely in the stalk region of thefilament. (c) NHE3-IRCs were located on the filaments. The cell membrane of apical pits ( ) of presumedMRCs was NHE3 immunoreactive. (d) In the gills of P. tanegasimae, NKA-immunoreactive MRCs weredistributed in the interlamellar regions of the filaments. (e), (f) VHA- and NHE3-IRCs ( ) were scat-tered on the lamellae and filaments. In the gill of E. yaeyamaensis, lamellae and filaments were thinand straight, respectively. (g) NKA-immunoreactive MRCs and (h) VHA- or (i) NHE3-immunoreactive( ) MRCs were observed between the lamellae on gill filaments. Analyses using serial (mirror image)sections of gills with antibodies against NKA and VHA indicated that only a few MRCs might be colo-calized on the filaments in the three blenny species: (a), (b) A. tetradactylus, (d), (e) P. tanegasimae and(g), (h) E. yaeyamaensis. Scale bars: (a), (b), (d), (e), (g) and (h), 20 μm; (c), (f) and (i), 50 μm.

(mirror image) sections of the gills with antibodies against NKA and VHA indicatedthat both NKA and VHA immunoreactivity were colocalized in only a few pre-sumed MRCs on the filaments of A. tetradactylus [Fig. 5(a), (b)], P. tanegasimae[Fig. 5(d), (e)] and E. yaeyamaensis [Fig. 5(g), (h)].

Immunohistochemistry with a heterologous antiserum to NHE3 showed NHE3immunoreactivity in the apical membrane of presumed MRCs with an apical pitand the basal membrane on the filaments of A. tetradactylus [Fig. 5(c)]. In thegills of P. tanegasimae and E. yaeyamaensis, NHE3-IRCs were detected on thecells of both the lamellae and the interlamellar region of the filaments [Fig. 5(f),(i)]. The frequency of NHE3-IRCs was higher than that of VHA-IRCs in the fila-ments of A. tetradactylus and P. tanegasimae but not in those of E. yaeyamaensis[Fig. 5(c), (f), (i)].


2246 M . U C H I YA M A E T A L .

G I L L U LT R A S T RU C T U R E

The gill epithelium is mainly composed of PVCs, MRCs, MCs and unidentifiedcells in the three species (Fig. 6); these cells are also observed in the gills of teleostsgenerally. PVCs are thin squamous or cuboidal cells that maintain contact with theirsurroundings through their apical surface [Fig. 6(a), (g), (i)]. These cells possessmany subapical cytoplasmic granules and vesicles. MRCs are ovoid large cells andhave high densities of mitochondria in the cytoplasm, with an extensive internaltubular system that is actually part of the basolateral membrane. MRCs occur singlyon the lamellae and in groups on the interlamellar region and multilayered filaments[Fig. 6(a)–(c), (e)]. These cells are surrounded by PVCs and accessory cells, andapical pits are present in multicellular complexes with other MRCs and accessorycells [Fig. 6(c)–(e)]. MRCs are of two types: electron-opaque and electron-densecells [Fig. 6(d)–(f), (i), (j)]. In this study, it could not be clarified whether these twotypes of MRCs were two morphological types of MRCs or different developmentalor degenerative stages of the same cell. MRCs were partially covered by PVCs.Large MCs were located on filaments but only a few were found on the lamellae inthe gills [Fig. 6(a), (i)].

DISCUSSION

Extant amphibious fishes leave the water for various reasons associated with degra-dation of their habitat or biotic factors (Sayer & Davenport, 1991). Their ability toadapt to air exposure led to the development and enhancement of new functions forfish gills. In this study, the general structure and localization of ion transporters inthe gills were observed and compared among blennid species that inhabit differentintertidal niches in vertical zones of rocky shores in subtropical seas. The resultsindicate that gill structure and distributions of NKA-, VHA- and NHE3-IRCs on thefilaments and lamellae differ among the three species.

G I L L S T RU C T U R E

The gills of the subtidal species E. yaeyamaensis have features similar to thoseof teleost. Two rows of gill filaments, each row comprising a hemibranch, radiateposteroventrally from the four pairs of gill arches (Wilson & Laurent, 2002). MCsstained by the PAS method were sparsely distributed on the tip and interlamellarregions of the filaments. The gills characteristically consisted of thick filaments andshort lamellae in P. tanegasimae inhabiting tide pools, which is similar to other air-breathing fishes that developed structural modifications of the gills to prevent lamellarcollapse during air exposure (Martin, 1995; Wilson & Laurent, 2002). Potentiallyhigh rates of mucus production by the gills indicate that mucus functions either asan antidesiccant or as an interlamellar lubricant on collapse (Low et al., 1988).In the supratidal species A. tetradactylus, the risk of desiccation in air is high.Their gills showed special arrangements of filaments on adjacent hemibranches ofthe gill arches; the filaments were thick and the lamellae were greatly convoluted.This type of gill structure may indicate that A. tetradactylus carried sea water tomaintain its body fluids in air and distends the branchial cavity to trap a quantity ofwater with air in the opercula and gill chambers. In this study, the lamella height



(a)

PVC

PVC

MC

MRC

MRC

MRC

MRC1

MRC2

AC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

MRC

(b)

(c) (d)

(g) (i)

( j)

(h)

(e)(f)

Fig. 6. Ultrastructure of the gills of the three blenny species: (a)–(f) Andamia tetradactylus, (g), (h) Praealticustanegasimae and (i), (j) Ecsenius yaeyamaensis. (a) A single layer of pavement cells (PVC) and cellpopulations of mitochondrion-rich cells (MRC) were observed on the apical and middle parts of thelamellae and on the base of the lamellae, respectively. (b)–(d) MRCs were located on multilayered fila-ments and under interlamellae. MRCs were surrounded by PVCs and accessory cells. (c) Large mucouscells were located close to the outer surface of the filaments but very few were found on the lamel-lae. (e), (f) Higher magnifications of MRCs indicate (e) electron-opaque MRC1 and (f) electron-denseMRC2. (g), (i) MRCs were moderately located on multilayered filaments and under interlamellae. Acces-sory mitochondrion-rich cells are less prominent in the gills of P. tanegasimae and E. yaeyamaensis.(h) Higher magnifications of the apical pit of MRC. (j) Higher magnifications of MRCs in the gills ofE. yaeyamaensis indicate electron-opaque and electron-dense MRCs as well as (e) and (f) in the gillsof A. tetradactylus. AC, accessory mitochondrion-rich cell; MC, mucous cell. *Apical pit. Scale bars:(a)–(d), (g) and (i), 10 μm; (e), (f), (h) and (j), 1 μm.


2248 M . U C H I YA M A E T A L .

LT−1 in A. tetradactylus was significantly shorter than in E. yaeyamaensis. It hasbeen reported that the number and length of gill filaments and gill surface area arereduced in several species of mudskipper Gobiidae (Low et al., 1988). As the lamellaheight per LT seems to relate closely to the gill surface area, these characteristics ofgill structure seem to be also applicable in the rockskipper A. tetradactylus, whichinhabits in supratidal zone where the wave spray falls on the rock. According tofield and laboratory observations, A. tetradactylus suspends opercular movementsand distends the brachial cavity when it moves onto land (N. Shimizu, pers. data).Thus, it is possible that A. tetradactylus carries water in the buccopharynx duringterrestrial sojourns. This system reduces the risk of desiccation and is convenient forboth aerial respiration and osmoregulation in A. tetradactylus in the supratidal zone.

N A C L S E C R E T I O N

To compensate for water loss, marine teleosts drink sea water and actively secretesalt through the gills as well as the kidneys while they are in the sea. MRCs in thegills are responsible for salt secretion in seawater fish, and the cellular mechanisms ofsalt secretion have been documented (Evans et al., 2005; Kaneko et al., 2008; Evans& Claiborne, 2009). Numerous MRCs were present in the gills of the three blennids,forming multicellular complexes with other MRCs and accessory cells. The multi-cellular complexes formed an apical crypt shared by the apical membranes of MRCsand accessory MRCs. These gill characteristics are similar to those of most seawaterteleosts (Wilson & Laurent, 2002; Evans et al., 2005). In addition to these aspects,the MRC population was a prominent feature of the gill filaments of A. tetradactylus.The NKA is a universal membrane-bound enzyme that provides the driving forcefor many transport systems in various osmoregulatory epithelia, including fish gills(Evans et al., 2005; Evans & Claiborne, 2009; Hwang et al., 2011). Immunocyto-chemical studies have demonstrated that NKA was mainly located on MRCs of gillepithelia in teleosts (Uchida et al., 2000; Hwang & Lee, 2007; Kaneko et al., 2008).Various different distributions of NKA-immunoreactive MRCs in the gills of eury-haline teleosts have been documented. MRCs on the filament were usually roundedand located at the base of the lamella. In contrast, MRCs on the lamellae were flatand appeared as protrusions above the lamellar surface (Kaneko et al., 2008). Inthe three blennid species studied, there seemed to be a relationship between habitatand the number and size of NKA-immunoreactive MRCs. Multiple cell populationsand large MRCs at the base of the lamellae were observed on the filaments ofA. tetradactylus and P. tanegasimae, but not on those of E. yaeyamaensis. The gillsof A. tetradactylus showed a marked tendency to develop these special features. Ithas been observed that marine intertidal fishes maintained out of water showed asignificant loss of body mass and increases in haematocrit and plasma electrolytelevels (Marusic et al., 1981). Andamia tetradactylus may need to excrete excessNaCl through NKA-IRCs because the blennid species inhabiting the rocky suprati-dal zone feeds on marine algae with high salt content during the daytime, underdirect sunlight at high temperature. On the contrary, NKA-IRCs of E. yaeyamaensisinhabiting subtidal regions were located individually in interlamellar regions of thefilaments. The distribution and density of NKA-IRCs in both P. tanegasimae inhab-iting intertidal level and in tidepools and A. tetradactylus inhabiting supratidal zonewere somewhat similar.



AC I D – BA S E R E G U L AT I O N

In marine teleosts, acid–base regulation under elevated CO2 levels leads to a tran-sient decline in extracellular and intracellular pH levels (Toews et al., 1983; Evanset al., 2005; Evans & Claiborne, 2009). Two mechanisms have emerged explainingthe net release of H+ from the organism through the gills: one involves VHA andthe other involves NHEs. In this study, both VHA- and NHE-3-immunoreactive cellswere observed in the gills of A. tetradactylus, P. tanegasimae and E. yaeyamaensis.

These immunohistochemical results demonstrated three different cell populationsin all three species: NKA-IRCs, NKA- and VHA-IRCs and VHA-IRCs in the fila-ments. These different cell populations have been reported in the gills of freshwaterair-breathing fishes (Huang et al., 2010). In freshwater fishes, apical electrogenic H+excretion is driven by VHA in combination with the passive uptake of Na+ throughepithelial sodium channels. Many studies in freshwater fishes have shown that VHAactivity increases at lower ionic strengths (Lin et al., 1994; Evans et al., 2005). Incontrast, VHA may play a lesser role in ion regulation in seawater teleosts. TheVHA immunoreactivity was observed mainly in the cytoplasm of a few branchialcells on the filaments of A. tetradactylus and P. tanegasimae. In E. yaeyamaensisinhabiting subtidal zone, VHA-IRCs were located in numbers similar to those ofNKA-IRCs in the interlamellar region of the filaments. The ratio of VHA-IRCsv. non-VHA-IRCs in the gills of E. yaeyamaensis was higher than those of A.tetradactylus and P. tanegasimae. As pH in ambient water of subtidal zone is rela-tively stable in comparison with that of tidepools, a main function of VHA-IRCs maybe other than H+excretion in the gills of E. yaeyamaensis. A study by Tresguerrerset al. (2005) showed that VHAs are localized mainly to the basolateral membrane inbase (NaHCO3)-infused dogfish Squalus acanthias L. 1758 and suggested that theyare involved in net base secretion. In a marine teleost Myoxocephalus octodecem-spinosus (Mitchill 1814), VHA immunoreactivity was also observed at the base ofthe lamellae; it might be involved in bicarbonate excretion rather than H+excretion(Catches et al., 2006). Furthermore, Katoh et al. (2003) suggested that VHA-IRCsare involved in the Na+ and Cl− absorption across the basolateral membrane in thegills of freshwater killifish Fundulus heteroclitus (L. 1766).

On the other hand, it is generally accepted that the primary gill acid secretionmechanism in marine fishes involves apical Na+/H+ exchange through specializedgill cells (Claiborne et al., 2002; Perry & Gilmour, 2006). Several studies haverevealed the presence of proteins in the gill that are similar to mammalian NHE3.NHE3-like proteins have been immunohistochemically detected in teleosts (Choeet al., 2005; Edwards et al., 2005) and several elasmobranchs (Edwards et al., 2002).Watanabe et al. (2008) suggested that the presence of NHE3-expressing MRCs ingills is related to acid–base regulation (H+ secretion) in seawater-adapted tilapiaOreochromis mossambicus (Peters 1852). In this study, NHE3-immunoreactive cellswere observed in the gill filaments of all three blennids. In rocky shore habitats,the subtidal zone is the most stable in water oxygen concentration, pH and salinity.On the contrary, large and rapid oxygen, carbon dioxide and pH changes occurin shallow-isolated tide pools in the intertidal zone when both animals and plantpopulations are present. The pH and ion composition of the water may have aprofound effect on fishes’ ability to regulate osmotic and acid–base disturbances viabranchial mechanisms. Some differences were observed in the immunohistochemicallocalization and distribution of the NHE3-IRCs in the epithelia of the gills, filaments


2250 M . U C H I YA M A E T A L .

and lamellae of the three blennid species. Further study is needed to interpret thedistribution and physiological significance of VHA- and NHE 3-IRCs in the gills ofthe three blennid species under natural conditions.

In summary, the structure and distribution of ion transporters in gills were exam-ined in relation to osmoregulation and acid–base regulation in the three blennidspecies inhabiting different parts of the vertical zonation of rocky intertidal areas.The supratidal rockskipper A. tetradactylus had a broad opercula space and well-developed lamellae and filaments, with no reduction in the number and size of gillfilaments. Many MCs and NKA-immunoreactive MRCs and a few VHA- and moreNHE3-immunoreactive MRCs were observed at the base of the lamellae and gillfilaments. In the gills of A. tetradactylus, numerous NKA-immunoreactive MRCsexcreted excess NaCl, and NHE3 was probably involved in an acid–base regulatorymechanism that secretes H+ to prevent respiratory acidosis. During the adaptationof A. tetradactylus to the supratidal zone, the gills of this species seem to havedeveloped primarily an osmoregulatory function in addition to respiration. MRCs inthe gills of the intertidal species P. tanegasimae appeared to have a role in ionicregulation as well as in acid–base regulation. In the subtidal species E. yaeyamaen-sis, VHA-IRCs were more prominent than NHE3-IRCs. The results of this studysuggested that the structure and distribution of ion transporters in the gills of thethree blennid species studied are associated with their habitat and lifestyle.

This study was supported in part by Grant-in-Aid for Scientific Research from the Ministryof Education, Culture, Sports, Science and Technology of Japan to M.U. The authors areindebted to M. Tsuboi of Hiroshima University for collecting fishes. The authors thank I.Shirasaki of Toyama University for their technical assistance as well as T. Kaneko of TokyoUniversity for providing tilapia NHE3 antibody.

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Documents

Structures and immunolocalization of Na+, K+-ATPase, Na+/H+ exchanger 3 and vacuolar-type H+-ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas