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Acta histochemica 112 (2010) 251—258

0065-1281/$ - sdoi:10.1016/j.

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Localization of aquaporin 1 and 3 in the gills of therainbow wrasse Coris julis

Elvira Brunellia,�, Angela Maucerib, Fasulo Salvatoreb, Alessia Giannettob,Maria Maisanob, Sandro Tripepia

aDepartment of Ecology, University of Calabria, Via P. Bucci, 4B I-87036 Rende, Cosenza, ItalybDepartment of Animal Biology and Marine Ecology, University of Messina, Contrada Sperone 31 I-98166 S. Agata,Messina, Italy

Received 17 September 2008; received in revised form 6 November 2008; accepted 19 November 2008

KEYWORDSGills;Na+/K+ ATPase;AQPs;Coris julis;Confocal microscopy

ee front matter & 2009acthis.2008.11.030

ing author. Tel.: +39 984986.ess: brunelli@unical.it

SummaryUltrastructural and immunohistochemical studies were conducted on the gillepithelium of the Mediterranean rainbow wrasse (Coris julis). We analysed theimmunolocalisation of aquaporin 3 (AQP3) and aquaporin 1 (AQP1) in the gills usingconfocal microscopy. The ultrastructural features of the gill were investigated usingtransmission and scanning electron microscopy. The C. julis gill apparatus showedstructural characteristics typical for Teleostei. Immunolocalization revealeddifferential localization of AQP1 and AQP3 in the gill epithelium. Doubleimmunolabelling for Na+/K+ ATPase with AQP1or AQP3 revealed that AQP1 islocalised in chloride cells, whereas AQP3 is localized in both the chloride cells andthe accessory cells. This result suggests an active role of these cells in water/glycerol transport in saltwater fish.& 2009 Elsevier GmbH. All rights reserved.

Introduction

Water movement across the cell membrane isregulated by aquaporins (AQPs) or major intrinsicproteins (MIPs), which constitute a family of highlyconserved transmembrane proteins (Agre, 1997;Borgnia et al., 1999). Aquaporins are expressed in a

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492975;

(E. Brunelli).

wide variety of tissues in plants, microorganismsand vertebrates and are required to transportwater and non-ionic compounds (Agre et al.,1993; Connolly et al., 1998; Heymann and Engel,1999). Different subgroups of aquaporins have beendescribed on the basis of transport specificity. Forexample, members of the water channel or channelforming integral protein (CHIP) group functionexclusively as water channels, whereas membersof the water/glycerol channels or glycerol intrinsicproteins (GLPs) are permeable to water, glycerol

rved.

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and urea (Zheng and Bollinger Bollag, 2003; Borgniaet al., 1999; Hatakeyama et al., 2001; Verkman andMitra, 2000; Shapiguzov, 2004). AQP1 was the firstmember of this family to be found in erythrocytesand renal tubules (Preston and Agre, 1991) and isthought to be highly selective for water, whereasAQP3 belongs to the aquaglyceroporin group and isexpressed in most types of epithelial cells of theurinary, digestive and respiratory tracts, as well asin the epidermis.

In teleost fish, the role of AQPs in the gill remainscontentious because of contradictory results, whichare attributable to either different organ-tissuepreparations and/or possible species specificities(Cutler et al., 2007; Giffard-Mena et al., 2007;Hirata et al., 2003; Lignot et al., 2002; Tse et al.,2006). Water transport mechanisms in the gills havebeen investigated in both freshwater and euryha-line fish (Cutler and Cramb, 2002; Giffard-Menaet al., 2007; Hirata et al., 2003; Lignot et al., 2002;Watanabe et al., 2005), but little is known aboutthe role of aquaporins in marine non-euryhalinefish. An integrated ion and water transport me-chanism in the gills is important to compensate forosmotic water loss and dehydration in marineteleosts.

The gill apparatus is one of the major osmor-egulatory organs in fish. Thus, in addition to theirrespiratory function, the gills represent an impor-tant site for osmotic and acid–base regulation(Claiborne, 1997; Evans et al., 1999; Laurent,1984). These different roles explain the structuralcomplexity of this organ (Evans et al., 2005; Wilsonand Laurent, 2002). The gill epithelium comprisesspecialised ion-transporting cells: the chloride cells(CCs) (Evans, 2002; Foskett and Scheffey, 1982;Karnaky, 1986), which are also called mitochondria-rich cells. CCs of marine fish and those of fresh-water fish differ in their function and morphology(Hirose et al., 2003; Laurent, 1984; Perry, 1997).Pisam et al. (1987) described two subtypes ofCCs – the b CCs and the a CCs – that are found infreshwater and seawater fish, respectively (for areview, see Pisam and Rambourg, 1991).

The structure of fish gills has been the subject ofseveral studies in different species (for recentreviews see Wilson and Laurent, 2002), but only afew studies have focused on marine species. In thework reported herein, we conducted preliminaryhistological and ultrastructural analyses to providedetailed anatomical information about the gillapparatus of the Mediterranean teleost Coris julis.We hypothesized that the localization of twomembers of the AQP family, an aquaporin and anaquaglyceroporin, would prove to be useful inassessing the role of this family of proteins in

maintaining the water balance of marine fish and indetermining the mechanisms of regulation andosmoreception in the gills.

Materials and methods

Animal collection and maintenance

We collected samples of wrasse, C. julis, fromthe Tyrrhenian coast (S. Lucido, Italy) using baitedtraps. Captured fish were kept alive in an aeratedwater tank, transferred to the laboratory, andplaced in two 150 l aquaria filled with naturalseawater and equipped with filter and oxygenationsystems. The animals were acclimatized for 8 daysat a temperature of 18–24 1C under a naturalphotoperiod (light/dark) cycle and were fed dailywith a commercial fish food (Tetramin). Waterquality parameters in the aquaria were determinedbefore and during the experimental period: salinity(35%), density (1.027–1.028 g/cm3), temperature(18–24 1 1C), nitrite and nitrate concentrations,dissolved oxygen (8.0–8.6mg/l), hardness (100mgCO3Ca/l) and the absence of heavy metals.

Sampling

Adult sea wrasses (n ¼ 8; weight 11–15 g; lengthranging from 12 to 16 cm) were randomly selectedas experimental animals. The animals were anaes-thetised with 2–4 g/l tricaine methane sulphonate(MS 222, Sandoz, Sigma, St. Louis, MO) and killed byspinal cord transection. Animal manipulation wasperformed according to Ethical Committee recom-mendations and under the supervision of authorisedinvestigators.

Light and electron microscopy

Tissue samples were fixed in 4% paraformalde-hyde (Electron Microscopy Sciences) in phosphate-buffered saline (PBS, pH 7.1, Electron MicroscopySciences) for 24 h, and then were postfixed in 1%osmium tetroxide (Electron Microscopy Sciences) inthe same buffer. The samples used for transmissionelectron microscopy (TEM) observation were dehy-drated in graded ethanol, soaked in propyleneoxide and embedded in Epon–Araldite. Semi-thinsections (1–2 mm) were stained with Grimley’s dyes(toluidine blue, malachite green and acid fuchsin)and observed using a light microscope (Leitz DialuxEB 20). Ultrathin sections were stained withuranyl acetate in ethanol and lead citrate, coatedin Edwards EM 400 and then examined and

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photographed with a Zeiss EM 900 electron micro-scope. For scanning electron microscope (SEM)observation, dehydrated specimens were subjectedto the progressive substitution of ethanol withhexamethyldisilazane (HMDS, Electron MicroscopySciences), removed by complete evaporation ac-cording to the methods reported by Nation (1983),then coated with gold in an Emitech K550.

Confocal microscopy

After dehydration in ethanol and clarification inxylene, the fixed specimens were embedded inparaffin wax with a mean fusion point of 56 1C. Ten-micrometer-thick tissue sections were cut, mountedon slides and processed according to the indirectimmunofluorescence technique (Coons et al., 1955).

We used the following primary antibodies in thisstudy: a monoclonal antibody (mAb) raised in mouseagainst Na+/K+ ATPase at working dilutions of 1:100(IgGa5; Developmental Studies Hybridoma Bank,Department of Biological Sciences, The Universityof Iowa, Iowa City, IA, USA); a polyclonal antibody(pAb) against the water channel aquaporin-3 (anti-water channel AQP3 developed in rabbit, IgGfraction of antiserum, diluted 1:100, Sigma-Aldrich);and a pAb against the water channel aquaporin-1(anti-water channel AQP1, developed in rabbit, IgGfraction of antiserum, diluted 1:100, Sigma-Aldrich).

The protocol entailed incubating with the firstprimary antibody (overnight at 4 1C), washing withphosphate buffered saline (PBS), incubating withthe labelled secondary antibody for 30min (TRITCconjugated anti-mouse IgG, developed in goat, andFITC conjugated anti-rabbit IgG, developed insheep, both diluted 1:50, Sigma-Aldrich), andwashing with PBS; the same procedure was re-peated for the second primary antibody. Incuba-tions with primary antibodies lasted for 4 h at roomtemperature, whereas incubations with secondaryantibodies lasted for 30min at room temperature.To check the specificity of the immunolabelling(negative control), the primary antiserum wassubstituted with non-immune goat serum at adilution of 1:200 in PBS. The sections were analysedusing a Leica TCS SP2 confocal laser scanningmicroscope (LSM).

Results

Morphological and ultrastructuralobservations

Four gill arches support the gill apparatus ofC. julis, as in other Teleostei; from each branchial

arch arises a double series of filaments givinginsertion to two series of lamellae (Figure 1a and b).In both filament and lamellae, the surface layerconsists mainly of pavement cells (PVCs), whichrepresent the more abundant cell type. Furtherenlargement revealed the irregular polygonalshape of PVCs and the system of superficialconcentric microridges; scattered among the pave-ment cells of the filament were the openings of theunderlying cells on the surface (Figure 1c). Histo-logical observations showed that the gill filament ismade up of a connective support and is vascular-ized by afferent and efferent arteries (Figure 1d).The multilayered epithelium that covers the gillfilament (primary epithelium) consists of basal cellsmucous cells (GCs), CCs, along with accessory cells,and PVCs. GCs occurred along the filament margin,whereas CCs were distributed in clusters close tothe onset of lamellae and in the filament inter-lamellar region (Figure 1d).

The ultrastructural features of epithelialcells were investigated using electron microscopy(Figure 1e). Both CCs and GCs are partially coveredby PVCs. Within a section, GCs are identifiable bytheir round-shape and their light cytoplasm, whichis filled with a large number of electronucleargranules. The CCs (also called mitochondria-richcells) have a typical apical crypt and are char-acterized by numerous mitochondria and an ex-tensive tubulo-vesicular system in their cytoplasm.The more internal layer consists of undifferentiatedbasal cells. A thin secondary epithelium covers therespiratory lamellae and consists of two layers: aninner layer of undifferentiated basal cells and anexternal layer of PVCs (Figure 1f).

Immunohistochemistry

Immunoreactivity for the AQP1 and Na+/K+

ATPase completely coincided: immunopositive cellswere regularly distributed along the main filamentand in the interlamellar areas but were not found inthe lamellar epithelium (Figure 2a–c). At highmagnification (Figure 2d) immunopositive cells,identified as CCs by colocalization with Na+/K+

ATPase, were easily recognizable by their shape andby their large dimension. AQP3 distribution exhib-ited a different pattern (Figure 3a–c); largeimmunopositive cells, identified as CCs by coloca-lization with Na+/K+ ATPase, were present along thefilament epithelium and in the interlamellar region.In addition, immunopositivity was also present insmall cells surrounding chloride cells (Figure 3a and b).Under higher magnification, it is possible todistinguish the AQP3 labelling of the putative

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Figure 1. Coris julis gill morphology. (a) Scanning electron micrograph of C. julis gill arch (ga). f ¼ gill filaments;sl ¼ secondary lamellae; bar ¼ 180 mm; (b) detail of a filament (f) showing a series of lamellae (sl); bar ¼ 25 mm; (c)scanning electron micrograph of the primary epithelium surface. Note the complicated system of concentricmicroridges on the pavement cell (PVC) surface and the apical crypt of chloride cells (arrows); bar ¼ 6mm; (d) alongitudinal section through a gill filament observed by light microscopy; note the chloride cells (*) and the mucous cell(arrows); bar ¼ 20 mm; (e) electron micrographs of primary epithelium showing pavement cells (pvc) with numerousmicroridges, a round-shaped mucous cell (GC) and chloride cells (CC), along with accessory cell (ac). CCs containnumerous mitochondria and a well-developed tubular system in the cytoplasm; note the apical crypt (*); bar ¼ 5mm; (f)transmission electron micrograph of a cross-section through a respiratory lamella. The external layer made up bypavement cells covers the underlying basal cells (bc); note the pillar cell (pc) interposed among blood vessels (c);bar ¼ 3.5 mm.

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accessory cells (Figure 3d). Adjacent control sec-tions incubated without the primary antibodyshowed no immunoreactivity (not shown).

Discussion

The structural characteristics of C. julis gillepithelium were typical for Teleostei (Laurent,1984; Wilson and Laurent, 2002). Morphologicalanalysis revealed the presence of four main cellulartypes: basal cells, pavement cells, goblet cellsand chloride cells. We identified only one type ofCC – the aCC – which is typical of saltwaterteleosts. This aCC always has an apical crypt andwas accompanied by accessory cells. Na+K+ ATPase,a key enzyme in ion transport, was used as markerfor CCs (McCormick, 1995; Watanabe et al., 2005)and showed the typical distribution previouslydescribed for saltwater species (Brunelli et al.,2007). We described here the immunolocalisationof AQP1, AQP3 and Na+K+ ATPase in the gills ofC. julis. All of these proteins were closely localizedalong the filament epithelium and in the inter-lamellar region.

Aquaporin 1

The role of AQP1 is not yet resolved. Giffard-Mena et al. (2007) discussed the function of AQP1 insea bass acclimated to both fresh- and seawater;they suggested that AQP1 plays a limited osmor-egulatory role in the gills. Cooper et al. (2002)suggested that AQP1 in the oocyte membrane ofXenopus laevis functions as a gas channel, and asimilar role was proposed in fish gills (Hirose et al.,2003).

Recent studies have demonstrated that AQP1transports ammonia in oocytes of the Africanclawed frog (Nakhoul et al., 2001). In marine fish,ammonia excretion appears to occur via passivediffusion as a consequence of the high perme-ability of the gills (Claiborne and Evans, 1988;Wilkie, 1997; Wilson and Taylor, 1992); ammonia

Figure 2. Confocal micrographs of gill sections labelledwith the mouse monoclonal antibody IgGa5 raised againstthe a subunit of the Na+/K+ ATPase (red) and rabbitpolyclonal antibody against AQP1 (green). Images areoptical sections at intervals of 0.3 mm along the z-axis.(a) Double immunolabelling revealed immunopositivecells distributed along the filament and in the inter-lamellar region (merged image), colocalization of Na+/K+

ATPase and AQP1 is visible as yellow labelling generatedwhere the colour images merge; bar ¼ 75 mm; (b) singleimmunolabelling with anti-AQP1; bar ¼ 75 mm; (c) singleimmunolabelling with anti-Na+/K+ ATPase as a marker ofchloride cells; bar ¼ 75 mm; (d) enlargement of a mergedimage showing the immunoreaction at level of CCs;bar ¼ 30 mm.

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accumulation is high in seawater fish, thus makingthem more vulnerable to ammonia toxicity (Wilkie,2002). Moreover, Verkman and Mitra (2000) demon-

strated that AQP1-mediated CO2 transport might beimportant in lung gas exchange. One may speculatethat AQP1 might be involved in ammonia transportin the gills of marine fish, but the data presentedhere do not address this potential functional role ofAQP1. Much remains to be learned about the role ofAQP1 in the fish gill and, in particular, its role in thegill of marine teleosts.

Aquaporin 3

At present, no consensus exists about AQP3localization in the fish gill epithelium; AQP3 wasimmunolocalised in both CCs and accessory cells inC. julis gills, whereas in the Osorezan dace(Tribolodon hakonensis), which inhabits extremelyacidic freshwater, AQP3 immunolocalisation wasreported only in the CCs (Hirata et al., 2003). Inboth freshwater- and saltwater-acclimated euryha-line fish, quantitatively similar AQP3 immunoposi-tivity was identified within the CCs (Hirata et al.,2003; Lignot et al., 2002), but more intenseimmunolabelling of AQP3 occurred in other epithe-lial cells in freshwater-acclimated European eel(Lignot et al., 2002) and in all cells of both primaryand secondary epithelium in Japanese eel (Tseet al., 2006). These contradictory results aboutAQP3 immunolocalisation could be related not onlyto different experimental protocols (e.g., hetero-logous/homologous antibodies), as suggested byCutler et al. (2007), but also to different species-specific physiological functions of CCs. According toSantos et al. (2004), because fish inhabit diverseaquatic environments, they could represent auseful group in which to study aquaporin evolution;however, relatively little is known about the exactphysiological role of AQP3 in freshwater and salt-water fish.

To our knowledge the present study is the first toexamine AQP3 immunolocalisation in marine non-euryhaline fish; interestingly, AQP3 is present in the

Figure 3. Confocal micrographs of gill sections labelledwith the mouse monoclonal antibody IgGa5 raised againstthe a subunit of the Na+/K+ ATPase (red) and rabbitpolyclonal antibody against AQP3 (green). Images areoptical sections at intervals of 0.3 mm along the z-axis.(a) Double labelling with anti-AQP3 and -Na+/K+ ATPase;colocalization is visible at the level of the CCs (yellow);AQP3 labelling (green) is also detectable in putativeaccessory cells; bar ¼ 75 mm; (b) single immunolabellingwith anti-AQP3; bar ¼ 75 mm; (c) single immunolabellingwith anti-Na+/K+ ATPase; bar ¼ 75 mm; (d) enlargementof a merged image showing the colocalization of AQP3and Na+/K+ ATPase; bar ¼ 30 mm.

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accessory cells that are typical of marine teleostas confirmed by colocalization experiments withNa+/K+ ATPase.

Several authors (Evans et al., 1999; Pisam andRambourg, 1991; Pisam et al., 1995) have sug-gested that accessory cells are the precursors ofmature CCs. The increase in CCs that occurred inresponse to higher salinity, temperature variations,variations in food availability and the presence ofpollutants (Boyd et al., 1980; Brunelli et al., 2007;Karnaky, 1986) seems to be attributable to acces-sory cell maturation. CCs also show great plasticityin their ion-transporting functions (Hiroi et al.,1999; Katoh and Kaneko, 2003; Watanabe et al.,2005). Autonomous functional differentiation fromfreshwater to saltwater, which is independent ofthe endocrine and nervous systems, has beendemonstrated in the yolk-sac membrane of Tilapiaembryos (Shiraishi et al., 2001; see also for reviewMancera and Fuentes, 2006).

We therefore suppose that a similar plasticitycould be responsible for the pattern of AQP3reported in this study. Our observations may beindicative of the involvement of accessory cells inwater/glycerol transport in seawater fish, while amajor role in ionic regulation could be an attemptat maturation in CC or as an adaptive response ofthe gill epithelium to environmental change.

Functional studies are needed to define the roleof aquaporins in fish osmoregulation.

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