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First published online March 12, 2009
Journal of Experimental Biology 212, 1003-1010 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.025957
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Morphological and functional classification of ion-absorbing mitochondria-rich cells in the gills of Mozambique tilapia

Mayu Inokuchi*,1, Junya Hiroi2, Soichi Watanabe1, Pung-Pung Hwang3 and Toyoji Kaneko1

1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
2 Department of Anatomy, St Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan
3 Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan


Figure 1
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  		Fig. 1. Scanning electron micrographs of gill filaments of tilapia acclimated to artificial freshwaters with different Na+ and Cl– concentrations: normal Na+/normal Cl– (Control; A); normal Na+/low Cl– (LowCl; B); low Na+/normal Cl– (LowNa; C); and low Na+/low Cl– (LowNa/LowCl; D). The apical structures of mitochondria-rich cells are classified into the following three types: a small apical pit (arrowhead); a concave apical surface (arrow); and a convex apical surface (asterisk). Small apical pits are narrow and deep, so that little or no internal structure can be observed. Concave apical surfaces are slightly dented, or sometimes flat, with a mesh-like structure on their surface. Convex apical surfaces are equipped with microvilli. Small apical pits predominate in Control fish (A). Concave and convex apical surfaces predominantly develop in LowNa and LowCl fish, respectively (B,C). Both concave and convex surfaces are frequently observed in LowNa/LowCl fish (D). Scale bar, 10 µm.

 

Figure 2
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  		Fig. 2. Densities of different apical types of mitochondria-rich cells (small apical pit, concave apical surface and convex apical surface) in the gills of tilapia acclimated to artificial freshwaters with different Na+ and Cl– concentrations: normal Na+/normal Cl– (Control); normal Na+/low Cl– (LowCl); low Na+/normal Cl– (LowNa); and low Na+/low Cl– (LowNa/LowCl). Data are expressed as the mean ± s.e.m. (N=8). Different letters indicate significant differences at P<0.05.

 

Figure 3
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  		Fig. 3. Relative mRNA expression levels of Na+/H+ exchanger-3 (NHE3; A) and Na+/Cl– cotransporter (NCC; B) in the gills of tilapia acclimated to artificial freshwaters with different Na+ and Cl– concentrations: normal Na+/normal Cl– (Control); normal Na+/low Cl– (LowCl); low Na+/normal Cl– (LowNa); and low Na+/low Cl– (LowNa/LowCl). The data are normalized to the expression levels of 18S rRNA. Data are expressed as the mean ± s.e.m. (N=8). Different letters indicate significant differences at P<0.05.

 

Figure 4
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  		Fig. 4. Triple immunofluorescence staining with anti-Na+/K+-ATPase (A,E,I,M, red), anti-NHE3 (B,F,J,N, green) and anti-NCC (C,G,K,O, blue) in gill filaments of tilapia acclimated to artificial freshwaters with different Na+ and Cl– concentrations: normal Na+/normal Cl– (Control; A–D); normal Na+/low Cl– (LowCl; E–H); low Na+/normal Cl– (LowNa; I–L); and low Na+/low Cl– (LowNa/LowCl; M–P). D,H,L,P, Merged images. Scale bar, 20 µm.

 

Figure 5
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  		Fig. 5. Horizontal sections (A–D) and cross-sections (E–H) of mitochondria-rich (MR) cells, stained immunocytochemically with anti-Na+/K+-ATPase (red), anti-NHE3 (green) and anti-NCC (blue). (A,C,E,G) Gill MR cells with small pits immunoreactive to NHE3 (A,E) and NCC (C,G) in tilapia acclimated to normal Na+/normal Cl– water. (B,D,F,H) MR cells with enlarged apical membranes immunoreactive to NHE3 (B,F) and NCC (D,H) in tilapia acclimated to low Na+/low Cl– and normal Na+/low Cl– waters, respectively. Scale bar, 5 µm.

 

Figure 6
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  		Fig. 6. Scanning electron microscopy immunocytochemistry for Na+/H+ exchanger-3 (NHE3) and Na+/Cl– cotransporter (NCC) in the gills of tilapia acclimated to artificial freshwaters with low Na+/low Cl– (A–C) and with normal Na+/low Cl– (D–F), respectively. (A,D) Scanning electron microscopic images. (B,E) X-ray signals specific for Ag indicating the presence of NHE3 (B) and NCC (E). (C,F) Merged images. NHE3 (B) and NCC (E) are confined to the concave (arrows) and convex (asterisks) apical surfaces, respectively. Scale bar, 10 µm.

 

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