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Immunolocalisation of aquaporin 3 in the gill and the gastrointestinal tract of the European eel Anguilla anguilla (L.)

Jean-H. Lignot^1,*, Christopher P. Cutler¹, Neil Hazon² and Gordon Cramb^1,

School of Biology, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland
¹ Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland
² Gatty Marine Laboratory, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland
^* Present address: Université Louis Pasteur, CNRS CEPE, 23 rue Becquerel, 67087 Strasbourg, Cedex 2, France

View larger version (100K): [in a new window]   Fig. 1. Western blot analysis of aquaporin 3 (AQP-3) in branchial tissues of silver eels maintained in freshwater (FW) or acclimated for 3 weeks to a seawater (SW) environment. The blot shows AQP-3 immunoreactivity in plasma membrane fractions isolated from the gills of SW-acclimated eels (lanes A-C) and FW eels (lanes D-F). Controls include the use of both pre-immune serum (FW eel membranes; PI, lane G) and peptide-negated antiserum (FW eel membranes; Pep., lane H), see Materials and methods. Individual lanes represent protein samples from separate membrane preparations from each experimental group. Gill tissue was removed from two fish for each membrane preparation. Size markers (right) are in kDa.

View larger version (16K): [in a new window]   Fig. 2. Quantitative analysis of branchial AQP-3 protein expression in freshwater (FW) and 3-week seawater (SW)-acclimated silver eels. Values are means ± S.D., derived from three different preparations each using two fish.

View larger version (166K): [in a new window]   Fig. 3. Immunolocalisation of AQP-3 in the gill. Typical immuno-histochemistry of the branchial epithelium in 3-week seawater (SW)-acclimated (A—C) and freshwater (FW; D—H) silver eels. (C,F) Control sections incubated with pre-immune serum. PF, primary filaments; SL, secondary lamellae; GA, gill arch; GAE, gill arch epithelium; CC, chloride cell; GC, goblet cell; PC, pillar capillary; RBC, red blood cell. Bars, 10 µm.

View larger version (136K): [in a new window]   Fig. 4. Immuno-localisation of AQP-3 in the intestinal and rectal epithelia. Typical immuno-histochemistry of both the intestinal (A—C) and rectal (D—F) epithelium of 3-week seawater (SW)-acclimated (A,D,F) and freshwater (FW; B,C,E) silver eels. (C,F) Control sections were incubated with pre-immune serum. IE, intestinal epithelium; RE, rectal epithelium; L, lumen; GC, goblet cell; RBC, red blood cell. Bars, 10 µm.

View larger version (96K): [in a new window]   Fig. 5. Confocal laser scanning microscopy: dual immuno-localisation of AQP-3 (false colour: red) and Na+, K+-ATPase (ß233 subunit) (false colour: green) in thin sections. Gill primary and secondary filaments of 3-week seawater (SW)-acclimated (A,B) and freshwater (FW; C,D) silver eels. Intestinal sections of SW-acclimated (E) and FW (F) silver eels. Rectal epithelium of a SW-acclimated silver eel (G). PF, primary filaments; SL, secondary lamellae; E, gut epithelium; L, gut lumen; CC, chloride cell; GC, goblet cell; M, macrophage-like body; RBC, red blood cell; AP, apical pit. Bars, 50 µm (A,E,F,G); 25 µm (B,C); 10 µm (D).

View larger version (183K): [in a new window]   Fig. 6. Transmission electron micrograph (A,B) and immuno-gold localisation of AQP-3 (C—E) in chloride cells of the branchial epithelium of seawater (SW)-acclimated silver eels. (E) Control section incubated with pre-immune serum. CC, chloride cell; AM, apical membrane; AP, apical pit; M, mitochondria; N, nucleus; PC, pavement cell; TS, tubular system; V, vacuole; VTS, vesiculo-tubular system; A, accessory cell; SA, sub-apical region; GP, gold particles (15 nm). Bars, 1 µm.