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Branchial expression of an aquaporin 3 (AQP-3) homologue is downregulated in the European eel Anguilla anguilla following seawater acclimation

Christopher P. Cutler^* and Gordon Cramb

School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland

View larger version (33K): [in a new window]   Fig. 1. Alignment of derived AQP3 amino acid sequences from the eel in comparison to those from other species. Dashes indicate identical residues, periods indicate spaces introduced to give an optimal alignment, bold underlinings indicate the approximate locations of membrane spanning domains (TM 1-6: Kyte and Dolittle, 1982) and wavy underlinings indicate the location of inter-transmembrane loops (A—E; Borgnia et al., 1999). Percentages represent the amino acid homology of each sequence to that of the eel. Sequences were aligned using GeneJockey II software (Biosoft). Dashes, underlining and periods were added after alignment. The EMBL/Swiss prot accession numbers for sequences were as follows: Eel (AJ319533), Xenopus (AJ131847), Rat (L35108), Mouse (AF104416) and Human (AB001325).

View larger version (46K): [in a new window]   Fig. 2. Northern blot using total RNA (10 µg) showing the tissue distribution of AQP3 mRNA expression in a seawater (SW)-acclimated yellow eel, with the exception of RNA samples from a freshwater (FW) yellow eel where labelled. RNA sizes (kb) were estimated from RNA standards (not shown).

View larger version (16K): [in a new window]   Fig. 3. Northern blots using total RNA (10 µg) showing the expression of AQP3 mRNA in the gill and intestine of freshwater (FW)- and 3-week seawater (SW)-acclimated yellow and silver eels. Each sample on the blot was taken from an individual fish such that N=6 for each group. RNA sizes (kb) were estimated from RNA standards (not shown). The gill blot autoradiograph was exposed for 3 h at -80°C and the intestine blot, 32 h at -80°C. The 7 kb band seen in the tissue blot in Fig. 2 was not visible on autoradiographs in this figure, although it was included in the quantification results (Fig. 4).

View larger version (16K): [in a new window]   Fig. 4. Quantification of the radiolabelled AQP3 DNA probe bound to northern blots of gill and intestinal RNA samples isolated from freshwater (FW)- and 3-week seawater (SW)-acclimated yellow or silver eels (as shown in Fig. 3). Values are means ± S.E.M. NS, not significant P>0.05; **P<0.01; ****P<0.0001; these values indicate FW to SW comparisons, where N=6 samples from different fish in each group.

View larger version (107K): [in a new window]   Fig. 5. Northern blots using total RNA (15 µg) showing the time course of changes in gill AQP3 mRNA expression during the seawater (SW) acclimation of yellow eels. Times indicate the period for either freshwater (FW) to SW acclimation or for FW to FW transfer of control eels. Time 0 fish were taken directly from the stock tank. The 7 kb band seen in the tissue blot (Fig. 2) was not visible on autoradiographs in this figure, although it was included in the quantification results (Fig. 6).

View larger version (14K): [in a new window]   Fig. 6. Quantification of the radiolabelled AQP3 DNA probe bound to the northern blot of yellow eel gill RNA samples following FW to FW and FW to SW transfer. FW, freshwater; SW, seawater. Values are means ± S.E.M. ****P<0.0001; these values refer to FW to SW comparisons for each time point, where N=6 samples from different fish in each group. The 6 hour control time point was significantly different from FW to FW controls at 1 day (P=0.049) and 21 days (P=0.038).

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