It has been known for

It has been known for some time that an oral salt load given to mammals induces a much greater natriuresis than the intravenous administration of an equivalent amount of saline (Carey et al., 1976, Singer et al., 1998). In addition, oral administration of salt to rats has been reported to augmented guanylin and uroguanylin mRNA levels in the intestine (Carrithers et al., 2002) and uroguanylin mRNA levels the kidney (Potthast et al., 2001). As a result of extensive research over the past 20–30 years and especially more recently with the development of new cellular and molecular techniques, a wealth of evidence has accumulated regarding the morphological, physiological and biochemical adaptations that are essential for successful osmoregulation and life in FW and SW environments (Marshall and Grosell, 2006; Takei, 2008). When eels are transferred to a SW environment a drinking response is induced to protect the fish from dehydration (Tierney et al., 1995). The initiation of the drinking response results in an increase in salt concentrations within the gastrointestinal tract; so the obvious question was, would these elevated luminal salt concentrations affect mRNA apiii of the guanylin-like peptides as does administration of an oral salt load to mammals? In addition, as uroguanylin has been found to be predominately located in the proximal small intestine, whereas guanylin mRNA levels peak in the distal small intestine and the large intestine in mammals (Qian et al., 2000, Carrithers et al., 2002), were there any regional differences in the expression of the guanylin peptides along the length of the eel intestine? The results suggest that, unlike mammals, mRNAs for all three of the eel guanylin-like peptides are expressed approximately equally along the length of the intestine. During the course of these experiments a variety of “housekeeping” genes were used to normalise the expression data. Like that reported previously for the Japanese eel (Yuge et al., 2003), SW acclimation induced profound changes in the expression of two potential “housekeeping” genes, GAPDH and β-actin. However, unlike in the Japanese eel where only transient increases in expression of these housekeeping genes was found, the increases in GAPDH and β-actin expression were sustained in the intestine of Anguilla anguilla for several months after SW transfer (unpublished observation). Transcripts which did not change included 18S and 28S rRNAs and the acidic riboprotein RPL-P0 (Weltzien et al., 2006, Lyng et al., 2008) and therefore both rRNAs were used to normalise the result from the Northern blot analyses and RPL-P0 gene was chosen as the reference “housekeeping” gene for expression normalisation in q-PCR. Acclimation of both yellow and silver eels to SW resulted in an increase in expression of uroguanylin and GC-C1 mRNAs in the intestine. The increases in uroguanylin expression were more marked in the migratory silver eels, which also showed some signs of pre-adaptive increases in expression compared to yellow eels when fish were still in FW. The abundance of guanylin, renoguanylin and GC-C2 mRNAs within the intestine were not significantly changed by SW acclimation, although small and in some cases statistically significant differences in expression were found between yellow and silver eels. In the kidney, no changes in expression were found between yellow or silver eels for any guanylin-peptide or cyclase receptor isoform either before or after SW transfer. These results are in contrast to those reported previously by Yuge et al., 2003, Yuge et al., 2006 for the Japanese eel, where all peptides and both receptor isoform mRNAs are upregulated in the intestine by 2- to 6-fold following SW transfer. Small increases in uroguanylin expression in the kidney were also reported (Yuge et al., 2003). The reasons for the discrepancies found between expression in the European and Japanese eels are unknown. Although we found high variability in gene expression between fish within the same experimental groups, it is unlikely that the SW-induced 6-fold increase in guanylin mRNA and over 2-fold increases in renoguanylin and GC-C2 mRNAs reported in the intestine of Anguilla japonica would have been masked in Anguilla anguilla by natural variations within the population. However, in this investigation the wild fish used were all much larger (>280g) and therefore presumably older than the aquaculture eels (<200g) used in Yuge’s study. It is possible that the salinity-induced changes in gene expression may be dependent upon the developmental stage of the eel. The experiments conducted in this study, and preliminary work reported previously (Cramb et al., 2005) suggest that some pre-adaptation is taking place in FW-acclimated silver eels, with respect to expression of uroguanylin in the intestine. Although this appears to be a minor response it is clear that much larger increases in expression are found in the migratory silver eels than yellow eels after movement to SW. Further investigations will be required to determine if earlier juvenile life-stages exhibit different responses to SW adaptation