Two closely related ureotelic fish species of the genus Alcolapia express different levels of ammonium transporters in gills

ABSTRACT Most fish excrete their nitrogenous waste across the gills as ammonia through the activity of the Rhesus glycoprotein ammonium transporters. In contrast, fish of the subgenus Alcolapia (Oreochromis) are the only vertebrates that survive the extreme conditions of the soda lakes of Natron and Magadi in East Africa and have evolved adaptations to the highly alkaline waters including the ability to excrete their nitrogenous waste as urea. Nevertheless, Alcolapia retain the Rhesus glycoprotein genes in their genomes and using two heterologous expression systems, we demonstrate that Alcolapia Rhbg is capable of moving ammonia. Comparing ammonia and urea excretion from two closely related Alcolapia species from the same aquarium, we found that while Alcolapia grahami remains fully ureotelic after many generations in lab conditions, Alcolapia alcalica excretes some of its nitrogenous waste as ammonia. Using in situ hybridisation, we demonstrate robust, localised gene expression of Rhbg, rhcg1 and rhcg2 in the gill tissue in both A. alcalica embryos and adults, similar to that in other ammoniotelic fish. In contrast, the expression of these genes in A. grahami gills is much lower than in A. alcalica, suggesting the rapid evolution of a molecular mechanism underlying the complete ureotelism of A. grahami.

. Gills were dissected from Alcolapia alcalica and Alcolapia grahami adults raised at the same facility under the same conditions over several generations; expression of Rhbg was analysed by in situ hybridasation. Antisense cRNA probes directed against the almost identical nucleotide sequence in the two species were used to detect the expression of Rhbg in the gill tissue in parallel using in situ hybridisation. The substrate was applied to the specimens at the same time and after 90 minutes the A.alcalica sample (above, left) showed robust expression of Rhbg, while no detectable expression was seen in A.grahami (above, right). By 16 hours the rhesus protein genes were detected in the gills of A.grahami but at a much lower level than in A.alcalica (see main text).

Fig. S3
. cDNA was prepared from RNA extracted from gills dissected from the same animals used for in situ hybridisations. Specific primers were designed to amplify rhbg, rhcg1, rhcg2 and, as a control, elongation factor alpha (eF1α). cDNA from two different samples of gill RNA (from two different animals) was prepared for A.alcalica (A1, A2) and for A.grahami (G1, G2). cDNA was replaced by water to control for any contamination of reagents, and a cDNA preparation done without the enzyme reverse transcriptase (no RT) was included to control for any genomic DNA contamination. The resulting products are visualised by gel electrophoresis (see top panel).
(A) In order to measure any differences in levels of rhesus protein gene expression detectable by RT-PCR, ImageJ plug-in was used to determine the relative intensity of the bands on each gel. There is a large difference in the relative gene expression of our control gene EF1a, which seems to be expressed to a much higher degree in A. grahami compared to A.alcalica. Non-stable reference gene expression may exaggerate the differences between A.alcalica and A.grahami, therefore in the main text, we present the data from the bottom graph that compares only the average raw intensity of each PCR product, not normalised to the expression of eF1a (so as not to exaggerate the difference). As the data derived from ImageJ is in arbitrary units and not comparable between different gels, the graph in the main text ( Figure 6) show the proportion of A.grahami expression of each gene compared to that in A.alcalica. Calculations are shown in A and the graphs derived from normalised and non-normalised data are presented in B.  Table S1. Results of branch models testing for variation in ω among branches of the phylogeny.
The following two ω models were tested compared to a model with a single ω among branches: Where two ω models are a statistically better fit to the data compared to a single ω model, these are indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. For rhag and rhcg1 sequences from only a single Alcolapia species were available, so we are unable to report results for both M1 and M2.

Rhcg2b -D. rerio
Forward -GTCTACCACTCCGATGTCTTTG 58 688 Reverse -CATCTTCAGGTACCTCCCAATAAA For semi-quantitative RT-PCR on RNA extracted from gill tissue from A.alcalica and A.grahami the following primers were used: