Expression levels of the tetratricopeptide repeat protein gene ttc39b covary with carotenoid-based skin colour in cichlid fish

Abstract

Carotenoid pigments play a major role in animal body colouration, generating strong interest in the genes involved in the metabolic processes that lead from their dietary uptake to their storage in the integument. Here, we used RNA sequencing (RNA-Seq) to test for differentially expressed genes in a taxonomically replicated design using three pairs of related cichlid fish taxa from the genera Tropheus and Aulonocara. Within each pair, taxa differed in terms of red and yellow body colouration, and high-performance liquid chromatography (HPLC) analyses of skin extracts revealed different carotenoid profiles and concentrations across the studied taxa. Five genes were differentially expressed in all three yellow-red skin contrasts (dhrsx, nlrc3, tcaf2, urah and ttc39b), but only the tetratricopeptide repeat protein-coding gene ttc39b, whose gene product is linked to mammalian lipid metabolism, was consistently expressed more highly in the red skin samples. The RNA-Seq results were confirmed by quantitative PCR. We propose ttc39b as a compelling candidate gene for variation in animal carotenoid colouration. Since differential expression of ttc39b was correlated with the presence/absence of yellow carotenoids in a previous study, we suggest that ttc39b is more likely associated with the concentration of total carotenoids than with the metabolic formation of red carotenoids.

Keywords: animal body colouration; carotenoids; cichlidae; colour genes; colour polymorphism; comparative transcriptomics.

Figure 1.

Cichlid taxa and skin regions analysed in the present study. (a) Schematic illustration of the phylogenetic relationships between the investigated cichlid taxa. (b) Ultra‐high‐performance liquid chromatography (UHPLC) chromatograms of carotenoid extracts of one typical skin sample per colour variant. Peaks after 2.5 min correspond to esterified carotenoids (except for free beta carotene at 3.8 min in A. baenschi). For a larger view, see electronic supplementary material, figure S1. (c) The skin regions sampled for paired comparisons between cichlid colour variants are marked by green boxes on the fish bodies and depicted in macroscopic photographs. Taxon codes as shown in (a).

Figure 2.

Differential gene expression. (a) Heatmap showing differential gene expression between yellow and red skin samples. Blue and orange shadings represent higher and lower relative expression levels, respectively. (b) qPCR validation of RNA-Seq expression patterns. Bars represent means and standard deviations of RQ in six biological replicates. Asterisks indicate significant differences in expression levels between the dorsal and ventral samples in within-population comparisons (t-tests; ***, p < 0.001; **, p < 0.01). Taxon codes as in figure 1a.