Comparative transcriptomics reveals candidate carotenoid color genes in an East African cichlid fish

Abstract

Background: Carotenoids contribute significantly to animal body coloration, including the spectacular color pattern diversity among fishes. Fish, as other animals, derive carotenoids from their diet. Following uptake, transport and metabolic conversion, carotenoids allocated to body coloration are deposited in the chromatophore cells of the integument. The genes involved in these processes are largely unknown. Using RNA-Sequencing, we tested for differential gene expression between carotenoid-colored and white skin regions of a cichlid fish, Tropheus duboisi "Maswa", to identify genes associated with carotenoid-based integumentary coloration. To control for positional gene expression differences that were independent of the presence/absence of carotenoid coloration, we conducted the same analyses in a closely related population, in which both body regions are white.

Results: A larger number of genes (n = 50) showed higher expression in the yellow compared to the white skin tissue than vice versa (n = 9). Of particular interest was the elevated expression level of bco2a in the white skin samples, as the enzyme encoded by this gene catalyzes the cleavage of carotenoids into colorless derivatives. The set of genes with higher expression levels in the yellow region included genes involved in xanthophore formation (e.g., pax7 and sox10), intracellular pigment mobilization (e.g., tubb, vim, kif5b), as well as uptake (e.g., scarb1) and storage (e.g., plin6) of carotenoids, and metabolic conversion of lipids and retinoids (e.g., dgat2, pnpla2, akr1b1, dhrs). Triglyceride concentrations were similar in the yellow and white skin regions. Extracts of integumentary carotenoids contained zeaxanthin, lutein and beta-cryptoxanthin as well as unidentified carotenoid structures.

Conclusion: Our results suggest a role of carotenoid cleavage by Bco2 in fish integumentary coloration, analogous to previous findings in birds. The elevated expression of genes in carotenoid-rich skin regions with functions in retinol and lipid metabolism supports hypotheses concerning analogies and shared mechanisms between these metabolic pathways. Overlaps in the sets of differentially expressed genes (including dgat2, bscl2, faxdc2 and retsatl) between the present study and previous, comparable studies in other fish species provide useful hints to potential carotenoid color candidate genes.

Keywords: BCO2; Body coloration; Carotenoids; Cichlidae; Color genes; Gene expression; Lipids; RNA-Seq; Tropheus.

Fig. 1

Adult males of two Tropheus duboisi populations used in this study. The red dashed lines specify the areas used for RNA, carotenoid and triglyceride analyses. M-d: Maswa, dorsal bar region; M-v: Maswa, ventral bar region; K-d: Kigoma, dorsal bar region, K-v: Kigoma, ventral bar region. Photographs by Wolfgang Gessl, Institute of Biology, University of Graz (www.pisces.at)

Fig. 2

Differential gene expression. a Heatmap showing differential gene expression between yellow (dorsal; M-d1 – M-d5) and white (ventral; M-v1 – M-v5) skin samples of T.duboisi Maswa. Red and green shadings represent higher and lower relative expression levels, respectively. b A Venn diagram showing the numbers of differentially expressed genes in the two populations. Only three genes, hsd3b1, zic1 and asip1, were differentially expressed in both populations

Fig. 3

Functional enrichment and functional associations among differentially expressed genes. a Gene ontology enrichment analysis (Manteia) for biological processes in the differentially expressed genes (T. duboisi Maswa). b and c Predicted functional associations between the differentially expressed genes (both variants of T. duboisi) based on zebrafish b and chicken c databases in STRING v10 (http://string-db.org/)

Fig. 4

Validation of RNA-Seq expression patterns using qPCR for 10 selected genes. Bars represent means and standard deviations of RQ in three biological replicates for each skin region and population (M-d: Maswa, dorsal bar region; M-v: Maswa, ventral bar region; K-d: Kigoma, dorsal bar region, K-v: Kigoma, ventral bar region). Asterisks indicate significant differences in expression levels between the dorsal and ventral samples in within-population comparisons (paired t-tests; ***, p < 0.001; **, p < 0.01; *, p < 0.05)