Bdh1l Gene Expression Is a Potential Molecular Factor in the Evolution of Carotenoid-Based Colour Diversity of Cichlid Fishes

Abstract

Carotenoids contribute substantially to animal body colour pattern diversity. While the ecological and evolutionary drivers of carotenoid coloration are reasonably well understood, the molecular mechanisms facilitating evolutionary transitions between red and yellow hues are less investigated. Here we leverage phylogenetically replicated red-versus-yellow colour contrasts in three pairs of closely related cichlid fishes (Tropheus and Aulonocara; Haplochromini) to investigate biochemical and genetic parallels in carotenoid colour differentiation. Red skin samples contained the ketocarotenoids rhodoxanthin, canthaxanthin, and astacene, the latter as likely saponification product of astaxanthin. A re-analysis of existing RNA-seq data using an improved bioinformatics pipeline identified consistent red-versus-yellow gene expression differences. Notably, transcripts of a gene coding for a 3-hydroxybutyrate dehydrogenase type 1 enzyme (bdh1l) and further known carotenoid genes (scarb1, bco2, ttc39b) were significantly more abundant in red than in yellow skin tissue in all taxon pairs. Homologues of Bdh1l have recently been discovered to mediate C4-ketocarotenoid biosynthesis in birds and fish, but only in the presence of a cytochrome P450 enzyme. We found no consistent differences in cytochrome P450 gene expression. Our results suggest that bdh1l expression regulation might operate as a molecular switch for C4-ketocarotenoid biosynthesis and colour pattern differentiation in different radiations of cichlid fish, apparently in the presence of a stably expressed and therefore inconspicuous P450 cytochrome enzyme. The divergent chemical structure of rhodoxanthin requires a different biosynthesis pathway than the C4-ketocarotenoids astaxanthin and canthaxanthin. Differential expression of hsd3b, encoding a dehydrogenase with a corresponding function in the steroid pathway, suggests a new candidate for rhodoxanthin biosynthesis.

Keywords: carotenoid metabolism; coloration; colour pattern evolution; comparative transcriptomics; diversification; fish.

FIGURE 1

Carotenoids detected in the fish skin samples. Fish photographs above the table represent the investigated taxa. Tissue samples were taken from the body areas indicated by the squares. Cichlid taxa are: TbR, T. sp. ‘black’ ‘Bulu Point’ (red); TbY, T. sp. ‘black’ ‘Ikola’ (yellow); TmR, T. moorii ‘Moliro’ (red); TmY, T. moorii ‘Mbita’ (yellow); AhR, A. hansbaenschi ‘Red Flush’ (red); AbY, A. baenschi (yellow). In the table, each column represents a sample of the corresponding cichlid taxon; integumentary carotenoids are represented in rows. Cell colours indicate the detection of a particular carotenoid (red for ketocarotenoids, yellow for yellow carotenoids) in a particular sample. Photos by Wolfgang Gessl, used with permission.

FIGURE 2

Expression patterns of carotenoid colour genes bdh1l, ttc39b, scarb1, hsd3b and bco2. (A) Transcript abundance (TPM, transcripts per million). See Table S5 for TPM values. Some dots overlap, n = 6 for each taxon. Cichlid taxa are: TbR, T. sp. ‘black’ ‘Bulu Point’ (red); TbY, T. sp. ‘black’ ‘Ikola’ (yellow); TmR, T. moorii ‘Moliro’ (red); TmY, T. moorii ‘Mbita’ (yellow); AhR, A. hansbaenschi ‘Red Flush’ (red); AbY, A. baenschi (yellow). (B) Pearson correlation coefficients calculated from VST normalised count data. Above diagonal: gene expression correlations in the red samples (AhR, TbR and TmR combined); below diagonal: gene expression correlations in the yellow samples (AbY, TbY and TmY combined). Asterisks indicate uncorrected significance levels: ***, p < 0.001; **, 0.001 < p < 0.01; *, 0.01 < p < 0.05.