(A) Wild-type fin and carotenoid profile, showing carotenoid absorbance at 455 nm in adult male proximal tissue (red) and distal tissue (yellow). Numbers indicate different carotenoid species, with the most abundant ketocarotenoid in erythrophore-containing tissue being astaxanthin (peak 3; Figure 1A; Figure 7—figure supplement 1). (B) Homozygous scarb1 mutants lacked red and yellow coloration and carotenoids were not detectable. (C) Homozygous mutant phenotypes of genes targeted from RNA-Seq comparisons. cyp2ae2 and bdh1a mutants were deficient for red color and astaxanthin. bco1 mutants had reduced red and yellow coloration and carotenoids. (D) Ratios of red to green autofluorescence for cells found within proximal erythrophore containing regions (red filled points) and distal xanthophore containing regions (yellow filled points) of wild-type males and females compared to mutant males. In the wild-type, erythrophores and xanthophores were segregated into different populations by R/G fluorescence, although differences in females were less marked. In males of each mutant, R/G ratios of erythrophores were reduced compared to wild-type, and lesser reductions were evident in xanthophores (ANOVA, genotype x region interaction, F4,736=310.82, p < 0.0001, after controlling for significant main effects and variation among individuals; N = 760 cells total from five individuals of each background). Plots show means ±95 % confidence intervals; means of groups not sharing the same letter differed significantly from one another (p < 0.05) in Tukey-Kramer post hoc comparisons. (E) Wild-type males and females, and mutant males, differed in total visible pigment, as measured by diameters of contracted pigment granules following epinephrine treatment (Saunders et al., 2019). (ANOVA, background x region interaction, F4,736=76.25, p < 0.0001, with significant main effects and variation among individuals; diameters were ln-transformed for analysis to control for increasing residual variance with means.). (F) Densities of erythrophores and xanthophores differed across backgrounds ( ANOVA, background x region interaction, F1,35=19.01, p < 0.0001). Each point represents the mean number of cells counted in three regions of 4 × 10–2 mm2 in proximal or distal regions with erythrophores or xanthophores, respectively, in each of 39 total fish. Scale bar: 50 μm.