Spatial expression pattern of melanin synthesis genes. (A) Heat map of the relative expression level of melanin synthesis/related genes and yellow family genes in P. xuthus. Red indicates positive values and green indicates negative values (color spectrum bar is shown to the right; N.D., not detected). Red circle indicates the gene examined by whole-mount in situ hybridization in (C). Stage and marking of each sample is shown above. The stage-specific co-expression cluster and marking specificity of each gene are also shown. See also Figures 2 to 4. (B) Neighbor joining tree of yellow family genes based on their amino acid sequences. The numbers at the tree nodes represent the bootstrap values. The scale bars indicate the evolutionary distance between the groups. Accession numbers are shown in parenthesis. Red boxes indicate the P. xuthus genes. Yellow-g gene has not been reported in lepidopteran species . (C) Spatial expression patterns of yellow-h3 mRNA in P. xuthus larva during the fourth molt. Numbers in each panel indicate molt stage-hours after head capsule slippage. (D) Biosynthetic pathway underlying the formation of melanin. Dopamine, N-beta-alanyldopamine (NBAD) and N-acetyldopamine (NADA)are used in the production of Dopamine-melanin (black and brown), NBAD pigment (yellow or reddish brown), and NADA pigment (transparent) in insects [5, 16, 45, 62]. Aa DCE: Aedes aegypti dopachrome conversion enzyme; BH4: tetrahydrobiopterin; Bm: Bombyx mori; DDC: dopa decarboxylase; Dm: Drosophila melanogaster; GTP: guanosine triphosphate; GTP-CH I: GTP cyclohydrolase I; Hm: Heliconius melpomene; NADA: N-acetyldopamine; NAT: arylalkylalamine-N-acetyltransferase; NBAD: N-beta-alanyldopamine; NBH2: dihydroneopterin triphosphate; Px: Papilio xuthus. TH: tyrosine hydroxylase.