Scheme of 20-hydroxyecdysone (20E) biosynthesis and a phylogenetic tree including Daphnia Halloween orthologs. A) Biosynthetic scheme showing the steroidogenic CYP enzymes encoded by genes in the Halloween family mediating steps in the conversion of cholesterol to 20E and the subcellular distribution of these enzymes. The Black Box denotes an uncharacterized series of oxidative modifications converting 7-dehydrocholesterol (7dC) to the first ecdysteroid-like molecule, namely the ketodiol (2,22,25-trideoxyecdysone: 2,22,25-dE) . Dashed arrow indicates that there is no direct evidence for the catalytic function of Spook (Spo) and Spookier (Spok), but several pieces of evidence point to a function of these enzymes in the Black Box . 2,22-dideoxyecdysone (2,22-dE). B) Maximum Likelihood phylogenetic tree showing relationships of the Daphnia sequences with orthologs of the insect steroidogenic CYP products of the Halloween genes Spo, Spok, Spookiest (Spot), Phantom (Phm), Disembodied (Dib), Shadow (Sad), Shade (Shd). Other selected vertebrate and C. elegans steroidogenic and non-steroidogenic CYPs are included to infer relationship to major classes of CYPs. Members of major metazoan CYP classes are represented in this analysis: the mitochondrial, CYP2-related, CYP3-related, CYP4s, CYP19 and CYP51. Numbers indicate support values obtained by bootstrapping 100 replicates and branches under the threshold value of 50 are shown as polytomies. Human CYP3A4 (AAI01632), Rat CYP4A1 (NP_787031), Crayfish CYP4C15 (AAF09264), Drosophila CYP4G15 (AAF76522) House fly CYP6A1 (AAA29293), Manduca CYP9A4 (AAD51036), Human CYP11A1 – P450scc cholesterol side chain cleavage enzyme (AAH32329), Bovine CYP17A1 – steroid 17α-hydroxylase/17,20 lyase (P05185), Rat CYP19A1 – aromatase converting androgens to estrogens (P22443), Human CYP21 – steroid 21-hydroxylase (AAB59440), Human CYP1A1 (AAH23019), Human CYP2U1 (NP_898898), Rat CYP51 – sterol 14α-demethylase (Q64654), and C. elegans DAF-9 (CYP22A1) – produces the steroid ligand for the DAF-12 nuclear receptor (AAL65132) . * Shows CYP enzymes from animals, other than arthropods, that are involved in steroidogenesis.