The role of P450 enzymes in ecdysteroid metabolism has been known for many years (review in Lafont et al., 2012) and several CYP genes have clearly been associated with ecdysteroid biosynthesis. These are the CYP2 clan CYP306A1 and CYP307 as well as the mitochondrial clan CYP302A1, CYP314A1 and CYP315A1.

nm-g/sro (non-molting glossy /shroud) is a short chain dehydrogenase/reductase that functions along CYP307 in the “Black Box” (Niwa et al., 2010)

These genes are often called by their confusing Drosophila mutant names, Cyp306a1 (phantom, phm), Cyp307a1 (spook, spo), Cyp307a2 (spookier, spok), Cyp302a1 (disembodied, dib), Cyp314a1 (shade, shd), and Cyp315a1 (shadow,sad). They were collectively dubbed Halloween genes, a term that does not appear in the original (1984) publications describing the mutants, and that obscures the relationship with CYP18. We will refer to them as ecdysteroidogenic P450 for convenience (see also review in Niwa and Niwa, 2014). This denomination is clearer and allows expansion to the P450s involved in ecdysteroid metabolism that remain to be characterized. There are also non-P450 genes in the “Halloween” series (e.g. neverland, nvd and shroud, sro), and this creates additional confusion, with some authors drawing phylogenies that include P450 and non-P450 sequences.

The reactions in ecdysteroid biosynthesis from sterol precursors are generally considered to be structured in a linear pathway:

In this pathway as classical depicted, the sterol precursor is the C27 cholesterol. In some insects, C28 or C29 sterols are used instead, leading to C24-methyl or ethyl-branched ecdysteroids. Furthermore, the product of the black box depicted here is a 3-hydroxysterol, but in some insects it may be a 3-ketosterol, with the 3 keto group reduced during or after ecdysone production.

Deviations from this scheme (see below with ecdysteroids identified in arthropods and their possible origin from a non-linear pathway) are rarely discussed and experimental evidence for the activity or specificity of the enzymes is limited to a few model species (see discussion in Dermauw et al., 2020). The most common deviation is the lack of C25 hydroxylation, as in spider mites (Grbic et al., 2011) leading to ponasterone A as major ecdysteroid.

CYP18 is another P450 involved in ecdysteroid metabolism, by hydroxylation and further oxidation at C26, which constitutes hormonal inactivation in Drosophila (Guittard et al., 2011).

The origin of the P450 genes involved in ecdysteroid biosynthesis is of great evolutionary importance, as one might expect “Ecdysozoa” to be capable of making a molting hormone, yet the presence of ecdysteroids, or indeed of the ecdysteroidogenic P450s, is clearly not a synapomorphy of “Ecdysozoa”. For instance the well-known nematode model Caenorhabditis elegans has no ecdysteroids and none of the P450s needed to make them.