C-2 hydroxylation occurs with retention of configuration in the ovaries of Schistocerca gregaria (Greenwood et al., 1984): The 2ß hydrogen of cholesterol is eliminated during ecdysteroid biosynthesis. This indicates that C-2 hydroxylation is direct as expected from a P450-catalyzed reaction. The hydroxylation of 2-deoxyecdysone to ecdysone in Locusta migratoria was observed in the prothoracic glands (PG) as expected, and in the Malpighian tubules, midgut, fat body and epidermal tissues as well (Kappler et al., 1986). It is now commonly observed that C-2 hydroxylation is not restricted to ecdysteroidogenic tissues such as PG and ovaries. The C-2 hydroxylation was characterized as a mitochondrial P450 in larval Malpighian tubules and in ovarian follicle cells of vitellogenic females (Kappler et al., 1986) as well as in the prothoracic glands (Kappler et al., 1988). This P450 activity is peculiar in its very low sensitivity to CO inhibition, but virtually stoichiometric incorporation of one atom of molecular oxygen was demonstrated (Kabbouh et al., 1987). The biochemical characterization of this P450 activity is supportive of the idea that the same gene product is responsible for C-2 hydroxylation in the Malpighian tubules and in the PG. The Malpighian tubule enzyme had a Km of 0.4 µM for 2-deoxyecdysone, and was inhibited by 2-deoxy-20-hydroxyecdysone, 2,22-dideoxyecdysone, 2,22-25-trideoxyecdysone and 3-epi-2-deoxyecdysone, but not by ecdysone or 20-hydroxyecdysone (Kappler et al.1986, Kabbouh et al. 1987).

CYP315A1 or shadow (sad) gene in Drosophila was shown to encode a C-2 hydroxylase (Warren et al., 2002). The gene is a mitochondrial CYP clan P450. CYP315A1 is expressed in embryos, as well as in the larval ring gland and follicle cells of adult ovaries. Transient expression of CYP315A1 in Drosophila S2 cells coupled with the use of radiolabeled 2-deoxyecdysone was used to identify the reaction (Warren et al., 2002).

The CYP315 gene is generally present in all arthropod species studied, but is not easily found in the copepods Tigriopus japonicus and T. californicus. However, it is found in other copepods, Paracyclopina nana and Lepeophteirus salmonis, and phylogenetic analysis reveals that the CYP315 clade does contains two Tigriopus japonicus genes, CYP3022A1 and CYP3023A1 (Dermauw et al. 2020). This suggests that these two P450s are in fact the products of a CYP315 duplication and rapid divergence. Whether or not 2-deoxyecdysteroids are found in the Tigriopus genus is not known, and the function of these two P450s merit examination.

Three CYP315 are apparent in the collembolan Folsomia candida, whereas there is just one gene in Sinella curviseta. In Limulus polyphemus, there are two pairs of CYP315 genes. The TSA of the millipede Chamberlinius hualienensis revealed no CYP315, but it was found in the genomes of Helicorthomorpha holstii and Trigoniulus corallinus. In centipedes, Strigamia maritima has two CYP315, which is intriguing given the apparent lack of CYP302.

Paradoxically, Descamps and Lafont (1993) were unable to show any 2-hydroxylase activity in Lithobius forficatus (Myriapoda, Chilopoda), despite the presence of a CYP315A1 sequence in the genome and of ecdysone and 20E in this species.

Schumann et al., (2018) reported the presence of three CYP315 genes, but no other ecdysteroidogenic gene in velvet worms (Onychophora). Dermauw et al. (2020) suggest that only two Euperipatoides CYP315 belong to a weakly supported monophyletic CYP315 clade, and whether they have ecdysteroid 2-hydroxylase activity is unknown.

Information on the specificity of CYP315A1 is scant. The conversion of 2-deoxyecdysone to ecdysone was shown to occur with heterologously expressed CYP315A1 from Drosophila melanogaster (Warren et al., 2002), Bombyx mori (Niwa et al., 2005), Manduca sexta (Rewitz et al., 2006), Anopheles gambiae (Pondeville et al., 2008) and Mamestra brassicae (Ogihara et al., 2017) as well as Nilaparvata lugens (Zhou et al., 2020).

The Drosophila and Anopheles gambiae CYP315A1 also converted 2,22-dideoxyecdysone to 22-deoxyecdysone (Warren et al., 2002; Pondeville et al., 2008).

Shi et al., 2022 reported a modest O-dealkylation activity of Helicoverpa armigera CYP315A1 towards 7-ethoxycoumarin (7EC). Apis mellifera CYP315A1 did not metabolite imidacloprid or thiacloprid (Alptekin et al., 2016).