Steroid hormones, regulate essential processes during development and reproduction, and are synthesized from cholesterol under the control of steroidogenic enzymes in the cytochrome P450 (CYP) family [1
]. In Caenorhabditis elegans
, insects and vertebrates, different steroids are produced to control developmental processes, suggesting that steroidogenic CYPs evolved and became functionally specialized in different lineages during evolution. In insects, a specific biosynthetic pathway yielding 20-hydroxyecdysone (20E), the arthropod molting hormone, evolved, whereas in the line leading to vertebrates, biosynthetic CYPs that produce the vertebrate-type steroids evolved [2
]. Since there is some evidence of the presence of vertebrate-type sex steroids in invertebrates such as echinoderms and mollusks, although no unequivocal evidence that they can synthesize these steroids [3
], the possibility remains that CYPs with the capacity to produce vertebrate-type sex steroids were present in the common ancestor even before the protostome-deuterostome split. Thus, the evolution of steroidogenic CYPs is still an open question.
Crustaceans are believed to represent the ancestral arthropods from which insects originated [4
]. The evolutionary relationship between these two groups is evident from the common growth strategy of insects and crustaceans that involves molting so that growth can occur. Molting is governed by periodic increases in the levels of 20E that elicit the programs that coordinate the developmental and metamorphic transitions [5
]. Although a great deal of evidence reveals that crustaceans, like insects, synthesize 20E from cholesterol [6
], the molecular details of steroidogenesis in crustaceans remain conjectural. In insects, steroidogenic CYPs are products of the Halloween genes phantom
) and shade
) and are responsible for the last four hydroxylations in the pathway leading to 20E [7
] that is biochemically similar to one that yields 20E in crustaceans [6
] (Fig. ). In Drosophila melanogaster
, mutations in these genes disrupt 20E production and cause the arrest of embryonic development and death. spook
) is another member of this CYP group which when mutated results in low 20E mutants [15
] and is believed to mediate a yet uncharacterized step (the Black Box) in the biosynthesis of 20E preceding those of Phm, Dib, Sad and Shd. In contrast to phm
for which each insect genome carries one ortholog, several paralogs of spo
) genes have been formed by duplications, which in turn have evolved lineage-specific complements of these genes [2
]. For example, Drosophila
has two spo
-like genes, spo
]. These two genes are close paralogs that are believed to mediate the same enzymatic reaction, although at different stages of development.
Figure 1 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 (more ...)
Although one would expect that orthologs of the insect Halloween genes are present in crustaceans, there is no molecular evidence for the existence of these genes in crustaceans. We have tried for several years to probe hexapod crustaceans for Halloween gene orthologs under various hormonal regimens using degenerate primers based on the Drosophila and Bombyx genes, but we have been unsuccessful (K.F. Rewitz, J.T. Warren, E. Chang and L.I. Gilbert). The development of the genome data base of the more primitive crustacean, Daphnia pulex, allowed us to survey this genome and conduct phylogenetic analyses that suggest strongly that orthologs of spo, phm, dib, sad and shd do exist in Daphnia and thus, in a crustacean i.e. the genes appeared in arthropods before the radiation of insects.