Review
Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization and functional conservation

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Abstract

The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1) and Shade (CYP314A1), the terminal hydroxylases. In Drosophila melanogaster, the Halloween gene spook (Cyp307a1) is required for the biosynthesis of 20E, although a function has not yet been identified. Unlike the other Halloween genes, the ancestor of this gene evolved into three paralogs, all in the CYP307 family, through gene duplication. The genomic stability of these paralogs varies among species. Intron–exon structures indicate that D. melanogaster Cyp307a1 is a mRNA-derived paralog of spookier (Cyp307a2), which is the ancestral gene and the closest ortholog of the coleopteran, lepidopteran and mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome–arthropod split. Conservation of putative substrate recognition sites of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway.

Introduction

Arthropods appeared more than 500 million years ago (Pisani et al., 2004) and the extant diversity of insects is evidence of their unprecedented evolutionary success. It is now clear that this success is due to several characteristics developed by insects including their ability to adapt their body plan to almost every habitat and ecological niche. The rigid cuticle has played an important role in this morphological adaptation and it serves as protection from desiccation and predators. However, the cuticle limits growth and hence must be digested and shed periodically in the process of molting. With the development of the ability to fly, insects expanded their habitats and the onset of metamorphosis allowed larvae and adults to exploit different niches (Truman and Riddiford, 2002). This tradeoff between larval growth and adult reproduction also reduced the competition for food resources. Thus, together the evolution of flight and metamorphosis probably created the foundation that led to the explosive evolutionary diversification of insects. Despite this great diversity, fundamental processes such as molting and metamorphosis were conserved during evolution. As a result of this, the insect Halloween genes have likely been highly conserved because they code for the P450 enzymes mediating the biosynthesis of the insect molting hormone, 20-hydroxyecdysone (20E) (Gilbert and Warren, 2005; Ono et al., 2006; see Rewitz et al., 2006a, Rewitz et al., 2006b).

Characterization of the Halloween genes emerged from molecular genetic studies of the fruit fly Drosophila melanogaster (Chávez et al., 2000; Warren et al., 2002, Warren et al., 2004; Petryk et al., 2003; Niwa et al., 2004; see Gilbert and Warren, 2005). The presence of these genes has been confirmed in other insect species (Niwa et al., 2004, Niwa et al., 2005; Warren et al., 2004; Sieglaff et al., 2005; see Rewitz et al., 2006c), although their functional conservation has only been demonstrated in D. melanogaster and lepidopterans. The primary structure of these P450s is highly conserved considering the period time since the divergence of the insects studied and the inherent, extensive diversification within the P450 family. The evolutionary constraint on the Halloween genes emphasizes their fundamental importance and only a few other P450s are evolutionarily more conserved, e.g. CYP51, the sterol 14-demethylase found in animals, plants, fungi and bacteria (Yoshida et al., 2000).

Fig. 1(A) shows the action of the P450 enzymes encoded by the Halloween genes, phantom (CYP306A1), disembodied (CYP302A1), shadow (CYP315A1) and shade (CYP314A1) in the biosynthetic pathway yielding 20E (see Gilbert and Warren, 2005). In the prothoracic glands (the Drosophila ring gland houses the prothoracic gland cells), CYP306A1, CYP302A1 and CYP315A1 catalyze the three sequential hydroxylations yielding ecdysone (E) from dietary cholesterol (see Gilbert and Warren, 2005; Rewitz et al., 2006c). In tissues peripheral to these glands, E is converted into the more active 20E by CYP314A1 also known as the ecdysone 20-monooxygenase. It should be mentioned that in addition to 20E, the principal insect molting hormone, E as well as other ecdysteroids such as makisterone are present at high levels depending on stage and species (see Lafont et al., 2005) and modulate developmental processes themselves. Each of the Halloween enzymes is believed to be the sole enzyme mediating one specific enzymatic step in the biosynthesis of 20E (also discussed by Ono et al., 2006) throughout embryonic and postembryonic development since: (1) disruption of the gene product results in low ecdysteroid levels and embryonic death; (2) in homozygous Cyp314a1 mutants conversion of E to 20E does not occur but providing CYP314A1 activity ectopically, using the GAL4-UAS system, rescues transgenic animals; (3) E is converted to 20E in the Malpighian tubules and fat body of wild type D. melanogaster larvae but, not in the GAL4-UAS rescued larvae where expression of Cyp314a1 is driven by a promoter that does not drive expression in these tissues; (4) these genes are expressed throughout embryonic and larval development and in the adult ovaries (Fig. 1(B)); (5) completely sequenced insect genomes contain only one ortholog of each of the Halloween genes and no very-close paralogs. Like Cyp306a1, Cyp302a1, Cyp315a1 and Cyp314a1, the D. melanogaster Halloween gene spook (Cyp307a1) was first identified as a low ecdysteroid mutant (Namiki et al., 2005: Ono et al., 2006). However, in contrast to the other Halloween genes, the function of CYP307A1 remains elusive and this gene is one of three related paralogs (all in the CYP307 family) that have acquired genomic stability depending on the insect species (Ono et al., 2006).

Multiple genome sequences are now available from different orders of holometabolous insects. This presents an opportunity to examine the evolutionary relationships and history of the Halloween gene family, changes associated with the evolution of higher and more complex insects, and perhaps establish possible evolutionary scenarios for these P450 genes. In this study, we use genome DNA sequence resources to identify orthologs of the Halloween genes for comparative analyses of phylogenetic relationships, gene organization with a discussion of Halloween genes in P450 phylogeny and their evolutionary conservation.

Section snippets

Annotation of Halloween genes

P450 protein sequences of characterized Halloween genes (Chávez et al., 2000; Warren et al., 2002, Warren et al., 2004; Petryk et al., 2003; Niwa et al., 2004, Niwa et al., 2005; Namiki et al., 2005; Ono et al., 2006; Rewitz et al., 2006a, Rewitz et al., 2006b) were used in tBlastn searches, to identify genome DNA sequences encoding P450 orthologs from holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. Using databases available at the National

Orthologous Halloween genes of P450s

Analysis of several insect genome sequences consistently suggests that each species contains only a single ortholog of CYP306A1, CYP302A1, CYP315A1 and CYP314A1. Reciprocal analyses demonstrate that each Halloween gene sequence is significantly more similar to its respective orthologs than to any other gene and that no closely related paralogs exist. This reflects a structural constraint on these genes that has preserved the ecdysteroid biosynthetic pathway through perhaps 400–500 million years

Conclusions and perspectives

The truly orthologous relationship among the Halloween genes clearly demonstrates the critical physiological function that has imposed constraining forces on their selection. So far, Halloween genes have only been identified and functionally characterized in holometabolous insects. Each of the insect genomes analyzed contain the same complement of the terminal hydroxylases (CYP306A1, CYP302A1, CYP315A1 and CYP314A1) whereas the presence of CYP307 paralogs varies depending on species. When

Acknowledgments

We thank members of the Gilbert and O’Connor laboratories for their consistent and excellent work on the Halloween gene project over the past several years as well as their collegiality. In addition we are indebted to Todd Vision (Bioinformaticist, University of North Carolina) and Rene Feyereisen (INRA) for helpful comments on the manuscript and Joseph Shaw (Dartmouth and Mt. Desert Island Biological Laboratory) for his help in gaining entry to the Daphnia database. We are also thankful to Ole

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    This paper is dedicated to Lynn Riddiford on her 70th birthday. She has been a most valued friend and colleague to LIG for half a century and it is hoped that this will continue for many years into the future.

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