Article

Rewitz KF, Gilber LI. Daphnia Halloween genes that encode cytochrome P450s mediating the synthesis of the arthropod molting hormone: Evolutionary implications. BMC Evolutionary Biology

Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280 USA.
BMC Evolutionary Biology (Impact Factor: 3.37). 02/2008; 8(1):60. DOI: 10.1186/1471-2148-8-60
Source: PubMed

ABSTRACT

In crustaceans and insects, development and reproduction are controlled by the steroid hormone, 20-hydroxyecdysone (20E). Like other steroids, 20E, is synthesized from cholesterol through reactions involving cytochrome P450s (CYPs). In insects, the CYP enzymes mediating 20E biosynthesis have been identified, but evidence of their probable presence in crustaceans is indirect, relying solely on the ability of crustaceans to synthesize 20E.
To investigate the presence of these genes in crustaceans, the genome of Daphnia pulex was examined for orthologs of these genes, the Halloween genes, encoding those biosynthetic CYP enzymes. Single homologs of spook-CYP307A1, phantom-CYP306A1, disembodied-CYP302A1, shadow-CYP315A1 and shade-CYP314A1 were identified in the Daphnia data base. Phylogenetic analysis indicates an orthologous relationship between the insect and Daphnia genes. Conserved intron/exon structures and microsynteny further support the conclusion that these steroidogenic CYPs have been conserved in insects and crustaceans through some 400 million years of evolution.
Although these arthropod steroidogenic CYPs are related to steroidogenic CYPs in Caenorhabditis elegans and vertebrates, the data suggest that the arthropod steroidogenic CYPs became functionally specialized in a common ancestor of arthropods and are unique to these animals.

Full-text

Available from: PubMed Central
BioMed Central
Page 1 of 8
(page number not for citation purposes)
BMC Evolutionary Biology
Open Access
Research article
Daphnia Halloween genes that encode cytochrome P450s
mediating the synthesis of the arthropod molting hormone:
Evolutionary implications
Kim F Rewitz
1
and Lawrence I Gilbert*
2
Address:
1
Department of Science, Systems and Models, Roskilde University, Postbox 260, DK-4000 Roskilde, Denmark and
2
Department of
Biology, University of North Carolina, Chapel Hill, NC 27599-3280 USA
Email: Kim F Rewitz - rewitz@ruc.dk; Lawrence I Gilbert* - lgilbert@unc.edu
* Corresponding author
Abstract
Background: In crustaceans and insects, development and reproduction are controlled by the
steroid hormone, 20-hydroxyecdysone (20E). Like other steroids, 20E, is synthesized from
cholesterol through reactions involving cytochrome P450s (CYPs). In insects, the CYP enzymes
mediating 20E biosynthesis have been identified, but evidence of their probable presence in
crustaceans is indirect, relying solely on the ability of crustaceans to synthesize 20E.
Results: To investigate the presence of these genes in crustaceans, the genome of Daphnia pulex
was examined for orthologs of these genes, the Halloween genes, encoding those biosynthetic CYP
enzymes. Single homologs of spook-CYP307A1, phantom-CYP306A1, disembodied-CYP302A1, shadow-
CYP315A1 and shade-CYP314A1 were identified in the Daphnia data base. Phylogenetic analysis
indicates an orthologous relationship between the insect and Daphnia genes. Conserved intron/
exon structures and microsynteny further support the conclusion that these steroidogenic CYPs
have been conserved in insects and crustaceans through some 400 million years of evolution.
Conclusion: Although these arthropod steroidogenic CYPs are related to steroidogenic CYPs in
Caenorhabditis elegans and vertebrates, the data suggest that the arthropod steroidogenic CYPs
became functionally specialized in a common ancestor of arthropods and are unique to these
animals.
Background
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 pro-
duced to control developmental processes, suggesting that
steroidogenic CYPs evolved and became functionally spe-
cialized in different lineages during evolution. In insects,
a specific biosynthetic pathway yielding 20-hydroxyecdys-
one (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 verte-
brate-type sex steroids in invertebrates such as echino-
derms and mollusks, although no unequivocal evidence
that they can synthesize these steroids [3], the possibility
remains that CYPs with the capacity to produce vertebrate-
Published: 25 February 2008
BMC Evolutionary Biology 2008, 8:60 doi:10.1186/1471-2148-8-60
Received: 15 November 2007
Accepted: 25 February 2008
This article is available from: http://www.biomedcentral.com/1471-2148/8/60
© 2008 Rewitz and Gilbert; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page 1
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 2 of 8
(page number not for citation purposes)
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 arthro-
pods 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 gov-
erned 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 evi-
dence reveals that crustaceans, like insects, synthesize 20E
from cholesterol [6], the molecular details of steroidogen-
esis in crustaceans remain conjectural. In insects, ster-
oidogenic CYPs are products of the Halloween genes
phantom (phm: CYP306A1), disembodied (dib: CYP302A1),
shadow (sad: CYP315A1) and shade (shd: CYP314A1) and
are responsible for the last four hydroxylations in the
pathway leading to 20E [7-14] that is biochemically simi-
lar to one that yields 20E in crustaceans [6] (Fig. 1). In
Drosophila melanogaster, mutations in these genes disrupt
20E production and cause the arrest of embryonic devel-
opment and death. spook (spo: Cyp307a1) is another mem-
ber of this CYP group which when mutated results in low
20E mutants [15,16] 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 con-
trast to phm, dib, sad and shd for which each insect genome
carries one ortholog, several paralogs of spo-like (CYP307)
genes have been formed by duplications, which in turn
have evolved lineage-specific complements of these genes
[2,16,17]. For example, Drosophila has two spo-like genes,
spo and spookier (spok: Cyp307a2) [16]. These two genes are
close paralogs that are believed to mediate the same enzy-
matic reaction, although at different stages of develop-
ment.
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 hexa-
pod crustaceans for Halloween gene orthologs under var-
ious 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.
Results and Discussion
By searching the Daphnia data bases [18,19] we obtained
candidate sequences for orthologs of the insect Halloween
genes in Daphnia. Single orthologs of phm, dib, sad and shd
were retrieved and only one sequence exhibited signifi-
cant similarity to the spo-like genes in the CYP307 family.
We also searched the genomes of non-arthropod inverte-
brates including the cnidarian Nematostella vectensis, the
nematodes C. elegans and Brugia malayi, the annelid
Capitella capitata, the mollusk Lottia gigantea, the echino-
derm Strongylocentrotus purpuratus [19,20] for Halloween
gene orthologs using TBLASTN. Analyses of these inverte-
brate genomes did not result in any significant hits indi-
cating the absence of Halloween orthologs and neither
have we been able to identify orthologs of these genes in
any of several vertebrate species.
The genes obtained from Daphnia encode approximately
500 amino acid open reading frames (ORFs) which is typ-
ically for proteins belonging to the CYP family [21]. Align-
ment of Daphnia sequences with orthologs of the insect
Halloween genes shows that the genes, in addition to
being conserved in areas that comprise canonical struc-
tural CYP motifs, exhibit considerable conservation in
regions that are believed to determine substrate specificity
(Additional file 1). This indicates that the genes are func-
tionally conserved in Daphnia as they are in insects [2].
The overall amino acid identity between deduced orthol-
ogous proteins from eight insects belonging to four differ-
ent orders and the Daphnia orthologs ranges from an
average of 55.2% ± 6.8 SD (standard deviation) for Spo-
like proteins to somewhat lower values for Sad proteins
(38.7% ± 8.8 SD). Thus, spo-like genes are the most highly
conserved of these genes and this appears to be true for
Daphnia like it is for insects. Conservation of these genes
from insect to crustaceans, which separated ~400 millions
years ago [4], shows that selection has preserved the genes
because of their function. The reason that spo-like genes
are more conserved than the other arthropod steroidog-
enic CYP enzymes is not known, although it may be
related to the possibility that Spo acts in the rate-limiting
Black Box reaction(s) [5,22]. If Spo is involved in control
of the flux through the pathway it may have been a partic-
ular target for selection because mutations altering its
enzyme activity would have had increased consequences
compared to mutations altering the activity of enzymes
that are less rate-limiting. A phylogenetic analysis was per-
formed using the Daphnia sequences.
Phylogenetic analysis of Daphnia candidate orthologs
A phylogenetic tree was constructed with Daphnia
sequences retrieved from the BLAST searches and
orthologs of the insect Halloween genes from Drosophila,
red flour beetle Tribolium castaneum and honey bee Apis
mellifera, which represent three different orders (Diptera,
Page 2
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 3 of 8
(page number not for citation purposes)
Scheme of 20-hydroxyecdysone (20E) biosynthesis and a phylogenetic tree including Daphnia Halloween orthologsFigure 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 medi-
ating 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) [1]. 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 [16]. 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 – aro-
matase 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) [35]. * Shows CYP enzymes from ani-
mals, other than arthropods, that are involved in steroidogenesis.
Page 3
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 4 of 8
(page number not for citation purposes)
Coleoptera and Hymenoptera, respectively) of holome-
tabolous insects. The Daphnia gene products separate with
the insect orthologs of Spo/Spok, Phm, Dib, Sad and Shd
in this phylogenetic tree with high bootstrap support (Fig.
1). In examining the genome of Daphnia for orthologs of
these genes, we noticed no closely related paralogs to the
obtained candidate orthologs. This supports the orthol-
ogy of the genes and indicates that the genes became func-
tionally specialized before the split between crustaceans
and insects and have been under heavy selection pressure
ever since. Only one spo-like gene was obtained and this
gene is phylogenetically a CYP307A subfamily gene i.e.
most closely related to spo/spok. No ortholog of spookiest
(spot: CYP307B1) was found indicating that this gene does
not exist in Daphnia, which implies that it is insect-specific
i.e. originating from a duplication occurring after insects
arose from crustaceans [4] or that it was lost in Daphnia as
it has been in some insects e.g. lepidopterans and Dro-
sophila species [2].
In the phylogenetic analysis, sequences of steroidogenic
CYPs from vertebrates and C. elegans were included to
probe ancestral relationships and the origin of CYPs
involved in steroid biogenesis (Fig. 1). Figure 1 includes
major groups of metazoans CYPs i.e. mitochondrial,
CYP2-related, CYP3-related, CYP4-related, CYP19s and
CYP51s. The steroidogenic CYPs of insects and their Daph-
nia orthologs are evolutionarily related to vertebrate and
C. elegans steroidogenic CYPs since they cluster in two
major groups, those related to CYP2 enzymes (Spo, Phm,
CYP17, CYP21 and DAF-9) and those that are mitochon-
drial (Dib, Sad, Shd and CYP11A1). Therefore, it is likely
that different steroidogenic CYP enzymes are derived from
common ancestors and were recruited for steroid biosyn-
thesis prior to the protostome-deuterostome split. Evolu-
tion of these ancestral CYPs in C. elegans, arthropods and
vertebrates, by gene duplication and divergence, likely
shaped the biosynthetic pathways yielding the different
groups of steroids in these animals.
Conservation of gene structure
Comparison of intron positions of the aligned Daphnia
protein sequences and the insect orthologs of the Hallow-
een genes shows that many introns are conserved (Fig. 2).
Each Daphnia ortholog contains conserved intron/exon
organization typical of their respective insect orthologs.
The conserved gene structure suggests that the Daphnia
genes have a common evolutionary origin with the insect
Halloween genes and support the inferred phylogenetic
relationship suggesting orthology (Fig. 1). In phm, five
introns are conserved among Daphnia and insects and
only two introns are unique to the Daphnia gene. Introns
that have no counterparts can be explained by intron gain
or alternatively these introns have been lost. Interestingly,
Daphnia spo has two introns whereas insect spo genes have
only one. The position and phase (the nucleotide position
in a codon) of introns in the Daphnia spo gene are equiva-
lent to those conserved in insect spot genes. However, the
Daphnia sequence is the closest ortholog of the insect spo/
spok genes phylogenetically, and only shares one of these
conserved introns. A likely evolutionary scenario is that
the ancestral gene was a spo gene containing both introns
(Fig. 3). In the insect lineage, after the split from crusta-
ceans, duplication of this gene resulted in two copies that
diverged into spo and spot paralogs after which one intron
was lost in the insect spo genes.
Although several introns are conserved among the mito-
chondrial dib, sad and shd, introns that are unique to each
group of orthologs (e.g. the first phase 1 intron in dib and
shd) indicate an orthologous relationship to the respective
groups. The conserved gene structure of these genes sug-
gests that they evolved from a common ancestor of mito-
chondrial CYPs by duplications and diverged into the
steroidogenic enzymes that are functionally specialized to
carry out the last three steps in which C22, C2 and C20
hydroxylations form 20E. This implies, at least in part,
that the biosynthetic pathway evolved step-wise by dupli-
cation and functional divergence of the biosynthetic
enzymes. In vertebrates, functional divergence and spe-
cialization of steroidogenic CYPs are known from studies
of the CYP11 paralogs. CYP11A1 is the enzyme that medi-
ates the removal of the side chain of cholesterol [23].
CYP11B1 and CYP11B2 are paralogs, from a recent dupli-
cation, that became functionally specialized as the steroid
11β-hydroxylase and the aldosterone synthase, respec-
tively [24]. dib, sad and shd also exhibit conservation of
gene structure when compared to vertebrate mitochon-
drial CYPs involved in steroidogenesis [2].
Microsynteny supports orthology
The conserved arrangement of genes on the chromosome
of different species (microsynteny) can be used to infer
phylogenetic relationships that support the orthology of
genes [2,17]. Analysis of local genome structures sur-
rounding the Halloween genes shows little conservation
of microsynteny between insects and crustaceans. This is
likely due to the relatively great evolutionary time separat-
ing insects from crustaceans. However, remnants showing
microsynteny can be found in at least two cases. CYP18A1
is a paralog of phm which is found in Drosophila and most
other insects [25]. In Drosophila and Apis, phm and
CYP18A1 are arranged tail-to-tail and adjacent to these
genes are CG6696 and fused (Fig. 2). In Daphnia, the
microsyntenic relationship of phm and CYP18A1 is con-
served, although CG6696 and fused appear to have been
rearranged to different chromosomal locations.
Comparison of gene organization in a region of approxi-
mately 100 kb surrounding the spo(k) locus reveals that
Page 4
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 5 of 8
(page number not for citation purposes)
spo is adjacent to neverland (nvd) [26] in Daphnia and the
mosquito Anopheles gambiae. Analysis of the genome
region of spo-like genes in species of Drosophila (available
at FlyBase [27]) shows that in Drosophila pseudoobscura,
Drosophila willistoni and Drosophila mojavensis nvd is
located within 60 kb of spok. This is in agreement with the
view that spok is the ancestral gene in Drosophilidae.
Intriguingly, Nvd is a conserved Rieske-like oxygenase
believed to be involved in the conversion of cholesterol to
7-dehydrocholesterol in insects and C. elegans [26,28]. In
insects, this is presumably the first step in the biosynthetic
pathway of 20E from cholesterol via 7-dehydrocholes-
terol, the latter being the substrate that enters the Black
Box, in which Spo/Spok likely participate (although at dif-
ferent developmental stages) to produce the substrate for
Phm, the ketodiol (Fig. 1). Although unequivocal evi-
dence for the exact function of Nvd and Spo/Spok is lack-
ing, experiments utilizing ecdysteroid precursors to rescue
mutant phenotypes (RNAi-induced for nvd and spok) sug-
gest that in Drosophila nvd and spo/spok genes function in
these two succeeding steps in the biochemical pathway
[16,26]. However, it must be emphasized that the Black
Box may hold more than one reaction and more than one
enzyme may be participating in this conversion [5].
Conclusion
Currently, it is clear that CYPs are involved in steroid bio-
synthesis in vertebrates and invertebrates. In C. elegans,
insects and vertebrates, the majority of steroidogenic CYPs
are related to one of two groups, the CYP2 enzymes and
Intron/exon structure and microsynteny of Daphnia and insect Halloween genesFigure 2
Intron/exon structure and microsynteny of Daphnia and insect Halloween genes. A) Introns are mapped on the
aligned protein sequences of Spo (CYP307A1), Spot (CYP307B1), Spok (CYP307A2), Phm (CYP306A1), Dib (CYP302A1), Sad
(CYP315A1), Shd (CYP314A1). Introns located at the same position and in the phase (the nucleotide position of the intron
within a codon: phase 0 between codons, phase 1 after the first base and phase 2 after the second base), on the aligned pro-
teins, are shown as conserved by connecting vertical lines. Insect introns represent introns found in species of insects previ-
ously described [2], except Tribolium shd which exhibits unique introns that are not shown. B) Preserved microsynteny in local
genome regions surrounding phm and spo(k). The arrangement of phm and its paralog CYP18A1 is conserved in insects and
Daphnia and so is the microsyntenic relationship of spo (spok in Drosophila) and neverland (nvd). Nvd is believed to be involved
in the conversion of cholesterol to 7-dehydrocholesterol (7dC), the step preceding the Black Box in which Spo/Spok may func-
tion [16,26]. The transcriptional direction of the genes is shown by arrowheads. The double arrowhead indicates that ApepP is
in opposite orientations in Drosophila and Anopheles.
Page 5
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 6 of 8
(page number not for citation purposes)
the mitochondrial CYPs. It is therefore likely that these
steroidogenic CYPs evolved from common ancestors
which had the capacity to modify cholesterol before the
split of these animals. This implies that the distinct bio-
synthetic pathways in these metazoans, which convert
cholesterol to different types of steroids, likely result from
lineage-specific evolution of ancestral steroidogenic CYPs.
The results suggest strongly that steroidogenic CYP
enzymes mediating the biosynthesis of 20E are present in
crustaceans and are relegated to the Arthropoda. There-
fore, these enzymes probably became functionally spe-
cialized in the line leading to arthropods. Although
functional characterization of the gene products, such as
that done for the insect Halloween genes [1,29], is
required to obtain unequivocal evidence of their role in
20E production in Daphnia, the present data provide the
first evidence for steroidogenic CYP genes in crustaceans
as well as the groundwork for future functional genomic
analyses in the field of crustacean endocrinology.
Methods
Data base search
The Daphnia pulex genome sequence (v1.0) made availa-
ble by the Daphnia Genomics Consortium and the DOE
Joint Genome Institute [19] was mined for orthologs of
the insect Halloween genes using TBLASTN searches. Apis
and Tribolium sequences, previously described in Rewitz et
al. [2], were used as queries to identify homologous
sequences in the Daphnia data base. Sequences of insect
Halloween genes, previously described in Rewitz et al. [2],
were gleaned from FlyBase [27] and Rene Feyereisen's
website for insect CYPs [30]. C. elegans and vertebrate CYP
protein sequences were acquired from NCBI [20].
Phylogenetic tree construction, intron positions and
microsynteny analysis
Predicted Daphnia orthologs of the insect Halloween
genes were analyzed using a phylogenetic tree with insect
Halloween genes from insects belonging to three different
Lineage-specific duplications and losses of spo-like genes in Daphnia and insectsFigure 3
Lineage-specific duplications and losses of spo-like genes in Daphnia and insects. An evolutionary scenario based on
the observed distribution of spo-like (CYP307) genes in arthropods. Since insects are believed to have evolved from crustaceans
living in freshwater environments [4] and the only spo-like gene observed in Daphnia belongs to the CYP307A subfamily, the
ancestral arthropod spo gene was likely a CYP307A gene. An early duplication, which probably occurred after insects diverged
from crustaceans, gave rise to spo (CYP307A1) and spot (CYP307B1). In Drosophila, the ancestral spo-like gene, referred to as
spok (Cyp307a2), underwent a second round of duplication in which an intronless retrogene arose. In Drosophila this gene is
referred to as spo. Note that another round of gene duplication, which occurred in the line of Drosophila evolution leading to
the subgenus Drosophila [17], is not shown.
Page 6
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 7 of 8
(page number not for citation purposes)
orders, Apis (Hymenoptera), Tribolium (Coleoptera) and
Drosophila (Diptera). To infer phylogenetic relationships
of these steroidogenic CYPs with other members of this
multigene family, steroidogenic and non-steroidogenic
CYPs representing some of the major groups of metazoan
CYPs were included in this analysis i.e. mitochondrial,
CYP2-related, CYP3-related and CYP4s, CYP19 and
CYP51. Deduced Daphnia protein sequences used in the
alignment can be found in Additional file 2. A multiple
alignment of the protein sequences was constructed with
ClustalX (1.83) [31] and manually edited using SeaView
[32]. A phylogenetic tree was constructed from this align-
ment using the Maximum Likelihood method under the
Jones-Taylor-Thornton (JTT) substitution model using
PHYML (v2.4.5) [33]. Support values were obtained by
bootstrapping 100 replicates. Branches with bootstrap
support below 50 were collapsed to form polytomies.
Gene structure, that is, intron position and phase (the
nucleotide position of the intron within a codon: phase 0
between codons, phase 1 after the first base and phase 2
after the second base) were predicted from manual anno-
tation of the Daphnia genes with support from expressed
sequence tags (ESTs) data and by homology to insect Hal-
loween genes for which gene structures are known. Intron
position and phase were mapped onto a multiple align-
ment to investigate conservation of intron/exon structure
in relation to phylogeny.
Additional support for orthology of Daphnia and insect
genes was sought by analyses of microsynteny (conserved
order of genes). Genome regions surrounding the candi-
date Halloween orthologs of Daphnia and several insects
were inspected for ORFs encoding conserved genes. Puta-
tive gene orthologs exhibiting conserved arrangement in
relation to the Halloween orthologs were tested for
orthology by reciprocal best hit Blast searches in which
orthologs of each species were BLAST searched against the
genome of the other species to look for best hits (putative
orthologs).
Abbreviations
20E (20-hydroxyecdysone); Dib (Disembodied-
CYP302A1); Nvd (Neverland); ORF (open reading
frame); Phm (Phantom-CYP306A1); Sad (Shadow-
CYP315A1); Shd (Shade-CYP314A1); Spo (Spook-
CYP307A1); Spok (Spookier-CYP307A2); Spot (Spooki-
est-CYP307B1); Spo-like (CYP307 family)
Authors' contributions
KFR conducted the actual phylogenetic and other analyses
in this paper and wrote the first draft. LIG came up with
the idea of identifying the Halloween genes in a crusta-
cean, gained access to the Daphnia data base, recom-
mended that KFR do the search analyses and helped write
the final draft of the manuscript. All authors read and
approved the final manuscript.
Additional material
Acknowledgements
We offer special thanks to John Colbourne and Joseph Shaw for their help
in gaining access to the Daphnia data base and Michael B. O'Connor and
James T. Warren for their continued collaboration on the Halloween gene
project. The Daphnia sequence data were produced by the US Department
of Energy Joint Genome Institute [19] in collaboration with the Daphnia
Genomics Consortium [34]. Our work benefits from, and contributes to,
the Daphnia Genomics Consortium. The present work was supported by
grant 0516623 from the National Science Foundation.
References
1. Gilbert LI, Warren JT: A molecular genetic approach to the bio-
synthesis of the insect steroid molting hormone. Vitam Horm
2005, 73:31-57.
2. Rewitz KF, O'Connor MB, Gilbert LI: Molecular evolution of the
insect Halloween family of cytochrome P450s: phylogeny,
gene organization and functional conservation. Insect Biochem
Mol Biol 2007, 37:741-753.
3. Lafont R, Mathieu M: Steroids in aquatic invertebrates. Ecotoxi-
cology 2007, 16:109-130.
4. Glenner H, Thomsen PF, Hebsgaard MB, Sørensen MV, Willerslev E:
The origin of insects. Science 2006, 314:1883-1884.
5. Gilbert LI, Rybczynski R, Warren JT: Control and biochemical
nature of the ecdysteroidogenic pathway. Ann Rev Entomol
2002, 47:883-916.
6. Lachaise F, Le Roux A, Hubert M, Lafont R: The molting gland of
crustaceans: localization, activity, and endocrine control. J
Crustacean Biol 1993, 13:198-234.
7. Chavez VM, Marques G, Delbecque JP, Kobayashi K, Hollingsworth
M, Burr J, Natzle JE, O'Connor MB: The Drosophila disembodied
gene controls late embryonic morphogenesis and codes for
a cytochrome P450 that regulates embryonic ecdysone lev-
els. Development 2000, 127(19):4115-4126.
8. Petryk A, Warren JT, Marques G, Jarcho MP, Gilbert LI, Parvy J-P,
Dauphin-Villemant C, O'Connor MB: Shade is the Drosophila
P450 enzyme that mediates the hydroxylation of ecdysone
to the steroid insect molting hormone 20-hydroxyecdysone.
Proc Natl Acad Sci USA 2003, 100:13773-13778.
9. Niwa R, Matsuda T, Yoshiyama T, Namiki T, Mita K, Fujimoto Y,
Kataoka H: CYP306A1, a cytochrome P450 enzyme is essen-
tial for ecdysteroid biosynthesis in the prothoracic glands of
Bombyx and Drosophila. J Biol Chem 2004, 279:35942-35949.
10. Niwa R, Sakudoh T, Namiki T, Saida K, Fujimoto Y, Kataoka H: The
ecdysteroidogenic CYP CYP302A1/disembodied from the silk-
worm,
Bombyx mori, is transcriptionally regulated by protho-
racicotropic hormone. Insect Mol Biol 2005, 14:563-571.
Additional File 1
Putative substrate recognition sites (SRSs).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2148-8-60-S1.pdf]
Additional File 2
Deduced protein sequences of Daphnia pulex Spo-CYP307A1, Phm-
CYP306A1, Dib-CYP302A1, Sad-CYP315A1 and Shd-CYP314A1.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2148-8-60-S2.pdf]
Page 7
Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
BMC Evolutionary Biology 2008, 8:60 http://www.biomedcentral.com/1471-2148/8/60
Page 8 of 8
(page number not for citation purposes)
11. Rewitz KF, Rybczynski R, Warren JT, Gilbert LI: Identification,
characterization and developmental expression of Hallow-
een genes encoding P450 enzymes mediating ecdysone bio-
synthesis in the tobacco hornworm, Manduca sexta. Insect
Biochem Mol Biol 2006, 36:188-199.
12. Rewitz KF, Rybczynski R, Warren JT, Gilbert LI: Developmental
expression of Manduca shade, the P450 mediating the final
step in molting hormone synthesis. Mol Cell Endocrinol 2006,
247:166-174.
13. Warren JT, Petryk A, Marques G, Jarcho M, Parvy J-P, Dauphin-Ville-
maont C, O'Connor MB, Gilbert LI: Molecular and biochemical
characterization of two P450 enzymes in the ecdysteroidog-
enic pathway of Drosophila melanogaster. Proc Natl Acad Sci USA
2002, 99:11043-11048.
14. Warren JT, Petryk A, Marques G, Jarcho M, Parvy JP, Shinoda T,
Itoyama K, Kobayashi J, Jarcho M, Li Y, O'Connor MB, Dauphin-Ville-
mant C, Gilbert LI: Phantom encodes the 25-hydroxylase of
Drosophila melanogaster and Bombyx mori: a P450 enzyme
critical in ecdysone biosynthesis. Insect Biochem Mol Biol 2004,
34:991-1010.
15. Namiki T, Niwa R, Sakudoh T, Shirai K, Takeuchi H, Kataoka H:
Cytochrome P450 CYP307A1/Spook: a regulator for ecdys-
one synthesis in insects. Biochem Biophys Res Comm 2005,
337:367-374.
16. Ono H, Rewitz KF, Shinoda T, Itoyama K, Petryk A, Rybczynski R, Jar-
cho M, Warren JT, Marques G, Shimell MJ, Gilbert LI, O'Connor MB:
Spook and spookier code for stage-specific components of the
biosynthetic pathway in Diptera. Dev Bio 2006,
298(2):555-5780. Epub 2006 Jul 29.
17. Sztal T, Chung H, Gramzow L, Daborn PJ, Batterham P, Robin C:
Two independent duplications forming the Cyp307a genes in
Drosophila. Insect Biochem Mol Biol 2007, 37:1044-1053.
18. Daphnia genome database [http://wFleaBase.org
]
19. DOE Joint Genome Institute [http://jgi.doe.gov/
]
20. NCBI [http://ncbi.nlm.nih.gov/
]
21. Feyereisen R: Insect cytochrome P450. In Comprehensive Molecu-
lar Insect Science Volume 4. Edited by: Gilbert LI, Iatrou K. Gill S: Else-
vier; Oxford; 2005:1-77.
22. Lafont R, Dauphin-Villemant C, Warren JT, Rees H: Ecdysteroid
chemistry and biochemistry. In Comprehensive Molecular Insect
Science Volume 3. Edited by: Gilbert LI, Iatrou K. Gill S: Elsevier;
Oxford; 2005:125-195.
23. Lisurek M, Bernhardt R: Modulation of aldosterone and cortisol
synthesis on the molecular level. Mol Cell Endocrinol 2004,
215:149-159.
24. Bülow HE, Bernhardt R: Analyses of the CYP11B gene family in
the guinea pig suggest the existence of a primordial CYP11B
gene with aldosterone synthase activity. Eur J Biochem 2002,
269:3838-3846.
25. Claudianos C, Ranson H, Johnson RM, Biswas S, Schuler MA, Beren-
baum MR, Feyereisen R, Oakeshott JG: A deficit of detoxification
enzymes: pesticide sensitivity and environmental response
in the honeybee. Insect Mol Biol 2006, 15:615-636.
26. Yoshiyama T, Namiki T, Mita K, Kataoka H, Niwa R: Neverland is
an evolutionarily conserved Rieske-domain protein that is
essential for ecdysone synthesis and insect growth. Develop-
ment 2006, 133:565-2574.
27. FlyBase [http://flybase.bio.indiana.edu/
]
28. Rottiers V, Motola DL, Gerisch B, Cummins CL, Nishiwaki K, Man-
gelsdorf DJ, Antebi1 A: Hormonal control of C. elegans Dauer
formation and life span by a Rieske-like oxygenase. Dev Cell
2006, 10:473-482.
29. Rewitz KF, Rybczynski R, Warren JT, Gilbert LI: The Halloween
genes code for cytochrome P450 enzymes mediating synthe-
sis of the insect moulting hormone. Biochem Soc Trans 2006,
34:1256-1260.
30. Rene Feyereisen's website for insect CYPs [http://p450.anti
bes.inra.fr/]
31. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins D: The
CLUSTALX windows interface: flexible strategies for multi-
ple sequence alignment aided by quality analysis tools.
Nucleic Acids Res 1997, 25:4876-4882.
32. Galtier N, Gouy M, Gautier C: SEAVIEW and PHYLO_WIN:
two graphic tools for sequence alignment and molecular
phylogeny. Comput Appl Biosci 1996, 12:543-548.
33. Guindon S, Gascuel O: A simple, fast, and accurate algorithm
to estimate large phylogenies by maximum likelihood. Syst
Biol 2003, 52:696-704.
34. Daphnia Genomics Consortium [http://daphnia.cgb.indiana.edu/
]
35. Motola DL, Cummins CL, Rottiers V, Sharma KK, Li T, Li Y, Suino-
Powell K, Xu HE, Auchus RJ, Antebi A, Mangelsdorf1 DJ: Identifica-
tion of ligands for DAF-12 that govern Dauer formation and
reproduction in C. elegans. Cell 2006, 124:1209-1223.
Page 8
    • "Nonetheless, ecdysteroids titer and its contribution for regulating the complex processes of the embryogenesis has not been addressed. In daphnids, recent studies have identified five Halloween genes in D. pulex (Rewitz and Gilbert, 2008), and nvd, shd and Cyp18a1 in D. magna (Sumiya et al., 2014 ). Additionally, D. magna EcR was reported to bind to 20E as in insects (Kato et al., 2007; Supplementary InformationTable 1). "
    [Show abstract] [Hide abstract] ABSTRACT: Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd.
    No preview · Article · Feb 2016 · Journal of Applied Toxicology
  • Source
    • "Curiously, the presence of ouib only in the Drosophilidae genomes is concordant with the Drosophilidae-specific duplication of Cyp307a P450 subfamily. While members of the Cyp307 P450 subfamily, which includes spok, are found in all arthropod species examined so far [20,21,57,58] , Drosophilidae Cyp307 genes have been duplicated within the Drosophila radia- tion [21,59]. In the case of D. melanogaster, the duplicated Cyp307 genes are Cyp307a1/spo and Cyp307a2/spok, which are sub-functionally divergent in terms of gene expression pattern; spo is expressed in early embryogenesis and oogenesis, while spok is expressed in the PG cells in late embryogenesis as well as the larval and pupal stages [20,21]. "
    [Show abstract] [Hide abstract] ABSTRACT: Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.
    Full-text · Article · Jan 2016 · PLoS Genetics
  • Source
    • "Other than in insects, their biosynthetic pathway has only been described in two crustaceans (Rewitz and Gilbert 2008; Sin et al. 2014) and a mite (Cabrera et al. 2015). In both insects and crustaceans, the ecdysteroid 20E is synthesized from dietary cholesterol via cytochrome P450 enzymes encoded by the Halloween genes (Rewitz et al. 2007; Rewitz and Gilbert 2008; Sin et al. 2014;fig. 2A ). "
    [Show abstract] [Hide abstract] ABSTRACT: The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the " Broad-Complex " was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor (" Methoprene-tolerant "). Furthermore, the gain of " Phantom " differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.
    Full-text · Article · Jun 2015 · Genome Biology and Evolution
Show more