Halloween genes and nuclear receptors in ecdysteroidbiosynthesis and signalling in the pea aphid

O. Christiaens*§, M. Iga*§, R. A. Velarde†, P. Rougé‡and G. Smagghe*

*Laboratory of Agrozoology, Department of CropProtection, Ghent University, Ghent, Belgium;†Department of Biology, Wake Forest University,Winston-Salem, NC, USA; and ‡Université de Toulouse,UMR 152 IRD-Université Paul Sabatier, Faculté desSciences Pharmaceutiques, Toulouse, France

Abstractimb_957 187..200

The pea aphid (Acyrthosiphon pisum) is the firstwhole genome sequenced insect with a hemi-metabolic development and an emerging modelorganism for studies in ecology, evolution anddevelopment. The insect steroid moulting hormone20-hydroxyecdysone (20E) controls and coordinatesdevelopment in insects, especially the moulting/metamorphosis process. We, therefore present here acomprehensive characterization of the Halloweengenes phantom, disembodied, shadow, shade, spookand spookiest, coding for the P450 enzymes thatcontrol the biosynthesis of 20E. Regarding the pres-ence of nuclear receptors in the pea aphid genome,we found 19 genes, representing all of the sevenknown subfamilies. The annotation and phylogeneticanalysis revealed a strong conservation in the classof Insecta. But compared with other sequenced insectgenomes, three orthologues are missing in theAcyrthosiphon genome, namely HR96, PNR-like andKnirps. We also cloned the EcR, Usp, E75 and HR3.Finally, 3D-modelling of the ligand-binding domain ofAp-EcR exhibited the typical canonical structuralscaffold with 12 a-helices associated with a shorthairpin of two antiparallel b-strands. Upon docking,20E was located in the hormone-binding groove,supporting the hypothesis that EcR has a role in20E signalling.

Keywords: ecdysteroid biosynthesis, nuclear recep-tors, ecdysteroid cascade, pea aphid, development,moulting, metamorphosis.

Introduction

The insect steroid hormone, 20-hydroxyecdysone (20E)controls and coordinates the development in insects. Apeak titer of 20E triggers the moulting/metamorphosisprocess, allowing insects to lose their old exoskeleton andenter into the next developmental stage. To produce thesteroid hormone 20E, the precursor hormone ecdysone (E)is synthesized in the prothoracic glands from dietary cho-lesterol or phytosterols as insects cannot synthesize thesteroid precursor cholesterol de novo. This E is thensecreted into the body cavity and converted into 20E invarious peripheral tissues predominantly in the insectmidgut and fat body (Petryk et al., 2003; Rewitz et al.,2006a; Iga & Smagghe, 2009). The whole traffickingmechanism of the ecdysteroid precursors has not beenelucidated yet in insects, but it may use similar mechanismsas the steroidogenesis in vertebrates (Rees, 1995; Gilbertet al., 2002; Lafont et al., 2005; Huang et al., 2008).

Cytochrome P450 (CYP) enzymes, well known fortheir monooxygenase activity, constitute one of thelargest families and are distributed throughout a widevariety of living organisms, from bacteria to mammals(Werck-Reichhart & Feyereisen, 2000). To date, fourP450 enzymes, namely CYP306A1 (Phantom, Phm),CYP302A1 (Disembodied, Dib), CYP315A1 (Shadow,Sad) and CYP314A1 (Shade, Shd), involved in the ecdys-teroid biosynthesis have been identified and character-ized. As shown in Fig. 1, the products of phm, dib andsad sequentially convert the precursor of E, 2,22,25-trideoxyecdysone (ketodiol), into 22,2-dideoxyecdysone(ketotriol), 2-deoxyecdysone and E (Chavez et al., 2000;Warren et al., 2002, 2004; Niwa et al., 2004, 2005; Rewitzet al., 2006b). Further on, the product of shd mediates thelast step of the conversion from E into 20E (Petryk et al.,2003; Rewitz et al., 2006a; Maeda et al., 2008). In addi-tion, CYP307A1 (Spook, Spo), the paralogue gene of Spo,CYP307A2 (Spookier, Spok) and CYP307B1 (Spookiest,

Correspondence: Guy Smagghe, Laboratory of Agrozoology, Departmentof Crop Protection, Ghent University, 9000 Ghent, Belgium. Tel.: +32 92646150; fax: +32 9 2646239; e-mail: guy.smagghe@ugent.be

§Equally contributed.

InsectMolecular

Biology

Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200 doi: 10.1111/j.1365-2583.2009.00957.x

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200 187

Spot), involved in the initial conversion process from7-dehydrochoresterol into ketodiol are identified, but theirbiochemical functions are not well understood (Namikiet al., 2005; Ono et al., 2006). Spok has so far only beenidentified in Drosophila, while spot is identified in mosqui-toes (Aedes aegypti and Anopheles gambiae), honeybees (Apis mellifera) and red flour beetles (Tribolium cas-taneum). Together, they are called the Halloween genes.The Halloween genes have been identified/predicted inmultiple insect species (Niwa et al., 2004, 2005; Warrenet al., 2004; Sieglaff et al., 2005; Rewitz et al., 2006a,b,2007; Iga and Smagghe, 2009) and the function of thesegenes is characterized in the fruitfly (Drosophila melano-gaster), the silkmoth (Bombyx mori) and the tobaccohornworm (Manduca sexta). In addition to insects, theHalloween genes are also identified in the crustaceangenome of Daphnia pulex (Rewitz & Gilbert, 2008), sug-gesting a high conservation for ecdysteroid biosynthesisin the Arthropoda phylum.

After secretion into the hemolymph, 20E will start themoulting/metamorphosis process by acting directly uponthe transcriptional activity of specific target genes throughchromosome puffing. Ashburner (1973) proposed aformal model to explain control of the transcription of thevast network of genes whose activity is induced by the

hormone. As shown in Fig. 1, the first step in this cascadeis the binding of 20E to a postulated receptor protein, aheterodimer formed by the ecdysone receptor (EcR) andUltraspiracle (Usp), which are both members of thenuclear receptor (NR) superfamily (Henrich, 2005; Billaset al., 2009). Activation of this receptor complex initiatesand mediates the transcription of a number of otherNRs, in a cascade with at first expression of ‘early’, then‘early-late’ and finally ‘late’ genes for a successfulmoulting/metamorphosis.

The NR superfamily is a group of ligand-activated tran-scription factors which are present in various animals.Studies of these NRs in both mammals and arthropodsrevealed seven distinct subfamilies (NR0-NR6) in whichthese NRs can be classified. All of them possess a highly-conserved DNA-binding domain (DBD), containing twoC4-type zinc finger regions, that is responsible for bindingof the transcription factor to the DNA. Except the NR0superfamily, all NRs also contain a less-conserved ligandbinding domain (LBD) with which the receptor is able tobind its ligand. Unlike most other transcription factors,NRs can be activated by binding of small lipophilic ligandssuch as hormones and fatty acids that are capable ofgoing through the cell membrane. Besides moultingand metamorphosis, NRs are involved in, e.g. embryonic

ketodiolBlack box

(2 22 25dE)Phm

20E biosynthesis

cholesterolketotriol

2-deoxyecdysone

(2,22,25dE)

(2,22dE)

(2dE)

Spo/Spok/SpotDib

ecdysone20E

( )

Shd Sad

20E signaling

EcR

‘early’ genes

Usp

E75 E74 E93BR

‘early-late’ genes HR3 HR4 KR-H1 HR38 E78

FTZ-F1

‘late’ genes

Figure 1. Summary of the biosynthesis of20-hydroxyecdysone (20E) and the 20E regulatorycascade. In the upper part, the biosynthetic schemepresents the functions of the Halloween genes (Spo/Spok/Spot, Phm, Dib, Sad and Shd) that are boxed,while intermediate products are mentioned in bold. Inthe lower part, binding of 20E to the EcR-Uspcomplex starts the ecdysteroid cascade with theexpression of the so called ‘early’ genes (EcR, E75,BR, E74 and E93) that will then be responsible forthe upregulation of a set of ‘early-late’ genes(including HR3, HR4, HR38 and E78). Via FTZ-F1,the signal will eventually be passed on to the ‘late’genes. The nuclear receptors are boxed. Redraftedafter Rewitz et al. (2007) and Bonneton et al. (2008).

188 O. Christiaens et al.

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

development (King-Jones & Thummel, 2005), cell differ-entiation (Siaussat et al., 2007), reproduction (Raikhelet al., 1999), and are therefore also considered as impor-tant novel targets in pest insect control (Palli et al., 2005;Billas et al., 2009).

Recent genome projects of both vertebrate and insectspecies contributed a lot in identifying the different NRs. Intotal, 48 NRs are known in humans (Robinson-Rechaviet al., 2001), over 284 are present in Caenorhabditiselegans (Gissendanner et al., 2004), 49 in mouse and 47in rat (Zhang et al., 2004). In insects on the other hand,the number of NRs found is surprisingly lower. In Droso-phila, only 21 NR genes have been identified, 20 inAnopheles, 22 in Apis, 19 in Bombyx and 21 in Tribolium(Adams et al., 2000; Holt et al., 2002; Velarde et al., 2006;Bonneton et al., 2008; Cheng et al., 2008). The latterstudies also showed that these NRs, especially the DBDand LBD, are highly conserved in holometabolous insects.So far, however, no complete set of NRs from hemime-tabolous insects, which are indirect developers that donot undergo a pupal metamorphosis stage, has beendescribed. The recent genome project on the pea aphid,Acyrthosiphon pisum (International Aphid Genomic Con-sortium, 2010) gives us the unique opportunity to presenta comprehensive identification and characterization of theNRs in this important hemipteran insect, which is anemerging model organism for ecological, developmentaland evolutionary studies (Brisson & Stern, 2006; Stern,2008).

In this paper, we will first focus on the Halloween genesthat control the ecdysteroid biosynthesis pathway to buildup a peak titer of 20E hormone. In a second part, we willunravel the presence of the NRs in the pea aphid with anemphasis on understanding and identifying the pathway ofhormone signalling by 20E through a regulatory cascadeof NRs – especially the functional receptor formed by theheterodimer EcR-Usp – and also on ‘early’, ‘early/late’and ‘late’ genes. We performed a phylogenetic analysisto confirm the annotation and to investigate evolutionarytraits of the pea aphid in the phylum of Arthropoda.Besides, we also cloned the EcR, Usp, E75 and HR3.Finally, we constructed a 3D-modelling of the LBD ofAp-EcR to evaluate if it exhibits the typical canonical struc-tural scaffold with 12 a-helices, and then performed aligand docking to support the theory that EcR has a role in20E hormone signalling.

Results and discussion

Phylogenetic analysis of Halloween genes

The candidates of the A. pisum Halloween genes wereobtained from AphidBase (http://www.aphidbase.com/aphidbase/) by TBLASTN using the amino acid sequence

of Apis mellifera and T. castaneum. We found three can-didates of A. pisum spook (Ap-spo1, Ap-spo2 andAp-spo3), one candidate for phantom (Ap-phm) and dis-embodied (Ap-dib) and also three candidates for shade(Ap-shd1, Ap-shd2 and Ap-shd3). The AphidBase ID andcross reference number are shown in Table 1. The expres-sion of these predicted sequences was confirmed byreverse transcriptase-PCR (RT-PCR) (data not shown).

The predicted sequences have an open reading frame(ORF) encoding the putative protein of Ap-Spo1 (518amino acids), Ap-Spo2 (528 amino acids), Ap-Spo3(507 amino acids), Ap-Phm (492 amino acids), Ap-Dib(493 amino acids), Ap-Sad (443 amino acids), Ap-Shd1(518 amino acids), Ap-Shd2 (518 amino acids) andAp-Shd3 (505 amino acids). The product size of thesecandidates is consistent with the character of the differ-ent CYP products (approx. 500 amino acids). Alignmentof the different A. pisum Halloween genes candidateswith those of other insect orders (Lepidoptera, Diptera,Hymenoptera and Coleoptera) show high conservation ofinsect P450 motifs (helix-C, helix-I, helix-K, PERF-motifand heme-binding domain) (Fig. S1A–E). For Spo, thehelix-C and helix-I structures that are usually so typicalfor P450 proteins were not well conserved but this isconsistent with the P450 proteins in other members ofthe class of Insecta. Only the heme-binding domain ofAp-Shd3 shows a significant difference compared withthat of other insect orthologs. The Ap-Shd3 completelylacked the sequence of the heme-binding domain whichmeans the protein may not be functional at all. RT-PCRshowed however, that the protein is expressed in the peaaphid (data not shown). This leads to the hypothesis thatthe protein might have other functions or another role inthe pea aphid than just the ones attributed to the Shdproteins so far.

The result of phylogenetic analysis shows two classes:the 2 Clan with Spo/Spok/Spot and Phm, and the MitoClan with Dib, Sad and Shd (Fig. 2). In the A. pisumgenome we detected two Spo-like products, Ap-Spo1 andAp-Spo2. Both are on the same branch as Spo ortho-logues of other species, and sequence comparison showsthat both Ap-Spo1 and Ap-Spo2 are quite similar to eachother (85% identity). Ap-Spo3, however, is very differentfrom Ap-Spo1 and Ap-Spo2, showing only 38% and 39%identity, respectively. When we compare with other Hal-loween genes, we notice that Ap-Spo3 shows 33–42%identity with Spo/Spok orthologues, and 44–47% identitywith Spot orthologues, suggesting Ap-Spo3 might be aSpot orthologue rather than a Spo orthologue. In addition,phylogenetic analysis confirms this hypothesis since bothAp-Spo3 and Spot orthologues are branched together(Fig. 2). Three Shd candidates were identified in A. pisum.Two of them, Ap-Shd1 and Ap-Shd2 show a high conser-vation (94% identity), suggesting these could be

Halloween and NR genes in the pea aphid 189

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

Tab

le1.

Hal

low

een

gene

sid

entifi

edin

Acy

rtho

siph

onpi

sum

;in

clud

ing

Gen

bank

IDs

ofD

roso

phila

mel

anog

aste

r,A

pid

mel

lifer

aan

dTr

ibol

ium

cast

aneu

mar

eal

soin

clud

ed

Nam

eF

unct

ion

D.

mel

anog

aste

r(D

m)

T.ca

stan

eum

(Tc)

A.

mel

lifer

a(A

m)

Aph

idB

ase

IDR

efse

q

iden

tity

%

Dm

Am

Tc

CY

P30

7A1/

2sp

ook

/sp

ooki

erU

nkno

wn

AF

4844

15/

NM

_001

1109

90A

AJJ

0100

0951

–A

CY

PI0

0151

9(A

p-sp

o1)

XM

_001

9457

2643

/45

–52

AC

YP

I002

012

(Ap-

spo2

)X

M_0

0194

6260

43/4

3–

51C

YP

307B

1sp

ooki

est

Unk

now

n–

AA

JJ01

0011

63A

AD

G05

0050

80A

CY

PI0

0071

6(A

p-sp

o3)

XM

_001

9486

80–

4746

CY

P30

6A1

phan

tom

25-H

ydro

xyla

seA

F48

4413

XM

_963

384

XM

_391

946

AC

YP

I006

623

(Ap-

phm

)X

M_0

0194

7839

3438

39C

YP

302A

1di

sem

bodi

ed22

-Hyd

roxy

lase

AF

2375

60X

M_9

6915

9X

M_0

0112

2832

AC

YP

I006

729

(Ap-

dib)

XM

_001

9482

6444

4849

CY

P31

5A1

shad

ow2-

Hyd

roxy

lase

AY

0791

70X

M_9

6502

9X

M_3

9536

0A

CY

PI0

0097

3(A

p-sa

d)X

M_0

0194

4148

3236

38C

YP

314A

1sh

ade

20-H

ydro

xyla

seA

F48

4414

XM

_967

606

DQ

2440

74A

CY

PI0

0822

8(A

p-sh

d1)

XM

_001

9485

7242

5143

AC

YP

I006

755

(Ap-

shd2

)X

M_0

0194

8633

4250

43A

CY

PI0

0981

3(A

p-sh

d3)

XM

_001

9424

7038

4438

Inth

eca

seof

T.ca

stan

eum

CY

P30

7A1/

2an

dC

YP

307B

1,co

ntig

sw

ere

give

non

whi

chth

ege

neis

foun

d.Id

emfo

rA

pis

mel

lifer

aC

YP

307B

1.O

nth

erig

ht,

iden

tity

perc

enta

ges

betw

een

A.

pisu

mse

quen

ces

and

the

resp

ectiv

eor

thol

ogs

are

also

pres

ente

d.

Ag-Shd

Aa-Shd

Dm-Shd

Ms-Shd

Bm-Shd

Am-Shd

100

100

97

68

100

Mito

Ap-Shd3

Ap-Shd2

Ap-Shd1

Tc-Shd

Aa-Dib

Ag-Dib

100

100

99

100

100

100

99

o Clan

Dm-Dib

Ms-Dib

Bm-Dib

Am-Dib

Tc-Dib

Ap-Dib

100

85

58

100

100

Am-Sad

Ap-Sad

Ag-Sad

Aa-Sad

Dm-Sad

Tc-Sad

Bm-Sad

100

94100

Bm-Sad

Ms-Sad

Am-Phm

Tc-Phm

Ms-Phm

Bm-Phm

Dm-Phm

100

100

63

92

2 Clan

Ag-Phm

Aa-Phm

Ap-Phm

Aa-Spot

Ag-Spot

Am-Spot

100

81

100

99

67100

n

Tc-Spot

Ap-Spo3

Ap-Spo2

Ap-Spo1

Tc-Spo

Ms-Spo

B S

100

100

52

100

100

Bm-Spo

Ag-Spo

Aa-Spo

Dm-Spo

Dm-Spok

100

71

68

77

0.2

Figure 2. Phylogenetic tree of the Halloween genes. This tree wasconstructed using the neighbour-joining method performed with theamino acid sequences of the whole sequences. Bootstrap values aspercentage of a 1000 replicates >50 are indicated on the tree. Aa:Aedes aegypti, Ag: Anopheles gambiae, Am: Apis mellifera, Ap:Acyrthosiphon pisum, Bm: Bombyx mori, Dm: Drosophila melanogaster,Ms: Manduca sexta, Tc: Tribolium castaneum.

190 O. Christiaens et al.

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

duplicated genes, while another Shd candidate, Ap-Shd3,only shows 66% identity with both Ap-Shd1 and Ap-Shd2.Ap-Shd1 and Ap-Shd2 show 38–51% and 38–50% iden-tity with Shd orthologues, respectively, while Ap-Shd3exhibits 34–44% identity to those same Shd orthologues.As we described before, Ap-Shd3 will probably not func-tion as a P450 enzyme since it lacks the necessary heme-binding domain which is important for P450 enzymeactivity. We can therefore assume that only Ap-Shd1and/or Ap-Shd2 are likely to be responsible for convertingE into 20E.

Identification of the nuclear receptors in the genome ofAcyrthosiphon pisum displays strong conservationin insects

All available Gene prediction sets (Gnomon, Augustus,Genscan, GeneID) and all available A. pisum sequencedata were used to identify the NRs in the pea aphidgenome. The in silico detection of NRs in the genome isgreatly facilitated by the strongly conserved DBD andLBD regions that characterize these NRs (Table 2). Blastsearches were performed using peptide sequences of allknown NRs from D. melanogaster, Apis mellifera and T.castaneum. As a result, an initial 20 NR sequences wereidentified in the A. pisum genome, representing all of theseven NR subfamilies. Predicted mRNA sequences andgene models were also manually edited if necessaryusing the Apollo Genome Annotation Curation Tool(Lewis et al., 2002). After further analysis, two NR0sequences turned out to be duplicated Knirps-like (Kni-like) genes, which bring the total set of different NRgenes to 19.

RT-PCR was used to confirm the presence of the pre-dicted NR mRNAs in the transcriptome of the pea aphid(data not shown). All NR mRNAs were picked up byRT-PCR, except for the HR83 gene. We did not manage toget a conclusive result for this gene despite using severaldifferent primer pairs. Some of them resulted in clearsingle bands, but the fragment size was not as would bepredicted based on the annotated gene. This means thateither we picked up a wrong fragment, or the exon/intronprediction of the gene is incorrect. Further sequencing ofthis fragment should give us more information about theidentity of this fragment and about the transcription of thisgene. These results prove that the complete set of NRsfound in the A. pisum genome, except HR83, is tran-scribed and none of them are pseudogenes.

Table 2 presents all the pea aphid orthologues for eachof the previously annotated D. melanogaster (Adamset al., 2000), Apis mellifera (Velarde et al., 2006), B. mori(Cheng et al., 2008) and T. castaneum (Bonneton et al.,2008) NRs. Similar numbers of NRs were found in thesefive insect genomes. All NRs are also structurally very

similar to their orthologues. All of them possess a DBDand LBD, except for the NR0 subfamily, which onlycontains a DBD. As could be predicted from previousanalyses of NRs, pairwise alignments of the conserveddomains of D. melanogaster and A. pisum NRs show avery high (71–99%) convergence for DBDs while theLBDs are more divergent (26–97%; with 77% identity forHR39 being the second highest). The most divergent NRsare HR83 (NR2E5) and TLL (NR2E2), while SVP (NR2F3)shows the least divergence.

In general, these results prove that NRs have a verystrong conservation among insects, also outside theholometabolous insect group. All pea aphid NRs showsimilar identity percentages for its orthologues as the iden-tity percentages which were reported in earlier NR anno-tation publications, where the NRs in T. castaneum andApis mellifera were compared with the NRs in D. melano-gaster, even though we would expect bigger differencesbased on the evolutionary distances of these species. TheNRs that are part of the 20E regulatory cascade, the‘early’ gene E75 (NR1D3) and the ‘early-late’ genes HR3(NR1F4), HR4 (NR2A4), HR38 (NR4A4), E78 (NR1E1)and FTZ-F1 (NR5A3) also show the same kind of conver-gence as reported with other species, demonstrating thatall the main NR members of this cascade are present inthe pea aphid. One remarkable observation was theextremely high conservation of the SVP-LBD amonginsects, much more than for the other NRs (97%, 96% and99% compared with D. melanogaster, T. castaneum andApis mellifera orthologues, respectively). The latter phe-nomenon may suggest that the structure of SVP, theinsect orthologue of the vertebrate chicken ovalbuminupstream transcription factor (COUP-TF), is critical to itsfunction and is under strong selective pressure againstamino acid replacements in the LBD of the molecule. In D.melanogaster, where two isoforms of this protein areexpressed, SVP has multiple reported functions. It isrequired for the development of four of the eight photore-ceptors that develop in the ommatidia of the eye (Hiromiet al., 1993; Begemann et al., 1995; Kramer et al., 1995),it is a key component in the control of cell proliferation inMalpighian tubules (Kerber et al., 1998) and it also has animportant role as a regulator in the development of neu-roblasts by acting upon the Hunchback/Krüppel switchnecessary for neuroblast differentiation (Kanai et al.,2005). In Ae. aegypti, this protein also has an effect on thevitellogenesis by acting as a negative regulator in theecdysone receptor complex-mediated transactivation inthe fat body (Miura et al., 2002).

Three NRs which were previously found in other insectspecies seem to be missing in the A. pisum genome:namely the NR1 subfamily member HR96, the NR0 sub-family member Knirps (Kni), and the NR2 family memberPNR-like (NR2E6).

Halloween and NR genes in the pea aphid 191

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

Tab

le2.

Nuc

lear

rece

ptor

sin

Acy

rtho

siph

onpi

sum

NuR

eBA

SE

Nam

eP

rodu

ctD

roso

phila

mel

anog

aste

rTr

ibol

ium

cast

aneu

mA

pis

mel

lifer

aA

phid

Bas

eID

Ref

seq

Dm

/Ap

iden

tity

%Tc

/Ap

iden

tity

%A

m/A

pid

entit

y%

DB

DLB

DD

BD

LBD

DB

DLB

D

NR

1D3

Ecd

yson

e-in

duce

dpr

otei

n75

E75

NP

_524

133

TC

_124

40X

P_3

9379

0A

CY

PI0

0777

3X

M_0

0194

6050

9558

9574

9577

NR

1E1

Ecd

yson

e-in

duce

dpr

otei

n78

E78

NP

_524

195

TC

_039

35X

P_3

9652

7A

CY

PI0

0230

7X

M_0

0195

2697

9651

94*

6093

60

NR

1F4

Hor

mon

ere

cept

orlik

ein

46H

R3

NP

_788

303

TC

_089

09X

P_3

9212

8.3

(LO

C10

0162

388)

9753

100

7110

072

NR

1H1

Ecd

yson

ere

cept

orE

cRN

P_7

2445

6T

C_1

2112

NP

_001

0916

85.2

AC

YP

I001

692

XM

_001

9426

3288

6397

7599

74N

R2A

4H

epat

ocyt

enu

clea

rfa

ctor

4H

NF

4N

P_4

7688

7.2

TC

_087

26–

AC

YP

I009

409

XM

_001

9468

9388

7492

7888

–†

NR

2B4

Ultr

aspi

racl

eU

sp/R

XR

NP

_476

781

TC

_140

27N

P_0

0101

1634

.1A

CY

PI0

0593

4X

M_0

0194

7055

9253

9572

9575

NR

2D1

Hor

mon

ere

cept

orlik

ein

78H

R78

NP

_524

203

TC

_045

98X

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192 O. Christiaens et al.

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

HR96 is an orphan receptor belonging to the NR1 sub-family (NR1J1). It is closely related to the EcR itself and isbelieved to be related to the vertebrate vitamin D-receptor(VDR), PXR and CAR, all of which bind a wide variety ofxenobiotics (Laudet, 1997). HR96 is proven to play arole in the response of D. melanogaster to xenobioticsas reported by King-Jones et al. (2006), but a functionregarding development or metamorphosis has not beenreported yet. This NR is a part of the 20E signallingcascade and it is known that this ecdysteroid-induced NRcan bind to the hp27 20E response element. This sug-gests that HR96 can compete with EcR-Usp for binding toa common set of target sequences (Fisk & Thummel,1995), but since this NR has no known hormone ligand, itis difficult to speculate on its actual function regarding theecdysteroid cascade. The absence of this gene suggestsits role in this signalling pathway is redundant in aphids orbeing taken over by another protein or NR.

A member of the NR2 group that was initially identifiedin the honey bee, the NR2E6, and an orthologue forvertebrate photoreceptor-cell-specific nuclear receptors(PNRs), is missing in the A. pisum genome. This gene isalso missing in the Drosophila genomes, although it hasbeen identified in the T. castaneum genome. The absenceof NR2E6 in the A. pisum and Drosophila genomes is asecondary loss in these lineages. A function for NR2E6 inthe development of the compound eye has been proposedbased on mRNA ‘in situ’ localizations in the Apis melliferadeveloping compound eyes (Velarde et al., 2006). Thefact that A. pisum shares with D. melanogaster the vastmajority of the genes involved in compound eye differen-tiation (Shigenobu et al., 2009) suggests this gene hasbeen retained potentially to regulate lineage specific dif-ferences in compound eye architectures.

The third missing NR in Acyrthosiphon is Knirps(NR0A1), while we identified two paralogues of Knirps-likeand one orthologue of Eagle. The T. castaneum, Apismellifera, B. mori genomes also seem to lack a Knirpsorthologue gene, as seen in the phylogenetic tree of theNR0 subfamily members. In Drosophila, Knirps has beencharacterized as encoding a transcriptional repressorimportant for the segmentation pathway (Nauber et al.,1988). The two other genes in the NR0 group Knirps-like(knrl) and Eagle (egon) are present in the A. pisumgenome. Analysis of these genes in the honey beesuggested no direct involvement during segmentation(Dearden et al., 2006), as is the case in Drosophila.However, in the case of T. castaneum, Knirps-like hasbeen characterized as having specific functions duringhead segmentation (Cerny et al., 2008). Our A. pisumanalysis supports the notion that these genes have beenindependently duplicated in different insect lineages. Atleast in the case of Dipterans and Coleopterans, Knirpsand Knirps-like have retained an ancestral role during

segmentation, which has been likely lost from honey beesand pea aphids. Knirps-like and Eagle may also functionas transcriptional repressors, but it remains to be deter-mined in which pathways they participate.

Further phylogenetic analysis for nuclear receptors ofpea aphid in phylum of Arthropoda

Besides the phylogenetic analysis of the NR0 subfamily(Fig. 3), NRs from the 6 other different subfamilies (NR1–NR6) were also examined by phylogenetic analysis andcompared with NRs from several different species repre-senting the major insect orders, such as Lepidoptera,Diptera, Hymenoptera, Coleoptera, and also from Crusta-cea and Arachnida (Fig. 4). This phylogenetic analysisshowed that many of the NRs of A. pisum show closerelationship with the NRs of the human louse (Pediculushumanus), as has been observed for genes throughoutthe genome. The T. castaneum NRs also showed veryhigh convergence with both the A. pisum and P. humanusNRs for a number of NRs, even though the red flour beetleis a member of the Endopterygota, while the pea aphidand the human louse belong to the infraclass of theParaneoptera.

When we look at these phylogenetic trees in Fig. 4 indetail, we notice that A. pisum NRs show a much higherresemblance to the T. castaneum and Apis mellifera ortho-logues than to the Diptera and Lepidoptera NRs, whichoften cluster together in a separate branch, even branch-ing off before the Crustacea and Arachnida. This deviationfrom normal topology, as shown in the trees of Fig. 4A andB for EcR and E78, respectively, is due to a long branchattraction caused by an acceleration of evolutionary rate inthe Mecopterida line (Diptera + Lepidoptera). This is con-sistent with the earlier findings of Bonneton et al. (2008)who have discovered that some NRs in Mecopteridaspecies (Diptera + Lepidoptera), including EcR and E78,have undergone an increase in evolutionary rate. Otherphylogenetic trees also confirm their results (data notshown).

In order to distinguish the different NR2 subfamilygenes found in the genome, we also constructed phylo-genetic trees for this entire subfamily. The NR2 subfamilytree (Fig. 5) clearly shows that the PNR-orthologue, foundin T. castaneum and Apis mellifera is missing in the peaaphid. Genes are clustered together according to thegroup (A–F) they belong to.

Elements of the 20-hydroxyecdysoneregulatory cascade

The 20E signalling cascade, as mentioned earlier in thiswork, is involved in moulting/metamorphosis and develop-ment. NRs play a very important role in this signalling

Halloween and NR genes in the pea aphid 193

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

pathway. Binding of 20E to the EcR-Usp heterodimer isthe start of this signal. This complex, after binding to thehormone, will act as a transcription factor, immediatelyinducing expression of a number of ‘early’ genes, includ-ing the NRs E75 and HR96. These ‘early’ gene productswill then be responsible for the upregulation of a set of‘early-late’ genes, including the NRs HR3, HR4, E78 andHR39. Through FTZ-F1, this signal will then be passed on

to induce expression of the ‘late’ genes (Fig. 1; Table 2).So far, most attention in this field has gone to holometab-olous insects, which undergo a pupal metamorphosisstage. No extensive set of NRs for a hemimetabolousinsect has been identified until now. Even though the peaaphid still undergoes several larval stages, it is possiblethat there are differences between the moulting processesof hemi- and holometabolous insects.

Most of the NRs involved in the 20E regulatory cascadeproved to be present, not only in the genome of pea aphid,but also in its transcriptome, indicating they are expressedcorrectly. Only the HR96 gene, which was discussedabove, is missing from this set of ecdysone-inducible NRs.And since its function in the moulting/metamorphosis pro-cesses is still unclear, speculation about the implicationsfor the entire pathway are very difficult to make.

EcR and Usp, two NRs that are at the basis of the 20Esignalling cascade, were cloned and sequenced in orderto confirm the annotation and sequence of both genes(Fig. S2A, B). Primers used to pick up the fragment span-ning most of the cDNA are listed in Table S1. The EcR inA. pisum has the typical DBD and LBD found in its ortho-logues in other insects. The EcR-DBD shows the typicalC4 zinc finger domains in this protein, as is the case forthe P-box, the D-box and the A/T-box. Regarding theEcR-LBD we score a strong conservation, with 63%, 75%and 74% identity compared with the Drosophila, Triboliumand Apis orthologues, respectively. Furthermore, thetypical structure with 12 a-helices is well conserved. Bothretrieved sequences confirmed the in silico analysis. Twoother important NRs in the 20E signalling cascade, the socalled ‘early’ genes, E75 and HR3 were also cloned andpartially sequenced in order to confirm their presence inthe transcriptome (Fig. S2C, D).

3D-modelling of the ligand binding pocket of Ap-EcRand ligand docking

The 3D model built for Ap-EcR-LBD exhibits the canonicalstructural scaffold of the EcR-LDBs, made of 12 a-helicesassociated with a short hairpin of two antiparallelb-strands (Fig. 6A). In addition, docking of 20E into thehormone-binding groove of Ap-EcR-LBD revealed abinding scheme similar to that found for other EcR-LBD(e.g. from the beetles Leptinotarsa decemlineata, Tene-brio molitor and Anthonomus grandis) (Billas et al., 2003;Soin et al., 2009). Upon docking, the alkyl chain of thehormone becomes inserted into one of the two pocketslocated at the bottom of the hormone-binding groove(Fig. 6B). A network of nine hydrogen bonds connects thehormone to residues Glu20, Met56, Thr57, Ala 112 andTyr122, forming the binding groove (Fig. 6C). Stackinginteractions with aromatic residues Phe111 and Trp238help to complete the interaction.

DmKNRL

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NR0B1 MOUSE

NR0B2 HUMAN

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0.05 changes

100

74

97

77

100

100

100

100

100

100

Figure 3. Phylogenetic tree of the insect NR0 subfamily members,showing the clustering of the novel Acyrthosiphon pisum genes withtheir respective orthologues. Novel A. pisum members of this group arehighlighted in bold. The tree was rooted using the NR0B1 and NR0B2vertebrate sequences as outgroup. The tree was constructed using theneighbour-joining method with the maximum length of sequence,resulting in 160 complete aligned sites. Support for the branches, whenpresent, is indicated as a percentage of 1000 bootstrap replicates ofneighbour-joining. Am: Apis mellifera, Ap: Acyrthosiphon pisum, Dm:Drosophila melanogaster, Dpse: Drosophila pseudoobscura, Tc:Tribolium castaneum.

194 O. Christiaens et al.

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Experimental procedures

Annotation of Halloween and nuclear receptor genes

The 1.0 release of the A. pisum genome was used as a basis forthe bioinformatic analysis. Putative Halloween genes sequenceswere searched and obtained by TBLASTN, using the knownorthologues from Apis mellifera and T. castaneum against thecomplete scaffold collection of the pea aphid genome.

NR protein sequences from D. melanogaster, T. castaneumand Apis mellifera were first used in BlastP searches against theNCBI Gnomon version 1 predicted protein sequences to findputative A. pisum orthologues. In case no orthologues were foundin the pea aphid Gnomon protein data set, we searched the Acyr1.0 assembly of the pea aphid genome (International AphidGenomic Consortium, 2010, main paper) for homologoussequences using TBLASTN. After identification and localization inthe genome, genes were examined and manually edited if nec-essary using the Apollo Genome Annotation Curation Tool (Lewiset al., 2002). This editing was done based on alignments ofD. melanogaster, T. castaneum and Apis mellifera orthologuestogether with several gene prediction programs (Gnomon, Augus-tus, Genscan and GeneID).

Phylogenetic analysis

Whole amino acid sequences for the Halloween gene ortho-logues in Apis mellifera, T. castaneum, D. melanogaster, Ae.aegypti, An. gambiae, M. sexta and B. mori and for the nuclearreceptors of D. melanogaster, T. castaneum and Apis melliferawere collected from the GenBank database. The LBD and DBDsequences of B. mori, An. gambiae, Ae. aegypti, Culex quinque-fasciatus, P. humanus corporis and Daphnia magna wereretrieved by Blast searches of A. pisum LBD sequences againstthe GenBank database or against the species’ sequencedgenome if no GenBank entry was present. The chosen NRsequences were then aligned by CLUSTALW2/CLUSTALX2(Larkin et al., 2007). The trees were made by the neighbour-joining method using MEGA4 software (Tamura et al., 2007).Bootstrap analysis with 1000 replicates for each branch positionwas used to assess support for nodes in the tree (Felsenstein,1985).

Confirmation of transcription of the Halloween and NR genes

Presence of these transcripts in the A. pisum RNA was examinedby RT-PCR. The pea aphids were taken from a continuous colony

A EcR Culex LBD

EcR Aedes LBD

92

99 EcR Aedes LBD

EcR Anopheles LBD

EcR Drosophila LBD

EcR Bombyx LBD

EcR Tribolium LBD

EcR Pediculus LBD

99

96

100

78

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EcR Acyrthosiphon LBD

EcR Ixodes LBD

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E78 Aedes LBD

E78 Anopheles LBD

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84

100

100

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E78 Tribolium LBD

E78 Pediculus LBD

E78 Acyrthosiphon LBD

E78 Ixodes LBD

96

E78 Daphnia LBD

0.05

Figure 4. Phylogenetic trees of EcR (A), E78 (B),HR39 (C) and ERR (D). This tree was constructedusing the neighbour-joining method performed withthe amino acid sequences of the LBD of the selectedsequences. Bootstrap values as percentage of a1000 replicates > 50 are indicated on the tree.

Halloween and NR genes in the pea aphid 195

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

in the Laboratory of Agrozoology at Ghent University. A mixture ofdifferent stages of A. pisum and a collection of newborn aphidsonly was used to extract total RNA using the TRI Reagent (Sigma,Bornem, Belgium), based on the single-step liquid phase sepa-ration method reported by Chomczynski & Sacchi (1987). Then,cDNA was synthesized from 1 mg of this RNA in a 20 ml reactionusing the First Strand cDNA synthesis kit (Roche, Berlin,Germany) according to the manufacturer’s instructions. BothcDNA samples (newborn only and a mixture of stages) were usedin these RT-PCR experiments. Primers were designed usingPrimer3 software (Rozen & Skaletsky, 2000) and are listed inTable S1.

Cloning and sequencing of EcR and USP, E75 and HR3 genes

Pea aphid EcR and Usp, E75 and HR3 were isolated byRT-PCR and afterwards sequenced. Same A. pisum cDNA asused in 2.3 was used for the initial PCR reactions. The PCRproducts were then purified using the Cycle Pure kit (OmegaBio-Tek, Norcross, GA, USA) and were ligated into a pGEM-Tvector (Promega, Madison, WI, USA) according to the manu-facturer’s instructions. Afterwards, plasmids were transformed incompetent Escherichia coli XL-1 Blue Cells by heat shock andthen plated out on a carbenicillin-containing LB agar plate. After16 h incubation, formed colonies were checked by colony PCR

and several of these positive colonies were then purified usingPlasmid mini prep kit (Omega Bio-Tek) and sent for sequencing(Agowa, Berlin, Germany).

3D-modelling of the ligand binding pocket of Ap-EcR andligand docking

Multiple amino acid sequence alignments were carried out withCLUSTAL-X (Thompson et al., 1997) using the Risler’s structuralmatrix for homologous amino acid residues (Risler et al., 1998).Molecular modelling of the EcR ligand-binding domain (EcR-LBD) from the pea aphid (Acces. NP_001152831.1), Ap-EcR-LBD, was performed on a Silicon Graphics O2 R10000workstation, using the programs InsightII, Homology and Dis-cover3 (Accelrys, San Diego, CA, USA). The atomic coordinatesof Tribolium Tc-EcR-LBD in complex with ecdysone (RCSBProtein Data Bank code 2NXX) (Iwema et al., 2007) were usedto build the 3D model of the receptor. The high percentages ofboth identity (~75%) and similarity (~90%) that ApEcR-LBDshares with the template Tc-EcR-LBD allowed us to build quitean accurate 3D model. Steric conflicts were corrected duringthe model building procedure using the rotamer library (Ponder& Richards, 1987) and the search algorithm of the Homologyprogram (Mas et al., 1992) to maintain proper side-chain orien-tation. An energy minimization of the final model was carried out

C HR39 Aedes LBD100

98 HR39 Culex LBD

HR39 Anopheles LBD

HR39 Drosophila LBD

HR39 Bombyx LBD

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98

89

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HR39 Acyrthosiphon LBD

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53

82

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ERR Drosophila LBD

96

100

83

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ERR Acyrthosiphon LBD

ERR Apis LBD

ERR Daphnia LBD

ERR Ixodes LBDERR Ixodes LBD

0.05 Figure 4. Continued.

196 O. Christiaens et al.

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by 150 cycles of steepest descent using the cvff forcefield ofDiscover. PROCHECK (Laskowski et al., 1993) was used toassess the geometric quality of the 3D model. In this respect,about 87% of the residues of the modelled Ap-EcR-LBD werecorrectly assigned to the best-allowed regions of the Ramachan-dran plot. The remaining residues were located in the gener-ously allowed regions of the plot except for three residues(Asn36, Glu39 and Glu42), which occur in the non-allowedregion (result not shown). Molecular cartoons were drawn withPyMol (W.L. DeLano, http://pymol.sourceforge.net). The fold rec-ognition program Phyre (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html) (Bennett-Lovsey et al., 2008), which also used 2NXXand structurally related proteins as templates, yielded a readilysuperposable 3D model for Ap-EcR-LBD. However, some dis-crepancies that essentially deal with the shape of the loops,

connecting the a-helical stretches, were observed with our lab-made, modelled structure. Importantly, these discrepanciesoccur far from the groove responsible for the binding ofecdysone.

Docking was performed with InsightII using Discover3 as aforcefield and we took TcEcR-LBD in complex with ecdysone asa template for docking. Clipping planes of Ap-EcR-LBD com-plexed to 20E were rendered with PyMol.

Acknowledgements

The authors are grateful for the support of the SpecialResearch Fund of Ghent University and the Fund ofScientific Research (FWO-Vlaanderen, Belgium) to GS.

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Figure 5. Phylogenetic tree of the NR2 subfamilymembers with NR2A, NR2B, NR2D, NR2E andNR2F. This tree was constructed using theneighbour-joining method performed with thefull-length protein sequences of NR2 subfamilymembers. The PNR-like NRs found in Triboliumcastaneum and Apis mellifera are also added,although no orthologue in the pea aphid could befound. Bootstrap values as percentage of a 1000replicates >50 are indicated on the tree. Am: Apismellifera, Ap: Acyrthosiphon pisum, Dm: Drosophilamelanogaster, Tc: Tribolium castaneum.

Halloween and NR genes in the pea aphid 197

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

PR acknowledges the financial support of Université PaulSabatier and CNRS.

References

Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne,J.D., Amanatides, P.G et al. (2000) The genome sequence ofDrosophila melanogaster. Science 287: 2185–2195.

Ashburner, M. (1973) Sequential gene activation by ecdysone inpolytene chromosomes of Drosophila melanogaster. Dev Biol35: 47–61.

Begemann, G., Michon, A.M., Van der Voorn, L., Wepf, R. andMlodzik, M. (1995) The Drosophila orphan nuclear receptorSeven-up requires the Ras pathway for its function in photo-receptor determination. Development 121, 225–235.

Bennett-Lovsey, R.M., Hebert, A.D., Stemberg, M.J.E.and Kelley, L.A. (2008) Exploring the extremes of se-quence/structure space with ensemble fold recognition in theprogram Phyre. Proteins Struct Funct Bioinformatics 70: 611–625.

Billas, I.M.L., Iwema, T., Garnier, J.M., Mitschler, A., Rochel, N.and Moras, D. (2003) Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor. Nature426: 91–96.

Billas, I.M.I., Browning, C., Lawrence, M.C., Graham, L.D.,Moras, D. and Hill, R.J. (2009) The structure and function ofecdysone receptors. In Ecdysone: Structures and Functions(Smagghe, G., ed.), pp. 335–360. Springer, Dordrecht.

Bonneton, F., Chaumot, A. and Laudet, V. (2008) Annotation ofTribolium nuclear receptors reveals an increase in evolution-ary rate of a network controlling the ecdysone cascade. InsectBiochem Mol Biol 38: 416–429.

Brisson, J.A. and Stern, D.L. (2006) The pea aphid, Acyrthosi-phon pisum: an emerging genomic model system for ecologi-cal, developmental and evolutionary studies. Bioessays 28:747–755.

Cerny, A., Grossmann, D., Bucher, G. and Klinger, M. (2008) TheTribolium ortholog of knirps and knirps-related is crucial forhead segmentation but plays a minor role during abdominalpatterning. Dev Biol 321: 284–294.

Chavez, V.M., Marques, G., Delbecque, J.P., Kobayashi, K., Holl-ingsworth, M., Burr, J. et al. (2000) The Drosophila disembod-ied gene controls late embryonic morphogenesis and codesfor a cytochrome P450 enzyme that regulates embryonicecdysone levels. Development 127: 4115–4126.

Cheng, D., Xia, Q., Duan, J., Wei, L., Huang, C., Li, Z. et al.(2008) Nuclear receptors in Bombyx mori: insights intogenomic structure and developmental expression. InsectBiochem Mol Biol 38: 1130–1137.

A

B CB C

Figure 6. (A) Ribbon diagram of the modelledAp-EcR-LBD. The 12 a-helices and the two b-strandsforming the 3D-structure are labelled and differentlycoloured. N and C indicate the N-terminal andC-terminal ends of the polypeptide chain,respectively. (B) Clipping plane across theecdysone-binding groove showing the insertion of thealkyl chain of 20-hydroxyecdysone (20E)(represented in pink stick) in one (black star) of thetwo pockets located at the bottom of the groove. (C)Network of hydrogen bonds (black dotted lines)anchoring 20E (pink stick) to amino acid residuesforming the hormone-binding groove of Ap-EcR-LBD.Aromatic residues involved in stacking interactionswith 20E are coloured orange.

198 O. Christiaens et al.

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

Chomczynski, P. and Sacchi, N. (1987) Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159.

Dearden, P.K., Wilson, M.J., Sablan, L., Osborne, P.W., Havler,M., McNaughton, E. et al. (2006) Patterns of conservation andchange in honey bee developmental genes. Genome Res 16:1376–1384.

Felsenstein, J. (1985) Confidence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783–791.

Fisk, G.J. and Thummel, C.S. (1995) Isolation, regulation, andDNA-binding properties of three Drosophila nuclear hormonereceptor superfamily members. Proc Natl Acad Sci USA 92:10604–10608.

Gilbert, L.I., Rybczynski, R. and Warren, J.T. (2002) Control andbiochemical nature of the ecdysteroidogenic pathway. AnnuRev Entomol 47: 883–916.

Gissendanner, C.R., Crossgrove, K., Kraus, K.A., Maina, C.V.and Sluder, A.E. (2004) Expression and function of conservednuclear receptor genes in C. elegans. Dev Biol 266: 399–416.

Henrich, V.C. (2005) The ecdysteroid receptor. In Comprehen-sive Molecular Insect Science, Vol. 3 (Gilbert, L.I., Iatrou, K.and Gill, S., eds), pp. 243–286. Elsevier, Oxford.

Hiromi, Y., Mlodzik, M., West, S.R., Rubin, G.M. and Goodman,C.S. (1993) Ectopic expression of seven-up causes cell fatechanges during ommatidial assembly. Development 118:1123–1135.

Holt, R.A., Subramanian, G.M., Halpern, A., Sutton, G.G.,Charlab, R., Nusskern, D.R. et al. (2002) The genomesequence of the malaria mosquito Anopheles gambiae.Science 298: 129–149.

Huang, X., Warren, J.T. and Gilbert, L.I. (2008) New players in theregulation of ecdysone biosynthesis. J Genet Genomics 35:1–10.

Iga, M. and Smagghe, G. (2009) Identification and expressionprofile of Halloween genes involved in ecdysteroid biosynthe-sis in Spodoptera littoralis. Peptides, in press.

International Aphid Genomics Consortium (2010) Genomesequence of the pea aphid Acyrthosiphon pisum. PLoS Biol,DOI 10.1371/journal.pbio.1000313.

Iwema, T., Billas, I.M.L., Beck, Y., Bonneton, F., Nierengarten, H.,Chaumot, A. et al. (2007) Structural and functional character-ization of a novel type of ligand-independent RXR-USP recep-tor. EMBO J 26: 3770–3782.

Kanai, M.I., Okabe, M. and Hiromi, Y. (2005) Seven-up controlsswitching of transcription factors that specify temporal identi-ties of Drosophila neuroblasts. Dev Cell 8: 203–213.

Kerber, B., Fellert, S. and Hoch, M. (1998) Seven-up, the Droso-phila homolog of the COUP-TF orphan receptors, controlscell proliferation in the insect kidney. Genes Dev 12: 1781–1786.

King-Jones, K. and Thummel, C.S. (2005) Nuclear receptors – aperspective from Drosophila. Nat Rev Genet 6: 311–323.

King-Jones, K., Horner, M.A., Lam, G. and Thummel, C.S. (2006)The DHR96 nuclear receptor regulates xenobiotic responsesin Drosophila. Cell Metab 4: 37–48.

Kramer, S., West, S.R. and Hiromi, Y. (1995) Cell fate control inthe Drosophila retina by the orphan receptor seven-up: its rolein the decisions mediated by the ras signaling pathway. Devel-opment 121: 1361–1372.

Lafont, R., Dauphin-Villemant, C., Warren, J.T. and Rees, H.(2005) Ecdysteroid chemistry and biochemistry. In Compre-

hensive Molecular Insect Science, Vol. 3 (Gilbert, L.I., Iatrou,K. and Gill, S., eds), pp. 125–195. Elsevier, Oxford.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGet-tigan, P.A., McWilliam, H. et al. (2007) CLUSTALW andCLUSTALX Version 2. Bioinformatics 23: 2947–2948.

Laskowski R.A., MacArthur M.W., Moss D.S. and Thornton J.M.(1993) PROCHECK: a program to check the stereochemistryof protein structures. J Appl Cryst 26: 283–291.

Laudet, V. (1997) Evolution of the nuclear receptor superfamily:early diversification from an ancestral orphan receptor. J MolEndocrinol 19: 207–226.

Lewis, S.E., Searle, S.M.J., Harris, N., Gibson, M., Iyer, V., Ricter,J. et al. (2002) Apollo: a sequence annotation editor. GenomeBiol 3: research0082.1–research0082.14.

Maeda, S., Nakashima, A., Yamada, R., Hara, N., Fujimoto, Y.,Ito, Y. et al. (2008) Molecular cloning of ecdysone20-hydroxylase and expression pattern of the enzyme duringembryonic development of silkworm Bombyx mori. CompBiochem Physiol 149B: 507–516.

Mas, M.T., Smith, K.C., Yarmush, D.L., Aisaka, K. and Fine, R.M.(1992) Modeling the anti-CEA antibody combining site byhomology and conformational search. Proteins Struct FunctGenet 14: 483–498.

Miura, K., Zhu, J., Dittmer, N.T., Chen, L. and Raikhel, A.S. (2002)A COUP-TF/Svp homolog is highly expressed during vitello-genesis in the mosquito Aedes aegypti. J Mol Endocrinol 29:223–238.

Namiki, T., Niwa, R., Sakudoh, T., Shirai, K., Takeuchi, H. andKataoka, H. (2005) Cytochrome P450 CYP307A1/Spook: aregulator for ecdysone synthesis in insects. Biochem BiophysRes Commun 337: 367–374.

Nauber, U., Pankratz, M.J., Kienlin, A., Seifert, E., Klemm, U. andJaeckle, H. (1988) Abdominal segmentation of the Drosophilaembryo requires a hormone receptor-like protein encoded bythe gap gene knirps. Nature 336: 489–492.

Niwa, R., Matsuda, T., Yoshiyama, T., Namiki, T., Mita, K.,Fujimoto, Y. et al. (2004) CYP306A1, a cytochrome P450enzyme, is essential for ecdysteroid biosynthesis in the pro-thoracic glands of Bombyx and Drosophila. J Biol Chem 279:35942–35949.

Niwa, R., Sakudoh, T., Namiki, T., Saida, K., Fujimoto, Y. andKataoka, H. (2005) The ecdysteroidogenic P450 Cyp302a1/disembodied from the silkworm, Bombyx mori, is transcription-ally regulated by prothoracicotropic hormone. Insect Mol Biol14: 563–571.

Ono, H., Rewitz, K.F., Shinoda, T., Itoyama, K., Petryk, A., Ryb-czynski, R. et al. (2006) Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway inDiptera. Dev Biol 298: 555–570.

Palli, S.R., Hormann, R.E., Schlattner, U. and Lezzi, M. (2005)Ecdysteroid receptors and their applications in agriculture andmedicine. Vitam Horm 73: 59–100.

Petryk, A., Warren, J.T., Marqués, G., Jarcho, M.P., Gilbert, L.I.,Kahler, J. et al. (2003) Shade is the Drosophila P450 enzymethat mediates the hydroxylation of ecdysone to the steroidinsect molting hormone 20-hydroxyecdysone. Proc Natl AcadSci USA 100: 13773–13778.

Ponder, J.W. and Richards, F.M. (1987) Tertiary templates forproteins. Use of packing criteria in the enumeration of allowedsequences for different structural classes. J Mol Biol 193:775–791.

Halloween and NR genes in the pea aphid 199

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Insect Molecular Biology (2010), 19 (Suppl. 2), 187–200

Raikhel, A.S., Miura, K. and Segraves, W.A. (1999) Nuclearreceptors in mosquito vitellogenesis. Am Zool 39: 722–735.

Rees, H.H. (1995) Ecdysteroid biosynthesis and inactivation inrelation to function. Eur J Entomol 92: 9–39.

Rewitz, K.F. and Gilbert, L.I. (2008) Daphnia Halloween genesthat encode cytochrome P450s mediating the synthesis of thearthropod molting hormone: evolutionary implications. BMCEvol Biol 8: 60.

Rewitz, K.F., Rybczynski, R., Warren, J.T. and Gilbert, L.I.(2006a). Developmental expression of Manduca shade, theP450 mediating the final step in molting hormone synthesis.Mol Cell Endocrinol 247: 166–174.

Rewitz, K.F., Rybczynski, R., Warren, J.T. and Gilbert, L.I.(2006b). Identification, characterization and developmentalexpression of Halloween genes encoding P450 enzymesmediating ecdysone biosynthesis in the tobacco hornworm,Manduca sexta. Insect Biochem Mol Biol 36: 188–199.

Rewitz, K.F., O’Connor, M.B. and Gilbert, L.I. (2007) Molecularevolution of the insect Halloween family of cytochrome P450s:phylogeny, gene organization and functional conservation.Insect Biochem Mol Biol 37: 741–753.

Risler, J.-L., Delorme, M.O., Delacroix, H. and Henaut, A. (1998)Amino acid substitutions in structurally related proteins. Apattern recognition approach. Determination of a new an effi-cient scoring matrix. J Mol Biol 204: 1019–1029.

Robinson-Rechavi, M., Carpentier, A.S., Duffraisse, M. andLaudet, V. (2001) How many nuclear hormone receptorsare there in the human genome? Trends Genet 17: 554–556.

Rozen, S. and Skaletsky, H.J. (2000) Primer3 on the WWW forgeneral users and for biologist programmers. Methods MolBiol 132: 365–386.

Shigenobu, S., Bickel, R.D., Brisson, J.A., Butts, T., Chang, C.-C.,Christiaens, O et al. (2009) Comprehensive survey of devel-opmental genes in the pea aphid, Acyrthosiphon pisum:frequent lineage-specific duplications and losses ofdevelopmental genes. Insect Mol Biol, in press.

Siaussat, D., Bozzolan, F., Porcheron, P. and Debernard, S.(2007) Identification of steroid hormone signaling pathway ininsect cell differentiation. Cell Mol Life Sci 64: 365–376.

Sieglaff, D.H., Duncan, K.A. and Brown, M.R., 2005. Expressionof genes encoding proteins involved in ecdysteroidogenesis inthe female mosquito, Aedes aegypti. Insect Biochem Mol Biol35: 471–490.

Stern, D.L. (2008) Aphids. Curr Biol 18: 504–505.Soin, T., Iga, M., Swevers, L., Rougé, P., Janssen, C.R. and

Smagghe, G. (2009) Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysoneactivity: development of EcR reporter assays using weevil(Anthonomus grandis)-derived cell lines and in silico analysisof ligand binding to A. grandis EcR ligand-binding pocket.Insect Biochem Mol Biol 39: 523–534.

Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4:Molecular Evolutionary Genetics Analysis (MEGA) softwareversion 4.0. Mol Biol Evol 24: 1596–1599.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F.and Higgins, D.G. (1997) The CLUSTAL-X windows inter-face: flexible strategies for multiple sequence alignmentaided by quality analysis tool. Nucleic Acids Res 15: 4876–4882.

Velarde, R.A., Robinson, G.E. and Fahrbach, S.E. (2006) Nuclearreceptors of the honey bee: annotation and expression in theadult brain. Insect Mol Biol 15: 583–595.

Warren, J.T., Petryk, A., Marques, G., Jarcho, M., Parvy, J.P.,Dauphin-Villament, C., O’Connor, M.B. and Gilbert, L.I. (2002)Molecular and biochemical characterization of two P450enzymes in the ecdysteroidogenic pathway of Drosophilamelanogaster. Proc Natl Acad Sci USA 99: 11043–11048.

Warren, J.T., Petryk, A., Marques, G., Parvy, J.P., Shinoda, T.,Itoyama, K. et al. (2004) Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: aP450 enzyme critical in ecdysone biosynthesis. InsectBiochem Mol Biol 34: 991–1010.

Werck-Reichhart, D. and Feyereisen, R. (2000) CytochromesP450: a success story. Genome Biol 1: reviews 3003.1–reviews 3003.1–9.

Zhang, Z., Burch, P.E., Cooney, A.J., Lanz, R.B., Pereira, F.A.,Wu, J. et al. (2004) Genomic analysis of the nuclear receptorfamily: new insights into structure, regulation, and evolutionfrom the rat genome. Genome Res 14: 580–590.

Supporting Information

Additional Supporting Information may be found in theonline version of this article under the DOI reference: DOI10.1111/j.1365-2583.2009.00957.x

Figure S1. Amino acid sequence alignment of Spo/Spok/Spot (A), Phm(B), Dib (C) Sad (D) and Shd (E). Residues in black are identities and ingray are similarities. The conserved P450 motifs are indicated: helix-C,helix-I, helix-K, PERF-motif and heme-binding domain.

Figure S2. Amino acid sequences of the cloned and sequenced fragmentsof EcR (A), Usp (B), E75 (C) and HR3 (D). For EcR and Usp, the entire DBDhas been sequenced and the P box (yellow), D box (red) and T/A box (teal)are indicated. The eight zinc-coordinating cysteines in the DBD are boxed inthe sequence. For E75, only a part of the DBD has been sequenced.

Table S1. Primers used for the RT-PCR detection in the transcriptome ofthe Halloween (upper part) and nuclear receptor genes and for the cloningof EcR, Usp, E75 and HR3 (lower part) in the pea aphid. The primers weredesigned to overlap at least one intron, except for the KNRL and EG genes,which mainly consist out of one big exon and only possess a very smallsecond exon. Expected fragment sizes are also indicated in the table

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

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