Báo cáo khoa học: Identification of an atypical insect olfactory receptor subtype highly conserved within noctuids potx

The OR18 gene was studied in details in the cotton leafworm, Spodoptera littoralis, where it presented all the characteristic features of an olfactory receptor encoding gene: its express

subtype highly conserved within noctuids

Isabelle Brigaud1, Nicolas Montagne´2, Christelle Monsempes1, Marie-Christine Franc¸ois1and Emmanuelle Jacquin-Joly1

1 INRA, UMR PISC, UMR-A 1272, Versailles, France

2 UPMC Universite´ Paris 6, UMR PISC, Paris, France

Introduction

The insect noctuid family includes devastating

agricul-tural pests As nocturnal animals, they depend strongly

on olfactory cues to detect food and mates Therefore,

their olfactory system is an attractive target for their

control Odour reception is primarily mediated by the

large family of olfactory receptors (ORs) that ensure

the specificity of the olfactory receptor neurone (ORN) responses ORs are expressed on the surface of ORN dendrites that are housed in morphofunctional units, distributed along the antennae – the olfactory sensilla Intense efforts to identify insect ORs are currently being undertaken, as their G-protein-coupled receptor

Keywords

Lepidoptera; Noctuidae; olfaction; olfactory

receptor; phylogeny

Correspondence

E Jacquin-Joly, INRA UMR 1272

INRA-UPMC PISC Physiologie de l’Insecte:

Signalisation et Communication, route de

Saint-Cyr, F-78000 Versailles, France

Fax: (33) 1 30 83 31 19

Tel: (33) 1 30 83 32 12

E-mail: jacquin@versailles.inra.fr

(Received 24 June 2009, revised 31 July

2009, accepted 3 September 2009)

doi:10.1111/j.1742-4658.2009.07351.x

Olfaction is primarily mediated by the large family of olfactory receptors Although all insect olfactory receptors share the same structure with seven transmembrane domains, they present poor sequence homologies within and between species As the only exception, Drosophila melanogaster OR83b and its orthologues define a receptor subtype singularly conserved between insect species In this article, we report the identification of a new subtype of putative olfactory receptors exceptionally conserved within noct-uids, a taxonomic group that includes crop pest insects Through homol-ogy-based molecular cloning, homologues of the previously identified OR18 from Heliothis virescens were identified in the antennae of six noc-tuid species from various genera, presenting an average of 88% sequence identity No orthologues were found in genomes available from diverse insect orders and selection pressure analysis revealed that the noctuid OR18s are under purifying selection The OR18 gene was studied in details

in the cotton leafworm, Spodoptera littoralis, where it presented all the characteristic features of an olfactory receptor encoding gene: its expression was restricted to the antennae, with expression in both sexes; its develop-mental expression pattern was reminiscent of that from other olfactory genes; and in situ hybridization experiments within the antennae revealed that the receptor-expressing cells were closely associated with the olfactory structures, including pheromone- and non-pheromone-sensitive structures Taken together, our data suggest that we have identified a new original subtype of olfactory receptors that are extremely conserved within noctuids and that might fulfil a critical function in male and female noctuid chemo-sensory neurones

Abbreviations

GPCR, G-protein-coupled receptor; OR, olfactory receptor; ORN, olfactory receptor neurones; PR, pheromone receptor; qPCR, quantitative real-time PCR.

(GPCR)-like structure may open up the way for the

design of agonist and⁄ or antagonist molecules based

on the pharmacological know-how accumulated on

GPCRs

ORs were first discovered in vertebrates in 1991 [1],

but, because of extreme sequence divergence, insect

ORs were not discovered until the Drosophila

melanog-aster genome was sequenced [2–5] Since then, insect

OR-encoding genes have been mainly identified

through bioinformatics analysis of complete or partial

available genomic databases Complete or partial sets

of OR genes are now available from various insects,

including 12 species of the Drosophila genus [6], the

mosquitoes Anopheles gambiae [7] and Aedes aegypti

[8], the lepidopterans Bombyx mori [9,10] and

Helio-this virescens [11,12], the honeybee Apis mellifera [13]

and the red flour beetle Tribolium castaneum [14,15]

All insect ORs identified so far share common features:

like GPCRs, they belong to the superfamily of seven

transmembrane domain receptors; they are exclusively

expressed in chemosensory organs; and the expressing

cells are located beneath the chemosensory sensilla

However, they present a pronounced intra- as well as

interspecific sequence diversity (20–40% sequence

iden-tity) [16] This poor sequence conservation has halted

industrial interest, precluding the elaboration of

broad-spectrum products for crop protection

As an exception, one particular set of insect ORs

defines a unique subtype of receptors [17] This subtype

groups receptors singularly conserved between insect

species, the so-called D melanogaster OR83b

(Dme-lOR83b) orthologues Their high conservation level

(60–90% sequence identity) has allowed the isolation

of their counterparts in various insect orders through

homology cloning [17–22], whereas this strategy has

failed for most other OR types The DmelOR83b

protein does not appear to encode an OR per se, but

heterodimerizes with conventional ORs, enabling their

correct trafficking and functionality [23,24]

Interest-ingly, DmelOR83b orthologues can retain their

func-tion when expressed in D melanogaster, although there

is as yet no direct proof of their function in vivo in any

other species apart from D melanogaster [25]

Although, at first glance, this receptor subtype could

appear to be the ideal universal target to disturb insect

olfaction, members are also found in beneficial insects

such as the honeybee [17], thus precluding the use of

molecules interfering with OR83b receptors

Another subfamily of insect ORs, restricted to

moths, can also be distinguished but with lower

con-servation This family consists of the pheromone

recep-tors (PRs) that share an average of 40% identity

Members have been identified in several moth species,

thanks to genomic data analyses [11,26,27], differential screening [9,28] and homology cloning strategies [29] These receptors are predominantly male specific, and some have been shown to respond to pheromones [9,29,30] As most of the moth species are severe crop pests, disruption of the moth pheromone communica-tion system, through the use of synthetic pheromones,

is currently an efficient strategy The design of com-pounds affecting PRs may allow the development of novel strategies, but their relatively high divergence in sequence will require a species-specific approach

In this article, we used homology cloning strategies to identify a new moth subtype of highly conserved candi-date ORs, the OR18 subtype Six full-length cDNAs encoding ORs highly related to the H virescens OR18 [11] were identified in representative noctuid species: the Amphipyrinae Spodoptera littoralis and Sesamia nonag-rioides, the Heliothinae Helicoverpa zea and Heli-coverpa armigera, the Noctuinae Agrotis segetum, and the Hadeninae Mamestra Brassicae (all crop pests) Interestingly, gene subtype members could be identified only in noctuids, and we present evidence that these receptors are under purifying selection

A detailed study of S littoralis OR18 revealed typi-cal features of insect ORs Its expression is restricted

to the antennae and it is expressed late in development and in association with olfactory sensilla Taken together, our data suggest that this new original sub-type of ORs might play a specific role related to noc-tuid ecology and its conservation may offer a single target for noctuid control

Results and Discussion

The cloning of six H virescens OR18 homologues

in noctuid species revealed a new highly conserved subtype of candidate ORs Through homology cloning strategies, six full-length cDNAs related to the H virescens OR HvirOR18, were identified from six species representative of the noctuid family The encoded proteins are 398–400 amino acids long and were named SlitOR18 (S litto-ralis), MbraOR18 (M brassicae), HzeaOR18 (H zea), HarmOR18 (H armigera), SnonOR18 (S nonagrio-ides) and AsegOR18 (A segetum) The Phobius tool revealed high probability for the occurrence of five to seven transmembrane domains depending on the pro-teins, but close examination of the hydropathy profiles and sequence alignment suggested the occurrence of seven transmembrane domains for all, occurring at similar positions (Fig 1A,B) OR18 sequences are characterized by an extraordinarily high sequence

I II

III

IV

V

VII

SlitOR18

Cell membrane

DmelOR83b

IC2

EC2

N-ter

C-ter

C-ter

N-ter

B

A

Fig 1 Noctuid OR18 sequences and predicted membrane topology (A) Alignment of amino acid sequences deduced from the HvirOR18, HzeaOR18, HarmOR18, SnonOR18, MbraOR18, AsegOR18 and SlitOR18 cDNAs Amino acids identical in the maximum sequences are marked with grey shading Arrows indicate the positions of the primers used in RT-PCR for gene fragment amplifications Transmembrane domains I–VII identified from Phobius [41] (http://phobius.sbc.su.se) are indicated (B) Representation of the transmembrane topology predic-tion for SlitOR18 compared with DmelOR83b Black, transmembrane domains; EC2, second extracellular loop; IC2, second intracellular loop.

identity, ranging from 82% (e.g SlitOR18⁄ SnonOR18

and SlitOR18⁄ AsegOR18 comparisons) to 98–99%

(e.g HvirOR18⁄ HzeaOR18, HvirOR18⁄ HarmOR18

and HarmOR18⁄ HzeaOR18 comparisons) Within the

OR18 sequences, the overall identity reached 77% and

even 92% homology Apart from the DmelOR83b

subtype, such a remarkable sequence conservation of

candidate ORs across species from different genera has

not been observed previously

A phylogenetic analysis was run using a

non-exhaus-tive repertoire of OR sequences identified in various

insect orders (Fig 2A) Three subtypes⁄ subfamilies

of insect ORs were clearly defined: the already

well-characterized OR83b subtype, the known moth PR

subfamily and a new subtype of insect ORs formed by HvirOR18 and its homologues identified in this study Like the members belonging to the OR83b and PR subfamilies, the OR18 candidates clustered into a monophyletic group, clearly distinct from the other insect ORs and supported by the bootstrap values (Fig 2A,B) These observations suggest that members

of the OR18 subtype are orthologues

OR18 orthologues were found only in noctuids and are under purifying selection

Interestingly, this well-supported group contained only lepidopteran sequences (in red, Fig 2A) This raised the

B

A

Fig 2 (A) Unrooted neighbour-joining tree

of ORs from Lepidoptera (in red) and from species representative of Hymenoptera (Apis mellifera, yellow), Diptera (Drosoph-ila melanogaster, green) and Coleoptera (Tribolium castaneum, blue) Bootstrap support values are based on 1000 repli-cates Nodes with high bootstrap support (over 90%) are marked by open circles and those with support between 70% and 90% are marked by filled circles The branch length is proportional to the genetic distance Three clades, grouping sequences from different species, are clearly visible: the already known moth pheromone recep-tor and OR83b clades, and a new clade grouping the OR18 sequences identified in this study (B) Detail of the clade containing the OR18 subtypes Note that all of these sequences only belong to the order Lepidoptera.

question of whether related receptor types may also exist

in insects other than noctuids To approach this

ques-tion, we used both RT-PCR experiments and blast

searches against available insect genomes Despite

several attempts, RT-PCR performed with total RNA

from the antennae of the Bombycidae B mori and the

Crambidae Ostrinia nubilalis – both representative

species of two other Lepidoptera families – gave no

amplification In blast searches against the National

Center for Biotechnology Information (NCBI) DNA

and protein databases, or directly towards partial or

complete sequenced genomes, the most significant

sequence matches did not exceed 36%, in accordance

with the absence of an amplicon in the B mori RT-PCR

experiments As complete OR repertoires are now

estab-lished in species representative of the major

holometab-olous insect orders (Diptera, Hymenoptera, Coleoptera,

Lepidoptera) and none of these ORs appears to share

significant identity with OR18 sequences, we concluded

that the OR18 subtype may be restricted to some

lepidopteran species, including the noctuids

Selection pressure on the OR18 gene subtype has

been studied by comparative analysis of synonymous

(dS) and nonsynonymous (dN) nucleotide divergence

This approach allows for the testing of evolutionary

selection scenarios, acting on protein coding sequences

Table 1 compares the dN⁄ dS values of the noctuid

OR18 and immediately related OR genes dN⁄ dS

val-ues are low for the OR18 gene subtype (0.009–0.131)

Assuming that all nucleotides have an equal

probabil-ity of changing over evolutionary time, the observation

of a disproportionate number of synonymous changes

(dN⁄ dS < 1) suggests purifying selection for noctuid

OR18s, confirmed by statistical analysis (P < 0.05)

This is consistent with the strong conservation of

OR18 genes across noctuids and also with a potential

essential functional role Indeed, such a low ratio

has also been observed for the OR83b gene, whose

essential function in insect olfaction is well established [23,24] Although dN⁄ dS ratios are relatively high for chemosensory genes across insects, such purifying selection has been proposed to be the main force gov-erning the evolution of chemosensory genes within the

D melanogaster group (reviewed in [31]) Thus, we extend such an evolutionary scenario within more dis-tantly related insect species To date, only a few OR genes have been identified in Lepidoptera Further OR gene identification within Lepidoptera, and particularly within noctuids, may reveal that purifying selection is

a more common force in the evolution of OR genes Like the OR18 subtype, other highly conserved OR subtypes may emerge

Detailed analyses of SlitOR18 revealed common features with insect ORs

The polyphagous S littoralis is an example of a crop pest The molecular characterization of its OR18 gene, SlitOR18, revealed expected features of genes belong-ing to the OR superfamily, thus confirmbelong-ing the newly identified genes as candidate OR encoding genes First, real-time PCR was used as a quantitative method to compare SlitOR18 expression levels in dif-ferent tissues (male and female antennae, brain, pro-boscis, abdomen and legs) As illustrated in Fig 3, SlitOR18 expression was restricted to the antennae, with negligible expression in the other tissues tested Within antennae, SlitOR18 was found to be almost equally expressed in males (55%) and females (43%) Second, the developmental expression pattern of SlitOR18 was established using RT-PCR on head⁄ antennal RNA from different stages of development

As shown in Fig 4A, SlitOR18 expression was detected only in late pupal stages (starting 2 days before emergence) and adulthood No expression was observed in embryos, fifth instar larvae heads and

Table 1 dN ⁄ dS in the OR18 subtype Values in bold italic indicate purifying selection (P < 0.05) that occurred in all the OR18 subtypes.

SlitOR18 MbraOR18 SnonOR18 AsegOR18 HzeaOR18 HarmOR18 HvirOR18 HvirOR20 BmorOR30 BmorOR33 MbraOR18 0.12

pupae antennae, collected 5 days before emergence.

This pattern of expression is similar to that of

previ-ously characterized olfactory genes in Lepidoptera

antennae [19,32,33] and coincides with the maturation

of the olfactory system

Third, in situ hybridization was performed to investi-gate more deeply the expression pattern of SlitOR18 in the adult male antennae In S littoralis, antennae are filiform and segmented The dorsal side is covered with scales, whereas the ventral side carries different morpho-logical⁄ functional types of sensilla, including the sensilla chaetica, the sensilla styloconica and the sensilla tricho-dea (Fig 5A) Two types of sensilla trichotricho-dea have been described: long and short, the latter being enriched in the middle of the ventral side These sensilla are devoted

to olfaction, the long ones being mainly tuned to phero-mone components and the short ones responding to both pheromone components and other chemicals [34]

A SlitOR18 sense strand probe gave no signal (Fig 5B) Antisense probe hybridizations were clearly restricted to the sensilla side of the antenna, with no signal on the scale side (Fig 5C) Labelled spots were clearly restricted to the bases of the olfactory sensilla of the trichodea type (Fig 5C,D) No staining could be observed at the base of either the sensilla chaetica (Fig 5E), known to be involved in mechano⁄ contact chemoreception [35], or the sensilla styloconica (Fig 5F), known to be involved in taste, suggesting that the expression of SlitOR18 should be confined to the olfactory sensilla As long and short sensilla trichodea are often intermingled in this species, their distinction was difficult in optical sections In some sections, entire long sensilla were visible, allowing us to clearly assign the expression of SlitOR18 to long sensilla trichodea (Fig 5C,D) In sections through the middle of the ventral surface (as in Fig 5F), the abundance and distri-bution of the labelled spots suggest SlitOR18 expression

in the short sensilla trichodea as well Thus, SlitOR18 seems to be expressed in different functional types of olfactory sensilla, including pheromone-sensitive and non-pheromone-sensitive Taken together, our data argue for a role of SlitOR18 in olfactory processes

Possible function for OR18 The relationship between OR sequences and functions

is the focus of intense research, and functional ortho-logues of ORs have been established only for the OR83b and PR subtypes Our data suggest that OR18 could play a critical role in olfaction within noctuids

To generate appropriate adaptive behaviours, insects need to sample salient features of the broad chemical environment Thus, the simplest hypothesis would be that OR18, expressed almost equally in the antennae

of both sexes, would respond to odorants particularly relevant to noctuid ecology Although OR18 sequences did not cluster within the PR clade (Fig 2A), the SlitOR18 expression pattern in male antennae suggests

0

0.5

1

1.5

Fig 3 Expression of SlitOR18 in different adult tissues using

real-time PCR Expression levels were calculated relative to the

expression of the rpL8 control gene, expressed as the ratio

ESlitOR18(DCT)SlitOR18 ⁄ E rpL8(DCT)rpL8 [45] and are reported relative to

the level in male antennae.

Eggs L5 E-5 E-2 E-1 E + 12 h E + 48 h

600 550

rpL8

650 Larvae

1000

500

2000

1500

cDNA gDNA

A

B

Fig 4 Temporal expression pattern of SlitOR18 using RT-PCR (A)

RT-PCRs were performed using the SlitOR18 primer pair and RNAs

isolated from the heads of fifth instar larvae (larvae L5), antennae

from pupae collected x days before emergence (pupae E-x) and

antennae from adults collected y hours after emergence (adult

E + yh) rpL8, control (B) PCRs were performed on adult antennal

cDNA (cDNA) and genomic DNA (gDNA), revealing different-sized

amplification products, as a control to exclude any gDNA

contami-nation in cDNA samples PCR products were analysed on agarose

gels and visualized by UV illumination after ethidium bromide

stain-ing The positions of marker bands (bp) are indicated.

expression in pheromone-sensitive sensilla Thus, their

function as pheromone receptors could not be

excluded Alternatively, OR18 may be expressed in an

ORN co-compartmentalized with a PR-expressing

ORN within the same sensillum, but unresponsive to

any pheromone-related odorants Indeed, such a

situa-tion has been described recently in H virescens male

antennae [36], giving new insights into the complex sex

pheromone detection system of moths [37]

Interest-ingly, OR18s share common features with the

noncon-ventional OR83b orthologues: they are highly

conserved; they are expressed at the bases of olfactory

sensilla with different functional properties; and their

protein structure exhibits a particularly long loop

between transmembrane segments IV and V (Fig 1B),

a feature not observed in conventional insect ORs

However, OR18 may not fulfil a general function in insect olfaction like OR83b, as the OR18 subtype is only found in noctuids In DmelOR83b, the long loop between transmembrane domains IV and V is intra-cytoplasmic (IC2) as a result of inverse topology of the receptor compared with classical GPCRs (Fig 1B), and it has been proposed to link OR83b to the intra-cellular transport machinery [24] The OR18 fourth loop presents no homology with DmelOR83b IC2, and

a PROSITE pattern search resulted in no hit with any known protein motif In addition, although the mem-brane topology of OR18 proteins has not been investi-gated experimentally to date, Phobius predicted an extracellular localization for this loop in all the OR18 sequences, defining it as the second extracellular loop (EC2, Fig 1B), which is incompatible with a function

sc

lst

lst

st

v

d

sch

C

lst

sc trachea

ssty sst

or

lst

Fig 5 Expression pattern of SlitOR18 in adult male antennae by in situ hybridization (A) Scheme of a longitudinal section of two antennal segments showing the distribution of scales (sc) on the dorsal side (d), and long and short sensilla trichodea (lst, sst), sensilla chaetica (sch) and sensilla styloconica (ssty) on the ventral side (v) or, ornamentations (B–F) Longitudinal sections of hybridized adult male antennae (B) Sense probe control (C) Consecutive antennal segments with antisense probe staining restricted to the ventral side carrying olfactory sen-silla, with no labelling on the dorsal scaled side (D) Detail of long sensilla trichodea showing intense labelling at the base (black arrows) Sensilla chaetica (E) and styloconica (F) are unstained (white arrows) Scale bars, 200 lm (A–C); 10 lm (D–F).

in intracellular trafficking In mammals, the EC2 loop

of certain types of GPCR may be critical for ligand

binding and affinity [38] Thus, if the classical GPCR

topology of OR18 were confirmed, OR18 EC2 might

also play an important role in ligand binding Further

functional analyses will help to answer these questions

and to define the exact role of the OR18 subtype

The discovery of an original subtype of extremely

conserved ORs opens up new routes for the

under-standing of insect OR evolution In particular, further

insect genome sequencing, some already under way,

may confirm that the OR18 subtype is restricted to

noctuids and may reveal other new conserved

types From an applied point of view, the OR18

sub-type offers a single target for the design of molecules

to interfere with the olfactory process in widespread

species – at least among the noctuids – but preserving

beneficial insects that lack such receptors

Materials and methods

Insect rearing and tissue collection

S littoralis, M brassicae, H armigera and S nonagrioides

were reared on a semi-artificial diet in the laboratory at

light : dark cycle until emergence A segetum, B mori and

(Lund University, Lund, Sweden), C Royer (UNS, Lyon,

France) and F Marion-Poll [Physiologie de l’Insecte:

Signali-sation et Communication (PISC), Paris, France],

respec-tively Antennae from H zea were generously provided by T

Baker (Pennsylvania State University, University Park, PA,

USA) Tissues from S littoralis whole embryos, L5 larvae

(heads), different pupal stages (male antennae) and adults

(male and female antennae and male brains, proboscis,

abdo-men, legs) were dissected and used for cDNA synthesis For

RNA extraction and cDNA synthesis

For RT-PCR, total RNAs were extracted from the different

USA) After a DNase1 step treatment (Promega, Madison,

WI, USA), single-stranded cDNAs were synthesized from

1 lg of total RNAs with 200 U of M-MLV reverse

trans-criptase (Clontech, Mountain View, CA, USA) using buffer

(Clontech)

For 5¢ and 3¢ rapid amplification of cDNA ends (RACE)

PCR, cDNAs were synthesized from 1 lg of male antennal

transcriptase (200 U, Gibco BRL, Invitrogen), using the 3¢-CDS primer (for 3¢ RACE) or the 5¢-CDS primer and the SMART II oligonucleotide (for 5¢ RACE), supplied

in the SMART RACE cDNA amplification kit (Clon-tech), following the manufacturer’s instructions

For quantitative real-time PCR (qPCR), RNAs were extracted from S littoralis male and female antennae (10 of each sex), brains (5), proboscis (5), abdomens (2) and legs

which included a DNase treatment Single-stranded cDNAs were synthesized from 1 lg of total RNAs as above

Molecular cloning of H virescens OR18 homologues

Two degenerate primers were designed from the H virescens OR18 amino acid sequence (accession number: AJ748333 [11]): OR18F (5¢-GTGCTYTRTTTCCTATTTATGCTGG-3¢) and OR18R (5¢-GTAATCAAAGTGAAGAARGARTAAG AAG-3¢) They were used for PCR amplifications of A sege-tum, H zea, H armigera, M brassicae, S nonagrioides,

templates with PCR Mastermix (Promega) through 40 cycles

for B mori and O nubilalis (no amplification) After gel purification (GenElute; Sigma-Aldrich, St Louis, MO,

plasmid (Invitrogen) Recombinant plasmids were isolated

by mini preparation (QIAprep Spin Miniprep Kit, Qiagen), and both strands were sequenced (Biofidal, Vaulx-en-Velin, France) The 3¢ and 5¢ regions of the cDNAs were amplified

by 3¢ and 5¢ RACE, using the Advantage 2 polymerase mix (Clontech) and the Universal Primer Mix versus the follow-ing gene-specific primers: 5¢RACE primer (used for all species), 5¢-GTGCTYTRTTTCCTATTTATGCTGG-3¢; 3¢RACE primers: Aseg3¢Race, 5¢-CTGGCATGGGGCTAGTCGTCTTCGAC ATGG-3¢; Mbra3¢Race, 5¢-CCGGGATGGGGCTCATCG TCTTCAATATGG-3¢; Snon3¢Race, 5¢-CCGGGATGGAT CTTGTCGTCTTTGACATGG-3¢; Harm3¢Race, 5¢-CCGG TATGGGGCTTGTGGTCTTCAACATGG-3¢; Hzea3¢Race, 5¢-CCGGCATGGGGCTTGTGGTCTTCAACATGG-3¢; Slit3¢Race, 5¢-CTGGGATGGGCATAGTGGTGTTTAAT ATGG-3¢ Touchdown PCRs were performed as follows:

on both strands and analysed as described above By merging overlapping 3¢, 5¢ and internal fragment sequences, six full-length cDNAs encoding putative open reading frames were generated and named SlitOR18 (S littoralis),

(H armigera), SnonOR18 (S nonagrioides) and AsegOR18

(A segetum) Nucleotide sequence data are available in the

GenBank database under the accession numbers: SlitOR18,

EU979124; MbraOR18, EU979123; HzeaOR18, EU979121;

HarmOR18, EU979122; SnonOR18, EU979119; AsegOR18,

EU979120

Sequence analyses and phylogenetic inference

Gene sequence analyses and database comparisons were

performed using the blast program [39] OR18 homologue

sequences were searched in GenBank and in insect available

genomes via the blastp program on protein databases

(Anobase: NCBI Map Viewer at http://www.ncbi.nlm.nih

gov/mapview/map_search.cgi?taxid=7165, RefSeq protein

data-base; Beebase: http://genomes.arc.georgetown.edu/beebase/

blast/blast.html, PreRelease2_protein database; Beetlebase:

protein sequences downloaded from ftp://bioinformatics.ksu

edu/pub/BeetleBase/3.0/; Butterflybase: http://www.butterfly

base.ice.mpg.de/, All species, Protein database; Flybase:

http://flybase.bio.indiana.edu/, Annotated Protein database;

Silkbase: http://silkworm.genomics.org.cn/silkdb/, Silkworm

Annotated Protein database) Alignment was performed

using clustalW2 [40] Transmembrane topology was

pre-dicted with the phobius tool [41], and protein motifs were

searched against all patterns stored in the PROSITE pattern

database For the phylogenetic analysis, the seven noctuid

OR18 amino acid sequences were included in a dataset

con-taining full-length lepidopteran OR sequences, together with

OR sequences identified from genomes of D melanogaster,

putative OR sequences, identified from this last species, only

the sequences of ORs expressed in adult tissues were included

[15] After alignment and removal of nonconserved residues,

the dataset contained 418 taxa and 378 characters An

un-rooted tree was inferred from this dataset by the

neighbour-joining method, with distance correction based on a Dayhoff

PAM matrix, as implemented in mega4 software [42] Node

support was assessed by a bootstrap procedure based on

1000 replicates

substitution calculations, nucleotide sequences were aligned

with their corresponding amino acid sequences using

Nei–Gojobori model [43] with Jukes–Cantor correction, as

implemented in mega4 [42] Evolutionary selection was

assessed using the Z-test (mega4) with the alternative

hypothesis of purifying selection (dN < dS)

Developmental studies in S littoralis

The temporal expression pattern of SlitOR18 was analysed

by RT-PCR carried out on cDNAs from whole embryos,

larvae heads, pupae antennae and adult antennae, using the

degenerated primer pair and the PCR conditions mentioned

above, generating a 750 bp band The ribosomal protein L8

gene (rpL8) was used as an RNA extraction control, as described previously [44], generating a 580 bp fragment In parallel, PCR was conducted with the same primer pair on genomic DNA, as a control to exclude contamination of the RNA preparations with genomic DNA This PCR led

to the amplification of a 1500 bp band (Fig 4B) The

750 bp bands obtained in the different cDNA samples then specifically reflected transcript amplification Amplification products were loaded on 1.5% agarose gels and visualized

by ethidium bromide staining

qPCR analyses in S littoralis tissues Gene-specific primers for SlitOR18 (SlitOR18F: 5¢-GCTG GGACCTTGATGAGTATTG-3¢; SlitOR18R: 5¢-CACGC ATTGGACGCAGTTATAG-3¢) and the endogenous con-trol rpL8 (rpL8F: 5¢-ATGCCTGTGGGTGCTATGC-3¢;

designed using Beacon Designer 4.0 software (Bio-Rad, Hercules, CA, USA), yielding PCR products of 150 and

210 bp, respectively qPCR mix was prepared in a total volume of 20 lL with 10 lL of Absolute QPCR SYBR Green Mix (ABgene, Epsom, UK), 5 lL of diluted cDNA (or water for the negative control, or RNA for controlling for the absence of genomic DNA) and 200 nm of each primer qPCRs were performed on S littoralis cDNAs pre-pared from male antennae, female antennae, male brains, proboscis, abdomen and legs using an MJ Opticon Monitor Detection System (Bio-Rad) The PCR programme began

hold, and allowed an assessment of the purity of the PCRs Standard curves were generated by a five-fold dilution series of a cDNA pool evaluating primer efficiency

control, two replicates of biological samples and dilution points SlitOR18 expression levels were calculated relative

to the expression of the rpL8 control gene and expressed as

SlitOR18 expression pattern in antennae Digoxygenin-labelled RNA sense and antisense probes (427 bp long) were reverse transcribed in vitro from PCR fragments amplified from the recombinant plasmid

Reverse primers, using T7 and SP6 RNA polymerases (Pro-mega) and following the recommended protocol SlitOR18 RNA probes were then purified on RNA G50 Sephadex columns (Quick Spin columns; Roche Applied Science, Indianapolis, IN, USA) The hybridization protocol was performed on whole-mount pieces of antennae, as described previously [46] After hybridization and embedding, longi-tudinal sections (6 lm) were prepared and counter-stained

with acridine orange and photographed Pictures were

Systems Inc., San Jose, CA, USA)

Acknowledgements

We thank Fabien Tissier (UMR PISC, INRA

Versailles, France) for help with insect rearing, Hadi

Quesneville (URGI, INRA Versailles, France) for help

with dN⁄ dS calculations, David Tepfer (Pessac, INRA

Versailles, France) for English improvement, and

Christer Lo¨fsted (Lund University, Lund, Sweden),

Corinne Royer (UNS, Lyon, France), Frederic

Marion-Poll (PISC, Paris, France) and Tom Baker

(Pennsylva-nia State University, University Park, PA, USA) for

providing insects or antennae from the different species

used in this work This work was supported by INRA,

Universite´ Paris VI, ACI JC5249 funding, as well as an

ACI JC doctoral fellowship to Isabelle Brigaud

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