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

Identification of an atypical insect olfactory receptor subtype highly conserved within noctuids ´ Isabelle Brigaud1, Nicolas Montagne2, Christelle Monsempes1, Marie-Christine Francois1 and ¸ Emmanuelle Jacquin-Joly1 INRA, UMR PISC, UMR-A 1272, Versailles, France ´ UPMC Universite Paris 6, UMR PISC, Paris, France 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) 30 83 31 19 Tel: (33) 30 83 32 12 E-mail: jacquin@versailles.inra.fr (Received 24 June 2009, revised 31 July 2009, accepted 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 noctuids, a taxonomic group that includes crop pest insects Through homology-based molecular cloning, homologues of the previously identified OR18 from Heliothis virescens were identified in the antennae of six noctuid 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 developmental 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 chemosensory neurones Introduction The insect noctuid family includes devastating agricultural 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 Abbreviations GPCR, G-protein-coupled receptor; OR, olfactory receptor; ORN, olfactory receptor neurones; PR, pheromone receptor; qPCR, quantitative real-time PCR FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6537 An atypical olfactory receptor subtype in noctuids I Brigaud et al (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 melanogaster 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 Heliothis 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 identity) [16] This poor sequence conservation has halted industrial interest, precluding the elaboration of broadspectrum 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 (DmelOR83b) 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] Interestingly, DmelOR83b orthologues can retain their function 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 conservation This family consists of the pheromone receptors (PRs) that share an average of 40% identity Members have been identified in several moth species, 6538 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 communication system, through the use of synthetic pheromones, is currently an efficient strategy The design of compounds 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 candidate 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 nonagrioides, the Heliothinae Helicoverpa zea and Helicoverpa 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 typical 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 subtype of ORs might play a specific role related to noctuid 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 littoralis), MbraOR18 (M brassicae), HzeaOR18 (H zea), HarmOR18 (H armigera), SnonOR18 (S nonagrioides) and AsegOR18 (A segetum) The Phobius tool revealed high probability for the occurrence of five to seven transmembrane domains depending on the proteins, 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 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS I Brigaud et al An atypical olfactory receptor subtype in noctuids A I II III IV V VI V VII B SlitOR18 N-ter DmelOR83b EC2 C-ter Cell membrane C-ter IC2 N-ter Fig 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 prediction for SlitOR18 compared with DmelOR83b Black, transmembrane domains; EC2, second extracellular loop; IC2, second intracellular loop FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6539 An atypical olfactory receptor subtype in noctuids I Brigaud et al 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-exhaustive repertoire of OR sequences identified in various insect orders (Fig 2A) Three subtypes ⁄ subfamilies of insect ORs were clearly defined: the already wellcharacterized 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 A B 6540 Fig (A) Unrooted neighbour-joining tree of ORs from Lepidoptera (in red) and from species representative of Hymenoptera (Apis mellifera, yellow), Diptera (Drosophila melanogaster, green) and Coleoptera (Tribolium castaneum, blue) Bootstrap support values are based on 1000 replicates 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 receptor 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 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS I Brigaud et al An atypical olfactory receptor subtype in noctuids question of whether related receptor types may also exist in insects other than noctuids To approach this question, 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 established in species representative of the major holometabolous 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 compares the dN ⁄ dS values of the noctuid OR18 and immediately related OR genes dN ⁄ dS values are low for the OR18 gene subtype (0.009–0.131) Assuming that all nucleotides have an equal probability 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 governing the evolution of chemosensory genes within the D melanogaster group (reviewed in [31]) Thus, we extend such an evolutionary scenario within more distantly 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 belonging to the OR superfamily, thus confirming 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 different tissues (male and female antennae, brain, proboscis, 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 days before emergence) and adulthood No expression was observed in embryos, fifth instar larvae heads and Table 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 BmorOR34 MbraOR18 SnonOR18 AsegOR18 HzeaOR18 HarmOR18 HvirOR18 HvirOR20 BmorOR30 BmorOR33 0.12 0.097 0.131 0.112 0.112 0.092 0.746 1.289 1.623 1.68 0.054 0.053 0.086 0.098 0.082 0.726 0.95 1.359 1.424 0.038 0.094 0.105 0.1 0.858 1.147 1.264 1.355 0.09 0.082 0.078 0.65 1.253 1.296 1.366 0.009 0.031 0.72 1.195 1.312 1.418 0.039 0.749 1.17 1.255 1.317 0.723 1.212 1.322 1.434 0.866 1.378 1.41 0.584 0.566 0.378 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6541 SlitOR18 transcript level relative to level in male antennae An atypical olfactory receptor subtype in noctuids I Brigaud et al 1.5 0.5 Fig 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 ⁄ ErpL8(DCT)rpL8 [45] and are reported relative to the level in male antennae Pupae Adult 48 E+ E+ E- E- E- L5 Eg gs 12 h h Larvae A 750 650 600 550 SlitOR18 gD cD NA B NA rpL8 2000 1500 1000 500 Fig 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 contamination in cDNA samples PCR products were analysed on agarose gels and visualized by UV illumination after ethidium bromide staining The positions of marker bands (bp) are indicated pupae antennae, collected days before emergence This pattern of expression is similar to that of previously characterized olfactory genes in Lepidoptera antennae [19,32,33] and coincides with the maturation of the olfactory system 6542 Third, in situ hybridization was performed to investigate 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 morphological ⁄ functional types of sensilla, including the sensilla chaetica, the sensilla styloconica and the sensilla trichodea (Fig 5A) Two types of sensilla trichodea 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 pheromone 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 distribution 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 orthologues 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 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS I Brigaud et al v d or An atypical olfactory receptor subtype in noctuids sst lst A B lst C lst sch ssty trachea Cuticule sc sc D sc E lst F st ssty sch Fig 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 sensilla, 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) 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 situation has been described recently in H virescens male antennae [36], giving new insights into the complex sex pheromone detection system of moths [37] Interestingly, OR18s share common features with the nonconventional 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 intracytoplasmic (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 intracellular 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 membrane topology of OR18 proteins has not been investigated 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 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6543 An atypical olfactory receptor subtype in noctuids I Brigaud et al 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 understanding 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 subtypes From an applied point of view, the OR18 subtype 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 20 °C and 60–70% relative humidity, under a 16 h : h light : dark cycle until emergence A segetum, B mori and O nubilalis pupae were generously provided by C Lofsted ¨ (Lund University, Lund, Sweden), C Royer (UNS, Lyon, France) and F Marion-Poll [Physiologie de l’Insecte: Signalisation et Communication (PISC), Paris, France], respectively 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, abdomen, legs) were dissected and used for cDNA synthesis For in situ hybridization, S littoralis male antennae were fixed overnight in 4% paraformaldehyde at °C, dehydrated in methanol and stored at )20 °C until use RNA extraction and cDNA synthesis For RT-PCR, total RNAs were extracted from the different tissues with TRIzolÒ reagent (Invitrogen, Carlsbad, CA, USA) After a DNase1 step treatment (Promega, Madison, WI, USA), single-stranded cDNAs were synthesized from lg of total RNAs with 200 U of M-MLV reverse transcriptase (Clontech, Mountain View, CA, USA) using buffer and protocol supplied in the Advantag RT-for-PCR kit (Clontech) For 5¢ and 3¢ rapid amplification of cDNA ends (RACE) PCR, cDNAs were synthesized from lg of male antennal RNA at 42 °C for 1.5 h, with SuperScriptÔ II reverse 6544 transcriptase (200 U, Gibco BRL, Invitrogen), using the 3¢-CDS primer (for 3¢ RACE) or the 5¢-CDS primer and the SMARTƠ II oligonucleotide (for 5¢ RACE), supplied in the SMARTÔ RACE cDNA amplification kit (Clontech), 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 (6) with the RNeasyÒ MicroKit (Qiagen, Hilden, Germany), which included a DNase treatment Single-stranded cDNAs were synthesized from 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 segetum, H zea, H armigera, M brassicae, S nonagrioides, S littoralis, B mori and O nubilalis antennal cDNA templates with PCR Mastermix (Promega) through 40 cycles at 94 °C for 30 s, 50 °C for 30 s and 72 °C for A single  740 bp fragment was amplified for all species, except for B mori and O nubilalis (no amplification) After gel purification (GenEluteÔ; Sigma-Aldrich, St Louis, MO, USA), fragments were cloned into the pCRÒII-TOPOÒ 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 AdvantageÔ polymerase mix (Clontech) and the Universal Primer Mix versus the following 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: at 94 °C, five cycles of 30 s at 94 °C and at 72 °C, then five cycles of 30 s at 94 °C, 30 s at 70 °C and at 72 °C, then 30 cycles of 30 s at 94 °C, 30 s at 68 °C and at 72 °C, followed by a final elongation step of 10 at 72 °C The PCR products were cloned, sequenced 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), MbraOR18 (M brassicae), HzeaOR18 (H zea), HarmOR18 (H armigera), SnonOR18 (S nonagrioides) and AsegOR18 FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS I Brigaud et al (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 database; 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 predicted 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 containing full-length lepidopteran OR sequences, together with OR sequences identified from genomes of D melanogaster, A mellifera and T castaneum Owing to the large number of 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 unrooted tree was inferred from this dataset by the neighbourjoining 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 For synonymous (dS) and nonsynonymous (dN ⁄ dS) substitution calculations, nucleotide sequences were aligned with their corresponding amino acid sequences using Tranalign (EMBOSS) dN ⁄ dS values were calculated using the 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 An atypical olfactory receptor subtype in noctuids 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 control rpL8 (rpL8F: 5¢-ATGCCTGTGGGTGCTATGC-3¢; rpL8R: 5¢-TGCCTCTGTTGCTTGATGGTA-3¢) were 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), 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 prepared from male antennae, female antennae, male brains, proboscis, abdomen and legs using an MJ Opticon Monitor Detection System (Bio-Rad) The PCR programme began with a cycle at 95 °C for 15 min, followed by 40 cycles of 20 s at 95 °C, 15 s at 55 °C and 20 s at 72 °C Melting curves were built every 0.2 °C from 50 to 95 °C with a s 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 E [E = 10()1 ⁄ slope)] All experiments included a no-template 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 the ratio ESlitOR18(DCT)SlitOR18 ⁄ ErpL8(DCT)rpL8 [45] 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 SlitOR18-pCRÒII-TOPOÒ with M13 Forward and M13 Reverse primers, using T7 and SP6 RNA polymerases (Promega) 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, longitudinal sections (6 lm) were prepared and counter-stained FEBS Journal 276 (2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6545 An atypical olfactory receptor subtype in noctuids I Brigaud et al with acridine orange and photographed Pictures were digitized and processed using AdobeÒ PhotoshopÒ 7.0 (Adobe 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 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(2009) 6537–6547 ª 2009 The Authors Journal compilation ª 2009 FEBS 6547 ... candidate olfactory receptor subtype highly conserved across different insect orders J Comp Physiol A 189, 519–526 18 Xia Y & Zwiebel LJ (2006) Identification and characterization of an odorant receptor. .. highly conserved and expressed in olfactory and gustatory organs Chem Senses 29, 403–410 22 Pitts RJ, Fox AN & Zwiebel LJ (2004) A highly conserved candidate chemoreceptor expressed in both olfactory. .. compilation ª 2009 FEBS I Brigaud et al An atypical olfactory receptor subtype in noctuids question of whether related receptor types may also exist in insects other than noctuids To approach this question,