An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research
© Legeai et al; licensee BioMed Central Ltd. 2011
Received: 16 April 2010
Accepted: 29 January 2011
Published: 29 January 2011
Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.
By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.
Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.
Olfaction serves to detect environmental chemical information. Nocturnal insects such as moths appear as ideal models to study the physiology of olfaction, since this sensory modality is essential for their survival and thus highly developed. In particular, the moth pheromone detection system is extremely sensitive: a male can smell and locate a female miles away for mating . It has been for long an established model to study the molecular bases of olfaction . In addition, moths include diverse and important pests of crops, forests and stored products. Olfaction underlies several behaviours critical for crop aggression, including sex pheromone-mediated reproduction, host selection and oviposition . It is thus an attractive target for pest control. For example, several olfactory-based strategies have been developed to control moth populations, such as mass trapping and mating disruption . Better knowledge on the molecular mechanisms by which an odour generates a neuronal signal could lead to the identification of targets for the development of new safe control strategies.
The olfactory signals are detected by the antennae, the peripheral olfactory organs, where they are transformed in an electrical signal that will be further integrated in the central nervous system. Located on the head, the antennae carry thousands of innervated olfactory structures, the sensilla, which house the olfactory receptor neurons. Within these sensilla, odour recognition relies on the expression of a diversity of olfactory genes involved in different steps (reviewed in ). First, volatile odours are bound by odorant-binding proteins (OBPs) in order to cross the aqueous sensillum lymph that embeds the olfactory neuron dendrites. The OBP family notably includes two sub-families: the pheromone-binding proteins (PBPs), thought to transport pheromone molecules, and the general odorant-binding proteins (GOBPs), thought to transport general odorants such as plant volatiles [6, 7]. Many other soluble secreted proteins are also found in abundance within the sensillum lymph, examples are the so-called chemosensory proteins (CSPs), the antennal binding proteins X (ABPX) and the sensory appendage proteins (SAPs) , but their role in olfaction remains elusive. After crossing the lymph, odorant molecules interact with olfactory receptors (ORs, called pheromone receptors or PRs when ligands are pheromones) located in the dendritic membrane of receptor neurons (reviewed in ). The chemical signal is then transformed into an electric signal that will be transmitted to the brain. Sensory neuron membrane proteins (SNMPs), located in the dendritic membrane of pheromone sensitive neurons [7, 10], are thought to trigger ligand delivery to the receptor . Signal termination may then be ensured by specific enzymes, the odorant-degrading enzymes (ODEs, called pheromone-degrading enzymes or PDEs when substrates consist of pheromones) (reviewed in ). Although we still lack a consensus on the exact function of each protein family, the occurrence of a large diversity within these families suggests they participate in the specificity of odour recognition . The combinatorial expression of these proteins within a sensillum may ensure the specificity and the sensitivity of the olfactory reception, defining the functional phenotypes of olfactory receptor neurons.
Complete or partial repertoires of putative olfactory genes have been established in insect species with an available sequenced genome. In other species for which no genomic data are yet available, such as crop pest moths, we still lack a global view of the olfactory genes. Homology-based cloning strategies led to the identification of conserved genes, such as OBPs , but failed to reliably identify divergent genes, in particular ORs. Insect ORs constitute an atypical family of seven transmembrane domain receptors exhibiting a pronounced intra - as well as inter-specific sequence diversity. As a result, OR repertoires have been established using the complete or partial genome databases of, among examples, the dipterans Drosophila melanogaster[13–15] and Anopheles gambiae, the hymenopterans Apis mellifera and Nasonia vitripennis, the coleopteran Tribolium castaneum and the lepidopteran B. mori[20, 21]. In other Lepidoptera, only few ORs and PRs have been identified to date [22–27]. Among them, one atypical subtype of ORs, defining the so-called D. melanogaster OR83b orthologue family, is required for the functionality of the other ORs [28, 29]. This subtype is highly conserved among insects and orthologues have been identified in numerous species, including a variety of moths [30, 31]. The identification of additional moth ORs and PRs is thus challenging. This will provide information on the evolution and diversification of this receptor family in this biodiverse group of insects and, in a context of plant protection, ORs appear as good targets for the design of molecules capable to interfere with the ligand and thus the receptor response and the associated insect behaviour.
Expressed Sequence Tag (EST) sequencing strategies are efficient in identifying a large number of genes expressed in a particular tissue, thus providing information on the physiological properties of this specific tissue. Such approaches are particularly relevant when no genomic data are available for the target species. EST collections are now established for various tissues in several Lepidoptera species, especially in B. mori, the only Lepidoptera for which the genome has been sequenced . However, only two EST strategies have been previously engaged on antennae. In 1999, Robertson et al  sequenced 300 ESTs from Manduca sexta antennae and identified a variety of candidate OBPs, but no ORs. In 2008, Jordan et al  sequenced 5739 ESTs from the antennae of the tortricid, Epiphyas postvittana, whose analysis revealed members of families implicated in odorant and pheromone binding (PBPs, GOBPs, ABPXs, CSPs) and turnover (putative ODEs). Only three genes encoding putative ORs were found, including one encoding an orthologue of the non-canonical odorant receptor OR83b from Drosophila.
In view of these difficulties in identifying ORs, we combined high-throughput sequencing and normalization of a cDNA library, prepared from the antennae of the cotton leafworm Spodoptera littoralis. This polyphagous noctuid species is one of the major pests of cotton, and much is known about its olfaction, thanks to previous behavioural and electrophysiological investigations: the sex pheromone, plant volatiles activating olfactory neurons, and various functional types of olfactory sensilla have been characterized . S. littoralis thus appears particularly well-suited to establish the molecular bases of olfactory and pheromone reception in a crop pest from the noctuid family, which groups some of the most aggressive herbivorous pests.
In this paper, we report the analysis and annotation of 20760 ESTs obtained from S. littoralis male antennae. First, this allowed us to establish the use of transcriptome sequencing to identify putative olfactory genes, and among them chemosensory receptor-encoding genes. We report on the identification of 31 candidate olfactory/gustatory receptor genes in a species for which no genomic data are available. Second, we provide evidence that the antennae express different non olfactory genes possibly involved in processes such as defense, plasticity and circadian rhythms. These EST resources will be invaluable for exploring the mechanism of olfaction and pheromone detection, but also other antennal processes, in a pest model species.
Results and discussion
EST statistics and unigene prediction
Identification of putative ORFs
The 678 sequences presenting no similarity with any known protein were analyzed using Interproscan  (Additional file 4). Interestingly, a sequence of this set presented a PBP/GOBP protein domain and appeared as a new original candidate OBP, in addition to the others we discovered (see paragraph below).
Specificity analysis using ESTs
Gene identification and functional annotation
Identification of putative enzymes and secretory proteins
Accession numbers of S. littoralis unigenes annotated as encoding Carboxylesterases (CXEs), Glutathione-S-transferases (GSTs), Cytochrome P450 (CYPs) and related enzymes.
NADPH CYP reductases
The olfactory process within the antennae is thought to be triggered by a large family of proteins, the so-called OBPs, secreted in the sensillum lymph . We thus found it relevant to search for secretory proteins through SignalP  in all the translated S. littoralis ORFs. A total of 636 translated ORFs (84.1%) were predicted to contain a signal peptide, among which 565 (88.8%) have matches with known proteins in the NR protein database. Among them, candidate binding proteins were found. A complete analysis of candidate OBPs found in the S. littoralis ESTs is detailed in the paragraph below. The remaining 11.2% of the putative S. littoralis secretory proteins did not share significant similarity with known proteins.
Identification of putative S. littoralis odorant-binding proteins
List of S. littoralis unigenes putatively involved in olfactory binding
Blastx best hit
gb|AAZ22339.1| pheromone binding protein 2 [Spodoptera litura]
gb|AAO16091.1| pheromone binding protein 3 [Helicoverpa armigera]
gb|ABQ84981.1| pheromone-binding protein 1 [Spodoptera littoralis]
General odorant-binding proteins
gb|ABM54824.1| general odorant-binding protein GOBP2 [Spodoptera litura]
gb|ABM54823.1| general odorant-binding protein GOBP1 [Spodoptera litura]
Other odorant-binding proteins
EZ983297 (EZ982153, FQ030234, FQ024265, FQ029312)
emb|CAC33574.1| antennal binding protein [Heliothis virescens]
gb|AAL60416.1|AF393491_1 antennal binding protein 2 [Manduca sexta]
emb|CAA05508.1| antennal binding protein X [Heliothis virescens]
FQ026764 (FQ031480, HO118380)
gb|AAL60413.1|AF393488_1 antennal binding protein 3 [Manduca sexta]
gb|AAL60425.1|AF393500_1 antennal binding protein 7 [Manduca sexta]
EZ981091 (FQ019778, FQ018991)
gb|AAX98168.1| pheromone binding protein 4 [Spodoptera frugiperda]
gb|AAO41344.1| Odorant-binding protein 59a [Drosophila melanogaster]
FQ021259 (FQ018920, FQ015705)
emb|CAR85645.1| odorant-binding protein 4 [Myzus persicae]
ref|NP_001140187.1| odorant-binding protein 3 [Bombyx mori]
ref|NP_001140191.1| odorant-binding protein 7 [Bombyx mori]
dbj|BAH79158.1| odorant binding protein [Bombyx mori]
EZ983259 (FQ026546, FQ020478)
dbj|BAH79159.1| odorant binding protein [Bombyx mori]
gb|AAK14793.1| sensory appendage protein-like protein [Mamestra brassicae]
gb|AAN63675.1|AF448448_1 chemosensory protein [Helicoverpa zea]
EZ983373 (GW825922 EZ982648 HO118345)
ref|NP_001037180.1| chemosensory protein CSP2 [Bombyx mori]
gb|ABM67686.1| chemosensory protein CSP1 [Plutella xylostella]
ref|NP_001037069.1| chemosensory protein 8 [Bombyx mori]
gb|AAV34687.1| chemosensory protein 2 [Heliothis virescens]
ref|NP_001037065.1| chemosensory protein 6 [Bombyx mori]
gb|ABM67689.1| chemosensory protein CSP2 [Spodoptera exigua]
ref|NP_001091779.1| chemosensory protein 11 [Bombyx mori]
Interestingly, using Interproscan, a new putative OBP type was found (FQ020630) that presented no sequence identity with any known insect OBPs (only 27% identity with its BLASTX best hit D. melanogaster OBP59a, e-value 0.27, unexpected for insect OBPs). The deduced encoded protein seemed to be complete (with start and stop codons) but SignalP analyses did not reveal the occurrence of a signal peptide, suggesting that this protein is not secreted or that we missed its N-terminal part. This latter hypothesis is supported by the fact that the amino acid sequence contains only five cysteine residues, one less than usually observed in insect OBPs. Alternatively, this sequence could encode a protein belonging to the Takeout or juvenile hormone-binding protein families, since these protein families also bind lipophilic molecules. However, alignment with Lepidoptera Takeout or juvenile hormone-binding proteins was not possible. Further molecular (eg localisation of the expressing cells) and functional (eg binding properties) analyses are now needed to definitely annotate this new protein as an OBP.
Although the best BLASTX hit for two unigenes, the contig EZ983259 and the singleton FQ014244, consisted of putative BmorOBPs, they could not be aligned with insect OBPs and both presented similarity with juvenile hormone-binding protein and Takeout-like proteins. Thus, they were not included in the following phylogenetic analyses.
Identification of S. littoralis candidate chemosensory receptors
31 candidate chemosensory receptors were identified in male antennae
The chemosensory receptor family includes ORs and gustatory receptors (GRs). In B. mori, the recently available sequenced genome offered the opportunity to identify the almost complete repertoires of ORs [20, 21] and GRs  in a lepidopteran species. 41 candidate B. mori ORs (BmorORs) previously identified  were compared to the unigenes using TBLASTN, leading to a first identification of 25 putative ORs in S. littoralis (SlitORs). Only six BmorORs gave no result when used as query sequences (BmorOR17, 18, 21, 22, 23 and 43). In parallel, the Interproscan result was scanned to retrieve sequences including one or more domains related to olfactory reception (IPR004117), resulting in the identification of seven putative ORs. Among them, three new sequences (EZ982994, EZ981047, FQ015038) were not identified during the precedent analysis using BmorORs. Additional searches using described insect OR families (D. melanogaster, A. gambiae, A. mellifera, T. castaneum, Aedes aegypti), as well as some isolated lepidopteran sequences [22–24, 26, 27] and additional BmorORs recently identified  led to the identification of a total of 35 candidate chemosensory receptor partial sequences: 33 olfactory receptors (SlitORs) and two gustatory receptors (SlitGRs). These S. littoralis sequences were in turn employed in searches to find more genes in an iterative process, which did not lead to the identification of additional candidates.
List of S. littoralis unigenes putatively involved in chemosensory reception
Blastx best hit
Candidate olfactory receptors
ref|NP_001103623.1| odorant receptor 33 [Bombyx mori]
ref|NP_001091790.1| candidate olfactory receptor [Bombyx mori]
dbj|BAH66326.1| olfactory receptor [Bombyx mori]
gb|ABQ82137.1| chemosensory receptor 2 [Spodoptera littoralis]
emb|CAG38117.1| putative chemosensory receptor 16 [Heliothis virescens]
EZ981960 (FQ025873, FQ021134)
emb|CAG38117.1| putative chemosensory receptor 16 [Heliothis virescens]
emb|CAD31853.1| putative chemosensory receptor 7 [Heliothis virescens]
ref|NP_001155301.1| olfactory receptor BmOr-60 [Bombyx mori]
dbj|BAH66361.1| olfactory receptor [Bombyx mori]
ref|NP_001103476.1| odorant receptor 35 [Bombyx mori]
dbj|BAG71423.2| olfactory receptor [Mythimna separata]
dbj|BAH66359.1| olfactory receptor [Bombyx mori]
emb|CAG38119.1| putative chemosensory receptor 18 [Heliothis virescens]
dbj|BAH66312.1| olfactory receptor [Bombyx mori]
emb|CAG38122.1| putative chemosensory receptor 21 [Heliothis virescens]
ref|NP_001104830.1| odorant receptor 41 [Bombyx mori]
gb|ACJ12929.1| odorant receptor 3 [Epiphyas postvittana]
tpg|DAA05977.1| TPA: TPA_ exp: odorant receptor 19 [Bombyx mori]
dbj|BAH66339.1| olfactory receptor [Bombyx mori]
emb|CAG38118.1| putative chemosensory receptor 17 [Heliothis virescens]
ref|NP_001104797.1| odorant receptor 30 [Bombyx mori]
gb|ACS45305.1| candidate odorant receptor 11 [Helicoverpa armigera]
emb|CAD31949.1| putative chemosensory receptor 8 [Heliothis virescens]
gb|ACM18061.1| putative odorant receptor OR3 [Manduca sexta]
ref|XP_ 312381.1| candidate odorant receptor (AGAP002558-PA) [Anopheles gambiae str. PEST
ref|NP_001091789.1| candidate olfactory receptor [Bombyx mori]
dbj|BAH66348.1| olfactory receptor [Bombyx mori]
ref|NP_001103476.1| odorant receptor 35 [Bombyx mori]
ref|NP_001116817.1| olfactory receptor-like receptor [Bombyx mori]
Candidate gustatory receptors
ref|XP_001848689.1| gustatory receptor 24 [Culex quinquefasciatus]
ref|XP_001654839.1| Gustatory receptor 21a, putative [Aedes aegypti]
Putative gustatory receptors expressed in adult antennae
The two GR candidates are, to our knowledge, the first identified in Lepidoptera antennae. It is not surprising to find candidate GRs since these organs are known to carry some taste sensilla . Interestingly, one of these GRs (GW825869) presented similarity with members of the GR21a family (Table 4). In Drosophila, GR21a forms a heteromeric receptor in combination with GR63a, which allows the detection of CO2[49, 50]. Putative CO2 receptors have been described in B. mori, one of which (BmGr2NJ as described by ) presented 78% identity with the partial sequence we obtained in S. littoralis. Since CO2 receptors are quite conserved among insects , this high sequence identity supports the annotation of this S. littoralis GR as a candidate CO2 receptor. Receptor cells to CO2 were found on the antennae of some insect species, such as the honey bee  and Drosophila, but up to now, moth receptor cells for CO2 have been only described on labial palps . Thus, annotation of this GR as a candidate CO2 receptor awaits further demonstration of CO2 detection by S. littoralis antennae.
Annotation of the S. littoralis putative ORs
In S. littoralis, 63 glomeruli could be identified in the antennal lobe . Considering the one receptor-one glomerulus paradigm [56, 57], by which the number of expected ORs in a given species should correlates with the number of glomeruli in the antennal lobe, we estimate that the 29 candidate OR genes identified represent half of the S. littoralis OR repertoire. 45% of the SlitOR amino acid sequences showed low sequence conservation with already known receptor proteins (less than 50% identity with the best hit, table 4). The remaining SlitORs presented higher conservation (up to 88% identity with the best hit), and eight SlitORs (26%) shared more than 70% identity with their respective best hits (Table 4). Among these conserved sequences, we could recognize SlitOR2 (the D. melanogaster OR83b orthologue, translated from EZ981047) and SlitOR18 (EZ983476), two S. littoralis receptors that we previously identified by homology cloning [25, 31]. The predicted translations of four unigenes exhibited a high conservation level (from 57 to 85% identity, table 4) with lepidopteran PRs previously described [21, 22, 26]. Since lepidopteran PRs form a relatively well conserved lineages , these unigenes could encode candidate PRs in S. littoralis.
qPCR analysis of S. littoralis chemosensory receptors
Since pheromone receptors are usually male-specific or male-enriched [22–24, 59, 60], we next compared SlitOR expression levels between male and female antennae (Figure 6B). Although this preliminary analysis is of limited value, we found that four unigenes were enriched in male antennae (EZ981394, EZ982621, EZ983328 and EZ982777) (Figure 6B). Only two of them (EZ983328 and EZ982777) corresponded to the SlitORs annotated as putative PRs after the BLAST and phylogenetic analyses (see paragraph above). The two additional putative ORs enriched in male antennae (EZ981394 and EZ982621) did not present high sequence identities with other moth PR candidates or functionally characterized PRs.
Other genes putatively involved in the olfactory process and its modulation
Ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs) and transduction
Recently, the ionotropic receptors (IRs), that constitute a family of ionotropic glutamate receptor-related proteins, have been identified as defining a new class of chemosensory receptors in D. melanogaster. The 61 described D. melanogaster IRs were used to search for homologues in the S. littoralis ESTs by TBLASTN. This led to the identification of five putative S. littoralis IRs. However, further studies are needed to annotate these candidates as IRs or classical glutamate receptors, such as obtaining the full length sequences for detailed examination of the binding site.
We also identified two unigenes encoding putative SNMPs, annotated as SNMP1 and SNMP2 in accordance with their best hit (accession numbers: EZ982816 and EZ982501, additional files 1 and 2). SNMPs were first identified in pheromone-sensitive neurons of Lepidoptera [10, 62] and are thought to play a role in pheromone detection, as demonstrated for the D. melanogaster SNMP1 homologue .
Our EST analyses (see above) revealed that the antennae appeared to be enriched in genes involved in metabotropic activity. Among examples, we annotated unigenes putatively encoding proteins such as G-proteins and G-protein related elements, second messenger-related enzymes and ions channels such as voltage-gated ion channels, calcium and chloride channels (additional files 1 and 2). Some of these genes have been previously described in detail, such as a diacylglycerol kinase  and a transient receptor potential channel , whose function in pheromone signal transduction was suspected. However, the way insect ORs transduce the signal is currently under debate and although a classical metabotropic pathway via G-protein was assumed , recent studies proposed an alternative or complementary ionotropic process [66, 67].
Unigenes were identified as encoding proteins putatively involved in modulation/regulatory process, such as hormone receptors (including ecdysone receptors EcR and USP), juvenile hormone-binding proteins, Takeout-like proteins and biogenic amine receptors (additional files 1 and 2). Consistent with the present data, we have previously characterized an octopamine/tyramine receptor expressed in the olfactory sensilla of an other noctuid, Mamestra brassicae. Biogenic amines act as neurohormones, neuromodulators or neurotransmitters in most invertebrate species , and evidence has been accumulated over the last decades that such biogenic amines participate in the modulation of olfactory reception [70, 71]. Ecdysone and juvenile hormone are key hormones involved in the maturation  and the plasticity  of the olfactory system.
Among our unigenes, we have also annotated putative circadian clock components. In addition to the previously described period and cryptochrome genes , we identified in S. littoralis antennae other fragments homolog to circadian clock encoding genes, such as timeless and vrille (additional files 1 and 2). These data support our previous finding that S. littoralis antennae house a peripheral circadian clock .
Lepido-DB (http://www.inra.fr/lepidodb) is a centralized bioinformatic resource for the genomics of major lepidopteran pests . This Information System was designed to store, organize, display and distribute various genomic data and annotations. Beside a BLAST search and a full text search facilities, the system was constructed using open source software tools from the Generic Model Organism Database (GMOD) including a Chado database. All the data, unigenes, ORFs and their annotation generated in this project have been included in LepidoDB. As a result, from the project page http://www.inra.fr/lepidodb/spodoptera_littoralis one can retrieve the whole sequence set, query with a keyword and retrieve the corresponding sequences.
The main objective of this study was to identify genes potentially involved in olfactory signal detection in a crop pest model, S. littoralis. We annotated a total of ~130 unigenes encoding putative proteins involved in all the steps of ligand detection (transport, docking, recognition, degradation). In particular, the normalization process, alongside with the high number of sequenced ESTs compared to previous antennal libraries, allowed enriching the EST collection in rare or low abundant transcripts. This strategy appeared to be particularly relevant for the identification of new insect chemosensory receptors in a species for which no genomic data are available. Concerning the pheromone detection process, we identified in this species three PBPs, two SNMPs, candidates PRs and many CXEs as putative PDEs, as a prerequisite to further identify which PBP/PR/SNMP/PDE act in concert to ensure the specificity of the recognition process within a given functional type of pheromone-sensitive sensilla. Their respective expression patterns remain to be elucidated to crack the code of their combinatorial expression.
Our analyses also suggest that the olfactory sensitivity may be modulated as early as the antennal level, before signal integration in the brain. Indeed, we annotated a long list of biogenic amine/hormone targets and circadian elements expressed in the antennae, as a first step toward understanding olfactory plasticity at the peripheral level.
Moreover, our study revealed that antennae express abundant defense-related elements involved in xenobiotic and pathogen protection. This observation could be explained by the fact that antennae, whose morphology is adapted to let odorant molecules enter the organism, represent an open space for harmful molecules.
Besides olfaction, insect antennae are involved in different non-olfactory processes, such as taste, balance/gravity, wind and sound sensing [76–78], and, as recently demonstrated, sun compass orientation . The availability of an antennal transcriptome is thus a valuable resource for olfaction and pheromone detection studies, but also for investigation of the molecular bases of other antennal functions.
Insect rearing and male antennae cDNA library construction
Insects originated from our inbred laboratory strain of S. littoralis. Insects were reared on semi-artificial diet , under 23°C, 60-70% relative humidity and 16:8 light:dark cycle. Pupae were sexed and males and females were kept separately. Antennae were collected from 1-2 day old naïve adult males and stored at -80°C until we obtained a total number of 12 000 antennae. Two mg of total RNA were isolated using the TriZol reagent (Invitrogen, Carlsbad, CA, USA), quantified in a spectrophotometer, and the quality verified by agarose gel electrophoresis. A custom normalized EvoQuest™ cDNA library was created by Invitrogen in the pSPORT 6.1 vector, using 1 mg of total RNA as starting material, without any amplification. The normalization step (performed by Invitrogen) introduced in the library construction consisted of a single stranded antisense DNA target-biotinylated sense RNA driver hybridization and a capture with streptavidin to remove target/driver hybrids and un-hybridized drivers, leftover DNA representing the normalized library. This led to a 20 fold reduction of abundant genes as measured for β-actin, while maintaining a good average insert size of 2.1 kb. The normalization procedure was used to minimize EST redundancy and to enrich the library for rare and low abundant genes, to allow new gene discovery.
The library was plated, and 2400 clones were randomly picked. Their 5' ends were sequenced using REV primer (Genome-express, Grenoble, France). 93% of the clones (2218) presented an insert, we thus undertook a high-throughput sequencing project (20000 sequences) in partnership with the Genoscope (Evry, France). The plated library was arrayed robotically and bacterial clones had their plasmid DNA amplified using phi29 polymerase. The plasmids were end-sequenced using BigDye Termination kits on Applied Biosystems 3730xl DNA Analysers. Adaptor and vector were localized using cross_match (http://www.phrap.org/) using default matrix (1 for a match, -2 penalty for a mismatch), with mean scores of 6 and 10, respectively. Sequences were then trimmed following three criteria: vector and adaptor, poly(A) tail or low quality (defined as at least 15 among 20 bp with a phred score below 12). Moreover, while submitting the unigenes to GenBank, the sequences were also compared to known vectors using the vecscreen software (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html). We finally obtained the 5' end sequences of 20760 ESTs.
Sequence processing, assembly, unigene and peptide generation
EST assembly was processed using the TGI Clustering tools (TGICL, http://compbio.dfci.harvard.edu/tgi/software) using the default parameters (minimum percent identity for overlaps: 94, minimum overlap length: 30 bp, maximum length of unmatched overhangs: 30), generating unigenes and singletons. Peptides were extracted from the unigenes using FrameDP 1.03 , with three training iterations and using Swissprot (398 181 entries, August 2009) as reference protein database.
Specificity analysis using EST
The unigenes were compared to the 550623 lepidopteran ESTs retrieved from the NCBI Entrez server (July 2009), using BLASTN with an e-value cut-off of 1e-10. The analysis of the enrichment of the EST library was performed with the help of the BLAST2GO application  using GOSSIP . In this application, GO terms are tested for enrichment in a test group when compared to a reference group using Fisher's exact test with multiple testing correction. The statistical tests were achieved with the R t-test and Chi2 methods.
Gene identification and functional annotation
The unigenes were compared to the NCBI non redondant protein database (7686184 entries, July 2009 version), the FlyBase translational database v5.16  (21 647 entries) and 14623 B. mori Glean peptide predictions from SilkDB (April 2008 release, ftp://silkdb.org/pub/current/Gene/Glean_genes/silkworm_glean_pep.fa.tar.gz)  using BLASTX, with a 1e-5 e-value threshold. The Gene Ontology mapping and distribution were done with the help of BLAST2GO (GO association done by a BLAST against the NCBI NR database). Finally, the functional domain protein profile and domain were predicted by queries against InterPro using InterproScan , running a batch of analyses (BLASTProDom, Coil, FprintScan, Gene3D, HMMPanther, HMMPfam, HMMPIR, HMMSmart, HMMTigr, PatternScan, ProfileScan, RNA-BINDING, Seg and Superfamily) on the predicted ORFs. The protein sequences were searched for the occurrence of a signal peptide using SignalP 3.0 .
Identification of odorant-binding proteins and chemosensory receptors
The S. littoralis antennal unigenes were searched with B. mori OBPs, CSPs, chemosensory receptors and all available insect ORs retrieved from Swissprot as queries using TBLASTN . Additionally, the Interproscan results were scanned for the Interpro accession IPR006170 (Pheromone/general odorant-binding protein, PBP/GOBP Molecular Function: odorant binding GO:0005549) and IPR004117 (Molecular Function: olfactory receptor activity GO:0004984). S. littoralis putative chemosensory receptor sequences were in turn employed in searches to find more genes in an iterative process.
We built OBP and OR neighbor-joining trees based on Lepidoptera data sets. The OBP data set contained the 43 complete amino acid sequences deduced from the genome of B. mori, together with the largest OBP repertoires characterized within noctuid moths (7 sequences from H. virescens and 5 from Spodoptera exigua) and outside noctuid moths (13 from M. sexta and 4 from Plutella xylostella) (Accession numbers available in additional file 6). Signal peptide sequences were removed following predictions of cleavage site location made by SignalP 3.0. The OR data set contained 58 amino acid sequences from B. mori (6 sequences were removed from the alignment because of their short length) and the 21 sequences characterized from H. virescens, completed with subsets of sequences characterized within noctuids (3 sequences from Mythimna separata) and outside noctuids (4 from P. xylostella, 3 from Diaphania indica and 3 from E. postvittana). Amino acid sequences were aligned using ClustalW2 . Unrooted trees were constructed by the neighbour-joining method, with Poisson correction of distances, as implemented in MEGA4 software . Node support was assessed using a bootstrap procedure base on 1000 replicates, and nodes supported by a bootstrap value under 70% were collapsed to an horizontal line when drawing cladograms.
Quantitative real-time PCR
Naïve males and females in the middle of their second scotophase were used in the following experiments. Male antennae, proboscis, legs (mixture of front, middle, and hind legs), brains, abdomens and female antennae were collected and stored at -80°C. Total RNAs were extracted with the RNeasy® MicroKit (Qiagen, Hilden, Germany) that included a DNase treatment. Single-stranded cDNAs were synthesized from 1 μg of total RNAs with 200 U of M-MLV reverse transcriptase (Clontech, Mountain View, CA, USA) using buffer and protocol supplied in the Advantage® RT-for-PCR kit (Clontech). Gene-specific primers for S. littoralis chemosensory receptors and the endogenous control rpL8 were designed using the Beacon Designer 4.0 software (Bio-Rad, Hercules, CA, USA), yielding PCR products ranging from 100 to 250 bp (see additional file 7). qPCR mix was prepared in a total volume of 20 μl with 10 μl of Absolute QPCR SYBR Green Mix (ThermoFisher Scientific, Epsom, UK), 5 μl 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 using a MJ Opticon Monitor Detection System (Bio-Rad). The PCR program began with a cycle at 95°C for 15 min, followed by 40 cycles of 20 s at 95°C, 15 s at 53 to 62°C (depending on the primer pair) and 20 s at 72°C. To assess the purity of the PCR reactions, a dissociation curve of the amplified product was performed by gradual heating from 50°C to 95°C at 0.2°C/s. Standard curves were generated by a five-fold dilution series of a cDNA pool evaluating primer efficiency E (E = 10(-1/slope)). All reactions were performed in duplicate. Chemosensory receptor expression levels were calculated relatively to the expression of the rpL8 control gene and expressed as the ratio = ESlitCR(ΔCTSlitCR)/ErpL8(ΔCT rpL8).
List of abbreviations
expressed sequenced tag
general odorant-binding protein
open reading frame
sensory neuron membrane protein
We thank Fabien Tissier and Pascal Roskam for insect rearing, David Tepfer for english improvement, the BioGenouest platform, especially Anthony Bretaudeau, Aurélien Roult and Olivier Collin for their bioinformatics support, and we thank reviewers for in depth comments on the manuscript. This work was supported by INRA AIP "Séquençage-génotypage", Université Pierre et Marie Curie (UPMC), ANR-07-NEURO-037-01 and ANR-09-BLAN-0239-01 funding.
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