Chemical Senses Advance Access originally published online on March 31, 2006
Chemical Senses 2006 31(5):453-465; doi:10.1093/chemse/bjj050
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Genome and EST Analyses and Expression of a Gene Family with Putative Functions in Insect Chemoreception
1 Rothamsted Research, Harpenden, AL5 2JQ, UK and 2 Nanyang-Rothamsted Joint Laboratory for Insect Biology, Nanyang Normal University, Nanyang, People's Republic of China
Correspondence to be sent to: Jing-Jiang Zhou, Biological Chemistry Division, Rothamsted Research, Harpenden, AL5 2JQ, UK. e-mail: jing-jiang.zhou{at}bbsrc.ac.uk
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Abstract |
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Odorant-binding proteins (OBPs) are thought to be responsible for the transport of semiochemicals across hydrophobic interfaces to olfactory receptors. In insects, a second class of OBPs with four conserved cysteines has been variously named as sensory appendage proteins, olfactory segment-D proteins, and chemosensory proteins (CSPs). The physiological functions of these proteins have remained elusive. Here we report a comprehensive survey of both genome and expressed sequence tags (EST) databases. This showed that CSPs are apparently only present in the phylum, Arthropoda, and in two subphyla, Crustacea and Uniramia. This is the first report of a putative CSP in Crustacea and suggests that the origin of these genes predates the divergence of Uniramia and Crustacea. For the Uniramia, we identified 74 new genes encoding putative CSPs of insect species from 10 different orders. Using tissue-specific EST libraries, we have examined the relative expression of putative CSP genes in many tissues from 22 insect species suggesting that the genes are expressed widely. One Drosophila CSPs is expressed sixfold higher in head than other CSPs. One Bombyx mori CSPs was found at a very high level in pheromone gland, and for the first time, six CSPs were identified in B. mori compound eyes. The different frequencies of CSP transcripts were observed between solitary and gregarious EST libraries of Locusta migratoria.
Key words: CSPs, OBPs, odorant binding, olfaction, semiochemicals
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Introduction |
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TopAbstractIntroductionExperimental proceduresResults and discussionConclusionsSupplementary materialAcknowledgementsReferences |
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Insects can sense a wide range of chemical signals (semiochemicals), such as pheromones and host odors, which they use to detect each other and to locate suitable food supplies. The semiochemicals are volatile hydrophobic molecules that enter the insects' antennae and other sensory organs via pores and travel across the hydrophobic space to the olfactory receptors (ORs), where the response of the insect to the signal is initiated.
It is currently assumed that the passage of semiochemicals to the ORs is facilitated by odorant-binding proteins (OBPs) which were first reported in Lepidoptera (Vogt and Riddiford, 1981
; Tsuchihara et al., 2005) and have since been characterized from a wide range of insect orders including Diptera (e.g., McKenna et al., 1994), Hemiptera (e.g., Dickens et al., 1998), Hymenoptera (e.g., Danty et al., 1998), Orthoptera (e.g., Ban et al., 2003a), Coleoptera (e.g., Wojtasek et al., 1998), Blattaria (Rivière et al., 2003), and Isoptera (Ishida et al., 2002). The transport role for OBPs is suggested by the findings that they are highly concentrated (up to 10 mM) in the lymph of chemosensilla, some are expressed specifically in antennae (Laue et al., 1994; Pikielny et al., 1994; Shanbhag et al., 2001; Ishida et al., 2002), and many can bind pheromones and other semiochemicals (Vogt and Riddiford, 1981; Du et al., 1994; Pelosi and Maida, 1995). Whether these OBPs act only as carriers or confer some specificity is still controversial; however, the idea of specificity is supported by the finding that many insect species have many different genes encoding putative OBPs. For example, studies of genome databases have identified 59 putative OBP genes in Drosophila melanogaster (Hekmat-Scafe et al., 2002) and 57 in Anopheles gambiae (Xu et al., 2003).
Based on sequence analyses, insect OBPs can be divided into four distinct groups: the "Classic" OBPs with six highly conserved cysteines, the "Plus-C" OBPs with more than six conserved cysteines, the "Atypical" OBPs found only in A. gambiae (Hekmat-Scafe et al., 2002; Xu et al., 2003; Zhou et al., 2004), and the chemosensory proteins (CSPs) with only four conserved cysteines (McKenna et al., 1994). The latter group of proteins are slightly smaller than other OBPs (100–120 residues) and have their four conserved cysteines linked in a noninterlocked fashion producing two small loops (Angeli et al., 1999). The first member of this group was reported as the protein induced in leg regeneration in Periplaneta americana (Nomura et al., 1992). A similar protein [olfactory segment-D protein (OS-D)] was demonstrated to be specifically expressed in sensilla coeloconica of D. melanogaster (McKenna et al., 1994). They were first named as CSPs by Angeli et al. in 1999 and have so far only been reported in insects where they show high levels of amino acid identity across species (Wanner et al., 2004).
For CSPs, there is some evidence of binding to semiochemicals, for example, in Mamestra brassicae, there is binding to pheromone components (Jacquin-Joly et al., 2001), a recombinant Apis mellifera protein binds to brood pheromone components (Briand et al., 2002) and a protein from Locusta migratoria binds an endogenous oleoamide (Ban et al., 2003b). The localization of some CSPs in the lymph of chemosensilla and the binding to semiochemicals strongly suggest that they are in some ways involved in insect chemoreception. However, there is no direct evidence for the involvement of CSPs in olfaction in vivo. Indeed, although many are expressed in the antennae, others are expressed in other tissues including legs (Mameli et al., 1996; Picimbon et al., 2001), labial palps (Maleszka and Stange, 1997), tarsi (Angeli et al., 1999), brain (Whitfield et al., 2002), proboscis (Nagnan-Le Meillour et al., 2000), pheromone gland (Jacquin-Joly et al., 2001), and wings (Ban et al., 2003b).
To date, genes encoding CSPs have been found mostly by conventional molecular techniques, and no genome-wide annotation or extensive expressed sequence tags (EST) analyses have been done. Also there is no universal naming system for these proteins, and they have been variously called sensory appendage proteins (SAPs) (Robertson, et al., 1999; Biessmann et al., 2002), OS-D (McKenna et al., 1994), CSPs (Angeli et al., 1999), and OS-D–like proteins (Wanner et al., 2004; Jacobs et al., 2005).
Although an increasing number of CSPs have been discovered in insects (Arthropoda; Uniramia), no study has extended the search to other subphyla of Arthropoda. Indeed, the genome-wide analysis of Arthropods is itself limited by the availability of completed, publicly accessible genome sequences which are only so far available for insect species. However, recent EST projects provide another resource for large-scale analyses (NCBI dbEST), and a large number of EST sequences for other subphyla of Arthropoda are available. In addition, ESTs are available for many insect species with some being assigned to specific tissues.
In this study, we report the genome annotation and EST analysis of CSPs. We have collated 58 previously identified genes and used this to build a CSP sequence motif, and to use such motif to search for protein sequences that contain such a motif in the completed genome sequences of 32 species from six kingdoms, and in the EST sequences of 23 species from the phylum Arthropoda. We have then compared gene expression profiles for the CSP genes within and between species using their presence in the EST libraries to quantify the relative tissue abundance for each gene.
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Experimental procedures |
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Structure of the CSP motif
Published CSP sequences were downloaded from the National Center for Biotechnology Information (NCBI) database using the keywords "CSP," "SAP," and "olfactory-specific protein." A total of 58 sequences were obtained in FASTA format and aligned using Clustal X (Thompson et al., 1997) with default gap penalty parameters of gap opening 10 and extension 0.2. The conserved cysteines were identified, and the number of residues between them was counted (in some cases, manual adjustment was necessary to align the cysteines), giving a "CSP Motif" of C1-X6–8-C2-X16–21-C3-X2-C4-X3. For this motif there are 18 (3 x 6 x 1 x 1) possible spacing combinations between the conserved cysteines, and the program "MotifSearch" takes "C1-X6-8-C2-X16-21-C3-X2-C4-X3" as the input, expands it to the 18 spacing combinations, and uses each combination to search the peptide sequences for the presence of the CSP motifs. The MotifSearch program is a home-developed window program and written in Delphi.
Identification of motif-containing sequences
The whole-genome peptide sequences of 32 species from six kingdoms (Metazoa, Plantae, Fungi, Protista, Archaea, and Bacteria) were downloaded from their genome databases in the FASTA format and searched for CSP motif–containing sequences using the MotifSearch program. For the EST libraries, the sequences were retrieved as FASTA files from the NCBI EST database (http://www.ncbi.nlm.nih.gov/dbEST/) using either the species or order name to search "text" and then translated in six frames into peptide sequences. The CSP motif–containing peptide sequences were used in Blast searches against the sequences in the NCBI database, and the sequences that had significant matches to known CSPs were selected. The EST id was used to retrieve full-length EST sequences that were used for assembly to get the consensus sequence. Briefly, all the sequences of CSP-encoding ESTs from different tissue-specific EST libraries of one species were combined into a FASTA file as the input file and then assembled with Vector NTI ContigExpress software (InforMax, Oxford, UK). The gaps and mismatching bases were corrected manually, and the consensus sequence of each contig was retrieved. For simplicity and to distinguish the retrieved sequences from published CSPs, they were not assigned specific names; instead, they were given four letters to represent the species and then EST followed by the contig number. CSP sequences from genome data were named using a four-letter species identifier and the letters CSP.
Calculation of the relative abundance of each CSP in each tissue type
This was calculated from NCSP-clones/Ntotal-clones, where NCSP-clones is the number of EST sequences in that tissue type which are assembled to give the consensus CSP sequence (i.e., the number of ESTs in one contig) and Ntotal-clones is the total number of EST sequences used in the assembly (i.e., total number of the motif-containing ESTs identified as CSPs from the EST libraries for one species) (see also Supplementary Tables 1–7).
Phylogenetic analyses
The sequences identified from genomic sequences and EST libraries were aligned using Clustal X (8.1) (Thompson et al., 1997) with default gap penalty parameters of gap opening 10 and extension 0.2. The phylogenetic trees were then constructed from these multiple alignments using MEGA3 software (Kumar et al., 2004). The final unrooted consensus tree was generated with 1050 bootstrap trials using the neighbor-joining method (Saitou and Nei, 1987) and presented with a cutoff bootstrapping value of 70.
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Results and discussion |
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Identification of CSPs in genome data
Using the MotifSearch program (see Experimental Procedures) in combination with Blast, we have searched for CSPs in the genome databases of 32 species in the six kingdoms: Archaea, Bacteria, Protista, Fungi, Plantae, and Metazoa. Many CSP motifs were found in representatives of all of the kingdoms, but only in members of the phylum Arthropoda did the motif-containing sequences show similarity to known CSP sequences (Table 1).
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We have further analyzed all CSP motif–containing sequences listed in Table 1. Insect sequences were not included in this analysis. Most of these CSP motif–containing proteins are at least twice as long as than insect CSPs. No single CSP hit during the Blast was obtained regardless of search score against insect CSPs using NCBI Blastp with the threshold of EXSPECT = 100 and DISPLAY = 50. However, this does not preclude the possibility that sequence divergence in more distant species prevents CSP detection. These CSP motif–containing sequences were then aligned with D. melanogaster CSP OS-D, and the sequences with the highest identity in each species were selected. The identities to OS-D (155 amino acid long) range from 7% of human intestine G-protein signaling regulator (accession no. BAC04934) to a maximum 21% of Arabidopsis thaliana transcription factor/zinc ion–binding protein (accession no. NP_200258). Furthermore, the CSP motif–containing sequences with similar size to known CSPs (between 100 to 300 amino acid) include human retinal pigment epithelium (RPE)–spondin (accession no. AAH42877) and keratin-associated proteins (accession no. CAA45283, CAF31639), pig plasmin trypsin inhibitor (accession no. NP_999036), as well as proteins that contain ring finger and CHY zinc finger domain, such as Danio rerio zinc finger protein 658 (accession no. XP_688998), human transcription elongation factor A and zinc finger protein (accession no. XP_528356 and AAL09356), pig transcription elongation factor A and zinc finger protein (accession no. XP_528356 and XP_517222), and A. thaliana transcription factor and ubiquitin–protein ligase (accession no. NP_200258, NP_194461 and NP_177614), and proteins that have a rabphilin-3A effector domain, such as Mus musculus Rab3-interacting molecule 2 (accession no. BAD32696) and Caenorhabditis elegans Rim (accession no. BAD32699). All bacterial proteins that contain CSP motifs are involved in energy production and conversion such as polyferredoxin and anaerobic dimethyl sulfoxide reductase chain B of Escherichia coli (accession no. BAB36804 and BAB36806, respectively). It was found that to each of all smaller CSP motif–containing proteins, there are Drosophila counterparts annotated with putative functions rather than chemoreception when these proteins were used in Blast against D. melanogaster sequences. For example, all plant proteins are similar to Drosophila zinc finger protein (accession no. AAF47094) and all E. coli proteins to Drosophila protein containing 4Fe-4S–binding domain (accession no. AAF50342). The mammal proteins (BAC04934, NP_999036, XP_5172222, and BAD32696) match to Drosophila fatty acid desaturase, extracellular matrix protein, zinc finger protein, and Rim (accession no. AAB17283, AAO84908, AAF52385, and AAF55479, respectively) with E values of 0.03, 9 x 10–12, 3 x 10–76, and 9 x 10–30, respectively. All these proteins also have no similarity to any of the known CSPs in both primary sequences and predicted tertiary structures (data not shown), suggesting that CSPs identified in the current study seem to be confined to the phylum Arthropoda.
Within the phylum Arthropoda, only insect (Uniramia) genomes are available, so it does not tell us how widespread these genes are in other subphyla of Arthropoda (see later). Indeed, the only available genomes are from insect species, that is, D. melanogaster, Drosophila pseudoobscura, A. gambiae, A. mellifera, and Bombyx mori, and from these 18,289, 18,331, 15,802, 24,640, and 21,302 peptide sequences were, respectively, searched giving a total of 102 motif-containing sequences of which 28 also showed similarity to known CSPs (Table 1). An alignment of these along with sequences identified from EST libraries (see later) is supplied as Supplementary Figure 1.
For D. melanogaster, four of the 24 motif-containing sequences showed similarity to CSPs (Table 1) and are presented as DmelCSP1-4. DmelCSP1 has not been annotated previously but is identical to CG30172 in the D. melanogaster genome annotation (FlyBase) and to D. melanogaster head ESTs (see Supplementary Table 1). DmelCSP4 was identified previously as a D. melanogaster OS-D (accession no. Q27377) (McKenna et al., 1994) or A10 (Pikielny et al., 1994). The deduced amino acid sequence of DmelCSP2 is identical to a virus-induced protein Pherokine-2 (Phk-2) (Sabatier et al., 2003) and has a very high identity (63.2%) to the ejaculatory bulb protein III (PEBmelIII) (Accession no. Q23981). The annotation of PEBmelIII as a possible CSP is interesting because this protein belongs to a family that is thought to be responsible for changes in the activity of D. melanogaster (in terms of movement, secretion, enzyme production, gene expression, etc.) or in responses to a virus stimulus (Brieger and Butterworth, 1970; Sabatier et al., 2003). A possible involvement of PEBmelIII in storing and delivering chemical messages has also been proposed on the basis of its expression in glands producing pheromones (Dyanov and Dzitoeva, 1995), and the high similarity of PEBmelIII to the OBP MbraOBP2 of M. brassicae has led to the suggestion that vaccenyl acetate could be a specific ligand for both proteins (Bohbot et al., 1998). DmelCSP3 resembles D. melanogaster Pherokine-3 (Phk-3) that was reported to be induced by virus and bacterial infections (Sabatier et al., 2003). For D. pseudoobscura, all four genome sequences identified as CSPs (from a total of 20 sequences with a CSP motif, Table 1) are homologues of the four D. melanogaster CSPs, with more than 75% amino acid identity, implying a very high conservation of CSPs in the genus Drosophila.
For the A. gambiae published genome annotation, the predicted peptide sequences are given as the longest open reading frames, most of them being without identified start codons (R. Holt, personal communication), so we used GenScan full-length coding sequences for each of the 20 CSP motif–containing sequences found by our MotifSearch. This showed that of the 20 motif-containing sequences, seven have significant similarity to known insect CSPs (Table 1), and these were compared with entries in GenBank and the mosquito Ensembl database and with three independent genome-wide annotations of A. gambiae OBPs (Vogt, 2002; Xu et al., 2003; Zhou et al., 2004). It was apparent that some of the annotated genes are from the same contig sequence and have almost identical sequences despite being given different names and accession numbers. One of our sequences, AgamCSP4, has been cloned previously and named as the SAP1, AgamSAP-1 (Biessmann et al., 2002), but the other six are newly identified as CSPs.
The 17 motif-containing sequences from the genome of the honeybee A. mellifera (Table 1) included five with similarity to published CSPs with one, AmelCSP4, being identical to a previously reported honeybee CSP (ASP3c) (Danty et al., 1998; Briand et al., 2002) and another, AmelCSP2, being identical to the worker antennal protein 1 (Kamikouchi et al., 2004). We also identified eight CSP genes in the B. mori genome, including two previously cloned CSPs, BmorCSP1 and BmorCSP2 (Picimbon, et al., 2000).
Identification of CSPs in EST libraries
As stated previously, within the phylum Arthropoda, the only organisms with genome sequences are insects in the subphylum Uniramia. We therefore used EST data to look for a wider distribution of CSPs in other subphyla of Arthropoda (Table 2). There are 96,276 EST sequences available for the subphylum Chelicerata, but although many were found to contain CSP motifs, there were no sequences with a significant similarity to any known CSPs. However, from 63,304 EST sequences of the subphylum Crustacea, 15 had CSP motifs including one (BQ563140
[GenBank]
) in the brine shrimp Artemia franciscana which had significant similarity to CSPs (24% identity to the Cactoblastis cactorum CSP CLP-1, 22% identity to the CSP-I of Schistocerca gregaria, and 18% identity to MbraSAP of M. brassicae). Thus, the origin of CSPs appears to predate the divergence of Crustacea and Uniramia, about 130 million years earlier than previously reported from the separation of the class Neoptera (Wanner et al., 2004).
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For the subphylum Uniramia, CSP motif–containing sequences were identified in 25 different species, and all of these had one or more sequences with significant similarity to known CSPs (Table 2) (Each CSP sequence was obtained by assembling large numbers of ESTs, and the information is provided in the Supplementary Tables 1–7.). For the species with genome sequences available, we were able to compare the genome sequence with the EST (see next section), but we also found CSPs in other insect orders for which the genome data are not available and some of which have not previously been found to have CSPs. Thus in the order Hemiptera, there are five CSPs in the pea aphid Acyrthosiphon pisum and four in the brown citrus aphid Toxoptera citricida. In Siphonaptera there is one CSP in the cat flea Ctenocephalides felis, in Phthiraptera one CSP in the human head-louse Pediculus humanus, in Coleoptera one from Biphyllus lunatus and two from Tribolium castaneum, and for Orthoptera a new group of CSPs with high similarity to CSPSgre-III-1 in the locust L. migratoria. For Lepidoptera where CSPs have been widely reported previously, we found additional six CSPs in the moth Manduca sexta and 10 in the silk moth B. mori of which five were found in both genome sequences and EST libraries, one only in genome sequences, and four only in EST libraries. We also found CSPs in species where they had not been reported previously, two in the butterfly Heliconius melpomene, and four in the diamondback moth Plutella xylostella. These data show that CSPs are extremely widespread throughout the insect species.
Comparison of CSP sequences in different insect species
An alignment of the deduced CSP sequences identified from genome data and EST libraries in this study is supplied as Supplementary Figure 1, and the data have been used to construct phylogenetic trees and to compare sequences for CSPs as given in Figure 1.
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For species in the order Diptera, the sequence analysis of CSPs identified from D. melanogaster, D. pseudoobscura, Drosophila yakuba, A. gambiae, and Glossina morsitans, as well as reported SAPs of Aedes aegypti and Culicoides sonorensis (Figure 1A) shows that many CSPs within each species are similar (e.g. AgamCSP4, 6, and 7) and that there are highly conserved genes between the three Drosophila species (e.g. DpseCSP2, DyakEST2, and DmelCSP2). Perhaps more surprisingly, some Drosophila and Anopheles sequences are very similar, for example, a group of three CSPs, DmelCSP1, DpseCSP1, and AgamCSP1, which are well segregated from other CSPs with a bootstrap value of 100. This suggests that they are a group of CSPs with a common ancestral protein and which have stayed the same because they perform similar vital functions in all three species. Despite some closely related groups of CSPs, there is clearly a diversity of sequences within these species, and all of this is consistent with the view that this family of genes arose by duplication events prior to the separation of the species and then diverged in each species. This would also seem to be true in the Lepidoptera where the CSPs from six species (B. mori, M. sexta, P. xylostella, H. melpomene, M. brassicae, and C. cactorum) are compared. For sequence comparison, two previously reported CSPs (CSP-MbraA6, CSP-MbraB3) of M. brassicae and CSPs identified in this study from Coleoptera, Siphonaptera, and Phithiraptera (Table 2) were also included. Figure 1B shows that moth CSPs, although the tree does not include all previously reported Lepidoptera CSPs, like those of Diptera species, form groups across species with a CSP from one species often being more similar to a CSP from a different species than to others of its own. In B. mori, eight of 12 CSPs have their homologues in other moth species, only four (BmorEST1, BmorEST4, BmorEST8, and BmorEST9) are separated from these clusters. However, their homologues would be found when more genome sequences of other moth species become available. Two B. mori CSPs (BmorCSP2 and BmorCSP3) were only found in the genome sequence, but their homologues (MsexEST8 and HmelEST1, respectively) were found in the EST libraries of other moth species (Figure 2B). MsexEST8 was identified from the male antennal ESTs of M. sexta, and HmelEST1 was found in the pupal EST library of Heliconius melpomene (Table 2).
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For Hemiptera, we have found CSPs in seven species, all of which are aphids (A. pisum, T. citridae, Aphis fabae, Nasanovia ribis-nigri, Metapolophium dirhodum, Myzus persicae, and Megoura viciae). There is a clear conservation of sequences; indeed, one sequence is almost identical in all seven species (Figure 1C and consistent with Jacobs et al., 2005
). This is also true for the order Orthoptera where we only have EST libraries from one species L. migratoria and where all CSPs are very closely related, presumably because they have arisen from a common gene fairly recently (Figure 1D). Furthermore, three homologues of recently reported CSP CSPSgre-III-1 in S. gregaria were identified from L. migratoria ESTs (Figure 1D). For Hymenoptera, EST libraries of two species (A. mellifera and Linepithema humile) were analyzed (Table 2). However, only three of five A. mellifera CSPs identified in the genome were found in the EST libraries. For sequence comparison, previously reported CSPs of other Hymenoptera species (Polistes dominulus, Vespa crabro, Camponotus japonicus) were included in the phylogenetic analysis. As shown in Figure 1E, apart from AmelEST1 and AmelEST5 there are two clusters, one of them contains CSPs from different species (AmelEST2, PdomCSP1, LhumEST1, CjapCSP), suggesting this CSP may perform a similar role in these insects. However, the wasp CSP VcraCSP1 was similar to other two honeybee CSPs AmelEST2 and AmelEST3 in another cluster.
Expression of CSP transcripts
Data from EST libraries not only allow us to identify sequences likely to be CSPs but also, when the origin of the tissues used to construct the libraries is known, we can deduce the relative expression of the CSP transcripts (see Experimental Procedure). This is shown for seven insect species in Table 2 and Figure 2. However, some of the libraries available in NCBI dbEST do not reflect the "natural" expression level of genes, according to the way the EST libraries have been constructed: some libraries are normalized, some are subtracted, or induced; that could interfere with the number of clones encoding one CSP (enriched or lowered). The data presented in Figure 2A–H were obtained from normalized EST libraries from A. gambiae and D. melanogaster and natural expressed EST libraries, and no subtracted and induced EST libraries were used to analyze relative expression level of CSPs. The data only represent the relative expression level of one CSP to other CSPs in one tissue-specific EST library of one insect species and is presented individually for each species in Figure 2A–H.
In the 21,916 normalized head ESTs and 10,284 normalized fat body ESTs of A. gambiae, there are seven CSPs, and the CSP expressed most frequently is AgamCSP4 which is expressed equally in both heads with antennae and in female fat body (Figure 2A). In contrast, in D. melanogaster (89,576 normalized head ESTs), one CSP is expressed most frequently (DmelCSP2) and here this is exclusively in heads with antennae which is more supportive of a possible role in Drosophila olfaction (Figure 2B). Phylogenetic analysis shows this CSP to be specific to the Drosophila genus (Figure 1A). The Drosophila CSP DmelCSP3 is expressed at low levels in all developmental stages including embryo (102,375 normalized ESTs), pupae (16,299 ESTs), and larvae (10,449 ESTs) (Figure 2B), and closely related genes are found in the embryo EST library of D. yakuba and in the genome of D. pseudoobscura (Figure 1A). Furthermore, DmelCSP3 has an identical sequence to Pherokine-3 (Phk-3), indicating its expression may be regulated normally at a very low level but that it is induced by viral and bacterial infections (Sabatier et al., 2003). The Drosophila CSP DmelCSP4 has an identical sequence to OS-D (A10) that has been shown to be an antennal-specific gene; however, we did not find it in the antennal EST libraries although this may be because of the relatively small numbers of ESTs available in the current database. But its homologue DpseCSP4 was identified in the genome sequence of D. pseudoobscura (Figure 1A).
In the honeybee, there are EST libraries, 202 from female antennae and 19,051 from female brain. Two CSPs AmelCSP2 and AmelCSP4 are present in female antennae with AmelCSP4 also being present in the female brain at a relatively high level (Figure 2C). The peptide sequence of AmelCSP4 is identical to a reported antennal-specific protein 3c that binds to brood pheromone components of A. mellifera (Briand et al., 2002), and AmelCSP2 was found to be expressed in the worker antennae (Kamikouchi et al., 2004).
For B. mori there are no EST libraries from heads or antennae, so our study for this species is limited to nonolfactory tissues (Figure 2D). Of the 10 CSPs identified, BmorEST1 is the most frequently expressed, mostly in the pupal and adult pheromone gland (8432 ESTs). It may be involved in the production and/or transportation and release of pheromones. Five out of the 10 B. mori CSPs (BmorEST3, 5, 6, 7, and 8) are expressed in the compound eyes, the first time that any CSPs have been reported in this tissue, and the functional role of these genes in eyes could not be olfaction. BmorEST6 is identical to the reported antennal CSP BmorCSP1. Two CSPs identified from the B. mori genome (BmorCSP3 and 5) and the previously reported BmorCSP2 were not found in the current EST libraries (a total of 65,389 ESTs but lacking antennae-specific ESTs). In fact, BmorCSP5 is identical to BmorCSP2. For another Lepidopteran species, M. sexta, our search found 11 CSP genes (from 1994 ESTs) of which only five have been reported previously from EST databases (SAP1, SAP2, SAP3, SAP4, and SAP5; Robertson et al., 1999). All 11 are expressed in antennae either in males, females, or both, suggesting a role in olfaction. However, the possibility of the expression in other parts of the insect can not be excluded from the current EST data.
Within the order Hemiptera, ESTs are available for only two species, both of which are aphids. For T. citricida, four CSP genes were identified in 2475 ESTs, but the library was constructed from whole insects, so it tells us nothing about expression patterns (Table 2). For A. pisum, all five CSP genes found are expressed in whole insects, and for three there is evidence of antennal expression (ApisEST2 and 4; Figure 2F) again supportive of a possible role in olfaction. It is interesting that extensive Blast searching and attempts at homology cloning in our laboratory have failed to identify any members of other OBP classes in aphids (Jacobs et al., 2005). The similar expression patterns were reported in the social wasps P. dominulus and V. crabro where CSPs are specifically expressed in the antennae, while OBPs are also expressed in legs and wings (Calvello et al., 2003).
So far the species with the most CSPs identified is the locust L. migratoria, where from 45,481 ESTs, 75 have CSP motifs and 19 have significant similarity with other CSPs (Table 2). For this species there are two recognized forms, the so-called "solitary" phase, where individuals live alone, and the "gregarious" phase, where they congregate and form swarms, and it is interesting that expression of many of the CSP transcripts is very different in the solitary (a total of 15,889 ESTs) and gregarious (a total of 17,175 ESTs) phases (Figure 2G and H). For example, the expression of LmigEST8 in heads is much higher in the solitary phase, whereas LmigEST12 is not expressed in the solitary phase but is at a high level in the gregarious phase. Most of the L. migratoria CSPs are expressed in heads (0.65% of 33,064 total ESTs), and the changes in expression during the developmental switch from solitary to gregarious is an interesting finding. Further experiments are needed to confirm the possible role of these CSPs in this phenomenon.
Overall, we have found that CSPs in D. melanogaster, A. gambiae, A. mellifera, M. sexta, A. pisum, and L. migratoria are expressed in antennae, or head material containing antennae, and could therefore possibly have a role in olfaction. It is also likely that in B. mori, one CSP may play roles in pheromone release and some in eye function, and in L. migratoria, some CSPs may be involved in the developmental transition between the solitary and gregarious life stages.
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TopAbstractIntroductionExperimental proceduresResults and discussionConclusionsSupplementary materialAcknowledgementsReferences |
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In this study, we have for the first time reported a CSP in a Crustacean species, suggesting that the origin of CSPs predates the divergence of Uniramia and Crustacea. We have found CSPs in many insect orders including for the first time in Siphonaptera and Phthiraptera. This demonstrates a wide occurrence of these proteins, and their high level of conservation suggests an important role in insect function. Furthermore, the presence of CSPs in different tissue-specific EST libraries has demonstrated a wide distribution for CSPs within each insect species, and they may involve in olfaction as well as in pheromone release and eye function. They are clearly an important family of insect proteins and many questions on their functions remain to be answered.
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Supplementary material |
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TopAbstractIntroductionExperimental proceduresResults and discussionConclusionsSupplementary materialAcknowledgementsReferences |
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Supplementary Figure 1 and Tables 1–7 can be found at http://www.chemse.oxfordjournals.org.
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Acknowledgements |
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TopAbstractIntroductionExperimental proceduresResults and discussionConclusionsSupplementary materialAcknowledgementsReferences |
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Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
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Accepted 6 March 2006
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