Chemical Senses Advance Access originally published online on October 10, 2007
Chemical Senses 2008 33(1):79-93; doi:10.1093/chemse/bjm067
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The Gr Family of Candidate Gustatory and Olfactory Receptors in the Yellow-Fever Mosquito Aedes aegypti
Department of Entomology, University of Illinois at Urbana-Champaign, 505 S. Goodwin, Urbana, IL 61801, USA
Correspondence to be sent to: Hugh M. Robertson, Department of Entomology, University of Illinois at Urbana-Champaign, 320 Morrill Hall, mc-118, 505 S. Goodwin, Urbana, IL 61801 USA. e-mail: hughrobe{at}life.uiuc.edu
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Abstract |
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The gustatory receptor (Gr) protein family contains most of the diversity in the insect chemoreceptor superfamily, including within it not only taste receptors but select olfactory receptors as well. Manual annotation of the Gr family in the genome sequence of the yellow-fever mosquito, Aedes aegypti, yielded a total of 114 potential proteins encoded by 79 genes. In the sequenced genome, 23 of these genes and protein isoforms are pseudogenic, leaving 91 putatively functional Grs. Comparison with our previously published set of 76 Grs encoded by 52 genes in the distantly related Anopheles gambiae mosquito revealed 13 new AgGrs encoded by 8 genes. Phylogenetic analysis reveals the conservation of carbon dioxide, sugar, and several orphan receptors in these 2 mosquitoes and Drosophila flies. On the other hand, most of these Grs are unique to mosquitoes and many are specific to the Aedes or Anopheles lineages, indicating their involvement in mosquito-specific aspects of both gustatory and olfactory perception. In particular, most instances of alternative splicing in orthologous loci appear to have evolved after the culicine–anopheline split ±150 million years ago.
Key words: chemoreceptor, gene annotation, gene phylogeny, molecular evolution, olfaction, taste
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Introduction |
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Aedes aegypti ranks high among the mosquitoes of most critical medical significance, being the primary vector of yellow fever and dengue fever, which are responsible for roughly 200 000 and 50–100 million cases of each disease, respectively, worldwide per year (Mackenzie et al. 2004
; Tomori 2004). Additionally, this mosquito is able to transmit a variety of other viral diseases as well as filarial worms, and it is also used as a laboratory model for avian malaria (e.g., Thathy et al. 1994; Morlais et al. 2003). In a reemergence and ongoing outbreak of chikungunya virus in India, in which A. aegypti is the presumed vector, 1.4 million cases of the disease were reported in 2006 alone (Pialoux et al. 2007). As a consequence of its anthropophily and facile adaptation to breeding in domestic environments, this mosquito has become an efficient disease vector, contributing to thousands of human deaths per year. In addition to its medical significance, A. aegypti has been frequently used as a model for physiological studies in insects—including chemoreception and phagostimulation research (e.g., Salama 1966; Lee 1974; Davis 1975; Werner-Reiss et al. 1999), thus laying the framework for application of molecular information unearthed by the recent sequencing of its genome (Nene et al. 2007).
The insect chemoreceptor superfamily is defined as the combination of the odorant receptor (Or) and gustatory receptor (Gr) families; however, the Ors are a single highly expanded lineage within the superfamily, whereas the Grs contain many highly divergent protein lineages that represent most of the diversity in the superfamily (Robertson et al. 2003). The Gr family was so named because most of those first identified in Drosophila melanogaster (DmGrs) are expressed in mouthparts and other structures with gustatory functions (Clyne et al. 2000); however, several of the DmGrs are expressed in olfactory organs such as the antennae and consequently are putative olfactory receptors (Scott et al. 2001; Suh et al. 2004; Fishilevich and Vosshall 2005). Indeed, the heterodimer of DmGr21a and DmGr63a was recently shown to be the carbon dioxide receptor in flies (Jones et al. 2007; Kwon et al. 2007). The Grs are defined by a conserved C-terminal motif, immediately after an ancient and common phase 0 intron (Clyne et al. 2000; Scott et al. 2001; Robertson et al. 2003). This motif is hh(G/A/S)(A/S)hhTYhhhhhQF, where "h" is a hydrophobic residue; however, even the highly conserved TY and QF positions are substituted in some Grs. The insect chemoreceptors are generally considered to have 7 transmembrane (7TM) domains and to be G-protein–coupled receptors (GPCRs) (e.g., Hill et al. 2002); however, hydropathy plots and study of 2 Ors indicate that the transmembrane domain arrangements might not be the same as most 7TM proteins (Benton et al. 2006). Furthermore, these proteins have no sequence similarity to other GPCRs, specifically the vertebrate and nematode chemoreceptors that are members of the rhodopsin superfamily (e.g., Bargmann 2006) and therefore at best are a completely independent class of GPCRs (e.g., Hill et al. 2002) or perhaps function in a completely different way.
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Materials and methods |
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The A. aegypti assembled genome sequence v1.0 available at VectorBase, the National Center for Biotechnology Information (NCBI), the Broad Institute, and The Institute for Genomic Research (Nene et al. 2007
) was searched using the TBLASTN algorithm (Altschul et al. 1997) for matches to all the AgGrs from Anopheles gambiae annotated by Hill et al. (2002), as well as all 68 DmGrs (Robertson et al. 2003) and the 10 AmGrs from the honey bee Apis mellifera (Robertson and Wanner 2006). Additional searches were performed in which the EXPECT significance value was relaxed to 1000 to find highly divergent genes sharing only weak statistically insignificant similarity in their TM7 C-terminal regions. Gene models were built manually in the text editor of PAUP* v4.0b10 (Swofford 2002), using the AgGrs as guides when appropriate. The splice site predictor using Neural Network at the Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/splice.html) was used to help predict splice sites. Regions causing pseudogenization of gene models were checked against the raw reads at the NCBI Trace Archive to establish whether they were polymorphic or misassemblies. Apparent pseudogenes were translated as best possible to produce a comparable protein product for phylogenetic analysis, for example, by ignoring in-frame stop codons in exons and judicious introduction of frameshifts to accommodate insertions and deletions. AaGr and AgGr proteins were aligned with CLUSTALX (Thompson et al. 1997), using multiple alignment mode with default settings. These alignments were used to detect potential problems with the gene models, which were then refined. All AaGr gene models were compared with those available in the 15 419 "high-confidence" gene set in Genebuild 1.0 (AaegL1.1) of the genome annotation as well as 15 396 "supplementary" gene models available at VectorBase (Nene et al. 2007). All amino acid translations are available as Supplementary material online. Note that although these genes and proteins are formally named as GPRgrs in Ensembl and VectorBase, and this convention was used in Hill et al. (2002), we will use the abbreviation Gr with the species suffices Aa and Ag for simplicity and in keeping with conventions in the insect chemoreceptor community (e.g., Dahanukar et al. 2001; Dunipace et al. 2001; Dobritsa et al. 2003; Robertson et al. 2003; Robertson and Wanner 2006).
The phylogenetic analysis employed a final CLUSTALX alignment with a few pseudogenic sequences removed due to their relative incompleteness (see Figure 1). In addition, extreme N- and C-termini were excluded, as a consequence of their highly divergent lengths and sequences. Three short internal regions of alignment gaps found in most sequences were excluded from the data set as well. Phylogenetic analysis of this large set of 200 often highly divergent proteins was performed using corrected distance analysis in TREE-PUZZLE v5.0 (Schmidt et al. 2002) and PAUP*v4.0b10 (Swofford 2002) (see Hill et al. 2002; Robertson et al. 2003; Robertson and Wanner 2006). Bootstrap analysis was performed using 10 000 replications of uncorrected distance analysis in PAUP*. Subtree analysis was performed using custom alignments and phylogenetics described in the Results.
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Results |
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AaGr and AgGr gene models
We identified 79 genes in the AaGr family. We interpret 9 of these genes to be alternatively spliced yielding a potential 114 encoded Gr proteins; however, 23 of these genes or alternatively spliced exons are pseudogenes in the sequenced genome, leaving 91 putatively functional Grs (Table 1, Figures 1 and 2). In A. gambiae (Hill et al. 2002) and in D. melanogaster (Robertson et al. 2003), some of the chemoreceptor pseudogenes in the sequenced strain are intact in other strains and sometimes even in alternative haplotypes within the sequenced strain. This is also true for some of the AaOrs (Bohbot et al. 2007). We, however, detected no examples of pseudogenes in the available A. aegypti assembly that were intact in alternative alleles, and we have not examined other strains of A. aegypti. We retained and named the pseudogenes in the AaGr gene set if they encoded more than 200 amino acids, which is roughly 50% of a typical Gr. The relevance of these retained pseudogenes lies in our expectation that some of them will be "flatliners" (Stewart et al. 2005) that are intact in other strains of A. aegypti, whereas the remainder provide evidence about how this gene family has evolved. We ignored 6 fragments of genes that encoded less than 200 amino acids.
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Unique characteristics of AaGr annotations
The AaGr gene models were often difficult to annotate in part because they commonly encode extraordinarily divergent proteins and many do not have simple AgGr orthologs. Most of the relatively conserved genes and some quite divergent genes were at least partially annotated in the AaegL1.1 Genebuild of the genome-wide automated annotation reported in the main genome paper (Nene et al. 2007) and deposited in VectorBase and Ensembl, which consists of 15 419 high-confidence gene models. Forty-one AaGr proteins are at least partially present in AaegL1.1, of which 11 agree with our models. The other 30 require at least some modification (see Table 1). In addition, another 36 gene models are partially represented in the 15 396 gene supplementary gene set available from VectorBase (Table 1). Gr predictions in AaegL1.1 and the supplementary gene set—particularly the alternatively spliced loci—are commonly concatenated to encode single large proteins, unlike the unique splice variants found in our annotations. Many of the genes contain long introns that include retrotransposons, which is a general feature of this genome (Nene et al. 2007). These were considered to be intact genes if the retrotransposons did not appear to affect the intron splice sites. The 23 pseudogenes are denoted by the suffix P in Table 1. All our gene models have been communicated to VectorBase for inclusion in the next version of the A. aegypti genome annotation, which will also be deposited in Ensembl.
There were several instances of highly divergent AaGrs and AgGrs that did not appear to have obvious orthologs in the other species. We searched intensively for possible orthologs using TBLASTN queries and also undertook extensive PSI-BLASTP searches at GenBank. The latter were executed starting with each AaGr and AgGr in turn as a query and reiterated until the only new proteins from A. aegypti or A. gambiae were the distantly related Ors (Robertson et al. 2003). This search method depends on the genes already being at least partially annotated in the automated AaegL1.1 Genebuild for A. aegypti or the Ensembl annotations for A. gambiae. Together these searches using the divergent AaGrs led to the discovery of 8 new AgGr genes, AgGr53–60, encoding 13 new AgGrs through alternative splicing of the AgGr56 locus (Figure 2; Table 2). These proteins are so highly divergent from other AgGrs that in TBLASTN searches they find no other matches in the genome, explaining why they were not discovered by Hill et al. (2002). Initially, gene models for only 2 of these new AgGrs were available from Ensembl. However, neither was complete for the C-terminus; hence, they could not be found by PSI-BLASTP searches at NCBI because the only conserved motif was absent. These raise our previously reported A. gambiae Gr repertoire of 76 Grs encoded by 52 genes (Hill et al. 2002) to 90 Grs encoded by 60 genes and suggest that there might be additional undetected highly divergent Grs in either or both species. In addition, comparisons of AaGr and AgGr gene models allowed refinement of 15 AgGr gene models. The 8 newly recognized and 15 refined AgGr gene models have been communicated to VectorBase for inclusion in the next release of the A. gambiae genome annotation, which will also be deposited in Ensembl.
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Orthologous Grs in the 2 mosquitoes
Phylogenetic analysis comparing the AaGrs with the entire set of AgGrs reveals several instances of highly conserved apparent orthologs, most prominently the AaGr1-3 and AgGr22-24 subfamily lineages which share 72–89% identity (Figure 1A). The orthologs of AaGr1:AgGr22 and AaGr3:AgGr24 in D. melanogaster are DmGr21a and DmGr63a, respectively (Hill et al. 2002), with an Aedes:Drosophila amino acid identity of 70% and 60%, respectively. DmGr21a and DmGr63a have recently been shown to function as a heterodimeric receptor for carbon dioxide (Jones et al. 2007; Kwon et al. 2007), which is a major chemical cue used by mosquitoes to find their vertebrate hosts.
Another prominent conserved subfamily is related to the trehalose receptor of D. melanogaster, DmGr5a (Chyb et al. 2003) (Figure 1A). There are 7 relatives of DmGr5a in the D. melanogaster genome, all of which are therefore candidate sugar receptors (Robertson et al. 2003). Anopheles gambiae also has 8 genes in this lineage (AgGr14-21) (Hill et al. 2002), and A. aegypti has apparently simple orthologs for each of these (AaGr4-13) ranging in amino acid identity from 39% to 65%, except that AaGr8 and AaGr13 are pseudogenes most closely related to AgGr19 (31% and 34% identity, respectively), and AaGr12 is a truncated pseudogenic copy of AaGr11 not shown in Figure 1A. If these are indeed all sugar receptors then it might be of biological interest that A. aegypti apparently does not have the sensory capacity conferred upon A. gambiae by AgGr19. These 8 mosquito candidate sugar receptors are not simple orthologs of the 8 in Drosophila (Hill et al. 2002; Robertson and Wanner 2006; Kent LB, Robertson HM, unpublished data). Consequently, drawing connections to the ligand specificity of these proteins in Drosophila, once established, will not necessarily be straightforward.
Other apparent simple 1:1 orthologs include AaGr73:AgGr53 (71% amino acid identity), AaGr34:AgGr25 (70%), AaGr14:AgGr2 (54%), AaGr64:AgGr46 (47%), AaGr48P/75P:AgGr35 (43%), AaGr31:AgGr38 (37%), AaGr77:AgGr42 (32%), and AaGr41:AgGr48 (28%) in Figure 1B/C. In addition, AaGr30P is a pseudogene relative of AgGr47 truncated by apparent loss of the last 2 exons (37% identity in shared N-terminal region). Of the ortholog pairs listed, only AaGr34:AgGr25 and AaGr14:AgGr2 show conservation to D. melanogaster, specifically with DmGr43a and DmGr66a, respectively (Hill et al. 2002; Robertson and Wanner 2006), having an Aedes:Drosophila amino acid identity of 40% and 37%, respectively. Interestingly, DmGr66a was recently demonstrated to be required for caffeine perception in Drosophila (Moon et al. 2006), suggesting a similar role for its orthologs in mosquitoes. Despite their conservation in mosquitoes at 71% amino acid identity, second only to the carbon dioxide subfamily gene conservation, Drosophila species do not have an ortholog of the AaGr73/AgGr53 gene pair, and this conserved gene is absent from the silk moth, flour beetle, and honey bee genomes. These 8 "orthologous" pairs are likely to have similar ligands and roles in the 2 mosquitoes, and sometimes even Drosophila, and AaGr73:AgGr53 is of particular interest as an apparently mosquito-specific receptor. Another noteworthy orthologous relationship is the many-to-one association of the alternatively spliced locus AaGr19:AgGr33, which also has an alternatively spliced ortholog in Drosophila, DmGr28b (Hill et al. 2002), and even distant relatives in honey bee (Robertson and Wanner 2006). Other orthologous relationships of alternatively spliced loci, including AaGr20:AgGr37, AaGr33:AgGr44, AaGr39/40:AgGr9, and AaGr67/68:AgGr56a-f, do not show conservation in Drosophila and are detailed further below.
Mosquito-specific Grs
There are many highly divergent AaGrs and AgGrs without obvious orthologs in the other species, and it is likely that for many of them the ortholog in the other species has been lost. The remaining genes in each species represent candidates for mediating species-specific behaviors. Expanded Gr subfamily lineages in Aedes and Anopheles are scattered throughout the phylogenetic tree and highlighted in Figure 1B,C. The largest of these in Aedes is a subfamily of 8 genes and in Anopheles, a subfamily of 5 genes (Figure 1C). These too likely mediate species-specific behaviors.
Several of the alternatively spliced genes also exhibit species-specific expansions (Figures 1 and 2), and for 3 of them (AaGr20:AgGr37, AaGr39/40:AgGr9, and AaGr67/68:AgGr56), the tree suggests that most or all the alternative splicing originated independently in each species lineage. The second locus is particularly complicated because it is duplicated in Aedes (AaGr39/40), and different alternatively spliced isoforms have become pseudogenes in the 2 loci (Figure 2), leaving a set of 6 intact Grs in Aedes compared with 14 in Anopheles. These alternatively spliced loci encode Gr isoforms that differ in their N-termini but share a common C-terminus (Figure 2), an architecture also shared by several DmOrs and DmGrs (Clyne et al. 1999, 2000; Robertson et al. 2003) and one AaOr locus (Bohbot et al. 2007). The extent of shared sequence varies from gene to gene, but the differing N-terminal regions are always at least one half of the protein, are usually encoded by a single long alternatively spliced 5' exon, and are presumed to confer distinct ligand-binding properties on the resultant protein isoforms. Because the isoforms from each locus cluster with each other by species in the Gr tree (Figure 1), it appears that they originated independently in each mosquito lineage over the past 
150 million years (Myr) since the split of the culicine (Aedes) and anopheline (Anopheles) subfamily lineages (Krzywinski et al. 2006). However, this conclusion could be biased by the necessary inclusion of the shared C-terminal sequences for each isoform in the alignments on which the Gr tree is based, which would tend to cause the isoforms from each species to cluster within the species rather than with potentially orthologous isoforms from the other species. Therefore, we do not include bootstrap support values for these branches in Figure 1.
To address this issue further, we undertook focused phylogenetic analysis of each set of alternatively spliced loci and after alignment of their encoded isoforms removed the shared C-terminal regions so that the phylogenetic analyses were based only on their differing N-terminal regions encoded by the long alternatively spliced exons. Ideally, these analyses should be rooted with the next closest Gr protein sequence in the tree declared as the outgroup. However, in the absence of the conserved C-terminal regions, these relatives are usually so highly divergent as to be ineffective as outgroups; hence, these subtrees are rooted at the midpoint, which still reveals how they evolved. These subtrees in Supplementary Figure 1 largely support the inference that at each locus most or all the alternatively spliced forms originated independently in each mosquito lineage. The only clear-cut exception is AaGr20:AgGr37 where 2 alternatively spliced N-terminal exons appear to have been present before these mosquito lineages split (purple branches in Supplementary Figure 1B), and they both were subsequently duplicated independently and repeatedly in each mosquito.
The argument for independent origin of splice forms is further supported by comparison of the AaGr39/40 and AgGr9 loci. In the former case, there is an upstream solo copy of a paralogous gene (AaGr37/38, see Figure 2A), whereas in the latter, there are 2 downstream paralogous solo loci (AgGr10 and 11, see Hill et al. 2002). It nevertheless seems more than coincidental that all 3 of these orthologous loci should have undergone such extensive duplication of their alternatively spliced 5' exons independently in each lineage, and we propose that in fact they were alternatively spliced in their common mosquito ancestor, but that each lineage lost all but one (or 2 for AaGr20 and AgGr37) of these alternative 5' exons independently and ended up with nonorthologous 5' exons which were subsequently duplicated again independently in each mosquito lineage. Such extensive loss of Gr-coding sequences is to be expected. Indeed as noted above, orthologs of many of the Grs in each species have clearly been lost from the other (Figure 1) and similar losses and duplications are seen even in close comparisons of 1- to 50-Myr-old Drosophila species (Guo and Kim 2007; Nozawa and Nei 2007; Robertson HM, unpublished data).
Finally, there are several AaGr pairs that seem to represent extremely recent duplication events within the Aedes lineage; however, an alternative explanation is that these are alternative assemblies of haplotypes or alleles. For example, AaGr27 is almost identical to AaGr28 but is encoded by a 7-kb contig that is >90% identical along >90% length to the equivalent region of the AaGr28 contig. Similarly, AaGr21/22 are very similar to AaGr58/59, and the 7-kb contig that encodes them is 95% identical to the start of the contig that encodes AaGr58/59. We excluded 4 other examples of gene fragments in short contigs entirely represented elsewhere within much larger contigs from the gene set as representing alternative haplotypes or alleles. The almost identical AaGr15/51 duplication appears to be real, however, in an inverted orientation in the same contig. Additionally, the AaGr48P/75P duplication copies are in tandem in neighboring contigs and 90% amino acid identical but with different stop codons inactivating them. Finally, the AaGr36/52P pair are clearly separate loci with equal read depths of about 8x each, yet AaGr52P has an insertion of a copy of the 6.3-kb Loner_Ele2 retrotransposon documented in the TEfam database (see Nene et al. 2007, supplementary information).
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Discussion |
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Using the AaegL1 assembly of the A. aegypti genome sequence, we report our annotation of this mosquito's 114 identifiable Grs and their phylogenetic relationships with previously annotated members of this gene family in other insects, primarily the African malaria vector mosquito, A. gambiae. We have chosen to number these AaGrs sequentially and independently of the AgGr numbering system, despite the fact that several of them are apparent orthologs of AgGrs (Figure 1 and Table 1). Despite the similarities between the genes, we believe that coordinating the naming schemes would introduce a level of ambiguity with respect to the inferred significance of the names. Confident orthology cannot be garnered at the exploratory level, and by prematurely extending putative orthology to a permanent name, we would in some cases lose clarity in defining where the orthology actually ends.
Our analysis reveals remarkable similarities between these distantly related mosquitoes. If we were to tally our final AaGr number in the same manner as done with the AaOrs (Bohbot et al. 2007), 6 pseudogene fragments and 4 excluded alternative haplotypes would bring the total to 124. However, when we take into account pseudogenes and possible alternate haplotype loci and focus on functional genes, there are 91 AaGrs. This is essentially the same as the AgGrs, now totaling 90. Remarkably, there are no apparent pseudogenes in Anopheles, although a few unannotated fragments may have indicated that some lost genes had already evolved beyond the scope of our identification. This pattern of additional pseudogenes in Aedes reflects the accumulation of transposons in more than 50% of the genome sequence (Nene et al. 2007).
One of our more noteworthy findings is that AaGr1:AgGr22 and AaGr3:AgGr24 are among the most highly conserved putative orthologs found in the mosquito Gr repertoire. These 2 proteins form a heterodimeric receptor for carbon dioxide perception (Jones et al. 2007; Kwon et al. 2007). The high amino acid conservation in these 2 orthologous pairs, extending to the D. melanogaster orthologs at 70% and 68% identity, respectively, presumably reflects stringent requirements for their structures both for heterodimerization and binding of carbon dioxide or a derivative, such as HCO3. These 2 proteins are also encoded by the silk moth, Bombyx mori, and flour beetle, Tribolium castaneum, genomes at 69/67% and 61/60% identity, respectively (unpublished results); however, the entire gene lineage is absent from the more basal honey bee A. mellifera genome (Robertson and Wanner 2006). Drosophila melanogaster has lost the third even more highly conserved gene in this subfamily, AaGr2:AgGr23 (89% identity, and 74% to B. mori and 63% to T. castaneum), and this gene loss occurred over 50 Myr ago because it is not present in the other 12 Drosophila species whose genome sequences are now available (see Robertson 2005). Ammonia is another gas detected by mosquitoes in their perception of mammalian hosts (Geier et al. 1999; Meijerink et al. 2001; Smallegange et al. 2005; Qiu et al. 2006); however, Drosophila flies can perceive this gas (Yao et al. 2005), so AaGr2:AgGr23 seem unlikely to be involved in its perception. Nevertheless the extraordinary conservation of AaGr2:AgGr23 argues for an important and widespread role in insect chemoperception.
At the other extreme, these 2 mosquito lineages exhibit several lineage-specific Grs, and in particular, almost all the alternatively spliced isoforms appear to have evolved subsequent to the culicine–anopheline split 150 Myr ago. Each of the Grs we have reported must ultimately be involved in the mosquito's ability to perceive chemicals in its environment, thus impacting the animal's behavior and ecology. Some of these behaviors are particularly relevant to vector and disease control. The obvious next step will be to define the ligands of these receptors (e.g., Chyb et al. 2003; Wanner et al. 2007). With this information in hand, we can begin to work on developing novel methods of disrupting mosquito ability to perceive its human host, improving our ability to control disease transmission.
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Supplementary material |
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Supplementary material can be found at http://www.chemse.oxfordjournals.org.
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Funding |
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National Institutes of Health (AI56081).
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Acknowledgements |
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We thank Catherine Hill, David Hogenkamp, and Larry Zwiebel for assistance in early stages of this project.
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References |
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Accepted 31 August 2007
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