Ionotropic glutamate receptors (iGluRs) are a highly conserved family of ligand-gated ion channels present in animals, plants, and bacteria, which are best characterized for their roles in synaptic communication in vertebrate nervous systems. A variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was recently identified as a new class of olfactory receptors in the fruit fly, Drosophila melanogaster, hinting at a broader function of this ion channel family in detection of environmental, as well as intercellular, chemical signals. Here, we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis. In marked contrast to the insect-specific Odorant Receptor family, we show that IRs are expressed in olfactory organs across Protostomia—a major branch of the animal kingdom that encompasses arthropods, nematodes, and molluscs—indicating that they represent an ancestral protostome chemosensory receptor family. Two subfamilies of IRs are distinguished: conserved “antennal IRs, ” which likely define the first olfactory receptor family of insects, and species-specific “divergent IRs, ” which are expressed in peripheral and internal gustatory neurons, implicating this family in taste and food assessment. Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences. Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family. Together, these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies. Furthermore, this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors.
Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels present in both eukaryotes and prokaryotes. By regulating cation flow across the plasma membrane in response to binding of extracellular glutamate and related ligands, iGluRs represent an important signalling mechanism by which cells modify their internal physiology in response to external chemical signals. iGluRs have originated by combination of protein domains originally encoded by distinct genes (Figure 1A) [1]–[2]. An extracellular amino-terminal domain (ATD) is involved in assembly of iGluR subunits into heteromeric complexes [3]. This precedes the ligand-binding domain (LBD), whose two half-domains (S1 and S2) form a “Venus flytrap” structure that closes around glutamate and related agonists [4]. Separating S1 and S2 in the primary structure is the ion channel pore, formed by two transmembrane segments and a re-entrant pore loop [5]. S2 is followed by a third transmembrane domain of unknown function and a cytosolic carboxy-terminal tail. Animal iGluRs have been best characterised for their essential roles in synaptic transmission as receptors for the excitatory neurotransmitter glutamate [1], [6]. Three pharmacologically and molecularly distinct subfamilies exist, named after their main agonist: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). AMPA receptors mediate the vast majority of fast excitatory synaptic transmission in the vertebrate brain, while Kainate receptors have a subtler modulatory role in this process. NMDA receptors require two agonists for activation, glutamate and glycine, and function in synaptic and neuronal plasticity. Representatives of these iGluR subfamilies have been identified across vertebrates [7], as well as invertebrates, such as the fruit fly Drosophila melanogaster, the nematode worm Caenorhabditis elegans and the sea slug Aplysia californica [8]–[10]. While most iGluRs have exquisitely tuned synaptic functions, identification of iGluR-related genes in prokaryotic and plant genomes provided initial indication of more diverse roles for this class of ion channel. A bacterial glutamate receptor, GluR0, was first characterised in the cyanobacterium, Synechocystis PCC6803 [11]. GluR0 conducts ions in response to binding of glutamate and other amino acids in vitro, suggesting a potential function in extracellular amino acid sensing in vivo. The flowering plant Arabidopsis thaliana has 20 iGluR-related genes, named GLRs [12]–[13]. Genetic analysis of one receptor, GLR3. 3, has implicated it in mediating external amino acid-stimulated calcium increases in roots [14]. We recently described a family of iGluR-related proteins in D. melanogaster, named the Ionotropic Receptors (IRs) [15]. Several lines of evidence demonstrated that the IRs define a new family of olfactory receptors. First, the IR LBDs are highly divergent and lack one or more residues that directly contact the glutamate ligand in iGluRs. Second, several IRs are expressed in sensory neurons in the principal D. melanogaster olfactory organ, the antenna, that do not express members of the other D. melanogaster chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs) [16]. Third, IR proteins localise to the ciliated endings of these sensory neurons and not to synapses [15]. Finally, mis-expression of an IR in an ectopic neuron is sufficient to confer novel odour-evoked neuronal responses, providing direct genetic evidence for a role in odour sensing [15]. The identification of the IRs as a novel family of olfactory receptors in D. melanogaster provides a potential link between the well-characterised signalling activity of iGluRs in glutamate neurotransmitter-evoked neuronal depolarisation and a potentially more ancient function of this family in environmental chemosensation. In this work, we have combined comparative genomics, molecular evolutionary analysis and expression studies to examine the evolution of the IRs. Four principal issues are addressed: first, when did olfactory IRs first appear? Are they a recent acquisition as environmental chemosensors in D. melanogaster, or do they have earlier origins in insect or deeper animal lineages? Second, what is the most recent common ancestor of IR genes? Do they derive from AMPA, Kainate or NMDA receptors, or do they represent a distinct subfamily that evolved from the ancestral animal iGluR? Third, what mechanisms underlie the expansion and diversification of this multigene family? Finally, do IRs function only as olfactory receptors or are they also involved in other sensory modalities? Through answers to these questions, we sought insights into IR evolution in the context of the origins of iGluRs, the appearance and evolution of other chemosensory receptor repertoires and the changing selective pressures during animal diversification and exploitation of new ecological niches.
iGluRs and IRs are characterised by the presence of a conserved ligand-gated ion channel domain (the combined Pfam domains PF10613 and PF00060 [17]) (Figure 1A). All iGluRs additionally contain an ATD (Pfam domain PF01094), which is discernible, but more divergent, in only two D. melanogaster IRs, IR8a and IR25a. Most IRs have only relatively short N-terminal regions preceding the LBD S1 domain (Figure 1A). To identify novel iGluR/IR-related genes, we therefore constructed a Hidden Markov Model (HMM) from an alignment of the conserved iGluR/IR C-terminal region, which is specific to this protein family. In combination with exhaustive BLAST searches, we used this HMM to screen raw genomic sequences and available annotated protein databases of 32 diverse eukaryotic species and 971 prokaryotic genomes (see Materials and Methods and Table S2 in Supporting Information). These screens identified all previously described eukaryotic iGluRs and all D. melanogaster IRs, as well as 23 prokaryotic iGluRs. Novel sequences were manually reannotated and classified by sequence similarity, phylogenetic analysis and domain structure as either non-NMDA (i. e. AMPA and Kainate) or NMDA subfamily iGluRs, or IRs (Figure 1B, Table S3, and Datasets S1 and S2). Like D. melanogaster IRs, newly annotated IRs have divergent LBDs that lack some or all known glutamate-interacting residues, supporting their distinct classification from iGluRs. iGluRs are widespread in eukaryotes, present in all analysed Metazoa (except the sponge, Amphimedon queenslandica [18]) and Plantae, but absent in unicellular eukaryotes (Figure 1B, Table S3, and Datasets S1 and S2). Analysis of iGluR subfamilies on the eukaryotic phylogeny suggests that NMDA receptors may have appeared after non-NMDA receptors, as we identified them in Eumetazoa but not in the placozoan Trichoplax adhaerens. Further support for this conclusion will require additional genome sequences. One member of the Eumetazoa, the sea urchin Strongylocentrotus purpuratus, may have secondarily lost NMDA receptors. Different species contain distinct numbers of each iGluR subfamily: vertebrates, for example, have more NMDA receptor subunits than invertebrates. Notably, IRs were identified throughout Protostomia, encompassing both Ecdysozoa (e. g. nematodes and arthropods) and Lophotrochozoa (e. g. molluscs and annelids) (Figure 1B, Table S3, and Datasets S1 and S2). There is substantial variation in the size of the IR repertoire, from three in C. elegans to eighty-five in the crustacean Daphnia pulex. Amongst insects, Diptera (i. e. flies and mosquitoes) generally had a larger number of IRs than other species. We did not identify IRs in Deuterostomia, Cnidaria or Placozoa. To explore the evolutionary origin of the IRs, we examined phylogenetic relationships of the identified protostome IRs. Reciprocal best-hit analysis using D. melanogaster sequences as queries revealed that a subset of this species' IRs was conserved in several distant lineages, allowing us to define putative orthologous groups. These include one group containing representatives of all protostome species (IR25a), one represented by all arthropods (IR93a), nine by most or all insects, and three by dipteran insects (Figure 2A and 2B). For most orthologous groups, a single gene for each species was identified. In a few cases, for example the IR75 group, certain species were represented by several closely related in-paralogues, some of which appeared to be pseudogenes (Figure 2A and 2B, Table S3, and Datasets S1 and S2). Consistent with its conservation in Protostomia, IR25a is the IR with the most similar primary sequence to iGluRs, suggesting that it is the IR gene most similar to the ancestral IR. Analysis of the phylogenetic relationship of IR25a and eukaryotic iGluRs locates it clearly together with the animal iGluR family, in the non-NMDA receptor clade (Figure 2C). To substantiate this conclusion, we asked whether the IR25a gene structure resembles more closely that of NMDA or non-NMDA receptors. Intron positions and numbers are extremely variable across IR25a orthologues, with multiple cases of intron loss, gain and putative intron sliding events by a few nucleotides (Figure 2D). Nevertheless, we identified eight intron positions that are conserved between at least subsets of IR25a orthologues and D. melanogaster non-NMDA receptor genes, some of which may represent intron positions present in a common ancestral gene. By contrast, only a single intron that was conserved in position (but not in phase) was identified between DmelIR25a (but not other IR25a orthologues) and DmelNMDAR1 (Figure 2D). A phylogram of intron positions in IR25a, non-NMDA and NMDA sequences reveals greater similarity of IR25a intron positions to those of non-NMDA receptors than NMDA receptors (Figure 2D). Together, these observations support a model in which IR25a evolved from a bilaterian non-NMDA receptor gene. The conserved D. melanogaster IRs encompass the entire subset of its IR repertoire that is expressed in the antenna [15]. Moreover, evidence for antennal expression of the three additional genes, DmelIR41a, DmelIR60a and DmelIR68a, has been obtained by reverse transcription (RT) -PCR analysis, although we have not yet been able to corroborate this by RNA in situ hybridisation (data not shown). These combined phylogenetic and expression properties led us to designate this subfamily of receptors the “antennal IRs”. We examined whether antennal expression of this subfamily of IRs is conserved outside D. melanogaster by performing a series of RT-PCR experiments on the honey bee, Apis mellifera, for all six putative antennal IR orthologues: IR8a, IR25a, IR68a, IR75u, IR76b and IR93a (see Materials and Methods for the nomenclature of newly-identified IRs). As in D. melanogaster, we could reproducibly amplify all of these bee genes from antennal RNA preparations but not in control brain RNA, except for AmelIR68a and AmelIR75u, which are also detected in the brain (Figure 2E). Thus, antennal expression of this subgroup of IRs is conserved across the 350 million years separating dipteran and hymenopteran insect orders [19], and therefore potentially in all insects. To investigate whether IRs are likely to have an olfactory function beyond insects, we examined expression of the IR repertoire from a representative of a distantly related protostome lineage, Aplysia molluscs, whose last common ancestor with D. melanogaster probably existed 550–850 million years ago [20]. We first used RT-PCR to analyse the expression of the ten Aplysia IR genes in a variety of sensory, nervous and reproductive tissues (Figure 3A). Notably, the Aplysia IR25a orthologue is predominantly expressed in the olfactory organs, the rhinophore and oral tentacle [21]. Two other Aplysia-specific IR genes, IR214 and IR217, are expressed in the rhinophore and oral tentacle, respectively, and not detected in other tissues, except for the large hermaphroditic duct (IR214) and skin (IR217). Five additional IRs are also expressed in the oral tentacle, but displayed broader tissue expression in skin and the central nervous system; both of these tissues are likely to contain other types of chemosensory cells [22]–[23]. Expression of two IR genes, IR209 and IR213, was not detected in this analysis (data not shown). To further characterise Aplysia IR25a, we analysed its spatial expression in the mature A. dactylomela rhinophore by RNA in situ hybridisation. An antisense probe for AdacIR25a labels a small number of cells in rhinophore cryosections. Their size and morphology is typical of neurons, although we lack an unambiguous neuronal marker to confirm this identification (Figure 3B–3D). These cells are found either singly or in small clusters adjacent or close to the sensory epithelial surface in the rhinophore groove, in a similar position to cells expressing other types of chemosensory receptors [21]. A control sense riboprobe showed no specific staining (Figure 3E). Together, these results are consistent with at least some of these molluscan IRs having a chemosensory function. The expression of putative IR25a orthologues has previously been reported in two other Protostomia. An IR25a-related gene from the American lobster, Homarus americanus, named OET-07, is specifically expressed in mature olfactory sensory neurons [24]–[25]. In C. elegans, a promoter reporter of the IR25a orthologue, GLR-7, revealed expression in a number of pharyngeal neurons [9], which might have a role in food sensing [26]. While both crustacean and nematode genes were classified in these studies as iGluRs, there is no evidence that they act as canonical glutamate receptors, and we suggest that they fulfil instead a chemosensory function. The antennal IR subfamily accounts for only a small fraction of the IR repertoire in most analysed insects and only 1–2 genes in other Protostomia. The remaining majority of IR sequences are - amongst the genomes currently available - largely species-specific, with low amino acid sequence identity (as little as 8. 5%) with other IR genes in either the same or different species. We refer to this group of genes here as the “divergent IRs”. Dipteran insects have particularly large expansions of divergent IRs (Figure 1B). Phylogenetic analysis revealed no obvious orthologous relationships of these genes either between D. melanogaster and mosquitoes or amongst the three mosquito species (Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae) (Figure 4). Instead, this subfamily of IRs displays a number of species-specific clades, perhaps reflective of the distinct ecological niches of these insects. By contrast to antennal IRs, divergent IR expression has not been detected in D. melanogaster olfactory organs [15], leading us to test whether these genes are expressed in other types of chemosensory tissue. As endogenous transcripts of non-olfactory chemosensory genes, such as GRs, are difficult to detect [27]–[28], we employed a sensitive transgenic approach to investigate divergent IR expression. We transformed flies with constructs containing putative promoter regions for these genes upstream of the yeast transcription factor GAL4 and used these “driver” transgenes to induce expression of a GAL4-responsive UAS-mCD8: GFP fluorescent reporter [29]. We sampled divergent IRs from several distinct clades, including IR7a, IR11a, IR52b, IR56a and IR100a (Figure 4). All IR promoter-GAL4 constructs were inserted in the same genomic location using the phiC31 integrase system [30], eliminating transgene-specific position effects on expression resulting from their site of integration. Expression of three of these divergent IR reporters was observed in highly selective populations of neurons in distinct gustatory organs (Figure 5A). In the adult, IR7a is expressed in at least eleven neurons in the labellum, a sense organ involved in peripheral taste detection (Figure 5B) [31]. Two reporters labelled neurons in internal sense organs in the pharynx: IR11a is expressed in one neuron in the ventral cibarial sense organ and IR100a is expressed in two neurons in the dorsal cibarial sense organ (Figure 5C and 5D). These internal pharyngeal neurons are thought to play a role in assessment of ingested food prior to entry into the main digestive system [16]. Expression was not detected in any other neurons or other cell types in the adult head (data not shown), although we cannot exclude expression in other regions of the body. IR52b and IR56a reporters were not detected in these experiments. We also examined expression of these reporters at an earlier stage in the D. melanogaster life cycle, third instar larvae, which display robust gustatory responses [16]. The same three IR reporters were exclusively detected in unique bilaterally-symmetric larval gustatory organs: IR7a was expressed in two neurons in the terminal organ at the periphery, IR11a in a single neuron in the ventral pharyngeal sense organ and IR100a in two neurons in the posterior pharyngeal sense organ (Figure 5E–5H). Notably, all of these neurons in both adult and larval tissues (except for a single IR7a-expressing cell in the terminal organ) co-express IR25a, as revealed by a specific antibody against this receptor (Figure 5) [15]. IR25a is also expressed in several other cells in each of the gustatory organs, which may express other divergent IRs not examined here. Together these results support a role for divergent IRs as taste receptors in distinct taste organs and stages of the D. melanogaster life cycle. To obtain more detailed insights into the processes underlying the expansion and diversification of IR repertoires, we investigated their evolution over a shorter timescale by comparative analysis of D. melanogaster with 11 additional sequenced drosophilid species [32]–[33]. The last common ancestor of these drosophilids is estimated to have existed 40 million years ago [34], by contrast to the ∼250 million years since the last common ancestor of D. melanogaster and the mosquito A. gambiae [35]. Certain species may have diverged much more recently, such as D. simulans and D. sechellia, whose last common ancestor may have existed only 250,000 years ago [36]. We used D. melanogaster sequences as queries in exhaustive BLAST searches of the drosophilid genomes. Retrieved sequences were manually reannotated to unify gene structure predictions across species and, in some cases, genes were partially resequenced to close sequence gaps or verify them as pseudogenes (see Materials and Methods, Table S3, and Datasets S1 and S2). Although predicted full-length gene sequences could be annotated for most genes, 28 sequences remain incomplete - but assumed in further analysis to be functional - because of a lack of sequence data or difficulty in precise annotation of exons in divergent regions of these genes. Of the 926 drosophilid sequences identified (including those of D. melanogaster), 49 genes were classified as pseudogenes because they consisted of only short gene fragments or contained frameshift mutations and/or premature stop codons. We clustered all genes into orthologous groups by examining their sequence similarity, phylogenetic relationships and, in the case of IR47a, IR47b, IR47c, IR56e and IR60f, their micro-syntenic relationships (Table S1 and Figure 6). For drosophilid species that are most distant from D. melanogaster, definition of precise orthologous relationships was not always possible, particularly for groups of closely related IR genes (e. g. IR52a–f, IR60b–f) (Table S1). Orthologous groups were named after their D. melanogaster representatives or a logical variant in groups where no D. melanogaster gene was identified (see Materials and Methods). This analysis identified 14 iGluR and 58–69 IR genes in each of the twelve drosophilid species (Figure 6A and Table S1). iGluRs are highly conserved, with a mean amino acid sequence identity of 89±1% s. e. m. , and a single representative for each species in every orthologous group. Antennal IRs are also well conserved (mean sequence identity = 76±2%) and amongst these genes we identified only a single pseudogenisation event, in D. sechellia IR75a, and a single gene duplication event, of D. mojavensis IR75d. By contrast, divergent IRs, though also largely classifiable into monophyletic groups, display a more dynamic pattern of evolution (mean sequence identity = 61±2%), with multiple cases of gene loss, pseudogenisation or duplication (Figure 6 and Table S1). We reconciled the gene phylogeny with the drosophilid species phylogeny to estimate the number of IR gene gain and loss events. While this analysis is necessarily constrained by our ability to accurately define gene orthology, we estimated across the entire phylogeny there to be sixteen gene gain events (gene birth rate, B = 0. 0006/gene/million years) and 76 gene loss events (gene death rate, D = 0. 0030/gene/million years) (Figure 7A, see Materials and Methods). Most (46/76) gene losses are pseudogenisation events, which indicates that many of these events must have occurred relatively recently, as drosophilid species appear to eliminate pseudogenes rapidly from their genomes [37]–[38]. Notably, 13 gene loss events – 12 of which reflect the presence of just one or a small number of premature stop codons or frameshift mutations – occur on the branch leading to the specialist D. sechellia. Consequently, the gene loss rate on this branch is remarkably high compared with its generalist sister species D. simulans (Figure 7A and 7B). We studied the selective forces acting on drosophilid iGluRs and IRs by calculating the ratio of nonsynonymous to synonymous nucleotide substitution rates (dN/dS, ω1) in these genes from all 12 species. All tested iGluR, antennal IR and divergent IR genes are evolving under strong purifying selection (ω1<<1) (Figure 7C, left and Table S4), suggesting that they all encode functional receptors. iGluRs have the lowest estimated dN/dS ratio (median ω1 = 0. 060), consistent with a conserved role in synaptic communication. Antennal IRs have an intermediate dN/dS ratio (median ω1 = 0. 107) and divergent IRs the highest (median ω1 = 0. 149), suggesting that divergent IRs have evolved under weaker purifying selection and/or contain more sites that have been shaped by positive selection. Amongst the IRs, IR25a has the lowest dN/dS ratio (ω1 = 0. 028), consistent with its high sequence conservation in and beyond drosophilids (Figure 2). To compare these properties with those of other insect chemosensory receptor families (ORs and GRs) [39], we also calculated dN/dS ratios for IR genes from only the five sequenced species of the melanogaster subgroup (D. melanogaster, D. sechellia, D. simulans, D. erecta and D. yakuba). For this subset of sequences, the relative differences between median dN/dS ratios (ω2) for the iGluR and IR gene subfamilies observed with all twelve species was reproduced (Figure 7C, right). The GR gene family has previously been noted to evolve under weaker purifying selection than ORs [39]. Notably, we found that the median dN/dS ratios for antennal IRs (ω2 = 0. 120) is statistically indistinguishable from that of ORs (ω2 = 0. 137) (p>0. 4, Wilcoxon rank-sum test), and that the median dN/dS ratio of divergent IRs (ω2 = 0. 176) is statistically indistinguishable from that of GRs (ω2 = 0. 217) (p>0. 5, Wilcoxon rank-sum test). Thus, the selective forces acting on the IR receptor gene subfamilies parallel those on the ORs and GRs and appear to correlate with their putative distinct chemosensory functions in olfaction and gustation (Figure 7C, right). The reason for this difference is unknown, but might reflect reduced evolutionary constraints on co-expressed and partially redundant taste receptor genes or selection for higher diversity in taste receptor sequences to recognise more variable non-volatile chemosensory ligands in the environment. Most residues of IR proteins can be expected to have evolved under purifying selection to maintain conserved structural and signalling properties, which may mask detection of positive selection (ω>1) at a small number of sites that contribute to their functional diversity. To obtain evidence for site-specific selection we applied site class models M7 and M8 in PAML to analyse 49 sets of orthologous IR genes of the six species of the melanogaster group. This test did not identify any sites significantly under positive selection after Bonferroni correction (Table S4), a result consistent with orthologous IR genes having the same function across drosophilids. Site-specific positive selection may be more easily detectable in relatively recent IR gene duplicates potentially undergoing functional divergence. We therefore analysed the sole duplication of an antennal IR, IR75d. 1 and IR75d. 2 in D. mojavensis. Assuming an estimated divergence time of 35 My between D. virilis and D. mojavensis [40], and based on analysis of dS of IR75d genes in these species (see Materials and Methods), we estimated this duplication to have occurred relatively recently, approximately 2. 6–5. 1 My ago. Using a branch-site test we identified two sites (p<0. 05) that have evolved under positive selective pressure, where DmojIR75d. 1 and DmojIR75d. 2 appear to contain the ancestral and derived residues, respectively: DmojIR75d. 2-S670 maps to the third transmembrane domain and DmojIR75d. 2-Q365 maps to the putative ligand binding domain. Functional characterisation of these variant receptors will be required to determine their significance. From potentially one ancestral IR, what genetic processes underlay the generation of large repertoires of IR genes? We initially sought evidence for these mechanisms through analysis of the D. melanogaster IR family. Several monophyletic groups of IR genes exist in clusters in the genome suggesting an important role of gene duplication by non-allelic homologous recombination. For example, eight divergent IRs of the IR94 orthologous groups are located in three close, but separate, tandem arrays on chromosome arm 3R (Figure 8A). Other genes in the same clade are also found scattered on other chromosome arms (X, 2R, 3L) (Figure 6 and Figure 8A), indicating that interchromosomal translocation has also occurred frequently, most likely both during and after formation of the tandem arrays. Similar patterns are observed in the orthologous/paralogous sequences of these IRs in other drosophilid species (Figure 8A), as well as for other IR clades (data not shown). These features are also observed in IR repertoires in other insects, although incomplete genome assembly prevented a more precise analysis. For example, in Aedes aegypti the 23 IR7 clade members are found in arrays of 1,1, 2,5, 7 and 7 genes on 6 different supercontigs (data not shown). We also noticed an unusual pattern in D. melanogaster IR gene structures, in which antennal IRs (as well as iGluRs) contain many (4–15) introns, while the vast majority of divergent IRs are single exon genes (Figure 8B). Drastic intron loss in multigene families is a hallmark of retroposition, where reverse-transcription of spliced mRNAs from parental, intron-containing genes and reinsertion of the resulting cDNA at a new genomic location may give rise to a functional, intronless retrogene [41]. The few introns that are present in these IRs in D. melanogaster have a highly biased distribution towards the 5′ end of the gene (19/25 introns in the first 50% of IR gene sequences) (Figure 8C), which is characteristic of recombination of partially reverse-transcribed cDNAs (a process which initiates at the 3′ end) with parental genes [42]. Sequence divergence of IRs prevented us from identifying parental gene-retrogene relationships. Nevertheless, these observations together suggest that divergent IRs arose by at least one, and possibly several, retroposition events of ancestral antennal IRs. Once “born”, single exon IRs could presumably readily further duplicate by non-allelic homologous recombination.
Our comprehensive survey and phylogenetic analysis of iGluR/IR-like genes permits development of a model for their evolution (Figure 9). The shared, unusual “S1-ion channel-S2” domain organisation of prokaryotic GluR0 and eukaryotic iGluRs is suggestive of a common ancestor of this family by fusion of genes encoding the separate domains that were present in very early life forms (Figure 9) [11]. However, we have found prokaryotic glutamate receptors in only a very small number of bacterial species. Thus, if an iGluR evolved in the common ancestor of prokaryotes and eukaryotes, it must have subsequently been lost in a large number of prokaryotic lineages. It is possible, therefore, that iGluRs only originated in eukaryotes and were acquired by certain prokaryotic species by horizontal gene transfer [43]. If the latter hypothesis is true, the presence of closely related iGluRs in both plants and animals implies their early evolution within eukaryotes, potentially in the last common eukaryotic ancestor [44]. However, the absence of iGluRs in sponges and all examined unicellular eukaryotes raises the alternative possibility that animal and plant receptors evolved independently, or were acquired by horizontal transmission, perhaps from prokaryotic sources. Whatever the precise origin of iGluRs in animals, their subsequent divergence into AMPA, Kainate and NMDA subfamilies also occurred early, although variation in the size of these subfamilies suggests continuous adaptation of the synaptic communication mechanisms they serve to nervous systems of vastly different complexities. Several outstanding issues regarding IR evolution can now be addressed. First, we have shown that the IRs were very likely to have been present in the last common ancestor of Protostomia, an estimated 550–850 million years ago [20]. IR25a represents the probable oldest member of this repertoire and conservation of chemosensory organ expression of IR25a orthologues in molluscs, nematodes, crustaceans and insects strongly suggests that this receptor may have fulfilled a chemosensing function in the protostome ancestor. Second, the apparent absence of IRs in Deuterostomia suggests the parsimonious model that IRs evolved from an animal iGluR ancestor rather than representing a family of chemosensing receptors that was present in a common ancestor of Animalia and lost in non-protostomes. Our phylogenetic and gene structure analysis suggests that IR25a may have derived from a non-NMDA receptor gene. The transition from an iGluR to an IR may not have involved drastic functional modifications: both receptor types localise to specialised distal membrane domains of neuronal dendrites (post-synaptic membranes and cilia, respectively) and, in response to binding of extracellular ligands, depolarise these domains by permitting transmembrane ion conduction which in turn induces action potentials [45]. Thus, it is conceivable that IRs arose simply by a change in expression of an iGluR from an interneuron (where it detected amino acid signals from a pre-synaptic partner) to a sensory neuron (where it could now detect chemical signals from the external environment). Third, our analyses of IR repertoires across both divergent and relatively closely related species provide insights into the mechanistic basis for the expansion and functional diversification of the IR repertoire. Gene duplication by non-allelic homologous recombination is a widespread mechanism for growth of most multigene families in chemosensory systems [46], and this is also true for the IRs. Our implication of retroposition as a second mechanism in the evolution of IR repertoires offers two advantages for functional diversification. First, by arising from random re-insertion of reverse transcribed copies of parental genes, retrogenes normally lack endogenous promoter sequences, and can therefore potentially acquire novel expression patterns from genomic sequences flanking their insertion site that are distinct from their parental ancestor [41]. Indeed, in D. melanogaster, retrogene or retrogene-derived IRs - the divergent IRs - are apparently no longer expressed in antennal neurons like their ancestors, but instead in gustatory (and perhaps other) tissues. Second, release from the evolutionary constraints of the preservation of splicing signals near exon boundaries may have contributed to the more rapid divergence of the protein sequences of these intronless IRs [47]. Analysis of IR repertoires across the well-defined drosophilid phylogeny provides clear evidence for a birth-and-death model of evolution, in which, following gene duplication, individual family members progressively diverge in sequence and, in some cases, are lost by pseudogenisation and/or deletion [48]–[49]. Differential rates of these processes will ultimately shape the precise IR repertoire of an individual species (discussed below). Our molecular evolutionary analysis has distinguished two subfamilies in the IR repertoire: conserved, antennal IRs and the species-specific, divergent IRs. Their distinct evolutionary properties may correspond to fundamental functional differences, as we provide here the first evidence, to our knowledge, for expression of divergent IR subfamily members in subsets of neurons in both peripheral and internal gustatory organs at both adult and larval stages of D. melanogaster. The selective and non-overlapping expression patterns observed in the small sample of IR genes examined indicate that a large fraction of the divergent IR repertoire may be expressed in gustatory neurons. It is also possible that some of these IRs may be expressed in non-chemosensory tissues. Although subsets of GR genes have been implicated in the detection of sweet or bitter compounds in peripheral taste bristles in D. melanogaster [31], a comprehensive understanding of the physiological breadth and molecular logic of taste detection is lacking. Our results introduce further complexity into the molecular mechanisms of taste detection and demand comprehensive and comparative expression and functional analysis of divergent IRs and GRs in this sensory system. Although many gustatory-expressed divergent IRs in D. melanogaster are recently derived in drosophilids, the ancestral chemosensory function of IRs is likely to be not in the detection of airborne volatiles but rather water-soluble, non-volatile compounds, as the last common ancestor of Protostomia was probably aquatic. Indeed, the strikingly similar expression of IR genes in internal pharyngeal neurons in D. melanogaster and C. elegans suggests a conserved role for these receptors in sensing chemical signals from ingested food. In this light, the derivation of IRs from receptors detecting amino acid-related neurotransmitters invites the attractive hypothesis that ligands for these gustatory IRs (as well as species-specific IRs in other protostomes) are also amino acids. Almost nothing is known about sensory responses to this class of chemical signals in D. melanogaster, despite their vital importance for normal insect physiology and metabolism [50], but amino acids are chemosensory stimulants in other insects, lobsters and molluscs [51]–[53]. Our evolutionary and expression studies have highlighted IR25a as an atypical member of the repertoire, displaying deep conservation and broad expression in many olfactory and gustatory neurons. While we cannot exclude the possibility that IR25a recognises a specific chemical ligand, co-expression of this receptor with other cell-type specific IRs favours a model in which this acts as a co-receptor, analogous both to the heteromeric assembly of iGluR subunits into functional complexes [1], as well as to the pairing of ligand-specific ORs with the common OR83b co-receptor [54]–[55]. An insect- and antennal-specific homologue of IR25a, IR8a, may play a similar role specifically for olfactory IRs. In addition to IR25a and IR8a, many other D. melanogaster antennal IRs are highly conserved in insects, both in sequence and expression pattern. These properties contrast starkly with the insect OR repertoires, which probably evolved only in terrestrial insects [56], and which contain only one member displaying orthology across multiple orders, the atypical OR83b co-receptor [57]. ORs are an expanded lineage of the ancestral GR repertoire whose evolutionary origins are unknown [56]. Homologues of GR genes exist in D. pulex and C. elegans [56], [58], but in the latter species these receptors may not be involved in chemosensation [59]–[60]. These observations suggest that, in insects, the IRs represent the first olfactory receptor family, whose members were fixed functionally early in their evolution to detect olfactory stimuli that are important for all species of this animal class. Consistent with this, the antenna of the mayfly Rhithrogena semicolorata – an insect belonging to the Paleoptera and not the Neoptera that encompasses all species described here – bears coeloconic sensilla (potentially housing IR-expressing neurons) but not trichoid or basiconic sensilla (which house OR-expressing neurons in all other insects examined) [61]. Available data on ligands for IR sensory neurons - and the role of specific IRs within these neurons - are limited, but include stimuli such as carboxylic acids, water and ammonia, which are known to be physiologically and behaviourally important in many insect species [62]. ORs, by contrast, may be primarily dedicated to detection of species-specific odour cues. In this light, the IRs are attractive molecular targets for novel, broad-spectrum chemical regulators of insect odour-driven behaviours, with applications in the control of disease vectors, such as mosquitoes, and agricultural pests. Given the general conservation of the antennal IRs, what is the significance of the more recently evolved, species-specific variation in this family of chemosensory receptors? It is particularly informative to consider this question in the evolutionarily closely related drosophilid species. These display prominent differences in their global geographical distribution and chemosensory-driven behaviours [63]–[64], and include both generalists, which feed and breed on a wide range of substrates, and specialists, which have highly restricted ecological niches. The chemical ecology is best-understood for D. sechellia, a species endemic to the Seychelles that utilises the acid-rich fruit of Morinda citrifolia as its sole food source and oviposition site, a remarkable specialisation as this fruit is repulsive and toxic for other drosophilids [64]–[65]. Genetic hybrids between D. sechellia and D. simulans indicate that host specialisiation is due to loss-of-function mutations, rather than gain of new chemosensory perception abilities [65]. The accelerated rate of IR gene loss in D. sechellia compared to its sibling D. simulans (and other drosophilids) bears the hallmark of genetic adaptation of this chemosensory repertoire to the restricted host fruit. Notably, one of the D. sechellia pseudogenes is IR75a, an antennal IR expressed in a neuron responsive to several acids [62]. Thus, DsecIR75a represents an interesting gene whose mutation may be directly linked to host specialisation of this species. Future study of this receptor, and other species-specific IRs, may offer novel models to link genetic changes with phenotypic adaptation during animal evolution. Finally, our results may shed light into the outstanding question of the evolutionary origin of animal olfactory systems. Common neuroanatomical features have long been appreciated in animal olfactory circuitry, notably glomeruli, which represent sites of synaptic connection of OSNs of identical molecular and physiological specificity with second order neurons [66]. Whether these represent homologous or analogous structures across phyla is unclear. Revelations of fundamental distinctions in the structure, function and regulation of mammalian and insect ORs support a theory of convergent evolution of the neuronal circuits in which these receptors act [67]–[68]. Our demonstration that most, if not all, insect olfactory systems comprise two molecularly distinct receptor families, the ORs and IRs, indicates that the evolution of receptor repertoires can be uncoupled from a presumed common origin of the OR and IR neuronal circuits within the insect ancestor. Thus, during a significantly greater timescale across animal phyla, profound molecular differences between olfactory receptor genes do not necessarily imply distinct evolutionary origins of the neuronal circuitry in which they are expressed. Our discovery of IRs in mollusc olfactory organs reveals this to be an interesting potential “hybrid” organism in olfactory system evolution. The A. californica rhinophore and oral tentacle also express a large family of GPCR-family candidate chemosensory receptors, belonging to the same Rhodopsin superfamily as vertebrate ORs [21]. The co-existence of both insect-like and vertebrate-like olfactory receptors in this species provides evidence for the occurrence of an evolutionary transition between these distinct olfactory receptor families. Thus, while extant animal olfactory systems display an enormous diversity in their receptor repertoires, there may remain - perhaps unexpectedly - a sufficient genetic trace within receptor gene families themselves to open the possibility of a common evolutionary origin of this sensory system.
IR genes were named according to a unified nomenclature system based upon a foundation of the cytologically derived D. melanogaster IR gene names [15]. Receptor names are preceded by a four-letter species abbreviation consisting of an uppercase initial letter of the genus name and three lower case initial letters of the species name (e. g. Anopheles gambiae = Agam; Daphnia pulex = Dpul). Orthologues of D. melanogaster sequences are given the same name (e. g. CquiIR25a, AcalIR25a). If multiple copies of an orthologue of a D. melanogaster gene exist for a species (based on sequence, not function), they are given the same name followed by a point and a number (e. g. ApisIR75d. 1, ApisIR75d. 2). If several in-paralogues exist both in D. melanogaster and other species, these are all given the same number (indicating their grouping within a common clade), but different final letterings. For novel, species-specific IRs, we defined new names numbering from 101 upwards to avoid confusion with D. melanogaster gene names, which number up to IR100a. For species-specific IRs that form monophyletic clades and had high (>60%) amino acid identity, we gave these the same name with an additional number suffix after a point (e. g. AaegIR75e. 1, AaegIR75e. 2). We did not rename genes with previously published names (e. g. C. elegans GLR-7 and GLR-8 [9]). For vertebrate iGluRs, we used the NC-IUPHAR nomenclature [81]: each species name is followed by “Glu”, a letter representing the subtype of the receptor (K for Kainate, A for AMPA and N for NMDA), and a number, reflecting predicted orthology with mammalian iGluRs. We did not name (or rename) invertebrate iGluRs in this study, except for newly predicted gene sequences (Table S3), where logical variants of NC-IUPHAR nomenclature were assigned. Genomic DNA was extracted from the sequenced drosophilid genome strains (obtained from the Drosophila Species Stock Center, University of California-San Diego) using a standard DNA extraction protocol. PCR primers were designed to amplify ∼500 bp regions covering putative nonsense or missense mutations or spanning gaps in the genome sequence (oligonucleotide sequences are listed in Table S5). PCR amplifications were performed using Taq DNA Polymerase (PEQLAB Biotechnologie GmbH) in a MasterCycler Gradient Thermocycler (Eppendorf) with the following programme: 95°C for 3 min, 35 cycles of (95°C for 30 sec, 55°C for 1 min, 72°C for 1 min) and 72°C for 10 min, with minor modifications of annealing temperature and elongation times for different primer pairs and amplicon sizes. Products were gel purified (Machery-Nagel) and sequenced with BigDye Terminator v3. 1 according to the manufacturers' protocols. Insects: total RNA was extracted from hand-dissected tissues of wildtype A. mellifera and D. melanogaster (w1118 strain) using the RNeasy Mini Kit (Qiagen), and reverse-transcribed using oligo-dT primers and the SuperScript III First-Strand Synthesis System (Invitrogen). Genomic DNA was extracted using standard procedures. Primers were designed to amplify short regions overlapping an intron, if possible at the 3′ end of the coding sequence (Table S5). PCR product amplification and purification were performed as described above and sequenced to verify their identity. Multiple independent cDNA preparations were analysed for each primer pair. Primers were designed to amplify putative promoter regions from Oregon-R D. melanogaster genomic DNA with flanking restriction sites, extending from immediately upstream of the predicted start codon to the following 5′ extents: IR7a (2318 bp), IR11a (2099 bp), IR52b (446 bp), IR56a (2400 bp) and IR100a (512 bp) (Table S5). Gel purified PCR products were T: A cloned into pGEM-T Easy (Promega), end-sequenced, and sub-cloned into a pGAL4-attB vector, comprising the GAL4 ORF-hsp70-3′UTR in the pattB vector [30]. These constructs were integrated into the attP2 landing site [88], by standard transformation procedures (Genetic Services, Inc.). IR-GAL4 transgenic flies were double-balanced and crossed with flies bearing a UAS-mCD8: GFP transgene [89] to visualise driver expression.
Ionotropic glutamate receptors (iGluRs) are a family of cell surface proteins best known for their role in allowing neurons to communicate with each other in the brain. We recently discovered a variant class of iGluRs in the fruit fly (Drosophila melanogaster), named Ionotropic Receptors (IRs), which function as olfactory receptors in its “nose, ” prompting us to ask whether iGluR/IRs might have a more general function in detection of environmental chemicals. Here, we have identified families of IRs in olfactory and taste sensory organs throughout protostomes, one of the principal branches of animal life that includes snails, worms, crustaceans, and insects. Our findings suggest that this receptor family has an evolutionary ancient function in detecting odors and tastants in the external world. By comparing the repertoires of these chemosensory IRs among both closely- and distantly-related species, we have observed dynamic patterns of expansion and divergence of these receptor families in organisms occupying very different ecological niches. Notably, many of the receptors we have identified are in insects that are of significant harm to human health, such as the malaria mosquito. These proteins represent attractive targets for novel types of insect repellents to control the host-seeking behaviors of such pest species.
evolutionary biology
genetics and genomics/comparative genomics
neuroscience/sensory systems
Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction