Detecting chemical cues can be a matter of life or death for insects, and many employ three families of receptor proteins to detect a broad range of odors. Members of one of these receptor families, the olfactory receptors, form a complex with another protein, the olfactory coreceptor that is essential for both positioning and stabilizing the receptor, as well as the actual function.

Crustaceans share a common ancestor with insects, and since they do not have olfactory receptors it has been proposed that these receptors evolved when prehistoric insects moved from the sea to live on land. According to this idea, olfactory receptors evolved because these ancestors needed to be able to detect odor molecules floating in the air rather than dissolved in water.

Previous research on insect olfactory receptors has focused on insects with wings. Missbach et al. have now used a wide range of techniques to investigate how evolutionarily older wingless insect groups detect scents. As all investigated groups evolved from a common ancestor at different times these experiments allow tracking of the historical development of olfactory receptors.

In the wingless species that is more closely related to the flying insects there was evidence of the presence of multiple coreceptors but not the olfactory receptors themselves. In the most basal insects no evidence for any part of the olfactory receptor-based system was found. This indicates that the main olfactory receptors evolved independently of the coreceptor long after the migration of insects from water to land. Missbach et al. suggest that olfactory receptors instead developed far later, around the time when vascular plants spread and insects developed the ability to fly.

All living organisms, including bacteria, protozoans, fungi, plants, and animals, detect chemicals in their environment. The sensitivity and chemical range of animal olfactory systems is remarkable, enabling animals to detect and discriminate between thousands of different odor molecules. Although there is a striking evolutionary convergence towards a conserved organization of signaling pathways in vertebrate and invertebrate olfactory systems (Hildebrand and Shepherd, 1997), the involved receptor gene families evolved independently. The molecular identity of olfactory receptors was first unraveled in vertebrates (Buck and Axel, 1991). In mammals, as many as 1000 heterotrimeric GTP-binding protein (or G protein)-coupled receptors are considered to be employed in olfactory discrimination (Buck and Axel, 1991). A similar number of chemoreceptors, with about 1300 receptor genes and 400 pseudogenes, have been hypothesized for Caenorhabditis elegans (Robertson and Thomas, 2006).

All data on insect olfactory receptors are based on studies investigating the neopteran insects (overview of insect order relationship is given in Figure 1). The identity of receptors involved in olfaction in the evolutionarily more ancient apterygote insects (Archaeognatha, Zygentoma) and paleopteran insects (Odonata and Ephemeroptera) is thus completely unknown. In neopteran insects (Polyneoptera, Paraneoptera, and Holometabola) most volatile stimuli are recognized by members of the olfactory receptor family (ORs). ORs are multitransmembrane domain proteins unrelated to nematode or vertebrate olfactory receptors (Mombaerts, 1999; Robertson, 2001; Hill et al., 2002), displaying a distinct membrane topology (Benton et al., 2006; Lundin et al., 2007). The number of functional OR genes varies from 10 in the human body louse Pediculus humanus humanus (Kirkness et al., 2010) to about 60 in Drosophila melanogaster (Clyne et al., 1999; Gao and Chess, 1999; Vosshall et al., 1999) and up to 350 OR genes in ants (Zhou et al., 2012). ORs have been suggested to be distantly related to the gustatory receptors of arthropods, with some proteins containing a signature motif in the carboxyl terminus (Scott et al., 2001).

Figure 1

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Insect olfactory receptors function as heteromultimers composed of at least one ligand-specific OR and the coreceptor Orco (Vosshall et al., 1999; Elmore et al., 2003; Krieger et al., 2003; Larsson et al., 2004; Sato et al., 2008; Wicher et al., 2008). Interestingly, while Orco (Vosshall and Hansson, 2011) is highly conserved among insects, the sequences of other olfactory receptor genes exhibit very little sequence similarity even within the same insect order (Krieger et al., 2003), complicating their identification. So far, Orco homologues have been identified in Lepidoptera, Diptera, Coleoptera, Hymenoptera, Hemiptera (Krieger et al., 2003; Pitts et al., 2004; Smadja et al., 2009), and Orthoptera (Yang et al., 2012). Neither Orco nor ORs are present in the genome of the crustacean Daphnia pulex, indicating that ORs are insect specific. However, GRs were found in Crustacea, just as in insects (Peñalva-Arana et al., 2009).

A second receptor family, the variant ionotropic glutamate receptors (IRs), is also involved in insect chemosensation (Benton et al., 2009). IRs act in combinations of up to three subunits; individual odor-specific receptors and one or two of the broadly expressed coreceptors IR25a, IR8a, and IR76b (Abuin et al., 2011). IRs are present in olfactory tissues across the Protostomia (Croset et al., 2010), for example two conserved members of this group were described in the Daphnia genome (Croset et al., 2010) and the coreceptor IR25a homologue is expressed in many, if not all mature OSNs of the American lobster Homarus americanus (Hollins et al., 2003) and the spiny lobster Panulirus argus (Tadesse et al., 2011). Since crustaceans are the closest relatives of insects (Friedrich and Tautz, 1995; Boore et al., 1998; Regier et al., 2010), IRs are most likely the ancient type of insect olfactory receptor.

But when and why did insect ORs evolve? Hexapods derived from an aquatic crustacean ancestor, probably in the Early Ordovician, approximately 483 mya (Rota-Stabelli et al., 2013). The transition from sea to land meant that molecules needed to be detected in gas phase instead of aquatic solution. Therefore, the olfactory system of a hexapod ancestor had to adapt to the terrestrial conditions and detection of volatile, air-borne chemicals. One proposed hypothesis has been that Orco and ORs of the insect type are an adaptation to this terrestrial lifestyle (Robertson et al., 2003; Krång et al., 2012). To reconstruct an evolutionary scenario for insect ORs, we investigated species belonging to different ancient insect orders, including Archaeognatha (jumping bristletails) and Zygentoma (silverfishes and firebrats), and a neopteran insect belonging to the Phasmatodea (leaf and stick insects) as so far not analyzed control group using morphological, electrophysiological and molecular techniques.

Our first step was to analyze the evolutionary ancestry of the insect olfactory system by assessing its complexity in each of three non-holometabolan insects.

To correlate OSN responses with type of sensillum (with pores and grooves) identified in SEM studies of the antennae, we investigated the morphological and physiological characteristics of olfactory sensilla and their olfactory sensory neurons.

Morphology and physiology

On the antennae of L. y-signata the only putative olfactory sensilla were porous olfactory basiconic sensilla (Figure 2B–E). These sensilla were arranged in a pattern that is highly stereotypical between antennal modules composed of 5–12 annuli, with annuli typically containing zero-to-four Sensilla basiconica (Missbach et al., 2011). Responses to all tested chemical classes of odors, including acids, alcohols, aldehydes, esters, and ketones, were recorded from OSNs housed in these sensilla using the single sensillum recoding measurements (SSR) (Figure 3, uppermost heat map). Based on the response profile, spontaneous activity, and colocalization inside the same sensillum, we identified 12 OSN types, present in five functional basiconic sensillum types. Out of the 12 OSN types, only seven responded to odors tested; two exclusively to acids, while five responded with a similar activity rate to acids or amines and to other odors. OSNs belonging to this second class were broadly tuned and exhibited relatively low spiking activity. In general, OSN classes displayed a low baseline activity with about 1 to 7 spikes/s, with Lys-ab2A that had a spontaneous activity of more than 25 spikes/s as the only exception. Only rarely was an increase in spiking rate of more than 60 spikes per second recorded, even for the best identified ligands (Figure 3—source data 1). No responses were obtained for ammonia or pyridine. Coeloconic-like sensilla, s-shaped trichoid sensilla, and chaetic sensilla did not display any morphological features indicating olfactory function and did also not respond to any odor tested (Missbach et al., 2011; data not shown). In conclusion, 7 OSN types that were all housed in basiconic sensilla responded to a wide spectrum of odor molecules.

Figure 2

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Figure 3

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Figure 3—source data 1

Excel file of mean responses and baseline firing rate of the different OSN classes of L.y-signata, T. domestica, P.siccifolium, and D. melanogaster.

https://doi.org/10.7554/eLife.02115.006

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The morphology of the zygentoman antenna and its sensilla was similar to that of L. y-signata, with the presence of grooved sensilla as the only exception (Figure 2G; Adel, 1984; Berg and Schmidt, 1997). Five different functional types of olfactory sensilla were present (Figure 3: three porous, two grooved s. basiconica, the latter are indicated by blue caption). In contrast to L. y-signata, a nascent functional and spatial separation of the detection of amines and acids, and ketones and alcohols appeared in T. domestica. The former primarily elicited responses in OSNs of grooved sensilla, while less polar ones were mainly detected by porous sensilla. However, most of the OSNs in porous sensilla exhibited broad tuning and responded to at least one of the tested acids or amines as well.

We then turned to a neopteran insect. Unlike the other analyzed species, the leaf insect P. siccifolium displayed a strong sexual antennal dimorphism, with males having very long antennae covered with trichoid sensilla (Figure 2L), and the females very short antennae without trichoid sensilla (Figure 2K). In comparison to the wingless insects, the response repertoire of the leaf insect was much more diverse, with a total of 23 different functional sensillum types as identified by SSR recordings (Figure 3). No responses were obtained from trichoid sensilla, but since they were only present on the male antennae they could be involved in detection of an unknown volatile pheromone. In all cases, reported detection of volatile pheromones in insects is dependent on very specific ORs. Taken together these data suggest that leaf insects have a much broader response repertoire with a higher number of different OSN types than the more basal species we analyzed; apparently the number of olfactory receptors has increased. It also seems likely that at least the leaf insect makes use of ORs in odorant detection.

No ORs or Orco were found in the transcriptome of L. y-signata

The transcriptome data sets were manually screened for genes encoding proteins putatively involved in insect olfaction, including ORs, Orco, GRs, and IRs (number of identified contigs are given in Table 2).

Table 2

Organism	Orco	ORs	GRs	IRs
Lepismachilis y-signata	–	–	7 (5 above 400 bp)	17 (16 above 400 bp)
Thermobia domestica	6 (1 above 400 bp)	–	9 (3 above 400 bp)	19 (9 above 400 bp)
Phyllium siccifolium	1 (1 above 400 bp)	30 (16 above 400 bp)	6 (2 above 400 bp)	32 (19 above 400 bp)

Neither OR- nor Orco-coding transcripts were identified in the transcriptomes of L. y-signata using BLAST and HMM domain profile searches as described in the ‘Material and methods’ section. Custom HMMR-profiles directed against conserved regions of Orco proteins also failed to identify any Orco-related sequences in the bristletail transcriptome. We discovered five GR candidates. MSA analysis of these together with ORs and GRs of various insect species and the Daphnia GRs always confirmed the position of the L. y-signata GR candidates within the GR and not the OR family (Figure 4A, Figure 4—source data 1, Figure 4—source data 2, Figure 4—source data 3, Figure 4—source data 4, Figure 4—source data 5). Since expression levels of gustatory receptors are very low even in gustatory tissue (Clyne et al., 2000; Scott et al., 2001), we argue that ORs or at least Orco should be represented in the large, sensory tissue-specific transcriptome data set of L. y-signata if they are indeed part of the olfactory system in the species.

Figure 4

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Figure 4—source data 1

Amino acid sequences of putative olfactory and gustatory receptors of L. y-signata, T. domestica, and P. siccifolium.

https://doi.org/10.7554/eLife.02115.010

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Figure 4—source data 2

Nucleotide sequences of putative olfactory and gustatory receptors of L. y-signata, T. domestica, and P. siccifolium.

https://doi.org/10.7554/eLife.02115.011

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Figure 4—source data 3

MAFFT-alignment of OR and GR candidates of L. y-signata, T. domestica, P. siccifolium and D. melanogaster (Clyne et al., 1999, Gao and Chess, 1999, Vosshall et al., 1999) and Apis mellifera (Robertson and Wanner, 2006) GR and OR proteins, as well as Daphnia pulex GRs done.

https://doi.org/10.7554/eLife.02115.012

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Figure 4—source data 4

FastTree file resulting from the MSA of Figure 4—source data 3 (can be opened with FigTree).

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Figure 4—source data 5

Tree file resulting from the MSA of Figure 4—source data 3 containing node support values (can be opened e.g., with Adobe Illustrator).

https://doi.org/10.7554/eLife.02115.014

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The three Orco-paralogues of T. domestica

In contrast to L. y-signata, three different Orco-related sequences were identified in the transcriptome of T. domestica. All candidates were cloned as full-length coding sequences using RACE-PCR. The three sequences displayed different similarities to the Orco sequence of D. melanogaster, one sequence shared 45.8%, one 35.1%, and the third 24.4% sequence similarity at the amino acid level. Orco was the protein most similar to all three Orco candidate sequences (Figures 4B and 5), although some of the key amino acids of the coreceptor are substituted at least in TdomOrco3 (Wicher et al., 2008; Sargsyan et al., 2011; Nakagawa et al., 2012; Kumar et al., 2013; highlighted in alignment Figure 5). Apart from the Orco variants, no OR-related sequences were identified, but 9 contigs for GR candidates were found that were assigned to seven GRs, including three candidates close to full length or full length and four additional fragments (Table 2 and Figure 4A).

Figure 5

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Orco expression in T. domestica

Considering that for all other insects analyzed so far one Orco is the norm, the appearance of three Orco candidates in T. domestica is highly unusual. We thus assessed the expression of the three candidates in different tissues using RT-PCR. For all three Orco types expression was limited to the antenna (Figure 6). To further assess the expression, we used in situ hybridization employing an antisense probe of one of the coreceptors. This led to staining of single cells below one or two basiconic sensilla of an antennal subsegment (Figure 7), suggesting that TdomOrco1 might indeed be expressed in OSNs. However, only one neuron per sensillum was stained. No signals were obtained when using a sense probe for TdomOrco1 (Figure 7—figure supplement 1).

Figure 6

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Figure 6—source data 1

Primers and their properties used in this study.

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Figure 7 with 1 supplement

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Figure 7—figure supplement 1

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Only IRs in L-y-signata

As none of the experiments gave a hint for the existence of any OR or Orco-related sequence in the bristletail transcriptome, we focused on the second olfactory receptor family of insects, the IRs. Although we could not identify any OR sequences in the transcriptome, a high number of putative glutamate receptor coding contigs was identified (Table 2). However, only five candidate iGluRs and 14 candidate IRs appeared to be real unigenes, possessing at least two of the three transmembrane domains. Some candidate sequences were extended in 3’-direction using RACE-PCR with antennal cDNA as template, allowing verification of unigene status and antennal expression. In MSA and phylogenetic analysis, the identified IRs grouped with DmelIRs (Croset et al., 2010). Among the identified putative LsigIRs were orthologues of the D. melanogaster coreceptors IR25a and IR8a, as well as one receptor similar to IR76b (Figure 8A, Figure 8—source data 1, Figure 8—source data 2, Figure 8—source data 3, Figure 8—source data 4, Figure 8—source data 5). As in other IRs (Benton et al., 2009) one or several key amino acids in the predicted glutamate binding domains were absent in the non-coreceptor IR candidates and LsigIR76b (Figure 8B). 7 out of 14 LsigIRs group close to a cluster of D. pulex IRs and the antennal IRs IR21a and IR68a of D. melanogaster, with no clear relationship to one or the other. None of the Lepismachilis IR candidates grouped with the ‘divergent’ Drosophila IRs.

Figure 8

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Figure 8—source data 1

Amino acid sequences of putative variant ionotropic glutamate receptors of L. y-signata, T. domestica, and P. siccifolium.

https://doi.org/10.7554/eLife.02115.021

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Figure 8—source data 2

Nucleotide sequences of putative variant ionotropic glutamate receptors of L. y-signata, T. domestica, and P. siccifolium.

https://doi.org/10.7554/eLife.02115.022

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Figure 8—source data 3

MAFFT amino acid alignment of iGluR and IR candidates of L. y-signata, T. domestica, P. siccifolium, D. melanogaster, and D. pulex (D. melanogaster and D. pulex sequences were sequences taken from Croset et al., 2010).

https://doi.org/10.7554/eLife.02115.023

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Figure 8—source data 4

FastTree file resulting from the MSA of Figure 4—source data 3 (can be opened with FigTree).

https://doi.org/10.7554/eLife.02115.024

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Figure 8—source data 5

Tree file resulting from the MSA of Figure 8—source data 3 containing node support values.

https://doi.org/10.7554/eLife.02115.025

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We then performed fluorescent in situ hybridization with RNA probes directed against the IR coreceptor candidates (Figure 9). Antisense probes of IR25a and IR8a led to labeling of one to three OSNs underneath basiconic sensilla (Figure 9—figure supplement 1). In control experiments with sense probes, or without any probe, no staining was obtained (Figure 9—figure supplement 2). The pattern of expression of IR coreceptors in OSNs of L. y-signata indicates that most OSNs are covered by this gene family.

Figure 9 with 2 supplements

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Figure 9—figure supplement 2

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Figure 9—figure supplement 1

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All experiments thus indicate that the olfactory system of this species employs other receptors like IRs or GRs, with no ORs or Orco present.

Insects provide us with an excellent opportunity to study groups of animals that have retained ancestral characteristics and understand how the specific building blocks in olfaction have evolved in both insects and other animals. Consequently, we selected insects at crucial positions of the phylogenetic tree with a functional olfactory system adapted to terrestrial conditions and detection of volatile chemicals. This species collection provides an excellent model to study the early evolution of the insect olfactory system.

To address which receptors are involved in odor detection in these insects and in basal insects in general, we applied several different approaches. Based on our transcriptome data sets, we suggest a stepwise evolution of the Orco/OR complex with Orco having evolved in the lineage of Dicondylia (Zygentoma + Pterygota) and the functional complex of Orco and ORs emerging within the pterygote insects (this study, Clyne et al., 1999; Gao and Chess, 1999; Smadja et al., 2009; Vosshall et al., 1999; Robertson and Wanner, 2006; Kirkness et al., 2010). Although it is impossible to completely rule out the presence of ORs, none of our extensive experiments led to the identification of either ORs or Orco in the bristletail L. y-signata. The well-established conservation of the Orco coding gene through evolution suggests that it is highly unlikely that we missed it. We did, however, identify a number of IRs, including the IR coreceptors IR25a, IR8a, and IR76b in the L. y-signata antennal transcriptome. FISH allowed us to visualize expression of the IR co-receptors in a large number of OSNs associated with basiconic sensilla. Based on these results we propose that the olfactory system of L. y-signata is not based on ORs.

In insects, different sensillum types house OSNs typically responding to different sets of odors. In D. melanogaster IRs are the functional receptor type of OSNs in double-walled coeloconic sensilla, and ORs are predominantly expressed in OSNs housed in single-walled basiconic and trichoid sensilla (Hallem et al., 2004; Silbering et al., 2011). It follows that this organization cannot exist with just one sensillum type present, as is the case in Archaeognatha (Berg and Schmidt, 1997; Missbach et al., 2011) and older hexapod taxa as the Collembola (Altner and Prillinger, 1980). The oldest insect taxon where double-walled sensilla were investigated is Zygentoma, which have both single-walled basiconic sensilla with pores and double-walled sensilla with spoke channels (Berg and Schmidt, 1997). Coeloconic sensilla differ dramatically from the single-walled trichoid and basiconic types in both wall structure and in internal environment. The coeloconic structure has been thought to be a prerequisite for IR function (Benton et al., 2009; Guo et al., 2014). However, in the Archaeognatha we find that IRs are most likely located in OSNs of Sensilla basiconica. IRs might thus have evolved in a single-walled sensillum and did not find their modern, coeloconic environment until neopteran insects evolved.

In the bristletail L. y-signata, we found that many of the OSNs are very broadly tuned, responding to volatiles with several different functional groups at higher doses. However, broadly tuned receptors might not have high affinities. By counting and integrating molecules over longer times, OSNs could include even low-probability binding events in generating their response (Firestein, 2001). This might also mean that the system does not have a high temporal resolution, which seems to be a fair trade-off for a walking insect that lives in its substrate.

The response spectrum of Drosophila IRs is much narrower than the responses we find in the bristletail. If IRs are the only olfactory receptor type in basal insects they should exhibit a broader spectrum of possible ligands, including acids, aldehydes, alcohols, but also esters and ketones, as revealed in our physiological measurements. One additional observation in the bristletail is that many of those neurons have a broad overlap in their response spectra. One hypothesis to explain an IR-based olfactory system in L. y-signata would be very broad tuning of single receptors, another that the selectivity of OSNs could be regulated by combinations of different IRs.

In D. melanogaster, one conserved IR (IR64a) is expressed in different subpopulations of sensilla in the third chamber of the sacculus (Silbering et al., 2011). Corresponding OSNs are activated either by free protons or organic acids and many other odors, including esters, alcohols, and ketones (Ai et al., 2010). Expression of this IR together with IR8a is both necessary and sufficient for sensitivity towards organic acids and other odors, but probably requires a different, until now unknown cofactor to mediate the specific response of OSNs to inorganic acids and CO₂ (Ai et al., 2010).

Alternatively, GR candidates could account for part of the non-neopteran olfactory setup, especially since it has been shown that GRs can add to the olfactory repertoire (Tauxe et al., 2013). Putative contact chemosensory sensilla are highly abundant on the antennae of L. y-signata (Missbach et al., 2011) and T. domestica (Adel, 1984). Both detection of sugars/amino acids (shown for T. domestica: Hansen-Delkeskamp, 2001) and a proposed contact-pheromone (Fröhlich and Lu, 2013) likely involve GRs, indicating that involvement of the limited set of GRs beyond this scope is unlikely.

However, these data do not explain the presence of three different Orco variants in the firebrat. So far only one Orco orthologue has been identified in each studied insect species (e.g., Krieger et al., 2003; Pitts et al., 2004; Smadja et al., 2009; Yang et al., 2012). All T. domestica variants were found to be expressed in antennae, suggesting their involvement in chemosensation. TdomOrco3 even has an amino acid exchange of a functional important residue from asparagine to glutamic acid at position 466. This residue was demonstrated as critical for the ion channel function in D. melanogaster, where substitution of D466 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity, but substitution to glutamic acid leads to an increase in sensitivity of the heteromeric receptor complex (Kumar et al., 2013). Additionally, this residue is highly conserved across insects (Kumar et al., 2013) including two of the three T. domestica Orcos (this study).

While the antennal expression argues for a potential involvement in chemosensation, the existence of three Orco types remains mysterious. It will be part of future studies to investigate if the Orco candidates form heterodimers with other receptors like GRs or with each other to build functional receptors or if they fulfill a channel function in other processes than olfaction.

Altogether our data suggests that ORs evolved in insects after the emergence of Archaeognatha and Zygentoma, and therefore long after insects transitioned to a terrestrial lifestyle. At the time when flying insects occurred, the vegetation on earth was rapidly spreading and diversifying. ORs might not only increase the diversity of detected chemicals, but also allow the olfactory system to rapidly assess airborne odors. This is especially important for insects for which stimulus contact is very short and a fast response time is critical (Getahun et al., 2012). The oldest flying insect orders Odonta (dragonflies and damselflies) and Ephemeroptera (mayflies) were traditionally considered to be anosmic, lacking both a glomerular antennal lobe and mushroom body calyces (Strausfeld et al., 1998; Farris, 2005). Recent studies have shown that at least dragonflies have an aerial sense of smell (Rebora et al., 2012). However the small antennae and the low number of olfactory sensilla will make it even more challenging to identify putative ORs and Orco in antennal transcriptomes. ORs were definitely present in the last common ancestor of ‘hemi’- and holometabolan insects at least 318–300 million years ago, with Orco present in both groups (this study, Krieger et al., 2003; Pitts et al., 2004; Smadja et al., 2009; Yang et al. 2012). The increasing dispersion of vascular plants together with the development of wings and a secondary wing articulation opened new and wider ranges of habitats and ecological niches for insects and the receptors to find them.

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Author details

1. Christine Missbach

   Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   CM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   cmissbach@ice.mpg.de

   Competing interests

   The authors declare that no competing interests exist.

2. Hany KM Dweck

   Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   HKMD, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Heiko Vogel

   Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   HV, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Andreas Vilcinskas

   Institute of Phytopathology and Applied Zoology, Justus-Liebig-Universität Gießen, Gießen, Germany

   Contribution

   AV, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Marcus C Stensmyr

   Department of Biology, Lund University, Lund, Sweden

   Contribution

   MCS, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Bill S Hansson

   Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   BSH, Conception and design, Drafting or revising the article

   Contributed equally with

   Ewald Grosse-Wilde

   Competing interests

   The authors declare that no competing interests exist.

7. Ewald Grosse-Wilde

   Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany

   Contribution

   EG-W, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Bill S Hansson

   For correspondence

   Grosse-Wilde@ice.mpg.de

   Competing interests

   The authors declare that no competing interests exist.

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